Biology of a duplicate gene system with glucose 6-phosphate dehydrogenase activity in Drosophila melanogaster: Genetic analysis and differences in fitness components and reaction to environmental parameters among Zw genotypes

There are two structural forms of glucose 6-phosphate dehydrogenase activity in Drosophila melanogaster. Whether one or the other or both show in vitro (and probably in vivo) activity depends on the genotype of a sex-linked locus (Zw). In this article, the relative fitnesses of heterozygotes (with both electromorphs active) and homozygotes (with activity demonstrable for only one or the other electromorph) for the Zw locus are described. It is shown that the relative fitness of heterozygotes increases with increase in population density, or degree of crowding and trophic stress, and that the mean development times of Zw heterozygotes are lower than those of the Zw homozygotes. In addition, and perhaps accounting for the fitness and viability excess of the heterozygotes, one set of evidence strongly suggests that they are better buffered against trophic stress than the homozygotes.     

Dosage compensation of sex-linked genes in Drosophila melanogaster. The activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in flies with normal and disturbed genetic balance

Summary The phenomenon of dosage compensation in Drosophila melanogaster which consists in doubling of the activity of the X-chromosome genes in males as compared to those in females was studied.The specific activities of 6-phosphogluconate dehydrogenase (6PGD) and glucose-6-phosphate dehydrogenase (G6PD) determined by the sex-linked structural genes Pgd and Zw respectively were studied in flies carrying duplications for different regions of the X-chromosome. The increase in dose of Pgd and Zw in females resulting from the addition of an extra X-chromosome or X-fragments leads to a proportional rise in the specific activities of 6PGD and G6PD. On the other had the addition to females of the X-chromosome carrying no Pgd gene or X-fragments lacking Pgd and Zw has no effect on the enzyme activities. Thus we failed to reveal in the X-chromosome any compensatory genes envisaged by Muller, which would repress sex-linked structural genes proportional to their dose.The 6PGD and G6PD levels in phenotypically male-like intersexes carrying two X-chromosomes and three autosome sets (2X3A) is 30% higher than in diploid (2X2A) or triploid (3X3A) females. However the specific activities of the enzymes in female-like intersexes are the same as in regular females. The levels of 6PGD and G6PD per one X-chromosome are 1.5–2.0 times higher in the intersexes than in the normal females and metafemales (3X2A). The results indicate that the level of expression of the X-chromosome is determined by the X:A ratio. It is suggested that the decreased X:A ratio in males is responsible for the hyperactivation of their X-chromosomes.     

The effect of astaxanthin and cadmium on rat erythrocyte G6PD, 6PGD,GR, and TrxR enzymes activities in vivo and on rat erythrocyte 6PGD enzyme activity in vitro

In this study, the effects of astaxanthin (AST) that belongs to carotenoid family and cadmium (Cd), which is an important heavy metal, on rat erythrocyte G6PD, 6PGD, GR, and TrxR enzyme activities in vivo and on rat erythrocyte 6PGD enzyme activity in vitro were studied. In in vitro studies, 6PGD enzyme was purified from rat erythrocytes with 2′,5′‐ADP Sepharose4B affinity chromatography. Results showed inhibition of enzyme by Cd at IC₅₀; 346.5 μM value and increase of 6PGD enzyme activity by AST. In vivo studies showed an increase in G6PD, 6PGD, and GR enzyme activities (P ? 0.05) and no chance in TrxR enzyme activity by AST. Cd ion inhibited G6PD, 6PGD, and GR enzyme activities (P ? 0.05) and also decreased TrxR enzyme activity (P ? 0.05). AST + Cd group G6PD enzyme activity was statistically low compared with control group (P ? 0.05). 6PGD and TrxR enzyme activities decreased without statistical significance (P ? 0.05); however, GR enzyme activity increased statistically significantly (P ? 0.05).     

Maternal effect for genes encoding 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase in Drosophila melanogaster

We studied the maternal effect for two enzymes of the pentose cycle, 6-phosphogluconate dehydrogenase (6PGD) and glucose-6-phosphate dehydrogenase (G6PD), using a genetic system based on the interaction of Pgd^? and Zw^? alleles, which inactivate 6PGD and G6PD, respectively. The presence and formation of the enzymes was investigated in those individuals that had not received the corresponding genes from the mother. We revealed maternal forms of the enzymes, detectable up to the pupal stage. The activities of “maternal” 6PGD and G6PD per individual increased 20-fold to 30-fold from the egg stage to the 3rd larval instar even in the absence of normal Pgd and Zw genes. Immunologic studies have shown that the increase in 6PGD activity is due to an accumulation of the maternal form of the enzyme molecules. We revealed a hybrid isozyme resulting from an aggregation of the subunits of isozymes controlled by the genes of the mother and embryo itself. These results indicate that the maternal effect in the case of 6PGD is due to a long-lived stable mRNA transmitted with the egg cytoplasm and translated during the development of Drosophila melanogaster.     

Polymorphism at the G6pd and 6Pgd loci in Drosophila melanogaster. IV. Genetic factors modifying enzyme activity

Different homozygous lines of similar genotype with respect to G6pd and 6Pgd were shown to have different enzyme activities for G6PD and 6PGD. Crosses between high and low lines suggested that there were modifying genes present on the autosomes, while others were probably located on the X chromosome. Allelic variation within each electrophoretic class of G6pd and 6Pgd might, however, also have contributed to this variation. An experiment on adaptation to sodium octanoate demonstrated that in adapted flies selection for lower enzyme activity had occurred, which provided further evidence for the existence of genetic differences in activity. Furthermore, a strong positive correlation between the activities of G6PD and 6PGD was found for each genotype. Since no correlation was found between MDH and the two enzymes G6PD and 6PGD, it could be concluded that this correlation was probably rather specific for G6PD and 6PGD. Interaction between genotypes with respect to activity was also found. It was shown that the variation at 6Pgd influenced the activity of G6PD within a genotype. The data are discussed in relation to fitness differences presented in foregoing articles.     

Autosomal Factors with Correlated Effects on the Activities of the Glucose 6-Phosphate and 6-Phosphogluconate Dehydrogenases in DROSOPHILA MELANOGASTER

Isogenic lines, in which chromosomes sampled from natural populations of D. melanogaster are substituted into a common genetic background, were used to detect and partially characterize autosomal factors that affect the activities of the two pentose phosphate pathway enzymes, glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD). The chromosome 3 effects on G6PD and 6PGD are clearly correlated; the chromosome 2 effects, which are not so great, also appear to be correlated, but the evidence in this case is not so strong. Examination of activity variation of ten other enzymes revealed that G6PD and 6PGD are not the only pair of enzymes showing a high positive correlation, but it is among the highest in both sets of lines. In addition, there was some evidence that the factor(s) affecting G6PD and 6PGD may also affect two other metabolically related enzymes, transaldolase and phosphoglucose isomerase.—Rocket immunoelectrophoresis was used to estimate specific CRM levels for three of the enzymes studied: G6PD, 6PGD and ME. This experiment shows that a large part of the activity variation is accounted for by variation in CRM level (especially for chromosome 3 lines), but there remains a significant fraction of the genetic component of activity variation that is not explained by CRM level.—These results suggest that the autosomal factors are modifiers involved in regulation of the expression of the X-linked structural genes for G6PD and 6PGD, but a role in determining part of the enzymes' primary structure cannot be excluded with the present evidence.     

Effect of astaxanthin and aluminum chloride on erythrocyte G6PD and 6PGD enzyme activities in vivo and on erythrocyte G6PD in vitro in rats

In this study, we investigated the effect of astaxanthin (Ast) and aluminum (Al) on the erythrocyte glucose‐6‐phosphate dehydrogenase (G6PD) and 6‐phosphogluconate dehydrogenase (6PGD) enzymes activities in vivo and on G6PD enzyme in vitro in rats. For in vitro studies, G6PD enzyme was purified from rat erythrocyte by using 2′,5′‐ADP‐Sepharose 4B affinity gel. The effects of Ast and Al³⁺ ion were investigated on the purified enzyme. It was determined that Ast increased the enzyme activity, whereas Al³⁺ inhibited the enzyme activity noncompetitively (IC₅₀ values; 0.679 mM, K_i values 1.32 mM). For in vivo studies, the rats were divided into the groups: control (Cont.), Al, Ast, and Al + Ast. The last three groups were compared with the control group. In Al group, a significant degree of inhibition was observed in the activity of G6PD and 6PGD enzymes when compared with the control group (P < 0.05), whereas there was an increase in the activities of G6PD and 6PGD enzymes in Ast and Al + Ast groups (P < 0.05).     

Dosage Compensation in Drosophila: Nadp-Enzyme Activities and Cross-Reacting Material

The relationships between gene dosage, enzyme activities and CRM levels have been determined for G6PD and 6PGD. Enzyme activities and CRM levels were directly proportional and increased in genotypes carrying duplications of the respective structural genes. When a duplication consisting of the distal 45% of the X chromosome was used to duplicate Pgd+, 6PGD activity and CRM increased and G6PD activity decreased. When the proximal 55% of the X chromosome was duplicated, G6PD activity and CRM increased whereas 6PGD activity and CRM levels decreased. These observations support the model of dosage compensation of X-linked genes that invokes an autosomal activator in limited concentrations for which X-linked loci compete. The distal 45% of the X chromosome, when duplicated, caused a significant increase in NADP-malic enzyme activity and CRM levels, as if a structural gene for NADP-ME is sex-linked.     

Quantitative Analysis of X Chromosome Effects on the Activities of the Glucose 6-Phosphate and 6-Phosphogluconate Dehydrogenases of DROSOPHILA MELANOGASTER

By combining 20 X chromosomes with five autosomal backgrounds, the relative importance of these factors with respect to the activity variations of G6PD and 6PGD in Drosophila melanogaster were investigated. Analysis of variance revealed that there exist significant X chromosome, autosomal background and genetic interaction effects. The effect of the X chromosome was due mainly to the two allozymic forms of each enzyme, but some within-allozyme effects were also detected. From the estimated variance components, it was concluded that the variation attributed to the autosomal background is much larger than the variation attributed to the X chromosome, even when the effect of the allozymes is included. The segregation of the allozymes seems to account for about 10% of the total activity variation of each enzyme. The variation due to the interaction between the X chromosome and the autosomal background is much smaller than variations attributed either to the X chromosome or to the autosomal background. The interaction effect is indicated by the change of the ranking of the X chromosomes for different autosomal backgrounds. Highly significant and positive correlation between G6PD and 6PGD activities was detected. Again, the contribution of the autosomal background to the correlation was much larger than that attributed to the X chromosome.     

IN VIVO Function of Rare G6pd Variants from Natural Populations of DROSOPHILA MELANOGASTER

From 1981 to 1983, 15,097 X-chromosomes were genetically extracted from a number of North American populations of D. melanogaster and were electrophoretically screened for rare mobility and activity variants of glucose-6-phosphate dehydrogenase (G6PD). Overall, 13 rare variants were recovered for a frequency of about 10^-3. Eleven variants affect electrophoretic mobility and are apparently structural, and two variants exhibit low G6PD activity. One low activity variant is closely associated with a P-element insertion at 18D12-13—all of the variants were subjected to the previously described genetic scheme used to identify relative in vivo activity differences between the two common electrophoretic variants associated with the global polymorphism. Most of the rare variants exhibit apparent in vivo activities that are similar to one or the other of the common variants, and these specific rare variants appear to be geographically widespread. Several variants have significantly reduced function. All of the variants were measured for larval specific activity for G6PD as a first measure of in vitro activity. It appears that specific activity alone is not a sufficient predictor for G6PD in vivo function.     

Comparison of in Vitro and in Vivo Activities Associated with the G6pd Allozyme Polymorphism in Drosophila Melanogaster

Earlier studies of the A and B allozymes at the G6pd locus show a differential ability of the genotypes to suppress the loss of viability associated with a low activity 6-phosphogluconate dehydrogenase mutation, 6Pgdlo1. This observation indicates a relatively lower activity for the A allozyme genotype, but it is not known if this level of suppression required a large difference in in vivo activity. To clarify this difference an analysis of the biochemical properties of the purified allozymes was carried out, as well as an analysis of the activity level associated with an original low activity P element-derived allele which had partially reverted and lost its suppression ability. G6PD activity and protein level were studied in 47 X chromosome lines from North America. The A genotype averages a 9% lower Vmax. From analysis of the correlation between G6PD activity and protein level it remains unclear whether the allozyme Vmax difference results from dissimilarity in protein level or kcat. At 25 degrees and physiological pH, comparative studies of the steady-state kinetics show the two purified allozyme variants differ significantly in their KM values for glucose-6-phosphate and NADP, and the K1 for NADPH. In aggregate these parameters predict the A genotype possesses a 20% lower in vitro catalytic efficiency. A partial revertant of a P element-derived low activity B variant, was shown to lose the ability to suppress 6Pgdlo1 low viability after acquiring only 60% of normal B activity. This last comparison shows the A genotype activity must be reduced in vivo by at least 40%.     

The Effect of Ddt on the Polymorphism at the G6pd and Pgd Loci in DROSOPHILA MELANOGASTER

For the degradation of DDT and other chlorohydrocarbon insecticides energy in the form of NADPH is needed which for the greater part is supplied by the pentose phosphate shunt. Therefore the influence of DDT on the polymorphism at the G6pd and Pgd loci in Drosophila melanogaster was investigated by studying its effect on egg to adult survival and adult survival. The results show the existence of significant differences in fitness between the different genotypes of the two loci for both components. It is found that the effect of DDT supplementation differs significantly from the effect of sodium octanoate addition. DDT treatment also increases the activity of the pentose phosphate shunt as measured by the activity of G6PD and 6PGD. In larvae a 50% increase in activity is found and in adults a 100% increase. As there is little doubt that the activities of G6PD and 6PGD are somehow correlated with the fitness of flies, the data are discussed in relation to the in vitro and in vivo differences in activity between the different allozymes of both G6PD and 6PGD.     

X-linked mutations that give rise to overproduction of glucose-6-phosphate dehydrogenase in Drosophila melanogaster

Two X-linked mutations that give rise to overproduction of glucose-6-phosphate dehydrogenase (G6PD) were found among the progenies of isogenic strains which had been subjected to selection for high G6PD activity. Mapping of the high-activity factor in these mutants was carried out using car Zw ^B sw males of low G6PD activity. As a result, the factor mapped 0.02–0.04 unit to the left of the Zw locus. The amount of the G6PD gene was also quantitated utilizing a cloned G6PD gene as a probe, but no significant difference was found between the mutants and low-G6PD activity flies which shared the same X, second, and third chromosomes with the mutants. These findings are consistent with our notion that the mutations might be regulatory mutations, possibly resulting from the insertion of a novel class of transposable genetic elements.This research was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.     

Evidence for linkage between glucose 6-phosphate dehydrogenase and hypoxanthine-guanine-phosphoribosyl transferase loci in Chinese hamster cells

G6PD and 6PGD activities were determined in diploid, hyperdiploid, tetraploid, and hybrid cells all originating from the same Chinese hamster cell line (the DON line). A relationship between gene multiplicity and enzyme activity has been observed. The same enzymes were studied in hybrid cells cultivated in selective media. Selection was carried out against and for the HGPRT⁺ locus. The differences in G6PD and 6PGD activities between the cell lines found under these conditions indicate a positive linkage of the G6PD and HGPRT loci and negative linkage of the 6PGD and HGPRT loci in these Chinese hamster cells.     

The electrophoretic patterns of glucose-6-phosphate, 6-phosphogluconate and glucose dehydrogenases in Amoeba proteus (Pallas, 1766) Leidy, 1878 cultured at different temperatures

• 1.1. The detection of G6PD and 6PGD in A. proteus can be used to investigate the functioning of the phosphogluconic (or pentose phosphate) pathway in these amoebae.
• 2.2. In amoebae cultured at 10°C compared with those kept at 25°C, no differences in the number of G6PD, 6PGD and GlcDH electromorphs are revealed.
• 3.3. The acclimation of amoebae cultured at 25°C to a relatively low temperature of 10°C is accompanied by an increase in the activities of total Triton-soluble G6PD, 6PGD and GlcDH per cell, a rise in the activity of GlcDH per unit cell protein, and a change in the activity and heat resistance of some G6PD electromorphs.

     

Developmental variation in effects of the second and third chromosomes on the activities of the glucose 6-phosphate and 6-phosphogluconate dehydrogenases in Drosophila melanogaster

Developmental profiles of the second- and third-chromosome modifiers of the activities of glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) in Drosophila melanogaster were investigated. Third-chromosome modifiers showed very strong effects on both enzyme activities at larval, pupal, and adult stages, whereas second-chromosome effects were detected mainly at larval and adult stages. For both enzyme activities and both chromosomes, the correlation over line means between larval and pupal stages was significantly positive, but the correlation between larval or pupal stage and adult stage was not significant. This result suggests that the actions of modifiers on G6PD and 6PGD activities are influenced by the change of developmental stages. Correlation between G6PD and 6PGD activities was positive and highly significant throughout the developmental stages for both sets of chromosomes, although third-chromosome correlations were slightly higher than second-chromosome correlations. The magnitude of the correlation between G6PD and 6PGD activities does not seem to be influenced by the change of development. Diallel crosses for both sets of chromosomes indicate that the action of activity modifiers is mainly additive for both sets of chromosomes, but dominance effects were detected in some cases in adult males. Significant maternal effects were detected for the third chromosome for both enzyme activities until the pupal stage. The change of the activity modifier action after emergence of the imago and the significant correlation between G6PD and 6PGD activities were also detected for diallel progeny.This work was supported by Public Health Service Grant NIH-GM11546.Paper No. 10211 of the journal series of the North Carolina Agricultural Research Service, Raleigh, North Carolina 27695.     

Regulation of Enzyme Activities in Drosophila: Genetic Variation Affecting Induction of Glucose 6-Phosphate and 6-Phosphogluconate Dehydrogenases in Larvae

The genetic basis of modulation by dietary sucrose of the enzyme activities glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) activities in third instar larvae of Drosophila melanogaster was investigated, using isogenic lines derived from wild populations. Considerable genetically determined variation in response was detected among lines that differed only in their third chromosome constitution. Comparison of cross-reacting material between a responding and a nonresponding line showed that the G6PD activity variation is due to changes in G6PD protein level. These differences in responses are localized in the fat body, with 300 mM sucrose in the diet resulting in a sixfold stimulation of G6PD activity and a fourfold one of 6PGD in the line showing the strongest response. In this tissue, the responses of the two enzymes are closely correlated with one another. Using recombinant lines, we obtained data that suggested the existence of more than one gene on chromosome III involved in the regulation of G6PD in the fat body, and at least one of these genes affects the level of 6PGD as well.     

Identification of Macrolepiota procera extract as a novel G6PD inhibitor for the treatment of lung cancer

Tumor metabolism, an emerging hallmark of cancer, is characterized by aberrant expression of enzymes from various metabolic pathways including glycolysis and PPP (pentose phosphate pathway). Glucose 6 phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD), oxidative carboxylases of PPP, have been reported to accomplish different biosynthetic and energy requirements of cancer cells. G6PD and 6PGD have been proposed as potential therapeutic targets for cancer therapy during recent years due to their overexpression in various cancers. Here, we have employed enzymatic assay based screening using in-house G6PD and 6PGD assay protocols for the identification of mushroom extracts which could inhibit G6PD or 6PGD enzymatic activity for implications in cancer therapy. For the fulfillment of the objectives of present study, nine edible mushrooms were subjected to green extraction for preparation of ethanolic extracts. 6xhis-G6PD and pET-28a-h6PGD plasmids were expressed in BL21-DE3 E. coli cells for the expression and purification of protein of interests. Using purified proteins, in house enzymatic assay protocols were established. The preliminary screening identified two extracts (Macrolepiota procera and Terfezia boudieri) as potent and selective G6PD inhibitors, while no extract was found highly active against 6PGD. Further, evaluation of anticancer potential of mushroom extracts against lung cancer cells revealed Macrolepiota procera as potential inhibitor of cancer cell proliferation with IC₅₀ value of 6.18 μg/ml. Finally, screening of M. procera-derived compounds against G6PD via molecular docking has identified paraben, quercetin and syringic acid as virtual hit compounds possessing good binding affinity with G6PD. The result of present study provides novel findings for possible mechanism of action of M. procera extract against A549 via G6PD inhibition suggesting that M. procera might be of therapeutic interest for lung cancer treatment.     

Natural and synthetic alleles provide complementary insights into the nature of selection acting on the Men polymorphism of Drosophila melanogaster

Two malic enzyme alleles, Men^113A and Men^113G, occur at approximately equal frequency in North American populations of Drosophila melanogaster, while only Men^113A occurs in African populations. We investigated the population genetics, biochemical characteristics, and selective potential of these alleles. Comparable levels of nucleotide polymorphism in both alleles suggest that the Men^113G allele is not recently derived, but we find no evidence in the DNA sequence data for selection maintaining the polymorphism. Interestingly, the alleles differ in both V_max and K_m for the substrate malate. Triglyceride concentration and isocitrate dehydrogenase (IDH) and glucose-6-phosphate dehydrogenase (G6PD) activities are negatively correlated with the in vivo activities of the Men alleles. We examined the causality of the observed correlations using P-element excision-derived knockout alleles of the Men gene and found significant changes in the maximum activities of both IDH and G6PD, but not in triglyceride concentration, suggesting compensatory interactions between MEN, IDH, and G6PD. Additionally, we found significantly higher than expected levels of MEN activity in knockout heterozygotes, which we attribute to transvection effects. The distinct differences in biochemistry and physiology between the naturally occurring alleles and between the engineered alleles suggest the potential for selection on the Men locus.