Genetic localization and sequential electrophoresis of glucose-6-phosphate dehydrogenase in Drosophila melanogaster

Studies were undertaken to investigate two critical aspects of the glucose-6-phosphate dehydrogenase polymorphism in Drosophila melanogaster. The first investigation unequivocally maps the genetic site of the G6PD locus to the X chromosome. The second study subjects a set of isochromosomal lines to sequential electrophoresis in an attempt to uncover common molecular heterogeneity within the global polymorphism, assuming that this variation may have gone undetected under conventional electrophoretic conditions. The genetic site was mapped following the segregation of the two common electrophoretic alleles, a so-called null allele, and two rare electrophoretic variants. From the pooled results, the Zw locus mapped to 62.9 on the X chromosome relative to the flanking markers car (at 62.5) and sw (at 64.7). A set of 126 iso-X chromosomal lines of diverse geographic origin was subjected to sequential electrophoresis under three different acrylamide conditions in addition to the conventional starch electrophoretic system. No additional variation beyond the common diallele polymorphism was seen.     

Genetic Limits of the Xanthine Dehydrogenase Structural Element within the Rosy Locus in DROSOPHILA MELANOGASTER

Experiments are described that provide an opportunity to estimate the genetic limits of the structural (amino acid coding) portion of the rosy locus (3:52.0) in Drosophila melanogaster, which controls the enzyme, xanthine dehydrogenase (XDH). This is accomplished by mapping experiments which localize sites responsible for electrophoretic variation in the enzyme on the known genetic map of null-XDH rosy mutants. Electrophoretic sites are distributed along a large portion of the null mutant map. A cis-trans test involving electrophoretic variants in the left- and right-hand portions of the map leads to the conclusion that the entire region between these variants is also structural. Hence most, if not all, of the null mutant map of the rosy locus contains structural information for the amino acid sequence of the XDH polypeptide. Consideration is given to the significance of the present results for the general problem of gene organization in higher eukaryotes.     

Hidden Electrophoretic Variation at the Xanthine Dehydrogenase Locus in a Natural Population of DROSOPHILA MELANOGASTER

Sixty-two isochromosomal lines of D. melanogaster were screened for cryptic electrophoretic variation at the xanthine dehydrogenase (XDH) locus. Sequential polyacrylamide vertical slab gel electrophoresis was performed using four electrophoretic criteria. A total of 15 classes of electromorphs were revealed. D. melanogaster appears to exhibit as much polymorphism at this locus as other extensively studied Drosophila species.--No evidence for loci on the X or second chromosomes which modified XDH mobility was found. Six of the electromorphs were mapped to the Xdh (ry) structural locus. Eight of the remaining nine classes exhibited mobility variation consistent with structural variation at the Xdh locus. The final class exhibited aberrant patterns and is under further study.     

Molecular and Phenotypic Variation of the Zw Locus Region in Drosophila Melanogaster

Restriction map polymorphism in a 13-kb region of the Zw locus in Drosophila melanogaster was investigated for 64 X chromosome lines with seven 6-cutter and ten 4-cutter restriction enzymes. A total of 203 restriction sites were scored, of which 20 were found to be polymorphic. The estimated nucleotide variation for this region for overall data (pi = 0.003 and 0.001, and theta = 0.003 and 0.002, for 4-cutter and 6-cutter studies, respectively) was smaller than that reported for most regions studied in D. melanogaster. It was found that the Slow allozyme has a larger nucleotide variation and haplotype diversity than the Fast allozyme. Results suggest the relatively recent divergence of the Fast allozyme from the Slow allozyme. Glucose 6-phosphate dehydrogenase (G6PD) activity was measured as a phenotype of the Zw locus. A significant difference in G6PD activity between allozymes was detected. The between-line effect was highly significant within the Slow allozyme, but was not significant within the Fast allozyme. Although a direct causative link could not be established, these results suggest an association between the amounts of quantitative and molecular genetic variation at the Zw locus region.     

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

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

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.     

Studies of esterase 6 in Drosophila melanogaster. XVIII. Biochemical differences between the slow and fast allozymes

Most natural populations of Drosophila melanogaster are polymorphic for two major electrophoretic variants at the esterase-6 locus. The frequency of the EST 6F allozyme is greatest in populations in warmer latitudes, whereas the EST 6S allozyme is predominant in colder latitudes. Latitudinal clines in electromorph frequencies are found on three continents. Purified preparations of the allozymes have been characterized for their pH optimum, substrate specificity, organophosphate inhibition, alcohol activation, thermal stability, and kinetic parameters. These and previous analyses of the EST 6 allozymes reveal that the two variants have differences in their physical and kinetic properties that may provide a basis for the selective maintenance of the polymorphisms and an explanation of the clinal variation observed in natural populations.      

Genetical Analysis of Chromosomal Interaction Effects on the Activities of the Glucose 6-Phosphate and 6-Phosphogluconate Dehydrogenases in DROSOPHILA MELANOGASTER

By combining ten second and ten third chromosomes, we investigated chromosomal interaction with respect to the action of the modifier factors on G6PD and 6PGD activities in Drosophila melanogaster. Analysis of variance revealed that highly significant chromosomal interaction exists for both enzyme activities. From the estimated variance components, it was concluded that the variation in enzyme activity attributed to the interaction is as great as the variation attributed to the second chromosome but less than attributed to the third chromosome. The interaction is not explained by the variation of body size (live weight). The interaction is generated from both the lack of correlation of second chromosomes for third chromosome backgrounds and the heterogeneous variance of second chromosomes for different third chromosome backgrounds. Large and constant correlation between G6PD and 6PGD activities were found for third chromosomes with any second chromosome background, whereas the correlations for second chromosomes were much smaller and varied considerably with the third chromosome background. This result suggests that the activity modifiers on the second chromosome are under the influence of third chromosome factors.     

Intracistronic Mapping of Electrophoretic Sites in DROSOPHILA MELANOGASTER: Fidelity of Information Transfer by Gene Conversion

A convenient method is described for the intracistronic mapping of genetic sites responsible for electrophoretic variation of a specific protein in Drosophila melanogaster. A number of wild-type isoalleles of the rosy locus have been isolated which are associated with the production of electrophoretically distinguishable xanthine dehydrogenases. Large-scale recombination experiments were carried out involving null enzyme mutants induced on electrophoretically distinct wild-type isoalleles, the genetic basis for which is followed as a nonselective marker in the cross. Additionally, a large-scale recombination experiment was carried out involving null enzyme rosy mutants induced on the same wild-type isoallele. Examination of the electrophoretic character of crossover and convertant products recovered from the latter experiment revealed that all exhibited the same parental electrophoretic character. In addition to documenting the stability of the xanthine dehydrogenase electrophoretic character, this observation argues against a special mutagenesis hypothesis to explain conversions resulting from allele recombination studies.     

Molecular Population Genetics of the Distal Portion of the X Chromosome in Drosophila: Evidence for Genetic Hitchhiking of the Yellow-Achaete Region

We have estimated DNA sequence variation and differentiation within and between Drosophila melanogaster and its sibling species, Drosophila simulans, using six-cutter restriction site variation at yellow-achaete (y-ac), phosphogluconate dehydrogenase (Pgd), and period (per). These three gene regions are of varying distance from the telomere of the X chromosome and range from very low to moderate rates of recombination in D. melanogaster. According to Tajima's test of neutrality, the Pgd region has been influenced by balancing selection in D. melanogaster. This is consistent with previous data suggesting the allozyme polymorphism at this locus is visible to selection. The Hudson, Kreitman, Aguadé test of neutrality reveals a significant departure from neutrality for the y-ac region compared to the per or rosy regions in D. simulans. There is also a significant departure for the y-ac region compared to the Adh 5' flanking region in D. melanogaster. In both species the departure appears to be due to reduced variation at y-ac compared to that expected from divergence between D. simulans and D. melanogaster. We conclude that recent hitchhiking associated with the selective fixation of one or more advantageous mutants in the y-ac region is the best explanation for reduced variation at y-ac.     

The Rosy Region of Drosophila Melanogaster and Drosophila Simulans. I. Contrasting Levels of Naturally Occurring DNA Restriction Map Variation and Divergence

A 40-kb region around the rosy and snake loci was analyzed for restriction map variation among 60 lines of Drosophila melanogaster and 30 lines of Drosophila simulans collected together at a single locality in Raleigh, North Carolina. DNA sequence variation in D. simulans was estimated to be 6.3 times greater than in D. melanogaster (heterozygosities per nucleotide of 1.9% vs. 0.3%). This result stands in marked contrast to results of studies of phenotypic variation including proteins (allozymes), morphology and chromosome arrangements which are generally less variable and less geographically differentiated in D. simulans. Intraspecific polymorphism is not distributed uniformly over the 40-kb region. The level of heterozygosity per nucleotide varies more than 12-fold across the region in D. simulans, being highest over the hsc2 gene. Similar, though less extreme, variation in heterozygosity is also observed in D. melanogaster. Average interspecific divergence (corrected for intraspecific polymorphism) averaged 3.8%. The pattern of interspecific divergence over the 40-kb region shows some disparities with the spatial distribution of intraspecific variation, but is generally consistent with selective neutrality predictions: the most polymorphic regions within species are generally the most divergent between species. Sequence-length polymorphism is observed for D. melanogaster to be at levels comparable to other gene regions in this species. In contrast, no sequence length variation was observed among D. simulans chromosomes (limit of resolution approximately 100 bp). These data indicate that transposable elements play at best a minor role in the generation of naturally occurring genetic variation in D. simulans compared to D. melanogaster. We hypothesize that differences in species effective population size are the major determinant of the contrasting levels and patterns of DNA sequence and insertion/deletion variation that we report here and the patterns of allozyme and morphological variation and differentiation reported by other workers for these two species.     

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.     

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.     

Targeted Selection Experiments and Enzyme Polymorphism: Negative Evidence for Octanoate Selection at the G6PD Locus in DROSOPHILA MELANOGASTER

Published studies have reported significant selection with respect to the G6pd locus for Drosophila melanogaster reared on Na-octanoate food. We have reexamined the selective effects of Na-octanoate on egg to adult viability with respect to the G6pd polymorphism using specially constructed X chromosomes. Four experiments were carried out using different 6Pgd backgrounds in two recombinant sets of chromosomes segregating for the G6pd locus but constructed so as to minimize variation over most of the X chromosome. In addition, two measures of viability were used, and the size of the experiments and their associated degrees of freedom are approximately double those reported in the former studies. Our results find no evidence for differential selection on G6pd genotypes (males and females) by Na-octanoate and, therefore, do not corroborate the positive results of selection reported by other investigators. The reasons for our different results are discussed.     

Chemical basis of the electrophoretic variation observed at the alcohol dehydrogenase locus of Drosophila melanogaster.

The amino acid substitution responsible for the different electrophoretic mobility of the ADHs alleloenzyme and the ADHf alleloenzyme of the alcohol dehydrogenase from a Nigerian population of Drosophila melanogaster has been established as lysine (ADHs) for threonine (ADHf). This result is discussed with reference to the charge state model of electrophoretic variation, in conjunction with other know substitutions at this locus. It is concluded that electrophoretic methods should be capable of distinguishing many alleloenzymes which have identical isoelectric points without recourse to explanations involving conformational variability.     

The circular dichroism of ribosomal ribonucleic acids.

1. The c.d. (circular dichroism) of Drosophila melanogaster rRNA (42% G+C) and of G+C-rich fragments (78% G+C) obtained by partial hydrolysis of rabbit L-rRNA (the largest RNA species isolated from the large subribosomal particle) were measured and found to differ substantially. 2. To interpret these spectra a relation between c.d. of bihelical RNA and % G+C was derived, namely delta epsilonfG = AFG2+bfG+c, where deltaepsilonfG is the c.d. of RNA characterized by a mole fraction, fG, of guanine nucleotides and a, b and c are constants. 3. A frame of reference was established by studying the c.d. of a range of rRNA species, including S-rRNA (the RNA species isolated from the smaller subribosomal particle) and L-rRNA of Escherichia coli. 4. It was found for the rRNA species studied that 0.60+/-0.05 of residues appear to form bihelical secondary structure. 5. A higher helical content, 0.66+/-0.05, was found for the G+C-rich fragment of L-rRNA. The difference in the c.d. of rabbit L-rRNA and of D. melanogaster rRNA is attributable to the dependence of c.d. of the bihelical parts on %G+C. 6. The minimum in c.d. at 295 nm increases with increasing %G+C. The c.d. of rRNA was compared with that of the parent subparticle in this region of the spectrum, where high precision may be attained.     

The protein of the ejaculatory bulb (PEB) in Drosophila melanogaster. 1. Its stage specificity and sex dimorphism

PEB is the major protein (35-39 kDa) of highly differentiated ejaculatory bulbs in D. melanogaster. A minor ejaculatory bulb protein (hPEB) of about 80 kDa was detected using immunoblotting technique. Both proteins exhibit parallel genetic variation in electrophoretic mobility. This suggests that they are coded by the same gene. The proteins are present in adult males and are not detected in virgin females. During development they are first detected in male pupa at the stage of eye pigmentation (that is shortly before imago eclosion). The quantities of PEB and hPEB increase and reach the constant level at 6-10 day of imago development.     

Spatial variation of biochemical and ecological phenotypes in Drosophila: Electrophoretic and quantitative variation

Electrophoretic variation at three enzyme loci-alcohol dehydrogenase (Adh), glycerophosphate dehydrogenase (Gpdh), triosephosphate isomerase (Tpi)- is compared in Australian Drosophila melanogaster populations at three levels of spatial heterogeneity; among breeding sites, within populations, and between populations at the geographic level. Heterogeneity at the breeding site level greatly exceeds that among adults within populations, indicating greater intermixing at the mobile adult stage than at the developmentally immature and less migratory larval stage. Heterogeneity at the microspatial level is large relative to the geographic level at two of these loci. Spatial patterns of variation in ecological phenotypes are also considered. It is argued that electrophoretic variants may contribute little to an understanding of this quantitative variation, and that a more useful approach in ecological genetics is to consider ecological phenotypes as primary data.     

Notes on individual sequence variation in humans: immunoglobulin kappa light chain.

Little is known concerning the magnitude of variability in the nucleic acid sequence of DNA at the individual level. We have collected a large set of sequence data from the human immunoglobulin kappa light-chain-locus constant region (10,444 bp) and subgroup IV variable region (18,580 bp). For the constant region, absolute conservation of sequence was observed, even in intron and coding-region silent sites, with the exception of one previously defined polymorphic site. For the variable region, 12 heterozygous positions were identified, giving a heterozygosity of 6 x 10(-4) per nucleotide site. The amount of nucleic acid sequence variation differs significantly (chi 2 = 4.88) between these two regions, and the observed variation is two orders of magnitude lower than that reported for two Drosophila melanogaster loci. These data suggest that, for at least some regions of the human genome, nucleic acid sequence may be less variable than previously estimated.