Purification and genetic control of NAD-dependent glutamate dehydrogenase from Drosophila melanogaster

Glutamate dehydrogenase has been purified to near-homogeneity from mature larvae of Drosophila melanogaster. The enzyme has a molecular weight of 347,000 measured by sucrose gradient sedimentation and 343,000 measured by variable-porosity acrylamide gel electrophoresis. Electrophoresis under denaturing conditions showed that the enzyme consists of six subunits of molecular weight 57,000. The structural gene for GDH has been mapped at 81.7±0.8 on the third chromosome by means of an electrophoretic variant.This work was supported by CNR Contract 76-01961-04.     

Rudimentary locus of Drosophila melanogaster: Partial purification of a carbamylphosphate synthase-aspartate transcarbamylase-dihydroorotase complex

Glutamine-dependent CPSase, ATCase, and DHOase from Drosophila, the first three enzymes in pyrimidine biosynthesis, show coordinate variation in activity throughout development. The three activities were highest in first instar larvae and decreased as development proceeded. The three activities cosediment in sucrose gradients as a single peak with a relative sedimentation coefficient of approximately 30S. CPSase, ATCase, and DHOase copurify during (NH₄) ₂SO₄ fractionation and during DEAE-cellulose and hydroxylapatite chromatography.This work was supported by a grant from the National Science Foundation (No. PCM75-22802). In addition, Stuart I. Tsubota was a NIH Predoctoral Trainee (No. 5T32-GM07 127) and Susan E. Germeraad was a Cystic Fibrosis Foundation Postdoctoral Fellow.     

Purification and properties of the enzymes from Drosophila melanogaster that catalyze the synthesis of sepiapterin from Dihydroneopterin triphosphate

Sepiapterin synthase, the enzyme system responsible for the synthesis of sepiapterin from dihydroneopterin triphosphate, has been partially purified from extracts of the heads of young adult fruit flies (Drosophila melanogaster). The sepiapterin synthase system consists of two components, termed enzyme A (MW 82,000) and enzyme B (MW 36,000). Some of the properties of the enzyme system are as follows: NADPH and a divalent cation, supplied most effectively as MgCl₂, are required for activity; optimal activity occurs at pH 7.4 and 30 C; the K _m for dihydroneopterin triphosphate is 10 µm; and a number of unconjugated pterins, including biopterin and sepiapterin, are inhibitory. Dihydroneopterin cannot be used as substrate in place of dihydroneopterin triphosphate. Evidence is presented in support of a proposed reaction mechanism for the enzymatic conversion of dihydroneopterin triphosphate to sepiapterin in which enzyme A catalyzes the production of a labile intermediate by nonhydrolytic elimination of the phosphates of dihydroneopterin triphosphate, and enzyme B catalyzes the conversion of this intermediate, in the presence of NADPH, to sepiapterin. An analysis of the activity of sepiapterin synthase during development in Drosophila revealed the presence of a small amount of activity in eggs and young larvae and a much larger amount in late pupae and young adults. Sepiapterin synthase activity during development corresponds with the appearance of sepiapterin in the flies. Of a variety of eye color mutants of Drosophila melanogaster tested for sepiapterin synthase activity, only purple (pr) flies contained activity that was significantly lower than that found in the wild-type flies (22% of the wild-type activity). Further studies indicated that the amount of enzyme A activity is low in purple flies, whereas the amount of enzyme B activity is equal to that present in wild-type flies.This work was supported by research grants from the National Institutes of Health (AM03442) and the National Science Foundation (PCM75-19513 A02). G. G. K. was supported as a predoctoral trainee by National Institutes of Health Training Grant GM00515.     

Selection at the alcohol dehydrogenase locus of Drosophila melanogaster: Effects of ethanol and temperature

Drosophila melanogaster larvae were subjected to 10 generations of selection on 6% ethanol at 17, 25, and 30°C. For each temperature there was a significant (P<0.01) increase in the frequency of the Adh isoallele. Controls with no ethanol showed no change in the frequency of the Adh ^F isoallele. Larvae subjected to stronger selection on 8% ethanol confirmed the results. When adults of various ages were subjected to 16 and 32°C, the ADH^F isoenzyme retained its twofold advantage in activity over ADH^S regardless of the temperature. The same result was obtained with larvae at 16 and 35°C. Although some effect of temperature was demonstrated, it was concluded that the effect was not strong enough for temperature to be a selective factor under the conditions studied. However, ethanol is a strong selective factor for laboratory populations.     

Pyrimidine biosynthetic enzymes in Babesia hylomysci

Gero A. M. and Coombs G. H. 1982. Pyrimidine biosynthetic enzymes in Babesia hylomysci. International Journal for Parasitology12: 377–382. The enzymes that catalyse the conversion of carbamylaspartate to UMP have been demonstrated in the rodent piroplasm, Babesia hylomysci and partially characterised. They were shown to be similar to the corresponding mammalian enzymes in that dihydroorotase, orotate phosphoribosytransferase and orotidine-5'-phosphate decarboxylase were soluble, whilst dihydroorotate dehyrogenase was membrane bound and appeared to be associated with a respiratory chain. Dihydroorotate dehydrogenase was found to have a K_m (l-DHO) of 21 μm and a pH optimum of 7.8. It was inhibited by analogs of ubiquinone and several pyrimidines, dihydroazaorotate being the most effective ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 17 μ}\text{m}$). It is concluded that Babesia parasites contain a functional de novo biosynthetic pathway for pyrimidines which provides a potential target at which to direct putative chemotherapeutic agents.     

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.     

Latitudinal variation in octanol dehydrogenase and acid phosphatase allele frequencies in Drosophila melanogaster

Summary The octanol dehydrogenase (Odh) and acid phosphatase (Acph) loci of Drosophila melanogaster are each polymorphic for two electrophoretically detectable alleles. The frequencies of the Odh and Acph alleles have been analysed in populations sampled from up to a 40 ° latitudinal range in each of Australasia, North America and Europe/Asia. Odh ^S frequency is found to be significantly negatively associated with distance from the equator in all three zones. The relationship of Acph ^S frequency to distance from the equator is significant and negative in Australasia but neither significant nor consistent in sign in North America and Europe/Asia.     

Effect of environmental alcohol on in Vivo properties of Drosophila alcohol dehydrogenase

The effects of environmental 2-propanol on the in vivo properties of Drosophila alcohol dehydrogenase (E.C. 1.1.1.1.) are presented. Exposed flies were found to exhibit a significant decrease in ADH specific activity with a concomitant increase in the enzyme's relative in vivo stability and concentration. The possible adaptive significance of the observed responses is discussed.This work was supported by NSF grant #DEB 7815466 to J.M. Journal Paper No. J-9979 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 2272.     

Analysis of the phenotypes exhibited by rudimentary-like mutants of Drosophila melanogaster

Flies mutant for one or both of the last two enzymes of de novo pyrimidine biosynthesis express a number of phenotypes that are also expressed by mutants of the first four pathway enzymes (r and Dhod-null mutants). However, r-1 flies also express two phenotypes, mottled eyes and poor viability, that are not usually expressed by r and Dhod-null flies. Chemical determinations show that orotic acid, a substrate for the fifth pathway enzyme, accumulates in r-1 individuals but not in r and wild-type individuals. Moreover, flies simultaneously mutant for r and r-1 do not express the mottled-eye phenotype, showing that r is epistatic to r-1 for this r-1-specific phenotype. When genotypically wild-type flies are cultured on a medium containing 6-azauracil, the base of a potent inhibitor of the last enzyme of de novo pyrimidine biosynthesis, phenocopies are obtained that include the mottled-eye as well as the wing phenotypes of r-1 flies. These results support hypotheses that the phenotypes common to r, Dhod-null, and r-1 flies are consequences of uridylic acid deficiency, whereas the r-1-specific phenotypes result from orotic acid accumulation in flies lacking either or both of the last two enzymes of de novo pyrimidine biosynthesis.This research was supported by NSF Research Grant PCM 78-14164, an NSF predoctoral fellowship award to T. Conner, and an NIH research career development award to J. Rawls.     

Genetic control of alcohol dehydrogenase levels in Drosophila

Among the progeny of Drosophila flies heterozygous for two noncomplementing Adh-negative alleles, two individuals were found that had recovered appreciable alcohol dehydrogenase activity, thereby surviving the ethanol medium used as a screen. The most likely explanation is that these Adh-positive flies are the product of intracistronic recombination within the Adh locus. Judging by the distribution of outside markers, one of the crossovers would have been a conventional reciprocal exchange while the other appears to have been an instance of nonreciprocal recombination. The enzymes produced in strains derived from the original survivors can be easily distinguished from wild-type enzymes ADH-S and ADH-F on the basis of their sensitivity to denaturing agents. None of various physical and catalytic properties tested revealed differences between the enzymes of the survivor strains except that in one of them the level of activity is 55–65% of the other. Quantitative immunological determinations of ADH gave estimates of enzyme protein which are proportional to the measured activity levels. These results are interpreted to indicate that different amounts of ADH protein are being accumulated in the two strains.This work was supported in part by NSF Grant PCM 76-19563.     

Segmental organisation of the tail region in the embryo of Drosophila melanogaster

Summary The segmental organisation of the tail region in the embryo of Drosophila melanogaster, which is defined here as the epidermal region posterior to the boundary between abdominal segments A7 and A8, has been investigated by means of ultraviolet (UV) laser fate-mapping and phenotypic analysis of embryonic mutants that alter the segmental pattern of the larval cuticle. Wild-type embryos were irradiated in the presumptive tail region with a UV- laser microbeam of 20 m diameter at the blastoderm stage. The ensuing defects were scored in the cuticle pattern of the tail region of the first-instar larva, which is described in detail in this paper. The spatial distribution of defect frequencies was used to construct a blastoderm fate-map of the cuticle structures of the larval tail region. The segmental origin of the larval tail structures was inferred from the phenotypic analysis of segmentation and homoeotic mutants, which revealed pattern repetition throughout the embryonic tail region corresponding to four segment anlagen, A8 to A11, and a non-segmental telson. These data enabled the transformation of the blastoderm fate-map of cuticle structures into a map of tail segment anlagen. The tail anlage occupies about 10% of the egg length (EL), bounded by segment A7 anteriorly at 20% EL and by the proctodaeum posteriorly at 10% EL, as measured from the posterior pole. The anlagen of segments A8 and A9 appear to be narrow dorso-ventral strips of blastoderm cells similar to the anlagen of the trunk segments, whereas the anlagen of A10 and A11 are smaller and produce fewer pattern elements. The telson is represented in the cuticle by the tuft which derives from a very dorsal posterior position. The antero-posterior axis of the entire tail anlage appears curved upward posteriorly. Differences in the mode of development between tail and trunk segments are discussed, as are similarities of larval and imaginal tail development in Drosophila. Comparison with tail development in other insects suggests that, during evolution, the transition from semi-long-germ to long-germ development modified the organisation of the tail region without affecting its primary subdivision into metameric units.     

Evolutionary genetics of the Drosophila alcohol dehydrogenase gene-enzyme system

Evolutionary genetics embodies a broad research area that ranges from the DNA level to studies of genetic aspects in populations. In all cases the purpose is to determine the impact of genetic variation on evolutionary change. The broad range of evolutionary genetics requires the involvement of a diverse group of researchers: molecular biologists, (population) geneticists, biochemists, physiologists, ecologists, ethologists and theorists, each of which has its own insights and interests. For example, biochemists are often not concerned with the physiological function of a protein (with respect to pH, substrates, temperature, etc.), while ecologists, in turn, are often not interested in the biochemical-physiological aspects underlying the traits they study. This review deals with several evolutionary aspects of the Drosophila alcohol dehydrogenase gene-enzyme system, and includes my own personal viewpoints. I have tried to condense and integrate the current knowledge in this field as it has developed since the comprehensive review by van Delden (1982). Details on specific issues may be gained from Sofer and Martin (1987), Sullivan, Atkinson and Starmer (1990); Chambers (1988, 1991); Geer, Miller and Heinstra (1991); and Winberg and McKinley-McKee (1992).Dedicated to Professor Billy W. Geer, because of his contributions to knowledge of the biochemical genetics of Drosophila.     

Variation in the biochemical properties of the Drosophila alcohol dehydrogenase allozymes

Thirteen Drosophila Adh variants have been characterized with respect to gene expression, substrate preference, thermostability, and specific activity. The results suggest that the variants may be grouped into two biochemical classes, typified by the properties of the two most common enzyme forms, ADH-F and ADH-S. Membership of these classes cannot be predicted from electrophoretic mobility, nor is any simple classification possible with regard to the characteristics of level of gene expression (in terms of ADH activity or ADH protein) or thermostability of the gene product.     

Nucleotide divergence of the rp49 gene region between Drosophila melanogaster and two species of the Obscura group of Drosophila

Summary A 2.1-kb SStI fragment including the rp49 gene and the 3 end of the -serendipity gene has been cloned and sequenced in Drosophila pseudoobscura. rp49 maps at region 62 on the tip of chromosome II of this species. Both the coding and flanking regions have been aligned and compared with those of D. subobscura. There is no evidence for heterogeneity in the rate of silent substitution between the rp49 coding region and the rate of substitutions in flanking regions, the overall silent divergence per site being 0.19. Noncoding regions also differ between both species by different insertions/deletions, some of which are related to repeated sequences. The rp49 region of D. pseudoobscura shows a strong codon bias similar to those of D. subobscura and D. melanogaster. Comparison of the rates of silent (K _S ) and nonsilent (K _a ) substitutions of the rp49 gene and other genes completely sequenced in D. pseudoobscura and D. melanogaster confirms previous results indicating that rp49 is evolving slowly both at silent and nonsilent sites. According to the data for the rp49 region, D. pseudoobscura and D. subobscura lineages would have diverged some 9 Myr ago, if one assumes a divergence time of 30 Myr for the melanogaster and obscura groups.Offprint requests to: C. Segarra     

Drosophila alcohol dehydrogenase activity in vitro and in vivo: Effects of acetone feeding

When adult Drosophila are placed on medium containing 0.5% acetone, their level of alcohol dehydrogenase activity drops rapidly. At the same time, the proportion of activity in the various electrophoretic forms of the enzyme shifts; most of the activity becomes localized in what is ordinarily a minor form of the enzyme. Moreover, the loss of enzyme activity occurs in vivo as well, as shown by sensitivity to ethanol poisoning, insensitivity to pentenol treatment, and inability to utilize ethanol as an energy source. These observations are discussed in light of a model advanced for the origin of the multiple forms of alcohol dehydrogenase in Drosophila.This research was supported by NIH Grants GM-18254 and ES-1527 and by DOE Contract No. EY-76-5-02-2965. Publication No. 1016 from the Department of Biology, Johns Hopkins University.     

Ether-induced segmentation disturbances in Drosophila melanogaster

Summary Drosophila embryos, exposed to ether between 1 and 4 h after oviposition, develop defects ranging from the complete lack of segmentation to isolated gaps in single segments. Between these extremes are varying extents of incomplete and abnormal segmentation. On the basis of both their temporal and spatial characteristics, five major phenotype classes may be distinguished: headless — unsegmented or incompletely segmented anteriorly; gap — interruptions of segmentation not obviously periodic; alternating segment gaps — interruptions with double segment periodicities; fused segments; and short segments — truncations with single segment periodicities. Many defects resemble known mutant phenotypes. The disturbances in segmentation are predominantly global and frequently accompanied by alterations in segment specification, such that the segments obtained show no resemblance to the normal homologues. These features, together with the distinctive spatiotemporal characteristics of the defects, all point to segmentation as a dynamic process. The regular spacing of the segments and the fact that the entire range of defects is inducible by ether are further consistent with the hypothesis that at least part of the segmentation process may consist of physicochemical reactions coordinated over the whole body. The relationship between our data and data from genetic and other analyses are briefly discussed.     

Ethanol tolerances of Drosophila melanogaster populations selected on different concentrations of ethanol supplemented media

Summary Eight recently collected Australasian populations of D. melanogaster were each divided into eight selection lines. Two of these lines from each population were maintained on one of four types of selection media: standard food supplemented with 0%, 3%, 6% and 9% ethanol. After 30 generations the selection lines were tested for tolerance to 9% ethanol medium and after another 20 generations adults were tested for tolerance to concentrated ethanol fumes. Significant differences in tolerance were found among lines selected on different media which were consistent across the eight populations. On the 9% test media, the 6% and 9% selection lines, as compared with the control lines selected on 0% ethanol, were more likely to survive as pre-adults or adults, faster to develop as preadults, and heavier and more productive as adults. However, the tolerance of the 3% lines to the 9% test media was less than that of the 0% control lines in preadult and adult survival, intermediate between that of the 0% and the 6% and 9% lines in productivities, and apparently superior to the 6% and 9% lines in development times and adult weights. The 3%, 6% and 9% lines showed similar tolerances to the ethanol vapour. Previous work showed that 3% ethanol can be a metabolic benefit to D. melanogaster but 6% and 9% are metabolic costs. The present results suggest that the phenotype selected on 3% to obtain a metabolic benefit differs in many respects from that selected on 6% and 9% to minimise their detrimental effects.     

A natural genetic polymorphism affects retroactive interference in Drosophila melanogaster

As environments change, animals update their internal representations of the external world. New information about the environment is learned and retained whereas outdated information is disregarded or forgotten. Retroactive interference (RI) occurs when the retrieval of previously learned information is less available owing to the acquisition of recently acquired information. Even though RI is thought to be a major cause of forgetting, its functional significance is still under debate. We find that natural allelic variants of the Drosophila melanogaster foraging gene known to affect rover and sitter behaviour differ in RI. More specifically, rovers who were previously shown to experience greater environmental heterogeneity while foraging display RI whereas sitters do not. Rover responses are biased towards more recent learning events. These results provide an ecological context to investigate the function of forgetting via RI and a suitable genetic model organism to address the evolutionary relevance of cognitive tasks.