Cloning and dosage compensation of the 6-phosphogluconate dehydrogenase gene (Pgd+) of Drosophila melanogaster

Using a heterologous rat cDNA probe, we have identified a 14.7 kbp Drosophila melanogaster genomic clone containing the X-linked gene Pgd+, which encodes the enzyme 6-phosphogluconate dehydrogenase (6PGD). We used in situ hybridization to larval polytene chromosomes, a somatic transient expression assay for enzyme activity, and the rescue of the lethal Pgd- phenotype by germline transformation to verify the identity of the gene. A 7.4 kbp fragment including the gene and approximately 1.2 kbp of upstream and 1.8 kbp of downstream sequences was relocated to autosomal ectopic sites by germline transformation; this transduced gene exhibits levels of enhanced activity in males comparable to those of the indigenous gene at its normal X chromosome locus. We conclude that the sequences responsible for dosage compensation of Pgd+ are included in this fragment.     

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.     

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.     

Segmental Aneuploidy and Enzyme Activity as a Method for Cytogenetic Localization in DROSOPHILA MELANOGASTER

A method of mapping genes which specify enzymes without the necessity of obtaining genetic variants has been explored. Three enzymes whose structural genes have known genetic positions were chosen to see if the relationship between gene dosage and enzyme activity could be used as a tool in cytological localization. Zw, the gene specifying G6PD, is located in the X chromosome region, 18D-18F. The structural gene for 6PGD, Pgd, maps in the X chromosome bands 2C1-2E1. Idh-NADP, the gene which specifies IDH-NADP, is found on the third chromosome, in bands 66B-67C.     

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.     

6-Phosphogluconate dehydrogenase (PGD) genetics in the mouse: Linkage with metabolically related enzyme loci

An electrophoretic polymorphism of 6-phosphogluconate dehydrogenase (PGD) has been observed in the subspecies Mus musculus musculus from northern Denmark. M. m. musculus is interfertile with inbred strains of mice, and F1 hybrids with C57BL/6J show a three-banded phenotype. This pattern is consistent with a dimeric enzyme structure with codominant expression of alleles. In backcrosses and the F2 generation, PGD segregated as a singly autosomal gene, designated Pgd, closely linked to Gpd-1 on chromosome 4(1.7 +/- 1.1%). Both gene products are dimers, both require NADP, and these enzymes catalyze sequential steps in metabolism.     

Gene order and recombination rates in the linkage group S-Phi-Hal-H-(Po2)-Pgd in pigs

Families of Czech Landrace (94 litters and 636 offspring) were tested for halothane sensitivity, A-O (S), H, PHI and PGD phenotypes. Informative matings for the estimation of recombination rates between marker loci were selected. The following recombination frequencies were established: S - Phi = 4.8% (2.5%-10.7%); S - H = 6.8% (4.3%-11.7%); Phi - H = 2.6% (0.9%-5.3%); H - Pgd = 4.4% (1.6%-8.0%). Cross-overs were observed also between S - Hal, Hal - H and Hal - Pgd, but were not found between Phi - Hal. On the basis of these results it has been possible to revise the position of the S locus in this linkage group. The most probable gene order would be: S - Phi - Hal (or Hal - Phi) - H - (Po2) - Pgd. A striking difference was found between the number of halothane-sensitive pigs (87) and HalnHaln genotypes determined by haplotyping (123). Segregation rates in 19 backcross matings and experimental matings of the animals proved that this difference is mostly due to incomplete penetrance or low expression of halothane sensitivity.     

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.     

A new allele at the Pgd locus in pigs

A new electrophoretic variant of porcine 6-phosphogluconate dehydrogenase (PGD) is described. The new variant, PGD C, has been shown to be controlled by a third allele, PgdC, at the Pgd locus.     

Expression and distribution of cytosolic 6-phosphogluconate dehydrogenase isozymes in maize

Maize (Zea mays L.) cytosolic 6-phosphogluconate dehydrogenase isozymes (EC 1.1.1.44; 6-PGD) are encoded by unlinked lociPgd1 andPgd2. Two families from a Robertson's Mutator line were isolated which have no detectable expression ofPgd2. ThesePgd2-null mutants and aPgd1-null line were used to generate plants homozygous for null alleles at both cytosolic 6-PGD loci. The specific activity of 6-PGD in the double-null mutant was between 20 and 30% of wild-type levels in root extracts. The double-null mutant was reproductively viable in a moderate environment, suggesting that wild-type levels of cytosolic 6-PGD activity are not essential for growth. Isozyme dimer ratios in roots, leaves, and scutellum were binomial and reflected the wild-type gene copy number. 6-PGD isozymes showed tissue- and cell type-specific expression. This research was supported by grants from the United States Department of Agriculture (Individual Postdoctoral Grant 89-37264-4837 to J.B.-S.) and the National Institutes of Health (Postdoctoral Grant 5-F32-GM11112-03 to J.B.-S. and Research Grant 2-R01-GM21734 to M.F.).     

Gene order and recombination rates in the linkage group S-Phi-Hal-H-(Po2)-Pgd in pigs

Families of Czech Landrace (94 litters and 636 offspring) were tested for halothane sensitivity, A-O (S), H, PHI and PGD phenotypes. Informative matings for the estimation of recombination rates between marker loci were selected. The following recombination frequencies were established: S-Phi = 4.8 % (2.5 % -10.7 %);S-H = 6.8 % (4.3 %-11.7 %); Phi-H = 2.6 % (0.9 %-5.3 9%); H-Pgd = 4.4 % (1.6 %-8.0 %). CCCC-overs were observed also between S- Hal, Hal-H andHal - Pgd, but were not found between Phi - Hal. On the basis of these results it has been possible to revise the position of the S locus in this linkage group. The most probable gene order would be: S - Phi - Hal (or Hal - Phi) -H- (P02) - Pgd.
A striking difference was found between the number of halothane-sensitive pigs (87) and HalⁿHal ⁿ genotypes determined by haplotyping (123). Segregation rates in 19 backcross matings and experimental matings of the animals proved that this difference is mostly due to incomplete CCC or low expression of halothane sensitivity.     

Growth-phase-dependent induction of 6-phosphogluconate dehydrogenase and glucose 6-phosphate dehydrogenase in the cyanobacterium Synechococcus sp. PCC7942.

In most cyanobacteria, the only known pathway for oxidation of stored carbohydrate in the dark or under energy-limiting conditions is the hexose monophosphate shunt. To determine whether the increased use of the shunt under these conditions derives from an increase in the activity level of the respective enzymes, we measured the effect of growth phase during the growth of batch cultures of Synechococcus sp. strain PCC7942 on the specific activity of 6-phosphogluconate dehydrogenase (6PGD) and glucose 6-phosphate dehydrogenase. The specific activities were constant during the exponential growth phase of the culture, but they increased about fivefold during the transition into stationary phase. As an approach to determining the level of expression at which the growth-phase-dependent regulation of 6PGD level is exerted, we constructed operon and gene fusions between the gnd gene, which encodes 6PGD, and the Escherichia coli lacZ gene, which encodes beta-galactosidase (beta Gal). Strains harboring the fusions integrated into the cyanobacterial chromosome were prepared, and the growth-phase dependence of beta Gal level was determined. The specific activity of beta Gal in cultures of both types of fusion strains increased during the transition into stationary phase, indicating that the growth-phase-dependent regulation is on the gnd mRNA level. Characterization of the growth-phase-dependent induction of 6PGD in strains carrying differing amounts of DNA upstream from the gnd structural gene led to the localization of the promoter and the regulatory site on the restriction map of the gene, whose sequence has previously been determined.(ABSTRACT TRUNCATED AT 250 WORDS)     

The main effect of chromosomal rearrangement is changing the action of regulatory genes

Effect of chromosomal rearrangements on the expression of mutations was studied in Drosophila melanogaster regulatory genes. These were facultative dominant lethals and recessive lethals on the X chromosome obtained by the classical Muller-5 method. Chromosomal rearrangements drastically changed the expression of regulatory gene mutations. Rearrangements either caused the lethal effect of mutations or suppressed the already present lethality. The action of rearrangements exhibited the maternal or paternal effect. Irrespective of the presence in the genome of mutations at regulatory genes, a rearrangement acted as a factor decreasing fertility of the organism. The rearrangement effect is identical to the expression of regulatory genes per se. It is concluded that the chromosomal rearrangement affects the examined regulatory genes indirectly through a change in the operation of regulatory genes located within the rearrangement. Thus, rearrangements gain great importance for the definition of the pattern of genome functional activity. Widespread distribution of rearrangements in individual genotypes and their effectivity in the process of speciation are thus explained.     

The Main Effect of Chromosomal Rearrangement Is Changing the Action of Regulatory Genes

Effect of chromosomal rearrangements on the expression of mutations was studied in Drosophila melanogaster regulatory genes. These were facultative dominant lethals and recessive lethals on the X chromosome obtained by the classical Muller-5 method. Chromosomal rearrangements drastically changed the expression of regulatory gene mutations. Rearrangements either caused the lethal effect of mutations or suppressed the already present lethality. The action of rearrangements exhibited the maternal or paternal effect. Irrespective of the presence in the genome of mutations of regulatory genes, a rearrangement acted as a factor decreasing fertility of the organism. The rearrangement effect is identical to the expression of regulatory genes per se. It is concluded that the chromosomal rearrangement affects the examined regulatory genes indirectly through a change in the operation of regulatory genes located within the rearrangement. Thus, rearrangements gain great importance for the definition of the pattern of genome functional activity. Widespread distribution of rearrangements in individual genotypes and their effectivity in the process of speciation are thus explained.     

Effects of nicotine and vitamin E on 6-phosphogluconate dehydrogenase activity in some rat tissues in vivo and in vitro

The aim of this study was to investigate whether nicotine affects 6-phosphogluconate dehydrogenase (6PGD) enzyme activity in some rat tissues, and to see the modulatory effects of vitamin E on this effect in vivo. In addition, the effects of nicotine and vitamin E on 6PGD activity were also tested in vitro. The groups were: nicotine [0.5 mg/kg/day, intraperitoneal (i.p.)]; nicotine + vitamin E [75 mg/kg/day, intragastric (i.g.)]; and control group (receiving only vehicles). There were eight rats per group and supplementation period was 3 weeks. The results of in vivo study showed that nicotine activated the muscle, lungs, and testicular 6PGD enzyme activity but had no effect on heart and liver 6PGD activity. Also, nicotine + vitamin E activated the muscle, testicle, and liver 6PGD enzyme activity, while this combination had no effect on heart, and lungs in vivo. When nicotine is administered with vitamin E the increase in 6PGD enzyme activity in muscle and testicles were lower. On the other hand the increase in 6PGD enzyme activity was eliminated by vitamin E in lungs, while 6PGD enzyme activity was increased by vitamin E, which was not affected by nicotine only. In vitro results correlated well with in vivo experimental results. Our results suggest that vitamin E may favourably increase 6PGD enzyme activity in liver in nicotine treated rats, while it has negligible effects on this enzyme activity in other tissues.     

Implications of the Hiroshima-Nagasaki genetic studies for the estimation of the human "doubling dose" of radiation

Since 1946 a continuous effort to evaluate the potential genetic effects of the atomic bombs has been sustained. Observations on children born in Hiroshima and Nagasaki include sex ratio, congenital malformations, stillbirths, survival of liveborn infants, chromosomal abnormalities (sex chromosomal abnormalities and balanced chromosomal rearrangements), mutations altering protein structure or activity, and physical growth and development. There are no statistically significant differences between the children of parents who received increased amounts of radiation at the time of the bombings and those whose parents did not. However, the difference between the two sets of children is consistent with the hypothesis of a genetic effect of the exposure, but its magnitude suggests humans are not as sensitive to the genetic effects of radiation as projected from the mouse paradigm.     

Effects of nicotine and vitamin E on 6-phosphogluconate dehydrogenase activity in some rat tissues in vivo and in vitro

The aim of this study was to investigate whether nicotine affects 6-phosphogluconate dehydrogenase (6PGD) enzyme activity in some rat tissues, and to see the modulatory effects of vitamin E on this effect in vivo. In addition, the effects of nicotine and vitamin E on 6PGD activity were also tested in vitro. The groups were: nicotine [0.5 mg/kg/day, intraperitoneal (i.p.)]; nicotine + vitamin E [75 mg/kg/day, intragastric (i.g.)]; and control group (receiving only vehicles). There were eight rats per group and supplementation period was 3 weeks. The results of in vivo study showed that nicotine activated the muscle, lungs, and testicular 6PGD enzyme activity but had no effect on heart and liver 6PGD activity. Also, nicotine + vitamin E activated the muscle, testicle, and liver 6PGD enzyme activity, while this combination had no effect on heart, and lungs in vivo. When nicotine is administered with vitamin E the increase in 6PGD enzyme activity in muscle and testicles were lower. On the other hand the increase in 6PGD enzyme activity was eliminated by vitamin E in lungs, while 6PGD enzyme activity was increased by vitamin E, which was not affected by nicotine only. In vitro results correlated well with in vivo experimental results. Our results suggest that vitamin E may favourably increase 6PGD enzyme activity in liver in nicotine treated rats, while it has negligible effects on this enzyme activity in other tissues.     

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.