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.     

Gene modulation in Drosophila: Dosage compensation of Pgd + and Zw + genes

The sex-linked Pgd ⁺ and Zw ⁺ genes of Drosophila melanogaster and their associated enzyme activities 6-phosphogluconate dehydrogenase and glucose 6-phosphate dehydrogenase were employed in an analysis of the relationship between dosage compensation and the location of genes in the genome. In the genotypes examined, the enzyme activity specified by each copy of the gene is twice in males what it is in females. This is true of normal, structurally rearranged, and duplication genotypes. Dosage compensation, therefore, is a regulatory function associated with single structural genes or small chromosomal segments and does not depend on the gene's physical location on the X chromosome.This research was supported by NIH Grant No. 5-R01-HD04859.     

Glucose 6-phosphate dehydrogenase in Drosophila: A sex-influenced electrophoretic variant

A variant of glucose 6-phosphate dehydrogenase (G6PD) in Drosophila melanogaster shows different electrophoretic migration in males and females. In heterozygotes, the variant influences the migration of G6PD produced by both chromosomes. Mixing of homogenates of males and females changes migration of the female-produced enzyme, suggesting that a protein produced in males is capable of altering the variant G6PD molecule. The hypothetical protein is also present in pseudomales and intersexes produced by sex transformation genes.This research was supported by NIH grants # 5-T1-GM 216-06 and GM 12768-01 and NSF grants GB 4587 and GB 4824.     

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.     

Heterochromatin as a factor influencing X-chromosome inactivation in hybrids of the common vole (Microtinae, Rodentia)]

Expression of X-linked genes for G6PD and alpha-GAL was studied in female interspecific hybrids of Microtus. The G6PD and alpha-GAL isozymes of Microtus arvalis were found to predominate in all cases when a species carrying a heterochromatin block on the X-chromosome served as one partner of hybridization and M. arvalis containing no heterochromatin block served as another. The proportions of G6PD and alpha-GAL parental forms were approx. equal in hybrid females when both species participating in hybridization contained heterochromatin blocks on X-chromosomes. Cytological analysis for revealing active and nonactive X-chromosomes on metaphase spreads of hybrid females supports the biochemical data. Non-random inactivation of X-chromosomes carrying the heterochromatin blocks in the interspecific hybrids with M. arvalis and a random one, when both parents contain heterochromatin blocks on the X-chromosomes are supposed to be the cause for the phenomenon observed. The study provided data supporting our previous hypothesis that heterochromatin affects the X-chromosome inactivation process in interspecific hybrid voles.     

Fine gentic structure of the 2D3-2F5 region of the X-chromosome of Drosophila melanogaster

Summary 97 lethal and semilethal mutations were induced by ethyl methanesulfonate, nitrosomethyl urea and -irradiation in the 2D3-F5 region of the X-chromosome of D. melanogaster. Approximately 1 per cent of the tested X-chromosomes carried a lethal in the 2D3-2F5 region. The mutation frequencies per band or DNA content in this region and the whole X-chromosome are equal.Complementation analysis revealed at least 10 functionally independent essential loci in this region including about 10 bands. The data presented in this study support the one bandone gene hypothesis.The Pgd locus coding for 6-phosphogluconate dehydrogenase (6PGD) is mapped in the 2D3 (or 2D4) band. Isolation of 11 lethal or semilethal point mutations with null or reduced 6PGD activity shows that the Pgd locus is a vital one.     

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.     

Viability Interactions, IN VIVO Activity and the G6pd Polymorphism in DROSOPHILA MELANOGASTER

Several biochemical studies have suggested that in Drosophila melanogaster the two common allozymes of G6PD differ in their in vitro activities and thermal stabilities. Yet, it remains to be shown that these characterizations reflect actual in vivo differences and are not artifacts of the biochemical approach. In this study it is shown that in vivo activity differences must exist between these two variants. This conclusion arises from the observation that the viability of flies bearing a low activity allele of 6PGD is strongly dependent on the genotype at the G6PD (Zw) locus, whereas no measurable difference in viability can be detected between Zw genotypes in a normal activity 6PGD background. These viability interactions are in the direction predicted by the reported in vitro activities of the allozymes and the proposed deleterious effects of 6-phosphogluconate accumulation.—In addition, a genetic scheme is used that uncouples and quantifies the effects of viability modifiers in the region of the Zw locus, while homogenizing 98% of the X chromosome. The viability of different Zw genotypes is measured by examining whole chromosome viabilities relative to the FM6 balancer chromosome. The advantages of this particular scheme are discussed.     

Investigations on the organization of genetic loci in Drosophila melanogaster: lethal mutations affecting 6-phosphogluconate dehydrogenase and their suppression.

Summary The molecular nature of lethal and semilethal mutations in the Pgd locus of D. melanogaster coding for 6-phosphogluconate dehydrogenase (6PGD) was studied. All the 11 mutations affect the structural gene of the Pgd locus: 3 semilethal mutations resulted in altered 6PGD molecules with decreased catalytic activities; the rest 8 lethals were null alleles characterized by mutant polypeptides capable of reacting with antisera against highly purified 6PGD.Null or low activity alleles for glucose-6-phosphate dehydrogenase induced by ethyl methanesulfonate were shown to be suppressors for the lethal mutations in the Pgd locus.A monocistronic type of organization of the Pgd locus is suggested taking into account the biochemical mechanism of suppression of the Pgd-lethals and their location in the structural gene coding for 6PGD.     

Primate red cell enzymes: glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase

Glucose-6-phosphate dehydrogenase (E. C.: 1.1.1.49) phenotypes and 6-phosphogluconate dehydrogenase (E. C.: 1.1.1.44) phenotypes were determined by starch-gel electrophoresis of red cell hemolysates of Galago crassicaudatus subspp., Propithecus verreauxi, Lemur spp., Hapalemur griseus, and Macaca mulatta. A single glucose-6-phosphate dehydrogenase (G6PD) phenotype was found in each species. A single 6-phosphogluconate dehydrogenase (6PGD) phenotype was found in Lemur spp., Hapalemur griseus, and Galago crassicaudatus argentatus. In a group of six Propithecus verreauxi, three 6PGD phenotypes, PGD A, PGD AB, and PGD B, were found. Three phenotypes, PGD A, PGD AB, and PGD B, were found in 38 G. c. crassicaudatus. The three phenotypes in each species are apparently the products of two codominant autosomal alleles, PGD^A and PGD^B. The frequency of PGD^A in G. c. crassicaudatus is 0.263. A population of 260 free-ranging macaques displays a polymorphism at the 6PGD locus. Three phenotypes, PGD A, PGD AB, and PGD B, were found. These also appear to be controlled by two codominant autosomal alleles, PGD^A and PGD^B the frequency of PGD^A = 0.913. Additional analysis of three well-defined troops within the macaque population indicated that there are no significant differences between the troops or within the population at the 6PGD locus.     

The hierarchical relation between X-chromosomes and autosomal sex determining genes in Drosophila

The classical balance concept of sex determination in Drosophila states that the X-chromosome carries dispersed female-determining factors. Besides, a number of autosomal genes are known that, when mutant, transform chromosomal females (XX) into pseudomales (tra), or intersexes (ix, dsx, dsx). To test whether large duplications of the X-chromosome have a feminizing effect on the sexual phenotype of these mutants, we constructed flies that were mutant for ix, dsx, dsx or tra and had two X-chromosomes plus either a distal or a proximal half of an X-chromosome. These or even smaller X-chromosomal fragments had a strong feminizing effect when added to triploid intersexes (XX; AAA). In the mutants, however, no shift towards femaleness was apparent. We conclude that enhancing the female determining signal is ineffective in flies that are mutant for an autosomal sex determining gene, and therefore, that these genes are under hierarchical control of the signal given by the X:A ratio. Parallels between sex-determining and homeotic genes are drawn.     

X-linked glucose-6-phosphate dehydrogenase (G6PD) and autosomal 6-phosphogluconate dehydrogenase (6PGD) polymorphisms in baboons

Electrophoretic polymorphisms of glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) were examined in captive colonies of five subspecies of baboons (Papio hamadryas). Phenotype frequencies and family data verified the X-linked inheritance of the G6PD polymorphism. Insufficient family data were available to confirm autosomal inheritance of the 6PGD polymorphism, but the electrophoretic patterns of variant types (putative heterozygotes) suggested the codominant expression of alleles at an autosomal locus. Implications of the G6PD polymorphism are discussed with regard to its utility as a marker system for research on X-chromosome inactivation during baboon development and for studies of clonal cell proliferation and/or cell selection during the development of atherosclerotic lesions in the baboon model.     

Starch-gel electrophoretic variants of erythrocyte 6-phosphogluconate dehydrogenase in asian macaques

Five alleles with eight electrophoretic phenotypes of 6-phosphogluconate dehydrogenase were found in 1,195 blood samples from fourteen populations of nine macaque species.Macaca fascicularis from Malaya showed the most polymorphism, with three Pgd alleles resulting in five phenotypes.Macaca mulatta, M. speciosa, M. nemestrina, andM. cyclopis had two alleles each (although the last two species showed a high percentage of homozygosity). The remaining four species (M. fuscata, M. radiata, M. maura, andM. nigra) were homozygous for the Pgd^a allele. The predominance of Pgd^a was observed in all macaque species, exceptM. speciosa which showed a high (57%) frequency of Pgd^d. The distinctive position ofM. speciosa with regard to 6PGD variants parallels observations that indicate that this species carries transferrin and carbonic anhydrase I alleles in different frequencies from those of the other macaque species. Other similarities between the patterns of transferrin and 6PGD variations include a tendency toward homozygosity at the Pgd locus in the insular macaque forms. However, in this case only the Pgd^a allele is involved, while some variation was found in the transferrin alleles fixed by the founder effect in the insular macaques.This research was supported by NSF grants GF 253, GB 7426, and GB 15060 of the U.S.-Japan Cooperative Science and Systematic Biology Programs.     

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).     

Stage and organ specificity of the degree of variegation of the 6-phosphogluconate dehydrogenase gene in Drosophila melanogaster

Summary The effects of chromosomal rearrangements pn2, pn3, TE100 and TE101 on variegation of the gene Pgd, which controls the synthesis of 6-phosphogluconate dehydrogenase (PGD), were studied in Drosophila melanogaster. The electrophoretic patterns of PGD activity were first examined at different developmental stages. The degree of variegation of Pgd caused by pn2 and pn3 was higher in adult flies (the calculated percentage of cells with inactive Pgd was 70%–80%) as compared with larvae (about 50%). This difference can be explained by the tissue-specific mosaicism of Pgd expression; variegation was high in the neural ganglia, imaginal discs, and posterior gut but relatively low in the salivary glands, fat bodies and Malpighian tubes. In the case of TE100, neither tissue specificity, nor marked differences in the degree of variegation between larvae and adults were found. None of the rearrangements examined had an effect on the expression of Pgd in the ovary cells, but repression was seen in some cells of the male gonads. The data obtained suggest that the timing of clonal initiation is influenced by the rearrangements studied. The possible mechanisms preventing changes in the expression of the Pgd gene in the nurse cells caused by these rearrangements are discussed.     

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.