Gpdh Locus Research Articles

Triglyceride pools, flight and activity variation at the Gpdh locus in Drosophila melanogaster.

We have created a set of P-element excision-derived Gpdh alleles that generate a range of GPDH activity phenotypes ranging from zero to full activity. By placing these synthetic alleles in isogenic backgrounds, we characterize the effects of minor and major activity variation on two different aspects of Gpdh function: the standing triglyceride pool and glycerol-3-phosphate shuttle-assisted flight. We observe small but statistically significant reductions in triglyceride content for adult Gpdh genotypes possessing 33-80% reductions from normal activity. These small differences scale to a notable proportion of the observed genetic variation in triglyceride content in natural populations. Using a tethered fly assay to assess flight metabolism, we observed that genotypes with 100 and 66% activity exhibited no significant difference in wing-beat frequency (WBF), while activity reductions from 60 to 10% showed statistically significant reductions of approximately 7% in WBF. These studies show that the molecular polymorphism associated with GPDH activity could be maintained in natural populations by selection in the triglyceride pool.

DNA polymorphisms at alpha-Gpdh locus of Drosophila melanogaster in Korean population.

Restriction site variation in 18-kb region including the Gpdh (sn-glycerol-3-phosphate dehydrogenase) locus has been assessed in 27 isofemale lines, 12 lines from the Chounan population and 15 lines from the Pusan population in Korea. Of the 25 restriction sites that were scored, 5 sites were polymorphic (20.0%): PstI (-2.35), XbaI (-1.7), HindIII (0.0), HindIII (+0.25), and PstI (+5.7). Two insertions (I) and three deletions (D) were detected. All the restriction sites and length polymorphisms were located in introns and outside regions of the Gpdh structure gene, the area from -7.0 site to -5.5 site. The proportion of segregating nucleotides, p, for all 27 lines was 0.019 and the estimated heterozygosity per nucleotide pair, was 0.013. Sixteen kinds of restriction map haplotypes of all lines analyzed were detected. Few lines were identical at the restriction map level. The estimated haplotype diversity, h, was 0.968. The haplotype diversity (0.997) of GpdhFF bearing chromosomes was slightly higher than that (0.982) of GpdhSS chromosomes. Analysis for a linkage disequilibria between the polymorphism also showed that HindIII (+0.25) vs. I (a) and PstI (-2.35) vs. D (a) have relatively high level of the linkage disequilibria. There were no significant relationship between restriction site polymorphism and GPDH activity in 27 second-chromosome lines; however, there was an apparent difference in the effect of length variation on GDPH activity. The activity of deletion-bearing lines was lower than that of deletion-free lines, while there was no effect of insertion on activity. Duplication of Gpdh was observed in about one-third of the 27 isofemale lines. The duplication-bearing lines in both populations had higher levels of GPDH activity and GPDH CRM than duplication-free lines. In a further investigation of the duplication structure, the duplication of Korean populations was shown to have a similar structure to other populations investigated (Symonds and Gibson, 1992; Takano et al., 1989).

Interaction between the Adh and Odh loci in response to ethanol in Drosophila melanogaster.

Six Drosophila melanogaster strains were constructed from two isofemale lines. The strains had four allele combinations at the alcohol dehydrogenase (Adh) and octanol dehydrogenase (Odh) loci, while all alpha-glycerophosphate dehydrogenase (alpha Gpdh), malate dehydrogenase (Mdh), and aldehyde oxidase (Aldox) alleles were identical. Second-instar and early and late third-instar larvae were exposed to different concentrations of ethanol (0, 5, and 7.5%) and 3 days later fresh weights and the activities of ADH, ODH, alpha GPDH, and MDH were measured. Activity differences were observed between the two Adh genotypes: ADHF allozyme had considerably higher activity than ADHS. Exogenous ethanol resulted in the highest increase in ADH activity in the second- and early third-instar stages. This ADH induction depended on the allele combination at the Adh and Odh loci; e.g., in the strain having the AdhS-OdhS allele combination, increased ADH activity was observed only after exposure to 7.5% ethanol. ODH activities differed according to the Odh genotypes, in that the ODHS allozyme had a higher activity than ODHF. ODH activities did not appreciably respond to different ethanol treatments. All six strains had identical alleles at the Mdh and alpha Gpdh loci, but nevertheless, the responses of these enzymes to ethanol depended on the allele combinations at the Adh and Odh loci. alpha GPDH activity followed that of ADH in all experiments. MDH activities were not influenced by exogenous ethanol in the strains homozygous for the AdhS allele. In AdhF strains, however, exposure to 7.5% ethanol resulted in a considerable decrease in MDH activity in the second-instar larvae. Correlations among the response variables showed that ODH activities were strongly associated with fresh weight and the activities of all other enzymes, except for ADH. ADH activity, however, showed a significant correlation only with alpha GPDH activity throughout the larval life. Both MDH and ODH activities were found to be in strong negative correlation with ADH activity in the second-instar larvae. At this most sensitive life stage, the metabolic response to ethanol is highly correlated.

Nucleotide Variation at the Gpdh Locus in the Genus Drosophila

The Gpdh locus was sequenced in a broad range of Drosophila species. In contrast to the extreme evolutionary constraint seen at the amino acid level, the synonymous sites evolve at rates comparable to those of other genes. Gpdh nucleotide sequences were used to infer a phylogenetic tree, and the relationships among the species of the obscura group were examined in detail. A survey of nucleotide polymorphism within D. pseudoobscura revealed no amino acid variation in this species. Applying a modified McDonald-Kreitman test, the amino acid divergence between species in the obscura group does not appear to be excessive, implying that drift is adequate to explain the patterns of amino acid change at this locus. In addition, the level of polymorphism at the Gpdh locus in D. pseudoobscura is comparable to that found at other loci, as determined by a Hudson-Kreitman-Aguadé test. Thus, the pattern of nucleotide variation within and between species at the Gpdh locus is consistent with a neutral model.

Excessive homoplasy in an evolutionarily constrained protein

The evolution of monomorphic proteins among closely related species has not been examined in detail. To investigate this phenomenon, the glycerol-3-phosphate dehydrogenase (Gpdh) locus was sequence in a broad range of Drosophila species. Although purifying selection to remove amino acid variation is the dominant force in the evolution of Gpdh, some replacements have occurred. The sequences were compared in the context of the phylogeny of the genus, revealing a high proportion of amino acid parallelism and reversal (homoplasy) at four sites. The level of homoplasy is significantly greater than that seen in other proteins for which multiple sequences are available, showing that Gpdh is strongly constrained by both the number of amino acid differences and the types of changes allowed. These four sites evolve at a much higher rate than do the other variable positions in the protein, accounting for half of the interspecific amino acid replacements. However, unlike typical hypervariable sites, where multiple changes to several different amino acids are seen, evolutionary 'flip-flopping' between two amino acid states defines this new class of hypervariable site.

Molecular analysis of Gpdh null mutations that arose in mutation accumulation experiments in Drosophila melanogaster.

In order to clarify the cause of null mutations in enzyme loci, the molecular structure of six null mutations in the Gpdh locus (encoding alpha GPDH: alpha glycerol-3-phosphate dehydrogenase (NAD+), E.C. 1.1.1.8; map position at 2-17.8) that arose in mutation accumulation experiments was examined. A restriction map analysis showed that five of the mutations are insertional mutations whereas the sixth is a deletion. The Gpdh regions of these null mutations were then cloned and sequenced. The inserted DNA fragments are all internally deleted P elements measuring 1.1 kb in length. Two are a KP element and two others are a HP element. All the insertions occur in the region near the initiation signal of transcription. The deletion encompasses the seventh and eighth exons over a length of 1.1 kb. These results therefore indicate that the null mutation rate at the Gpdh locus is largely influenced by P elements.

Starvation resistance in Drosophila melanogaster in relation to the polymorphisms at the Adh and α Gpdh loci

In view of the world-wide latitudinal cline of the Adh and α Gpdh allozyme frequencies of Drosophila melanogaster and the interactions between these loci, experiments were performed to study the phenotypic effects of these loci. Starvation resistance, oxygen consumption, body weight, protein content and triglycerides content were measured in flies with all possible combinations of Adh and α Gpdh alleles. Genotypic differences were found for survival time under food deprivation, body weight, protein content and triglycerides content. Oxygen consumption did not differ significantly between genotypes. A significant positive correlation was observed between triglycerides content and starvation resistance. Body weight, protein content and triglycerides content decreased significantly during starvation but no rate differences between genotypes could be found. It is argued that genotypic differences in starvation resistance and triglycerides content can play a role in the maintenance of the world-wide cline of Adh and α Gpdh allele frequencies.

Defective P element insertions affect the expression of sn-glycerol-3-phosphate dehydrogenase alleles in natural populations of Drosophila melanogaster.

A distinctive and geographically widespread category of low-activity variant at the Gpdh locus in Drosophila melanogaster is shown to have defective P elements inserted between the TATA box and the transcription start site. In four examples the insertion was a single 1.15 kilobase (kb) KP element, whereas in another variant there were two KP elements in tandem. A sixth example contained a 0.61 kb defective P element. The target site for all of the insertions is GTGCAAAC. There was no sequence variation either between the insertions or in comparison with two other KP elements previously described from natural populations. The insertions cause a reduction in GPDH mRNA, and are the most likely cause of the low GPDH activity.

Restriction site variation, gene duplication, and the activity of sn-glycerol-3-phosphate dehydrogenase in Drosophila melanogaster.

Restriction site variation in a 25-kb region including the sn-glycerol-3-phosphate dehydrogenase (Gpdh) locus has been assessed in 29 single female D. melanogaster lines from the Cardwell (Australia, QLD) population. The Gpdh locus was duplicated in about one-third of the lines, although the duplication was incomplete and lacked exons 1 and 2. There was no restriction site variation in the duplicated region. Three insertions were found in the gene region but only one affected GPDH activity. The lines with the duplication had higher levels of GPDH activity and protein amount than did nonduplicated lines. This effect was also observed in lines extracted from two other Australian populations. The duplication is shown to have a similar structure in each population investigated and is also present in populations from China and Africa. It is suggested that the effect of the duplication on GPDH activity, which might be due to structural factors affecting transcription at the Gpdh locus, could account for the worldwide distribution of the duplication.

Changes in relative fitness with temperature among second chromosome arrangements in Drosophila melanogaster.

Development time and body weight of In(2L)t, R (a putative short inversion on the left arm of the second chromosome) and ST (standard) karyotypes of Drosophila melanogaster were measured at different temperatures. Frequency changes were followed in populations polymorphic for In(2L)t and ST and kept under different environmental conditions. These experiments were carried out in order to explain the worldwide latitudinal clines for In(2L)t and other inversions. To avoid interactions with the Adh and alpha Gpdh loci, which also have latitudinal clines, all karyotypes were homozygous AdhS alpha GpdhF. In(2L)t homokaryotypes had a longer development time and a lower weight than the other karyotypes at all temperatures. R/ST heterokaryotypes had the shortest development time and ST/ST had the smallest weight decrease with increasing temperature. The differences among the In(2L)t and ST karyotypes in development time were further analyzed in an experiment where the age at which 50% of the larvae were able to become adults, without further food ingestion, was determined. In polymorphic populations at 20 degrees and 25 degrees a significant decline of In(2L)t frequencies was observed. At 29.5 degrees and 33 degrees there was no change in In(2L)t frequencies but a significant excess of heterokaryotypes occurred. On ethanol-supplemented food the most drastic decline in In(2L)t frequency was observed. Populations transferred at 2- and 3-week intervals at 25 degrees exhibited large differences in final In(2L)t frequencies. The frequency changes could in part be attributed to the differences in development time and to previously observed differences in high temperature resistance. The experiments prove that the karyotypes are under selection. The results are discussed in relation to the geographic distribution of In(2L)t.

Structural characterization of the alpha-glycerol-3-phosphate dehydrogenase-encoding gene of Drosophila melanogaster.

In Drosophila, multiple isoforms of alpha-glycerol-3-phosphate dehydrogenase (sn-glycerol-3-phosphate: NAD+ 2-oxidoreductase, EC 1.1.1.8) are produced in a tissue- and stage-specific manner. To understand the underlying molecular basis of these isoforms, we have sequenced a 5.8-kilobase region of the Drosophila genome that contains the entire Gpdh locus. Primer-extension and RNase protection assays show that the gene consists of eight exons and has a single transcription-start point. RNase protection mapping and comparison of the genomic sequence from three different cDNA clones reveal that three protein isoforms of glycerol-3-phosphate dehydrogenase are produced by alternative processing of 3' exons. Two of the isoforms differ from the third by the addition of either three or ten amino acids to their C-terminal ends. Transcripts corresponding to two of the isoforms are expressed during both larval and adult stages, while the third isoform is produced only in adults.

Stress, altered energy availability and larval fitness in Drosophila melanogaster.

This paper reports some effects of temperature variation, nutritional stress and a novel alteration in energy availability upon larval fitness in Drosophila melanogaster. The cofactor nicotinamide adenine dinucleotide (NAD) has been chosen as a novel energy source to be supplemented in food during larval development. The effects have been assessed at the phenotypic level for larval survival and development time and at the genotypic level for the alcohol dehydrogenase (Adh) and glycerol-3-phosphate dehydrogenase (Gpdh) loci. Supplemented NAD was found to increase survival at the lower temperatures and decrease survival at the higher temperatures. Further, for each temperature, NAD decreased development time although this effect diminished as temperature increased. There were no significant effects at the genotypic level. Hence a phenotypic approach studying the effects of environmental stresses and novel energy availability may be useful in understanding fitness variation in Drosophila populations.

Dominance for sn-glycerol-3-phosphate dehydrogenase activity in Drosophila melanogaster: Evidence for differential allelic expression mediated via a trans-acting effect

Two second chromosomes (H31 and T198) which when homozygous give relatively low α glycerol-3-phosphate dehydrogenase (GPDH) activity have been isolated from natural populations of Drosophila melanogaster. In heterozygotes between H31 and T198 and chromosomes bearing standard Gpdh alleles there is dominance for GPDH activity and for the amount of GPDH protein. The increased level of activity in the heterozygotes appears to be due to the differential expression of the standard alleles rather than to increased heterodimer activity. Recombination analyses have shown that if the effect is due to a modifier it must be less than 0·04 cM from the Gpdh locus.

Genetic variability of flight metabolism in Drosophila melanogaster. III. Effects of Gpdh allozymes and environmental temperature on power output.

The effect of allozyme variation at the sn-glycerol-3-phosphate dehydrogenase (Gpdh) locus on variation in the mechanical power output of the flight muscles of Drosophila melanogaster was investigated. The influence of different rearing and flight temperatures and of their interactions with the Gpdh allozymic genotypes (allotypes) on flight ability also were analyzed. Populations from three continents were used, and Gpdh allotypes were generated from crosses between randomly paired isofemale lines made autozygous for each of the two alleles by inbreeding. Measurements made during tethered flight, together with wing morphology, were used to estimate power output using both Weis-Fogh's and Ellington's formulas. Analyses of variance (ANOVA) indicated significant main effects for both environmental components (rearing and flight temperatures) but for only one of the three genetic components (genetic backgrounds within continent); Gpdh allotypes and populations (continent of origin) were not significant. The interaction between rearing and flight temperature was highly significant, indicating some physiological adaptation. The effect of Gpdh allozymes depended on both rearing and flight temperature and was either significant or marginally so, depending on which set of formulas was used. In either case, the S/S allotype showed a 2-4% greater power output than the F/F allotype at low temperature for both interactions. In addition, the S/S allotype showed significantly greater power output than the F/F allotype among flies raised at 15 degrees and flown at 15 degrees, whereas the reverse was true for flies raised at 30 degrees and flown at 30 degrees. Significant differences among the three allotypes for GPDH activity level were found in general, with S/S having the highest, F/S intermediate and F/F the lowest activity, and an inverse relationship existed between rearing temperature and activity. The temperature effects on power output are consistent with the geographical and seasonal variation observed at the Gpdh locus in nature. In general, the results show that Gpdh can be considered a minor polygene affecting quantitative variation in the power output during flight and that genotype-by-environment interaction is an important component of that effect.

Developmentally regulated alternate modes of expression of the Gpdh locus of Drosophila.

Immunoblot analyses have been performed on extracts prepared from Drosophila melanogaster. Those analyses have revealed two subunit forms of enzyme glycerol 3-phosphate dehydrogenase (GPDH) in larval tissues and in adult abdominal tissues. Thoracic tissue, which accounts for the bulk of the adult GPDH, has only one subunit form, the smaller. The two subunit forms differ by approximately 2400 daltons. In agreement with previous genetic and biochemical data indicating that this enzyme is encoded by a single structural gene, analyses of extracts prepared from a strain carrying a GPDH null mutation detect no GPDH polypeptides in larvae or adults. Similarly, analyses of extracts prepared from a strain carrying a mutation which produces a GPDH polypeptide that differs in size from wild-type reveal a change in the adult thoracic GPDH polypeptide as well as a change in both GPDH polypeptides found in larvae. Total Drosophila RNA prepared from larvae or newly eclosed adults has been translated in a mRNA-dependent cell-free system. GDPH was immunoprecipitated from the translation products and analyzed. Two subunit forms of GPDH were immunoprecipitated from translation products whose synthesis was directed by larval RNA and only one was detected in the polypeptides synthesized from adult RNA. The GPDH polypeptides synthesized in vitro are approximately the same size as the corresponding polypeptides found in vivo. The relative proportion of total GPDH represented by each subunit form synthesized in vitro is similar to those found in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

RNA directed synthesis of catalytically active Drosophila sn-glycerol-3-phosphate dehydrogenase in Xenopus oocytes.

Xenopus laevis oocytes injected with poly(A)+ RNA isolated from Drosophila melanogaster direct the synthesis of catalytically active glycerol-3-phosphate dehydrogenase (NAD+) (EC 1.1.1.8). The de novo synthesized enzyme reflects the electrophoretic properties appropriate to the stock of flies from which the injected RNA was isolated and is electrophoretically distinct from endogenous Xenopus activity. Immunoprecipitation of 35S-labeled translation products has demonstrated two immunologically related proteins with molecular masses of 32- and 34kDa which are encoded by two separate mRNA molecules. The 32-kDa protein is identical in size and charge properties to the protein purified from the fly and possesses the catalytic activity observed in the Xenopus translational assay. Poly(A)+ RNA isolated from a strain of flies bearing a CRM- null mutation at the GPDH locus does not contain translatable RNA for the 32-kDa protein. These results suggest that the two immunologically related proteins are the translational products of two separate transcripts derived from either two related loci or from differential transcription and/or processing of the same genetic locus.

Selection at the alpha-Gpdh locus in experimental populations of Drosophila melanogaster.

Three sets of experiments have been conducted in order to evaluate the role of natural selection at the alpha-Gpdh locus in Drosophila melanogaster. (1) The evolution of the F-allele frequency has been followed for many generations in 13 experimental populations having different genetic backgrounds. (2) Egg-to-adult viability has been measured in synthetic populations derived from one locality (Brouilly) and the results have been compared with those of a previous experiment involving a different local population (Tostes). (3) The effects of sodium octanoate on egg-to-adult viability have been measured on the genotypes FF, FS, SF, and SS. The results demonstrate that selection operates on a small block of genes which includes the alpha-Gpdh locus.

Maintenance mechanism of polymorphism at the ?-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 in Natural Populations.

1. Study of Selection.- Estimation of mating cycle components of selection in plants.- Population genetics of Zoarces viviparus (L.), a review.- A study of sexual selection in natural populations of the milkweed beetle, Tetraopes tetraophthalmus.- How does the genome congeal?.- Attempts to measure selection by altering gene frequencies in natural populations.- Genetic variance for viability and linkage disequilibrium in natural populations of Drosophila melanogaster.- Sexual selection and the evolution of territoriality in birds.- Functional aspects of genetic variation.- On conditional inference for deviation from Hardy-Weinberg distribution.- 2. Study of Polymorphism.- Selection and neutrality.- Protein evolution: Nonrandom patterns in related species.- Polymorphism, selection, and multi-locus heterozygosity in the plaice, Pleuronectes platessa L..- Hidden heterogeneity among electrophoretic alleles.- Natural selection and the ?-GPDH locus in Drosophilidae.- Nonrandom associations between allozymes in natural populations of Drosophila melanogaster.- Polymorphism for the number of genes coding for salivary amylase in the bank vole, Clethrionomys glareola.- A geometric formulation of the stability condition at a triallelic locus.- A general model to account for enzyme variation in natural populations. IV The quantitative genetics of viability mutants.- 3. Sex and Evolution.- The sex habit in plants and animals.- Evolution of the sex ratio in the wood lemming, Myopus schisticolor.- Population genetics, demography and the sex ratio.- A comparative study on enzyme polymorphisms in sympatric diploid and polyploid populations of Lumbricillus lineatus (O.F.M.), Enchytraeidae, Oligochaeta.- Selection and genetic differentiation in parthogenetic populations.- 4. Ecology and Evolution.- Cactus-breeding Drosophila - A system for the measurement of natural selection.- Test of the hypothesis that migration balances selection in differentiated subpopulations of Spirorbis borealis.- The selection regime of Philaneus spumarius (L.) (Homoptera).- Effects of a virus on competition and selection in barley.- Selection and interspecific competition.- Coevolution in ecological systems: Results from loop analysis for purely density-dependent coevolution.- 5. Human Evolution.- Selection and non-Mendelian variability.- Estimation of the characteristics of rare variants.- The genetics of HLA and disease associations.

Origin of ?-glycerophosphate dehydrogenase isozymes in Drosophila melanogaster and their functional relationship in the ?-glycerophosphate cycle

The basis for the differentiation of L-glycerol-3-phosphate dehydrogenase (alpha-GPDH) into larval and adult isozymes in Drosophila melanogaster was investigated by the correlation of a lack of appearance of each isozyme during development within Drosophila bearing alpha-GPDH "null" alleles and by the study of a putative conversion factor. Conversion studies indicate the presence of a heat-labile RNase-resistant conversion factor present in crude larval extracts with the ability to convert GPDH-1 to GPDH-2 and GPDH-3 but not vice versa. In addition, "null" mutations at the Gpdh locus obliterate all isozymatic species of alpha-GPDH in all developmental stages. These observations suggest that all alpha-GPDH isozymes are the product of a single structural gene and that the multiple forms of this enzyme arise during successive developmental stages through an epigenetic modification of the primary Gpdh+ polypeptide. Finally, observations are reported which bear on the functional divergence of the alpha-glycerophosphate cycle in the adult and larval stage of development.