synergistic effect of adh alleles in drosophila melanogaster

Synergistic Effect of Adh Alleles in Drosophila melanogasterAuthor(s): Xinmin LiSource: Proceedings: Biological Sciences, Vol. 247, No. 1318 (Jan. 22, 1992), pp. 9-16Published by: The Royal SocietyStable URL: http://www.jstor.org/stable/49766 .

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Synergistic effect of Adh alleles in Drosophila melanogaster

XINMIN LIt Department of Genetics, Queen's Medical Centre, University of Nottingham NG7 2UH, U.K.

SUMMARY

In laboratory cultures of Drosophila melanogaster derived from an African population, the quantities of six out of seven enzymes (G6PD, IDH, GPDH, ME, MDH, PGM and ADH) were higher in Adh-FF homozygotes than they were in Adh-SS. In crosses between Adh-FF and Adh-SS flies, the differences segregated as co-dominant alleles of a single Mendelian gene closely linked, or identical, to the Adh locus. The generality of these associations was suggested by the study of a French population with a very different history and genetic background. The possibility that the associations were caused by artefacts of the immunodiffusion techniques, or to a linked inversion (In(2L) t), was excluded. Possible ways by which the Adh locus may affect the quantities of other enzymes are discussed.

1. INTRODUCTION

Early in the history of genetics it was found that a single gene can affect many different characters. Genes with multiple phenotypic consequences have been called pleiotropic. It has now become evident that pleiotropy is 'virtually universal' (Wright 1968). Pleiotropic gene action has been found in all organisms from viruses (Welham & Wyke 1988) to bacteria (Dias et al. 1986; Klug & Cohen 1988; Van Dyk et al. 1987; Yang et al. 1986), and from plants (Estelle & Somerville 1987; Epperson & Clegg 1988; Gottlieb et al. 1988) to animals (Allendorf 1983; Hodson et al. 1983; Jinks et al. 1985). In Drosophila melanogaster Morgan et al. (1915) mentioned that a mutation of a single gene can have multiple effects on the phenotype. The phenomenon has now proved to be widespread. The alcohol dehydrogenase (Adh) locus is one of the best studied loci in Drosophila melanogaster. Natural populations generally contain two common electrophoretic alleles: Adh-Fast (F) and Adh-Slow (S). Amino acid sequencing has shown that Adh-F differs from Adh-S by only a threonine-lysine substitution at residue 192. This single change leads to many differences in biochemical properties (Day et al. 1974). The major function of ADH is the utilization and detoxification of dietary alcohol. The presence of large quantities of ADH (up to 1-2 % of the total protein) in Drosophila melanogaster allows the possibility of functions other than alcohol detoxification alone (Heinstra et al. 1983; Delden & Kamping 1988; Geer et al. 1985; Middleton & Kacser 1983). Here I report apparent synergistic effects of the Adh locus on the quantities of several other enzymes. I ask whether they can truly be attributed to the Adh locus itself, rather than to other loci, or to linked inversions, and whether the phenomenon is a general one or specific to one population.

t Present address: Department of Plant Science, The Waite Agricultural Research Institute, Glen Osmond, South Australia 5064, Australia.

2. MATERIALS AND METHODS

Four independent experiments were done: (i) immunological and electrophoretic assays of Adh-FF and SS flies derived from the Kaduna population (including parental, F and F2 crosses); (ii) spectrophotometric assays of the Kaduna population (experiments A and B); (iii) immunological and electrophoretic assays of a French population; and (iv) chromosome inversion (In(2L) t) analysis of the Kaduna population using crosses with a mutant stock.

(a) Drosophila strains and culture conditions

Three Drosophila melanogaster populations have been used, the Kaduna population (Kaduna-FF, Kaduna-SS, and Kaduna-wild type containing both FF and SS flies), a French population and a mutant stock. Kaduna-FF and SS were extracted from a population collected at Kaduna, Nigeria in 1949 and have been maintained since then in plastic cages at 25 ?C and approximately 70 % relative humidity under conditions of 18 h illumination and 6 h dark. The Kaduna flies were grown on a standard dead-yeast medium with the following composition: 75 g maize meal, 11 g dried flaked yeast, 75 g black treacle, 2.5 ml propionic acid, 10 g agar, 2.5 g nipagin, and 1 1 distilled water. The French population was kindly provided by Professor J. R. David. The flies were collected in a wine cellar at St Gepy, near Montpellier, France, in October 1988. As soon as the flies arrived, they were immediately etherized and frozen at -80 ?C until required. The mutant stock with black body and dumpy wings were generously sent by Dr M. Ashburner (De- partment of Genetics, University of Cambridge) in February 1990. Since then the flies have been maintained in half-pint bottles under the standard culture conditions.

(b) Experimental design

(i) Crossing strategy. In experiment 1, parental, F, and F2 generations were produced by the crossing procedures shown in figure 1.

In experiment 4, the mutant stock, Kaduna Adh-FF and Kaduna Adh-SS flies were used to estimate the percentages of recombination between the black (b) and dumpy (dp) loci on the left arm of chromosome 2. Because the phenotypes of black and dumpy are recessive, the crossing procedures were

Proc. R. Soc. Lond. B (1992) 247, 9-16 Printed in Great Britain

9



10 Xinmin Li Synergistic effect of Adh alleles in Drosophila melanogaster

Adh -FF cage Adh -SS cage

40 flies in each bottle 20 of them were females

Flies were shaken out after 2 days

Newly emerged flies were FF I 1 FF 2< SS I SS2transferred to the bottles

with fresh media After 5 days

F 0K Kj Four bottles were put to I

1 1 ~~~~~~~~~~~~back into the fly-room to

Flies etherized collect virgins for crossing and stored in 4

-80? C freezer

20 FF virgin females FXSI IFX SF X S and 20 males in each

I_______________ bottle Flies discarded after 2 days -

40 flies in each bottle I IF _ _ _

20 of them were virgin females

Newly emerged flies I were transferred e-

Fl 1 Newly emerged flies were

Flies etherised after 5 days and stored in Flies etherized after -8(PC freezer 1F22 5 days and stored in

to -800C freezer Figure 1. Crossing strategy.

simple. Virgin mutant females were first crossed with Kaduna Addh-FF and Adh-SS males, and their virgin females in the Fl generation were back-crossed to mutant males. Rates of recombination were estimated from the phenotypes of the offspring.

(ii) Experimental procedures. The procedures in experiments 1 and 3 are the same. The mass of a fly randomly taken from the freezer was found on a Beckman electronic microbalance (the numbers of males and females showed in figures 2, 3, and 4), then the fly was homogenized in an Eppendorf with 22 jtl NADP solution (2 mg ml-'). The homogenates were centrifuged at 5500 r.p.m. for 16 min. The supernatant was immediately used for immunodiffusion and electrophoresis. A feature of this experiment was that single flies were treated as the units of study. The supernatant from a fly was used not

only to measure the quantities of seven enzymes, but also to monitor their corresponding genotypes (the seven enzymes studied are given in table 1).

(c) Electrophoresis

Three hundred Kaduna flies in experiment 1, and 360 French flies in experiment 3, were analysed electro- phoretically. Seven loci that code the enzymes were investigated. Two electrophoretic gels were used throughout the experiment: either 11 0% TEB or 11 0% TEMM starch gels (the compositions of the buffer solutions for 1 1: TEB, 1.9376g tris, 0.6183g boric acid, and 0.1117g EDTA, pH 8.6; TEMM, 1.211 g tris, 1.161 g maleic acid, 0.3722 g EDTA, and 0.2033 g MgCl2, pH 7.4). Electrophoresis was

Proc. R. Soc. Lond. B (1992)



Synergistic elfect of Adh alleles in Drosophila melanogaster Xinmin Li 11

Table 1. Enzymes assayed in this study

abbrevi- E. C. map enzyme ation number position

glucose-6-phosphate G-6-PD 1. 1 . 1 .49 1-63 dehydrogenase

alcohol dehydrogenase ADH 1.1.1.1 2-50.1 NAD-malate MDH 1.1.1.37 2-37 .2 dehydrogenase

ot-glycerophosphate GPDH 1. 1. 1 .8 2-20 .5 dehydrogenase

phosphoglucomutase PGM 2.7.5. 1 3-43.4 NADP-malic enzyme ME 1. 1. 1 .40 3-53. 1 NADP-isocitrate IDH 1.1 .1 .42 3-27 .1 dehydrogenase

done at 190 V and 25 mA for 4 h with the TEB gel and at 80 V and 60 mA for 4.5 h with the TEMM gel. After electrophoresis, each gel was sliced into three sections. The sections of the TEB gel were stained for G-6-PD, ME, ADH, and GPDH (ADH and GPDH were stained on the same section of the gel, using a GPDH staining mixture with an ADH substrate). The three sections of the TEMM gel were stained for IDH, MDH, and PGM respectively (see table 2 for staining mixtures).

(d) Immunological assays

Radial immunodiffusion (RID) assays of enzymes were done in agarose using methods described by Clarke & Whitehead (1984). The loaded gels were incubated in plastic boxes containing damp tissue at 4 'C for 70 h. The corrected radial immunodiffusion area (RIDA.), used as the measure of the quantity of an enzyme, was the area of the precipitin ring divided by the average areas of the two standard samples on the same gel. Standard samples were obtained from a mass homogenate of Kaduna wild type. After centrifugation, the supernatants were put into small Eppendorf tubes and then stored at -80 'C. Standard samples were never reused. Once an Eppendorf was taken out of the freezer, any sample remaining in it was thrown away.

(e) Spectrophotometric assays

(i) Flies. The procedures for obtaining the flies (Kaduna FF and Kaduna SS) for electrophoresis and for the study of enzyme activity in experiment 2 were exactly the same as those used to obtain parental flies in experiment 1.

(ii) Preparation of extracts. Two independent subsets of experiment 2 (A and B) were done, differing only in the ways that the flies were prepared. In experiment A, 0.5842 g each of the Kaduna FF and Kaduna SS flies were taken from the freezer, and were ground with 6 ml of the following solution: 0.01 M K2HPO 4-KH2PO4, pH 7.4, 0.002 M Na2EDTA, 0.2 mm DTT, 0.07 mm PTU (recommended by McKechnie (1984)). In experiment B, 400 Kaduna FF and 400 Kaduna SS flies were each ground with 4 ml of the same solution. The rest of the procedures were the same in both subsets. The homogenate was centrifuged at 8000 r.p.m. for 18 min. The supernatants were removed and put into small Eppendorfs, each of which contained 0.2 ml, and then stored at -80 'C until used.

(iii) Enzyme assay methods. Enzymes were assayed by following the change of absorption in the reaction mixture at 28 ?C, using a Cecil CE515 double beam spectrophotometer. The activities of enzymes were expressed as 0D340 per min per mg of fly in experiment A, and as 0D340 per min per fly in

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experiment B. Different amounts of enzyme preparation were tested to ensure that an increase in enzyme content was accompanied by a corresponding increase in optical density at 340 nm. Four enzymes were analysed, following the protocols of Clarke & Keith (1988) for G-6-PD and ME, McKechnie (1984) for ADH, and Barnes & Laurie-Ahlberg (1986) for GPDH.

3. RESULTS (a) Quantities of the enzymes in the Kaduna population

Although polymorphic at the Adh locus, the Kaduna population is monomorphic at all of the other six enzyme loci.

(i) Differences in the quantities of enzymes between Adh-FF and Adh-SSflies in the parental and F2 generations. Figure 2

3

0 0 2 A B C D E F

Figure 2. Average quantities of different enzymes among Adh genotypes in the parental and F1 generations: A, ADH; B, ME; C, G-6-PD; D, IDH; E, GPDH; F, PGM. Number of flies tested of genotype FF (unshaded bar) = 58 (38 female, 20 male); of genotype FS (striped bar) = 60 (40 female, 20 male); of genotype SS (dark bar) = 62 (42 female, 20 male). *p < 0.05 (compared with SS); **p < 0.01; ***p < 0.001.

3

20 A B C D E F G Figure 3. Average quantities of different enzymes among Adh genotypes in the F2 generation: A, ADH; B, ME; C, G-6-PD; D, IDH; E, GPDH; F, PGM; G, MDH. Number of flies tested of genotype FF (unshaded) = 57 (31 female, 26 male); of FS (striped) = 55 (34 female, 21 male); of SS (dark bar) = 8 (4 female, 4 male). Probabilities as figure 2.

Table 3. Average quantities of enzymes adjusted by average body mass in Kaduna population

genotypes FF(P1) SS(P2) FF(F2) SS(F2)

ADH 1.9621 1.5086 1.8795 1.2253 PGM 1.8276 1.4941 1.9659 1.2707 ME 1.0952 1.1722 1.1980 1.2370 GPDH 1.4817 1.4193 1.6406 1.4002 G-6-PD 1.2145 1.1513 1.2545 1.2956 MDH 2.0267 1.2788 average 1.1424 Htg 1.0268 Htg 0.8865 Htg 0.7825 Htg body mass

shows the mean quantities of six enzymes from the P1 (FF) and P2 (SS) groups. Five of the six enzymes show greater quantities in Adh-FF flies than in Adh-SS (the Adh-FF flies were 1.45 times higher for ADH, 1.36 for PGM, 1.16 for GPDH, 1.17 for G-6-PD, and 1.04 for ME). Three of these differences are significant at the 0.001 level (ADH, G-6-PD and PGM), and one at the 0.05 level (GPDH). To investigate the inheritance of the quantities of enzymes, F1 (FFx SS) and F2 (Fl x F1) flies were produced. The results showed that the quantities of all enzymes except IDH in F, flies were intermediate between those in the parental strains (figure 2). In the F2, the differences among Adh genotypes were substantially in agreement with those in the parental and F1 generations (figure 3). The Adh- FF flies have larger quantities of seven enzymes than Adh-SS flies. For four enzymes (ADH, MDH, GPDH and PGM) the differences are statistically significant. The quantities in FS flies were intermediate between those of FF and SS flies, once again excepting IDH. These observations suggest that the differences were not haphazard, and that the segregations of the quantities for most of the enzymes were in parallel with those of the Adh genotypes.

(ii) Differences in quantity between Adh-FF and Adh-SS flies after adjustment for body mass. In making any necessary adjustment for body mass we have to consider several points: (i) the relations between body mass and the quantities of enzymes are different for different enzymes (e.g. coefficient of correlation is 0.87 between MDH and body mass, and only 0.51 between GPDH and body mass); (ii) these relations may change under different environmental conditions (e.g. the coefficient of correlation between ADH and G-6-PD increased from 0.36 in standard medium to 0.64 in the same medium with 25 g extra lipid per litre); and (iii) there is little information whether these relations represent cause and effect, or are both related to some other factors. It is therefore sensible to investigate these points and then to decide whether or not to make adjustments, and how to make them. The data, however, are insufficient to estimate accurately the effects of body mass on individual enzymes. As an alternative, relative quantities (average quantities of different enzymes adjusted by their corresponding average body mass) have been calculated, and are shown in table 3. Clearly, the same pattern is found. Although the differences between Adh-FF and Adh-SS flies are reduced to some degree, AdIh-FF flies still have greater quantities of enzymes than do AdIh-SS flies in eight out of eleven cases. These results argue against the differences being solely a result of secondary effect of body size.

(b) Spectrophotometric results with Kaduna population

It might be that the differences are only an artefact of the immunodiffusion technique. To address this issue, four enzymes (ADH, GPDH, G-6-PD, and ME) were studied spectrophotometrically, because differences in quantity should produce differences in activity. In choosing the few enzymes, two criteria were used: (i) chromosome position; the structural genes of the




Synergistic effect of Adh alleles in Drosophila melanogaster Xinmin Li 13

Table 4. The activities of enzymes compared between Adh-FF and Adh-SS (expressed as OD340 per min per mg fly) in experiment A

enzymes ADH GPDH G-6-PD ME

no. of tests 15 15 18 17 oD340 FF 0.1240a 0. l OOOa 0.0288a 0.0406a

SS 0.0513a 0.0900a 0.0235a 0.0356a s.e. FF 0.0018 0.0014 0.0006 0.0004

SS 0.0009 0.0014 0.0006 0.0007 ODFF/ODss 2.419 1.11 1.224 1.14

a Significant difference (p < 0.001).

3-

.o 2-

s0 A B C D E F G Figure 4. Average quantities of different enzymes among Adh genotypes in the French population of Drosophila. A, ADH; B, ME; C, G-6-PD; D, IDH; E, GPDH; F, PGM; G, MDH. Number of flies tested of genotype FF (unshaded) = 342 (180 female, 162 male); of FS (striped) = 14 (5 female, 9 male); of SS (dark bar) = 4 (2 female, 2 male). Probabilities as Figure 2.

In(2L)t

22D 34A

Adh

dp b l ,(2-50.1) (2-13.0) (2-48.5)

Figure 5. Approximate locations of dp (dumpy), b (black), Adh and inversion (Jn(2L) t) on the left arm of the chromosome.

enzymes studied scatter over three chromosomes; and (ii) cofactors; both NAD- and NADP-dependent enzymes were included. The results of experiment A are given in table 4. The Adh-FF group had, on the average, about 2.5-fold more ADH activity than the Adh-SS group. The FF group had consistently higher activities of GPDH, G-6-PD, and ME. Experiment B was designed to determine the differences in activity without considering the body mass. Similar results were obtained (not shown here). Thus the spectrophotometric results agreed with the immunological ones.

(c) Quantities of enzymes in a French population

To test the generality of the differences in quantities of enzymes between Adlh-FF and Adlh-SS flies, 360 flies

from a French population were analysed. The mean quantities of seven different enzymes are shown in figure 4. Six out of the seven enzymes showed larger quantities in Adh-FF than in Adh-SS flies. On average, there was about a 26 0 difference in quantity between the two genotypes. As with the Kaduna population, the quantities in the FS flies usually fell between those in the FF and SS.

(d) Chromosomal inversion results with mutant stock

There remains one possibility that needs to be excluded. Many natural populations are polymorphic for an inversion on the left arm of chromosome 2 (In(2L) t), and this inversion is in linkage disequilib- rium with Adh. In natural populations, the inversion is usually associated with Adh-S gene. The Kaduna population was chosen because of its supposed absence of inversions. However, this population has been kept in the laboratory for a long time. It is not impossible that the Kaduna stock became at some stage contaminated with the In(2L) t inversion, and that the parallel results in the Kaduna and the French population were the result of a common association between loci determining the quantities of the enzymes and the inversion. It seemed therefore desirable to test for the presence of the inversion in the Kaduna population. As shown in figure 5, most of the In(2L) t inversion is located between the marker genes dp (dumpy) and b (black). These locations provide the opportunity for measuring recombination between the marker genes and, thus, for detecting the inversion. If the Kaduna population was contaminated at some stage with the In(2L) t inversion, we expected to observe dramatically reduced recombination in a back- cross between F1 females, from a cross of mutant female x Kaduna SS male, and mutant males. The results show no significant difference in the rate of recombination between the cross with Kaduna Adh-FF flies and the cross with Kaduna Adh-SS flies (in the cross of F1 (mutant female x Kaduna FF male) female x mutant male, the flies with normal phenotypes, 461; with the black body, 125; with the dumpy wings, 156; with the black body and the dumpy wings, 441; the recombination rate is 23.8 %. In the cross of F1 (mutant female x Kaduna SS male) female x mutant male, the flies with normal phenotypes, 429; with the black body, 118; with the dumpy wings, 164; with the black body and dumpy wings, 392; the recombination rate is 26 %). Therefore the possibility of contamination with the In(2L) t inversion in the Kaduna population can be excluded. The parallel results in the Kaduna and the French population are most likely to result from a common factor, the Adh locus, or a gene closely linked to it, rather than to an association between loci determining the quantities of enzymes and the In(2L) t inversion.

4. DISCUSSION

The quantities of seven enzymes have been assayed in Kaduna Ad/i-FF and Ad/i-SS flies, and in F1 and F2 flies generated from a cross between them. The results





clearly show that Adh-FF flies have greater quantities of most of the enzymes -than Adh-SS flies. These differences segregate as if they were caused by a single Mendelian gene with codominant alleles. To test the generality of this phenomenon, a wild-collected French population, with a totally different genetic background and history, was studied. The results corroborate the evidence for differences in quantities between Adh-FF and Adli-SS. Spectrophotometric data suggest that the systematic differences in enzyme quantities between the two lines are not caused by the immunodiffusion technique. A study of recombination rates excluded the possibility that the parallel behaviour of the two populations resulted from a common association between the loci determining the quantities of the enzymes and the In(2L) t inversion.

The results presented here immediately lead to the suggestion that the changes in the quantities of enzymes are in blocks rather than individual units, which is essentially in agreement with the model of Kacser & Burns (1981), and throw light into the mystery of how a single enzyme could have a significant effect on fitness (Allendorf et al. 1983).

(a) Is the Adh locus responsible for the di/ferences in quantity between Adh-FF and Adh-SS flies?

Consistent associations between the genotypes at the Adh locus and the quantities of other enzymes in different generations and populations could have two categories of causes: genetic and environmental. For the purpose of the discussion, it is convenient to consider the two classes separately.

(i) Environmental causes. Early studies (Clarke et al. 1979, 1984; Geer et al. 1988) have shown that environmental factors, such as sampling and breeding conditions, can greatly influence the quantities of the enzymes. In these experiments, the methods of taking samples and the actual samples tested make this possibility slight. Throughout the experiment great care has been taken to minimize variation resulting from environmental differences (e.g. by keeping con- stant temperature, thoroughly mixing the yeast up in the medium, and randomizing the samples on the gels). We can not imagine any systematic artefactual bias being responsible for the differences. In addition, F2 flies were raised together, and differences observed among Adh genotypes will actually eliminate this possibility.

(ii) Genetic causes. A priori, the observed differences could have several different causes: (i) many factors throughout the genome might affect the quantities of the enzymes; (ii) factors located on the first and the third chromosome might be responsible; (iii) factors located on the second chromosome might be responsible; (iv) a factor located on the second chromosome, reasonably far away from the Adh locus, might be responsible; and (v) the Adh locus or a locus strongly linked to it might be responsible. If the differences were caused by the possibility (i), after random segregation in the F2 generation, the differences in quantity between Adh-FF and Adh-SS flies would become smaller. The results shown in figure 3 show that they

did not. If the second possibility were the case, there should be no differences between FF and 8S flies in the F2 generation. As to possibility (iii), it seems rather unlikely that the Kaduna population carries many independent mutations on the second chromosome, each responsible for the quantity of a different enzyme, and arranged so that each mutation associated with the Adh-F allele increases the quantity of the enzyme. If possibility (iv) was responsible for the differences, there should be some recombinants in the F2. The quantities of the four enzymes (MDH, ADH, PGM and GPDH) in single flies have been plotted (figure not shown here). There are no signs of recombinants. A more plausible hypothesis is possibility (v), that the Adh locus, or a locus closely linked to it, has pleiotropic effects on the quantities of the other enzymes. This hypothesis has two basic requirements: (i) that the quantities of other enzymes must segregate with the Adh alleles; and (ii) that no genetic recombination (or very little) should be observed. These conditions appear to have been met. The flies used in this experiment have been maintained in plastic cages containing about 3000 individuals for about 750 generations. During this long history they have become highly inbred. This assumption is supported by the electrophoretic data. All the loci investigated were monomorphic except for Adh. In addition, we have electrophoresed about 400 individuals from the population cages from which FF and SS lines were separated, and the same results were obtained. We can reasonably suppose that the flies are nearly isogenic. All the differences in quantity between Adh-FF and Adh-SS flies would then presumably be the consequences of the F-S allelic substitution.

In this study, as in others, it is not possible to say whether the effects observed at the enzymic level are directly caused by the Adh locus or to a tightly linked locus. I consider the latter possibility to be unlikely, but cannot exclude it.

(b) Mechanisms by which the Adh gene might act

(i) Does ADH, as a transcription factor, regulate the gene expression of a set of genes? The work shows that the level of ADH is associated with the level of other enzymes. This association raises the possibility that ADH can also act as a transcription factor, and regulate the expression of other genes by binding to certain DNA sequences. This hypothesis requires two conditions: (i) that some homologous DNA sequences exist in the vicinities of the genes concerned, and (ii) that there is a structural motif within the ADH molecule which can bind to these sequences.

This kind of interaction between proteins and DNA has been the subject of intensive study. Some transcription factors have been identified in both eukary- otes and prokaryotes. Structural studies on steroid receptors (Evans 1988), TFIIIA (Ginsberg et al. 1984), SPI (Kadonage et al. 1987), and APl1/c-jun (Bohmann 1987), have shown the presence of conserved domains. We have checked ADH amino acid sequences carefully (the sequence data were kindly provided by Dr Richard H. Thomas) and did not find a specific region




Synergistic effect of Adh alleles in Drosophila melanogaster Xinmin Li 15

The level of ADH (common factor)

NAD Ikinases

The levels of NAD NADP / The levels of NAD-dependent NADH NADP-dependent enzymes enzymes

Alkaline phosphatase Figure 6. Double directional regulation mechanism.

comparable to the DNA-binding domains so far discovered. This finding does not exclude the possibility that a new and different structural motif for a DNA binding protein exists.

(ii) Does NAD: NADH ratio, mediated by ADH level, regulate the gene expression at other loci ? NAD is a molecule with broad metabolic and physiological functions. Under normal conditions, an organisms's NAD- NADH balance is carefully regulated. Because ADH uses NAD to remove a hydrogen from ethanol in the first step of alcohol catabolism, the amount of ADH must affect the level of the NAD, and hence the NAD: NADH ratio. The levels of other NAD- dependent enzymes should also influence this ratio. However, ADH in Drosophila is the most abundant enzyme. It is realistic to assume that ADH has a major role in regulating the NAD :NADH ratio. A plausible explanation for the observed differences in quantity between two lines could then be proposed as shown in figure 6. This mechanism might produce a bi- directional regulatory interaction between the NAD: NADH ratio and the expression of genes (whose products are NAD-dependent enzymes). Similarly, the regulation between the NADIP:NADPH ratio and the expression of the genes (whose products are NADP- dependent enzymes) follows exactly the same pattern. Because of the existence of NAD kinase and alkaline phosphatase, communication between the NAD: NADH ratio and the NADP: NADPH ratio can be done. Adh-FF flies have about 1.5 times more ADH than do Adh-SS flies. The significantly increased level of ADH in FF flies could change the NAD :NADH ratio, which could then positively trigger the expression of the genes whose products are NAD-dependent enzymes. The NADP: NADPH ratio will be affected in the same direction through the NAD kinase. As a consequence, similar changes will happen to the expression of the genes whose products are NADP- dependent enzymes. In short, the level of ADH concentration is able to affect indirectly the quantities of other enzymes by affecting the NAD: NADH and NADP NADPH ratios, which are presumably able to influence either the synthesis of the enzymes or their stability.

(iii) A novel function for 'ADH'? One striking dis- covery that may be important as far as the explanation of the current finding is concerned is that Drosophila ADH is different in structure from the ADH of other animals (Thatcher 1980). The ADH of D. melanogaster

consists of two identical subunits, each 254 residues in length and of molecular mass 27400. The enzyme is much smaller in size than the ADH from horse liver (374 residues per monomer) and yeast (347 residues per monomer). The Drosophila enzyme shows no significant similarity in sequence to these two mol- ecules, and contains less cysteine than both. Secondary structure predictions suggest that the enzyme is similar in secondary structure to other dehydrogenases. How- ever, the nucleotide-binding domain is N-terminal, in contrast with the horse liver and yeast enzymes, which have a C-terminal nucleotide-binding fold. Such large differences in primary and secondary structures suggest that the enzyme had an independent origin. One of the most likely possibilities is that ADH came from the modification of another enzyme which is still working with an undiscovered function. This unknown function may be responsible for the phenomena observed in this study. Molecular studies have revealed a gene very closely linked to the Adh locus in D. melanogaster, with a DNA sequence that is very similar to Adh. But nobody knows what the gene is doing (personal communication with Dr Ashburner). Is it the gene that is responsible for all these enzyme differences? If this is the case, more questions will be raised about the mechanism by which differences between Adh-FF and Adh-SS flies come about.

It is important to recognize that, although such mechanisms might be plausible, discriminatory evidence is lacking. Direct molecular genetic analysis is needed to discover the mechanism by which Adh locus has its role at the enzymic level. A high priority for future work therefore lies in elucidating the molecular basis of the phenomenon.

I am deeply grateful to Professor B. C. Clarke for many invaluable suggestions at all stages of the experiments and for constructive comments on the manuscript. I also wish to thank Professor J. R. David and Dr M. Ashburner for providing wild and mutant flies. This work was partly supported by the Shanxi province, People's Republic of China.

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Received 9 September 1991; accepted 3 October 1991




synergistic effect of adh alleles in drosophila melanogaster

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