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Developmental studies in Drosopnila melanogaster Holden, Jeanette Jeltje 1973

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DEVELOPMENTAL STUDIES IN DROSOPHILA MELANOGASTER by JEANETTE JELTJE HOLDEN B.Sc, University of British Columbia, 1969 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in Genetics We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March v, 1973 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C olumbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p urposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f Genetics The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date March 27, 1973 i ABSTRACT The study of development and d i f f e r e n t i a t i o n has been greatly f a c i l i t a t e d through the use of mutations which a f f e c t t h i s process at various d i s c r e t e stages. The gross morphological e f f e c t s produced by the mutations are i n d i c a t i v e of t h e i r r o l e i n development. The condi-t i o n a l expression of, f o r example, a l e t h a l phenotype, permits an exami-nation of the e f f e c t s of the mutation at d i f f e r e n t times during develop-ment—even a f t e r an i n i t i a l l e t h a l phase has been passed. In t h i s respect, a temperature-sensitive expression of developmental mutations i s a u s e f u l property which i s subject to r e l a t i v e l y simple yet extremely v a r i a b l e experimental procedures. Thus, dominant temperature-sensitive (DTS) l e t h a l s were induced i n Drosophila i n order that the nature, i . e . the molecular b a s i s , of dominant l e t h a l mutations, as w e l l as t h e i r genetic and developmental c h a r a c t e r i s t i c s , could be in v e s t i g a t e d . I t was hoped, a l s o , that l o c i w i t h i n heterochromatin might be detectable among such mutations. Ten DTS-lethal mutations on chromosome 3 which are l e t h a l when heterozygous at 29°C but survive at 22°C were recovered. Eight of the mutations were mapped, 7 were tested f o r complementation; these mutations probably define eight l o c i . Only DTS-2 survived i n homozygous condition at 22°C; homozygous DTS-2 females expressed a maternal e f f e c t on embryonic v i a b i l i t y . Two of the mutant-bearing chromosomes, DTS-1 and DTS-6, ex-h i b i t e d dominant phenotypes s i m i l a r to those associated with Minutes. i i Each of the seven mutants examined exhibited a characteristic pheno-type with respect to the time of death at 29°C and the temperature-sensitive period(s) during development. Only DTS-4 expressed a dominant lethal phenotype in t r l p l o i d females. In addition to the DTS-lethals, a special class of recessive tem-perature-sensitive mutations, which are lethal when homozygous or in combination with the multiply-inverted TM2 chromosome, were recovered, Only one combination of the four mutations i n this category failed to complement at 22°C whereas a l l trans-heterozygotes were lethal at 29°C. These were found to be closely linked to one another and to differ only slightly in their developmental characteristics. Together they provide an excellent model system for genetic fine structure analysis. Although such studies provide an indication of the morphological changes occurring during development, and the use of temperature-sensi-tive mutations gives some suggestion as to the time of action of the gene product, a determination of the precise biochemical defect i s not possible short of exceedingly extensive biochemical analyses. Alterna-tively, then, development may be viewed as a series of precisely regulated changes in the species of macromolecules present within the organism. Thus, differences in the amounts of certain RNAs- during the c e l l cycle or with specific developmental stages provide a measure of transcriptional changes. Since tRNAs represent primary gene products which can be readily analyzed qualitatively and quantitatively, their role(s) in development may be indicated by an absence or reduced amount of a specific tRNA species. The extensive array of mutations available in Drosophila i i i provides the material from which tRNA mutations might be detected. However, i t i s f i r s t e s s e n t i a l to e s t a b l i s h a pattern f o r the synthesis of s p e c i f i c isoaccepting tRNAs during the development of wild-type Drosophila so that a comparison can be made with those from various mutants. The reversed-phase chromatographic system (RPC-5) was used to compare the 20 aminoacyl-tRNAs from f i r s t i n s t a r , t h i r d i n s t a r , and adult f l i e s . While some of the aminoacyl-tRNAs remain e s s e n t i a l l y un-changed throughout these stages, others show marked qu a n t i t a t i v e changes. One group of tRNAs, i n c l u d i n g tRNA A s p, tRNA A s n, tRNA H l S, and tRNA T y r> showed s i m i l a r d i f f e r e n c e s i n the r e l a t i v e proportions of c e r t a i n chromatographically d i s t i n c t ( i . e . isoaccepting) forms. In a d d i t i o n , these four tRNAs show a f u r t h e r q u a n t i t a t i v e change i n f l i e s of the 2 su(s) v; bw genotype. Two-dimensional t h i n - l a y e r chromatograms of pancreatic RNase digests of the two major tRNA A s p chromatographic forms revealed almost i d e n t i c a l patterns whereas such chromatograms of p i p e r i -dine hydrolysates revealed a d i f f e r e n c e of one a l t e r e d nucleotide. I t i s suggested that these chromatographic forms are homogeneic ( i . e . they are transcribed from the same gene) but that they d i f f e r only i n the ex-2 tent of the modification of a s i n g l e nucleotide. The su(s) v; bw mu-tant would, then, appear to be d e f e c t i v e i n some aspect of t h i s modifica-t i o n process. P o s s i b l e models d e s c r i b i n g the a c t i v i t y of the modifica-t i o n enzyme are discussed. iv ACKNOWLEDGEMENTS The s i x years I have spent in David Suzuki's-Laboratory have, indeed, been a unique experience -- f o r which I am t r u l y g r e a t f u l . To David Suzuki and each of the past and present members of the "Fly Gang", my warmest "Thanks" --not only for your help and many discussions during the years, but also f or your continual f r i e n d s h i p and understanding. In p a r t i c u l a r , I wish to thank Misses Rachel Pratt, Diana Combes, and Barbara Banner for t h e i r help in the recovery of the mutations studied in Part I. Without t h e i r persistence, there would have been no t h e s i s . And to Bradley White, I extend a s p e c i a l "Thanks" --his patience and exc e l l e n t i n s t r u c t i o n have been responsible for Chapters 2 and 3 of Part II of t h i s thesis.- I thank him and Dr. Tener for t h e i r allowing me to p a r t i c i p a t e i n some of the experiments. And "Thank you" Carolyn Kiceniuk for growing ALL those f l i e s . And my Committee Members: Drs. J . Berger, D. Holm, J . M i l l e r , C. 0. Person, H. F. S t i c h , D. T. Suzuki, G. Tener, and R. A J . Warren. Thank you. And I would l i k e to express my deepest, most e s p e c i a l "Thanks" to my Family and Friends who.have been so patient, help-f u l , and understanding throughout my studies. Some of the Chapters are from p u b l i c a t i o n -- e i t h e r in press or submitted for p u b l i c a t i o n . In Part I, Chapter 3 i s Holden and Suzuki (Genetics, in press); Chapter 4 i s Holden (Developmental Biology, submitted f o r p u b l i c a t i o n ) ; Chapter 5 i s Holden (Mutation Research, in preparation). In Part I I , Chapter 2 i s White, Tener, Holden, and Suzuki (Developmental Biology, in press); Chapter 3 i s White, Tener, Holden, and Suzuki (Journal of Molecular Biology, in press). I wish also to acknowledge with g r e a t f u l "Thanks" f i n a n c i a l support from Gulf O i l Canada ( i n the form of a Gulf O i l Canada Graduate Fellowship for 1969-70, 1970-71) and the U n i v e r s i t y of B r i t i s h Columbia ( i n the form of an H. R. MacMillan Family Fellowship for 1971-72, and 1972-73). v i TABLE OF CONTENTS ABSTRACT i ACKNOWLEDGEMENTS iv LIST OF TABLES ix LIST OF FIGURES x i i - : GENERAL INTRODUCTION 1 PART I: TEMPERATURE-SENSITIVE MUTATIONS IN DROSOPHILA CHAPTER 1 THE PROBLEM OF HETEROCHROMATIN 5 CHAPTER 2 THE NATURE OF DOMINANT LETHALITY 19 CHAPTER 3 THE NATURE OF DOMINANT LETHALS I. Introduction 27 I I . Materials and Methods 29 I I I . Results 35 IV. Discussion 52 CHAPTER 4 THE DEVELOPMENTAL PROPERTIES OF DOMINANT TEMP-ERATURE-SENSITIVE LETHALS ON CHROMOSOME 3 OF DROSOPHILA I. Introduction 56 I I . Materials and Methods 59 I I I . Results 63 IV. Discussion 114 CHAPTER 5 CHARACTERIZATION OF A SPECIFIC CLASS OF TEMPERA-TURE-SENSITIVE LETHALS I. Introduction 122 v i i CHAPTER 5, continued I I . Materials and Methods 123 I I I . Results 128 IV. Discussion 161 PART I I : tRNAs DURING THE DEVELOPMENT OF DROSOPHILA INTRODUCTION TO PART II 167 CHAPTER 1 tRNA CHANGES DURING DEVELOPMENT 169 CHAPTER 2 ANALYSIS OF tRNAs DURING THE DEVELOPMENT OF DROSOPHILA I. Introduction 185 I I . Materials and Methods 187 I I I . Results 189 IV. Discussion 205 CHAPTER 3 ACTIVITY OF A tRNA MODIFYING ENZYME DURING THE DEVELOPMENT OF DROSOPHILA AND ITS RELATIONSHIP TO THE su(s) LOCUS I. Introduction 211 II. Materials and Methods 213 II I . Results 218 IV. Discussion 243 PART I I I : SUMMARY AND CONCLUSIONS 251 LITERATURE CITED 257 APPENDICES 1 A MODEL FOR CHROMOSOME STRUCTURE 276 2 WHAT CONSTITUTES TEMPERATURE-SENSITIVITY? 332 3 DESCRIPTION OF MUTATIONS AND REARRANGEMENTS USED IN THE PRECEDING STUDIES 350 v i i i APPENDICES, continued! 4 LOCALIZATION OF Ubx-ts-1 and Ubx-ts-4 WITH RESPECT TO THE RECESSIVE MARKER red 357 5 METHOD FOR THE LOCALIZATION OF A RECESSIVE LETHAL MUTATION ON THE TM2 EHROMOSOME IN THE REGION OF THE Ubx-ts-lethals 364 6 INTERACTIONS OF THE Ubx-ts-lethals WITH MUTATIONS AND DELETIONS IN THE VICINITY OF THE rv_ LOCUS 368 7 THE RECOVERY OF DTS-5 "HOMOZYGOTES" 370 8 CONDITIONS FOR THE AMINOACYLATION OF DROSOPHILA tRNAs " 373 LIST OF TABLES i x TABLE DTS 1 R e l a t i v e v i a b i l i t i e s of f l i e s bearing + or L(3)X chromo-somes at d i f f e r e n t temperatures 36 2 Cross over r e s u l t s from t e s t c r o s s e s of + and DTS-bearing females at 22° and 29°C 40 3 Ratios of DTS-a/DTS-b to t o t a l progeny from the cross DTS-a/TM3 x DTS-b at 22"C 41 4 Determination of the e f f e c t i v e l e t h a l phase of homozygotes f o r the DTS-lethals at 22°C 45 5 Progeny from the Cross XX, y ^ s j c w ^ e _ c or y_w fan°/FM6, y"^^ s c ^ dm B; 3A,+ x +/Y; 3*/TM3, Sb Ser 46 6 The E f f e c t i v e L e t h a l Phase of DTS-lethal mutations i n h e t e r o -zygoses at d i f f e r e n t temperatures 51 7 V i a b i l i t i e s of DTS-1, DTS-6, and Two Standard Minutes w i t h Ly_, J , D l ^ and w_ at 22 C 65 8 L o c a l i z a t i o n of the s i t e a f f e c t i n g maternal f e r t i l i t y on the DTS-2 chromosome: Method I 78 9 L o c a l i z a t i o n of the s i t e a f f e c t i n g maternal f e r t i l i t y on the DTA-2 chromosome: Method I I 82 10 Determination of the LPs and corresponding TSPs f o r DTS-3 heterozygotes 88 11 E c l o s i o n times and percentages of s u c c e s s f u l e c l o s i o n s of DTS-4/ TM3 heterozygotes exposed to 29 C f o r 24 hours during d i f f e r e n t developmental stages 95 12 K i n e t i c s of temperature-sensitive female s t e r i l i t y a s s o c i a t e d w i t h DTS-7/TM3 heterozygotes 107 13 L o c a l i z a t i o n of the s i t e ( s ) on the DTS-5 and DTS-7 chromosomes causing l e t h a l i t y of the DTS-5/DTS-7 transheterozygotes 109 14 C h a r a c t e r i z a t i o n of crossover chromosomes obtained from DTS-5 and DTS-7 females heterozygous f o r 1. t h sjt pj> p__ or 2. t h s t S_b e 111 15 Determination of the L e t h a l Phase of DTS-5/DTS-7 trans-hetero-heterozygotes at 22 C 113 16 R e l a t i v e V i a b i l i t i e s of the U b x - t s - l e t h a l s w i t h v a r i o u s t h i r d chromosomes at 29 C 124 X TABLE 17 Genetic localization of Ubx-ts mutations by crossing over 129 18 Determination of the Effective Lethal Phase of Ubx-ts-lethal hooozygotes at 22°C 133 19 Survival of Ubx-ts-bearing cross overs between Ly_ and Sb as homozygotes at 22°C 134 20 Results of tests of a l l possible combinations of Ubx-ts mutations in trans-heterozygotes 136 21 Tests for crossing over between different Ubx-ts-mutations and Ubx-ts-1 138 22. Relative viabilities of flies heterozygous for + or Ubx-ts-X and TM2 at different temperatures 139 23 Lethal Period of progeny after increasing exposure of Ubx-ts/ TM2 parents to 29°C 148 24 A comparison of the viabilities of Ubx-ts-X and bx individuals heterozygous for various bx-Pseudoalleles at 220~and 29 C 151 25 A comparison of the viabilities and phenotypes of individuals heterozygous for Ubx-ts-2 or various bs-alleles and one of several bx-pseudoalleles 152 26 Summary of exceptional progeny recovered in the crosses with Ubx-ts-lethals and bx-pseudoalleles 155 27 Viabilities of Pm-1-bearing chromosomes at 23° and 29° C 157 28 Ratios of Pm-l-X/Pm-l-Y to total progeny from the Cross Pm-l-X/ SM5 males x Pm-1-Y/SM5 females at 22 C 158 29 Complementation tests of Cy-lethal mutations at 17°C 160 30 A comparison of the number of peaks of isoaccepting tRNAs in Drosophila as determined by BD-cellulose and RPC 5 chromatography 183 31 Acceptance of amino acids by tRNA from fi r s t and third instar larvae and adult Drosophila. 190 32 Number of peaks on RPC-5 chromatographic oolumns for i t 191 x i TABLE 33 Amino a c i d acceptor a c t i v i t y of tRNA from f i r s t and t h i r d i n s t a r larvae and a d u l t Drosophila' 219 34 Q u a n t i t a t i v e a n a l y s i s of <5 and (Y forms of tRNAs 233 35 D e s c r i p t i o n of mutations and rearrangements used i n the s t u d i e s 351 36 L o c a l i z a t i o n of Ubx-ts-1 and Ubx-ts-4 w i t h respect to red 360 37 Progeny t e s t i n g of cross over males from Cross 1 f o r the presence of the Ubx-ts-1 t h a l s 361 38 L o c a l i z a t i o n of Ubx-ts-1 and Ubx-ts-4 w i t h respect to r e d ; Cross 3: D Ubx-ts Sb/red ? X red/redo* 362 39 Progeny t e s t s of cross over males from Cross 3 f o r the presence of the Ubx-ts-mutations 363 40 V i a b i l i t y of heterozygotes f o r a U b x - t s - l e t h a l or TM2 and v a r i o u s mutations and d e f i c i e n c i e s i n the v i c i n i t y of the rosy. locus 369 41 Conditions f o r the aminoacylation of D r o s o p h i l a tRNAs 374 LIST OF FIGURES x i i FIGURE 1 Genetic crosses f o r the detection of dominant temperature-s e n s i t i v e l e t h a l mutations on chromosome 3 31 2 Genetic p o s i t i o n s of the DTS-lethal.mutations on chromosome 3 38 3 The E f f e c t i v e Lethal Phase and temperature-sensitive period of each DTS-lethal mutation 44 4 Determination of temperature-sensitive period f o r DTS-1/TM2 heterozygotes 69 5 Determination of temperature-sensitive-period f o r DTS-6 heterozygotes 71 6 Determination of temperature-sensitive period f o r DTS-2 homo-zygotes and heterozygotes 74 7 Method I for the l o c a l i z a t i o n of the maternal e f f e c t associated with DTS-2 77 8 Method II for the l o c a l i z a t i o n of the maternal e f f e c t associated with DTS-2 80 9 Determination of the temperature-sensitive period f o r adult v i a b i l i t y of DTS-3/TM2 heterozygotes 85,87 10 Lethal phases of DTS-4/TM3 heterozygotes a f t e r s h i f t s to 29°C at various developmental stages 91 11 Percentage of DTS-4/TM3 adults e c l o s i n g a f t e r 2- and 6-hour heat pulses at various times during development 93 12 E f f e c t of age and temperature on the egg-laying capacity of DTS-4/TM3 females 98 13 Determination of the temperature-sensitive period f o r DTS-5/TM2 heterozygotes 100 14 Determination of the temperature-sensitive period f o r adult v i a b i l i t y of DTS-7 heterozygotes 103,105 15 Genetic p o s i t i o n s of the Ubx-ts-lethal mutations on chromosome 3 131 16 The E f f e c t i v e Lethal Phases (LPs) and temperature-sensitive periods (TSPs) of the Ubx-ts-lethals 142 x i i i 17 E f f e c t of age and temperature on the egg-laying c a p a c i t y of a. Samarkand, b. Gl Sb es/TM2, c. Ubx-ts-2/TM3, and d. Ubx-ts-4/ TM3 females 145 18 E f f e c t of age and temperature on the egg- l a y i n g c a p a c i t y of Ubx-ts-2/TM2 and Ubx-ts-4/TM2 females 147 19 A comparison of the d i s t r i b u t i o n of i s o a c c e p t i n g tRNA peaks of D r o s o p h i l a by B D - c e l l u l o s e and RPC-5 chromatography 179,181 20 Chromatography of ^ C a l a n y l - , a r g i n y l - , phenylalany 1-, and tryptophanyl-tRNAs. 194 14 21 Chromatography of C g l u t a m i n y l - , g l u t a m y l - , l y s y l - , and glycyl-tRNAs 196 14 22 Chromatography of C h i s t i d y l - , t y r o s y l - , a s p a r t y l - , and asparaginyl-tRNAs 198 14 23 Chromatography of C s e r y l - , t h r e o n y l - , m e t h i o n y l - , and cy s t e i n y l - t R N A s 201 14 24 Chromatography of C i s o l e u c y l - , l e u c y l - , p r o l y l - , and valyl-tRNAs 203 25a Chromatography of phenylalany1-tRNA from w i l d - t y p e a d u l t s of mixed ages 221 25b Chromatography of h i s t i d y l - t R N A from w i l d - t y p e a d u l t s of mixed ages 221 25c Chromatography of tyrosyl-tRNA from w i l d - t y p e a d u l t s of mixed ages 221 25d Chromatography of aspartyl-tRNA from w i l d - t y p e a d u l t s of mixed ages 221 25e Chromatography of asparaginyl-tRNA fom the w i l d - t y p e a d u l t s of mixed ages 221 26 Comparison of tyrosyl-tRNA from developmental stages o type and su(s) v; Jbw mutant of Drosophi l a f the w i l d -224 27 Comparison of ^ i s t i d y l - t R N A from developmental stages of the w i l d -type and su(s) v; t>w mutant of Drosophi l a 227 28 Comparison of aspartyl-tRNA from the developmental stages of the w i l d - t y p e and su(s) v; bw mutant of Drosophila 229 xiv 29 Comparison of asparaginyl-tRNA from the developmental stages of the wild-type and s u ( s ) ^ v; bw mutant of Drosophila 231 3Q RPC-5 chromatography of wild-type tRNA A s p 235 31 Two-dimensional t h i n - l a y e r chromatograms of pancreatic RNase digests of t R N A ^ and tRNAAsp 237 32 Two-dimensional t h i n - l a y e r chromatograms of piper i d i n e hydrol-ysates of tRNA A s p and tRNA A s p and p i p e r i d i n e treated E. c o l i Qp 240 A o n 33 U l t r a v i o l e t absorption spectra of Q p from Drosophila tRNA£ and Qp from E. c o l i 242 34 Proposed models ;for the tRNA modifying enzyme 247 35 Model f o r chromosome structure and a m p l i f i c a t i o n 285 36 Model f or the regulation of gene a c t i v i t y 291 37 Model f o r the generation of chromosome bridges one generation post i r r a d i a t i o n 298 38 Model f o r variegated p o s i t i o n e f f e c t -- based on the proposed model f o r chromosome structure 303 39 Model f o r exchange within heterochromatin 311 40 Crosses made for the l o c a l i z a t i o n of Ubx-ts-1 and Ubx-ts-4 with respect to the recessive marker red 359 41 Outline of crosses for the localization of a recessive lethal mutation on the TM2 chromosome in the region of the Ubx-ts-lethals 366 GENERAL INTRODUCTION In a short twenty years, the significance of bacteria and bac-teriophages as genetic tools for the elucidation of the molecular aspects of gene structure and function has clearly been demonstrated (see, for example, Hayes, 1969; Watson, 1970). These organisms provide an easily accessible system for the investigation of such diverse cellular processes as DNA replication (Delbruck and Stent, 1957; Meselson and Stahl, 1958a, 1958b) and control mechanisms of gene function (Ippen, et a l . , 1968; Yanofsky, 1960; Ames and Hartman, 1962). The profound extent to which these studies have contributed to an understanding of basic molecular events i n c e l l biology owes this success primarily to the analysis of conditional mutations (i.e. those which confer v i a b i l i t y under a specific set of "permissive" growth conditions but which are lethal when "rest r i c -tive" conditions are imposed). The large spectrum of auxotrophic, tempera-ture-sensitive, and suppressible amber, ochre, and topaz mutations, coupled with the phenomenal array of biochemical lesions detected through them, have led to the establishment of the fundamental principles of DNA replication , the "genetic code", transcription, translation, and various control mechanisms of gene activity. We must not, however, i n this fascination for the microbial tools, overlook the role of eukaryotes, whose early investigations led to the discovery of the nature of gene products and the significance of mutations. As early as 1902 Garrod attributed the accumulation of specific metabolites 2 i n persons a f f l i c t e d w i t h c e r t a i n g e n e t i c metabolic d i s o r d e r s to the s p e c i f i c a f f e c t of mutations i n t e r m i n a t i n g a step-wise b i o c h e m i c a l process ( r a t h e r than t h e i r e f f e c t i n g the production of a f o r e i g n sub-stance) . His r e c o g n i t i o n that unique g e n e t i c b l o c k s i n the c a t a b o l i s m of phenylalanine were r e s p o n s i b l e f o r the disease symptoms of a l b i n i s m and a l k a p t o n u r i a provided the groundwork f o r the development, by other workers, of d i e t a r y c o n t r o l s f o r i n d i v i d u a l s s u f f e r i n g from a l k a p t o n u r i a and phenylketonuria ( B r a i d and Hickmans, 1929; Armstrong and T y l e r , 1955). Studies i n Neuropora formed the prototype f o r the i n t e n s i v e a n a l y -s i s of b i o c h e m i c a l mutants i n a v a r i e t y of lower organisms. Beadle and Tatum, as e a r l y as 1941, had performed a systematic a n a l y s i s of mutants unable to grow on minimal medium but capable of growth on media supple-mented w i t h a p a r t i c u l a r amino a c i d , v i t a m i n , or n u c l e o t i d e which was apparently d e f i c i e n t i n the mutant s t r a i n . Joshua Lederberg (1950), a student of Tatum and pioneer i n b a c t e r i a l g e n e t i c s , subsequently a p p l i e d , these methods to s t u d i e s i n E_. c o l i , l a y i n g the foundation f o r the enormity of i n v e s t i g a t i o n s w h i c h , taken as a whole, form the g r e a t e r p a r t of our present understanding of molecular b i o l o g y . The s t o r y does not end h e r e , however, but r a t h e r b e g i n s . The a t t e n t i o n of m u l t i c e l l u l a r e u k a r y o t i c systems, "momentarily" d i v e r t e d to e s t a b l i s h the b a s i c b i o c h e m i c a l p r o p e r t i e s of c e l l u l a r f u n c t i o n s i n p r o k a r y o t e s , has again been aroused—probably i n view of the impending e l u c i d a t i o n of the few remaining problems i n u n i c e l l u l a r organisms:; w i t -ness the "conversion" of such prominent molecular b i o l o g i s t s as S. Benzer 3 now studying behavior i n Drosophila 1970, S. Brenner (who has i n i t i a t e d an in t e n s i v e study of neural anatomy i n nematodes), and D.S. Hogness (whose a t t e n t i o n has been d i r e c t e d to the problem of eukaryotic chromo-some structure) . Thus the problems of chromosome structure and func-t i o n , determination, d i f f e r e n t i a t i o n , c e l l communication, and behaviour - c h a r a c t e r i s t i c of a l l higher organisms—provide new avenues to the mo-l e c u l a r b i o l o g i s t . Questions concerning these various phenomena have been posed for many years, but the a p p l i c a t i o n of methods established for lower organisms allows f o r new and v a r i e d approaches to these pro-blems. A few of these problems are discussed here i n d e t a i l and form a basis f o r the experimental approaches which follow. In order of t h e i r d i s c u s s i o n , these are: 1) the structure and genetic (or other?) functions of heterochromatin; 2) the nature of dominance ( i n p a r t i c u l a r with reference to the causes of dominant l e t h a l i t y ) ; 3) the po s s i b l e mechanisms r e s u l t i n g i n the s e n s i t i v i t y of mutants to changes i n environmental temperatures; and A) the possible r o l e s of s p e c i f i c macromolecules, i n p a r t i c u l a r the t r a n s f e r RNA's, i n c o n t r o l l i n g gene a c t i v i t y or the f u n c t i o n of gene products. A d i s c u s s i o n of the fundamental, established mechanisms of temperature-s e n s i t i v i t y as w e l l as a t h e o r e t i c a l treatment of the problem i s found * 4 in Appendix 2. Also, number 4 above is enlarged upon in Part II of this thesis, where i t is more applicable to the studies undertaken in that section. In most of the studies discussed in Chapters 2 and 3, Drosophila has been used as the experimental organism. Thus, mention of the or-ganism is made only when exception to this generalization applies. CHAPTER 1 THE PROBLEM OF HETEROCHROMATIN Let us first begin with a quotation from Schultz (1956) for i t beautifully summarizes the purpose to which several years of intensive investigation of this "anomalous substance" have been dedicated: The attraction which the heterochromatic regions of the chromosomes holds for investigators resides in the fact that they are differentiated chromosome regions. This fact provides the possibility that by their study, one might gain insight into some quality which could itself serve as a guide to generalities of the nature and function of a l l genes. Henking, as early as 1891, termed the sex chromosomes of certain insects "heterochromosomes", reflecting their different staining pattern as compared to that of the autosomes. This remained a mere cytolo-gical observation with l i t t l e attention as to the functional or structur al relationships until 1928 when the subsequent identification of dif-ferentially staining segments of Drosophila neuroblast chromosomes led Heitz (see Schultz, 1947) to postulate the existence of two types of chromatin: the heterochromatin, which stains darkly and is presumably condensed throughout the nuclear cycle, and the euchromatin, which be-comes more diffuse than the former during interphase. The prospective differences in the structural and behavioral properties of the two types of chromatin stimulated research from genetic, cytological, and biochemi cal standpoints. Attempts were made to show that the different major heterochromatic regions are homologous and that these contain redundant gene sequences; that they contain genetic information and are therefore 6 not inert; that unique chromosomal functions may be attributed to them; and f i n a l l y that a l l chromatin possesses the potential for heterochroma-tization, thereby indicating that euchromatin and heterochromatin merely represent different "states" of the same basic material. Chapters 3, 4 and 5 are concerned with an investigation into the biological significance of "heterochromatin".' Part II of this thesis considers the spectrum of tRNA molecules present during the development of Drosophila and may be also linked to the question of heterochromatin (see Appendix 1 for a theoretical discussion). Although the problem is not a new one, modern analytical techniques make these approaches some-what unique, and so, i n order to establish a groundwork for the experi-mental approaches described herein, a brief review of the main character-i s t i c s of heterochromatin i s f i r s t presented. The discussion w i l l be restricted mainly to a consideration of the large heterochromatic "blocks of Feulgen-positive material" found adjacent to the centromeres (centric heterochromatin or heteropycnotic regions—Schultz, 1947). It must, however, be kept in mind that smaller, intercalated regions, shar-ing the properties attributed to heterochromatin, appear to be distribu-ted throughout the chromosome (Prokofyeva-Belgovskaya, 1939; Kaufmann, 1939). Early studies indicated that the hetero- and euchromatic regions are distinguishable not only in terms of their cytological appearance, but also with respect to genetic a c t i v i t y — t h e latter having an abundance of genes (as determined by mutation localization) and the former having 7 a p a u c i t y of genetic i n f o r m a t i o n ( M u l l e r and P a i n t e r , 1932). Whereas the c e n t r i c heterochromatin c o n t r i b u t e s very l i t t l e to the l e n g t h of the s a l i v a r y gland chromosomes, these regions can represent as much as one h a l f of the m i t o t i c chromosome l e n g t h , as i n the case of the X chromosome (Kaufmann, 1934). This d i f f e r e n c e i s accounted f o r by the observation that the heterochromatic regions i n polytene c e l l s show severe u n d e r - r e p l i c a t i o n compared to other p o r t i o n s of the chromosomes. Heterozygous d e f i c i e n c i e s of heterochromatin a c c o u n t i n g , f o r example, f o r as much as t w o - f i f t h s o f the m i t o t i c chromosome l e n g t h are v i a b l e ( H i n t o n , 1942), w h i l e d e f i c i e n c i e s of t h i s dimension w i t h i n euchromatin have never been observed to s u r v i v e ( c f . L i n d s l e y and G r e l l , 1968, s e c -t i o n s on d e f i c i e n c i e s and i n v e r s i o n s , and the f o l l o w i n g chapter on domi-nant l e t h a l s ) . Such observations suggest a very fundamental d i f f e r e n c e i n the f u n c t i o n a l p r o p e r t i e s of the two chromatin types. As e a r l y as 1916, s p e c u l a t i o n as to the f u n c t i o n of heterochromatic segments, derived from the obs e r v a t i o n of Bridges that the Y chromosome i n D r o s o p h i l a i s not e s s e n t i a l f o r v i a b i l i t y , l e d to an assumption of t h e i r g e n e t i c i n e r t n e s s . However, the dis c o v e r y of the bobbed locus w i t h i n the heterochromatic p o r t i o n of the X chromosome ( S t u r t e v a n t , 1920; B r i d g e s , 1926) as w e l l as i n the short arm of the Y chromosome ( S t e r n , 1926; Cooper, 1959) and the r e g u l a r m e i o t i c d i s j u n c t i o n of these two chromosomes are i n d i c a t i v e of at l e a s t a p a r t i a l homology of the re s p e c -t i v e heterochromatic r e g i o n s . The r e c o g n i t i o n of the importance of the Y chromosome i n c o n f e r r i n g the f e r t i l i t y of males ( B r i d g e s , 1916; SIvertzev-Dobzhansky and Dobzhansky, 1933) and i t s d i v i s i o n i n t o d i s c r e t e f e r -t i l i t y f a c t o r s (Brosseau, 1960) gave f u r t h e r support f o r the g e n e t i c a c t i v i t y 8 of t h i s wholly heterochromatic chromosome. F e r t i l i t y f a c t o r s have also been implicated on the Y chromosome of the plant, Rumex t h y r s i f l o r u s , (Zuk, 1970), supporting the c y t o l o g i c a l evidence f o r i t s a c t i v i t y i n premeiotic stages of microsporogenesis (Zuk, 1970). The chromocentric configuration formed by the condensation and as s o c i a t i o n of the c e n t r i c heterochromatin of the various chromosomes ( i n s a l i v a r y gland n u c l e i ) has been taken by Prokofyeva-Belgovskaya (1935) to i n d i c a t e the homologous nature of these c e n t r i c regions. A meiotic r e l a t i o n s h i p f o r non-homolo-gous chromosomes has also been suggested by many workers (Schultz and Re d f i e l d , 1951; Li n d s l e y , 1955a,b). Studies on the s p e c i f i c homologies of the X and IV*"*1 chromosomes, using the extent of X-IV d i s j u n c t i o n as a measure of p a i r i n g a f f i n i t i e s , i n d i c a t e that i n s o f a r as p a i r i n g i s con-cerned these are indeed homologous (Gershenson, 1940; Parker, 1954). Since these experiments involved X chromosome rearrangements which may have i n t e r f e r e d with normal X-X p a i r i n g , Sandler and N o v i t s k i (1956) repeated these experiments using normal X chromosomes and t r i p l o - I V ' s , obtaining s i m i l a r r e s u l t s . R.F. G r e l l (1962, 1967) has, however,in a se r i e s of elegant experiments, shown that whereas chromosome recognition at exchange p a i r i n g i s r e l a t e d to the extent of homology, that at d i s t -r i b u t i v e p a i r i n g i s correlated with t o t a l s i z e and i s independent of homology. E.H. G r e l l ' s studies (1963) do, on the other hand, implicate some p a i r i n g r o l e f o r the c e n t r i c heterochromatic regions. Further corroboration of the "homology concept" comes from the analogous behaviour of the d i f f e r e n t heterochromatic regions f o r a 9 spectrum of rather unique properties. These include "genetic inertness" position effect, temporary genetic inactivity, absence of template ac-tivity in vitro, late replication in S-phase, high content of redundant DNA, specific breakage with maleic hydrazide, abnormal mitotic coiling after cold treatment, and specific staining with fluorochromes such as quinacrine mustard (Ris and Kubai, 1970). Some of these are discussed In more detail in the following paragraphs. Not only are few mutations recovered within heterochromatin, but linkage of the few disclosed within a single heterochromatic block is also very tight (X chromosome: su(f) at 65.9 and bb_ at 66.0; chromosome 2: i t in the left arm at 55.0 and both rl_ and stw in the right arm are located at 55.1, Cooper, 1959). That this apparent close linkage reflect the low frequency of crossing over within heterochromatin, as opposed to the fortuitous recovery of mutations in genes which are very close together, is substantiated by the tight linkage relationship of genes outside of heterochromatin but spanning the centromere (Hannah, 1951). This is in direct contrast to the observations on mitotic recombination which Stern (1936) and Kaplan (1953) found to occur predominantly within the centric heterochromatin. Discussion of the possible significance of this observation is reserved until the section concerned with the bio-chemical properties of chromatin in Appendix 1. Another aspect of the differential genetic behaviour of euchromatin versus heterochromatin has been implicated from numerous observations on the influence of inversions on meiotic recombination within heterolo-10 gous chromosomes. In 1915, Sturtevant posed the question: "Does crossing over i n one chromosome have any effect upon the other chromo-somes in the same c e l l ? " — t h i s might, supposedly, be indicative of interference operating between non-homologues. In the following year he presented evidence suggesting that the decrease i n exchange fre-quency of one chromosome pair i s complemented by an increase i n the linkage of another pair. Since that time, extensive studies have been made attempting to correlate the factors involved in what has come to be called the "interchromosomal effect". The following summary, taken from Lucchesi and Suzuki (1968), points out the prominent features of this phenomenon and i s based on experimental evidence provided by many workers (see, for example, Ward, 1923; Steinberg and Fraser, 1944; Schultz and Redfield, 1951; Levine and Dickinson, 1952; Welshons, 1955; Suzuki, 1963; Procunier and Suzuki, 1967; and review by Lucchesi and Suzuki, 1968): (a) Causative factors: most chromosome rearrangements (inver-sions , translocations) when present i n heterozygous condition; some homozygous chromosome rearrangements (inversions, translocations), es-pecially i f they involve the relocation of heterochromatic segments; chromosome rearrangements which attach homologues or arms of homologues to the same centromere (compounds); aneuploid changes involving the pre-sence of extra elements in the same genome, providing that these ele-ments are totally heterochromatic or exhibit a greater hetero/euchromatin ratio than their normal genomic counterparts; and mutations of a few "major" genes or the accumulation of a specific set of polygenic modifiers. (b) Effects: these can be interchromosomal but also intrachromo-somal (inter- or intrabrachial); most often they consist of an increase in levels of recombination with concommitant changes in chromosome inter-ference; and they do not bear a simple correlation with the change i n crossing over pattern within the causative chromosome pair or pairs. 11 Not only do structural anomalies influence the rate of exchange i n heterologues, but their effect i s restricted primarily to the centric heterochromatin, although often the di s t a l tips of the chromosomes may be similarly affected. This common mode of response lends strong sup-port to the structural, functional, and behavioural homology of the different heterochromatic segments. The concept f i r s t suggested by Wenrich (1916) , that the hetero-chromosomes and isochromosomes differ i n their temporal "states" rather than i n their basic biochemical make-up was revived in later years by, among others, Poulson and Metz (1938), Prokofyeva-Belgovskaya (1941, 1947) , and Pontecorvo (1944), and i t i s now generally accepted that "heterochromatization" i s a feature inherent to a l l chromatin—and that the "state" of a particular chromatin segment i s dependent on nuclear and chromosomal environmental conditions. The evidence i n support of this comes from both genetic and cytological observations and i s discussed below. The phenomenon of variegated- or V-type position effect, whereby a change in gene action i s associated with a change i n the position of that gene with respect to the normal gene sequence on the chromosome, has been attributed to the heterochromatization of a euchromatic gene upon i t s juxtaposition to heterochromatin (Schultz, 1936, 1941). Thus when a euchromatic gene, which i s normally metabolically active,comes into association with heterochromatin that gene becomes inert, permitting the phenotypic expression of the a l l e l e located on the homologue. Since 12 this "inactivation" or heterochromatization is necessarily dependent upon other metabolic activities within the cells, somatic mosaicism or a variegated phenotype is usually produced (Schultz, 1936; Lewis, 1950). Cytological observations revealed the disorganization of the newly lo-cated euchromatin in the immediate vicinity of the heterochromatin, detracting from its normal morphological appearance of distinct banding (Prokofyeva-Belgovskaya, 1947) and supporting this contention. In the g same study, the effects of the sc inversion, in which breaks near the tip of the X and close to the centromere resulted in the inversion of most of the chromosome with respect to the distal and proximal reference points, were also examined. Here, the novel disposition of a hetero-chromatic segment in apposition to euchromatin causes the former to become "almost euchromatic"—so that a characteristic banding pattern is acquired (Prokofyeva-Belgovskaya, 1947). Thus, a correlation between the phenotypic manifestations and chromosomal properties associated with the V-type position effect (Schultz, 1941; Prokofyeva-Belgovskaya, 1947) has been established, pointing to the importance of the structural integrity and organization of the chromosome. Further support for the reversibility of the hetero- and euchroma-tic phases comes from the observations of Pavan and Breuer (1952) on the tissue specificity of heterochromatization in the polytene chromo-somes of Rhynchosciara. It would seem that certain regions take on a pronounced heterochromatic character in Malpighian tubule cell nuclei whereas in salivary gland nuclei these same regions have a distinctively 13 euchromatic appearance. The role of environmental stimuli i n determin-ing the "state" of a given chromosome segment i s further implicated by the observation that the Y chromosome of Drosophila busckii may be either heterochromatic or euchromatic or a combination of both i n poly-tene cells (Krivshenko, 1952). From these and similar studies (Brown, 1966), i t has become apparent that heterochromatin i s not uniform in i t s behaviour. The terms facultative and constitutive have been introduced to distinguish between two distinct types of heterochromatin (Sanders and Pavan, 1972), which have been defined as follows: "There i s facultative heterochromatin which, during c e l l l i f e , i s metabolically more active than inactive in the produc-tion of nucleic acids and which is probably composed primarily of heterochromatized euchromatin. Constitutive heterochromatin, i s , i n general, considered more inactive than active i n nucleic acid production. We have come a long way i n our discussion, providing evidence which i n i t i a l l y suggested a subdivision of chromatin into two distinct types but which upon further investigation brought about a realization of the basic structural uniformity of chromatin. The behavioural differences between the constitutive heterochromatin (as opposed to the "induced" or facultative type described above) and euchromatin are, however, real. Thus the problem remains: to what may these differences be attributed? It i s , perhaps, instructive at this time to examine the biochemical na-ture of these two regions with a view to determine whether nucleotide composition might confer these distinguishing properties and,if so, what the evolutionary significance of such a distinction might be. Early suggestions as to the function of heterochromatin contested that i t was responsible for the nucleic acid metabolism of the c e l l 14 (Muller and Gershenson, 1935; Schultz, 1936). Such a role would ac-count for its apparent genetic inactivity and the organism's tolerance i to greater or lesser amounts of heterochromatin. These observations, coupled with the homology concept of heterochromatin (Prokofyeva-Belgovskaya, 1947X led to the suggestion that tandem duplications with-in heterochromatin were responsible for conferring these unique properties (Pontecorvo, 1944; Suzuki, 1963, 1973). The recent demonstration by Ritossa and Spiegelman (1965) of the relationship between the nucleolus organizer and DNA complementary to ribosomal RNA provided the first evidence that redundant DNA sequences were, in fact, present within heterochromatin. Reassociation kinetics of dissociated DNA obtained from several different higher organisms revealed a rapidly annealing fraction (the proportion of the total DNA of which was dependent on the species), in-dicating that some of the nucleotide sequences are frequently repeated within the genome (see review by Britten and Kohne, 1968). This was identified as satellite DNA, having a buoyant density in an alkaline-CsCl gradient which is distinct from that of the main DNA. This has been attributed to the A-T rich nature of the repetitive sequences (Britten and Kohne, 1968). The technique of in situ hybridization has been used to localize the highly repetitive DNA sequences in the polytene chromosomes of many Diptera (in Rhynchosciara: Eckhard and Gall, 1971 and in Drosophila: Par due e_t al . , 1970; Rae, 1970; Jones and Robertson, 1970; Hennig et al., 1970; and Botchan e_t a l . , 1971); these have 15 i n v a r i a b l y been found to l o c a l i z e w i t h i n heterochromatin. Further, more recent r e s u l t s on r e i t e r a t e d DNA sequences and t h e i r r e l a t i o n s h i p to the metabolic a c t i v i t i e s of the chromosomes are given i n Appendix I. Recently, a model f o r gene r e g u l a t i o n i n v o l v i n g repeated DNA se-quence has been postulated by B r i t t e n and Davidson (1969). This i s also discussed i n more d e t a i l i n Appendix I, i n conjunction with a model proposed f o r chromosome structure. According to the B r i t t e n and Davidson theory, r e p e t i t i v e sequences are important at two l e v e l s of the regulatory process. The basic features of the model may be summar-ized as follows: " i n t e g r a t o r " genes respond to "sensor" gene induction by forming " a c t i v a t o r " RNA. This RNA then combines with "receptor" genes, thus permitting the t r a n s c r i p t i o n of s t r u c t u r a l or "producer" genes. As an example of how t h i s might operate, we can suppose that an inducing hormone e i t h e r binds d i r e c t l y to a s p e c i f i c sensor gene or i n -duces a p r o t e i n which i s capable of undergoing t h i s r e a c t i o n . Many hor-mones are known to e l i c i t a response c o n s i s t i n g of the synthesis of several d i f f e r e n t RNA and p r o t e i n sequences. This may be accomplished i n one of two ways: f i r s t l y , the r e a c t i o n with the sensor gene may a c t i v a t e a family of i n t e g r a t o r genes, each sharing a s p e c i f i c nucleotide sequence f o r part of t h e i r length but d i s t i n g u i s h a b l e from one another by other non-matching nucleotides; secondly, the a c t i v a t o r RNA (the pro-duct of a s p e c i f i c i n t e g r a t o r gene) may combine with a s e r i e s of r e l a t e d receptor genes which again show regions of homology and non-homology. Such systems ensure the response required to give "an integrated a c t i v a -t i o n of a very large number of noncontiguous genes" ^ ( B r i t t e n and 16 Davidson, 1969). This model not only suggests a r e l a t i o n s h i p between d i f f e r e n t regions of a chromosome but a l s o between d i f f e r e n t chromosomes. Indeed .examples o f both types have been presented: the interchromosomal e f -f e c t s on c r o s s i n g over and the intrachromosomal V-type p o s i t i o n e f f e c t s s a t i s f y these requirements. Numerous other i n s t a n c e s of chromosomal i n t e r r e l a t i o n s h i p s have been r e p o r t e d ; a few i n s t a n c e s of Y-mediated e f f e c t s can b r i e f l y be mentioned h e r e . The presence of supernumerary Y's has been shown to reduce or enhance the amount of v a r i e g a t i o n c h a r a c t e r i s t i c a l l y produced by a s p e c i f i c rearrangement ( S c h u l t z , 1936; Baker and S p o f f o r d , 1959). A d i r e c t e f f e c t of the Y chromosome on the behaviour of the other chromosomes i s evident from the observations that males bearing d e f e c t i v e Y chromosomes produce sperm c o n t a i n i n g n u c l e i which f a i l to complete the e l o n g a t i o n of the sperm head, the f i n a l stage i n sperm maturation ( S c h u l t z , 1947). The mechanism of t h i s e f f e c t may be r e l a t e d to a s i m i l a r i n f l u e n c e of the d i s t a l ends of a l l s a l i v a r y gland chromosomes ( S c h u l t z , 1947). The o b s e r v a t i o n that c e r t a i n non-homologous regions of s a l i v a r y gland chromosomes r e g u l a r l y a s s o c i a t e d ( e c t o p i c p a i r i n g ) may f u r t h e r suggest a r o l e of the chromosome i n the r e g u l a t i o n of genetic a c t i v i t y (Barr and E l l i s o n 1972) and i n p a r t i c u l a r that type which " i s expressed c y t o l o g i c a l l y by p u f f i n g of the s a l i v a r y gland chromosome". Although a number of c h e m i c a l , s t r u c t u r a l , and b e h a v i o u r a l proper-t i e s of heterochromatin have been presented h e r e , the f u n c t i o n a l aspects 17 remain s t i l l very obscure. Of the limited number of genes apparently located "within" heterochromatin a few have striking phenotypes which are suggestive of their regulatory importance. These are the so-called homoeotic mutations, a number of which are located i n the proximal heterochromatin of chromosome 3 (Lindsley and Grell, 1968). These mu-tations result i n the replacement of one organ structure by another structure from an homologous organ (Bateson, 1894). But these do not account for a l l the DNA present within the heterochromatic regions; seg-ments remain with as yet unidentified roles. One approach to obtaining mutations within heterochromatin has em-ployed a highly specific technique for generating deficiencies i n the centromeric region. X-irradiation was used f i r s t to generate and sub-sequently to detach the homologous arms of compound-3-chromosomes in order to obtain lethals (presumably deficiencies) i n the heterochromatic region (Baldwin and Suzuki, 1971). Two major complementation groups were obtained, probably representing groups on either side of the centro-mere. It may be argued that the i n a b i l i t y to recover such lethals through mutagenesis with an agent causing point mutations supports the contention that these lethals are deficiencies for l o c i which are tan-demly duplicated. If, indeed, this region i s composed of redundant sequences, then the detection of specific l o c i requires the generation of dominant muta-tions. And i f , as alluded to, the heterochromatic segments contain l o c i important i n gene regulation or affecting basic cellular processes, i t i s expected that dominant mutations would surely be lethal. And so we 18 are faced with a dilemma: perhaps the only means of detecting mutations w i t h i n the region of i n t e r e s t i s by the recovery of dominant l e t h a l mutations—the very presence of which eliminates i t from genetic analy- ' ses. With the successful recovery of temperature-sensitive mutations i n Drosophila (Suzuki e_t a l . , 1967) i t became obvious that a c o n d i t i o n a l dominant l e t h a l would allow detection of the postulated v i t a l functions and furthermore would allow a developmental c h a r a c t e r i z a t i o n of t h e i r e f f e c t s . ^ Although i t may at f i r s t appear somewhat s u p e r f i c i a l , we must f i r s t consider what the p r o b a b i l i t y of recovering such dominant l e t h a l s might be. In t h i s context a b r i e f review of the nature of dominant l e t h a l i t y i s presented i n the next s e c t i o n f o r , as w i l l become apparent, the pre-vious l i t e r a t u r e suggests that the p o s s i b i l i t y of obtaining dominant l e t h a l s which are point mutations in higher organisms (and there-fore temperature-sensitive by v i r t u e of an amino acid s u b s t i t u t i o n ) i s very remote indeed -- i f not impossible. 19. CHAPTER 2 THE NATURE OF DOMINANT LETHALITY The r a p i d i t y with which genetics has developed as a science i s quite phenomenal: one can hardly say that t h i s i s due to the many-faceted "milestones" established on the w a y — i t would be more appropriate to c a l l them "metrestones". In 1900, the long-neglected works and concepts of Mendel (1866) were thrice-over r e d i s c o v e r e d — i n France (H. de V r i e s ) , Germany (C. Correns), and A u s t r i a (E. von Tschermak-Seysenegg). The next few decades saw numerous confirmations and extensions of Mendelism, but perhaps one of the most important contributions was made by Muller i n 1927. At that time he reported a successful means of " a r t i f i c i a l l y " producing mutations at a rate f a r exceeding the spontaneous rate (by "15000%"). His use of X - i r r a d i a t i o n to induce an abundant supply of mu-tations with which to carry out genetic analyses led him to the discovery of dominant l e t h a l mutations (to which he had a t t r i b u t e d the p a r t i a l s t e r i l i t y of i r r a d i a t e d males). This l a t t e r observation has l e d to many years of intensive i n v e s t i g a t i o n i n t o the nature and causes of dominant l e t h a l i t y and i s where we w i l l begin our survey. Nineteen hundred and twenty-seven then, marked the beginning of an era i n which several agents were to be examined f o r t h e i r a b i l i t y to induce dominant l e t h a l s — w i t h a view to determining the nature of muta-t i o n s , e s p e c i a l l y those producing dominant l e t h a l i t y . I t was hoped that 20 such s t u d i e s would e s t a b l i s h whether such mutations r e s u l t e d from a s p e c i f i c a c t i o n at the chromosomal o r , perhaps, the genie l e v e l . Nu-merous chemicals were t e s t e d ( f o r example, n i t r o g e n mustard i n Habrobracon, Whiting and von B o r s t e l , 1954; i n V i c i a faba; d i e p o x i d e , ffi^, and t e r t i a r y b u t y l hydroperoxide, R e v e l l , 1952 and maleic h y d r a z i d e , McLeish, 1952; i n D r o s o p h i l a : 1:2, 3:4-diepoxybutane and 2 : 4 : 6 - t r i ( e t h y l e n e a m i n o ) - l : 3 : 5 - t r i a z i n e , Fahmy and B i r d , 1952) and a l l were found to produce c h a r a c t e r i s t i c frequencies of l e t h a l zygotes. Not only were chemical agents and X - i r r a d i a t i o n capable of t h i s a c t i o n , but UV and v a r i o u s types of i o n i z i n g r a d i a t i o n s a l s o had t h i s e f f e c t (see, f o r ex-ample, Gray, 1952). By f a r the most ex t e n s i v e s t u d i e s on dominant l e t h a l i t y i n D r o s o p h i l a have employed X-rays as the mutagenic agent. D i s c r e p a n c i e s i n the ob-served frequencies of dominant l e t h a l s aroused concern as to the b i o l o g i -c a l f a c t o r s which might be i n f l u e n c i n g the mutation r a t e . Undoubtedly considerable v a r i a t i o n i s introduced to experimental procedure through the l a r g e number of i n v e s t i g a t o r s and by t h e i r f a c i l i t i e s , et c e t e r a . Thus Bonnier and Luning (1951) c a r r i e d out s e v e r a l s t u d i e s o f the p h y s i o -l o g i c a l and g e n e t i c f a c t o r s r e g u l a t i n g mutation frequency. They f i r s t examined the e f f e c t of storage of t r e a t e d sperm (by a l l o w i n g d i f f e r e n t groups of males to mate a f t e r i n c r e a s i n g l y longer times post i r r a d i a t i o n ) and found t h a t storage r e s u l t e d i n the production of a higher frequency of dominant l e t h a l s . These workers a l s o observed a d i f f e r e n c e i n the s e n s i t i v i t y of c e l l s i n d i f f e r e n t stages of spermatogenesis and 21 spermiogenesis. Since i t had e a r l i e r been established that X - i r r a d i a -t i o n i s instrumental i n d i s r u p t i n g chromosome i n t e g r i t y (among others, Muller, 1940 and Pontecorvo, 1942), they suggested that chromosomal movement i s involved i n determining the p r o b a b i l i t y of r e s t i t u t i o n a f t e r breakage and that f e r t i l i z a t i o n i s a stimulus to chromosome move-ment. Presumably, i f t h i s occurs soon a f t e r i r r a d i a t i o n , there i s a higher p r o b a b i l i t y that the two broken ends w i l l r e j o i n . A l t e r n a t i v e l y one might suppose that some fac t o r s w i t h i n the egg are capable of r e -p a i r i n g the i r r a d i a t i o n damage but only soon a f t e r i t i s i n c u r r e d — a n d that t h i s a b i l i t y i s c h a r a c t e r i s t i c of eggs and not sperm. Perhaps a system analogous to (or i d e n t i c a l with) the r e j o i n i n g of chromosomes which have undergone a crossover i s operative here, thus accounting f o r the lack of such a system i n males. In any case, p h y s i o l o g i c a l condi-tions do appear to have an e f f e c t on the production frequency of dominant l e t h a l s . An i n t r i g u i n g r e l a t i o n s h i p between dominant and recessive l e t h a l s has been dis c l o s e d through studies comparing t h e i r induction frequencies and factors i n f l u e n c i n g these rates. That a genetic component i s of importance i n determining the s u s c e p t i b i l i t y of a s t r a i n to i r r a d i a t i o n damage has been shown through studies comparing the v i a b i l i t i e s of the wild-type Oregon-R and Swedish-b stocks, whereas the Oregon-R stock was apparently more s e n s i t i v e to the induction of dominant l e t h a l s , the inverse r e l a t i o n s h i p was evident when the frequency of recessive l e t h a l s was measured. While t h i s observation i s indeed of i n t e r e s t , an analysis of the p r e c i s e mechanism whereby mutation frequencies are influenced i s 22 impeded by the large number of f a c t o r s c o n t r i b u t i n g to species and, as i n t h i s case, s t r a i n d i f f e r e n c e s . That t h i s r e l a t i o n s h i p between dominant and recessive l e t h a l s i s a v a l i d conclusion has been substan-t i a t e d by studies i n which the v a r i a b l e s have been severely r e s t r i c t e d — a s , for example, the age of males during the treatment or v a r i a t i o n s i n the type or dosage of mutagen tested. A study by Hanson and Heys (1935), i n which males were aged f o r d i f f e r e n t lengths of time p r i o r to i r r a d i a t i o n , i n d i c a t e d that the s e n s i t i v i t y to the induction of recessive l e t h a l mutations decreases with the age of the male while that of dominant l e t h a l s increases with aging of the males (Dempster, 1941; Bonnier and Luning, 1950). Dempster (1941) also noted e f f e c t s on the production of mutations i n these two categories by neutrons and by X-rays. The former was l e s s e f f i c i e n t i n producing recessive l e t h a l s (about 0.75 times as many as produced by X-rays) but more e f f e c t i v e i n inducing dominant l e t h a l s (about 1.5 times as many as with X-rays), although the t o t a l frequency of l e t h a l s ( i . e . dominant plus recessive) was the same for the two mutagenic agents. Thus, a reverse r e l a t i o n s h i p between dominant and recessive l e t h a l induction i s implicated. Although i n eukaryotes the pr e c i s e molecular bases f o r the two mutational types are unknown the above considerations suggest that the d i s t i n c t i o n i s dependent, at l e a s t to some extent, on the "degree" or s e v e r i t y of the mutation. From the work of Demerec et  a l . (1938) i t would not seem unreasonable to suggest that what appears to be a recessive l e t h a l i n one stock might w e l l give a dominant l e t h a l 23 phenotype upon introduction into a different genetic background. Indeed there have been many such instances ("synthetic" lethality) reported (Dobzhansky, 1946) in which a trans-heterozygote for two unrelated (i.e. nonallelic) recessive lethals i s lethal. Examples of such interactions have also been demonstrated for certain Minutes which are lethal with other specific dominant phenotypic mutations (see discussion on Minutes in Lindsley and Grell, 1968). Having established the possi b i l i t y that recessive and dominant lethals differ only i n "degree", one may ask how this may be recognized i n molecular terms. One might suppose that a hypermorphic or hypomorphic mutation (ones which result in either the over-production or under-pro-duction of a specific gene product) would be recessive while an amorphic mutation (one producing no gene product) would be dominant. This i s , of course, an over-simplification since such factors as the specific gene locus and dosage compensation have not been taken into account. Since the protein products of very few genes have been established i n Drosophila, i t i s not possible at this time to determine whether such a model might be operative in even a few cases. Even i f these were known, the task of developing microanalysis systems allowing isolation and characterization of a specific protein from individual eggs or larvae (to which the tests might be limited since dominant lethals cannot be propagated) makes such an approach even more unrealistic at this time. Since a large proportion of the studies on dominant lethals has involved their induction by X-rays and since recessive lethals can also be induced by this mutagen, then a comparison of the cytological effects 24 might provide some i n s i g h t into why i n one case l e t h a l i t y occurs with one dose of the mutation whereas i n another case two doses are required o f o r t h i s e f f e c t . An extensive study i n which breakpoints of i n v e r s i o n and t r a n s l o c a t i o n chromosomes produced through the action of X-rays were determined, i n d i c a t e d that breaks may occur i n any part of the chromosomes and r e c o n s t i t u t i o n y i e l d s v i a b l e heterozygotes (Kaufmann and Demerec, 1937). Thus i t i s u n l i k e l y that the l o c a t i o n of the breaks i s of primary importance i n e s t a b l i s h i n g the dominant-recessive r e l a t i o n s h i p . C y t o l o g i c a l observations of zygotes bearing dominant l e t h a l muta-tions have provided considerable information about the e f f e c t s of X-rays. A d e t a i l e d examination of l e t h a l embryos has led Sonnenblick (1940) to suggest that the l e t h a l i t y r e s u l t s from a v a r i e t y of disturbances which may occur i n e i t h e r the meiotic or the m i t o t i c d i v i s i o n s . Disturbances i n spindle formation, r e s u l t i n g i n multipolar spindle configurations, were a common cause of very early embryonic l e t h a l i t y . In a d d i t i o n , "chromosome fragmentation, chromatin bridges, assymmetrical d i v i s i o n s , clumping of chromosomes, ...and cytoplasmic v a c u o l i z a t i o n have been ob-served", i n d i c a t i n g that X - i r r a d i a t i o n produces considerable damage to the normal chromosome structure. Sonnenblick also found c e r t a i n zygotes which appeared normal with respect to c e l l p r o l i f e r a t i o n but which showed no evidence of d i f f e r e n t i a t i o n at a time when normal zygotes have considerable s t r u c t u r a l organization. These observations suggest that the mechanism of X-ray-produced dominant l e t h a l i t y involves the same kind of a c t i o n on the chromosome as has been shown to be involved i n the production of v i a b l e rearrangements 25 (which are generally recessive l e t h a l s ) . The breakage e f f e c t s of X-rays and the e f f e c t s of dose ( i n which low doses appear to give one-h i t - k i n e t i c s and high doses give two-hit-kinetics f o r the production of dominant l e t h a l s ) suggested that two mechanisms might be involved i n the i n d u c t i o n of dominant l e t h a l s by X-rays (as w e l l as other chromosome breaking agents). Muller (1940) and l a t e r Pontecorvo (1942) suggested that s i n g l e breaks which are not closed by r e s t i t u t i o n may lead to the formation of d i c e n t r i c and a c e n t r i c chromosomes i f the broken chromatids undergo s i s t e r union. M u l t i p l e breaks could lead to s i m i l a r chromosome types when r e j o i n i n g of the chromosome fragments i s asymmetric (Lea and Catcheside, 1945; Catecheside and Lea,. 1945a, 1945b; Fahmy and Fahmy, 19540. That large d e f i c i e n c i e s are often produced by X-rays (these would nor-mally be l e t h a l i f recovered i n a male) was shown by Liming (1952). His experimental design allowed recovery of hyperploid males which received a greatly d e f i c i e n t - X chromosome from t h e i r fathers and a normal X from t h e i r mothers, thus v e r i f y i n g the chromosome breakage e f f e c t of X-rays on chromosomes as w e l l as substantiating the fact that dominant and r e -cessive l e t h a l i t y are dependent on the r e s i d u a l genome. The model proposed by Muller (1940) and Pontecorvo (1942) f o r single-break-induced dominant l e t h a l i t y was tested by what has come to be known as g e n e t i c a l l y - c o n t r i v e d dominant l e t h a l i t y . Thus, f o r example, approximately one-half of the zygotes produced by translocation-hetero-zygote females mated to wild-type males are l e t h a l since they bear duplications and d e f i c i e n c i e s f o r l o c i present on the translocated chromosomes. N o v i t s k i (1951) examined the zygotes produced by tandem 26 metacentric compound-X-bearing females in which approximately one-half of the single and three-strand-double exchanges within this chromosome generate dicentric bridges during anaphase II of meiosis. About one-quarter of these embryos die within a few nuclear divisions, a stage reminiscent of the lethal phase of embryos which apparently carried dominant lethals resulting from a single break (i.e. low X-ray dose). As mentioned, eggs laid by females heterozygous for a translocation may carry duplicated or deleted segments. Death of such an embryo has been shown to occur much later than in the previous situation, i.e. usually towards the end of embryonic development or shortly after hatching. A third lethal phase characteristic of X-ray-induced dominant lethality occurs after the blastula stage but before hatching. This has been mimicked by eggs produced by triploid females—thus indicating that a somewhat "more" abnormal chromosome complement, with varying degrees of monosomy and trisomy, causes death at a slightly earlier stage than is produced by single, duplicated and deleted segments. We may conclude, then, that the different types of irradiation - produced dominant lethality are very similar to those characteristic of a chromosomal imbalance. In each of the aforementioned studies, there has been no suggestion that dominant lethality might result from the production of point muta-tions. Since the direct genetic analysis of dominant lethals is impeded by their very inviability, the detection of point mutant dominant lethals is impossible. The following two chapters discuss the genetic and developmental properties of a class of dominant lethal mutations which are lethal only under special environmental conditions. Chapter 5 is concerned with studies on a cluster of recessive conditional lethal mutations. 27 CHAPTER 3 THE GENETIC AND DEVELOPMENTAL CHARACTERISTICS OF DOMINANT LETHALS ON CHROMOSOME 3 I . Introduction Dominant mutations have been described for both higher organisms and micro-organisms at a gross morphological level. However, the dis-tinction between dominance and recessiveness becomes less clear as one attempts to define them at the molecular level. Bernstein and Fisher (1968) have attributed the molecular basis for dominance in micro-organ-isms to the polymerization of wild-type and mutant polypeptide monomers resulting in either the restoration of biological activity (if the wild-type is dominant) or the inactivation of the polymeric hybrid complex (if the mutant is dominant). As mentioned, H.J. Muller (1927) was the first to attribute the marked increase in the mortality of developing embryos after treatment of Drosophila males with X-rays to the production of dominant lethal muta-tions in the sperm. Several other workers (e.g., Pontecorvo, 1942; Haldane and Lea, 1947) found similar results and, in the majority of cases, cytological observations and genetical experimentation suggested that dominant lethality results from abnormal chromosomal complements. Genetically-contrived dominant lethality resulting from the presence of deletions or duplications was found to yield lethal phenotypes similar to the dominant lethal phenotypes obtained after X-irradiation (Von Borstel and Rekemeyer, 1959). 28 The nature of dominant lethal mutations precludes their direct genetic analysis since successive generations cannot be propagated. How-ever, the recovery of temperature-sensitive lethals in Drosophila (Suzuki et a l . , 1967) suggested a possible means of screening for conditional mutants which would behave as dominant lethals only under restrictive conditions. Dominant temperature-sensitive lethals i n Drosophila melanogaster have been recovered in chromosomes 2 and 3 which, i n heterozygous condition, die at 29°C but are completely viable at 22°C ( B a i l l i e et a l . , 1968; Suzuki and Procunier, 1969; Holden and Suzuki, 1968). In this chapter the genetic properties and basic developmental patterns of seven dominant temperature-sensitive lethals recovered on chromosome 3 are discussed. .29 II. Materials and Methods Screening Procedure: Within 48 hours of eclosion, males of isogenic Oregon-R and Samarkand stocks were collected and fed 0.025M ethyl methanesulfonate (EMS) dissolved i n 1% sucrose for 24 hours (Lewis and Bacher, 1968). The mutagenic effectiveness of each treatment was assayed by crossing ten of the EMS-treated males to attached-X-bearing females at room tempera-ture (RT, 22° + 1°C) and scoring the sex ratio. Since the sex-linked lethal frequency reflects the frequency of autosomal lethals (Wallace, 1951), crosses to detect autosomal mutants were made only i f the sex ratio (males/females) of offspring i n the attached-X test was less than 0.50. The complete screening protocols for the detection of dominant temperature-sensitive (DTS) lethal mutations on chromosome 3 are shown in Figure 1. This procedure i s similar to the protocol used for the detection of DTS-lethals on chromosome 2 (Suzuki and Procunier, 1969) with the substitution of chromosome 3 markers, TM2/CxD, Sb Ser/Xa, or G1/TM3. These various balancers have dominant visible phenotypes and are lethal when homozygous (for a complete description of the mutants and chromosomes used, see Lindsley and Gre l l , 1968). The important proper-ties of mutations and rearrangements used in the studies included i n this and i n following chapters are summarized in Appendix 3 (for a complete description see Lindsley and Grell, 1968). FIGURE 1 Genetic crosses f o r the detection of dominant temperature-s e n s i t i v e l e t h a l s on chromosome 3 30 •/+<f (0-025M EMS)x TM2/CxD or I •7CxD<TxTM2/Cxp? i Bo SbUbx/Xa i or Co GtSbes/TM2 I +VSbUbx^xSb/TM2? +«/GlSbes<? x Gl/TM3,Sb$ery 1 I at 22°C for 4 days - then transfer parents to fresh vials at 29°C 22°C ^ score for presence of non-CxD 29°C — progeny score for absence of non-CxD progeny 22°C 22°C score for presence of progeny 29°C 29°C score for absence of progeny Repeat Steps © and © Repeat Cross at 17°C 32 Several lines were maintained for each confirmed DTS-lethal and these were tested for lethality at 29°C every one or two generations. In addition, for each experiment, control crosses were made to test for the presence of the DTS's. For example, in the mapping crosses, DTS/III- ple and DTS/steroca males were testcrossed at both 22° and 29°C. In this way, any contamination or modification of the DTS effect (due to change in residual background) could be detected. Only lines showing 100% lethality at 29°C were retained. DTS The mutations recovered are designated as L(3)X (where X i s the specific number of the mutation). For this and subsequent chapters, reference to specific mutations w i l l be made by the abbreviation of DTS-X. Each of the confirmed DTS-lethals was also tested for v i a b i l i t y and homozygosis at 17°C. Unexpectedly, four temperature-sensitive mu-tations which appeared to be lethal at 29°C only when individuals were heterozygous for TM2 were recovered and confirmed. In crosses at 22°C, such heterozygotes survive. Flies heterozygous for one of these chromo-somes and CxD, TM3 or a wild-type chromosome are viable at 29°C. Studies on these four mutations w i l l be discussed in Chapter.5. Genetic Localization of Confirmed DTS-lethals; The genetic positions of the DTS-lethals were determined relative to the recessive markers carried by the " I l l - p i e " (ru h. s_t jr_ ss_ e ) and "steroca" (st e s ro ca) stocks (Lindsley and Grell, 1968). Females heterozygous for each DTS-lethal and either III-ple or steroca were test-crossed at 22°C and 29°C. At least 40 vials containing single females and three males were set up for each DTS-lethal. 33 V i a b i l i t y of the DTS-lethals i n t r i p l o i d s : In order to determine whether two doses of a wild-type a l l e l e are s u f f i c i e n t to overcome the l e t h a l e f f e c t of a DTS mutation at 29°C, DTS-bearing males were mated to t r i p l o i d females carrying an attached-X chromosome (homozygous f o r e i t h e r y_^ w^_ e£ sc_ or y_ w f a 1 1 0 ) and the multiply-inverted rod X chromosome, FM6 (marked with y_ B_) . This cross was: XX, sc wf_ ec or y_ w fa n°/FM6, y 3 1 d s c 8 dm B;3A+ ? x +/Y; DTS/TM3, Sb Ser d* . The expression of Sb_ i n t r i p l o i d s permitted the d i s t i n c t i o n of DTS-bearing from TM3-bearing progeny. The absence of DTS-bearing d i p l o i d progeny at the r e s t r i c t i v e temperature confirmed the presence of the DTS-lethal i n the male parent. The presumed t r i p l o i d females r e -covered at 29°C were i n d i v i d u a l l y tested f o r the presence of the attached-X chromosome i n order to confirm t h e i r t r i p l o i d c o n s t i t u t i o n . Determination of the E f f e c t i v e L e t h a l Phase (LP) and Temperature-Sensitive  Period (TSP) of the DTS-lethals; growth curves of the DTS-lethals at  22°C and 29°C. The e f f e c t i v e l e t h a l phase, that i s , the developmental stage at which death occurs at 29°C, was determined for each of the DTS-lethal stocks. One to two hundred p a i r s of DTS-bearing f l i e s from each stock were placed i n empty quarter-pint milk b o t t l e s inverted over p e t r i plates containing standard Drosophila medium. Eggs were c o l l e c t e d w i t h i n 1-2 hour i n t e r v a l s . Duplicate cultures were simultaneously c o l l e c t e d and maintained at 22°C and 29°C and inspected every 12 hours f o r developmen-t a l stages reached and the onset of death. 34 In order to determine the temperature-sensitive period (TSP) , the developmental interval during which the organism is irrevocably committed to death by the restrictive temperature, a series of reciprocal shift-up and shift-down experiments were performed (Suzuki and Procunier, 1969; Tarasoff and Suzuki, 1970). Preliminary experiments were initiated with eggs 0-2 hours old and a shift interval of 12 hours. More accurate de-terminations of the TSP were made by initiating experiments with cultures synchronized at discrete developmental stages - newly hatched/molted f i r s t , second or third instar larvae, late third instar larvae, or pre-pupae. During the latter stage, the larvae become immobilized, the an-terior spiracles evert but the larval skin is s t i l l white. Prepupae were removed from the side of the dish using a paint brush which had been moistened in a semi-liquid mixture of Drosophila medium and distilled water. Careful manipulation and transfer of the prepupae to the side of a vial resulted in complete survival. For shifts later than the prepupal stage, pupal development was determined by the appearance of specific adult appendages and pigmentation. The earliest time at which a shift-down fails to yield adults delineates the initiation of the TSP, where-as the first culture to yield adults in a shift-up marks the end of the TSP. 351 III. R e s u l t s Ten confirmed DTS-lethal mutations were recovered from 25,000 c h r o -mosomes t e s t e d . This frequency of DTS-lethals (0.04%) c o n t r a s t s s h a r p l y w i t h the frequency of 0.34% f o r DTS-lethals on chromosome 2 (Suzuki and P r o c u n i e r , 1969). Three of the mutations were l o s t and of t h e s e , two (DTS-9 and DTS-10) rendered females s t e r i l e at both 17°C and 22°C and the r e f o r e would not have been mappable. The other DTS (DTS-8) was l o s t a f t e r i t was found to k i l l i n the second i n s t a r and g e n e t i c a l l y l o c a l i z e d to the l e f t of ss_ ( s p i n e l e s s - 56.7). The present assignment of numbers to each mutation was made a f t e r the completion of the crossover experiments. The r e l a t i v e v i a b i l i t y of each of the DTS-lethals was determined by the r a t i o of the number of DTS/A (where A r e f e r s to e i t h e r TM3 or Gl) or DTS/B (where B_ r e f e r s to e i t h e r Gl Sb ef_ or TM2) o f f s p r i n g to the t o t a l number of progeny recovered from a cross of DTS/TM3 (or DTS/TM2) males by G1/TM3 (or Gl Sb e /TM2) females. The values obtained f o r c o n t r o l crosses i n which males heterozygous f o r TM3 or TM2 and e i t h e r an Oregon-R or g Samarkand chromosome were crossed to G1/TM3 or G l Sb e /TM2 females, r e -s p e c t i v e l y , were very c l o s e t o the 0.67 r a t i o of + - be a r i n g progeny to the t o t a l p r e d i c t e d by Mendelian expectations (Table 1 ) . The v i a b i l i t i e s of the DTS-lethals were determined r e l a t i v e to the observed c o n t r o l values f o r the w i l d - t y p e stock from which the DTS's were d e r i v e d . A l l of the DTS mu-t a t i o n s are comp]etely l e t h a l at the r e s t r i c t i v e temperature and have a v i a -b i l i t y g reater than 90% tha t of the w i l d - t y p e under pe r m i s s i v e c o n d i t i o n s . The approximate g e n e t i c p o s i t i o n s of the DTS-lethals are shown i n Figure 2 and the data used i n these determinations are summarized i n Table TABLE 1 Relative Viabilities of Flies Bearing • or L(3)X' Chromosomes aC Different Temperatures Cro3S A: + or DTS/TK3 <* x G1/TM3 2 Cross B: + or DTS/TM2 o* x Cl Sb WTM2 ? DTS Stock of Origin Cross Made 17° C 22°C 29°C Total Progeny Progeny Ratio" 7. of wild type Total Progeny Progeny Ratio * 7.o f wild type Total - Progeny Progeny Ratio* 7. of wild type Oregon-R A 898 0.63 1846 0.67 3718 0.72 Oregon-R B '396 0.65 846 0.70 969 0.65 Samarkand A 927 0.64 1527 0.67 1608 0.72 Samarkand B 532 0.66 1072 0.66 1063 0.68 D T S - l Oregon-R B 888 0.70 107.5 2389 0.62 92.5 858 - 0 DTS-2 Samarkand A 174 0.65 101.6 1011 0.62 92.5 582 m 0 DTS-3 Samarkand 3 908 0.64 97.0 3449 0.66 100.0 496 0 DTS-3 Samarkand A 473 0.71 106.0 890 - 0 DTS-4 Samarkand B 992 0.63 95.5 1869 0.65 98.5 593 m o BTS-4 Samarkand A 1419 0;66 98.5 319 m 0 DTS-5 Samarkand B 688 0.62 93.9 950 0.63 95.5 723 m 0 DTS-5 Samarkand A 285 0.60 89.6 261 - 0 DTS-6 Samarkand B 159 0.65 98.5 985 0.65 98.5 1163 0 DTS-7 Samarkand A 1369 0.64 95.5 427 m 0 *Prog«ny ratio: ratio of + or pTS-bearlng progeny to total offspring. FIGURE 2 Genetic positions of the DTS-lethal mutations on chromosome 3 • - centromere 37 DTS-lethals Genetic position 0 Markers ru 756 8 1 3 IJL_—h 1—L—L 1—>j 1 • • • i h -44 48 58-5 707 911 1007 st pp ss e ro ca 39 2. For both the wild-type and DTS-chromosomes, there was a large v a r i a -t i o n i n the values obtained f o r the map distances between the markers of the I I I - p l e and steroca chromosomes. I t has been shown, however, that d i f f e r e n c e s i n genetic background can greatly a f f e c t crossover frequencies (Suzuki, 1963). Since the DTS-stocks contained X, 2 and 4 chromosomes carry i n g p o s s i b l e mutations and/or chromosomal abnormalities, there are no adequate control values with which the DTS's can be compared. The DTS-lethals which could be mapped are seen to be d i s t r i b u t e d throughout the t h i r d chromosome i n eight d i s t i n c t p o s i t i o n s , a pattern very d i f f e r e n t from the highly c l u s t e r e d d i s t r i b u t i o n of mutants on chromosome 2 (Suzuki and Procunier, 1969). In a d d i t i o n , the Minute phenotype associated with DTS-1 could be seen to segregate with t h e - l e t h a l phenotype suggesting that a s i n g l e mutation was responsible f o r the two c h a r a c t e r i s t i c s . DTS-6 females are almost s t e r i l e at 22°C but s h i f t i n g of eggs l a i d by DTS-6/III- ple females mated to I I I - p l e males at 22° to 29°C permitted approximate l o c a l i z a t i o n of the mutation. However, too few progeny were obtained to determine whether the Minute phenotype was also associated with the locus determining l e t h a l i t y . Although the genetic p o s i t i o n s of the DTS-lethals were found to be d i s t i n c t from one another, i t was decided to carry out complementation tests i n order to e s t a b l i s h whether any of the mutations might be f u n c t i o n -a l l y r e l a t e d . Crosses were made to generate a l l p o s s i b l e heterozygotes f o r two d i f f e r e n t DTS mutations at 22°C. These studies revealed that a l l but one of the combinations survived under permissive conditions (Table 3). Trans-heterozygotes f o r DTS-5 and DTS-7 d i d not survive at e i t h e r TABLE 2 Crossover results from testcroases of +- atad DTS-bearing females at 22°C and 29°C Stock Temp. ru • /+ h h + /+ st. % S t + /• j£ % p P *•/• ss % 1 s „ ss •/+ e_ % ro'*/* ca % Scored Map Position Oregon-R 220 29° 22° 467/316 W*/150 22.3 20.7 400/335 l98/178 20.9 22.6 76/69 49/55 4.1 6.3 184/215 H9/156 11.3 16.6 201/298 101/138 14.2 14.4 250/229 23.2 10*/104 10.1 3518 1661 2063 Samarkand 22° 29° 22° , 29° 354/230 257/149 24.7 22.7 313/197 181/178 21.2 20.1 *»/33 13/10 3.4 1.3 Jtl/140 61/73 11.7 7.5 128/215 U2/131 14.3 13.6 210/264 5^9/413 23.1 24.9 109/106 236/252 13.0 11.3 2397 1789 3767 1904 DTS-1 22° 29° 25/89 47/43 16.3 18.9 51/79 31/59 18.6 18.9 8/11 2/14 2.7 3.4 25/42 V65 9.6 14.5 28/70 7/90 14.0 20.4 693 475 78 DTS-2 22° 29o 158/173 18/234 20.4 24.3 233/136 215/80 22.1 28.5 52/29 44/2 4.9 4.4 118/105 107/31 13.4 13.3 106/ii8 103/so 13.4 14.8 1663 1026 33.4 DTS-3 220 29° 205/l64 63/131 21.7 23.4 124/117 78/0 14.1 9.4 1704 330 84.5 DTS-4 22° 29° 215/205 17/209 24.2 23.5 305/X26 291/4 24.9 26.8 41/21 I8/2 3.6 2.1 128/92 105/27 12.7 13.7 124/35 13.2 16.5 1734 961 24.5 DTS-5 22° 29° 604/568 36/361 21.6 17.0 535/471 5/595 18.9 25.6 104/82 62/5 3.5 2.9 436/308 303/e 13.9 13.3 329/397 34I/17 13.6 15.2 5336 2340 44.3 DTS-6 29° 0/30 27.5 0/22 20.2 O/o < 1 12/0 11.0 I8/2 18.3 109 »t-pP DTS-7 22» 290 266/229 u/121 25.5 23.8 238/178 13/U8 21.4-23.6 40/36 23/3 3.9 4.7 146/U9 72/5 13.6 13.9 124/i59 72/8 14.6 14.4 1944 554 42.3 DTS-8 29° TS/ib9 26.8 2b/lS6 20.0 3/t4 1.9 14.6 13.4 910 55.5 * X of t o t a l progeny TABLE 3 Ratios of DTS-a/DTS-b to t o t a l progeny from the cross DTS-a/TM3 X DTS-b/TM3 at 22°C DTS-b DTS-a DTS-1 DTS-2 DTS-3 DTS-4 DTS-5 DTS-6 DTS-7 DTS-1 0/197 DTS-2 43/114 79/159 DTS-3 119/331 31/92 0/375 DTS-4 15/81 35/87 34/89 0/427 DTS-5 30/85 50/125 33/111 57/141 0/306 DTS-6 30/102 68/214 45/150 69/225 71/281 — DTS-7 51/140 76/198 62/181 77/217 0/596 55/182 0/327 42 17 C or 22 C, although these mutations probably are not a l l e l i c since DTS-7 maps 1.7 units to the l e f t of sjt (based on 132 recombinants between h and st) and DTS-5 maps 0.3 units to the right of s_t (based on 67 recom-binants between s_t and p p). Although both mutations have lethal phases in the pupal stage, these are phenotypically distinguishable. In addition the TSP for DTS-7 extends from the end of the late third instar stage u n t i l shortly after pupation, whereas that for DTS-5 ends abruptly with pupation (Figure 3). The developmental properties of each DTS-lethal-bearing chromosome i n homozygotes are shown i n Table 4. DTS/Inv males were crossed to DTS/Inv (where Inv refers to either TM2 or TM3) females and the rates of hatching of the eggs and of eclosion of their offspring were determined. Eggs from the latter crosses were collected at 22°C as described. First i n -star larvae, within two to six hours of hatching, were collected from plates and placed into v i a l s . The extent of egg hatchability could be calculated from the ratio of larvae to eggs. The number of pupae and fi n a l l y the number of adults eclosing provided an indication of the sur-vival of the larvae collected. Since TM2 homozygotes died as larvae and TM3 homozygotes died during the egg stage, i t was possible to determine the lethal phase of the DTS homozygotes from the above information. Only DTS-2 survived i n the homozygous condition, although failure of the other DTS-homozygotes to survive may have been caused by recessive lethals i n -duced elsewhere on the chromosomes. The v i a b i l i t i e s of triploids carrying the DTS-lethals are shown in Table 5. Although the yields per v i a l were low, tests at 22°C of the FIGURE 3 The effective lethal phase (LP) and temperature-sensitive period(s) (TSP) of each DTS-lethal mutation - LP 43 r — r -jggsssss c o —> JZ .3 I cn r~ -|rJL^[i i^i..inmii!UMia 1 1st 1 2nd j 3rd 1 *~ EGG LARVAL IN STAR PUPA Developmental Stage |dies[survtves IMAGO TABLE 4 Determination of the E f f e c t i v e Lethal Phase of homozygotes f o r the DTS-lethals at 22°C Stocks Eggs l s t s Adults % l s t s : Eggs % Adults: l s t s % Adults: Eggs Homozygous DTS: Oregon-R/TM3 851 590 540 69.3 91.5 63.5 Viable Samarkand/TM3 700 467 396 66.7 84.8 56.6 Viable DTS-1/TM3 985 670 428 68.0 63.9 43.5 Egg l e t h a l DTS-2/TM3 624 468 437 75.0 93.4 70.0 Viable DTS-3/TM2 1586 519 436 32.7 84.0 27.5 Egg l e t h a l DTS-4/TM3 1092 590 535 54.0 90.7 49.0 Egg l e t h a l DTS-5/TM2 409 363 146 88.8 40.2 35.7 1st i n s t a r l e t h a l DTS-5/TM3 877 555 358 63.3 64.5 40.8 1st i n s t a r l e t h a l DTS-7/TM3 415 285 179 68.7 62.8 43.1 1st i n s t a r l e t h a l TABLE 5 Progeny from the cross XX, y£ sc ec or £ w fa n o/rMS. y 3 1 d sc£ dm B;3A,+ 8 x +/Y; .3*/TM3,. Sjb Ser 4 at 22°C and 29°C (where 3* represents a chroino3orae 3 carrying either a wlld-type or DTS lethal allele) 3N ? . 2N ? 2N <f 220 29 0 22° 29° 22® 29© •3* TK3 3* TM3 3*. TM3 3* TM3 3* TM3 3* TM3 Oregon-R 27 23 A5 37 44 48 65 52 39 29 AO 21 Samarkand Al 30 39 15 57 45 40 32 42 31 22 23 DTS-1 13 20 2 29 - 14 DTS-2 3 6 • - 14 m 5 DTS-3 23 26 - 18 - . .29 DTS-4 A4 35 1 28 49 46 - 26 m 17 1 9 - ; 14 - 9 DTS-5 20 13 m 16 9 17 14 - 16 m 7 DTS-6 2 7 - 10 m 6 DTS-7 42 35 33 27 64 37 1 26 40 27 (3)t 8 10 13 1 19 9 fthese males had a characteristic phenotype of rare "escaper" oV from this stock which are recovered at 29° -very heavy wing venation, abnormal leg segmentation, rough eyes. 47 t r i p l o i d progeny recovered at 29 C proved the 3N genotype of these fe-males. It can be seen that DTS-4 has a very reduced v i a b i l i t y even i n tr i p l o i d females at 29°C, whereas the other DTS-lethals appear to sur-vive at a rate comparable to their sibling non-DTS bearing triploids and triploids bearing a wild-type third chromosome derived from an Oregon-R or Samarkand stock. The LPs and TSPs delineated for each of the DTS-lethals studied are > o summarized i n Figure 3. DTS-1, which is lethal at 29 C during the late pupal to early adult stages, has a TSP extending for about 100 hours prior to death. The large spread i n the TSP for this and other DTS-lethals may be attributable to asynchrony i n the cultures. In terms of developmental stages, the TSP coincides with the interval following ever-sion of the cephalic complex u n t i l the fully-developed imago i s formed. The LP of DTS-2 heterozvgotes occurs during the early stages of pupa-tion, as indicated by the successful eversion of the leg and wing discs but failure of the cephalic complex to evert. Although this phenotype resembles that of the mutation l(2)crc (Fristrom, 1965) , individuals heterozygous for both l(2)crc and DTS-2 were viable at 22°C, indicating an absence of a synergistic effect between the two mutations. The LP i s preceded by a TSP persisting for about 15 hours. In crosses of DTS-2/TM3 f l i e s , the TSPs for lethality of the homozygotes and heterozygotes are identical. However, in the homozygous DTS-2 stock, only 0-5% of the eggs la i d at 29°C hatch. Those ' escapees' (Hadorn, 1961) that do hatch at the restrictive temperature continue to develop u n t i l pupation, at which time death occurs. These observations suggest an embryonic 'boundary' for lethality which is overcome by the presence of a wild-type a l l e l e in the parents. Therefore, crosses were carried out to determine the nature of the parental influence on embryonic leth a l i t y . DTS-2/DTS-2 males were crossed to +/+ females at 29°C and of 1786 eggs collected, 1554 hatched and 1321 died as pupae. On the other hand, of 2316 eggs collected from the reciprocal cross of DTS-2/DTS-2 females by +/+ males, only 52 hatched, thereby indicating that the embryonic le t h a l i t y of heterozygotes as well as of homozygotes i s maternally determined. Moreover, preincubation of DTS-2/DTS-2 females at 29°C for 6 hours prior to egg collection results in complete egg le t h a l i t y , suggesting that the maternally deposited factor for v i a b i l i t y i s thermolabile. The lethal phase of DTS-3 occurs during the third larval instar. At 29°C, this stage may be prolonged for some fourteen days before death f i n a l l y ensues. As these individuals age, fat deposits become depleted and the salivary glands degenerate. This suggests that the larvae prepare for metamorphosis since histolysis of the larval salivary glands normally takes place immediately prior to and during this transition stage. By using cultures synchronized at the f i r s t larval instar, i t was possible to establish a TSP extending for about f i f t y hours beginning just prior to the second to third instar molt and ending about midway through the third instar stage. Inspection of cultures of DTS-4 heterozygotes indicates that the LP for cultures initiated at 29°C occurs during the egg and very early f i r s t instar stages. Eggs collected immediately after the parents are shifted to the restrictive temperature yield about 15% hatch. The larvae which 49 do emerge never assume the elongated shape of normal f i r s t instar larvae, but rather remain the approximate size and shape of eggs. After twelve to sixteen hours, these larvae cease movement and death follows. When parents are preconditioned at 29°C for twenty-four hours and eggs are then collected, no hatching occurs although some embryonic development may be detectable. The shifts-up reveal the sensitivity of the mutant to the high temperature throughout the embryonic and larval stages u n t i l midway through the pupal stage. At 29°C, DTS-5 heterozygotes die as pupae which phenotypically resem-ble the mutant halfway (Lindsley and Grell, 1968). Within the pupal case, the anterior portions of the adult are f u l l y differentiated but the abdo-minal region f a i l s to develop and histolyzes. The TSP begins during the third larval instar and ends with puparium formation - a period lasting approximately 36 hours. The lethal phase for DTS-6 also occurs during the pupal stage. At 22°C the developmental time of DTS-6 heterozygotes i s longer than that of the wild-type by four to six days. Although the shift experiments did not allow a precise delineation of the TSP for this mutation, i t would appear that the sensitive interval occurs during the larval period. When f l i e s carrying DTS-7 are maintained at 29°C, death occurs in the puparium. Dead late pupae (those which appear to have completed the development of the imago) as well as early pupae in which extensive his-tolysis has occurred, appear to be the expression of the LP of DTS-7. The TSP for this mutant immediately precedes the lethal phase, as indicated in Figure 3. so; The extent of development of some of the DTS-lethals was examined at different temperatures (Table 6). For each DTS-lethal, a set of ten v i a l s , each containing one pair of adults heterozygous for the DTS-lethals, was placed at 22°, 25°, 26°,27°, 28° and 29°C. After four days, the parents in each set were removed. Cultures were periodically examined for the developmental stages reached and the time of death of the DTS-lethals at each temperature by the method described previously (Tarasoff and Suzuki, 1970). For DTS-1 and DTS-2, complete v i a b i l i t y was attained for cultures raised at 26°C or lower, whereas DTS's 5_ and 7_ were viable even at 27°C. In addition, the LPs of DTS's 5_ and 6_ were similar at 28° and 29°C, but those of DTS's 1_, 1_ and 3_ differed with the temperature - with further development occurring at the lower temperatures. TABLE 6 The e f f e c t i v e l e t h a l phases of DTS-lethal mutations in heterozygotes at d i f f e r e n t temperatures DTS # i 24°C 25°C 26° C 27°C 28°C 29°C + extreme Minute phenotype l a t e pupa, e a r l y adult l a t e pupa 2 heterozygous parents semi-viable adult l e t h a l l a t e pupa c r c - l i k e c r c - l i k e 2 homozygous parents pupa larva egg egg late pupa, pseudopupa pseudopupa t h i r d i n s t a r halfway-like, l a t e pupa halfway-like, late,pupa h i s t o l y t i c & l a t e pupae h i s t o l y t i c & l a t e pupae +* : denotes v i a b i l i t y 52 I V . D i s c u s s i o n The i s o l a t i o n , i n D r o s o p h i l a melanogaster,of a c l a s s of dominant mutations which i s c o n d i t i o n a l l y l e t h a l has permitted t h e i r g e n e t i c and developmental a n a l y s e s . Studies on the nature of dominant l e t h a l s had p r e v i o u s l y been r e s t r i c t e d t o the p r o d u c t i o n of gross chromosomal abnor-m a l i t i e s by I r r a d i a t i o n ( M u l l e r , 1927), chemicals (Von B o r s t e l and Rekemeyer, 1959) and g e n e t i c manipulation ( N o v i t s k i , 1951). Death of the i n d i v i d u a l s c a r r y i n g the dominant l e t h a l thereby prevented f u r t h e r charac-t e r i z a t i o n of that mutation. Consequently, "dominant l e t h a l i t y " has been a d e s c r i p t i v e category, probably encompassing a wide v a r i e t y of g e n e t i c and chromosomal changes. Suzuki and P r o c u n i e r (1969) and the present study i n d i c a t e that a low p r o p o r t i o n of mutations induced by EMS may a l s o behave as dominant l e t h a l s . Although EMS i s known to produce a preponder-ance of missense mutations i n phages ( K r i e g , 1963) and few chromosomal r e -arrangements i n Dr o s o p h i l a (Kim and Snyder, 1968), EMS-induced d e l e t i o n s have been recovered ( W i l l i a m s o n , 1970, E.B. L e w i s , unpubl.). Although some t e m p e r a t u r e - s e n s i t i v i t y could be the consequence of EMS-induced d e l e -t i o n s ( F r i s t r o m , 1970), c y t o l o g i c a l and g e n e t i c s t u d i e s i n d i c a t e that f l i e s heterozygous f o r d e l e t i o n s of extensive p o r t i o n s of the genome are v i a b l e (Lefevre and Green, 1971, L i n d s l e y et a l . , 1972), whereas even s m a l l homozygous d e f i c i e n c i e s may be c e l l l e t h a l s (Demerec, 1934). Moreover, the v i a b i l i t y of DTS-2 as a homozygotes i n d i c a t e s that t h i s mutation i s not a d e f i c i e n c y . F i n a l l y , the g e n e t i c p r o p e r t i e s of s e x - l i n k e d EMS-induced t s - l e t h a l s support a missense b a s i s f o r the mutation ( S u z u k i , et a l . , 1967, S u z u k i , 1970, and Suzuki and P i t e r n i c k , manuscript submitted f o r p u b l i c a t i o n ) . That i s , t s mutations are much more frequent among EMS-53 mutations than among those induced by mitomycin C or Y-rays: a l l EMS-induced ts-lethals map as point mutations whereas rearrangements have been found among EMS-induced non-ts-lethals and Y-ray-induced ts-lethals. In addition, a ts mutation of vermilion which affects the activity of tryptophan pyrrolase has been recovered (Camfield and Suzuki, 1972). X-irradiation-induced and genetically-contrived dominant lethality resulting from duplications and deletions invariably results in early embryonic death (Von Borstel and Rekemeyer, 1959). Our studies, on the other hand, yielded dominant lethals which permitted considerable develop 3 ment beyond the embryonic to the larval and pupal stages. Although the relative rarity of DTS-lethals requires large screening programs for their recovery, l o c i which are extensively duplicated may only be genetically detectable among DTS mutations. The continued lethality of DTS-4 in triploids at 29°C shows that a mutation can be dominant over at least two wild-type alleles. The possible molecular basis for dominant lethality merits brief mention, even though our experiments do not permit any definitive inter-pretation. Genetically-controlled cellular functions which tolerate l i t -t le deviation from wild-type levels of product might be detectable only by the recovery of DTS mutations. Similarly, mutations which would cre-ate a high level of cellular "noise", such as defects i n polymerases or in the transnational apparatus could be dominant lethals. Indeed, Guthrie ejt a l . (1969) found that cold-sensitive lethal mutations i n E_. c o l i which affected ribosome assembly, were dominant. Such defects might be expected to show a continuous temperature-sensitivity similar to that 54, of DTS-4. In addition, genes controlling regulatory molecules, such as repressors, have been found to have dominant alleles (Sadler and Novick, 1965). The aggregation of defective polypeptides in enzymic or structur-al polymers can result in loss of biological activity of the complex i n spite of wild-type subunits (Garen and Garen, 1963; Bernstein and Fisher, 1968). A distinction among these alternatives w i l l be possible only upon detection of the primary lesion imposed by the DTS-lethals. The discrepancy in recovery frequency of DTS-lethals on chromosomes 2 (0.34%) and 3 (0.04%) becomes less dramatic upon inspection of the gene-t i c distributions of the mutations. A high frequency of dominant lethals of both heat- and cold-sensitive types on chromosome 2 occur i n a "hot spot" within a small genetic segment to the right of dp_ (Suzuki and Pro-cunier, 1969; Rosenbluth et a l . , 1972). DTS-lethals on chromosome 3, on the other hand, were distributed throughout the chromosome. In terms of different sites, the frequency on chromosome 2 i s 0.16%. Two mutants, DTS-1 and DTS-6, are worth special mention. Heterozy-gotes for either mutation at 22°C are phenotypically identical to the Minute class of mutations; i.e., the developmental period i s retarded and bristles on the thorax are very short and slender. Furthermore, DTS-1 enhanced the dominant effect of j3£ and resulted in synthetic semi-lethality with Dl_ (see Lindsley and Grell, 1968, for a description of Minutes). Both of these effects are characteristic of Minute mutations. K.C. Atwood (cf Lindsley and Grell, 1968) has suggested that Minutes may represent mutations in sites of tRNA synthesis. Support for this proposal has re-sulted from the demonstration by iri situ tRNA hybridization to salivary 55 gland chromosomes that hybridization does appear to occur near bands known to be associated with Minutes (Steffensen and Wimber, 1971). Re-cently, Smith et a l . , (1970) showed that ts mutations could occur in tRNA genes. The pupal TSP of DTS-1 provides an intriguing incentive for determining whether the locus does, in fact, produce a thermolabile tRNA. This is currently under investigation. The utility of ts mutations in analyses of development has been re-cently reviewed (Suzuki, 1970). The recovery of DTS-lethals provides another tool in the extensive array available for chromosome mechanics in Drosophila. Thus, translocations between the Y chromosome and an egg lethal DTS allow the early elimination of male zygotes. Wright (1970) has utilized DTS-lethals to select specific classes of heterozygotes. In cultures of mammalian cells, the recovery of ts-lethals may include DTS-lethals that could allow analyses of cell cycles in vitro (Thompson et a l . , 1970). Finally, the selection of DTS-lethals with continuous sensitivity to high or low temperatures encountered in the wild might be a useful genetic tool for pest control. 5.6.. CHAPTER 4 THE DEVELOPMENTAL PROPERTIES OF DOMINANT TEMPERATURE-SENSITIVE LETHALS ON CHROMOSOME 3 OF DROSOPHILA I. Introduction Since Muller, i n 1927, f i r s t suggested that the partial s t e r i l i t y of irradiated Drosophila males resulted from the production of dominant lethals in the sperm, the biological basis for dominant lethality has been the subject of extensive investigation and speculation. The results of these studies have led to the postulate that dominant lethals arise pre-dominantly through major changes in chromosomal structure (Fahmy and Fahmy, 1954). Pontecorvo and Muller (1940) have suggested that since both small deficiencies and symmetrical exchanges are viable i n hetero-zygotes, dominant lethality must involve more drastic changes, in the form of large deletions and asymmetrical exchanges. Dominant zygotic lethality was seen to occur mainly during the egg stage for lethals induced both by X-irradiation (Muller, 1927; Hanson, 1928; Sonnenblick, 1940) and by chemicals (Fahmy and Fahmy, 1954). In the latter studies, cytological observations of unhatched eggs f e r t i l i z e d by mutagenized sperm revealed cleavage disturbances resulting from abnormal mitotic configurations. The kinetics of X-ray induced dominant let h a l i t y suggests that both single and multiple chromosome breaks are instrumental in causing heterozygous zygotic death: the former by sister chromatid fusion (Muller, 1940) and the latter by asymmetrical exchanges between heterologues, leading to the formation of dicentric chromosomes and acentric fragments (Fahmy and 57 Fahmy, 1954). Such alterations in chromosome structure result i n mitotic disruptions which may be accompanied by chromosome loss and arrest of embryonic development. More recently an examination of the molecular basis for dominance i n bacteriophage T4D has led Bernstein and Fisher (1968) to suggest that the polymerization of mutant and wild-type polypeptides in enzymic or structural polymers i s responsible for the production of dominance in micro-organisms. It is feasible that such a mechanism might also be operable in higher organisms, thus accounting for some instances of dominant lethality. It i s clear, however, that such cannot be the case in those instances in which deletions are known to be involved. The i n a b i l i t y to propagate dominant lethals makes them inaccessible to direct genetic analysis and thus the extent to which chromosomal aber-rations contribute to the production of dominant lethals cannot be determined. The recovery of a class of conditional dominant lethals (dominant temperature-sensitive (DTS) lethals) (Suzuki and Procunier, 1969; Holden and Suzuki, 1973) provides a tool by which the genetic characterization and developmental analysis of dominant lethals can be made. The basic properties of DTS-lethals on chromosome 3 i n Drosophila have been reported i n the previous chapter. Eight of ten such lethals have been localized to specific regions of the chromosome and probably define eight distinct l o c i . Complementation tests revealed a functional relatedness between DTS-5 and DTS-7 since these mutations f a i l to 58 complement under permissive growth conditions. Only one mutant-bear-ing chromosome (DTS-2) survives i n homozygous condition and, i n addi-t i o n , such females express a maternal e f f e c t on embryonic v i a b i l i t y . DTS-1 and DTS-6 have phenotypes reminiscent of the Minute c l a s s of mutations. Each of the seven mutants examined was found to e x h i b i t a c h a r a c t e r i s t i c phenotype with respect to the time of death under r e -s t r i c t i v e conditions (29°C) and the temperature-sensitive period(s) during development. This chapter discusses the d e t a i l e d developmental e f f e c t s of each DTS mutation as w e l l as other genetic studies c a r r i e d out to f u r -ther characterize some of the mutations. 59 II. Materials and Methods The dominant temperature-sensitive (DTS) l e t h a l mutations on chromosome 3 were induced by eth y l methanesulfonate and characterized by the v i a b i l i t y of DTS/+ heterozygotes at 22°C and t h e i r death at 29°C. De t a i l e d genetic analysis and a b r i e f d e s c r i p t i o n of developmental e f f e c t s of these mutations were presented i n Chapter 3. DTS The mutations studied herein are designated as L(3)X where X r e f e r s to the s p e c i f i c number of the mutation. For the purpose of sim-p l i c i t y , we w i l l r e f e r to a mutation by the abbreviated form of DTS-X. Determination of the E f f e c t i v e Lethal Phase (LP) and Temperature-Sensitive  Period (TSP) of the DTS-lethals; growth curves of the DTS-lethals at 22°C  and 29°C. The e f f e c t i v e l e t h a l phase, that i s , the developmental stage at which death occurs at 29°C, was determined f o r each of the DTS-lethal stocks. One to two hundred p a i r s of DTS-bearing f l i e s from.each stock were placed i n empty quarter-pint milk b o t t l e s inverted over p e t r i plates containing standard Drosophila medium. Eggs were c o l l e c t e d with-i n 1-2 hour i n t e r v a l s . Duplicate cultures were simultaneously c o l l e c t e d and maintained at 22° and 29°C and inspected every 12 hours for develop-mental stages reached and the onset of death. The developmental stages of l i v e and dead larvae were determined by p l a c i n g larvae i n a drop of s a l i n e on a microscope s l i d e and squashing with a c o v e r s l i p . The l a r v a l mouthparts and the presence or absence of the s p i r a c u l a r openings pro-vided a means of i d e n t i f y i n g each l a r v a l i n s t a r (Bodenstein, 1950). 60 Since u n f e r t i l i z e d eggs remain opaque and white, dead embryos could be c l a s s i f i e d when they turned brown, an i n d i c a t i o n that some develop-ment had occurred p r i o r to death. Larvae were scored as dead when gentle probing f a i l e d to produce any evidence of external or i n t e r n a l movement. The death of pupae was determined by the f a i l u r e to develop past a d e f i n i t e stage i n pupation, the stages s p e c i f i e d being prepupa, formation of yellow then brown pupal case, eversion of the cephalic complex, eye pigment accumulation, wing pigmentation, presence of thor-a c i c b r i s t l e s , and f i n a l l y , abdominal pigmentation. Each of these stages could be r e a d i l y detected by inspection through the pupal case. In order to determine the temperature-sensitive period (TSP), the developmental i n t e r v a l during which the organism i s i r r e v o c a b l y committed to death by the r e s t r i c t i v e temperature, a s e r i e s of r e c i p r o c a l " s h i f t " experiments was performed. Eggs were c o l l e c t e d i n the manner described for the determination of the LP. A rough estimation of the TSP was made by taking blocks of medium containing 50-80 eggs and t r a n s f e r r i n g them to v i a l s which were e i t h e r l e f t at 22°C or immediately s h i f t e d to 29°C. At successive i n t e r v a l s of approximately 12 hours, three v i a l s of each l e t h a l were s h i f t e d from 22 to 29 C (shift-up) and from 29" to 22°C (shift-down), and inspected p e r i o d i c a l l y f o r developmental stages reached a f t e r the s h i f t . The e a r l i e s t time at which a shift-down f a i l s to y i e l d adults delineates the i n i t i a t i o n of the TSP, whereas the f i r s t c u lture to y i e l d adults i n a s h i f t - u p marks the end of the TSP (Tarasoff and Suzuki, 1970). More accurate determinations of the TSP were made by synchronizing cultures at d i s c r e t e developmental stages. In order to obtain newly en hatched f i r s t i n s t a r l a r v a e , eggs were c o l l e c t e d w i t h i n short i n t e r v a l s of from one-half to one hour's duration. A f t e r the onset of hatching, the cultures were inspected for new f i r s t i n s t a r larvae approximately every half-hour, although more frequent c o l l e c t i o n s had to be made f o r some experiments. Larvae were then gently removed from the plate by means of a probe and placed i n a v i a l containing fresh medium. In general, f i f t y f i r s t i n s t a r larvae, timed w i t h i n a half-hour of hatching i n t h i s way were d i s t r i b u t e d to each v i a l . Although such c o l l e c t i o n s of f i r s t i n s t a r larvae l e d to remarkably well-synchronized cultures throughout development, more r i g o r o u s l y timed cultures were e s s e n t i a l i n the study of c e r t a i n mutants. In such cases, second and t h i r d i n s t a r larvae had to be c o l l e c t e d soon a f t e r moulting. Synchronizing second i n s t a r larvae was f a c i l i t a t e d by p l a c i n g l a t e f i r s t i n s t a r larvae (distinguished by t h e i r size) on a p e t r i d i s h containing standard Drosophila medium. These were then observed every half-hour for the presence of second i n s t a r larvae, which were removed from the plate and placed i n v i a l s , e i t h e r t h i r t y or f i f t y per v i a l (depending on the experiment). Young t h i r d i n s t a r larvae were selected s i m i l a r l y . Late t h i r d i n s t a r larvae are defined as those which ascend the side of the pla t e i n preparation f o r pupation, and may be c o l l e c t e d at that time. With some experience, i t i s possible to c o l l e c t l a rge numbers of i n t e r -moult larvae which represent perhaps the best stage for synchronizing, since i t i s possible to miss some of the l a t e r stage larvae which bore i n t o the medium. 62 The f i n a l stage used to obtain synchronously maturing i n d i v i d u a l s was the prepupal stage. At t h i s time, the larvae become immobilized, the a n t e r i o r s p i r a c l e s evert but the l a r v a l skin i s s t i l l white. Pre-pupae were removed from the side of the dis h using a paint brush which had been moistened i n a semi-liquid mixture of Drosophila medium and d i s t i l l e d water. Ca r e f u l manipulation and transference of the prepupae to the side of a v i a l r e s u l t e d i n complete s u r v i v a l . For s h i f t s l a t e r than the prepupal stage, pupal development was determined by the appear-ance of s p e c i f i c adult appendages and pigmentation. Other Experimental Procedures: The s p e c i f i c d e t a i l s of analyses p e c u l i a r to each of the DTS-lethals are discussed i n the next se c t i o n . 63 I I I Results DTS-1 and DTS-6: Minute-like DTS mutations Two mutations, DTS-1 and DTS-6, when heterozygous f o r t h e i r respect-ive w i l d - type a l l e l e s , exhibited a mutant phenotype of slender b r i s t l e s at 22°C a c h a r a c t e r i s t i c of a c l a s s of mutations c a l l e d Minutes which are found on a l l four chromosomes. This i s of considerable i n t e r e s t i n view of the suggestion that Minute l o c i may represent s i t e s coding for t r a n s f e r RNA (Ritossa e_t a l . , 1966) , a p o s s i b i l i t y f o r which Steffensen and Wimber (1971) have reported supporting evidence based on i n s i t u h y b r i d i -zation of tRNA with s a l i v a r y gland chromosomes. Consequently, DTS-1 and DTS-6 were examined further f o r phenotypic e f f e c t s c h a r a c t e r i s t i c of Minutes. Two t h i r d chromosome Minutes, M(3)S34 (map p o s i t i o n 44.3) and 124 M(3)w (map p o s i t i o n 79.7), are g e n e t i c a l l y located close to the p o s i t i o n s determined for DTS-6 and DTS-1, re s p e c t i v e l y . However, s u r v i v a l of DTS-6/ 10/ M(3)S34 and DTS-1/M(3)w heterozygotes at 22°C indicated that the DTS's were at s i t e s d i s t i n c t from the s p e c i f i e d Minutes. The properties of 12 A M(3)S34 and M(3)w were thus used as controls f o r comparison with the various phenotypes and i n t e r a c t i o n s of DTS-6 and DTS-1. Since some Minutes are known to produce dominant l e t h a l e f f e c t s when combined with the mutations DJL, J or D (see L i n d s l e y and G r e l l , 1968, for discu s s i o n of Minutes), the Minute and DTS stocks were tested f o r such synthetic l e t h a l i t y . Single v i r g i n females from each of the test stocks (and males from DTS-6 stocks) were crossed i n separate v i a l s at 22°C with 3 two males (or females) of the following genotypes: Ly/D , J/ln(2L)NS, and 64 D^ "/TM2, Ubx^"^ (for a complete description of the mutations and rearrange-ments used, consult Lindsley and G r e l l , 1968). A l l progeny emerging from replicate vials were examined for enhancement of the dominant mutant 3 1 phenotypes (D , J_ or Dl ) or for their le t h a l i t y . Preliminary studies 124 indicated that whereas DTS-1 and M(3)w reduced the v i a b i l i t i e s of Dl and J_ heterozygotes, respectively, both M(3)S34 and DTS-6 appeared to 3 reduce the v i a b i l i t y of heterozygous D_ progeny - although results from 2 out of 7 vials for DTS-6 were not consistent with this trend (Table 7). Thus, no positive correlations could be made between the effects of the Minutes and the DTS's i n question on their a b i l i t y to interact with these dominant mutations. Minutes are often located i n the heterochromatic regions associated with deletions. The genetic location of DTS-6 between s_t and pp,which span the centric heterochromatin, makes i t of special interest i n this regard. Since i t is known that small heterochromatic deficiencies can enhance variegation (Prokofyeva-Belgovskaya, 1947), the DTS and Minute m4 124 mutations were tested with the inversion, w , Although M(3)w signi-ficantly increased the eye-mottling phenotype of w /Y males, neither of the DTS's nor M(3)S34 had any effect (Table 7). Most Minutes also induce dominance of recessive mutations affecting venation (such as px and net) or bristles (sc). Flies heterozygous for such recessive mutations and a Minute often express a phenotype charac-t e r i s t i c of homozygous px, net, or sc. DTS-1 was tested for i t s a b i l i t y to produce a S£ phenotype in heterozygous individuals. v_ s_c v f car males were mated to DTS-1/TM3, Sb Ser females (Experimental) and wild-type Oregon-R females (Control). In a l l , three bottles of experimentals and TABLE 7 V i a b i l i t y of Minute and DTS-lethal mutations i n trans-heterozygotes with dominant mutations Mutation Tested: 124 ?-parent: mutation: M(3)w DTS-1 M(3)S34 DTS-6 # %* # %* # %* #** %* #*** % Ly_/p_^  Ly. 121 25.3 55 23.5 80 29.5 78 38.6 17 18.3 D 3 111 23.2 65 27.8 58 21.4 31 15.3 25 26.9 t o t a l progeny 478 234 271 202 93 J/In(2L)NS J 10 8.5 54 29.0 27 35.5 26 18.3 I.n(2L)NS 35 29.7 27 14.5 10 13.2 36 25.3 t o t a l progeny 118 186 76 142 D11/TM2 1 Dl 73 19.4 51 18.5 49 27.1 42 20.7 TM2 82 21.8 79 28.6 41 22.7 48 23.6 t o t a l progeny 376 276 181 203 enhancement of M(3)w w mot t l i n g : +++ ++ * the r a t i o of progeny bearing the s p e c i f i e d Minute chromosome and the t e s t e r chromosome to the t o t a l progeny recovered in the crosses -- expressed as a percentage ** v i a l s 2, 3, 5, 6, 7 * * * v i a l s 1, 4 66 one of c o n t r o l s , each containing ten females and twenty males, were established. A l l cultures were kept at 22°C and the progeny scored f o r the absence, d u p l i c a t i o n , or i r r e g u l a r l o c a t i o n of thoracic and head b r i s t l e s . Although a l l DTS-1 heterozygotes have very reduced b r i s t l e s i z e , a c e r t a i n percentage (5.25%) of the progeny express an extreme Minute phenotype, characterized by small body s i z e and f a i l u r e of the wings to unfold. In the experimental crosses, the parental females were selected according to t h e i r s i z e . Thus, i n b o t t l e 1, the parental f e -males were normal i n body s i z e and wing morphology, whereas the females used i n b o t t l e s 2 and 3 exhibited the extreme Minute phenotype. The progeny of the two types of females were s t r i k i n g l y d i f f e r e n t i n the extent of sc^ enhancement. In b o t t l e 1, about 35% of the female progeny showed enhancement, whereas almost 95% of the progeny from b o t t l e s 2 and 3 had at l e a s t one thoracic b r i s t l e missing, with the majority missing three or four. The TM3 sibs showed no i r r e g u l a r i t i e s i n the th o r a c i c b r i s t l e pattern but often one or more of the o r b i t a l s were absent. This l a t t e r c h a r a c t e r i s t i c was never observed i n non-TM3 progeny. In the c o n t r o l crosses, only 6% of the female progeny had disturbances i n the ; thoracic b r i s t l e pattern and i n a l l cases only a sin g l e b r i s t l e was mis-sing from any p a r t i c u l a r f l y . Here, DTS-1 c l e a r l y behaved as a Minute. Synthetic l e t h a l i t y with c e r t a i n dominant mutations, enhancement m4 of w v a r i e g a t i o n , and induction of dominance of c e r t a i n recessive muta-t i o n s , are properties c h a r a c t e r i s t i c of some, but not a l l , Minutes. There are, however, three properties diagnostic of every mutation i n t h i s c l a s s : slender thoracic b r i s t l e s and prolonged developmental time of 67 heterozygotes and the l e t h a l i t y of homozygotes. As mentioned, both DTS-1 and DTS-6 have the t y p i c a l Minute b r i s t l e phenotype, thus s a t i s -fying the f i r s t requirement. In a d d i t i o n , both are l e t h a l when homozy-gous (although the p o s s i b i l i t y of a second s i t e recessive l e t h a l induced elsewhere on the DTS-6 chromosome has not been c r i t i c a l l y r u l e d out). Observation on t h e i r developmental rates at the permissive tem-perature revealed delays of 2 to 3 days and 4 to 6 days i n e c l o s i o n times f o r DTS-1 and DTS-6 heterozygotes, r e s p e c t i v e l y . DTS-1 heterozygotes ra i s e d continuously at 29°C died during the l a t e pupal and e a r l y adult stages and were also delayed i n the time of puparium formation. S h i f t experiments delineated a TSP f o r t h i s mutant extending f o r about 120 hours p r i o r to death, a period coinciding with the i n t e r v a l following eversion of the cephalic complex u n t i l the f u l l y developed imago i s formed, (Figure 4 ). The l e t h a l phase f o r DTS-6 also occurred during the pupal stage. Although the s h i f t experiments did not allow a p r e c i s e d e l i n e a t i o n of the TSP f o r t h i s mutation, preliminary studies i n d i c a t e a TSP during the l a t e second and e a r l y part of the t h i r d i n s t a r s (Figure 5 ). In order to t e s t whether s e n s i t i v i t y to 29°C was c h a r a c t e r i s t i c of several or a l l Minute l o c i , several stocks carrying Minutes on chromo-some 2 or 3 were tested i n t h i s respect. Of 23 Minute stocks tested, s i x were completely v i a b l e at 29°C while eleven showed from 0 to 10% l e t h a l l a t e pupae at t h i s temperature. Since many laboratory stocks have been found to be s i m i l a r l y susceptible to 29°C, t h i s low degree of l e t h a l i t y FIGURE 4 Determination of temperature-sensitive period (TSP) for DTS-1/TM2 heterozygotes. S h i f t s of 100 to 150 eggs, larvae, or a s p e c i f i c stage, or pupae were made from 22°C to 29°C (x ) and from 29° to 22°C ( ) at the times indicated in order to define the TSP. Since DTS-1/DTS-1 and TM2/TM2 eggs are present i n the early s h i f t s , and TM2 homo-zygotes survive to the l a r v a l stage, a determination of the number of adults eclosing -- expressed as a percentage of the number of pupae -- was made in order to e s t a b l i s h a TSP for the late pupal/ e a r l y adult LP c h a r a c t e r i s t i c of DTS-1 heterozygotes. The times . of the s h i f t s from 29°C to 22°C were adjusted to account f o r the d i f f e r e n t rates of development at the two temperatures by comparing the growth curves at these temperatures. In t h i s way a TSP extending over approximately 120 hours, beginning s h o r t l y a f t e r pupation, was defined f o r t h i s mutant. 68 TSP 4 > EGG LARVAL INSTAR PUPA ADULT FIGURE 5 Determination of temperature-sensitive period (TSP) for DTS-6 heterozygotes Shifts were made using 50 to 100 eggs, larvae (synchronized at the first larval instar), or pupae (synchronized at the prepupal stage) obtained from the following cross: DTS-6/TM2 °"x Gl Sb eS/TM2 ? The ratio of DTS-6 bearing to Gl Sb es/TM2 progeny was used to determine the TSP. The 29° to 22°C shifts were plotted according to the developmental stages shifted (rather than absolute times of the shifts) to take into account the different developmental rates at 22° and 29°C. x x shifts from 22° to 29°C . shifts from 22° to 29°C 70 71 72 i s probably not a r e f l e c t i o n of the Minute character per se. Of the s i x remaining, two had 15 to 25% pupal l e t h a l i t y , two had about 50% v i a b i l i t y with a l e t h a l phase again i n the pupal stage f o r the remaining 3 i n d i v i d u a l s , and the other two, M(2)S2 /SM2 and M(2)S5/SM2, showed very high degrees of pupal l e t h a l i t y . One of these gave f i f t e e n adults out of 70 pupae, and the other showed only 6% (out of 160 pupae) v i a b i l i t y with a LP i n the l a t e pupal stage. I t i s u n l i k e l y that the SM2 balancer chromosome of the l a t t e r two stocks was responsible f o r the temperature-s e n s i t i v i t y since i t i s v i a b l e i n combination with other 2nd chromosomes. If the l e t h a l i t y of these two Minute stocks r e f l e c t s the temperature e f -f e c t on the Minute l o c i themselves, then the s i m i l a r l e t h a l phases of these and DTS' s 1_ and 6_ may i n d i c a t e that they belong to a sub-class of Minutes showing temperature-sensitivity. DTS-2 The LP of DTS-2 heterozygotes occurs i n pupae and i s characterized by the successful eversion of the l e g and wing discs but f a i l u r e of the cephalic complex to evert. In cultures i n i t i a t e d by DTS-2/TM3 parents, the TSP's for l e t h a l i t y f o r both heterozygous and homozygous DTS-2 pro-geny are i d e n t i c a l , p e r s i s t i n g f o r about 15 hours p r i o r to the LP (Figure 6). When DTS-2/DTS-2 parents are used, however, only 0-5% of the eggs l a i d at 29°C hatch. Therefore, embryonic death i s maternally deter-mined, with both heterozygous and homozygous DTS-2 zygotes dying at 29°C when produced by homozygous females (see Chapter 3 ). Since t h i s maternal e f f e c t could be a property of the DTS-2 muta-t i o n i t s e l f or the consequence of a second mutation induced elsewhere on FIGURE 6 Determination of temperature-sensitive period (TSP) for DTS-2 homozygotes and heterozygotes Shifts of 100 to 200 eggs, larvae (synchronized at the first instar), or pupae (synchronized as prepupae), from a cross of heterozygous females to heterozygous males, were made as indicated (29° to 22°C shifts were plotted according to the developmental stages shifted as for Figure 1). Since only 75% of the eggs hatch, for shifts involving eggs an estimate 61 the first instar larvae hatching was made using this figure. Virtually a l l of the first instar larvae eclose at 22°C and reach the LP at 29°C, thus the ratio of adults/ firsts per culture (expressed as a percentage) was used in the determination of the TSP. The results indicate that shifts to 29° (x x) after pupation and from 29° to 22°c.(. .) after puparium formation led to viability and death respectively, thereby delineating the TSP for both heterozygotes and homozygotes. 73 TSP EGG L A R V A L I N S T A R pupa P U P A ADULT 75 the DTS-2 bearing chromosome, two methods were employed to d i s t i n g u i s h between these p o s s i b i l i t i e s : Method 1: Homozygous DTS-2 males were mass-mated to I I I - p l e (ru h_ s_t p^ s o ss e ) females at 22 C and t h e i r heterozygous v i r g i n female progeny c o l -l e c t e d (Figure 7). These were then mated i n b o t t l e s to homozygous DTS-2 males and a l l v i r g i n female progeny from t h i s cross were c o l l e c t e d . Single females were crossed to three I I I - p l e males i n v i a l s at 22°C f o r four days a f t e r which time the parents were transferred to fresh v i a l s incubated at 29°C. Progeny from both the 22°C and 29°C r e p l i c a t e v i a l s were scored i n order to e s t a b l i s h the o r i g i n a l genetic composition of the females i n the step I I I cross (Figure 7). Those v i a l s containing few or no progeny at 29°C were presumed to have a female parent homozygous f o r the maternal f a c t o r . Progeny i n the corresponding 22°C cul t u r e r e -vealed her genetic c o n s t i t u t i o n with respect to the I I I - p l e markers. Three hundred and ninety step I I I females were tested and a second locus, d i s t i n c t from that determining the DTS-lethal e f f e c t was found between st and pj_. These data are summarized i n Table 8. Method 2: More p r e c i s e genetic l o c a l i z a t i o n was obtained using the standard method for mapping recessive mutations (Figure 8). In t h i s case, homozygous DTS-2 males were mated to th s_t p_b p^/TM3 females. A l l pro-geny from eighteen b o t t l e s containing s i n g l e th s_t pb_ p^/DTS-2 females mated to three I I I - p l e males at 22°C were scored. The s_t p ^ + and s t + p ^ males from each b o t t l e were c o l l e c t e d and i n d i v i d u a l l y mated to G1/TM3 and Gl Sb e /TM2 v i r g i n females, r e s p e c t i v e l y (Step I I I ) . Stocks carrying the crossover chromosome balanced over e i t h e r TM3 or TM2 were maintained FIGURE 7 Method I for the localization of the maternal effect associated with DTS-2 - indicates chromosome which undergoes crossing over with the III-ple chromosome 76 STEP I DTS-2*/DTS-2* 0*0* STEP II DTS-2*/III-ple ? STEP III crossover DTS-2 /DTS-2 ? STEP IV score progeny III-ple ?? mass-mated: in bottles DTS-2/DTS-2 0*0* s ingle females: in bottles III-ple 0*0* single females: in vials 4 days, then transfercparents to fresh vials at 29 C .78 TABLE 8 L o c a l i z a t i o n of the s i t e a f f e c t i n g maternal f e r t i l i t y on the DTS-2 chromosome CHARACTERISTICS: phenotype at s t e r i l e very few c r c - only c r c - l i k e c r c - l i k e and l i v e 29 C: l i k e progeny progeny progeny genotype: 1 maternal _ 1 • . ' ts t s + +• e f f e c t : mat mat mat mat 2 DTS-2: DTS-2 or DTS-2 or DTS-2 DTS-2* DTS-2+ DTS-2+ # chromosomes w i t h above c h a r a c t e r i s t i c s : 1 s t £ 3 1 0 3 2 s t * p £ 2 1 5 1 ts map p o s i t i o n of mat usi n g the above data: 46.5 1 t s mat : temperature-sensitive maternal e f f e c t FIGURE 8 Method II for the l o c a l i z a t i o n of the maternal e f f e c t associated with DTS-2 79 80 FIGURE 8 STEP I: DTS-2/DTS-2 a* X th st_ pb pP/TM3 ? 22°C STEP II: DTS-2/th st pb pP ? X III-ple o" ^ / 2 2 ° X X Score al l progeny; collect st pp + and st pp efd* / ~ K STEP III: st pP+/III-plecTX G1/TM3 ? stWlII-pleo*X Gl Sb eS/TM2 ? STEP IV: st pP+/TM3 d" X 2 sjt__ pP/TM2 cf X ? STEP V: a) st pb+ pP/st pb+pp+o*x g, a) st* pb* p P/st + pb+ pP d 1 X ? (maintained as homozygous stock) OR b) st_ p_b_ pP*/st pb pP* , b) st* pb pP/st+ pb pP , (maintained as balanced stock over TM3 or TM2) STEP VI: a) Test homozygous cross-over stocks for fe r t i l i t y at 29°C. b) Homozygosis of the maternal effect factor is generated by crossing cross-over males to homozygous DTS-2 females and collecting the non-TM2 or non-TM3 female progeny. These are then tested as are the homozygous cross-over stocks. and homozygous female progeny were subsequently t e s t e d f o r the presence of the maternal e f f e c t l o c u s by p u t t i n g homozygous c u l t u r e s to 29°C and examining f o r embryonic l e t h a l i t y . Since pb_ i s present i n many of the s t o c k s , homozygous maternal e f f e c t females o f t e n had to be generated (Step VIb) by c r o s s i n g heterozygous males from the crossover stocks to homozygous DTS-2 females and c o l l e c t i n g the non-TM3 or non-TM2 female progeny. These were then t e s t e d i n the manner described above. Table 9 shows the r e s u l t s of t h i s study and again i n d i c a t e s that the mutation expr e s s i n g a t s maternal e f f e c t i s separable from DTS-2 and l o c a t e d ap-proximately 3.4 u n i t s to the r i g h t of st_. Thus, the e f f e c t of DTS-2 i s t o cause l e t h a l i t y of both homozygotes and heterozygotes d u r i n g pupa-t i o n w i t h a s i n g l e TSP preceding death. DTS-3 The l e t h a l phase of DTS-3 occurs during the t h i r d l a r v a l i n s t a r , a stage which may be prolonged at 29°C f o r some f o u r t e e n days before death f i n a l l y ensues. As these 29°C i n d i v i d u a l s age, f a t d e p o s i t s become d e p l e -ted and the s a l i v a r y glands degenerate. Since h i s t o l y s i s of the l a r v a l s a l i v a r y glands normally takes place p r i o r to and during t h i s t r a n s i t i o n s t a g e , t h i s suggests that the l a r v a e prepare f o r metamorphosis. Examina-t i o n of the im a g i n a l d i s c s i n d i c a t e s that the growth o f the anlagen i s not i n h i b i t e d , thus producing g r e a t l y enlarged d i s c s i n the o l d e r l a r v a e . Moreover, there i s a n o t i c e a b l e enlargement of the r i n g gland i n DTS-3/TM2 l a r v a e from 29°C when compared to w i l d - t y p e l a r v a e maintained at t h i s temperature or heterozygous DTS-3 l a r v a e kept at 22°C. I t w i l l be of i n t e r e s t to determine whether a normal r i n g gland implanted i n t o mutant 82 TABLE 9 Localization of the site affecting maternal f e r t i l i t y on the DTS-2 chromosome Genotype: 1 ratio + p st E l 4:0 45 3 3:1 4 0 2:2 1 2 1:3. 2 6 0:4 8 31 Total: 60 42 ts map position of mat using the above data: 47.4 ratio: four vials were established at 22°C and at 29°C for each of the cross over-stocks using homozygous parents. The ratio refers to the number of sterile vials/crc-like and progeny-containing vials for a a particular set of four v i a l s . A ratio of 4:0 indicates the presence of the maternal effect mutation on the cross over chromosome, whereas a ratio of 0:4 indicates the presence of the wild-type a l l e l e . 83 larvae under restrictive conditions w i l l allow pupation and what be-haviour the imaginal discs show upon implantation into wild-type larval hosts. Detailed shift experiments involving DTS-3 heterozygotes have yielded a complex picture for the sensitivity of this mutation to the restrictive temperature. Between 100 and 250 larvae were shifted at the times indicated (Figure 9). Maximal adult v i a b i l i t y occurred only when cultures were kept at 22°C from the late second to mid-third instar stages. Double shift experiments in which cultures were maintained at 29°C except for pulses of 22°C for 24, 48 and 96 hours at various times confirmed this sensitive interval (Figure 9b). A complication arises, however, from the observation that exposure to 29°C at any stage during development led to a high degree of lethality during the late pupal and early adult stages - so that several individuals died during eclosion while others died within 24 hours of successful eclosion (Table 10). This late l e -thality could reflect a second site semi-lethal DTS mutation which i s sensitive throughout development and has an LP during the late pupal and adult stages. If this were the case, however, the two DTS-lethals would have to be closely linked, since the mapping studies exposed a single locus responsible for the temperature-sensitivity. DTS-4 For cultures initiated at 29°C, the LP for DTS-4 heterozygotes occurs during the egg and very early f i r s t instar stages. About 15% of the eggs collected immediately after the parents are shifted to the FIGURE 9a Determination of the temperature-sensitive period (TSP) for adult viability of DTS-3/TM2 heterozygotes The TSP for DTS-3 heterozygotes was determined by shifting 100 to 250 larvae synchronized to within 1/2 hour (i.e. + 15 minutes) at the times indicated. In this figure the absolute times are plotted for shifts initiated both at 29° (. .) and 22°C (x x). The viability Is expressed as a percentage of firsts reaching the adult stage. These studies indicate that shifts to 29° or from 29° made during the second larval instar result in lethality (often at the second to third instar moult). 84 85 FIGURE 9b Double shift experiments (29° to 22° to 29°C) of DTS-3 heterozygotes Shifts of 75 to 150 larvae (collected as first instar larvae within 1/2 hour of hatching) were made at the times indicated as follows: x x to 22°C for 96 hours, . . to 22°C for 48 hours, . . to 22°C for 24 hours. These results confirm a sensitive interval extending from the late second larval until late in the third larval instar. 86 % of larvae eclosing 88/ TABLE 10 Determination of LPs and corresponding TSPs for DTS-3 heterozygotes ' Age at Shift Stage at Shift Teiaperature-Sansitive Period f o r : (hours) „ „ ~ n ( l a r v a l instar) Adult V i a b i l i t y Larval pupal and Pseudo-Lethality V i a b i l i t y after Late Pupal Stage 22"-29u 29-22 22u-29° 29°-22 u 22°-29 o 29 u-22° 22°-29° 29°-22° 0 0 f i r s t 0 59 84 6 0 69 43 f i r s t 0 91 0 0 46 24 second 0 45 94 11 1 52 60 39 second 0 39 91 3 2 56 73 48 second 1 38 88 1 1 47 84 73 second and t h i r d 1 29 88 1 6 35 96 t h i r d 61 16 84 third 12 6 20 120 t h i r d 28 18 52 129 t h i r d 26 24 50 96 third 9 31 19 141 t h i r d 26 19 45 154 l a t e t h i r d 32 16 51 108 late t h i r d 1 . 62 3 172 120 prepupae and pupae 43 2 2 82 65 4 185 130 pupae 27 0 3 80 61 1 196 144 pupae 28 0 9 94 60 2 228 158 pupae 34 0 3 87 59 0 240 168 pupae 37 0 4 82 59 0 183 pupae 0 88 1 197 pupae 0 93 1 -% of surviving adults: f i r s t instar larvae shifted 2% of pseudopupae: f i r s t instar larvae shifted of individuals surviving past the late pupal stage: f i r s t instar larvae shifted 89 restrictive temperature hatch. Those larvae which do emerge never assume the elongated shape characteristic of wild-type f i r s t instar larvae but rather remain the approximate size and shape of eggs. Pre-incubation of the parents at 29°C for twenty-four hours prior to egg collection re-sults in complete egg l e t h a l i t y , although some embryonic development can be discerned. Shifts-up revealed a sensitivity of the mutant to 29°C throughout the embryonic and larval stages u n t i l early i n the pupa. A more precise analysis of the temperature effects during the larval period was made by shifting up at various times batches of 150 to 200 larvae collected as f i r s t instars within fifteen minutes of hatching. Shifts made every twelve to fifteen hours exposed sensitive periods which resul-ted i n death in the embryo, during specific larval instars, and even at specific times during the development of the imago (Figure 10). Exposure to 29°C from the early part of the third larval instar u n t i l the onset of puparium formation allowed eclosion of a low percentage of progeny but these adults died within 36 hours. Shifts made during the early pupal stage led to a higher eclosion frequency but again early death occurred. Many of the adults from cultures shifted to 29°C from the late third instar u n t i l the early pupal stages became paralyzed at the restrictive temperature, a phenomenon partially reversed upon returning these adults to 22°C. Although completely normal activity was never observed, some movements and attempts to walk were noticed. A closer analysis of the temperature effects during the embryonic stage, made by administering 2- and 6-hour heat shocks at two-hour inter-vals, revealed a very sensitive interval extending from 16 to 18 hours post egg deposition (Figure 11). Unfortunately, there was some asynchrony FIGURE 1 0 Lethal phases of DTS-A/TM3 heterozygotes after shifts to 29°C at various developmental stages Shifts of 100 to 200 larvae collected within 15 minutes of hatching were shifted to 29°C at the times indicated. The stages during-which death occurred were recorded and are indicated on the ordinate. designates the limits of stages reached in the cultures represents the stage at which at least 85% of the individuals die about equal numbers reached these stages in these shifts 90 developmental stage a t t a i n e d ! eclose and sur v i v e eclose and die l a t e pupa pupa 3rd i n s t a r 2nd i n s t a r elongated 1st i n s t a r "egg-shaped" 1st i n s t a r Growth curve at 22°C Z.0 1st T T O < i a f t ja T 1 9 MO 60 eo tOO IfcO I80 -COS-2nd 3rd -8*-climbing head begins eversion pre-pupa . . //-Z O O ? Z O time of shift to 19*C thouts) Jf— L A R V A L I N S T A R PUPA ADULT FIGURE 11 Percentage of DTS-4/TM3 adults eclosing after 2- and 6-hour heat pulses at various times during development. Batches of 50 to 100 eggs were heat-shocked for 2 or 6 hours at the times indicated. The effect of the heat treatment can be seen as a decrease in the percentage of adults eclosing; a maxi-mum of 50% indicates complete survival of the DTS-4 heterozygotes. It would appear that the most sensitive interval occurs from 16 to 20 hours of development at 22°C. indicates the duration of the heat treatment (short dashes: 2-hour pulses; long dashes: 6-hour pulses to 29°C) 92 93 94 in the eggs used and heat shocks of two hours duration never induced complete lethality but did cause a considerable reduction i n the per-centage of successful eclosions. The effects of pulse shifts to the restrictive temperature were studied by exposing batches of at least 90 larvae, synchronized to with-i n 1/2 hour of hatching, to 29°C for 24 hours at successive 12-hour intervals. Observations were made at the time of the shifts from low temperature to high and again upon return of the cultures to low temper-ature. Apparently the pulse at 29°C arrests or slows down development since larvae remained i n the same developmental stage during the 24 hour period at 29°C. Progeny were scored every six to twelve hours after the emergence of the f i r s t adults from the control cultures only for the f i r s t three and a half days. Table 11 summarizes the results of this experiment. There appear to be specific intervals during which there i s a greater sensitivity to the heat treatments. Whereas twenty-four hours at 29°C during the f i r s t twenty-four hours of larval l i f e was sufficient to k i l l a large proportion of the larvae, such treatment was less detri-mental at later stages. A l l DTS stocks were examined for the effects of age and temperature on the egg-laying capacity of heterozygous DTS-bearing females. For each stock, one v i a l containing five virgin females and males heterozygous for the same DTS chromosome was established at both 22° and 29°C. The adults were transferred to new medium every 12 hours for 16 days and the number of eggs laid recorded at the time of each transfer. Whereas the egg-laying patterns of the other DTS-lethal stocks did not differ markedly TABLE 11 Eclosion times and percentages of successful eclosions of DTS-4/TM3 heterozygotes exposed to 29°C for 24 hours during d i f f e r e n t developmental stages. Age (hours) at e c l o s i o n Age at i n i t i a t i o n develop-mental < 293 294-307 • ' r 308 313 314 317 318 331 332 341 342 354 355 362 363 494 % of Is eclosec of heat pulse: stage at s h i f t : # % # 7. # % # % #: % # % # % # % # % . 0 l s t s 0 12 l s t s 2 2 3 3 1 1 4 4 11 24 l s t s 1 1 3 3 40 42 49 36 lsts/2nds 4 4 2 2 2 2 2 2 41 41 57 48 2nds 1 1 0 0 7 8 3 3 5 5 1 1 23 25 44 60 2nds 1 1 4 4 6 6 5 5 2 2 15 16 37 72 2nds 2 2 0 0 8 9 1 1 7 8 3 3 23 25 49 84 3rds 5 4 3 3 29 27 17 16 5 5 5 5 24 22 73 96 3rds 1 1 2 2 19 20 27 29 16 17 3 3 13 14 90 108 3rds 2 7 6 19 2 7 10 33 7 23 1 3 0 0 2 7 100 120 3rds 3 11 1 3 9 30 10 33 4 13 0 0 1 3 93 132 3rds 10 17 1 2 7 12 24 41 7 12 1 2 0 0 2 3 87 Controls 47 16 114 32 42 12 108 30 12 3 2 1 4 1 8 2 94 CO 96 from the Oregon-R and Samarkand controls, that of the DTS-4/TM3 females was unique. After 4 days at 29°C, the number of eggs l a i d decreases dramatically to about 40% that prior to this time and that of the 22°C cultures (Figure 12). Thus a spectrum of effects may be produced by the DTS-4 mutation, depending on the developmental stage during which exposure of heterozygotes to the restrictive temperature i s made. DTS-5 and DTS-7 Trans-heterozygotes for DTS-5 and DTS-7 f a i l to complement, i.e., do not survive, at 22°C; yet DTS-5 and DTS-7 map unambiguously at different sites, DTS-5 being located to the right of st at 44.3 and DTS-7 to the l e f t at 42.3 (see Chapter 3 :, page 36 '. In addition, the developmental properties conferred by these two mutations differ. Pupae phenotypically resembling the mutant, halfway (hwy - Lindsley and Gre l l , 1968), characterize the lethal phase of DTS-5 heterozygotes developing under restrictive conditions. Although the abdominal region f a i l s to develop and histolyzes, the anterior portions of the adult are ful l y differentiated within the pupal case. The TSP for this lethal phase begins during the third larval instar and ends with pupal formation - a period lasting approximately 36 hours (Figure 13). The shift experiments revealed a second lethal phase i n the late pupa and a TSP for that l e t h a l i -ty extending from the late third instar u n t i l early i n pupation, thus partly overlapping the earlier TSP. Adults emerging from shifts made late i n this TSP had a very characteristic phenotype: wings were much hairier i n appearance and the venation pattern resembled that of the mutant FIGURE 12 Effect of age and temperature on the egg-laying capacity of DTS-4/TM3 females Five pairs of virgin males and females, collected within 12 hours of eclosion at 22°C, were either kept at that temperature or transferred to 29°C. These parents were transferred to fresh vials every 12 hours for 16 days, and the number of eggs laid was deter-mined at the time of transfer. Since there is a great difference in the number of eggs laid during the daytime versus that laid dur-ing the night, the following calculation was made for purposes of graphing the data: two consecutive egg lays were added together and divided by two, the number derived therefrom was plotted as the number of eggs laid for the earlier time interval. . . eggs laid per 12 hour interval at 22°C x x eggs laid per 12 hour interval at 29°C 97 FIGURE 13 Determination of the temperature-sensitive period (TSP) for DTS-5/TM2 heterozygotes Shifts of cultures containing approximately 150 eggs, 100 to 200 larvae (synchronized as first instar larvae), or 30 to 60 pupae (synchronized as prepupae) from 29°C (. .) or to 29°C (x x) were made at the times indicates. Again the shifts from 29°C were adjusted to take into account different develop-mental rates at 22° and 29°C. Since DTS-5/DTS-5 and TM2/TM2 zygotes survive to the larval stage, but only DTS-5/TM2 individuals reach pupation, an estimate of the TSP for the heterozygotes was made taking the ratio of adults/pupae (expressed as a percentage). The TSP was thus shown to extend from the mid-third larval instar U n t i l early In pupation. 99 TSP 1st 2nd 3rd egg l a r v a l i n s t a r pupa adult io 1 px; the eyes were rough and somewhat smaller than wild-type; and the legs were often deformed. In addition, many of these f l i e s showed a duplication or an absence of thoracic or head b r i s t l e s , although no clear pattern of b r i s t l e type affected was noted. Pulsing of 22°C cultures for a 48 hour period within the TSP also produced the abnormal br i s t l e pattern and wing phenotype. Histolytic,as well as late, pupae(those which appear to have com-pleted the development of the imago) characterize the lethal phase of DTS-7. The TSP for adult v i a b i l i t y immediately precedes the lethal phase as indicated i n Figure 14a. However, i t i s noticed that although exposure of male larvae to 29°C as much as 100 hours prior to the TSP does not prevent their eclosion, they die within 24 hours of emergence (Figure 14b). Female survival, on the other hand, i s not at a l l affected by the same treatment. This i s another example of a sexually dimorphic expression of a locus which has been noted for other ts-lethals (Tarasoff and Suzuki, 1970; Mayoh and Suzuki, 1973). DTS-5 and DTS-7 can be distinguished further by the effects of ex-tended exposure to the restrictive temperature on heterozygous females of the two stocks. Although DTS-5 females remain f e r t i l e even after 10 days at 29°C, DTS-7 females become ste r i l e after three days of exposure. A c r i t i c a l analysis of the time required to cause failure of egg deposition was made in the following manner. Sixty non-virgin DTS-7/TM3 females were placed individually in v i a l s at 29°C with three G1/TM3 males. For six days, the adults were kept at 29°C and transferred to fresh vials every 24 hours. Control vials containing one DTS-7/TM3 male and five G1/TM3 FIGURE 14a Determination of the temperature-sensitive period (TSP for adult viability of DTS-7 heterozygotes Shifts of 150 to 200 eggs or larvae (synchronized as first instar larvae) or 30 to 50 pupae (synchronized as prepupae), at the times indicated revealed a short TSP beginning just prior to puparium formation and ending with pupation. Since DTS-7/DTS-7 zygotes survive to the larval stage, this TSP was delineated using the -ratio of adults/pupae (expressed as a percentage). The shifts initiated at 29°C were adjusted as outlined in Figure 4. x x shifts from 22° to 29°C . shifts from 29° to 22°C 102 FIGURE 14b Details of eclosion of adults from shifts of DTS-7 heterozygotes from 22° to 29°C from Figure 14a Different TSPs are indicated for the heterozygous males and females, as shown by the following plots: . . total viable adults (i.e. those which survived more than 24 hours post eclosion, x x total individuals eclosing (including adults which died within 24 hours post eclosion), The area between the two curves represents dead males. 104 Hps 80 70 time of shift to 29°C (hours after hatching from egg) 106 females were treated i n a s i m i l a r way and a f t e r ten days the v i a l s were checked f o r the presence of adult progeny, unhatched pupae and dead larvae. Cultures having no larvae were examined f o r the presence of eggs. Table 12 summarizes the r e s u l t s obtained. I t can be seen that a f t e r two days at 29°C, the average number of eggs l a i d per DTS-7/TM3 female was d r a s t i c a l l y reduced to about 11% of that of the f i r s t day. A f t e r 3 days, only four females l a i d any eggs. Heterozygous DTS-7-bear-ing eggs deposited wi t h i n the f i r s t 3 days a f t e r s h i f t i n g to 29°C can develop u n t i l the LP i n the l a t e pupal stage. The co n t r o l cultures a l l contained several G1/TM3 progeny and numerous dead pupae which were DTS-7 heterozygotes. Females carrying crossovers between DTS-7 and th s_t p_b _pj_ were tested f or the presence of the D T S - l e t h a l i t y and the t s - s t e r i l i t y . The r e s u l t s show that 29°C-induced s t e r i l i t y occurs only when DTS-7 i s present. We conclude, therefore, that the two phenotypes r e s u l t from the DTS mutation. Although a double mutation at two very c l o s e l y l i n k e d s i t e s i s not c r i t i c a l l y ruled out, that p r o b a b i l i t y i s remote. The developmental and genetic properties c l e a r l y d i s t i n g u i s h DTS-5 from DTS-7, yet complementation studies at both 17° and 22°C suggest that these mutations are f u n c t i o n a l l y r e l a t e d (see Chapter I I I , page 40). L e t h a l i t y of DTS-5/DTS-7 f l i e s could be due to a genuine i n t e r a c t i o n be-tween the two DTS-lethals or the r e s u l t of homozygosis f o r a second s i t e recessive l e t h a l c a r r i e d by both chromosomes. This was tested i n two ways. The f i r s t procedure attempted to l o c a l i z e the f a c t o r s responsible f o r the l e t h a l i n t e r a c t i o n to a region delineated by the markers on the TABLE 12 K i n e t i c s of temperature-sensitive female s t e r i l i t y associated with DTS-7/TM3 females as determined by s u r v i v a l a f t e r condi^ tioning of parents to 29°C f o r d i f f e r e n t lengths of time stage attained by F^ progeny at 29°C Days at # # adults dying # # % 29°C: l i v e at eclosion or l e t h a l dead t o t a l : of adults: soon afterwards: pupae: larvae: Day 1: 1 0 30 916 193 1139 100.0 2 1 11 684 164 860 75.5 3 0 0 104 26 130 11.4 4 0 0 8 1 9 0.8 5 0 0 0 1 1 < 0.1 6 0 0 0 0 0 0 .108 I I I - p l e chromosome. I n d i v i d u a l DTS-5 (or DTS-7) v i r g i n females hetero-zygous f o r the I I I - p l e chromosome were testcrossed to three males i n v i a l s at 22°C and the progeny were scored a f t e r 14 and 17 days. A l l male progeny carrying a crossover were c o l l e c t e d , t h e i r phenotype noted and i n d i v i d u a l l y mated to two v i r g i n females of the other DTS stock ( i . e . DTS-7/TM3 females were crossed to DTS-5 crossover males and DTS-5/ TM3 females were crossed to DTS-7 crossover males) at 22°C. A f t e r four days, the parents were tr a n s f e r r e d to f r e s h v i a l s and placed at 29°C to test f o r the presence of the DTS on the crossover chromosome. I t was expected that i f the crossover chromosome c a r r i e d the l e t h a l i n t e r a c t i o n f a c t o r , the r a t i o of non-TM3 to TM3-bearing progeny at 22°C would be 1:2, whereas i f the l e t h a l f a c t o r were not present t h i s r a t i o would be 1:1. In a l l cases, i t could be then checked whether the DTS i t s e l f were present by inspecting the 29°C c u l t u r e s . A t o t a l of 341 crossover males of DTS-5 and 221 crossover males of DTS-7 of various genotypes was tested. Un-f o r t u n a t e l y , a d i s t i n c t i o n between a 1:1 and 1:2 r a t i o was not always p o s s i b l e , thus these cases were not included i n the f i n a l a n a l y s i s . In a d d i t i o n , c e r t a i n crossover chromosomes had a phenotype i n d i s t i n g u i s h a b l e from III-ple/TM3 heterozygotes and i t was, therefore, d i f f i c u l t to deter-mine whether the DTS was present i n these cases. However, i n a l l cases where the DTS's c l e a r l y were present on the crossover chromosome, the l e t h a l i n t e r a c t i o n p e r s i s t e d , i n d i c a t i n g that i f there i s a second l e t h a l s i t e shared by the two stocks, i t must map very c l o s e l y to one of the DTS-l e t h a l s . Table 13 summarizes the data for these experiments. The second method employed was d i r e c t e d towards obtaining crossovers very close to the DTS l o c i . The th st_ pjb £ ^ chromosome was used f o r TABLE 13 Method 1: Localization of the site(s) on the DTS-5 and DTS-7 chromosomes causing lethality of the DTS-5/DTS-7 trans-heterozygotes genotype of cross-over chromosome Cross-over Chromosome JDTS-5  1 DTS"1" 1 DTS 1+DTS 1+DTS+ ' DTS-7  1 DTS+ 1 DTS 1+DTS 1+DTS"*" ru h + + + + 55(2)* ru h st + + + 1(1) 6(1) ru h + + SS S e 2 + + st p p + + 5 + + st p p SS + 1 ru h + + + s e KD + h st + + + 1 + + + p p + + 2 + + St + + + 1 ru h st + + s e 1 ru h St p p + + 7 + + + + SS s e 6(4) ru + + + SS s e 28(14)* 1 (1) K D Total 68(8) 21(1) (1) 29(17) 3(13) 5(2) (2) 6(2) 7(2) 2(1) (3) (1) 24(26) *numbers in brackets refer to numbers of chromosomes tested which show definite 1_ or 1+ interaction, but the presence of the DTS was not confirmed (i.e. s t e r i l e at 29°C or insufficient progeny at 29°C). 1 : lethal interaction present 1+ : lethal interaction absent DTS : DTS present on cross-over chromosome DTS+: DTS not present on cross-over chromosome o co 110 this. Individual heterozygous DTS/th st pb p virgin females were mated to either two III-ple or two th st_ j>b_ cu kar su(Hw)^ j v l ss bx sr  gl/TM6 males in vials at 22°C and the progeny were scored. Males of the following phenotypes were collected and mated to G1/TM3 or Gl Sb e /TM2 females i n order to obtain balanced stocks of the crossover chromosomes: st p^ +, s t + , th s t + pb + p^ + and t h + st pb p^. Males from each cross-over stock were then separately mated to two virgin females from the DTS-5 and DTS-7 stocks at both 22° and 29°C, thus testing for the pre-sence of the synthetic lethal factor as well as for homozygosis of the individual DTS lethals. Again, the results of testing 11 crossover stocks from DTS-7 and 18 crossover stocks from DTS-5 indicated that the interaction between the DTS's i s probably due to the DTS-lethals them-selves, although the possibility of a second site recessive lethal on the DTS-5 chromosome located near the DTS-7 locus could not be excluded by the second method (this possibility seems remote in view of the results obtained by the f i r s t method of study in which one crossover between st and pj^ yielded a ru h st chromosome which showed both the DTS—5 mutation and the lethal interaction with DTS-7). This procedure also permitted the localization of DTS-7 between th and st. In addition, i t was found that two recessive lethal sites are present on the original DTS-7 chromo-some, one located to the right of sst_ and the other between th and st. The following observation makes i t highly probable that the second site recessive lethal i s at the DTS-7 si t e : of three crossover chromosomes no longer carrying the f i r s t recessive lethal ( i . e . , t h + st Sb - from Table 14), two have both the DTS-7 mutation and the recessive lethal mutation while the other has both wild-type alleles. I l l TABLE 14 Characterization of crossover chromosomes obtained from DTS-5 and DTS-7 females heterozygous for 1. th st pb p P, or 2. th st Sb ef_ DTS Genotype: Cross stock homozygous # lethal presence of DTS: synthetic2 lethality DTS-•5 th + + + 1 none th st + + 1 11-2b 1 + DTS+ 1 + 11-lb-l 1 + DTS+ 1 + l l - 3 a N.T.3 DTS+ 3 N.T. 4a-l 1 + DTS+ 1 + + + pb p p 1 II-2b-l 1 1 II-2b-2 1 DTS 1 4a-2 1 DTS 1 + + + P p 1 l a 1 DTS 1 2c 1 DTS 1 DTS--7 th + + + 1 3:2/20 1 DTS+ 1 + 3a-l 1 DTS + 1 + th st + + 1 4a-l 1 DTS+ 1 + + + pb p p 1 l a 1 DTS 1 + st Sb 2 lb-1 1 DTS 1 3b-l 1 + DTS+ 1 + 5b-l 1 DTS 1 Hiomozygous lethal: 1 - lethal; 1 + - viable 2 + synthetic l e t h a l i t y : 1 - lethal; 1 viable N.T.: Not tested 112 Since both DTS-5 and DTS-7 are homozygous first instar lethals at 22°C, i t was decided to examine the lethal phase for the DTS-5/DTS-7 trans-heterozygote to determine whether a unique lethal phase character-ized the combined mutant. A series of crosses was made as outlined in Table 15, utilizing crossover DTS- and DTS+-bearing chromosomes. Those which did not bear the DTS-mutations served as controls. It would seem from crosses 1 and 4 that the trans-heterozygotes are lethal during the larval stage - and more specifically in the first instar larva as in-spection of the cultures indicated. TABLE 15 Determination of the Lethal Phase of DTS-5/DTS-7 trans-heterozygotes at 22°C Cross Made: Hatchability and Eclosability Viability %lsts %Adults %Adults Ratio* #Eggs #lsts //Adults eggs lsts eggs "DTS-5"/"DTS-7" 1) DTS-5 pP/TM3o* x DTS-7 pb. pP/TM3g 0/241 345 94 57 27.2 60.7 16.5 larval lethal 2) th st DTS-5+ pP/TM3g x th st DTS-7+/TM3o* 55/185 243 85 74 34.9 37.9 30.5 viable 3) th st DTS-5+ pP/TM3g x DTS-7 pb pP/TM3c/ 92/277 172 118 108 68.7 91.4 62.8 viable 4) DTS-5 pb pp/TM3? x th st DTS-7/TM3 o* 0/271 89 67 42 75.3 62.6 47.2 larval lethal 5) DTS-5 pb pp/TM3 g x th st DTS-7+/TM3 a* 79/219 181 97 91 53.6 93.8 50.4 viable * Viability ratio DTS-5/DTS-7 : total progeny. Data are from crosses made independent of hatchability and eclosability studies. 114 IV. Discussion The genetic and developmental analyses of the seven DTS-lethals reported here i l l u s t r a t e the potential usefulness of such mutations i n the study of the regulation of development. It i s now possible to begin an objective comparison of the developmental and genetic properties of an extensive collection of heat- and cold-sensitive dominant and reces-sive lethal mutations (Procunier and Suzuki, 1969; Rosenbluth et a l . , 1972; Holden and Suzuki, 1973; Mayoh and Suzuki, 1973). Whether a model for the mechanism of dominant lethal effects emerges remains to be seen. Originally, DTS-lethals were sought as one class of mutations which might allow the genetic detection of biologically important redundant regions of the chromosome. Examination of the genetic regulation of cross-ing over led Suzuki (Suzuki, 1967, 1973) to postulate that genes con-t r o l l i n g many steps i n c e l l division are located i n proximal heterochroma-t i n . Since this region i s known to be highly redundant (Ritossa and Spiegelman, 1965; Berendes and Keyl, 1967; Gall and Pardue, 1969), i t was thought that the absence of genetically detectable l o c i i n this region could reflect the masking of recessive mutations by wild-type duplicates or the lethality of dominant mutations affecting c e l l division and other v i t a l processes. The multiplicity of cistrons for ribosomal and transfer-RNA suggests that other l o c i important i n basic cellular functions may also be tandemly duplicated. In this context, i t i s interesting to note the continued dominance of DTS-4 i n tr i p l o i d s , thereby showing that DTS lethality can be expressed over at least two wild-type alle l e s . i 115 The two mutants, DTS-1 and DTS-6 are of interest since phenotypical-ly they resemble the large class of mutations called Minutes. Hetero-zygotes for both DTS mutations have very extended developmental periods under permissive growth conditions and very short, slender thoracic bristles. In addition, both mutations are recessive lethals. We feel, therefore, that they are legitimate Minute mutations. The suggestion by K.C. Atwood (cf. Lindsley and Grell, 1968) that Minute mutations represent lesions in the structural genes for transfer RNAs and the observation of in situ hybridization of tRNA to salivary gland chromosomes near Minute sites (Steffenson and Wimber, 1971), provide an incentive for biochemical analysis of these two DTS-lethals. The demonstration that the amounts of certain iso-accepting forms of several species of tRNA molecules change during the development of Drosophila (Chapter 2, Part II) correlates with the larval and pupal TSPs of DTS-6 and DTS-1, respectively. The patterns of tRNA synthesis in these mutations is currently under investigation (B. White, personal communication). Although these studies were undertaken to demonstrate that point mutations could also behave as dominant lethals (Suzuki and Procunier, 1969; Chapter 3 ) , i t soon became apparent that the developmental proper-ties of the mutants recovered were of considerable interest. It is ex-pected that mutations in genes governing steps in protein synthesis or those important in cell division would have continuous TSPs. The sensi-tivity of DTS-4 to the restrictive conditions throughout the larval stage and early into pupation provides an intriguing possibility of a mutation in such a basic process. 116 An alternative interpretation of the defect i n DTS-4 individuals raised at 29°C i s indicated by the phenotypic manifestations of this mutation. Virtually a l l developmental stages are affected, to lesser or greater degrees, by exposure of DTS-4 heterozygotes to 29°C. The observations made during the shift experiments suggest a possible defect either in the muscle protein or i n the synthesis of energy-rich compounds (which may be required i n great quantity by the muscle c e l l s ) . Only a small proportion of the eggs l a i d by DTS-4/TM3 females at 29°C are seen to hatch. The successful larvae, however, never become elongated, but rather die i n a "contracted" form. Although they are able to move, few do so unless they are encouraged by a gentle probing. Also, very few eggs containing larvae which had developed to within one hour of hatch-ing do hatch when shifted to 29°C at this stage. This i s presumably a very c r i t i c a l time, since the trauma of the temperature shock prevents escape from the egg chamber. Later larval stages are also dramatically affected by temperature treatments. The 24-hour pulse experiments i n d i -cated that larvae were capable of survival at the restrictive temperature and although development was severely disrupted, this effect can be over-come by return to 22°C within twenty-four hours. Acclimatization does not occur, however, since larvae die when kept at 29°C for extended periods. One further comment before continuing to the discussion of later stages affected by temperature: the lethal phases are determined by specific TSPs which, during the larval stages, do not overlap. Thus, a shift of young f i r s t or young second instar larvae to 29°C results in lethality during those stages respectively, whereas shifts midway through the 117 f i r s t or second instars result i n death during the following larval => instars (i.e. second and third instars, respectively). This would suggest specific intervals of synthesis or requirement of a gene product restricted to defined times within each of the developmental stages. Lethality may also occur during the early or late pupal stages, or even in the adult. The f i r s t two of these latter lethal phases coin-cide with times of extensive muscle synthesis and innervation (see Demerec, 1951). The lethality i n the adult stage, resulting from shifts made during the late third instar and early pupal stages, i s characterized by a paralysis which i s partially reversible upon return of the f l i e s to 22"C. It i s of int erest that the f l i e s which survived these later shifts were hyperactive when etherized - perhaps indicating lack of neural or muscular coordination. And f i n a l l y , the reduced egg-laying a b i l i t y of females exposed to 29°C for extended periods may reflect some affect on the muscle system involved with oviposition. Whereas most females of the several mutant and wild type stocks tested were induced to oviposit upon anesthetization with CC^, none of the DTS-4/TM3 females which had been at 29°C for four days showed this response. Control DTS-4/TM3 females kept at 22°C showed a positive egg-laying response to this treat-ment, although not a l l females could be induced to lay with successive hourly Ct^-treatments. A dissection of DTS-4/TM3 females preconditioned to 29°C for five days revealed the presence of well-developed ovaries with mature eggs. Thus the ,low rate of egg-laying by 29°-conditioned DTS-4/TM3 females does not appear to be due to a lack of eggs but must reflect some other, perhaps mechanical, defect in egg-laying behaviour. 118 One of the foremost problems i n the developmental biology of i n -sects has been the hormonal control of metamorphosis. The control of molting, metamorphosis, organ growth, and also some general metabolic activity has been attributed to hormones produced within the "ring gland" (Hadorn and Neel, 1938; Bodenstein, 1943; Vogt, 1942). Several mutations interfering with metamorphosis have been reported (l(2)gl; Scharrer and Hadorn, 1938; l(2)gd: Bryant and Schubiger, 1971). DTS-3 heterozygotes are lethal during the third larval instar at the restrictive temperature. The fact that the ring gland of such individuals i s substantially en-larged may provide some clue as to the possible lesion produced by this mutation. In order for metamorphosis to successfully occur, there must be sufficient ecdysterone production by the larval ring gland. One may ask, then, whether the large size of the ring gland of the mutant at 29°C reflects either the i n a b i l i t y to transport this hormone to target organs or, perhaps, the absence of the stimulus for transport, thereby causing unlimited production and accumulation of the hormone. Alternatively, the corpus allatum may overproduce juvenile hormone, thus preventing ecdysis. These possibilities are currently under investigation. Mutations which show a sexual dimorphism have been detected and studied i n Drosophila. Goldschmidt (1953) and Muller and Kaplan (1966) have discussed the concept of dosage compensation and the mechanism of sex-specific differences in gene expression for some mutations on the X chromosome. If a recessive or dominant lethal factor, represented in equal dose i n both sexes, manifests differences in penetrance according to sex, the factor i s said to produce a sex-limited let h a l i t y . A number of 119 ; sexually dimorphic temperature-sensitive mutations have been recovered which affect the v i a b i l i t y or f e r t i l i t y of males or females. A recessive X chromosome dimorphic gene (Tarasoff and Suzuki, 1970) gives a sex ratio varying between 0.33 and 0.67 (females;males) at room temperature but at 29°C. 100% of the pupae formed contain male imagoes. The females were shown to be sensitive to the restrictive conditions at a l l times whereas the males were sensitive during a short interval immediately preceding their lethal phase. An X-2 translocation has been found to cause the death of males at 29°C and their survival at 22°C, yet females homozygous for the translocation are f e r t i l e and viable at both temperatures (T.C. Kaufman and D.T. Suzuki, i n preparation). Although several X-chromosomal and autosomal mutations are known which affect the f e r t i l i t y of either males or females, few autosomal lethal or phenotypic mutations have been reported which are sexually d i -morphic. Thus, the observation that heterozygous DTS-7 males and females have different TSPs for lethality makes this mutation somewhat unique, and perhaps presents clues as to the physiological differences (other than those involved i n reproduction) between the two sexes. In this connection i t w i l l be of interest to examine the interaction of this mution with the third chromosomal mutation tra (transformer) which trans-forms females into male-like intersexes. A l l tests of interactions of tra with other sex-dependent mutations indicate that tra females are really physiologically or developmentally females, although they resemble males externally (Sturtevant, 1945). 120 The somewhat more prominent c l a s s of sexual dimorphic genes a f f e c t -ing f e r t i l i t y also has some representatives i n the temperature-sensitive category of mutations. Recently, Yarger and King (1971) reported studies on the dominant female s t e r i l e , FS(2)D, which causes a reduction i n o v i -p o s i t i o n . In a d d i t i o n , there i s an e f f e c t on the wing morphology which i s temperature-dependent. Two DTS-lethals have been demonstrated to have dominant female s t e r i l i t y e f f e c t s . Heterozygous DTS-6 females are s e m i - s t e r i l e at 17° and 22°C and completely s t e r i l e at 29°C, whereas DTS-7 females are f e r t i l e at the lower temperatures but an exposure of three days duration to the higher temperature causes i n h i b i t i o n of o v i -p o s i t i o n . The recovery of a DTS-female s t e r i l e mutant which was mappable implies that a d d i t i o n a l s i m i l a r mutations may be detectable. A spectrum of such mutants would, perhaps, provide a means f o r determining the primary biochemical l e s i o n s associated with female s t e r i l i t y (Garen and Gehring, 1972). The f i n a l type of sexual dimorph which arose" i n our studies was the maternal e f f e c t mutation induced simultaneously with DTS-2. The proper-t i e s of t h i s mutation resemble those of the recessive t s - l e t h a l , E25 (Tarasoff and Suzuki, 1970) i n that rescue of eggs of mutant females i s possible by the i n t r o d u c t i o n of a wild-type a l l e l e by the male parent. A number of s i m i l a r non-ts mutations have been extensively studied ( f u : Counce, 1956b; dor: M e r r e l l , 1947 and Lucchesi, 1968; r_: Counce, 1956a), ts and i t w i l l be of i n t e r e s t to determine whether homozygous mat ovaries develop autonomously i n mosaics. 121 An interesting property of DTS-2 i s i t s characteristic LP under restrictive conditions, a phenotype strikingly reminiscent of the non-ts-lethal mutant, crc (cryptocephal). This mutation , (crc), which permits pupation, prevents eversion of the cephalic complex. The phenotype re-flects directly a metabolic error which has been shown to cause an ab-normally r i g i d integument resulting from increased incorporation of glutamine during the synthesis of glucosamine-6-phosphate (Fristrom, 1965). Biochemical studies of a similar nature may help to identify a temperature-sensitive polypeptide i n DTS-2 individuals. Kinetic studies on such proteins would be invaluable in establishing the molecular basis for dominant lethality. The recovery of DTS-lethals provides a tool for examining the molecular and biological bases for dominance and may be a means of prob-ing the genetic contributions of redundant l o c i such as those in proximal heterochromatin. Thus, the detection of genes with functions fundamental to v i a b i l i t y and f e r t i l i t y may be possible by the induction of dominant temperature-sensitive mutations within these l o c i . Coupled with bio-chemical studies, this technique of selection of specific DTS-lethals may provide a means of studying those reactions that lead to the death of an organism or to the alteration of phenotypic characteristics. The localization of DTS-5 between j 3 t and th eliminated the possibility that i t l i e within heterochromatin, although i t may s t i l l be under the i n -fluence of this region. Mapping of DTS-6 was not extensive enough to deter-mine i t s relationship with the centromere. The intriguing possibility that the maternal effect ts l i e s i n heterochromatin i s of interest i n view of i t s proximity to the homoeotics mapping to the l e f t of pink. 122 CHAPTER 5 CHARACTERIZATION OF A SPECIFIC CLASS OF TEMPERATURE-SENSITIVE LETHALS I. Introduction Several temperature-sensitive and non-temperature-sensitive mutations, which are l e t h a l only when combined with s p e c i f i c second and t h i r d chromosomes, were recovered during the search for dominant temperature-sensitive (DTS) l e t h a l s on chromosomes 2 (Suzuki and Procunier, 1969) and 3 (Holden and Suzuki, 1973). Mutations were obtained which were l e t h a l or semi-lethal in combination with e i t h e r VI 13 Iri(2LR)SM5t Cy_ or Irt(2LR)bw , Pm on chromosome 2 or In(3LR)TM2, Ubx or Lv_ on chromosome 3. It is assumed that l e t h a l a l l e l e s of the respective marked chromosomes had been induced. These were of inte r e s t since few condit i o n a l l e t h a l mutations of previously known l o c i have been studied. A d e t a i l e d examination of the genetic and developmental properties of four temperature-sensitive mutations which are l e t h a l i n combination with TM2 was made and the r e s u l t s of these studies are reported here. •123 II. Materials and Methods Screening Procedure: The temperature-sensitive (ts) mutations reported here were re-covered in a screen for dominant temperature-sensitive (DTS) lethal mutations of chromosome 3, according to methods B and C of Chapter;3. They are characterized by lethality of the mutagenized chromosome over TM2 at 29°C but their survival and f e r t i l i t y at 22°C. In cultures initiated at 29°C, individuals heterozygous for TM2 and one of these mutations die during the early larval stages (predominantly as first and second instar larvae). These mutations are a l l viable with other balancer chromosomes tested at both 22° and 29°C. Table 16 summarizes Ubx-ts the properties of these mutations, which are designated as 1(3)JHX  where JH are the initials of the discoverer, X is the specific number of 130 the mutation, and Ubx-ts points to the interaction with the TM2, Ubx chromosome (allelism with the Ubx-locus is not implied). In order to simplify the nomenclature, the abbreviation Ubx-ts-X will be used here-after. The important properties of other mutations and the rearrangements used in these studies are summarized in Appendix 3 (for a complete description see Lindsley and Grell, 1968). The various crosses made to determine the map positions and com-plementation patterns of the Ubx-ts-lethals are discussed in the Results section. TABLE 16 Relative v i a b i l i t i e s of the Ubx-ts-at lethals with 29°C various th i r d chromosomes Cross made: ratio: Ubx-ts-1 Ubx-ts-2 Ubx-ts-3 Ubx-ts-4 Ubx-ts-5 Ubx-ts-6 Ubx-ts-X/lnv Ubx-ts-X/Gl : 52:76 77:126 371:283 37:48 N.T. N.T. X G1/TM3 Ubx-ts-X/TM3 Ubx-ts-X/lnv X Xa Rs(pbx)/T(2;3)ap A a Ubx-ts-X/Rg'(pbx) : Ubx-ts-X/T(2;3)apXa 70:72 35:46 22:40 21:39 N.T. N.T. Ubx-ts-X/lnv x Ubx-ts-X/TM2 : 0:3564 0:3198 0:2763 0:3075 0:128 0:237 Gl Sb eS/TM2 Ubx-ts-X/Gl Sb e S Ubx-ts-X/lnv X Ubx-ts-X/TM2 : 0:125 0:263 0:168 0:229 0:73 0:135 CxD/TM2 Ubx-ts-X/CxD Ubx-ts-X/lnv X Ubx-ts-X/TM2 : 0:69 0:103 0:125 0:57 N.T. N.T. TM2/red Ubx-ts-X/red *discarded N.T.: Not Tested 125 Temperature E f f e c t s : The e f f e c t i v e l e t h a l phase of the Ubx-ts-X homozygotes and hetero-zygotes at the r e s t r i c t i v e temperature of 29°C and the temperature-s e n s i t i v e periods (TSPs) of the various mutants were determined as des-cribed i n Chapter 4. In view of known parental e f f e c t s on the LP of some non-ts-lethals (Hadorn, 1961), eggs were c o l l e c t e d from parents incubated at 22° and 29°C f o r varying lengths of time p r i o r to egg c o l l e c t i o n and the subse-quent development of t h e i r progeny at these temperatures was examined. Males and females from each of the Ubx-ts-lethal stocks were c o l l e c t e d at 22°C wit h i n twelve hours of emergence from the pupal case. Five males and f i v e females of each mutant stock were mated at both 22° and 29°C and transferred at twelve hour i n t e r v a l s to f r e s h v i a l s kept at the two temperatures. T r a n s f e r r i n g continued f o r sixteen days, a f t e r which time the parents were discarded. At the time of each parental t r a n s f e r , the eggs l a i d during the previous twelve hour period were counted. F i f -teen to seventeen days a f t e r the 22°C cultures were i n i t i a t e d and twelve to fourteen days a f t e r those at 29°C were st a r t e d , the number of adults that emerged, the number of pupae (and the developmental stage a t t a i n e d ) , and the number of pseudopupae ( i . e . larvae which ascend the sides of the v i a l s and prepare for metamorphosis, but never form a puparium) present were determined f o r each v i a l . The numbers of males and females were scored at 22°C i n order to determine the temperature-effect on the sex g r a t i o . Controls, i n c l u d i n g a wild-type Samarkand stock, Gl Sb e /TM2 f l i e s , and each of the Ubx-ts-lethals balanced over TM3, were treated s i m i l a r l y . 1?6 i Interactions of the Ubx-ts-lethals with other Chromosomes: The possibility existed that each Ubx-ts-lethal was in fact an 130 allele of a lethal fortuitously carried on Ubx Otherwise the l e -thality could be due to a specific interaction of the Ubx-ts-allele with 130 the Ubx locus. Therefore, several crosses were carried out to deter-mine whether the Ubx-ts-lethals interacted with any of the other mutations in the bithorax complex. The bithorax pseudoallelic series is comprised of six genetically separable groups of mutants, each having characteris-tic phenotypes which are readily distinguishable from those of the other groups. This cluster of homeotic mutations (i.e. mutations which result in the replacement of one organ structure by another structure from an homologous organ—Bateson, 1894) is concerned with the transformation of the meso- and metathoracic as well as the first abdominal segments. Depend-ing on the mutation under study, these segments may be converted to mesothorax (bx and pbx which change the haltere into a wing-like struc-ture) or metathorax (Cbx and Cbxd which cause the wing-bearing segment to be transformed in the direction of the haltere-bearing segment; bxd which results in the transformation of the first abdominal segment toward a thoracic state; and Ubx which combines the properties of bx, bxd, and pbx) (Lewis, 1967). Trans-heterozygotes of the Ubx-ts-lethals with several other third chromosomes were generated by mating single virgin females of the Ubx-ts-X/TM3 stocks to males from the tester stocks at both 22° and 29°C. The progeny were scored for enhancement of the various phenotypes asso-ciated with the bithorax complex for abnormal legs, antennae, or •127 genitalia, and for synthetic lethality or semi-lethality. Since the number of individuals recovered in some of the crosses was not very large, considerable fluctuation in recovery frequencies of the various chromosomes must be expected. The distinction between synthetic le-thality and semi-lethality was thus based on the ratio of Ubx-ts (or tester mutation)-bearing progeny heterozygous for a bithorax pseudo-allele to their sibs bearing the balancer chromosome from the bithorax-pseudoallele stocks. Ratios of less than 1/30 were arbitrarily desig-nated as synthetic lethals and those greater than 1/30 but less than 1/8 as synthetic semi-lethals. •128 I I I . Results Among 25,000 chromosomes tested for the induction of DTS-lethals, s i x mutations were recovered which are v i a b l e at 22°C but behave as l e t h a l s at 29°C when maintained as heterozygotes with TM2, Ubx^"3^. Whereas DTS-lethals are l e t h a l when heterozygous f o r any t h i r d chromo-some under r e s t r i c t i v e conditions, these mutations (Ubx-ts-lethals) are completely v i a b l e with CxD, TM3, and several other t h i r d chromosomes at 29°C (Table 16). The f i r s t two Ubx-ts-lethals recovered i n the screen were discarded, but the subsequent induction of four more muta-tions with s i m i l a r c h a r a c t e r i s t i c s prompted us to carry out a study of the genetic and developmental properties of t h i s unique c l a s s of tempera-t u r e - s e n s i t i v e mutations. Genetic Properties of the Ubx-ts-lethals. Approximate genetic l o c a l i z a t i o n s of the Ubx-ts-lethals were made r e l a t i v e to the markers Lv_ (map p o s i t i o n 40.5) and Sb (map p o s i t i o n 58.2) i n the following manner: r e p l i c a t e v i a l s containing s i n g l e v i r g i n females heterozygous f o r one of the Ubx-ts-lethals and the Ly_ Sb chromo-some and three Gl_ Sb eS/TM2 males were maintained at both 22° and 29°C. Progeny were scored three and seven days a f t e r the onset of emergence at the two temperatures. Ubx-ts-X/Ly Sb males were mated to Gl Sb e /TM2 females at 29°C i n order to confirm the presence of the Ubx-ts-lethals while s i b l i n g Ubx-ts-X/Ly Sb females were tested for crossing over. Table 17 summarizes the r e s u l t s obtained i n these studies and Figure 15 shows the po s i t i o n s of the Ubx-ts-lethals r e l a t i v e to other markers on TABLE 17 Genetic localization of Ubx-ts mutations by crossing-over Cross made: A: Ubx-ts-X/Ly Sb 2 x Gl Sb eS/TM2 <S B: Ubx-ts-X/Ly SW x Gl Sb eS/TM2 2 Genotypes of Progeny Cross Temp. Ly_ Sb/TM2 + +/TM2 + +/G1 Sb ef_ Ly +/TM2 Ly_ +/G1 Sb ej_ + Sb/TM2 Total Progeny Map Posi-tion Ubx-ts-1 A 22 1105 1030 1113 170 248 261 3827 A 29 1407 20 1431 150 208 79 3295 46.6 B 22 664 645 599 0 0 0 1908 B 29 2189 0 2302 0 0 0 4491 Ubx-ts-2 A 22 515 536 425 107 117 168 1868 A 29 712 6 185 164 130 32 1229 43.4 B 22 389 437 415 0 0 0 1241 B 29 1595 0 652 0 0 0 2247 Ubx-ts-3 A 22 365 421 339 43 68 110 1346 A 29 516 3 564 57 99 32 1271 47.2 B 22 96 99 112 0 0 0 307 B 29 833 0 847 0 0 0 1680 Ubx-ts-4 A 22 653 567 713 115 175 151 2374 A 29 1671 31 1745 273 380 104 4304 B 22 204 218 237 0 ' 0 0 659 B 29 1379 0 1543 0 0 0 2922 FIGURE 15 Genetic pos i t i o n s of the Ubx-ts-lethal mutations on chromosome 3 130 Markers Genetic positions 0 Ubx-ts- lethals Genetic positions 434 •132 the third chromosome. The four Ubx-ts mutations can be seen to map within 4.6 map units of each other. These observed differences i n map position may be real or they may reflect differential effects of the Ly_ deficiency on crossing-over with the Ubx-ts-bearing chromosomes as well as the genetic heterogeneity of each stock. Ubx-ts-1 and Ubx-ts-4 were also mapped with respect to the marker red, (map position 53.6). The crosses made and the results of these studies are outlined in Appendix 4. Whereas the map positions, as determined by the Ly-Sb studies, indicated that Ubx-ts-1 and -4_ were approximately 1.4 map units apart (46.6 and 48.0 respectively), those determined by the second method suggested they are only 0.5 map units apart and located at 45.0 and 44.5 respectively. Thus the four Ubx-ts-lethals may be substantially closer to one another than indicated in these studies. The developmental properties of homozygous Ubx-ts-bearing progeny are shown i n Table 18. Ubx—ts—X/TM3 males were crossed to Ubx-ts—X/TM3 females and the rates of hatching of the eggs and of eclosion of the progeny at 22°C were determined. Only Ubx-ts-1 survived i n the homozy-gous condition, although such males were ste r i l e at both 17° and 22°C. In order to test whether the homozygous lethality of the other Ubx-ts's was due to recessive lethals present elsewhere on the chromosomes, a number of cross-over chromosomes (from the 22°C mapping crosses—Table 17) containing the Ubx-ts's and either Ly_ or Sb were tested for via-b i l i t y with the original Ubx-ts chromosomes. Table 19 summarizes the ^ results of these tests and shows that Ubx-ts-3 i s also viable in homo-zygous condition. The tests with Ubx-ts-2 and Ubx-ts-4 were not TABLE 18 Determination of the Effective Lethal Phase of Ubx-ts-lethal homozygotes at 22°C Number in each developmental stage % lsts/eggs % adults/lsts %adults/eggs Parental Genotype Eggs lsts Adults Homozygous-Ubx-ts Samarkand/TM3 700 467 396 66.7 84.8 56.6 Ubx-ts-l/TM3 400 272 259 68.0 95.2 64.8 Viable Ubx-ts-2/TM3 374 259 152 69.2 58.7 40.6 Larval le-thal Ubx-ts-3/TM3 585 361 218 61.7 60.4 37.3 Larval le-thal Ubx-ts-4/TM3 392 259 175, 66.0 67.5 44.6 Larval le-thal TABLE 19 Survival of Ubx-ts-bearing cross-overs between Ly_ and Sb as homozygotes at 22UC. Cross made: Ly_ Ubx-ts-X/ or Ubx-ts-X Sb/TM2 </ x Ubx-ts-X/TM3 ? Ubx-ts-X genotype of number cross-over tested chromosome 2 2 ^ 29°C: Number of Cross-over stocks showing indicated Temperature Response  viable viable viable lethal lethal lethal homozygous Ubx-ts-X at 22UC Ubx-ts-1 '+ Sb 17 3 14 0 viable Ly + 0 — - — Ubx-ts-2 + Sb 19 3 0 16 lethal Ly + 0 - — — Ubx-ts-3 + Sb 7 0 6 1 viable Ly + 3 1 2 0 Ubx-ts-4 + Sb 6 0 0 6 lethal Ly + 3 3 0 0 •135 extensive enough to determine whether secondary recessive l e t h a l s were responsible f o r t h e i r homozygous l e t h a l i t y . In view of t h e i r phenotypic s i m i l a r i t i e s ( l e t h a l i t y with TM2 at 29°C) and t h e i r genetic proximity, complementation te s t s were c a r r i e d out to determine whether the four Ubx-ts-lethals were indeed function-a l l y r e l a t e d . Trans-heterozygotes f o r a l l combinations of the Ubx-ts-l e t h a l s were generated by crossing heterozygous Ubx-ts-X/Inv (where Inv i s e i t h e r TM2 or TM3) males to Ubx-ts-Y/lnv females at both 22° and 29°C. At 22°C, only the Ubx-ts-2/Ubx-ts-4 trans-heterozygotes f a i l e d to sur-vive whereas a l l combinations were i n v i a b l e at 29°C. (Table 20). The s u r v i v a l of most heterozygote combinations under permissive growth con-d i t i o n s r u l e s out the p o s s i b i l i t y of a common second s i t e recessive l e t h a l induced on a l l four ts-containing chromosomes as being responsible for the l e t h a l i t y at 29°C. These r e s u l t s suggest that the mutations are independent changes wit h i n a sing l e locus, which are genuine ts a l l e l e s that survive at 22°C. Since each Ubx-ts was independently induced, t h e i r f a i l u r e to complement at 29°C strongly suggests that they a l l share a common l e t h a l locus. Since they a l l i n t e r a c t with TM2 i t i s not un-reasonable to suppose that the i n t e r a c t i n g locus i s the causative s i t e . Attempts were made to determine the r e l a t i v e p o s i t i o n s of the other Ubx-ts's with respect to Ubx-ts-1 i n the following manner: females, heterozygous f o r D Ubx-ts-1 red and one of the other Ubx-ts-lethal chromosomes marked with Sb, were i n d i v i d u a l l y mated to three D red Sb/TM2 males i n v i a l s at 29°C. Note that a l l non-crossover chromosomes produce 136 T A B L E 20 Results of tests of a l l possible combinations of Ubx-ts mutations in trans-heterozygotes Cross made: Ubx-ts-X/TM3 °* x Ubx-ts-Y/TM3? Ubx-ts-X Ubx-ts-Y Temperature °C Ubx-ts-1 Ubx-ts-2 Ubx-ts-3 Ubx-ts-4 Ubx-ts-1 22° 29° 243/520 0/826 '> Ubx-ts-2 22° 191/253 0/361 - 29° 0/141 0/791 Ubx-ts-3 22° 140/245 224/357 0/287 29° 0/238 0/154 0/465 Ubx-ts-4 22° 195/280 0/292 138/214 0/296 29° 0/230 0/214 0/174 0/512 Numbers represent the ratio of Ubx-ts-X/Ubx-ts-^: Ubx-ts/TM3 progeny. •137 l e t h a l combinations with the paternal chromosomes. Moreover, only wild-type crossovers between the Ubx-ts a l l e l e s survive with TM2 at 29°C and such crossovers between D and Sib survive with the D red Sb chromosomes. A l l progeny r e s u l t i n g from t h i s cross were scored and estimates of the number of zygotes ( l e t h a l and viab l e ) from each cross were made i n order to e s t a b l i s h linkage distances between the four t s -l e t h a l s (Table 21). Although i n s u f f i c i e n t progeny were obtained to permit an ordering of the other t s ' s r e l a t i v e to Ubx-ts-1, i t can be seen that the mutations are a l l t i g h t l y l i n k e d and with i n a maximum of 0.17 map units of Ubx-ts-1. This distance may be further reduced upon examination of l a r g e r numbers of progeny. These data f u r t h e r suggest that the four chromosomes carry mutations i n a common locus. Temperature E f f e c t s . In order to determine the r e l a t i v e v i a b i l i t i e s of the Ubx-ts-l e t h a l s , a cross of Ubx-ts-X/TM2 males by Gl_ Sb_ eS/TM2 females was made at 17 , 22 and 29 C. In con t r o l crosses, Ubx-ts-X was replaced by a Samarkand chromosome. The r a t i o of Ubx-ts-X/TM2 progeny to the t o t a l number of f l i e s provides a measurement of the r e l a t i v e v i a b i l i t y of Ubx-ts f l i e s at d i f f e r e n t temperatures. The r e s u l t s are shown i n Table 22. A l l of the Ubx-ts-lethals are completely l e t h a l with TM2 at 29°C and have a v i a b i l i t y ranging between 68 and 84% that of the wild-type at 17° and 22°C. A comparison of the growth curves of the Ubx-ts-lethal/TM2 heterozygotes at 22°C reveals a high degree of s i m i l a r i t y among them, TABLE 21 Tests for Crossing Over Between Different Ubx-ts Mutations and Ubx-ts-1 Cross Made: D Ubx-ts-1 red + 0 D red Sb , Qo_ + Ubx-ts-X + Sb + TM2 + Genotypes of Progeny: D Sb/TM2 red/TM2 red/D red Sb +/D red Sb Total ; Estimate of Maximum distance Progeny Total # of between zygotes sara- Ubx-ts-1 + Ubx-ts-X pled Crossover Region: ts -ts x 1 ts -ts 1 x D - red red - Sb Expected crossover frequency: Unknown Unknown 12.9% 4.6% Transheterozygote: Ubx-ts-l/Ubx-ts-2 0 0 128 26 154 880x2=1760 .11/2=.06 Ubx-ts-l/Ubx-ts-3 0 0 40 12 52 297x2=594 .33/2=.17 Ubx-ts-l/Ubx-ts-4 0 0 83 29 112 640x2=1280 .15/2=.08 03 09 Temperature TABLE 22 Relative V i a b i l i t i e s of Flies Heterozygous for + or Ubx-ts-X and TM2 at Different Temperatures Cross: + or Ubx-ts-X/TM2 x Gl Sb eS/TM2 $ 17°C 22°C 29°C Total Progeny % of Total Progeny % of Total Progeny % of Progeny Ratio* Control Progeny Ratio* Control Progeny Ratio* Control Ratio Ratio Ratio Samarkand 532 0.31 1072 0.32 1063 0.34 Ubx-ts-1 196 0.26 84.0 231 0.26 80.4 926 - 0 Ubx-ts-2 241 0.25 80.6 195 0.24 75.0 1127 - 0 Ubx-ts-3 148 0.23 74.2 204 0.22 68.8 1329 - 0 Ubx-ts-4 217 0.25 80.6 227 0.22 68.8 , 821 — 0 * Ratio of + or Ubx-ts-X/TM2 bearing progeny to total offspring. Y40 each having larval development prolonged by about 18 to 24 hours over the wild-type. In addition, a l l four have a polyphasic lethal phase, with most of the individuals i n a culture maintained at 29°C dying i n the embryonic and f i r s t and second larval stages, although very rare third instar larvae and early pupae are also seen. Since the cultures contain both homozygous Ubx-ts and TM2 as well as Ubx-ts/TM2 larvae, the various LPs may reflect their diverse zygotic genotypes. Conditioning of the parents of each stock at 29°C for four days prior to egg collection resulted i n death of the progeny exclusively during the embryonic and f i r s t instar larval stages. Temperature shift experiments were carried out to determine the temperature-sensitive periods for each of the mutants. Again, the heterogeneity of larval genotypes does not allow a precise delineation of the TSPs. Shifts to high temperature revealed three additional lethal phases: as hist o l y t i c pupae, late pupae, and young adults (Figure 16). The TSPs for the latter two LPs overlapped considerably, suggesting that the distinction between lethality during the late pupal stage as opposed to the early part of the adult phase may reflect variables not controlled i n these experiments (for example, residual genotype and heterogeneity of developmental stages at each s h i f t ) . The TSP and LP patterns for the four Ubx-ts-lethals are remarkably alike, thus adding further support for their allelism (Shannon, Kaufman, and Judd, 1972). Although the possible influence of residual background cannot be ruled out, the minor variations i n duration of the TSPs may be FIGURE 16 The effective lethal phases (LPs) and temperature-sensitive periods (TSPs) of the Ubx-ts-lethals TSP LP 1,2,3 represent respective TSPs and LPs x time (in hours) that first and second instar larvae may be maintained at 29°C before shifting to 22°C and giving at least 15% viability to the adult stage. 1 4 1 I I J X X> 4 1 4 C N I tn j-i i X 1=1 i Ul I X Xi v t I (0 4-1 I X X) 92 hr 60 hr 80 hr HS3SS 52 hr EGG 1 1st 1 2nd J 3rd 1 L A R V A L I N S T A R PUPA j dlesjsurvives ADULT 143 an i n d i c a t i o n that each a l l e l e represents a d i f f e r e n t p o s i t i o n of the l e s i o n w i t h i n the locus . Another i n t e r e s t i n g aspect of the Ubx-ts's i s an extensive prolongation of the f i r s t and second l a r v a l i n s t a r stages at 29°C, yet s u r v i v a l to adulthood upon return to 22°C (Figure 16). Maintenance of Ubx-ts-4/TM2 heterozygotes as f i r s t and second i n s t a r larvae f o r more than about 52 hours p r i o r to the s h i f t down f a i l s to y i e l d s u r v i v i n g adults. On the other hand, more than 15% of Ubx-ts-l/Ubx-ts-1 and Ubx-ts-l/TM2 larvae develop to adulthood a f t e r a 90-hour exposure to the r e s t r i c t i v e temperature. The other mutants show intermediate s u r v i v a l times, although again the r e s i d u a l genotype may be responsible f o r these d i f f e r e n t i a l e f f e c t s . A s t r i k i n g d i f f e r e n c e among the four Ubx-ts-lethals i s the e f f e c t of prolonged exposure to 29°C on the number of eggs l a i d w i t h i n succes-sive twelve hour periods. Although the number of eggs oviposited by con t r o l (Samarkand, Gl Sb eS/TM2 and Ubx-ts-X/TM3) females decreased a f t e r about twelve days at both 22° and 29°C, eggs were l a i d throughout the experimental time (Figure 17a-d). No eggs were deposited at 29°C by Ubx-ts-2/TM2 females a f t e r twelve days (Figure 18a) and by Ubx-ts-4/TM2 females a f t e r s i x days (Figure 18b). Moreover, prolonged parental expo-sure to 29°C r e s u l t e d i n e a r l i e r death of t h e i r o f f s p r i n g (Table 23). Interactions of the Ubx-ts-lethals with Other Chromosomes. Three possible explanations accounting f o r the temperature-sensitive synthetic l e t h a l combination of the Ubx-ts mutations and the TM2 chromo-some are as follows: (1) there may be a recessive mutation at the Ubx-ts locus on the TM2 chromosome and the combination of t h i s mutation with FIGURE 17 E f f e c t of age and temperature on the egg-laying capacity of females of the following genotypes: a. Samarkand, b. Gl Sb eS/TM2 c. Ubx-ts-2/TM3 d. Ubx-ts-4/TM3 The procedure followed was the same as f o r Figure 12. . . number of eggs l a i d at 22°C x x number of eggs l a i d at 29°C 144 145 -[eA-isquj anoq n J 3 d p t e x s2Sa jo jaqinnN FIGURE 18 Effect of age and temperature on the egg-laying capacity of Ubx-ts-2/TM2 and Ubx-ts-4/TM2 females The procedure followed was as for Figure 12. upper graph: Ubx-ts-2/TM2 parents lower graph: Ubx-ts-4/TM2 parents . . number of eggs laid at 22°C x x number of eggs laid at 29°C 1 4 6 TABLE 23 Lethal Period of Progeny after Increasing Exposure of Ubx-ts/TM2 Parents to 29°C Lethal Phase Time at 29 C Prior To Initiation of Egg Collection (hrs.) Ubx-ts-1 b* d* Ubx-ts-2 a b e d Ubx-ts-3 a b e d Ubx-ts-4 a b c d 12 24 36 48 60 . 72 84 96 108 120 132 144 144 hr. pattern maintained until: new pattern: + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + • + + + + 240 hr. + + + + + + + + + + + + + + + + + + + + + + + + + + 180 hr. + + + + + + + + + .+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 276 hr. + + + + + + + + + + + + + + • + + • + 156 hr. + + * a) eggs, no development apparent on gross examination * b) dead embryos * c) dead 1st instar larvae * d) dead 2nd instar larvae 149 any one of the t s - a l l e l e s on the mutant chromosome produces l e t h a l i t y at 29°C. This i s testable by recovering cross-overs between the TM2 chromosome and a multiply-marked chromosome bearing c l o s e l y l i n k e d mutations i n the region where Ubx-ts-lethals map. Thus, e i t h e r a TM2-chromosome which no longer i n t e r a c t s with the Ubx-ts-X a l l e l e s or a r e c i p r o c a l marked chromosome which does, w i l l be expected. The pro-cedure, f o r t h i s t e s t i s o u t l i n e d i n Appendix 5 . (2) There may be a temperature-affected p o s i t i o n e f f e c t on l o c i within the TM2 chromosome (Hartmann-Goldstein, 1967) so that v a r i e g a t i o n of the wild-type a l l e l e of the Ubx-ts gene (on the TM2 chromosome) allows the expression of the Ubx-ts-lethal at 29°C, thus giving a synthetic l e t h a l phenotype. However, temperature-sensitivity of the Ubx-ts-X a l l e l e s themselves i s suggested by the ts combinations of the a l l e l e s (Table 20). A test f o r t h i s would be to look f o r v a r i e g a t i o n of v i s i b l e mutations which are g e n e t i c a l l y located near the Ubx-ts-locus. I f t h i s explanation i s correct then one should obtain f l i e s which are mosaic for mutant and wild-type a l l e l e s even though they are heterozygous f o r the mutant a l l e l e s . The r e s u l t s of such a t e s t would, however, be d i f f i c u l t to assess i n view of the temperature-sensitivity of many mutations located i n the region of the c e n t r i c heterochromatin of chromosome 3. (3) There may indeed be a 130 s p e c i f i c i n t e r a c t i o n of the Ubx-ts-lethals with Ubx ,a recessive l e t h a l pseudoallele of the bx_ locus, which i s associated with the TM2 chromosome. This a l t e r n a t i v e was tested by generating trans-heterozygotes f o r the Ubx-ts-lethals and several other Ubx a l l e l e s . These combinations •150 were examined for l e t h a l i t y or enhancement of the mutant phenotypes at both the permissive and r e s t r i c t i v e temperatures. In a d d i t i o n , other members of the bithorax complex were tested with the t s - l e t h a l s . The r e s u l t s of these crosses are summarized i n Tables 24 and 25. Neither the point mutant Ubx a l l e l e s nor those associated with i n v e r -sions showed any consistent e f f e c t with a l l four Ubx-ts-lethals, although Ubx-ts-1 and -2_ exhibited reduced v i a b i l i t y with Ubx^^ at both 22° and 29°C. Two other i n t e r a c t i o n s are evident i n Table 24, 3 namely l e t h a l i t y and s e m i — l e t h a l i t y of Ubx—ts—2 with bx and bx Cbx Ubx bxd pbx r e s p e c t i v e l y at both 22° and 29°C. The Ubx-ts-2 b x H 2 Sb chromosome which shares these properties was recovered i n the cross-H2 " over studies (Table 17) and i s associated with a new bx a l l e l e , bx H2 The complete l e t h a l i t y of the Ubx-ts-2 bx Sb/bx Cbx Ubx bxd pbx may be due to the e l i m i n a t i o n of a modifier from the r i g h t arm of the Ubx-ts-2 chromosome or to the presence of the new b x - a l l e l e . A compari-son of the i n t e r a c t i o n s of the two Ubx-ts-2-bearing chromosomes with those of various bx-containing chromosomes i s given i n Table 25. 3 Again, of the b x - a l l e l e s tested, bx acts most l i k e Ubx-ts-2, although P15 v i a b i l i t y with Ubx , f o r example, i s d i f f e r e n t f o r the two mutations. Since i t might be expected that the extent of i n t e r a c t i o n between d i f f e r e n t mutations would depend on the degree of mutant expression of the a l l e l e s tested, one might expect the Ubx-ts-lethals to be more 130 v i a b l e with other Ubx-alleles since TM2, Ubx i s known to be the 130 "strongest" U b x - a l l e l e : Ubx produces greater transvection phenotypes TABLE 24 A Comparison of the Viabilities of Ubx-ta-X and bx Individuals Heterosygoua for Various bx-pseudoalleles at 22° and 29°C. Cross i Ubx-ts-X/Inv; x a/b , where a and b are the various chromosomea tested. Ratio* •/b lfbx-ta-1 •ta-2 •ta-3 Ubx-ta-•4 . o _ .Vbx-tn-1 bx8* ** a 22"C 29°C 22°C 29°C 22°C 29 °C 22°C 29 C 22 C — j»°e J2CC *»°C by Cbx Pbx bxd pbx/T(2{3)ap _ 45:43 88: S3 3a:49 3*:33 59:70 44:60 91:97 61:49 0:20 S.T. 0b:62 0b:83 Vbx 6 1 d/K 3 7« 103:131 72:95 48:71 75:133 N.T. 80:98 38:35 98:87 N.T. 3:3 N.T. N.T. 123:94 100:77 147:105 84:80 149:124 68:67 92:55 136:120 N.T. N.T. 147:105 N.T. I V 1 e'/InOMPayna 198:171 50:47 N.T. 73:5a 59:39 109:68 139:18 N.T. N.T. 106:48c lll:38 e :nC3«)Ubit101/Sb 129:136 161:174 39:53 54:84 N.T. N.T. N.T. N.T. 1*:5> J0'i21 115:132 88:82 7(2;3)Ub*105/CxD 3:9 33:39 17:7 26:31 10:6 21:26 9:10 17:24 4:9 7:10 N.T. N.T. 7H6. UbxP15/T«i, Ma 43:42 46:15 27:40* 31,0* 23:29 16:24 71:58 2:20 N.T. 43:0b 31iOb PJ!(pb»)/T(2i3)<.p'" 23:23 20:26 10:14 13:18 22:46 13:10 21:39 9:12 N.T. N.T. N.T. N.T. bx3/T(2;3)aD" N.T. 87:87 0b:60 0b:51 18:21 43:73 18:17 46:40 N.T. N.T. 0b:102 0b:63 •u(Hu)2 b* bxd/TMl. Ma 110:103 82:32 74:57* 78:8* N.T. N.T. N.T. 86:36 11:3 N.T. 76:0b J»i0b ss bud V e'/T(2i3)a«X* 63:63 N.T. 92:100 N.T. 40:46 N.T. 70:61 81:62 N.T. N.T. 50:51 94:99 pbx/T(2;3)«pX* 33:44 51:33 21:30 37:46 133:132 121:100 160:123 132:131 6:12 N.T. 9:5 22:16 •ratio: Ubx-ts-X or bx^ /a i Ubx-ts-X or bx^ /b ft-ae=i-lethal combination ^-tenperfcure-aeneitive lethal combination ^-lathal combination *-teraper«4tur«-aenaitive serai-lethal combination C-seai-viable combination 152 153 with bx-pseudoalleles than Ub_x_^____, Ubx"^^, and Ubx P^ (Lewis, 1954, 1967; and observations i n Table 25). The recent observation that z_ enhances the bithorax phenotype of bx/Ubx"*" (Kaufman, e_t a l . , 1973) giving a phenotype strongly resemb-ling the transvection phenotype of TM2/bxP prompted a study of the via-b i l i t y of the z ; Ubx-ts-X/Ubx (where p and q refer to specific bx and Ubx a l l e l e s , respectively) combination at 29°C. Both Ubx-ts-3 and Ubx-ts-4 were tested with Ubx"*"^  and Ubx^"^. A l l combinations were viable. The halteres of the male za;Ubx-ts-3/Ubx"*"^ progeny appeared to be slightly larger than those of the female FMA3; Ubx-ts-3/Ubx^^ sibs. If the break-point of the TM2 inversion near the Ubx-ts locus i s important in the synthetic temperature-sensitivity, one might expect other chromosomes with breakpoints i n this v i c i n i t y to have an effect on the Ubx-ts's v i a b i l i t y . The TM2 breakpoint which i s closest to this region occurs in salivary band 74 (Lindsley and Gr e l l , 1968). The TMl inversion also has a breakpoint i n this v i c i n i t y , at 72E1-2 (Lindsley and Gr e l l , 1968). It i s of interest that Ubx-ts-2 i s lethal with TM1 at 29°C but viable at 22°C, although none of the other Ubx-ts-lethals share this property. I t i s possible that a second ts which interacts with TM1 i s present on the Ubx-ts-2 chromosome but the probability of this i s remote. It is more l i k e l y that the genetic position of Ubx-ts-2, which is closer to the 72E1-2 breakpoint than those of the other Ubx-ts's, i s important i n determining whether a viable or lethal interaction results. Ubx-ts-2 i s also semi-lethal with In(3LR)Payne at 29°C (via-154 b i l i t y at 22 C not tested), whereas the Ubx-ts-2 bx Sb chromosome Is A viable (about 50% the viability of Ubx e_ sibs). The viability of the latter suggests that a locus to the right of the Ubx-ts locus is re-sponsible for the lethal interaction with the Payne inversion. Finally, a number of exceptional progeny were recovered in the interaction studies. These are summarized in Table 26. Most of these were mosaics having distorted legs (or missing legs) or enlarged (or missing) halteres. The enlarged, or wing-like, halteres were similar to those regularly produced by flies heterozygous for a Ubx and bx allele (in trans). Since only one exception of this type was found among flies heterozygous for a wild-type chromosome and either a Ubx-or bx-allele (but not both), but several Ubx-ts-X/Ubx^ or bxP flies had this phenotype, the possibility of a relationship between the Ubx-ts-lethals and the bithorax locus cannot be completely eliminated. Other Second and Third Chromosome "synthetic" lethals. In the process of screening for DTS-lethals, mutations other than the Ubx-ts's were recovered and found to interact with specific chromo-somes. In a search for DTS-lethals on chromosome 3, two mutations were recovered which are lethal only when combined in trans with the small deficiency, Ly, or when homozygous. The two mutations are designated Ly-1-1 and Ly-1-2. In complementation tests, Ly-l-l/Ly-1-2 flies sur-vived at both 22° and 29°C. Crosses of Ly-l-l/Ly-1-2 females by Ly/CxD males failed to yield + + recombinants among an estimated 1000 progeny examined. Thus, the two mutations are very closely linked and may represent defects in loci uncovered by the L^ deficiency. 155 TABLE 26 Exceptional progeny from the crosses of the Ubx-ts-lethals to the various stocks containing the bithorax pseudoalleles Genotype: temp. °C. Description: Ubx-ts-1: /Ubx61d /Xa /In(3LR)Ubxl01  Ubx-ts-2: /Ubx e£ /bx Cbx Ubx bxd pbx m i /Ubx e 4 /Ubx Ubx-ts-2 bx H 2 Sb: /Ubx e 4 /Ubx eT /Ubx e* /Ubx6TlT /In(3LR)Ubxl01 /bx3 /bx Cbx Ubx bxd pbx Ubx-ts-3: 22 1 d 1 no external genitalia 22,29 several hemithorax 29 8 have 1 large wing-like haltere 29 1 o* : 1 leg missing 22,29 semi=lethal 29 ts-pupal lethal 29 1 individual with 4 legs 29 1 individual with 1 haltere missing 22,29 large, wing-like halteres 22 1 ' : ie£t haltere missing 29 1 o* : front leg missing 22,29 slightly enlarged halteres 22,29 wing-like halteres, no extra thoracic tissue 22,29 lethal 22,29 lethal /bxd /Ubx61d /Ubx e 4 Ubx-ts-4: 22 hemithorax (1/9 progeny) 29 slightly enlarged halteres, dropping at sides 29 1 ? : large, wing-like halteres /Ubx e4 29 1 ? : 1 haltere partly wing-like 156 The screen for heat-sensitive DTS-lethals on chromosome 2 yielded nine mutations which were apparently lethal only with In(2LR)bw^ (hereafter called Pm). These were designated as Pm-l-X. Table 27 summarizes the viability of these mutations at 23 + 2°C and 29°C. In crosses of Pm-l-X/Cy x Cy/Pm, for three of the eight stocks Pm-l-X/Pm flies emerge two to six days after the last +/Cy and Cy/Pm flies com-plete elcosion. The viability of Pm-l-X/Pm flies is very poor but a l l have a common phenotype reminiscent of the mutant ds_ (dachous having short, broad wings which are more rounded distally and bear short cross-veins; "chunky" legs and abdomen; short legs, with tarsi four-jointed; body foreshortened). This phenotype suggests that the mutant chromo-somes carry lethal alleles of the ds_ locus. Indeed, the Pm chromosome, which has one break point at salivary gland band 21C8-D1, also carries the ds mutation located at or near the break point. Another four Pm-l's are lethal during the early pupal stage when heterozygous for Pm. The Pm chromosome also has a breakpoint in 40F and several known lethals map close to this region of the second chromosome. Among these is the pupal lethal l(2)crc(cryptocephal). The ts Pm-lethal (Pm-1-4) has a LP at 29° early in pupation and the remaining Pm-lethal is a late pupal lethal. Tests have not been carried out to determine whether the pupal Pm-lethals f a i l to complement the crc mutation. Complementation tests were carried out for six of the Pm-l's and the results are tabulated in Table 28. Two of the Pm-l's having the ds-phenotype and the late pupal Pm-1 produced low numbers of trans-heterozygotes a l l having the ds-phenotype, whereas each of these when combined with a pupal Pm-1 was TABLE 27 Via b i l i t i e s of Pm-l-bearing chromosomes at 23° and 29°C Cross made: Pm-1/SM5, Cy d* x Pm-1/SM5, Cy ?' Pm-l-stock PROGENY GENOTYPES Phenotype of Pm-l/Pm Pm-l/Cy 23° 29 23° Cy/Pm Pm-l/Pm 29 23° 29> Pm-1-1 179 96 207 132 0 0 23°C late pupal lethal; very rare escapers have ds-phenotype Pm-1-2 423 137 450 161 0 0 23°, 29° early pupal lethal Pm-1-3 189 56 255 102 17 0 23°C ds ; 29°C lethal Pm-1-4 329 36 341 89 345 0 23°C viable, wild-type; 29°C early pupal lethal Pm-1-5 354 114 281 167 158 0 23°C ds; 29°C early pupal lethal Pm-1-6 ; 168 59 190 114 29 0 23°C ds_; 29°C early pupal lethal Pm-1-7 291 48 327 136 0 0 23°C early pupal lethal; 29°C early pupal lethal Pm-1-8 251 * 318 * 0 0 23°C early pupal lethal. Pm-1-9 110 45 139 100 0 0 23°C early pupal lethal; 29°C early pupal lethal * not tested TABLE 28 Ratios of Pm-l-X/Pm-l-Y to total progeny from the cross Pm-l-X/SM5 x Pm-1-Y/SM5 at 22°C. Pm-l-Y Pm-1-1 Pm-1-2 Pm-1-3 Pm-1-4 Pm-1-5 Pm-1-6 Pm-1-1 • early pupal lethal 0/465 Pm-1-2 • early pupal lethal 7/452 0/206 Pm-1-3 • • late pupal lethal 190/715 95/231 0/225 Pm-1-4 • ds-like 154/432 26/226 34/242 0/231 Pm-1-5 • ds-like 158/654 69/266 8*/230 ll*/252 0/250 Pm-1-6 • • ds-like 159/634 49/298 6*/224 5*/272 20*/285 0/296 * have ds-like phenotype (for description, see text) '159 more viable (approaching the expected frequency) and wild-type in appearance. In contrast, the trans-heterozygote bearing two pupal Pm-lethals was semi-lethal (1.6% of total progeny). The final class of synthetic lethals was obtained in the screen for dominant cold-sensitive lethals on chromosome 2 (Rosenbluth, et a l . , 1971). In a l l , twenty mutations were recovered a l l of which appeared to be lethal only with SM5, Cy. Nothing is known about their genetic positions and only very limited complementation studies have been car-ried out for a few of the mutants at 17°C. These are summarized in Table 29 and indicate that at least three and possibly four Cy-l's are functionally related. TABLE 29 Complementation Tests of Cy-1 Mutations at 17°C Cross made: Cy-l-X/Pm cf x Cy-l-Y/Pm ? Trans-heterozygote Cy-l-X/Cy-l-Y Cy-l-X + Cy-l-Y/Pm # progeny # progeny 2/1 12 10 2/2 0 21 2/4 22 36 2/6 0 31 2/9 0 30 2/11 26 2/12 13 23 2/14 14 31 2/16 7 21 8/15 0 17 17/2 0 40 17/4 12 22 17/6 0 24 .161 IV. Discussion A number of apparent "synthetic" lethal systems, comprised of two mutations (each of which i t s e l f i s viable) which when combined i n trans produces l e t h a l i t y , has been described for Drosophila. One of the earliest reports of such a system involved the very much reduced v i a b i l i t y of the cv fu double mutant, even though each mutation i s perfectly viable in the homozygous condition (Sturtevant, 1929). This effect i s not restricted to recessive mutations: several Minutes have been reported to be lethal in combination with other dominant visible mutations such as Dl_, >J, and D (cf. Lindsley and Gre l l , 1968) or to enhance the expression of other dominant (L^ , B_ - Dunn and Coyne, 1935; 3 A Ly, Ser, Bx - Bryson, 1940) and recessive (fa, ap - Bryson, 1940) mutations. The latter two categories may be termed "synthetic" visible systems. An extensive study by Dobzhansky (1946), in which recombina-tion between two wild-type chromosomes led to the production of synthe-t i c lethals, semi-lethal, and visible combinations, led him to suggest that chromosomes consist of gene complexes, the components of which may be dispersed throughout one or more chromosomes. He argued that such a system would allow for considerable v a r i a b i l i t y , attainable with re-lative ease since recombination between the constitutents might be ex-pected to occur more frequently in a "dispersed" gene complex than in complex l o c i which are very tightly linked (ex. bithorax locus, Lewis, 1954, 1967). •162 The problem of determining the mechanism of lethal "synthesis" arises from the fact that most systems involve components having pheno-types which do not appear to be related, so that "doubly" reduced v i a b i l i t y (i.e. the summation of two characteristics which independently reduce v i a b i l i t y ) i s not indicated. For example, f l i e s bearing the pn eye colour mutation are specifically k i l l e d when made heterozygous for the K-pn mutation which by i t s e l f has no phenotype and does not alter v i a b i l i t y of otherwise wild-type f l i e s (Sturtevant, 1955, 1956). The dor-ry synthetic lethal combination may, however, provide an indication of how this lethality arises (Lucchesi, 1968). The interaction of dor and rjf, both of which affect pteridine levels, i s highly specific since the substitution of ma-1 for ry_ (both mutants lack the enzyme xanthine dehydrogenase (Glassman and Mitchell, 1959) permits the v i a b i l i t y of dor individuals. It has been suggested (Lucchesi, 1968) that the synthetic lethality of the dor-ry and other mutant combinations (dor-cn bw; dor-pd; fu-ry) i s somehow related to pteridine metabolism (since a l l but one of these mutations affect pigment synthesis or deposition). In this study the genetic and developmental properties of four re-lated temperature-sensitive mutations which are lethal when heterozygous for TM2 (and as trans-heterozygotes with one another) at 29°C but viable at 22°C have been examined in detail. The failure of two of these muta-tions (Ubx-ts-2 and Ubx-ts-4) to complement at 22°C prompted a further characterization of a l l four mutations i n an effort to determine what the relationship between the Ubx-ts-lethal's and the TM2 chromosome i s 163 that produces the temperature-sensitive synthetic l e t h a l i t y . Several experiments were undertaken to determine whether four Ubx-ts-lethal's have i d e n t i c a l genetic and developmental p r o p e r t i e s . The r e s u l t s pointed to d i f f e r e n c e s i n v i a b i l i t y at 22°C as w e l l as t h e i r responses to 29°C (as r e f l e c t e d i n d i f f e r e n c e s i n the f e r t i l i t y of females a f t e r prolonged exposure to t h i s temperature and i n the duration of c e r t a i n TSPs). In a d d i t i o n , the mutations could be distinguished on the basis of the i n t e r a c t i o n s with several other mutant l o c i , i n c l u d i n g some of the bithorax-pseudoalleles and other i n v e r s i o n chromosomes. In a separate study (see Appendix 6 ), v i a b i l i t y at 29°C of the four Ubx-ts's with several ry_ point mutations and d e f i c i e n c i e s was examined. Again the r e s u l t s i n d i c a t e that Ubx-ts-2 and Ubx-ts-4 are indeed d i f f e r e n t from one another. The problem remains, however, that mutations induced e l s e -where on the Ubx-ts-bearing chromosomes may be responsible f o r some or a l l of these v a r i a t i o n s . Although the v a r i e t y of t e s t s c a r r i e d out have served to e l u c i d a t e a spectrum of properties f o r each of the Ubx-ts-lethals, the question as to the genetic mechanism whereby the synthetic temperature-sensitive l e t h a l i t y i s achieved i s as yet to be answered. The r e s u l t s do not e x c l u s i v e l y support one or another of the mechanisms suggested—the genetic l o c a t i o n s of the Ubx-ts's i n the v i c i n i t y of one of the break-points of the TM2 chromosome suggest that l e t h a l i t y at 29°C r e s u l t s from pseudo-dominance of the Ubx-ts's, whereas the i n t e r a c t i o n s with other bithorax pseudoalleles(albeit with low expressivity) seem to implicate :164 some r e l a t i o n s h i p with the bithorax complex locus. Although we have not been able to e s t a b l i s h the operative mechanism, the a l t e r n a t i v e s do suggest that the s e l e c t i o n of mutations such as these may prove an i n -valuable t o o l i n the study of genetic f i n e structure and complex l o c i . In t h i s l i g h t , a more d e t a i l e d discussion of the a l t e r n a t i v e mechanisms for generating the synthetic l e t h a l s and t h e i r s i g n i f i c a n c e i s warranted. As mentioned, the genetic p o s i t i o n s of the Ubx-ts-lethals and the break-points of the TM2 chromosome suggest the p o s s i b i l i t y of pseudo-dominance i n creating the t s - l e t h a l e f f e c t . This i s strengthened by the recovery of s p e c i f i c " l e t h a l s " which i n t e r a c t with the Pm chromosome, three of which have been shown to carry the dachous mutation. The ds phenotype i s expressed by v i r t u e of the presence of t h i s mutation on the Pm chromosome. In a d d i t i o n , the two L y r a - l e t h a l s , which might be ex-pected to map at the Ly-locus c e r t a i n l y does lend support to the notion that a large proportion (and perhaps even a l l ) of the s p e c i f i c a l l y i n t e r -a c t i n g mutations recovered i n our studies owe that s p e c i f i c i t y to pseudo-dominance of a recessive mutation induced at the break-point of the balancer chromosome. In view of the large proportion of these mutations that were recovered, i t must be postulated that an excessive number of l e t h a l s i s produced upon treatment of the sperm with EMS - such a high frequency, i n f a c t , that most treated chromosomes would harbor several recessive l e t h a l mutations. A l t e r n a t i v e l y , i f the mutagen were to produce i t s e f f e c t l a t e r (say, a f t e r f e r t i l i z a t i o n - Auerbach, 1951 and Auerbach and K i l b e y , 1971), then a s e l e c t i v e action might r e s u l t . There .165 may be a greater opportunity for the mutagen to act in regions which are not tightly paired, such as where the breakpoint of an inversion attempts to pair with a normal homologue. A delayed action of the mutagen implies that gonadal mosaics should be produced, and indeed one such case has been conclusively demonstrated (see Appendix 7 ). Also, during the screen several putative DTS-lethals were recovered which, when tested further, yielded mixed progeny—those bearing a putative DTS-lethal and those free of the ts. In recovery of both heat and cold DTS-lethals on chromosome 2, the majority of which map i n the same position (Suzuki and Procunier, 1969; Rosenbluth et a l . , 1972) suggest that there is some specificity in the action of EMS or that the regions affected contain l o c i which are normally susceptible to tem-perature-regulation (see Appendix 2 ). Furthermore, of recessive heat-sensitive and recessive cold-sensitive lethals on chromosome 3 90% and 8 8 %, respectively, map in the same region as the Ubx-ts-lethals (E. Tasaka and D.T. Suzuki, submitted for publications). In any event, the screening procedure has yielded a very interest-ing class of mutations which are amenable to fine structure analysis. The recovery of mutations which behave similarly in both of the major autosomes suggests that a large number of l o c i can be studied i n terms of their fine structure by the Isolation of ts mutations of a specific type. It remains possible, however, that either the inversion, TM2, i s important in conferring l e t h a l i t y at 29°C of the Ubx-ts heterozygotes '166 or that there i s a specific interaction with the bithorax-locus which confers the i n v i a b i l i t y . The former situation i s reminiscent of the position effect phenomenon described by many authors for many complex l o c i (see, for example, Lewis, 1967; Carlson, 1959; Green, 1955). It might be supposed that disruption of the normal gene sequence on the homologue allows the expression of the temperature-sensitive Ubx-lethals under appropriate conditions. Several indications of interactions (of low penetrance) have been mentioned. An important observation which has not yet been discussed, but may be of considerable relevance, is that during the cross-over studies with the L^ Sb chromosome, two new spontaneous bx-alleles were recovered. One of these arose simultaneously with a cross-over to yield H2 a Ubx-ts-2 bx Sb recombinant while the other arose in the Ubx-ts-4 chromosome and was not associated with a cross-over. These may have been new bx mutations occurring at the time they were detected or they may even have been present in the original chromosomes but somehow phenoty-pically suppressed. It i s of interest that the induction of the new bx-alle l e in Ubx-ts-2 was accompanied by the loss of semi-lethality with In(3LR) Payne. Whatever the origin of the new bx-mutations they do seem to strengthen the notion of a relationship between the Ubx-ts's and the bithorax locus. If such i s the case, then this would i l l u s t r a t e an i n -teresting example of the components of a complex locus being dispersed throughout the chromosome. Lewis (1968) has previously shown the rela-tionship of a locus, Rg(pbx), separable from the bithorax-locus by about five map units, which interacts with the wild-type al l e l e of the pbx-pseudo-allele. '167 INTRODUCTION TO PART II M u l t i c e l l u l a r organisms are capable of d i f f e r e n t i a t i n g several morphological and p h y s i o l o g i c a l l y d i s t i n c t c e l l types, organizing them int o b i o l o g i c a l l y f u n c t i o n a l t i s s u e s , and co-ordinating these into i n -t r i c a t e organ systems. Such a task, of necessity, requires an " i n f a l l i b l e " means of ensuring the unerring i n t e g r a t i o n of the funda-mental components at each of these l e v e l s of organization. Elegant analyses of gene reg u l a t i o n i n prokaryotes have provided an i n s i g h t into the p o s s i b l e mechanisms governing the sophisticated d i f f e r e n t i a t i o n processes c h a r a c t e r i s t i c of eukaryotic organisms. C l e a r l y , s p e c i a l i z e d "macro-regulatory" ( L u r i a , 1970) systems have been superimposed to pro-vide for generalized c o n t r o l during s p e c i f i c stages i n the development of organisms with complex l i f e cycles marked with periods of d i s t i n c t l y d i f f e r e n t metabolic a c t i v i t y ( i . e . d i f f e r i n g e i t h e r q u a l i t a t i v e l y or q u a n t i t a t i v e l y ) . Examples of t h i s type of "macroregulation" can be found i n organisms as diverse as b a c t e r i a (for example, those with sporulating phases), insec t s ( i n which l a r v a l and imaginal stages are prevalent), plants (gametophytic and sporophytic generations), and so on. C l a s s i c a l -l y , we are aware of the importance of various hormones i n c o n t r o l l i n g s p e c i f i c c e l l functions both i n plant and i n animal systems. Numerous other l e v e l s of control have also been proposed, and undoubtedly, as more i s known about the s p e c i f i c mechanisms of t r a n s c r i p t i o n and t r a n s l a -t i o n , other means of c o n t r o l w i l l become apparent. A study of possible regulatory r o l e s of s p e c i f i c macromolecules i s f a c i l i t a t e d by determining the e f f e c t of an absence or reduced quantity of the s p e c i f i c macromolecules 168 on the subsequent development of the organism. One of the few primary gene products amenable to analysis both quantitatively and qualitatively is tRNA. By the production of mutations within tRNA genes it is possible to study the specific effects of altered or reduced amounts of specific tRNAs both in vivo and in vitro. The studies presented in the following Chapters 2 and 3 form a basis for the study of tRNAs in Drosophila melanogaster. These chapters are taken from papers in press in Developmental Biology and Journal of Molecular Biology, respectively, and are co-authored by Bradley N. White, Gordon M. Tener, David T. Suzuki, and myself. My interest in Drosophila tRNAs arose during the developmental studies on the DTS-lethals showing a Minute phenotype during the permissive conditions. The studies were initiated in order that K. C. Atwood's hypothesis might be tested. My role in these studies was to help establish conditions for optimal amino-acylation of the various tRNA species and to examine differences in isoaccepting species present in adult and third instar larvae. The various "special techniques" (i.e. thin-layer chromatography of pancreatic A S D RNas digests and piperidine hydrolysates of purified tRNA , UV absorption spectra, detailed analyses of isoaccepting species from carefully aged stages of Drosophila by RPC 5 chromatography, et cetera} were contributions of Dr. Bradley White. 169 CHAPTER i 1 tRNA CHANGES DURING DEVELOPMENT (1) P r e r e q u i s i t e to the ra p i d rate of nuclear d i v i s i o n following the f e r t i l i z a t i o n of an egg, i s the presence of large q u a n t i t i e s of DNA, RNA, and prote i n . Rates such as the 15-minute cleavage cycle of the Culex pipiens egg ( I d r i s , 1960) and the remarkably rapid 8-minute nuclear d i v i s i o n time f o r the Drosophila egg (Schneider-Minder, 1966) necessitate some mechanism whereby f e r t i l i z a t i o n i s followed immediately by the rapid turnover of the appropriate components with a minimum of energy expenditure. (2) The l a r v a l and adult forms of an insec t might w e l l be c l a s s i -f i e d as two d i s t i n c t and unrelated organisms i n the absence of the ob-served metamorphosis of the former i n t o the l a t t e r . The v i s u a l and locomotory organs of the adult, f o r example, f i n d no analogous structures i n the l a r v a . Thus i t would appear that d r a s t i c a l t e r a t i o n s i n c e l l u l a r metabolism accompany d i f f e r e n t i a t i o n : a large proportion of the proteins i s no longer produced while an e n t i r e l y new spectrum of macromolecules appears. (3) The t r u l y dramatic process whereby an organism with a very d i s -t i n c t i v e morphology metamorphoses to a wholly new and unrelated form v i a a reorganization i n v o l v i n g the simultaneous breakdown of s p e c i f i c t i s s u e s and formation of new ones, demands an i n t r i c a t e and p r e c i s e l y c o n t r o l l e d set of regulatory events. £70 Some or a l l of the above s i t u a t i o n s are met i n p r i n c i p l e by many and diverse organisms at some time during t h e i r l i f e c y c l e . The ob-vious property shared by a l l three types of events i s the r a p i d i t y with which a change i n metabolic s t a t e — e i t h e r from a r e l a t i v e l y i n e r t to an inte n s e l y a c t i v e or from one a c t i v e to an a l t e r n a t i v e a c t i v e state—must take place. The co-ordinated induction or repression of a large number of genes must then involve some sort of "macroregulatory" ( L u r i a , 1970) process. Although r e g u l a t i o n of the type described by Jacob and Monod (1961) undoubtedly plays a r o l e i n some aspects of d i f f e r e n t i a l p r o t e i n synthesis, one might imagine that such a mechanism would require more than, for example, the eight b r i e f minutes a l l o t t e d p r i o r to the onset of the f i r s t nuclear d i v i s i o n i n the f e r t i l i z e d Drosophila egg. Two a l t e r -native l e v e l s of r e g u l a t i o n (corresponding to the l a t e r steps i n the pro t e i n synthetic process) , coupled with t h i s i n i t i a l t r a n s c r i p t i o n a l c o n t r o l , might be expected to play major r o l e s during the t r a n s i t i o n phases described above. The f i r s t of these i s at the l e v e l of t r a n s l a -t i o n , and might involve the presence of any one of a number of "stage-or t i s s u e - s p e c i f i c f a c t o r s " . Thus, any molecules involved with, f o r ex-ample, messenger RNA s t a b i l i z a t i o n or some event leading to the success-f u l a c t i v a t i o n of a transfe r RNA and i t s subsequent i n t e r a c t i o n with the messenger RNA and ribosomes, may be a candidate f o r co n t r o l at t h i s l e v e l . The t h i r d l e v e l , then, would be p o s t - t r a n s l a t i o n a l , i n v o l v i n g the a c t i v a -t i o n of previously synthesized proteins. Without negating the po s s i b l e importance of each of these regulatory mechanisms, the present discus-sion w i l l be r e s t r i c t e d to the possible r o l e s of tr a n s f e r RNAs as 171 regulatory molecules. The f i r s t indication that tRNAs might be important i n regulating protein synthesis came from studies on the mechanism of suppression in bacteria (Yanofsky et a l . , 1961) and bacteriophages (Benzer and Champe, 1961). Since then, tRNAs have been found to play a major role i n the suppression of nonsense mutations (Engelhardt et a l . , 1965; Smith et a l . , 1966). Several mechanisms by which tRNAs may regulate protein synthesis have been suggested and w i l l be briefly discussed i n the following para-graphs, in the chronological order of their proposal. The f i r s t of these, the "modulation hypothesis", supposes that par-ticular isoaccepting tRNAs are either present in low amounts or have a lower a f f i n i t y for ribosomes. During translation, then, a ribosome has . a low probability of completing synthesis of the protein corresponding to the message with which i t i s associated once i t reaches a "modulating t r i p l e t " on the messenger (Ames and Hartman, 1963). This concept was formulated to account for the observation made by Itano, i n 1963, that in heterozygous individuals different hemoglobin alleles produce di f f e r -ent amounts of their respective hemoglobins. In 1964, Sueoka and Kano-Sueoka suggested that the genetically-controlled chemical modification of specific isoaccepting tRNAs would eliminate their functioning i n protein synthesis, thus controlling the spectrum of proteins synthesized at a particular time. Since that time Peterkofsky et a l . (1966, 1968) have shown that i n E_. c o l i methylation of one isoaccepting leucyl-tRNA confers specificity i n i t s a b i l i t y to recognize certain codons, giving support for this role of modification of tRNAs. ,172' The third means of control has been termed the "abundancy hypothe-s i s " by Sueoka and Kano-Sueoka (1970) but i s , i n fact, an extension of the proposal of Ames and Hartman (1963). In a series of experiments Anderson (1969) showed that high tRNA concentrations (i.e. high relative to the ribosome population size) stimulate the rate of polypeptide syn-thesis, suggesting to him that specific "rate-limiting" tRNAs within cells are responsible for the post-transcriptional control of the rate of protein synthesis. Subak-Sharpe and Hay, i n 1965, suspected that a new tRNA species might be produced upon infection of c e l l s with Herpes virus. Since the base composition of the v i r a l DNA differed markedly from that of i t s host, i t was supposed that the host's translational apparatus would be inefficient in synthesizing v i r a l proteins.Although these workers later re-tracted their claim of a v i r a l - s p e c i f i c iso-accepting arginyl-tRNA being synthesized upon infection, this possibility remains an interesting one. In such cases, i t is expected that the new tRNA species is involved i n effect-ing the synthesis of new proteins during differentiation (to which the presence of the virulent virus within i t s host c e l l might be likened). The f i n a l mechanism of control by tRNA has been referred to as the "inhibitor hypothesis" (Sueoka and Kano-Sueoka, 1970) and assumes the presence within cells of a particular tRNA inhibitor which prevents i t s functioning i n protein synthesis. The recent evidence by Twardzik et a l . (1971) and Chapter 3 of this section suggest that the inactivation of Tyr tryptophan pyrrolase by a specific tyrosyl-tRNA (tRNA^ ) may represent such a system, for this tRNA apparently remains complexed with the enzyme, thus preventing i t from participating i n translation. .173 Several mechanisms have been described by which tRNA molecules may be involved i n the c o n t r o l of the p r o t e i n synthesis at the t r a n s l a -t i o n a l l e v e l . Evidence of systems i n which some of these might be operative has also been presented. However, i f such r o l e s are of im-portance i n the types of events presented at the beginning of t h i s sec-t i o n , then one might suppose that an analysis of trans f e r RNAs produced at d i f f e r e n t times during c e l l d i f f e r e n t i a t i o n or, subsequently, during the development of the organism might produce a spectrum of changes de-pendent on the system investigated. Some of these w i l l be pointed out here as a prelude to the study of tRNA changes during the development of Drosophila melanogaster. In the past decade several developing systems have been examined for tRNA changes occurring at d i s t i n c t developmental stages. An accurate analysis of tRNA-species f l u c t u a t i o n s i s dependent upon a s e n s i t i v e means of i d e n t i f y i n g not only each of the aminoacyl-tRNAs, but also a l l of the isoaccepting, as w e l l as chemically modified, forms. Since the i n i t i a -t i o n of these studies, a number of d i f f e r e n t chromatographic systems have been developed, and, as i s to be expected, the methods applied i n the e a r l i e r studies were not as s e n s i t i v e as those currently applied. Thus, the r e s u l t s , seemingly perhaps, are not as s t r i k i n g as they might be i f a 100% separation of isoaccepting forms were possi b l e . In several cases, then, the observed p i c t u r e i s one of minimal changes occurring i n the stages examined. This w i l l be commented upon again a f t e r the evidence i n support of tRNA changes i s presented. 174 Perhaps the simplest model-system for differentiation is the virus-or bacteriophage-infected c e l l . In a l l such "host-parasite" systems, dramatic changes in the cell's metabolism can be detected soon after infection. In many such relationships the new "tenant" assumes a "directing role", utilizing the cell's machinery to reproduce its e l f . For many viruses, this process involves two distinct virally-determined phases: the "early" and "late" phases of infection. The so-called "early" proteins are those involved in the synthesis of new viral DNA, although there may also be specific enzymes produced for the dissolution of the host DNA (as in T-even infection—Kellenberger, 1961). A compari-son of the profiles from MAK column chromatography of tRNA from T^-inf ected and non-inf ected E_. coli revealed a new, phage-specif ic leucyl tRNA which hybridizes to the T^ -genome (Weiss et a l . , 1967, 1968). There is some suggestion that this new tRNA is formed prior to the synthesis of the late proteins (i.e. those forming the head and t a i l of the phage), implicating the necessity for this new species in the translation of certain late virus-specific proteins. The induction by Herpes virus of a new arginyl-tRNA has already been mentioned. The spore and vegetative stages of Bacillus subtills represent two very different states—both morphologically and metabolically. Elution of valyl-tRNAs from MAK column revealed two species which change in re-lative amounts from the vegetative to the early sporulation phase, revert-ing back to the vegetative pattern late in sporulation (Doi e_t a l . , 1966, 1968). »7S Numerous other studies with B_. su b t i l i s have been made investigat-ing the role of nutrients on tRNA species available for translation. Differences, both quantitative and qualitative, were found (Lazzarini, 1966; Sueoka et a l . , 1966, 1969), possibly suggesting an interrelation-ship between amino acid a v a i l a b i l i t y and the presence of corresponding tRNAs. An extensive survey of aminoacyl-tRNAs from several organs of numerous species has been made by Holland et a l . (1967a, 1967b, and 1968) using MAK column chromatography. Some differences were noted be-tween elution profiles from different organs (for example, seryl-tRNA from l i v e r contained a minor species, not present i n kidney or muscle tRNA, for both the rabbit and mouse) and between elution profiles of tRNA from the same organ but different species (as an example, tyrosyl-and leucyl—tRNAs from avian and mammalian sources are different). Other workers (Hatfield and Caicuts, 1967), using the RPC (reversed-phase chromatographic) system, found differences for methionyl-, arginyl-, and seryl-tRNAs from the brain as compared to other organs examined. Several studies have been made of tRNAs present during early and later stages of differentiation as well as at various times during the development of multicellular organisms. Yang and Comb (1968) studied lysyl-tRNA from the unfertilized sea-urchin egg and the two-cell stage, and found differences i n the distribution of two isoaccepting species between the particulate (i.e. containing mitochondria and ribosomes) and soluble fractions. Quantitative differences in two methionyl-tRNAs from immature red blood cells and mature reticulocytes have also been demon-strated, using both the MAK column and the reversed-phase chromatographic •m methods (Lee and Ingram, 1967). Ilan (1969) and Ilan et a l . , (1970) have presented evidence for translational control i n Tenebrio pupae. Their studies indicate that preparations from 5-day-old pupae contain both a new isoaccepting tRNA species (tyrosyl-tRNA) and a new aminoacyl-tRNA synthetase which are able to effect the synthesis of adult cuticular protein. Dewitt (1971) has found differences both in the methionyl- and arginyl-tRNAs from larval and adult bullfrogs. Studies on several other systems similarly indicate that both qualitative and quantitative changes in tRNA acceptor activity may occur during development (brine shrimp: Bagshaw at a l . , 1970; wheat seedlings: Void and Sypherd, 1968; soybean hypocotyls and cotyledons: Anderson and Cherry, 1969). The observations that certain viruses are able to induce the syn-thesis of viral-specific tRNAs prompted an investigation into possible tRNA differences between normal and transformed c e l l lines. By reversed phase chromatography, Yang and Novell! (1968) found large differences in seryl-tRNAs from two plasma c e l l tumors. Mach et a l . (1967) also found differences between aminoacyl-tRNA patterns from plasmocytoma tumors as compared to those from normal c e l l s . Differences i n leucyl-and threonyl-tRNAs were noted for different tumors. Taylor et a l . (1967), by comparing tRNA elution profiles from Ehrlich ascites tumor and mouse sarcoma-1 cells and from normal rabbit and mouse tissues, found differences in some of the tRNAs examined (phenylalanyl-, glycyl-, tyrosyl-, and seryl-tRNAs). The pattern of differences suggested to the workers that the normal tRNA had undergone some form of modification which may or may not be related to the neoplastic—as opposed t o — 177; normal c e l l types. We have seen, then, that very diverse systems may show a l t e r a t i o n s i n tRNA patterns depending on t h e i r stage i n development or d i f f e r e n t i a -t i o n . I t thus seems reasonable to expect some changes to occur during the development of Drosophila. The importance of i n v e s t i g a t i n g tRNA changes i n yet another organism i s not simply one of the confirmation that most or a l l higher organisms share t h i s property of tRNA frequency f l u c t u a t i o n s , f or Drosophila o f f e r s unique t o o l s f o r i n v e s t i g a t i o n be-yond t h i s b a s ic biochemical c l a s s i f i c a t i o n . I t ' s well-characterized genetics o f f e r s an opportunity to examine the action of mutant tRNA molecules with a view to determining the s p e c i f i c r o l e ( s ) of tRNAs i n c o n t r o l l i n g developmental processes. t Two chromatographic methods were employed i n the study of tRNAs i n Drosophila. The f i r s t of these employed the BD-cellulose column developed by Gillam et a l . (1967). Using the f i l t e r paper technique (a modification of the method introduced by Cherayil et_ al_. (1968)), 14 f r a c t i o n s of Drosophila tRNA were assayed f o r C amino a c i d acceptor a c t i v i t y (White and Tener, submitted). The various tRNAs were d i s t r i b u -ted throughout the column as shown i n Figure 19. Seryl-tRNA showed maximal r e s o l u t i o n , giving f i v e d i s t i n c t peaks. Although f o r most or-ganisms examined phenylalanyl-tRNA i s present i n the ethanol f r a c t i o n , Drosophila phe-tRNA appears unique i n t h i s respect, thus resembling more the b a c t e r i a l Phe-tRNA. The second method made use of the reversed-phase chromatographic column (RPC) of Pearson eit a l . (1971) and i s the method used f o r separating tRNAs f o r the studies presented i n the next FIGURE 19a A comparison of the d i s t r i b u t i o n of i s o a c c e p t i n g tRNA peaks of D r o s o p h i l a by B D - c e l l u l o s e and RPC-5 chromatography The f o l l o w i n g tRNA s p e c i e s a r e compared: o r d i n a t e v a l u e s f o r b: a l a n i n e c t s x l O " 3 / m i n / f r a c t i o n i s o l e u c i n e c t s x l O " 3 / m i n / f r a c t i o n g l u t a m i c a c i d c t s x l O " 2 / m i n / f r a c t i o n a s p a r a g i n e c t s x l O " 2 / m i n / f r a c t i on c y s t e i n e c t s x l O " 2 / m i n / f r a c t i o n t y r o s ine c t s x l O " ^ / m i n / f r a c t i o n t h r e o n i n e c t s x l O " 2 / m i n / f r a c t i o n a s p a r t i c a c i d c t s x l O " 2 / m i n / f r a c t i o n p r o l i n e c t s x l O " 3 / m i n / f r a c t i o n p h e n y l a l a n i n e c t s x l O " 3 / m i n / f r a c t i o n Column a: e l u t i o n p r o f i l e on B D - c e l l u l o s e column ( o r d i n a t e -1 v a l u e : p moles x 10 of amino a c i d e s t e r i f i e d per m i l l i l e t r e of e l u a t e ) Column b: e l u t i o n p r o f i l e on RPC-5-column 178 179 30 20 10 15 10 15 10 20 10 ALANINE . ISOLEUCINE GLUTAMIC ACIDl ASPARAGINE CYSTEINE -TYROSINE -THREONINE _ l _ ASPARTIC ACID PROLINE PHENYLALANINE 50 100 150 200 ALANINE ai9 ISOLEUCINE 4J 2-^  -GLUTAMIC AGED, -ASPARAGINE dsn "CYSTEINE 1 L , lb i 14> n. TYROSINE thr THREONINE^ 14 (4 pro ASPA3RTIC AC I EM PROLilNE PHENYLALANINE FRACTION 50 NUMBER 100 FIGURE 19b A comparison of the distribution of isoaccepting tRNA peaks of Drosophila by BD-eellulose and RPC-5 chromatography The following tRNA species are compared: ordinate values for b: glyc ine cts xlO" "Vmin/f racti on methionine cts xlO" 2 /min/fraction lys ine .cts xlO" 3 /min/fraction tryptophan cts xlO" 2 /min/fraction leucine cts xlO" "Vmin/fraction va1ine cts xlO" 3 /min/fract ion serine cts xlO" 2 /min/fraction histidine cts xlO" 3 /min/fracti on glutamine cts xlO" 2 /min/fract i on arginine cts xlO" 2 /min/fraction Column a: elution profile cm BD-cellulose column (ordinate -1 value: p moles xlO of amino acid esterified per millilitre of eluate) Column b: elution profile on RPC-5 column 180 181 60 40 20 30 20 10 15 10 20 10 GLYCINE METHIONINE LYSINE TRYPTOPHAN LEUCINE —•-VALINE SERINE . HISTIDINE i GLUTAMINE .mos*. ARGININE 50 100 T50" 9>i M ii ' ii i ' •4 ! ! ? I ') 1 M ' 1 t 1 fit 1 GLYCINE METHIONINE mgt| LYSINE to-TRYPTOPHAN LEUCINE leu — VALINE ™l i b •HISTIDINE "GLUTAMINE ARGININE" 100 150 arg FRACTION NUMBER 182 two chapters. Figure 19 shows the elution profile for tRNAs using the RPC-5 system. An analysis of isoacceptor activity shows that many of the single peaks resolved on BD-cellulose do, in fact, separate into several isoaccepting forms with the RPC-5 column. For example, aspara-ginyl-tRNA produces up to eleven peaks on an analytical RPC-5 column where BD-cellulose is capable of resolving only one symmetrical peak. Figure 19 illustrates this dramatic difference. Table 30 summarizes the major and minor peaks obtained using the BD-cellulose and RPC-5 systems, thus demonstrating the "power of the technique". In a study of the lysyl-tRNA species from vegetative cells and spores of B_. subtilis, Void (1970) found differences in the resolving power of the methylated albumin-kieselguhr (MAK) and RPC columns, the latter again being the more powerful. Although the RPC-5 system was used for the studies in the following chapters on Drosophila tRNAs and its resolving power appears to be far superior to other methods of separation currently employed, i t must be remembered that the differences obtained for different developmental stages or for different genetic strains represent the minimum number, for i t is s t i l l possible that some species are not resolved even by this method. Although this possibility cannot be ruled out, i t is of interest to note that the RPC-5 column is able to resolve two aspartyl isoaccept-ing tRNAs which appear to differ only by one nucleotide (in actual fact, the difference may be as l i t t l e as a modification of a specific nucleo-tide in one of the species and the absence of that modification in the 183 TABLE 30 Comparison of the peaks :on BD - c e l l u l o s e and RPC-5 columns Number of peaks Amino . 1 , 2 major minor a c i d : B D - c e l l u l o s e RPC-5 BD-c e l l u l o s e RPC-5 a l a n i n e 3 5 1 0 a r g i n i n e 2 3 2 2 asparagine 1 9 0 2 a s p a r t i c a c i d 2 2 2 3 c y s t e i n e 1 4 1 0 glutamic a c i d 3 4 0 2 glutamine 2 2 0 3 g l y c i n e 2 3 " 0 0 h i s t i d i n e / 1 2 2 1 0 i s o l e u c i n e 2 0 2 l e u c i n e 2 3 2 1 l y s i n e 2 2 3 4 methionine 1 2 1 1 phenylalanine 1 1 0 2 p r o l i n e 2 2 0 1 s e r i n e 6 5 2 2 threonine 3 5 1 3 tryptophan 1 1 1 2 t y r o s i n e 1 2 1 3 v a l i n e _2 * _4 _1 * _3 T o t a l : 40 63 18 36 more than 10% of major peak 2 l e s s than 10% of major peak * some of these are shoulders to major or minor peaks 184 other species) (see Chapter 3 ). Chapter 2 , then deals with the pattern of tRNAs found during the development of Drosophila and Chapter 3 extends the studies on four particular tRNAs, correlating differences in a wild-type strain 2 and the mutant su(s) strain patterns with changes normally occurring during the development of Drosophila. J185 CHAPTER 2 ANALYSIS OF tRNAs DURING THE DEVELOPMENT OF DROSOPHILA I. Introduction It is now well-documented that during the development of various organisms there are both qualitative and quantitative alterations in the chromatographic elution profiles of certain isoaccepting tRNAs (Lee and Ingram, 1967; Yang and Comb, 1968; Molinaro and Mozzi, 1969; Bagshaw, Finamore and Novelli, 1970; Dewitt, 1971). In few i f any cases, however, have these changes been correlated with any form of metabolic control of development. Several workers (Ames and Hartman, 1963; Stent, 1964; Sueoka and Kano-Sueoka, 1964) have suggested a role for tRNA in the regulation of protein synthesis at the translational level, but this has as yet to be demonstrated in vivo. There exists in Drosophila a class of mutations called Minutes, which K.C. Atwood (see Lindsley and Grell, 1968) has suggested represent the sites of tRNA transcription. Minute loci are found on a l l Drosophila chromosomes and are characterized by their recessive lethality and the prolonged developmental time and slender bristles of heterozygous adults. Defective tRNA seems a likely candidate for the cause of the Minute pheno-type for not only do the presently determined 50 to 60 loci approximate the predicted number of tRNA genes but the physiological effects can also be correlated with a slow rate of protein synthesis at certain stages of development. Hybridization of a crude tRNA preparation to salivary gland chromosomes in situ has been interpreted as supporting the tRNA -Minute hypothesis (Steffensen and Wimber, 1971). However, no evidence for or against the hypothesis has been found at the molecular level. 186 The existence of a large number of mutations i n Drosophila o f f e r s a wide opportunity f o r analyzing tRNAs with a view to determining whether tRNAs play any s p e c i f i c r o l e i n the c o n t r o l of developmental processes. I t becomes very important to have a well-characterized analysis of tRNAs from the d i f f e r e n t developmental stages of w i l d type f l i e s i n order to provide a c o n t r o l pattern against which any p o t e n t i a l mutant phenotype can be compared. This chapter, then, i s concerned with such an a n a l y s i s of the tRNAs of the Samarkand s t r a i n of Drosophila  melanogaster during development. ,187 II. Materials and Methods Materials: Labeled amino acids were obtained from New England Nuclear Corpora-tion and Amersham/Searle. Aquasol was obtained from New England Corp. Adogen 464, a trialkylmethylammonium chloride with the predominant chain length of the alkyl groups being Cg-C^Q, w a s a gift from Ashland Chemical Co., Columbus, Ohio. Plaskon CTFE 2300 powder was a gift from Allied Chemical Corp., Morristown, N.J. Growth of Drosophila: A Samarkand strain of Drosophila melanogaster was grown at 22°C in pint glass bottles and in plastic boxes with ventilation ports. Growth medium was prepared according to the method of Lewis (1960). Collected organisms were stored at -40°C until required. Isolation of tRNA and Preparation of Aminoacyl-tRNA Synthetases: The methods used are described fully elsewhere (Chapter 3, Part II), and are modifications of the procedures described by Twardzik et a l . (1971). Aminoacylation of tRNA: Transfer RNA was aminoacylated at 22°C in a final reaction volume of 0.2 ml. Each ml of the reaction mixture contained: Tris-HCl, 50 ymoles (pH 7.5 or 8.0); 2-mercaptoethanol, 5 „imoles; 19 unlabeled amino acids, 14 50 ymoles of each; MgC^, ATP and C amino acid, (6-50 nmoles) in the amounts reported elsewhere (see Appendix 8 for details). 1-5 An/,n zoU units of tRNA and enough crude aminoacyl-tRNA synthetases to completely charge a l l the tRNA in less than 20 minutes. il 88 ; Preparation of labeled aminoacyl-tRNA; 14 3 Reaction mixtures for the preparation of C or H labeled amino-acyl-tRNA were increased proportionately from those described above. The reaction mixtures were incubated for 30 minutes at 22°C and applied to DEAE-cellulose columns as described by Yang and Novell! (1968). The aminoacyl-tRNA prepared in this way was stored at -20°C until used. Reversed-phase chromatography of aminoacyl-tRNA: The RPC-5 system of Pearson, Weiss and Kelmers (1971) was used. The Plaskon CTPE was coated with Adogen 464 as described elsewhere (Chapter 3, Part II). The 0.9 x 14 cm columns were developed at 22°, 30° or 37°C with a linear 100 ml NaCl gradient containing 0.1 M-MgCl2, 0.01 M-sodium acetate (pH 4.5) and 1 mM-2-mercaptoethanol. Radioactivity was determined in the fractions by the addition of 5 volumes of Aquasol and counting in a scintillation counter. 189 III. Results 14 Acceptance of C amino acids by tRNA from different developmental stages. Approximately 50% of the tRNA fraction has amino acid acceptor activity. Unpublished results from the chromatography of this crude tRNA fraction on Sephadex G-100 columns showed 50% of the ultraviolet absorb-ing material to be in the 4S region, 9% in the 5S region and the remaining material to be of higher molecular weight. With this purity, the acceptance of about 800 pmoles of amino acid per ^fro ° f this crude tRNA (Table 31) is expected on the basis of an uptake of approximately 1700 pmoles per A-26Q u n^- t f ° r a P u r e tRNA. A comparison of the acceptance of the 20 amino acids by the tRNAs isolated from the first and third instar larvae showed no difference greater than 30% (Table 32) . A few differed by more than 15%; these i n -clude increases in the acceptance of cysteine, isoleucine, leucine and methionine and decreases in glutamine, glutamic acid and glycine. Dif-ferences in acceptance between tRNA from third instar and adults likewise showed only minor changes. Those differing by more than 15% include i n -creases in acceptance for glutamic acid, glycine and proline and decreases in cysteine, methionine and serine. Chromatography on RPC-5 columns of aminoacyl-tRNAs from the different  developmental stages. The chromatographic profiles of the 20 radioactively labeled amino-acyl-tRNAs for the adult and first and third instar larvae are shown in Figures 20 to 24. 190 TABLE 31 Acceptance of amino acids by tRNA from first and third instar larvae and adult Drosophila Amino Acid pmoles amino acid accepted per unit of tRNA and percent of total acceptor activity first instar third instar adult pmoles % pmoles % pmoles % alanine 41 5.2 44 5.4 39 5.1 arginine 42 5.3 40 4.9 39 5.1 asparagine 35 4.4 33 4.1 35 4.6 aspartic acid 38 4.8 40 4.9 39 5.1 cysteine 15 1.9 19 2.4 13 1.7 glutamic acid 36 4.6 31 3.8 34 4.5 glutamine 41 5.2 34 4.2 35 4.6 glycine 74 9.4 60 7.4 69 9.1 histidine 35 4.4 36 4.5 35 4.6 isoleucine 30 3.8 37 4.6 31 4.1 leucine 38 4.8 46 5.7 39 5.1 lysine 58 7.3 57 7.1 58 7.6 methionine 42 5.3 54 6.7 43 5.7 phenylalanine 34 4.3 31 3.8 32 4.2 proline 28 3.5 24 3.0 29 3.8 serine 74 9.4 84 10.4 67 8.8 threonine 31 3.9 34 4.2 33 4.4 tryptophan 26 3.3 28 3.5 24 3.2 tyrosine 23 2.9 20 2.5 21 2.8 valine 50 6.3 57 7.1 49 6.5 TOTAL 791 809 759 191: TABLE 32 Number of tRNA peaks on RPC-5 columns . . ., Number of peaks Amino acid r major"'' minor ^ alanine 5 0 arginine 3 2 asparagine 9 2 aspartic acid 2 3 cysteine 4 0 glutamic acid 4 2 glutamine 2 3 glycine 3 0 histidine 2 0 isoleucine 2 2 leucine 3 1 lysine 2 4 methionine 2 1 phenylalanine 1 2 proline 2 1 serine 5 2 threonine 5 3 tryptophan 1 2 tyrosine 2 3 valine 4 3 TOTAL 63 36 more than 10% of the major peak less than 10% of the major peak 192 Alanyl-, arginyl-, phenylalany1- and tryptophanyl-tRNAs (Figure 20). Although resolution from peak 3 is poor, there appear to be quanti-tative changes in the shoulders labeled 1 and 2 in the alanyl-tRNA profile during development. Similarly there are quantitative changes in peaks 4 and 5 of arginyl-tRNA. There are also small quantitative changes in the minor peak 3 in both the phenylalany1- and tryptophanyl-tRNA profiles. Glutaminyl-, glutamyl-, lysyl- and glycyl-tRNAs (Figure 21). There appears l i t t l e change in the glutaminyl-tRNA profile during development. Peak 4 of glutamyl-tRNA, however, shows a marked change between the developmental stages. The most significant change in the lysyl-tRNA profile is the increase in peak 6 by the third instar larval stage. Cyanogen bromide treatment of adult Drosophila tRNA completely inhibits the amino acid acceptance of tRNA^n, tRNA^U, and tRNA^S, indicating that these tRNAs in Drosophila contain 2-thiouracil derivatives in the anticodons as do certain isoacceptors of these three amino acids in E_. coli (Saneyoshi and Nishimura, 1970). Peaks 1 and 2 of glycyl-tRNA are substantially reduced at the third instar larval stage. Histidyl-, tyrosyl-, aspartyl- and asparaginyl-tRNAs (Figure 22). The tRNA changes associated with these isoacceptors are described in more detail in Part II, Chapter 3 . The relative proportions of the 6 and y forms of these four tRNAs change in a similar manner during development. These changes are related to the activity of a tRNA modify-ing enzyme, which is probably involved in the modification of a nucleotide in the first or "wobble" position of the anticodons of these tRNAs. The 5 and Y peaks of the different isoaccepting tRNAs appear to have the same FIGURE 20 Chromatography of C a l a n y l - , a r g i n y l - , phenylalany1-, and tryptophanyl-tRNAs. The a l a n y l - , a r g i n y l - , and phenyl-alanyl-tRNAs were chromatographed at 37°C and tryptophanyl-tRNA at 30°C with l i n e a r 100 ml NaCl gradients. The flow rate was 15 ml/hour with 0.5 ml f r a c t i o n s being c o l l e c t e d . 193 194 4 - i r ADULT (Ala) i r 0-70-0-65-0-5S-3rd INSTAR 5 -c .2 4 -•I- AA. < U D km I 1st INSTAR 1 o x 2 -U 1 -S ADULT (Phe) _1_ NoCI(M) 0-70 0-65H 0-60-0-55-3rd INSTAR 1 hi INSTAR 0-50-3 1 70 SO 110 130 1S0 Fraction No. 120 140 100 180 Fraction No. 12 ADULT I* (Ar9) " 1 NoCI(M) 0-70-0-65. 0-60-0-55-0-50-3rd INSTAR j. hi INSTAR UV-i . 12 c o u o > 8 O U 1 1 i ADULT - (Trp) 1 1 j NaCIM i -70-u 3rd 1 -60- hi INSTAR - 1 -55-1 1 -SO- J t0 100 ' 120 140 'Fraction No. to 80 100 120 Fraction No. «o F I G U R E 2 1 Chromatography of C glutaminyl-, glutamyl-, l y s y l - , and glycyl-tRNAs. Lysyl-tRNA was chromatographed at 3 G°C. while glutaminyl-, glutamyl-, and glycyl-tRNAs were chromatographed at 3 7°C as described in the legend of Figure 2 0 . 195 196 T 1 1 1 r ADULT (Gin) C 16 u o c o x V? O 12 NoCIlM) 0-70-0-5S-0-60-0-5S-3rdl INSTAR 1st INSTAR l i 60 80 100 120 140 Fraction No. —r 1 1 1—r ~ i r ADULT (Glu) 16 e o E *o 8 K w? u NoCI(M) 0-70-0-65-0-60-0-55-0-50-3rd INSTAR lit INSTAR 60 60 100 120 140 160 Fraction No. 70 SO 110 130 ISO Fraction No. c .2 6 c o X. D T r ADULT (Gly) 0-70-0-65-oco-0-5S-0-50-3rd INSTAR CO 80 100 120 Fraction No. FIGURE 22 Chromatography of C h i s t i d y l - , t y r o s y l - , a s p a r t y l - , and asparaginyl-tRNAs. Asparaginyl- and histidy1-tRNAs were chromatographed at 22°C and t y r o s y l - and asparty1-tRNAs were chromatographed at 37°C as described in the legend to Figure 20. 197 198' 1C 8 s A-o r— K *t u 1 T~ ADULT (His) J L NoCI(M) 0-65-0-60' 0-55-0-50-3rd INSTAR h i INSTAR l i <C 60 £0 100 Fraction No. IE a c E 'o _ ADULT (Tyr) _ , . , — 1 3rd INSTAR -NoCI(M] 0-70-i i ^ 0-65-0-00- 1 W ; INSTAR - 0-55-/ J t > 1 3 N 20 16 c o o • c ' o • X U ADULT (Asp) " i IP NoC'l lMj 0 65-0-55-0-50-•I 3rd INSTAR A Ut INSTAR 16 c E •> 'o x 120 140 160 180 Fraction No. 1 )— T T ADULT (Asn) 31 31 31 3rd INSTAR NoCI(M) 0-60 0-50 Ut INSTAR 1 (0 SO 100 120 Fraction No. 60 80 100 120 Fraction No. base sequences and are presumably products of the same gene. The propor-tions of the 6'and y forms of tRNA^8^ differ from those characteristic of the other three tRNAs. In addition peak 4 of tyrosyl tRNA is quite substantial during the first instar but becomes much reduced in the later stages. Seryl-, threonyl-, methionyl- and cysteinyl-tRNA (Figure 23). Several of the major peaks of seryl-tRNA show quantitative altera-tions during development. While peak 4 of threonyl-tRNA decreases from the first instar until the adult, peak 5 dramatically increases by the third instar stage and decreases in the adult. The latter peak appears to be composed of three small peaks in the first instar and adult stages, but the shoulders are no longer detectable in third instar larvae. Changes in peak 8 resemble those of peak 4 while peak 9 is apparent only in the first larval instar. The two major methionyl-tRNA peaks undergo quite significant changes throughout development, with peak 3 probably substantially increasing at the third instar stage. Peak 2 of cysteinyl-tRNA is reduced by the third instar, while peak 4 is much increased in first instar larvae. Isoleucyl-, leucyl-, prolyl- and valyl-tRNAs (Figure 24) The isoleucyl-, leucyl-, prolyl- and valyl-tRNA profiles remain essentially constant throughout development. Quantity of peaks and the number of changes. An arbitrary grouping of isoaccepting peaks into those with heights greater than 10% (major peaks) and those with peak heights less than 10% FIGURE 23 Chromatography of C s e r y l - , threonyl-, methionyl-, and cysteinyl-tRNAs. Methionyl-tRNA was chromatographed at 22°C, cysteinyl-tRNA at 30°C and s e r y l - and threonyl-tRNAs were chromatographed at 37°C as described in the legend of Figure 20. 200 201 c t i 6 p c £ b x •> * • U ADULT (Ser) NoCI (M) J L 3rd INSTAR ii \f U l INSTAR 100 120 140 160 1S0 Fraction No. 12 c o o > I E o x ADULT (Cys) ~i r ~ NoCI w 0-70-0-65-0-60-i v I 0-5S-o-so-3rd INSTAR h i INSTAR c 36 o u O c E 24 'o X 0 12 SO 70 90 110 130 Fraction No. 40 . 60 80 100 . 120 Fraction No. r T 1 r ADULT (Thr) 3rd INSTAR CO 80 100 120 140 Fraction Na FIGURE 24 Chromatography of C i s o l e u c y l - , l e u c y l - , p r o l y l - , and valyl-tRNAs. Prolyl-tRNA was chromatographed at 30 and i s o l e u c y l - , l e u c y l - , and valyl-tRNAs were chromato-graphed at 37°C as described i n the legend of Figure 20. 202 203' c .2 10 U ' O E I 9- 5 X «! 1 1 — ADULT (He) J N'oCI(M) 0-70 H , M 5 -0-60-0-55-0-50-3rd INSTAR U l INSTAR c o '•5 1 0 c £ I o X u 100 120 140 160 Fraction No. 80 100 120 140 Fraction No. , r ADULT a (Val) 1 r NoCI(M) 0-65-,0-60' 0-55-i 0-50-3rd INSTAR Ul INSTAR 80 100 120 140 Fraction No. <—r ADULT (Pro) i r NaCI(M) 0-6S-,0-60" 0-55-0 50-J L 3rd INSTAR Ul INSTAR 60 , 80 100 120 140 Fraction No. 204 (minor peaks) of the largest peak reveals that there are 63 major peaks and 36 minor ones (Table 32). Since normalization of peaks for the dif-ferent developmental stages for each aminoacyl-tRNA is di f f i c u l t , the determination of the precise number of peaks subject to change also be-comes difficult. However, approximately one third of the peaks undergo some change during development. Relatively few changes involve the ap-pearance or complete loss of a peak but those that do invariably concern a minor peak. 205: IV. Discussion In order to study the role of a specific macromolecule during development i t is often necessary to determine the effect of the absence or drastically altered amounts of that molecule in the development of the organism. One of the few primary gene products that can be qualita-tively and quantitatively analyzed is tRNA. Mutations in tRNA genes can provide an important tool for determining specific roles of these mole-cules in development. Drosophila remains unique among multicellular or-ganisms in having a well-characterized genetic system with a wealth of developmental mutants (Hadorn, 1955; Goldschmidt, 1958; Wright, 1970; Fristrom, 1970). The advent of the RPC-5 columns (Pearson, Weiss and Kelmers, 1971), which allow rapid chromatography of aminoacyl-tRNAs, makes i t possible to analyze many mutants systematically for altered tRNA patterns and hopefully to correlate these changes with specific phenotypes. Conditions have been established for the satisfactory amino-acylation of Drosophila tRNA with the 20 radioactively labeled amino acids (White and Tener, in preparation) and for the chromatography of these aminoacyl-tRNAs on RPC-5 columns. However, i t should be noted that the aminoacyl-tRNA synthetases used in this study were derived from adult f l i e s . If there are tRNA species in the larvae which are aminoacylated only by synthetases unique to that stage of development, these tRNAs would have been missed in the current study. The developmental stages of Drosophila are readily divided into the embryo (egg), larva (three instars separated by moults), pupa and adult. 206 Transfer RNAs from first and third instar larvae and adults were chosen for the i n i t i a l analyses for several reasons. It has been established that the cytoplasm of an egg carries material reflecting the maternal genotype and that elements such as ribosomes endure for a long period during early embryogenesis. Consequently the first instar was selected as an arbitrary point at which zygotic nuclei would be functioning. The three larval instars reflect the growth phase of larval and imaginal structures, with l i t t l e apparent differentiation occuring at this time. Transition from the larval stage to the imago represents a complete change in cell structure and function and tissue composition. It is during the switch from larval to adult tissue that the most significant changes in the regulation of the translational apparatus may occur (Fristrom,1970). Consequently the differences in the tRNA profiles between the third instar and adult stages are of greatest interest. At this point i t is impossible to interpret most of the observed changes in peaks of the different isoaccepting tRNAs. One major problem is that the tRNA is extracted from the whole organism and changes that may be dramatic within a single tissue may appear minor when examined with tRNA from other tissues. A further problem arises from the uncertainty as to whether the different peaks represent products of different genes or the same gene product modified to different extents in different tissues. Both types of change may have some regulatory importance. There exists two likely control mechanisms by which tRNA changes could exert a regulatory effect. These are the control of protein synthesis ,207: at the translational level (Ames and Hartman, 1963; Stent, 1964; Sueoka and Kano-Sueoka, 1964) and the possible direct regulation of enzyme activity (Jacobson, 1971). Evidence for translational control in Tenebrio pupae arises from the observations that preparations from 5-day-old pupae contain a new aminoacyl-tRNA synthetase and a new species of tRNA that affects the translation of pre-existing messenger (Ilan, 1969; Ilan et a l . , 1970). The observed tRNA changes in Drosophila may reflect similar situa-tions. Quantitative alterations may be as significant as qualitative ones for control of the rate of translation of certain mRNAs by limiting tRNAs may have as profound an effect as the absolute requirement of a par-ticular tRNA for the translation of a mRNA. It has been demonstrated that the amounts of minor tRNA species can regulate the rates of translation of certain mRNAs in vitro (Anderson, 1969; Anderson and Gilbert, 1969). The Minute phenotype in Drosophila may represent such a genetically-induced mechanism in vivo. If an essential tRNA is quantitatively reduced by 50% in Minute heterozygotes, this could markedly slow down the rate of protein synthesis, especially during those periods when the translational machinery is functioning maximally. The report by Twardzic, Grell and Jacobson (1971) that suppression of the vermilion phenotype involves the loss of a tyrosine tRNA peak pro-vided the first indication that tRNAs may play a direct role in the regula-tion of enzyme activity in Drosophila. Jacobson (1971) found that in the vermilion mutant a tRNA^^ (labeled tRNA^r in this study) binds to trypto-phan pyrrolase, thus inhibiting its activity. The loss of this tRNA in 208 the flies carrying the suppressor of sable and vermilion mutations allows some tryptophan pyrrolase activity. In a following chapter (Chapter 3 ) , T y r i t is seen that this marked reduction in tRNA^. is probably the result 2 of the altered activity of a tRNA modifying enzyme. The su(s) mutant also shows marked reductions in a l l the peaks denoted by the "Y subscript in Figure 22. This tRNA modifying enzyme appears to be active only during a part of the l i f e cycle of Drosophila and therefore two forms of the same gene product are present in different proportions during development. This enzyme appears to be involved in the modification of a base in the first or "wobble" position of the anticodons of these tRNAs making i t conceivable that i t is involved in some regulatory mechanism of translation. Further-Tvr Tyr more, i t is possible that the relative proportions of tRNA., i and tRNA.,:;. lo IT regulate the activity of wild-type tryptophan pyrrolase activity during the different developmental stages. Of the 99 peaks catalogued in this study, at least seven are probably differently modified forms of other peaks. Therefore i t is felt that there is a maximum of 92 different genes for tRNAs in Drosophila of which 56 give rise to peaks that have been arbitrarily designated as major. It has been calculated from data obtained by hybridization techniques (Ritossa, Atwood and Spiegelman, 1966; Tartof and Perry, 1970) that 0.015% of the genome or about 750 cistrons represent tRNA genes. This would provide for an average of 8 copies of the gene for each peak observed. There may be some relationship between the number of genes for a tRNA and the amount of that tRNA present within the c e l l . Analysis of the peak heights of dif-ferent tRNAs indicates that the relative proportions of isoacceptors are 209 really too complex to be explained simply in terms of gene dosage. How-ever, gene dosage may play a role in determining the amount of tRNA trans-cribed and i t will be of interest to analyze mutants associated with chromosome deletions and duplications for alterations in the amounts of specific tRNAs. This could also provide some genetic data on the gross localization of tRNA genes in the Drosophila genome. There is as yet no indication as to the mechanism of control of the transcription of tRNA genes in any organism. If there are multiple copies of tRNA genes as there are for the ribosomal RNA genes in Drosophila, then presumably there must be some mechanism whereby only one unique sequence is maintained and transcribed, otherwise a much greater heterogeneity of peaks would have been found in this study. It seems unlikely that 8 copies of a gene, permitted indepen-dent mutation and evolution, would give rise to a single tRNA peak. The interspecific heterogeneity of isoaccepting tRNAs argues against the suggestion that a specific sequence is essential for a functional tRNA. This indicates that either only one copy of the tRNA gene is transcribed or a master gene-slave gene relationship assures that only one gene se-quence is retained (Callan, 1967; Whitehouse, 1967). It is possible that the hybridization techniques have led to over-estimates of the percentage of the genome representing tRNA gene sequences, possibly as a result of the impurity of the tRNA used. That hybridization results can lead to completely misleading estimations of genetic redundancy is amply demonstrated by the work of Paul and co-workers (Williamson, 210 Morrison and Paul, 1970; Harrison, Hell, Birnie and Paul, 1972) whose in i t i a l estimate of 50,000 copies of a globin gene in the mouse genome was subsequently changed to a single copy. A previous estimate of between 130 and 140 tRNA genes by the in situ hybridization technique (Steffensen and Wimber, 1971) was probably also an overestimate due to impure tRNA preparations. These workers explained the results by suggesting that genes for the same tRNA were located on different chromosomes or on dif-ferent parts of the same chromosome. Such a situation would require a very complicated master gene-slave gene mechanism to assure the maintenance of the intraspecific homogeneity of the tRNA genes. By analyzing the tRNAs from mutant Drosophila the direct products of about 90 genes can be examined. Estimates in the order of 5000 genes (Judd, Shen, and Kaufman, 1972) for Drosophila indicate that such an analysis would result in the study of about 2% of them. This takes into account also the tRNA modifying enzymes which would be indirectly examined. It is hoped that such analyses of mutants will not only map tRNA genes on the Drosophila chromosomes but also help to explain the role of some of the tRNA changes during development observed in this study. j21! CHAPTER 3 ACTIVITY OF A tRNA MODIFYING ENZYME DURING THE DEVELOPMENT OF DROSOPHILA AND ITS RELATIONSHIP TO THE su(s) LOCUS I. Introduction It is now well established that the passage of a cell from one physiological state to another is accompanied by both qualitative and quantitative alterations in chromatographic elution profiles of certain isoaccepting tRNAs. Such changes occur in sporulating Bacillus subtilis (Kaneko and Doi, 1966), in Escherichia coli infected with bacteriophage T2 (Sueoka and Kano-Sueoka, 1964; Waters and Novell!, 1967a and 1968) as well as in virus-infected hamster cells (Subak-Sharpe, Shepherd and Hay, 1966; Holland, Taylor and Buck, 1967). Similar changes have been observed at different developmental stages of various organisms (Lee and Ingram, 1967; Yang and Comb, 1968; Molinaro and Mozzi, 1969; Bagshaw, Finamore and Novelli, 1970; Dewitt, 1971). In few i f any cases, however, have these changes been correlated with any form of metabolic control. It has long been postulated that tRNA might play an important role in the regulation of protein synthesis at the translational level (Ames and Hartman, 1963; Stent, 1964; Sueoka and Kano-Sueoka, 1964), but this has s t i l l to be adequately demonstrated in vivo. Recently i t has been shown that a specific tRNA inhibits tryptophan pyrrolase activity in the vermilion (v) mutant of Drosophila (Jacobson, 1971; Twardzic, Grell and Jacobson, 1971), suggesting that tRNAs may act directly as enzyme regulators. 212 Drosophila is a good organism for the study of developmental changes in tRNAs because of the large number of characterized developmental mu-tants and the marked morphological, physiological and biochemical changes that occur in well-defined stages from the egg through the f i r s t , second and third instar larva, to the pupa and finally to the adult f l y . In this paper we provide evidence that a specific group of tRNAs undergoes similar post-transcriptional modifications during the development of Drosophila. y This is the same group that was recently shown to contain the minor nucleoside, Q, in E. coli (Harada and Nishimura, 1972). Fur-ther, i t is the same group that has altered elution profiles in flies 2 carrying the mutation, suppressor of sable (su(s) ). The results pre-sented here suggest that the difference between these chromatographically separable isoacceptors is the degree of modification of a nucleoside ana-logous to Q of E_. coli. 213 ; II. Materials and Methods Materials: 3 14 The ( H) and ( C) amino acids and Aquasol were obtained from New England Nuclear Corp. Adogen 464, a trialkylmethylammonium chloride with the predominant chain length of the alkyl groups being Og-C^ Q, w a s a gift from Ashland Chemical Co., Columbus, Ohio. Plaskon CTFE 2300 powder was a gift from Allied Chemical Corp. , Morristown, N.J. E_. coli Q and Qp were gifts from Dr. S. Nishimura. Strains of Drosophila: 2 The inbred wild-type (Samarkand) strain and the su(s) v; bw strain of Drosophila melanogaster were obtained from the California Institute of 2 Technology. Both the v (vermilion eye color) and the su(s) (suppressor of sable body color) loci are located on the X chromosome, while the bw (brown eyes that result from absence of red pigments) locus is on the second chromosome. Growth of Drosophila: Drosophila were grown at 22°C in pint glass bottles and in plastic boxes with ventilation ports. Growth medium was prepared according to the method of Lewis (1960). Collected organisms were stored at -40°C until required. Isolation of tRNA: Transfer RNA was prepared by the phenol method of Kirby (1956) and DEAE-cellulose chromatography procedures of Kelmers, Novelli and Stulberg 2,4 (1965). Whole Drosophila of a particular developmental stage were homogenized in equal volumes of 88% phenol and buffer A, containing 0.01 M-Tris-HCl (pH 7.5), 0.1 M-NaCl, 0.01 M-magnesium acetate and 1 mM-2-mercaptoethanol. The homogenate was centrifuged at 10,000 g_ for 15 minutes and the aqueous layer removed. The nucleic acids in this solu-tion were precipitated by the addition of 2 volumes of 95% ethanol and left overnight at -20°C. The precipitate was recovered by centrifugation, resuspended in buffer A and applied to a DEAE-cellulose column, previously equilibrated with buffer A. The column was successively eluted with buf-fer A containing 0.3 M-NaCl and 1.0 M-NaCl. The RNA eluted by 1.0 M-NaCl was taken as the tRNA fraction. The tRNA was precipitated with 2 volumes of ethanol, stored overnight at -20°C and the precipitate sedimented. This was then dissolved in distilled water, dialyzed extensively against dis-tilled water and freeze-dried. Preparation of aminoacyl-tRNA synthetases: Crude synthetases were prepared from adult flies by a modification of the method of Twardzic et a l . (1971). A l l procedures were performed at 0-4°C. Frozen Drosophila were added to 4 volumes (w/v) of buffer B, con-taining 0.01 M-Tris-HCl (pH 7.5), 0.01 M-2-mercaptoethanol, 0.01 M-magne-sium acetate, 1 mM-phenylmethylsulphonyl fluoride (Pelley and Stafford, 1970) and 10% glycerol and homogenized in a Virtis homogenizer. The homo-genate was centrifuged at 10,000 j> for 20 minutes and the supernatant fur-ther centrifuged at 105,000 £ for 2 hours. The supernatant was applied to a DEAE-cellulose column and eluted with buffer B, containing 0.3 M-NaCl. The peak fractions of material absorbing at 280 nm were pooled, dialyzed 215 overnight against buffer B containing 50% glycerol and stored at -50°C. The glycerol was removed from the preparation immediately prior to use by Sephadex G-25 chromatography. Aminoacylation of tRNA; Transfer RNA was aminoacylated at 22°C in a final reaction volume of 0.20 ml. For the aminoacylation of tRNA with phenyl-alanine, tyrosine, asparagine or histidine each ml contained Tris-HCl (pH 7.5), 50 ymoles; 14 3 2-mercaptoethanol, 5 ymoles; ( C) or ( H) amino acid, 10-25 nmoles; MgC^, 4 ymoles; ATP, 10 ymoles; 1-5 units of crude Drosophila tRNA and enough crude amino-acyl-tRNA synthetases to completely charge a l l the tRNA. For the aminoacylation of tRNA with aspartic acid, the reaction mixture contained the same components as above except there were 14 3 MgC^, 20 ymoles; ( C) or ( H) aspartic acid, 50 mymoles; and ATP, 2 ymoles per ml of reaction mixture. The rate of the amino-acylation reaction was followed by pipeting 50-yl samples on f i l t e r paper discs and removing acid insoluble radioactive amino acids (Bollum, 1959). Amino-acyl-tRNA for reverse phase chromatography was isolated from the components of the reaction mixture by DEAE-cellulose chromatography as described by Yang and Novelli (1968). Reverse phase chromatography: The RPC-5 system of Pearson, Weiss and Kelmers (1971) was used. The solid support is Plaskon CTFE 2300 coated with Adogen 464. Plaskon (300 g) was initially passed through a 200 mesh sieve to break clumps to the p r i -mary 10 ym size. This was then mixed with Adogen 464 (12 ml), dissolved 216. in chloroform (600 ml), and vigorously agitated on a wrist action shaker for 1.5 hours. The slurry of Plaskon was placed in a glass tray and the chloroform allowed to evaporate while the mixture was stirred with a glass rod. This material was then suspended in buffer C, containing 0.01 M-MgC^j 0.01 M-sodium acetate (pH 4.5), 0.45 M-NaCl and 0.001 M-2-mercaptoethanol and again passed through a 200 mesh sieve to remove the clumps formed dur-ing the coating process. The slurry was degassed and poured into a jacketed column that was partially f i l l e d with aqueous buffer. The packing was compacted in the column under maximum aqueous flow (2 ml/min) to reduce the aqueous void volume. Radioactively labeled aminoacyl-tRNA (100-500 yg total tRNA) was loaded onto a 13 x 0.9 cm column, which was eluted at 15 ml/hr using a 100 ml gradient of NaCl containing 0.01 M-sodium acetate (pH 4.5), 0.01 M-MgC^ and 1 mM-2-mercaptoethanol, with a fraction size of 0.5 ml. Radioactivity was determined in the fractions by addition of 5 volumes of Aquasol and counting in a scintillation counter. For preparation of tRNA^p and At) Aso tRNA.2.y , a jacketed 2.4 x 45 cm RPC-5 column was used and eluted by a 2-1 gradient of NaCl. Pooled fractions were dialyzed extensively against dis-tilled water and the tRNA isolated by freeze-drying. Aso Isolation of tRNA by the naphthoxyacetylatibn procedure: The general procedure used was that described by Gillam et a l . , (1967). Crude Drosophila tRNA or fractions containing tRNA^Sp were charged with aspartic acid in large scale incubation mixtures increased proportionately from that described above. The aspartyl-tRNA and crude tRNA were isolated from the incubation mixture by DEAE-cellulose chromatography (Yang and 217 and Novelli, 1968). The aspartyl-tRNA was derivatized and isolated by BD-cellulose chromatography. The chromatographically distinct forms of tRNA^15 were separated by RPC-5 chromatography as described above. Transfer RNA degradations: For pancreatic RNase digestion, 10 u n^t s ° ^ tRNA were incubated with 100 yg RNase in 0.05 M-triethylammonium bicarbonate, (pH 7.5) for 18 hours at 37°C. For alkaline hydrolysis 10 ^ (,0 un^ts ° f tRNA in 10% piperidine were sealed in a capillary tube and heated in a boiling water bath for 2 hours (Sedat and Hall, 1965). Thin layer chromatography of nucleotides and oligonucleotides: The tRNA hydrolysates were applied directly to thin layer plates and run in the first dimension in a solution composed of 5 volumes isobutyric acid and 3 volumes 0.5 N-NH^ OH. Oligonucleotides were then chromatographed in the second dimension in a solution of 1 volume _t-butanol and 1 volume 0.5 M-formic acid (pH 3.8). Nucleotides were separated in the second dimension in a solution of 70 volumes t_-butanol, 15 volumes concentrated HCl and 15 volumes water (Nishimura, 1972). 2.18 III. Results Quantitative analyses of tRNAs from the developmental stages of wild-type 2 and the su(s) v; bw mutant. Transfer RNA prepared from developmental stages of the wild-type and 2 the su(s) v; bw mutant were aminoacylated with the wild-type adult enzyme preparation. The amino acid acceptor activity for these preparations is shown in Table 33. The results are expressed both as pmoles/A2gQ unit and pmoles/pmole phenylalanine accepted because of the different amounts of contaminating material in the preparations. No appreciable differences in the acceptance of (^C) histidine, asparagine, aspartic acid, and tyro-sine were noted in any of these preparations. Chromatographic studies of phenylalanyl-, asparaginyl-, aspartyl-, histidyl-, and tyrosyl-tRNAs. Aminoacy1-tRNAs prepared from adult flies (Samarkand) of mixed age were resolved into chromatographically distinct forms of RPC-5 columns. The conditions used for eluting these columns were determined by the proximity of the various peaks and the stability of the individual aminoacyl-tRNAs. Each of the aminoacyl-tRNAs was resolved into several peaks (Figures 25a-e), which are labeled according to their elution position and their probable relationship to one another, as determined by a comparison of the profiles from the various developmental stages. One major and two minor peaks of phenylalanyl-tRNA were resolved (Figure 25a). Histidyl-tRNA chromatographed as two major peaks (Figure 25b). Tyrosyl-tRNA was resolved into two major TABLE 33 Amino acid acceptor activity of tRNA from developmental 2 stages of the wild-type and su(s) v;bw mutant of Drosophila Source of tRNA pmoles/A2gg unit pmoles/pmole Phe His Tyr Asp Asn Phe His Tyr Asp Asn wild-type egg 35.7 24.1 52.1 46.9 43.4 0.82 0.56 1.20 1.08 first instar larva 22.6 15.7 37.5 29.5 27.6 0.82 0.57 1.36 1.07 second instar larva 31.0 23.3 52.7 43.6 39.3 0.79 0.59 1.34 1.11 early third instar larva 23.0 13.5 28.0 25.6 25.3 0.91 0.53 1.11 1.01 late third instar larva 18.7 13.1 31.9 24.7 23.3 0.80 0.56 1.37 1.06 prepupa 24.2 17.0 36.4 30.2 30.6 0.79 0.56 1.19 0.99 early pupa 20.6 15.4 30.3 26.5 27.1 0.76 0.57 1.12 0.98 late pupa 25.6 17.9 45.2 35.3 31.7 0.81 0.56 1.43 1.11 mixed age adult 29.0 19.6 44.1 36.6 36.1 0.80 0.54 1.22 1.01 two-week old adult 24.9 15.8 40.8 34.9 31.1 0.80 0.51 1.32 1.12 2 su(s) v;bw mutant third instar larva 32.6 21.6 44.4 40.5 39.3 0.83 0.55 1.13 1.03 mixed age adult 26.8 17.3 46.1 35.0 32.7 0.82 0.53 1.41 1.07 FIGURE 25 Chromatography of phenylalanyl-tRNA from wild-type adults of mixed ages. E l u t i o n of (^C) phenylalany!-tRNA (57,215 cts/min) was by a 100 ml l i n e a r gradient from 0.5 to 0.7 M-NaCl at 37°C, as described i n Methods. Chromatography of histidyl-tRNA from wild-type adults of 3 mixed ages. E l u t i o n of ( H)histidyl-tRNA (251,421 cts/min) was by a 100 ml l i n e a r gradient from 0.50 to 0.65 M-NaCl at 22°C. Chromatography of tyrosyl-tRNA from wild-type adults of 14 mixed ages. E l u t i o n of ( C)tyrosyl-tRNA (26,400) cts/min) was by a 100 ml l i n e a r gradient from 0.5 to 0.7 M-NaCl at 37°C. Chromatography of aspartyl-tRNA from wild-type adults of 14 mixed ages. E l u t i o n of ( C)aspartyl-tRNA (23,250 cts/min) was by a 100 ml gradient from 0.50 to 0.65 M-NaCl at 37°C. Chromatography of asparaginyl-tRNA from the wild-type 14 adults of mixed ages. E l u t i o n of ( C)asparaginyl-tRNA (47,908;cts/min) was by a 100 ml l i n e a r gradient from 0.5 to 0.6 M-NaCl at 22°C. 220 221 and three minor peaks at 37°C (Figure 25c), but at 22°C peaks (2) and (3) chromatographed as one. Two major and three minor peaks of aspartyl-tRNA were found although peak 1 was not always resolved (Figure 25d). Eleven isoaccepting forms of asparaginyl-tRNA could be demonstra-ted when maximum resolution was achieved (Figure 25e). In other runs the smaller peaks appeared as shoulders or were not detectable. The best resolution was always achieved using freshly packed RPC-5 columns, as a slow deterioration was noticed with continued use. Comparison of aminoacyl-tRNAs from developmental stages of the wild-type' 2 and the su(s) y; bw mutant. In order to correlate the peaks found at developmental stages of the 2 14 wild-type and the su(s) v; bw mutant, ( C) aminoacyl-tRNAs from wild-3 type adults of mixed age were co-chromatographed with ( H) aminoacyl-2 tRNAs from the first and third instar larvae and the su(s) v; bw adults of mixed age. This was not done for asparaginyl-tRNA because of the dif-3 ficulty in obtaining ( H) asparagine. In a l l cases, the positions of the peaks labeled in Figure 25 were exactly the same and therefore the pro-files obtained from adults are not shown. Phenylalanyl-tRNA gave similar chromatographic profiles for the 2 wild-type first and late third instar larvae and su(s) v; bw adults. Peaks 1 and 3 were somewhat larger in the first and late third instar larval profiles (Figure 25a). During development from the egg to the late third instar larva, Tyr Tvr tRNAjg decreases while tRNA^ increases (Figure 26). At the prepupal FIGURE 26 Comparison of tyrosyl-tRNA from developmental stages of the wild 2 type and su(s) v; bw mutant of Drosophila. E l u t i o n was as in Figure 25c, except for the second and e a r l y t h i r d i n s t a r p r o f i l e s o which were run at 22 C. 223 wt. 099 W.t. e a r l y p u p o -8 w.t. 1st instar w.t late pupa V-H .—-tv w.t. 2nd instar w.t mixed age adult wt. early 3rd instar w.t. 2wks. old adult -w.t. late 3rd instar late 3rd instar wt. pre pupa -a 1_Z L mixed age adult S Frdcf ion no. 225 Tyr Tyr stage this trend ceases and tRNA^ begins to increase and tRNA^ to decrease. This pattern continues through adult l i f e until the propor-tions of these two chromatographic forms are approximately equal at two Tyr weeks after eclosion. The content of tRNA^  is fairly substantial in the egg but becomes progressively smaller until i t is virtually absent in the adult. The two chromatographically distinct histidyl-tRNAs change in Tyr Tyr proportion during development in the same manner as tRNA^ g and tRNA^ (Figure 27). Although the major chromatographic forms of aspartyl-tRNA (Figure 28) are not present in the same proportions as the tyrosyl- and histidyl-tRNAs, their relative proportions do change in a similar way. Transfer RNAA!P dec reases until the late third larval instar and then increases zo until the adult is two weeks old, whereas tRNAA^P does the reverse. The asparaginyl-tRNA profile is so complex that i t is difficult to Asn Asn correlate a l l of the peaks (Figure 29). Transfer I^^g , tRNA^ g and Asn tRNA(.g clearly decrease from the egg to the late third instar larva and Asn Asn thereafter increase until the two week old adult, while tRNA2^ , tRNA^ Asn and tRNA^ do the reverse. There is a marked change in the relative Asn As n proportions of tRNA^^ and tRNA^ ,^ from the egg to prepupal stage. Similar changes in the relative proportions of the 6 and Y forms of tRNATyr, tRNAHlS, tRNAAsp and tRNAAsn were observed throughout the develop-ment of wild-type Drosophila. In the late third instar larval stage of 2 the su(s) v; bw mutant the 6 forms are substantially greater than in the FIGURE 27 Comparison of histidy1-tRNA from developmental stages of the wild 2 type and su(s) vj bw mutant of Drosophila. E l u t i o n was as in Figure 25b. 226 —«7f~ aorly 3rd instar —wJ. - late 3rd instar w.t. egg w.t 1st- instar 1 v.t. 2nd instar ^ l_ v*t. prepupa A L W.t.early pupa 1 w.i late pupa wt mixed age odwlt 2 week old adwlr 1,1 /i S Ai Sulsfv; bw Late 3 rd instar Sufsfv; bw M i x e d age adu l t 6 1 i \ H i / A F r a c t i o n no. ( FIGURE 28 Comparison of asparty1-tRNA from the developmental stages of 2 the wild-type and su(s) v; bw mutant of Drosophila. E l u t i o n was as in Figure 25d. 228 F r a c t i o n no. FIGURE 29 Comparison of asparaginyl-tRNA from the developmental stages 2 the wild-type and su(s) v; bw mutant of Drosophila. E l u t i o n for l a t e pupa and two week old adult of the wild-type and the 2 t h i r d i n s t a r larva and adult of the su(s) v; bw mutant were as i n Figure 25e, while the others were eluted by a l i n e a r gradient from 0.50 to 0.65 M-NaCl. 230 _ W.t. egg C o O c to w.+. .Ur instar w.t early pupa 3* M •wt 2 n d instar 3 * clearly 3 rd instar 3* wf. late pupa 3Jf wt 2 week adult 36 fu($)*v; bw Late 3rd instar s u ( s ) ' v ; b w Mixed aae adult 3 S | Fraction no. 232 wild-type (Figures 26 to 29). This difference from the wild-type is more apparent in the profiles obtained from the mutant adults of mixed age where the Y forms are markedly reduced in tRNA7^, tRNA^^S and tRNAAsn and virtually abs ent in tRNAAsp. The reproducibility of RPC-5 columns allows approximate quantita-tion of peaks from peak heights. The percentage of the peak heights of the 6 forms are given in Table 34. The changes are very similar for the four aminoacyl-tRNAs, clearly indicating their relationship during a l l 2 developmental stages of the wild-type and the su(s) v; bw mutant. Purification of tRNA^^ and tRNA^^ . In order to determine the relationship between the <5 and Y tRNAs, these forms of tRNAAsp were purified. A fraction of adult wild-type tRNA (5000 A 2 6 Q units) that had been enriched for tRNAAsp by prior chromatography was charged with aspartic acid, naphthoxyacetylated and isolated by BD-cellulose chromatography as described in the Methods. The 6. and Y forms were separated by RPC-5 chromatography as shown in 14 Figure 30. Fractions were assayed for ( C) aspartic acid acceptor ac-tivity. The peaks of tRNA^ and tRNA^ accepted 1,611 and 1,677 pmoles/A2gQ unit respectively. Chromatographic analysis of pancreatic RNase digests of tRNAA^P and tRNAAyP A comparison of the two dimensional chromatographic maps of digests illustrated in Figure 31 reveals only one minor difference, namely the area labeled (A). The areas (A), (B), and (C) were eluted and the TABLE 34 Quantitative"*" analysis of 6 and Y forms of tRNAs Source of tRNA percentage of form Tyr 1 Asp 2 Asn 2 Asn3 Asn,. wild-type egg 22 25 57 24 29 26 first-instar larva 15 12 32 26 21 19 second-instar larva 11 12 31 8 10 10 early third-instar larva 7 7 20 7 10 11 late third-instar larva 5 4 12 6 5 15 prepupa 10 7 25 9 10 12 early pupa 10 7 24 10 9 14 late pupa 27 29 62 36 34 32 mixed age adult 40 36 72 36 29 39 two-week old adult 49 47 74 48 55 47 2 su(s) v;bw mutant late third instar larva 52 49 79 31 51 49 mixed age adult 88 88 99 65 88 91 peak heights were so reproducible on RPC-5 columns that they were used for approximate quantitation of peaks. FIGURE 30 As p As p RPC-5 chromatography of wild-type tRNA . Transfer RNA was prepared by; the naphthoxyacetylation procedure described i n the Methods. The tRNA (188 uni t s ) was applied to a 50 O x 2.5 cm RPC-5 column maintained at 37 C. E l u t i o n was by a 2-1 . gradient from 0.50 to 0.65 M-NaCl at 120 ml/hour. Absorbancy at 260nm of the f r a c t i o n s (10 ml) was determined. The f r a c t i o n s 14 were assayed for ( C)aspartic acid acceptor a c t i v i t y as described i n the Methods. Op t i c a l density at 260 nm ( • • ). Acceptance of (*^C) as p a r t i c a c i d (125 mCi/mmole) - cts/min/ml 14 ( O-TO ). S p e c i f i c a c t i v i t y - pmoles ( C)aspartic acid accepted per A 2 6 Q unit ( A ). 234 235 Jieoo< Fraction no. FIGURE 31 Two-dimensional thin-layer chromatograms of pancreatic RNase digests of tRNA^y15 and tRNA^P. The tRNAs (10 A ^ units) were hydrolyzed with 100 ug pancreatic RNase, applied directly to 20 x 20 cm plates and chromatographed. Under U. V. light the open areas were fluorescent in acid, while the solid ones were not. The areas delineated by the incomplete lines were very weak. 236 Asp 28 c s 12 C D o r i g i n 0 An o second dimension 238; oligonucleotides recovered were subjected to hydrolysis in 10% piperi-dine. Only Up and Gp could be detected from these spots, although Gp from (A) was weak. Chromatographic analysis of piperidine hydrolysates of tRNAAgP and tRNA^y^ The only consistent difference between the maps of nucleotide com-position of hydrolysates of the two whole tRNAs (Figure 32) is the area marked Qfip in the hydrolysate of tRNAAgP . No spot is present in this area in the hydrolysate of tRNA^^. In this system the nucleotide Qp from tRNA of E_. coli chromatographed in the position shown in Figure 32c. Both Q6p and Qp were eluted from the thin layer plates and their spectra determined at pH 2 and 10 and in 1 N-HC1 (Figure 33). Although the spectra are very similar they are not identical. FIGURE 32 Two-dimensionalthin-layer chromatograms of piperidine hydrol-ysates of tRNA^^ and tRNA^y P and pi p e r i d i n e treated E. c o l i Qp. The tRNAs (10 A n ^ A u n i t s ) and Qp (0.1 A„,_ un i t ) i n 10% zoU 2oU p i p e r i d i n e were sealed in c a p i l l a r y tubes, b o i l e d in a water bath f o r two hours and applied d i r e c t l y to t h i n - l a y e r plates (20 cm x 20 cm). Under U. V. l i g h t the open areas were f l u o -rescent and the s o l i d areas were not fluorescent. The area in (b) delineated by the incomplete l i n e represents pGp which was not apparent on t h i s chromatogram but was found on other chrom-atograms of hydrolysates of tRNA^f . 239 second dimension to o FIGURE 33 Asp Ultraviolet absorption spectra of Q$p from Drosophila tRNA^ and Qp from E. co l i . 1 N-HC1 ( ) ; pH 2.0 ( ) pH 12.0 ( ) 241 1 1 r—i 1 1 1 1 1 r W a v e l e n g t h (nm) 243 IV. Discussion Reverse phase chromatography of radioactively-labeled tyrosyl-, aspartyl-, asparaginyl-, and histidyl-tRNAs from developmental stages of wild-type p_. melanogaster has revealed similar quantitative altera-tions in their major isoaccepting forms. These four tRNAs show further 2 quantitative changes in flies of the genotype su(s) v; bw. No similar changes were observed for phenylalanyl-tRNA or the remaining fifteen aminoacyl-tRNAs (Part II, Chapter 2). From the wild-type adult two major chromatographically distinct Tyr Asp His forms of each of tRNA - , tRNA and tRNA can be resolved while six A. STI major forms of tRNA are apparent. Comparison of Figures 26 - 29 re-veals that only the later eluting (Y) forms of these tRNAs are present in significant proportion in the late third larval instar stage. A quantitative examination of 6 and Y forms (Table 3 4 ) shows that at the prepupal stage the 6 forms increase and reach maxima two weeks after eclosion. The 5 forms are of significant proportion in the egg but gra-dually decrease until the late third instar. Although there is a dis-tinctive increase in the 6 forms from the late third-instar larva to the adult, there is no marked increase in the overall acceptance of tyrosine, histidine, asparagine and aspartic acid compared to phenyl-alanine in this period. 2 Both late third instar larvae and adults of the genotype su(s) v;bw have significantly reduced amounts of the Y forms of these tRNAs. This mutant has been previously shown to lack one chromatographic form of 244 tRNATyr (Twardzic, Grell and Jacobson, 1971). It appeared more likely that the difference between the S and Y forms was related to post-transcriptional modifications of the same gene products rather than a simultaneous "turning off" of genes for the Y forms and "turning on" of genes for the S forms. The uniformity of the change for the four tRNAs suggested a similar modification to a l l of them making the <5 forms elute from the columns at an almost equally re-duced ionic strength. These four tRNAs form a natural group in that they respond to the codons XAy and, in JE. c o l i , this group exclusively contains the modified nucleoside Q, of unknown structure, in the first position of the anti-codon (Harada and Nishimura, 1972). Q is thought to be a guanosine deri-vative, presumably formed by modification after synthesis of the tRNA chain (Nishimura, 1972). Two-dimensional chromatograms of the two forms of tRNAAsp, previously subjected to piperidine hydrolysis, revealed one altered nucleotide in the tRNA^ g as the only detectable difference. This nucleotide, which had a mobility similar to Qp in the chromatographic system used was isolated and its U.V. spectra at various pH's compared to an authentic sample of Qp from E_. coli tRNA which had been treated with piperidine and chromatographed at the same time as the tRNA^^ and tRNA^^ hydro-lysates. From Figure 33 i t can be seen that the spectra of the altered nucleotide (Q<5p) at the various pH's were similar but not identical to those of the standard Qp. 245 Twardzic et a l . , (1971) showed that the 3'-0H terminal oligo-2 nucleotide obtained by RNase digestion of wild-type and su(s) v;bw Tyr 14 3 tRNA 3 , labeled respectively with ( C) and ( H) tyrosine, chromato-graphed identically on a DEAE-cellulose column when eluted by an ammonium formate gradient. This indicates, then, that the same 3'-0H terminal oligonucleotides are released from the major chromatographic Tyr forms of tRNA upon RNase T^ hydrolysis. Chromatographic analysis of pancreatic RNase digests of the two purified forms of tRNAAsp produced almost identical patterns. Although this does not prove their identical sequence, i t strongly suggests that this is the case. The evidence presented herein argues very strongly in favour of the 5 and Y forms of the tRNAs being identical, save for the degree of modi-fication of a nucleoside analogous to Q or E_. coli. There are two models which most simply explain the present data (Figure 34). (1) An enzyme that modifies specific tRNAs by converting a nucleotide (termed GYp for convenience) to Q6p, is normally not active in the period from the egg to late third instar larva. Thus tRNAs synthe-tized during this time are present only in the Y form. At the pre-pupal stage this enzyme becomes active and the tRNAs synthesized after this time undergo the modification to Q5p. The Y forms syn-thesized prior to the prepupal stage are not subject to this modifi-cation. This may be for one of several reasons: (a) the enzyme recognizes only precursor tRNA; (b) the enzyme may be confined to the nucleus and therefore cannot modify tRNAs already in the FIGURE 34 Proposed models for the tRNA modifying enzyme 246 247 Egg | Late third Wild type su (s)V; bw mutant 1. Enzyme YP 8P Wild type suisfv'jbw mutant 2.Enzyme Wild type su(s)2v; bw mutant 248 cytoplasm, or (c) the enzyme may be switched on only in specific cells at the prepupal stage. Partial activity of this enzyme in 2 the su(s) v;bw flies from the egg to the late third instar would account for the high level of the § form during these stages in the mutant. (2) A tRNA modifying enzyme responsible for the conversion of the nucleotide Q6p to GYp, is active from the egg until the prepupal 2 stage after which time i t is inactive. The mutant su(s) v;bw can produce this enzyme only in a partially active form, leaving a considerable amount of tRNA in the unmodified <5 form. It is further suggested that the failure to detect the minor nucleo-tide GYp (which is implicated in both schemes) by thin-layer chromatography of the piperidine hydrolysates of tRNA^^reflects either its Gp (i.e. unmodified) nature or its obscurance by a major nucleotide. Both models explain the changes in the tRNA peaks equally well. They also provide a possible explanation for the difference in the pro-portions of tRNAA^P and tRNAA!*P as compared to the <5 and Y forms of tRNA^ "*"S, tRNAAsn and tRNATyr. By assuming an increased rate of the transcription of tRNA^15 at the prepupal stage, i t follows that there will be an increase in the proportion of the 6 to the Y form since at this stage newly synthesized Y form is converted to 6 form. If tRNA^r and tRNA^ are, respectively, tRNA^yr and tRNA^ of Twardzic e_t al . (1971) (this seems reasonable considering the results 2 with the su(s) v; bw mutant), the results presented here lead to certain 249 predictions concerning the mechanism of suppression of vermilion in Tvr Drosophila. Jacobson (1971) found that tRNA2 binds to the tryptophan pyrrolase of the vermilion mutant, thus inhibiting its activity. If Tyr Tyr the only difference between tRNA, ~ and tRNA, lies in the modifica-J 1 6 ly tion of a nucleotide in the anticodon loop, as suggested above, i t follows that the postulated nucleotide, GyP, in this part of the tRNA molecule is responsible for inhibiting tryptophan pyrrolase from the mutant. If Gyp is the same in tRNAAsp, tRNA?1S, tRNAAsn, tRNAAsn and r r 2y ' ly 2y 3y Asn tRNA,.y , as our results suggest, i t also follows that there must be some mechanism by which the mutant's tryptophan pyrrolase discriminates the different tRNAs, since these do not inhibit the enzyme (Jacobson, 1971). It is likely that tryptophan pyrrolase from the vermilion mutant has an allosteric regulatory site (possibly identical with that to which inhibitors such as pteridines and allopurinol bind in the wild-type enzyme (Ghosh and Forest, 1967)) which interacts with Gyp in a suitable tRNA molecule. As G^ p is probably located in the anticodon of the tRNA i t would be well-exposed for such interactions. This type of mechanism is essentially the same as that proposed by Baillie and Chovnick (1971) for the regulation of tryptophan pyrrolase in vivo by positive and nega-tive allosteric effectors. It has as yet to be demonstrated whether Tyr tRNA^ y interacts with wild-type tryptophan pyrrolase, but i f i t does, as was suggested by Jacobson and Grell (1971) , i t is likely that the ^ Tyr Tyr relative proportions of tRNA^ g and tRNA^ may control its enzymatic activity during the development of Drosophila. This would be one of the first instances in which changes in tRNA levels could be correlated with a control mechanism. 250 The su(s) locus suppresses a l l e l e s of the purple, speck, and sable l o c i as well as vermilion. I t i s pos s i b l e that the reduction of the forms of tRNA A s p. tRNA A s n' and tRNA H l s as well as tRNA T y r during the development of Drosophila may be responsible f o r t h i s suppression. I t i s not known whether the Q^p^Gyp modification plays any r o l e i n the con t r o l at the t r a n s l a t i o n a l l e v e l . The codon recogni-t i o n properties of the 6 and Y forms w i l l be of i n t e r e s t because the E. c o l i tRNAs with Qp i n the f i r s t (wobble) p o s i t i o n of the a n t i -codon bind with U p r e f e r e n t i a l l y ofer C i n the t h i r d p o s i t i o n of the 2 codon (Harada and Nishimura, 1972). As the su(s) v; bw mutant appears to grow normally i t would seem that the Y forms present i n reduced amounts, are not e s s e n t i a l f o r growth and development although the 6 forms may be e s s e n t i a l f o r development of prepupa to adult. The r e l a t e d S and Y forms of tRNA A s p, tRNA A s n, tRNA H i s, and tRNA T y r probably have the same nuclotide sequence and are only d i s t i n g u i s h a b l e by the extent of mo d i f i c a t i o n of one minor nucleoside i n the anticodon. As these tRNAs are presumably products of the same gene, we propose to c a l l them homogeneic tRNAs instead of l a b e l l i n g them as i n d i v i d u a l i s o -accepting species. 251 SUMMARY AND CONCLUSIONS A number of problems concerned with the genetic control of develop-ment have been investigated: (1) the genetic properties of dominant lethal mutations, (2) a developmental characterization of dominant temperature-sensitive lethal mutations, (3) the genetic and developmental properties of a clustering group of recessive temperature-sensitive mutations which do not complement under non-permissive growth conditions, (4) the relation-ship between changes in a specific group of macromolecules, the tRNAs, and development, and (5) the role of a specific group of isoaccepting tRNAs during the development of Drosophila melariogaster. The recovery of dominant temperature-sensitive (DTS) lethal mutations provided a means for investigating the nature of dominant lethality. In this respect, i t was necessary to establish the genetic properties and developmental characteristics for each of the DTS-lethals. In the ab-sence of any gross distortions in the genetic map during the cross over studies, i t would appear that each of the DTS-lethals maps as a point mutation. This observation supports that of Suzuki and Procunier (1969) that dominant lethals need not be associated with chromosomal aberrations as was indicated in earlier studies. The complementation studies indicate that a l l of these mutations are functionally distinguishable, with the exception of DTS-5 and DTS-7 which f a i l to survive as trans-heterozygotes both at 17° and 22°C. A detailed genetic analysis of this synthetic lethal interaction revealed a true synergistic effect of the two DTS-lethals. A study of the effects of two wild-type alleles on the 252 v i a b i l i t y of DTS-bearing i n d i v i d u a l s was made by introducing the DTS-l e t h a l s i n t o a t r i p l o i d stock. The r e s u l t s indicated that only DTS-4 retained i t s dominant phenotype at 29°C i n t r i p l o i d females (3X;3A), metafemales (2X;3A), and supermales (X;3A). The developmental properties of each of the DTS-lethals revealed t h e i r c h a r a c t e r i s t i c temperature-sensitive periods (TSPs) and l e t h a l phases (LPs). Detailed analyses established fundamental di f f e r e n c e s i n the r e l a t i o n s h i p s between the TSPs and LPs; a corresponding TSP may immediately precede the LP or there may be a time separation of several days. In addition, mutations such as DTS-2 show a very short TSP f o l -lowed by a d i s t i n c t i v e LP, while DTS-4 shows a continuous s e r i e s of TSPs and LPs extending from the egg stage u n t i l mid-way through pupation f o r the TSPs and u n t i l the adult stage f o r the LPs. The response to d i f f e r e n t temperatures also revealed behavioural di f f e r e n c e s among the DTS-lethals. In addition to the developmental d i f f e r e n c e s , a number of properties were uncovered f o r s p e c i f i c mutations during the studies. Sexual d i -morphisms were evident with respect to both f e r t i l i t y and v i a b i l i t y . DTS-7-bearing males showed a much shorter TSP f o r adult e c l o s i o n than did females of t h i s genotype, although the TSPs f o r the v i a b i l i t y of adults corresponded. On the other hand, exposure of DTS-7 females to 29°C f o r three days r e s u l t e d i n t h e i r s t e r i l i t y , whereas s i b l i n g males were unaffected. A recessive t s maternal-effect mutation, recovered simultaneously with DTS-2, was found to cause embryonic l e t h a l i t y of both heterozygous and homozygous progeny (although a low degree of rescue 253 was possible through the introduction.of a wild-type allele via the sperm). Another class of mutations, the Minutes, was also represented in these studies. Thus, the phenotypic properties of DTS-lethals at the permissive temperature are reminiscent of similar mutations in the non-ts category. The spectrum of developmental properties character-istic of these DTS-lethals provides additional evidence that dominant lethality need not result from abnormal cell-division as had previously been thought. In order to test whether structural or enzymic proteins confer this dominant lethality, DTS-lethals in specific functions must be studied. Several of the DTS-lethals described here may provide clues as to their biochemical lesions through the examination of their charac-teristic phenotypes and TSP-LP relationships. For example, DTS-4 shows a behaviour suggesting its candidacy as a muscle mutant, and biochemical studies in this respect may serve to elucidate the specific defect. DTS-2 shows a characteristic LP closely resembling that of cryptocephal homozygotes. The latter have been shown to have an abnormally rigid integument resulting from an increased incorporation of glutamine during the synthesis of glucosamine-6-phosphate. Tests comparable to those carried out by Fristrom (1965) may help to identify the nature of the defect. Kinetic studies on the proteins involved in this pathway would be invaluable in establishing the molecular basis for dominant lethality. The third problem was concerned with a genetic and developmental characterization of four ts non-complementing recessive mutations. These were shown to behave similarly, but not identically, in the many tests for interactions with mutations of the bithorax pseudoallelic 254 series. All showed multiple TSPs and LPs, and.although for each of the mutations the LPs were essentially identical, the TSPs varied some-what, depending on the mutant studied. Although these observed differ-ences may reflect differences in their residual genotypes, the interest-ing possibility exists that the four Ubx-ts-lethals represent lesions in different parts of the same gene. These related mutations provide a model system for genetic fine structure analysis. The second part of this study was concerned with an analysis of the changes in species of macromolecules during the development of Drosophila. Transfer RNAs were chosen for this study because of the ease with which they may be qualitatively and quantitatively analyzed. In addition, the suggestion that Minutes represent mutations in transfer RNA genes (K. C. Atwood, see Lindsley and Grell, 1968) or, less specifi-cally, that they are defective in some aspect of protein synthesis, provided an intriguing incentive for the examination of Drosophila tRNAs and changes in isoaccepting species during development. Reversed-phase 5 chromatography of radioactively labeled Drosophila aminoacyl tRNAs isolated from fir s t and third instar larvae and adults yielded elution profiles which remained unchanged throughout the develop-mental stages examined for certain aminoacyl tRNAs, while others showed marked quantitative differences. A specific pattern emerged for four of the aminoacyl tRNAs; tRNAAsn, tRNAAsp, tRNAHls, and tRNATyr, suggest-ing that certain of the chromatographically distinct forms were altered in a similar manner. This relationship formed the basis for the inves-tigations carried out in the last chapter. 255 The inclusion of tRNA 1 in this group showing an ordered speci-ficity in the alteration of peak heights with development, coupled with the observation of Jacobson (1971) and Twardzic et_ al_. (1971) that a Tyr specific tRNA 3 inhibits tryptophan pyrrolase activity in the vermilion mutant, prompted a more detailed analysis of the four above-mentioned tRNAs during discrete stages of development. The lack of one of the Tyr isoaccepting tRNA J 's, is apparently, responsible for suppression of the vermilion eye colour by the suppressor of sable mutation, and thus 2 developmental studies on the su(s) v; bw stock were also included. In addition, the two major tRNAAsp peaks were purified and subjected to (a) pancreatic RNase digestion, and (b) to piperidine hydrolysis. The former enzyme produced almost identical patterns on two-dimensional thin-layer chromatograms whereas the latter revealed a difference of a single a l -tered nucleotide. It was concluded that these chromatographic forms are homogeneic and that their difference lies only in the extent of the modification of a single nucleoside. Furthermore, i t was apparent that 2 the su (s) y_; bw mutant is altered in some aspect of this modification process. The suggestion that the altered nucleotide was related to the Q-base isolated from E_. coli (Harada and Nishimura, 1971) was supported by its chromatographic properties and U.V. spectra. The ability to purify large amounts of a specific isoaccepting tRNA species suggests that i t should be possible to localize tRNA genes on the salivary gland chromosomes by in situ hybridization. Hybridiza-tion of single species should give unambiguous results and, in this way, mutations mapping close to sites where specific tRNAs localize could be 256 screened for defects or reduced amounts of these specific tRNAs. In order to study more exactly the roles of tRNAs in relation to differen-tiation and development, an analysis of events occurring when a defec-tive tRNA is present in the genome should give a better understanding to its role during normal development. These studies have shown the adaptation of Drosophila to studies of development from both genetic and biochemical approaches. The use of temperature-sensitive lethals has allowed the genetic and develop-mental characterization of a class of mutations which are otherwise not amenable to such studies--namely, the dominant lethals. And the detailed studies of a specific group of macromolecules, the tRNAs, and their changes during development, provide a handle for the investigation of the roles of these molecules in controlling various aspects of develop-ment. 257 LITERATURE CITED Ames, B.N. and Hartman, P.E. (1962), Genes, enzymes, and control mechanisms in histidine biosynthesis. In "The Molecular Basis of Neoplasia", pp. 322-345. University of Texas Press, Austin. Ames, B.N., and Hartman, P.E. (1963). The hisitidine operon. Cold Spring Harb. Symp. Quant. Biol. 28: 349-Anderson, M.B. and Cherry, J.H. (1969). Differences in leucyl-transfer RNA's and synthetase in soybean seedlings. Proc. Nat. Acad. Sci. U.S.A. 62: 202-209. Anderson, W. F. (1969). The effect of tRNA concentration on the rate of protein synthesis. Proc. Nat. Acad. Sci. U.S.A.62^ : 566-573. Anderson, W.F., and Gilbert, W.F. (1969). tRNA-dependent translational control of in vitro haemoglobin synthesis. Biochem. Biophys. Res. Commun. 36_: 456-462. Armstrong, M.D., and Tyler, F.H. (1955). Studies on phenylketonuria. I. Restricted phenylalanine intake in phenylketonuria. J . Clin. Invest. 34_: 565-580. Atwood, K.C. (1968). In "Genetic Variations of Drosophila melanogaster" (Lindsley, D.L., and Grell, F.H.). Carnegie Inst. Wash. Publ. 627, p. 152. Auerbach, C. (1951). Inductions of changes in genes and chromosomes. Cold Spring Harb. Symp. Quant. Biol. 16^ : 199-213. Auerbach, C , and Kilbey, B.J. (1971). Mutation in Bukaryotes. Annual Rev. Genet. 5_: 163-218. Bagshaw, J.C, Finamore, F.J., and Novelli, G.D. (1970). Changes in transfer RNA in developing brine shrimp. Develop. Biol. 23_: 23-55. Baillie, D., and Chovnick, A. (1971). Studies on the genetic control of tryptophan pyrrolase in Drosophila melanogaster. Moi. Gen. Genet. 112: 341-353. Baillie, D., Suzuki, D.T., and Tarasoff, M. (1968). Temperature-sensi-tive mutations in Drosophila melanogaster. II. Frequency among second chromosome recessive lethals induced by ethyl methanesul-fonate. Can. J . Genet. Cytol. 10: 412-420. Baker, W.K., and Spofford, J.B. (1959). Heterochromatic Control of Position-Effect Variegation in Drosophila. Biological Contri-butions, The University of Texas, Publ. No. 5914: 135-154. 258 Baldwin, M.C. , and Suzuki, D.T. (1971). A Screening Procedure for Detection of Putative Deletions in Proximal Heterochromatin of Drosophila. Mutat. Res. 11:203-213. Barr, H.J., and Ellison, J.R. (1972). Ectopic Pairing of Chromosome Regions Containing Chemically Similar DNA. Chromosoma 39: 53-61. Bateson, W. (1894). "Materials for the Study of Variation, Treated with Especial Regard to Discontinuity of the Origin of Species." MacMillan and Co., London and New York. Beadle, G.W., and Tatus, E.L. (1941). Genetic control of biochemical reactions of Neurospora. Proc. Nat. Acad. Sci. U.S.A. 2_7: 499-506. Benzer, S., and Champe, S.P. (1961). Ambivalent r__ mutants of phage T4. Proc. Nat. Acad. Sci. U.S.A. 4J7: 1025-1038. Benzer, S., and Hotta, Y. (1970). Genetic dissection of the Drosophila nervous system by means of mosaics. Proc. Nat. Acad. Sci. U.S.A. 67_: 1156-1163. Berendes, H., and Keyl, H.G. (1967). Distribution of DNA in heterochro-matin and euchromatin of polytene nuclei of Drosophila hydei. Genetics S7_: 1-13. Bernstein, H., and Fisher, K.M. (1968). Dominance in bacteriophage T4D. Genetics 5_8: 307-318. Bodenstein, D. (1943). Factors influencing growth and metamorphosis of the salivary gland in Drosophila. Biol. Bull. 84_: 13-33. Bodenstein, D. (1950). The Postembryonic Development of Drosophila. In "Biology of Drosophila", (M. Demerec, ed.) pp. 275-367. Hafner, New York. Bollum, F.J. (1959). Thermal Conversion of Nonpriming Deoxyribonucleic Acid to Primers. J. Biol. Chem. 234: 2733-2734. Bonnier, G., and Luning, K.G. (1950). X-ray induced dominant lethals in Drosophila melanogaster. Hereditas 36: 445-456. Bonnier, G., and Luning, K.G. (1951). Spontaneous and X-ray induced gynandromorpha in Drosophila melanogaster. Hereditas 37: 469-487. Bonnier, G., and Luning, K.G. (1952). Interaction of centromere and cytoplasm in chromosome elimination in Drosophila melanogaster. Hereditas 38: 339-344. 259 Botchan, M., Kram, R., Schmid, C.W., and Hearst, J.E. (1971). Isola-tion of chromosomal localization of highly repeated DNA sequences in Drosophila melanogaster. Proc. Nat. Acad. Sci., U.S.A. 68: 1125-1129. Braid, F., and Hickmans, E.M. (1929). Metabolic study of an alkaptonuric infant. Arch. Dis. Childhood 4_: 389-398. Bridges, C.B. (1916). Non-disjunction as Proof of the Chromosomal Theory of Heredity. Genetics 1_: 1-52, 107-163. Bridges, C.B. (1926). In "Genetic Variations of Drosophila melanogaster" Lindsley and Grell, pp. 26-28. Britten, R.J., and Davidson, E.H. (1969). Gene Regulation for Higher Cells: A Theory. Science (Washington) 165: 349-357. Britten, R.J., and Kohne, D.E. (1968). Repeated Sequences in DNA. Science (Washington) 161: 529-540. Brosseau, G.E. (1960). Genetic analysis of the male fe r t i l i t y factors on the Y chromosome of Drosophila melanogaster. Genetics 45: 257-274. Brown, S.W. (1966). Heterochromatin. Science (Washington), 151: 417-425. Bryant, P.J., and Schubiger, G. (1971). Giant and Duplicated Imaginal Discs in a New Lethal Mutant of Drosophila melanogaster. Develop. Biol. 24: 233-263. Bryson, V. (1940). The modifying effect of Minutes. Genetics 2_5: 113 (Abstract). Callan, H.G. (1967). The organization of genetic units in chromosomes. J . Cell Sci. 2: 1-7. Camfield, R.G., and S^uzuki, D.T. (1972). A temperature-sensitive vermil-ion mutant (AT ) in Drosophila melanogaster. Can. J . Genet. Cytol. in press. Carlson, E.A. (1959). Comparative genetics of complex lo c i . Quart. Rev. Biol. 34: 33-67. Catcheside, D.G., and Lea, D.E. (1945). The rate of induction of domi-nant lethals in Drosophila melanogaster sperm by X-rays. J . Genet. 47: 1-9. Catcheside, D.G., and Lea, D.E. (1945b). Dominant lethals and chromo-some breaks in ring-X-chromosomes of Drosophila melanogaster. J . Genet. 47: 25-40. 260 Cherayil, J.D., Hampel, A., and Bock, R.N. (1968). Rapid Microscale Assay for sRNA. Methods Enzymol. XII, Part B. 166-169. Cooper, K.W. (1959). Cytogenetic analysis of major heterochromatic elements (especially Xh and Y) in Drosophila melanogaster and the theory of "heterochromatin". Chromosome 10_: 535-588. Counce, S.J. (1956a). Studies on female-sterility genes in Drosophila melanogaster. II. The effects of the gene deep orange on embryonic development. Z. indukt. Abstamm. -u. VererbLehre 87: 443-461. Counce, S.J. (1956b). Studies on female-sterility genes in Drosophila  melanogaster. I. The effects of the gene fused on embryonic development. Z. indukt. Abstamm. -u. VererbLehre 87_: 462-481. Delbruck, M., and Stent, G.S. (1957). On the mechanism of DNA replica-tion. In "The Chemical Basis of Heredity" (W.D. McElroy and B. Glass, eds.) pp. 699-736. Johns Hopkins Press, Baltimore. Demerec, M. (1934). Biological action of small deficiencies of X-chromosomes of Drosophila melanogaster. Proc. Nat. Acad. Sci. U.S.A. 20_: 354-359. Demerec, M. (1939). Chromosome structure as viewed by a geneticist. Amer. Natur. 73_: 331-338. Demerec, M., and Kaufmann, B.P. (1938). X-ray-induced chromosomal alter-ations in Drosophila melanogaster. Genetics 23_: 610-630. Dempster, E.R. (1941). Dominant versus recessive lethal mutation. Proc. Nat. Acad. Sci. U.S.A. 27_: 247-250. Dewitt, W. (1971). Differences in methionyl- and arginyl-tRNAs of larval and adult bullfrogs. Biochem. Biophys. Res. Commun. 42: 266-270. Dobzhansky, Th. (1946). Genetics of natural populations. XIII. Recom-bination and variability in populations of Drosophila pseudo-ob- scura. Genetics 31_: 269-290. Doi, R.H., Kaneko, I., and Igarashi, R.T. (1966). Regulation of a serine transfer RNA of Bacillus subtilis under two growth condi-tions. Proc. Nat. Acad. Sci. U.S.A. 56_: 1548-1551. Doi, R.H., Kaneko, I., and Igarashi, R.T. (1968). Pattern of Valine Transfer Ribonucleic Acid of Bacillus subtilis under Different Growth Conditions. J. Biol. Chem. 243: 945-951. Dunn, L.C., and Coyne, J. (1935). The Relationship between the Effects of Certain Mutations on Developmental Rate and on Adult Charac-ters. Biol. Zentrglbl. 55: 385-389. 261 Eckhard, R.A., and Gall, J.G. (1971). Satellite DNA associated with heterochromatin in Rhynchosciara. Chromosoma (Berlin) 32_: 407-427. Engelhardt, D.L., Webster, R.E., Wilhelm, R.C., and Zinder, N.D. (1965). In vitro studies on the mechanism of suppression of a nonsense mutation. Proc. Nat. Acad. Sci, U.S.A. 54_: 1791-1797. Fahmy, O.G., and Bird, M.J. (1952). Chromosome breaks among recessive lethals induced by chemical mutagens in Drosophila melanogaster. Heredity, supplement to Vol. 6_: 149-162. Fahmy, O.G., and Fahmy, M.J. (1954). Cytogenetic analysis of the action of carcinogens and tumor inhibitors in Drosophila melanogaster. II. The mechanism of induction of dominant lethals by 2: 4: 6-tr i (Ethyleneimino)-1: 3: 5-Triazine. J. Genet. 52_: 603-619. Fristrom, J.W. (1965). Development of the morphological mutant cryptoce- phal of Drosophila melanogaster. Genetics 52_: 297-318. Fristrom, J.W. (1970). The developmental biology of Drosophila. Ann. Rev. Genet. 4_: 325-346. Gall, J.G., and Pardue, M.L. (1969). Formation and detection of RNA-DNA hybrid molecules in cytological preparations. Proc. Nat. Acad. Sci. U.S.A. 63_: 378-383. Garen, A., and Garen, S. (1963). Complementation in vivo between struc-tural mutants of alkaline phosphatase from E_. c o l i . J. Moi. Biol. 7: 13-22. Garen, A., and Gehring, W.H. (1972). Repair of the Lethal Developmental Defect in Deep Orange Embryos of Drosophila by Injection of Normal Egg Cytoplasm. Proc. Nat. Acad. Sci. U.S.A. 69: 2982-2985. Garrod, A.E. (1902). The Incidence of Alkaptonuria: a study in chemical individuality. Lancet i_i_: 1616-1620. Gartland, W.J., Ishida, T., Nirenberg, M., and Sueoka, N. (1969). Coding Properties of Two Conformations of Tryptophanyl-tRNA in E_. coli. J . Moi. Biol. 44: 403-413. Gershenson, S.M. (1940). The nature of so-called genetically inert parts of chromosomes. Vid. Acad. Nauk. URRS 3+116 (Ukrainian with English summary). Ghosh, D., and Forest, H.S. (1967). Inhibition of Tryptophan Pyrrolase by Some Naturally Occurring Pteridines. Arch. Biochem. 120: 578-582. 262 Gillam, I.C., Millward, S., Blew, D., von Tigerstrom, M., Wimmer, E., and Tener, G.M. (1967). The separation of soluble ribonucleic acids on benzoylated Diethylamino-ethylcellulose. Biochemistry 6: 3043-Glassman, E., and Mitchell, H.K. (1959a). Mutants in Drosophila melano- gaster deficient in xanthine dehydrogenase. Genetics 44_: 153-162. Glassman, E., and Mitchell, H.K. (1959b). Maternal effect of ma-l* on xanthine dehydrogenase of Drosophila melanogaster. Genetics 44: 547-554. Goldschmidt, R.B. (1953). Heredity within a sex-controlled structure of Drosophila. J. Exp. Zool. 122: 53-95. Goldschmidt, R.B. (1958). Theoretical Genetics. University of Califor-nia Press, Berkeley. Gray, L.H. (1952). Characteristics of chromosome breakage by different agents. Heredity, supplement to Vol. 6_: 311-315. Green, M.M. (1955). Pseudoallelism and the gene concept. Amer. Natur. 89_: 65-71. Grell, E. H. (1963). Distributive pairing of compound chromosomes in females of Drosophila melanogaster. Genetics 48: 1217-1229. Grell, R. F. (1962). A new model for secondary nondisjunction: The role of distributive pairing. Genetics 47: 1737-1754. Grell, R. F. (1967). Pairing at the chromosomal level. J. Cell. Physiol. 70 (Suppl. 1): 119-146. Guthrie, C, Nashimoto, H., and Nomura, M. (1969). Structure and func-tion of E_. coli ribosomes, VIII. Cold-sensitive mutants defective in ribosome assembly. Proc. Nat. Acad. Sci. U.S.A. 6_3_: 384-391. Hadorn, E. (1955). "Developmental Genetics and Lethal Factors". John Wiley and Sons, Inc., New York. Hadom, E. (1961). "Developmental Genetics and Lethal Factors". (Engl, trans.). Methuen, London. Hadom, E., and Neel, J. (1938). Der hormonale Einfluss der Ringdruse (corpusallatum) auf die Pupariumbildrung bei Fliegen. Wilhelm Roux Arch Entwicklungsmech. Organismen 138: 281-304. 2639 Haldane, J.B.S., and Lea, D.E. (1947). A mathematical theory of chromosomal rearrangements. J . Genet. 4_8: 1-10. Hannah, A. (1951). Localization and function of heterochromatin in "Drosophila melanogaster. Advan. Genet. 4_: 87-125. Hanson, F.B., (1928). The effects of X-rays on productivity and the sex-ratio of Drosophila melanogaster. Amer. Natur. 62: 352-362. Hanson, F.B., and Heys, F. (1935). The relation of the induced mutation rate to different physiological states in Drosophila melanogaster IV. The effect of age. Amer. Natur. 6j8: 166-167. Harada, F., and Nishimura, S. (1972). Possible Anticodon Sequences of tRNAHis, tRNAAsn, and tRNAAsp f r o m Escherichia coli B. Universal Presence of Nucleoside Q in the First Position of the Anticodons of These Transfer Ribonucleic Acids. Biochemistry 11_: 301-308. Harrison, P.R., Hell, A., Birnie, G.D., and Paul, J. (1972). Evidence for single copies of globin genes in the mouse genome. Nature (London), 239: 219-221. Hartmann-Goldstein, I.J. (1967). On the relationship between hetero-chromatization and variegation in Drosophila, with special refer-ence to temperature-sensitive periods. Genet. Res. (Cambridge) 10_: 143-159. Hatfield, D., and Caicuts, M. (1967). Specificity of tRNAs in mammalian tissues. Fed. Proc. 28_: 349. Hayes, W. (1969). "The Genetics of Bacteria and Their Viruses". John Wiley and Sons, Inc., New York. Henking, H. (1891). Untersuchungen uber die ersten Entwicklungsvorgange in den Eiern der Insekten. II. Z. wiss, Zool. 51: 685-736. Hennig, W., Hennig, I., and Stein, H. (1970). Repeated sequences in the DNA of Drosophila and their localization in giant chromosomes. Chromosoma, 32_: 31-63. Hinton, T. (1942). A comparative study of certain heterochromatic re-gions in the mitotic and salivary gland chromosomes of Drosophila  melanogaster. Genetics, 27_: 119-127. Holden, J., and Suzuki, D.T. (1968). Dominant temperature-sensitive (DTS) lethal mutations on chromosome 3 of Drosophila melanogaster. Genetics 60: 188-189. (Abstract). 263b Holden, J.J., and Suzuki, D.T. (1973). Temperature-sensitive mutations in Drosophila melanogaster. XII. The Genetic and Developmental Characteristics of Dominant Lethals on Chromosome 3. Genetics, in press. Holland, J.J., Taylor, M.W., and Buck, CA. (1967). Chromatographic differences between tyrosyl transfer RNA from different mammalian cells. Proc. Nat. Acad. Sci. U.S.A. 58_: 2437-2444. Idris, B.E.M. (1960). The development of separated parts of eggs of —' p i p ie n s• Wilhelm Roux' Archives Entwichmech. Organismen. 152: 230-262. Ilan, J . (1969). The role of tRNA in translational control of specific mRNA during insect metamorphosis. Cold Spring Harb. Symp. Quant. Biol. 34_: 789-791. Ilan, J., Ilan, J., and Patel, N. (1970). Mechanism of gene expression in Tenebrio molitor. Juvenile hormone determination of transla-tional control through transfer ribonucleic acid and enzyme. J. Biol. Chem. 245: 1275-1281. Ippen, K., Muller, J.H., Scaife, J., and Beckwith, J . (1968). A new controlling element in the lac operon of E_. coli. Nature 217: 825-827. Itano, H.A. (1963). In "Abnormal Hemoglobins in Africa" (J.H.P. Jonxis, ed.) pp. 3 - No. 1. Oxford University Press, London and New York. Jacob, F., and Monod, J. (1961). On the regulation of gene activity. Cold Spring Harb. Symp. Quant. Biol. 26_: 193-211. Jacobson, F.B. (1971). Role of an isoacceptor transfer ribonucleic acid as an enzyme inhibitor. Effect of tryptophan pyrrolase of DrosOphila. Nature, New Biology, (London) 231: 17-19. Jacobson, K.B., and Grell, E.H. (1971). The Role of a Specific Isoaccep-tor tRNA in Genetic Suppression, and in Enzyme Regulation in Drosophila. In "Nucleic Acid-Protein Interactions - Nucleic Acid-Synthesis in Viral Infection" (D.W. Ribbons, J.R. Woessner, and J . Schultz, eds.); North Holland Publishing Company, Amsterdam and London, pp. 204-214. Jones, K.W., and Robertson, F.W. (1970). Localization of reiterated nucleotide sequences in Drosophila and mouse by iii situ hybridiza-tion of complementary RNA. Chromosome (Berlin) 31_: 331-345. Judd, B.H., Shen, M.W., and Kaufman, T.C, (1972). The anatomy and function of a segment of the X chromosome of Drosophila melanogas-ter. Genetics 71: 139-156. 264 Kaneko, I., and Doi, R.H. (1966). Alteration of Valyl-sRNA during Sporulation of Bacillus subtilis. Proc. Nat. Acad. Sci. U.S.A. 55: 564-570. Kaplan, W.D. (1953). The influence of Minutes upon somatic crossing-over in Drosophila melanogaster. Genetics 38_: 630-651. Kaufman, T.C., and Suzuki, D.T. Temperature-sensitive mutations in Drosophila melanogaster. XIX. The Properties of Three X-Auto-some Translocations, in preparation. Kaufman, T.C., Tasaka, E.S., and Holden, J.J. (1972). The interaction of two pseudoallelic series zeste and bithorax in Drosophila  melanogaster. Canad. J. Genet. Cytol. Kaufmann, B.P. (1934). Somatic mitoses of Drosophila melanogaster. J . Morphol. 56: 425-455. Kaufmann, B.P. (1939). Distribution of induced breaks along the X chromosome of Drosophila melanogaster. Proc. Nat. Acad. Sci. U.S.A. 25_: 571-577. Kaufmann, K.B., and Demerec, M. (1937). Frequency of induced breaks in chromosomes of Drosophila melanogaster. Proc. Nat. Acad. Sci. U.S.A. 23_: 484-488. Kellenberger, E. (1961). Vegetative bacteriophage and the maturation of the virus particles. Adv. Virus. Res. 8_: 1-61. Kelmers, A.D., Novelli, G.D., and Stulberg, M.P. (1965). Separation of Transfer Ribonucleic Acids by Reverse Phase Chromatography. J. Biol. Chem. 240: 3979-3983. Kim, J.K., and Snyder, L.A. (1968). The mutagenic effects of two mono-functional alkylating chemicals on mature spermatosoa of Drosophila. Mutat. Res. 6_: 129-137. Kirby, K.S. (1956). A New Method for the Isolation of Ribonucleic Acids from Mammalian Tissues. Biochem. J . 64_: 405-408. Knox, W.E. (1958a). The detection in the heterozygote of the metabolic effect of the recessive gene for phenylketonuria. Amer. J. Hum. Genet. 10_: 53-59. Knox, W.E. (1958b). Sir Archibald Garrod's "Inborn Errors of Metabolism". II. Alkaptonuria. Amer. J . Hum. Genet. 10_: 95-124. 265 Krieg, D.R. (1963). Ethyl methanesulphonate-induced reversion of bacteriophage T4rII mutants. Genetics 48_: 561-580. Krivshenko, J. (1952). A cytogenetic study of the Y-chromosome in Drosophila buski. Genetics 3_7: 500-548. Lazzarini, R.A. (1966). Differences in lysine-sRNA from spore and vegetative cells of Bacillus subtilis. Proc. Nat. Acad. Sci. U.S.A. 56_: 185-190. Lea, D.E., and Catcheside, D.G. (1945). The relation between recessive lethals, dominant lethals, and chromosome aberrations in Drosophila melanogaster. J. Genet. 47_: 25-40. Lederberg, J. (1950). Isolation and characterization of biochemical mutants of bacteria. Methods Med. Res. 3_: 5-22. Lee, J.C, and Ingram, V.L. (1967). Erythrocyte transfer RNA change during chick development. Science (Washington) 158: 1330-1332. Lefevre, G., and Green, M.M. (1971). Interactions of deficiencies in the 3C region. Drosophila Information Service 46: 141. Levine, R.P., and Dickenson, J.I. (1952). The modification of recombina-tion by naturally occurring inversions in Drosophila pseudo-obscura. Rec. Genet. Soc. Amer. 21_: 43-44 (Abstract). Lewis, E.B. (1950). The Phenomenon of Position Effect. Advan. Genet. 3_: 73-115. Lewis, E.B. (1954). The Theory and Application of a New Method of Detecting Chromosomal Rearrangements in Drosophila melanogaster. Amer. Natur. 88_: 225-239. Lewis, E.B. (1960). A new standard food medium. Drosophila Information Service 34_: 117. Lewis, E.B. (1967). Genes and Gene Complexes. Heritage from Mendel pp. 17-47. Lewis, E.B. (1968). Genetic control of developmental pathways in Drosophila melanogaster. Proc. X I I Int. Congr. Genet., Vol. I I , 96-96. Lewis, E.B., and Bacher, F. (1968). Method of feeding ethyl methane-sulfonate (EMS) to DrosOphila males. Drosophila Information Service 43: 193. 266 Lindsley, D.L. (1955a). Spermatogonial exchange between the X and Y chromosomes of Drosophila melanogaster. Genetics 40_: 24-44. Lindsley, D.L. (1955b). Heterochromatic exchange between a reversed acrocentric compound X chromosome and the Y chromosome. Drosophila Information Service 29: 134. Lindsley, D.L., and Grell, E.H. (1968). "Genetic Variations of Drosophila  melanogaster". Carnegie Inst. Wash. Publ. 627. Lindsley, D.L., Sandler, L., Baker, B.S., Carpenter, A.T.C., Denell, R.E., Hall, J.C, Jacobs, P.A., Miklos, G.L.C, Davis, B.K., Gethemann, R.C, Hardy, R.W., Hessler, A., Miller, S.M., Nozawa, H., Parry, D.M., and Gould-Somero, M. (1972). Segmental aneuploidy and the genetic gross structure of the Drosophila genome. Genetics 71: 157-184. Lucchesi, J.C. (1968). Synthetic lethality and semi-lethality among functionally related mutants of Drosophila melanogaster. Genetics 59_: 37-44. Lucchesi, J.C, and Suzuki, D.T. (1968). The Interchromosomal Control of Recombination. Annu. Rev. Genet. 2_: 53-86. Luning, K.G. (1952a). X-ray induced dominant lethals in different stages of spermatogenesis. Hereditas 3_8_: 91-107. Luning, K.G. (1952b). X-ray induced chromosome breaks in Drosophila  melanogaster. Hereditas 38: 321-338. Luria, S.E. (1970). Phage, colicins, and macroregulatory phenomena. Science (Washington) 168: 1166-1170. McLeish, J. (1952). The action of maleic hydrazide in Vicia. Heredity, supplement to Vol. 6_: 125-148. Mach, B., Koblet, H., and Gros, D. (1967). Biosynthesis of Immunoglobu-lin of a cell-free system. Cold Spring Harb. Symp. Quant. Biol. 32_: 269-275. Mayoh, H., and Suzuki, D.T. (1973). Temperature-sensitive mutations in Drosophila melanogaster. XVII. The genetic properties of sex-linked recessive cold-sensitive mutants. Can. J . Genet. Cytol. (in press). Mendel, G. (1866). Versuche uber Pflanzenhybriden. Verh. naturforsch. Verein Brunn. 4: 3-47. 267 Merrell, D.J. (1947). A mutant in Drosophila melanogaster affecting fe r t i l i t y and eye colour. Amer. Natur. 81_: 399-400. Meselson, M., and Stahl, F.W. (1958a). The replication of DNA. Cold Spring Harb. Symp. Quant. Biol. 23: 9-12. Meselson, M., and Stahl, F.W. (1958b). The replication of DNA in Escherichia c o l i . Proc. Nat. Acad. Sci. U.S.A. 44_: 671-Molinaro, M., and Mozzi, R. (1969). Heterogeneity of tRNA during embryonic development of the sea urchin Paracentrotus lividus. Exp. Cell Res. 56_: 163-167. Muller, H.J. (1927). Artificial transmutation of the gene. Science (Washington) 66_: 84-87. Muller, H.J. (1940). An analysis of the process of structural change in chromosomes of Drosophila. J. Genet. 40: 1-66. Muller, H.J., and Gershenson, S.M. (1935). Inert regions of chromosomes as the temporary products of individual genes. Proc. Nat. Acad. Sci. U.S.A. 21_: 69-75. Muller, H.J., and Kaplan, W.D. (1966). The dosage compensation of Drosophila and mammals as showing the accuracy of the normal type Genet. Res. 8_: 41-59. Muller, H.J., and Painter, T.S. (1932). The differentiation of the sex chromosomes of Drosophila melanogaster into genetically active and inert regions. Z. indukt. Abstamm. -u. VererbLehre 3_: 316-365. Nierlich, D.P., Lamfrom, H., Sarabhai, A., and Abelson, J . (1973). Transfer RNA Synthesis in vitro. Proc. Nat. Acad. Sci. U.S.A. 70: 179-182. Nishimura, S. (1972). Minor components in Transfer RNA: Their Charac-terization, Location, and Function. Progr. Nucl. Acid Res. Moi. Biol. 12; 49-85. Novitski, E. (1951). Non-random disjunction in Drosophila. Genetics 36_: 267-280. Novitski, E. (1952). The genetic consequences of anaphase bridge forma-tion in Drosophila. Genetics 37_: 270-289. Pardue, M.L., Gerbi, S.A., Eckhardt, R.A., and Gall, J.G. (1970). Cyto-logical localization of DNA complementary to ribosomal RNA in polytene chromosomes of Diptera. Chromosoma 29: 268-290. 268 Parker, D.P. (1954). Radiation-induced exchanges in Drosophila females. Proc. Nat. Acad. Sci. U.S.A. 40: 795-809. Pavan, C., and Breuer, M.F. (1952). Polytene chromosomes in different tissues of Rhynchosciara. J . Heredity 43: 151-157. Pearson, P.L., and Babrow, M. (1970). Definitive evidence for the short arm of the Y chromosome associating with the X chromosome during meiosis in the human male. Nature (London) 226: 959-961. Pearson, R.L., Weiss, J.F., and Kelmers, A.D. (1971). Improved separa-tion of transfer RNAs on polychlorotrifluoroethylene-supported reversed-phase chromatography columns. Biochim. Biophys. Acta 228: 770-774. Pelley, J.W., and Stafford, D.W. (1970). Studies on the Enzymatic Bind-ing of Aminoacyl Transfer Ribonucleic Acid to Ribosomes in a Drosophila in vitro System. Biochemistry 9_: 3408-3414. Peterkofsky, A., and Capra, J.D. (1968). Effect of in vitro methylation on the chromatography and coding properties of methyl-deficient leucine tRNA. J. Moi. Biol. 33_: 591-607. Peterkofsky, A., Jesensky, C , and Capra, J.D. (1966). The Role of Methy-lated Bases in the Biological Activity of E_. coli leucine tRNA. Cold Spring Harb. Symp. Quant. Biol. 31_: 515-524. Piternick, L. and Suzuki, D.T. Temperature-sensitive mutations in Droso- phila. melanogaster. XVI. Genetic positions and viability indices of sex-linked recessive lethals. Submitted to Mutat. Res. Pontecorvo, G. (1942). The problem of dominant lethals. J . Genet. 45: 295-300. Pontecorvo, G. (1944). Structure of Heterochromatin. Nature (London) 153: 365-367. Pontecorvo, G., and Muller, H.J. (1940). The lethality of dicentric chromosomes in Drosophila. Genetics 26: 165 (Abstract). Poulson, D.F., and Metz, C.W. (1938). Studies on the structure of nucleolus-forming regions and related structures in the giant salivary gland chromosomes of Diptera. J . Morphol. 63: 363-395. Procunier, D., and Suzuki, D.T. (1967). Interchromosomal effects of crossing over in Drosophila melanogaster. IV. All Compound X Chromosomes. Can. J . Genet. Cytol. 9: 874-879. 269 Prokofyeva-Belgovskaya, A.A. (1935). The structure of the chromo-center. Cytologia (Tokyo) 6_: 438-443. Prokofyeva-Belgovskaya, A.A. (1939). Inert regions in the inner part of the X chromosome of Drosophila melanogaster. Bull. Acad. Sci. U.R.S.S., No. 3, pp. 362-370. Prokofyeva-Belgovskaya, A.A. (1941). Cytological properties of inert regions and their bearing on the mechanisms of mosaicism and chromosome rearrangement. Drosophila Information Service 15: 34-35. Prokofyeva-Belgovskaya, A.A. (1947). Heterochromatization as a change of chromosome cycle. J. Genet. 48_: 80-98. Rae, P.M.M. (1970). Chromosomal distribution of rapidly reannealing DNA in Drosophila melanogaster. Proc. Nat. Acad. Sci. U.S.A. 67_: 1018-1025. Revell, S.H. (1952). Chromosome breakage by X-rays and radiometic sub-stances in Vicia. Heredity, Supplement to Vol. 6_: 107-124. Ris, H., and Kubai, D.F. (1970). Chromosome structure. Annu. Rev. Genet. 4: 263-294. Ritossa, F.M., Atwood, K.C., and Spiegelman, S. (1966). On the redun-dancy of DNA complementary to amino acid transfer RNA and its absence from the nucleolar organizer region of Drosophila melano-gaster. Genetics 54_: 663-676. Ritossa, F.M., and Spiegelman, S. (1965). Localization of DNA comple-mentary to ribosomal RNA in the nucleolus organizer region of Drosophila melanogaster. Proc. Nat. Acad. Sci. U.S.A. 5_3: 737-745. Rosenbluth, R., Ezell, D., and Suzuki, D.T. (1972). Temperature-sensitive mutation in Drosophila melanogaster. IX. Dominant cold-sensitive lethals on the autosomes. Genetics 70_: 75-86. Sadler, J.R., and Novick, A. (1965). The properties of repressor and the kinetics of its action. J. Moi. Biol. 12_: 305-327. Sanders, P.F., and Pavan, C. (1972). Heterochromatin in Development of Normal and Infected Cells. In "Cell Differentiation", Vol. 2: Sandler, L., and Novitski, E. (1956). Evidence for genetic homology between chromosomes I and IV in Drosophila melanogaster, with a proposed explanation for the crowding effect in triploids. Genetics 41: 189-193. 270 Saneyoshi, M., and Nishimura, S. (1970). Selective modification of 4-thiouridylate residue oh Escherichia coli transfer RNA with cyanogen bromide. Biochim. Biophys. Acta 204? 389-399. Scharrer, B., and Hadom, E. (1938). The structure of the ring-gland (corpus allatum) in normal and lethal larvae of Drosophila  melanogaster. Proc. Nat. Acad. Sci. U.S.A. 24_: 236-242. Schneider-Minder, A. (1966). Cytologische Untersuchung der Embyonal-entwicklung von DrOsophila melanogaster nach Roentgenbestrahlung in fruehen Entwicklungsstadien. Int. J. Radiat. Biol. 11: 1-20. Schultz, J. (1936). Variegation in Drosophila and the inert chromosome regions. Proc. Nat. Acad. Sci. U.S.A. 22: 27-33. Schultz, J. (1941). The evidence of the nucleoprotein nature of the gene. Cold Spring Harb. Symp. Quant. Biol. 9_: 55-65. Schultz, J. (1947). The Nature of Heterochromatin. Cold Spring Harb. Symp. Quant. Biol. 12_: 179-191. Schultz, J. (1956). The relation of the heterochromatic chromosome re-gions to the nucleic acids of the c e l l . Cold Spring Harb. Symp. Quant. Biol. 21_: 307-328. Schultz, J., and Redfield, H. (1951). Interchromosomal effects on cros-sing over in Drosophila. Cold Spring Harb. Symp. Quant. Biol. 16: 175-197. Sedat, J.W., and Hall, J.B. (1965). Gramicidin S Messenger RNA. II. Physical and Chemical Properties. J . Moi. Biol. 1_2: 174-182. Shannon, M., Kaufman, T.C., Shen, M.W., and Judd, B.H. (1972). Lethality patterns and morphology of selected lethal and semi-lethal mutations in the zeste-white region of Drosophila melanogaster. Genetics 72: Sivertzev-Dobzhansky, N.P., and Dobzhansky, Th. (1933). Deficiency and duplications for the gene bobbed in Drosophila melanogaster. Genetics 18_: 173-192. Smith, J.D., Abelson, J.N., Clark, B.F.C., Goodman, H.M., and Brenner, S. (1966). Studies on Amber Suppressor tRNA. Cold Spring Harb. Symp. Quant. Biol. 31_: 479-485. Smith, J.D., Barnett, L., Brenner, S., and Russell, R.L. (1970). More mutant tyrosine transfer ribonucleic acids. J . Moi. Biol. 54: 1-14. ~ 27» Sonnenblick, B.P. (1940). Cytology and development of the embryos of X-rayed adult Drosophila melanogaster. Proc. Nat. Acad. Sci. U.S.A. 26: 373-381. Steffensen, D.M., and Wimber, D.E. (1971). Localization of tRNA genes in the salivary chromosomes of Drosophila by RNA:DNA hybridiza-tion. Genetics 69: 163-178. Steinberg, H., and Fraser, G. (1944). Studies on the effect of X chromosome inversions on crossing over in the third chromosome of Drosophila melanogaster. Genetics 29_: 83-104. Stent, G.S. (1964). The Operon: on its third anniversary. Modulation of transfer RNA species can provide a workable model of an operator-less operon. Science (Washington) 144: 816-820. Stem, C. (1926). An effect of temperature and age on crossing over in the fir s t chromosome of Drosophila melanogaster. Proc. Nat. Acad. Sci. U.S.A. 12; 530-532. Stern, C. (1936). Somatic crossing over and segregation in DrOsOphila  melanogaster. Genetics 21_: 625-730. Sturtevant, A.H. (1915). The behaviour of the chromosomes as studies through linkage. Z. indukt. Abstamm. -u. Vererb. 1_3: 234-287. Sturtevant, A.H. (1920). The Vermilion Gene and Gynandromorphism. Proc. Soc. Exp. Biol. Med. 27; 70-71. Sturtevant, A.H. (1929). The genetics of Drosophila simulans. Carnegie Inst. Wash. Publ. 399: 1-62. Sturtevant, A.H. (1945). A gene in Drosophila melanogaster that trans-forms females into males. Genetics 30_: 297-299. Sturtevant, A.H. (1956). A highly specific complementary lethal system in Drosophila melanogaster. Genetics 41: 118-123. Subak-Sharpe, H., Shepherd, W. M., and Hay, J . (1966). Studies on sRNA Coded for by Herpes Virus. Cold Spring Harb. Symp. Quant. Biol. 31: 583-594. Sueoka, N., and Kano-Sueoka, R. (1964). A specific modification of leucyl-sRNA of Escherichia coli after phage T2 infection. Proc. Nat. Acad. Sci. U.S.A. 52_: 1535-1540. Sueoka, N., and Kano-Sueoka, R. (1970). Transfer RNA and cell differen-tiation. Progr. Nucl. Acid Res. 10_: 23-55. Sueoka, N., Kano-Sueoka, R., and Gartland, W.J. (1966). Modification of sRNA and regulation of protein synthesis. Cold Spring Harb. Symp. Quant. Biol. 31: 571-580. 272 Suzuki, D.T. (1963). Interchromosomal affects on crossing over in Drosophila melanogaster. II. A re-examination of X chromosome inversion effects. Genetics 48_: 1605-1617. Suzuki, D.T. (1970). Temperature-sensitive mutations in Drosophila  melanogaster. Science (Washington) 170: 695-706. Suzuki, D.T. Temperature-sensitive mutations in Drosophila melanogaster Submitted to Scientific American. Suzuki, D.T., Piternick, L., Hayashi, S., Tarasoff, M., Baillie, D., and Erasmus, U. (1967). Temperature-sensitive mutations in Drosophila melanogaster. I. Relative frequencies among gamma-ray and chemically induced sex-linked recessive lethals and semi-lethals. Proc. Nat. Acad. Sci. U.S.A. 57_: 907-912. Suzuki, D.T., and Procunier, D. (1969). Temperature-sensitive mutations in Drosophila melanogaster. III. Dominant lethals and semilethals on chromosome 2. Proc. Nat. Acad. Sci. U.S.A. 6_2_: 369-376. Tarasoff, M., and Suzuki, D.T. (1970). Temperature-sensitive mutations in Drosophila melanogaster. VI. Temperature effects in develop-ment of sex-linked recessive lethals. Develop. Biol. 23_: 492-509. Tartof, K.D. (1969). Interacting gene systems. I. The Regulation of Tryptophan Pyrrolase by the vermilion suppressor of vermilion sys-tem in Drosophila melanogaster. Genetics 6_2: 781=790. Tartof, K.D., and Perry, R.P. (1970). The 5S genes of Drosophila melano- gaster. J . Moi. Biol. 51_: 171-183. Tasaka, E.S., and Suzuki, D.T. Temperature-sensitive mutations in Drosophila melanogaster. XVIII. Heat- and Cold-Sensitive Lethals on Chromosome III. Submitted to Genetics. V. Taylor, M.W., Buck, CA., Granger, G.A., and Holland, J.J. (1968). Chromatographic Alterations in Transfer RNA's accompanying Specia-tion, Differentiation, and Tumor Formation. J . Moi. Biol. 33: 809-828. Taylor, M.W., Granger, G.A., Buck, CA., and Holland, J.J. (1967). Simi-larities and Differences Among Specific tRNA's in Mammalian Tis-sues. Proc. Nat. Acad. Sci. U.S.A. 57: 1712-1719. Thompson, L.H., Mankovitz, R., Baker, R.M., T i l l , J.E., Siminovich, L., and Whitmore, G.F. (1970). Isolation of temperature-sensitive mutants of L-cells, Proc. Nat. Acad. Sci. U.S.A. 66: 377-384. 273 Twardzic, D.R., Grell, E.H., and Jacobson, K.B. (1971). Mechanism of suppression in DrOsOphila: A change in tyrosine transfer RNA. J. Moi. Biol. 57_: 231-245. Vogt, M. (1942). Induction von Metamorphoseprozessen durch implantierte Ringdrusen bei Drosophila. Wilhelm Roux' Arch. Entwicklungsmech. Organismen, 141: 131-182. Void, B.S. (1970). Comparison of lysyl-transfer ribonucleic acid species from vegetative cells and spores of Bacillus subtilis by methylated albumin-kieselguhr and reversed-phase chromatography. J . Bacteriol. 102: 711-715. Void, B.S., and Sypherd, P.S. (1968). Modification in Transfer RNA during the Differentiation of wheat seedlings. Proc. Nat. Acad. Sci. U.S.A. 59_: 453-458. Von Borstel, R.C, and Rekemeyer, M.L. (1959). Radiation-induced and genetically-contrived dominant lethality in HabrQbracon and Drosophila. Genetics 44_: 1053-1074. Wallace, B. (1951). Dominant lethals and sex-linked lethals induced by nitrogen mustard. Genetics 36_: 364-373. Ward, L. (1923). The genetics of curly wing in Drosophila, another case of balanced lethal factors. Genetics 8_: 276-300. Waters, L.C, and Novelli, CD. (1967a). A new change in leucyl-transfer RNA observed in Escherichia coli infected with bacteriophage T2. Proc. Nat. Acad, Sci. U.S.A. 57: 979-985. Waters, L.C, and Novelli, CD. (1967b). The early change in E_. coli leucine tRNA after infection with bacteriophage T2. Biochem. Bionhvs. Res. Commun. 32: 971-976. Watson, J.D. (1965). "The Molecular Biology of the Gene". New York, Amsterdam. Weiss, B., and Richardson, C.C (1967). Enzymatic breakage and joining of deoxyribonucleic acid: I. Repair of single strand breaks in DNA by an enzyme system from Escherichia coli infected with T4 bacteriophage. Proc. Nat. Acad. Sci. U.S.A. 5_7: 1020-1028. Weiss, B., Thompson, A., and Richardson, C.C. (1968). Enzymatic breakage and joining of deoxyribonucleic acid: II. Properties of the enzyme-adenylate intermediate in the polynucleotide ligase reaction. J . Biol. Chem. 243: 4556-4563. 274 Welshons, W.J..(1955). A comparative study of crossing over in at-tached-X chromosomes ih Drosophila melanogaster. Genetics 40: 918-936. Wenrich, D.H. (1916). The spermatogenesis of Phfyriotettix magrius with special reference to synapses and the individuality of the chromosomes. Bull. Mus. Comp. Zool. 60: 57-134. White, B.N., Tener, G.M., Holden, J., Suzuki, D.T. (1973). Activity of a tRNA modifying enzyme during the development of Drosophila and its relationship to the su(s) locus. J. Moi. Biol., in press. White, B.N., Tener, G.M., Holden, J.J., and Suzuki, D.T. (1973). Analysis of tRNAs during the development of Drosophila. Develop. Biol. (in press). Whitehouse, H.L.K. (1967). A cycloid model for the chromosome. J. Cell Sci. 2_: 9-22. Whiting, A.R., and von Borstel, R.C. (1954). Dominant lethal and in-activation effects of nitrogen mustard oh Habrobracon sperm. Genetics 39_: 317-325. Williamson, J.H. (1968). The induction of sterile X chromosomes in Drosophila melanogaster with ethyl methanesulphonate. Genetics 60: 238. Williamson, R., Morrison, M.R., and Paul, J. (1970). DNA-RNA hybridiza-tion of 98 messenger RNA for mouse globin. Biochem. Biophys. Res. Commun. 40: 740-745. Wright, T.R.F. (1970). The genetics of embryogenesis in Drosophila. Adv. Genet. 1_5: 261-395. Yang, S.S., and Comb, D.G. (1968). Distribution of multiple forms of lysyl transfer RNA during early embryogenesis of sea urchin, Lytechinus variegatus. J. Moi. Biol. 31_: 139-142. Yang, W.K., and Novelli, G.D. (1968). Isoaccepting tRNAs in mouse plasma cell tumors that synthesize different myeloma proteins. Biochim. Biophys. Res. Commun. 31: 534-539. Yanofsky, C. (1960). The tryptophan synthetase system. Bacteriol. Rev. 24: 221-245. 275 Yanofsky, C , Helinski, D., and Maling, B. (1961). The effects of muta-tion on the composition and properties of the A protein of Escherichia coli tryptophan synthetase. Cold Spring Harb. Symp. Quant. Biol. 26_: 11. Yarger, R.J., and King, R.C. (1971). The phenogenetics of a temperature sensitive, autosomal dominant, female sterile gene in Drosophila  melanogaster. Develop. Biol. 24_: 166-177. Zuk, J . (1970). Function of Y chromosomes iri Ruriiex thyrsifldrus. Theor. Appl. Genet. 40_: 124-129. 276 APPENDIX 1 A Model for Chromosome Structure and Behaviour The analysis of chromosome structure seeks to des-cribe the spatial relationships of the various molecular components of chromosomes and to relate changes in these configurations to chromosome functions such as replication, transcription, and genetic recombination. At present, we are s t i l l far from this goal. For one, the chromosome is a very large structure from a molecular point of view and many of its components are s t i l l poorly known. In addition, chromosomal functions affect many aspects of cell l i f e , and thus information on chromosomes is scattered over many fields of study. It has, therefore, become practically impossible for one individual to evaluate all the avail-able data. One result is that the field of chromosome analysis has been inundated with naive models based on a limited perspective. With this introduction from Ris and Kubai (1970), i t should be clear why so many models for chromosome structure have been proposed. Several of the models for chromosome organization that have been suggested during the past several years can only be described as "con-fusing contradictions" of one another. It seems remarkable that we can so easily accept such phenomena as the fidelity of chromosome replication The exceedingly large number of references relating to chromosome struc-ture and behaviour precludes the inclusion of a l l pertinent references in this Appendix. Instead, a few aspects of the problem are presented to illustrate the basic features and "use" of the model. What I have tried to do is to evaluate a l l the relevant literature which I have come across during the past five years, in terms of this model. Any inconsistencies have led to modification of the original proposals—resulting in the pre-sent form. There are aspects of the model which are assumed, but, in general, there is strong evidence for the proposed situations in either eukaryotic or prokaryotic systems. 277 and the precision of exchange between homologous chromosomes, having such a limited knowledge of the basic structure of the chromosome. It was for these reasons that I decided to look into the problem of chromosome structure. The approach made was a simple one: taking the basic observations of cytologists, I tried to f i t them into some arrangement which would satisfy also concepts derived from genetic investigations. With a basic model in mind, I sought support from studies involving both eukaryotic and microbial systems. Thus, a wide variety of aspects con-cerning chromosome behaviour have been considered and incorporated into the model. As in Schultz' quotation on Page 5 of this thesis, heterochromatin is viewed as a "differentiated chromosome region", and thus much of the experimental evidence described in this Appendix will be concerned with the activity of heterochromatin. The discussion has been divided into several sections as follows; 1) early studies which provided the groundwork for this and a number of other models for chromosome structure; 2) a description of the basic features of the model; 3) the biochemistry of heterochromatin and its relationship to the model; 4) explanations of several observations on the behaviour of chromo-somes based on various aspects of this model (additional features of i t are presented in connection with some specific observations); 278 5) examples of gene systems having properties which might best be.described in terms of the proposed model are then presented with a brief discussion of the relevance of such systems. 1. The Many Discrepancies Which Led to the Formulation of Many Models Since organismal complexity and diversity, with respect to both biochemistry and structure, are dependent to a certain extent upon the nature and amount of chromosomal DNA, i t is perhaps not surprising that the genomes of higher organisms contain a greater amount of DNA than do those of the lower evolutionary forms. However, two major problems arise when the nucleus must contend with such copious amounts of DNA. The first concerns the regulation of genetic activity, which undoubtedly invokes mechanisms far more elaborate and extensive than those operative in bacteria. The recent studies of Szybalski (1970) indicate that even the relatively simplistic bacteriophage lambda possesses control mechan-isms of a highly intricate and complex nature, making the feasibility of determining the precise control and regulatory systems employed by eukaryotes so much more remote. The second problem involves the organization of the DNA into units which may be easily replicated and distributed to daughter cells. Among the factors operative here appear to be the specific nucleotide sequence of certain chromosome segments and the complexing of the DNA with various protein moieities. The very large size of chromosomes, whether they be mitotic or the specialized polytene chromosomes found in certain tissues of Diptera, 279 reflects an enormous length of DNA complexed with specialized proteins. Three alternative means of achieving this substantial length of DNA have been proposed: the first envisages a single, very long and contin-uous molecule, the second supposes that several shorter molecules (tan-demly arranged?) make up the required length, and the third requires several molecules of identical sequence and has been called the multi-stranded chromosome model. A major objection to the second suggestion is that it would appear that during the cell cycle, the integrity of the chromosome is somehow maintained, and how this might be achieved when several short molecules exist is not immediately obvious. One possible mechanism invokes the presence of single-stranded permuted ends on the various segments. In reality, then, a linear molecule with single-strand nicks on alternate strands of the DNA double helix is constructed. Dur-ing replication and perhaps also at other discrete times during the cell cycle, a ligase-type reaction could restore the continuity of the mole-cule. The recent demonstration of permuted ends for the lambda genome and the single-strand nicks in the T7 genome (see review by Cohen, 1967) make such a supposition plausible. Indeed evidence obtained by Lett et a l . (1970) indicates that chromosomes may be made up of single-stran-ded subunits joined by alkali-labile linkers. Upon centrifugation of mammalian cell lysates on alkaline sucrose gradients, these workers ob-tained single-stranded subunits of uniform molecular weight. The third possibility of chromosome multi-strandedness will be discussed further shortly. 280 Studies on the replication and segregation of chromatids have provided another line of evidence in support of the subunit hypothesis. That chromatids replicate semi-conservatively was demonstrated by Taylor et a l . (1957), one year prior to Meselson and Stahl's classic experiment. Several workers (Hsu, 1962; Howard and Plaut, 1965; and Mulder et al_., 1968) have since shown the existence of many replicating units per chromosome by pulse-labelling with tritiated-thymidine, and the term "replicon" has been applied to them (Plaut et a l . , 1966). Whether these are, in fact, analogous to the replicons of prokaryotes remains to be determined. One extremely controversial aspect of chromosome organization con-cerns the variation in DNA content per genome for even closely related species. Since it is highly unlikely that this may be attributable to large differences in gene numbers, the variations in DNA per genome may reflect differences either as tandem duplications extending lengthwise along the chromosome or as amplifications formed parallel to the long axis of the chromosome. Tandem repeats have been demonstrated both genetically and cytologically in Drosophila (Bar: Bridges, 1936; Dp(l;l)zl: Gans, 1953) and other Diptera (for example, in Chironomus: Keyl, 1965). In these cases the variation is relatively small, never approaching a doubling of the entire genome. There are, however, instances wherein differences among related species may be described as entire genome duplication, quadruplication, or even higher order magnification. Polynemy would best account for the latter situation (see Ris and Kubai, 1970) since tandem duplication of all (or almost all) genes simultaneously 281 and to the' same extent appears a rather difficult evolutionary maneuver. However well one set of evidence supports a particular model, another line equally strongly opposes i t . One obvious difficulty with the polynemic chromosome model arise from recombination studies. The precision of exchange between two DNA double helices is already a re-markable accomplishment, but between four or eight or sixteen or even more duplexes staggers the imagination! The awkwardness of such a model is not, however, sufficient to negate the possibility of the multi-stranded chromosome. Indeed, some experimental results support this mo-del better than the single-stranded models. It would seem that the isolabeling pattern found after replication in the presence of H-thymidine followed by one round of replication in unlabeled medium is better accounted for i f one assumes a polynemic chromosome structure (Peacock, 1963), although folding of a single molecule might also account for this observation. Ris and Kubai (1970) consider the appearance of chromosome breaks two divisions subsequent to irradiation as perhaps the strongest evidence for chromosomal polynemy. And the multi-stranded cytological appearance of chromosomes has led to such hypotheses as the "folded fibre theory" (although i t must be kept in mind that light microscope observations cannot resolve the precise arrangement of the DNA strands). Any model for chromosome structure must take into account, at the very least,the above features. In addition, many aspects of chromosome 282 behaviour should be accountable for by i t . The presentation of the model will therefore be followed by interpretations, according to pre-dictions made by the model, of results from several different experi-mental systems. Before continuing with the discussion of the model, a few words about heterochromatin terminology. As one might anticipate, the large number of varied approaches—genetic, cytological, histochemical, bio-chemical --to the study of chromatin have led to a variety of descriptive terms identifying specific fractions of the genetic material. Whether direct correlations of, for example, regions genetically defined as "heterochromatin" and regions defined cytologically as "heterochromatin" can be made with 100% accuracy at present remains a matter of opinion. An attempt to relate some of these terms is presented here. Heitz, in 1934, distinguished two types of centric heterochromatin: the ofheterochromatin was considered to represent the fused centromere regions of the chromosomes, and the ^-heterochromatin, which was more lightly staining and diffuse , contained the remaining proximal hetero-chromatin. Using Brown's (1966) terminology, these together form the constitutive heterochromatin. It is probable that the few genetic loci located within heterochromatin, actually l i e in p-heterochromatin. The rapidly annealing satellite DNA's have been shown to hybridize primarily to the cC-heterochromatin, whereas rapidly annealing non-satellite DNA hybridizes to /3-heterochromatin, indicating that lower repeat sequences are present in the latter (Rae, 1972). 2B3 2) The Model A diagrammatic representation of the model is given in Figure 35. It must be kept in mind that only the basic features are presented here, additional aspects are discussed in the sections which follow. The model combines the single- and multi-stranded models in an attempt to explain why certain experimental results implicate a single-stranded chromosome while others support the multi-stranded chromosome model. Basically, i t assumes a single-stranded chromosome (and thus crossing over remains a less difficult accomplishment), segments of which may undergo varying amounts of amplification at specific times during the cell cycle or during development. The manner in which this amplifi-cation occurs is important and is represented in Figure 35. The inte-grity of the original double helix is never disturbed, but rather an uncoiling facilitates replication in the specified regions. The newly synthesized copies may initiate new rounds of replication before they themselves are complete, resulting in a cascade type of replication. Not a l l genes undergo such lateral duplication, but for those that do there are specific times during the cell cycle when such genetic activi-ty persists—different genes having possibly different times of ex-pression. It is further supposed that such replication may occur for both euchromatic and heterochromatic lo c i , but that there may be some differences in the end products of the event. For euchromatic loci (although the restriction to euchromatic versus heterochromatic differ-ences is not an absolute one) amplification is transitory and required for the increased synthesis of specific gene products, whereas for FIGURE 35 Model for Chromosome Structure -- and Amplification Amplification of the DNA sequences takes place at certain designated regions: ? a. heterochromatic regions: amplification is of a "stable nature; segments are adjacent to "spacer" sequences (see text); b. euchromatic regions: amplification is of a "transitory nature -- sequences are then used as templates for RNA and f i n a l l y protein synthesis. original helix f i r s t amplification strands second amplification strands A/ 284 285 286 heterochromatic loci there may be no transcription (or a transcription only of limited sequences) and the excess DNA (or its complementary RNA) may serve a regulatory function. The time of amplification is generally restricted to shortly before cell division (and possibly also after i t in some cell types) for heterochromatic loci and to short, discrete intervals during the remainder of the cell cycle for euchromatic loci. Furthermore the amplified DNA of heterochromatic regions may be relative-ly "stable" with the possibility of transmission to daughter cells while that of euchromatic loci is transitory. The significance of these differences is discussed in the following sections. 3) Biochemical Aspects of the Chromosome. The association of heterochromatin with nucleic acid synthesis in the cell was made in the mid-1930's (Muller and Gershenson, 1935; Schultz, 1936) and the possibility was seriously investigated by Schultz and Caspersson in the following two decades (see, for example, Schultz and Caspersson, 1949; Schultz, 1956). It remained until more recent years before this interest was renewed and serious attempts to analyze the biochemistry of chromosomes made. Two important developments of the early 1960's gave way to inten-sive investigations into the biochemical nature of the chromosome. The first of these was the dissociation and re-association of double-stranded DNA upon incubation under appropriate conditions (Marmur et a l . , 1963); and the second was the hybridization of viral RNA with viral DNA (Hall and Spiegelman, 1961; Bautz and Hall, 1962). With these tools, and the technical improvements which followed, some very surprising and 287 intriguing results were obtained despite the expected slow rate of reannealing of dissociated DNA (since individual DNA sequences would be diluted by the large quantity of DNA in metazoan cells), some nucleotide sequences were found to reanneal very rapidly (indicating their frequent repetition in the genome)(Britten and Waring, 1965) . This 'satellite' DNA, when spun to equilibrium in a CsCl density gra-dient, forms a band slightly separate from the main peak (for example, see Kit, 1961). In situ hybridization of mouse satellite DNA to the chromosomes localized i t to the centromeric region of all but one (the Y?) chromosome (Pardue and Gall, 1970). Since then the localization of satellite DNA to the centric heterochromatin has been determined for several organisms (in Microtus by Arrighi et a l . , 1970; in RhynchOsciara by Eckhardt and Gall, 1971; in Drosophila by Gall eJTal., 1971, Hennig et al_., 1970, Jones and Robertson, 1970, and Rae, 1970; in the mouse by Yasmineh and Yunis, 1969, and Yunis e£ al_., 1971; see also review by Yunis and Yasmineh, 1972). DNA from several organisms often show one or more minor bands on either side of the main band when centrifuged to equilibrium in a CsCl density gradient (see reviews by Britten and Kohne, 1968 and Rae, 1972). These minor "satellite" bands represent fractions exhibiting a base composition sufficiently distinct from the bulk of the DNA that they can be separated on the basis of the differential buoyant densities. Gall et^ al_. (1971) have detected a prominent light density satellite (i.e. G + C rich) in Drosophila and Travaglini et a l . (1972a, 1972b, and 1972c) and Kram et^ al_. (1972) have both reported the isolation of heavier satellites from Drosophila DNA. Guinea pig DNA has a heavy 288 shoulder which Flamm et a l . (1969) have separated into four bands on alkaline CsCl. Decapods have been shown to have extremely "heavy" satellites, in which a large portion of the sequence is a d(A-T) co-polyner having about 3% G + C (Smith, 1964). Although the functional significance of the satellite DNA's is unknown, the studies of Travaglini et^ al_. (1972a, 1972b, 1972c) indicate that changes in their proportion relative to the main DNA occur during the early development of the embryo. In addition, the studies of Kram and his co-workers (1972) suggest that "spacer" DNA sequences are associated with the highly reiterated centric heterochromatin. Although i t has generally been accepted that satellite DNA is transcriptionally inert, Georgiev et_ al_. (1972) have provided some evidence for an i n i t i a l transcription of what they call "acceptor sites" (corresponding to the reiterated base sequences) in conjunction with transcription of the structural gene. The 'non-informative1 (i.e. from the reiterated portion) RNA is then degraded, so that i t does not direct-ly contribute to the synthesis of new proteins. Several workers have suggested a role for poly A in determining those species of RNA which may act as functional mRNA's (for example, see Darnell et a l . , 1971), either through active selection (since the sequences appear to be added after completion of their transcription) prior to, or by, the activity of the "polyA polymerase" enzyme, or through a protection mechanism against the action of nucleases (so that unprotected RNA's never reach the cytoplasm) (Philipson et^ al_. :t 1971) . The model presented in Section 2 provides for different nucleotide sequences within the heterochromatic regions; in addition, a means for 289 the selective amplification of certain regions (with respect to time and also in terms of the degree of amplification) is implicated. It is further suggested that the "spacer" sequences of Kram et a l . (1972) represent a heterogeneous group of molecules interspersed among polyAT regions within the heterochromatin, and that these "spacer" sequences have an important regulatory role in selecting specific DNA sequences for transcription. How this might be achieved is outlined in Figure 36. Amplification of specific "spacer" and adjoining polyAT regions is itself regulated—either through the nucleotide sequence of the "spacer" segment itself or through an additional promoter-for-DNA-poly-merase site. Further aspects of chromosome biochemistry and their relationship to the model--with particular emphasis on the present status of the ribosomal cistrons in Drosophila and Amphibia--are discussed in Section 5. 4) The Behaviour of Chromosomes and Chromosome Segments—Possible Explanations Based on the Model To both the geneticist and cytologist, chromosome behaviour has presented a most intriguing problem. Many facets of those functions associated with the heterochromatic portions of chromosomes have been described in Chapter 1 of Part I. Hypothetical schemes for the biochemi-cal and/or genetic bases for some of these and other related phenomena are presented here: The following aspects are discussed: FIGURE 36 Model for the regulation of gene a c t i v i t y a Segment of genome showing spacer DNA and adjacent polyAT-r i c h sequence (both of which amp l i f i y at s p e c i f i c times during the c e l l cycle -- as i l l u s t r a t e d i n b); b a m p l i f i c a t i o n in heterochromatic regions; c enlargement of amplified DNA of b: now the amplified segments are engaged in RNA synthesis; d segment of genome: 'active* RNA sequences nearby ( i l l u s t r a t e s the r e l a t i o n s h i p of RNA with i t s complementary DNA sequences); e "spacer RNA" now associates with complementary "receptor" sequence which i s adjacent to the " s t r u c t u r a l " gene; f mRNA synthesis proceeds; g a second i n i t i a t i o n of mRNA synthesis may begin before the f i r s t i s complete; h mRNA and "spacer" RNA with polyA sequence are joined by a, " l i g a s e - l i k e " reaction; t h i s "complex" can now be transported to the cytoplasm for t r a n s l a t i o n (poly-A-rich sequence serves to ei t h e r protect mRNA from nuclease a c t i o n or f a c i l i t a t e s i t s transport across the nuclear membrane). - DNA double h e l i x w^fHtai^ - "spacer" RNA sequence polyA-rich RNA sequence sp redun s t r rec s t r u c t u r a l gene receptor sequence spacer sequence redundant DNA sequences 290 291 292 a. species differences in DNA content of bands and puffs; b. the case for polynemy; c. intrachromosomal effects or position effect variegation; and d. interchromosomal and intrachromosomal effects on crossing over a. Species differences in DNA content of bands and puffs. A number of instances of asymmetric (heterozygous) bands in the salivary gland chromosomes have been reported for interspecific hybrids of both Chiroriomus (Keyl and Strenzke, 1956) and Drosophila (Patau, 1935), as well as for individuals of Chirbnomus thummi thummi and Ch. th. piger (Keyl, 1965) and Twiririia sp. (Rothfels and Freeman, 1966), among others (see also Rae, 1972). In addition, differences in puffing extent have been reported for both RNA (in Drosophila: Asburner, 1967a, 1967b) and DNA (see Perondini and Dessen, 1969) puffs. A detailed study of the DNA content of several corresponding bands in the salivary chromosomes of the two Chironomus subspecies mentioned above revealed a localization to approximately 30 bands of the extra DNA present in Ch_. th_. thummi (Keyl, 1965). Since the affected bands are distributed throughout the genome, i t would be of interest to deter-mine whether this difference in DNA content (amplification?) is main-tained for some or a l l of these loci when different cell types are compared. If this were the case, it would provide strong evidence for the co-ordinate control of amplification of certain l o c i . Also, since in the hybrid of the two subspecies the respective bands retain their •'thummi" and "piger" DNA content, a • cis-dominant' type of regulation must exist. Assuming that experimental results confirmed the relationship 293 between several of the 30 bands discussed above, then one might invoke a control mechanism for the extent of amplification such as suggested earlier: i.e. control by "spacer" DNA or RNA sequences (either directly or through a "spacer RNA"-protein complex acting on specific sites adjacent to the genes under their control. There are several attractive properties about this control by "spacer" sequences. First, the genomes of higher organisms generally have a substantial amount of excess DNA (i.e. there is much more than is needed on the basis of the number of genetic functions required for the maintenance of the organism) and this would give a function to a large proportion of i t . Second, i t might be expected that during the course of the evolutionary divergence of two subspecies, control systems would be relatively unaltered, at least so far as those systems under the control of enzymes or other proteins are concerned (since, undoubted-ly, changes in these entities would, for the most part, be deleterious). However, mutation of even a few bases in the "spacer" sequence or in its complementary sequence adjacent to the genes under its control, might reduce the affinity for the two nucleotide sequences--but only very slightly, since several hundred nucleotides are involved. On the other hand, accumulated changes in, for example, the "spacer" sequence might reduce or even enhance the affinities sufficiently to cause a noticeable change in the extent of gene amplification. That even closely related species have very different populations of reiterated sequences has been demonstrated for Drosophila (Robertson et_ al_., 1969, compared D. 294 melanogaster and D_. siimilans) and closely related rodents (McLaren and Walker, 1968). Since closely related species would not be expected to differ greatly in the number of functions performed during development, this DNA must have similar functions in the different species despite these variations. To continue the speculation for a moment, one might even evoke an evolutionary significance to this type of regulation by specific DNA sequences. Since, during the evolution of organismal complexity and differentiating systems, one of the most important factors to be consi-dered was a means for co-ordinately controlling the expression of speci-f i c genes at certain times during development, a simple—yet unerring-means for control-must have been established. It would seem to me that given the molecular components of a cell and asked to devise a means of establishing precise control over several genes, I would be at a loss trying to design a suitable protein but could probably come up with several nucleotide sequences which, i f transcribed and translated, would never act as anything more than a mixture of amino acids. Probably their only useful property would be that given the right conditions, dissociated molecules would reanneal or complex with complementary RNA and that's a l l that this system of regulation requires. Perhaps a better system for investigating the co-ordinated regula-tion of amplification would involve a study of the puffing pattern of DNA puffs i n , for example, Sciara. Studies of the degrees of amplifica-tion can be made in several ways: by autoradiography after a short pulse 3 with H-thymidine (Perondini and Dessen, 1969) or by various staining 295 procedures which allow a quantitative estimation of the reacting substance (for example, the Feulgen method used by Rudkin, 1969). Examination of changes in several puffs during the development of a specific tissue, as well as a comparison of the puffing patterns of several tissues at a specific time in development, should reveal whe-ther a system such as the one described above might exist. b. The Case for Polynemy Ris and Kubai (1970) have listed the production of chromosomal bridges one generation after X-irradiation as perhaps the best evidence in favour of a polyrieme chromosome. However, the model presented here also provides a means by which such bridges might be generated. It assumes only that the position of the X-ray-induced damage occurs at a region of amplification. More specifically, the model predicts that such events might occur when damage is incurred in the heterochromatic regions. Here amplification has been described as relatively "stable" and any structural change induced during the irradiation (such as bridge formation between two homologous amplified segments) has a certain pro-bability of persisting until the following generation. The situation visualized at the chromosomal level by Ris and Kubai is that bridges appearing coincident with irradiation are formed by a sister chromatid fusion event. The same type of process may be suggested to occur in the amplification model. In this case, a sister helix (i.e. between homologous amplified segments) fusion may first occur upon irradiation. This would go unnoticed unless there is a subsequent exchange between 296 these sister helices and the parent chromatids (See Figure 37). During meiosis, then, a dicentric bridge is formed. On the other hand, further exchange between sister amplified segments would simply result in the transposition of the fused helices with no consequence to later segrega-tion. The somewhat restricted segregation of amplified segments (by virtue of their relationship to the parent molecule) usually protects against the formation of chromosomal and (possibly to a slightly lesser extent) chromatid dicentric bridges, but the occasional exchange between the parental DNA strands and amplified copies, with subsequent segrega-tion, would result in the formation of such a bridge and an aberrant meiosis. The isolabelling pattern described earlier is hard to envisage for single-stranded chromosomes, and has been used in defense of a multi-stranded organization. However, the seemingly rare occurrence of iso-labeled segments has caused some investigators (for example, Thomas, 1971) to dismiss such patterns as artifacts. The amplification model does, however, provide a simple interpretation for such isolabeling. Again the occasional segregation of sister helices as well as exchange between non-sister helices (see Figure 40) could easily account for Peacock's (1963) observations. One last point concerning species variation in DNA content might be discussed in relation to the question of polynemy. As mentioned earlier there are several instances of closely related species or sub-species having widely differing DNA contents per genome, often reflecting FIGURE 3 7 Model for the generation of chromosome bridges one generation post irradiation a. Breakage occurs in one strand of .each of two amplified seg-ments -- with subsequent 'joining' of strands with 'opposite' polarity; b. consequence of a; c. replication of amplified segments (as for Figure 3 5 ) -- a 'hairpin' loop is formed; d. i f c is followed by an exchange between the amplified seg-ment and sister chromatids (or chromosomes), as outlined in Figure 3 9 , then e. there is a generation of fragments (left portions of chromatids or chromosomes) and a dicentric bridge (right portion of chromatids or chromosomes). 297 298 299 a duplication or higher order magnificiation of DNA content in one of the species. In the absence of an incremental increase in chromosome numbers such observations suggest strongly that the higher order DNA content results from a polyneme situation. If this were indeed the case, then there would be no valid argument against a l l chromosomes being multi-stranded. But perhaps there is an alternative explanation for the apparent polynemy of different species. The problem with the polyneme model arises mainly from genetic studies, but i f the germ line were to have single-stranded chromosomes (at least until after pachytene of meiosis I), then it would not matter to what extent the chromosomes became polynemic . This could, feasibly, begin as early as during the following interphase. Then, for the most part, the organism has poly-nemic chromosomes-except for the relatively few pregonial cells, which have the true diploid DNA content. c. Intrachromosomal Effects or Position Effect Variegation. The phenomena of intra- and interchromosomal effects were described in Chapter 1, Part I and will be discussed in sections c. and d. re-spectively, with suggestions as to their biochemical bases. It will be recalled that position effect variegation reflects a change in the behaviour of a gene in response to its juxtaposition to foreign chromatin (i.e. either a euchromatic gene is placed near hetero-chromatin or a heterochromatic gene becomes associated with euchromatin). Several factors have been found to influence the degree of variegation 300 produced by such a rearrangement of the genetic material—a few of these will be mentioned here. Not only does the amount of heterochromatin in the genome have a profound effect on the expression of the variegated phenotype but a detailed examination of the effects of different Y chro-mosome fragments has established that certain portions of the Y chromo-some are particularly potent modifers (Baker and Spofford, 1959). In addition parental genotypes have been found to influence the variegation of several characteristics (see for example, Spofford, 1959; Hessler, 1961). Whether enhancement of variegation (i.e. an increase in the pro-portion of mutant tissue) or its suppression occurs in the presence of one or another of the modifying factors, appears to be dependent on the locus showing the variegation. For example, variegation of lt_ (normally a heterochromatic gene), caused by its relocation to euchromatin, is en-hanced in the presence of an extra Y chromosome (Schultz, 1936), whereas variegation of a euchromatic gene (such as white) by transposition to heterochromatin is suppressed by the addition of a Y to the genotype (Schultz, 1936; and review by Lewis, 1950). Precisely the opposite ef-fects are produced when the amount of heterochromatin per genome is reduced (i.e. either by heterochromatic deficiencies or by the lack of a Y chromosome in males—see Lewis, 1950). Two general hypotheses as to the mechanism of position effect have been proposed. The first of these, the kinetic hypothesis, relates the phenotypic effects to events interfering with the immediate or early gene products, but assumes no alteration in the gene itself (Sturte-vant, 1925, among others). The second takes on the opposing view, 301 suggesting that the gene is altered-but in a reversible manner (Ephrussi and Sutton, 1944). The model for chromosome organization presented earlier offers an alternative means by which the apparent gene insta-bilities might be produced, and is diagrammed in Figure 38. The hypo-thesis assumes only that in many somatic cell types heterochromatic loci are, in general, actively engaged in gene amplification, and that this amplification is necessary for the genetic functioning of the loci (whether i t be through the synthesis of a specific protein or through the regulation of several other genes via the mechanism outlined earlier). Euchromatic l o c i , on the other hand, do not become extensively amplified, i f at a l l , (any amplification is of the transitory type and reflects only a means of producing additional gene copies for transcription), thus establishing functional as well as behavioural differences for the two types of loci. Furthermore, the regulation for heterochromatization (which is either equivalent to or results in gene amplification) is a feature of specific segments within the centric heterochromatin for Drosophila. Position effect variegation of a euchromatic gene, then, reflects its inactivation through the process of gene amplification suggesting that amplified DNA i s , therefore, unable to act as a trans-criptional template (possibly because it lacks the polymerase attachment site or that site is protected against interaction with this enzyme), and thus no gene product (or an amount insufficient for the production of a wild-type phenotype) is produced. Alternatively the temporal differences in gene activity for heterochromatic and euchromatic loci may result in euchromatic genes being "inactive" at the critical times FIGURE 38 Model for variegated position effect -- based on the proposed model for chromosome structure. a. Normal state of chromosome activity in the c e l l : heterochromatic loci amplify (to greater or lesser extents; at specified times during the cell cycle) while euchromatic loci are transcribed and translated. b. An inversion causes the eu-2 locus to become juxtaposed to heterochromatin, resulting in its heterochromatization and consequent inactivation. c. An inversion with a breakpoint within heterochromatin trans-poses a part of that heterochromatic block to another position in the chromosome where it is surrounded by euchromatic loci and thus becomes (so to speak) euchromatized. •v^' - double stranded DNA helix; short segments are amplified DNA /^y^y - messenger RNA <»»;<ftffe - proteins h - heterochromatin eu-1 - euchromatic locus-1 eu-2 - euchromatic locus-2 302 .303 304 or in specific tissues when those genes are juxtaposed to heterochroma-tin. Variegation of a heterochromatic gene might, then, reflect the lack of amplification which is necessary to the functioning of these loc i . One might expect that variegation of loci having the postulated regulatory functions would be lethal—indeed, the Ubx-ts-lethals studied in Chapter 5, Part I, do have a lethal phenotype at 29°C either when homozygous or when heterozygous for the TM2 inversion. If the Ubx-ts's represent temperature-sensitive mutations within a regulatory gene, then variegation of the wild-type allele on the TM2 inversion (which is enhanced by the high temperature) might be expected to give a lethal phenotype, as i t does. The effects of the heterochromatic elements on the enhancement or suppression of the different variegation situations can be accounted for as follows. It is suggested that there is some type of a "sensor" system within each cell which is capable of regulating the amount of heterochromatin per nucleus (the "sensor" system may be dependent on the cell type) as exemplified by the extent of amplification. Evidence in support of such a system for rDNA will be discussed in the following section. Given that such a system is general, then the suppression of variegation of euchromatic loci by the addition of a Y chromosome would be expected i f , in fact, that variegation were due to amplification at the effected locus. Similarly, the presence of the Y chromosome, which presumably carries some of the 'regulators of amplification', in indivi-duals bearing a euchromatized heterochromatic gene would be expected to restore the heterochromatic state of the gene—as, indeed, appears to 305 be the case. The opposite effects of the reduced heterochromatin complement can be explained similarly. The existence of variegation implies that the "sensor" system is not capable of detecting small changes in the nuclear heterochromatin content. d. Interchromosomal and Intrachromosomal Effects on Crossing Over. Another effect of structural heterozygosity--the interchromosomal effect--has been recognized by several workers (see review by Lucchesi and Suzuki, 1968), and has been briefly discussed in an earlier Chapter on heterochromatin. The term 'interchromosomal effect' has been used to describe the effect of one chromosome (usually one, having one or more structural aberrations) on recombination frequencies in heterologous chromosomes. The genetic behaviour of heterochromatin has been shown to differ markedly in meiotic and mitotic cells. In the former crossing over is strongly reduced in the heterochromatic regions while in the latter recombination takes place almost exclusively in this same area. One effect of chromosomes having at least one breakpoint within the centric heterochromatin is to increase crossing over in this otherwise almost refractile region in non-homologous chromosomes during meiosis (see re-view by Lucchesi and Suzuki, 1968). A similar effect has been demonstra-ted for mitotic cells by Ronen (1964). In his studies, the combined presence of the Cy_ and D inversions (on chromosomes 2 and 3, respectively) substantially increased crossing over on the X chromosome, while there was no appreciable increase with either inversion when tested alone. 306 Furthermore his results indicate that this increased exchange rate occurs primarily, i f not exclusively, in the proximal region of the chromosome. The first demonstration of an intrachromosomal effect on recombina-tion—that is to say, an increase in the frequency of crossing over on chromosomes which themselves bear a rearrangement--was made by Dobzhansky and Sturtevant in 1931. Their results indicated that the presence of an inversion on one of the arms of the translocation in translocation heterozygotes greatly increased crossing over in the other arm. Similar studies were carried out by Grell in 1962, in which she showed a very dramatic decrease in recombination frequency in the immediate vicinity of the inversion breakpoints while distal regions exhibit an increased rate. Such effects have, unfortunately, not been studied for somatic exchange so that no correlation can be made in this case. Another important aspect of the 'induced' increase in recombination is the influence of extra heterochromatin which, as in the case of position effect variegation, can either enhance or suppress the effects brought about by the presence of an inversion. Schultz and Redfield (1951) and later Suzuki (1968) demonstrated that the addition of a large segment of X heterochromatin to the genome substantially increases ex-change on chromosome 3. The influence of the Y chromosome on intra-chromosomal exchange has been reported by Grell (1962), and indicates that the. presence of the Y reduces the proximal increase observed when a distal inversion is present on the X-chromosome, whereas the Y enhances 307 the increased distal frequency obtained with a proximal inversion. In another line of study, Walen (1964) has shown that the amount of heterochromatin in the X chromosome is important in determining the extent of somatic recombination on that chromosome, whether i t be a rod-X or ring-X chromosome. Duplicated heterochromatic segments have a profound effect in increasing proximal exchange frequencies. Her studies also revealed an effect of the shape of the chromosome (the ring chromosome itself giving an increase in somatic crossing over). In his studies on the effects of eight different X chromosome inversions, Suzuki (1963) found increases in third chromosome meiotic exchange for all eight. Six of these also showed increases when tested in the homo-zygous condition--and, of these, five had relocations of part of the proximal heterochromatin to the tip of the X, a factor he considered to reflect the importance of the distal tips in establishing interchromo-somal relationships. With this brief review of some of the factors influencing somatic and meiotic exchange frequencies, one can recognize the importance of chromosome organization in mediating interrelationships between the various chromosomes. Several hypotheses (see Lucchesi and Suzuki, 1968) have been put forth to account for the interchromosomal effects: the "limited chiasmata" theory of Mather (1936): the "position effect" model of Steinberg and Fraser (1944) : the "non-homologous-induced stress on homologously paired chromosomes which results in frequent breakage of the latter" model of Schultz and Redfield (1951) : the "selective tetrad elimination through non-disjunction of non-homologous chromosomes" model 308, of Cooper et a l . (1955); the "limited recombinase" theory of Yost and Benneyan (1957); the "bouquet" model of Okasala (1958); the "suppres-sion of centromere repulsion" model of Thompson (1964); and the "exten-ded pairing interval" model of Lucchesi and Suzuki (1968). These are all briefly discussed in the review of the latter authors, who have included evidence in support of, as well as objections to, the various theories in their discussion. On the basis of the model presented for chromosome structure some comments can be made as to how the interchromosomal effects might be mediated. The point which is of greatest importance in this respect is the difference in the recombination frequency within heterochromatin for somatic and meiotic cells. We know that heterochromatin forms a large portion of the mitotic chromosome length (Kaufmann, 1934) whereas this region is genetically very short. It may be supposed that the cytolo-gical appearance of heterochromatin in somatic cells reflects a degree (possibly extensive for some loci) of amplification of the DNA-sequences found there, and that in meiotic cells there is a very much reduced state of amplification (or none at all) during pachytene when crossing over occurs. It is suggested that the amplified DNA segments are re-sponsible for the apparent high exchange frequency in the heterochromatin of mitotically dividing cells. The free ends of the amplified DNA would provide one of the ends required for a recombinational event. Since a single cross-over between a short piece of DNA and the chromosome would yield two fragments of the chromosome, a further restriction of only double cross-overs must be imposed for each or any of the amplified 309 DNA segments. This is not as severe a restriction as i t might fir s t appear, for i t has been well-established that recombination between the bacterial genome and a newly introduced chromosome segment (after con-jugation) must always involve a double cross-over (single exchanges would lead to linearization of the bacterial chromosome). In addition there are certain organisms in which crossing over in one arm of a metacentric chromosome is complemented by an exchange in the other arm at approximate-ly the same distance from the centromere as in the first cross over event. Although in our system the first exchange may take place between the chromosome and an amplified segment, there is no guarantee that the se-cond one will restore the chromosome's integrity. Exchange may occur with a different amplified segment, with the chromosome involved in the original exchange, with the sister chromatid, or with one of the chroma-tids from the homologous chromosome. These alternatives are illustrated in Figure 39. Only in the latter case would the event be detectable--as a mitotic exchange. The observed restriction of somatic exchange to the heterochromatic regions can easily be accounted for by this model. As for the interchromosomal effect, the effect of an inversion is sup-posedly this model, to increase the amount of heterochromatin (extent of amplification) in other parts of the genome while simultaneously de-creasing the amount of heterochromatin in the chromosome (or chromosome arm) bearing the inversion. For inversions having one breakpoint within heterochromatin (these show the greatest interchromosomal effect) the discontinuity of the heterochromatic block can be envisaged as being responsible for the decreased amplification of that region, suggesting FIGURE 3 9 Model for Exchange within Heterochromatin 1. Exchange between one s i s t e r chromatid-and an amplified segment -- no apparent genetic exchange; 2. exchange between one s i s t e r chromatid and an amplified segment ; a d d i t i o n a l exchange between two amplified segments --no apparent genetic exchange;. 3 . exchange between one s i s t e r chromatid and an amplified segment; a d d i t i o n a l exchange between amplified segment and other s i s t e r chromatid -- no apparent genetic exchange ; 4. exchange between one s i s t e r chromatid and an amplified segment; a d d i t i o n a l exchange between amplified segment and one chromatid from homologous chromosome -- genetic exchange _i£ apparent. sc—1 . s i s t e r chromatids of d i f f e r e n t chromosomes sc-2 a, a' amplified DNA segments represents double-stranded h e l i x 310 311 312, that there may be some means of co-ordinated regulation of the hetero-chromatic regions. That this does not extend from one side of the centromere to the other is evident from the intrachromosomal effects. The effects of the Y chromosome on suppressing or enhancing the "induced" recombination parallel those effects on the variegated position effects described in the previous section. The addition of this heterochromatic element suppresses the stimulated extra amplification in heterologous centromeric regions, thus decreasing the opportunity for exchange. In both types of events, the addition of heterochromatin apparently "corrects" the defect imposed by the chromosomal aberration. 5. Gene Systems Behaving in Accordance with the Model Two gene systems, the bobbed locus and the rosy cistron, have been extensively studied both genetically and biochemically. Many of the properties characteristic of these two systems are very similar to those which have been described in the previous sections in connection with the proposed model for chromosome structure. Thus a brief descrip-tion of their genetic behaviour, with possible explanations as to the mechanisms involved, is presented here. The rosy cistron is discussed in terms of gene conversion and a possible model for this phenomenon,derived from features of the model for chromosome structure, is also given. The bobbed locus, on the other hand, is discussed in relation to the ques-tions of gene amplification and genetic instability. a. Gene conversion and rOsy. Lindegren, in 1953, in his studies in Saccharoroyces, observed aber-rant segregation ratios of mutant and wild-type characters in the 313 spores. These abnormal segregations showed 1:3 and 3:1 ratios for mutant to wild-type character (instead of the expected 2:2) and was termed gene conversion for its apparent conversion of one allele to an alternate state during meiosis. Later, in 1955, Mitchell observed similar abnormal segregations iii NeurOspdra, which she attributed to mutant sites within the chromosome rather than to the behaviour of the entire chromosome. Since then, gene conversion, or non-reciprocal recombination, has been detected and studied at great lengths in numer-ous organisms including Drosophila (Fogel and Hurst, 1967; Kitani and Olive, 1969; Lamb, 1969; and Wildenberg, 1970). The studies in Drosophila have been restricted to events occurring at a few loci, the rosy and marobn-like cistrons having been most ex-tensively examined. The work on these latter two l o c i , both of which affect xanthine dehydrogenase, has been carried out by Chovnick and his co-workers (see recent publication, Chovnick et a l . , 1971). Their re-sults may be summarized as follows. Conversion occurs only in mutant heterozygotes and never in individuals homozygous for one mutant allele. It may occur associated with or without recombination for outside mar-kers, and takes place only in female, never in male, Prosophi1a. The frequency of conversion may be suppressed by the presence of heterozy-gous inversions when a break occurs in the vicinity of the locus in study, thus i t is affected in much the same way as is normal reciprocal recombination. Conversion leads to the production of a wild-type allele which is indistinguishable from that present in wild-type populations and, in addition,all wild-type alleles generated in this manner are 314 identical to one another regardless of the genotype of the parent molecules. Conversion is seen only in cases of extremely tight linkage. And finally, there appears to be a polarization of frequency, so that an allele at one end of the gene undergoes conversion more frequently than an allele at the other end. Between the two outermost alleles there is a continuous distribution of frequencies. On the basis of the above information Chovnick et_ al_. (1971) pro-posed a model to account for gene conversion as well as conventional recombination. They suggest that exchange occurs as a result of repair of spontaneous breaks within a limited region on two 'non-sister' DNA double helices" and that such exchange may yield reciprocal conversions occurring with or without exchange for adjoining markers. They report also on one exceptional progeny (of which there are now 5 or 6 examples, A. Chovnick, personal communication) which survived their screening pro-cedure (and therefore must have had ry+ gene product), but which when progeny-tested yielded ry_ progeny. This suggested to the authors the possibility that in such cases a hybrid DNA intermediate, consisting of one strand of the double helix containing the sequence for the rosy mutation-- the other having a 'conversion' sequence, exists. If this hybrid DNA is not corrected, then segregation at the first mitotic division after fertilization would yield a mosaic individual whose gonadal tissue may be primarily ry*, ry, or ry+/ry (i.e. hybrid), and whose progeny would reflect these genotypes. An alternative explanation for conversion may be suggested, taking into account the model presented earlier for chromosome structure. In 315 this case i t must be assumed that the cistron undergoing conversion (in this case rosy) is slightly amplified during the time when con-version occurs, presumably at the beginning of Meiosis I. A conver-sion event, then, is a recombinational event (which may occur through a process such as suggested by Chovnick et_ al_., 1971) between one DNA double helix and either an amplified segment of the non-sister double helix or the non-sister strand double helix itself. The models (i.e. this and the model presented by Chovnick ejt al_., 1971) differ only by the stipulation of amplified segments in the former--and the ability of these units to enter into the conversion process. This is analogous to the method suggested for somatic exchange, but, unlike the latter, con-version is restricted to females. The model suggested here does account for two observations which are somewhat difficult to reconcile by the model presented by Chovnick and his co-workers. The first of these is the instability for ry+ found in a few individuals. By a segregation of amplified segments with non-sister chromatids i t is possible to obtain individuals which may express the ry+ allele 'for only a limited number of cell divisions (depending on the segregation and 'half-life' of the amplified segments that are not incorporated, by exchange, into the parent DNA duplex). Transcription, followed by translation,of both the parent rosy cistron and the amplified rosy DNA results in the ry* phenotype through complementation of polypeptide subunits which form the functional multimeric enzyme, xanthine dehydrogenase. If the amplified segment later undergoes exchange (meiotic or premeiotic) with the parent molecule, this expression will become stabilized (i.e. there will be a 316 a delayed conversion). Since the evidence for the heteroduplex model is only inferential from results in bacteria and bacteriophage, i t is possible that amplified DNA might also account for conversion instabili-ties in yeast, Neurospora, Sordaria, etc. The second feature of this model which makes i t attractive is that i t can account for the observed polarity in conversion frequencies. To illustrate, one can suppose that at the time of conversion the amplifica-tion is not complete and that the situation resembles that illustrated in Figure 35, #3--at one end of the 'amplifying unit' (in this case a cistron) there are several copies of the amplified DNA, while at the other end there are very few. It follows that the more copies there are of a particular segment, the greater is the probability that that segment will be involved in a particular conversion event. Another feature of the model presents an interesting possibility as to how the limits of the "limited region" (i.e. the 'repair unit* of Chovnick et^al_.) might be delineated. If polarity does reflect different levels of amplification and their subsequent probability for exchange, then the interval from one initiation of amplification until the next might provide this divi-sion into 'units'. b. The 'problem' of the bobbed locus. A reduction in the number of ribosomal DNA cistrons has been shown to be the cause of the bobbed phenotype in Drosophila. This phenotype is characterized by a delayed developmental time, short, fine bristles, and abdominal etching (Ritossa et a l . , 1966). Different bobbed mutants •3 1l have different amounts of rDNA and individuals heterozygous for two bobbed mutations have an rDNA complement equal to the sum provided by each of the mutations (Ritossa and Spiegelman, 1965). Furthermore, i t has been shown that, in general, the more extreme the bobbed phenotype the fewer the copies of rDNA cistrons present in the genome. An interesting property of the bobbed stocks has been described as their "long term instability"--that i s , in the absence of con-tinued selection for the bobbed phenotype there is a progressive modi-fication towards the wild-type. In addition, wild-type stocks have been shown to accumulate weak bobbed alleles (Lindsley and Grell, 1968). Ritossa et_al_. (1966) have suggested that the instability results from unequal crossing over within this highly tandemly duplicated region, a mechanism which provides for stepwise increases or decreases in the num-ber of rDNA copies. In this connection, Schalet (1967) has shown that crossing over within the bobbed region yields recombinants which differ in their expression of the bobbed character. His studies were carried 8 out using females which were homozygous for an inversion (In(l)sc ) which removes the bobbed locus from its normal location in the proximal region of the X chromosome to the distal end. Since i t could be argued that this displacement resulted in the instability of the bobbed region, further tests were made to determine whether, in fact, crossing over does occur within bobbed when this locus is in its normal proximal posi-tion. Schalet (1969) tested this possibility using two deficiencies in the bobbed region and obtained progeny differing in their phenotype from the parents, in some cases stronger bobbed and in other cases weaker 318 bobbed, phenotypes had been generated. These were not recombinant for outside markers—indicating that some form of intrachromosomal exchange had occurred. Atwood (1969) examined this problem using a different approach: he selected for newly generated bobbed alleles from a stock bearing the wild-type allele at this locus. It was expected that i f interhomologue crossing over were the means by which bobbed muta-tions are generated, then such mutants would be recombinant for outside markers, according to his scheme. This was not found to be the case. Although i t was first thought that a given bobbed mutation was stable in terms of its rDNA content, several instances of "instability' (possibly related to the low frequency 'instability' discussed above) have been reported. These will be described here briefly, for their characteristics are of great interest in terms of lending support to the model suggested for chromosome organization. bb 1 Atwood (1969) reported studies on apparent reversion of a Y when combined with X chromosomes bearing other bobbed-lethals (bb*) or defi-ciencies for the bobbed locus. When appropriate crosses are made to generate males of the genotype Y^^/bb*, their survival rate is 10 to 20%. Yet when these males are used as parents and the cross is set up to generate male progeny of this same genotype, their survival is 80 to 90% and their phenotype is now medium bobbed (in contrast to the strong bobbed phenotype of the previous generation). A repeat of this cross gives a survival of 100% for these males in G3 and their phenotype is nearly wild-type. That this 'reversion' does not result from inter-homologue exchange was shown when the same pattern is achieved in the 319.-presence of a complete deficiency for the bobbed region on the X chromosome. A bb+ allele on the X chromosome inhibits these events, and the Y^* appears stable. Another example of such a 'reversion* type phenomenon was first observed by Ritossa (1968). In this and subsequent studies (Ritossa and Scala, 1969), Ritossa has shown that X chromosomes bearing deficien-cies for the bobbed region, when carried in males having a Y chromosome with a nonfunctional bobbed locus, become magnified in terms of their rDNA content. Again, after three generations the bobbed phenotype is almost wild-type, but this reversion occurs in steps. If Gl or G2 bb"** males are mated to females carrying a Y , then the male progeny, when tested for the magnified bobbed phenotype which they had apparently acquired, are again bobbed. That i s , most of these males which had shown an increase in rDNA cistrons (as suggested by their less mutant phenotype and subsequently confirmed biochemically), after one or two generations in the presence of a Y^ , reverted to their original type phenotype when a bb+ allele was present for even one generation. bb After three generations in the presence of a Y " the accumulated rDNA cistrons apparently stabilize (Henderson and Ritossa, 1970). This sequence of events is restricted to the male. A final example of such 'magnification' of the rDNA cistrons is evident from studies by Tartof (1971).. He noted that single doses of the bobbed locus (either in males or in females) tend to increase the number of rDNA genes in a compensatory manner. Again phenotypic changes 320 with the generation were paralleled by increases in ribosomal DNA sequences. Tartof suggested that some system capable of sensing a deficiency in the number of rDNA sequences existed and that this system permitted a disproportionate replication of the rDNA when deficiences were apparent. Perhaps such a function could be attributed to the abo (abnormal-oocyte) locus (chromosome 2) described by Sandler (1970). Homozygotes for abo apparently decrease the viability of heterozygous bobbed individuals. I would like to suggest that the means by which the magnification occurs in each of the above systems is through the intervention of am-plified rDNA's. For the 'magnifiable' bobbed mutations, some or several (depending on the severity of the bobbed mutations) of the amplified rDNA segments remain in association with the chromatids during cell division—although they may no longer be contiguous with the parent DNA double helix through the replication system described earlier. It is further supposed that there is some means of 'sensing' the deficiency in the number of rDNA cistrons (as compared to the wild type number de-termined by the bb+ allele), and that for these mutations the "extra-chromosomal" copies are retained. The greater susceptibility of some bobbed mutations to this magnification might reflect, for example, the reduced activity (or absence) of an enzyme required for the degradation of the amplified segments. Alternatively the 'receptor' site for the enzyme on the segments might be absent or modified,thus preventing their degradation. Whatever the reason for their retention, i t most probably reflects a similar mechanism for all of the "magnifiable" mutations. 32V For the first two generations amplified rDNA cistrons remain essentially "loosely" associated with parent molecule. After this there is an increasing probability that some of the amplified segments become in-corporated into the chromosome (either through somatic recombination or through a "conversion" type of event), thus accounting for the sta-bi l i t y of the phenotype after three generations. The reversion of the magnified bobbed phenotype to the mutant phenotype in the male progeny from crosses of Gl or G2 males (Ritossa, 1968; Ritossa and Scala, 1969; and Ritossa and Henderson, 1970) to females having a bb+ Y chromosome can be explained on the basis of the "sensing* mechanism having detected sufficient rDNA copies in the genome (thus amplified segments were not retained). I would like to further suggest a possible relationship be-tween the abo* gene, mentioned in the previous paragraph, and bobbed. It might be supposed that abo* is responsible for "sensing" and conse-quently regulating the number of rDNA copies (either directly or indirect-ly through a control on the ribosomal DNA synthesis or ?) and that the mutant allele, abo, is no longer functional in this respect. If abo* regulates the extent of amplification or the retention of the amplified segments, then perhaps homozygous abo females bearing a magnifiable bobbed mutation might no longer show this magnification. If the magni-fication regulation is different for the different systems mentioned above, then one might expect a positive relationship in some cases and a negative relationship in others. If the effect of abo* is on several or a l l amplifying systems, then perhaps the conversion model suggested earlier could also be tested. In this case, individuals must be made 322 homozygous for abo and heterozygous for two rosy alleles. If conversion no longer occurs-, then this would lend support to the interpretations given for these two mutations. Before summarizing this section, I would like to comment briefly on the multiplicity of genes for a different set of molecules, the transfer RNAs. It has been calculated for Drosophila (Ritossa et a l . , 1966; Tartof and Perry, 1970) that 0.015% of the genome, or about 750 cistrons represent tRNA genes, providing for an average of 8 copies of the gene for each peak observed in the studies of Chapter 2, Part II. If this is correct, then there must be some mechanism whereby only a single gene is transcribed for each isoacceptor tRNA, or a master-gene : slave-gene relationship (Callan, 1967; Whitehouse, 1967) assures that only one sequence is retained since i t is unlikely that eight copies of a gene, permitted independent mutation and evolution, would give rise to a single tRNA peak. In this context, the 'amplification' model is attractive, for i t provides for extra DNA copies of the tRNA genes which have the same sequence as the parent tRNA gene and so, when trans-cribed, should give rise to a single peak (as observed in our studies). In addition, it provides a means whereby a certain isoaccepting tRNA might be produced in exceptionally large amounts in specific cell types, according to the demands of the cell and the types of proteins being synthesized. Most studies on amplification of ribosomal DNA have utilized the amphibian oocyte as the experimental organism , and amplification 323 of these cistrons is now a well-established phenomenon in both Xenopus (see review by Macgregor, 1972) and Acheta (Lima-de-Faria et a l . , 1969). It does not seem unreasonable, then, to suppose that similar amplification occurs also in Drosophila. Several hypotheses as to the mechanism of gene amplification have been proposed in recent years. Tocchini-Valentini and Crippa (1970; Crippa and ToccMni-Valentini, 1971) and Ficq and Brachet (1971) suggest that amplification occurs through a ribosomal RNA 'intermediate', on which the reverse transcriptase apparently acts in order for the amplified rDNA to be synthesized. Opposing this view, Brown and Blackler (1972) feel that amplification proceeds through a chromosome copy mechanism and Wallace et a l . (1971) suggest that the ribosomal DNA genes (i.e. non-chromosomal) are restricted to the germ line. Whatever the mechanism, it is possible that a similar means is followed for amplification of other cistrons, such as the tRNA cistrons mentioned above. To summarize, then, a model for chromosome structure has been pre-sented which is designed to account for the behaviour of different segments of the chromosome under varying conditions. Variegated posi-tion effects and the interchromosomal effect on crossing over can be described in terms of specific features of the model. These features may be summarized as follows: 324 1) the chromosome is basically composed of a single DNA double helix (with associated proteins, et cetera); 2) specific regions of the chromosome may, at specified times during the cell cycle or during the l i f e of the organism, undergo an amplifica-tion process; 3) this amplification process is not restricted to heterochromatic lo c i , but the behaviour of heterochromatic and euchromatic loci in this respect may be very different. It is suggested that amplification of euchroma-tic loci is of a transitory nature, i.e. not long-lived, whereas that of heterochromatic loci may, under appropriate genetic or environmental conditions, persist through a few or several cell generations, being renewed when necessary; 4) the biochemical properties of heterochromatin have suggested a pos-sible means for regulating other loci through "spacer" sequences, which are highly specific in their nucleotide sequence and their reaction with complementary sequences (through an intermediate RNA) present in the genome. These "spacer" sequences are adjacent to poly T regions which when transcribed form a "spacer-RNA-polyA" complex which is im-portant in regulating the transcription of specific loci. This sequence oovalently bonds with the messenger RNA after the synthesis of the mRNA and facilitates transfer of the latter to the cytoplasm (possibly by protecting it from nuclease attack); 325 6) amplified DNA has been suggested to play a role in gene conversion, and magnification at the bobbed locus. It must be remembered that this phenomenon need not be restricted to the bobbed locus, and may, in fact, occur at several loci; 7) other specific details too numerous to elaborate here are mentioned throughout the discussion. 326 L I T E R A T U R E C I T E D Arrighi, F.E., H'su, T.C., Saunders, P., and Saunders, G.F. (1970). Localization of repetitive DNA in chromosomes of Microtus  agrestis by means of in situ hybridization. Chromosomal 32: 224-236. Ashburner, M. (1967a). Patterns of puffing activity in the salivary gland chromosomes of Drosophila. I. Autosomal puffing patterns in a laboratory stock of Drosophila melanogaster. Chromosoma 21_: 398-428. Ashburner, M. (1967b). Gene activity dependent on chromosome synapsis in the polytene chromosomes of Drosophila melanogaster. Nature (London) 214: 1159-1160.. Atwood, K.C. (1969). Some aspects of the bobbed problem in Drosophila. Genetics 61_ (Suppl.): 319-328. Baker, W.K., and Spofford, J.B. (1960). Heterochromatic Control of Position Effect Variegation in Drosophila. Biol. Contributions, The University of Texas No. 5914: 135-154. Bautz, E.K.F., and Hall, B.D. (1962). The Isolation of T4-specific RNA on a DNA-Cellulose Column. Proc. Nat. Acad. Sci. U.S.A. 48: 400-408. Bridges, CB. (1936). The Bar "Gene" - A Duplication. Science (Wash-ington) 83_: 210-211. Britten, R.J., and Kohne, D.E. (1968). Repeated Sequences in DNA. Science (Washington) 161: 529-540. Britten, R.J., and Waring, M. (1965). Carnegie Inst. Wash. Year Book 1964: 316. Brown, D.D., and Blackler, A.W. (1972). Gene amplification proceeds by a chromosome copy mechanism. J . Moi. Biol. 63: 75-84. Chovnick, A., Ballantyne, G.H., and Holm, D.C (1971). Studies on gene conversion and its relationship to linked exchange in Drosophila melanogaster. Genetics 69_: 179-209. Cohen, G.N. (1967). "Biosynthesis of Small Molecules" (Harper and Row) New York. * only references not appearing in the main Bibliography are cited here. '327 Crippa, M., and Tocchini-Valentini, G.F. (1971). Synthesis of Amplified DNA That Codes for Ribosomal DNA. Proc. Natl. Acad. Sci. U.S.A. 68: 2769-2773. Darnell, J.E., Philipson, L., Wall, R., and Adesnik, M. (1971). Poly-adenylic Acid Sequences: Role in Conversion of Nuclear RNA into Messenger RNA. Science (Washington) 174: 507-510. Dobzhansky, Th., and Sturtevant, A.H. (1931). Translocations between the second and third chromosomes of Drosophila and their bearing on Oenothera problems. Carnegie Inst. Wash. Publ. 421: 29-59. Ephrussi, B., and Sutton, E. (1944). A reconsideration of the mechanism of position effect. Proc. Nat. Acad. Sci. U.S.A. 30: 183-197. Ficq, A., and Brachet, J. (1971). RNA-Dependent DNA Polymerase; Possible Role in the Amplification of Ribosomal DNA in Xeridpus oocytes. Proc. Nat. Acad. Sci. U.S.A. 68: 2774-2776. Fogel, S., and Hurst, D.D. (1967). Meiotic Gene Conversion in Yeast Tetrads and the Theory of Recombination. Genetics 57_: 455-481. Gall, J.G. (1969). The Genes for Ribosomal RNA during Oogenesis. Genetics (Suppl.) 61_: 121-132. Gall, J.G., Cohen, E.W., and Polan, M.L. (1971). Repetitive DNA se-quences in Drosophila. Chromosoma 33: 319-344. Gans, M. (1953). Etude genetique et physiologique du mutant de Drosophila melanogaster. Bull. Biol. France et Belgique, suppl. 38: 1-90. Georgiev, G.P., Ryskov, A.P., Coutelle, C , and Mantieva, V.L. (1972). Properties of Newly Formed dRNA and the Structure of Transcription-al Unit in Eukaryotic Cells. In "Cell Differentiation", pp. 233-237, Copenhagen. Hall, B.D., and Spiegelman, S. (1961). Sequence complementarity of T2-DNA and T2-specific RNA. Proc. Nat. Acad. Sci. U.S.A. £7: 137-146. Henderson, A., and Ritossa, F. (1970). On the Inheritance of rDNA of Magnified bobbed Loci in D_. melanogaster. Genetics 66_: 463-473. Hennig, W., Hennig, I., and Stein, H. (1970). Repeated sequences in the DNA of Drosophila and their localization in giant chromosomes. Chromosoma 32: 31-63. Hessler, A.Y. (1961). A Study of Parental Modification of Variegated Position Effects. Genetics 46: 463-484. 328 Howard, E.F., and Plaut, W. (1965). Ordered chromosomal DNA synthesis in Drosophila melanogaster. J . Cell. Biol. 35_: 59A. H'su, T.C. (1962). Differential Rate in RNA Synthesis Between Euchro-matin and Heterochromatin. Exp. Cell Res. 27_: 332-334. Keyl, H.G. (1965). A demonstrable local and geometric increase in the chromosomal DNA of ChirOnOmus. Experientia 21_: 1-7. Keyl, H.G., and Strenzke, K. (1956). Taxonomie und Cytologie von swei Subspecies der Art Chironomus thummi. Z. Naturforsch. lib: 727-735. Kit, S. (1961). Equilibrium sedimentation in density gradients of DNA-preparations from animal tissues. J. Moi. Biol. 3_: 711-716. Kitani, Y., and Olive, L.S. (1969). Genetics of Sordaria fimicola. VII. Gene Conversion at the G Locus in Interallelic Crosses. Genetics 62_: 23-66. Krara, R., Botchan, M., and Hearst, J.E. (1972). Evidence for Spacer DNA Sequences Associated with the Highly Reiterated DNA in the Centric Heterochromatin of DrOsophila melanogaster Chromosomes, In "Cell Differentiation", pp. 250-255, Copenhagen. Lamb, B.C. (1969). Evidence from Sordaria that recombination and con-version frequencies are partly determined before meiosis, and for a general model of the control of recombination frequencies. Genetics 6_3: 807-820. Lett, J.V., Klucis, E.S., and Sun, C. (1970). On the size of the DNA in the mammalian chromosome: structural subunits. Biophysical J. 40_: 277-292. Lima-de-Faria, A., Birnstiel, M., and Jaworska, H. (1969). Amplifica-tion of Ribosomal Cistrons in the Heterochromatin of Acheta. Genetica 61 (Suppl.): 145-160. Lindegren, C.C. (1953). Concepts of gene structure and gene action derived from tetrad analysis of Saccharomyces. Experientia 9_: 75-80. Macgregor, H.C. (1972). The Nucleolus and its Genes in Amphibian Oogenesis. Biol. Rev. 47_: 177-210. McLaren, A., and Walker, P.M.B. (1968). The comparison of closely re-lated rodents by DNA/DNA annealing. Genetica!Res. 12_: 117-124. 329. MarmuT, J., Rownd, R., and Schildkraut, CL. (1963). Denaturation and Renaturation of Deoxyribonucleic Acid. Progr. Nucl. Acid. Res. 1_: 231-300. Mitchell, M.B. (1955). Aberrant recombination of pyridoxine mutants of Neurospora. Proc. Nat. Acad. Sci. U.S.A. 41_: 215-220. Mulder, M.P., Duijn, P. van and Gloor, H.J. (1968). The replicative organization of DNA in polytene chromosomes of Drosophila hydei. Genetica 39_: 385-428. Pardue, M.L., and Gall, J.G. (1970). Chromosomal Localization of Mouse Satellite DNA. Science (Washington) 168: 1356-1358. Patau, L. (1935). Chromosomenmorphologie bei Drosophila melanogaster und Drosophila simulans und ihre genetische Bedentung. Natur-wissenschaften 23: 537-543. Peacock, W.L. (1963). Chromosome duplication and structure as deter-mined by autoradiography. Proc. Nat. Acad. Sci. U.S.A. 49: 793-801. Perondini, A.L.P., and Dessen, E.M. (1969). Heterozygous puffs in Sciara ocellaris. Genetics 61 (Suppl.): 251-260. Philipson, L., Wall, R., Gluckman, G., and Darnell, J.E. (1971). ; i Addition of Polyadenylate sequences to Virus-Specific RNA during Adeno Virus Replication. Proc. Nat. Acad. Sci. U.S.A. 68_: 2806-2809. Plaut, W., Nash, D., and Fanning, T. (1966). Ordered replication of DNA in polytene chromosomes of Drosophila melanogaster. J. Moi. Biol. 16_: 85-93. Rae, P.M.M. (1972). The distribution of repetitive DNA sequences in Chromosomes. Adv. Cell Moi. Biol. 2\ 109-149. Ritossa, F.M. (1968). Unstable Redundancy of Genes for Ribosomal RNA. Proc. Nat. Acad. Sci. U.S.A. 60: 509-516. Ritossa, F.M., and Scala, G. (1969). Equilibrium Variations in the Redundancy of rDNA in Drosophila melanogaster. Genetics 61 (Suppl.)t305-318. Robertson, F.W., Chipchase, M., and Man, N.T. (1969). The comparison of differences in reiterated sequences by RNA-DNA hybridization. Genetics 63: 369-385. 330 Ronen, A. (1964). Interchromosomal effects on somatic recombination in Drosophila melanogaster. Genetics 50: 649-658. Rothfels, K., and Freeman, M. (1966). The salivary gland chromosomes of three North American species of Twinnia. Can. J. Zool. 44: 937-945. Rudkin, G.T. (1969). Non replicating DNA in Drosophila. Genetics 61 (Suppl.): 227-238. Sandler, L. (1970). The Regulation of Sex Chromosome Heterochromatic Activity by an Autosomal Gene in Drosophila melanogaster. Genetics, 64_: 481-493. Schalet, A.:: (1967). The generation of a variety of alleles associated with exchange within the bobbed locus of Drosophila melanogaster. Genetics 56_: 587 (Abstract). Schalet, A. (1969). Exchanges at the bobbed locus of Drosophila melano-gaster. Genetics 63_: 133-153. Schultz, J., and Caspersson, T. (1949). Nucleic Acids in Drosophila eggs, and Y-chromosome effects. Nature (London) 163: 66-67. Sturtevant, A.H. (1925). The effects of unequal crossing over at the Bar locus in Drosophila. Genetics 10_: 117-147. Suzuki, D.T. (1962). Interchromosomal effects on crossing-over in Drosophila melanogaster. I. Effects of compound and ring X chromosomes on the third chromosome. Genetics 47: 305-319. Szybalski, W. (1970).. Transcriptional controls in bacteriophage lambda. In "RNA-polymerase and Transcription: (L. Silvestri, ed.) pp. 209-217. Amsterdam and London. Spofford, J.B. (1959). Parental Control of Position-Effect Variegation. I. Parental Heterochromatin and Expression of the white locus in Compound-X Drosophila melanogaster. Proc. Nat. Acad. Sci. U.S.A. 45_: 1003-1007. Tartof, K.D. (1971). Increasing the Multiplicity of Ribosomal RNA Genes in Drosophila melanogaster. Science (Washington) 171: 294-296. 331 Taylor, J.H., Woods, P.S., and Hughes, W.L. (1957). The organization and duplication of chromosomes as revealed by autoradiographic studies using tritium-labeled thymidine. Proc. Nat. Acad. S c i . U.S.A. 43: 122-128. Thomas, CA. (1971). The Genetic Organization of Chromosomes. Annu. Rev. Genet. 5_: 237-256. Tocchini-Valentini, G.P., and Crippa, M. (1970). On the Mechanism of Gene Amplification. In "The Biology of Oncogenic Viruses" Lepetit Colloquia on Biology and Medicine, pp. 237-243, Amsterdam and London. T r a v a g l i n i , E.C, Petrovic, J . , and Schultz, J . (1972b). A Character-i z a t i o n of the DNA i n Drosophila melanogaster. Genetics 72: 419-430. — Tr a v a g l i n i , E.C, Petrovic, J . , and Schultz, J . (1972c). S a t e l l i t e DNA's i n the embryos of various species of the genus Drosophila Genetics 72_: 431-439. T r a v a g l i n i , E.C, and Schultz, J . (1972c). C i r c u l a r DNA molecules i n the genus Drosophila. Genetics 72_: 441-450. Wallace, H., Morray, J., and Langridge, W.H.R. (1971). Alternative model for gene amplification. Nature, New B i o l . (London) 230: 201-203. Walen, K.H. (1964). Somatic crossing over i n re l a t i o n s h i p to hetero-chromatin i n Drosophila melanogaster. Genetics 49_: 905-923. Wildenberg, J . (1970). The r e l a t i o n of mi t o t i c recombination to DNA r e p l i c a t i o n i n Yeast pedigrees. Genetics 66_: 291-304. Yasmineh, W.G., and Yunis, J. (1969). S a t e l l i t e DNA i n mouse autosomal heterochromatin. Biochem. Biophys. Red. Commun. 35_: 779-782. Yunis, J . J . , Roladan, L., Yasmineh, W.G., and Lee, J.C (1971). Stain-ing of s a t e l l i t e DNA i n metaphase chromosomes. Nature (London) 231: 532-533. Yunis, J . J . , and Yasmineh, W.G. (1970). S a t e l l i t e DNA i n Constitutive Heterochromatin of the Guinea Pig. Science (Washington) 168: 263-265. Yunis, J . J . , and Yasmineh, W.G. (1972). Model f o r mammalian const i t u -t i v e heterochromatin. Adv. C e l l Moi. B i o l . 2: 1-46. 332 APPENDIX 2 What Constitutes Temperature-Sensitivity? The interaction between organisms and their environ-ment is an old but very important problem for biologists. Organisms respond to environmental change in different ways according to the time during which the environmental change persists and according to the magnitude of the stress. C. Ladd Prosser The study of morphogenetic and biochemical processes has been greatly facilitated with the introduction of conditional lethal mutations. These may be defined as mutations causing death under one set of defined (restrictive) conditions but permitting viability in a different (per-missive) environment. As mentioned earlier, temperature-sensitive lethals represent one such class of conditional mutations in that sur-vival of individuals carrying these mutations is dependent upon the ex-ternal temperature. Such lethals, then, may be perpetuated under per-missive conditions, while their biological defects may be examined during growth at a restrictive temperature. Temperature-sensitive muta-tions have been recovered in a variety of micro-organisms, including bacteriophages (Epstein et_ al_., 1963; Edgar and Lielausis, 1964; Naono and Gros, 1967), bacteria (Leupold and Horowitz, 1952; Maas and Davis, 1952; Nishihara and Ronig, 1964), yeast (Hartwell, 1967), Tetrahymena (Byfield et^ al_., 1969), and Paramecium (Igarashi, 1966). In addition, temperature-sensitivity has been a recognized property of several muta-tions recovered in higher organisms such as Arabidopsis (Langridge, 1965), a Southdown breed of sheep (see Hadorn, 1961), and Drosophila (P.T. Ives, 333 unpublished manuscript; Dobzhansky and Spassky, 1944; Dobzhansky, 1946, and Rizki, 1955). Evidence for the molecular basis of temperature-sensitivity has been provided through extensive studies on micro-organisms. Naono and Gros (1967) suggested that the temperature-sensitivity of a mutant lambda prophage was the consequence of a thermolabile repressor protein, rather than the result of temperature-dependent transcriptional or translational events. From a ts-strain of E_. c o l i , Maas and Davis (1952) were able to isolate a pantothenate-synthesizing enzyme which was highly sensitive to temperature changes (whereas the wild-type enzyme was shown to be stable for the f u l l range of physiological temperatures). In addi-tion, the temperature-sensitivity of some tobacco mosaic virus strains is attributable to the temperature-dependent aggregation property of the mutant virus coat proteins as demonstrated both in_ vivo and in_ vitro (Jockusch, 1966). These particular mutants have been shown to differ from the wild-type by a single amino acid substitution in the coat pro-tein (Wittmann ejt al_. , 1962) . Although systematic screening procedures for the detection of temperature-sensitive mutations in bacteria were successfully employed as early as 1951 (Horowitz and Leupold), application of these methods to higher organisms was not initiated until the late 1960's (in Drosophila: Suzuki et al_., 1967; and yeast: Hartwell, 1967). The studies by Suzuki and his co-workers indicate that a high percentage of ethyl methane-sulfonate (EMS)-induced lethals are temperature-sensitive. 334 Since EMS is known to cause a preponderance of missense mutations in micro-organisms, representing transitions of guanine to adenine (Krieg, 1963), i t is reasonable to suppose that a large fraction of temperature-sensitive mutations in higher organisms also result from single base changes in the DNA. Indeed, as mentioned in a previous section, the genetic properties of both the recessive and dominant ts-lethals in Drosophila support such an interpretation. In the studies presented in Chapters 3, 4 and 5 of Part I, the molecular basis for the TSP's and LP's is of vital important to the interpretation of the biological defects leading to the developmental arrests and phenotypic expressions under restrictive conditions. Again, the studies in micro-organisms have served to elucidate some of the relationships between the transcription of a gene and the activity of that gene's protein product. Studies by Hotta and Stern (1963) and later by Sussman (1966) have shown that in many cases gene product ac-tivity appears shortly after transcription. Spirin (1966), on the other hand, has demonstrated the synthesis of an inactive form of certain pro-teins prior to their utilization, in which case the temperature-sensi-tive period would appear long after activity of the gene has ceased. In contrast to this latter situation, masked messengers have been sug-gested to be translated long after their i n i t i a l formation. These three variations between the time of transcription and the eventual utilization of the gene product, then, provide for distinctly different TSP-LP relationships. 335 A large number of temperature-sensitive mutations, affecting a spectrum of cellular functions, have been recovered in micro-organisms. These include mutations affecting DNA replication (Kohiyami et^ al_., 1966; Yoshikawa, 1970), RNA synthesis (Neidhardt, 1964; Igarashi, et a l . , 1970a, 1970b), ribosome function (Byfield et a l . , 1969; Kang, 1970), and a host of proteins having either structural or enzymatic roles (Epstein et^ al_., 1963; Kohiyami et a l . , 1966). In each of these cases, i t would appear that the absence of a functional macromolecule is the cause of death. One might suppose that an altered amino acid sequence might also render a polypeptide susceptible to degradation by an enzyme which would normally have no effect. This might in itself result in lethality, although the further possibility that a noxious substance is formed as a result of the cleavage could also account for the inviability. Recent studies by Smith et_ al_. (1970) indicate that not only may pro-teins be rendered temperature-sensitive, but so may certain types of RNA. Undoubtedly some or all of the above-mentioned mechanisms are also operative in conferring temperature-sensitivity in higher organisms. There i s , however, a fundamental difference between micro-organisms and higher multicellular organisms: the former have only to coordinate metabolic functions within a single c e l l , whereas the latter must extend this to a large number of diverse cell types. I would now like to con-sider the possibility that temperature-sensitive lethality in higher organisms (and I will limit the discussion more specifically to Drosophila and other insects) might also result from an impairment of 336 the regulatory system for adaptation to different temperatures and/or temperature fluctuations. The observation which stimulated my concern about temperature-controls in Drosophila came from studies made on the temperature-sensi-ts tive paralytic (para ) mutation discovered in our laboratory by Tom Grigliatti and Rodney Williamson in 1969. This mutant, when kept at 22°C, appears wild-type in terms of its activity, yet on exposure to 29°C becomes paralyzed within five seconds. This is reversible by re-turn to 22°C, normal activity being resumed within two seconds. This remarkably rapid rate of first paralysis, then activity, cannot reflect the inactivation with subsequent re-synthesis of a particular enzyme--for the responses are far too rapid for de novo macromolecule synthesis. The conclusions from several studies on this mutation led Suzuki and the above-workers (1970a, 1970b, 1971, 1972) to conclude that parat s affects the central nervous system. ts One of the extremely interesting properties of the para mutation is that adults bearing this mutation, when exposed to 29°C for more than 10 to 15 minutes, begin to kick their legs and after an hour do walk around--that i s , they acclimatize (Suzuki et al_., 1971). On the basis of these observations one might ask what kind of a metabolic disruption could have occurred which, given enough time, corrects it s e l f . Is this really a "delayed" response, indicating that wild-type flies make this adaptation without the intermittent period of paralysis? And is there any need for Drosophila (or other insects) to adapt to temperature 337 differentials of such small increments (a form of paralysis also occurs when parat s individuals grown at 17°C are exposed to 22°C, Grigliatti et a l . , 1972)? My suggestion is that Drosophila (and other poikilotherms) do respond to temperature differences by altering their metabolism to compensate for different rates of reactions as well as for new functions imposed by the new conditions, and that these changes are actively regulated and co-ordinated. If this is true, then one might predict a number of behavioural characteristics indicative of such a system. These might include the following: (1) If temperature changes influence the metabolism of the insect, then the insect must be capable of antici-pating a temperature change, and this, in turn, requires some system whereby the anticipation might occur. (2) If metabolic changes accompany some of the adaptations to temperature, then different products should be formed upon transfer from one temperature to another. (3) If there is some means of detecting temperature fluctuations, then such fluctuations must be important to the development of the organism, and one should be able to find evidence for such a role. (4) A requirement for such a regulatory and co-ordinating system is justified only i f there is not a linear relationship between the activities of several enzymes and the temperature. (5) If temperature adaptation is truly essential to the viability of the organism, then i t might be expected that closely related species, inhabiting different ecological niches, would have different re-sponses to temperature variances. (6) If temperature is an effective modifier of an insect's activity and metabolic state, then seasonally-related differences in these two aspects might be expected. 338 With these thoughts in mind, an investigation of literature relating temperature and development proved most valuable. In particular, I came across a book entitled "Thermobiology" which contained several chapters of immediate concern here. Each of the above points, as well as others,was to some extent discussed in a chapter by K.U. Clarke; some of the examples cited by Clarke are presented in the following para-graphs . 1) Anticipation of Temperature Changes and Temperature Perception. The sensillae are important in conveying information to the CNS about the temperature. Geist has shown that the antennae of the grass-hoppers, Melanoplus femur-rubrum and Dissosteira Carolina, are particu-larly sensitive to temperature while other appendages are less so, while in the cricket Herter showed the importance of the labial palps in sen-sing temperature differences. Clarke further discusses numerous insect species which have apparently adapted different organs for temperature perception. In addition, behavioural differences have been detected for insects exposed to slight temperature variations: Aedes aegypti was found to be sensitive to temperature changes of 0.5 to 1°C, the ant (Formica  ruf a) to 0.25°C fluctuations, and the louse (Pediculus humanis corporis) was sensitive to changes of +4°C. Not only can temperature changes be detected, but this message is conveyed to the CNS where interpretation and subsequent adjustments to behaviour are initiated. Electrophysiological studies on the isolated nerve cord of Periplaneta americana have demonstrated four kinds of nerve cord units differing in their response to temperature. Clarke (1967) 339 has summarized their characteristics as follows: (a) those that show an increase in frequency of impulses with an increase in temperature; (b) those that show transient changes in frequency with changes in temperature and soon resume their original level of activity; (c) those that show activity over a restricted part of the tempera-ture range; and (d) one unit appears relatively unaffected by temperature.... One very interesting example of the co-ordination of behaviour and temperature is illustrated by the ants. In many species the workers apparently control the rate of development of the larvae and pupae by placing the developing individuals in warmer or cooler parts of the nest depending on the needs of the colony (see Clarke, 1967). The ants must be able to anticipate the results in the two cases in order to make this workable scheme. 2) Temperature-dependent Gene Products. Changes in body composition have been noted for a species of ant, Campanotus permsilvahicus penrisilvariicus, which accumulates glycerol in its tissues only when exposed to temperatures of 0 to 5°C for six days. This disappears with slow warming of the insects (see Clarke, 1967). Clarke (1967) suggests that hormones are important in regulating the temperature response at the gene level, either by controlling the rate at which the system can function or by selecting and inducing spe-cif i c genes. 3) Importance of Temperature Fluctuations to the Development of Insects. An interesting experiment with Tribolium castaneum, carried out in a temperature-choice apparatus, revealed that the larvae prefer 29°C 340 during the greater part of this stage. However, shortly before pupation they spend a l i t t l e time at 35°, followed by pupation at 30-31°C (see Clarke, 1967). This preference for a specific temperature sequence may reflect optimal temperatures for specific metabolic events. Temperature fluctuations have also been implicated as important in determining the hatching and eclosion times of the bee, Megachile rotundata. Whereas eclosion times are random when these insects are kept at a con-stant temperature (either 26°, 29°, 32°, or 3 5°), i t occurs 6 hours after a return to high temperature following alternate pulses of 29° and 22°C (see Clarke, 1967). This may very well reflect the situation in the wild, so that eclosion would occur during the afternoon. It is not difficult to imagine the importance of eclosion times--there may be predators seek-ing the young at a particular time of day, and i t would be best to avoid eclosion during that time, or there may be specific food substances re-quired by the young adults which would be difficult to find during the night, and so on. 4) Non-linearity Between Enzyme Activity and Temperature To quote Clarke (1967) : At different temperatures, the state of the insect is not just that of an identical system working faster or slower under one set of conditions than under another. Changes occur in the state constants which need information to effect and time to accomplish. Several instances of non-linear relationships between enzyme activi-ty and temperature have been noted (Clarke, 1967; McWhinnie, 1967). More specifically, i t has been found that in Trypanosoma sp. specific synthetic enzyme systems are destroyed by a pulse to 31° while systems involved in 341 energy release are not affected (see McWhinnie, 1967). 5) Species Differences in Temperature Response. Timofeeff-Ressovsky reported findings that the several morphologi-cally distinguishable races of Drosophila funebris show different tempera-ture tolerances which are apparently related to their own environments. Other studies have indicated that Drosophila populations from Lebanon depend on a lower and wider temperature range for viability than do popu-lations from Uganda which experience a much narrower but higher tempera-ture range (see Clarke, 1967). One further example sited in Clarke is of particular interest, and concerns two species of wasps thriving in a sand dune in Minnesota. The adults from one of these dig deep into the sand providing burrows for the larvae to live. The adults of this species are inactive below 25° and are killed above 42°C, even though they are able to work on the sand which reaches 52°C during hot summer days. This is accomplished through their frequent escapes to the cooler air layer only 1-2 feet above the desert surface. There is another species, however, which is wingless and cannot escape the hot desert surface. This species is not affected by tempera-tures below 52°C. Beyer (1972), in his review on cold-sensitive enzymes, points out that the same enzyme may show different temperature inactivation proper-ties for different organisms, even though the organism themselves adapt similarly to cold temperatures. 342 6) Seasonally-related Changes in Metabolic State, Several insects are capable of existing in one of two metabolically very different states: a normal active state, and an inactive one (diapause). For some insects the change from one state to the other is an integral part of the li f e cycle, while for others it occurs only under certain environmental conditions. Several features distinguish these alternative states: the energy requirements of a dispausing insect are very much less than those of the active insect; the rate of turnover of specific metabolites is" much greater in the active insect; many metabo-lites are required in the active insect which are unnecessary to the diapausing insect. Somero (1972) has reviewed some of the molecular mechanisms of tem-perature compensation in aquatic poikilotherms, outlining the several means by which adaptation to seasonal changes apparently occurs. In some cases alternative isozymes are synthesized during warm and cold seasons, while in other instances i t is apparent that enzyme concentrations change to compensate for differential enzyme activities. An interesting means of adaptation has been demonstrated for pyruvate kinase in the Alaskan king crab (Somero, 1967). Although only one protein appears to be present, this protein undergoes an interconversion from one active form to another as the temperature of the organism changes (i.e. within the physiological range of temperatures). At low temperatures the enzyme is predominantly in a 'cold' variant form, while at higher temperatures the 'warm' variant persists. Somero compared this system with the 'warm' and 'cold' pyruvate kinase variants in the rainbow trout, pointing out that in the trout 343 system (where two distinct proteins have been detected) a change from one variant to the other apparently takes days or weeks, but that a similar change in the crab can occur almost instantaneously in response to temperature changes. The above discussion, then provides us with a l l of the features mentioned earlier that would be expected to occur i f indeed there were some means of regulating metabolic functions in response to temperature changes. Both Clarke (1967) and McWhinnie (1967) and undoubtedly many others would agree with this suggestion. If we accept this suggestion, i t is not difficult to imagine that mutations in any of the anticipatory or response systems would appear as temperature-sensitive mutations when exposure to different temperatures is made. In addition, mutations, which inactivate enzymes which are synthesized specifically in response to a change in temperature, might also confer temperature-sensitive lethality on the organism. This would be the case i f one of the two (or more) alternative enzymes were present in a given temperature range (as is evident for pyruvate kinase in the trout). The obvious differences be-tween this latter means of conferring temperature-sensitive lethality and that in which the enzyme itself is rendered temperature-sensitive (by an amino acid substitution) is that all genetic changes affecting the active portions of the first type of enzyme would give the ts-lethal phenotype, whereas only specific alterations at this site in the second system would produce ts-lethality (many more changes would probably result in strict lethality). In this context i t is interesting to note the cluster of DTS-lethals on chromosome 2 (Suzuki and Procunier, 1969) and 344 the four Ubx-ts-lethals reported in Chapter 5, Part I. The ability of an enzyme (pyruvate kinase in king crab—Somero, 1967) to exist in two kinetically distinct forms provides an interesting model system for the behaviour of certain of the ts-lethals. The first ts is the para mutation described earlier in this section. The rapidity ts o with which para individuals become immobile upon exposure to 29 C and the even quicker recovery on transfer to 22°C may be explained i f a system such as that for the crab pyruvate kinase were operative. If the mutation ts causing the para phenotype affected the interconversion to the 'warm' variant, then exposure to the high temperature might be expected to result in a heat coma. The acclimatization noticed after a period at the restrictive temperature may reflect the functioning of the 'cold' variant, albeit at a reduced efficiency for the system. In view of its role in the production of ATP, the enzyme pyruvate kinase might itself be a likely ts candidate for the defect in para individuals. The induced paralysis, then, may reflect a metabolic state which is characteristic of, say, dia-pause. Clarke explains such a relationship as follows: The changes which occur in an insect in response to a change in temperature indicate that, for any given temperature, the insect is in a definite state with regard to the rates of its metabolic reactions, the concentrations of metabolic products and intermediates, and the amount of genetic information in use. It has been pointed out that this state is that which requires the least consumption of energy to maintain itself; any other state at that temperature would call for a greater expenditure of energy. The achievement of this state, and its maintenance is the poikilotherm equivalent to the work that homeotherms have to do to maintain a constant body temperature. The second example of temperature-sensitive mutations which might be explained on the basis of a protein which is functional at different 345 temperature 'extremes' is the class of 'heat and cold'-sensitive muta-tions. Several of these have been reported for the X-chromosome (Baillie et_ al_., 1967) and the third chromosome (Tasaka and Suzuki, sub-mitted for publication) ih Drosophila, and a l l render mutant flies lethal at both 17° and 29°C. These might be explained as follows: i f the func-tional unit of the mutant protein were a multimer, and the functioning of both the 'warm' and 'cold' variants were dependent in part on an identical sequence, then the complex would be incapable of functioning at either of the temperature extremes i f a l l the molecules were present in either the 'warm' or 'cold' variant forms (i.e. the form present is dependent on the temperature). On the other hand, at an intermediate temperature, viability could be accounted for by a complementation of 'warm' and 'cold' variants both of which exist at 22°C. (It should be pointed out that genetic tests have not conclusively eliminated the pos-sib i l i t y of double mutations causing the heat-sensitivity on the one hand and the cold-sensitivity on the other; however, this is unlikely for a l l of the mutations of this category). One final point of discussion regarding temperature-controlled regulation of gene activity concerns the genetic location of genes which might in one way or another be involved in this system of temperature adaptation. If a number of different proteins are to be synthesized in response to a temperature change, then a regulatory mechanism similar to the one outlined by Britten and Davidson (1969) or that outlined in (3) of this Appendix might be operative. An interesting feature of such a system is that heterochromatization appears to be sensitive to tempera-r ture (Hartman-Goldstein, 1967, among others). Whether different sequences 346 within heterochromatin are differentially susceptible to modifications by different temperatures remains to be seen, but would provide for an attractive model for the temperature-controlled regulation suggested herein. If such were the case one might expect to find a clustering of genes affecting various aspects of this control in such a heterochroma-tic block. The location of 87% of the third chromosome recessive ts-lethals in the region st^ to Sb_ (Tasaka and Suzuki, manuscript submitted for publication) present an intriguing possibility for such mutations. In addition, many of the mutations in the centromeric region of chromo-some 3 are temperature-sensitive. Of these a relatively large proportion belong to the homeotic class of mutations which may be thought of as regulatory mutants. 347 Beyer, R.E. (1972). Effects of Low Temperature on Cold Sensitive Enzymes from Mammalian Tissues. In "Hibernation and Hypothermia, Perspectives and Challenges", (F.E. South, J.P. Hannon, J.R. Willis, E.T. Pengelley, N.R. Alpert, eds.) pp. 17-54: Amsterdam, New York. Byfield, J.E., Lee, Y.C., and Bennett, L.R. (1969). Thermal Instability of Tetrahymena Ribosomes: Effects on Protein Synthesis. Biochem. Biophys. Res. Commun. 37: 806-812. Clarke, K.U. (1967). Insects and Temperature. In "Thermobiology", (A.H. Rose, ed.), pp. 293-352. Academic Press, London. Dobzhansky, Th., and Spassky, B. (1944). Genetics of natural populations, XI. Manifestation of genetic variants in Drosophila pseudoobscura in different environments. Genetics 29: 270-290. Edgar, R.S., and Lielausis, I. (1964). Temperature-sensitive mutations in T4D: Their isolation and genetic characterization. Genetics 49_: 649-662. Epstein, R.H., Bolle, A..-, Steinberg, CM., Kellenberger, E., Boy de la Tour, E., Chevalley, R., Edgar, R.S., Susman, S. , Denhardt, CH., and Lielausis, A. (1963). Physiological studies of conditional lethal mutations of bacteriophage T4D. Cold Spring Harb. Symp. Quant. Biol. 28_: 375-392. Grigliatti, T., Suzuki, D.T., and Williamson, R. (1972). Temperature-sensitive mutations in Drosophila melanogaster. X. Developmental analysis of the paralytic mutation parat^i Develop. Biol. 28: 352-371. Grigliatti, T., Williamson, R., and Suzuki, D.T. (1970b). A temperature-sensitive mutation (para s) causing paralysis in Drosophila melano-gaster. Genetics 64_: 527 (Abstract). Hartwell, L.H. (1967). Macromolecule synthesis in temperature-sensitive mutants of yeast. J . Bacteriol. 93: 1662-1670. Hotta, Y., and Stern, H. (1963). Molecular facets of mitotic regulation. I . Synthesis of thymidine kinase. Proc. Nat. Acad. Sci. U.S.A. 49: 648-654. Igarashi, S. (1966). Temperature sensitive mutation in Paramecium aurelia. I.. Induction and inheritance. Mutat. Res. 3: 13-24. 348 Igarashi, S.J., E l l i o t t , J.F., and Bissonnette, R.P. (1970a). Tempera-ture-sensitive mutation. IV. Induction and characterization of a ts-mutation in RNA-containing bacteriophage R17. Can. J. Micro-biology 16_: 165-172. Igarashi, S.J., E l l i o t t , J.F., and Bissonnette, R.P. (1970b). Tempera-ture-sensitive mutation. V. Infection of ts-mutant of RNA phage R17 and host cell metabolism. Can. J. Biochemistry 48_: 47-53. Ives, P.T. A review of the literature on temperature-effective periods in Drosophila. Unpublished manuscript obtained from Dr. D.T. Suzuki. Jockusch, H. (1966). Relations between temperature sensitivity, amino acid replacements, and quaternary structure of mutant proteins. Biochem. Biophys. Res. Commun. 24_: 577-583. Kang, Soo-Sang. (1970). A Mutant of Escherichia coli with Temperature-Sensitive Streptomycin Protein. Proc. Nat. Acad. Sci. U.S.A. 65; 544-550. Kohiyama, M., Cousin, D., Ryter, A., and Jacob, F. (1966). Mutants thermo-sensibles d'Escherichia coli K12. I. Isolement et characterisation rapide. Ann. Inst. Pasteur 110: 465-486. Langridge, J . (1965). Temperature-sensitive vitamin-requiring mutants of Arabidopsis thaiiana. Aust. J. Biol. Sci. 18: 311-321. Leupold, U., and Horowitz, N.H. (1952). Uber Temperaturmutanten bei Escherichia coli und ihre Bedeutung fur die "Ein Gen-Ein Enzym"-Hypothese. Zeitschrift fur Vererbungslehre 84: 306-319. McWhinnie, M.A. (1967). The Heat Responses of Invertebrates (Exclusive of Insects). In "Thermobiology", (A.H. Rose, ed.), pp. 353-373. Academic Press, London. Maas, W.K., and Davis, B.D. (1952). Production of an altered pantothenate-synthesizing enzyme by a temperature-sensitive mutant of Escherichia  c o l i . Proc. Nat. Acad. Sci. U.S.A. 38_: 785-797. Naono, S., and Gros, F. (1967). On the mechanism of transcription of the lambda genome during the induction of lysogenic bacteria. J. Moi. Biol. 25: 517-536. Neidhardt, F.C. (1964). The regulation of RNA synthesis in bacteria. Progr. Nucl. Acid Res. Moi. Biol. 3_: 145-181. Nishihara, M., and Romig, W.R. (1964). Temperature-sensitive mutants of Bacillus subtilis Bacteriophage SP3. I. Isolation and Characteriza-tion. J . Bacteriol. 88: 1220-1229. 349; Rizki, M.T.M. (1954). Genetic Modification of a Temperature-Sensitive Lethal in Drosophila willistbni. Genetics 40_: 130-136. Somero, G.N. (1972). Molecular Mechanisms of Temperature Compensation in Aquatic Poikilotherms. In "Hibernation and Hypothermia, Per-spectives and Challenges" (F.E. South, J.P. Hannon, J.R. Willis, E.T. Pengelley, N.R. Alpert, eds). pp. 55-80. Amsterdam, New York. Somero, G.N., and DeVries, A.L. (1967). Temperature-sensitive tolerance of some Antartic fishes. Science (Washington) 156: 257-258. Spirin, A.S. (1966). On "masked" forms of messenger RNA in early embryo-genesis and in other differentiating systems. In "Current Topics in the Developmental Biology", Vol. 1_: 2-38. (A.A. Moscona, A. Monroy, eds), Academic Press, New York. Sussman, M. (1966). Some genetic and biochemical aspects of the regula-tory program for slime mould development. In "Current Topics in Developmental Biology," vol. 1_: 61-83. (A.A. Moscona and A. Monroy,edsJ Academic Press, New York. Suzuki, D.T. (1970). Temperature-Sensitive Mutations in Drosophila  melanogaster. Science (Washington) 170: 695-706. Suzuki, D.T., Grigliatti, T., and Williamson, R. (1971). Temperature-sensitive mutations in Drosophila melanogaster. VII. A mutation (parats) causing reversible adult paralysis. Proc. Nat. Acad. Sci. U.S.A. 68: 890-893. Williamson, R., Grigliatti, T., and Suzuki, D.T. (1970a). A temperature-sensitive mutation (parats) causing paralysis in Drosophila  melanogaster. Can. J. Genet. Cytol. 12_: 395 (Abstract). Wittmann, H.G. (1962). Proteinuntersuchungen an Mutanten des Tabakmosaik-virus als Beitrag zum Problem des genetischen Godes. Z. indukt. Abstamm.-u VererbLehre 93: 491-530. Yoshikawa, H. (1970). Temperature-Sensitive Mutants of Bacillus subtilis, I . Multiforked Replication and Sequential Transfer of DNA by a Temperature-Sensitive Mutant. Proc. Nat. Acad. Sci. U.S.A. 65: 206-213. 350 APPENDIX 3 Description of Mutations and Rearrangements Used in the Preceding Studies The following l i s t of mutations and rearrangements (Table 35) is accompanied by a brief description of their important and/or relevant characteristics, as outlined in "Genetic Variations of Drosophila  melanogaster" (Lindsley and Grell, 1968). For complete descriptions, this reference should be consulted. Mutations described in connection with studies by other workers are not included here but, again, these may be found in the above reference. The order of the mutations described reflects their order of appearance in the text. 351 TABLE 35 Description of Mutations and Rearrangements Used in the Studies Mutation or Rearrangement: Symbol: Genetic Location: Description: Chapter 3: Inversion(3LR) Third Multiple 2 Crossover sup-pressor Dichaete Stubble Serrate Xasta Translocation (2;3)apterous Glued Inversion(3LR) Third Multiple 3 multiply marked chromosome 3 roughoid hairy scarlet pink-peach TM2 CxD Sb Ser Xa or T(2;3)ap Xa Gl TM3 III-ple ru h st multiple breakpoints multiple breakpoints 3:58.2 3:92.5 multiple breakpoints 3:41.4 multiple breakpoints 3:0.0 3:26.5 3:44.0 3:48.0 balancer chromosome for chromosome 3; mutant for Ubx150 and reduces crossing over on most of chromosome 3, mu-tant for D_ short, thick bristles on thorax; homozygous lethal wings notched at tip; Ser/ Ser:lethal balancer for right arm of chromosome 3; mutant for Xa-irregularly reduced wing length eyes rough, small, oblong; G1_/G1_: lethal balancer chromosome for chromosome 3; mutant for Sb, Ser, y+ r i pP sep bx34e es marked with ru_ h s_t p_P_ ss_ es eyes small and rough extra hairs on scutellum eyes bright vermilion eyes dull brown-orange, be-comes brown with age 352 Table 35 (cont'd) Mutation or Rearrangement: Symbol Genetic Location: Description: spineless ebony-sooty multiply marked chromosome 3 rough claret marked attached-X 'yellow scute white-apricot echinus marked attached-X facet-notchoid Inversion(X): First Multiple- 6 scute-8 diminutive ss s e sterOca  ro ca / v XX 2 3:58.5 3:70.7 sc a w ec A XX fa1 no FM6 SC dm 3:91.1 3:100.7 X:0 .0 X:0 .0 X:1 .5 X:5 .5 X :3 .0 multiple breakpoints inversion X:4 .6 bristle very short and fine body colour dusty colour marked with st e ro ca eyes rough, slightly smaller than wild-type eye colour dull brown reversed metacentric, marked with 'y£.'sc_ wj* ec yellow body colour, hairs and bristles black; y_ allele reduced number of thoracic bristles eyes yellowish-orange colour, w allele eyes large, bulging; wings short and broad reversed metacentric, marked with v_ w f a n o wing notched; fa allele balancer chromosome for chromosome 1, marked with y 3 1 d sc 8 dm B slight s£ phenotype, asso-ciated with inversion bristle, body small, slender; females sterile S53 Table 35 (cont'd) Mutation or Genetic Rearrangement: Symbol: Position: Description: Bar Minutes lethal-cryptocephal vermilion M l(2)crc X:57.0 eyes reduced to a narrow bar; associated with small tandem duplication all four homozygous lethal; hetero-chromosomes zygotes have short, thin bristles; prolonged develop-mental time 2:55. homozygous pupal lethal -cephalic complex fails to evert: abnormally rigid in-tegument X:33.0 eye colour bright scarlet Chapter 4: Minute on chromo-some 3 Minute on chromo-some 3 M(3)S34  M(3)w124 3:44.3 medium Minute phenotype 3:79.7 strong Minute phenotype Delta-1 Jammed Dichaete Dl Lyra Inversion on chromosome 2 D til In(2L)NS 3:66.2 2:41.0 3:40.7 3:40.5 break-points : 23E2-2; 35F1-2 veins thickened and broadened into deltas at margin junc-tions; homozygous lethal wings often compressed into narrow strips; homozygote semi-viable wings uniformly extended at 45 from body axis; dorsocen-trals reduced; D/D_: lethal lateral margins of wings ex-cised; homozygous lethal relatively good balancer for 2L 3 5 4 Table 35 (cont'd) Mutation or Rearrangement: Symbol: Genetic Position: Description: Inversion on chromosome X plexus net carnation In(l)w or w"H P2L net m4 multiply marked X b-210 forked f car breakpoints: 3C1-2; 20A 2:100.5 2:0.0 X:56.7 X:62.5 variegated for white wings have network of extra veins wing veins form plexus like net marked with y_ sc_ y_ f_ car bristles shortened, gnarled, bent eye colour dull brownish red Chapter 5: Inversion on chromosome 2 Curly Inversion on chromosome 2 bithorax In(2LR)SM5 9L In(2LR)bwV1 bx bx bx 34e multiple breakpoints 2:6.1 multiple breakpoints 3:58.8 most complete balancer for chromosome 2; marked with Cy_ wings curled upward balancer for chromosome 2: marked with ds_; varie-gated for It, bw, mi, and abb anterior half of metathor-ax becomes mesothoracic; halteres appear winglike; rudimentary ant. mesothor-acic elements between scu-tellum and first abdominal segment very strong bjx allele slightly stronger allele than bx 3 S 5 Table 35 (cont'd) Mutation or Rearrangement: Symbol: Genetic Positions: Description bx H2 bithoraxoid bxd Contrabithorax Cbx Contrabithoraxoid Cbxd Ultrabithorax Ubx Ubx 61d Ubx 105 Ubx 101 Ubx P15 Posterior-bi thorax red pbx red allele which arose on Ubx-ts-2 chromosome; expression as bx^ 3:58.8 posterior portion of metathor-ax becomes mesothoracic; hal-teres appear enlarged and disclike; first abdominal seg-ment: ant. is metathoracic-like while post, is mesothoraciclike -may get abdominal legs in ex-treme cases 3:58.8 post, mesothorax transformed into post, metathorax; wings become haltere-like 3:58.8 metathorax becomes similar to the first abdominal segment. 3:58.8 haltere slightly enlarged; homo-zygous lethal-combines the pro-perties of bx, bxd, and pbx not associated with an inver-sion; associated with T(2;3)Ubx105, strong Ubx-phenotype multiple breakpoints multiple breakpoints multiple breakpoints 3:58.8 101 3:53.6 associated with In(3LR)Ubx strong Ubx-phenotype associated with In(3LR)TM6, strong Ubx-phenotype posterior metathorax trans-formed into post, mesothorax adult and larval Malpighian tubules red; eye colour brown 356 Table 35 (cont'd) Mutation or Rearrangement: Symbol: Genetic Position Description Attached-X-chromosome Inversion on chromosome 3 Inversions on chromosome 3 dachsous FMA3 TM3 In(3LR)P ds Regulator of pbx Rg(pbx) multiple breakpoints Inversions also present multiple balancer for chromosome 3; breakpoints carries Me, r i , and sbd multiple breakpoints 2:0.3 3:54+ balancer for chromosome 3 wings shorter, blunter, broader, with crossveins uniformly close together; abdomen and legs are chunky interacts with the wild-type allele of pbx to give a pbx phenotype 357 APPENDIX 4 Localization of* Ubx-ts-1 and Ubx-ts-4 with Respect to the Recessive Mutation red Two of the Ubx-ts-lethals, Ubx-ts-1 and Ubx-ts-4, were mapped with respect to the recessive marker red (map position 53.6). The crosses made to establish the appropriately marked chromosomes are out-lined in Figure AO. A total of 301 (4.2%) out of 7113 and 107 (3.4%) out of 3174 Cross 1 progeny for Ubx-ts-1 and Ubx-ts-4,respectively,were phenotypically D Sb/Gl_ (Table 36). From previous mapping data (Chapter 5, Table 17), i t was expected that 19 (of 57 fertile males) and 10 (of 25 fertile males) D Sb chromosomes would carry their respective Ubx-ts-lethals. Table 37 indicates that instead these figures are 50 (91% for Ubx-ts-1) and 20 (80% for Ubx-ts-4) --thus far more cross overs between D and Sb were recovered bearing the Ubx-ts-lethals than anticipated from earlier mapping data. The map distances D--red (12.9%) and D--Sb (17.5%) are 100% in agreement with the distances in Lindsley and Grell (1968), a l -though the positions for the Ubx-ts-lethals are not consistent. This is likely due to sampling error since the results of progeny testing Cross 3 males again gives map positions for the Ubx-ts-lethals consistent with the Ly—Sb mapping data, establishing the positions of Ubx-ts-1 at 44.7 and Ubx-ts-4 at 44.6. FIGURE 40 Crosses made for the l o c a l i z a t i o n of Ubx-ts-1 and Ubx-ts-4 with respect to the recessive marker red 358 ass-Cross 1 Ubx-ts-X Sb/D^ ? x G1/TM3 a" at 22°C score a l l progeny and collect a l l D Sb/Gl males (Table 36) Cross 2 . D Jb/Gl x Gl Sb eS/TM2?at 22°C for 4 days, then transfer adults to fresh vials at 29°C. score 22 and corresponding 29 C vials: presence of D Sb/TM2 progeny at 22°C but their absence at 29°C is indicative of the presence of the Ubx-ts mutation on the D Sb chromosome. (Table 37). Cross 3 D Ubx-ts Sb/red g x red/red a" at 22°C score a l l progeny and collect a l l cross over males (Table 38). Cross 4 cross over males from Cross 3: D red/red D red /red red+ Sb/red red Sb/red cf x Gl Sb e S/TM2£ at 22°C for 4 days, then trans-fer adults to fresh vials at 29°C. score 22° and corresponding 29°C vials: presence of cross over progeny at 22°C but their absence at 29 C is indicative of the presence of the Ubx-ts mutation on the cross over chromosome (Table 39) 360 TABLE 36 Localization of Ubx-ts-1 and Ubx-ts-4 With Respect to red Cross 1: Ubx-ts-X Sb /D3? x Gl /TM3V Stock used: Sb/Gl D^/Gl -Genotyp D /TM3 6 , * D Sb/Gl +/G1 +/TM3 Total Ubx-ts--1 1) 633 523 468 73 58 79 1834 D) 585 469 370 86 57 81 1648 E) 713 580 512 99 71 114 2089 F) 518 438 389 43 72 62 1522 Sum of Above: 2449 2010 1739 301 258 336 7113 Ubx-ts--4 G) 482 525 432 33 59 53 1589 H) 520 439 426 74 58 68 1585 Sum of Above: 1002 964 863 107 117 121 3174 *Males of this genotype were collected and progeny-tested for the presence of the Ubx-ts mutations (Cross 2, Table 37). 361 TABLE 37 Progeny testing of cross over males from Cross 1 for the presence of the Ubx-ts lethals Stock Tested: ts Temperature sensitivity ts sterile Ubx-ts-1: l j 3 D: 0 E: 2 F: 0  Total: 5 52 %of fertile males bearing Ubx-ts-1: 91% 21 3 21 7 4 3 5 6 18 Ubx-ts-4: G: H: Total: 0 5 8 20 3 3 28 hof fertile males bearing Ubx-ts-4: 803 TABLE 38 Localization of Ubx-ts-1 and Ubx-ts-4 with respect to red Cross 3: D Ubx-ts Sb/red? x red/red o" Genotype Stock used: D Sb/red red/red D/red p_ red/red Sb/red red Sb/red* +/red D red Sb/red tt Except-ions Ubx-ts-1 10-1 1187 1260 74 204 178 76 6 1 2D-2 641 778 34 99 94 38 0 3 4E-3 1033 1071 73 211 218 56 5 1 13F-1 1097 1291 52 154 145 58 3 3 Sum of Above: 3958 4400 233 668 635 228 14 8 Total progeny: 10143 Ubx-ts-4 14G-1 980 1203 60 173 195 68 4 2 5H-2 1389 1396 91 209 201 66 2 2 red/red :1 Sum of above: 2369 2599 161 382 396 134 6 4 1 Total progeny: 6052 *males of these genotypes were collected and progeny-tested for the presence of the Ubx-ts lethals (Cross 4, Table 39). TABLE 39 Progeny tests of cross over males from Cross 3 for the presence of the Ubx-ts mutations Cross 4: Cross over males X Gl Sb eS/TM2 ? (from Table 38) Stock Used D Ubx-ts-1 Sb: Genotype of cross over chromosome D red* Sb* D red St/ D* red* Sb D* red Sb + + + + ts ts sterile ts ts sterile ts ts sterile ts ts sterile 10-1 10 1 1 26 14 3 13 29 3 0 15 0 2D-2 9 0 1 13 2 4 8 19 3 2* 7 1 4E-3 14 1 3 '29 14 8 17 32 6 0 15 2 13F-1 8 0 1 19 11 1 8 5 0 0 9 1 Above 41 2 6 87 41 16 46 85 12 2* 46 4 D Ubx-ts-4 Sb: 14G-1 15 0 4 28 12 5 10 32 3 0 15 0 E-2 20 _0 _3 l i 19_ _7_ 11 34 _ 1 _ 2 19 _1_ Sum of Above 35 0 7 70 31 12 27 66 7 2 34 1 u cn * progeny test gave few of the expected Gl_ Sb to interpret. e /cross over chromosome sibs; thus these results are difficult 364 APPENDIX 5 Method for the Localization of a Recessive Lethal Mutation on the TM2 Chromosome in the Region of the Ubx-ts-lethals One possible explanation for the specificity of the interaction of the Ubx-ts-lethals with the TM2 chromosome is that the latter contains a recessive lethal mutation (either a point mutation or a small deletion) in the region of the Ubx-ts's, which permits the pseudo-dominant expres-sion of the ts's under appropriate conditions. The breakpoint in salivary region 74 in the TM2 chromosome is suggestive of a small deficiency in this part of the chromosome. In order to test this possibility, the cross outlined in Figure is being carried out. The markers used map very close to the Ubx-ts-le-thals. If, in fact, the TM2 chromosome carried a recessive lethal in the region spanned by these phenotypic markers, it is expected that rare cross-overs between the TM2 inversion and the multiply marked chromosome would yield a marked TM2 chromosome (which is then viable with the Ubx-ts-lethals at 29°C) and a th s_t tra cp in r i p_^_ derivative bearing a wild-type allele of, or a deletion for one of these mutations (and which is then lethal with the Ubx-ts-lethals at 29°C) would be recovered. A cross-over transposing a small deletion from the TM2 chromosome but covering one of these mutations would be phenotypically indistinguishable from a non-cross-over; thus, progeny tests of a l l phenotypically non-cross-over males must be made. This procedure is shown in Figure 38b, but has thus far not been carried out. 365 Of 627, IM2 and 526 th st tra cp in r i pj^ progeny examined j no marked TM2 chromosomes have been recovered but possible cp* and i n+ cross-overs have been obtained and retained for further study. Since cp_ is itself temperature-sensitive the apparent cp_+ individuals may well be ones in which the clipped phenotype has not been expressed. This is testable by making a cross of such flies by females bearing the multiply marked chromosome at 25°C at which temperature the mutant phenotype is invaria-bly expressed. cp_ presents an additional problem since often its expres-sivity is so great that a large fraction of the wing, including vein L2 (which is interrupted in ri) is missing; thus these individuals cannot be established as either ri^ or r i+. Furthermore the transformer gene has no phenotypic expression in normal XY males and is therefore not de-tectable in the male offspring. These properties of the mutations reduce the recoverability of cross-overs in the region under study but scoring of a minimum of 10000 progeny should yield a few cross-overs. The fre-quency of crossing-over may be increased through the introduction of in-versions on the X and 2nd chromosomes. FIGURE 41 Outline of crosses for the localization of a recessive lethal mutation on the TM2 chromosome in the region of the Ubx-ts-lethals 366 367 a. th st tra cp in r i pF/TM2 ? x th st tra cp in r i pp/TM3 d* score progeny as follows: i . TM2-bearing (non-TM3): look for presence of one or more of the phenotypes associated with the multiply marked chromosome; i i . homozygous th st tra cp in r i pp: look for "reversion" of one or more of the phenotypes; i i i . th st_ tra cp in r i pp/TM3, r i p^: the presence of ri_ and p_^_ on the TM3 chromosome permit the detection of cross-overs involving these l o c i . b. phenotypically th st tra cp_ in ri_ p^ d* x Ubx-ts-X/TM3 ? 22°C for four days, then transfer to fresh vials at 29°C score progeny as follows: i . i f a deletion is present on the multiply marked chromosome which might have undergone a cross-over with the TM2 chromosome in the previous generation, and i f that deletion covers the Ubx-ts-re-gion, then the ratio of TM2 to non-TM3 offspring at 29°C should be 2:1; i i . in the event that such a lethal is recovered, tests should be made to determine whether i t is lethal with TM2 at 22° and 29°C; i i i . the extent of the deletion should also be established: crosses to additional mutant stocks and a pseudo-dominant expression of phenotypes associated with mutations within the th-pP interval would allow the limitations of the deletion to be determined. 368 APPENDIX 6 Interactions of the Ubx-ts-lethals with Mutations and Deletions in the Vicinity of the ry locus After the first cross of Appendix 4 was carried out, and in view of the ambiguous mapping data for the Ubx-ts-lethals, i t was decided to examine the interaction of the Ubx-ts-lethals with various rosy deficien-cies and other mutations near the rosy locus. The results of the tests with a number of mutations are summarized in Table 40. All crosses were carried out at 29°C, thus lethality may be due to the temperature sensi-tive mutation or to a second recessive lethal site not previously detected on the Ubx-ts-2, 3 or 4 chromosomes. The results indicate that only Ubx-ts-2 shows an interaction with 2 75 2 two of the chromosomes tested: kar ry and kar l(3)26d. That the le-thality of these heterozygotes is due to an interaction with some property 75 associated with the ry and 1(3)26d mutations is substantiated by the via-2 75 bi l i t y of Ubx-ts-2/kar l(3)26c individuals. Since ry is associated with 75 a deficiency, Df(3R)ry , with unknown breakpoints but not extending to 1(3)26, i t is possible that the latter is also a small deletion which 75 partially overlaps the ry deletion. It might then be possible that the lethal interaction of these mutations in combination with Ubx-ts-2 is attributable to the postulated common deficiency. Unfortunately complemen-75 tation tests between ry and 1(3)26d have not been carried out. Although the nature of the interaction is unknown, i t does reveal a further distinguishing feature of the non-complementing Ubx-ts-2 and Ubx-ts-4 mutations. 369 TABLE I 40 Viability of Heterozygotes for a Ubx-ts-lethal or TM2 and various Mutations and Deficiencies in the Vicinity of the rosy locus Ubx-ts-1 Ubx-ts-2 t, 2 75 kar ry MKRS 116 -100 0 61 kar2126d DcxF 94 67 0 21 76 m MRS 72 -52 13 -18 85 m MRS 18 17 26 -28 kar2126c DcxF 84 72 16 26 74 MRS 157 -108 44 -52 81 MRS 95 85 30 44 Ubx-ts-3 Ubx-ts-4 TM2, Ubx" 111 81 78 -100 -67 70 65 44 11 48 42 6 68 47 65 -61 -36 47 32 52 16 -42 '-40 15 64 65 38 57 46 51 93 71 90 -69 -60 88 112 98 69 80 97 80 — approximation: i n c e r t a i n of these crosses, a d i s t i n c t i o n between two of the progeny genotypes was not possi b l e , thus the figures given represent 1/2 of the progeny with the ambiguous phenotype Crosses were made at 29°C 370 APPENDIX 7 The Recovery of DTS-5 "Homozygotes" Once mutant stocks are characterized as "confirmed" DTS-lethals, attempts are made to generate homozygous mutant-bearing stocks at both 17° and 22°C. In the case of DTS-5, three bottles containing DTS-5/TM2 parents were established at 22°C and a l l progeny which emerged were scored. In one of the bottles, nine apparently homozygous progeny (two females and seven males) were recovered from a total of 360 f l i e s . Mat-ing of these individuals indicated their fertility at 22°C, thus enabling a "homozygous" stock to be maintained. Interestingly enough, in a cross of "homozygous" DTS-5 males (or females) to Oregon-R (wild-type) females (or males) at 29°C, approximately one-half of the zygotes develop to the pupal stage and die, whereas the remaining zygotes develop into viable pro-geny. We can see, then, that the recovery of apparent "homozygotes" de-pended on the loss of the DTS-5 characteristic or at least occurred simul-taneously with i t . That the non-DTS-5 bearing derivative chromosome con-tains a recessive lethal mutation has been demonstrated in two ways. Firstly, tests of the "homozygous" stock, repeated at 29°C at various times during the past four years, have always yielded lethality at 29°C or eclosa-bility to a degree comparable to that rarely observed for independent DTS-5/  TM2 lines. Secondly, the results of hatchability tests using "homozygous" parents revealed that only 70% of the eggs from these crosses hatch at 22°C, a value somewhat lower than expected i f the homozygous DTS-5/DTS-5 zygotes are completely viable at this temperature. Since true DTS-5 homozygotes 371 have been shown to die almost exclusively during the larval stage (see Chapter 4), the lethality of approximately one-quarter of the zygotes from "homozygous" parents must be due to homozygosis of a recessive lethal present on the non-DTS-5 bearing chromosome. Since the recovery of "homozygous" progeny occurred only once, i t would suggest that a single mosaic parent gave rise to the nine progeny observed during the early stages of the characterization of the DTS-5 mutation. The gonadal mosaicism of this individual may have arisen in one of three ways: 1) There may have been a pre-meiotic cross-over remov-ing the DTS-5 mutation from the mutant chromosome and replacing i t with a recessive lethal mutation (which may have resulted simply via the cross-over process per se or the mutation may have been present on the TM2 chro-mosome and removed from i t by a cross-over event). 2) A cross-over between the DTS-5 chromosome and the TM2 chromosome may have removed the Ubx muta-tion from TM2, thus giving the appearance of a wild-type chromosome. 3) There may have been a pre-mutation induced simultaneously with the DTS-5 mutation which later stabilized in a few cells in which the "appropriate" environmental conditions had been provided (Auerbach, 1951; Auerbach and Kilbey, 1971). In the latter case i t is difficult to imagine how the sta-bilization of a pre-mutation might occur at the same as the DTS-5 mutation disappears - unless they are somehow related. The fir s t and third alternatives cannot be distinguished experimen-tally, but the following test allows confirmation or negation of the second proposal. If the Ubx mutation has been removed from the TM2 chromo-some, then other recessive mutations characteristic of the inverted 372 chromosome should, i n a l l probability s t i l l be present on i t . Two proper-g ties can be examined i n this respect: the f i r s t i s whether the e_ a l l e l e i s s t i l l present and the second i s whether the interaction with the Ubx-ts-lethals reported i n Chapter 5 remains. 373 APPENDIX 8 Conditions for the Aminocylation of Drosophila tRNAs The conditions for the aminoacylation of Drosophila tRNAs were established during earlier experiments in which the Bd-cellulose sys-tem was used for the separation of the various isoaccepting species. These have been summarize