Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Genetic and developmental studies of proximal segments of chromosome 3 of Drosophila melanogaster Sinclair, Donald A. R. 1977

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1977_A1 S55.pdf [ 12.97MB ]
Metadata
JSON: 831-1.0094310.json
JSON-LD: 831-1.0094310-ld.json
RDF/XML (Pretty): 831-1.0094310-rdf.xml
RDF/JSON: 831-1.0094310-rdf.json
Turtle: 831-1.0094310-turtle.txt
N-Triples: 831-1.0094310-rdf-ntriples.txt
Original Record: 831-1.0094310-source.json
Full Text
831-1.0094310-fulltext.txt
Citation
831-1.0094310.ris

Full Text

C-  \  GENETIC AND DEVELOPMENTAL STUDIES OF PROXIMAL SEGMENTS OF CHROMOSOME 3 OF DROSOPHILA MELANOGASTER by DONALD A.R. SINCLAIR B.Sc.(Hons), University of Manitoba, 1969 M.Sc, University of Manitoba, 1972 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  i n the Department of Zoology We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA March, 1977  ©  Donald A.R. S i n c l a i r , 1977  In presenting this thesis in partial  fulfilment of the requirements for  an advanced degree at the University of B r i t i s h Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives.  It  is understood that copying or publication  of this thesis for financial gain shall not be allowed without my written permission.  Department of The University of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5  Date  ABSTRACT The present work deals with several approaches to the study of regions near the centromere of chromosome 3 of Drosophila melanogaster. The goals of this research were: ( i ) to examine spontaneous crossing over near the centromere i n d e t a i l ; ( i i ) to locate the Deformed locus genetically and  to determine whether this l e s i o n i s a recessive l e t h a l ;  ( i i i ) to test the e f f i c a c y of radiation-induced method of producing proximal aberrations; and  crossing over as a  (iv) to g e n e t i c a l l y and  developmentally characterize a temperature-sensitive  (ts) a l l e l e of a  Minute locus, located near the centromere. CHAPTER 2 describes a study of recombination, which deals with short genetic regions near the centromere of chromosome 3, using intervals, s t - i n - r i - e g ^ - K i - p ^ and Gl-p^-Sb-H.  The  the  following generali-  zations have emerged: ( i ) an excess of multiple crossover chromosomes was  recovered,  and the intervals which immediately span the centromere  showed the highest negative  interference; ( i i ) a positive c o r r e l a t i o n  of simultaneous exchange within c l o s e l y - l i n k e d i n t e r v a l s , was for many of the multiple crossovers; and  ( i i i ) several classes of  reciprocal crossover products were not recovered possible explanations  noted  equally.  for these results are: pre-meiotic  chromatid interference and gene conversion.  Three exchange,  The results of one  experi-  ment also indicated that the interchromosomal effects of C(1)M3 are most pronounced within the s t - i n and Ki-p^ i n t e r v a l s . CHAPTER 3 describes a genetic study of the Deformed locus.  The  mapping results confirmed that Dfd i s closely linked to K i . Genetic analysis of the crossover chromosomes suggested that Dfd i s homozygous viable and this was confirmed by the synthesis of homozygous Dfd stocks. This indicates that the Dfd locus i s not located within section 84F i n proximal 3R. CHAPTER 4 deals with experiments involving the use of radiation to produce crossovers near the centromere of chromosome 3, i n males. Crossovers originating from exchange nearest the centromere, were associated with clusters more frequently than those originating from exchange within other proximally-adjacent segments. was frequently accompanied  Induced exchange  by mutation and/or chromosome damage, at  or near the s i t e of exchange.  This was p a r t i c u l a r l y true for cross-  overs resulting from exchange i n wholly euchromatic segments.  It is  suggested that many of the radiation-induced crossovers arise through asymmetrical exchange, and that this approach w i l l permit the i s o l a t i o n of proximal aberrations. CHAPTER 5 describes the genetic and developmental analysis of a ts Minute.  As a ts a l l e l e of a proximally-located Minute locus, Q-III  exhibits the c l a s s i c a l dominant M t r a i t s , recessive l e t h a l i t y , and a highly p l e i o t r o p i c phenotype, at 29°C.  This phenotype was analysed i n  d e t a i l through the use of various temperature s h i f t experiments. Q-III possesses a polyphasic temperature-sensitive period (TSP) for l e t h a l i t y extending from the f i r s t l a r v a l instar to late pupation. Shorter heat pulses defined discrete l a r v a l , larval/pupal, and pupal TSPs for l e t h a l i t y .  In addition, homozygous Q-III females exhibit ts  s t e r i l i t y and maternal effects, indicating that the Q-III gene product is essential throughout  development.  iv  Heat-pulse experiments revealed a number of adult developmental abnormalities, involving derivatives of eye-antennal, leg, wing and genital imaginal discs.  Many defects, for example, those involving  the eye or antenna (eye-antennal d i s c ) , male g e n i t a l i a (genital d i s c ) , and scutellum (wing d i s c ) , have l a r v a l TSPs; whereas others, such as b r i s t l e or sex comb t r a i t s , have pupal TSPs.  I t i s suggested that the  former defects may be related to c e l l death i n the l a r v a l anlagen; while the l a t t e r are more l i k e l y due to blockages i n d i f f e r e n t i a t i o n during pupation. Q-III also interacts i n ts fashion with several n o n - a l l e l i c mutations . Thus, at 29°C, Q-III i s l e t h a l when combined with DI, Ly_ and Dfd; suppresses the sex comb phenes of Msc and Pc; and produces wing nicking effects when combined with vg or Sex. TSPs were defined for the vg, Dl and Sex interactions.  I t i s suggested that many of these  interactions arec:metabolic rather than s p e c i f i c . The fact that Q-III phenotypically resembles bobbed and ts suppressor of f o r k e d — , strengthens the notion that Minute gene products are active i n translation.  I t i s concluded that translational  defects can f u l l y account for the pleiotropy of Q-III.  V  ACKNOWLEDGEMENTS I would l i k e to express my gratitude to David Suzuki for his guidance, encouragement and patience throughout the course of this work.  Above a l l I am thankful for his friendship.  I would also l i k e to thank Thomas Kaufman for his help, encouragement and friendship.  His creative approach has helped me  immeasurably. As long as I have been i n the lab, Shirley Macaulay has been the centre of strength to nearly everyone, a truly beautiful friend to whom I am indebted for so much. I thank David Holm for his helpful and c r i t i c a l l y  knowledgeable  discussions over the past few years. I am very grateful to Deborah Koo who did a beautiful job of the Scanning E. M. work and the photography. I would l i k e to express my thanks to my patient and accomplished typists Mrs. Thorpe and Helen Curro. F i n a l l y , I would l i k e to thank a l l of the friends who have affected me both s c i e n t i f i c a l l y and personally throughout the years in the f l y lab.  I w i l l treasure the memories.  I am grateful for the f i n a n c i a l support of the National Research Council and MacMillan Fund during the course of this work. Parts of CHAPTER 2 are published i n Genetical Research.  vi  TABLE OF CONTENTS  ABSTRACT  i i  ACKNOWLEDGEMENTS  v  LIST OF TABLES  ix, x  LIST OF FIGURES  xi, xii  CHAPTER 1 I.  GENERAL INTRODUCTION Background 1. 2.  3.  Heterochromatin The Genetic Importance of Proximal Regions  1  The search for heterochromatic l o c i The genetic study of proximally-adjacent segments of chromosome 3  2  Other Genetic Properties of Proximal Regions Crossing over  II. CHAPTER 2  6  The Present Work  8 12  CROSSING OVER BETWEEN CLOSELY LINKED MARKERS SPANNING THE CENTROMERE OF CHROMOSOME 3  I.  Introduction  14  II.  Materials and Methods  15  III.  Results  22  IV.  Discussion  38  CHAPTER 3  A GENETIC STUDY OF THE DEFORMED LOCUS  I.  Introduction  44  II.  Materials and Methods  47  III.  Results  48  IV.  Discussion  61  vii CHAPTER 4  A STUDY OF INDUCED CROSSING OVER NEAR THE CENTROMERE OF CHROMOSOME 3  I.  Introduction  64  II.  Materials and Methods  68  III.  Results 1.  Radiation-Induced Crossing Over i n Males Numbers of progeny and frequency of crossing over Numbers of crossover events Recovery of mutants amongst crossovers Regional comparison of crossovers and mutants  2.  Analysis of Lethal Stocks Inter se complementation, pseudodominance and additional tests Cytological analysis Other complementation tests  IV. CHAPTER 5 - '  70 72 73 75  Discussion  77 81 83 85  A GENETIC AND DEVELOPMENTAL STUDY OF Q-III, A TEMPERATURE-SENSITIVE MINUTE MUTATION  I.  Introduction  88  II.  Materials and Methods  92  1.  Genetic Analysis Genetic mapping Complementation of Q-III with other mutations on chromosome 3  2.  93 proximal  Developmental Analysis Tests for s t e r i l i t y and maternal e f f e c t s Regular temperature s h i f t studies Pulse s h i f t studies Scanning electron microscopy Q-III interactions Tests of other Minutes with homeotics  III.  94  94 95 98 99 99 100  Results 1.  Genetic Analysis Viability Mapping  101 105  viii  Tests of Q-III i n t r i p l o i d s Complementation tests  108 108  Developmental Analysis Duration of developmental periods Tests for s t e r i l i t y and maternal effects Stage d i s t r i b u t i o n of l e t h a l i t y of Q-III homozygotes and heterozygotes Temperature-sensitive periods for l e t h a l i t y of Q-III Temperature-sensitive periods for dominant rough eye and b r i s t l e t r a i t s Phenotypes revealed by s h i f t experiments Interactions displayed by Q-III and other Minutes Scx-ts67 interactions Summary of TSPs involving Q-III IV.  114 117 125 128 160 175 178  Discussion 1.  The Nature of Minutes and Q-III Is Q-III a temperature-sensitive Minute? The function of Minute genes Is Q-III a single s i t e lesion?  2.  Developmental Characteristics of Q-III Pattern defects and c e l l death Defects r e s u l t i n g from heat treatment of Q-III during pupation The interactions of Q-III Additional uses f o r Q-III i n studies of development  CHAPTER 6  112 112  OVERVIEW  BIBLIOGRAPHY  182 183 187 189 190 195 197 199 201 207  APPENDICES 1.  Crossover Data and Estimates of Crossing Over  218  2.  Summary of Results of 24-hour Exposure of Q-III Homozygotes to 29°C at Specific Times During Development  221  Summary of Results of 48-hour Exposures of Q-III Heterozygotes (TMl/Q-III) to 29°C at Specific Times During Development  223  3.  ix LIST OF TABLES 1.  Summary of A H  Third Chromosome Mutations Used  16  2.  Summary of Special'Mutant Chromosomes Used  3.  Crossover Frequencies i n the _st to JD Interval i n  18  P  Chromosome 3  23  4.  Types and Numbers of Recombinant Chromosomes Recovered  24  5.  Coefficients of Coincidence Computed From A l l Multiples Recovered Results of Mapping Experiments Using The Markers Gl Sb H/p_  25  6.  27  P  7. 8. 9.  I n t e r v a l - S p e c i f i c Examination of Data of Females Producing Double Crossovers  29  Crossover Values i n Progeny of Females Producing Multiple Crossovers Compared to Those of Females Producing None  31  Analysis of Crossing Over i n Those Females Which Produced T r i p l e Crossover Chromosomes  10.  Tetrad Analysis of The Crossover Data  11.  Comparison of Observed With Expected Numbers of  32 33  Double Exchanges  34  12.  Types and Numbers of Recombinant Crossovers Recovered  13.  Results of The Cross of Dfd/Ki p Females to K i p / K i p Males Results of Inter Se and Deficiency Complementation Tests With Recombinant Lethals D i s t r i b u t i o n of Crossovers i n Proximal and Non-Proximal Intervals for sjz to _p_ Exchanges  14. 15.  P  P  P  16. 17.  35 P  49 53 71  Regional Summary of Lethals and S t e r i l e s Present on Crossover Chromosomes  74  Relative Occurrence of Crossovers and Lethal Events for Proximal and Non-Proximal Intervals  76  X  18.  19.  20. 21.  Results of Inter Se and Deficiency Complementation Crosses Involving Crossover Lethals  78  Summary of Pseudodominance and Complementation Tests Between Selected Crossover Mutants and Known Proximal Mutations  80  Phenotypic Description and Cytological Analysis of Crossover Mutants  83  Relative V i a b i l i t i e s of Q-III Homozygotes and Heterozygotes at Different Temperatures  22.  Crossover Data From Crosses Designed to Localize Q-III  23.  Relative V i a b i l i t y of Q-III i n Combination With Various Mutations at 22° or 29°C The Lengths of the Developmental Periods From Egg Deposition to Eclosion i n Different Classes at Different Temperatures  24.  25. 26.  27. 28.  102 106  109  111  L e t h a l i t y of Control and Q-III-Bearing Progeny at Different Developmental Stages at Various Temperatures  115  Lethal and V i s i b l e Phenotypes of Heterozygotes for Q-III and Dl, vg_, Ly_, Sex, Msc and P_c at Different Temperatures  161  Interactions of Known Minutes With Different Homeotic Mutations A f f e c t i n g Sex Combs  164  Effect of ts67 on Sex Expression  177  xi  LIST OF FIGURES  1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.  14. 15.  16.  Schematic representation of proximal regions of the third chromosome  19  Schematic representation of possible r e l a t i v e arrangements of Ki and Dfd  50  Complementation pattern emerging from inter se and deficiency complementation crosses (Dfd)  54  Proposed arrangements of l e t h a l s i t e s i n proximal 3R i n the o r i g i n a l Dfd/Ki pP female  56  Scanning electron micrographs showing the eye development of Dfd adults  60  Results of the s h i f t study to delineate a TSP for l e t h a l i t y of Q-III homozygotes  118  Results of 48-hour pulse s h i f t s to delineate TSPs for l e t h a l i t y of Q-III heterozygotes and homozygotes -  120  Results of 24-hour pulse s h i f t s to delineate TSPs for l e t h a l i t y of Q-III homozygotes  123  Results of the s h i f t study to delineate TSPs for rough eyes and reduced b r i s t l e s of Q-III heterozygotes  126  Scanning electron micrographs showing the effects of Q-III on macrochaete development  129, 131  Scanning electron micrographs showing the effects of Q-III on eye development  134, 136  Scanning electron micrographs showing the eyeantennal pattern defects of a Q-III homozygote Scanning electron micrographs showing the effects of Q-III on the development of the scutellum at 17°C  139 142, 144 146, 148  A scanning electron micrograph showing a wing-like duplication i n a Q-III/TM3 f l y  151  Scanning electron micrographs, showing the effects of Q-III on sex comb development  154  Scanning electron micrographs showing foreleg fusion of a Q-III/TM3 f l y  157  xii  17. 18. 19. 20. 21.  Results of the s h i f t study to delineate a TSP for the vg-Q-III interaction  168  Results of the s h i f t study to delineate a TSP for the Dl-Q-III interaction  170  Results of the s h i f t study to delineate a TSP for the Scx-Q-III interaction  174  TSPs for l e t h a l , s t e r i l e and adult morphological effects of Q-III  179  A schematic diagram of a mature eye-antennal imaginal disc  191  1  CHAPTER 1 GENERAL INTRODUCTION I.  Background  For many years, geneticists have studied segments that l i e near the centromeres of chromosomes.  Attempts to characterize these  proximal segments have been marked by considerable  speculation  (see  Cooper, 1959), as well as by d e f i n i t i v e experimentation. 1.  Heterochromatin Long ago, Heitz (1933) reported  that s p e c i f i c chromosome segments  possess special c y t o l o g i c a l properties, i n that they remain permanently condensed.  Such segments are p a r t i c u l a r l y prominent i n regions near  the centromeres of chromosomes of eukaryotes, and more recently they have been called constitutive heterochromatin (Brown, 1966) . When c y t o l o g i c a l length i s considered, includes 20 to 25 percent X chromosome and The  constitutive heterochromatin  of the two autosomes, 35 to 50 percent  the entire Y chromosome of Drosophila melanogaster.  previous discovery of highly redundant s a t e l l i t e DNA  in Drosophila and  the important study of Peacock e_t a_l. (1974). 7 species of s a t e l l i t e DNA  DNA  sequences  their preliminary l o c a l i z a t i o n within heterochromatin  through _in s i t u hybridization (Gall e_t a_l., 1971)  repetitive.  of the  has recently led to  These workers detected  and determined that they were highly  In s i t u analysis of l a b e l l e d RNA  complementary to these  sequences revealed that the l a t t e r are located mainly i n the  chromocentre i n s a l i v a r y gland chromosome preparations.  Nucleotide  analysis of 3 of the s a t e l l i t e s showed that the basic repeating units  are r e l a t i v e l y short and simple, thereby supporting the idea that most of this r e p e t i t i v e DNA  is incapable of coding for a gene product of  average complexity. 2.  The Genetic Importance of Proximal Regions Directly or i n d i r e c t l y , several workers have given valuable i n -  formation concerning the nature of proximally-located genes.  This  information provides ample j u s t i f i c a t i o n for continued study of this region of the chromosome. The search for heterochromatic  loci  Few genes capable of being mutated have been l o c a l i z e d within proximal heterochromatin  i n any of the chromosomes of Drosophila.  This observation led to the hypothesis that these chromosome regions are genetically inert (Muller and Painter, 1932).  To test t h i s , sev-  eral approaches have been adopted to attempt to genetically dissect the various heterochromatic regions i n the genome of this organism. Since the only phenotype which accompanies the lack of the entire Y chromosome i s male s t e r i l i t y (Bridges, 1916), i t seemed that no v i t a l genes resided on this chromosome. f u l l y exploited by Brosseau 7, Y-linked male f e r t i l i t y  Such a s t e r i l e phenotype was  (1960), who  success  provided a minimum estimate of  factors.  Muller jst al_. (1937) used the method of selecting for reciprocal products of meiotic exchange within overlapping inversions to recover heterochromatic deletions and duplications on the X-chromosome.  They  found no i n v i a b i l i t y associated with large duplications or deficiencies and concluded  that aside from bobbed (bb), no essential genes are  3 present i n the major blocks of heterochroma t i n near the centromere. This has recently been corroborated by Schalet and Lefevre However, the l a t t e r workers mentioned that the suppressor locus may  (1973). of forked  be located within heterochromatin.  The bobbed (bb) phenotype (short, fine b r i s t l e s and delayed eclosion) has been mapped to a proximal l o c a t i o n .  Quantitative c o r r e l a -  tions between the dosage of bb and b r i s t l e length (Stern, 1929), as well as the existence of a l e t h a l a l l e l e , pointed to a probable hypomorphic basis for bb a l l e l e s .  High frequency mutation and reversion of  bb had been noted i n several studies. Nucleolus Organizing (Cooper, 1959). was  (NO) region was  The c y t o l o g i c a l position of the located close to the bb locus  Consequently, a combined genetic-biochemical  approach  i n i t i a t e d to see i f the bb locus and the NO region were i d e n t i c a l . F i r s t , Ritossa and  Spiegelman (1965) used a nucleic acid h y b r i d i -  zation technique to measure the proportion of t o t a l DNA which codes for the 2 species of ribosomal  (r)RNA (18S and 28S) and  they found that  each X chromosome carried about 130 copies of both types. they showed that f l i e s bearing d i f f e r e n t numbers of NO  Furthermore,  regions  possessed d i f f e r e n t numbers of rRNA genes, thereby i n d i c a t i n g that the NO region and the s i t e ( s ) of these genes are either i d e n t i c a l or closely linked.  F i n a l l y , Ritossa et a l . (1966a) compared phenotypically  d i f f e r e n t stocks of hb and  found that the numbers of rDNA copies varied  inversely with the phenotypic i n t e n s i t y . The i n s t a b i l i t y of bb i s attributed to the propensity of such a highly redundant locus to cause asymmetrical pairing of homologues, because v i a crossing over this would generate deficiency and duplication products. The apparent lack of l o c i within proximal heterochromatin of the  4 autosomes has stimulated a great deal of i n t e r e s t .  Since highly repe-  t i t i v e s a t e l l i t e DNA had been l o c a l i z e d to constitutive heterochromatin and because of the existing evidence which equated bb with the tandemly redundant rRNA genes, Suzuki (1970; 1974a) suggested that genes cont r o l l i n g v i t a l functions such as c e l l d i v i s i o n might be highly redundant and reside i n heterochromatin.  Thus, the loss of one or even a  few copies of such genes through recessive mutation could be normalized by the presence of many wild-type duplicates. Two alternative methods of testing the hypothesis were formulated by Suzuki and his co-workers.  F i r s t they used Ethyl methanesulphonate  (EMS) to screen for Dominant temperature-sensitive (DTS) l e t h a l mutations, following the rationale that any unconditional dominant mutation in such essential genes would of necessity be l e t h a l or s t e r i l e .  Sub-  sequent screens yielded several such mutants, both on the second (Suzuki and Procunier, 1969) and the third (Holden and Suzuki, 1973) chromosomes.  However, none was d e f i n i t e l y l o c a l i z e d to proximal  heterochromatin, although DTS-6 mapped to a proximal position i n chromosome 3. The second approach involved attempts  to synthesize extensive  deletions within heterochromatin through the method of attaching and detaching compound third chromosomes (Baldwin and Suzuki, 1971).  The  rationale was based on the idea that formation of compounds i s f r e quently accompanied by asymmetrical exchange within the proximal segments of the chromosome.  This method enabled these workers to i s o l a t e  a large number of recessive lethals whose complementation suggested that many of these lesions were d e f i c i e n c i e s .  pattern Many also  displayed the dominant Minute phenotype (Minutes are recessive l e t h a l  5 mutations having the dominant v i s i b l e t r a i t s , thin b r i s t l e s and delayed development).  However, as with the PTS l e t h a l s , none of the above  mutations could be unequivocally assigned to heterochromatin (although in this instance many were mapped to proximal p o s i t i o n s ) . The lack of known heterochromatic mutations, as well as the r e l a tive dearth of lesions i n proximally-adjacent euchromatic segments, has made work with this region of chromosome 3 extremely d i f f i c u l t .  In  contrast, r o l l e d ( r l ) and l i g h t ( l t ) have been genetically l o c a l i z e d near or within the proximal heterochromatin of chromosome 2 and Pf(2R)M(2)S210 lacks a l l of the heterochromatin i n 2R (see Lindsley and G r e l l , 1968).  H i l l i k e r and Holm (1975) used a method involving  detachment of compound second chromosomes to i s o l a t e a large number of recessive lethals i n both 2L and 2R heterochromatin.  Several of these  were shown to be deletions through pseudodominance tests with rJL and l t and complementation  tests with Pf(2R)M(2)S210.  H i l l i k e r (1976)  next isolated EMS-induced a l l e l e s of some of these deficiencies and he was able to resolve a t o t a l of 13 heterochromatic l o c i (including It and r l ) i n this chromosome.  He argues that the r e l a t i v e l y high muta-  b i l i t y of these genes i s inconsistent with the idea that they are redundant. The above genetic evidence, coupled with previously mentioned biochemical evidence indicating that the basic r e p e t i t i v e units of PNA i n heterochromatin are simple, would suggest that heterochromatic genes are l i k e l y s t r u c t u r a l l y and functionally d i s t i n c t from the surrounding simple sequence PNA.  However, i t would be interesting to genetically  probe the constitutive heterochromatin of chromosome 3 i n a s i m i l a r fashion and to extensively study such uniquely-placed l o c i .  The genetic study of proximally-adjacent  segments of chromosome 3  Several approaches have proved successful in helping to g e n e t i c a l l y characterize regions near the constitutive heterochromatin of chromosome 3.  Three studies involved the d i r e c t s e l e c t i o n for chromosome  aberrations  near the centromere, while an additional study  relevant information even though i t was  provided  not i n i t i a l l y designed to  investigate proximal l o c i . Lindsley _et al_. (1972) exploited the segregation  properties of a  large number of Y-autosome translocations to survey the v i a b i l i t y  and  phenotypic effects of aneuploidy for s p e c i f i c segments of the autosomes.  They v e r i f i e d the existence of two previously  identified,  proximal Minutes (M) on chromosome 3, M(3)S34 i n 3L and M(3)S39 i n 3R; and  they discovered  a new M s i t e , M(3)LS4 i n 3L and an additional haplo-  i n s u f f i c i e n t locus, Splayed (Spl) which is located between the heterochromatin and M(3)S39.  3R  The existence of a T r i p l o - l e t h a l (Tpl)  locus in a proximal part of 3R did not allow the characterization of an appreciable  portion of this segment, since this locus i s inviable  when present i n either haploid or t r i p l o i d doses.  A major drawback of  this method i s the fact that the duplications and deficiencies synthesized do not exist as stable stocks, but must be re-synthesized time they are to be used.  each  However, this characterization of the  dosage-sensitivity of the genome is extremely valuable.  In p a r t i c u l a r ,  the findings concerning the proximal Minutes i n chromosome 3 are important since many workers are presently attempting to study these l o c i with a view to determining their primary functions. A group of so-called homeotic l o c i has been l o c a l i z e d within proximal segments of the right arm of chromosome 3.  Since mutations  i n these l o c i cause switches i n developmental fates of imaginal discs r e s u l t i n g i n the production of structures normally derived from other discs, such l o c i have generated considerable interest (for a review see Postlethwait and Schneiderman, 1973). One of the homeotic genes, Nasobemia (Ns) was morphic (Muller, 1932).  suspected  to be  I t had been previously shown that the  neo  neo-  morph, K i l l e r of prune (K-pn) could be phenotypically reverted through the use of r a d i a t i o n and the genetic evidence suggested that such revertants were often deficient for the K-pn 1969).  Denell (1972, 1973)  and recovered  locus (Lifschytz and  Falk  extended this method to the study of Ns,  several revertants including a putative deletion.  In a  subsequent study, Duncan and Kaufman (1975) used this approach to isolate a number of deletions i n proximal 3R, 2 involving Ns and -3 involving doublesex (dsx).  They corroborated  revertants  Denell's findings  which indicated that Antennapedia (Antp), Ns and Extra sex comb (Sex) are mutations i n the same locus (possibly including Multiple sex comb) Their experiments have also provided a number of stable d e f i c i e n c i e s in proximal 3R which have been very useful for mapping other mutants, especially since the r e s t r i c t i v e nature of proximal makes the map  positions of proximal  In a study that was  crossing over  l o c i less meaningful.  designed to select for  temperature-sensitive  lethals along the entirety of chromosome 3, Tasaka and Suzuki (1973) obtained  interesting results.  Ninety percent of the ts mutations were  mapped to the proximal region between scarlet (st) and  Stubble  (Sb).  Genetically the sj: to Sb i n t e r v a l is small, although c y t o l o g i c a l l y i t includes nearly 40 percent of the chromosome.  One  of the mutants  l e t h a l at 29°C but viable at 25°C and at the l a t t e r temperature,  was  8  heterozygotes displayed a phenotype similar to that of Spl, which i s located near the heterochromatin i n the r i g h t arm.  Some of the  aforementioned Ns_ and dsx deficiencies have been used to map at least five of these ts lethals (T. C. Kaufman, personal  communication),  thereby supporting the results of the recombination mapping. The basis for the apparent p r e f e r e n t i a l selection of ts mutations i n proximal l o c i of chromosome 3 i s not known.  However, the eventual  cytological mapping of a l l of the ts lethals may provide information concerning the genetic organization of these l o c i .  The  well-documented  u t i l i t y of ts mutations for investigating developmental properties of a given gene (see Suzuki, 1970; Hartwell, 1974), should provide the impetus 3.  to determine how  these proximal l o c i function during development.  Other Genetic Properties of Proximal Regions For  decades, geneticists have observed numerous properties of  chromosome segments residing near the centromere, p a r t i c u l a r l y i n Prosophila.  The following category i s the most relevant to my work.  Crossing over The area of investigation involving the centromeric regions which has produced the most s t r i k i n g results, i s that of crossing over. Several approaches  to the study of crossing over near the centromere  have been adopted, and i n most cases, the results are descriptive i n nature. Spontaneous meiotic crossing over i n females i s the type of exchange most frequently studied.  The work of several people has provided  a number of interesting observations.  For example, i t was found that  while the most proximal segment makes up about 20 to 25 percent of the  9 mitotic length of chromosome 3 c y t o l o g i c a l l y , i t constitutes only 1 percent  of the t o t a l genetic length (Dobzhansky, 1930;  Thus, the obvious suggestion was  Painter, 1935).  that for meiotic crossing over i n  females, exchange occurs only within euchromatin.  The idea that no  crossing over occurs within heterochromatin has been supported by the findings of Baker (1958) and Roberts (1965), and of p a r t i c u l a r importance i n this regard is the work of H i l l i k e r (1975). this, however, i t has long been recognized  In addition to  that crossing over between  markers spanning proximal regions i s severely r e s t r i c t e d r e l a t i v e to comparable regions i n more d i s t a l locations. appropriate  chromosome aberrations to displace markers from d i s t a l to  more proximal positions and was them was  Beadle (1932) used  able to show that crossing over between  reduced, thereby suggesting  that some sort of i n h i b i t o r y  e f f e c t of the centromere on crossing over e x i s t s .  More recently,  Thompson (1963a,b) proposed that i f exchange pairing of centromeric intervals was  rapidly followed by l o c a l i z e d centromeric repulsion just  prior to exchange, this may  explain the observed decrease i n crossing  over between proximal l o c i . Another i n t e r e s t i n g observation  concerning  spontaneous crossing  over is that while p o s i t i v e interference usually governs double exchange i n adjacent  i n t e r v a l s of the same chromosome arm  (Morgan et a l . ,  1925), studies of Drosophila have shown that simultaneous exchange within closely linked intervals which span the centromere i s independent (Graubard, 1934;  Stevens, 1936).  coincidence values of 1.3 chromosome 3.  In fact, Morgan et a l . (1925) found  for crossing over near the centromere of  10 The above information poses some interesting questions with respect to spontaneous crossing over near the centromere.  Does negative inter-  ference d e f i n i t e l y appear i n multiple exchange between proximal l o c i ? I f so, i s i t r e s t r i c t e d to one arm of the chromosome?  Besides causing  mapping d i f f i c u l t i e s i n work with proximally-located mutant l o c i , could centromeric i n h i b i t i o n obscure the nature of a given gene by maintaining close linkage of accumulated  lethals to that gene?  Induced crossing over has been demonstrated  i n both females  (reviewed by Schultz and Redfield, 1951; and see W h i t t i n g h i l l , and males (Friesen, 1933; h i l l , 1937).  1955)  1937a,b; Patterson and Suche, 1934; Whitting-  I t has long been known that i n females,  recombinagenic  agents increase crossing over p r e f e r e n t i a l l y near the centromere of the chromosome (see Schultz and Redfield, 1951; and Lucchesi and Suzuki, 1968).  Similarly, i t has been reported by several people that  induced exchange i n males occurs mainly within proximal regions (Friesen, 1937b; W h i t t i n g h i l l , 1937;  Puro, 1966; Hannah-Alava, 1968).  However, no published study has conclusively established that induced crossing over i n proximal regions occurs p r i n c i p a l l y within heterochromatin. Friesen (1933) was the f i r s t to report that radiation could induce crossing over i n males of Drosophila.  From h i s data he concluded that  radiation-induced exchange i n males closely resembles spontaneous meiotic exchange i n females.  Contemporaries  i n this f i e l d (Patterson  and Suche, 1934; W h i t t i n g h i l l , 1937) agreed with this conclusion and f e l t that induced exchange was r e l a t i v e l y precise, since crossover chromosomes were not usually associated with l e t h a l i t y .  However,  Muller (1954, 1958) has argued that induced crossing over i n both  11 sexes occurs by a mechanism similar to that which produces  transloca-  tions, and thus, crossover chromosomes formed i n such an asymmetrical manner could be associated with aberrations. Recently, studies have shown that radiation-induced crossover chromosomes are frequently associated with l e t h a l i t y or s t e r i l i t y and i n some cases chromosome aberrations (Hannah-Alava, 1968; Mglinets, 1972). I f induced crossing over does involve asymmetrical exchange, and i f such crossing over occurs p r e f e r e n t i a l l y within or near heterochromatin, the selection of induced crossovers might prove to be an important way of enriching for stable proximal d e f i c i e n c i e s .  12  II.  The Present Work  These background studies have established an important  foundation  for subsequent investigations of the proximal regions of the chromosome.  Since many of the features described above are i n some ways  related, I decided to adopt a multifaceted approach i n my work with these regions.  This thesis represents a report of the results of  such an approach. CHAPTER 2 describes a series of experiments designed to explore proximal recombination amining:  i n chromosome 3 of females with a view to ex-  (a) the degree and nature of negative interference i n  d i f f e r e n t genetic intervals near the centromere and interchromosomal  (b) the extent of  effects within these i n t e r v a l s .  CHAPTER 3 represents a genetic study of the Deformed locus, i n cluding both an experiment designed to map  this gene r e l a t i v e to other  proximal markers i n 3R and the subsequent analysis of r e s u l t i n g l e t h a l crossover chromosomes.  Since evidence exists which suggests that the  l e t h a l i t y formerly ascribed to this locus i s due to a closely-linked but separate mutation,  the main aim of this study was  the Dfd lesion i s i t s e l f a recessive l e t h a l .  to determine i f  The potential use of Dfd  i n future crossover studies, as well as the developmental  interest of  this mutant stemming from i t s temperature-sensitivity and i t s effects on the derivatives of the eye-antennal  imaginal disc, provided the  impetus for this study. CHAPTER 4 describes the use of radiation-induced, male crossing over to recover proximally-located aberrations and l e t h a l s .  The r a -  tionale for this method arises from recent evidence which argues that  many induced crossovers may be the result of asymmetrical exchange events. F i n a l l y , CHAPTER 5 i s a report concerned with the genetic and developmental investigation of a ts a l l e l e of a Minute locus, located proximally i n chromosome 3.  By studying the mutant I hoped to provide  more information about (a) the basis of the Minute phenotype and (b) the effects of such a lesion on the development  of the organism.  14  CHAPTER 2 CROSSING OVER BETWEEN CLOSELY LINKED MARKERS SPANNING THE CENTROMERE OF CHROMOSOME 3  I.  Introduction  Recently, the correct location of the centromere of chromosome 3, r e l a t i v e to the position of l o c i known to be t i g h t l y linked, has been ascertained.  Thus, radius incompletus and inturned have been assigned  to the l e f t arm (Arajarvi and Hannah-Alava, 1969) along with Polycomb (Puro and Nygren, 1975) and eagle (Holm, et aj.., 1969), while Kinked (Merriam and Garcia-Bellido, 1969) and Deformed (Holm et a l . , 1969) have been positioned i n the right arm. The unequivocal l e f t and right l o c a l i z a t i o n of these genes makes i t possible to more accurately interpret crossover data from this region and therefore an intensive study of crossing over i n the intervals adjacent to the centromere of chromosome 3 was i n i t i a t e d .  15  II.  Materials and Methods  Tables 1 and 2 represent summaries of a l l third chromosome mutations and special chromosomes used i n these experiments.  A l l third  chromosome balancers that have been used are described f u l l y i n Lindsley and G r e l l (1968).  The balancer referred to as TM3 should  henceforth be considered as equivalent to TM3, otherwise indicated.  Stubble Serrate unless  A l l experiments were performed at 22 i 0.5°C  unless otherwise s p e c i f i e d . Recombination was measured i n the proximal region of chromosome 3 using the following markers (for a complete description, see Table 1 and consult Lindsley and G r e l l , 1968):  s_t - s c a r l e t (44.0), in -  2 inturned (47), _ri - radius incompletus (47.0) , eg Ki - Kinked (47.6), and £ ^ - pink peach (48.0).  - eagle-2 (47.3),  Figure 1 i s a sche-  matic representation of the map positions of the markers along the chromosome (Lindsley and G r e l l , 1968).  The centric blocks of hetero-  chromatin are believed to be immediately flanked by eagle (Holm et. a l . , 1969) and Kinked (Merriam and Garcia-Bellido, 1969) on the l e f t and right respectively. The s_t - in i n t e r v a l was designated as 1, jLn 2 2 p r i as' 2, r i - eg-' as 3, eg - K i as 4 and K i - pj_ as 5. Note that the centromere l i e s i n i n t e r v a l 4. Three d i f f e r e n t major studies of crossing over were performed: Experiment I .  100 st i n r i eg^ Ki p / + + + + + + females were testP  crossed for 5 consecutive 3-day broods and their progeny were scored. 2 Experiment I I . + + + + Ki p  P  Crossing over was s i m i l a r l y measured i n 213 s t i n r i eg /  females; 108 of these were studied for five 3-day broods  (Expt. I l a ) and 105 for one 3-day brood (Expt. l i b ) .  Experiment I I I .  16 Table 1 Summary of A l l Third Chromosome Mutations Used  Mutant  Symbol  Genetic Position  Phenotype  40.5  Excised wing margins (rec. l e t h a l )  ,G1  41.4  Rough small eyes  scarlet  st  44.0  Bright red eyes  transformer  tra  45  Transformation of females into s t e r i l e males  inturned  in  47  Thoracic hairs and b r i s t l e s directed towards midline  radiusincompletus  ri  47.0  Interruptions i n L2  Polycomb  Pc',  47.7  A l l legs of male possess sex combs (rec. l e t h a l )  . 2 eagle  2 eg.  47.3  Wings spread  Deformed  Dfd  47.5  Reduced eyes (rec. lethal)  Deformedrecessive  Dfd  47.5  Recessive a l l e l e of Dfd  Kinked  Ki  47.6  Short and twisted b r i s t l e s and hairs  roughenedeye  roe  47.6  Eyes rough  proboscipedia  pb  47.7  Transformation of oral lobes into tarsus or arista  Extra sexcomb  Sex  47  A l l legs of male possess sex combs (rec. l e t h a l )  Lyra Glued  1  17  Table 1 (continued)  Mutant  Symbol  Antennapedia  Antp  48  Transformation of antenna into leg structures (rec. lethal)  Multiplesex comb  ,Msc  48.0  A l l legs of male possess sex combs, associated with ::in(3R)84B;85F (rec. l e t h a l )  Nasobemia  Ant£  48.0  Transformation of antenna into leg structures  double sex  dsx  48  Males and females intersexual  pink peach  £  48.0  Dull ruby eyes  Stubble  Sb  58.2  Short and thin b r i s t l e s (rec. l e t h a l )  Delta  DI  66.2  Termini of wing veins thick and broad (rec. lethal)  Hairless  H  69.5  Missing p o s t v e r t i c a l and abdominal b r i s t l e s (rec. l e t h a l )  70.7  Black body  ebonysooty  P  Ns  Genetic Position  Phenotype  18  Table 2 Summary of Special Mutant Chromosomes Used  Chromosome  Abbreviation  +R2 Df(3R)Antp  +R2 Antp-  Ns+R21 Df (3R)Antp-  +R21 Ns'  EH-R2 Df (3R)dsx-  D+-R2 dsx-  D+-R5 Df (3R)dsx-  dsx  T(3;Y)P92  DP-P92  Dfd kar ry  Dfd-rk  DH-R5  Cytology Df(3R)84B3;84Dl-2  Reference Duncan and Kaufman, 1975  Df(3R)84A-B;84D-E  "  Df(3R)84D9-12;84F16  1  Df(3R)84F2-3;84Fl6  11  Insertion of 3R(84D10-ll;85Al-3) into Y " — Chovnick et a_l., 1971  FIGURE 1  Schematic representation of proximal  regions  of the third chromosome showing the genetic markers used.  Published map positions are  given below the symbols with numerically designated crossover intervals indicated above the l i n e .  / —I 4 4 . 0  2 H 4 7  3 ir 47.0  4 t^mmmxBnmm  47.3  5 i  47.6  1 —  48.0  0  21  Crossing over was measured i n 25 C(1)M3/Y; s t i n r i eg Ki p  P  females for five 3-day broods.  + + /+ + + +  Expt. I l l females were tested i n  order to determine any interchromosomal effects of the inversions contained i n each arm of the compound X (Lucchesi and Suzuki, 1968). A l l females tested were mated i n d i v i d u a l l y (within 40 hours of eclosion at 22 i 0.5°C) with 2 or 3 males homozygous for 2 st i n r i eg  o s Ki p  e .  The t h i r d chromosomes of a l l females were  isogenized prior to use i n order to minimize the presence of lethals i n the stocks.  However, the other chromosomes were not made co-  isogenic and the K i _p_^ chromosomes of Expt. l i b females were of d i f f e r e n t origins than those of Expt. H a  females.  Progeny i n each  v i a l were scored d a i l y u n t i l the eighteenth day a f t e r the parents had been introduced.  III.  Results  A summary of the numbers of progeny examined and the crossover values for the region studied (including published map distances, Lindsley and G r e l l , 1968) are given i n Table 3.  Data for females  carrying normal X chromosomes (columns 2 and 3, Table 3) reveal that recombination was consistently higher i n Expt. I than i n Expt. I I 2 (X  = 108.9, P = 0.05).  These differences probably r e f l e c t random  differences i n genetic backgrounds i n the two s e r i e s .  The i n s e r t i o n  of C(1)M3 into test females (columns 4 and 5, Table 3) noticeably augmented recombination, thereby reconfirming i t s interchromosomal effects on crossing over near proximal heterochromatin. were more prominent  These effects  for the distalmost i n t e r v a l s ; for example, re-  combination i n regions 1 and 5 increased 3- and 4-fold, respectively. Table 4 summarizes the number of d i f f e r e n t crossover chromosomes recovered.  A t o t a l of 3,603 single, 85 double and 20 t r i p l e crossover  chromosomes was scored.  The most frequent class of doubles occurring  i n Expts. I and II involved regions 1 and 5 (nearly a third of the total) and 3 and 4 (more than a t h i r d of the t o t a l ) .  Other doubles  frequently recovered were 3, 5 and 1, 4. Double crossovers involving exchange i n intervals known to be on the same side of the centromere were never recovered.  However, 1, 2,  4 and 1, 3, 4 t r i p l e crossovers, which included exchange i n two of these i n t e r v a l s , were recovered. Since double crossover chromosomes involving exchange i n the two distalmost intervals (1 and 5) were frequently encountered, these data  23.  Table 3 Crossover Frequencies i n The s_t to _p_£ Interval i n Chromosome 3  Experiment Genetic Region  Reference Values  I  Ila rod X  Number III C(1)M3  Ratio III/IIa  1  3.0  3.99  1.77  5.39  3.05  2  0.06  0.25  0.20  0.30  1.50  3  0.30  0.21  0.08  0.17  2.13  4  0.30  0.43  0.22  0.64  2.90  5  0.40  0.65  0.25  1.11  4.44  Number of Fertile Females  -  92  108  25  Number of Progeny  -  36,948  33,139  4,063  Table 4 Types and Numbers of Recombinant Chromosomes Recovered  Experiment Number Ha  lib  III  1393 71 27 101 183  574 63 17 50 64  217 12 4 24 41  547 13 30 58 114  1775  768  298  762  1,2 1,3 1,4 1,5 , 2,3 2,4 2,5 3,4 3,5 4,5  0 0 3 14 0 2 4 22 5 0  0 0 2 8 0 0 0 6 2 3  0 0 0 1 0 0 0 1 2 0  0 0 1 2 0 1 1 4 1 0  Totals  50  21  4  10  Region  I  SINGLES 1 2 3 4 5 Totals DOUBLES  TRIPLES  •  1,2,3 1,2,4 1,2,5 1,3,4 1,3,5 1,4,5 2,3,4 2,3,5 2,4,5 3,4,5  0 3 0 2 0 4 0 0 0 1  0 0 0 2 0 4 0 0 0 0  0 0 0 0 0 2 0 0 0 0  0 0 0 0 0 2 0 0 0 0  Totals  10  6  2  2  25  Table 5 Coefficients of Coincidence Computed From A l l Multiples  Recovered  Experiment Number Intervals  I  Ha  III  1.2  0.81  1.3  0.64  4.25  1.4  1.90  6.21  1.43  1.5  1.90  8.17  1.23  2.3  -  2.4  12.50  2.5  6.75  3.4  75.20  137.16  22.36  3.5  11.90  30.00  49.20  4,5  4.83  38.40  6.93  26  support the contention of other work i n Drosophila (Graubard,  1934;  Stevens, 1936), i n Neurospora (Bole-Gowda et a l . , 1962) and yeast (Hawthorne and Mortimer, 1960), that positive interference does not extend to regions i n d i f f e r e n t arms of the chromosome.  This i s fur-  ther emphasized by the fact that most of the t r i p l e crossovers (12 of 20) involved exchanges i n intervals 1, 4 and 5.  However, i t should  also be noted that a l l of the t r i p l e crossovers involved the most 2 proximal i n t e r v a l (eg  to K i ) .  Coefficients of coincidence were calculated for doubles i n a l l three experiments (excluding Expt. l i b ) .  In a l l cases but 1, 2 and  1, 3 doubles of Expt. I, these values exceeded unity (Table 5).  Ex-  tremely high values for 3, 4 and 4, 5 exchanges (and 3, 5 exchanges for Expt. I l l ) indicate a very high negative interference i n these intervals.  Therefore, i n spite of very tight linkage between s_t and  p , the recovery of multiple crossover chromosomes greatly exceeds P  conventional expectations.  I t i s noteworthy that while single ex-  changes were increased i n a l l intervals by C(1)M3, a  concomitant  increase i n the occurrence of multiple exchanges (except for 3, 5 doubles) did not occur, as shown by coincidence values. In order to further test d i f f e r e n t proximal intervals for i n t e r ference, two other mapping experiments were carried out with the markers: Glued  (Gl), Stubble (Sb), Hairless (H) and  and consult Lindsley and G r e l l , 1968  (see Table 1  for descriptions of these l o c i ) .  Heterozygous Gl + Sb H/+ _p_ + + females were crossed to j> /p_ males P  P  P  (20 males and 20 females per quarter pint bottle) and crossing over was measured for the three i n t e r v a l s : (a) Gl and  (c) Sb to H.  to p_ ; P  (b) _p_ to Sb; P  Note that the (a) i n t e r v a l spans the centromere.  27  Table 6 Results of Mapping Experiments Using The Markers Gl Sb H/p  Experiment Number  IV  Number of Progeny  1148  Map Distances (percent)  Single Crossovers  a  b  68  98  7.67  9.93  Map Distances Coefficients of Coincidence  909  57  73  8.36  9.57  Multiple Crossovers  c  a,b  a,c  b,c  a,b,c  123  10  9  5  1  1.26  0.95  0.44  9  9  4  1.37  1.20  0.53  12.02  Coefficients of Coincidence  V  P  84 10.78  1  28  Table 6 summarizes the data from Experiments  IV and V.  The map  dis-  tances for the three intervals show good agreement with book values (see Lindsley and G r e l l , 1968) although (a) i s s l i g h t l y larger i n both experiments.  Coincidence values were calculated for the d i f f e r e n t  interval combinations: a, b (1.3 to 1.4); a, c (0.95 to 1.2); and b, c (0.44 to 0.53).  I t i s apparent that more multiple crossovers  than expected occurred for the combination involving the centromeric and the immediately adjacent intervals (a, b), while that involving the most d i s t a l and centromeric intervals (a, c) showed about the expected number of multiple crossovers.  However, interference was  positive when the most d i s t a l intervals are considered.  The l e v e l of  negative interference for this series of experiments was also much less marked than that of the major series which had involved more proximal markers.  Thus, these data further support the idea that  multiple exchanges are more common i n adjacent intervals more c l o s e l y associated with the centromere, and that although positive interference does not extend to both sides of the centromere, negative interference does. Previous workers have suggested that some rare multiple exchange chromosomes could, i n fact, r e s u l t from successive single crossover events.  Thus, a mitotic crossover i n a gonial c e l l could be followed  by a meiotic exchange to produce an apparent double crossover chromosome (Whittinghill, 1955;  Suzuki et a l . , 1966).  Such a Two-Step model pre-  d i c t s that the gonial exchange could be amplified through mitotic d i v i sions, thereby generating doubles amidst a cluster of single crossovers (Suzuki et a l . , 1966).  The progeny of individual females y i e l d i n g  29  Table 7 Interval-Specific Examination of Data of Females Producing Double Crossovers (Experiment I)  Female  Type of Double  Number of Doubles  Singles Occurring i n Either Interval  Total Number of Progeny  J  1,5  1  23 (16.8)*  391 (360)  K  3,4  2  2 ( 1.3)  388 (360)  L  2,4  1  3 ( 1.6)  465 (360)  M  3,5  2  2 ( 2.6)  466 (360)  N  1,4  1  13 (15.6)  429 (360)  *Numbers i n parentheses represent mean values of comparable data for 52 non-multiple females  30  double exchanges (but not t r i p l e s ) were examined for evidence of clustering of single crossovers i n the regions where the doubles had occurred.  A sample of 5 such females (Expt. I) i s given i n Table 7  (along with mean values for females y i e l d i n g no multiple exchange progeny).  No noticeable clusters of singles appeared to accompany  doubles for the regions i n question. I f multiple recombinant chromosomes are generated  by a Two-Step  mechanism, then crossover values for multiple-producing females would be expected  to be higher than the values from females producing  multiples.  These subsets of data were s i g n i f i c a n t l y d i f f e r e n t  no (Table  8) and i n both experiments, crossover values for regions 3 and 4 were higher i n those females producing multiple crossovers.  However, when  the crossover data of the 9 females of Expt. I that had produced t r i p l e recombinant progeny (within the sjt to j )  P  interval) were examined, i n  each case the d i s t r i b u t i o n of crossover types followed a Poisson d i s t r i b u t i o n (Table 9). A tetrad analysis as inferred from single strand recovery (Weinstein, 1936), was  i n i t i a t e d with a view to distinguishing between  a meiotic and a gonial o r i g i n of the t r i p l e exchanges (Table 10). Two-Step production of rare multiple exchange chromosomes was  The  inferred  from an i n s u f f i c i e n t number of double exchange chromosomes predicted from a tetrad analysis of the multiple exchange chromosomes (Suzuki et a l , , 1966). Ha  In every case examined i n present tests (Expts. I,  and l i b ) , the number of t r i p l e exchange tetrads was equivalent to  or exceeded that of the double exchange tetrads.  This supports a  .31  Table 8 Crossover Values i n Progeny of Females Producing Multiple Crossovers Compared to Those of Females Producing None  Female Type Multiples Expt I Number of Females  42  Number of Progeny  19,284  No multiples  Expt  Ha  24  7,111  Expt I  Expt  52  85  15,378  26,029  Ha  Crossover Values  > U  cu  4.09  1.65  3.71  1.76  2  0.28  0.22  0.17  0.18  3  0.26  0.21  0.07  0.05  4  0.53  0.36  0.24  0.15  5  0.56  0.40  0.59  0.20  +J  ci  M  S i g n i f i c a n t difference for X of both experiments at P =  was  0.01  indicated for a subset  comparison  32  Table 9 Analysis of Crossing Over i n Those Females Which Produced T r i p l e Crossover Chromosomes (Experiment I)  Types of exchange (st to p^) 0  Female  1  2  3  *Chi-Square Values  A  431  28  0  1  0.002  B  438  35  1  1  0.329  C  460  27  0  1  0.060  D  482  25  0  1  0.081  E  423  15  1  1  0.046  F  163  10  0  2  2.200  G  467  23  0  1  0.427  H  401  29  2  1  2.280  I  295  21  1  1  1.333  0 = no exchange 1=1  exchange  2=2  exchanges  3=3  exchanges  *In each case recombination -was found to approximate a Poisson d i s t r i b u t i o n at P = 0.05  33  Table 10 Tetrad Analysis of The Crossover Data (Inferred From Recovery of Single Strands)  Experiment Number Tetrad  Distribution  I  Ha  lib  T r i p l e Exchange  80  48  16  Double Exchange  80  12  16  Single Exchange  3,410  1,536  734  No Exchanges  33,378  31,543  10,088  Total Tetrad Sample  36,948  33,139  10,854  34  Table 11 Comparison of Observed With Expected (From Meiotic T r i p l e Exchange Tetrads) Numbers of Double Exchanges  Experiment Number Classes of Doubles  Expected  I  Observed  Ha Expected Observed  lib Expected Observed  1,2  3  0  0  0  0  0  1,3  2  0  2  0  0  0  1,4  9  3  6  2  2  0  1,5  4  14  4  8  2  1  2,3  0  0  0  0  0  0  2,4  3  2  0  0  0  0  2,5  0  4  0  0  0  0  3,4  3  22  2  6  0  1  3,5  1  5  0  2  0  2  4,5  5  0  4  3  2  0  35  Table 12 Types and Numbers of Recombinant Chromosomes Recovered  Experiment I  Experiment  Ha  Number Region  ic  Genotype Recovered  SINGLES  2 D i n r i eg K i p 667 st i n  56  2 „. P r i eg K i p  15  st i n r i  16  2 .  st K i p 1:1  Ki p,P  2  st i n r i eg  24  st i n r i K i p  2.5:1 12  st i n r i eg^ K i p  P  31  1.5:1  K i 75  +  +  +  +  +  19  st i n r i eg2 pP  19  Ki  45  st  2  2 „. P i n r i eg K i p  0  st K i  0  2 p i n r i eg p  8  st i n  0  1.5:1 108  1.5:1  5  2  + +  1:1  39  r i eg  58 44  f  272  st i n K i p* 3.5:1  11  st i n r i eg  302  F  2 in r i eg  1.5:1  P  v  R  R  726  st  Number Recovered  Genotype  1.5:1  2.5:1  DOUBLES st K i p  3  F  1,4  3:0  2  0  i n r i eg 1,5  st p  7  P  2 „. in r i eg K i 2,4  st i n K i p 2 n eg st i n p  2,5  2 „. r i eg K i  7  1:1  1  P  1  1:1  2  2 rr. P r i eg K i p  0  st i n K i  0  2 p eg P  0  F  2  2:0  1:1 r  L  F  8:0  36  Table 12 (continued)  Experiment I Region  Experiment H a  Number Recovered  Genotype  R  Genotype  Number Recovered  R  DOUBLES st i n r iK i p 3,4  3,5  19  P  2 eg_ st i n r iP 2  P  V  eg K i 4,5  st i n r i eg Ki  st i n r i  6  6 :1  2  P p r  6:0  3  eg  1  st i n r i K i 4 :1  2  2  Ki  p P  p p  0 2 0  4  eg  A  st i n r i eg K i  3  2!  0  0 0  r  2:0  3:0  * R = Ratio of reciprocal classes  (  Two-Step explanation for the o r i g i n of the multiples.  Comparison of  the minimum expected numbers of doubles ( i . e . , doubles generated by t r i p l e exchange tetrads) with the actual numbers recovered (generated by both double and t r i p l e exchange tetrads) reveals (Table 11) that in a few cases (1, 2; 1, 3 and 4, 5) the observed numbers were appreciably less than expected, while i n most of the remaining classes the observed numbers exceeded  or approximated  the expected.  Table 12 shows a summary of the reciprocal crossover classes r e covered from Expts. I and 11a females, along with the numbers of each class obtained.  In several cases ( p a r t i c u l a r l y for the more proximal  i n t e r v a l s ) , these classes do not appear to be equally represented, despite the apparent lack of any obvious selective advantage  for non-  mutant a l l e l e s . In order to rule out high r e v e r t a b i l i t y of the markers studied as a contributive factor to some of the multiple exchange chromosomes, homozygous stocks were screened for revertants and none was found among 1.1 X 10^ st i n r i e g and 2.0 X 10^ s t i n r i e g K i p 2  2  P  e  S  chromosomes.  38  IV.  Discussion  Although genetically small, the region studied i n these experiments represents a large portion of the physical length of chromosome 3. Unexpectedly, these experiments have revealed evidence for the e x i s t ence of non-classical, non-reciprocal recombination events i n this region of the chromosome. of coincidence)  Thus, interference (expressed  for these intervals was  high and  as c o e f f i c i e n t s  negative.  The present work i s concerned s o l e l y with intergenic recombination near the centromere of one of the autosomes of Drosophila.  Previously,  i t had appeared that exchange within genetically short regions i n this organism was  generally accompanied by high p o s i t i v e interference  (Morgan et a l . , 1925).  Exceptions to this i n Drosophila  involved intragenic crossing over (e.g. Hexter, 1958; 1960), although Sturtevant  usually  Green,  (1951) reported that negative  1959,  interference  could be detected i n the study of intergenic crossing over i n the fourth chromosome of t r i p l o i d females. ence was 1957;  S i m i l a r l y , negative  interfer-  seen i n intergenic crossing over i n Aspergillus (Calef,  Pritchard, 1960)  and barley (S^gaard, 1974).  One  explanation  offered to account for such negative interference i s equivalent to the idea of e f f e c t i v e pairing i n bacteriophage (see Chase and Doermann, 1958), which e s s e n t i a l l y assumes that short localized regions of pairing e x i s t , and within these regions recombination i s highly probable. Both Calef (1957) and Pritchard (1960) used this hypothesis to explain coincidence values exceeding 100 loci.  for exchange between t i g h t l y linked  However, S(6gaard (1974) discounted  any explanation  invoking  39 l o c a l i z e d pairing for his work with the eciferum in this case he was  l o c i of barley, since  dealing with r e l a t i v e l y large interlocus i n t e r v a l s .  Recently, workers have promoted another p o s s i b i l i t y to account for the recovery of multiple crossovers at unexpected frequencies.  Thus,  the occurrence of successive gonial and meiotic exchange to produce rare multiple crossovers (Whittinghill, 1955;  ( i . e . the Two-Step model), has been postulated  Suzuki et a l . , 1966).  In the present study some  evidence supports this idea, v i z . the r e l a t i v e lack of double compared to t r i p l e exchange tetrads and  the higher levels of recombina-  tion i n those females producing multiple exchange progeny.  However,  examination of the data for individual females f a i l e d to show the clustering phenomenon that would be predicted for the females generating multiples.  Furthermore, the types of double crossover chromo-  somes encountered were not d i s s i m i l a r to those that would be predicted to a r i s e from the d i f f e r e n t types of meiotic t r i p l e exchange tetrads. In most cases, the numbers of the recovered those of the expected (see Table 11).  double crossovers  exceeded  However, the Two-Step model  should be considered as a possible contributive factor to the results of these experiments, p a r t i c u l a r l y since C. Sharpe (personal communication) finds high coincidence values  for proximal recombination near  the centromere of chromosome 2 and claims that gonial exchange i s involved. As previously mentioned, work i n several organisms has indicated that exchange across the centromere i s marked by a lack of positive chromosome interference and, negative  i n the case of chromosome 3 of  Drosophila,  interference has been observed (Morgan e_t a_l., 1925).  In the  40 present study, the occurrence of negative  chromatid interference for  crossing over near the centromere could provide an explanation high negative  chromosome interference that was  detected,  for the  since an excess  of two-strand doubles would generate more double r e l a t i v e to single crossover chromatids thereby i n f l a t i n g coincidence values. regard,  In this  i t i s noteworthy that Strickland (1961) and Bole-Gowda et a l .  (1962) found evidence of chromatid interference i n Neurospora, p a r t i c u l a r l y with respect to centromeric crossing over.  Hawthorne and  Mortimer (1960) have mentioned a similar situation i n yeast. (1956) and  Stadler (1956) had repudiated  Howe  e a r l i e r claims that this  phenomenon occurs i n Neurospora. Welshons (1955) reported  that negative  chromatid interference  could be seen i n the study of crossing over i n short genetic intervals in attached-X chromosomes of Drosophila.  However, Baldwin and Chovnick  (1967) found no chromatid interference in exchange i n compound third chromosomes.  Davis (1974) also f a i l e d to detect chromatid interference  when using the meiotic mutant mei-s332 for the recovery tetrads.  of h a l f -  I t should be mentioned that neither of the l a t t e r 2 studies  examined crossing over i n proximal regions and  therefore chromatid  interference cannot be eliminated as a c h a r a c t e r i s t i c of exchange in these segments of the chromosomes. The demonstration of conversion invoked as an explanation intragenic exchange. Green (1959, 1960)  i n Drosophila  has previously been  for the occurrence of exceptional events i n  For example, i n his study of the white locus,  could account for some exceptional chromosomes by  assuming that rare true double exchange was  involved.  However, i n  41 crossover studies of d i f f e r e n t white-apricot pseudoalleles, four exceptions appeared which could be explained by gene conversion, while no single crossovers were recovered.  Recently, i t has been argued  that recombination and conversion may be manifestations of the same homologous exchange event, p a r t i c u l a r l y i n l i g h t of the findings i n work with maroon-like (Smith e_t al_., 1970) and i n yeast (Hurst et: a l . , 1972), which revealed that half of the convertants were associated with exchange of flanking markers. In this study, conversion must be considered as a possible mechanism for the frequent production of multiple recombinant chromosomes. 2 For example, simple conversion of eg  to i t s wild-type a l l e l e and v i c e  versa, would result i n apparent 3, 4 double crossovers.  Triples i n -  volving 3,4 exchange might be explained by the conversion of eagle accompanied by exchange of either of the most d i s t a l markers.  Extend-  ing this l o g i c , 1, 4, 5 t r i p l e s could r e s u l t from conversions of K i or Ki*~ with a crossover i n region 1, while 1, 2, 4 t r i p l e s could be generated by conversion of ri_ or ri"^" and an exchange i n region 4.  I t must  be emphasized that previous f a i l u r e to detect intergenic exchange events which resemble conversion i n Drosophila i s l i k e l y related to the effects of high positive interference. The absence of interference across the centromere might permit the appearance of such a phenomenon. Indeed, evidence from other organisms suggests  that when t i g h t l y - l i n k e d  markers are studied, crossing over produces multiple exchange chromosomes at inordinately high frequencies (see Calef, 1957; and S^gaard, 1974).  Conversion has been mentioned as a possible contributor.  Crossover  frequencies i n this present work indicate that the region  from in to K i i s p a r t i c u l a r l y small g e n e t i c a l l y .  42  The use of the inverted attached-X chromosome (C(1)M3) a c t u a l l y resulted i n lower coincidence values ( i n most cases), suggesting that fewer multiple r e l a t i v e to single crossovers occurred. demonstration  Previous  of i n t r i n s i c a l l y (Schultz and Redfield, 1951)  and  e x t r i n s i c a l l y (Suzuki and Parry, 1964) mediated recombinagenesis i n Drosophila were marked by decreased  positive interference.  Therefore,  the present data are consistent with the suggestion that conversion may  be contributing to the appearance of multiple crossover chromosomes,  since one would expect recombinagenic agents to e f f e c t similar increases i n the occurrence of true multiple crossovers as well as of s i n g l e s . Green (1975) has reported similar results from his work with this region of the chromosome.  Puro and Nygren (1975) also observed a double  crossover involving radius incompletus  when they were mapping Polycomb.  These workers raise the p o s s i b i l i t y that conversion i s involved. Other support for the conversion-based  explanation for the multi-  ple crossovers i s provided by the i n e q u a l i t i e s of r e c i p r o c a l crossover classes (Table 12),- even though spontaneous reversion of these l o c i not  was  observed. The d i f f e r e n t p o s s i b i l i t i e s discussed above might be distinguished  by using females carrying t h i r d chromosome p e r i c e n t r i c inversions which include a l l of the l o c i used i n this study.  Recombinants recovered  from such heterozygotes would almost c e r t a i n l y a r i s e from even-numbered crossovers, since odd-numbered crossovers would produce inviable progeny bearing extensive duplications or d e f i c i e n c i e s .  Comparisons of  the control frequencies of multiple crossovers with the number of progeny produced by these females should provide information about the o r i g i n of multiple crossover chromosomes.  43 It might also be possible to use meiotic mutants which e f f e c t i n creased levels of non-disjunction but do not a l t e r recombination and i n this way capture half-tetrads to test for r e c i p r o c a l i t y and chromatid interference for crossing over i n proximal regions of this chromosome. However, given the low rates of recombination i n these regions, this would be a formidable project. F i n a l l y , i t i s noteworthy  that the interchromosomal  effects of  C(1)M3 were more marked for the d i s t a l crossover intervals that were examined.  This raises the p o s s i b i l i t y that most of the proximal i n -  creases noted previously (see Lucchesi and Suzuki, 1968) may have occurred near, but not i n heterochromatin.  In fact, since several  lines of evidence suggest that no crossing over occurs within heterochromatin (Baker, 1958; Roberts, 1965; H i l l i k e r , 1975), i t i s possible that this sort of recombinagenesis  i s wholly euchromatic.  It would be interesting to study this further.  For example, one  could compare these effects to the a r t i f i c i a l induction of crossing over, which also occurs p r e f e r e n t i a l l y near the centromere males and females (see Schultz and Redfield, 1951).  i n both  Since i t appears  that both l_t and xl_ l i e within heterochromatin ( H i l l i k e r and Holm, 1975), chromosome 2 would probably be more useful for this  purpose.  This type of approach w i l l t e l l us a great deal about the properties of heterochromatin, p a r t i c u l a r l y with respect to crossing over.  44 CHAPTER 3 A GENETIC STUDY OF THE DEFORMED LOCUS I.  Introduction  Deformed (Dfd) i s a mutation mapping i n the proximal part of chromosome 3 at 47.5.  Dfd mutants express a dominant phenotype re-  s u l t i n g i n ventral and l a t e r a l reduction of ommatidial tissue i n the eyes of the adult.  Concomitantly, tufted vibrissae are often observed.  The penetrance of the eye phenotype  i s variable and sometimes Dfd/+  f l i e s are indistinguishable from wild-type. Recessive a l l e l e s (e.g. r Dfd—) have been described, with a l l e l i s m based on the observation that TO  r  *  r  Dfd/Dfd— f l i e s are phenotypically more extreme than Dfd—/Dfd—.  Pre-  viously, i t had been thought that the Dfd lesion was also l e t h a l when homozygous, although the recovery of aberrant homozygotes at a very low frequency has been reported (Lindsley and G r e l l ,  1968).  This locus i s of particular interest from a developmental standrL point.  A temperature-sensitive a l l e l e (Dfd-—) has permitted the de-  l i n e a t i o n of a TSP for the Dfd gene i n the f i r s t to second l a r v a l instars (Vogt, 1947).  The eye phenotype could be due to the promo-  tion of localized c e l l death within the eye-antennal disc (Fristrom, 1969).  This may explain the frequent occurrence of mirror-image d u p l i -  cations of antennae of Dfd f l i e s .  This i s supported by the observation  that at 29°C, Dfd interacts s y n e r g i s t i c a l l y when heterozygous with a temperature-sensitive Minute, causing l e t h a l i t y by preventing the .formation of eye-antennal structures (see CHAPTER 5).  The occurrence of  extensive p r o l i f e r a t i o n of vibrissae, which i s also diagnostic of the  45  Dfd phenotype, may  be due  to a repatterning of surviving c e l l s within  the disc following c e l l death (see Postlethwait and Schneiderman, 1973). Furthermore, Dfd i n t e r a c t s with ophthalmontera to produce homeotic wing tissue i n the eye  (Ouweneel, 1969).  Thus, further study of this locus  with respect to patterns of c e l l death i n the eye d i s c during development, should prove i n t e r e s t i n g . The c y t o l o g i c a l l o c a t i o n of Dfd has not been determined. genetic l o c a t i o n does not define the chromosome arm resides.  Its  i n which this locus  However, Holm et a l . (1969) found that the synthesis of com-  pound chromosomes heterozygous for Dfd (Dfd/+) from normal homologues, resulted i n the high frequency a s s o c i a t i o n of this gene with the  3R  elements, while no 3L element containing Dfd was  thereby  proving that Dfd i s i n the r i g h t arm  ever recovered,  of this chromosome.  Duncan and Kaufman (1975) synthesized an array of proximal somal aberrations i n 3R by s e l e c t i n g for radiation-induced  chromo-  revertants  of the homeotic mutant Nasobemia (Ns) as w e l l as of the dominant a l l e l e of double sex observable  (dsx^).  Three revertants of the l a t t e r are c y t o l o g i c a l l y -  d e f i c i e n c i e s , with the smallest (dsx^" ^) lacking the R  material i n the 84B-F i n t e r v a l .  3R  A l l three are l e t h a l i n combination  with either of two d i f f e r e n t chromosomes bearing Dfd. r  However, none r  of these d e f i c i e n c i e s exposes the phenotype of Dfd—, that i s , Dfd—/ d e f i c i e n c i e s are wild-type.  Moreover, they were able to show that the  l e t h a l i t y associated with both o r i g i n a l Dfd stocks i s covered by Dp-P92, which includes the. region of 3R between 84D and 85A 2).  They offered two possible explanations  (see Tables 1 and  for these r e s u l t s : ( i ) The  Dfd locus i s a c t u a l l y located i n 84B-F, but Dfd— i s not an a l l e l e of  46  Dfd  ( i . e . the recessive l e t h a l i t y jLs associated with the Dfd locus),  ( i i ) The l e t h a l mutation i s d i s t i n c t from the Dfd locus, and therefore Dfd i s located elsewhere i n proximal 3R and i s v i a b l e when homozygous. A minimal genetic characterization of a locus i s a prerequisite to any analysis of i t s developmental properties.  Therefore,  the pre-  sent study was i n i t i a t e d to accomplish two things: (a) to map Dfd with respect to Kinked i n the hope that their correct r e l a t i v e positions might a i d future genetic studies i n this region of the chromosome; and (b) to g e n e t i c a l l y analyse any recombinant chromosomes derived from this mapping study, i n order to determine which of the aforementioned p o s s i b i l i t i e s concerning  the nature of the locus i s correct.  47  II. One hundred Dfd/Ki p  Materials and Methods females were collected within 40 hours of  P  eclosion and mass mated to homozygous s t i n r i e g K i p males (hence2  P  forth I w i l l refer only to the markers scored, Dfd, K i and p ) i n s i x P  quarter-pint milk bottles (20 females and 20 males per b o t t l e ) .  Every  three days, the parents were transferred to fresh bottles and a f t e r nine days these f l i e s were discarded.  Progeny were scored u n t i l the  f i f t e e n t h day a f t e r the introduction of the parents. ment using Dfd/Ki p  P  females crossed  An early experi-  to Dfd/TM3 males was abandoned  because of poor penetrance of Deformed i n Dfd/TM3 f l i e s . several Dfd p  P  and K i as well as one  However,  recombinant chromosomes from  the l a t t e r were saved for genetic a n a l y s i s .  A l l recombinant and one  parental chromosomes were balanced over either TM3 or CxD and examined for recessive  lethality.  A l l of the lethal-bearing recombinant chromosomes were tested for complementation inter se as well as with the following stocks: the Dfd parental stock; Dfd-rk (kindly contributed by Dr. D. G. Holm); and j • *. i M four deficiency stocks, Ns +  R  2  1  A Antp +  R  2  Adsx D+-R2 dsx D+R5 A  ( a l l kindly  contributed by Dr. T. C. Kaufman, see Table 2 for c y t o l o g i c a l descriptions) .  48  III.  Results  The results of the Deformed mapping experiment are presented i n Table 13.  A total of 5822 progeny was scored and 28 confirmed cross-  overs between Dfd and _p_ were recovered (17 Dfd p P  P  and 11 K i types) .  I n i t i a l l y , 34 progeny were scored as wild-type ( i . e . Dfd^/Ki p ) P  recombinants  but upon subsequent testing, 32 of these proved to be  carrying Dfd ( i . e . were parentals) and the remaining 2 (males) were sterile.  I f the l a t t e r 2 were genuine crossovers, this would argue  that Dfd i s proximal to K i i n 3R (Figure 2a). The computed map d i s tance (excluding the two unconfirmed wildtypes) from K i (or Dfd) to p  P  i s 0.48 percent, a value reasonably close to that predicted from  standard book values (Lindsley and G r e l l , 1968). Fourteen Dfd p  P  and seven K i recombinant  chromosomes were balanced.  Recessive lethals were present on a l l of the former while 6 of 7 of the l a t t e r carried recessive l e t h a l s . stocks including s i x Dfd p p  P  P  In addition, 8 recessive l e t h a l  and one K i recombinants  along with a single  type were found i n the early crossover experiment.  of the JD£ recombinant 2b).  The existence  argues that K i i s proximal to Dfd i n 3R (Figure  For complementation purposes, the lethal-bearing  recombinant  stocks were designated as follows: K i , lethals 1 to 7; Dfd p , lethals P  8 to 27; and  l e t h a l 28.  The results of the complementation tests are summarized i n Table 14 and Figure 3.  Since a l l of the l e t h a l crossover chromosomes were  inviable i n combination with the o r i g i n a l Dfd parental chromosome, a l l must share a l e t h a l s i t e ( s ) i n common with the parental stock. A l l  Table 13 Results of The Cross of Dfd/Ki p  P  Females to K i p / K i p P  P  Males  Progeny Number of Parentals  Genotype Dfd/Ki p  P  Ki p /Ki p P  P  Dfd p / K i p P  Ki/Ki p  Unknowns  Totals  2789  2789  3003  3003  P  P  Dfd /Ki p +  Number of Recombinants  P  17  17  11  11 2 (sterile)  2 5822  Map distance Dfd to j> = 0.48 percent  FIGURE 2  Schematic representation of possible r e l a t i v e arrangements of K i and Dfd i n the proximal portion of the right arm of chromosome 3.  In a, Dfd i s  proximal, K i d i s t a l ; i n b, K i i s proximal and Dfd distal.  o  Dfd  K i +  Dfd'  r Ki  K  r  Dfd  I  L  p  p+  52  i n t e r se combinations of the Dfd p ( p ) stocks as well as those of the P  P  Ki stocks were inviable, indicating that at least one common l e t h a l i s present on a l l of the chromosomes of a given recombinant c l a s s .  How-  ever, when members of the d i f f e r e n t recombinant classes were tested together i n turn, and also each with the Dfd-rk and deficiency stocks, a d i f f e r e n t i a l pattern of complementation emerged (Figure 3). A l though the results are not unequivocal, i . e . the deficiency or Dfd-rk stocks may contain more l e t h a l s i t e s , the simplest explanation i s that the o r i g i n a l Dfd parental chromosome contained a minimum of 3 b  st  c  l e t h a l s i t e s designated as m , m , m , i n addition to the Dfd locus. One possible arrangement of these lethals on the o r i g i n a l Dfd chromosome i s shown i n Figure 4. generate a Dfd p  A single crossover i n region 2 would  recombinant carrying m  (Group IV also including b c  l e t h a l 28) and r e c i p r o c a l l y a K i recombinant carrying both m  and m  (Group I ) . Similarly, a single exchange i n region 3 would provide Dfd p  P  recombinants carrying both m  reciprocals bearing m  (Group I I ) .  2 and 4 could produce Dfd p  P  3  and  (Group III) and the Ki  A double exchange involving regions  chromosomes carrying m  3  accounting for Group V types.  and m , C  thereby  F i n a l l y , i f K i were proximal to Dfd, a  crossover between them could generate a j 3 _ recombinant bearing m (lethal 28, Group IV).  Thus, i t i s proposed that  i s the l e t h a l s i t e  which i s carried by the Dfd-rk chromosome and that this s i t e i s exposed Df"R by both dsx deficiencies. The above proposal s a t i s f i e s the requirement that a l l K i recombinc p ants carry a common l e t h a l (m i n Groups I and II) as do Dfd p types (m  i n both Group III and IV).  and I I I are non-complementing  Furthermore, the members of Groups I  by v i r t u e of the m  3  site.  Table 14 Results lethals  3 4  of I n t e r Se and D e f i c i e n c y Complementation Tests With Recombinant  1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Dfd-  ----•++ _ _ _ +  5  Lethals  +  + +  +  + +  +  + +  +  _ _ _ + + - - .  6 7 8 9 10 11  + +  - - + + + + + + + + . . . -  + +  - -  + + +  +  +  +  + +  + + + + + + + + + + + + + + + +  - +  + -  +  + - -  + + -  + + - + + + + + + + + + +  .  .  .  .  -  .  -  -  -  .  -  -  -  -  " - -  -  -  15 16 17 18 19 20 21 22  -  + +  - " - -  -  -  + -  -  " " .  -  -  " "  " " " "  -. . -  - . - -  23 24  "  25 26 27  . - - - -  .  + + + + +  "  "  "  +  -  -  -  + +  28  +  D4-R2 dsx-—— IH-R5  + + + +-  -+ -+  + -  -  -  -  dsx  + + + +- -+ -+  +  Antpr^+R21 Ns Dfd-par  + + + + + + + ++  +  + + + + + + + + + + + + + + - - - - - - - - - - - -  *Dfd-rk = Dfd r v stock Dfd  +  -  -  +  +  . + + + + + + -  -  12 13 14  + +  par. = o r i g i n a l  +  +  +  +  -  +  +  - +  + -  +  +  - + + + + +-  + + +-  +  +  +  +  +  +  +  + + + + + + - - - - - - -  + -  +  +  +  +  +  +  +  +  + + + + + + + - - - - - - - -  (see Table 2)  Dfd p a r e n t a l chromosome  + -  +  +  +  +  +  +  FIGURE 3  Complementation pattern emerging from inter se and deficiency complementation crosses.  The numbers  1 to 7 represent K i recombinant l e t h a l stocks while 8 to 27 are the Dfd p  P  lethals and 28 i s the  lethal.  a b , c m , m and m represent proposed l e t h a l sites o r i g i n a l l y present i n Dfd parental stock. ^ ^dsx , D+R2 , , D4-R5 Def = dsxand dsx Def-rk = Dfd ry stock I, I I , I I I , IV and V are d i s t i n c t complementation groups.  Dfd  Parental  Dfd-rk  5,6  I  1,2,3,4,7 III  8, 11,12,13,14,15,17,23, 27  IV  V  9,10,16,18,19, 21, 22, 25,26, 28  20 24  FIGURE 4  Proposed arrangement of lethal sites i n proximal 3R i n the o r i g i n a l Dfd/Ki p combination was studied, the l e t h a l s i t e s . possible crossover  P  female i n which re-  m , m  and m  represent  Regions 1, 2, 3 and 4 are intervals.  67  m  Dfd i  _L_  O  a+  —rKi  m'  _i  m  b+  pP  mc+  58 As represented i n Figure 3, a l l Group IV Dfd p viable i n combination with Dfd-rk.  P  recombinants are  However, a l l of the heterozygotes  (except l e t h a l 28/Dfd-rk) displayed a very extreme form of the Deformed phenotype, which suggested that Dfd i s viable when homozygous.  The  v e r i f i c a t i o n of the explanation for l e t h a l i t y of Dfd chromosomes would be the synthesis of Dfd homozygotes and such a stock was isolated i n the following manner:  Dfd p /+ + (the lethal-26 stock was used) females P  were crossed to Dfd p /TM3 males (from the lethal-26 stock, presumably P  carrying only m ) i n bottles and the progeny scored. Dfd p /Dfd p P  m  a+  Dfd p  P  P  Surviving  progeny should a r i s e only from the f e r t i l i z a t i o n of a  recombinant oocyte by a m  3  Dfd p  P  sperm.  Three such re-  combinant f l i e s (one male and two females) and a single JD^ (non-Dfd) individual were scored i n a t o t a l of 2772 progeny. Dfd p  P  Stocks of the three  recombinant chromosomes were produced and individual lines were  established for each. gotes were produced.  In a l l three cases f e r t i l e Dfd p /Pfd p P  homozy-  Figure 5 i s a scanning electron microscope picture  of Dfd p /+ + and Dfd p /Dfd p P  P  P  P  adults.  The l a t t e r c h a r a c t e r i s t i c a l l y  possess extremely reduced eyes (sometimes antennal structures are also missing) with extensively tufted v i b r i s s a e .  The eye phenotype appears  to be even more extreme than that of Dfd/Dfd—.  In addition, mirror  image duplications of the antennae (or i n some cases aristae only) are frequently observed. That the Dfd locus does not appear to be located within the D1"R  cytological i n t e r v a l 84A,B to 85A (which i s delineated by the d s x — deficiencies) i s suggested by the observation that combining the newly isolated Dfd p  P  chromosome with the deficiency chromosomes, does not  expose the more extreme phenotype which i s c h a r a c t e r i s t i c of the homozygote.  v>  FIGURE 5  Scanning electron micrographs showing the eye development of Dfd heterozygotes (a, Dfd  p /+) P  and homozygotes (b, Dfd p /Dfd p ) , (magnification P  about x400).  P  61  IV.  Discussion  This study has shown that the Dfd locus i s g e n e t i c a l l y separable from a l e t h a l s i t e present i n the 84F i n t e r v a l i n the proximal part of 3R of Dfd stocks (Duncan and Kaufman, 1975).  Kinked and Deformed are  very t i g h t l y linked and their r e l a t i v e positions are not d e f i n i t e and await further c l a r i f i c a t i o n through crossover studies.  This should  be  f a c i l i t a t e d through the use of Dfd i n the homozygous condition,thereby eliminating the problem of m i s c l a s s i f i c a t i o n of heterozygotes of incomplete  penetrance.  Thus recombination  using females of the c o n s t i t u t i o n s t i n r i eg unequivocal  because  experiments, for example, 2  Dfd p /Ki,should P  allow  ordering of the two genes i n question and shed further  l i g h t on the nature of proximal recombination  i n general.  The question of the exact functional defect of the Dfd mutation remains unresolved, as does that of the c y t o l o g i c a l l o c a t i o n of the locus.  Deformed may  mutation may  be an amorph (Muller, 1932)  i n that the i n i t i a l  produce the Dfd phenotype and the homozygote would simply  exhibit an extreme version of the phenotype.  A l t e r n a t i v e l y , Dfd  could be an hypomorph, that i s , haplo-abnormal or dosage-sensitive. This i s c l e a r l y possible since i n their study of segmental  aneuploidy  i n Drosophila, Lindsley et a l . . (1972) were unable to synthesize  flies  heterozygous for a deficiency spanning the region 82CD to 83EF, an interval which includes the T r i p l o - l e t h a l segment.  This unique locus  i s l e t h a l i n either a haploid or t r i p l o i d condition. s i t u a t i o n , +/deficiency  In a hypomorphic  would presumably be equivalent to Dfd/+.  The p o s s i b i l i t y that Dfd i s an hypermorph i s more remote, since no Dfd phenotype was  produced i n f l i e s bearing i n t e r s t i t i a l duplications for  62  most of the proximal regions of 3R,and the only region resistant to trisomy was  the 83DE (Tpl) i n t e r v a l (Lindsley et a l . , 1972).  Dfd  could be interpreted as an antimorph, p a r t i c u l a r l y since there is evidence that i t i s dominant i n t r i p l o i d s (Lindsley and G r e l l , 1968). This could be tested d i r e c t l y by determining i f Dfd/+/+ f l i e s are phenotypically less mutant than Dfd/+ i n d i v i d u a l s . F i n a l l y , Dfd could be a neomorph.  I f the l a t t e r is the case, i t  should be possible to induce d e f i c i e n c i e s of the locus as has been done previously (Lifschytz and Falk, 1969; Kaufman, 1975).  Denell, 1973;  Duncan and  However, i t should be noted that such attempts to  revert Ki have not been successful (Duncan and Kaufman, 1975). radiation-induced  If  revertants of either of these mutations are pheno-  t y p i c a l l y indistinguishable from their respective heterozygotes, i t may  not be possible to use such an approach to acquire proximal chromo-  somal aberrations. This present study emphasizes the need to be cautious when one i s assessing the v i a b i l i t y of a given mutation.  Dfd has been known and  studied for more than f i f t y years and a l l reports have assumed that l e t h a l i t y i s a property of the Dfd mutation i t s e l f .  This  cautionary  note i s p a r t i c u l a r l y important i n the case of mutants isolated through the use of chemical or radiation mutagenesis, since their potent mutagenicity enhances the p r o b a b i l i t y that double lesions w i l l be induced in single chromosomes.  The lack of completely correct knowledge about  the v i a b i l i t y of an a l l e l e is apt to mislead workers' attempts to genetically dissect a locus.  Furthermore, the use of lethal flanking  markers i n recombinant systems demands precise knowledge of whether or  63  not an a l l e l e i s l e t h a l .  F i n a l l y , studies of the developmental  effects  of s p e c i f i c genes would be d i f f i c u l t unless their v i a b i l i t y characteri s t i c s are well-defined.  64  CHAPTER 4 A STUDY OF INDUCED CROSSING OVER NEAR THE CENTROMERE OF CHROMOSOME 3 I.  Introduction  The lack of duplications and d e f i c i e n c i e s for s p e c i f i c i n t e r v a l s has been one of the p r i n c i p a l factors l i m i t i n g the complete genetic dissection of Drosophila.  The dosage dependent expression of many  segments of the genome (Lindsley et a l . , 1972), p a r t i c u l a r l y those where the s t r u c t u r a l genes of enzymes have been l o c a l i z e d ( G r e l l , 1962; Stewart and Merriam, 1974; Hodgetts, 1975), has permitted  the cyto-  l o g i c a l mapping of several functions where aberrations which include them e x i s t .  However, segmental aneuploidy,  produced by combinations  of d i f f e r e n t translocations involving the Y chromosome and the autosomes (Lindsley et a l . , 1972), suffers from l i m i t a t i o n s stemming from the variety of disjunctional p o s s i b i l i t i e s .  Therefore, workers have  attempted to i s o l a t e heritably stable aberrations. stable aberrations has permitted  The use of small,  important genetic analyses.  For example,  a study of the i n t e r v a l between zeste and white on the X chromosome led to the conclusion that one functional unit exists i n each polytene chromosome band (Judd e_t al_., 1972) . A recently reported method for the production and recovery of l o calized aberrations involved s e l e c t i o n of crossover chromosomes, where the crossovers were induced by r a d i a t i o n (Mglinets, 1972, 1973).  Since  normal meiotic crossing over occurs i n females, i t follows that crossovers a r i s i n g from i r r a d i a t e d females would include both induced and  65 meiotic types.  However, since no meiotic crossing over occurs i n  Drosophila melanogaster males, a l l exchange chromosomes recovered must be of the induced variety. The question of the o r i g i n of induced crossovers has previously centered on two d i s t i n c t proposals:  (a) i n both sexes, induced ex-  changes r e s u l t from intimate pairing of homologues coupled with precise crossing over (breakage and r e j o i n i n g ) , a s i t u a t i o n analogous to the production of meiotic crossovers i n females; or (b) i n both sexes, induced exchange occurs i n a manner similar to that of the production of translocations and therefore may involve mispairing and d i f f e r e n t breakpoints on each homologue and the potential for asymmetrical exchange. According to (a), induced crossover chromosomes should not be pref e r e n t i a l l y associated with l e t h a l s and/or chromosome aberrations at or near the s i t e s of exchange. several studies.  Evidence for this has been provided by  Patterson and Suche (1934) found that of a t o t a l of 59  t h i r d chromosome crossover progeny of X-irradiated males, only 9 carried recessive l e t h a l s .  Moreover, most of these l e t h a l s were mapped to s i t e s  other than where exchange had occurred.  They suggested that radiation  might promote crossing over i n males by releasing the normal constraints on meiotic crossing over.  Friesen (1937a) found a similar s i t u a t i o n  for crossovers involving both chromosomes 2 and 3 as did w h i t t i n g h i l l (1937) for heat-induced crossovers from males.  Ives and Fink (1962)  found that while only a low frequency of crossover progeny produced by gamma-irradiated males carried recessive l e t h a l s , translocations involving non-crossover chromosomes were r e l a t i v e l y frequent.  Finally,  Raytnayake (1970) found that the majority of crossover chromosomes of progeny produced by formaldehyde-treated males could be homozygosed.  66 Muller (1954, 1958) favoured the second alternative (b) and argued that the production of radiation-induced crossover chromosomes resembles the formation of translocations. In support of this idea, Herskowitz and Abrahamson (1957) found that radiation-induced exchange i n centromeric regions i n X chromosomes of females, conformed to a two-hit k i n e t i c situation.  O l i v i e r i and O l i v i e r i (1964) confirmed this i n males and  also showed that dose fractionation or administration i n a nitrogen atmosphere decreased crossing over, while delivery df radiation i n oxygen enhanced exchange.  Recently, Williamson et a l . (1970) found  that induced exchange involving the fourth chromosomes of females, followed two-hit k i n e t i c s . After studying her own data and those of other workers, HannahAlava (1968) concluded that there i s a c o r r e l a t i o n between r a d i a t i o n induced crossing over and the occurrence of recessive l e t h a l s and dominant s t e r i l i t y , p a r t i c u l a r l y for meiotic broods sampled from male parents.  She also found that crossovers involving intervals outside  the centromeric region (the centromeric region i s loosely defined as that region spanned by the most proximal markers used), were p r e f e r e n t i a l l y recovered i n intermediate broods, while proximal crossovers ( i . e . those occurring within the centromeric interval) were detected i n l a t e r broods and were primarily associated with large c l u s t e r s .  Apparently,  this  brood pattern i s a r e f l e c t i o n of the meiotic and pre-meiotic origins of so-called non-proximal and proximal crossovers, respectively.  She  claims that e a r l i e r studies (e.g. Patterson and Suche, 1934; W h i t t i n g h i l l , 1937)  f a i l e d to compensate f o r the occurrence of crossovers i n clusters  when estimating l e t h a l frequencies amongst crossover stocks.  She  concluded that induced crossovers frequently arise i n a translocationl i k e fashion or less l i k e l y , that exchange chromosomes must somehow be predisposed towards the possession of l e t h a l s i t e s .  67  Mglinets (1972) provided d e f i n i t i v e support for the asymmetricalexchange proposal through his finding that about 20 percent of recombinant third chromosomes from gamma-irradiated  males, possessed  chromosome aberrations (especially duplications and d e f i c i e n c i e s ) . Furthermore, nearly a l l of the aberrations had at least one breakpoint at or near the point of exchange.  The majority of the crossover re-  arrangements were present i n 'meiotic' broods and few occurred i n 'gonial' broods, a finding which p a r a l l e l s the results of Hannah-Alava (1968),  He also found a s i g n i f i c a n t correlation between sites of  chromosome damage and sites of exchange i n recombinant third chromosomes derived from irradiated females.  Thus, his data have raised the  p o s s i b i l i t y that aberrations involving particular regions of the chromosome can be recovered through the selection of appropriate crossover progeny.  i  The aim of this present study was to use radiation to induce crossovers within proximal intervals i n chromosome 3 and to analyse the resulting crossover chromosomes genetically.  I hoped that i t would  be possible to determine i f such a method could provide a source of useful proximal d e f i c i e n c i e s .  68  II.  Materials and Methods  Three separate experiments were performed. 2 st i n r i eg  p Ki p  Heterozygous  s e /+ + + + 4- + males, collected within 30 hours of,  eclosion, were irradiated i n g e l a t i n capsules (the source of radiation was a cobalt-60 Gammacell i n the U.B.C. Chemistry Department) and then 2 p s mass mated to homozygous st i n r i eg  Ki p  e  v i r g i n females (these  virgins were maintained for at least s i x days prior to mating) i n quarter-pint milk bottles bottle).  (using 8-10 males with 10 to 15 females per  At the end of various intervals (depending upon the experi-  mental protocol),  new v i r g i n females along with the irradiated males  were added to fresh bottles.  Wicks of f i l t e r paper were added to each  bottle i n order to maximize freedom of movement and mating a b i l i t y . The mass mating technique, coupled with observed differences  in via-  b i l i t y of female parents, precluded the use of a standardized d e f i n i tive brooding procedure for any of the experiments. Two-hundred forty and 80 males were irradiated with 1000 R (Expt. I) and 2000 R (Expt. II) respectively and test crossed to females for six  successive intervals of 3, 4, 4, 5, 5 and 5 days, for a t o t a l of  26 days. Two-hundred f i f t y males were irradiated with a dose of 3000 R and mated to females for five successive intervals of 3, 3, 6, 5 and 5 days, for a t o t a l of 22 days.  Because the female parents frequently became  mired i n the wet food at the beginning of the third brood i n t e r v a l of this experiment, a new i n t e r v a l was started on the seventh day (the 55 non-crossover progeny recovered i n the short i n t e r v a l were not included in the t o t a l ) .  69  As a control, 90 males of the heterozygous genotype were test crossed for five 3-day i n t e r v a l s . The crossover (between s_t and p ) and non-crossover progeny were P  scored u n t i l the twenty-fourth day a f t e r the parents had been introduced.  Although crossing over was not s t r i c t l y monitored between  g  and e_, several crossovers for this region were noted. 2 A l l crossovers and 30 st i n r i eg  p Ki p  s e /+ + + + + + +  non-  crossover males (the l a t t e r were selected at random from cultures at days 12-22  i n Expt. I l l ) were balanced with one of TM1,  TM3 or CxD and  tested for recessive l e t h a l i t y and each of the crossovers was  coded  according to i t s genotype. Sixteen of the l e t h a l - and semi-lethal-bearing chromosomes (henceforth the mutants w i l l a l l be referred to as lethals unless otherwise s p e c i f i e d ) , collected from these experiments, were tested for complementation inter se and with 4 c y t o l o g i c a l l y - i d e n t i f i a b l e deletions lacking s p e c i f i c proximal segments i n the right arm of chromosome 3 (see Table 2).  In addition, some of the mutants were tested for  complementation  with the following dominant mutations: J?c, K i , Msc,  Q  Sex and Antp ; and for pseudodominant expression of the recessive r v i s i b l e mutations: t r a , pb, Dfd , roe and dsx.  .All of these l o c i are  known to be genetically located i n the proximal regions of chromosome 3 (see Table 1). Salivary gland chromosomes of 11 of the 16 mutant stocks were inspected by Dr. T. C. Kaufman using a standard technique.  70  III.  1.  Results  R a d i a t i o n - I n d u c e d C r o s s i n g Over i n Males The c r o s s o v e r i n t e r v a l s w i l l h e r e a f t e r be r e f e r r e d to as  follows:  2 s t to i_n - r e g i o n 1; i n to r i - r e g i o n 2; rji to eg K i - r e g i o n 4; K i to p_^_ - r e g i o n 5; and r e g i o n 6.  Note t h a t the centromere  i n r e g i o n 4 (see F i g u r e 1 ) .  2 - r e g i o n 3, eg  for reference,  to e ^ -  and p r o x i m a l h e t e r o c h r o m a t i n l i e  H e r e a f t e r , r e g i o n 4 w i l l be known as  P r o x i m a l i n t e r v a l w h i l e r e g i o n s 1, 2, 3 and 5 w i l l be known as the Non-Proximal i n t e r v a l .  S i n c e t h i s study was  r e c o v e r c r o s s o v e r s near the centromere  the  collectively  P  overs may  to  designed to s  and because some p_ to e_ c r o s s -  not have been s c o r e d , data i n v o l v i n g r e g i o n 6 w i l l be c o n s i d e r e d 2 s  only b r i e f l y .  Although phenotypes such as eg , s t o r e_ c o u l d a r i s e  from  mutation a t a low frequency, a l l were s c o r e d as c r o s s o v e r s . Numbers o f progeny and  frequency o f c r o s s i n g  over  The c o n t r o l and e x p e r i m e n t a l c r o s s o v e r data f o r the progeny o f t r e a t e d and u n t r e a t e d males are p r e s e n t e d i n APPENDIX 1. experiments  For a l l t h r e e  i t i s e v i d e n t (APPENDIX l a ) t h a t the s i n g l e most f r e q u e n t  type o f c r o s s o v e r o c c u r r e d i n the P r o x i m a l i n t e r v a l as has been p r e v i o u s l y noted  (see Hannah-Alava, 1968).  of P r o x i m a l c r o s s o v e r s was crossovers  In Expt. I, the t o t a l number  s l i g h t l y l e s s than t h a t o f Non-Proximal  (19 v e r s u s 22), w h i l e f o r both E x p t s . I I and I I I the numbers  of P r o x i m a l types g r e a t l y exceeded those o f the Non-Proximal types (Expt. I I , 13 v e r s u s 3; Expt. I l l , were d e t e c t e d and ( M g l i n e t s , 1972).  63 v e r s u s 12).  Double c r o s s o v e r s  these have been r e p o r t e d i n an e a r l i e r  study  Table 15 D i s t r i b u t i o n of Crossovers i n Proximal and Non-Proximal* Intervals for st t o ^ Exchanges  Crossovers (Crossover Events)** Treatment of Male Parent  Intervals 1  Expt. I 1000 R  11(7)  _3 2(2)  4 20(8)  Expt. II 2000 R  3(3)  -  13(5)  Expt. I l l 3000 R  4(4)  -  61(19)  * Proximal = Exchange i n region 4 Non-Proximal = Exchange i n region 1, 3 or 5 ** Crossover Event = Each cluster counted as one event  5_  3,4  5,6  2(2)  1(1)  9(6)  5(4)  72 APPENDIX lb i s a summary of the t o t a l crossover progeny for the entire st^ to p^_ and p_^_ to e^ regions as well as an estimate of crossing over between s_t and p^_ i n Expt. I I I .  Since i t i s thought that gametic  samples of e a r l i e r broods are mainly post-meiotic ( i . e . with respect to i r r a d i a t i o n , see Hannah-Alava, 1968), the crossover frequency includes only progeny recovered from day 7 to 22 i n c l u s i v e .  Furthermore,  only the progeny of fourteen randomly selected cultures which had produced a minimum of 25 progeny per brood i n t e r v a l , were used for this estimate.  Thus, the estimated frequency of induced crossing over i n  males treated with 3000 R i s 0.59 percent, while the comparable l e v e l for untreated males i s 0.078 percent.  A similar analysis of Puro's  (1966) data gives a crossover frequency of 0.65 percent.  The fact that  he used a single male technique probably accounts for the frequency difference.  Numbers of crossover events Presumably, radiation-induced crossovers can arise either i n germ c e l l s which have stopped d i v i d i n g ( i . e . , those that have reached late gonial or early meiotic stages), or i n germ c e l l s which are s t i l l dividing.  In the former case, one would expect to recover single  crossover progeny of a given type (or of each reciprocal type); while i n the l a t t e r case, one could recover clusters of i d e n t i c a l crossovers (and/or reciprocals) that arose from a single exchange event.  The size  of the c l u s t e r would depend upon the number of gonial divisions occurring after i r r a d i a t i o n . In the present work, the crossover frequency i s low (about 1 i n 150 to 200 progeny).  Therefore, the probability that 2 (or more) pheno-  t y p i c a l l y i d e n t i c a l crossover progeny i n the same culture had arisen from independent crossover events, i s even lower.  Consequently, when  73 2 2 or more of the same (e.g., st i n r i eg ) or reciprocal (e.g., 2 st i n r i eg  p and K i p  s e ) types were seen i n the same culture, they  could be c o l l e c t i v e l y scored as a single crossover event. D  ters were observed, with the largest including 26 K i p  Many clusS  e  progeny i n  two consecutive brood intervals (Expt. I I I ) . Table 15 gives the total numbers of crossover progeny, along with the numbers of crossover events ( i n parentheses) for the three experiments.  These data underscore the fact that i n Expt. I, Non-Proximal  and Proximal crossovers occurred at the same frequency and i n Expts. II and I I I , the Proximal types were scored much more frequently. However, when crossover events are considered, the Non-Proximals are about twice as frequent as the Proximals i n Expt. I, while the d i f f e r e n t i a l between the two types i s markedly reduced i n both Expts. II and I I I (Expt. I I , 13 Proximal:3 Non-Proximal crossovers versus 5 Proximal:3 Non-Proximal crossover events; Expt. I l l , 63 proximal:12 Non-Proximal crossovers versus 19 Proximal:ll Non-Proximal crossover events).  Thus,  i t seems that clustering i s more c h a r a c t e r i s t i c of crossovers occurring within the region which includes heterochromatin than of those occurring outside of this region.  This may indicate that Proximal crossovers are  primarily gonial i n o r i g i n , while Non-Proximal crossovers are not and this would support the findings of Hannah-Alava (1968). Recovery of mutants amongst crossovers Table 16 summarizes the d i s t r i b u t i o n of recessive lethals  (includ-  ing 3 semi-lethal v i s i b l e s ) and putative dominant s t e r i l e s amongst the various types of crossover chromosomes.  The appropriate numbers of  l e t h a l or s t e r i l e events (after correcting for clusters) are included in parentheses.  A total of 16 independently-induced lethals (or semi-  lethals) was recovered amongst 52 tested crossover chromosomes which  Table 16 Regional Summary of Lethals* and Steriles Present on Crossover Chromosomes Experiment Number  _1  Lethals  1(1)*"*  Steriles  2(2)  Lethals  1(1)  Steriles Lethals  _3 -  _4  _5  _6  1(1)  5(3)  1(1)  3,4  Total Proximal  -  1(1)  6(4)  I  Total Non-Proximal"'  1(1)  -  -  -  1(1)  3(3)  -  1(1)  -  -  -  1(1)  1(1)  1(1)  _  _  3(3)  -  3(3)  2(2)  -  2(2)  1(1)  1(1)  II  III  Steriles  -  _  _  _ 2(1) -  _ 1(1)  3(3)  6(6)  1(1)  2(2)  2(2)  * Includes semi-lethals ** Not including i n t e r v a l 6 *** Number i n parentheses = Number of lethal or s t e r i l e events (each c l u s t e r counted as one event)  75  arose from independent  exchanges between sj: and pjj 5 i n Expt. I, 2 i n  Expt. I I and 9 i n Expt. I I I .  The totals show that o v e r a l l , most of  the lethals occurred i n Non-Proximal crossover chromosomes (11 of 16). A t o t a l of 9 of the crossover progeny were s t e r i l e (3 Proximals and 6 Non-Proximals).  Since the mass mating technique makes i t d i f f i c u l t to  define these as genuine s t e r i l e s , they w i l l not be mentioned further. Regional comparison of crossovers and mutants. Table 17 shows the r e l a t i v e occurrence (as percent of total crossover events) of Proximal versus Non-Proximal crossover events for the three experiments  as well as the frequencies ( i n percent) of l e t h a l s  amongst the crossover events. the percentage of independent  The l a t t e r frequencies were based upon crossover stocks which carried lethals  (since s t e r i l e s could not be tested, they were omitted).  These data  (Table 17) reveal that i n Expt. I most of the independent  crossovers  occurred outside the Proximal interval, while this trend was reversed at the higher doses of radiation (although i n Expt. I I only 2 NonProximals were tested since 1 was s t e r i l e ) . tested independent  While 14 percent of the  Proximal crossovers were associated with lethals i n  Expt. I, these frequencies were 20 percent i n both Expts. II and I I I . In contrast, the l e t h a l frequencies for Non-Proximal crossover events were 33.3, 50 and 66.7 percent i n Expts. I, I I and I I I , respectively (in Expt. II only two stocks could be tested). determined  For reference, i t was  that 16.7 percent (5 of 30) of wild-type parental chromo-  somes from day 7 to 22 i n Expt. I l l ,  carried recessive l e t h a l s .  The above results support the idea that there i s a preferential association of l e t h a l i t y with proximal crossovers that are induced i n  Table 17 Relative Occurrence of Crossovers and Lethal Events for Proximal and Non-Proximal Intervals  Crossover Events (Percent of Total) Experiment Number  Proximal  Non-Proximal  Number of Lethal-Associated Crossovers (Percent of Region-Specific T o t a l ) * Proximal  Non-Proximal  I  8(35.0)  15(65.0)  1(14.3)  4(33.3)  II  5(62.5)  3(37.5)  1(20.0)  1(50.0)  III  19(63.3)  11(36.7)  3(20.0)  6(66.7)  *Percent L e t h a l i t y =((number of l e t h a l events)/(number of crossovers - number of steriles)) x 100  the euchromatic portions of the chromosome i n the region between st and p^_.  It would also appear from these results that more induced  crossing over occurs (or at least i s detected) outside of the Proximal i n t e r v a l at 1000 R, while at higher doses of radiation ( i . e . 2000 and 3000 R), this trend i s reversed and Proximal crossover events are more frequently observed.  The question of whether the l a t t e r difference  (between Expts. I, and II and III) i s r e a l or i s due to a r t i f a c t s of the technique, must await more analyses involving precise brood and single male studies.  2.  Analysis of Lethal Stocks  Inter se complementation,  pseudodominance, and additional tests  The results of the inter se and deficiency complementation ing the 16 crossover mutants are shown i n Table 18.  involv-  The mutants have  been coded according to their genotypes and where more than 1 member of a given class occurred as a l e t h a l , each was assigned a d i f f e r e n t 2 number.  The mutant stocks st-3, st-4 and st i n r i eg -4 actually  possessed recessive semi-lethal s i t e s and i n each case less than 10 percent of the expected number of homozygotes survived to adulthood and these were extremely small (about 1/3 to 1/2 of normal s i z e ) . It i s clear from the inter se complementation mutant combinations were v i a b l e .  results that most  The non-complementing exceptions are:  (a) st-1, st-2; (b) st i n r i eg^-2, st i n r i eg^-3; (c) i n r i eg^ K i p 2 2 st i n r i eg -2; and a group of f i v e (d) st-3, st-4, st i n r i eg -4, 2 2 st i n r i eg Ki-1 and st i n r i eg Ki-2. Note that i n groups a and b, non-complementation occurred between d i f f e r e n t l e t h a l s associated with crossovers within the same region, whereas i n group c, crossing over  P  e , S  Table 18 Remits of Inter Se and Deficiency Complementation Crosses InvolvlnR Crossover Lethals  (A)  (B)  (C)  (D) St-4  ( E) In r l e g K l pP e 2  8  (F) s t in r l eg -l 2  (G) st in r l es -2 2  (H) st i n r l en -3 2  st in r i eft -4 z  (J) Kl p e p  (D (K) , st l nr l en K l - l s t i n r l eg Ki-2 2  s  +  (M) P e -l s  P  (N) pP e -2 s  (0) pP e -3 s  2-  (P  +  +  +  +  +  •t-  +  +  +  +  +  +  +  +  +  (A)  +  +  +  +  +  +  +  +  (B)  +  +  +  +  +  +  +  +  (0  _  +  +  +  +  +  -  -  +  +  -  +  +  +  +  +  +  +  +  +  +  +  +  (D)  +  +  (0  +  (F)  +  fo) ,  (H)  -  -  + +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  -  -  +  f  +  +  +  +  +  +  +  -  +  +  +  +  +  +  +  +  +  +  +  +  +  (I)  +  (J) <->  (L) (M) (N)  +  (0) (P) •HUI +R2 DfR2 "&4-R5  +  + +  +  +  +  +  + +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  +  + +  + +  +  +  +  +  -  + + + +  00  79  occurred i n d i f f e r e n t regions for the 2 lethals ( i t i s possible that 2 st i n r i eg -2 carries two lethal s i t e s ) .  Group d presents a hetero-  geneous situation i . e . non-complementation occurred between similar as well as d i f f e r e n t crossovers. 2 l a t t e r group, both st i n r i eg  I t should be mentioned that i n the  Ki mutant stocks were l e t h a l as homo-  zygotes and i n combination with each other, but i n combination with any of the other 3 members of this group, produced  the c h a r a c t e r i s t i c  small f l i e s at low frequencies. Most of the combinations  between the crossover mutants and the  proximal deficiencies were viable (Table 18). Four exceptions to this ,. p s,, EH-R2 p s,, EH-R5 p s ... ,_ +R2 , p s Ki p e /dsx , K i p e /dsx , p e -3/Antp and p e -3/ N s — — S i n c e a l l of these deficiencies lack chromosome material  w  e  r  e  T  between K i and _p_P_, these data reveal that both the K i p  P  e  S  and p  chromosomes possess lethals near or at the sites of exchange. ~f~R21 f a i l u r e of the Ns 2  P  e  S  The  D4"R2 and dsx deficiencies to complement with  st i n r i eg -1 w i l l be dealt with l a t e r . Table 19 represents a summary of pseudodominance and additional complementation tests involving four of the crossover l e t h a l stocks and known proximal mutations were non-mutant.  i n chromosome 3.  Most of the  combinations  However, a l l tra/st-1 progeny were males, thereby  indicating that the st-1 stock f a i l s to complement the tra mutation. D  In addition, p  S  e -3 was  l e t h a l when combined with Msc, Ns  Sex and Antp.  There i s good evidence that Sex and A n t p — are a l l e l e s (see Denell, 1973;  Duncan and Kaufman, 1975), while the r e l a t i o n of both of these  with Msc i s unclear.  F i n a l l y , the Ki/p  e -3 combination was  viable  but the f l i e s displayed an enhanced Kinked phenotype reminiscent of Ki/Ki f l i e s .  80  Table 19 Summary of Pseudodominance and Complementation Tests Between Selected Crossover Mutants and Known Proximal Mutations Proximal Mutants t r a Pc  r  pb  Ki  Dfd—  Msc  Sex Antp  roe dsx  Crossover Mutants st-1  -*+  +  +  +  +  +  +  +  +  2 st i n r i eg -1  +  +  +  + not  +  +  +  +  +  +  Ki p  +  +  +  done  +  +  +  +  +  +  +  e  +  -**  +  +  p  P  P  e  e -3 S  S  * A l l tra/st heterozygotes were males ** Lethal e = K i appeared to be enhanced  +  + -**  -**  81 Cytological analysis The results of the c y t o l o g i c a l analysis of 11 of the 16  crossover  mutants are given i n Table 20 along with some phenotypic descriptions. Eight of the 11 stocks examined displayed no obvious c y t o l o g i c a l d i s ruptions.  However, the remaining 3 were a l l abnormal and displayed  following aberrations:  st-1 = Df(3L)72F-73A;74BC, i n r i e g  2  Ki p  P  the e  S  2 = In(3L)70C;74A, st i n r i eg -1 = Df(3L)79E5-6;80 heterochromatin. Note that a l l of these stocks show low v i a b i l i t y as heterozygotes and 2 i n addition, the basal deficiency (st i n r i eg -1) includes a Minute, probably M(3)LS4 (Lindsley et a l . , 1972).  The extent of the  hetero-  chromatic deficiency of this stock i s not known. The low v i a b i l i t y of 2 -HR21 D+R2 heterozygotes involving the st i n r i eg -1 and the Ns_ or dsx d e f i c i e n c i e s , may each case.  be due to the combined e f f e c t of the d e f i c i e n c i e s i n  It i s worthy of mention that since the recessive l e t h a l i t y of  Pc i s not exposed by this basal deficiency (see Table 19) and because this gene has recently been l o c a l i z e d to the i n t e r v a l between 75A-B to 80 (Puro and Nygren, 1975), these present r e s u l t s further narrow the i n t e r v a l containing Pc_ to 75A-B to 79D or E i n proximal 3L. The fact that st-1 i s a deletion explains i t s lack of complementation with both st-2 and t r a . Thus, i n the case of a l l 3 aberration-bearing  crossover chromosomes,  the s i t e of damage for at least one of the breakpoints the region where crossing over had p  corresponded to  occurred.  u s e -3- and Ki p^ e -bearing f l i e s were weakly viable and  P S  since  genetic evidence supports the idea that the former i s a deletion which Ns exposes the Antp  -Scx-Msc homeotic region as well as K i , i t i s sur-  p r i s i n g that neither of these stocks possess chromosome abnormalities.  Table 20 Phenotypic Description and Cytological Analysis of Crossover Mutants Crossover  Phenotype (other than markers) Low v i a b i l i t y  st--1  -  st--2  Low frequency recovery of very small s t e r i l e homozygotes  st-•3 i n r i eg st i n r i Ki P  P  P  P  P  P  P  I  Ki P  P  s e  2 -2 eg -  s  s -1 e s -2 e s •3 e -  eg'  2  e  st i n r i P  (as heterozygote)  2 eg  Ki-1  _ Df(3L)72F-73A; 74BC +* +  Low v i a b i l i t y (as heterozygote) and infrequent haltere enlargement  In(3L)70C;74A  Minute, low v i a b i l i t y (as heterozygote), low female f e r t i l i t y , thick aristae, rough eyes  Df(3L)79E5,6; 80 heterochromatin  Low v i a b i l i t y , gaps i n wing vein L2  +  Low frequency recovery of very small homozygotes  +  -  +  -  +  Low v i a b i l i t y , sex combs have fewer teeth (5-7) than wild type  +  Slight broadening i n wing vein L2 near t i p s , rough eyes  +  83 Other complementation tests 2 Since the mutant stock eg /TM3  displayed a Delta-like phenotype, i t 2  was  tested with an a l l e l e of DI. 2  thereby indicating that the eg  No DI/eg  heterozygotes were scored,  chromosome carries an a l l e l e of DI.  Tasaka and Suzuki (1973) recovered an EMS-induced ts v i s i b l e mutation on the t h i r d chromosome, which although normal at 22°, when grown at 29° C produced homozygotes that were undersized.  When this mutant  (1 (3)ET* '*"^) was retested and crossed to the st i n r i e g 1S  2  Ki-1 stock,  normal heterozygotes were produced at both 22° and 29° C.  It i s  possible that the small body mutation which i s c h a r a c t e r i s t i c of the inter se complementation group (d) i s due to contamination. 2 the recessive l e t h a l i t y carried by both st i n r i eg  However,  K i members may  be  due to a second s i t e which could s t i l l correspond to the regions of exchange. Of the f i v e recessive l e t h a l s detected amongst the 30  non-crossover  chromosomes tested (from Expt. I l l ) , four were d i s t i n c t ( i . e . two did not complement).  None of these four was exposed by any of the s i x  deficiencies which were at my disposal (the four described i n Table 2 and the two newly synthesized deletions from the present study). In summary:  I detected a t o t a l of 16 associated mutations  amongst 52 independently-occurring crossovers.  Three of the 16  possessed s t r u c t u r a l abnormalities coinciding with the s i t e s of ex2 s 2 change (st-1, i n r i eg K i p e , and st i n r i eg -1) and the lesions p s p s of three additional l e t h a l s (st-2, p e -3 and K i p e ) were cytoP  S  l o g i c a l l y mapped to s i t e s close to or at the regions of exchange.  Thus,  at least 6 of the 16 mutants show correlations between the position of  84  l e t h a l sites and sites of crossing over.  The f a i l u r e of st i n r i eg^-2  2 and st i n r i eg -3 to complement each other may indicate a similar correlation  for these 2 lethal crossover chromosomes, thereby further  increasing this t o t a l to 8 of 16.  85  IV.  Discussion  It i s clear from the present study that s e l e c t i o n of induced  cross-  overs between proximal markers i n i r r a d i a t e d males, can enrich for proximally-located l e t h a l s and more s p e c i f i c a l l y , for aberrations. Furthermore, the more frequent types of aberrations found i n this case were d e f i c i e n c i e s . Since the c y t o l o g i c a l positions of both the aberrations and many of the non-aberrant crossover l e t h a l s were very s i m i l a r to the regions of exchange, these data support  the contention that at  least some of the crossover chromosomes originated from asymmetrical exchange events. The r e s u l t s of this study reveal that crossovers occurring within proximally-adjacent  euchromatic segments are more frequently associated  with mutations, than are crossovers occurring within the more proximal segment which spans the heterochromatin.  This observation i s consistent  with the findings of Hannah-Alava (1968) and Mglinets  (1972).  At least 3 factors which could contribute to the apparent lower s u s c e p t i b i l i t y of proximal mutations are:  exchanges to the concomitant occurrence of  ( i ) r e l a t i v e l y few s i t e s capable of being mutated to  l e t h a l i t y exist i n heterochromatin as compared to euchromatin (Muller et a l . , 1937;  H i l l i k e r and Holm, 1975;  H i l l i k e r , 1976), ( i i ) a mechanism  which exists i n the t e s t i s could allow for the s e l e c t i v e elimination of pre-meiotic stem c e l l s possessing chromosome breaks (Puro, 1966), and ( i i i ) stage-specific differences i n p a i r i n g of homologous chromosomes during spermatogenesis could allow for more complete p a i r i n g of homologues i n gonia and therefore promote r e c i p r o c a l crossing over (HannahAlava, 1968;  Mglinets, 1972).  86  The relevance of ( i ) i s self-evident.  Puro (1966) has found, evi-  dence for a regenerative stem c e l l mechanism i n the testes of Drosophila. Therefore i t i s possible that where breakage of unpaired homologous chromosomes does occur i n stem c e l l s , resulting i n crossovers which involve chromosome damage, these c e l l s would be lethal, thereby accounting for the r e l a t i v e dearth of crossovers i n broods derived from such c e l l s (see Hannah-Alava, 1968).  The results of this present study  corroborate those of e a r l i e r workers i n showing that proximal crossovers are more frequently associated with c l u s t e r s .  Such a selective  mechanism as described above would l i k e l y lead to a situation where fewer proximal crossover chromosomes contained l e t h a l s . F i n a l l y , both Hannah-Alava (1968) and Mglinets (1972) have referred to evidence which suggests that homologous pairing occurs to d i f f e r e n t degrees during gonial stages and meiosis.  Thus, more complete  somatic  pairing prior to meiosis would permit more exact homologous interchange to take place following radiation-induced chromosome breakage, producing a s i t u a t i o n analogous  thereby  to that of meiotic exchange i n females.  The stable deficiencies isolated i n the course of this study are potentially very useful.  Thus, st-1 (Df(3L)st) has permitted the cyto2  l o g i c a l l o c a l i z a t i o n of tra and st i n r i eg -1 (Df(3L)M(3)LS4)  has  further c l a r i f i e d the position of Pc i n the l e f t arm of the chromosome. It i s worthwhile  to emphasize that the l a t t e r deficiency probably lacks  some of the heterochromatin i n 3L, although the extent of this heterochromatic deletion i s unknown.  The existence of such a mutant w i l l be  an important tool for use i n the continuing search for genes within heterochromatin.  87  The induced  crossover technique offers a unique approach for the  study of proximal  regions of chromosome 3.  To increase the resolution  of this method, i t should be possible to construct a genetic whereby induced eliminated.  screen  exchanges would be selected for and non-crossovers  For example, males containing two ts mutations  (spanning  the region of interest) linked i n trans, could be irradiated and crossed to females homozygous for both ts mutations at 22°C.  I f the  r e s u l t i n g cultures were raised at 29°C, only wild-type crossover  pro-  geny would be expected to survive, thereby enriching the selection system for radiation-induced crossover chromosomes.  CHAPTER 5 A GENETIC AND  DEVELOPMENTAL STUDY OF Q-III,  A TEMPERATURE-SENSITIVE MINUTE MUTATION I. The phenotypic suppressor  Introduction  s i m i l a r i t i e s between bobbed, some a l l e l e s of  of forked and Minutes have been used to argue that they a l l  represent defects i n protein synthesis, since protein synthesis is abnormal i n both bb (Ritossa et a l . , 1966a) and 1 ( l ) s u ( f ) (Dudick e_t a_l., 1974;  t s 6 7  8  Lambertsson, 1975b).  No evidence has been presented which unequivocally i d e n t i f i e s a common mode of action for the various Minute l o c i .  In fact, i t has  been proposed that although a l l of these mutants probably do i n h i b i t protein synthesis, mutants of d i f f e r e n t l o c i could be acting at d i f f e r e n t levels i n the overall process  (White, 1974).  Thus, while  some Minute genes could code for d i f f e r e n t iso-accepting species of tRNA, others could code for processing enzymes for tRNA or other molecules,  amino-acyl synthetases, ribosomal proteins or any of the  multitude of structural and  functional components which comprise this  system. The hypothesis 1966b; Atwood, 1968)  that Minute l o c i code for tRNA (Ritossa et a l . , has attracted considerable interest because i t  provides a plausible explanation for the i d e n t i c a l phenotype of many different l o c i .  Since tRNA genes are redundant, this hypothesis also  explains why mutations at M l o c i are often deletions.  Furthermore, the  number of l o c i corresponds roughly to the expected number of tRNA  89  species.  Experimentally, the tRNA-Minute relationship can be explored  by correlating biochemical studies ( v i z . i_n s i t u hybridization, chromatographic i s o l a t i o n and nucleotide sequencing etc.) with genetic tests such as segmental aneuploidy (Lindsley et a l . , 1972) and fine structure mapping. The importance of further genetic study of Minutes therefore, cannot be overemphasized.  In this regard, the recovery of EMS-induced  Minutes (Holden and Suzuki, 1973; J. Stone, unpublished r e s u l t s ; Huang and Baker, 1975) i s p a r t i c u l a r l y noteworthy,as EMS i s assumed to induce single base t r a n s i t i o n s .  The assessment of developmental anomalies  of Minutes which are associated with cytologically-observable deletions, i s fraught with d i f f i c u l t i e s .  However, temperature-sensitive EMS-  induced Minutes which are presumed to be point mutations (Suzuki, 1970), should permit more d e f i n i t i v e and accurate examination of the ontogenic basis of defects produced by such lesions, e s p e c i a l l y during embryogenesis  i n Minute homozygotes.  Moreover,  there is evidence of  the existence of recessive l e t h a l sites which have no dominant v i s i b l e effects and do not complement standard Minute a l l e l e s (A. Datagupta, personal communication).  This presents the p o s s i b i l i t y that Minute  loci  are complex and that the analysis of d i f f e r e n t a l l e l e s and their biochemical properties w i l l provide an insight into the function(s) of Minutes. A s i g n i f i c a n t proportion of EMS-induced mutations i s temperaturesensitive i n Drosophila (Suzuki, 1970).  The relevance of such mutants  to genetic, biochemical and developmental analysis of this organism should considerably expand the scope of Minute investigations.  For  90  example, ts a l l e l e s of Minutes could allow construction of recombination selective systems, thereby increasing the resolving power i n fine structure studies of such l o c i .  Furthermore, i f d e f i n i t i v e  evidence  concerning the primary gene products of Minutes i s forthcoming, existence of a conditional a l l e l e could allow the unequivocal  the  identi-  f i c a t i o n of the potentially thermolabile gene product and i t s _Ln vivo and i_n v i t r o biochemical characterization. Temperature s h i f t experiments during development have provided a wealth of information about the i n t e r v a l ( s ) when the gene product of a given locus i s u t i l i z e d 1970,  1974b).  ( i . e . temperature-sensitive period, see Suzuki,  Furthermore, heat pulse experiments involving ts mutants  or tests of such stocks at middle temperature ranges, have resolved phenotypes that are usually masked by l e t h a l i t y i n orthodox TSP studies (Poodry e_t ajL., 1973;  Tasaka and Suzuki, 1973).  shift  Heat pulse  studies of a ts a l l e l e of a Minute locus with respect to the attenuated b r i s t l e phenotype and TSPs for any other M e f f e c t s , would help to delineate the extent of Minute function during development. I t is noteworthy that i n their search for DTS lethals on chromosome 3, Holden and Suzuki (1973) isolated two mutants, DTS-1  and DTS-6,  which displayed the dominant Minute phenotype at 22°C as well as dominant l e t h a l i t y at 29°C.  Neither could be made homozygous at 22°C.  Owing to s e m i - s t e r i l i t y of DTS-6 females at 22°C, an accurate genetic location could not be found. to map  proximally.  However, the ts dominant l e t h a l i t y seemed  Crossover studies involving DTS-1  placed the M and  dominant lethal phenotypes at an i d e n t i c a l s i t e i n a d i s t a l segment of  91  the  right arm of the chromosome.  Furthermore, the l e t h a l phase (LP)  of DTS-1 homozygotes i s embryonic and this is also true for other known Minutes (see Brehme, 1939; Farnsworth, 1957a,b).  I t appears  therefore that temperature-sensitive Minutes can indeed be induced and recovered. Since the autonomous c e l l - l e t h a l nature of Minutes has been demonstrated (Stern and Tokunaga, 1971) and because many important features of pattern formation i n imaginal discs have been described through the study of X-linked, ts autonomous c e l l - l e t h a l s (Russell, 1974; Simpson and Schneiderman,  1975; Arking, 1975), i t i s l i k e l y that a ts Minute  could provide similar as well as unique information about the development of Drosophila. The existence of a ts mutation that interacts phenotypically with a d i s t i n c t , non-ts mutant has been used to define the time of a c t i v i t y of the non-ts mutant (Dudick et a l . , 1974). su(f)  Thus, the ts a l l e l e of  was used to determine the time of a c t i v i t y of the forked gene.  Since i t i s known that Minutes interact phenotypically with several n o n - a l l e l i c genes (see Schultz, 1929; Lindsley and G r e l l , 1968), a ts Minute might be exploited i n a s i m i l a r fashion and the time of the interaction between the Minute locus and the product of another nonts locus determined. This chapter is a preliminary report on such genetic and developmental studies of a ts Minute, located near the centromere of chromosome 3, which I recovered.  The study was i n i t i a t e d to determine the  potential u t i l i t y of the mutation i n exploring both Minute function during development and i t s interaction with other l o c i .  92  II.  Materials and Methods  The temperature-sensitive Minute, Q-III was  recovered by chance  in a screen for EMS-induced, ts a l l e l e s of a known third chromosome mutation.  The retarded development of Q-III/4- f l i e s at 29°C i n i t i a l l y  resulted i n the m i s c l a s s i f i c a t i o n of this mutant as a l e t h a l a l l e l e of the test chromosome.  Crosses of Q-III with Oregon-R or _ _ / j _ P  in the production of heterozygotes at 29°C.  P  resulted  This led to the prelimin-  ary c l a s s i f i c a t i o n of Q-III as a ts Minute. Henceforth, I w i l l simply refer to the temperature-sensitive Minute as Q-III.  I t should be noted that a l l experiments involve  Q-III linked to j> . P  CxD, and TM3. TM3,  Chromosomes used to balance Q-III include:  A l l references to TM3  throughout  Sb Ser unless otherwise indicated.  these balancers, see Lindsley and G r e l l  TMl,  the text s i g n i f y  For f u l l descriptions of (1968).  Unless specified otherwise, a l l mapping, complementation and other crosses for assessing the properties of Q-III were carried out in quarter-pint milk bottles with standard Drosophila medium.  Ten  pairs of parents were introduced into each bottle and these were usually sub-cultured at least once on fresh medium.  Where tests at  29°C were made, the females were allowed to lay for 1 or 2 days at 22°C before they were removed and then the culture bottles were shifted up to 29°C. A standard method of egg c o l l e c t i o n on p e t r i dishes was used for the developmental Suzuki, 1970).  and some of the genetic studies (see Tarasoff and  Generally, the f i r s t two 2-hour batches of eggs l a i d  were discarded and the t h i r d was used either by counting the eggs and  93 s h i f t i n g the plates d i r e c t l y or more usually, by picking required numbers of eggs along with some of the medium and placing them i n pre-incubated v i a l s or p e t r i dishes for s h i f t s or other analyses. To test for v i a b i l i t y , l e t h a l phases and lengths of developmental periods of Q-III homozygotes and heterozygotes, 22° and 29°C batches of eggs were collected i n two control crosses:  I j3 Ap_ x £ /£ '» P  P  II CxD/TM3 males x ,£ /p_ females; and three experimental P  P  (A) Q-III/TM3 females x £ / £ P  (C) Q-III/TM3 x Q-III/TM3.  P  P  P  crosses:  males, (B) Q-III/TM1 x Q-III/TM1,  The eggs were counted and transferred to  pre-incubated 50mm p e t r i plates on fresh medium (50 to 100 eggs per plate) which were then placed at 17°, 22° or 29°C.  No attempt  made to distinguish between f e r t i l i z e d and u n f e r t i l i z e d eggs 1973) was  during this procedure.  estimated  was  (Wright,  Stage-specific d i s t r i b u t i o n of l e t h a l i t y  for the control and 2 of the experimental  crosses by  inspecting cultures intermittently and computing the proportions of expected  progeny which successfully survived the egg, l a r v a l and  pupal  stages.  In some cases, lengths of developmental periods were estimated  by inspecting the cultures at various intervals and noting the time when at least h a l f of the t o t a l surviving progeny had eclosed, thus providing the period of time (in hours) from oviposition to eclosion.  1.  Genetic Analysis  Genetic mapping Q-IIIp /TM3 males were crossed to Gl Sb H/Payne (see Table 1) or P  2 2 o st i n r i eg /st i n r i eg females at 22 C. st i n r i eg^/Q-IIIp males.  P  F l Gl Sb H/Q-III p  P  and  females were then crossed separately to £ /p_  A l l matings were at 22°C.  P  P  <  In the former cross, six bottles of  94 3-day cultures (3 o r i g i n a l s , then sub-cultured) were kept at 22°C and 6 were transferred to 29°C.  In the l a t t e r cross, the 3 o r i g i n a l s and  3 subcultures were shifted to 29°C, while two more broods were retained at 22°C.  Progeny of a l l bottles were scored u n t i l the twentieth day  a f t e r the parents had been introduced.  As many as possible of the  Q-III-bearing recombinants (or putative multiples) recovered at 29°C, and a l l recombinants from 22°C cultures, were tested to v e r i f y their genotypes with respect to Q-III or other recessive markers.  In addi-  tion, the 22° recombinants were crossed at 29° and 22°C so that their progeny could be scored for other phenotypic t r a i t s associated with the Q-III chromosome.  Complementation of Q-III with other proximal mutations on chromosome 3 Df(3L)M(3)LS4 i s a Minute mutation associated with a c y t o l o g i c a l l y observable deletion i n a proximally-located segment of the l e f t arm of chromosome 3 (see CHAPTER 4 ) .  Thus, males from this stock were crossed  to Q-III/TM3 females to test for complementation at 22° and 29°C. Q-III heterozygotes sometimes display reduced eyes at 29°C.  Since  Ns this i s also a t r a i t c h a r a c t e r i s t i c of Dfd and A n t p — , males from the stocks Dfd p /Dfd p , Ns/Ns and ru h Ki Antp P  P  G  e /TM3 were crossed to S  Q-III/TM3 females to test for complementation at 22° and 29°C. 2.  Developmental Analysis  Tests for s t e r i l i t y and maternal effects Fifteen homozygous Q-III/Q-III females (0 to 24 hours i n age) were i n d i v i d u a l l y mated wi th 5 __ /p_ males i n s h e l l v i a l s and transferred P  P  for four consecutive 2-day broods to fresh v i a l s at 22°C.  Subsequent-  l y , fresh males were added and the cultures exposed to 28°C for  95 two additional 3-day broods. brood.  New males were added for the second 28°C  The v i a l s were l e f t at t h e i r respective temperatures and l a t e r  examined for the appearance of any developmental study involving 15 Q-III/Q-III males was  stages.  A similar  initiated.  To test for any maternal effects, Q-III/Q-III females were mass mated to homozygous _£ /_£ males at 22°C and 322 eggs were collected P  over a 2-hour period.  P  Of these, 102 were shifted to 29°C while the  remaining 220 were kept at 22°C.  A l l eggs were p e r i o d i c a l l y examined  for signs of development. Regular temperature  s h i f t studies  For detailed descriptions of the rationale and experimental procedures for determining TSPs, see Tarasoff and Suzuki (1970) and Suzuki (1970).  For the present study, the beginning of the TSP was  usually  defined as the f i r s t point when a culture that was shifted to the permissive temperature produced s i g n i f i c a n t numbers of mutant animals (or decreases i n v i a b i l i t y ) , and the end of the TSP was defined as the f i r s t point when a culture that was s h i f t e d to the r e s t r i c t i v e temperature produced non-mutant animals (or s i g n i f i c a n t levels of v i a b i l i t y ) . Developmental  stages present i n the cultures were determined either  by inspecting cultures at 12-hour intervals (or i n a few cases, at 6hour intervals) for the duration of development at 22° and 29°C or by scoring one of the cultures at the time of each s h i f t . experiments  Since a l l s h i f t  involved crosses producing a minimum of two classes, at  least 20 (usually 30 to 40) progeny were staged at a given i n t e r v a l . The standard method of scoring l a r v a l mouthparts was used to distinguish between the d i f f e r e n t l a r v a l stages (Bodenstein, 1950).  96 Cultures were shifted from permissive (22°C) to r e s t r i c t i v e temperatures (29°C) and vice versa at 12-hour i n t e r v a l s .  A l l cultures were  inspected every 12 to 18 hours and progeny scored for up to 25 days after the eggs were c o l l e c t e d . (a)  TSP for recessive l e t h a l i t y of Q-III  A t o t a l of 400 to 500 eggs (50 to 60 eggs per v i a l ) was at each 12-hour i n t e r v a l a f t e r o v i p o s i t i o n .  shifted  Developmental stages  reached i n the cultures at the time of s h i f t s were assessed by scoring the  progeny i n one extra v i a l .  Furthermore, a detailed assessment of  stages reached i n cultures kept at 22° or 29°C was provided by inspection at 6-hour intervals during the l a r v a l stages. (b)  TSP for dominant eye and b r i s t l e phenotypes  of Q-III  Since Q-III heterozygotes at 29°C possess rough and less frequently, reduced eyes as well as short and thin b r i s t l e s , the TSPs for these phenotypic t r a i t s were studied.  The reciprocal crosses Q-III/TM3  females x J_ /__ males and Q-III/TM3 males x _ _ / j _ P  P  to provide eggs.  P  females were used  Since results i n the two lines were similar, the  samples were pooled.  A t o t a l of 400 to 500 eggs was shifted (50 to  60 eggs per v i a l ) at each i n t e r v a l . the  P  Developmental stages present i n  samples were determined at the time of s h i f t .  The l a r v a l stages  present were further assessed by 6-hour inspection of p a r a l l e l cultures at 22° and 29°C.  The adult f l i e s were scored for the occurrence of  roughened eye surfaces and b r i s t l e disruptions. (c)  TSP for vg-Q-III i n t e r a c t i o n  The recessive mutation v e s t i g i a l (vg), on chromosome 2 (at 67.0, see Lindsley and G r e l l , 1968) causes a marked reduction of wing s i z e . Since i t i s known that i n the presence of Minutes, vg/-fr f l i e s exhibit  97 wing scalloping  (Green and Oliver, 1940), a vg_ stock was crossed to  Q-III and the resulting 22° and 29°C progeny examined (see the part on Q-III i n t e r a c t i o n s ) .  At 29°C a high frequency of the double heterozy-  gotes displayed nicked wing margins  ( p a r t i c u l a r l y d i s t a l l y ) , while a t  22°C, e s s e n t i a l l y no interaction was apparent. decided to study the TSP of this i n t e r a c t i o n . ments was performed.  I t was therefore Only one set of experi-  Eggs were collected from the cross +/+; Q-III/  TM3 females x vg/vg;+/+ males, and 100 to 200 (50 to 100 per p e t r i plate) were shifted at 12-hour i n t e r v a l s .  P a r a l l e l 22° and 29°C c u l -  tures were examined every 12 hours to determine the developmental stages present.  The adult f l i e s were scored for the scalloped wing  phenotype. (d)  TSP for Dl-Q-III interaction  Since Schultz (1929) reported that some Minutes exhibit lower v i a b i l i t y when combined with d i f f e r e n t a l l e l e s of Delta (see Table 1 and Lindsley and G r e l l , 1968), i t was thought that Q-III might 2 interact s i m i l a r l y with DI.  Preliminary crosses of a JJ1 stock (eg DI  see CHAPTER 4) with Q-III produced no Dl/Q-III heterozygotes at 29°C, while normal heterozygotes survived at 22°C (see Q-III i n t e r a c t i o n s ) . I t was therefore decided to study the TSP of t h i s i n t e r a c t i o n .  Eggs  were collected from the cross Q-IH/TM3 females x D1/TM3 males, and 250 to 300 of these were shifted at 12-hour i n t e r v a l s .  Developmental  stages present at the time of s h i f t were determined by inspecting the culture i n an extra v i a l for each s h i f t at both temperatures.  Q-III/D1  f l i e s could be distinguished by their extremely retarded development at 29°C.  The survival of adult f l i e s was scored at both temperatures.  98  (e)  TSP for Scx-Q-IU  interactions  In testing for Scx-Q-IH  interactions (these were primarily de-  signed to see i f the sex comb phenotype of Sex could be suppressed, see the part on Q-III interactions), i t was discovered that Sex/Q-III heterozygotes exhibit low v i a b i l i t y at 29°C as well as marked scalloping or nicking of the posterior wing margin, while exhibiting no such phenotypic i n t e r a c t i o n at 22°C. this i n t e r a c t i o n .  I t was decided to study the TSP of  Eggs were collected from the cross, Q-III/TM3  females x Scx/TM3 (ru h st r i Sex p  P  e /TM3) males, and 200 to 300 of S  these (50 to 60 per v i a l ) were shifted at 12-hour i n t e r v a l s .  Develop-  mental stages present i n cultures at the time of s h i f t were determined as for (d). The adult f l i e s were scored for the presence of nicks i n the wing margin. I t should be mentioned that the d i s t i n c t i o n between Q-III and other heterozygous progeny classes was considerably f a c i l i t a t e d by the retardation of growth of Q-III larvae at 29°C. Q-III/Q-III homozygotes at 22°C.  This was also true of  Furthermore, both the Q-III homozy-  gotes at 22° and heterozygotes at 29°C frequently possess diagnostic internal melanization which provided an additional marker.  Pulse s h i f t studies Eggs of Q-III/TM1 x Q-III/TM1 matings at 22°C were collected at 2-hour intervals and at various times during development, shifted to 29°C for a period of 24 to 48 hours.  The numbers of eggs tested  varied for d i f f e r e n t intervals (see APPENDICES 2 and 3).  P e t r i plate  cultures were used for the s h i f t s (50 to 100 eggs per plate) and owing to the d i f f e r e n t i a l rate of development of Q-III, no synchronization  99  was attempted.  Several plates were allowed to develop continuously at  22° and 29°C and the developmental stages reached i n these cultures were assessed every 12 hours.  The progeny were scored daily u n t i l at  least 20 to 25 days a f t e r the eggs had been collected.  The numbers of  survivors and the occurrence of various phenotypic t r a i t s were noted. In some cases, imagoes incapable of emerging from the pupal cases, were dissected and inspected. The TSP of a p a r t i c u l a r mutant defect was defined as the developmental interval when exposure to 29°C would e l i c i t the mutant phene i n a s i g n i f i c a n t proportion of the progeny.  Scanning electron microscopy Selected f l i e s were anaesthetized with CO^, mounted on chucks using s i l v e r conductant paint and examined a l i v e i n a scanning electron microscope (SEM, Cambridge Instruments, Cambridge, England). Q-III interactions I t has been found recently that some Minutes suppress the expression of the extra sex comb phenotypes of the various sex comb homeotic mutations (R. Denell, personal communication).  In order to test whe-  ther Q-III i s capable of effecting similar suppression, groups of Q-III/ s TM3 females were crossed separately to ru h Msc e /TM3, ru h st r i Sex p  P  e /TM3 and Pc /TM3 males i n bottles (this and a l l S  3  subsequent Q-III tests involved 5 to 10 bottles at 22°C, sub-cultured to 29°C), and the progeny grown at 22° or 29°C.  The adults were scored  for v i a b i l i t y and the occurrence of v i s i b l e phenotypes,  particularly  the presence of sex combs on the second pair of legs of males.  Prelim-  inary tests revealed that none of the recessive markers present on the  100 test chromosomes (singly or i n combination)  interacts with Q-III.  Henceforth the stocks tested w i l l simply be referred to as Msc,  Sex  and Pc. A l e t h a l i n t e r a c t i o n has been reported for the combination of Lyra and M ( 3 ) h — ( L i n d s l e y and G r e l l , 1968).  To test for a similar i n t e r -  action between Q-III and Ly_, Q-III/CxD females were crossed to Ly/CxD males at 22° and 29°C and the offspring were scored for s u r v i v a l . Tests of other Minutes with homeotics In order to assess interactions of other Minutes with the sex comb homeotics,  Df(3L)M(3)LS4/TM3 males were crossed to females carrying  Sex, Msc or Pc.  Five bottles of each cross were established and a f t e r  3 days, subcultured for another 3 days.  Similarly, M(2)173/SM5 males  were crossed to Sex and Msc i n two bottles which were then each subcultured once a f t e r 3 days.  The adult male progeny of a l l crosses were  scored for the presence of sex combs on the second pair of legs.  Also,  v i a b i l i t y effects and the occurrence of any other phenotypes were noted in the a d u l t s .  101  III.  Results  Only the numbers of eclosing adults were recorded i n the studies of v i a b i l i t y .  In other tests, cultures were examined at least every  24 to 48 hours and the numbers of animals reaching key stages instar larva, pupa and adult) were noted.  (first  The amount of l e t h a l i t y for  each stage was estimated on the basis of the expected frequency of each class, r e l a t i v e to the number of eggs for a given cross.  Simi-  l a r l y , percent v i a b i l i t i e s were calculated using the expected frequencies from the following crosses: £ / £ ; I I 0.5 CxD/p , 0.5 TM3/p . P  P  P  P  Controls, I a l l progeny  Experimentals  (A) 0.5 Q-III/p  P  0. 5 TM3/p ; (B) 0.5 Q-III/TM1, 0.25 Q-III/Q-III, 0.25 TM1/TM1 (egg P  l e t h a l ) ; (C) 0.5 Q-III/TM3, 0.25 Q-III/Q-III, 0.25 TM3/TM3 (egg lethal).  Percent v i a b i l i t i e s were calculated as:  l i v e progeny/Expected  (Observed number of  number of l i v e progeny) x 100, where Expected  number = Expected proportion of eggs x Total number of eggs. 1.  Genetic Analysis  Viability The crosses to assess Q-III v i a b i l i t y (Table 21A,B and C) show that r e l a t i v e to the controls, Q-III i s e s s e n t i a l l y f u l l y viable at 22°C when heterozygous with p_ , TM3 (also at 17°C) or TM1, while at P  29°C the percent v i a b i l i t i e s for these classes were 49, 13 and 0 respectively (where applicable, homozygotes for TMl, TM3 and CxD have been considered as egg l e t h a l s ) .  The exact reason for the low v i a -  b i l i t y of Q-III i n combination with the two balancers i s not known. Two of the inversions present i n TM3 have the following breakpoints  102  Table 21 Relative V i a b i l i t i e s of Q - I I I Homozygotes and Heterozygotes at Different Temperatures  Crosses  Temperature  Number of eggs  Progeny Genotypes  Number of Adults  Percent Viability  151  77.4  149  74.5  CxD/_p_£  38  72.4  TM3/p  42  80.0  CxD/j^  54  74.5  TM3/p  55  75.9  Q-m/Z  309  83.6  TM3/p  291  81.5  Q-IH/Z  192  49.0  TM3/p  329  84.0  Q-III/TM1  256  76.3  44  27.4  Q-III/TM1  0  0  Q-III/Q-III  0  0  Control I  Z/Z  xZ / Z  22°C  195  Z / Z  29°C  200  Z / Z  22°C  105  T\  T"\  Control I I  P  CxD/TM3 x  Z/Z n  29°C  145 P  (A) Q  22 C  739  P  Q-III/TM3 x p / p P  P  n  29°C  783  P  (B) 22°C  642 Q-III/Q-III  Q-III/TM1 x Q-III/TM1 29°C  900  103  Table 21 (continued) Crosses  Temperature  Number of eggs  Progeny Genotypes  Number of Adults  Percent Viability  Q-III/TM3  335  88.8  (C) 17°C  800 Q-III/Q-III  62*  31.0  Q-III/TM3 x Q-III/TM3 Q-III/TM3 22°C  29°C  357  86.6  0-111/Q-III  51  24.8  Q-III/TM3  69  13.4  0  0  825  1034 Q-III/Q-III  *22/62 showed s c u t e l l a r disruptions  104  (see Lindsley and G r e l l , 1968):  In(3LR)79E;100C and In(3LR)76C;93A.  It i s noteworthy that M(3)LS4 resides i n the segment 79D to 80 or 81, while M(3)S34 i s located within the segment 75D to 76C.  Thus, both  proximal Minute l o c i could be under the influence of some sort of position effect which potentiates the l e t h a l effects of Q-III.  Alterna-  t i v e l y , the dominant markers Sb and Ser carried by TM3 could be i n t e r acting with Q-III.  However, the l a t t e r idea i s less l i k e l y , since  tests of TM3 without these dominants resulted i n s i m i l a r low frequencies of Q-III/TM3 progeny at 29°C. breakpoints.  TMl has no comparable inversion  However, i t i s possible that Moire, a recessive l e t h a l  marker carried by this balancer,  interacts with Q-III to produce .  synthetic l e t h a l i t y of TMl/Q-III progeny at 29°C.  This p o s s i b i l i t y  was not pursued. In a l l crosses the heterozygotes which survived continuous exposure to 29°C, displayed small, thin b r i s t l e s (see Figure 10), a roughened eye surface (sometimes reduced or malformed eyes), s l i g h t l y pale body colour and occasionally, leg deformities. In contrast to complete v i a b i l i t y of Q-III heterozygotes, to 31 percent of the expected number of Q-III homozygotes  only 25  (crosses B  and C) survived to adulthood at 22° and 17°C, while none survived continuous exposure to 29°C throughout development.  The homozygous adults  surviving at 22° and 17°C had b r i s t l e s with a thickness that appeared to be intermediate between those of Q-III/+ and +/+ at 29°C.  In addi-  tion, they exhibited s l i g h t l y roughened eyes, pale body colour and frequently, internal melanotic masses ( p a r t i c u l a r l y within the abdomen). At 17°C, one third or more of the homozygotes also displayed a disrupted  105  thorax phenotype.  In the least severe cases this t r a i t consists of  extra or misplaced b r i s t l e sockets on the scutellum and i n the most severe cases, grossly distorted or malformed s c u t e l l a .  The prolonged -  development c h a r a c t e r i s t i c of Minute heterozygotes was also seen for Q-III/+ f l i e s at 29°C and Q-III/Q-III f l i e s at 17° and 22°C.  Clearly  the mutation Q-III i s very p l e i o t r o p i c with a complex of d i f f e r e n t phenotypic e f f e c t s .  Mapping The results of the mapping experiments are presented i n Table 22. They show that Q-III i s located between Gl and _p_ (Table 22a), indeed p  i t i s between s_t and j>  P  (b) .  Owing to the v a r i a b i l i t y i n survival of  Q-III-bearing f l i e s , unequivocal l o c a l i z a t i o n was d i f f i c u l t but a tentative position based on the dominant semi-lethal effect was computed.  Thus, Q-III maps to 45.4 r e l a t i v e to the recessive markers.  The location of Q-III cannot be unambiguously assigned.  Its most  l i k e l y position i s between _st and i n , but the in to jp_ i n t e r v a l cannot P  be completely ruled out. st Q-IIl" " p 1  P  The ambiguity arises from the recovery of  ( i . e . st i n r i +  and st i n Q-III p  P  +  p ) , st i n r i Q-III p P  ( i . e . st i n r i  P  ( i . e . st i n r i p  p —M) recombinants  +  P  at 29°C.  P  —M)  If  Q-III l i e s between s_t and in, the l a t t e r two classes could be generated only by t r i p l e crossovers, and t r i p l e crossovers are again required to explain the occurrence of st Q - I I I in and _p_P.  +  p  P  recombinants,  i f Q-III i s between  In either case, the observation of putative multiple ex-  change classes extends the report that multiple crossovers occur within short genetic intervals i n proximal regions of chromosome 3 ( S i n c l a i r ,  Table 22 Crossover Data From Crosses Designed to Localize Q-III a)  Gl Sb H/Q-III p  Females x  P  • Number of Progeny in Each Class  Genotype/p?  Type  Males  Gl Sb H  22°C  29°C  861  562  748  30  Parentals SCO  1  Gl p  SCO  2  Gl  SCO  3  Gl p^  SCO  4  Gl  SCO  5  Gl p** Sb H  SCO  6  Gl  Sb  DCO  1  Gl p ^ Sb  DCO  2  Gl  +  DCO  3  Gl  +  DCO  4  Gl p  DCO  5  Gl p  DCO  6  Gl  +  p ^ Sb  +  TCO  1  Gl  +  p ^ Sb  +  Totals  Sb  P  p**  +  P  35  Sb H  51  H  +  138  +  Sb H  123  2 (IM)* 25 (IM) 60 4 (M)  +  92  85  +  H  138  1  +  H  2  1  2  1 (M)  r Sb..H  5  2  P  Sb  2  0  P  Sb H  9  0  11  3  1  0  2218  776  p  +  +  Sb  p  +  H  +  P  1  p  P  P + P  1  Sb H  +  +  +  H  H H  +  107  b)  st i n r i eg /Q-III p  Type  Females x st i n r i p / s t i n r i p  Genotype/st i n r i p  Parentals  SCO  1  SCO  2  SCO  3  SCO  4  SCO  5  st i n r i  . + .+ p st i n r i p -+ • • P+ st i n r i p .+ p st i n r i p st i n r i p st  + . + .+ p+ in r i p  Totals Map p o s i t i o n of Q-III:  Males  Number of Progeny i n Each Class  22°C  29°C  1315  1405  1345  402  10 (M) 6  8 (5M) 23  0  1 (M, s t e r i l e )  1  9 (IM)  4 (M)  4 (M)  2681  1852  45.4 map units  M = Minute phenotype (delayed eclosion, thin, small b r i s t l e s )  108  1  1975).  I f Q-III i s located between .st and in,  i t i s surprising that  no Q-III i n r i crossovers were recovered at 22°C. Progeny tests of the recombinants recovered at 22°C (included i n Table 22b) support the suggestion that Q-III l i e s closely linked to in between _st and i_n. I t i s important to mention that a Minute locus exists between s_t and i_n, while two are located between r i and j> ,  one  P  on either side of the centromere. In addition to the i n i t i a l progeny tests, several recombinant chromosomes were cloned at 29° and 22°C. phenotypes  Almost a l l of the p l e i o t r o p i c  (to be referred to l a t e r ) attributable to Q-III, segregated  with the mutation. Two Q-III p  P  Sb  H recombinants that had been generated at 22 C  were used to make stocks of Q-III for use in the s h i f t studies.  Thus,  the marker H was removed from the chromosome v i a crossing over and with i t a second s i t e cold-sensitive mutation that rendered Q-III l e t h a l at 17°C. Test of Q-III i n t r i p l o i d s In a preliminary test of Q-III i n t r i p l o i d s , Q-III/TM3 males were 2 crossed to C(1)RM, y C(1)RM, y  2  sc w  a  a sc w  ec/FM6;3A females i n s h e l l v i a l s and  ec/X;Q-IH/+/+ females isolated at 29°C.  females were scored and none displayed the M phenotypes,  Five such thereby i n d i -  cating that Q-III i s recessive i n t r i p l o i d s . Complementation  tests  The complementation parameter.  data have been converted to a v i a b i l i t y index  Thus, the v i a b i l i t y index, V.I. = Number (Q-IIl/mutant)/  109  Table 23 Relative V i a b i l i t y of Q-III i n Combination With Various Mutations at 22° or 29°C  Mutant V.I.*  22°C Total Progeny  Df(3L)M(3)LS4  1.95  428  0  231  Dfd  1.25  234  0  200  Ns  1.18  804  0.09  246  1.44  814  0.19  38  Antp 0  V.I.  . _ ,„. , . - . _ -r , s Number of Q-Ill/mutant * V.I. ( V i a b i l i t y Index) = - — ,. 2... , ^—-— Number of TM3/mutant TI  J  29°C Total Progeny  110 Number (mutant/TM3).  Remember that at 29°C, a l l Q-III heterozygotes  are half as viable as wild-type and therefore some decrease i n the v i a b i l i t y of heterozygotes i s expected.  However, the V.I. estimates  should not be lower than 0.5, i f Q-III i s viable with these mutations. The results of the complementation Table 23.  crosses of Q-III are shown i n  In each case, Q-III was viable i n combination with the  mutations tested at 22°C, while i t was l e t h a l when combined with either Dfd or Df(3L)M(3)LS4 at 29°C.  Furthermore, Q-III was semiQ  l e t h a l i n combination with Ns (V.I. = 0.09), and Antp- (V.I. = 0.19) at 29°C (although i n the l a t t e r case, very few progeny of any class resulted).  Any uneclosed Q-III/Dfd pupae (at 29°C) which were dissected  showed a complete lack of head  ( i . e . eye-antennal disc) structures,  and sometimes uneverted pigmented  ommatidia within their thoraces.  The phenotype of these f l i e s resembled Arking's (1975) i l l u s t r a t i o n of 1 (1)ts480, a sex-linked ts autonomous c e l l - l e t h a l .  No  Df(3L)M(3)LS4/Q-III pupae were found at 29°C, thereby showing that an e a r l i e r l e t h a l phase exists for these heterozygotes. Since the most l i k e l y map position of Q-III i s d i s t a l to M(3)LS4, their l e t h a l i t y i n combination may r e f l e c t the propensity of Q-III to interact with d i f f e r e n t l o c i such as Dfd, rather than a l l e l i s m .  Fur-  thermore, the r e l a t i v e l y normal v i a b i l i t y of Df(3L)M(3)LS4/Q-III  flies  at 22°C argues against a l l e l i s m .  However, i t should be noted that  ts6 7g 1 (l)su(f)  &  (Dudick est al_., 1974) i s viable when heterozygous with  a deficiency for the su(f) locus at 18° and 25°C, but no heterozygotes of this type survive at 29°C.  Table 24 The Lengths of the Developmental Periods From Egg Deposition to Eclosion i n Different Classes a t Different Temperatures Data From Different Temperatures Genotypes of Parents  Genotypes of Progeny  Total Eggs  17°C Number Eclosing  T 1/2* (hrs.)  Total Eggs  22°C Number Eclosing  195  151  300±6  38  312±6  T 1/2 (hrs.)  Total Eggs  29°C Number Eclosing  T 1/2 (hrs.)  200  149  192±6  54  192±6  Controls  I I . CxD/TM3  CxD/p  P  105  145  TM3/p  42  312±6  55  192±6  Q-III/p*  75  312±6  72  240+6  P  Experimentals A. Q-III/TM3  200 TM3/p  P  335  Q-III/TM3 C. Q-III/TM3 x Q-III/TM3 Q-III/Q-III  600±6  276 87  312±6  125  192+6  180  324±6  10  300±6  500  800 62  672±6  500 53  372±6  = Time i n hours from o v i p o s i t i o n to eclosion of h a l f of l i v e progeny. *T 1/2 (hours)  112  These data underscore the problems inherent i n deciding whether particular mutants are a l l e l i c , since the p o s s i b i l i t y of synthetic l e t h a l i t y should always be considered. 2.  Developmental Analysis  Duration of developmental periods Table 24 summarizes the lengths of developmental periods of controls and Q-III heterozygotes and homozygotes.  This i n t e r v a l was de-  fined as the time ( i n hours) from oviposition (+ 2 hours) to the time when half the progeny of a given class had eclosed (since cultures were observed every 12 hours, each time period had an error of about 6 hours). are  Cross A provides an additional control since TM3/p  P  generated along with Q-III/p  P  progeny  individuals.  The results of cross C show that the Q-III homozygotes took considerably longer to eclose than did the controls, while the Q-IH/TM3 heterozygotes developed a t the same rate as the controls.  At 29°C  only a few heterozygotes survived and the l a t t e r took a long time to eclose r e l a t i v e to the controls. Development of Q-III/p  P  progeny i n cross A i s normal at 22°C  but greatly prolonged a t 29°C.  Thus, i n addition to the conditional  b r i s t l e and eye phenotypes, Q-III/+ heterozygotes also develop more slowly at 29°C than at 22°C.  At 22°C the homozygous individuals take  longer to eclose than do the controls.  Tests for s t e r i l i t y  and maternal effects  Of 15 female homozygotes brooded at 22°C, 5 produced no progeny or eggs while the remainder produced a t o t a l of 139 progeny (an average  113 of  14 o f f s p r i n g per f e r t i l e f e m a l e ) .  the  Examination o f the 28°C v i a l s  few white (and some brown) eggs.  eggs were d e t e c t e d . for  t o 28°C, two o f  above 10 f e r t i l e females d i e d d u r i n g the f i r s t brood, w h i l e the  other 8 produced no progeny. a  Upon s h i f t i n g  revealed  A f t e r the second 28°C brood, no  Seven females that had been mated to p / p P  males  P  f i v e days a t 28°C were d i s s e c t e d i n D r o s o p h i l a R i n g e r ^ s s o l u t i o n  and t h e i r  o v a r i e s examined w i t h a compound m i c r o s c o p e .  abundant i n the seminal r e c e p t a c l e s .  Sperm were  The o v a r i e s c o n t a i n e d degenerate  oocytes which appeared to be heterogeneous f o r e a r l y stages o f oogenesis and i n few cases, p o l y t e n i c n u c l e i were d e t e c t e d . . The l a t t e r were presumably undegenerated nurse c e l l s The study o f f e r t i l i t y  When such homozygotes  eggs were l a i d but f a i l e d  1950).  o f Q - I I I / Q - I I I males was abandoned  o n l y 2 o f 15 showed f e r t i l i t y poor.  (Miller,  a t 22°C, and t h a t were mated t o j 5 / p _ P  fertility P  when  was v e r y  females a t 28°C,  to d e v e l o p , thereby s u g g e s t i n g that both  homozygous males and females a r e c o m p l e t e l y s t e r i l e a t 28°C.  When  homozygous males and females a r e c r o s s e d a t 22°C, white eggs a r e d e p o s i t e d but no development  occurs.  Of the 102 eggs which were produced by homozygous Q-III females (mated to j^/pj' males) and t r a n s f e r r e d  to 29°C, 70 remained white  w h i l e 30 turned dark a f t e r one or two days but development ceased. In the  o n l y 2 cases d i d any l a r v a l development ensue and i n both cases, l a r v a e turned b l a c k and d i e d s h o r t l y a f t e r h a t c h i n g .  Of the 220  eggs kept a t 22°C, 107 e x h i b i t e d v a r y i n g degrees o f d a r k e n i n g , 42 r e mained white and 71 hatched as l a r v a e . as  p h e n o t y p i c a l l y normal a d u l t s .  Of the 71 l a r v a e , 60 e c l o s e d  I t i s worthy o f mention t h a t  eggs  114  which remain white could be either u n f e r t i l i z e d eggs or embryos i n which development was blocked at very early stages.  On the other hand,  eggs which turn dark a f t e r a few days are assumed to be embryonic lethals (Wright, 1973) .  I t therefore appears that Q-III can exert a  ts maternal effect, since eggs produced by homozygous Q-III females mated to normal males are e s s e n t i a l l y incapable of supporting normal development at 29°C, while at least a s i g n i f i c a n t proportion of such eggs incubated at 22°C, develop normally.  Stage d i s t r i b u t i o n of l e t h a l i t y of Q-III homozygotes and heterozygotes Even as early larvae the Q-III/Q-III progeny could be distinguished from the Q-III/TM1 or Q-III/TM3 heterozygotes due to their slower development and internal melanization at 17° and 22°C.  At 29°C, since the  homozygotes never progressed past the f i r s t l a r v a l instar, this dist i n c t i o n was also possible. Most Q-III/pP progeny developed considerably more slowly than p_ / P  TM3 types at 29°C (but not at 22°C) and therefore, Q-IH/p  P  larvae  could be unequivocally c l a s s i f i e d on this basis (as well as on that of internal melanization).  However, some overlap of classes did e x i s t .  For this reason, stage-specific l e t h a l i t y was determined for a l l progeny of the cross of Q-III/TM3 females to j_ /p_ males, without attemptP  P  ing to separate the classes, on the assumption that most ts l e t h a l i t y would be due to the death of Q-III/p individuals which would be reP  flected i n the r e s u l t s . Table 25 i l l u s t r a t e s the results of the analysis of stage by stage d i s t r i b u t i o n of l e t h a l i t y during the embryo, l a r v a l and pupal stages  Table 25 L e t h a l i t y of Control and Q-III - Bearing Progeny at Different Developmental Stages at Various Temperatures Percent Mortality  Controls P/ P CxD/p  Number of Eggs  29°C  22°C  17°C Genotype of Progeny  L  E  Number of Eggs  P  _E  L  P  Number of Eggs  E  L  P  -  195  14.4  5.6  2.6  200  8.5  16.5  0.5  -  105  12.4  4.8  6.7  145  8.3  10.3  6.2  -  300  0.5  5.5  450  21.6  71.9  6.5  -  268  8.7  5.0  250  15.0  23.1  12.1  200  2.0  225  52  48(L1)*  P  CxD/TM3 Experimentals Q-III/TM1 j£/TM3j Q-III/p  P  Q-III/Q-III  100  *L1 = F i r s t l a r v a l instar  3.0  26.0  E = Embryo  40.0  L = Larva  20.5  9.4 44.0  P = Pupa  22.0  0  116  of the controls and of the various progeny a r i s i n g from the Q-III/TM3 x p_ /£ P  P  crosses:  (22° and 29°C); Q-III/TMl x Q-III/TMl (22° and  and Q-III/TM3 x Q-III/TM3 at 17°C  (see Table 21).  A l l Q-III/TMl heterozygotes died when exposed continuously More than 70 percent died as larvae. for Q-III/p  P  progeny at 29°C.  29°C);  to 29°C.  Larval death was also most common  At 22°C, again a high proportion of  Q-III/TMl types died as larvae, while mortality was buted between the three stages for Q-III/p  P  more evenly  progeny.  distri-  In comparison,  Q-III homozygotes died equally as frequently as embryos or f i r s t i n stars at 29°, while at 22°C the l e t h a l i t y shifted more towards the l a t e r l a r v a l and pupal stages with very l i t t l e egg l e t h a l i t y . more, at 17°C l e t h a l i t y of Q-III homozygotes was frequent l a r v a l l e t h a l i t y s t i l l occurred.  c h i e f l y pupal, although  ,Thus, at progressively lower  temperatures, Q-III homozygotes survived to l a t e r stages. Q-III/Q-III pupae at 17° and 22°C contained legs.  This may  Further-  Most of the  pale imagoes with bent  indicate that l e t h a l i t y results from defective  s c l e r o t i z a t i o n of the c u t i c l e which leads to desiccation and lack of the muscular control necessary for eclosion. At 29°C, homozygous Q-III/Q-III larvae died as f i r s t i n s t a r s . though this death was  often not immediate (they sometimes survived for  up to 4 or 5 days), no growth or molting showed internal discolouration and Considerable stages was  Al-  took place.  They eventually  died.  v a r i a t i o n i n patterns of l e t h a l i t y i n d i f f e r e n t  observed i n the controls at a l l temperatures and  l a r v a l death increased upon exposure to higher  temperatures.  generally,  117 Temperature-sensitive periods for l e t h a l i t y of Q-III Percent v i a b i l i t y was computed for a l l cultures on the following basis. 22°,  Since only 25 to 30 percent of Q-III/Q-III progeny survived at  the 22°C l e v e l of v i a b i l i t y (see cross B, Table 21) was normalized  to 100 percent.  Thus, for example, the expected number of Q-III/Q-III  progeny at 22°C would be:  0.274 x (total number of eggs x 0.25).  This kind of analysis was used whenever c a l c u l a t i o n of such percent v i a b i l i t i e s was necessary. The TSP of Q-III recessive l e t h a l i t y can be inferred i n Figure 6. This TSP extends continuously from the l a t t e r part of embryogenesis to  up  the second h a l f of pupation, and therefore the Q - I I I gene product +  is c l e a r l y required throughout most of development. Since long exposures to the r e s t r i c t i v e temperatures can mask successive, but separate TSPs (Poodry ejt ajL., 1973; Suzuki, Kaufman, Falk e_t ajL., 1976), 24 and 48-hour pulse s h i f t s were performed. Although a s i g n i f i c a n t number of Q-III/TM1 heterozygotes usually survived most 48-hour heat pulses, three discrete temperature-sensitive intervals were resolved (Figure 7).  One spans the l a t t e r half of the  f i r s t instar and the f i r s t half of the second instar, another covers most of the third instar, and the t h i r d TSP of l e t h a l i t y spans the middle of pupation.  On the other hand, the Q-III homozygotes appear  to have a single, continuous TSP.  These results re-emphasize  importance of the Q - I I I gene product. +  the  A lack of this substance for  even r e l a t i v e l y short periods of development produces death. Forty-eight-hour heat pulses of homozygotes from the f i r s t to mid second l a r v a l instar, often resulted i n sluggish larvae, many of which did  FIGURE 6  Results of the s h i f t study to delineate a temperature-sensitive period (TSP) for l e t h a l i t y of  Q-III  homozygotes.  given as percent eclosion of that were shifted from 22  The data are  Q-III/QTTII  progeny  to 29 C (closed  c i r c l e s ) , or from 29° to 22°C (open c i r c l e s ) at various times during development.  Temporal  estimates of the d i f f e r e n t developmental stages at 22°C, are indicated below; E = eclosion.  Time of Shift (in Hours) Developmental Stages Egg a y  1  .1st I instar  at 22°C  2nd 1 instar  57d instar  I  Pu~^a~ H  u  p  1  a  E  FIGURE 7  Result of 48-hour pulse s h i f t s to delineate temperature-sensitive periods (TSPs) for l e t h a l i t y of Q-III heterozygotes gotes.  and homozy-  The data are given as percent eclosion  of Q-III/TMl (open triangles) or  Q-III/Q-III  (closed triangles) progeny, that were heat-pulsed for 48 hours, at various times during development (pulses were shorter during embryogenesis).  The  horizontal bars indicate the duration of the heat pulses to 29°C.  Temporal estimates of the  d i f f e r e n t developmental stages of the heterozygotes  (het.) and homozygotes (hom.) at 22°C, are  indicated above and below, respectively; E = eclosion.  Developmental at  I gg E  1st  I  instar  1  2nd  22°C  Stages het.  3rd  I  instar  I  P  u  p  a  instar  125}  Time of  Developmental at 22°C I  ggg  I  1st instar  I  2nd instar  I  Shift (in  Hours)  Stages horn. 3rd instar  I  P  u  p  a  1  122  not grow.  These larvae exhibited d i f f e r e n t degrees of internal d i s -  colouration.  They sometimes survived for several days a f t e r the s h i f t ,  but most eventually died.  Later s h i f t s ( i . e . early to late third i n -  star) frequently produced sluggish larvae that never metamorphosed but some also formed incomplete pupae, while s t i l l others reached various stages of pupation.  The l a t e r s h i f t s (late t h i r d instar and  through-  out the f i r s t h a l f of pupation) produced pupae which reached various stages including the pharate stage, but never eclosed. Q-III/TMl heterozygotes survived 24-hour heat pulses at d i f f e r e n t stages.  However, d i f f e r e n t l e t h a l TSPs of Q-III homozygotes could be  distinguished (Figure 8).  The pattern of TSPs i s s t r i k i n g l y s i m i l a r  to that r e s u l t i n g from 48-hour heat pulses to Q-III/TMl heterozygotes (see Figure 7), although the actual position of each TSP i s d i f f e r e n t . Again, l e t h a l i t y from e a r l i e r pulses was  primarily l a r v a l .  For  example, a heat pulse from 84 to 108 hours of development (early second instar) produced many dead larvae displaying considerable  internal  discolouration (probably diagnostic of generalized disruptions). Some pupae were formed but i n most cases successful metamorphosis did not occur, although a few managed to complete pupation.  Later l a r v a l  pulses produced progressively more advanced development at the time of lethality.  In some cases (for example a pulse from 108 to 132  a f t e r surviving homozygous adults had eclosed and were scored, third instar larvae began to appear i n the cultures and pupated (after several days), but never eclosed. eclose may  hours), other  these eventually  This f a i l u r e to  have been due to poor leg d i f f e r e n t i a t i o n , since the legs  of dissected pharates were often bent i n appearance.  FIGURE 8  Results of 24-hour pulse s h i f t s to delineate temperature-sensitive periods (TSPs) for l e t h a l i t y of Q-III homozygotes.  The data are  given as percent eclosion of Q-III/Q-III progeny that were heat-pulsed  for 24 hours, at  various times during development.  The h o r i -  zontal bars indicate the duration of the heat pulses to 29°C.  Temporal estimates of the  different developmental stages at 22°C are indicated below; E = eclosion.  125  The occurrence of this second wave of pupation raises the i n t e r e s t ing p o s s i b i l i t y that l a r v a l growth can be reversibly blocked  i n homozy-  gous Q-III individuals by a b r i e f 29°C exposure during development. This could be further studied by exposing large numbers of synchronously developing  larvae to 29°C for even shorter intervals, or a l t e r -  natively, by employing very short intervals coupled with higher temperatures (e.g. 30° or 31°C). F i n a l l y , much of the l e t h a l i t y induced  by 24-hour heat pulses  during pupation occurred at the pharate stage, with some of the eclosed f l i e s possessing poorly d i f f e r e n t i a t e d legs.  un-  A l l of these were  l i g h t i n colour and many seemed to have been rapidly desiccated  (as  indicated by collapsed abdomens). Since only about 30 percent of Q-III homozygotes usually survive at 22°C, conclusions about the patterns of l e t h a l i t y should be viewed with caution.  Nevertheless,  i t does appear that 24-hour heat pulses  have resolved three discrete TSPs for i n v i a b i l i t y :  one early i n the  second instar, one i n the late third instar and the early part of pupation, and another just a f t e r mid-pupation. of. pulse survivors w i l l be dealt with  Temperature-sensitive  Phenotypic descriptions  later.  periods for dominant rough eye and b r i s t l e  traits  Since heterozygous Q-III (Q-III/+, Q-III/p , Q-III/TM3) f l i e s P  which survive continuous exposure to 29°C have rough eyes and  short,  thin b r i s t l e s , TSPs for these phenotypes could be defined (Figure 9). The TSP for the eye phene extends from about a third of the way the second instar up to the end of this i n s t a r .  This TSP was  into  also  FIGURE 9  Results of the s h i f t study to delineate temperature-sensitive periods (TSPs) for rough eyes and reduced b r i s t l e s gotes.  of Q-III heterozy-  The eye data are given as percent ex-  pression of rough eyes i n Q-III/p  P  progeny  that were shifted from 22° to 29° (closed c i r c l e s ) , or from 29° to 22°C (open c i r c l e s ) , at various times during development.  The TSP for the  b r i s t l e phene was also provided and i s indicated by the horizontal bar.  Temporal estimates of the  different developmental stages at 22° and 29°C, are indicated above and below, respectively; E = eclosion.  Developmental at  ' E g g ' 1st ' instar  100  —c •  2nd instar  i  Stages 29°C  r  7Z 3rd instar  •••o \•  Pupa  p-O-O-O-O-O-O-O-O-O-O"  o-o-o  \ c oV  75 A  1 ' 11 ( 1 I  >> UJ  £  I  |> 50 0  \  Bristle \  TSP  f  OC  c  9)  o  $  2 5  0.  jo  120  60  iTt instar  180  Time of Shift (in  Hours)  Developmental  Stages  at £99  > t nf  1 — « f  -•• O O - O - O - Q  2ndT r instar  240  »•o•y 300  22°C  3rd instar  Pupa  1  E  128  observed a f t e r 24 and 48-hour pulses (see APPENDICES 2 and 3) and i t overlaps the e a r l i e r TSP for i n v i a b i l i t y that was also resolved by pulse s h i f t s .  It i s noteworthy that this TSP also coincides with the  developmental i n t e r v a l during which intense mitotic a c t i v i t y has been reported for the eye imaginal discs (see Nothiger, 1972).  The  probable  basis for this phenotype w i l l be dealt with l a t e r . The TSP for the b r i s t l e phene was  i n i t i a l l y delineated to a 34-  hour period during the f i r s t h a l f of pupation.  The 24-hour pulse  studies of heterozygous Q-III/TMl progeny allowed  further resolution  of this TSP to a 16-hour i n t e r v a l prior to mid pupation time when yellow pigment i s deposited i n the eyes).  (before the  Moreover, i t was  possible to v e r i f y this TSP i n homozygotes surviving 24-hour heat pulses (although the TSP of the b r i s t l e t r a i t i n the homozygote d i f f e r s somewhat from that i n the heterozygote, see Figure 20).  Scanning  electron micrographs of the b r i s t l e phenotype can be seen i n Figure 10. Note the reduction i n or absence of thoracic macrochaetae i n the homozygote  (c), whereas the phenotype i s less severe i n heterozygotes  The abdominal b r i s t l e s were also s l i g h t l y reduced zygote.  (b).  in size i n the homo-  I t i s noteworthy that the b r i s t l e TSP corresponds  to the pupal  interval during which the thoracic and abdominal b r i s t l e s are formed (Bodenstein, 1950).  Phenotypes revealed by s h i f t experiments Since nearly a l l heterozygous phenotypes observed  i n the s h i f t  experiments (both pulse and regular TSP s h i f t studies) were also seen (and were usually more extreme) i n Q-III homozygotes, only the phenotypes of the l a t t e r w i l l be dealt with i n d e t a i l .  However, reference  FIGURE 10  Scanning electron micrographs showing the effects of Q-III on macrochaete development; (a) a control ( p / p ) f l y , grown at 22°C (magnification about P  P  x400);  /30  FIGURE 10 (continued)  (b)  a Q-III/p  P  heterozygote, grown continuously  at 29 C; (c) a Q-III homozygote, heat-pulsed at 252-276 hours post oviposition (magnification about x400) .  133  w i l l be made to similar phenotypes of heterozygotes, and any unique t r a i t s w i l l be referred to s p e c i f i c a l l y . APPENDIX 2 summarizes the types and frequencies of the phenotypes observed i n surviving homozygotes a f t e r 24-hour heat pulses.  These  w i l l be dealt with on the basis of the individual imaginal discs from which the affected structures are derived. (a)  pattern defects of the eye-antennal disc  In addition to the roughened eye phenotype, heat-pulsed heterozygous and homozygous (and i n the case of heterozygotes from regular s h i f t experiments, those surviving s h i f t s to 29° or 22°C) Q-III f l i e s displayed moderate to severe loss of ommatidial tissue.  Figure 11a, b,  c and d are scanning electron micrographs of such i n d i v i d u a l s .  Note  that there i s considerable ventral displacement of eye tissue i n the heterozygote (a).  High power magnification (b) reveals that although  inter-ommatidial b r i s t l e s are absent i n the displaced portion, they are duplicated for many of the other ommatidia.  These duplications  could be responsible for the rough eye phenotype mentioned e a r l i e r (see Poodry et al_., 1973).  Note also the presence of extra vibrissae  which extend across the eye at three o'clock. In the homozygote, the l e f t eye has been severely reduced (c), while no v i s i b l e reduction of the right eye has taken place.  Thus,  although this reduction frequently can be extreme in'the homozygote, i t does not necessarily always occur for both eyes.  Note also that  this f l y completely lacks the third segment (including the a r i s t a ) of the l e f t antenna.  The highest frequency (83 percent, APPENDIX 2) of  ommatidial deficiencies of homozygotes was induced by a heat pulse at  FIGURE 11  Scanning electron micrographs showing the effects of Q-III on eye development;  (a) a  Q-III/p heterozygote, shifted to 29°C at 24 P  hours post oviposition (magnification about x400); (b) (the same f l y ) , one arrow ( i ) points out a duplicated interommatidial b r i s t l e , while another ( i i ) points out the enlarged vibrissae (magnification about x800);  Z35-  FIGURE 11 (continued)  (c) a Q-III homozygote, heat pulsed at 72-96 hours post oviposition (magnification about x400); (d) (the same homozygote), an arrow points out the second antennal segment on the f l y ' s (magnification about x800).  left  138  96 to 120 hours (this phenotype was also observed i n pulses on either side of this i n t e r v a l ) .  Therefore, the TSP of this phene spans most  of the second and the early part of the third instar:. . Less often, deficiencies and duplications of antennae as well as palps and o c e l l i occurred within the l i m i t s of this TSP.  In two cases where the palps  were absent, the i p s i l a t e r a l antennae were also missing.  Thick, fleshy  aristae were also often induced by heat pulses during this TSP.  Since  a l l of the above structures are derived from the eye-antennal disc, the correspondence i n TSPs for the d i f f e r e n t defects i s not surprising, but the spectrum of phenotypes  i s noteworthy.  Dissection of several pharate pupae resulting from heat pulses at 84 to 120 hours revealed that i n some cases almost no eye-antennal structures had formed i n such pupal l e t h a l s .  In a few cases, severe  deficiencies of the head region were accompanied duplication. this.  by complete antennal  A pulse experiment was i n i t i a t e d to further investigate  Two hundred larvae from a Q-III/TM3 x Q-III/TM3 cross were syn-  chronized i n the early part of the second instar and heat-pulsed from about 100 to 140 hours (mid second to early third instars) post oviposition.  Only four homozygotes eclosed.  Of these, one displayed  extreme eye deficiencies on both sides, while the other three exhibited u n i l a t e r a l antennal duplications along with less severe eye d e f i c i e n c i e s . Figure 12 i s a scanning electron micrograph of one of the l a t t e r . following were observed i n this i n d i v i d u a l :  The  (a) mirror image duplica-  tion of the l e f t antenna along with t r i p l i c a t i o n of the a r i s t a (compare with the normal right antenna and a r i s t a and note also the reduction of the ventral surface of the l e f t eye) and (b) a jointed  FIGURE 12  Scanning electron micrographs showing the eyeantennal  pattern defects of a Q-III homozygote,  heat pulsed at 100-140 hours post oviposition; (a) arrows point out the duplicated l e f t antenna with t r i p l i c a t e d a r i s t a ; note the ventral reduction of the eye; (b) (the same f l y ) , arrows point out the unidentified jointed structure (magnification about x400) .  141  structure of unknown o r i g i n extending from the proboscis and positioned adjacent to the l e f t palp.  Undefined structures have also been fre-  quently observed at the periphery of reduced eyes i n other  experiments  involving Q-III as well as with Dfd (D. S i n c l a i r , personal observations). Of twenty-one dissections of dead pupae present i n the cultures of this heat-pulse experiment,  17 possessed few or no head structures  (in many cases, only the proboscis was  found) and sometimes i n these  individuals, eye pigment globules could be seen within the thorax. This phenotype was also observed tinuously at 29°C.  for Dfd/Q-III heterozygotes grown con-  The other four dead pupae had severely reduced  eyes and, in one case, b i l a t e r a l duplication of the second and third antennal segments (along with the aristae) occurred. Thus, deficiencies of eye-antennal structures ( p a r t i c u l a r l y reduction of the eyes) occur frequently i n Q-III/Q-III f l i e s heat-pulsed for b r i e f intervals during the second i n s t a r . this phene resembles  In i t s most extreme form,  the phenotype of a sex-blinked autonomous c e l l -  l e t h a l , 1(l)ts480, which was  i l l u s t r a t e d and described by Arking  (1975).  In heterozygotes, ommatidial deficiencies resembling (but less severe than) those of Q-III homozygotes were found i n survivors of 48hour pulse s h i f t s (see APPENDIX 3) and survivors of s h i f t s up or down. Again the TSP for this phene appears to occur within the second  larval  instar. (b)  pattern defects of the dorsal mesothoracic  (wing) disc  At least t h i r t y percent of homozygotes that were continuously exposed to 17°C, possessed pattern defects of the thorax, p a r t i c u l a r l y of the scutellum.  As previously mentioned, expressivity of this  trait  FIGURE 13  Scanning electron micrographs showing the effects of Q-III on the development of the scutellum at 17°C (the effects are similar to those produced by heat-pulsing); (a) a p / p P  (magnification  about x400);  P  f l y grown at 22°C  )4S  FIGURE 13 (continued)  (b) a Q-III homozygote (magnification about x400); (c) (the same f l y ) , at a higher power with an arrow pointing out the hairs i n the region of the scar (magnification about x800);  145  FIGURE 13 (continued)  (d) a Q-III homozygote (magnification about x400); (e) (the same f l y as i n d), at a higher power (magnification about x800);  FIGURE 13 (continued)  (f) a Q-III homozygote with an arrow pointing out a duplicated b r i s t l e (magnification about x800).  A l l Q-III homozygotes were grown continu-  ously at 17°C, while (a) was  grown at 22°C.  150  ranged from the appearance of extra b r i s t l e sockets, randomly placed on the scutellum,  to duplicated b r i s t l e s and even b i f u r c a t i o n of the  scutum and prescutum (Figure 13 a,b,c,d,e, and  f) . Note the normal  scutellum and b r i s t l e arrangement on the control f l y (a). Q-III f l y reared at 17°C tellum.  A  Q-III/  (b and c) had marked indentation of the scu-  Higher magnification  (c) shows that disarrangement of the  small s c u t e l l a r hairs has occurred  i n the v i c i n i t y of the scar.  The  f l y shown i n d and e exhibited an analogous disruption, but i t i s uncertain whether this represents a duplication of the posterior or anterior s c u t e l l a r b r i s t l e s .  F i n a l l y , the f l y shown i n (f) possessed  a s i m i l a r s c u t e l l a r indentation, as well as what appears to be a non mirror-image duplication of a posterior s c u t e l l a r b r i s t l e . By heat pulsing Q-III/Q-III larvae, s i m i l a r phenotypes could be induced.  When the pulse was  applied from 108 to 132 hours a f t e r ovi-  position, a l l of the progeny displayed the s c u t e l l a r phenotype, with a minority having only extra b r i s t l e sockets at unusual positions on the' scutellum  (APPENDIX 2).  scutellum were seen.  Very rarely, large deficiencies of the  Their r a r i t y may  r e f l e c t that any more severe  pattern defect i n this disc produces death.  The TSP  for this anomaly  thus encompasses the eye TSP i n early to late second i n s t a r , although the peak for the former was  attained just a f t e r the middle of this  instar. Heterozygotes (Q-III/TMl, Q-III/TM3 and Q-IH/p ) also displayed P  the thoracic pattern phenotype at low frequencies, either when heatpulsed _£ ). P  (Q-III/TMl) or when shifted up during the TSP  (Q-III/TM3, Q-III/  Occasionally, Q-III/TM3 heterozygotes surviving s h i f t s down  possessed wing-like or haltere duplications (Figure 14).  FIGURE 14  A scanning electron micrograph showing a wingl i k e duplication (arrow) i n a Q-III/TM3 f l y that was s h i f t e d from 29° to 22°C at 156 hours post oviposition (magnification about x800).  153  It should be mentioned that the above Q-III-bearing  heterozygotes  which survived s h i f t s up to 29°C also displayed disruption of wing venation, p a r t i c u l a r l y for vein L2.  However, this phenotype was  observed i n homozygotes pulsed to 29°C and no attempt was  not  made to  define i t s TSP. C e l l death within imaginal discs followed by d i f f e r e n t degrees of pattern reconstruction, could provide an explanation above phenotypes. the Discussion. (c)  for most of the  This idea w i l l be more s p e c i f i c a l l y dealt with i n '  thoracic macrochaetae  To r e i t e r a t e , thoracic ( p a r t i c u l a r l y s c u t e l l a r ) macrochaetae were severely reduced i n homozygotes by heat pulses administered f i r s t h a l f of (d)  during  the  pupation.  defects involving the leg discs  Twenty-four hour heat pulses of Q-III homozygotes during late second or early third instars induced a low frequency of either missing legs ( p a r t i c u l a r l y mesothoracic) or legs with shortened t a r s i (APPENDIX 2).  This was also observed i n heterozygotes that had  devel-  oped continuously at 29°C or i n the s h i f t cultures which had been used to define the eye, b r i s t l e and l e t h a l i t y TSPs. Male homozygotes heat-pulsed  from 228 to 276 hours (during pupa-  tion) exhibited gaps i n their sex combs (Figure 15).  Note that the  sex combs appear to be incompletely rotated, i n that neither the upper nor the lower halves of the comb are aligned with the axis of the leg. Although the TSP  for this phenotype corresponds roughly  leg disc eversion and elongation (Bodenstein,  to the time of  1950), the r e l a t i o n s h i p  between these phenomena and sex comb d i f f e r e n t i a t i o n i s unknown.  FIGURE 15  Scanning electron micrographs showing the effects of Q-III on sex comb development; (a) the basitarsus of a normal male ( p / p ) , P  P  grown at 22°C; (b) the basitarsus of a Q-III homozygote, heat-pulsed at 228 to 252 hours post oviposition (magnification about x2000).  156  A fused foreleg phenotype was observed only i n s p e c i f i c s h i f t s down (or heat pulses at s p e c i f i c times during l a r v a l development) i n volving Q-III/TM3 heterozygotes. This phene was marked by a progressive but variable fusion of the forelegs as seen i n Figure 16. At most, only about one third of a l l heterozygotes i n a culture exhibited this phenotype.  Although such fusion was seen i n s h i f t s down and heat  pulses within the i n t e r v a l 84 to 144 hours, i t was never observed i n f l i e s shifted up during this i n t e r v a l (even i n dissections of dead pharate pupae).  Furthermore, although the phenotype was repeatedly  seen i n several independent heat pulse tests using the balancer TM3, SbSer, when a TM3 balancer lacking the _Sb and Ser markers was used, leg fusion was never observed. the  Thus, i t appears that this t r a i t may be  result of some ts interaction between Q-III and these or other  mutant a l l e l e s on the balancer. It i s noteworthy that of the three pairs of discs producing legs, only the pair giving r i s e to the forelegs remain closely juxtaposed throughout development  (Bodenstein, 1950).  The fusion may r e f l e c t non-  autonomous disc overgrowth, resulting from rapid c e l l p r o l i f e r a t i o n at the  medial edges of both discs.  Simpson and Schneiderman (1975)  described a s i m i l a r phenotype for l ( l ) t s 5 4 0 , an X-linked autonomous cell-lethal. (e)  defects involving the genital disc  A variable number of heterozygous (Q-III/TM3 or Q-III/p ) male P  progeny displayed malformation of their external g e n i t a l i a when shifted up or down during development.  This phene involved either incomplete  rotation of the terminalia (see M i l l e r , 1950) or i n extreme cases,  FIGURE 16  Scanning electron micrographs showing the forelegs of: (a) a normal f l y ( p / p ) , grown P  P  at 22°C; and (b) a Q-III/TM3 f l y , shifted from 29° to 22°C at 120 hours post oviposition, an arrow points out the proximal fusion of the legs (magnification about x400).  159  terminalization or lack of the g e n i t a l i a .  The TSP of this phene occurs  during the second and part of the third l a r v a l i n s t a r s . When homozygotes are heat-pulsed, higher proportions (see APPENDIX 2) of males treated during the l a r v a l intervals ( p a r t i c u l a r l y from 108 to 132 hours, 62 percent) exhibited the genital phene. of this phenotype can be more accurately defined.  Thus, the TSP  It lies  predominantly  within the second h a l f of the second instar (although i t spans the entire second as well as extending into the early third instar) and i t corresponds to the TSPs of many of the other phenotypes discussed heretofore.  M i l l e r (1950) claims that c e l l s forming the external male  g e n i t a l i a ( i . e . the terminal abdominal segments) normally undergo rotation during development.  Whether the lack of Q-III gene pro+  duct d i r e c t l y prevents this normal process i s unclear.  Since female  terminalia are not known to rotate, i t i s not surprising that a comparable phenotype was not observed i n female (f)  progeny,  defects involving the abdominal histoblasts  Although no homozygotes survived 48-hour heat pulses at 204 to 252 hours (during pupation),many of the dead imagoes (dissected from their cases) exhibited consistent abdominal anomalies  including  tergite malformation (the segments were missing, uneven, or etched) and incomplete pigmentation.  In a few cases, whole patches of tergites  were missing from the abdomen.  This phenotype was observed at a lower  frequency i n homozygous survivors of shorter heat pulses at 204 to 228 hours.  A few of the dead pharate pupae resulting from a 24-hour pulse  showed tergite deficiencies or etching (see APPENDIX 2).  160  Interactions displayed by Q-III and other Minutes Some Minutes interact with n o n - a l l e l i c mutations to reduce v i a b i l i t y or to enhance the expression of certain phenotypic abnormalities.  As previously mentioned, some of the more well known interacting  l o c i include: Delta (Schultz, 1929), v e s t i g i a l (Green and Oliver, and Lyra (Lindsley and G r e l l , 1968).  1940)  More recently i t has been observed  that Minutes suppress the sex comb effects on the second or third pairs of legs of some homeotic mutations (R. Denell, personal communication). Consequently, possible interactions between Q-III and some of these mutants were tested for temperature-sensitivity.  Although these i n t e r -  actions have both genetic and developmental significance, I have chosen to deal with the results of these tests here. Heterogeneity of genetic backgrounds  i n d i f f e r e n t stocks prevented  a meaningful detailed assessment of differences i n expressivity of the sex comb phenotypes.  Therefore, a l l male f l i e s were scored s t r i c t l y  for presence or absence of the supernumerary  combs (any number of  teeth) on either member of the second pair of legs. Table 26 summarizes the phenotypes and v i a b i l i t y  of heterozygotes  carrying Q-III and DI, V £ , Ly_, Sex, Msc, or Pc^ when raised at 22° or 29°C.  While Q-III/Dl, Q-III/Ly, Q-III/Sex and Q-III/Msc f l i e s were  f u l l y v i a b l e a t 22°C (crosses 1,3,4  and 5), they were either t o t a l l y  inviable (Q-III/Dl, Q-III/Ly) or weakly viable (Q-III/Scx, at 29°C. the  In addition, a l l Q-III/Sex f l i e s displayed a scalloping of  posterior wing margin at 29°C but not at 22°C.  were f u l l y viable at both temperatures Q-III/p  P  Q-III/Msc)  vg/+;Q-III/+ f l i e s  (cross 2, when compared with  at 29°C) and more than 80 percent possessed nicked wing  Table 26 Lethal and V i s i b l e Phenotypes of Heterozygotes for Q-III and Dl, vg_, Ly_, Sex, Msc and _Pc at Different Temperatures V i s i b l e Phenotypes (Percent Expression)  Surviving Progeny Number Parental Genotype  Progeny Genotype  22°C  29°C  22°C  1. Q-III/TM3 and D1/TM3  Q-III/TM3  119  5  normal (100)  M (100)  D1/TM3  121  175  s l i g h t Delta (100)  s l i g h t Delta (100)  Dl/Q-III  127  0  236  172  normal wings (100)  M (100); nicked wings (81.4)  220  359  ebony (100)  ebony (100)  Q-III/CxD  40  4  Dichaete (100)  severe M (100); Dichaete (100)  Ly/CxD  30  139  Lyra; Dichaete (100)  Lyra; Dichaete (100)  Ly/Q-III  50  0  2. +/+;Q-III/TM3  vg/+;Q-III/e  S  n  29°C  II  and s s vg/vg;e /e 3. Q-III/CxD  vg/-£;TM3/e  S  and CxD/Ly  Lyra (100)  Table 26 (continued) V i s i b l e Phenotypes (Percent Expression) Number Parental Genotype  Progeny Genotype  22°C  4. Q-III/TM3 and Scx/TM3  Q-III/TM3  5. Q-III/TM3 and Msc/TM3  29°C  22°C  29°C  249  61  normal (100)  M (100)  Scx/TM3  269  970  *extra sex combs (51.6)  extra sex combs (99.6)  Sex/Q-III  292  10  normal wings (100); extra sex combs (48.2)  M (100); nicked wings (100); extra sex combs (50)  Q-III/TM3  202  7  normal (100)  M (100)  Msc/TM3  197  893  extra sex combs (58.3)  extra sex combs (91.1)  Msc/Q-III  203  102  Q-III/TM3  77  4  Pc/TM3  69  291  Pc/Q-III  83  143  "  (68.4)  M (100): extra sex combs (10)  6. Q-III/TM3 and Pc/TM3  normal (100) M (100)  * Presence of sex combs on second legs of males  extra sex combs (96.4) (80)  extra sex combs (100) M (100); extra sex combs (4.4)  163 margins  ( p a r t i c u l a r l y i n d i s t a l margins) at 29°C, but none displayed  such a phenotype at 22°C.  This ts i n t e r a c t i o n resembles the non-ts  effects of Minutes on vg. reported by Green and Oliver (1940).  N. Dower  (unpublished) discovered that EMS-induced Minutes also enhance' the expression of vg i n heterozygotes. Eight of the 10 Sex/Q-III survivors at 29°C were males and 4 of these males had extra sex combs.  Owing to the small numbers of these  survivors, no further mention w i l l be made of them with respect to the extra sex comb phene. Q-III/Pc f l i e s were r e l a t i v e l y viable at both temperatures.  Amongst  the male progeny of the Q-III/Pc and Q-IH/Msc constitutions, there was a marked reduction i n the frequency of individuals displaying the extra sex comb t r a i t at 29°C compared with that at 22°C (Pc/Q-III: 4 percent, down from 80 percent; Msc/Q-III: 10 percent, down from 68 percent). In summary, Q-III has the following interactions:  (1) ts l e t h a l -  i t y with 1)1 and Ly. (2) reduced v i a b i l i t y with Msc and Sex at 29°C (3) ts scalloping of the wing margin with vg. and Sex and (4) ts suppression of the sex comb phenotypes of JPc and Msc. The ts suppression of homeotics by Q-III prompted an appraisal of combinations involving other Minutes and these l o c i .  To this end,  Df(3L)M(3)LS4 (hereafter abbreviated as M(3)LS4) and M(2)173 were tested with Sex, Msc and, i n the case of the former M, JPc. sults of the crosses performed are shown i n Table 27. mentioned  The re-  I t should be  that M(3)LS4 f l i e s are poorly viable (undoubtedly due to the  deletion), yet M(3)LS4/Scx f l i e s were even less viable, thereby resembling the combination, Sex/Q-III at 29°C.  In contrast, v i a b i l i t y of  Table 27 Interactions of Known Minutes With Different  o t i c Mutations A f f e c t i n g Sex Combs Number Showing V i s i b l e Phenotypes (Percent Expression)  Surviving Progeny  Male Sex Comb Parental Genotype  Progeny Genotype  M(3)LS4/TM3 and Scx/TM3  M(3)LS4/TM3 and Msc/TM3  M(3)LS4/TM3 o and Pc /TM3  Wing s  Number  (Percent) (Viability)  M(3)LS4/TM3  252  (34.5)  0  136 (100)  0  252 (100)  Scx/TM3  357  (48.9)  80(44.4)  100 (55.6)  0  357 (100)  M(3)LS4/Scx  121  (16.6)  3 (3.4)  85 (96.6)  121 (100)  0  M(3)LS4/TM3  129  (15.9)  0  70 (100)  -  -  Msc/TM3  373  (46.1)  140 (70)  60 (30)  -  -  M(3)LS4/Msc  307  (37.9)  21 (12)  154 (88)  -  -  M(3)LS4/TM3  6  (6.5)  0  4  (100)  -  -  4  (16.7)  -  -  15 (88.2)  -  -  Extra  Normal  Scalloped  Normal  Pc/TM3  50  (54.4)  20 (83.3)  M(3)LS4/Pc  36  (39.1)  2 (11.8)  M(2)173/+;TM3/+  63  (21.6)  0  32 (100)  0  63 (100)  SM5/+;Scx/+  82  (28.1)  24 (61.5)  15 (38.5)  0  82 (100)  M(2)173/+;Scx/+  70  (24.0)  11 (29.7)  26 (70.3)  35 (50)  35 (50)  SM5/+;TM3/+  77  (26.3)  0  35 (100)  0  77 (100)  M(2)173/ SM5;+/+ and.+/+; Scx/TM3  Table 27 (continued) Number Showing V i s i b l e Phenotypes (Percent"Expression)  Surviving Progeny  Male Sex Comb Parental Genotype  Progeny Genotype  Number  (Percent) (Viability)  Extra  Wings Normal  Scalloped  Normal  M(2)173/ SM5;+/+ and +/+;Msc/TM3  M(2)173/+;TM3/+ ' SM5/+;Msc/+ M(2)173/+;Msc/+ SM5/+;TM3/+  82  (21.5)  0  39 (100)  -  -  107  (28.1)  51 (76 .1)  16 (23.9)  -  109  (28.6)  20 (37 .7)  33 (62.3)  -  83  (21.8)  0  46 (100)  -  -  -  166 M(2)173/+;Scx/+ f l i e s was not decreased.  Also no s t r i k i n g v i a b i l i t y  effects were seen for M(3)LS4/Msc, M(2)173/+;Msc/+ or M(3)LS4/Pc progeny (although the cross which produced  the l a t t e r yielded  few  progeny from 4 c u l t u r e s ) . Both M(3)LS4/Scx and M(2)173/+;Scx/+ f l i e s displayed a wing margin phenotype c h a r a c t e r i s t i c of Q-III/Sex survivors at 29°C, with penetrance levels of 100 and 50 percent respectively.  Expressivity  of this phenotype was also greater i n progeny of the former c l a s s . It i s clear from the data (Table 27, columns 5 and 6) that M(3)LS4 suppresses expression of the sex comb phenotypes of Sex and Msc.  Thus,  44 percent of Scx/TM3 males had extra sex combs while only 3 percent of Scx/M(3)LS4 d i d .  Similarly, while 70 percent of Msc/TM3 males had  extra sex combs, only 12 percent of M(3)LS4/Msc types d i d .  Penetrance  of _Pc i s also affected, with more than 80 percent of Pc/TM3 males having had the mutant phene, while only 12 percent of M(3)LS4/Pc displayed i t .  These results indicate that the other third chromosome  Minute acts just l i k e Q r l l l .  However, i t should be remembered that  Q-III might be an a l l e l e of M(3)LS4. The second chromosome Minute, M(2)173 also suppressed the sex comb t r a i t s of Msc and Sex (Table 27, columns 5 and 6).  Thus, while 62 per-  cent of the SM5/4-; Scx/+ males had extra sex combs, only 30 percent of M(2)173/+;Scx/+ males d i d .  Further inspection shows that 76 per-  cent of the SM5/+;Msc/+ males showed the t r a i t , compared to 38 percent of M(2)173/+;Msc/+ males.  While suppression of Ms_c and Sex by  M(2)173 i s less e f f i c i e n t than by M(3)LS4, i t i s nevertheless apparent that Minutes  i n general have a similar e f f e c t .  Furthermore,  Minutes  167  appear to interact with Sex to produce a phenotype which i s characterized by scalloping of the posterior wing margin i n both males and females.  This further strengthens the c l a s s i f i c a t i o n of Q-III as a  genuine Minute mutation. The existence of interactions between Q-III and vg_, DI and Sex suggested the p o s s i b i l i t y that s p e c i f i c TSPs could be determined for these interactions. for  Consequently, regular s h i f t studies were i n i t i a t e d  this purpose. (a)  vg-Q-III wing scalloping  vg/+;Q-III/+ progeny a r i s i n g from the cross +/+;Q-III/TM3 x vg/vg; +/+.were scored for wing scalloping a f t e r s h i f t s (Figure 17).  I f the  beginning of the TSP i s taken at the point where a s h i f t down f i r s t gives mutant f l i e s , second instar.  then this i s c l e a r l y about or just a f t e r mid-  The end of the TSP should coincide with the point where  a s h i f t up f i r s t yields non-mutants.  These data indicate a very short  TSP that terminates about two-thirds of the way through the second instar.  Harnly (1936) exploited the suppression of the vg_ phenotype  by high temperature to determine that the "temperature-effective period" for  this gene extends from late second (about the second molt) to the  early third instar, a l a t e r TSP estimate than that provided by this present study. (b)  Dl-Q-III v i a b i l i t y  Adult progeny a r i s i n g from the cross Q-III/TM3 x D1/TM3 were counted a f t e r s h i f t studies (Figure 18).  I t can be seen that the re-  ciprocal s h i f t s are not symmetrical, i n that i n the s h i f t s down, a gradual reduction i n v i a b i l i t y of DI/Q-III f l i e s occurred i n cultures  FIGURE 17  Results of the s h i f t study to delineate a temperature-sensitive period for the vg-Q-III interaction.  The data are given as percent  expression of wing nicking i n vg/Q-III progeny that were shifted from 22° to 29°C (closed c i r c l e s ) , or from 29° to 22°C (open c i r c l e s ) , at various times during development.  Temporal  estimates of the d i f f e r e n t developmental stages at 29° and 22°C, are indicated above and below, respectively; E = eclosion.  Developmental Stages at 29°C  Time  J  ___ ____^ i  I rr„„ I 1st I  of Shift (in  Developmental at 22°C  2nd  I  3rd  Hours)  Stages  T  FIGURE 18  Results of the s h i f t study to delineate a temperature-sensitive period for the Dl-Q-III interaction.  The data are given as percent  eclosion of Dl/Q-III progeny that were shifted from 22° to 29°C (closed c i r c l e s ) , or from 29° to 22°C (open c i r c l e s ) , at various times during development.  Temporal estimates of the d i f f e r e n t  developmental stages at 29° and 22°C, are i n d i cated above and below, respectively; E = eclosion.  Developmental Stages Egg  at 29°C I 3rd  I 1st I  2nd instar instar  Pupa  instar  100- •O-Q  75 A  O O  c o o 50u  Ul  c o o  I 25• c60» e  /20  180  o  t>.  240  300  Pupa  1  Time of Shift (in Hours)  Egg 1  Developmental Stages at 22°C 1st I 2~nd T 3rd T instar instar instar  E  172 shifted progressively l a t e r , while i n the s h i f t s up, normal v i a b i l i t y was  attained f a i r l y r a p i d l y .  In this case, i f the beginning of the  TSP i s a r b i t r a r i l y taken as the point where 75 percent  of DI/Q-III  progeny survived s h i f t s down (since s h i f t s down thereafter generally produced more l e t h a l i t y ) and s i m i l a r l y , i f the end of the TSP i s set at the point where a s i g n i f i c a n t increase i n v i a b i l i t y followed a s h i f t up, then an estimate of the TSP would be from about mid second to mid  or late third i n s t a r .  One  of the problems encountered when c o l l e c t i n g data for the  above experiments was  that Dl/Q-III progeny which emerged i n cultures  that were shifted down at 12-hour i n t e r v a l s from 92 to 192 hours (post oviposition), did so i n a bimodal fashion i n that, i n i t i a l l y , for a given s h i f t only a few of the f l i e s eclosed, but a f t e r 3 or 4 days the bulk of the survivors were scored.  This observation, along with  the fact that DI/Q-III larvae showed extremely slow development at 29°C, indicates that even though prolonged exposure of these heterozygotes to 29°C greatly retards their growth, i t does not necessarily k i l l them. Most of the l e t h a l i t y of Q-III/Dl at 29°C was  i n cultures kept  continuously  l a r v a l , although a few dead pharate individuals were seen  amongst a s i g n i f i c a n t number of heterogeneous (with respect to development) pupae.  Schultz (1929) mentioned that some M-Dl  died mainly as larvae. some of the Q-III/Dl  combinations  Although the Dl wing phenotype was  severe i n  individuals surviving s h i f t s down during the  no consistent pattern emerged.  TSP,  Possibly a more precise TSP could be  derived from studying Q-III-mediated enhancement of the wing phenotype,  173  using other DI a l l e l e s that are less susceptible to Q-III-induced lethality. (c)  Scx-Q-III wing scalloping  Wings of Scx/Q-III survivors from the cross Scx/TM3 x Q-III/TM3 were scored  for the presence of nicks and the results are shown i n  Figure 19. I t should be mentioned that the pattern of semi-lethality of Sex/Q-III individuals i s s l i g h t l y e a r l i e r than the TSP for wing nicking.  However, this l e t h a l i t y did create a problem.  For example,  few Sex/Q-III f l i e s survived s h i f t s up at 24, 60 and 72 hours and none survived s h i f t s up at 12, 36, 48 and 84 hours.  Nevertheless, the  s h i f t s up immediately preceding the end of the TSP (96, 108, and 120hour s h i f t s ) produced s i g n i f i c a n t numbers of Scx/Q-III f l i e s , a l l of which displayed  the scalloped phenotype.  the TSP for this interaction i s confined  nearly  I t can be seen that  to a small developmental  period i n the early part of the third i n s t a r . Qualitative differences i n expression  of this phenotype were  noted, e s p e c i a l l y for s h i f t s down at various times during the TSP. For example, many of the Scx/Q-III survivors from e a r l i e r s h i f t s down showed anterior and/or posterior scalloping, while those shifted down progressively l a t e r (including the 132-hour s h i f t ) exhibited d i s t a l to proximal i n c i s i o n s i n the wing blade.  Indeed, some progeny pheno-  t y p i c a l l y resembled f l i e s carrying a less extreme form of a p t e r o u s — (see Lindsley and G r e l l , 1968). Scx-ts67 interactions Since wing scalloping was displayed by f l i e s carrying Sex along with Q-III, M(3)LS4 or M(2)173, i t could be postulated  that i t i s a  FIGURE 19  Results of the s h i f t study to delineate a temperature-sensitive period for the Scx-Q-III interaction.  The data are given as percent ex-  pression of wing nicking i n Sex/Q-III progeny that were shifted from 22° to 29°C (closed c i r c l e s ) , or from 29° to 22°C (open c i r c l e s ) , at various times during development.  Temporal  estimates of the d i f f e r e n t developmental stages at 29° and 22°C, are indicated above and below, respectively; E = eclosion.  176 r e f l e c t i o n of some general metabolic or developmental effect of Minutes.  In that case, other non-Minute mutations such as the ts su(f) ts67g  allele, l(l)su(f)  &  (henceforth known as ts67) (Dudick e_t a l . ,  1974) with similar b i o l o g i c a l a c t i v i t y , might also interact with Sex. To test t h i s , a stock of ts67 (the X chromosome also bore a suppressg  i b l e a l l e l e of forked (f ) ) was obtained from T. Wright, and males of the genotype v f  su(f)/Y;Scx/+ were constructed. These were crossed  to ts67/ts67;+/+ females i n bottles and the progeny allowed to develop at either 28° or 22°C (3 bottles at each temperature).  Since ts67/Y  males and ts67/ts67 females die at 30°C (Dudick e_t al_., 1974), cultures were maintained at 28°C i n the hope that the males might survive, but at the same time display the 'deficiency' t r a i t s (small b r i s t l e s , rough eyes, see Dudick et a l . , 1974), phenotypes which are s i m i l a r to those of Minutes.  This would permit a test for interactions between Sex and  ts67 to be measured. The results of this test are summarized i n Table 28.  At 28°C a l l  (183) of the male progeny were non-forked and displayed the deficiency phenotype.  F i f t y - f i v e of the 183 males possessed extra sex combs,  diagnostic of Sex and 31 of these 55 exhibited s l i g h t incisions i n the posterior wing margin.  In contrast,,at 22°C a l l males (136) had forked  b r i s t l e s ( i . e . normal with respect to the 'deficiency' b r i s t l e phene) and 52 of these had extra sex combs. at 22°C possessed scalloped wings.  However, none of the males reared  A l l of the females (207) reared at  28°C were non-forked, and 58 of these displayed the deficiency phenotype, but only 5 of the 58 had scalloped wings.  In contrast, a l l of  the females (150) produced at 22°C had forked b r i s t l e s .  F i n a l l y , no  Table 28 Effect of ts67 on Sex Expression i n Progeny of the Cross ts67/ts67;+/+ x v f  Sex of Progeny (X Chromosome Genotype) Males  Scalloped  S  su(f)/Y;Scx/+ at 22° and 28°C  V i s i b l e Phenotypes of Surviving Progeny Non-Scalloped Extra Sex Combs Normal Sex Combs  22°C  0  136  52  22°C  0  150  -  84  Def.  Non-Def.  0  136  0  150  Females (ts67/v f  S  su<f) )  2 g  o  c  5  202  Def. = Deficiency phenotype i . e . small, thin b r i s t l e s Non-Def. = Normal b r i s t l e s  -  -  58  149  178  consistent reduction i n the penetrance of the extra sex comb phene was noted i n ts67/Y;Scx/+ males at 28°C. These data suggest that the wing scalloping interaction between Minutes and Sex i s probably not s p e c i f i c to M l o c i , since a s i m i l a r , a l b e i t less marked interaction exists between Sex and s u ( f ) . Summary of TSPs involving Q-III Figure 20 i s a summary of the TSPs that have been provided by the s h i f t experiments of the present study.  I t i s clear that most of the  TSPs a f f e c t i n g structures derived from the eye-antennal or wing discs, occur about the second instar (or early third) and that these overlap with the early l e t h a l - s e n s i t i v e as well as Dl-mediated i n v i a b i l i t y TSPs.  In contrast, the TSPs associated with the d i f f e r e n t i a t i o n of  b r i s t l e derivatives ( i . e . macrochaetae and sex combs) occur during early pupation, a t almost the same time as the second TSP for l e t h a l i t y . This d i f f e r e n t i a l undoubtedly r e f l e c t s a d i f f e r e n t basis for the o r i g i n of these defects.  The TSP for female s t e r i l i t y (actually male s t e r i l i t y  may be added as well) has been included for reference.  The actual ex-  tent of this TSP could include pre-eclosion stages, since such females were not tested for f e r t i l i t y . and  F i n a l l y , an embryonic TSP was included  i t i s l i k e l y that the l a t t e r could be more accurately delineated  by using shorter s h i f t s coupled with higher  temperature.  FIGURE 20  Temperature-sensitive periods (TSPs) for l e t h a l , s t e r i l e and adult morphological effects of Q-III. Stippled bars indicate the extents of the TSPs r e l a t i v e to developmental stages given below. TSPs for recessive t r a i t s are summarized above the dashed l i n e and include those for: embryonic l e t h a l i t y ( l e ) ; l a r v a l and pupal l e t h a l i t y (1); s t e r i l i t y (s); defects involving aristae, scutella and male g e n i t a l i a (a, s and g); eye reduction (e); sex comb rotation (c); and macrochaete reduction (ma); TSPs for dominant t r a i t s are summarized below the dashed l i n e and include those for: rough eyes (E); macrochaete reduction (Ma); the l e t h a l i n t e r action with DI (L(D1)); and the wing nicking i n t e r actions with vg (W(vg)) and Sex (W(Scx)).  le  a, s and g  W(v3) W(Scx)  Ma  L(QI)  Developmental I  I  TTt  I  2nd~l  3rd  Stages I  I  181  IV.  Discussion  A temperature-sensitive mutation, Q-III has been found to produce a variety of ts phenes when homozygous and heterozygous.  Furthermore,  when Q-III i s heterozygous with Dfd, vg, Sex, Pc, Msc and DI, ts i n t e r actions s p e c i f i c for each of these mutations are detectable.  Unique  or overlapping TSPs were determined for several of these a t t r i b u t e s . Of the ts mutations known to have multiple, complex patterns of TSPs (Poodry et a l . , 1973; G r i g l i a t t i and Suzuki, 1970; Foster, 1973; Shellenbarger and Mohler, 1975; Holden and Suzuki, 1973), none has been found to be as highly p l e i o t r o p i c as Q-III. The pattern  of recessive l e t h a l i t y and extreme s e n s i t i v i t y of  Q-III homozygotes to 48-hour heat pulses argues that the Q-III locus i s indispensible for most of the l a r v a l as well as the pupal stage. However, the fact that the organism i s r e l a t i v e l y refractory to 24hour exposures to 29°C (except for s p e c i f i c intervals) suggests that i r r e v e r s i b l e death i s not mandatory unless a more prolonged period of deprivation of Q - I I I product i s involved. +  Thus, we might expect that  larvae for example, might have two types of temperature-sensitivity, one i n which a short heat pulse leads d i r e c t l y to death and others where such a pulse stops development  but i n a semi-reversible fashion.  This i s supported by the waves of pupation after heat treatment at d i f f e r e n t times.  The temperature-sensitivity (of l e t h a l i t y ) of Q-III  contrasts with that of shibere, since the l a t t e r i s extremely susceptible to very short exposures to high temperatures  (Poodry et a l . ,  1973).  Preliminary observations show that the presence of functional Q-III  +  gene product i s v i t a l also for embryogenesis  and for adult  182 fertility.  Eggs l a i d by Q-III/Q-III females do not survive at 29°C.  Even when Q-III/Q-III females are crossed to normal males, the Q-III/+ embryos f a i l to survive at the higher temperature, but do survive at 22°C.  In contrast, many homozygous Q-III embryos produced by hetero-  zygous females are i n i t i a l l y resistant to the l e t h a l effects of high temperature  (eventually succumbing  after continuous exposure).  This  difference could indicate that functional gene product i s supplied by the mother to the oocyte during oogenesis. The relationship of oogenesis v i s - a - v i s embryogenesis ing here, with reference to the nature of the Q-III l e s i o n .  i s interestIf the  process of oogenesis usually demands a minimum supply of Q - I I I gene +  product, then i t i s not surprising that p a r t i a l or complete s t e r i l i t y i s manifested at 22° and 29°C.  The following 2 alternatives could be offered  to explain why Q-III/+ progeny of homozygous Q-III females f a i l e d to develop at 29°C, while at least an appreciable proportion developed normally at 22°C:  (a) the homozygous females can package enough essential  molecules into the egg to support development  at 22°C, but not at 29°C  and (b) the Q-III gene product i s thermolabile.  Obviously the l a t t e r i s  the more a t t r a c t i v e p o s s i b i l i t y , p a r t i c u l a r l y with respect to eventual biochemical analysis of the Q-III gene product. 1.  The Nature of Minutes and Q-III  Is Q-III a temperature-sensitive Minute? Certainly Q-III possesses the basic phenotypic c h a r a c t e r i s t i c s of Minute mutations.  At the r e s t r i c t i v e temperature, the Q-III hetero-  zygote has lowered v i a b i l i t y , lengthened developmental period, and dominant expression of thin, small b r i s t l e s , rough eyes, and other  183  less penetrant dominant phenotypes. recessive l e t h a l at 29°C.  In addition, the Q-III lesion i s  These properties have been reported for  other mutations called Minutes (Lindsley and G r e l l , 1968). at 22°C no dominant effects are seen.  However,  The interactions of Q-III with  Dl, Ly and vg have been described for other Minutes (see Schultz, Green and Oliver, 1940).  1929  At 29°C, Q-III/+/+ t r i p l o i d s have wild-type  b r i s t l e s and normal developmental periods, a result which has been reported for other Minutes i n t r i p l o i d s (Schultz, 1929).  Probably the  strongest evidence i s that the Q-III interactions led to predictions of Minute interactions that were indeed f u l f i l l e d . Although Q-III i s l e t h a l i n combination with Df(3L)M(3)LS4, genetic mapping positions Q-III i n a region between s_t and in which does not include the deleted portion of this proximal deficiency. There i s a p o s s i b i l i t y that a Minute point mutant or a c y t o l o g i c a l l y i n v i s i b l e deficiency of the Q-III locus i s present on the deficiency chromosome.  At any rate, Q-III amply f u l f i l l s the properties of a  genuine temperature-sensitive Minute. The function of Minute genes The burning question to be answered i s , what i s the primary function of Minute l o c i ?  This study has not provided any d e f i n i t i v e  answers concerning the s p e c i f i c nature of Minutes.  The proposal that  some or many of these l o c i code for tRNA (K.C. Atwood, 1968) i s s t i l l tenable, although preliminary genetic evidence argues against the idea that they are redundant  (Huang and Baker, 1975).  Temperature-  TYR sensitive mutations of the tRNA  locus have been reported for E.  c o l i (Smith et a l . , 1970: Nomura, 1973).  In one case, i n vivo  184  experiments suggested that conformational  changes i n the mutant tRNA  species l e d to i r r e v e r s i b l e i n a c t i v a t i o n of the molecule due to degradation at high temperatures (Nomura, 1973).  In yeast, a ts nonsense  suppressor has been i d e n t i f i e d and i t s genetic properties indicated TYR that a mutant tRNA 1973) .  was l i k e l y involved (Rasse-Messenguy and Fink,  The i n v i t r o a b i l i t y of tRNA isolated from a  super-suppressor  mutant i n yeast to translate nonsense codons, argues that at least f o r lower eukaryotes,  nonsense suppression occurs by tRNA-mediated transla-  tional defects (Capecchi et a l . , 1975).  These studies caution us  against assuming that thermolabile gene products must be proteins, thereby leaving the p o s s i b i l i t y open that Q-III i s a ts mutation i n a tRNA gene. The p l e i o t r o p i c phenotype of Q-III i s c l e a r l y compatible  with the  'tRNA hypothesis', especially i n view of the quantitative and qualitative changes that occur for d i f f e r e n t tRNA species during development in Drosophila (White et a l , , 1973).  The disruptive effects of tRNA  abnormalities on protein synthesis which might be regulated d i f f e r e n t l y i n various c e l l types and discs, could adequately account for the pleiotropy, although  the additional contribution of translational  (Ilan, 1968) and post-translational (Jacobsen,  1971) control, i s also  possible. In s i t u h y b r i d i z a t i o n to salivary gland chromosomes permits  cyto-  l o g i c a l mapping of l o c i specifying tRNA transcripts ( G r i g l i a t t i et a l . , 1974) .  I f i t can be shown that an iso-accepting species maps near the  Minutes i n proximal 3L, the possession of such a mutant w i l l be invaluable (especially since the Q-III a l l e l e can be made homozygous at 22°C)  185  for  the i s o l a t i o n and biochemical study of this gene product.  It  may  be possible for example, to study the a b i l i t y of the homozygote to i n corporate l a b e l l e d amino acids into protein at the r e s t r i c t i v e temperature (see Farnsworth, 1970), thereby d i r e c t l y testing for disruption of protein synthesis  i n Q-III.  Another idea that i s mentioned i n the l i t e r a t u r e but seldom considered i n d e t a i l i s that Minute l o c i might code for ribosomal proteins. Previous evidence suggesting that the basal region of the X chromosome in Drosophila  contained  (Steffensen, 1973;  a cluster of genes specifying ribosomal proteins  Finnerty et a l . , 1973)  has been recently  (Lambertsson, 1975b; Vaslet and Berger, 1976). some of the Minute mutations could represent  Therefore,  disputed at least  lesions i n s t r u c t u r a l  genes for these proteins. Berger and Weber (1974) found almost no polymorphic electrophoretic variants i n ribosomal proteins of several d i f f e r e n t mutants including su(f) and various Minutes, as well as wild-type strains of melanogaster.  One  protein from the small ribosomal sub-unit  Drosophila in flies  bearing a t h i r d chromosome Minute did show altered electrophoretic migration.  However, such an observation  mutation i n a non-Minute locus.  could a r i s e from a second s i t e  The authors did not indicate whether  quantitative differences i n protein patterns  could be measured.  The  Minutes i n which no differences i n ribosomal proteins were noted could have been deficiencies or hypomorphs, i n which case no q u a l i t a t i v e differences would be expected. Lambertsson (1975a) analysed ribosomal protein patterns e l e c t r o phoretically at various stages of development i n Drosophila.  He  was  186 able to detect from 69 ( i n pupae and adults) to 74 ( i n larvae) d i f f e r ent proteins, numbers which far exceed the estimated number of Minute l o c i (Lindsley e_t ajL.,  1972).  In addition, he found q u a l i t a t i v e and  quantitative changes i n some of these proteins during development, p a r t i c u l a r l y i n the third i n s t a r .  He (Lambertsson,  1975b) was  unable  to find evidence for the existence of a mutant ribosomal protein i n ts67g the l ( l ) s u ( f )  6  strain.  However, he did find that pattern changes  c h a r a c t e r i s t i c of the l a r v a l to pupal t r a n s i t i o n i n the wild-type were delayed i n ts67 at higher temperatures, and he concluded that the mutation probably causes a severely reduced l a r v a l a b i l i t y to synthesize imaginal  ribosomes.  Sussman (1970) proposed a model involving quantitative and q u a l i tative control exerted by ribosomes on translation during development. DeWitt and Price (1974) found differences in ribosomal protein patterns which corresponded  temporally with stage-specific appearance of immature  erythrocytes i n Rana catesbeiana, and they suggested that Sussman's model may have merit.  Thus, absence or abnormality of a ribosomal pro-  tein could d i r e c t l y (through aberrant translational control), or i n d i r e c t l y (through generalized disruptions i n protein synthesis) p r e c i pitate a large array of developmental defects. Future considerations of the nature of Minutes should be  concerned  with the fundamental genetic organization and control of these genes as well as their biochemical properties. Indeed, i t w i l l l i k e l y be possible to genetically answer many of the outstanding questions, before their biochemical dissection has been accomplished.  187  I s Q-III a s i n g l e s i t e  lesion?  The m u l t i p l e phenes d i s p l a y e d by Q-III c o u l d have a t r i v i a l such as s e v e r a l d i f f e r e n t mutations on the Q-III chromosome.  basis  Cytologi-  c a l examination of the s a l i v a r y gland chromosomes o f Q-III by T.  C.  Kaufman, r e v e a l e d no c y t o l o g i c a l l y - v i s i b l e a b e r r a t i o n i n chromosome 3. While a l l EMS-induced  t s mutations u s u a l l y map  sites  the o c c u r r e n c e of m u l t i p l e mutations must be  (Suzuki, 1970),  c o n s i d e r e d as a p o s s i b i l i t y .  genetically at single  Since a l l experiments of the p r e s e n t  study were performed u s i n g a recombinant  s t o c k i n which  the d i s t a l  3R  r e g i o n o f the Q-III chromosome, from H a i r l e s s ( 6 9 . 5 ) to the t i p was r e placed, this l i k e l i h o o d  i s diminished.  However, t h i s i s o n l y a  rela-  t i v e l y s m a l l segment o f the chromosome and second s i t e mutations reside  could  elsewhere.  Three main f i n d i n g s support the n o t i o n t h a t d e f e c t s a s s o c i a t e d with Q-III r e p r e s e n t e x p r e s s i o n o f a s i n g l e mutant (1)  site.  I n no case i n e i t h e r o f the mapping experiments  22) were the rough eye, b r i s t l e or l a t e - e c l o s i n g each o t h e r by r e c o m b i n a t i o n .  (see Table  phenes s e p a r a t e d from  For example, a l l 7 l a t e - e c l o s i n g recom-  b i n a n t s between G l and H possessed s m a l l b r i s t l e s and d i d a l l 11 l a t e - e c l o s i n g c r o s s o v e r s i n the s_t to p_  P  rough eyes as  interval.  more, w h i l e many o f the l a t e e c l o s i n g , e y e - b r i s t l e recombinants hibited  l e g anomalies  (e.g. shortened t a r s i , g n a r l e d l e g s ) and  Furtherexwing  v e i n d i s r u p t i o n , as w e l l as absence o f p o s t - v e r t i c a l s , none o f the recombinants w i t h normal b r i s t l e s ,  eyes and developmental p e r i o d s  d i s p l a y e d any o f these d e f e c t s . T h i s was between s_t and £  P  also verified  f o r recombinants  r e c o v e r e d a t 22°C and r e t e s t e d a t 29°C.  While a l l  188  late-eclosing recombinants which displayed rough eyes and b r i s t l e phenes produced some progeny with d i f f e r e n t combinations of eye, wing, leg, and g e n i t a l i a defects, none of these phenotypes was ever seen i n  + recombinant progeny that were M . (2)  Some of the ts phenotypes of Q-III such as ommatidial d e f i -  ciencies and rough eyes have also been observed for ts67 (Dudick et a l . , 1974).  Furthermore, the effects of the combination of Q-III and Sex  on the wings at 29°C were also apparent for ts67 males (and a few females) carrying Sex at 28°C.  Since Dudick e_t al_. claimed that most  of the o r i g i n a l X chromosome containing this mutation was ts67 i s l i k e l y due to a single l e s i o n .  replaced,  Thus, a single s i t e ts Minute  such as Q-III, could mimic the known p l e i o t r o p i c effects of ts67. (3)  I f other third chromosome mutant sites are responsible for  some of the mutant phenes expressed by Q-III, either: ( i ) the second s i t e ( s ) must be a ts a l l e l e of a separate locus or ( i i ) the other s i t e ( s ) must i n t e r a c t with Q-III at 29°C but not at 22°C.  The proba-  b i l i t y of ( i ) would be low since the frequency of EMS-induced third chromosome Minutes i s 1 i n 3000 tested chromosomes (0.00033) and that of third chromosome recessive ts lesions i s 0.055 (including both lethals and v i s i b l e s ) at the same dose of EMS 1973).  (Tasaka and Suzuki,  The probability of such a double mutant would be 0.00033 x  0.055 = 1.8 x 10 ^ or about 1 i n more than 55,000 chromosomes.  Still,  such a p o s s i b i l i t y cannot be dismissed e n t i r e l y because Simpson and Schneiderman  (1975) reported the recovery of a doubly mutant X chromo-  some containing a ts a l l e l e of scalloped ,as well as a ts autonomous c e l l - l e t h a l mutation.  189 The second p o s s i b i l i t y ( i i )  i s d i f f i c u l t to rule out.  The best  way to counter this i s to separate the smallest segment of the o r i g i n a l chromosome which s t i l l carries the ts Minute locus, or a l t e r n a t i v e l y , to map this locus c y t o l o g i c a l l y using d e f i c i e n c i e s .  Thus, while i t i s  possible that more than one mutant s i t e i s involved i n Q-III, i t i s extremely  2.  unlikely.  Developmental Characteristics of Q-III With a few exceptions, shorter ( i . e . 24-hour) exposures of Q-III  cultures to 29°C did not k i l l the animals.  However, such  resulted i n the production of many imaginal defects.  treatment  The type of  defects produced, depended on when during development the cultures were pulsed to 29°C.  Therefore, the pulse experiments helped to de-  fine several TSPs for the pleiotropic phenotype of Q-III. While most of the adult defects resulted from exposure of Q-III larvae (usually during the second or third ins tars) to 29°C, a few resulted from exposure during pupation.  The former phenes w i l l here-  a f t e r be referred to as pattern defects.  These are p a r t i c u l a r l y ex-  emplified by d e f i c i e n c i e s , duplications or other abnormalities which involve derivatives of the eye-antennal Such defects were observed alone, or i n combination (dorsal mesothoracic  and dorsal mesothoracic  discs.  i n heat-treated progeny which bore Q-III  with Dfd (eye-antennal d i s c ) , vg or Sex  disc) .  Pattern defects involving derivatives of  other imaginal discs, such as the genital and leg discs were also seen, but at lower frequencies.  Since nearly a l l of the mitotic a c t i v i t y of  the imaginal discs of Drosophila i s r e s t r i c t e d to the l a r v a l stages (see Nothiger, 1972), i t i s not surprising that the pattern defects of Q-III are associated with heat treatment during these stages.  190 Pattern defects and c e l l death C e l l death i n d i f f e r e n t parts of an imaginal disc, followed by varying degrees of p r o l i f e r a t i o n could explain most of the eye-antennal phenes of Q-III homozygotes and  heterozygotes.  Deficiencies, p a r t i c u l a r l y of eye tissues, were the most frequently encountered pattern defects i n derivatives of the eye-antennal discs of Q-III-bearing  flies.  eye-antennal d i s c .  Figure 21 i s a schematic representation of an  I f Q-III-induced c e l l death occurred more frequently  in region a, lack of adequate c e l l replacement would produce ommatidial deficiencies .  Less frequently, extensive c e l l death could embrace  several regions simultaneously, concomitant absence of eye etc.  thereby accounting  (a)antennal  for the occasional  (b), and head (d) structures,  (assuming that the presumptive c e l l s were not replaced).  More  r e s t r i c t e d c e l l death involving s p e c i f i c regions, for example, b, c, and d, could lead to d e f i c i e n c i e s of i n d i v i d u a l antennal structures, o c e l l i or palps, respectively. According  to Bryant (1971), l o c a l i z e d c e l l death i n an  imaginal  disc could produce d e f i c i e n c i e s , as well as regeneration of the missing structures, or mirror-image duplication of structures already The type of result depends on the repatterning of the new  present.  cells.  Bryant suggests that there i s a "developmental" gradient i n which c e l l s at the  'high' end are totipotent, whereas c e l l s at the 'low'  are more l i m i t e d .  Thus, i f c e l l death occurred and was  end  mitotically  compensated for i n a region of the disc that is nearer to the top of the developmental gradient, the new  c e l l s could be re-patterned  to  produce structures normally derived from portions of the disc that are  FIGURE 21  A schematic diagram of a mature eye-antennal imaginal disc showing the following structures: presumptive f i r s t  (AI), second ( A l l ) and third  (AIII) antennal segments; presumptive a r i s t a (Ar); presumptive eye (E); presumptive head (H); presumptive palpus (P); and the proposed ocellar (0).  region  Regions a, b, c, d, e and f are hypothetical  zones of c e l l death; (after Gehring, 1966).  193 further down the gradient and regeneration would r e s u l t .  However,  c e l l death and subsequent replacement i n a section of the disc that i s low i n the gradient would allow only for the 'regeneration' of lower structures and as a r e s u l t , mirror-image duplication would occur. In  the case of the schematic eye-antennal disc (Figure 21), i f  c e l l death \was. induced i n region b to obliterate the antennal portion of the disc, adequate replacement of c e l l s at region e could lead to regeneration of the antenna  (Gehring, 1966; Bryant, 1971).  On the  other hand, i f c e l l death i n the area of the disc between e and f was followed by c e l l divisions near the antennal anlagen ( i . e . at f ) , this could result i n mirror-image duplication of the antenna.  Finally, i f  c e l l death i n this same section was followed by simultaneous p r o l i f e r a tion at both e and f, concomitant regeneration and duplication could take place, thereby giving r i s e to a t r i p l i c a t i o n of antennal structures.  This present study has demonstrated  that a l l of these eye-  antennal pattern defects occur i n Q-III-bearing f l i e s exposed to 29°C during the l a r v a l period. V e r i f i c a t i o n of ts induction of c e l l death by Q-III w i l l depend upon histochemical tests of d i s c s .  Such investigations could i n d i -  cate whether c e l l death i s random or l o c a l i z e d .  In this regard, the  temperature-sensitivity of Q-III should prove amenable to jLn v i t r o analyses of c e l l death i n isolated d i s c s . I f we can assume that Q-III i s an autonomous c e l l - l e t h a l  like  other Minutes (Stern and Tokunaga, 1971), then this i s the f i r s t report of a ts autonomous c e l l - l e t h a l mutation on an autosome.  Since  Minutes have been shown to e f f e c t cell-autonomous reduction i n mitotic rate (Morata and R i p o l l , 1975), this may explain the low frequency of  194  duplications and t r i p l i c a t i o n s found i n this study.  Thus, even b r i e f  exposure of the disc to high temperature could prevent c e l l replacement that might normally follow c e l l death. In l i g h t of the above, i t would not be surprising i f some sort of c e l l death i n the wing disc i s also responsible for the s c u t e l l a r pattern defects observed i n Q-III-bearing i n d i v i d u a l s .  According to Bryant  (1975), fragments isolated from the mesothoracic (wing) discs which include the anlagen of the scutellum, frequently give r i s e to structural duplications.  However, since misplaced b r i s t l e s or sockets are f r e -  quently scattered over the s c u t e l l a of Q-III f l i e s , clear pattern disruptions, as well as what appear to be duplications, are occurring. While wing scalloping was not seen i n Q-III f l i e s , i n adults bearing  Q-III i n concert with Sex or vg at 29°C, such wing margin effects  are  quite s t r i k i n g , p a r t i c u l a r l y i n Sex/Q-III f l i e s .  Fristrom (1969)  showed that the wing phenotypes of y j * , Beadex (Bx), cut (ct) and Xa a p t e r o u s — are the result of c e l l death either during the third l a r v a l instar, or early pupation ( i n the case of c t ) .  Santamaria and Garcia-  B e l l i d o (1975) used an elegant approach involving clonal analysis to show that the c e l l death i n Bx occurs at about the middle of the third instar.  In the present work, the TSP for the Q-III-Scx wing phene  occurs during the f i r s t h a l f of the third instar and therefore i t i s l i k e l y that c e l l death occurs at that time or shortly thereafter.  It  i s worthy of mention however, that the TSP for the Q-III-vg interaction seen i n this study i s considerably e a r l i e r than the time when c e l l death purportedly takes place. the  Even i f Harnly's (1936) TSP for vg_ at  end of the second instar is quite precise, Fristrom (1969) has  195 reported that c e l l death i s not detectable u n t i l the late part of the third instar i n wing discs of vg f l i e s . C e l l death routinely occurs i n wild-type discs of Drosophila.  For  example, Spreij (1971) observed degenerating c e l l s i n the wing pouch and other sections of the dorsal mesothoracic disc, at about mid instar.  third  In addition, Fristrom (1969) detected similar necrosis i n the  wild-type eye-antennal discs, p a r t i c u l a r l y near the area between the eye and antennal portions, i n both the second and third i n s t a r s . Since c e l l death has been offered as a potential contributor to morphogenesis i n normal discs (see Nothiger, 1972), one hypothetical Xa  mechanism by which mutants such as vg, a p — ,  Bx and cJ: could produce  wing defects, would involve an extension of the boundaries of normallyr e s t r i c t e d c e l l death which i s assumed to occur during the formation of the wing margin (Bryant, 1975).  A similar explanation could apply  to eye mutants such as Bar, eyeless, and Dfd with regard to extensive destruction of presumptive  facet tissue promoted by these mutants.  Presumably, Q-III alone, or i n combination with mutants l i k e Sex (wing disc) and Dfd (eye-antennal disc) might s i m i l a r l y extend the boundaries of c e l l death upon exposure to 29°C by making more c e l l s susceptible to the genetically programmed, r e g i o n a l l y - s p e c i f i c death.  Further study of these Q-III-mediated  cell  interactions should pro-  vide considerable information about c e l l death during development i n imaginal discs, and i t s relationship to pattern phenomena such as duplication formation and regeneration. Defects resulting from heat treatment of Q-III during pupation Whereas most of the pattern defects of Q-Ill-bearing f l i e s (homozygotes) were produced by b r i e f l y exposing larvae to 29°C, other imaginal  196 defects were expressed by Q-III individuals that had been heat-pulsed during pupation.  For example, the c l a s s i c a l M phene of short b r i s t l e s ,  as well as the comb gap phene, were observed i n Q-III adults which emerged from cultures treated i n this manner.  This raises the possi-  b i l i t y that these l a t t e r abnormalities are d i r e c t l y related to the disruptive e f f e c t s of the Q-III lesion on protein synthesis at the time of d i f f e r e n t i a t i o n .  This s i t u a t i o n contrasts with that of the pattern  defects, which probably  originate from imaginal c e l l death at much  e a r l i e r stages than d i f f e r e n t i a t i o n ( i . e . , during the l a r v a l stages). The finding that the TSP for the Minute b r i s t l e phenotype occurs at the time of b r i s t l e formation during the f i r s t h a l f of (Bodenstein,  1950)  pupation  i s consistent with the idea that rapid accumulation  of protein i s a prerequisite to b r i s t l e synthesis (Howells, that any disturbance  i n the t r a n s l a t i o n process  Minutes) d i r e c t l y results i n attenuated  1972)  ( i . e . bb, s u ( f )  bristles.  agreement with those of Dudick e_t a l . (1974) who  My  t s (  and ^  Q  r  findings are i n  determined that ts67  suppression of forked occurs at the time of b r i s t l e formation.  It  should be possible to use this phenotype to select for dominant and recessive mutations that a f f e c t t r a n s l a t i o n . While the TSP of the comb gap phenotype also occurs during f i r s t h a l f of pupation,  the  the s i t u a t i o n here with respect to the effects  of Q-III on protein synthesis, i s unclear.  C e l l lineage studies of  the legs of Drosophila males (Tokunaga, 1962)  have revealed that c e l l s  i n the region of the basitarsus which contain the presumptive sex comb tissue (the combs are a c t u a l l y modified macrochaetae) undergo a chara c t e r i s t i c s h i f t and r o t a t i o n of about 90 degrees, so that the transverse row becomes l o n g i t u d i n a l l y placed.  formerly  Tokunaga makes no mention  197 of when this s h i f t actually occurs i n the presumptive sex comb tissue. I f rotation occurs during the early part of pupation, then presumably the lack of Q-III~*~ gene product could prevent successful rotation.  For  example, the effects of Q-III on translation could k i l l key c e l l s i n volved i n the rotation.  On the other hand, rotation might occur  e a r l i e r and removal of the Q - I I I  +  product at, or just p r i o r to leg  eversion could d e - s t a b i l i z e the sex-comb alignment.  Remember that  rotation of the terminalia i n males i s also blocked by exposing Q-III progeny to 29°C.  However, i n this case, the TSP i s l a r v a l .  Further  study of this ts Minute w i l l c l e a r l y increase our knowledge about such morphogenetic processes.  The interactions of Q-III This study has shown that lesions such as Q-III can be useful for the developmental  study of other genes for which ts a l l e l e s are un-  a v a i l a b l e (see Dudick et a l . , 1974). yg, Dl and Sex.  Thus, TSPs have been defined for  The TSP for the vg-Q-III interaction occurs i n the  second half of the second instar, while that for the Scx-Q-III a c t i o n f a l l s i n the f i r s t h a l f of the third i n s t a r .  inter-  The Dl-Q-III TSP  for l e t h a l i t y extends from the mid second to mid third i n s t a r s .  It is  interesting that the TSP for Sex i s l a r v a l since this may also be true for i t s homeotic e f f e c t .  However, i t should be emphasized that more  than one TSP may exist for a given gene product ( G r i g l i a t t i and Suzuki, 1970; Mglinets, 1975).  Therefore, the p o s s i b i l i t y of additional TSPs  should be considered for the above genes. Similar use of Q-III could permit the delineation of the time of action of the gene products of Dfd and Ly_.  The suppression of the sex  198  comb phenotypes  of some of the homeotics by Minutes has been demonstrated.  Q-III might also provide an estimate of the interval i n development when the gene product of Polycomb (the most f u l l y penetrant of the sex comb homeotics) i s u t i l i z e d . I t i s noteworthy that of the mutants affected by Q-III at 29°C, both vg_ and _p_l are dosage-sensitive (Lindsley et a l . , 1972).  The basis  for the Dfd l e s i o n i s unknown and there i s some evidence which suggests that at least Pc i s an antimorph (Puro and Nygren, 1975).  Thus, f l i e s  heterozygous for deficiencies for y_g_ and Dl display the respective phenotypes  (Lindsley et a l . , 1972).  The interaction of Minutes with  these mutations could therefore be due to the i n h i b i t o r y effects of Minutes on protein synthesis.  In other words, the Minutes could be  producing a synthetic hypomorphic s i t u a t i o n by decreasing the amount of vg_  or 1)1  product.  I f this i s true then we might expect that the  expression of heterozygous deficiencies for these l o c i would be enhanced by Q-III at 29°C and this would e f f e c t i v e l y rule out the possib i l i t y that Q-III i s interacting with mutant yj» or D_l gene products. Other dosage-sensitive l o c i such as Ultrabithorax, Intersex and Hairless on chromosome 3, and Star, black and Plexate on chromosome 2 (Lindsley e_t ajL., 1972) could be tested to see i f they are also enhanced by Q-III. The interaction of Q-III with the homeotics i s c l e a r l y complex. While Q-III suppresses the sex comb phenes of JPc and Msc, i t decreases the v i a b i l i t y of Msc-,  Sex-, Antp-, and possibly Ns-bearing f l i e s .  Furthermore, i t interacts with Sex to produce a new mutant phene, wing scalloping.  The l a t t e r phene i s not s p e c i f i c to Minutes, suggesting  199 that this interaction i s metabolic rather than s p e c i f i c .  I t might be  argued that the retarding effects which Minutes have on development a c t u a l l y allow the accumulation of the antimorphic gene products of (some of) the sex comb homeotics, new phenotypes or l e t h a l i t y .  thereby leading to the production of  The suppression of the sex comb phenes  is puzzling since Q-III does not i n h i b i t sex comb formation on the forelegs.  This phenomenon c l e a r l y merits further study.  The fact that ts67 interacts with Sex i n a fashion similar to Q-III, underscores the contention that many of the phenotypic i n t e r actions described for d i f f e r e n t l o c i i n Drosophila ( i . e . suppression or enhancement) may be metabolic rather than s p e c i f i c (Kaufman et a l . , 1973) .  Therefore, i t i s important to exercise caution when attempting  to interpret interactions i n s p e c i f i c terms as has been done with the proposed nature of the suppression of forked by ts67 (Dudick jet a l . , 1974) .  I t may  be that this suppression i s due to generalized decreases  in protein synthesis rather than to ribosomally-mediated, informational suppression. Additional uses for Q-III i n studies of development Several potential uses for Q-III i n the study of development emerge from the fact that Minutes lower mitotic rates i n a fashion (Morata and R i p o l l , 1975).  cell-autonomous  For example, i t should be possible  to produce clones of Q-III homozygous c e l l s i n a Q-III heterozygous background at 22°C by somatic crossing over.  Since the background c e l l s  would be wild-type at 22°C, such an approach may allow more information to be gleaned from studies which exploit this phenomenon.  In one  + instance, Garcia-Bellido _et _al. (1973) used the tendency for M /M  +  200  clones to overgrow their heterozygous background to investigate developmental compartmentalization i n the wing d i s c .  The possession  of  Q-III should considerably expand the scope of similar investigations, p a r t i c u l a r l y those involving the eye-antennal d i s c . The detrimental effects of Q-III on v i a b i l i t y and growth of larvae are amply demonstrated i n this study.  Some of the Q-III-induced l a r v a l  l e t h a l i t y could be due to the i n a b i l i t y of the larvae to metamorphose, which i n turn could be related to lack of competence of imaginal disc c e l l s (see Nothiger, 1972)  because of death or slow mitosis.  However,  l a r v a l death i s undoubtedly also due to the direct effects of Q-IIImediated disruptions i n protein synthesis.  I t i s known that l a r v a l  c e l l s grow by increases i n c e l l size, while imaginal disc grow m i t o t i c a l l y (Bodenstein,  1950).  I t should therefore be possible to speci-  f i c a l l y probe the growth of imaginal v i s a v i s that of l a r v a l tissue.  201  CHAPTER 6 OVERVIEW  The research described i n this thesis was designed to investigate regions near the centromere  of chromosome 3 of Drosophila melanogaster,  with the following aims: (a) to investigate proximal recombination i n females with a view to examining interference and interchromosomal effects (b) to determine i f Deformed, a mutation which maps near the centromere,  i s recessive l e t h a l and to map this locus r e l a t i v e to  Kinked (c) to see i f selecting for radiation-induced, proximal crossovers w i l l enrich for deletions i n proximal segments and (d) to genetically and developmentally characterize Q-III, a ts a l l e l e of a proximally-located Minute.  For the most part these objectives have  been r e a l i z e d . The experiments described i n CHAPTER 2 have helped to further characterize crossing over near the centromere  of chromosome 3.  F i r s t , some results suggested that much of the increase i n proximal crossing over caused by the inverted attached-X chromosome C(1)M3, p r e f e r e n t i a l l y takes place within centrically-adjacent  euchromatin.  Since i t i s l i k e l y that l i t t l e or no spontaneous crossing over occurs in heterochromatin, such recombinagenesis proximal euchromatic segments.  may be confined solely to  Second, this study has provided results  suggesting that multiple (double and t r i p l e ) crossovers are detectable at higher than expected frequencies i n proximal i n t e r v a l s . The l a t t e r r e s u l t s are of p a r t i c u l a r interest since they mark a s t r i k i n g departure from two c l a s s i c a l rules of intergenic recombination: (a) positive chromosome interference within a given region varies  202 inversely with the genetic size of that region and (b) crossing over within d i f f e r e n t arms of the same chromosome i s independent, i . e . positive interference does not extend across the centromere. possible explanations  Three  were offered to account for these r e s u l t s :  (1) the Two-Step model (mitotic followed by meiotic  crossovers)  (2) chromatid interference and (3) gene conversion. The  following experimental evidence supports the f i r s t p o s s i b i l i t y :  ( i ) females producing multiples also showed higher frequencies of crossing over than did those females producing no multiples and ( i i ) fewer double r e l a t i v e to t r i p l e exchange tetrads were observed i n a tetrad analysis of the data.  However, the predicted clustering of  single crossovers which would l i k e l y accompany a Two-Step production of multiple crossovers, did not occur for females producing double crossovers. Two lines of evidence were presented i n support of the idea that gene conversion may be involved i n the production crossovers.  of putative multiple  F i r s t , equal numbers of reciprocal crossover classes were  not recovered.  Second, when crossing over was measured i n females  carrying C(1)M3, negative  interference i n most proximal intervals  showed a r e l a t i v e decrease, i n spite of the fact that crossing over was increased i n a l l i n t e r v a l s , high negative  whatever the cause of this observed  interference, these findings have introduced a t o t a l l y  new dimension to the consideration of linked exchange i n this organism. CHAPTER 3 described a genetic study of the Dfd locus.  The results  of the mapping experiment suggest that K i and Dfd are very close, genetically.  The results of the analysis of a large number of recombin-  ant crossover chromosomes and successful synthesis of a homozygous Dfd  203 stock, confirm the notion that the Dfd lesion i s by nature homozygous viable.  Therefore, most Dfd stocks must carry at- least 1 l e t h a l s i t e  in addition to the Dfd mutation.  However, the Dfd stock examined i n  the present study probably carried a minimum of 3 extra l e t h a l s . This information w i l l allow a more complete assessment of developmental and genetic studies of Dfd.  I t also emphasizes the fact that  cytological mapping i s preferable to crossover mapping, p a r t i c u l a r l y when the mutation(s) i n question l i e s near the centromere.  Thus, the  need for a wider inventory of stable proximal deficiencies and duplications i s obvious. The results of the study described i n CHAPTER 4 agree with the idea that a large proportion of induced crossovers recovered from irradiated males, a r i s e v i a asymmetrical  exchange.  Some of the  results also support other workers i n their claim that lethals are more common to crossover chromosomes, when the crossovers are produced by induced exchange within proximally-adjacent euchromatic  segments.  In future, similar investigations w i l l not only be sources of proximal aberrations, but w i l l also provide considerable information about induced crossing over.  Of p a r t i c u l a r interest w i l l be the com-  parison of exchange within heterochromatic and proximally-adjacent euchromatic  segments of the chromosome.  CHAPTER 5 was a detailed description of the genetic and  develop-  mental properties of Q-III, a ts a l l e l e of a Minute which i s located near the centromere of chromosome 3.  This represents the f i r s t report  of a t r u l y conditional Minute a l l e l e i . e . one which e l i c i t s the dominant M t r a i t s under r e s t r i c t i v e conditions, but produces no such effects under permissive conditions.  204  The phenotypic s i m i l a r i t i e s between Q-III, ts67 ( s u ( f ) — " ) and bobbed, favour the idea that Minute l o c i are involved at some l e v e l ( s ) in the process of translation.  No conclusions about the exact nature  of M gene products were reached i n this study.  In the case of Q-III,  the primary product may be thermolabile. However, this would not eliminate the p o s s i b i l i t y that Minutes code for products as diverse as tRNA. species or ribosomal proteins.  Indeed, the assortment of M  gene products may not be homogeneous with respect to the d i f f e r e n t components of t r a n s l a t i o n . The pleiotropy of Q-III i s impressive.  By exposing cultures to  29°C at various developmental i n t e r v a l s , i t was possible to k i l l Q-III homozygotes as well as to produce homozygous (and heterozygous) adults with a large spectrum of imaginal defects.  While the TSP of l e t h a l i t y  i s polyphasic, TSPs of the d i f f e r e n t phenotypes are generally monophasic.  The patterns of l e t h a l i t y and s t e r i l i t y show that the Q-IIl"*"  product i s essential for nearly a l l stages of development.  However,  i f the organism i s deprived of this substance for shorter periods, v i a b i l i t y i s more normal, but the phenotypic anomalies are seen.  The  observation that homozygous Q-III individuals (produced by heterozygous mothers) frequently reach the f i r s t l a r v a l instar before dying at 29°C, suggests that considerable Q - I I I developing oocyte.  +  product i s supplied to the  This i s further supported by the observed ts  maternal effects of Q-III. S e n s i t i v i t y of Q-III larvae to heat-induced l e t h a l i t y i s marked by either a reversible blockage of growth (usually followed by death), or more frequently by f a i r l y rapid death.  On the other hand, pupal  205 s e n s i t i v i t y appears to be related to blockages i n d i f f e r e n t i a t i o n .  A  p a r a l l e l difference i s apparent for some of the imaginal defects. Thus, most pattern defects,for example, those involving eye-antennal or wing disc derivatives, were produced by exposure of larvae to 29°C (usually during the second or third i n s t a r ) , whereas other defects such as attenuated b r i s t l e s or abnormal sex combs, resulted from heat exposure during pupation.  I t i s l i k e l y that the former phenes are  caused by c e l l death within the respective anlagen i n larvae, while the TSPs of the l a t t e r defects imply that they are due to the d i r e c t disruption of d i f f e r e n t i a t i o n i n Q-III i n d i v i d u a l s . The Q-III-mediated translational d i f f i c u l t i e s can e a s i l y account for a l l of the phenotypic t r a i t s of this mutant.  A l l developmental  stages of Q-III homozygotes which require protein synthesis would inevitably succumb to heat-induced death.  At 29°C, an embryo produced  + by a Q-III female could survive only as long as i t s supply of Q-III product lasted.  C e l l death, which i s l i k e l y responsible for pattern  defects exhibited by imagoes heat-treated as larvae, could be the r e s u l t of the cell-autonomous f a i l u r e of t r a n s l a t i o n .  On the other  hand, imaginal defects s,uch as the small b r i s t l e and comb gap phenes, which are seen i n individuals heat treated during pupation, could be produced by impaired d i f f e r e n t i a t i o n due to translational collapse at c r i t i c a l intervals i n Q-III pupae.  F i n a l l y , a lack of protein syn-  thesis would probably r e s u l t i n the s t e r i l i t y of Q-III (homozygous) adults at 29°C. Several i n t r i g u i n g ts interactions between Q-III and n o n - a l l e l i c genes were documented i n this study.  Some of these interactions (but  206  non ts) had been previously reported for Minutes, v i z . synthetic l e t h a l i t y with DI and Ly_, production of wing scalloping with vg_ and suppression of the sex comb phenes of some of the homeotic  mutants.  In addition, I was able to show that Q-III i s l e t h a l i n combination with Dfd at 29°C and that this s i t u a t i o n i s due to the f a i l u r e of eyeantennal disc derivatives to develop.  Q-III/Scx and Q-III/Msc com-  binations are less viable than controls at 29°C and the former i n d i viduals possess variable nicking of the posterior wing margin. It was possible to determine TSPs of the gene products of vg, Dl and Sex. In each case the TSP i s l a r v a l and i n the case of Sex and vg, c e l l death i s probably involved i n the production of these traits. The notion that these interactions are s p e c i f i c rather than metab o l i c i n nature, i s challenged by the observations that ts67 and other Minutes interact s i m i l a r l y with Sex a t 29°C, and also that a t least one additional Minute mutation suppresses the sex comb phene of Msc. These findings imply that the reduced t r a n s l a t i o n a l capacity of Q-III-bearing individuals i s s u f f i c i e n t to account for the observed interactions, without invoking the idea of gene-product  interactions  involving Q-III and the other l o c i . The wide spectrum of Q-III properties has thus provided additional scope f o r the analysis of Minutes i n general, and this proximallylocated Minute i n p a r t i c u l a r . the  That Q-III possesses great potential for  study of developmental processes peculiar to i t s e l f or other non-  a l l e l i c mutations, has been amply demonstrated by these experiments.  \  207  BIBLIOGRAPHY A r a j a r v i , P., and Hannah-Alava, A. (1969). and r i . Dros. Inf. Serv. 44: 73.  Cytogenetic mapping of in  Arking, R. (1975). Temperature-sensitive c e l l - l e t h a l mutants of Drosophila: i s o l a t i o n and characterization. Genetics 80: 519-537. Atwood, K. C. (1968). In "Genetic Variations of Drosophila melanogaster" (D. L. Lindsley and E. H. G r e l l , eds.) Carnegie Inst. Wash. Publ. 627. Baker, W. K. (1958). 92: 59-60.  Crossing over i n heterochromatin.  Amer. Natur.  Baldwin, M., and Chovnick, A. (1967). Autosomal half-tetrad analysis i n Drosophila melanogaster. Genetics S5: 277-293. Baldwin, M. C , and Suzuki, D. T. (1971). A screening procedure for the detection of putative deletions i n proximal heterochromatin of Drosophila. Mutation Res. _11: 203-213. Beadle, G. W. (1932). A possible influence of the spindle fiber on crossing over i n Drosophila. Proc. Natl. Acad. S c i . (U.S.A.) 18: 160-165. Berger, E. M., and Weber, L. (1974). The ribosomes of Drosophila. I I . Studies on i n t r a s p e c i f i c v a r i a t i o n . Genetics _78: 1173-1183. Bodenstein, D. (1950). The postembryonic development of Drosophila, In "Biology of Drosophila" ( (M. Demerec, ed.) pp. 175-367. John Wiley and Sons, New York. Bole-Gowda, B. N., Perkins, D. D., and Strickland, W. M. (1962). Crossing over and interference i n the centromere region of linkage group I of Neurospora. Genetics 4_7: 1243-1252. Brehme, K. S. (1939). A study of the effect on development of Minute mutations i n Drosophila melanogaster. Genetics 24: 131-161. Bridges, C. B. (1916). Non-disjunction as proof of the chromosomal theory of heredity. Genetics 1: 1-52. Brosseau, G. E., J r . (1960). Genetic analysis of the male f e r t i l i t y factors on the Y chromosome of Drosophila melanogaster. Genetics 45: 257-274. Brown, S. W. (1966). 417-425.  Heterochromatin.  Science (Washington)  151:  208  Bryant, P. J . (1971). Regeneration and duplication following operations i n s i t u on the imaginal discs of Drosophila melanogaster. Devel. B i o l . 26: 606-615. Bryant, P. J . (1975). Pattern formation i n the imaginal wing disc of Drosophila melanogas ter: fate map, regeneration and duplication. J . Exp. Zool. 193: 49-78. Calef, E. (1957). Effect on linkage maps of selection of crossovers between closely linked markers. Heredity 1_1: 265-279. Capecchi, M. R., Hughes, S. H., and Wahl, G. M. (1975). Yeast supersuppressors are altered tRNAs capable of translating a nonsense codon i n v i t r o . C e l l 6_: 269-277. Chase, M., and Doermann, A. H. (1958). High negative interference over short segments of the genetic structure of bacteriophage T4. Genetics 43: 332-353. Chovnick, A., Ballentyne, G. H., and Holm, D. G. (1971). Studies on gene conversion and i t s relationship to linked exchange i n Drosophila melanogas ter. Genetics 6S>: 179-209. Cooper, K. W. (1959). Cytogenetic analysis of major heterochromatic elements (especially Xh and Y) i n Drosophila melanogaster, and the theory of "heterochromatin". Chromosoma (Berlin) 10: 535-588. Davis, B. K. (1974). Chromatid interference i n Drosophila melanogaster. Genetics 7_7: 16s. Denell, R. E. (1972). 48: 45.  Reversion studies of Nasobemia.  Denell, R. E. (1973). Homeosis i n Drosophila. studies with revertants of Nasobemia.  Dros. Inf. Serv.  I. Complementation Genetics 7_5: 279-297.  DeWitt, W., and Price, R. P. (1974). Structural changes i n ribosomes during development. Biochem. Biophys. Res. Comrnun. 5_6: 593598. Dobzhansky, T. (1930). Cytological map of the second chromosome of Drosophila melanogaster. Biolog. Zent. 50: 671-685. Dudick, M. E., Wright, T. R. F., and Brothers, L. L. (1974). The developmental genetics of the temperature-sensitive a l l e l e of the suppressor of forked, 1 ( l ) s u ( f ) — — ^ i n Drosophila melanogas ter. Genetics _76: 487-510. Duncan, I. W., and Kaufman, T. C. (1975). Cytogenetic analysis of chromosome 3 i n Drosophila melanogaster: mapping of the proximal portion of the right arm. Genetics 80: 733-752.  209  Farnsworth, M. W. (1957a). Effects of the homozygous Minute - IV deficiencies on the development of Drosophila melanogaster. Genetics 42: 7-18. Farnsworth, M. W. (1957b). Effects of homozygous f i r s t , second and third chromosome Minutes on the development of Drosophila melanogas ter. Genetics 42: 19-27. Farnsworth, M. W. (1970). Uptake and incorporation of amino acids in Minute mutants of Prosophila. J . Exp. Zool. 175: 375-382. Finnerty, V. G., Barton, L. M., Schalet, A., Elmer, W. A., and Smith, P. D. (1973). The suppressor of forked mutation: a putative protein synthesis mutant i n Drosophila melanogaster. Genetics 74: 579. Foster, G. G. (1973). Temperature-sensitive mutations i n Drosophila melanogaster. XIII. Temperature-sensitive periods of the l e t h a l and morphological phenotypes of selected combinations of Notch-locus mutations. Devel. B i o l . 32: 282-296, Friesen, H. (1933). A r t i f i c i a l l y induced crossing-over i n males of Drosophila melanogaster. Science (Washington) 78: 513-514. Friesen, H. (1937a). A r t i f i c i a l release of crossing-over i n meiosis and mitosis. Nature 140: 362. Friesen, H. (1937b). Mechanism of crossing-over i n males of Pros ophila melanogaster. J . Genetics 35: 141-150. Fristrom, D. (1969). C e l l u l a r degeneration i n the production of some mutant phenotypes i n Drosophila melanogaster. Moi. Gen. Genet. 103: 363-379. G a l l , J . G., Cohen, E. H., and Polan, M. L. (1971). Repetitive DNA sequences i n Drosophila. Chromosoma (Berlin) j}3: 319-344. Garcia-Bellido, A., R i p o l l , P., and Morata, G. (1973). Developmental compartmentalisation of the wing disk of Drosophila. Nature N.J.B. 245: 251-253. Gehring, W. J . (1966). Uebertragung und Aenderung der Determinationsqualita'ten i n Antennen-Scheiben-Kulturen von Drosophila melanogaster. J . Embryol. Exp. Morphol. _15: 77-111. Graubard, M. A. (1934). crossing over.  Temperature effect on interference and Genetics 19: 83-94.  Green, M. M. (1959). Double crossing over or gene conversion at the white locus i n Drosophila melanogaster? Genetics 45: 15-18.  210  Green, M. M. (1960) . Apparent double crossing over i n a short genetic i n t e r v a l i n Drosophila melanogas ter. Nature 126: 990-991. Green, M. M. (1975). Conversion as a possible mechanism of high coincidence values i n the centromere region of Drosophila. Moi. Gen. Genet. 139: 57-66. Green, M. M., and Oliver, C. P. (1940). The action of certain mutants upon the penetrance of heterozygous v e s t i g i a l wing i n Drosophila melanogaster. Genetics 2_5: 584-592. G r e l l , E. H. (1962). The dose effect of ma-l+ and r y on xanthine dehydrogenase a c t i v i t y i n Drosophila melanogaster. Genetics 47: 956. +  G r i g l i a t t i , T., and Suzuki, D. T. (1970). Temperature-sensitive mutations i n Drosophila melanogaster. V. A mutation a f f e c t i n g concentrations of pteridines. Proc. Natl. Acad. S c i . (U.S.A.) 67: 1101-1108. G r i g l i a t t i , T. A., White, B. N., Tener, G. M., Kaufman, T. C , Holden, J. H., and Suzuki, D. T. (1974). Studies on the tRNA genes of Drosophila. Cold Spring Harbor Symp. Quant. B i o l . 38: . 461-474. Hannah-Alava, A. (1968). Induced crossing-over i n the p r e s t e r i l e broods of Drosophila melanogaster males. Genetica 39_: 94-152. Harnly, M. H. (1936). The temperature-effective periods and the growth curves for length and area of the v e s t i g i a l wings of Drosophila melanogaster. Genetics 2JL: 84-103. Hartwell, L. (1974). Saccharomyces cerevisiae c e l l cycle. Rev. 38: 164-198.  Bacteriol.  Hawthorne, D. C , and Mortimer, R. K. (1960). Chromosome mapping i n Saccharomyces: centromere linked genes. Genetics 45: 10851110. Heitz, E. (1933). Die somatische Heteropyknose bei Drosophila melanogaster und ihre genetische Bedenteung. Z. Z e l l f o r s c h 20: 237-287. Herskowitz, I. H., and Abrahamson, S. (1957). Induced changes i n female germ c e l l s of Drosophila. IV. Dependence of induced crossoverl i k e exchanges i n oocytes and oogonia upon X-ray i n t e n s i t y . Genetics 42: 444-453. Hexter, W. M. (1958). On the nature of the garnet locus i n Drosophila melanogaster. Proc. Natl. Acad. S c i . (U.S.A.) 44: 768-771. H i l l i k e r , A. J . (1975). Genetic analysis of the proximal heterochromatin of chromosome 2 of Drosophila melanogaster. Ph.D. thesis, University of B r i t i s h Columbia.  211  H i l l i k e r , A. J . (1976). Genetic analysis of the centromeric heterochromatin of chromosome 2 of Drosophila melanogas ter: d e f i ciency mapping of EMS-induced l e t h a l complementation groups. Genetics &3: 765-782. H i l l i k e r , A. J . , and Holm, D. G. (1975). Genetic analysis of the proximal region of chromosome 2 of Drosophila melanogas ter. I. Detachment products of compound autosomes. Genetics 81: 705-721. Hodgetts, R. B. (1975). The response of dopa decarboxylase a c t i v i t y to variations i n gene dosage i n Drosophila: a possible location of the structural gene. Genetics 7_9: 45-54. Holden, J . J . , and Suzuki, D. T. (1973). Temperature-sensitive mutations i n Drosophila melanogaster. XII. The genetic and developmental effects of dominant lethals on chromosome 3. Genetics 7_3: 445-458. Holm, D. G., Baldwin, M., Duck, P., and Chovnick, A. (1969). The use of compound autosomes to determine the r e l a t i v e centromeric position of chromosome 3. Dros. Inf. Serv. 44: 112. Howe, H. B. (1956). Crossing over and nuclear passing i n Neurospora crassa. Genetics 41: 610-622. Howells, A. J . (1972). Levels of RNA and DNA i n Drosophila melanogas ter at d i f f e r e n t stages of development: a comparison between one bobbed and two phenotypically non-bobbed stocks. Bioch.i Genetics 6: 217-230. Huang, S. L., and Baker, B. S. (1975). The mutability of the Minute l o c i of Drosophila melanogaster with Ethyl methanesulfonate. Mutation Res. 34: 407-414. Hurst, D. D., Fogel, S., and Mortimer, R. K. (1972). Conversionassociated recombination i n yeast. Proc. Natl. Acad. S c i . (U.S.A.) 69: 101-105. Ilan, J . (1968). Amino acyl incorporation and aminoacyl transfer i n an insect pupal system. J . B i o l . Chem. 243: 5859-5866. Ives, P. T., and Fink, G. R. (1962). Comparison of translocation and crossover chromosomes produced by y i r r a d i a t i o n of Drosophila males. Genetics 47: 963. Jacobsen, K. B. (1971). Role of an isoacceptor transfer ribonucleic acid as an enzyme i n h i b i t o r : e f f e c t on tryptophan pyrrolase of Drosophila. Nature N. B. 231: 17-19. Judd, B. H., Shen, M. W., and Kaufman, T. C. (1972). The anatomy and function of a segment of the X chromosome of Drosophila melanogaster. Genetics 7_1: 139-156.  212  Kaufman, T. C , Tasaka, S. E., and Suzuki, D. T. (1973). The interaction of two complex l o c i , zeste and bithorax i n Drosophila melanogaster. Genetics 7_5: 299-321. Lambertsson, A. (1975a). The ribosomal proteins of Drosophila melanogaster. IV. Characterization by two-dimensional gel electrophoresis of the ribosomal proteins from nine post-embryonic developmental stages. Moi. Gen. Genet. 139: 133-144. Lambertsson, A. (1975b). The ribosomal proteins of Drosophila melanogaster . V. Analysis by two-dimensional gel electrophoresis of the ribosomal proteins of the temperature-sensitive l e t h a l a l l e l e of suppressor of forked, l ( l ) s u ( f ) ^ : a putative ribosomal protein mutant. Moi. Gen. Genet. 139: 145-156, t s  7 g  Lifschytz, E., and Falk, R. (1969). A system for screening of rare events i n genes of Drosophila melanogaster. Genetics 63: 353-352. Lindsley, D. L., and G r e l l , E. H. (1968). "Genetic Variations of Drosophila melanogaster." Carnegie Inst. Wash. Publ. 627. Lindsley, D. L., Sandler, L., Baker, B. S. B., Carpenter, A. T. C , Denell, R. E., H a l l , J . C , Jacobs, P. A., Miklos, G. L. G., Davis, B. K., Gethmann, R. C , Hardy, R. W., Hessler, A., M i l l e r , S. M., Nozawa, H., Parry, D. M., and Gould-Somero, M. (1972). Segmental aneuploidy and the genetic gross structure of the Drosophila genome. Genetics 7JL: 157-184. Lucchesi, J . C , and Suzuki, D. T. (1968). The interchromosomal control of recombination. Annu. Rev. Genet. 2_:. 53-86. Merriam, J . R., and Garcia-Bellido, A. (1969). Linkage data, melanogaster. Dros. Inf. Serv. 44: 51.  D.  Mglinets, V. A. (1972). Irradiation-induced crossing-over i n Drosophila males. Cytological investigation of crossovers. Genetika 8: 193-201. Mglinets, V. A. (1973). Cytological analysis of crossovers induced by i r r a d i a t i o n i n females of Drosophila melanogaster. Genetika 2: 323-328. Mglinets, V. A. (1975). Time of gene a c t i v i t y i n the ontogenesis of Drosophila. I. Temperature-sensitive period for the spineless e f f e c t i n aristapedia mutants. Ontogenez j5: 416-421. M i l l e r , A. (1950). The internal anatomy and histology of the imago of Drosophila melanogas ter, In "Biology of Drosophila" (M. Demerec, ed.), pp. 420-534. John Wiley and Sons, New York. Morata, G., and R i p o l l , P. (1975). Minutes: mutants of Drosophila autonomously a f f e c t i n g c e l l d i v i s i o n rate. Devel. B i o l . 42: 211-221.  213  Morgan, T. H., Sturtevant, A. H., and Bridges, C. B. (1925). genetics of Drosophila. B i b l . genet. 2: 1-262.  The  Muller, H. J . (1932). Further studies on the nature and causes of gene mutations. Proc. 6th Int. Cong. Genet. 1: 213-255. Muller, H. J . (1954). The nature of the genetic effects produced by radiation, In "Radiation Biology" (A. Hollander, ed.) v 1, pp. 351-474. McGraw-Hill, New York. Muller, H. J . (1958). Pseudo-crossingover near centromere of the third chromosome induced i n late oocytes by X-rays. Dros. Inf. Serv. 32: 140-141. Muller, H. J . , and Painter, T. S. (1932). The d i f f e r e n t i a t i o n of the sex chromosomes of Drosophila into genetically active and inert regions. Mu. indukt. Abstamm.-vererb. Lehre 62_: 316-365. Muller, H. J . , R a f f e l , D., Gershenson, S. M., and ProkofyevaBelgovskaya, A. A. (1937). A further analysis of l o c i i n the so-called "inert region" of the X chromosome of Drosophila. Genetics 22.: 87-93. Nomura, Y. (1973). I r r e v e r s i b l e i n a c t i v a t i o n at high temperature of temperature-sensitive mutant tRNA^^ In vivo. Nature N. B. 2-42: 12-14. Nothiger, R. (1972). The l a r v a l development of imaginal disks, In "Results and Problems i n C e l l D i f f e r e n t i a t i o n : The Biology of Imaginal Disks" (H. Ursprung and R. Nothiger, eds.) v 5, pp. 1-34. Springer-Verlag, New York, Heidelberg and B e r l i n . O l i v i e r i , G., and O l i v i e r i , A. (1964). Evidence for the two-hit nature of Xfray induced crossing over i n the centromeric region of Drosophila males. Mutation Res. 1: 279-295. Ouweneel, W. J . (1969). Morphology and development of loboidophthalmoptera, a homeotic s t r a i n i n Drosophila melanogaster. wllhelm Roux Arch. Entwicklungsmech. Organ. 178: 247-275. Painter, T. S. (1935). The morphology of the third chromosome i n the s a l i v a r y gland of Drosophila melanogaster and a new c y t o l o g i c a l map of this element. Genetics 2_0: 301-326. Patterson, J . T., and Suche, M. L. (1934). Crossing over induced by X-rays i n Drosophila males. Genetics 1_9: 223-236. Peacock, W. J . , Brutlag, D., Goldring, E., Appels, R., Hinton, C. S., and Lindsley, D. L. (1974). The organization of highly r e p e t i t i v e DNA sequences i n Drosophila melanogas ter chromosomes. Cold Spring Harbor Symp. Quant. B i o l . _28: 405-416.  214  Poodry, C. A., H a l l , L., and Suzuki, D. T. (1973). Developmental properties of shiberp*-^: a p l e i o t r o p i c mutation a f f e c t i n g l a r v a l and adult locomotion and development. Devel. B i o l . 32: 373-386. Postlethwait, J . H., and Schneiderman, H. A. (1973). Developmental genetics of Drosophila imaginal d i s c s . Annu. Rev. Genet. 7_: 381-433. Pritchard, R. H. (1960). Localized negative interference and i t s bearing on models of gene recombination. Genetical Res. 1: 1-24. Puro, J . (1966). Mutational response of the premeiotic germ-cell stages of adult Drosophila melanogaster males to X - i r r a d i a t i o n . Ann. Zool. Fenn. 3: 99-126. Puro, J . , and Nygren, T. (1975). Mode of action of a homeotic gene i n Drosophila melanogaster: l o c a l i z a t i o n and dosage effects of Polycomb. Hereditas 81: 237-248. Rasse-Messenguy, R., and Fink, G. R. (1973). Temperature-sensitive nonsense suppressors i n yeast. Genetics 7j>: 459-464. Raytnayake, W. E. (1970). Studies on the relationship between induced crossing-over and mutation i n Drosophila melanogaster. Mutation Res. 9: 71-83. Ritossa, F. M., Atwood, K. C., and Spiegelman, S. (1966a). A molecular explanation of the bobbed mutants of Drosophila as p a r t i a l deficiencies of "ribosomal" DNA. Genetics .54: 819-834. Ritossa, F. M., Atwood, K. C., and Spiegelman, S. (1966b). On the redundancy of DNA complementary to amino acid transfer RNA and i t s absence from the nucleolar organizer region of Drosophila melanogaster. Genetics .54: 663-676. Ritossa, F. M., and Spiegelman, S. (1965). L o c a l i z a t i o n of DNA complementary to ribosomal RNA i n the nucleolus organizer, region of Drosophila melanogaster. Proc. Natl, Acad. S c i . (U.S.A.) 53: 737-745. Roberts, P. A. (1965). Difference i n the behavior of eu- and heterochromatin: crossing over. Nature 205: 725-726. Russell, M. A. (1974). Pattern formation i n the imaginal discs of a temperature c e l l - l e t h a l mutant of Drosophila melanogaster. Devel. B i o l . 40: 24-39. Santamaria, P., and Garcia-Bellido, A. (1975). Developmental analysis of two wing scalloping mutants c t ^ and Bx^ of Drosophila melanogaster. Wilhelm Roux Arch. Entwicklungsmech. Organ. 178: 233-245.  215  Schalet, A., and Lefevre, G., J r . (1973). The l o c a l i z a t i o n of "ordinary" sex-linked genes i n section 20 of the polytene X chromosome of Drosophila melanogaster. Chromosoma (Berlin) 44: 183-202. Schultz, J . (1929). The Minute reaction i n the development Drosophila melanogaster. Genetics 14: 366-419.  of  Schultz, J . , and Redfield, H. (1951). Interchromosomal e f f e c t on crossing over i n Drosophila. Cold Spring Harbor Symp. Quant. B i o l . 16: 175-197. Shellenbarger, D. L., and Mohler, J . D. (1975). Temperature-sensitive mutations of the Notch locus i n Drosophila melanogaster. Genetics _81: 143-162. Simpson, P., and Schneiderman, H. A. (1975). I s o l a t i o n of temperaturesensitive mutations blocking clone development i n Drosophila melanogaster, and the effects of a temperature-sensitive c e l l l e t h a l mutation on pattern formation i n imaginal d i s c s . Wilhelm Roux Arch. Entwicklungsmech. Organ. 178: 247-275. S i n c l a i r , D. A. (1975). Crossing over between closely linked markers spanning the centromere of chromosome 3 i n Drosophila melanogaster . Genetical Res. 11: 173-185. Smith, J . D., Barnett, L., Brenner, S., and Russell, R. L. (1970). More mutant tyrosine transfer ribonucleic acids. J . Moi. B i o l . 54: 1-14. Smith, P. D., Finnerty, V. G., and Chovnick, A. (1970). Gene conversion i n Drosophila: non-reciprocal events at the maroon-like c i s t r o n . Nature 228: 442-444. S^gaard, B. (1974). The l o c a l i z a t i o n of eciferum l o c i i n barley. I I I . Three point test of genes on chromosome I i n barley. Hereditas 76: 41-47. Spreij, T. E. (1971). C e l l death during the development of the imaginal disks of Calliphora erythrocephala. Neth. J . Zool. 2_1: 221264. Stadler, D. R. (1956). 41: 623-630.  Double crossing over in Neurospora.  Genetics  Steffensen, D. M. (1973). Mapping genes for the ribosomal proteins of Drosophila. Nature N. B..244: 231-234. Stern, C. (1929). Uber die additive Wirckung multipler A l l e l e . Zent. 49: 261-290.  Biolog.  Stern, C , and Tokunaga, C. (1971). On c e l l lethals i n Drosophila. Proc. Natl. Acad. S c i . (U.S.A.) 68: 329-331.  216  Stevens, W. L. (1936). 51-64.  The analysis of interference. J . Genetics 32:  Stewart, B. R., and Merriam, J . R. (1974). Segmental aneuploidy and enzyme a c t i v i t y as a method for cytogenetic l o c a l i z a t i o n i n Drosophila melanogaster. Genetics 76: 301-309. Strickland, W. N. (1961). Tetrad analysis of short chromosome regions of Neurospora crassa. Genetics 46: 1125-1141. Sturtevant, A. H. (1951). A map of the fourth chromosome i n Drosophila melanogas ter based on crossing over i n t r i p l o i d females. Proc. Natl. Acad. S c i . (U.S.A.)37: 405-407. Sussman, M. (1970). Model for quantitative and q u a l i t a t i v e control of mRNA translation i n eukaryotes. Nature 225: 1245-1246. Suzuki, D. T. (1970). Temperature-sensitive mutations i n Drosophila melanogaster. Science (Washington) 170: 695-706. Suzuki, D. T. (1974a). Genetic analysis of crossing over and i t s relation to chromosome structure and function i n Drosophila melanogaster. Oregon State University Genetics Lectures 3^: 7-32. Suzuki, D. T. (1974b). Temperature-sensitive Mutations i n Drosophila melanogaster, In "Handbook of Genetics" (R. C. King, ed.) p. 653-668. Plenum, New York. Suzuki, D. T., B a i l l i e , D., and Parry, D. (1966). The o r i g i n of multiple crossing over chromatids i n short genetic intervals in Drosophila melanogaster. Genetics 5_4: 1359-1370. Suzuki, D. T., Kaufman, T. C , and Falk, D. R. (1976). Conditionally expressed mutations i n Drosophila melanogaster, In "The Genetics and Biology of Drosophila" (M. Ashburner and E. Novitski, eds.) v a, pp. 207-263. Academic Press, London, New York, and San Francisco. Suzuki, D. T., and Parry, D. M. (1964). Crossing over near the centromere of chromosome 3 i n Drosophila melanogaster females. Genetics 50: 1427-1432. Suzuki, D. T., and Procunier, D. (1969). Temperature-sensitive mutations i n Drosophila melanogaster. I I I . Dominant lethals and semilethals on chromosome 2. Proc. Natl. Acad. S c i . (U.S.A.) 62: 369-376. Tarasoff, M., and Suzuki, D. T. (1970). Temperature-sensitive mutations in Drosophila melanogaster. VI. Temperature effects on development of sex-linked recessive l e t h a l s . Devel. B i o l . 23: 492509.  217 Tasaka, S. E., and Suzuki, D. T. (1973). Temperature-sensitive mutations i n Drosophila melanogaster. XVII. The genetic properties of heat- and cold-sensitive lethals on chromosome 3. Genetics 74: 509-520. Thompson, P. E. (1963a). Centric pairing and crossing over i n Drosophila melanogaster. Genetics 48: 697-701. Thompson, P. E. (1963b). Evidence on the basis of the centromere e f f e c t i n the large autosomes of Drosophila melanogaster. Genetics 49: 761-769. Tokunaga, C. (1962). C e l l lineage and d i f f e r e n t i a t i o n on the male foreleg of Drosophila melanogaster. Devel. B i o l . _+: 489-516. Vaslet, C. A., and Berger, E. M. (1976). Electrophoretic i d e n t i t y among r-proteins i n s i b l i n g and mutant species of D. melanogaster. Genetics _83: 79s. Vogt, M. (1947). Zur l a b i l e n Determination der Imaginalscheiben von Drosophila. I I I . Analyse der Manifestierungsbedingungen sowie der Wirkungsweise der zu Antennen und Palpusrerdoppel-^ ungen fuhrenden Genmutation Deformed-recessive-Luers (Dfd——) . Biolog. Zent. _66: 81-105. Weinstein, A. (1936). The theory of multiple-strand crossing over. Genetics 2_L: 155-199. Welshons, W. J . (1955). A comparative study of crossing over i n attached-X chromosomes of Drosophila melanogaster. Genetics 40: 918-936. White, B. N. (1974). An analysis of tRNAs i n five Minutes and two suppressors. Dros. I n f . Serv. 51_: 58-59. White, B. N., Tener, G. M., Holden, J . , and Suzuki, D. T. (1973). Analysis of tRNA's during the development of Drosophila. Devel. B i o l . 33: 185-195. W h i t t i n g h i l l , M. (1937). Induced crossing over i n Drosophila males and i t s probable nature. Genetics 22: 114-129. W h i t t i n g h i l l , M. (1955). Crossover v a r i a b i l i t y and induced crossing over. J . of C e l l . Comp. Physiol. 45: 189-220. Williamson, J . H., Parker, D. R., and Manchester, W. G. (1970). Xray-induced recombination i n the fourth chromosome of Drosophila melanogaster females. I. Kinetics and brood patterns. Mutation Res. 9_: 287-297. Wright, T. R. F. (1973). The recovery, penetrance, and pleiotropy of X-linked, cold sensitive mutants i n Drosophila. Moi. Gen. Genet. 122: 101-118.  APPENDIX 1 Crossover Data and Estimates of Crossing Over a)  Crossover Data from Irradiated "Males and Controls  Treatment of Male Parents Control Mo Radiation  Brcod Interval (davs) 0-3 3-6 6-9 9-12 12-15  Totals Expt I 1000R  0-3 3-7 7-11 11-16 16-21 21-26  Totals Expt I I 2000R  0-3 3-7 7-11 11-16 16-21 21-26  Totals Expt I I I 3000R  Totals  0-3 3-6 6-12 12-17 17-22  Pa rental Genotypes 2 ... p s in + r i eg K i p e 1,584 1,565 623 665 707 638 858 822 429 384 4,201 4,074 1,585 1,518 544 552 920 918 3,710 3,699 8,293 5,391 1,716 1,678 13,768 13,756 141 140 109 97 549 589 303 309 1,703 1,693 760 732 3,565 3,560 1,142 1,11.3 680 628 848 805 3,586 3,614 3,981 3,881 10,208 10,070  st  Single Crossover Progeny Region 3 Region 1 2__.ps 2 „. p s eg K i p e in r i eg K i p e  1 3 4  1 2  1 1  ~~8  ~3  ~2  2  1  ~2  1  I 1 1  1  ~~3  -  APPENDIX l a (continued) Treatment of Male Parents Control No Radiation  Brood Interval (days) 0-3 3-6 6-9 9-12 12-15  Region 4 o  st i n r i eg  Single Crossover Progeny Region 5  Ki D  P  e  S  st i n r i 2 eg K i  1000R  2000R  0-3 3-7 7-11 11-16 16-21 21-26 0-3 3-7 7-11 11-16 16-21 21-26  Totals Expt I I I 3000R Totals  s  2 D st i n r i eg Ki p  0-3 3-6 6-12 12-17 17-22  s e1  ^ P eg_ p _  "T  ~3  Totals Expt I I  P P e 3  Totals Expt I  Doubles 3,4 5,6  Region 6  1  1  6 6  1  6 2 5 13  1 1 2 4  2  1 1 5  1 1  ~T  ~2  1 1  7 ~~8  3 5  5 5 8 18  5 12 26 43  1 1  2 1  2  ~3  ~2  2 ~2  2  APPENDIX 1 (continued) b) Summary of Male Crossover Data and Estimation of Crossing Over Between st and p . P  Treatment of Male Parents  Number of Crossovers p ^ c p P to st to p 1 3  Number of Parentals  s  Experiment Totals 8,279  Control  8,275  Expt I  27,524  41  3  27,568  Expt II  7,125  16  -  7,141  Expt I I I  20,278  73  3  20,354  Experiment Totals  54,927  130  6  55,063  Control  3,835  3  -  10,796  64  4  Expt I I I * Estimations of Crossing Over  Control = Expt I I I =  0.08 Percent (3838 Progeny) 0.59 Percent  * Progeny of fourteen cultures, days 7 to 22  10,864  APPENDIX 2 Summary of Results of 24-Hour Expiosures of Q-III Homozygotes to 29°C at Specific Times During Development Hours of Pulse  Number of Adults  0-24 12-36 24-48 36-60 48-72 60-84 72-96 84-108 96-120 108-132 120-144 132-156 144-168 156-180 168-192 180-204 192-216 204-228 228-252 252-276 276-300 300-324 324-348  14(300)* 12(242) 28(300) 22(285) 25(515) 34(600) 37(504) 12(500) 35(500) 34(500) 20(200) 17(225) 13(250) 42(300) 29(300) 27(200) 14(300) 20(300) 6(300) 16(360) 13(342) 8(360) 22(300)  Corrected Percent Viability  * Number i n parentheses  58.3 63.2 116.0 95.7 60.6 70.8 93.8 30.0 87.5 85.0 125.0 106.0 65.0 120.0 120.8 130.0 58.3 47.6 25.0 55.6 47.5 27.7 91.7  Percent Expression of Various Phenotypes of Surviving Progeny Palp Antennal Thick Antennal Ommatidial Deficiencies Duplications Aristae Deficiencies Deficiencies 0 0 0 0 0 26.1 53.3 72.3 83.1 58.3 20.0 5.9 0 0 0 0 0 0 0 0 7.6 0 0  = Number of Eggs Shifted  0 0 0 0 0 8.8 0 0 0" 8.8 0 0 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 8.3 0 8.8 0 0 0 0 0 0 0 0 0 0 0 •0 0  0 0 0 0 4.0 11.8 13.5 25.0 22.9 58.8 50.0 5.9 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 8.3 2.9 2.9 5.0 5.9 0 0 0 0 0 0 0 0 0 0 0  APPENDIX 2  Hours of Pulse 0-24 12-36 24-48 36-60 48-72 60-84 72-96 84-108 96-120 108-132 120-144 132-156 144-168 156-180 168-192 180-204 192-216 204-228 228-252 252-276 276-300 300-324 324-348  Ocellar Duplications or Deficiencies 0 0 0 0 0 0 5.4 0 8.6 14.7 0 0 0 0 0 0 0 0 0 0 0 0 0  (continued)  Percent Expression of Various Phenotypes of Surviving Progeny Misrotated Wing Thoracic Sex Small Leg Male Pattern Vein Comb Thoracic Disruptions Terminalia Deformities Disruptions Macrochaetae Gaps 0 0 0 0 0 0 0 0 0 0 0. 0 0 0 0 0 0 0 16.5 100.0 7.7 0 0  0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 100.0 (males) 100.0 0 0 0  0 0 0 0 12.0 32.4 21.6 41.7 94.3100.0 65.0 23.5 33.3 0 0 0 0 0 0 0 23.1 0 0  0 0 0 0 0 0 0 10.0 39.1 5.0 0 0 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 4.0 10.0 20.0 50.0 33.3 61.5 37.5 50.0 33.3 0 0 26.6 25.0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 0 8.8 30.0 0 0 0 0 0 0 0 0 0 0 0 0  Etched Tergites 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 5.0 16.7 0 0 0 0  APPENDIX 3 Summary of Results of 48-Hour Exposures of Q-III Heterozygotes  (TMI/QIII)  to 29°C at Specific Times During Development Hours of Pulse 0-48 24-72 48-96 72-120 96-144 120-168 144-192 168-216 192-240 216-264 204-252 252-300 300-348 348-396  Number of Adults  Corrected Percent Viability  191 152 93 308 244 58 30 26 129 204 265 300 270 304  86.6 82.6 42.9 53.0 90.2 85.3 21.8 28.0 70.1 100.0 123.8 56.2 126.0 120.0  Percent Expression of Various Phenotypes Ommatidial Roughened Antennal Deficiencies Deficiencies Eyes 0 0 98.8 98.9 98.4 16.4 0 0 0 0 0 0 0 0  0 0 53.1 63.3 3.2 11.5 0 0 0 0 0 0 0 0  0 0 0 1.0 0 0 0 0 0 0 0 0 0 0  APPENDIX 3  Hours of Pulse 0-48 24-72 48-96 72-120 96-144 120-168 144-192 168-216 192-240 216-264 204-252 252-300 300-348 348-396  (continued)  Percent Expression of Various Phenotypes Thoracic Small Antennal Wing Pattern Thoracic Duplications Duplications Disruptions Macrochaetae 0 0 0 0.7 0.4 0 0 0 0 0 0 0 0 0  0 0.7 0 0 0 0 0 0 0 0 0 0 0 0  '  0 0.7 0 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 20.0 100.0 98.4 62.0 25.0 0 0 0  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0094310/manifest

Comment

Related Items