An independent non-linear latitudinal cline for the sn-glycerol-3-phosphate (alpha- Gpdh ) polymorphism of Drosophila melanogaster from eastern Australia

Latitudinal variation of the polymorphic sn-glycerol-3-phosphate (alpha-Gpdh) locus in Drosophila melanogaster has been characterized on several continents; however, apparent clinal patterns are potentially confounded by linkage with an inversion, close associations with other genetic markers that vary clinally, and a tandem alpha-Gpdh pseudogene. Here we compare clinal patterns in alpha-Gpdh with those of other linked markers by testing field flies from eastern Australian locations collected in two separate years. The alpha-Gpdh variation exhibited a consistent non-linear cline reflecting an increase in the alpha-GpdhF allele at extreme latitudes. This pattern was not influenced by the In(2L)t inversion wherein this locus is located, nor was it influenced by the presence of the alpha-Gpdh pseudogene, whose presence was ubiquitous and highly variable among populations. The alpha-Gpdh pattern was also independent of a cline in allozyme frequencies at the alcohol dehydrogenase (Adh) locus, and two length polymorphisms in the Adh gene. These results suggest clinal selection at the alpha-Gpdh locus that is partially or wholly unrelated to linear climatic gradients along the eastern coast of Australia.     

Maintenance mechanism of polymorphism at the α-Gpdh locus in Drosophila melanogaster

Polymorphism at the alpha-Gpdh locus was studied in Drosophila melanogaster. Using two different lines, one marked by the F allele (FF line) another by the S allele (SS line), four populations were initiated, two in which the initial frequency of F was 0.1 and two in which it was 0.9. They have been observed for 34 generations. From the fifth generation on, the equilibrium frequency in the four cages was about 0.60. Viability has been measured during the evolution of te populations while F frequencies changed and recombinations between the FF and SS lines occurred. It has been evaluated in synthetic populations built with different frequencies: (1) from the original FF and SS lines and (2) from FF and SS lines extracted after 34 generations of joint evolution. In all three cases, the FF viability depended on the frequency of the F allele. The similarity of the three linear regressions implies that alpha-Gpdh locus or other closely linked loci is the target of the selection in the populations analyzed here.     

Measuring Selection Coefficients Affecting the Alcohol Dehydrogenase Polymorphism in DROSOPHILA MELANOGASTER

This paper describes a perturbation experiment on the frequency of the F and S Alcohol dehydrogenase (Adh) alleles of D. melanogaster. Fifty-four iso-female lines set up from three wild populations and with initial F frequencies of either 0.25, 0.50 or 0.75 were maintained on standard laboratory food medium at 22 degrees. At generations 4, 12 and 20 the lines were again scored for Adh gene frequencies. Maximum likelihood procedures were used to estimate selection coefficients for the Adh genotypes. An analysis of deviance was used to compare the coefficients against expectations under the hypotheses of neutrality and of constant values for the three base populations, and for the three initial gene frequency classes. Highly-significant departures from neutrality were observed; over all 54 lines, the set of relative fitnesses for S/S:F/S:F/F was estimated as 1.00:1.08:1.08. In addition, there were significant differences between lines in the outcome of selection which were not attributable to differences between base populations or initial F frequencies. These residual between-line differences, as well as some between-generation, within-line differences are discussed in terms of linkage disequilibria with background genes and electrophoretically cryptic variation at the Adh locus.     

On Recombination among In(2L)t, α-Gpdh and Adh in Drosophila melanogaster

The occurrence and patterns of linkage disequilibrium between an inversion and allozymic loci within it or nearby have been used in attempts to discriminate among different hypotheses for the maintenance of variability. The interpretation of the data on the best-documented case, that of the nonrandom association between In(2L)t and alpha-Gpdh or Adh in the second chromosome of Drosophila melanogaster, has been done on the basis that recombination between alpha-Gpdh and Adh is almost entirely due to the recombination between In(2L)t and the locus within it (alpha-Gpdh), the recombination between the inversion and the nearby locus (Adh) being negligible. In this paper, we show that the pattern of recombination is just the opposite.     

Structural analysis of glycerol-3-phosphate dehydrogenase from severalDrosophila species

This report describes preliminary protein structural studies of glycerol-3-phosphate dehydrogenase (alpha-GPDH) from Drosophila spp. and an important innovative feature of our enzyme purification protocol. The scheme involves the coupling of substrate (alpha-glycerophosphate) elution from CM-Sephadex and cofactor (NADH) elution from Affi-Gel blue resin. Using this method a 32.7% yield and a 111-fold purification were obtained from a D. melanogaster line carrying the alpha-GpdhS allele at the alpha-Gpdh locus. The product obtained from 0 to 3-day-old adult flies was electrophoretically homogeneous and consisted mainly of the adult alpha-GPDH-1 isozyme. The method was used to obtain alpha-GPDH protein from D. melanogaster (two lines), D. hydei, D. immigrans, and D. mercatorum. Peptide mapping revealed structural differences among the enzymes from the different species, and amino acid sequencing showed many similarities between D. melanogaster alpha-GPDH and the rabbit muscle enzyme.     

Conditioning to ethanol in the fruit fly-a study using an inhibitor of ADH

To identify processes involved in the choice of ethanol by adult Drosophila, flies homozygous Adh(F), reared in the absence of alcohol were placed in contact with: a) an ethanol-free medium, b) a medium containing ethanol, c) a medium supplemented with 4-methylpyrazole (4-MP, an inhibitor of the ADH pathway), d) a medium containing ethanol and 4-MP. The choice of ethanol over a medium without ethanol was evaluated by measuring the duration of extension of the proboscis of the flies in each of the media. A slight preference for the ethanol-supplemented medium was observed in the naive flies, which was enhanced by previous exposure to ethanol. Exposure to ethanol and 4-MP, however, led to an avoidance of ethanol. There was a reduction in ADH activity on treatment of the flies with 4-MP, and signs of malaise (reduced locomotor activity, loss of balance) were observed in the flies who ingested both ethanol and inhibitor. We concluded that the preference for ethanol stems from an associative learning related to ethanol utilization. Inhibition of enzymes of ADH pathway led to a conditioned aversion due to disturbance of ethanol metabolism giving rise to malaise.     

An electrophoretically cryptic alcohol dehydrogenase variant in Drosophila melanogaster. I. Activity ratios, thermostability, genetic localization and comparison with two other thermostable variants

The alcohol dehydrogenase (ADH) variant ADH-FCh.D. has a secondary alcohol/primary alcohol activity ratio characteristic of ADH-S although it has an electrophoretic mobility inseparable from ADH-F. ADH-FCh.D. is distinguished from these two common ADH variants by being much more thermostable. Genetic analysis suggests tht ADH-FCh.D. is specified by an allele at the Adh locus. Biochemical comparisons show that ADH-FCh.D. has the same electrophoretic mobility, activity ratio and thermostability as the two other heat-resistant variants which have been reported, ADH-F71K in Europe and ADH-Fr in North America. The geographically widespread distribution of a thermostable ADH variant within the ADH-F electrophoretic class indicates that it should be considered in attempts to explain the Adh polymorphism in natural populations.     

Identification of a genetic element that controls the organ-specific expression of adh1 in maize

Allozyme balances serve as markers of quantitative behavior of electrophoretically distinguishable alleles. By the use of ADH Set I allozyme balances, it is demonstrated that all Adh1-S/Adh1-F individuals from more than 20 diverse S/F families exhibit a reciprocal correlation between Adh1 quantitative behavior in two maize organs: the scutellum and primary root. Within an electrophoretic mobility class, the Adh1 allele that is relatively underexpressed in the scutellum is relatively overexpressed in the primary root, and vice versa. Segregation tests prove that this "reciprocal effect" is the property of a cis-acting site that is closely linked to or within the Adh1 structural gene, and it is not affected by diverse genetic backgrounds. Immunological and [(3)H]-leucine incorporation experiments establish that Adh1 quantitative variants differ in ADH1.ADH1 synthetic rates in the anaerobic primary root. The reciprocal-effect phenomenon suggests that the cis-acting loci controlling Adh1 quantitative expression in each respective organ are at least in close proximity, or may share common DNA sequences. We discuss the possibility that the reciprocal-effect locus is a regulatory component of the Adh1 cistron.     

Alcohol dehydrogenase allele frequencies in Bactrocera oleae (Dipt., Tephritidae): effect of ethanol addition in larval diet

Abstract: The effect of ethanol in larval medium on Bactrocera oleae larvae was examined at four concentrations. Ethanol exerted a differential effect on the three alcohol dehydrogenase allele frequencies. While originally being at equilibrium under laboratory conditions, after three generations of larval development in a diet containing ethanol at 1% concentration, Adh -F allele frequency increased, that of Adh -I dropped significantly and the frequency of Adh -S remained unaltered. Adh -S allele seems to be adapted in nature where only minor quantities of alcohol are present in the insects' natural host, while Adh -I is best adapted in the alcohol-free laboratory culture medium. The frequency of Adh -F allele remains unaltered when feral populations are introduced in the laboratory.     

Genetic Divergence under Uniform Selection. II. Different Responses to Selection for Knockdown Resistance to Ethanol among DROSOPHILA MELANOGASTER Populations and Their Replicate Lines

We have tested the hypothesis that genetic differences among conspecific populations may result in diverse responses to selection, using natural populations of Drosophila melanogaster. Selection for ethanol tolerance in a tube measuring knockdown resistance was imposed on five West Coast populations. In 24 generations the selected lines increased their mean knockdown times, on average, by a factor of 2.7. An initially weak latitudinal cline was steepened by selection. The two southernmost populations showed the same increases in the selected character, but differed consistently in their correlated responses in characters related to ethanol tolerance. This result indicates that the populations responded to selection by different genetic changes. Selection decreased female body weight and increased resistance to acetone, suggesting components of the response unrelated to ethanol metabolism. The Adhs allele was favored by selection in all populations at the onset, but increased in frequency only in the selected lines of the southernmost population. There was a correlation between latitude and Adh frequency changes, suggesting that fitnesses of the Adh alleles were dependent on the genetic background. Genetic background also had a large effect on the loss of fitness due to selection. Genetic drift between replicate lines caused more variation in selection response than initial genetic differences between populations. This result demonstrates the importance of genetic drift in divergence among natural populations undergoing uniform selection, since the effective population sizes approached those of small natural populations. Drift caused greater divergence between selected replicates than control replicates. Implications of this result for the genetic model of selection response are discussed.     

Genetic sexing in the mediterranean fruit fly,Ceratitis capitata,using the alcohol dehydrogenase locus

Summary Using the alcohol dehydrogenase (ADH) locus a genetic sexing system is being developed in the Mediterranean fruit fly Ceratitis capitata based on the sensitivity of ADH null mutations to environmental ethanol. A series of null mutants have been induced at this locus, however, none proved viable as homozygotes. One of these null mutants was translocated to the male determining chromosome and this line can be used for genetic sexing. When larvae from this line were reared on larval medium containing various concentrations of allyl alcohol, 97% of the emerging adults were males; in the absence of the allyl alcohol the sex ratio in the line is distorted in favour of the females. It is proposed that the higher ADH activity of the females (homozygous positive) in comparison with the males (heterozygous null) is responsible for their lower survival in larval medium containing allyl alcohol. ADH converts the allyl alcohol to the lethal ketone. The possible use of this line to sex large populations of medflies for use in sterile insect release programmes is discussed.     

Change in the genetic structure of an experimental population of Drosophila by electrophoretic locus under the effect of ecological conditions]

Dynamics of the genetic structure of experimental Drosophila populations being kept over 50 generations at 25 and 17 degrees C6 and on the medium with ethanol (12%) was studied. In all experimental populations alteration in allele frequencies of Adh, Gpdh, Hex and Est-6 loci took place in the first 20-25 generations on monitoring under the influence of the temperature and ethanol. Later on, an equilibrium relation between allele frequencies will be established.     

Studies of Esterase-6 in DROSOPHILA MELANOGASTER. II. the Genetics and Frequency Distributions of Naturally Occurring Variants Studied by Electrophoretic and Heat Stability Criteria

Measurements of the electrophoretic mobility and thermostability of esterase-6 allozymes have been used to determine the amount of allelic variation at the esterase-6 locus in Drosophila melanogaster. We studied 398 homozygous lines obtained from four natural populations. Use of a spectrophotometric assay for esterase-6 activity has allowed precise quantitation of heat-stability variants. Using these methods, eight putative alleles were detected within the two most common electrophoretic classes. Analyses of F1 and F2 progeny show that the behavior of stability variants is consistent with the hypothesis that this variation is due to allelic variation at the Est-6 locus. Analyses of the gene-frequency distributions within and between populations show (1) that observed allele-frequency distributions do not deviate significantly from those expected for neutral variants, and (2) that there is little evidence for an increase in apparent divergence of the different populations at the genotypic or phenotypic levels when the additional variation detected is considered. These findings suggest that gene-frequency analysis alone is unlikely to resolve the question of the selective significance of allozyme variation.     

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.     

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.     

Molecular Variation of Adh and P6 Genes in an African Population of Drosophila Melanogaster and Its Relation to Chromosomal Inversions

Four-cutter molecular polymorphism of Adh and P6, and chromosome inversion polymorphism of chromosome II were investigated in 95 isogenic lines of an Ivory Coast population of Drosophila melanogaster, a species assumed to have recently spread throughout the world from a West African origin. The P6 gene showed little linkage disequilibrium with the In(2L)t inversion, although it is located within this inversion. This suggests that the inversion and the P6 locus have extensively exchanged genetic information through either double crossover or gene conversion. Allozymic variation in ADH was in linkage disequilibrium with In(2L)t and In(2R)NS inversions. Evidence suggests either that inversion linkage with the Fast allele is selectively maintained, or that this allele only recently appeared. Molecular polymorphism at the Adh locus in the Ivory Coast is not higher than in North American populations. New haplotypes specific to the African population were found, some of them connect the ``Wa(s)-like' haplotypes found at high frequencies in the United States to the other slow haplotypes. Their relation with In(2L)t supports the hypothesis that Wa(s) recently recombined away from an In(2L)t chromosome which may be the cause of its divergence from the other haplotypes.     

Latitudinal Adh allozymic variation and ethanol tolerance in Indian populations of Drosophila melanogaster

Ten Indian geographical populations of D. melanogaster were assayed electrophoretically for Adh genic variation. The Indian geographical populations of D. melanogaster revealed significant clinal variation (3 % for 1 d? latitude) at Adh locus and Adh^F allelic frequency correlated significantly with increase in latitude. It was suggested that the abundance of secondary alcohols in the southern Indian tropical and humid environment might exert selective pressure favouring higher frequency of Adh^S allele. Patterns of ethanol utilization as well as ethanol tolerance were analyzed in larval and adult individuals of six geographical populations of D. melanogaster. Latitudinal variation in ethanol tolerance was observed in D. melanogaster populations from India. The parallel occurrence of latitudinal variation it Adh locus as well as ethanol tolerance in Indian geographical populations of D. melanogaster could be maintained by balancing natural selection varying spatially along the north-south axis of the Indian sub-continent.     

ADH enzyme activity and Adh gene expression in Drosophila melanogaster lines differentially selected for increased alcohol tolerance

In Drosophila melanogaster, alcohol dehydrogenase (ADH) activity is essential for ethanol tolerance, but its role may not be restricted to alcohol metabolism alone. Here we describe ADH activity and Adh expression level upon selection for increased alcohol tolerance in different life-stages of D. melanogaster lines with two distinct Adh genotypes: Adh(FF) and Adh(SS). We demonstrate a positive within genotype response for increased alcohol tolerance. Life-stage dependent selection was observed in larvae only. A slight constitutive increase in adult ADH activity for all selection regimes and genotypes was observed, that was not paralleled by Adh expression. Larval Adh expression showed a constitutive increase, that was not reflected in ADH activity. Upon exposure to environmental ethanol, sex, selection regime life stage and genotype appear to have differential effects. Increased ADH activity accompanies increased ethanol tolerance in D. melanogaster but this increase is not paralleled by expression of the Adh gene.     

Ethanol-resistant mutants of Nicotiana plumbaginifolia are deficient in the expression of pollen and seed alcohol dehydrogenase activity

Summary Six independent mutant lines ofNicotiana plumbaginifolia resistant to ethanol, designated E3, E8, E101, E112, E144 and E251, were isolated as germinating seedlings on selective medium. In all cases, resistance to ethanol was conferred by a single recessive nuclear mutation at the same locus. Mutant seeds and pollen lacked detectable ADH activity, with the exception of E251 where a residual activity was detected. An antiserum directed againstArabidopsis thaliana ADH detected an ADH-related polypeptide of 44 kDa present in wild-type seeds and, to a lesser extent, in the seeds of the leaky mutant E251. No ADH-related polypeptide could be detected in seeds of the other mutants. However, all of them had a nearly normal level of ADH mRNA except one which did not synthesize any mRNA. These results suggest that these ethanol-resistant mutants are impaired in one of the structural genes coding for alcohol dehydrogenase. The corresponding locus has been designatedAdh1.Abbreviations ADH alcohol dehydrogenase - EMS ethyl methane-sulfonate - MTT dimethyl thiazol tetrazolium - NAD nicotinamide adenine dinucleotide - NBT nitro blue tetrazolium - p-cells protoplast-derived cells - PMS phenazine methosulfate - SDS sodium dodecyl sulfate