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Developmental and genetic analysis of a purported new class of sex-lined mutations in Drosophila melanogaster. Pratt, L. Rachel 1971

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DEVELOPMENTAL AND GENETIC ANALYSIS OF A PURPORTED NEW CLASS OF SEX-LINKED MUTATIONS IN DROSOPHILA MELANOGASTER by L. Rachel Pratt B.A., Reed College, 1964 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Genetics i n Zoology We accept th i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1971 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t he L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s tudy . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d tha t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . The U n i v e r s i t y o f B r i t i s h Co lumb ia V a n c o u v e r 8, Canada Department o f ABSTRACT During the screening process 5,20 8 X chromosomes of -Drosophila melanogaster were analyzed for the presence of temperature-sensitive (ts) l e t h a l mutations ( i . e . mutants which die at 29°C but are viable at 22°C) i n short proximal and d i s t a l segments of the chromosome. Seven ts and 16 non-ts le t h a l s were recovered i n both regions combined. A new class of mutations (class-3), which f a i l e d to survive at 29°C with either proximal or d i s t a l duplication and showed ts l e t h a l i t y with one, was found and extensively analyzed. These mutants were i n i t i a l l y interpreted to be dominant t s ' s , although the heterozygotes of each mutant showed t h i s not to be so. I t was decided that these might more probably be chromosomes carrying a l e t h a l mutation covered by the d u p l i -cation, and a ts l e t h a l mapping elsewhere. By masking the non-conditional l e t h a l with a d u p l i -cation, developmental studies of the ts mutant were made. The temperature-sensitive period (TSP) and l e t h a l phase (LP) were characterized for each. A l l TSP 1s spanned the early pupal i n t e r v a l , though an" i n d i v i d u a l TSP might extend to either side of t h i s i n t e r v a l . The pattern of temperature-sensitivity of C3-3 sug-gested that once formed at permissive temperature, i t s product i i i was not affected by 29°C. The experiments suggest that the temperature-sensitive process occurs at t r a n s c r i p t i o n or t r a n s l a t i o n . A l e t h a l a l l e l e of the dor locus was recovered, and, i n analysis of t h i s mutant with other dor a l l e l e s and several variegating duplications, dor i t s e l f was found to be a ts l e t h a l . "Warped" wing, a new phenotype of the dor locus which occurred only with the variegating duplications, was described. This paper further describes a method for developmental analysis of non-ts l e t h a l mutations, involving the use of variegating rearrangements. i v TABLE OF CONTENTS Page INTRODUCTION 1 METHODS AND MATERIALS 4 RESULTS 22 DISCUSSION 75 SUMMARY 92 BIBLIOGRAPHY 93 APPENDIX 99 V LIST OF TABLES TABLE PAGE I. A Synopsis of Gene Symbols and Chromosomes used i n the Text 5 I I . Results of the Screening Procedure for Lethal Mutations covered'by'Duplications of the X Chromosome 23 I I I . Pattern of Survival of Male Progeny for the Cross yw/Bs Y £ x X*/Dp Y ( f x yf:=/67g Y 9 for Each Class of Lethals 24 IV. V i a b i l i t y Properties of Different Stocks at 22°C and 29°C 26 V. Test for Dominance of Class 3 Stocks with Wild-type X-chromosome 28 VI. Homozygosis and Complementation Between the Class 3 Chromosomes 29 VII. Survival of the Class 3 Mutants i n the Presence of Different Y Chromosomes 32 VIII. Summary of the Tests of Class 3 Mutations with Different Y Chromosomal Constitutions 33 IX. Test of C3-6(S) Males which Survived at 29°C "for the Presence of the Lethal Mutation. . . . 36 X. Genetic L o c a l i z a t i o n of Class 3 Mutants Relative to y, f and car 38 XI. Egg Hatchability and Adult V i a b i l i t y of C3-n Cultures at 22°C and 29°C (from s h i f t studies) . 42 XII. Cross of Putative C3-5/C3-5/67g Y Females to FM-6/Y Males . 56 XIII.. Variegation and Interaction of dor A l l e l e s i n Males with Different Y Chromosomes 61 XIV. Summary of Table XIII: Male V i a b i l i t y and Variegation of dor a l l e l e s 62 v i TABLE PAGE XV. Trends of dor a l l e l e s i n Males with Different Y's, Summarized as Temperature Sensitive (ts) , Lethal (1) or Non-lethal ( + ) 63 XVIa.• • Interactions of dor a l l e l e s i n Females . . . . 67 XVIb. Summary of Table XVI(a): L e t h a l i t y (1) and Temperature S e n s i t i v i t y (ts) of dor female Interactions, with V i a b i l i t y Index at 22° given with Phenotype 6 7 v i i LIST OF FIGURES FIGURE PAGE 1. X chromosome l o c i present on duplications attached to the Y chromosome 7 2. Screening protocol for the detection of ts l e t h a l mutations at the t i p of the X chromosome . 9 3. Genetic l o c a l i z a t i o n of leth a l s by the use of overlapping duplications 11 4. Scheme for the genetic positioning by a 3-point testcross of leth a l s located d i s t a l l y 13 5. Method for generation of X/X/Y f l i e s by non-disjunction of sc^sc^ 14 6. Method to generate ts/ts/67g Y females using the mutation, mei-S332 15 7. Protocol for r e c i p r o c a l " s h i f t " experiments to determine TSP and LP of ts mutants. ....... . . .19 8. Scheme to test v i a b i l i t y of C3-6 with Su, S_u+ and E of variegation. . 21 9. Comparison of double-hit and o r i g i n a l theories to explain v i a b i l i t y pattern 41 10. Relative v i a b i l i t i e s of f l i e s from the cross yw/B_S Y? x. C3-1/Bs Y <f aft e r s h i f t s at d i f f e r e n t times during development 45 11. Relative v i a b i l i t i e s of f l i e s from the cross yf :=/Bs Y? x C3-2/Bs Y i? a f t e r s h i f t s at d i f f e r e n t times during development 46 12. Relative v i a b i l i t i e s of f l i e s from the cross yf:=/B s Y ? x C3-2/Bs J a f t e r double s h i f t s at d i f f e r e n t times during development 47 13. Relative v i a b i l i t i e s of f l i e s from the cross yw/BS Y? x C3-3/BS Y <f a f t e r s h i f t s at d i f f e r e n t times during development 48 v i i i FIGURE PAGE 14. Relative v i a b i l i t i e s of f l i e s from the cross yf:=/B s Y? x C3-3/BS Y<? afte r double s h i f t s at d i f f e r e n t times during development 50 15. Relative v i a b i l i t i e s of f l i e s from the cross C3-4/KFM-6) ? x C3-4/67g Y <?* afte r s h i f t s at d i f f e r e n t times during development 52 16. Relative v i a b i l i t i e s of f l i e s from the cross yf :=/67g Y j? x C3-5/67g Yd" after s h i f t s at d i f f e r e n t times during development. . . 54 17. E f f e c t i v e l e t h a l phases and temperature sensitive periods of each stock 58 18. Relative v i a b i l i t i e s of f l i e s from the cross". C3-6/T(FM-6) $ x y dor/T2E Y D cf a f t e r s h i f t s at d i f f e r e n t times during development 6 8 19. Relative v i a b i l i t i e s of f l i e s from the cross y dor/FM-6 $ x y dor/Y cf after s h i f t s at d i f f e r e n t times during development. 69 20. Relative v i a b i l i t i e s of f l i e s from the cross y dor/FM-6 ? x y dor/Y cf a f t e r double s h i f t s at d i f f e r e n t times during development 72 ACKNOWLEDGEMENT I wish to express my gratitude for Dr. D. T. Suzuki's encouragement and stimulation throughout t h i s work. I am also thankful for the excitement generated by a l l the people i n the lab who helped make thi s experience so worthwhile. I am p a r t i c u l a r l y indebted to Michael Schewe and Jeanette Holden for t h e i r patience and help. INTRODUCTION Much work has been done with lower organisms to determine whether c l o s e l y linked genes exhibit a functional or temporal a c t i v a t i o n r e l a t i o n s h i p . The c l a s s i c example of such a rela t i o n s h i p was elucidated for cistrons of the lac operon by Jacob and Monod (1961) and extended to the operons governing the h i s t i d i n e pathway i n Salmonella (Ames and Hartman, 1963), the tryptophan operon i n E. c o l i (Yanofsky, 1960), and numerous other operon systems (Ames and Martin, 1964) . • The most'striking example of c o r r e l a t i o n between genetic p o s i t i o n and function was detected i n phage T4D by Epstein et a l . (1963), who found that genes with early functions c l u s t e r i n one region of the genophore and those which function late group elsewhere. Moreover, within the c l u s t e r s , subsets of l o c i a f f e c t i n g a common morphological event, such as t a i l - f i b r e construction, head assembly, DNA synthesis, and the assembly of the parts into a functional phage, were noted to map together. In-higher organisms, the number of known operons, or gene complexes, i s much smaller. In maize, a number of mutant series believed to represent gene complexes have been reported (Lewis, 1967). In spite of extensive knowledge of biosynthetic pathways i n Neurospora, few operons are known 2 (Giles et a l . , 1967) and i n contrast to the b a c t e r i a l system, very few cistons of the h i s t i d i n e pathway are linked (Lewis, 1967) . The most extensively studied m u l t i c e l l u l a r eukaryote, Dorsophila, has several examples of f u n c t i o n a l l y related gene complexes (or pseudoallelic series as they are often c a l l e d ) . Their r e l a t i o n s h i p to operons i s not known. The dumpy (dp) complex contains at l e a s t 8 g e n e t i c a l l y separable l o c i , lozenge (lz) has 4, and bithorax (bx) has 5 (Lindsley & G r e l l , 1968). The Notch locus comprise a- f u n c t i o n a l l y diverse series of at least eleven l i n e a r l y ordered mutant s i t e s (Welshons, 1965). The complexity of interactions (e.g. lack of complementation) between the Notch mutants, even i f they do l i e i n separate c i s t r o n s , makes t h e i r c l a s s i f i c a t i o n as a gene complex more tentative than the more c l e a r l y defined examples given above (Lewis, 1967). In Drosophila melanogaster, conditional l e t h a l muta-tions of the temperature-sensitive type provide a useful class for the analysis of gene action during development (Suzuki, 1970). Temperature-sensitivity of l e t h a l mutations permits observation of the e f f e c t s of temperature changes at d i f f e r e n t times i n development (Tarasoff & Suzuki, 1970). Thus, cultures s h i f t e d from permissive or r e s t r i c t i v e temp-erature (and vice versa) permit a delineation of the actual developmental i n t e r v a l affected by temperature (Suzuki, 1970). 3 In view of the current paucity of information on developmental e f f e c t s of t i g h t l y linked l o c i , the recovery of a number of ts l e t h a l s located within a short genetic region was considered an important undertaking. A detailed developmental analysis of such mutants might provide us with information,on ,:the •role of genetic p o s i t i o n i n development. This report describes experiments designed to satur-ate a short genetic segment i n Drosophila with ts l e t h a l mutations and the analysis of mutants recovered. METHODS AND MATERIALS a) Detection of temperature-sensitive l e t h a l mutations within s p e c i f i c regions of the X chromosome. A synopsis of the important properties of the mutants and chromosomes used i s seen i n Table I; for a more complete description consult Lindsley & G r e l l (1968). One to three day old wild type (Oregon-R^gg or Samar-kand) males were placed on f i l t e r papers saturated with 0.025M ethyl methane sulfonate (EMS) dissolved i n a 1% sucrose solution (Lewis and Bacher,' 1968) . After 2 4 hours the treated males were mated i n quarter pint milk bottles to 2 + either yf:=/y sc 59k9-Y#4 or yf:=/67g24 Y v i r g i n females (10 males and 20 females per b o t t l e ) , at 22°C ± 1°C. The females ca r r i e d Y chromosomes to which a duplication of the d i s t a l portion of the X chromosome was attached. The extent of the -duplications can be seen i n Figure 1. The t i p duplications were synthesized.by Dr. Rayle, who kindly supplied them, and provided the map (Fig. 1) describing them. Individual F 1 males carrying a treated X chromosome and duplication-bearing Y chromosome derived from t h e i r mothers were then mated i n -d i v i d u a l l y at 22°C i n s h e l l v i a l s to two yw/Bs•Y and two yf:=/59k9 or 67g24 Y females, depending on which Y the male car r i e d . A f t e r 4 days at 22°C, the f l i e s were transferred 5 Table I A Synopsis of Gene Symbols and Chromosomes Used i n the Text Symbol or Chromosome Chromosome and Location Phenotype or Description Oregon-R 3 g 9 Samarkand C(1)DX, y f (= .yf : = ) C(1)RM, y w (= yw) B SY 67g24 Y y f car rb dor spl sc^sc^ FM-3 Y Y 1 - 0.0 1 -56.7 1 -62.5 1 - 7.5 1 - 0.3 1 - 3.0 1 wild type stock wild type stock -reversed acrocentric attached - X chromosome, marked with y and f reversed metacentric attached -X chromosome, marked with y and w a Y chromosome derivative marked with B£ a Y chromosome derivative carrying the t i p of the X chromosome. For a com-plete description of a l l Dp. Y's used, see F i g . l Yellow body colour B r i s t l e s shortened and bent eye colour dark ruby eye colour pure ruby eye color orange, female s t e r i l e eyes small and rough Inversion of the X chrom-osome, d e f i c i e n t for the bb locus Multiply inverted X chromosome, used to suppress crossing over Marked with y and B. 6 Table I (Continued) Symbol or Chromosome Chromosome and Location Phenotype or Description FM-6 1 Similar to FM-3 1(FM-6) 1 Similar to FM-3, but carri e s a l e t h a l murS = ^  mei-S332 2 Causes non-disjunction at 2nd miotic d i v i s i o n (Sandler et a l . , 1968) SM-1, QyCcn 2 Multiply inverted II chromosome, used to suppress crossing over. Marked with cn and Cy. 1 , or ts Any recessive point mutants which are l e t h a l at 29°C, but viable at 22°C. to fresh vials.and placed at 29°C. After 5 days at 29°C, the adults were discarded. The presence of both white and wild-type eye-coloured females indicated that the male had mated successfully with both types of females and only these cultures were scored. Preliminary tests showed that 80% of the males successfully mated with both types of females within 4 days. Each 29°C r e p l i c a t e culture was scored by inspection through the v i a l for the absence of Y-bearing F i g u r e 1: X-chromosome l o c i p r e s e n t on d u p l i c a t i o n s a t t a c h e d to t h e Y chromosome. G e n e t i c L o c a t i o n .0 . 0 , .01 L o c i .1 .3 .3 .3 v_f. s c ^ s u ( s ) ' r su(W a )+ s t a * d o r * hfw* D u p l i c a t i o n m > W///////A T ( U Y ) 2 E mm 59k9(4) W////////////A «*"<» Y/////J////////////////////A BS 64.8 65.9 + c e n t r o -maj s u ( f )*** mere Y' W7777m\ V////////////////7A  V///J//////////7A  W////////////77JX 8 males and the s u r v i v a l of males carrying the X-duplicated-Y, thereby i n d i c a t i n g the presence of a recessive l e t h a l muta-tio n covered by the duplication on the Y chromosome. Such cultures, along with the corresponding 22°C culture, were kept and a l l f l i e s were etherized and c l a s s i f i e d phenotypi-c a l l y . The presence of both classes of males at 22°C led to the c l a s s i f i c a t i o n of the stock as a putative t s . Seven duplication-bearing males of each 22°C culture retained were then mated i n d i v i d u a l l y , as had been done i n the cross, i n order to v e r i f y the temperature s e n s i t i v i t y . The complete screeing procedure can be seen i n Figure 2. The ts stocks were retested p e r i o d i c a l l y during the course of the develop-mental studies. The same procedure can be used to i s o l a t e l e t h a l mutations located around the B region. b) Genetic l o c a l i z a t i o n s I t can be seen (Figure 1) that each Y chromosome carri e s proximal as well as d i s t a l X chromosomal material. Thus, l e t h a l s covered by the duplication could be located i n either the d i s t a l or proximal region. Consequently, each l e t h a l mutation retained was l o c a l i z e d g e n e t i c a l l y with res-pect to the following recessive v i s i b l e markers (followed by t h e i r symbol and genetic p o s i t i o n ) : yellow (y, .0), forked (f, 56.7) and carnation (car, 62.5). For a complete description of the markers used, consult Lindsley and G r e l l Figure 2: Screening protocol for the detection of ts lethal mutations at the tip of the X chromosome. * X* denotes a mutagen!zed chromosome. ** DpY denotes either the 59k9Y or 67g24Y duplication. 9. X/Yef (0.025 M EMS) x yf:=/Dp Y** ? I yf t»/DpY ? x X*/DpY d" x yw/ BSY ? 4 days at 22°C transfer parents to 29°C F2 yf :=/DpY? X*/DpY 4 X*/BSY <? vw/ DpY ? - score of v i a b i l i t y of dtf at 22°C and 29 °C - retest as in cross - make stock by mating to v_f :=•/ DpY 10 (1968). Males carrying the l e t h a l chromosome were mated to y f car females and t h e i r female progeny were t e s t -crossed (20 females and 20 males per bottle) i n quarter pint milk bottles at 29°C. A l l male offspring were then counted. The d i s t a l p o s i t i o n of a l e t h a l was indicated by the excess recovery of y males, whereas proximally situated l e t h a l s yielded an excess of f car o f f s p r i n g . Lethals at the t i p of the X could be located more accurately by t h e i r s u r v i v a l or l e t h a l i t y i n the presence of duplications of d i f f e r e n t s i z e s . Thus, the duplications 67g, 59k, 60d and T2E delineated 5 regions into which lethals might f a l l (Figure 1). Ten lethal-bearing males were mated to 20 v i r g i n females carrying an attached-X chromosome and each of the X-duplicated Y's at 22°C and 29°C. The cultures were examined for s u r v i v a l of male progeny. The pattern of v i a b i l i t y expected for l e t h a l s located i n d i f f e r e n t positions can be seen i n Figure 3. As additional controls, each l e t h a l was tested for s u r v i v a l at 22°C and 29°C with and without a normal Y chromosome. Those mutants located within either a proximal or a d i s t a l region were tested i n trans heterozygotes for s u r v i v a l at 29°C. This was determined by mating ts.^/FM-6 to ts^/Pj? Y <T a n c ^ scoring for v i a b i l i t y of heterozygous ts^/tS2 females at 22° and 29°C. A l l possible combinations were produced. The l o c i were located r e l a t i v e to each other by a three point testcross. Chromosomes bearing the d i s t a l l e t h a l s Figure 3: Genetic localization of lethals by the use of overlapping duplications. * +» lethal mapping in this region w i l l live ** lethal mapping in this region w i l l die. 12 were marked with rb (7.5) and the proximal let h a l s were marked with car (62.5). The method of l o c a l i z a t i o n can be seen i n Figure 4. This method allows the ready ordering of very c l o s e l y linked mutants, such as d i f f e r e n t a l l e l e s of the same locus, so long as heterozygous females survive and are f e r t i l e . Figure 4 i l l u s t r a t e s the scheme for the d i s t a l l e t h a l s ; the proximal ts's were marked by a similar procedure with carnation which i s 5 map units to the ri g h t of B +. c) Production of females homozygous for ts leth a l s In order to generate females homozygous for the l e t h -a l s , ts/FM-6 £ were crossed to ts/Dp Y (f at 2 2°C. Where such females were recovered, they were tested for f e r t i l i t y at both 22°C and 29°C. Of those mutants which could not be made homozygous, an attempt was made to generate ts/ts/Dp Y females by two methods. The scheme shown i n Figure 5 u t i -4 8 l i z e d the high frequency of non-disjunction i n sc so /Y males (Sandler and Braver, 1954) to generate X/Y/Y males. In turn, the X/Y/Y males could produce X/X/Y females. The possible f a i l u r e of t h i s method to provide the desired females could be either the f a u l t of the method i t s e l f or the i n a b i l i t y of such females to l i v e . Therefore, a second scheme was devised. The mutant mei-S332 causes non-disjunction of s i s t e r chroma-ti d s at the second meiotic d i v i s i o n i n both males and females (Sandler et a l . , 1968). The complete procedure can be seen i n Figure 6. F i g u r e 4$ Scheme f o r t he g e n e t i c p o s i t i o n i n g by a 3 - p o i n t t e s t c r o s s o f l e t h a l s l o c a t e d d i s t a l l y . 13 . wa rb/Y 4 x l t s/FM-6 ? wa r b / l t s S x +/67gY 4 I Select rb/67gY 44, 10 per lethal. Pair mate to XX/67gY S 4 days at 22°C, then transfer to 29 °C 22°C 29°C Score for lethal Then cross each marked lethal to the other two unmarked. its. 1 2 10 bottles 29°C - Score male survivors for rb or r b + — Count females Rationale: i f l t S 1 to l e f t of l t s 2 l j + rb x — surviving 44 + + rb if l t s 1 to right of l t S 2 + 1± rb lo + + + + r b + Figure 5: Method for generation of ts/ts/67gY females by non-disjunction of sc^sc 8. * T2EYD variegates for eye colour with ts lethal C3-6, under consideration here. 67gY does not variegate and is dominant. t Lethal class. 14. STEP 1. 2. 3 i y m s c 4 s c 8 / BSY 4 x XX/67gY ? i y w sc 4sc 8/B SY/67gY 4 x FM-6/ts ? Select non-B ?? (either y w sc^sc 8/ts of y w sc 4sc 8/ts/67gY) Pair mate non-B ?? from step 2; y w sc 4 s c 8 / t s ? x ts/T2EYD* 4 x y w sc 4sc 8/ts/67gY ? y w sc^sc 8/ts f ts/ts 1/2 y w sc^sc8/T2EYD 1/2 ts/T2EYD phenotype y w - non-w - variegates y w sc^sc 8/ts t ts/ts y w sc^sc 8/ ts/67gY ts/ts/67gY 44 fy w sc^sc8/T2EY 1 / 2 ( y w sc*sc8/T2EY/67gY 1/4 ts/T2EY 1/4 ts/T2EY/67gY phenotype y w -non-w -variegates -non-w -does not variegate 4,' Pair mate a l l females from vials yielding "B" results to ts/67gY 4. 5. Test a l l vials yielding no y w sc^sc 8 male progeny for ts lethality of a l l male progeny. Figure 6: Method to generate ts/ts/67gY females using the mutation, mei-S332. 67gY * t s cn me i -S332 67gY' cn me i -S332 s e l e c t y . + f + f ema le s t e s t f o r t e m p e r a t u r e s e n s i t i v i t y o f the X chromosome - t e s t f o r p r e s e n c e of 67gY 1 6 d) Determination of the E f f e c t i v e Lethal Phase (LP) and Temperature Sensitive Period (TSP) of each ts l e t h a l . The a b i l i t y of f l i e s which carry ts le t h a l s to survive at permissive temperature, but to die at r e s t r i c t i v e temper-atures, permits a determination of t h e i r developmental properties. The "e f f e c t i v e l e t h a l phase" (LP), that i s , the time at which death occurs, could be shown by growing con-tinuously at 29°C. The temperature sensitive period (TSP) i s the developmental i n t e r v a l during which the organism i s irrevocably committed to death by r e s t r i c t i v e temperature. If possible, an attempt was made to recover females homozygous for each l e t h a l by crossing ts/Dp Y £ x ts/FM-6 <f at 22°C. F a i l i n g t h e i r recovery or f e r t i l i t y , developmental studies were made as follows. 50 - 75 ts/FM-6 were mated with 30 - 50 ts/Dp Y <y , allowed to mate for several days and then placed i n quarter pint milk bottles at 29°C. After.2 hours, the adults were removed, the number of eggs were counted, and the culture was retained at 29°C for observation at regular d a i l y i n t e r v a l s . Since synchrony of the cultures i s highly desirable, and f e r t i l i z e d eggs can be held i n the uterus for up to 20 hours, the f i r s t egg c o l l e c t i o n was d i s -carded. The number of eggs hatching was counted and l a r v a l i n s t a r stages were determined by examination of mouthparts and eversion of trachea (Bodenstein, 19 50). Larval death 17 was indicated by the i n a b i l i t y of larvae to move af t e r gentle prodding...Death i n the pupal stage could be detected by the f a i l u r e to eclose, the absence of re a d i l y observable organs (eyes, wing pads), or the detection of gross h i s t o l y -s i s . The LP i n males was studied by mating ts/Dp Y males to XX/Dp Y females. However, although one quarter of the progeny were Y/Y zygotes which died i n the egg, another A quarter were XX/ts_ metafemales which died over a prolonged i n t e r v a l up to eclosion. Accurate determination of separate LP 1s between the sexes could have been achieved by.sexing 1st i n s t a r larvae (Bodenstein, 1950) and studying separate cultures of each sex. When cultures are not separated as to sex, and i f two separate LP's are evident, one can sex the animals s t i l l a l i v e a f t e r the f i r s t distinguishable LP. The LP defines only the time at which s u r v i v a l of ts-bearing f l i e s i s no longer possible at 29°C, but does not delineate the actual developmental i n t e r v a l during which the r e s t r i c t i v e temperature imposes death. The temperature sensitive period (TSP) can be determined by re c i p r o c a l "shift-ups" and."shift-downs". Cultures established at 22°C, each having at least' 75 eggs, were transferred to 29°C at d i f f e r e n t successive time i n t e r v a l s (shift-ups). The r e c i -procal shift-down involves transferring 29°C cultures to 22°C at d i f f e r e n t times. The e a r l i e s t shift-up which y i e l d s viable ts adults signals the end of the TSP, whereas the 1 8 f i r s t shift-down which does not permit ts adults to emerge indicates the s t a r t of.the TSP. The procedure i s i l l u s t r a t e d i n Figure 7a. These experiments were carried out i n s h e l l v i a l s i n which 40 pairs of parents were allowed to deposit eggs over a one-two hour period at 22°C and 29°C. The number of eggs l a i d and hatched were then counted. For the f i r s t 4-5 days, s h i f t s were made at 12-hour i n t e r v a l s . After that, s h i f t s were made at 24 hour i n t e r v a l s . The TSP determined by the s h i f t s i s the r e s u l t of two separate r e c i p r o c a l experiments. A c r i t i c a l t e s t of the v a l i d i t y of the i n t e r v a l so defined i s the "double s h i f t " experiment. In thi s t e s t , cultures established at 22°C were allowed to grow e n t i r e l y at 22°C except for a short period, presumably encompassing the TSP, during which they were s h i f t e d up to high temperature and then back down. This should y i e l d no viable ts_ adults. Conversely, cultures grown e n t i r e l y at 29°C, except for a short time (the puta-t i v e TSP) at 22°C, should y i e l d viable adults. The procedure for the double s h i f t experiments i s i l l u s t r a t e d i n Figure 7b. e) Modification of variegation of the mutants Several of the mutants were quite "leaky" at 29°, that i s , a large number of progeny (up to 20%) survived at the r e s t r i c t i v e temperature. E r r a t i c expression of the wild type a l l e l e i n the duplication ( i . e . , variegation) could be Figure 7: Procedure for reciprocol " s h i f t " experiments to determine TSP and LP of ts mutants. a) Procedure for single shifts to determine start and end of TSP. b) Procedure for TSP delineating double s h i f t . a.) Single shifts > ADULTS > ADULTS 1 2 3 4 5 6 7 DAYS AT INITIAL TEMPERATURE B E F O R E SHIFT 10 11 12 b.) Double shif ts 29°C 22°C 1 2 3 4 5 6 DAY OF S H I F T - U P AND SH IFT-DOWN 8 >ADULTS 10 i 20 responsible for t h i s . This p o s s i b i l i t y could be tested by determining and comparing v i a b i l i t y of the ts mutants i n the presence of a suppressor of variegation, (Su(var)) (Spofford, 1969), and an enhancer of variegation (E(var)) (Lindsley and G r e l l , 1968). Both Su(var) and Su +(var)must be tested as d i f f e r e n t l o c i are affected by either one or the other (Spofford, personal communication). A negative r e s u l t i s not meaningful since i t i s known that Su(var) and Su +(var) do not act on a l l variegating l o c i . E(var) i s also locus and a l l e l e s p e c i f i c (Schultz, personal communica-tion) . The crosses are shown i n Figure 8, but i t should be noted that while E(var) i s a complete dominant, the suppres-sors show incomplete dominance. Thus, had time permitted, crosses to obtain"ts/Dp Y; Su(var)/Su(var), and the Su +(var) complement, would have given more c l e a r l y defined r e s u l t s . Figure 8: Scheme to test v i a b i l i t y and variegation of C3-6 at 22°c and 29°C with Suppressor-, Suppressor**"-, ana Enhancer of variegation. * Both Su(var) and Su(var)* w i l l be tested in these crosses. ** Both 67gY and T2EYD w i l l be u t i l i z e d . Su(var)* XX/67gY**? x TM-3/G1 4 i XX/Y;Su(var)/Su(var)? x X/67gY;TM-3/+ x XX/67gY ? XX/67gY;TM-3/Su(var)? x C3-6/67gY;+/+ x XX/67gY; TM-3/+ set at 22°C and 29°C C3-6/67gY;Su(var)/+ <f C3-6/67gY; +/+ Score v i a b i l i t y and variegation at 22°C and 29°C. E(var) XX/67gY ? x Cy/Pm X/67gY; Pm/+ d x X/X; Cy/E(var) ? C3-6/FM-6; +/+ ? x X/67gY; Pm/E(var) <f set at 22°C and 29°C C3-6/67gY; E(var)/+ <? Score v i a b i l i t y and variegation at 22°C and 29°C. RESULTS Out of 5,208 X-chromosomes tested, 7 temperature-sensi t i v e (ts) and 16 non-ts let h a l s were recovered (Table I I ) . Of the non-ts l e t h a l s , 12 mapped at the t i p of the X chromosome and 4 were located i n the proximal region around the B locus. Of the ts l e t h a l s , two were covered by g 67gDp Y and the other 5 by B_ Y. Since 67gDp Y c a r r i e s at least. 0.3 map units of X chromosomal material which includes a minimum of 42 known l o c i of which only 2 were h i t , i t appeared that at least 100,000 chromosomes would have had to be tested i n order to approach saturation of the l o c i covered by the duplication. Consequently, screening was terminated. In the o r i g i n a l screening process, a t h i r d class of let h a l s (Class 3 mutants abbreviated C3-1 etc.) which yielded s u r v i v a l patterns d i f f e r e n t from the non-ts and ts reces-sive l e t h a l s was detected. Six such mutations were con-g firmed, 3 being covered by B_ Y and 3 by 67g Y. The d i s t i n c t pattern of su r v i v a l of class 3 mutants i n contrast to the recessive l e t h a l s can be seen i n Table I I I . The r e l a t i v e v i a b i l i t y of f l i e s carrying a class 3 chromosome could be measured by the r a t i o of X*/Dp Y £ (where X* denotes the mutant X chromosome and Dp Y either 67g Y or S B Y) to t h e i r attached-X s i s t e r s . I f no l e t h a l i t y i s e v i -dent, the r a t i o w i l l be close to the Mendelian expecta-23 Table II Results of the Screening Procedure for Lethal Mutations covered by Duplications of the X Chromosome Stock Number.of Lethals ts Lethals Class 3 Chromosomes Tested No Q, No. Q. *o No. % Amherst 1,848 5 .3 4 .2 2 .1 Samarkand 3,360 11 .3 3 .1 4 .1 Totals 5,208 16 .3 7 .1 6 .1 Mutants Number of Lethals ts Lethals Class 3 Covered by Chromosomes by Tested No o. No. Q. P No. % B!Y 5,208 4 <.l 5 .1 3 .05 67g Y 5,208 12 .2 2 <.l 3 .05 24 Table III Pattern of Survival of Male Progeny of the Cross yw/BS Y % x X*/Dp Y / x yf:=/6 7g Y ? for Each Class of Lethals a) D i s t a l mutations Non-ts Lethal ts-Lethal Class 3 29°C 22°C 29°C 22°C 29°C 22°C X*/B^ Y - + -X*/67g Y + + + + + b) Proximal mutations X*/B^ Y + + + + + X*/67g Y - + -25 tion of 1.0. Indeed, control r a t i o s for wild-type Samar-kand X chromosomes varied only s l i g h t l y from the predicted value at both 22°C and 29°C (Table IV). The r e l a t i v e v i a -b i l i t i e s of each class 3 chromosome at. both temperatures can be seen i n Table IV. A chi-square analysis of sur v i v a l of mutants C3-5 and C3-6 under permissive conditions ( i . e . 22°C with 67g Y) revealed a s i g n i f i c a n t increase over the expected 1:1 ration of cT:? ; accordingly, the v i a b i l i t y r a t i o at 22°C was designated as 1.0 and the 29°C r a t i o adjusted r e l a t i v e to i t . Most of the mutants obviously gave reduced s u r v i v a l even under permissive conditions. In addition, "leakiness," that i s , s u r v i v a l under r e s t r i c t i v e conditions, was considerable, p a r t i c u l a r l y at 22°C for those mutants covered by B_ Y and at 2 9°C for those covered by 67g Y. Thus, mutants of t h i s class exhibited v a r i a b i l i t y i n su r v i v a l patterns that would normally have caused t h e i r r e j e c t i o n from further characterization (Suzuki et a l . 1967). In contrast to the others, class 3 chromosomes survive only i n the presence of one of the duplications at 22°C; thus, class 3 mutants behaved as dominant ts (DTS) l e t h a l s . Their f a i l u r e to survive with the other duplication resembles the known recessive l e t h a l i t y of DTS let h a l s at permissive temperatures (Suzuki and Procunier, 1969) . Since DTS le t h a l s on the X-chromosome had not been detected before (Suzuki, 1970), i t was decided to study class 3 mutations i n some d e t a i l i n order to compare t h e i r properties with known autosomal DTS l e t h a l s . 26 Table IV V i a b i l i t y Properties of Different Stocks at 22°C and 29°C Stock Temp, yf := ? X*/67g Y ^aSo** X V ^ - Y r a t i o * (C) Samarkand 22° 820 814 .99 766 1.27 602 29° 529 431 .81 881 1.07 822 C3-1 22° 418 43 .1 417 .64 648 29° 302 1 .00 24 .04 571 C3-2 22° 541 45 .08 447 .67 668 29° 423 0 .00 8 .02 362 C3-3 22° 302 33 .11 221 .46 505 29° 316 1 .01 4 .01 441 C3-4 22° 607 367 .6 8 .02 300 29° 526 27 .05 4 .01 311 C3-5 22° 431 518 1.00*** 0 .00 454 29° 461 119 .20 0 .00 326 C3-6 22° 206 343 1.0*** 0 .00 405 29° 165 18 .06 0 .00 342 * designates treated chromosome = C3 o^/non-lethal female sibs * * * Adjusted r a t i o , see text for explanation 27 The actual DTS nature of the class 3 mutants"could be shown i f females heterozygrbus for.each mutant were i n -viable at 29°C. Consequently, X*/Dp Y males of each class 3 stock were crossed simultaneously to wild-type (Samarkand) and XX/Dp Y females and cultures established at 22°C and 29°C. The results (Table V) showed that none of the chrom-osomes was dominant i n females at either temperature. The results of the complementation tests (Table VI) showed that a l l of the class 3 stocks survived as trans-heterozygotes and therefore are n o n - a l l e l i c . The previous detection of sexually r e s t r i c t e d ts l e t h a l i t y (Suzuki, personal communication) or sexually d i -morphic expression of temperature-sensitivity (Tarasoff & .Suzuki, 1970) suggested that class 3 mutants might act only i n males. Crosses to homozygose each chromosome (Table VI) showed that 4 of the mutants behaved as recessive ts lethals i n females. Of those, females i n only two stocks.were f e r -t i l e . I t was not determined whether s t e r i l i t y resulted from a second mutation on. the class 3 chromosome. The two re-maining stocks, C3-5 and C3-6 could not be homozygosed at either 22°C or 17°C and therefore, i n females behaved as recessive l e t h a l s . Interestingly, these two mutants yielded excess numbers of X*/67g Y rfV'at 22°C. Further attempts to make these two mutants homozygous w i l l be discussed l a t e r . Thus, there appear to be three subgroups of class 3 mutants 28 Table V Test for Dominance of Class 3 Stocks with Wild-type X-chromosome Cross: +/+ £ x C3/Dp Y / x XX/Dp Y ? Stock +/C3 ? P r o g e n y XX/Dp Y ? C3-1 22°C 156 219 103 29°C 142 173 59 C3-2 22°C 95 170 68 29°C 117 139 104 C3-3 22°C 125 108 66 29°C 84 94 47 C3-4 22°C 55 - 54 11 29°C 31 27 21 C3-5 22°C 123 205 102 29°C 141 115 93 C3-6 22?C 107 69 91 29°C 136 102 47 Table VI Homozygosis and Complementation Between the Class 3 Chromosomes C3-1 C3-2 FM-6/C3-X C3-x/C3-y V.I.* f e r t i l . FM-6/C3-X C3-x/C3-y V.I. f e r t i l , C3-1 22°C 380 210 .55 + 35 45 1.29 + 29°C 90 14 .15 146 82 .56 290/22°** .27 .43 C3-2 22°C 520 425 .81 29°C 280 0 .00 29°/22° .00 C3-3 CTj 4 C3-1 22°C 120 130 1.08 + 29°C 220 195 .88 29°/22° .81 C3-2 22°C 165 175 1.06 + 29°C 206 180 .87 29°/22° .82 C3-3 22°C 449 286 .63 29°C 648 14 .02 29°/22° .03 to Table VI. .(Continued) C3-4 C3-5 FM-6/C3-X C3-x/C3-y V.I. f e r t i l . FM-6/C3-X C3-x/C3-y V.I. f e r t i l , C3-4 22° 420 380 .9 + 125 410 3.28 + 29° 145 0 .00 145 370 2.55 29°/22° .00 .77 C3-5 22° 1,166 4 .00 29° 647 0 .00 29°/22° .00 C3-6 C3-4 22° 340 + 29° - 260 29°/22° .76 C3-5 22° 240 220 .91 + 29° 230 130 .56 .61 C3-6 22° 911 0 .00 29° 656 0 .00 .00 * V.I. = v i a b i l i t y index: </V? at one temperature ** 29°/22° = r a t i o of 29° r a t i o to 22° r a t i o o 31 based on t h e i r properties i n females: a) non-ts l e t h a l (C3-5, C3-6), b) ts l e t h a l , s t e r i l e (C3-2, C3-3), and ts l e t h a l , f e r t i l e (C3-1, C3-4). It may be.asked whether the expression of class 3 defects .might also be variable i n males. Consequently, three types of Y'chromosome alte r a t i o n s were tested with a l l class 3 mutants at 22°C and 29°C: 1) a series of Y chromosomes bearing duplications of d i f f e r i n g sizes of the t i p of the X chromosome; '2Y Y chromosomes derived from three d i f f e r e n t stocks, and 3) the absence of any Y chromosome element. The results of these combinations are shown i n Table VII and summarized i n Table VIII. I f one (momentarily) ignores the l a s t column, the v i a b i l i t i e s of nullo-Y males carrying class 3 chromosomes support the conclusions drawn from the female data: mutants C3.-1, C3-2, CS-^ and C3-4 are recessive ts leth a l s whereas C3-5 and C3-6 are non-ts l e t h a l s . There i s a noticeable v a r i a t i o n i n the results of the Y experiments, depending on the source of the Y chromosome. The d i s t a l C-3 mutants are l e t h a l with a l l 3 Y's. C3-4 may be sexually dimorphic, appearing as a ts recessive l e t h a l i n females and as a non-ts l e t h a l i n males. The mutants of the B region vary considerably i n t h e i r v i a b i l i t y with the d i f f e r e n t Y's. C3-3 survived only at 22°C with Y's from the Samarkand and XX stocks. C3-1 survived exceedingly well with the XX de-rived Y at 22°C and was "leaky" at 29°C. At both 22°C and Table VII Survival of the Class 3 Mutants i n the Presence of Dif f e r e n t Y Chromosomes 67g2y Y 59k9 Y 60dl9 Y T(1:Y)2E YD Y Class 3 Stock' ? <? V] I. * • " • < ? V.I. ? V.I. ? V.I. ? V.I C3-1 22° 418 43 .10 176 0 .00 172 15 .08 16 8 0 .00 648 417 .64 29° 292 7:~1 .00 164 0 .00 135 19 .14 95 0 .00 571 23 .04 C3-2 22° 631 55 .08 150 10 .06 121 0 .00 172 30 .17 958 627 .65 29° 513 0 .00 148 0 .00 141 0 .00 89 0 .00 527 8 .02 C3-3 22° 302 33 .11 791 476 .60 29° 466 1 .00 658 4 .01 C3-4 22° 1437 629 .44 664 273 .41 794 440 .55 1015 87 .08 372 11 .03 29° 1161 56 .04 612 41 .06 665 209 .31 769 0 .00 220 4 .02 C3-5 22° 601 518 .86 419 208 .49 422 293 .69 678 4 .01 748 6 .01 29° 656 119 .18 257 10 .03 295 117 .39 461 7 .01 552 0 .00 C3-6 22° 426 343 .80 422 0 .00 307 13 .04 572 529 .92 683 0 .00 29° 240 18 . .08 306 0 .00 168 32 .19 504 167 .19 522 0 * V.I. = v i a b i l i t y index; <**/? Samarkand Y ^ (XX) Oregon R 3 g 9 (XX) (XY) J b y nullo-Y nullo-Y Y ^ V.I. o ^ V . l . o ^ 160 24 .15 142 0 .00 306 0 .00 328 0 .00 V.I. | , V.I. ? ^ V.I. ? ^ V.I 1 3 8 23 .16 212 174 .82 145 14 .09 457 286 .63 162 40 .24 95 27 .28 68 21 .30 132 14 .10 276 23 .08 102 7 .06 1 3 8 12 .08 510 323 .63 147 48 .32 688 504 .73 153 24 .16 1 3 5 i .01 229 3" .01 112 0 .00 505 2 .04 139 0 .00 245 210 .85 483 290 .60 233 222 .95 180 0 .00 107 0 .00 242 0 .00 462 57 .12 138 17 .12 366 114 .31 145 38 .26 303 11 .04 129 3 .02 268 1 .00 123 0 .00 3 1 7 o .00 626 4 .01 230 0 .00 47 o .00 376 1 .00 98 0 .00 298 0 .00 219 0 .00 215 0 .00 601 3 .00 228 2 .01 218 0 .00 68 0 .00 158 0 .00 335 . 0 .00 128 0 .00 Table VIII Summary of the Tests of Class 3 Mutations with Different Y Chromosomal Constitutions 67g Y 59k Y 60d Y T2E Y B_f Y Sam. Y (XX) Y Ore.-R Y (XX) nullo-Y (XY) nullo Y C3-1 22°C _* - — - +++ + ++++ — +++ + 29°C - - + - - + + - - -C3-2 22° 29° - - - + +++ - +++ + ++++ + C3-3 22° 29° + +++ ++++ +++ ++++ C3-4 22° ++ ++ +++ - - + + + ++ + 29° - - ++ - - - - - - -C3-5 22° ++++ ++ +++ - - - - - -29° + - ++ - - - - - -C3-6 22° ++++ - - ++++ - - - - - -29° — — + + — — — — — -• * V i a b i l i t y index of males: - £.10 v i a b i l i t y + = .11 - .30; ++ = .31 - .50; +++ = .51 - .70; ++++ = .71 - .90 34 29°C, C3-1 was very leaky with the Samarkand Y. " C3-2 sur-vived well only at 22°C with" the' (XX) Y, and' was leaky at 22°C with the Ore-R Y. Heterogeneity of genetic backgrounds, and hence of modifying'elements, i s a convenient but hardly s a t i s f y i n g explanation for the v a r i a b i l i t y , p a r t i c u l a r l y when the results of mutants 4, 5 and 6 were so uniform. Interestingly, the parental source of mutants C3-1 and C3-2 seemed to a f f e c t t h e i r s u r v i v a l with Y (regardless of i t s source) or nullo-Y at 22°C. The Samarkand and Oregon-R Y's were contributed by the male parent whereas the (XX)Y came from the mother (Table VII). The l a s t column, (XY) nullo-Y, was added afte r t h i s observation to compare the s u r v i v a l of i d e n t i c a l X*/0 males whose nullo-Y condition was contributed by the female parent ((XX)/0 column) and i n the other by the father ((XY)/0 column). These experiments indicate that when the mutant came from a female, s u r v i v a l of X*/Y or X/0 males was 25% or l e s s . On the other hand, i f the mutant came from the father, s u r v i v a l of such males was 60% or greater. This leads me t e n t a t i v e l y to propose that C3-1 and C3-2 have some sort of dominant maternal e f f e c t on s u r v i v a l , a proposal supported somewhat by the observation that homozygous C3-2 females are s t e r i l e . The,survival.patterns of the t i p class 3 l e t h a l s with the four d i s t a l l y duplicated Y's support the suggestion that i n males the mutants act as dominant ts l e t h a l s . Moreover, 35 the patterns of s u r v i v a l with the d i f f e r e n t duplications could be interpreted i n a manner consistent with a genetic positioning of the mutations. Referring back to Figure 3, we can see that C3-4 and C3-5 mapped around su(s)'in region 2 of the d i s t a l portion of the X and that C3-6 f e l l i n region 5, which spans dor and hfw. The "leakiness" of C3-4 and C3-5 at 29° with the 60dl9 Y cannot be explained. Dr. Mel Green, who supplied the stock indicated that i t exhibited aberrant be-havior, but did not expand on the statement. The e r r a t i c s u r v i v a l of C3-6/T2E Y might be accounted for by the known variegation of T2E Y for dor, the locus i n which C'3-6 mapped. This w i l l be considered i n greater d e t a i l i n the discussion of C3-6. I t was also possible that C3-6 i t s e l f was unstable and reverted at a high frequency. This p o s s i b i l i t y was tested by simultaneously mating three C3-6/  T2E Y males which had survived at 29°C (this chromosome i s indicated by C3-6 (S)) to y dor/FM-6 and yf:=/67g Y females. (As w i l l be discussed l a t e r , one t e s t of the temperature-s e n s i t i v i t y of C3-6 i s i t s l e t h a l i t y i n C3-6/y dor females at 29°C.) The t o t a l l e d r e s u l t s of the matings are seen i n Table IX,. The r a t i o of C3-6 (S)/67g Y males to t h e i r C3-6 (S)/FM-6 s i s t e r s was .13 at 29°C. These results show that the s u r v i v a l of the C3-6(S)/T2E male parents was not due to a change i n the temperature-sensitive properties of the mutant and there-fore probably r e f l e c t s i t s i n t e r a c t i o n with the duplication Y. Table IX Test of C3-6(S) Males which Survived at 29°C for the Presence of .the Lethal Mutation C3-6(S)/y dor C3-6(S)/FM-6 yf:=/T2E Y y dor/T2E Y C3-6 (S)/67g Y FM-6/T2E Y 22°C 76 91 70 73 54 49 29°C - 70 35 85 9 28 u> 37 The use of the d i f f e r e n t d i s t a l duplications permitted the l o c a l i z a t i o n of mutants. However, a degree of ambiguity exists owing to the fact that both the t i p Dp Y's and the S B Y car r i e d d i f f e r i n g amounts of proximal X heterochromatin which might, i n fact, produce the s u r v i v a l pattern observed. Consequently, a recombination te s t of each mutation with prox-imal and d i s t a l markers was car r i e d out. A l l male progeny of Y_ £ car/X* females were c l a s s i f i e d and counted at 22°C and 29°C. The results are shown i n Table X. The greatly reduced proportion of the + car recombinants compared with the f+ class at 29°C indicated that C3-1 and C3-2 mapped close to f (which i s only .3 map units from B). The crossover products from C3-4-, C3-5-, C3-6-/y car confirmed that these mutants mapped near y. C3-4 apparently was separable from and to the r i g h t of y. Thus, the crossover tests confirmed the proximal and d i s t a l positions of the class 3 mutants. The complementation tests reported e a r l i e r (Table VI) showed that we are dealing with six d i f f e r e n t functional regions. Up u n t i l the time of ac t u a l l y writing the thesis, I assumed that the mutants being analyzed were single mutations. I t was then suggested that the pattern of male l e t h a l i t y and su r v i v a l summarized i n Table VIII might be explained i f i t was assumed that each mutant chromosome actually c a r r i e d two i n -duced mutations, one non-ts l e t h a l covered by the respective duplication Y and the other ts l e t h a l mapping elsewhere on 38 Table X Genetic L o c a l i z a t i o n of Class 3 Mutants Relative to y, f and car f car ++ + car f + C3-1 22°C 138 192 234 117 8 9 19 9 29°C 313 329 371 86 20 3 23 29 C3-2 22°C 227 138 211 171 18 9 20 12 29°C 161 110 207 34 23 1 15 12 y car ++ + car y + ? <r ? <? ? ? i C3-4 22°C 149 196 255 39 141 16 187 240 29°C 314 465 393 0 218 7 496 364 C3-5 22°C 29°C 149 250 185 0 130 0 179 172 C3-6 22°C 29°C 162 246 230 0 165 0 177 242 39 the chromosome. The ts l e t h a l could cause a l l class 3 mutant males to die at 29° and the non-ts l e t h a l could cause a l l males not carrying the 1 + a l l e l e on the Y to die at 22°. Evidence for t h i s came from r e s u l t s obtained while attempting to cross s p l or rb onto the chromosomes carrying mutant 4_, 5_ and 6_. When mutants 4_, 5 and 6_ were made heterozygous for y_ spl'or y rb; ;y* s p l ' (and rb) /67g Y recombinant males were tested for temperature-sensitivity. When the proximal portion of the o r i g i n a l mutant 4 and 6 chromosome was crossed o f f , the y + s p l (and rb)/67g Y males tested (6-8 for each stock) were no longer temperature-sensitive. One ts stock of C3-5 rb/67g Y was obtained out of 10 tested, in d i c a t i n g that the ts""" maps between s p l and rb, but closer to rb. Analysis of the y f car crossover data and the 3-point testcross was hopelessly confused by several factors: s u r v i v a l at 29°C of the proximal mutants, C3-1, C3-2 and C3-3; lack, i n some cases, of 22°C data for comparison to separate l e t h a l i t y due to the non-ts l e t h a l mutation from l e t h a l i t y due to the ts l e t h a l ; and f i n a l l y , the problem of dealing (at 29°C) with two l e t h a l factors on a single chromosome crossing over with very few markers. Hence, a more accurate genetic positioning of the l e t h a l and' ts* l e t h a l s cannot be given. The hypothesis that C-3 mutants are double mutants holds very well for mutants 4_, 5_ and (5, but i t cannot explain the high degree of s u r v i v a l of mutants 1_, 2_ and 3_ i n the nullo-Y condition and with at least one of the 3 wild type 40 Y's. A further complication was the s u r v i v a l of females homozygous for mutants 1_, 2_, 3_ and 4_. This, however, could be accounted for by considering that l e t h a l s 1, 2_, 3_, and 4_ might be hypomorphs that could not dosage compensate i n a normal manner. Hence on each mutant X chromosome the l e t h a l gene might produce half the amount of product required for s u r v i v a l , and therefore X^/X^ females produce just enough products to survive, but X"*"/Y males do not. A comparison of the two hypotheses proposed to explain the s u r v i v a l patterns of class 3 mutants i s seen i n Figure 9. The developmental properties of the class 3 mutants could be determined because of t h e i r conditional l e t h a l i t y . The developmental stage i n which l e t h a l i t y occurred was studied for each mutation. I t can be seen that only one (C3-5) of the class 3 mutations i n males or homozygous females appeared to die i n s i g n i f i c a n t numbers i n the egg stage (Table XI). In crosses of XX females, the expected 75% egg hatch (owing to embryonic l e t h a l i t y of the one quarter Y/Y zygotes) was observed. Although egg hatches varied consider-ably (from 76% to 93%), the difference i n a given cross at the two temperatures was not great. Since the TSP was determined by comparing the v i a b i l -i t y of class 3 hemi- or homozygous f l i e s r e l a t i v e to the v i a b i l i t y of t h e i r non-lethal s i s t e r s , i t was necessary to demonstrate that s u r v i v a l of f l i e s i n the l a t t e r class was / F i g u r e : 0 : Compar i son of d o u b l e - h i t and o r i g i n a l (DTS) h y p o t h e s e s to e x p l a i n v i a b i l i t y p a t t e r n o f c l a s s 3 m u t a n t s . * " m " i s m u t a t i o n somewhere on chromosome. 4 1 . Original Pattern of Via b i l i t y 32--+- 7 29°C — 22°C -+-Possible Mechanisms ORIGINAL Observed Survival DOUBLE HIT DfS 29°C lethal 22°C + A t „ DTS DTS 29°C leth. leth. leth. 22°C leth. ster- + i l e J? - 4 -£5 Table XI Egg Hatchability and Adult V i a b i l i t y of C3-n Cultures at 22°C and 29°C (from s h i f t studies) Cross Number Number Percentage Expected Observed no. Adjusted of eggs of eggs eggs no.of non- of non- v i a b i l i t y l a i d hatched hatched l e t h a l females l e t h a l females of non-lethal . - - females* C3-1/BS Y/ X yw/B"5" Y? 22°C 29° 1719 1011 1383 766 80% 76 461 255 449 177 .97 .70 C3-2/BS Y<? 22° 1125 766 68 191 184 .96 x yf:=/B SY? 29° 1278 903 70 301 306 1.00 C3-3/BS y / 22° 1574 1190 75 396 337 .85 x yw/BS Y? 29° 1241 934 75 311 193 .62 C3-4/67g'-Y<f 22° 1034 824 79 274 278 1.00 x yf:=/67g'rY 29° 345 243 70 81 55 .67 C3-5/67g Y<? 22° 439 390 89 97 110 1.00 x C3-5/FM-6? 29° 621 475 76 158 179 1.00 l(FM-6)/C3-6'r,220 946 799 85 236 251 1.00 x y dor/T2E Y/29° 910 753 82 227 209 .87 y dor/FM-64? x y dor/Y V* 22° 29° 1384 2083 1284 1929 93 93 ' 320 482 482 609 1.00 .84 ""When v i a b i l i t y . a a standard of 1. 6 W W ? . greater than 1.0, v i a b i l i t i e s at 29°C were adjusted to 43 not greatly affected by temperature s h i f t s . This could be determined by comparing the observed number of adult non-lethal females with the number deduced from the number of eggs which hatched (Table XI). A reduced su r v i v a l of non-lethal females could d i s t o r t the apparent r e l a t i v e v i a b i l i t y of t h e i r class 3 s i b l i n g s . Non-lethal females of the crosses involving C3-1, C3-3 and C3-4 did show reduced , v i a b i l i t i e s at 29° (Table XI). This would make the su r v i v a l of t h e i r l e t h a l s i b l i n g s appear to be greater than i t actually was, but i n fact the r e l a t i v e l y clean separation of l e t h a l and non-lethal periods i n the s h i f t experiments of these mutants indicates that t h i s problem did not confuse our elucidation of the beginning and end of the TSP. I t should be mentioned here that each point i n the figures graphing r e l a t i v e v i a b i l i t i e s of f l i e s s h i f t e d at d i f -ferent times i n development represents a single v i a l , which produced at least 40 f l i e s . The prolonged (1st l a r v a l i n s t a r through late pupa) l e t h a l phase of the XX/X females generated i n a l l crosses with attached-X females makes i t d i f f i c u l t to define the LP when the LP of the mutation was also during a l a r v a l stage or spanned the pupal period. A d i s t i n c t early pupal l e t h a l could be discerned when the.number of undeveloped pupae cor-responded c l o s e l y with the number of class 3 f l i e s predicted on the basis of egg counts. - In crosses of attached-X females, dead class 3 males could often be distinguished i n the pupal cases. The LP of C3-1/B Y males extended from early pupal development through v i s i b l e wing pad formation. The TSP pre-ceded the onset of death by 2 4 hours and continued u n t i l eye pigment formation (Figure 10). I t was not determined whether those f l i e s exposed to r e s t r i c t i v e temperature early i n the TSP died e a r l i e r than those exposed l a t e r . c The LP of C3-2/B Y males was si m i l a r to that of g C3-1/B Y. The TSP was very short, spanning the end of the 3rd l a r v a l i n s t a r and prepupal formation, possibly the time when the larvae crawl up onto the glass (Figure 11). The up-and-down double s h i f t s of C3-2 were carr i e d out at d e f i n i t e times i n development, before, during, and af t e r the TSP which had been defined by the single s h i f t experiments. After 1 day at 29°C, male v i a b i l i t y did not decrease; aft e r 2 days v i a -b i l i t y dropped by 20%, and 3 or more days at 29°C produced almost complete l e t h a l i t y (Figure 12a). In the reverse experi-ment, a s h i f t down-and-up, 2 or more days at 22° during the TSP was not s u f f i c i e n t to allow s i g n i f i c a n t s u r v i v a l (Figure 12b). These results showed that during the TSP, reversal of temperature e f f e c t s required a considerable time i n t e r v a l . Only prolonged growth at a given temperature irrevocably fixed s u r v i v a l or death. The TSP of C3-3/B Y was d i s t i n c t i v e (Figure 13). Single s h i f t studies suggested a continuous TSP which might be an i n d i c a t i o n that the function of C3-3 i s indispensible. Figure 10: Relative v i a b i l i t i e s of f l i e s from the cross C3-l/BSYd* x yw/lifYS after shifts-up and shifts-down at different times during development. Via b i l i t y ratio is number of lethal bearing males or females to their non-lethal sisters (FM-6 or XX). a) shifts- up b) shifts-down * Abbreviations of developmental stage at time of shift (used throughout shift study figures): e - egg I - 1st instar larva II - 2nd instar larva III - 3rd instar larva PP - pre-pupa pep - pre-eye pigment deposition ep - eye pigment deposition wp - wing pads darkening Throughout the shift study figures the following w i l l be used: A O o lethal males/FM-6 or XX female sibs A • • lethal females/FM-6 or XX female sibs a ) shift-up shift-down DAYS AT 29°C BEFORE SHIFT -DOWN Figure 11: Relative v i a b i l i t i e s of f l i e s from the cross C3-2/BSYcf x VJ^=/B£Y$ after r;?; a) shifts-up b) shifts-down at different times during development. a) shift-up Figure 12: Relative v i a b i l i t i e s of f l i e s from the cross C3-2/BSYd' x after a) shifts-up-and-down b) shifts-down-and-up at different times during development. o o 1 day pulse at 22°C or 29°C • greater than 1 day at 22°C or 29°C (as indicated at point) a) shift-up -and-down 1.0--TIO 0,8,--< or >- 0.6--h-_i cn 0 .4- -< > 0.2+ •4 -*—1—• 1 2 I ' I i h I 3 4 i n •3,5 •4 + 6 7 ft HI PP 9 10 11 ep - wp 12 DAYS AT 22 C BEFORE SHIFT-UP b) shift-down-and-up 1.0--o S o&--£ 0.6-5 0.44-0.2 + o > r »3 o • 4 » t » \ M i I 1 f-2 3 4 5 6 7 8 "DT PP ep DAYS AT 29°C BEFORE SHIFT-DOWN Figure 13: Relative v i a b i l i t i e s of f l i e s from the cross C3-3/BSY<f x yw/B^Y? after a) s h i f t s * up b) shifts-down at different times during development. a) shift-up b) shift-down 1.0-o 0£>-or > 0.6-CD < 0.4-> 0.2-1 2 3 4 5 6 7 8 9 10 e. I DAYS AT 29°C BEFORE SHIFT-DOWN 49 Shifts-up at any time before eclosion resulted i n death of a l l males. Eggs l a i d at 29°C and sh i f t e d down only 12 hours l a t e r s t i l l resulted i n male l e t h a l i t y . These results could be obtained i f there was a TSP i n the early stages of embry-onic development and a late TSP just before eclosion. Unfor-tunately, the stock was l o s t before shifts-down could be made at one hour i n t e r v a l s a f t e r egg lay. This would have d e l i n -eated the s t a r t of the TSP. In order to determine whether C3-3 exhibited a continuous TSP or, i n fa c t , has successive TSP 1s separated by i n s e n s i t i v e i n t e r v a l s , pulse s h i f t s either up-and-down or down-and-up, at d i f f e r e n t times were carr i e d out. Following the 12 hour i n t e r v a l a f t e r egg c o l l e c t i o n at 22°C, a 24 hour pulse at 29°C at any time i n development did not produce l e t h a l i t y (Figure 14a). The only exception to this was during embryogenesis i n the f i r s t day when v i a b i l i t y was reduced to 75%. Forty-eight hour pulses of 29°C up to the early pupal stage reduced v i a b i l i t y greatly, and a 3 day exposure to 29°C reduced s u r v i v a l even further. These re s u l t s suggested that thermal denaturation of a preformed C3-3 pro-duct occurred very slowly or not at a l l . This i s supported by the requirement for prolonged 29°C exposure of f l i e s l a i d at 22° before death i s in e v i t a b l e . However, 2 day pulses at 29° at any time i n the l i f e cycle showed the continuous nature of the TSP. These observations suggest that temperature-Figure 14: Relative v i a b i l i t i e s of f l i e s from the cross C3-3/BSY</ x v_f :=/B£Y$ after a) shifts-up- and-down b) shifts-down-and-up at different times during development. o 0 o 1 day at 29 °C • • • 2 days at 29°C A A- • • -A 3 days at 29°C * o shifts-down of 3,4,5, and 6 days at 22°C *"*"• shifts-down of 1,2 and 3 days at 22°C a) shitt-up-and-down i I i h I 1 I i I 1 1 1 1 1 1 h 1 2 3 4 5 6 7 8 9 10 11 12 TJI pp ep . wp DAYS AT 22°C BEFORE SHIFT-UP b) shift-down-and-up 1.0--o •< o.&--£ 0.6--1 0.4--> 0.2--f i i > i i T i—f—i—t-1 2 3 4 5 6 7 8 DAYS AT 29°C BEFORE SHIFT-DOWN 51 s e n s i t i v i t y occurs at the time that the thermolabile gene product i s made. Once made at 22°C, the product i s not sen-s i t i v e to 29°C. The C3-3 gene product has about a two-day turnover rate, by which time the amount of normal product f a l l s below the threshhold necessary for v i a b i l i t y . In other words, the f l y can survive at 29°C on preformed product for one day but dies by the end of 2 days because the product has not been replaced i n s u f f i c i e n t form or quantity. The shift-down-and-up (Figure 14b) experiment indicated that the f l i e s could never recover from the i n i t i a l production of defective gene products or. from the absence of the products at high temperature. The LP of C3-3 .began as early as 3rd l a r v a l i n s t a r and continued u n t i l eclosion. From the single s h i f t studies i t was noted that there was a d e f i n i t e c o r r e l a -tion between time of a shift-up and the onset of death. F l i e s died 2-3 days aft e r a shift-up u n t i l the end of the TSP when a shift-up resulted i n death almost immediately. The function controlled by C3-3 i s needed only during development; adult C3-3 f l i e s are viable when put at 29° for 4 days. The LP for C3-4 males and females occurred from early through wing-pad pupa. The s t a r t of the TSP could not be determined, even aft e r repeated experiments, but the TSP for male l e t h a l i t y ended at the time of eye-pigment deposition and either then or l a t e r for females (Figures 15a and b). Three d i f f e r e n t crosses were made for the s h i f t studies, Figure 15: Relative v i a b i l i t i e s of f l i e s from the cross C3-4/67gY<f x C3-4/KFM-6) ? after a) shifts-up b) shifts-down at different times during development. a) shift-up DAYS AT 29°C BEFORE SHIFT-DOWN 53 C3-4/C3-4 4 x C3-4/6 7g Y <f , (FM-6) /C3-4 ? x C3-4/67g Y ^ and yf :=/6 7g Y ° x C3-4/67g Y o*, and a l l yielded results similar to those shown i n Figure 15. Tests of C3-5 were made i n two crosses: C3-5/FM-6 $ x C3-5/6 7g Y <? and yf :=/67g Y ? x C3-5/6 7g Y £. Both crosses yielded s i m i l a r r e s u l t s showing that the male LP occurred during pupation p r i o r to eye-pigment formation and through late wing-pad production. The re s u l t s of s h i f t studies were highly e r r a t i c and did not permit any demon-st r a t i o n of a c l e a r l y defined TSP (Figure 16). The highly e r r a t i c v i a b i l i t y of males i n both the shift-ups and s h i f t -downs could indicate that several times i n the f l y ' s develop-ment the shock of changing temperature rather than actual temperature could i n i t i a l l y a f f e c t v i a b i l i t y . There i s pre-cedent for such an in t e r p r e t a t i o n . F l i e s carrying the ts mutation para are not affected by temperature during develop-ment, but immediate paralysis of adults occurs a f t e r a s h i f t -up (Suzuki, G r i g l i a t t i and Williamson, 1971). Prolonged incubation at 29°C permits gradual recovery of a c t i v i t y of ts ,., . para f l i e s . Females homozygous for C3-5 did not survive at any temperature. From counts, of egg hatches i n the cross C3-5/FM-6 £ x C3-5/67g Y cf / i t appeared that homozygous f l i e s died as eggs at 29°C, since only 76% of the eggs hatched (Table XI). At 22°C some C3-5/C3-5 appeared to hatch Figure 16: Relative v i a b i l i t i e s of f l i e s from the cross C3-5/67aY <f x yf :°/67gY ? after a) shifts-up b) shifts-down at different times during development. 54. a) shift-up 1.0 + g o.&+ < cc > 0.6 + _ l ca 0.4 -< > 0.2-1 e 2 I 3 1 4 6 7 8> 9 10 11 12 HL pp pep ep wp DAYS AT 22 C BEFORE SHIFT-UP DAYS AT 29°C BEFORE SHIFT-DOWN 55 and died during the l a r v a l stages, without reaching the pupal stage. An attempt to generate C3-5/C3-5/67g Y females was made using second-division non-disjunctive e f f e c t s of mei-S332 (Figure 6). The following cross was made at 22°C: C3-5/67g Y; > 2 mei-S332/mei-S332 6 x yf:=/67g Y, y From th i s cross, the 2 * following progeny were recovered: 337 y f_ °., 456 C3-5 o and 57 + ? . No y f females r e s u l t i n g from nullo-XY sperm were detected because of a bb deficiency i n the yf: = chromosome. The wild type females were presumed to be C3-5/C3-5/67g Y, and 14 such females were i n d i v i d u a l l y crossed to C3-5/6 7g Y from the o r i g i n a l stock for 4 days at 22°C and then trans-ferred to fresh v i a l s at 29°C for 5 days. Of these, a l l were s t e r i l e at 29°C, although 11/14 l a i d 50 or more eggs. At 22°C, 10 of these females l a i d viable eggs. The progeny of 3 of the females which did lay eggs at 2 9°C and whose eggs hatched at 22°C were subjected to further t e s t s : 1) putative C3-5/67g Y males were crossed to yf:=/67g Y ? to v e r i f y the presence of the mutation. At 22°C, 31d*:52 ? were recovered to give a r a t i o of 0.6. At 29°C, 10f/:64? hatched for a r a t i o of 0.16. This showed that the ts e f f e c t s t i l l persisted. 2) 16 more putative C3-5/C3-5/6 7g Y females were i n -d i v i d u a l l y mated; a l l were s t e r i l e at 29°C and 11 were f e r t i l e at 22°C. Thus, the 29°C s t e r i l i t y was indeed passed on. 56 3) s t e r i l i t y of C3-5/C3-5/6 7g Y females at 29°C could be maternally-affected condition. Consequently, such females were crossed to FM-6/Y males to determine whether some zygotes could be "rescued" by the presence of a wild-type a l l e l e of C3-5 on the FM-6 X chromosome. The re s u l t s can be seen i n Table XII and show that, i n f a c t , the maternal genotype appears to commit a l l zygotes to death unless rescued i n time by a wild-type a l l e l e . The factor responsible for female Table XII Cross of Putative C3-5/C3-5/67g Y Females to FM-6/Y Males Progeny ? FM-6 ? C3-5 £ a* C3--5 cT FM-6 C3-5 67g Y ? 1) 22° 29° 3 s t e r i l e 0 0 0 2) 22° 29° 63 s t e r i l e 7* 16 1 3) 22° 29° 86 5 0 0 15 0 2 0 * From non-virgin mother 57 s t e r i l i t y at 29° might e x i s t at a s i t e other than the C3-5 locus (possibly the putative ts l e t h a l site) and hence might not be covered by the Y duplication. A tes t for t h i s could be made by homozygosis o f . d i f f e r e n t crossover products i n which the C3-5 mutation chromosome i s replaced by d i f f e r i n g amounts of wild-type chromosome. 4) The presence of 6_7g Y i n the females could be tested for by using the fact that the duplication c a r r i e d •+• a su (w ). Non-disjunction i n the females could generate an egg carrying,no X and only 6 7g Y. C3-5/C3-5/67g Y females 2 a a were mated to XY, y su (w )w /0 males so that the 67g Y could be detected by the occurrence of w_ males. Three d i f f e r e n t crosses were made, and out of 63 offspring (38 ?, 25 c?) no w males were detected. Obviously larger numbers of progeny are needed i n order to prove the presence of 67g Y i n the female. The LP's and TSP's of a l l six mutants are summarized in Figure 17. I t is . i n t e r e s t i n g that a l l six appear to have TSP 1s that cover the i n t e r v a l from 3rd l a r v a l i n s t a r to pupa. This corroborates Hadorn 1s (1961) observations that a large proportion of lethals act during t h i s i n t e r v a l . Considerable morphogenetic a c t i v i t y i n thi s period requires a c t i v a t i o n of numerous genes. Mutant C3-6 was found to be a l l e l i c to deep "-orange (dor 1-.3). This was discovered while testing C3-6 with the Figure 17: Effective lethal phase and temperature sensitive period of each C3 stock. temperature sensitive Y/tttttrmtm p e r r o d X 'X lethal phase C ~ _ T " " j TSP unclear 00 sped -uatnSid ednd -t V--aad ^ 1 V- -4 ^ ^ P U J 3SX 8S3 IB3U9UI _ J J ( , -dO|9A3(I t W///////////////////////X x x w/mvszz X * r-iii!)ni/in!u/inniiiinn/////i////////////////////////i/////////7zn7i X X X £ X -zip. W77777\ W///////////A W777ZA - X 9 - £D Z'ZO T - £ D jop 59 T2E Y stock. C3-6 died at 29° with a l l Y. chromosomes prev-iously tested,, but survived (0.3. v i a b i l i t y ) with T2E Y. The eyes of a l l the survivors were variegated orange and many showed a "warped" wing phenotype. Fortuitously, the T2E Y chromosome was. maintained with an X chromosome carrying a dor a l l e l e . In the. cross, C3-6/FM-6 ? x y dor/T2E Yd", y dor/C3-6 females had orange eyes at 22°C and at 29°C, such females were never recovered. T2E Y was already known to variegate for dor. Therefore, I tested a l l available dor a l l e l e s i n d i f f e r e n t combinations i n females at both high and low temperatures. Moreover, a l l of the dor a l l e l e s were tested i n males carrying d i f f e r e n t Dp "Y1s which might a f f e c t a dor mutant. Since so few ts a l l e l e s of known mutants e x i s t , the relationship of C3-6 to the dor locus was of con-siderable i n t e r e s t . The stock 1(1)7A kindly supplied by Dr. John Lucchesi ca r r i e s a dor"*". 1 (1) 7A males died 90 - 100 hours a f t e r hatching and bear large melanotic tumors. 11-B22 was picked up i n my screening as a non-ts t i p l e t h a l and i d e n t i f i e d as a l e t h a l dor a l l e l e by Dr. Lucchesi and had the same l e t h a l phase as 1(1)7A. T2E Y chromosomes received from stock centres at Davis and Pasadena (labelled T2E Y D and T2E Y P, respectively) were tested separately. TIE Y involves s i m i l a r break points as T2E Y but the differences are not c l e a r l y defined i n the l i t e r a t u r e (Masterson, 1968). TE Y, received 60 from Dr. Lucchesi, covered 1(1)7A i n males and did i n t e r a c t with the other dor a l l e l e s . The results of the interactions of the d i f f e r e n t dor a l l e l e s with the Y's tested are presented i n Table XIII and summarized in.Tables XIV and XV. Note that a l l four dor a l l e l e s can be distinguished by t h e i r unique interactions with the various Y's. The dor a l l e l e i t s e l f was found.to be a recessive ts l e t h a l with a Y, nullo-Y and TIE. C3-6.on the other hand, behaved as a dor 1 i n combination with a Y, nullo-Y or TIE. However, i t did show temperature-sensitivity with 67g Y and both of the T2E' s. If we assume that C3-6_ i s a double mutant chromosome, the temperature-sensitivity could r e f l e c t the second h i t , while the dor mutation, i t s e l f , i s a non-ts l e t h a l . U n t i l the two-hit hypothesis of class 3 mutant behavior was adopted, i t was d i f f i c u l t to reconcile the fact that while 67g Y completely covered l e t h a l i t y of the 2 known dor le t h a l s at 29°, i t seemed to variegate for l e t h a l i t y with C3-6, but did not variegate for any of the known morphological phenotypes. The dor l e t h a l s , 1(1)7A and 11-B22 can be distinguished from each other by t h e i r interactions with TE. 1(I)7A was temperature-sensitive, whereas 11-B22 was completely l e t h a l at both temperatures. The l e t h a l i t y of both was covered completely by 67g Y and both the T2E's, while they.were com-plete l e t h a l s at both, temperatures with Y, nullo-Y and TIE. The temperature s e n s i t i v i t y of the dor locus appears to be Table XIII Variegation and Interaction of dor A l l e l e s i n Males with Different Y Chromosomes A l l e l e 67g Y Y nullo -Y T2E YD T2E YP TIE Y. TE Y 22°C 29°C 22°C 29°e 22°C 29°C 22°C 29°C 22 °.C 29°C 22°C 29°C 22°C 29°C . ? 434 352 632 444 601 335 318 338 431 417 200 85 178 128 C3-6 392 66 0 0 0 0 242 119 342 87 0 0 10 0 V.I.* .9 .2 .0 .0 .0 .0 .76 .3 .8 .2 .0 .0 .05 .0 variegation* * NV NV MV MV NV NV EV 234 208 162 124 J58 42 137 63 82 62 46 42 74 48 dor 138 71 111 0 58 0 101 52 61 24 66 0 42 40 V.I. .6 .3 .7 .0 1.0 .0 .7 .8 .7 .4 1.+ .0 .6 .8 variegation NV NV NV NV LV HV MV NV LV EV 0 +• 58 49 330 296 86 46 120 136 150 118 123 58 1(1) 7A <? 44 46 0 0 155 261 157 151 0 0 74 6 V.I. .8 .8 .0 .0 1.+ 1.+ 1.+ 1.+ .0 .0 .6 .1 variegation NV NV LV HV NV NV LV EV ? 49 100 314 217 58 71 80 127 123 127 103 119 11-B22 9 64 95 0 0 153 237 127 152 0 0 0 0 V.I. 1.+ .9 .0 .0 1.+ 1.+ 1.+ 1.+ .0 .0 .0 .0 variegation NV NV NV NV NV NV *V.I. = v i a b i l i t y index, at a given temperature. ** Variegation (V) i s recorded as none (N); l i g h t (L); moderate (M); heavy (H); extreme (E). 6 2 Table' XIV Summary of-Table XIII: Male V i a b i l i t y and Variegation of dor a l l e l e s dor a l l e l e 67g Y TIE Y -Y— nullo-Y T2E YD T2E YP TE Y C3-6 22°C .9 NV* .0 .8 MV .8 NV .05 EV 29°C .2 NV .0 .3 MV .2 NV .0 dor 22° .6 NV 1.0 dor ,NV.7 LV .7 NV .6 LV 29° .3 NV .0 .8 HV .4 NV .8 EV 1(1)7A 22° .8 NV .0 1.0 LV 1.0 NV .6 LV 29° .9 NV .0 1.0 HV 1.0 NV .1 EV 11-B22 22° 1.0 NV .0 1.0 NV 1.0 NV .0 29° .9 NV .0 1.0 NV 1.0 NV .0 * Variegation (V) designated as: none (N), l i g h t (L), moderate (M), heavy (H) or extreme (E). 63 Table XV Trends of dor a l l e l e s i n Males with D i f f e r e n t Y's, Summarized as Temperature Sensitive ( t s ) , l e t h a l (1) or non-lethal (+) dor a l l e l e 67g Y Y nullo-Y T2E Y TE Y C3-6 ts 1 ts 1 dor ts + ts + + 1(1)7A + 1 + ts 11-B22 + 1 + 1 a l l e l e s p e c i f i c , a property comparable to a l l e l e s of the Notch locus. Among the many a l l e l e s of Notch which a f f e c t several d i f f e r e n t phenes, two are known to be temperature-se n s i t i v e : N 6 0 g l l ^ s ^ a t sensitive for wing phenotype, cold sensitive for eye phenotype (Foster and Suzuki, 1970), and -.264-103 , , . ._ . .. N i s heat sensitive for the wing (Foster, personal communication). Not only can the dor, a l l e l e s be distinguished from each other (Table XIII), the unique properties of each of the 64 X-duplicated Y's can be seen. Obviously, the stock l a b e l l e d 4. as TIE Y (from Pasadena) no longer c a r r i e s the dor a l l e l e as was o r i g i n a l l y reported, since i n a l l cases i t gives the same su r v i v a l pattern with d i f f e r e n t dor a l l e l e s as Y. and nullo-Y. The o r i g i n a l hypothesis that 67g Y might be v a r i e -gating for l e t h a l i t y . w i t h C3-6 was rejected when i t was found to cover the 2 dor l e t h a l s completely at 29°; however,, .it did show a d i s t i n c t temperature-sensitive e f f e c t with dor which s i s d i f f i c u l t to explain. Hence, i t may well. be. variegating-for l e t h a l i t y with C3-.6 also. To. distinguish. T2.E YD, T2E Y P and TE from one another, we must consider t h e i r non-lethal, as well as l e t h a l , properties with, each mutant. The Pasadena (T2E Y P) chromosome appears to have accumulated modifiers of i t s . o r i g i n a l , variegating e f f e c t since i t does not show any phenotypic variegation with any of the mutants. T2E YD, on the other hand, does show l i g h t (22°C) to moderately heavy (29°C) eye and wing variegation with C3-6, dor and 1(1)7A, but none with 11-B22. I n i t i a l l y , the "warped wing" phenotype of C3-6/T2E Y D was considered to be the result- of a second s i t e h i t , since the phenotype has not previously been reported for dor a l l e l e s i n the l i t e r a t u r e . I now believe i t to be t r u l y another phenotypic e f f e c t of the dor locus, since.dor i t s e l f exhibited the same phenotype with T2E Y D. and TE Y as did 1(1.) 7A/TE Y. The variegating proper-t i e s of T2E Y P, T2E Y D and TE varied considerably, the f i r s t showing no, the second moderate, and the t h i r d extreme va r i e -65 gation for both the eye and wing phenotypes. In the case of T2E Y D and TE, 29°C d e f i n i t e l y enhanced variegation. The e f f e c t of temperature on variegation has been well documented (Gersh, 1949; Hartmann-Goldstein, 1967; Baker, 1968; Van Breugel, 1970) and w i l l be discussed i n d e t a i l l a t e r . The basis for variegation i s very poorly understood (Baker, 1968). Of considerable relevance to the problem i s the observation that the extent of variegation for the eye and wing phenotypes of the dor a l l e l e s .did not correlate with the extent of l e t h a l i t y . The clearest example of t h i s can be seen by comparing v i a b i l i t i e s and degree of variegation of dor/T2E Y P and dor/TE Y males at 22° and 29°C (Table XIV). dor/T2E Y P males were poorly viable at 29°C, thereby sugges-ting that the dor + .allele f a i l e d to function i n a v i t a l t issue, yet the eyes and wings of survivors were completely wild-type, thereby r e f l e c t i n g the .activity of d o r + i n these d i s c s . This could also r e f l e c t a lower requirement of the eye and. wing discs for d or + substance for wild-type expression. dor/TE Y males on the other hand were almost completely viable at 29°C yet showed the highest degree of eye and wing variegation observed. This w i l l be examined more clos e l y i n the discus-sion. I t should be.added here though, that a clear under-standing of the interaction" between any dor a l l e l e and a s p e c i f i c Y i s complicated by the f a c t that either one may ex-h i b i t d i f f e r e n t s e n s i t i v i t i e s to high temperature. 66 The interactions between d i f f e r e n t dor a l l e l e s i n females are shown i n Tables.XVIa.and b. The l e t h a l i t y of C3-6, 1(1)7A and 11-B22 was shown i n a l l combinations with each other. dor, on the other hand, i s s u f f i c i e n t l y "leaky'1 i n i t s l e t h a l phenotype to allow s u r v i v a l i n combination with the other a l l e l e s at 22°, although a l l survivors have dor coloured eyes. The LP of y dor/y dor, C3-6/C3-6 and y dor/C3-6 females did not occur i n the egg stage" (Table XI) . Like males carry-ing the dor and C3-6 a l l e l e s , most of the females died i n the early pupal stage. This i s i n contrast to the l e t h a l action of C3-6 and dor with either I(1)7A or 11-B22 where numerous dead 3rd i n s t a r larvae with melanotic inclusions were noted. The TSP of C3-6/y dor females began i n the late 3rd l a r v a l i n s t a r and ended 'abruptly around wing-pad formation (Figure 18). The TSP of y dor/y dor females was more r e s t r i c t e d , s t a r t i n g i n the 3rd i n s t a r but ending before detectable eye-pigmentation (Figure 19). I t must be remembered that the C3-6/y dor females carry three d i f f e r e n t l e t h a l s i t e s -the C3-6 (dor) l e t h a l , the dor, and the ts l e t h a l associated with the C3-6 chromosome. This may account for i t s longer TSP. The LP of C3-6 and dor males with any of the 29° sen-s i t i v e Y's occurred primarily i n the very early pupal stage. At t h i s time, gross h i s t o l y s i s was v i s i b l e i n the dead pupae but many f l i e s developed r i g h t up to eclosion. I t was f i r s t 67 Table XVI(a) Interactions of dor a l l e l e s i n Females C3-6 dor .1(1)7A 11-B22 FM-6? Mutant? FM-6? Mutant? FM-6? Mutant? FM-6? Mutant? C3-6 22° 1,215 0 583 382(.6) 245 0 178 0 (dor) 29° 1,036 0 629 1 (EVT 182 0 128 0 dor 22° 104 84(.8) 245 137(.6) 294 131(.4) (dor) (dor) (dor) 29° 35 0 221 152 0 . Table XVI.(b) Summary of Table XVI(a): l e t h a l i t y (1) and Temperature S e n s i t i v i t y (ts) of dor" female interactions, with v i a -b i l i t y index at 22° given with phenotype C3-6 dor 1(1)7A 1T-B22 C3-6 1 ts .6 dor dor ts ts ts .8 .6 .4 dor dor dor Figure 18: Relative v i a b i l i t i e s of f l i e s from the cross C3-6/KFM-6) ? x v. dor/T2EY D J after a) shifts-up b) shifts-down at different times during development. a) shift-up DAYS AT 29°C BEFORE SHIFT-DOWN Figure 19: Relative v i a b i l i t i e s of f l i e s from the cross y_ dor/FM-6 ? x v_ dor/Y o* after a) shifts-up b) shifts-down at different times during development. 70 thought that the pupal l e t h a l phase of C3-6/67g Y f l i e s was due to the second mutation, but i t was carefully, noted that only a few dor/Y and C3-6/Y males died as 3rd i n s t a r larvae. This observation adds another phene, pupal l e t h a l i t y , to the wide variety of dor a l l e l e phenotypes. Clancy (196 4) also observed a v a r i a t i o n of the pupal l e t h a l i t y phene. Testing two d i f f e r e n t stocks of 1 (1)7, -1 and -2, he found that while each stock homo- or hemizygous died as 3rd i n s t a r larvae without melanotic tumors, the 1(1)7-1/1(1)7-2 heterozygote developed normally up to puparium formation; then melanotic tumors appeared and death ensued within 36 hours. The TSP of dor males was e s s e n t i a l l y i d e n t i c a l to dor/  dor females (Figure 19). The TSP of C3-6/T2E Y D males was less c l e a r , the results- shown in- Figure 18 being repeatedly obtained with T2E Y D and 67g Y. The ambiguity probably re-f l e c t s the two separate mutations. The early TSP, from 12 hours through 2nd i n s t a r , i s l i k e l y that of the second s i t e t s - l e t h a l . The l a t e r TSP, which i s approximately the.same as dor males and females, i s l i k e l y that of the C3-6 (dor) l e t h a l . But note that t h i s must also r e f l e c t the time that the dor + duplication shuts, o f f by i t s e l f , and/or i s sensitive to high temperature. Thus the duplication of males s h i f t e d to 29° on day 6 seems not.to be sensitive to temperature and by staying.turned on allows the males to get through the TSP. These data indicate that the duplication i s sensitive to change i n temperature on days 8 - 1 0 . Double s h i f t studies 71 were run only with dor, since i t seemed easier to pinpoint i t s TSP than that of C3-6. The r e s u l t s of the shift-up-and-down for males was surprising (Figure 20a). They indicate two TSP 1s, one around the late 3rd i n s t a r , and a second, less c r u c i a l i n t e r v a l , around eye-pigmentation. The dor/dor female results showed one TSP, s i m i l a r to that obtained from the single s h i f t studies (Figure 20b). The shift-down-and-up study on both males and females (Figure 20c) supports the existence of two TSP's, since no single period spent at 22° (up to 3 days) w i l l enable f l i e s to survive the l e t h a l e f f e c t s of 29°C. The dor a l l e l e s have many p l e i o t r o p i c e f f e c t s on eye color, melanotic tumor formation, l e t h a l i t y , wings shape, and pigmentation i n testes and Malpighian tubules (Counce, 1957; Clancy, 1964, Lucchesi, 1968; Lucchesi and Bischoff, 1969). Furthermore, dor females are s t e r i l e owing to a maternal e f f e c t on egg h a t c h a b i l i t y (Counce, 1956). One test for escapers of the maternal-effect was made by crossing C3-6/y dor $ to +/Y d*. Of 1,619 eggs c o l l e c t e d , none hatched. In a comparable cross using dor/dor females Counce (1956) noted several survivors. This shows the greater defect of C3-6. Attempts to recover females homozygous for C3-6 by adding a 67g Y a l l f a i l e d . None was recovered when the non-4 8 disjunctive properties of sc so (Sandler and Braver, 1956) were used (Figure 5). S i m i l a r l y , the meiotic e f f e c t s of Figure 20: Relative v i a b i l i t i e s of f l i e s from the cross y_ dor/FM-6 ¥ x y_ dor/Y <f after a) shifts-up-and-down for females b) shifts-up-and-down for males, and c) shifts-down-and-up for males and females at different times during development. For figures a) and b): o o 1 day at 29 °C • • 2 days at 29 °C A 3 days at 29°C For figure c ) : • • Shift down on each day for 1,2 and 3 days a) shift-up-and-down,©57 DAYS AT 22°C BEFORE SHIFT "UP b) shift-up-and-down, 9 f — • DAYS AT 22^0 BEFORE SHIFT-UP c) shift-down-and-up, cf&$ o ^ 0 . 2 + >-d 0.1 — QQ < T - " T " T - - - T " - f - " t - " t - - ^ T " - T - - - T 1 2 3 4 5 6 7 - 8 9 10 DAYS AT 29°C B E F O R E SHIFT-DOWN 73 mei-S332 were used i n the cross: C3-6/67g Y; mei-S322/mei-S332 d* 2 x yf:=/67g Y ?(Figure 6). From that cross, 215 y f ? , 354 + c? and 36 + °. were recovered. 20 + females, presumed to be C3-6/C3-6/67g Y, were ^ i ndividually mated to C3-6/67g Y cf at 29 °C and no l e t h a l i t y , of the male progeny was noted. Since the technique worked with C3-5, the f a i l u r e to produce C3-6/  C3-6/67g Y females probably r e f l e c t s the nature of the l e t h a l dor i t s e l f , but i t cannot be ruled out that the l e t h a l i t y i s not the r e s u l t of a second mutation. One y dor/C3-6/T2E Y D female may have been recovered by chance from the cross.: FM-3/y dor ? x C3-6/T2E Y D at 29°C. One non-FM-3 female with variegated eyes and warped wings was recovered among 196 female o f f s p r i n g . Primary non-disjunction i s increased i n the presence of multiple-break rearranged chromosomes(Dr. D. Holm, personal communication); t h i s may have generated a FM-3/y dor/T2E Y D female which was used i n the cross. The attempt to modify the variegation of C3-6/67g Y and C3-6/T2E Y D with possible enhancers and suppressors A (Figure 8a and b) f a i l e d just at the f i n a l cross. The XX chromosome of one of the suppressor stocks broke down, and from another cross",' the required' femalesTwere" found' to have a very low v i a b i l i t y and too few were generated to make a cross that would produce . s u f f i c i e n t progeny to compare s t a t -i s t i c a l l y . An alternative and probably more r e l i a b l e , experi-ment would have u t i l i z e d the general suppression of variegation 74 e f f e c t of extra heterochromatin (Baker and Spofford, 1959; Brosseau, 1964). Hence, generation of C3-6/67g Y/Y males would have avoided the locus and a l l e l e s p e c i f i c i t y problems of Su(var). DISCUSSION A b r i e f review of clustered genes having related functions i n lower organisms was given i n the Introduction, and a few examples of those known i n Drosophila were men-tioned. One of those mentioned was the Notch s e r i e s . The Notch locus may actually.be a duplication of a single gene (Foster, personal communication), with dominant N mutants flanking both sides of the region (as well as lying within i t ) ; a l l known mutants within t h i s region are f u n c t i o n a l l y related. Another example i s zeste (z_) which l i e s about 0.5 map units to the l e f t of white (w). z i s considered to be a duplication of the white locus which was evolved to a d i f -ferent function. Phenotypic interactions between zeste and white implies a functional r e l a t i o n s h i p . Furthermore, the w locus i t s e l f i s part of a pseudo-allelic series containing 3-5 s u b - l o c i . Two to three of the sub-loci l i e between z_ and w, and while f u n c t i o n a l l y related to w, do not i n t e r a c t with z. This led Judd (1962) and Kaufman (1970) to analyze the region lying between z_ and w. This region i s comparable i n genetic size to the t i p of the X analyzed i n my research, but the t i p region, has about f i v e times more DNA than does the z-w region (Rudkin, 1965). Kaufman and Judd recovered 117 point mutations i n that region which formed 14 complemen-tatio n units, none of which were found to be f u n c t i o n a l l y 76 related to each other or to z_ or w. E a r l i e r , attempts had been made to determine the func-t i o n a l r e l a t i o n s h i p between genes within a small chromosome region. Demerec and Hoover (1936) and Poulson (1940, 1945) worked on small deletions of the t i p of the X-chromosome. The deletions extended to ac (8 bands), S £ (10 bands) and svr (13 bands). When the f i r s t 4 d i s t a l bands, were deleted, a wild-type phenotype resulted. However, larger deletions caused si m i l a r l e t h a l phenotypes, namely, gross malformation of the embryo. Most were early.embryonic l e t h a l s involving massive p r o l i f e r a t i o n of nervous tissue and l i t t l e or poor musculature. This work did not necessarily indicate a functional r e l a t i o n s h i p between genes at the t i p of the X-chromosome, rather i t i s probable that a common v i t a l locus had been deleted i n a l l of the d e f i c i e n c i e s . Preliminary studies (Suzuki and Duck, 1967) showed no gross c o r r e l a t i o n between genetic p o s i t i o n and TSP or LP. Extensive mapping of sex-linked and autosomal ts le t h a l s revealed two gene clusters (Suzuki, 1970). One cl u s t e r of 12 recessive ts leth a l s occurred within 3 map units of crossveinless (cv, 1-13.7). When 4 of the mutants were analyzed extensively, each exhibited a d i s t i n c t i v e pattern of TSP's and LP's (Tarasoff and Suzuki, 1970). A second clus t e r of 11 DTS mutants mapping near dumpy was found on chromosome II (Suzuki and Procunier, 1969). Their functional 77 relatedness was indicated by t h e i r f a i l u r e to complement each other's recessive l e t h a l i t y and t h e i r i d e n t i c a l TSP 1s. I t cannot be stated at the present time whether they, i n f a c t , constitute a polyfunctional region with a single c o n t r o l l i n g element or a single functional region. Rosy (ry, 3-52.0) and deep orange (dor, 1-0.3) i n Drosophila are f u n c t i o n a l l y related, i n that both are i n -volved i n hypoxanthine synthesis and when combined r e s u l t i n synthetic l e t h a l i t y . However,' the two l o c i are linked to d i f f e r e n t chromosomes (Lucchesi, 1968). Thus, the evidence already available i n Drosophila suggests that even i f the t i p region had.been successfully saturated with ts l e t h a l s , the results might have indicated no functional r e l a t i o n s h i p between.them. Therefore, since small regions of the genome that are f u n c t i o n a l l y related are rare i n higher organisms, they are not l i k e l y to be found by random s e l e c t i v e procedures. Moreover, i n higher organisms pleiotropy of many genes, and extensive phenotypic differences.between many a l l e l e s within the same gene render the detection of f u n c t i o n a l l y related s i t e s within small regions even more remote. While numerous non-ts l e t h a l mutations have, been characterized, determination of the time or tissue of t h e i r action i s d i f f i c u l t . Hadorn (1961) reviewed the e f f e c t i v e l e t h a l phase, i . e . time of death, of several non-ts l e t h a l s . He i n f e r r e d polyphasic gene a c t i v i t y on the basis of "durch-78 brenner" (breakthroughs, or escapers). He found that with some mutations an i n i t i a l LP could be defined by death at a p a r t i c u l a r time; the few surviving that b a r r i e r would then die at a l a t e r LP. However, as he pointed out, the separ-ation between the actual.time of gene action and the end phenotype of death may be considerable. Moreover, the v i s i b l e manifestations of abnormal development.may not necessarily r e f l e c t the tissue i n which the primary gene defect i s i n i -t i a t e d . The time or tissue of action of action of. non-ts let h a l s can be obtained on the basis of somatic mosaics generated by spontaneous ring-X loss or somatic recombination i n i t i a t e d at selected times by the experimenter's use of X-rays (Becker, 1957). The considerable separation between the TSP and LP of some mutations indicates.that temperature-s e n s i t i v i t y provides a means for detecting defective gene action long before any phenotypic manifestation of abnormal-i t y or death. The u t i l i t y of temperature-sensitivity as a to o l for the dissection of numerous problems i n genetics and a l l biology has been reviewedrrecently (Suzuki, 1970). I t was pointed out that ts mutations at s p e c i f i c , l o c i or a f f e c t i n g a s p e c i f i c function provide the best means of exploring, the b i o l o g i c a l events involved. Few ts a l l e l e s of known l o c i have been characterized, although expressivity of many mutants i s known to be temperature dependent, e.g. t e t r a l t e r a (3-48.5) i s 35% penetrant at 25°C, 1% at 17°C (Lindlsey and G r e l l , 79 1968); Curly (2-6.1) penetrance i s also greatly reduced by low temperature (Ibid.); r o l l e d (2-55.1) shows i t s most extreme phenotype at 26°C; above and below t h i s temperature the eye becomes more wild-type (Hackman and Lakovaara, 1966). The d i s t i n c t i o n between ts mutants as t h i s lab has character-ized them, and temperature dependent ones may be purely semantic i n many.cases. The concept of a temperature-sensi-t i v e change ( i . e . thermolability of a gene product) was unknown'when the temperature dependent mutants, were, described. On the other hand, the difference may be real,.one.class (the ts mutants) r e f l e c t i n g an actual biochemical change i n a gene product which makes i t thermolabile, the other class r e f l e c t i n g "an interference with the k i n e t i c s of one process i n r e l a t i o n to other simultaneous ones" (Goldschmidt, 1958, p. 366). Examples of ts a l l e l e s found so far include raspberry ( G r i g l i a t t i , 1970), homeotics ( G r i g l i a t t i and Suzuki, in.press), Notch (Foster and Suzuki, 1970), Ver-m i l l i o n (Camfield, unpublished), Minutes (Holden and Suzuki, i n preparation) and 1(1). myo (Wright, 1968). The detection of.temperature-sensitivity at the dor locus, therefore, was of considerable i n t e r e s t , especially... i n l i g h t of i t s known p l e i o t r o p i c e f f e c t s (Lucchesi and. Bischoff, 1969). The analysis of dor and C3-6 presented i n t h i s study has revealed two hitherto undescribed properties of the dor locus. F i r s t , the dor a l l e l e i t s e l f i s a temperature-sensitive l e t h a l . Given hindsight, there was some in d i c a t i o n 80 of t h i s c h a r a c t e r i s t i c i n Lucchesi's (1968) report involving the synthetic l e t h a l i t y of dor with ry. He mentioned that while, dor/dor; ry/ry f l i e s were l e t h a l at room temperature, some were observed to. l i v e when grown at low temperatures. The new dor, phenotype,"warped" wing, was f i r s t observed i n C3-6/T2E Y D males raised at 29°C. I t was i n i t i a l l y thought that t h i s phenotype resulted from a second-site h i t (possibly at dwarp (1-0.) which has a wing effect) unmasked by "spreading-effect" variegation of the T2E Y D chromosome (Cohen, 1962; Hartman-Goldstein, 1967). However, the detec-ti o n of thi s phenotype i n dor and 1(1)7A with both T2E Y D and TE Y, c l e a r l y indicates that i t i s indeed associated with the dor locus and therefore constitutes a new dor phenotypic e f f e c t . A t h i r d new phene of the dor locus was confirmed i n th i s study. Pupal l e t h a l i t y / with melanotic inclusions, was observed by Clancy (196 4) i n the trans-heterozygote female of two dor l e t h a l s . C3-6 males showed pupal l e t h a l i t y , but without melanotic inclusions; instead, gross h i s t o l y s i s of the pupa was evident. In. addition to these new findings, the research on: dor a l l e l e s reported here suggests the u t i l i t y of variegating rearrangements in.genetic analysis. This includes the e f f e c t of temperature on variegation, the timing mechanisms of 81 variegation, and tissue s p e c i f i c i t y for the e f f e c t . Each w i l l be discussed separately. In the?majority of cases described i n the l i t e r a t u r e , temperatures lower than room temperature (22°C - 26°C) en-hance variegation (Gowen and Gay, 1933; Schultz, 1947; Gersh, 1949; Baker, 1968). However, there are many exceptions to this general rul e . Prokofieva-Belgovskaya (19 47) observed maximum heterochromatinization (which is.generally related g to variegation (Baker, 1968)) of In(1) sc .at 25°C; above and below this temperature, heterochromatinization was 8 greatly reduced. Studying the same In(1)sc chromosome, Gersh (19 49) found that variegation for both the scute and Hairy-wing.phenotypes was minimal at 25°C, maximal at 18°C and 30°C; comparing these results with Prokofieva-Belgovskaya's cy t o l o g i c a l data we see evidence for tissue s p e c i f i c i t y of variegation. The dor a l l e l e s studied i n my research showed enhancement of variegation for eye colour and wing warp and i n some cases, for l e t h a l i t y at high temperature. The e f f e c t of 17°C on these phenes i s currently being tested. Thus i t appears unimportant whether high or low temperature affects a p a r t i c u l a r variegating locus. So long as the temperature at which enhancement of variegation i s known, use of the e f f e c t can become a tool for c r i t i c a l analysis of the events controlled by the locus. 82 Baker (196 8) pointed out that a problem i n under-standing the mechanism of variegating p o s i t i o n - e f f e c t s i s the i n a b i l i t y to distinguish between determinative and d i f f e r e n t i a t i v e events; the determinative event i s the decision that a chromosome and a l l of i t s m i t o t i c derivatives w i l l be turned on or o f f ; the d i f f e r e n t i a t i v e event i s the time when the determined state i s phenotypically expressed. The d i s t i n c t i o n between the two events w i l l become clearer from a few examples. Chen (1948) and Becker (1961) (cited by Baker (196 8)) have shown that enhancement of eye v a r i e -gation by temperature occurs i n the pupal period (the d i f f e r -e n t i a t i v e event). S h i f t studies done on C3-6/T2E Y D and dor/T2E Y D males confirmed the temperature-sensitivity of variegation i n the late 3rd l a r v a l and mid-pupal periods. These observations by no means imply that t h i s i s the only sensitive period. Baker's (1965) studies.provided " i n i t i a l evidence that the pigment p o t e n t i a l i t i e s of the developing variegated eye anlage are determined during the end of the f i r s t l a r v a l i n s t a r . " This finding of early s e n s i t i v i t y i s supported by Hartmann-Goldstein's (1967) work on a rearrange-ment which variegated both for Notch and-white. Exposure to low temperature for as l i t t l e as 12 hours i n the early embryo increased variegation of both l a r v a l Malpighian tubules and adult .wings. Using clonal transfer through 8 generations of trans-determining d i s c s , Hadorn, G s e l l and Schultz (1970) have shown that once the decision to variegate ( i . e . , allow 83 recessive expression) or not (i.e.,. + a l l e l e remains on) has been made, i t i s i r r e v e r s i b l e . Indeed, the poten t i a l s u s c e p t i b i l i t y to enhancers of eye variegation (e.g., tem-perature) occurs very early (1st l a r v a l i n s t a r ) , thus, eyes induced to variegate by a temperature change i n the late 3rd inst a r exhibit patterns of variegation s i m i l a r to the c e l l lineage relationships set i n the 1st l a r v a l i n s t a r (Baker, 1968). Whatever the mechanism of variegation (and t h i s i s Baker's concern for distinguishing between determinative and d i f f e r e n t i a t i v e events), these examples indicate that there are at least 2 periods during which temperature can enhance variegation. Less conclusive evidence for possible s p e c i f i c times of variegation ( i . e . , turning on-and-off of a variega-t i n g rearrangement) comes from the shift-up studies of C3-6 with either 67g Y or T2E Y D (Figure 18). I t was noticed that many of these f l i e s survived when shi f t e d up on day 6 of development even though they subsequently passed through a TSP at 29°C (days 8 - 10)'.'' This might indicate that i f shi f t e d to 29°C on day 6, the X-duplicated Y does not switch off thereafter. On days 8 - 1 0 , the duplication Y i s sen-s i t i v e to change i n temperature and.switches off upon trans-fer to 29°C. Another p o s s i b i l i t y i s that the duplication i s already switched o f f , but the change of temperature ad-versely affects the temperature-sensitive mutation i t s e l f , 84 as i s the case with para (Suzuki, et a l . , 1971). Thus the use of variegating rearrangements for genetic analysis could follow the procedures defined for analysis of temperature-sensitive mutations discussed i n Methods and Materials. The study of a ts a l l e l e of raspberry showed the tissue s p e c i f i c i t y and time of action of a gene ( G r i g l i a t t i , ts 1970). I t was shown that the various phenes of ras , l e t h a l i t y , and pigmentation of eyes, Malpighian tubules and testes, had d i f f e r e n t and separable TSP 1s. In studies of g the e f f e c t of temperature on variegation of scute i n I n ( l ) s c , Gersh (19 49) found an i n t e r e s t i n g tissue s p e c i f i c i t y . Sever-a l d i f f e r e n t b r i s t l e s are affected by the scute phenotype, but the p a r t i c u l a r b r i s t l e s affected varied at d i f f e r e n t temperatures. Thus, the s c u t e l l a r s were l o s t i f f l i e s were raised at 30°C but present at any of the 3 lower temperatures tested; the anterior/'supraalars, on the other hand, were l o s t at 18°C but not affected at 25°C. We know that there i s no d i r e c t r e l a t i o n s h i p between phenotypically.visible variegation and variegation i n t i s -sues necessary for v i a b i l i t y . This was c l e a r l y demonstrated with dor, which i s a temperature-sensitive l e t h a l and yet i s almost 100% viable with. TE Y, which gives the greatest amount of eye variegation. In contrast to t h i s , dor/T2E Y P males had low v i a b i l i t y at 22 9c, thereby in d i c a t i n g variegation i n the v i t a l tissues, but showed no eye or wing variegation whatsoever. Further evidence for tissue differences i n 85 variegation of a duplication can be seen by comparing the e f f e c t of TE Y on the two dor l e t h a l s , 11-B22 and 1(1)7A. 1I-B22 could not be compensated by TE Y at a l l whereas 1(1)7A survived with TE Y at 22°C, but died at 29°C. The simplest explanation of these observations i s to propose that temperature affects variegation i n v i t a l tissues d i f f e r -ently from eyes and wings. Mutation 11-B22 i s assumed to be so defective (perhaps an amofph) that even extensive dor + function (on the Y.chromosome) at 22°C could not compensate for i t s defect. On the other hand, 1(1)7A i s s u f f i c i e n t l y "leaky", to get by at 22°C, but the decrease i n d or + a c t i v i t y i n TE Y becomes l e t h a l at 29°C. I was very g r a t i f i e d to f i n d upon reviewing the l i t -erature that Clancy's (1964) and Lucchesi and Bischoff's (1969) e a r l i e r work on the phenotypic relationships between various dor a l l e l e s and two variegating duplication Y's agreed completely with my findings. While they studied other phenes of dor i n addition to eye variegation and l e t h a l i t y (e.g. melanotic tumor formation, and female s t e r i l i t y ) , a l l of t h e i r work was done at room temperature. A s i m i l a r analysis, has also been done by Kauffman (19 70) . He covered complete lethals i n the z-w region with variegating d u p l i -cations and found many that survived, but exhibited mutant phenotypes of semi-lethal a l l e l e s mapping i n the same l o c i . This does not t e l l us the actual time of action of the mutant and l e t h a l phenes, but does indicate that they are 86 separate and that variegation occurs only at the time of a c t i v i t y of the gene for the mutant, but non-lethal, phene. The developmental features of the determination of var i e -gation s t i l l poses a fascinating problem (Baker, 1968). The extent of variegation of some chromosomal re-arrangements has been shown to be affected by temperature. The results of these experiments prompt me to suggest that the Y-linked duplications a f f e c t i n g d i f f e r i n g expression of dor phenes may, i n f a c t , be caused by temperature e f f e c t s on the i r variegation. Indeed the proposed temperature e f f e c t permitted a d i s t i n c t i o n between two otherwise i n d i s t i n g u i s h -able dor l e t h a l s . I suggest that temperature-affected variegating rearrangements may permit the delineation of an i n t e r v a l comparable to the TSP for non-ts l e t h a l s . The procedure for such experiments would be to give temperature shocks (high or low according to the known properties of the rearrangement) at various times to animals heterozygous for the l e t h a l and the lethal" 1" rearrangement. One problem arises i n t h i s type of experiment involving the co-ordination of variegating and mutant gene expression. Consider the following: C3-6/T2E Y D - t s - l e t h a l ; variegates for eye and wing, 1(I)7A/T2E Y D - non-lethal; variegates, 11-B22/T2E Y D - non-lethal; does not variegate. 87 Assuming T2E Y D behaved s i m i l a r l y i n each genotype, these results indicate multiple, a l l e l e s p e c i f i c times of action for the v i t a l and pigmentation properties of the three dor a l l e l e s . Thus a p o s i t i o n - e f f e c t rearrangement may switch on and of f several times, but we can. observe only those times which coincide with the time of action (either v i t a l or phenotypic) of the mutant gene. Conversely, temperature dependent variegation of l o c i which have detectable gene products may elucidate the nature of c e l l s p e c i f i c functions of variegating rearrangements. The use of such variegating rearrangements may be indicated i n the report of Lindsley, e t a l . (1960) on an inte r e s t i n g class of Y-suppressed X chromosome l e t h a l s . They found that most of them were associated with chromosomal rearrangements (T(X:A)),. were variegated, and were not bb . These f l i e s died i n the X/0 condition, but survived as X/Y and X/X adults. In our lab, an X-ray-induced X-autosome translocation was found to be a ts l e t h a l i n males. When made homozygous i n females, the arrangement was found to be non-lethal and non-ts (Suzuki, unpublished). Some class-3 mutants exhibited a sim i l a r sexual dimorphism, l e t h a l i n X/Y males at 22°C, but viable i n homozygous females. Other sexually dimorphic mutants are known: dor/dor and dor/+ females have greater than wild-type . leve l s of isoxanthopterin, whereas dor/Y males have less isoxanthopterin than wild-type (Counce, 1957); dor progeny of dor/dor (female s t e r i l e ) 88 mothers have sexually d i s t i n c t i v e l e t h a l phases, the female progeny are early embryonic l e t h a l s , the males die l a t e r (Counce, 1956) and as a f i n a l example, z/z females have orange coloured eyes while z/Y males have wild-type eyes. The b i o l o g i c a l basis for t h i s sexual dimorphism may provide an insight into sexually r e s t r i c t e d genes or dosage compen-sation. The group of mutants exhibiting the class 3 phenotype (Table II) was of i n t e r e s t because of the apparent dominance of the mutation at r e s t r i c t i v e temperature; further tests for dominance i n females yielded r e s u l t s c o n f l i c t i n g with the in t e r p r e t a t i o n , thereby suggesting a sexual dimorphic expression of t h i s c l a s s . Consequently, a great deal of e f f o r t was exerted i n pursuing t h i s i n t e r p r e t a t i o n of the mutants. Upon intensive examination of the data i t became clear that a double mutational event.provided a simpler explanation of the class 3 patterns (see Figure 9 for the two hypotheses). The DTS model was abandoned i n favour of the 2 h i t when attempts were made to mark the t i p mutants with v i s i b l e markers. When the proximal portion of the mutant chromosome was eliminated (by crossing o f f ) , y + recombinants no longer exhibited ts l e t h a l i t y . In spite of my preference for the double h i t hypothesis, the data s t i l l confront us with d i f f i c u l t i e s . Interpretation of female data cannot be simply explained by either hypothesis, nor 89 can the s u r v i v a l of B_ C_3 mutants with Y and nullo-Y. Type "a" mutations (Figure 9) are r e a d i l y explainable by either hypothesis. The d e f i n i t i v e d i s t i n c t i o n between the two hypotheses can be read i l y made by recombination analy-s i s , but th i s has not yet been done. We might s t i l l be l e f t with the problem of a sexually dimorphic l e t h a l (e.g. C3-4). The above discussion does not a f f e c t the meaningful-ness of the s h i f t studies for the' determination of TSP. Since most of the s h i f t studies for ts l e t h a l i t y were done on males carrying the duplication which covered the.presumed non-ts l e t h a l , temperature s e n s i t i v i t y was measured for the ts mutation. EMS i s known to produce single base changes i n micro-organisms (Jockusch, .1966). Thus temperature s e n s i t i v i t y i s due to a single amino acid change. The polypeptide thus produced i s thermolabile; t e r t i a r y changes at high temper-ature r e s u l t i n i n a c t i v a t i o n . The use of such mutants i s well demonstrated i n phage T4D (Epstein, et a l . , 1963; Epstein and L i e l a u s i s , 1964). In Drosophila melanogaster, the genetic properties of most of the mutants analyzed con-forms to the expectation of single base change substitutions (Suzuki, 1970). I t has been assumed that the TSP corresponds to the time at which, the ..primary gene ..product i s b i o l o g i c a l l y active. The s h i f t .studies of C3-3 are p a r t i c u l a r l y i n t e r e s t i n g i n interpreting the basis of thermolability. Single s h i f t 90 studies established a prolonged TSP; however, up-and-down pulse s h i f t s during the TSP c l e a r l y suggest a requirement for prolonged maintenance at the r e s t r i c t i v e temperature before l e t h a l i t y resulted (Figures 13 and 14). The simplest interpretation of these results i s that the gene product once formed at the permissive temperature remains b i o l o g i c a l l y active. The requirement for long exposure to r e s t r i c t i v e temperature indicates that the time required to drop below the threshold l e v e l of wild-type product for v i a b i l i t y i s 2-3 days, and that the temperature-sensitive stage probably occurs at the time of t r a n s c r i p t i o n or t r a n s l a t i o n . The resistance to temperature denaturation of pre-formed products resembles the observations of assembled ts phage T4D par-t i c l e s (Edgar and L i e l a u s i s , 1964). Cline and Hastings (1971) have i s o l a t e d a temperature-sensitive l u c i f e r a s e which i s sensitive' only during synthesis. Once formed at permissive temperatures', the l u c i f erase i s not thermolabile. Other p o s s i b i l i t i e s for interpretation of the molecu-l a r basis for thermosensitivity e x i s t and cannot be d i s -tinguished by s h i f t experiments. For example, reversible thermodenaturation of the primary gene product would give a TSP which corresponds to the time of action of the gene product. This mechanism i s undoubtedly the basis of ts temperature-sensitivity for para (Suzuki, et a l . , 1970). The heterogeneity for the basis of temperature s e n s i t i v i t y i s indicated by the studies of Cline and Hastings (1971) 91 with ts mutations for bioluminescence i n bacteria. They had three classes of ts bioluminescent mutants: 1) an inactive l u c i f e r a s e i s produced at 36°C; at 26°C the luc i f e r a s e syn-thesized i s wild type but loses a c t i v i t y upon transfer to 36°C; 2) a wild-type l u c i f e r a s e i s produced at 36°C but lacks a co-factor necessary for luminescence, and 3) no -1-uciferase i s synthesized at 36°, but the product made at 26°C i s not thermolabile. The rest of the TSP's i n t e r e s t i n g l y indicated a common in t e r v a l of s e n s i t i v i t y during the pupal period. This i s not surprising since early metamorphosis i s a time of con-siderable genetic a c t i v i t y . Compared ..with non-ts mutations, our determination of TSP's and LP's more, f i n e l y d i f f e r e n t i a t e s the actual event of primary.gene action leading to death. Note that i n most cases (Figure 17) the TSP i s approximately co-incident with the LP, i n 2-3 cases i t precedes i t . SUMMARY In the course of screening the X chromosome of Drosophila melanogaster for ts leth a l s mapping i n a short genetic region, a new class of mutants (class 3) was observed. These were i n i t i a l l y thought to be dominant t s - l e t h a l s because i n the presence of a duplication they survived at 22°C but died at 29°C. After intensive analysis i t was decided that they were more readi l y explained as double-mutation chromosomes. One mutation, covered by a duplication was a non-ts l e t h a l , and the second was a ts l e t h a l . Recombination studies were not completed to v e r i f y t h i s .proposal. The developmental e f f e c t s of the ts le t h a l s were characterized by s h i f t i n g cultures from 22°C to 29°C, and vice versa, a t . d i f f e r e n t successive times. In t h i s way, the temperature.sensitive.period (TSP) and e f f e c t i v e l e t h a l phase (LP) were determined for each stock. A l l TSP's were found to encompass a common i n t e r v a l during the early pupal period. A l e t h a l a l l e l e of dor was recovered and comparative studies were made on several dor a l l e l e s i n combination with variegating duplications. The a l l e l e dor was i t s e l f 93 found to be temperature sensitive with i t s TSP occurring from the pre-pupal period u n t i l eye pigmentation. In addi-t i o n , a-new dor phenotype "warped wings" was described. A method for developmental analysis of non-ts l e t h a l mutations, involving the use of variegating rearrangements, was presented. BIBLIOGRAPHY Ames, B.N. and P.E. Hartman, 1963. The h i s t i d i n e operon. Cold Spr. Harb. Symp. Quant. B i o l . 28:349-356. Baker, W.K., 1965. A method for the developmental timing of the pattern of variegation. Dros. Inform. Serv. 40:61. , 1968. P o s i t i o n - e f f e c t variegation. Adv. Genetics. 14:133-169. _, and J.B. Spofford, 1959. 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