Biology of a Duplicate Gene System with Glucose

8/7/2019 Biology of a Duplicate Gene System with Glucose

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Biochemical Genetics, Vol. 14, Nos. 9[10, 1976

Biology of a Duplicate Gene System with Glucose6-Phosphate Dehydrogenase Activity inDrosophila melanogaster: Genetic Analysis andDifferences in Fitness Components and Reactionto Environmental Parameters Among Zw GenotypesJ a m e s T . G i e s el IReceived 27 May 1975--Final 5 Apr. 1976

There are two structural fo rm s o f glucose 6-phosphate dehydrogenase activityin Drosoph i l a melanogas te r . Whether one or the other or both show in v i t ro(and probably in v ivo) activity depends on the genotype o f a sex-lin ked locus(Zw). In this article, the relative fitnesses o f heterozygotes (with both electro-morphs active) and homozyg otes (with activity demonstrable fo r only one or theother electromorph) fo r the Z w locus are described. It is shown that the relativefitn ess o f heterozygo tes increases with increase in population density, or degreeo f crowding and trophic stress, an d that the mean development times o f Z wheterozygotes are lower than those of the Z w homozygotes. In addition, andperh aps accounting fo r the fitne ss a nd viability excess o f the heterozygotes, oneset of evidence strongly suggests that they are better buffered against trophicstress than the homozygotes.KEY WORDS : fitness; Zw genotypes; genetics.

I N T R O D U C T I O NDupl ica te gene sys tems a re qu i t e common in po ik i lo therms , where the i rf itness value is tho ugh t to be re la ted to the abi l i ty o f thei r possessors toacc l imate and main ta in near -o p t im um leve ls o f metabo l i c func t ion in sp it e o fchanges in phys ica l pa ram ete r s o f the env i ronment . Fo r example , the re appea rto be tw o dis t inct gene-enzymes wi th ace tylchol inesterase activ i ty in the s teel-t Department of Zoology, University of Florida, Gainesville, Florida.

823 I976 Plenum Publishi ng Corpor ation , 227 West 17th Street, New York, N.Y. 1001 I. No p art o f this publica-tion may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic,mechanical, photocopyi ng, microfilming, recording, or otherwise, wi thout written permission of the publisher


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824 Giesel

head t rou t . One o f the se , w i th a h igh tempe ra tu re op t im um , is thou gh t to beinduced o r a c t iva ted wh en the fi sh en te r wa rm wa te r ; the o the r , w i th a lowerop t imum tempe ra tu re , r ep lace s the h igh- tempe ra tu re gene -enzyme in co ldenv i ronments ( see Som ero and H och achk a , 1973). I t appea r s tha t a ll m emb ersof the popu la t ion have th i s ab i l i ty to swi tch f rom one gene -enzyme fo rm tothe o the r . The phenomenon i s no t l im i ted to th i s enzyme o r spec ie s bu tappea r s to be qu i te gene ra l among po ik i lo the rms .

In D ro so p h i l a m e l a n o g a s t e r the re ex i s t two d i f fe ren t fo rm s o f g lucose6-phosph a te dehydrogenase . One o f the se migra tes s lowly up on e lect ro -phores is , has a mo lecula r w eight of 317,000, a re la tive ly low Km G6 P, a nd are la t ive ly h igh K , , NADP. The o the r migra te s much fa s te r , ha s a molecu la rweigh t of 150,000, a h igher Km G6 P, an d a low er / , , N A D P (Steel et al . , 1968;bu t s ee a l so Ko m m a , 1968) . These two fo rm s a re the a ll eli c p roduc ts o f Z w ,an X- l inked gene . Z w has been de f ined a s the s t ruc tu ra l gene fo r G6PDac t iv i ty (Young et al . , 1964) . Ho wev e r , when e lec trophore s i s is c a r r ied ou tfo r a longe r t ime than u sua l and und e r a va r ie ty o f cond i t ions , a ll de signedto minimize subuni t d issoc ia t ion (see Stee l et al . , 1968) , one f inds tha t bothfo rm s segrega te in the i r ow n r igh t fo r s eve ra l e lec t romo rphs .

I t i s the purpo se o f th is a r t ic le to p re sen t gene t ic da ta w hich dem ons t ra tetha t the s low- and fa s t -migra ting fo rms o f G 6P D a re the p rod uc ts o f d i f fe ren t(dupl ica te ) s t ruc tura l genes and tha t Z w mu s t be a modi f ie r locus which ac t sto de te rm ine the r e la t ive concen t ra t ions o f the two s t ruc tu ra l gene p roduc ts .In addi t ion da ta wil l be presented which speak to the re la t ive f i tness of Z whom ozyg o te s and he te rozygo te s and p oss ib le r ea sons fo r the r e su l t s ob ta inedwill be discussed.

M A T E R I A L S A N D M E T H O D SG e n e t i c I n f e r e n c e s

In o rde r to inves t iga te the gene tic s o f th is sys tem, a map p ing expe r imen t w asdone on the s low fo rm (G6PDs) , s eg rega t ion ana lys i s was done on the f a s tfo rm (G 6P D 0, and gene t ic d i st inc tnes s o f the loci was inves t igated by us ingove r 100 f li es to f ind whe the r e lec t romorph ic phe no typ es o f the tw o loc i a rei n d e p en d e n t . M a p p i n g o f G 6 P D s w a s d o n e b y c ro s si n g h - , r i - , es - , I D H s,A O ~ , G 6 P D ~ with Oregon- r (h + , r i + , e ~+, I D H ~, A O y , G6PDZ~), backc ros s ingthe i r p roge ny to h - , r i - , e ~ - , I D H ~, A O ~, G 6 P D ] , and ana lyzing game te s fo rr e c o m b i n a t i o n f r e q u e n c y b e t w e e n t h e k n o w n m a r k e r l o c i a n d G 6 P D ~ .S e g r eg a ti o n a n a ly s is w a s d o n e o n G 6 P D f b y c ro s si n g p u t a ti v e ly h o m o z y g o u sfemales (1 /1) wi th puta t ive ly he te rozygous males (1 /3) and inves t iga t ing theprog eny geno type f r equenc ie s fo r f it to M ende l ian expec ta t ion b y X~ dr.


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Biology of Dupl icate Ge ne System 825

Gen e t ic independ ence o f the loc i was inves t iga ted by Z2 ana lys is o f over 100f l i e s t aken f rom a cage popu la t ion in which bo th we re segrega t ing fo r twoalleles.

In the mapp ing and segrega t ion ana lys i s expe r imen ts , e le c t rophore s i swas don e on 11% s ta rch ge ls m ade w ith 0.025 M his tidine-HC1 (p H 7.5) towhich 0 .005 M T P N had been added . E lec t rophore s is was ca r r ied o u t in a0 .47 M Na -c i t r a te (p H 8 .0 ) b r idge bu f fe r fo r 18 h r a t 25 m A/ge l a t 5 C . G e lswere sp l i t and s ta ined fo r G6PD, i soc i t r a te dehydrogenase , and a ldehydeoxidase fo l lowing the methods expla ined by Brewer (1970) . The f l ies used inthe independence ana lysi s we re t r e a ted a s exp la ined be low.

F i t n e s s A n a l y s i sTo col lec t the d a ta presen ted here , indiv idua l f lies were hom oge nized in 1 .5 /~1o f a 2 0 % s o l u ti o n o f su c r o se in 0 .1 M T B E D T A ( p H 8 .6 ). T h e s a m p l es w e r ecentr i fuged a t h igh speed in a Misco re f r igera ted centr i fuge , a f te r whichsupe rna tan t s we re loa ded in to the pock e ts o f ve r ti c a l 5 ~ po lyac ry lam ide gelsm a d e w i t h 0.3 M T B E D T A ( p H 8 .6 ) t o w h i c h T P N h a d b e e n a d d e d . T h e g el swe re e lec t ropho re sed in the s ame b r idge a t 300 V fo r 5 h r. Phen o types we rere so lved by the s ta in ing m e tho d o f Brewer (1970) fo r G 6P D in Drosophila.G 6 P D s m i g r at e d u n d e r t h es e c o n d it i o n s a t a b o u t h a l f th e r a t e o f G 6 P D f (s eeF ig . 1 ), bu t bo th fo rm s s ta ined wi th the same t ime cons tan t . Pheno typ es we reread a f te r 45 ra in o f s tain ing a t room temp e ra tu re ( abo u t 25 C) . Bo th fo rmsw e r e p o l y m o r p h i c .

The da ta p re sen ted he re a re f ro m th ree se ts o f expe r imen ts, o n ly the l a sto f wh ich wa s des igned spec if ical ly to inves t iga te d if fe rences be tw een geno type so f t h e Z w locus in the com po nen ts of fi tness as re la ted to leve ls of temp era tu rea n d f o o d .

In the f ir s t expe r imen t , th ree l a rge cage pop u la t ions we re kep t a t cons tan tfood and tempe ra tu re and censused a t month ly in te rva l s and s imul taneous lya s sayed fo r geno types o f s eve ra l gene -enzymes , inc lud ing the tw o G 6P D loc i.Us ing the se da ta , r e la tionsh ips be tw een pop u la t ion dens i ty and the a lle lea n d g e n o t y p e f r e q u e nc i es o f th e Zw locus could be s ta t is t ica l ly inves t iga ted .

In the second se t o f expe rimen ts , aga in d one fo r ano th e r pu rpose , f li eswere co l le cted f rom a l a rge , long-s tand ing cage popu la t io n a s eggs by a l lowingthe popu la t ion ' s f ema le s to ov ipos i t on f r e sh cups o f m ed ium fo r 1 h r .Sepa ra te s ample s we re co l le c ted sequen t ia l ly on Ca ro l ina Bio log ica l Supp lyins tan t Drosophila m e d i u m ( C B S I D M ) , C B S I D M d i lu t ed 5 0 ~ b y w ei gh t w i thFu l le r ' s e a r th (which Thro ckm or ton , p e r sona l com mu nica t ion , be l ieves i si n g e s t e d b u t n o t m e t a b o l i z e d ) , a n d C B S I D M f o r t i f i e d w i t h g l u c o s e - - 5 % b yweight . The f l ies were a l lowed to deve lop and la te r were ana lyzed for Z wg e n o t y p e . E a c h s e t o f d a t a w a s in v e s ti g a te d f o r d e v i at io n s f r o m H a r d y -


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826 Giesel

Fig. 1. Electropherogram showing som e variants of G 6PDgene-enzymes.Colum ns 1, 2, and 9-12 are Zw a homozygotes;the flies in columns 3-8 are Zw ~ homozygotes. The loci are asfollows: slow, G6PDs; fast = G6PDf.

Weinberg expec t ion by compar ing observed wi th expec ted geno type f r e -quencies by 7~ dr.Th e th i rd se t of exper iments was in tended to pro vide data on the develop-ment t imes , average ages a t r ep roduc t ion , and mea n num ber s o f eggs perday per f emale o f Zw he te rozygo tes and homo zygo tes when r a ised under theenvironm ental co ndi t ions presented in Table I . F or th is purpose , l ines of f liesh o m o z y g o u s f o r t h e Z w allele giving onl y G6PD ~ activi ty (Z w BB) and fo r theZ w allele giving only G 6P D f activi ty (Zw AA) were isolated. Th ese were thenoutc ross ed to give 15 non identic al F~ l ines of each Zw geno type . These matedpairs of f lies were al low ed to ovipo sit for nine successive 8-hr periods. Ea ch


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828 GieselTable II . Results of Mapping E xperiments of G6PD~"

ri G6PD l ' l 106 ID H s's G6P D 1'1 69ri G6P D 1'2 49 ID H ~'s G6P D 1'2 35+ G6PD 1'I 55 ID H s'e G6 PD 1'I 30+ G6P D 1'2 114 ID H ~'f G6P D 1'2 83Percent crossover = 0.321 +0 .0 53 Percent crossover = 0.29+0.062Z~ as = 41.2 4 Z~ df = 36.92

"AO-G6PD percent crossover indistinguishable from 50Y o; eS-G6PDpercent crossover indistinguishable from 50% .

I n t h e t e s t f o r g e n e t ic in d e p e n d e n c e , 1 04 f li es t a k e n f r o m a c a g e p o p u l a -t i o n w e r e s c o r e d f o r g e n o t y p e s o f t h e l o c i . T h e d a t a w e r e o r g a n i z e d i n t o a3 x 3 m a t r i x o f g e n o t y p e s . T h i s r e v e a l e d t h a t a ll n i n e p o s s ib l e g e n o t y p e s w e r ep r e s e n t. M o r e o v e r , w h e n g a m e t e f r eq u e n c i e s w e r e t a k e n i t w a s f o u n d t h a tc o u p l i n g g a m e t e s w e r e s i g n if i ca n t ly in e x c e ss o f r e p u l s i o n g a m e t e s (X ] af =1 7.5 8), w h i c h s u g g e s ts t h a t t h e l o c i m a y b e l i n k e d a n d w e r e i n t h i s c a s e i n as t a t e o f l i n k a g e d i s e q u i l ib r i u m . S p e c if ic d a t a o n t h e e x a c t l i n k a g e r e l a t i o n s h i p so f t h e t w o l o c i w i l l b e p u b l i s h e d e l s ew h e r e .

T h u s i t b e c o m e s n e c e s s a r y t o r e d e f in e t h e f u n c t i o n o f th e Z w l o c u s . T h i sl o c u s a c ts t o d e t e r m i n e t h e in vitro, a n d p r o b a b l y in vivo, r e l a t ive a c t iv i t i e s o f

i

t h e G 6 P D d u p l i c a t e g e n e - e n z y m e s . Z w h e t e r o z y g o u s f e m a l e s s h o w r o u g h l ye q u a l a c t i v i t y f o r t h e s e e n z y m e s , w h i l e Z w h o m o z y g o t e s a n d h e m i z y g o u sm a l e s d i s pl a y, u p o n e l e ct r o p h o re s i s, p r im a r i l y t h e a p p r o p r i a t e g e n e - e n z y m e(Z w bb i s p r i m a r i l y a c t i v e f o r G 6 P D s , Z w a" f o r G 6 P D f ) . H o w e v e r , b o t h Z wh o m o z y g o t e s c a n , w i t h i n b a s i c li m i ts s e t b y t h e Z w g e n o t y p e , e x h i b i t a c t i v i t yf o r b o t h s t r u c t u r a l g e n e -e n z y m e s . W o r k d e s ig n e d t o . d e fi n e t h e r e a s o n s f o rt h i s v a r i a b i li t y o f e x p r e s s i o n o f Z w g e n o t y p e s i s in p r o g r e s s .

C o m p o n e n t s - o f - F i t n e s s A n a l y s i sD a t a f r o m t h e f i r s t e x p e r i m e n t r e v e a l e d , u p o n i n s p e c t i o n , t h a t t h e h e t e r o -z y g o t e g e n o t y p e o f t h e Zw l o c u s w a s a l w a y s in e x c es s o f H a r d y - W e i n b e r ge x p e c t a t i o n i n t h e c a g e p o p u l a t i o n s - - e v e n t h o u g h a l l e l e f r e q u e n c i e s w e r er e l a ti v e l y c o n s t a n t a t abou tp Zw ~ = 0 . 4 5- 0 .5 2 . T h e d a t a w e r e t h e n e x a m i n e dg r a p h i c a l l y t o d e t e c t e v i d e n c e o f a s s o c i a t i o n b e t w e e n e x t e n t o f h e t e r o z y . g o t ee x ce s s a n d p o p u l a t i o n r a t e o f i n c re a s e f o r t h e c o r r e s p o n d i n g p e r i o d . A p o s i ti v ec o r r e l a t i o n w a s r e v e a l e d b e t w e e n e x t e n t o f ex c es s a n d p o p u l a t i o n d e n s i ty f o rt h e e i g h t c a s e s i n w h i c h s a m p l e s i z e s w e r e l a r g e a n d t h e n o n r a n d o m f r e -q u e n c ie s o f g e n o t y p e s c o u l d b e c o n f i r m e d b y X2 ( F ig . 2 ) . Th i s sugge s t s tha t( s o m e c o m p o n e n t o f ) h e t e r o z y g o t e r e la t iv e fi tn e ss m u s t i n c re a s e w i t h p o p u l a -t i o n d e n s i t y , o r , s i n c e i n t h e s e e x p e r i m e n t s f o o d l e v el w a s c o n s t a n t w h i l e


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Biology of Duplicate Gene System 829

4 0

,m 3o.~XLidLUo~

2o

t.Ol - -OdI I 0 -

I I I i I

0 0

D

o ~o .'oo ; ~; ioPOPULATION 51ZE (THOUSANDS}Fig . 2 , Associa t ions be tween popula t ion dens i ty andZw heterozygote excess (observed f requency minusexpected f requency).

population density varied, with degree of crowding or competit ion foravailable food resources (see Discussion for another possible reason for theheterozygote excesses).

The second set of data were analyzed by comparing observed and ex-pected frequencies o f the three Z w genotypes of flies taken as egg samples atthe same time from the base population and raised at 25 C with food main-tained at high, normal, and low levels. The results of these com pariso ns a represented in Table III . The only significant deviation from expected frequen-cies is found in the sample raised under the most trophically deprived condi-tions. Th e 2 6 ~ excess of hete rozy gote s is highly significant. Since the fliesused w ere derived from egg samples, this hetero zygote excess must have be endue to the larval viabilities of the heterozyg otes being higher than those o f

Tab le I II . F r equenc i e s Zw Pheno typ e o f F li e s Taken f rom the SameBase Popu la t i on bu t Deve loped on Low, Norma l , o r Fo r t i f i ed Food

P h e n o t y p eCo ndi t io n ss s f f f Heterozyg ote excess

H F 0 .13 0 .46 0 .4 Equ i l ibr iumM F NO 30 87 63 0 .08 (N.S.)N E 38 73 70LF NO 29 148 16NE 56 96 41 .3 0 .2 6;p < 0 .05


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830 Giesele i t h e r h o m o z y g o t e cl as s . A g a i n , t h e r e s e e m s t o b e a d i r e c t r e l a t i o n s h i p b e t w e e n( a c o m p o n e n t o f ) h e t e r o z y g o t e r el a ti v e fi tn e ss a n d e x t e n t o f t r o p h i c d e p r i v a -t ion ( se e Disc uss ion ) .

T h e t h i r d s e t o f e x p e r i m e n t s w a s d e s i g n e d s p e c i fi c al l y t o i n v e s t i g a t eg e n o t y p i c p e r f o r m a n c e s i n t e rm s o f d e v e l o p m e n t t im e , a v e r a g e a g e a t r e p r o -d u c t i o n , a n d a v e r a g e n u m b e r o f eg g s l a id p e r f e m a l e p e r d a y i n e n v i r o n m e n t sw h i c h d i ff e re d i n f o o d q u a l i t y a n d ( c o n s t a n t ) t e m p e r a t u r e .

D e v e l o p m e n t T i m eT h e d a t a w e r e fi rs t a n a l y z e d b y t h r e e - w a y a n a ly s is o f v a r ia n c e t o d e t e r m i n et h e r e l at i v e i m p o r t a n c e o f th e i n d e p e n d e n t v a r i a b l e s - - f o o d l e v el , t e m p e r a t u r e ,a n d g e n o t y p e - - a n d t h e i r i n t e r a c ti o n s in e x p l a in i n g th e v a r i a n c e o f th e d e p e n -d e n t v a r i a b l e - - e g g t o a d u l t d e v e l o p m e n t ti m e . R e s u l ts o f t h is a n a l y si s a r ep r e s e n t e d i n T a b l e I V . H e r e w e s e e t h a t a l l t h r e e i n d e p e n d e n t v a r i ab l e s a r ei m p o r t a n t i n t he e x p l a n a t i o n o f s a m p le v a r i a n c e in d e v e l o p m e n t t im e a n d t h a ts i g n i f i c a n t i n t e r a c t i o n s e x i s t b e t w e e n t h e t w o e n v i r o n m e n t a l v a r i a b l e s - -t e m p e r a t u r e a n d g e n o t y p e - - a n d t h e e n v i ro n m e n t a l v a ri a bl es a n d g e n o t y p eo f t h e Zw l o c u s . A s e x p e c t e d f o r a p o i k i l o t h e r m , f o o d l e v e l i s r e l a t i v e l yu n i m p o r t a n t b y i t se l f b u t i n t e r a c t s s t r o n g l y w i t h t e m p e r a t u r e . S i n ce it is o fm a j o r i n t e re s t t o t h is s t u d y t o d e fi n e t h e r e a c t i o n s o f t h e h e t e r o z y g o t e s vs .t h o s e o f t h e h o m o z y g o t e s t o t h e e n v i r o n m e n t v a r ia b l e l ev e ls , m e a n d e v e l o p -m e n t t i m e s o f th e t w o b r o a d g e n o t y p i c c l a ss e s -- -h e t e ro z y g o t e s a n d h o m o z y -g o r e s - - w e r e c a l c u l a te d f o r t h e t h r ee d i f f er e n t t e m p e r a t u r e s a n d f o r t h e f o o dl e v e l s . I n T a b l e V w e s e e t h a t a t l o w t e m p e r a t u r e , w h i c h , f o r a p o i k i l o t h e r m ,

Table IV. Results o f the T hree-W ay Analysis of V ariance of Fo od Level,ZwGeno type, and T emperature and Their Interaction with Respect to E gg-Ad ultDevelopment TimeaComp onent of variance ss ~s s F val ue

Fo od level 17,945 0.49 10.27dTem perature 649,711 17.8 248aGe notyp e 7,284 0.2 2.78bFo od level x temperature 19,838 0.54 3.78cFo od level genotype 4,211 0.1 N.S.Tem perature genotype 20,610 0.56 2.26bFo od level temperature genotype 43,194 1.2 2.91 bTotal 3,667,947a Sample siz e 1245.bp


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Biology of Du plicate Gen e System 831Ta b le V . Me a n He te r o z y g o te a n d Ho m o z y g o te De v e lo p m e n tT ime a t 2 5 , 2 8 , a n d 1 9 C a n d Hig h , Me d iu m, a n d Lo wF o o d L e v el s

G e n o t y p eHo m ozyg otes s ign if icance Hete rozyg otesAt 19 CL F 301 P < 0 .01 286M F 289 N.S . 283H F 2 64 N .S . 2 69At 2 5 CH F 253 N.S . 247M F 2 70 N .S . 2 30L F 269 N.S. 271At 2 8 CH F 2 10 P < 0 . 0 1 185M F 216 P < 0.05 205L F 225 P


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8 3 2 G i e s e l

average ages at reproduction were calculated and then collected over thetreatments in which all gentotypes were represented, to form genotypic meansand variances over all experimental treatments. Results of this treatment ofthe data are presented in Table VI. They are remarkably clear. There are nogenotypic differences in either average egg production per day or averageage of reproduction. However, genotypic differences in variance of these meanvalues engendered by experimental conditions are striking and highly signi-ficant (F ratio, P


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Biology o f Duplicate Gene System 833Somero , G. H. , and Hochachka, P . W. (1973) . Strategies of Biochemical Adaptation,Saunders, Philaddphia.Steel, M . W ., Y oun g, W. J., an d Childs, B. (1968). G ene tic regulation of glucose 6-phosphatedehydrogenase in Drosophila melanogaster: Starch gel electrophoretic variat ion d ue tomolecular instability. Biochem. Genet. 2:159.Young, W. J., Porter, J. E., and Childs, B. (1964). Glucose 6-phosphate dehydrogenase inDrosophila: X-linked electrophoretic variants. Science 143:140.

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Biology of a Duplicate Gene System with Glucose