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Dominant temperature-sensitive lethal and semilethal mutations on chromosome 2 of Drosophila melanogaster. Procunier, James Douglas 1968

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DOMINANT T E M P E R A T U R E - S E N S I T I V E LETHAL AND SEMI LETHAL MUTATIONS ON CHROMOSOME 2 OF DROSOPHILA MELANOGASTER by JAMES DOUGLAS PROCUNIER B . S c , 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 , 1966 A T H E S I S SUBMITTED IN P A R T I A L FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n t h e D e p a r t m e n t o f ZOOLOGY We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i pfed - s t a n d a r d THE U N I V E R S I T Y OF B R I T I S H COLUMBIA J u l y , 1968 In presenting th is thesis in pa r t ia l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i sh Columbia, I agree that the Library sha l l make i t f ree ly ava i lab le for reference and Study. I further agree that permission for extensive copying of th is thesis for scholar ly purposes may be granted by the Head of my Department or by h.ils representat ives. It is understood that copying or publ icat ion of th is thesis for f inancia 1 .gain shal l not be allowed without my wri t ten permission. Department The Univers i ty of B r i t i sh Columbia Vancouver 8, Canada ABSTRACT C o n d i t i o n a l ' l e t h a l m u t a n t s w h i c h d i e u n d e r r e s t r i c t i v e c o n d i t i o n s b u t a r e v i a b l e i n a p e r m i s s i v e e n v i r o n m e n t p r o v i d e u s e f u l t o o l s f o r t h e g e n e t i c a n d d e v e l o p m e n t a l a n a l y s i s o f c e r t a i n l o c i . One c l a s s o f c o n d i t i o n a l m u t a n t s , t e m p e r a t u r e -o o s e n s i t i v e r e c e s s i v e l e t h a l s w h i c h d i e a t 29 C b u t s u r v i v e a t 23 C h a s b e e n r e p o r t e d f o r an e x t e n s i v e number o f l o c i i n Drosophi1 a  m e l a n o g a s t e r . H o w e v e r , l o c i may e x i s t w h i c h a r e h i g h l y r e d u n d a n t , o r c o n c e r n e d w i t h f u n c t i o n s r e q u i r i n g t h e t o t a l o u t p u t o f b o t h w i l d t y p e a l l e l e s o r w i t h s y n t h e s i s o f a s t r u c t u r a l c o m p o n e n t w h i c h w o u l d p r e - e x e m p t t h e i r r e a d y d e t e c t i o n a s r e c e s s i v e m u t a t i o n s . M u t a t i o n s w i t h i n s u c h r e g i o n s c o u l d , h o w e v e r , be r e c o v e r e d b y s e l e c t i n g t e m p e r a t u r e - s e n s i t i v e m u t a t i o n s w h i c h b e h a v e a s d o m i n a n t l e t h a l s a t t h e r e s t r i c t i v e t e m p e r a t u r e . In a d d i t i o n , s u c h m u t a n t s w o u l d p e r m i t t h e f i r s t g e n e t i c c h a r a c t e r i z a t i o n s o f d o m i n a n t l e t h a l s . Ethy1 m e t h a n e s u 1 f o n a t e - i n d u c e d d o m i n a n t t e m p e r a t u r e - s e n s i t i v e l e t h a l a n d semi l e t h a l m u t a t i o n s w e r e i n d u c e d i n ch r o m o s o m e 2. T w e n t y - o n e l e t h a l s o f t h i s t y p e w e r e i s o l a t e d f r o m 6,130 t e s t e d c h r o m o s o m e s a n d s i x t e e n w e r e c h a r a c t e r i z e d w i t h r e s p e c t t o t h e i r g e n e t i c l o c a l i z a t i o n a n d d e v e l o p m e n t a l e f f e c t s . . U n e x p e c t e d l y , e l e v e n o f t h e m u t a n t s w e r e f o u n d t o be c l o s e l y l i n k e d t o t h e dumpy, d p , l o c u s . A l l e l e v e n w e r e r e c e s s i v e l e t h a l s a t room t e m p e r a t u r e a n d w e r e f u n c t i o n a l l y a l l e l i c . The t e m p e r a t u r e - s e n s i t i v e p e r i o d ( T S P ) was s i m i l a r f o r a l l c l u s t e r m u t a n t s a l t h o u g h t h e e f f e c t i v e l e t h a l p h a s e ( L P ) a t 29°C d i f f e r e d . I t was c o n c l u d e d t h a t t h e c l u s t e r l e tha ls are, in fac t , genet ica l ly a l l e l i c . Three other loc i were demonstrated by genetic recombination and each had a cha rac te r i s t i c TSP and LP. In add i t ion , two mutants caused s t e r i l i t y of females and could not be l o c a l i z e d . The recovery of mutations which map genet ica l ly wi th in a segment and are dominant l e t ha l s , proves that dominant l e t ha l i t y need not re f l ec t gross chromosomal a l te ra t ions in higher organisms. ACKNOWLEDGEMENTS I am indebted to Dr. D.T. Suzuki for his helpful d iscussion and c r i t i c i s m throughout the work and preparation of the manuscript. I would l i ke to thank Dr. Leonie P i te rn ick for her valuable assistance in the laboratory. I wish to acknowledge Misses Rachel Pra t t , Jeanette Holden and J i l l Cameron for the i r excel lent technical help in the i so la t ion of these mutants. iv TABLE OF CONTENTS Page INTRODUCTION 1 METHODS AND MATERIALS 6 RESULTS 11 DISCUSSION 18 SUMMARY 23 LITERATURE CITED 32 V LIST OF FIGURES Figure Page Screening protocol for the detect ion of dominant temperature-sensit ive lethal mutations in chromosome 2. 28 2 The rat ionale for genetic l oca l i za t i on of dominant temperature-sensit ive le tha ls by reciprocal crossing over. 29 3 Protocol for reciprocal " s h i f t " experiments to del ineate the temperature-sensit ive period and e f fec t i ve lethal phase of dominant temperature-sens i t i ve l e tha l s . 30 k Genetic pos i t i on , temperature-and e f fec t i ve lethal phase of temperature-sensit ive l e t h a l . sens i t ive period each dominant 31 1 INTRODUCTION Dominance of an a l l e l e is defined when, in the presence of two or more d i f fe rent a l l e l e s of a par t i cu la r locus within a s ing le nucleus, the phenotype of one of the a l l e l e s is expressed. Dominance is readi ly determined at a gross morphological level in higher organisms or even in microorganisms (e.g. streptomycin s e n s i t i v i t y ) ; however, as the phenotypic assay approaches the molecular level of gene product determination, the d i s t i nc t i on between dominance and recessiveness becomes more ambiguous and must be defined opera t iona l ly . The molecular basis for dominance in microorganisms has been at t r ibuted to the polymerization of mutant and wi ld type monomeric polypeptides with a consequent restorat ion of b io log ica l a c t i v i t y to the hybrid polymer (wild type dominant) or inact ivat ion of the ent i re multimer (mutant dominant) (Bernstein and F isher , 1968). Inact ivat ion of such "hybr id" polymers resul ts in a net decrease in enzyme a c t i v i t y and may be analogous to the appearance of mutant phenotypes in higher organisms heterozygous for one wi ld type gene and a point mutant for the locus in the homologous chromosome. Another c lass of dominant a l l e l e s in microorganisms is that involving regulator genes (Jacob and Monod, 1961). The d ip lo id condit ion for a regulator locus can be generated in bacter ia and i t is found that the wi ld type a l l e l e of a regulator gene ( i + ) is dominant to a cons t i tu t i ve mutant ( i -) for that locus. 2 Dominant l e t h a l i t y was f i r s t suggested by H .J . Mul ler (1927) to explain the marked increase in s t e r i l i t y of Drosophila males af ter X - i r r a d i a t i o n . The ef fect of X-ray treatment was i n i t i a l l y thought to be an inact ivat ion of sperm, thereby preventing f e r t i l i z a t i o n . However, Stancati (1932) proved that X-ray-induced dominant l e t h a l i t y in Habrobracon was not the consequence of sperm inac t i va t ion ; He found that a f ter X - i r r ad ia t i on of Habrobracon males, the frequency of biparental females (diploids a r i s ing from f e r t i l i z e d eggs) decreased while the frequency of matrocl inous, uniparental males (haploids a r i s ing from un fe r t i l i zed eggs) was not changed. Sperm inact ivat ion would have elevated the frequency of matroclinous males. X-ray-induced dominant le thals in Drosophila melanogaster and Habrobracon have been examined extensively by von Borstel (1960) . He has dist inguished a number of c lasses of dominant le thals on the basis of the time and phenotypic e f fects of l e t h a l i t y in zygotes recovered from heavi ly i r radiated females. Three d i f fe rent types of dominant l e t h a l i t y were recognised by cy to log ica l observation of number of cleavages and egg hatchabi1 i ty . Type I dominant l e t ha l i t y occurs most frequently and is characterized by an i n i t i a l depression of the mi to t ic rate followed by a complete cessat ion of mitosis af ter the second or th i rd nuclear d i v i s i o n . Type II dominant l e t ha l i t y is defined by zygot ic death shor t ly af ter hatching from the egg (diploids) or before hatching but af ter b las tu la formation (haploids). Type III dominant l e t h a l i t y occurs af ter b las tu la formation but before 3 egg hatching whether the egg is haploid or d i p l o i d . In Drosophila melanogaster, both the Types I and II c lasses of dominant l e t h a l i t y were demonstrated when e i ther eggs or sperm were i r rad ia ted . However, Type III dominant l e t ha l i t y is d i f f i c u l t to demonstrate since Drosophi1 a melanogaster eggs must be f e r t i l i z e d in order to develop. Since indiv iduals carry ing dominant le thals do not surv ive, the nature of the l e t ha l i t y cannot be examined by c l a s s i c a l genetic techniques. Therefore, genet ica l ly contr ived dominant le thals were constructed in order to determine whether they mimic dominant le thals induced by X- rays . The p o s s i b i l i t y that X-ray-induced Type I dominant le thals resul t from chromosome breakage followed by s i s t e r strand fusion and formation of d icen t r i c bridges was investigated by creat ing a genetic system which generates d icen t r i c bridges in Drosophi la. The compound X chromosome, the tandem metacentric (TM) was u t i l i z e d for such a purpose. Half of the s ing le and three strand double exchanges wi th in the TM generate chromosome bridges at second anaphase of meiosis (Novi tsk i , 1951). Therefore, approximately 22% of the to ta l eggs la id by TM-bearing females should contain pronuclei bearing chromatids produced by breakage of the br idges; indeed, cy to log ica l examination of the embryos establ ished that approximately 25% died within a few nuclear d i v i s i o n s . This c i rcumstant ia l evidence was taken as support for the suggestion that X-ray-induced Type I dominant l e t ha l i t y may resul t from a breakage-fusion-bridge cyc le . Approximately one-half o f the eggs la id by an organism, heterozygous for a t ranslocat ion w i l l carry a delet ion for one chromosome segment and a dup l ica t ion for another caused by adjacent-1 segregation. The time of death of embryos resu l t ing from unfer t i1 ized and f e r t i l i z e d eggs from Habrobracon females heterozygous for f i ve d i f ferent t ranslocat ions exhib i ted a close resemblance to radiat ion-induced Type II l e t h a l i t y . Thus, von Borstel a t t r ibuted Type II dominant l e t ha l i t y to chromosome imbalance resu l t ing from dupl icat ions or de f i c i enc ies . A cer ta in proport ion of eggs recovered from Habrobracon and Drosophila t r i p l o i d females which were s t e r i l i z e d by normal sperm showed a dominant lethal phenotype which resembled radiat ion-induced Type III dominant l e t h a l i t y . T r ip lo id females produce eggs which contain chromosome numbers varying from the haploid to d ip lo id number and therefore, whether f e r t i l i z e d or not can have an abnormal chromosome complement in the zygote. Thus, the pattern of radiat ion-induced Type III dominant l e t ha l i t y is s im i la r to the l e t h a l i t y caused by loss or dup l ica t ion of ent i re chromosomes. Therefore, i t has been concluded that the d i f fe rent types of dominant l e tha l i t y induced by radiat ion are s imi la r to those generated by chromosomal imbalance or breakage. The frequency of dominant lethal induction by X-rays is dependent upon the dose administered and the ploidy of the organism (Mortimer, 1958). Mortimer demonstrated an inverse re lat ionship between radioresistance and p lo idy , from d ip lo idy to hexaploidy in yeast. Haploid cul tures were more rad io-sens i t i ve than d ip lo id 5 cul tures owing to the expression of induced recessive l e t ha l s . These observations are consistent with the suggestion that most dominant le tha ls resul t from chromosome breakage of Type I (von Bors te l , I960). Direct genetic analys is of dominant le tha ls has not been possib le in past studies because of the i n a b i l i t y to propagate any mutant through successive generations. The recent recovery of condi t ional lethal mutations in higher organisms (Suzuki et al . , I967) now suggests a means of screening for mutants expressed as dominant le tha ls under r e s t r i c t i v e condit ions but v iab le in a permissive environment. Hartwell (I967) found that V^OO temperature-sensit ive le tha ls recovered in haploid yeast c e l l s behaved as dominant l e t ha l s . Thus, dominant temperature-sensit ive le tha ls which die in a heterozygote at a r e s t r i c t i v e temperature (29°C) but/v iable under permissive condit ions (22 C) were screened for in Drosophila me 1anogaster. Their successful detect ion and genetic character izat ion demands a rev is ion in the concept of dominant l e t ha l i t y in higher organisms and may provide a tool for the detect ion of classes of mutants not otherwise demonstrable. 6 METHODS AND MATERIALS (a) Screening protocol for detect ion of dominant temperature-sensit ive (Dts) le tha ls on chromosome 2. Wild type (Oregon-R^q and Samarkand) males maintained by mass t ransfer were placed for 2k hours in quarter p int bot t les containing a pad of f i l t e r paper saturated with 1 ml of 0.025M ethyl methane-sulfonate (EMS) dissolved in 1% sucrose. They were then mass mated in quarter p int bot t les at 22- 2°C (RT) to Cy/Pm v i rg i n females (10 males and 20 females per b o t t l e ) . Cy_ and Pm are d i f fe rent inversions of chromosome 2 associated with the dominant v i s i b l e phenotypes of curled wings and dark variegated eyes, respect ive ly . Both chromosomes behave as recessive l e tha l s , i . e . are lethal when homozygous. _Cy_ car r ies two paracentr ic inverions (one in each arm) and Pm is a pe r i cen t r i c inversion (for a complete descr ip t ion see Bridges and Brehme, 19^*0. Individual Fj males, heterozygous for a treated second chromosome and ei ther Cy_ or Pm, were mated in shel l v i a l s to 3 Cy/Pm v i rg ins for h days at RT. The f l i e s in each v ia l were then t ransfer red, without e ther izat ion to fresh v i a l s which were then o ° placed at 29 C. Af ter 10 days, f l i e s in each 29 C cul ture v i a l were inspected without e the r i za t ion , for the absence of non-Curly winged f l i e s ( i . e . Pm/+* where +* represents the treated second chromosome). Such cul tures were then etherized and scored for the presence of any Cy/+* and Pm/+* f l i e s . A l l f l i e s in the RT cul ture from which 29°C v i a l s carrying no +* -bearing f l i e s were derived were then inspected 7 for the presence of +* -bearing f l i e s . The stock was designated as a Putat ive Dts l e t h a l , i f the +* -bearing ind iv iduals were present. From the RT cul ture of each Putat ive Dts stock, seven males heterozygous for the treated chromosome and _Cy_ were mated ind iv idua l l y to 3 Cy/Pm v i rg i n females at RT for k days and transferred to fresh v i a l s at 29°C. The fa i l u re -o f +•' -bearing f l i e s to survive at 29°C and thei r survival at RT was taken as a confirmation of dominant temperature-sensi t iv i ty and the stock was then maintained at RT with _Cy_ as a balancer. The ent i re screening protocol is out l ined in Figure 1. Each confirmed Dts lethal was tested for i t s v i a b i l i t y at 17°C in order to determine the optimum temperature for maximal surv ival . Second chromosomes from untreated Oregon-R^g and Samarkand males were a lso screened as out l ined in Figure 1 in order to provide control values in ca lcu la t ing re la t i ve v i a b i l i t i e s of each Dts lethal . (b) Genetic l oca l i za t i on of confirmed Dts l e tha l s . The genetic posi t ions of the Dts le tha ls were determined re la t i ve to the. markers contained in the " a l l " stock (for a complete descr ip t ion see Bridges and Brehme, 19^). This stock contains, seven markers which mark the ent i re length of chromosome 2. The recessive markers from le f t to r ight in " a l l " are (followed by thei r genetic symbols and linkage pos i t i ons ) : ar i stal ess-a l 0.0, dumpy-djp_ 13.0, bl ack-b kS .5, purple-pr 5*4.5, curved-c 75 • 5, plexus-£x 100.5, speck-s£ 107.0. The regions delineated by the markers were numbered from le f t to r ight so 8 that the al-dp region is 1, dp-b is 2, b-pr is 3, pr-c is k, c-px is 5 and px-sp is 6. Note that region k (pr-c) spans the centromere. V i rg in D t s / " a l l " females were col 1ected within k8 hours of eclosion and mated to C y / " a l l " males in quarter p int bot t les at 29°C. An average of 10 males was crossed to 20 females per bo t t l e . If Dts l e t h a l i t y resul ts from single mutants, crossover classes in a l l but one of the regions are expected to show non-reciproci ty since one of the reciprocal crossover products in each region w i l l carry the Dts mutant. The region in which reciprocal crossover classes are recovered is the segment in which the Dts lethal l i e s , the two classes representing crossovers on each side of the l e t h a l . The frequency of the two reciprocal c lasses provides a measure of the genetic pos i t ion of the lethal wi th in a segment (Figure 2). (c) Determination of the Ef fec t ive Lethal Phase (LP) and Temperature^Sensitive Period (TSP) of each Dts l e t h a l . The time at which f l i e s heterozygous for the Dts lethal die when raised at 29°C (ef fect ive lethal phase) was determined for each stock.. F i f t y to one hundred Cy/Dts adults were placed, in empty quarter pint bot t les inverted over petr i p lates which contained standard Drosophila medium, coloured with grape j u i c e . Eggs were recovered in th is way wi th in a 1 to 2 hour laying period at RT. Zygotes formed in th is cross have the genetic const i tu t ion of Cy/Cy ( lethal in the embryo), Cy/Dts or Dts/Dts. Differences in the LPs of the Dts heterozygotes and homozygotes might be detected by noting the time and number of deaths at d i f fe rent time in te rva ls . This was not determined in these experiments. A possib le d i f ference between the LP of homozygotes and heterozygotes was shown by egg counts, as described in Section (d) The plates were sh i f ted immediately to 29°C and checked at 12 hour in terva ls for the onset of death. Death was determined by arb i t ra ry c r i t e r i a such as f a i l u r e of eggs to hatch, d is in tegra t ion of larvae and cessat ion of development at d iscrete stages of pupation. The LP r only defines the period at which the temperature ef fects manifests i t s e l f phenotypical ly but does not necessar i ly del ineate the actual period at which the primary b io log ica l processes are affected by the high temperature. This temperature-sensit ive period 0"SP) can be determined by reciprocal "upsh i f t s " and "downshi f ts" . Cultures establ ished at RT were transferred to 29°C at d i f fe rent successive time intervals (upshi f t ) . The reciprocal downshift involves t ransfer r ing 29°C cul tures to RT at d i f fe rent t imes. The e a r l i e s t upshif t which y ie lds v iab le adults del ineates the end of the TSP, whereas the f i r s t downshift which does not permit adults to emerge indicates the i n i t i a t i o n of the TSP. The protocol for the sh i f t experiments is i l l us t ra ted in Figure 3. (d) Complementation tests and egg counts. Each Dts lethal was tested for v i a b i l i t y in the homozygous condit ion at RT by crossing males and females heterozygous for each mutant. The f a i l u r e of the homozygotes to survive does not necessari indicate recessive l e t ha l i t y of the Dts mutant since the high level of mutagenesis by EMS w i l l resul t in the recovery of Dts lethals on chromosomes carrying recessive le tha ls induced elsewhere. Di f ferent Dts le tha ls were crossed to each other at RT, the v i a b i l i t y of the trans heterozygotes ind icat ing complementation of the mutants. Lethals which f a i l ed to complement in such a trans test were tested further at RT for the LP of the homozygotes. Eggs were co l lec ted within a 2 hour interval from crosses of ind iv idua ls carrying _Cy_ and a par t i cu la r Dts lethal or an Oregon-R or Samarkand chromosome. Af ter k8 hours, the number of unhatched and hatched eggs was recorded. The number of adults which emerged from each egg sample was also determined. 11 RESULTS (a) Dts lethal v i a b i l i t y . Twenty-one mutants which behaved as Dts le thals and semilethals were detected among 6,130 chromosomes tes ted. Henceforth, "D ts -l e t h a l " w i l l be used in reference to both lethal and semi lethal mutants. In crosses of Cy/+*'*dX Cy/Pm 2 , the Cy. homozygotes die and the expected ra t io of +* -bearing progeny to the total survivors is 0.67 i f no lethal is present. It can be seen that control crosses, using untreated Oregon-R and Samarkand chromosomes yielded rat ios at RT and 29°C which were very c lose to the expected 0.67 value (Table l ) . These control rat ios were used as the standard v i a b i l i t y index against which the v i a b i l i t y of Dts lethal heterozygotes could be compared. Thus, the control rat ios were designated as indicat ing 100% v i a b i l i t y and the rat ios of each Dts lethal adjusted accordingly to the respective wi ld type chromosome control value from which the mutant was i so la ted . The re la t i ve v i a b i l i t i e s of each Dts lethal hetero-zygote at 29°C and RT are shown in Table I. Of the 20 Dts le thals tested, 8 which behaved as complete le thals at 29°C had a v i a b i l i t y of 3k% or greater at RT. None of the Dts mutants was less than 51% v iab le at RT or more than 5% at 29°C (Table l ) . The re la t i ve v i a b i l i t i e s of twenty of the Dts lethal heterozygotes at 17°C were approximately the same as the corresponding v i a b i l i t i e s at 23°C (Table l ) . A s t a t i s t i c a l analysis was not carr ied out to show any s i gn i f i can t d i f ference in the v i a b i l i t i e s at these two temperatures. 12 (b) Genetic posi t ions of Dts l e tha l s ; The regional l oca l i za t i ons of 15 mutants are shown in Table I. The crossover data are shown in Table I I I . F l i e s homozygous for o the " a l l " chromosome or for several mutant markers at 29 C show extreme i n v i a b i l i t y , as seen in the control cross of Oregon-R/"a l1" 2X C y / " a l l " ^ . Since map distances for each region are d i s to r ted , such loca l i za t i ons permit only a crude pos i t i on ing . The re la t i ve pos i t ion of the Dts lethal wi th in a region could be estimated roughly by the ra t io of the reciprocal crossover c lasses . Surpr is ing ly , 11 of the Dts le tha ls appear to reside to the r ight of d_£ in region 2 . The control map distance for region 2 was 12.9 un i t s , a considerable reduction from the standard map distance of 35 map uni ts and re f lec ts the extreme weakness of f l i e s carrying several markers at 29°C. The re la t i ve posi t ions of the le tha ls near d_£ with respect to each other could not be determined in th is study. Henceforth, these II le thals loca l i zed to the r ight of djp_ shal l be referred to as the c lus ter and w i l l be discussed l a te r . The other Dts le tha ls were mapped in several other segments of chromosome 2 (Table I ) . Two Dts l e t ha l s , Dts-Ll and D t s - L 1 5 were loca l ized in region 5, (c to p_x) , approximately 1 -2 uni ts to the le f t of £x. The posi t ions of these two le tha ls re la t ive to each other could not be determined. Three mutants were not l oca l i zed . The genetic map of the Dts le tha ls re la t i ve to the "al1" markers is seen in Figure k. Tests of Dts-L.8 y ie lded ambiguous resul ts which did not permit l o c a l i z a t i o n . Two Dts l e tha l s , Dts-L9, and, D t s - L 2 0 , produced s t e r i l e females and could not be located. Therefore, out of the 16 Dts le tha ls that were tested, 15 were readi ly loca l ized wi th in a spec i f i c region. Thus, Dts le tha ls behave genet ica l ly as point mutants. (c) Dts lethal LP and TSP. Since eggs heterozygous or homozygous for a Dts lethal could not be d i f f e ren t ia ted , the LP determined for each mutant corresponds to the time of death of e i ther Cy/Dts-L or Dts-L/Dts-L ind iv idua ls . In the sh i f t experiments, on the other hand, the TSP was determined by survival of Dts- le tha l heterozygotes and therefore is of relevance only to the heterozygote. A l l developmental stages were corrected and correspond to the time scale of RT cu l tu res . Of the 11 c lustered mutants, 8 had an LP in the late 3rd instar larvae stage (Figure k). The LP of two others, D ts -L l l and Dts-Ll6, was la ter in the 3rd larval instar and ear ly pupal stages. Dts-L7, on the other hand, k i l l e d in the egg stages, since 89% of eggs la id from the cross Cy/Dts -L7 X Cy /Dts-L7 f a i l ed to hatch at 29°C. The remainder died in la ter larval stages. The TSP of a l l the mutants in the c luster was found during the egg stage, approximately ]8-2k hours af ter the eggs were la id (eggs normally hatch wi th in 2k hours af ter la id at 23 C) . The LP of Dts-L6 occurred in the late pupal stage (210 hours), af ter wing pad formation and eye pigmentation. The TSP of th is mutant preceded the LP occurr ing about ]kk to 192 hours af ter depos i t ion. Dts-L19 f l i e s also died as late pupae (210 hours) with the TSP occurr ing 2 or 3 days beforehand at 125-150 hours. It is worthy of note that death of f l i e s carrying th is mutant occurs during the process of emergence. Both Dts-Ll and Dts -L15 exhibi ted an LP as ear ly pupa (115-130. hours) before the onset of wing pad formation or eye pigmentation. The TSPs of both of these mutants were just pr ior to or coincident with the LP at 110-140 hours. Dts-L8, which could not be mapped gene t i ca l l y , had an LP and TSP at 205 and 120-1M+ hours respect ive ly . (d) Complementation test and egg counts. No adults homozygous for the Dts lethal chromosomes hatched at RT from crosses of Cy/Dts-L heterozygotes. This may be due to the recessive l e t ha l i t y of the Dts lethal i t s e l f or to other recessive le tha ls induced elsewhere on the treated chromosome. For three c lus ter Dts l e tha l s , Dts-L2, Dts-L3, and Dts-L5, the Dts lethal was / 1 ' removed from the o r ig ina l EMS'treated chromosome by crossing D t s - L / " a l l " v i rg ins to Cy/al 1 males and se lect ing crossovers in region 2 which contain the Dts l e t h a l . Thus the Dts lethal was crossed onto an untreated chromosome. None of the three le tha ls tested in th is way survived in the homozygous condit ion at RT thereby suggesting that the recessive l e t ha l i t y of these Dts lethal chromosomes is due to the Dts lethal i t s e l f . In RT crosses of Cy /Dts -L x X Cy_/Dts-Ly of d i f fe rent Dts l e tha l s , the ra t io Dts -L x /D ts -Ly ind iv iduals to Cy/Dts-L heterozygotes is 0.50 i f the homozygotes are completely v iab le at RT. The v i a b i l i t i e s of d i f fe rent combinations of Dts lethal chromosomes in the trans conf igurat ion were estimated by th is r a t i o . The resu l ts of the complementation tests of d i f ferent Dts le tha ls at 23°C demonstrated that a l l le tha ls which mapped genet ica l ly in d i f fe rent regions complemented with one another (Table I I ) . Al1 of the Dts le thals in regi.on 2 were tested for survival with two c lus ter mutants, Dts-L2 and Dts-L3, chosen a r b i t r a r i l y . Dts-L2 y ie lded a maximum ra t io of 0.13 in combination with Dts-L l6 (Table I I ) . Dts-L3 fa i l ed to complement with any other Dts le tha ls in the c lus te r . The survival of Dts-L2 and Dts-L3 with Dts le tha ls located elsewhere in the chromosome indicates that these par t i cu la r mutants are not necessar i ly lethal in combination with any Dts l e t h a l . Thus i t appears that the f a i l u r e of trans heterozygotes of the c lus ter mutants to survive at RT demonstrates their funct ional relatedness. In very prel iminary tes ts , crosses were made at 17°C between d i f fe rent mutants in the c lus ter and v iable trans heterozygotes were recovered. This should permit a crossover test between d i f ferent a l l e l e s . Both the Dts-Ls loca l i zed in region 5, Dts-Ll and Dts-Ll 5^/al so f a i l ed to complement with each other ind icat ing a l l e i ism of these two mutants. The LP at RT of the homozygous Dts le tha ls of the c luster was estimated by co l l ec t i ng eggs from a cross between Cy/Dts-L heterozygotes Seventy-f ive percent of the eggs la id are expected to hatch i f the homozygotes for the Dts-L chromosome are not lethal in the egg. The control cross of Cy/+ heterozygotes using an Oregon-R or Samarkand wi ld type chromosome yielded a hatch frequency of 0.62, a value much lower than the expected (Table IV). The reduction in egg hatching 16 o f the controls obscures any interpretat ion of hatch frequencies in the cross of Dts -Ls . The average egg hatch in crosses of the c lus ter Dts le tha ls was 0.41, Dts-L2 y ie ld ing a maximum of 0.50 and Dts-L7 a minimum of 0.30. Average hatchab i l i t y was reduced by 0.21 for any of the c lus ter Dts le tha ls and is presumed to re f lec t the f a i l u re of eggs homozygous for the lethal to hatch. Unfortunately, the presence of unrelated recessive le tha ls with LPs in the egg stage elsewhere in the chromosome render these resu l ts subject to a l ternat ive in terpre ta t ions . (e) Female-ster i le Dts l e tha l s . Two mutants, Dts'-L9, and Dts-L20 yielded v iab le heterozygous females at RT which were s t e r i l e . Owing to th is sexual dimorphism ( i . e . f e r t i l e males, s t e r i l e females) the le tha ls could not be mapped genet ica l ly nor the i r LPs and TSPs determined. The mutants were maintained by crossing heterozygous males to Cy/Pm females each generation. Males and females heterozygous for Dts-L20 had a v i s i b l e phenotype of abnormal abdominal te rg i te formation which may have interfered with f e r t i l i t y in females. Each female s t e r i l e Dts lethal was tested for complementation with a l l other d i f fe rent Dts lethal mutants. In crosses of males heterozygous for the female s t e r i l e Dts lethal and _Cy_ to female heterozygous for 0y_ and other Dts l e tha l s , the expected ra t io of the trans heterozygotes for both mutants to the Cy heterozygotes is 0.50 at RT i f the mutants complement. The resul ts (Table II) show that the female s ter i 1 e Dts l e t ha l s , Dts-L9 and Dts-L20 y i e l d e d a r a t i o of .01 and .06 in trans conf igura t ion with Dts-I_3 and Dts-L6 r e s p e c t i v e l y . This may ind icate that these female c s t e r i l e Dts l e t h a l s are a l l e l i c to the respect ive Dts l o c i . Three Dts l e t h a l s , D t s - L 2 l , D t s - L l 8 , and Dts-L17 out of the 21 i s o l a t e d in th is report were not c h a r a c t e r i z e d . 18 DISCUSSION Previous studies in higher organisms on the nature of dominant l e t ha l i t y u t i l i z e d heavy i r rad ia t ion and special genetic constructs to produce gross chromosome breakage or imbalance (von B o r s t e l , 1960). The present report of condit ional l e t ha l i t y of f l i e s heterozygous for le tha ls induced by EMS indicates that mutants may also behave as dominant l e t ha l s . EMS has been shown to induce point mutants of the missense type in microorganisms (Jockusch, 1966) and very few chromosome aberrat ions in Drosophila (E.B. Lewis, personal communication; Suzuki, 1968). A l l sex- l inked recessive ts le tha ls induced in Drosophila by EMS map genet ica l ly as point mutants and a l l but one of the mappable EMS-induced Dts le tha ls were readi ly loca l ized wi th in a genetic region. The non- local ized mutant appears to be a synthet ic dominant lethal ( i . e . , a synerg is t ic e f fec t of subvital mutants when l inked in the ci s arrangement), which accounts for i t s ambiguous loca t ion . The observation that Dts le tha ls map wi th in a region shows that dominant l e t ha l i t y that is temperature/ dependent is not the resul t of mutations at a number of d i f fe rent loc i whose cumulative ef fect on v i a b i l i t y is temperature/dependent. Of the 15 Dts le tha ls mapped gene t i ca l l y , only four d i s t i n c t s i t es on chromosome 2 were found. The paucity of d i f ferent loc i occupied by the mutants resul ts from the unexpected c lus ter ing of 11 le tha ls wi thin a s ingle region. Functional a l l e l i s m of the genet ica l ly clustered mutants was demonstrated by the fa i l u re of trans heterozygote combinations of d i f ferent mutants to survive at 19 room t e m p e r a t u r e . F u r t h e r p r o o f o f t h e r e l a t e d n e s s o f t h e m u t a n t s comes f r o m t h e o b s e r v a t i o n t h a t t h e T S P s o f a l l e l e v e n c l u s t e r l e t h a l s w e r e i d e n t i c a l . T h u s , g e n e t i c , f u n c t i o n a l a n d d e v e l o p m e n t a l s t a g e s p e c i f i c i t y c r i t e r i a a l l p o i n t t o t h e a l l e i i s m o f t h e c l u s t e r e d D t s l e t h a l s . . The r e c o v e r y o f a l l e l e s i n t h e c l u s t e r f r o m b o t h O r e g o n - R a n d S a m a r k a n d s t r a i n s p r e c l u d e s a n y u n u s u a l l o c u s ' s e n s i t i v i t y p e c u l i a r t o a g e n e t i c s t o c k . T h a t t h e a l l e l e s r e p r e s e n t d i f f e r e n t c h a n g e s w i t h i n t h e l o c u s m i g h t b e e x p e c t e d f r o m t h e i r i n d e p e n d e n t i n d u c t i o n a n d r e c o v e r y . M o r e o v e r , t h e L P s v a r y w i t h e a c h m u t a n t . The c o i n c i d e n c e o f t h e LP a n d TSP o f m u t a n t D t s - L 7 s u g g e s t s t h a t o t h e r m u t a n t s w i t h L P s a t l a t e r t i m e s a r e l e a k y ( h y p o m o r p h s ) . We h a v e f o u n d t h a t t r a n s h e t e r o z y g o t e s f o r d i f f e r e n t a l l e l e s w i t h i n t h e l o c u s s u r v i v e a n d . a r e f e r t i l e when r a i s e d a t 17°C, t h u s p e r m i t t i n g a means o f d e t e r m i n i n g w h e t h e r r e c o m b i n a t i o n b e t w e e n a l l e l e s may o c c u r . The f r e q u e n c y o f r e c o v e r y o f D t s l e t h a l s i n d i f f e r e n t g e n e t i c l o c i i s ( .065%), w h i c h d i f f e r s b y 2 o r d e r s o f m a g n i t u d e f r o m t h e ( r e c o v e r y o f r e c e s s i v e t s m u t a n t s ( 10 .5 - 12%) ( S u z u k i e_t a_l_., 1967) . The r a t i o o f r e c e s s i v e t o d o m i n a n t l e t h a l s i n d u c e d by X - r a y s i n y e a s t was 15:1 ( M o r t i m e r , 1955) , w h e r e a s H a r t w e l l ( 1 9 6 7 ) f o u n d t h a t h o u t o f M-00 t s m u t a n t s i s o l a t e d i n h a p l o i d y e a s t b e h a v e d a s d o m i n a n t l e t h a l s . The u n e x p e c t e d r e c o v e r y o f 11 D t s l e t h a l s i n a s i n g l e l o c u s d e m o n s t r a t e s p r e f e r e n t i a l s u s c e p t i b i l i t y o f c e r t a i n chromosome r e g i o n s t o m u t a t i o n . S e x - l i n k e d r e c e s s i v e l e t h a l s i n d u c e d by EMS and m i t o m y c i n C a l s o t e n d t o be g e n e t i c a l l y c l u s t e r e d ( S u z u k i , I 9 6 8 ) . C a r l s o n ( I962) h a s shown t h a t t h e dumpy l o c u s i s h i g h l y s e n s i t i v e t o 20 mutation, and i t may be of importance that the c lus ter Dts le tha ls are c lose ly l inked to dp. Two Dts l e tha l s , Dts-L9 and Dts-L20, which were not mapped genet ica l ly but may represent d i f fe rent l o c i , behaved as female s t e r i l e s at RT. The sexual dimorphism for f e r t i l i t y under permissive condit ions is of in te res t . Dts-L20 exh ib i ts abnormal ch i t i n formation in both males and females and th is phenotypic abnormality is probably associated with the i n f e r t i l i t y of the females. However, the nature of the s t e r i 1 i t y , whether the resul t of a st ructura l defect or abnormal physio logical process such as a disturbance in a meiot ic event, merits further examination. Temperature-sensit iv i ty of point mutants in microorganisms has been shown to be the resul t of s ingle amino acid subst i tu t ions which cause thermolabi1 i ty of a protein (Epstein e_t a_l_, 1963; Edgar and- L i e l a u s i s , \S6k; Nishihara and Romig, 1967) - Bernstein 1(1968) Q^ L t - , has also found ts dominant le tha ls in Tk phages which appear to have a s imi la r bas i s . The propert ies of sex- l inked recessive ts le tha ls in Drosophila conform to the expectations of such a mechanism. If temperature 'sensi t iv i ty of dominant le tha ls resul t from single amino — / acid subs t i tu t ions , then the molecular nature of dominant l e t ha l i t y may be the resul t of : (a) a threshold requirement of gene products for v i a b i l i t y in which the c r i t i c a l level is not reached in a nucleus heterozygous for a mutant a l l e l e ; (Welshons and von Ha l l e , 1962) . 21 (b) loss of b io log ica l a c t i v i t y of an enzyme due to the polymerization of mutant and wi ld type monomers, which impedes the a c t i v i t y of the ent i re protein multimer; (Sundaram and Fincham, 1967). (c) incorporation of defect ive polypeptides spec i f ied by the mutant a l l e l e into a c e l l u l a r structure which renders that structure defect ive at r e s t r i c t i v e temperatures; (Bernstein and F isher , 1968). (d) an abnormal repressor with al tered binding proper t ies ; (Jacob and Monod, 1961). The TSP of a Dts-L defines the period at which a spec i f i c b io log ica l process regulated by that locus is thermojlabi 1e. One K c lass of such processes is t ranscr ip t ion and t rans la t i on , which are essent ia l steps for the formation of b i o l o g i c a l l y ac t ive constituents of the c e l l . However, i f these processes were made thermolabi le, one would not expect a f i n i t e TSP or LP during otogeny. Ca ta l y t i c reactions in metabolytic pathways required at spec i f i c developmental stages, on the other hand, might demonstrate a f i n i t e TSP and LP. Thermolabi1 i ty of an enzyme would prevent the u t i l i z a t i o n of the gene product and the phenotypic ef fect would u l t imate ly manifest i t s e l f in the LP. Dominant temperature-sensit ive l e tha l s , which permit maintenance of the mutant under permissive condit ions through progeny, enable the 22 character izat ion of cer ta in loc i that were not previously amenable to genetic and developmental ana lys i s . One c lass of mutants which " V - ' J. may now be analyzed are those involved in v i t a l functions such as A-*"1.-. eel 1 d i v i s i on or oxidat ive metabolism. These genetic loc i may be redundant and therefore only dominants would be detectable. Since a dominant mutation in such v i t a l loc i is probably l e t h a l , a Dts lethal provides a tool for the further maintenance and character izat ion of such a mutation. 23 SUMMARY EMS'\nduced dominant temperature-sensit ive lethal and / semi lethal mutations (Dts-L) were recovered in chromosome 2 in Drosophi1 a melanogaster. Twenty-one of the mutants (0.34% of chromosomes tested) had a v i a b i l i t y greater than 50% at 22± 2°C and less than 5%.at 29- 0.5°C Of the 16 Dts-L loca l ized gene t i ca l l y , 15 mapped as point mutants. The e f fec t i ve lethal phases and temperature-sensit ive periods were determined for 16 of the mutants. Eleven Dts-Ls were t i gh t l y l inked to the dumpy locus and found to be funct iona l ly a l l e l i c . TABLE I. Relat ive v i a b i l i t y at 17°C, RT and 29°C and genetic pos i t ion of a l l confirmed dominant temperature-sensit ive l e tha l s . Dts- L Stock of o r i g i n Locat ion 17° % Viabi1 i ty (%) re la t i ve to wi ld type C #* 23°C % (RT). # 29°C % Control (0regon-R369) 0.67* 477 0.67 493 0.64 385 Control (Samarkand) - - 0.65+ 209 0.62 238 0.68 177 1 Oregon-R c - px 90.6 344 73.9 93 1 .5 104 2 11 d£ - b 97.5 509 100.0 353 0.0 138 3 11 d£ - b 98.3 433 94.9 159 0.0 151 4 11 d£ - b 101 .0 527 93.8 156 4.5 103 5 11 d£ - b 101.5 427 97.7 465 0.0 180 6 Samarkand b - pr 105.0 493 96.4 702 0.0 156 7 11 djp_ - b 90.4 187 105.5 447 2.1 65 8 11 ambi guous 93.6 266 88.0 311 1.3 107 9 11 s t e r i l e 67.0 364 93.1 683 0.0 156 10 11 d£ - b 94.7 764 107.7 781 0.0 140 11 11 d£ - b 96.7 529 100.0 690 2.7 161 12 11 d£ - b 95.6 548 103.2 1723 1.7 244 13 11 d£ - b 97.3 535 107.2 1750 0.50 276 14 11 dp_ - b 93.0 390 101.7 850 0.0 114 15 11 c - px 74.1 392 68.2 575 2.1 130 16 11 d£ - b 100.1 477 104.2 527 1 .7 86 17 11 - - - - • - -18 11 - 95.8 565 110.2 453 2.9 149 19 11 pr - £ 98.3 624 107.4 605 3.9 149 20 11 ster i1e 89.2 293 105.5 711 0.0 I69 21 11 -. 74.6 317 51.5 236 0.0 112 * Total progeny scored t- Rat io of + -bearing progeny to total o f fspr ing TABLE I I. Complementation tests temperature-sens i t i ve of dominant le tha ls at RT. 25 Dts - L 3 2 9 20 1 6 19 15 Cluster Dts-Ls: 3 0.0 0.0 0.01 *256 232 316 2 0.6 0.0 _ 232 201 5 0.0 0.0 _ 125 334 4 0.0 0.04 _ 212 236 16 0.0 0.13 480 381 13 0.0 0.0 17^  224 14 0.0 0.0 _ 302 334 12 0.0 0.0 250 198 11 0.0 0.05 _ 193 248 10 0.0 0.0 _ 148 344 7 0.0 0.02 _ 93 183 2 ster i1es 9 0.01 - -316 20 0.45 • _ _ 455 other 1 0.32 0.33 0.45 192 300 162 6 0.28 0.40 0.37 200 218 339 17 0.32 0.41 0.40 439 434 423 15 0..44 - 0.25 291 401 0.45 0.32 0.28 0.32 0.44 455 192 200 437 291 _ 0.33 0.40 0.41 0.26 300 318 434 356 - 0.45 0.37 0.40 0.25 162 339 423 401 _ 0.48 0.06 0.25 0.30 313 271 336 413 0.48 0.0 0. 25 _ 0.0 313 251 298 348 0.06 0.25 0.0 - 0.11 271 298 172 330 0.25 - - 0.0 -336 201 0.30 0.0 0.11 - 0.0 413 348 330 260 * number of progeny scored in each cross TABLE I I I . Crossover resul ts in genetic l oca l i za t i on of dominant temperature-sensit ive l e t ha l s . Classes Control (Oregon-f^fcg) 1 1 Dts 10 • L 16 13 14 15 19 parental Cy. " a l l " 1500 52 wi Id 171 reci proca 1 crossovers in Dts-•L region region 1 al + 47 + dp 8 reg i on 2 dp +• 85 + b 108 regi on 3 b + 55 + pr regi on 4 pr + 124 + c 109 reg ion 5 c + 65 + px 127 region 6 px + 9 •+ sp 20 2360 2660 1560 1840 670 770 850 870 1070 930 900 570 1250 1260 900 4 13 4 6 2 3 1 6 1 2 4 10 4 22 5 4 1 13 0 0 1 0 17 5 3 1 17 0 8 7 114 0 29 45 22 28 20 23 8 13 18 2 39 3 70 5 29 16 33 14 84 7 44 6 14 106 7 20 17 24 7 50 TABLE IV. Determination of the e f fec t ive lethal phase at RT of homozygous c lus ter mutants. Dts-L Number of Eggs Hatchabi1i ty Control (Oregoh-Rj^g and Samarkand) 1049 0 . 62 2 961 0.50 3 1032 0.39 4 801 0.43 5 448 0.46 7 419 0.30 10 468 0.42 11 900 0.43 12 451 0.47 13 535 0.40 14 280 0.39 16 378 0.39 Screening protocol for the detect ion of dominant temperature-sensit ive lethal mutations in chromosome 2. +/+ d (0.025 EMS) x Cy/Pm? RT Cy/+* d score for presence of +*-bearing f l i e s Cy/Pm ? RT for h days then transfer parents to a fresh v ia l score for absence of +* -bearing f l i e s repeat steps 2 and 3. repeat cross 2 at 17 C. The rat ionale for genetic l oca l i za t i on of dominant temperature-sensit ive le tha ls by reciprocal crossing over. Protocol for reciprocal " s h i f t " experiments to del ineate the temperature-sensit ive period and e f fec t i ve lethal phase of dominant temperature-sensit ive l e tha l s . m 29°C *- RT ^1 7 L P 7 -O Adults —X .0 1 2 T I M E 3 4 5 O F S H I F T IN (CORRECTED TO RT) 6 7 D A Y S 8 O Genetic pos i t i on , temperature-sensit ive period (TSP) and e f fec t i ve lethal phase (LP) of each dominant temperature-sensit ive l e t h a l . 32 LITERATURE CITED Bernste in, H. and K. F isher , 1963 . Dominance in bacteriophage T^D. Genetics 58: 307-318. Br idges, C B . and K.S. Brehme, 1944 . The mutants of Drosophi1 a  melanogaster. Carnegie Inst. V/ash. Publ . 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