Genetic and Biochemical Basis of Enzyme Activity Variation in Natural Populations. I. Alcohol Dehydrogenase in DROSOPHILA MELANOGASTER

Recent studies by various authors suggest that variation in gene regulation may be common in nature, and might be of great evolutionary consequence; but the ascertainment of variation in gene regulation has proven to be a difficult problem. In this study, we explore this problem by measuring alcohol dehydrogenase (ADH) activity in Drosophila melanogaster strains homozygous for various combinations of given second and third chromosomes sampled from a natural population. The structural locus (Adh) coding for ADH is on the second chromosome. The results show that: (1) there are genes, other than Adh, that affect the levels of ADH activity; (2) at least some of these "regulatory" genes are located on the third chromosome, and thus are not adjacent to the Adh locus; (3) variation exists in natural populations for such regulatory genes; (4) the effect of these regulatory genes varies as they interact with different second chromosomes; (5) third chromosomes with high-activity genes are either partially or completely dominant over chromosomes with low-activity genes; (6) the effects of the regulatory genes are pervasive throughout development; and (7) the third chromosome genes regulate the levels of ADH activity by affecting the number of ADH molecules in the flies. The results are consistent with the view that the evolution of regulatory genes may play an important role in adaptation.     

Genetical variation for enzyme activity in a population of Drosophila melanogaster. VI. Molecular variation in the control of alcohol dehydrogenase (ADH) activity

Four characters, ADH activity at 25 degrees, immunologically determined ADH protein level, total protein and body weight were measured upon 72 hour old adult female and male Drosophila melanogaster from 16 highly inbred lines, derived from the laboratory population, "Texas" (established 1966). The highest levels of ADH activity and ADH protein level were observed in the 2 lined homozygous for the AdhF allele. Amongst the 14 AdhS/S lines variation for ADH protein level was associated with genetical variation for ADH activity (r = 0.6). The genetical association between ADH activity or ADH protein level and either body weight or total protein in the 16 inbred lines was not statistically significant. A study of ADH activity, ADH protein and total protein in 8 lines representing all homozygous combinations of chromosomes I, II and III and derived from two inbred AdhS/S lines, chosen for their respective high and low ADH activities, showed that ADH activity was considerably modified by a post-translational event controlled from chromosome III. Total protein was controlled by different chromosomal effects from those controlling ADH activity. Michaelis constants for crude fly extracts of the two AdhF/F and the above two AdhS/S lines showed clear differences in affinity for isopropanol.     

Linkage disequilibrium at the ADH2 and ADH3 loci and risk of alcoholism

Two of the three class I alcohol dehydrogenase (ADH) genes (ADH2 and ADH3) encode known functional variants that act on alcohol with different efficiencies. Variants at both these genes have been implicated in alcoholism in some populations because allele frequencies differ between alcoholics and controls. Specifically, controls have higher frequencies of the variants with higher Vmax (ADH2*2 and ADH3*1). In samples both of alcoholics and of controls from three Taiwanese populations (Chinese, Ami, and Atayal) we found significant pairwise disequilibrium for all comparisons of the two functional polymorphisms and a third, presumably neutral, intronic polymorphism in ADH2. The class I ADH genes all lie within 80 kb on chromosome 4; thus, variants are not inherited independently, and haplotypes must be analyzed when evaluating the risk of alcoholism. In the Taiwanese Chinese we found that, only among those chromosomes containing the ADH3*1 variant (high Vmax), the proportions of chromosomes with ADH2*1 (low Vmax) and those with ADH2*2 (high Vmax) are significantly different between alcoholics and controls (P<10-5). The proportions of chromosomes with ADH3*1 and those with ADH3*2 are not significantly different between alcoholics and controls, on a constant ADH2 background (with ADH2*1, P=.83; with ADH2*2, P=.53). Thus, the observed differences in the frequency of the functional polymorphism at ADH3, between alcoholics and controls, can be accounted for by the disequilibrium with ADH2 in this population.     

Recessive genetic variants affecting mating activity of males in natural populations ofDrosophila melanogaster

Mating activity of 115 wild males was compared with 88 homozygotes and 42 heterozygotes for their second chromosomes. Wild males, 48–96 hours old, inseminated on the average, 4.4±0.1 females per 24 hours. The hetero- and homozygotes for their second chromosomes (other chromosomes being randomly combined with those from the laboratory strain), inseminated on the average 2.8±0.2 and 2.0±0.2 females/24 h. respectively. There is no correlation between homozygotes and heterozygotes for the second chromosome and their wild ancestors which carried these chromosomes. Wild second chromosomes which in homozygous condition produced total sterility of their carriers, and some others which made for an unusually high activity in homozygous males, had on an average similar effects in wild carriers.This ariicle is warmly dedicated to Professor Theodosius Dobzhansky.     

Chromosome Interactions in DROSOPHILA MELANOGASTER. II. Total Fitness

In a large experiment, using nearly 200 population cages, we have measured the fitness of Drosophila melanogaster homozygous (1) for the second chromosome, (2) for the third chromosome, and (3) for both chromosomes. Twentyfour second chromosomes and 24 third chromosomes sampled from a natural population were tested. The mean fitness of the homozygous flies is 0.081 ± 0.014 for the second chromosome, 0.080 ± 0.017 for the third chromosome, and 0.079 ± 0.024 for both chromosomes simultaneously. Assuming that fitnesses are multiplicative (the additive fitness model makes no sense in the present case because of the large selection coefficients involved), the expected mean fitness of the homozygotes for both chromosomes is 0.0066; their observed fitness is more than ten times greater. Thus, it appears that synergistic interactions between loci are considerable; and that, consequently, the fitness function substantially departs from linearity. Two models are tentatively suggested for the fitness function: a "threshold" model and a "synergistic" model.—The experiments reported here confirm previous results showing that the concealed genetic load present in natural populations of Drosophila is sufficient to account for the selective maintenance of numerous polymorphisms (of the order of 1000).     

The intra-acinar distribution patterns of alcohol-dehydrogenase activity in the liver of juvenile, castrated and testosterone-treated rats

Rat liver alcohol dehydrogenase shows characteristic sex-differences with respect to activity and heterotopy. For the recognition of gonadal influences on the intra-acinar distribution patterns luminometric determinations of ADH activity were carried out on 50-150 ng lyophilized liver tissue samples which had been microdissected along the sinusoidal length. Juvenile rats of both sexes showed equally high alcohol dehydrogenase activity, which surpassed the adult values by a factor of 2 in males and 1.3 in females. The distribution pattern was rather flat, with a weak maximum at the beginning of the last third of the sinusoid. Castration of adult male and female rats resulted in an increase of alcohol dehydrogenase activity to around the prepubertal values. The intra-acinar profiles showed a gradual increase in activity from low periportal values to a peak near the perivenous zone. Only the hepatocytes directly adjacent to the efferent venule showed an even lower activity. Administration of testosterone to castrated animals had no effect on the ADH activity in males and resulted in only a slight decrease of enzyme activity in females. The intra-acinar distribution patterns showed an intermediary peak at the end of the second third of the sinusoidal length in males and a gradual increase of activity, beginning periportally, in the direction of the perivenous zone in females. The present findings on total activity of ADH and its distribution patterns in the liver are considered to be the result of complex hormonal alterations rather than a specific effect of testosterone.     

ADH, α-GPDH and SOD enzyme activities of second and third chromosomal genotypes from two geographically different populations of Drosophila melanogaster

ADH, α-GPDH and SOD enzyme activities have been measured in lines of Drosophila melanogaster homozygous and/or heterozygous for chromosomes extracted from two different populatioi Globally the results demonstrate that factors other than structural genes are determining the observed pattern of enzyme activities. ADH and α-GPDH activities are, however, more affected than SOD by these factors. Geographic origin, sex, chromosome, genetic background of the lines, containing regulatory genes in a broad sense, can be mentioned as the more relevant factors that influencing enzyme activities. A high and significant correlation is detected between ADH and α-GPDH enzyme activities and it can be interpreted as due to linkage disequilibrium among these two loci. SOD activity shows a lesser correlation with ADH and α-GPDH because it is less variable within population, i.e. it is a more canalized character. Finally, a principal component analysis, using the three enzyme systems shows that both populations are clearly separated, with a first principal component explaining 71.1 percent of the observed variance.     

Genetical variation for enzyme activity in a population of Drosophila melanogaster. VII. Genotype-environment interaction for alcohol dehydrogenase (ADH) activity

Genotype-environment interaction was detected for ADH activity amongst a set of 18 highly inbred lines of Drosophila melanogaster which had been extracted from the laboratory population, "Texas". The genotype-environment interaction for ADH activity was not wholly associated with genotype-environment interaction for body weight or total protein level. Detailed analyses of the responses of the individual inbred lines in ADH activity in relation to the environmental index, ej and following the procedure of Jinks and Pooni (1979), showed substantial diversity in the form of response. Lines homozygous for the AdhF allele were more environmentally sensitive than AdhS/S lines. Amongst the 16 AdhS/S lines, models of linear, quadratic or two intersecting-straight-lines were used to illustrate the varied responses of genotypes to the environment. The heterogeneity in the response characteristics of the inbred lines was attributed to variations in the conditions of culture media normally present within populations and laboratories. Moreover the non-linear responses shown by some lines to the environment are consistent with a model of genotype-environment interaction for ADH activity mediated by varied genotype-specific sensitivities to different environmental factors.     

Evidence for Complex Genic Interactions between Conspecific Chromosomes Underlying Hybrid Female Sterility in the Drosophila Simulans Clade

F(1) hybrid females between the sibling species Drosophila simulans, Drosophila mauritiana and Drosophila sechellia are completely fertile. However, we have found that female sterility can be observed in F(2) backcross females who are homozygous for D. simulans X chromosomes and homozygous for autosomal regions from either D. mauritiana or D. sechellia. Our results indicate that neither D. mauritiana autosome (2 or 3) can cause complete female sterility in a D. simulans background. The simultaneous presence of homozygous regions from both the second and third chromosomes of D. mauritiana, however, causes nearly complete female sterility which cannot be accounted for by their individual effects. The two autosomes of D. sechellia may show a similar pattern. From the same crosses, we also obtained evidence against a role for cytoplasmic or maternal effects in causing hybrid male sterility between these species. Taken with the results presented elsewhere, these observations suggest that epistatic interactions between conspecific genes in a hybrid background may be the prevalent mode of hybrid sterility between recently diverged species.     

The Genetic Factors Altered in Homozygous abo Stocks of DROSOPHILA MELANOGASTER

Females homozygous for the maternal-effect mutation abo (2-44.0) produce a large fraction of eggs which arrest during embryogenesis. Increasing doses of defined heterochromatic regions inherited by offspring of abo mothers from their fathers function zygotically to bring about a partial rescue of the abo-induced embryonic lethality. Another property of the abo mutation is that the severity of the maternal effect decreases when an abo stock is maintained in homozygous condition for a number of generations. Here, we show that the factors which change in homozygous abo stocks to result in the decrease in maternally induced embryonic lethality, act zygotically, dominantly and additively. More importantly, we show that the X and second chromosomes, but not the Y and third chromosomes, derived from homozygous abo stocks are, when inherited from males, more effective in promoting zygotic rescue of the abo-induced lethality than are the equivalent chromosomes derived from an abo stock maintained in heterozygous condition. The chromosomal locations of the factors maintained in the homozygous condition. The chromosomal locations of the factors altered in homozygous stock, as well as their behavior, strongly suggest that the same heterochromatic elements that are responsible for rescuing embryos from the abo-induced maternal effect are altered in homozygous abo flies in such a way that the maternal effect itself is less severe.     

Effect of acetone feeding on alcohol dehydrogenase activity in the olive fruit fly,Bactrocera oleae

The purpose of this study is to demonstrate a clear connection between the presence of acetone in larval diet and alcohol dehydrogenase (ADH) activity in laboratory raised populations of Bactrocera oleae. ADH activity of B. oleae is depressed in acetone-impregnated diets. At the same time the change of activity is accompanied by a change in the relative proportions of the multiple forms of ADH. The bulk of activity in the most cathodally migrating form is lost, and all the activity becomes localized in the less cathodally migrating forms of the enzyme. Moreover, ADH activity, expressed in vivo, appears to drop after exposure to acetone, as shown by the fact that larvae become less sensitive to pentenol poisoning. Our results show clear selective differences imposed by acetone on three homozygous genotypes involving the ADH alleles F, S and I in B. oleae. The directions of these differences were found to vary with the fitness component under test. Acetone treatment seems to affect developmental time and larva's viability as well as allele frequencies of ADH under artificial rearing. The effect of acetone on the maintenance of ADH polymorphism in artificially reared populations of B. oleae is further discussed.     

Effects on ADH activity and distribution, following selection for tolerance to ethanol in Drosophila melanogaster.

Strains of Drosophila melanogaster homozygous for either the AdhF or the AdhS allele were kept on food supplemented with ethanol for 20 generations. These strains (FE and SE) were tested for tolerance to ethanol and compared with control strains (FN and SN). The E strains showed increased tolerance to ethanol both in the adult and in the juvenile life stages. In adults the increase in tolerance was not accompanied by an increase in overall ADH activity. However, there were changes in the distribution of ADH over the body parts. Flies of the FE strain possessed significantly more ADH in the abdomen, compared with FN. Another set of FN and SN populations were started both on standard food and on ethanol food with reduced yeast concentrations. After 9 months ADH activities were determined in flies from these populations which had been placed on three different media: the food the populations had been kept on, regular food and regular food supplemented with ethanol. The phenotypic effects of yeast reduction on ADH activity were considerably, but longterm genetic effects were limited.     

Comparison of alcohol dehydrogenase expression in Drosophila melanogaster and D. simulans

Alcohol dehydrogenase (ADH) gene expression was analyzed in Drosophila melanogaster and its sibling species D. simulans. The levels of ADH activity, ADH-cross-reacting material (CRM), and ADH-mRNA were analyzed for several strains of each species, which derive from diverse geographic locations around the world. There is considerable quantitative variation in ADH activity, CRM level, and RNA level among strains within species at all developmental stages. However, the only consistent differences between the two species are in pupal RNA level and in late-adult activity and CRM level. Late-adult melanogaster flies that are homozygous for the Slow allozyme have approximately twice the level of ADH activity and CRM as do simulans flies. The regression of activity on CRM over strains is highly significant and essentially the same for each species, which means that most, if not all, of the activity difference between the species is due to a difference in concentration of the ADH protein. In contrast, there is no significant regression of CRM level on mRNA level in adults of either species; nor is there a significant difference in RNA level between species. Therefore, the difference in ADH protein concentration is not due to RNA template availability. Thus, the interspecific difference in ADH level in adults must be due either to a difference in the rate of translation of the two RNAs or to a difference in protein stability.     

Selection for ethanol tolerance in two populations of Drosophila melanogaster segregating alcohol dehydrogenase allozymes.

Selection for ethanol tolerance was equally successful in two populations of D. melanogaster in both of which the frequency of AdhF was 0.5 at the start of the experiment. Increased tolerance to ethanol was not invariably associated with increased frequencies of AdhF. In one population alcohol dehydrogenase (ADH) activity was significantly higher in three of the four selected sublines compared with their controls but there was no difference in activity between the selected and control sublines in the second population. The level of ADH activity in the control and selected lines was significantly correlated with the frequency of AdhF, but not with ethanol tolerance. These results show that adaptation to environmental alcohols in populations of D. melanogaster can be independent of the ADH system.     

Genetic variation in the expression of ADH in Drosophila melanogaster

Several chromosomes derived from natural populations have been identified that affect the expression of alcohol dehydrogenase (ADH). Second chromosomes, which also carry the structural gene Adh, show a great deal of polymorphism of genetic elements that determine how much enzyme protein accumulates. The level of enzyme was measured in third instar larvae, 6-to-8-day-old males and in larval fat bodies and alimentary canals. In general, activities in the different organs and stages are highly correlated with one another. One line was found, however, in which the ADH level in the fat body is more than twice the level one would expect on the basis of the activity in alimentary canal. We have also found evidence of third-chromosome elements that affect the level of ADH.     

Alcohol dehydrogenase polymorphism in populations of Drosophila melanogaster. II. Relation between ADH activity and adult mortality

Eight Drosophila melanogaster strains, seven homozygous for Adh ^F alleles and one for an Adh-null mutant, were compared for ADH activity in males and adult mortality on ethanol-supplemented food. The strains differed considerably in these qualities. A positive correlation was found between ADH activity and ld ₅₀. The relevance of this finding is discussed in relation to the differential selection acting on Adh genotypes kept on ethanol-supplemented food.     

Chromosome Interactions in DROSOPHILA MELANOGASTER. I. Viability Studies

The nature of fitness interactions is an important, yet unsolved, question in population genetics. We compare the egg-to-adult viability of individuals homozygous for either a second or a third chromosome with the viability of individuals homozygous for both chromosomes simultaneously. On the average, the viability of the two-chromosome homozygotes is somewhat greater than expected assuming that the fitnesses of the single-chromosome homozygotes interact in a multiplicative fashion. This result differs from previous observations that indicate either no significant deviations from the expectation or lower-than-expected average fitnesses for the double homozygotes.     

Studies of the effects of ethylene on yeast alcohol dehydrogenase activity

The temperature at which incubation with ethylene takes placehas a significant effect on the purified alcohol dehydrogenase(ADH) activity subsequently determined at room temperature.Ethylene can be separated completely from ADH on a Sephadexcolumn. Factors, such as the ionic strength of the buffer andthe presence of gelatin and NAD during the incubation with ethylenecan modify the effects of the gas. In yeast cells the effectsof ethylene on ADH activity are similar to those in the purifiedsystem. The presence of cyloheximide in the incubation mediumdid not suppress the effects of ethylene on ADH activity. Ethylenemay induce its effect, directly, through involvement in hydrophobicbonding in enzymes. (Received March 4, 1974; )     

Effects of differential alcohol dehydrogenase activity on the developmental toxicity of ethanol in Drosophila melanogaster

The role of alcohol dehydrogenase (ADH) activity in ethanol toxicity was investigated in Drosophila melanogaster. Flies from three congenic Adh strains (high, medium, and low ADH activity) were allowed to deposit eggs on medium containing 0, 4, or 8% ethanol. The resulting larvae were allowed to complete their development in the medium, and emerging flies were examined for defects. Flies with high ADH activity had malformation incidences of 0.8, 2.4, and 5.2% at 0, 4, and 8% ethanol, respectively. The comparable incidences for the low ADH strain were 1.0, 4.1, and 8.4%, while those for the medium ADH strain were intermediate in value. These results indicate that ethanol teratogenesis may be inversely related to ADH activity. When larvae were treated with ethanol for different lengths of time during development, the incidence of defects in flies from the high ADH strain was 3.9% when exposure started at the first instar and 3.09% when exposure started at the third instar. Results of the same exposures for the intermediate ADH strain were 5.2 and 3.4%, respectively, while those for the low ADH strain were 6.9 and 5.5%, respectively. Thus, length of ethanol exposure was directly related to the increased incidence of malformations in all tested Drosophila strains. For all tested strains, defect incidences appeared to be dose-related as well, regardless of length of exposure. ADH in Drosophila has a dual function and thus can catalyze oxidation of both ethanol and its toxic metabolite, acetaldehyde. This suggests that ethanol is the proximate teratogen in Drosophila.     

Genetical Analysis of Chromosomal Interaction Effects on the Activities of the Glucose 6-Phosphate and 6-Phosphogluconate Dehydrogenases in DROSOPHILA MELANOGASTER

By combining ten second and ten third chromosomes, we investigated chromosomal interaction with respect to the action of the modifier factors on G6PD and 6PGD activities in Drosophila melanogaster. Analysis of variance revealed that highly significant chromosomal interaction exists for both enzyme activities. From the estimated variance components, it was concluded that the variation in enzyme activity attributed to the interaction is as great as the variation attributed to the second chromosome but less than attributed to the third chromosome. The interaction is not explained by the variation of body size (live weight). The interaction is generated from both the lack of correlation of second chromosomes for third chromosome backgrounds and the heterogeneous variance of second chromosomes for different third chromosome backgrounds. Large and constant correlation between G6PD and 6PGD activities were found for third chromosomes with any second chromosome background, whereas the correlations for second chromosomes were much smaller and varied considerably with the third chromosome background. This result suggests that the activity modifiers on the second chromosome are under the influence of third chromosome factors.