Genetics and ontogeny of α-glycerophosphate dehydrogenase isozymes in Drosophila melanogaster

-Glycerophosphate dehydrogenase (GPDH) occurs in Drosophila melanogaster in three isozymic forms. These are separable by starch gel electrophoresis and have been tentatively numbered 1, 2, and 3. GPDH-1 is most concentrated in the adult thorax and GPDH-3 in the abdomen; 1 and 3 are in approximately equal amounts in the head. GPDH-2 is relatively weak in all preparations. In larvae, only GPDH-3 is present. Purified GPDH-1 has optimal activity at pH 6.7–7.0. GPDH-3 at pH 7.5, and GPDH-2 is intermediate. Changes in total GPDH activity parallel larval growth, pupal histolysis, and differentiation of adult tissues. In the latter period the ratio of activity at pH 6.7 to pH 7.6 increases, reflecting the shift from GPDH-3 to GPDH-1. Two types of homozygous GPDH patterns which differ in the electrophoretic mobilities of all three isozymes have been found in inbred strains. In heterozygous adults six bands, the parental forms of GPDH-1 and GPDH-3 and hybrid forms of each, can be resolved. Analysis of F₂ and backcross progeny suggests that a single genetic locus affects all three isozymes. Heterozygous embryos have only the maternal form of GPDH-3 until just before they hatch as first instar larvae. At this stage they have maternal and paternal GPDH-3 plus an intermediate band.This project was supported in part by National Institutes of Health research grant GM-15597.     

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

The basis for the differentiation of l-glycerol-3-phosphate dehydrogenase (-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 -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 -GPDH in all developmental stages. These observations suggest that all -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 -glycerophosphate cycle in the adult and larval stage of development.This investigation was supported in part by NIH Research Grant No. GM-15691 and Genetics Training Grant No. 2 TI GM-685 at the University of North Carolina and by NIH Research Grant No. GM-11546 at North Carolina State University.Paper No. 5054 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, North Carolina.     

Polymorphism at the α-glycerophosphate dehydrogenase locus in Drosophila melanogaster. I. Properties of adult allozymes

A biochemical comparison was made between alpha-glycerophosphate dehydrogenase allozymes from Drosophila melanogaster. Enzymes extracted from the three major genotypes were indistinguishable in terms of their pH optima and thermal stabilities. Distinctive differences were observed for three parameters; temperature dependence of specific activity, temperature dependence of K-m, and reaction rate constancy over a physiological temperature range. These results are discussed in terms of a model of balancing selection and the existence of spatial and temporal allele frequency clines in natural populations.     

Thermal stability of α-glycerophosphate dehydrogenase in Drosophila

Thermal stability of -glycerophosphate dehydrogenase-1 (-Gpdh-1) in nine Drosophila species was studied at pH's ranging from 6.4 to 8.5. This was done by measuring the changes in the activity of enzymes during the heat denaturation process. In addition to temperature, the rate of denaturation is highly dependent on the pH of the incubation buffer. The results of this study show that the thermal stability of enzyme molecules is different in different species. This holds true also in the species in which the enzymes have been found to be identical by other means. The differences between species of the Drosophila virilis group are discussed.This study was supported by funds from the National Research Council of Sciences of Finland.     

Heat stability studies at the α-glycerophosphate dehydrogenase locus in populations of Drosophila melanogaster

The level of hidden variation in populations of Drosophila melanogaster at the Gpdh ⁺ locus was determined by thermal stability studies of the protein. The results indicate a lack of variation using these methods both in and between the two common electrophoretic variants. It is suggested that -GPDH is conserved in primary structure, which may be related to its critical role in flight muscle metabolism.This investigation was supported by NIH Research Grants No. GM-11546 and GM-23617. Paper No. 5262 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, North Carolina 27607.     

Relationship between α-glycerophosphate dehydrogenase activity and metabolic rate during flight in Drosophila melanogaster

Measurements of wing-beat frequency (WBF) have been used to characterize flight muscle metabolic rate in Drosophila melanogaster during tethered flight. Progeny of crosses between 17 X-chromosome substitution lines and three null-activity stocks have been studied in order to determine the effect on flight metabolism of sharply reduced activity of -glycerophosphate dehydrogenase (GPDH). It was found that flies with an approximate 50% reduction in GPDH activity have a metabolic rate that is, in most cases, indistinguishable from that of wild-type flies and, in the most extreme cases, reduced by only 4%. These results demonstrate that Gpdh is not a major gene for flight metabolism, in the quantitative genetic sense of the term. These results are in agreement with the Kacser and Burns (1973, 1979, 1981) theory of flux, which postulates that the activity of an enzyme embedded in a multienzyme pathway can sometimes vary from wild-type to very low levels (perhaps 5–10% wild type) with no significant effect on flux through the total pathway.This research was supported by several grants to JWC: NSF Grant 8211667, a grant from the Graduate School, University of Minnesota, and a Research Career Development Award from the NIH.     

Repeated stress exposure results in a survival–reproduction trade-off in Drosophila melanogaster

While insect cold tolerance has been well studied, the vast majority of work has focused on the effects of a single cold exposure. However, many abiotic environmental stresses, including temperature, fluctuate within an organism''s lifespan. Given that organisms may trade-off survival at the cost of future reproduction, we investigated the effects of multiple cold exposures on survival and fertility in the model organism Drosophila melanogaster. We found that multiple cold exposures significantly decreased mortality compared with the same length of exposure in a single sustained bout, but significantly decreased fecundity (as measured by r, the intrinsic rate of increase) as well, owing to a shift in sex ratio. This change was reflected in a long-term decrease in glycogen stores in multiply exposed flies, while a brief effect on triglyceride stores was observed, suggesting flies are reallocating energy stores. Given that many environments are not static, this trade-off indicates that investigating the effects of repeated stress exposure is important for understanding and predicting physiological responses in the wild.     

A comparative study of enzyme activity variation between α-glycerophosphate and alcoholdehydrogenases in Drosophila melanogaster

An intraspecific comparison of -glycerophosphate (-GPDH: E.C.1.1.1.8) and alcohol dehydrogenase (ADH: E.C.1.1.1.1) enzyme activity levels was carried out in Drosophila melanogaster. The results indicate that (1) -GPDH is a relatively conservative and ADH a relatively variable enzyme system with regard to structurally determined activity variation but that (2) the conservative nature of -GPDH activity variation does not extend to the intra-genotypic level. The results are consistent with the view that different kinds of selective pressures are being exerted on the enzyme's structural and modifier gene loci.     

The Mechanisms Underlying α-Amanitin Resistance in Drosophila melanogaster: A Microarray Analysis

The rapid evolution of toxin resistance in animals has important consequences for the ecology of species and our economy. Pesticide resistance in insects has been a subject of intensive study; however, very little is known about how Drosophila species became resistant to natural toxins with ecological relevance, such as α-amanitin that is produced in deadly poisonous mushrooms. Here we performed a microarray study to elucidate the genes, chromosomal loci, molecular functions, biological processes, and cellular components that contribute to the α-amanitin resistance phenotype in Drosophila melanogaster. We suggest that toxin entry blockage through the cuticle, phase I and II detoxification, sequestration in lipid particles, and proteolytic cleavage of α-amanitin contribute in concert to this quantitative trait. We speculate that the resistance to mushroom toxins in D. melanogaster and perhaps in mycophagous Drosophila species has evolved as cross-resistance to pesticides, other xenobiotic substances, or environmental stress factors.     

The distribution of monoamine oxidase and α-glycerophosphate dehydrogenase in pig brain

Activities of the enzymes monoamine oxidase (EC 1.4.3.4), alpha-glycerophosphate dehydrogenase (EC 1.1.99.5) and cytochrome oxidase (EC 1.9.3.1) were determined in homogenates and in the mitochondrial fraction prepared from individual regions of pig brain. The variation in the activity of alpha-glycerophosphate dehydrogenase paralleled that of cytochrome oxidase, but this was not the case with monoamine oxidase. The differences in the activities of the enzymes among homogenates of the various regions of the brain persisted in mitochondria prepared from these homogenates. The purification of these three enzymes paralleled each other when mitochondria were prepared, suggesting that the three enzymes are bound to the same particles.     

Enzyme specificity: “pseudopolymorphism” of lactate dehydrogenase in Drosophila melanogaster

An electrophoretic variant in the LDH (l-lactate:NAD oxidoreductase, E.C.1.1.1.27) of Drosophila melanogaster was observed on starch (or polyacrylamide) gels. This variant was found to exhibit an identical isozymic pattern (three isozymes with a decreasing staining density) on starch gel and map position as the Adh locus. On the other hand, anodal polyacrylamide gel electrophoresis in crude extracts has shown LDH to consist of nine bands and ADH of four bands. We have shown that ADH (Alcohol:NAD oxidoreductase, E.C.1.1.1.1) also oxidizes l(+)-lactate or d(–)-lactate with the NAD, while LDH oxidizes ethanol. By using various genetic and biochemical techniques, we have shown that the observed Ldh electrophoretic variant was not a real one and could be attributed to the presence of ADH. We have called this phenomenon pseudopolymorphism, and the problem of enzyme specificity has been examined. The appearance of a band in an assay using lactic acid as a substrate is not sufficient evidence for the presence of LDH. Hence, caution is called for before characterizing an electrophoretic band on a gel as being equivalent to the presence of a genetic locus. Out of the nine electrophoretic zones of activity observed on polyacrylamide gel (or out of the six previously observed) using crude extract, only two (one major and one minor) belong to LDH, as revealed by purified enzyme preparations. Furthermore, purified LDH exhibits activity in two bands on starch gel (out of three observed in crude extracts), which appear in different positions as compared with those of ADH. Finally, one band which responds to the presence of d(–)-lactate but not to l(+)-lactate has been revealed.     

Extensive variation at the α-glycerophosphate dehydrogenase locus in species of waterstriders (Gerridae: Hemiptera)

Variation at the -glycerophosphate dehydrogenase (-Gpdh; EC 1.1.1.8) locus was surveyed in 11 species of waterstriders (Gerridae: Hemiptera) and five other species of aquatic Hemiptera. Species of waterstriders exhibited considerable inter- and intraspecific variation in degree of winglessness. Average heterozygosity (0.401±0.090) and average number of observed electromorphs (5.36±0.96) for the 11 gerrid species were well above values reported for nearly all other insect species surveyed to date. Wing-monomorphic and wing-polymorphic species did not differ in average -Gpdh heterozygosity. Of the three wing-polymorphic species surveyed geographically, two species exhibited marked variation in wing-morph frequencies but homogeneous -Gpdh allele frequencies. The third species exhibited geographically homogeneous -Gpdh and wing-morph frequencies, but no significant association between -Gpdh phenotype and wing morph was observed in any surveyed population. These results are consistent with hypotheses evoking either relaxed purifying selection at the -Gpdh locus in species of Gerridae due to the apparent reduced importance of flight, or selective maintenance of common -Gpdh electromorphs.This work was supported by NSF Grant DEB 76-20967 to Alan H. Brush, funds from the Research Foundation of the University of Connecticut to Carl W. Schaefer, and USPHS Grant GM 21133 to Richard K. Koehn.     

Functional significance of amylase polymorphism in Drosophila melanogaster. III. Ontogeny of amylase and some α-glucosidases

Changes in amylase (E.C. 3.2.1.1), maltase (E.C. 3.2.1.20), sucrase, and PNPGase activities in relation to changes in wet weight and protein content were studied during the development of larvae and adult flies from two strains of Drosophila melanogaster, homozygous for different amylase alleles. All -glucosidase activities increase exponentially during a large part of larval development, parallel to the increase in weight, and drop at the end of the third instar. Amylase activity of the Amy ¹ strain follows the same pattern. In contrast, amylase activity of the Amy ^4,6 strain continues its exponential increase longer. In the third larval instar amylase activity in the Amy ^4,6 strain becomes much higher than in the Amy ¹ strain. During the first hours of adult life amylase activity of the two strains does not differ. Then Amy ^4,6 activity starts to rise and becomes much higher (4–5 times) than Amy ¹ amylase activity, which remains approximately constant. All adult enzyme activities are much higher than in larvae. Comparison of enzyme activity of amylase and -glucosidases in larvae and adults confirms that differences in amylase activities can become important only when starch is a limiting factor in the food.The investigations were supported by the Foundation for Fundamental Biological Research (BION), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).     

On Recombination among In(2L)t, α-Gpdh and Adh in Drosophila melanogaster

The occurrence and patterns of linkage disequilibrium between an inversion and allozymic loci within it or nearby have been used in attempts to discriminate among different hypotheses for the maintenance of variability. The interpretation of the data on the best-documented case, that of the nonrandom association between In(2L)t and alpha-Gpdh or Adh in the second chromosome of Drosophila melanogaster, has been done on the basis that recombination between alpha-Gpdh and Adh is almost entirely due to the recombination between In(2L)t and the locus within it (alpha-Gpdh), the recombination between the inversion and the nearby locus (Adh) being negligible. In this paper, we show that the pattern of recombination is just the opposite.     

Molecular polymorphism in Drosophila melanogaster and D. simulans: what have we learned from recent studies?

We present a review of recent studies of molecular polymorphism in Drosophila melanogaster and D. simulans. The availability of African and non-African samples for these species makes it possible to compare microsatellite and DNA sequence polymorphism between these species, both inside and outside their native regions. There are four main results: (i) variability is larger in African populations; (ii) variation is usually higher on the autosomes, except for African D. melanogaster; (iii) DNA sequence variation is higher on D. simulans than on D. melanogaster autosomes; (iv) the ratio of replacement to silent polymorphisms is higher for D. melanogaster autosomal loci. We summarize the main hypotheses put forward to explain these results.     

The stimulatory Gα(s) protein is involved in olfactory signal transduction in Drosophila

Seven-transmembrane receptors typically mediate olfactory signal transduction by coupling to G-proteins. Although insect odorant receptors have seven transmembrane domains like G-protein coupled receptors, they have an inverted membrane topology, constituting a key difference between the olfactory systems of insects and other animals. While heteromeric insect ORs form ligand-activated non-selective cation channels in recombinant expression systems, the evidence for an involvement of cyclic nucleotides and G-proteins in odor reception is inconsistent. We addressed this question in vivo by analyzing the role of G-proteins in olfactory signaling using electrophysiological recordings. We found that Gα_s plays a crucial role for odorant induced signal transduction in OR83b expressing olfactory sensory neurons, but not in neurons expressing CO₂ responsive proteins GR21a/GR63a. Moreover, signaling of Drosophila ORs involved Gα_s also in a heterologous expression system. In agreement with these observations was the finding that elevated levels of cAMP result in increased firing rates, demonstrating the existence of a cAMP dependent excitatory signaling pathway in the sensory neurons. Together, we provide evidence that Gα_s plays a role in the OR mediated signaling cascade in Drosophila.     

Genetic polymorphism of α-L-fucosidase in brittany (France)

Summary The authors studied the phenotypic distribution of -L-fucosidase in a random sample of the population of the area of Rennes (France). The frequencies of Fu ¹ (0.64) and Fu ² (0.36) genes are significantly different from the frequencies observed in New York whites and blacks.