Alcohol dehydrogenase polymorphism inDrosophila: Enzyme kinetics of product inhibition

Summary Because natural populations ofDrosophila melanogaster are polymorphic for different allozymes of alcohol dehydrogenase (ADH) and becauseD. melanogaster is more tolerant to the toxic effects of ethanol than its sibling speciesD. simulans, information regarding the sensitivities of the different forms of ADH to the products of ethanol degradation are of ecological importance. ADH-F, ADH-S, ADH-71k ofD. melanogaster and the ADH ofD. simulans were inhibited by NADH, but the inhibition was relieved by NAD⁺. The order of sensitivity of NADH was ADH-F<ADH-71k, ADH-S<ADH-simulans with ADH-F being about four times less sensitive than theD. melanogaster enzymes and 12 times less sensitive than theD. simulans enzyme. Acetaldehyde inhibited the ethanolto-acetaldehyde activity of the ADHs, but at low acetaldehyde concentrations ethanol and NAD⁺ reduced the inhibition. ADH-71k and ADH-F were more subject to the inhibitory action of acetaldehyde than ADH-S and ADH-simulans, with ADH-71k being seven times more sensitive than ADH-S. The pattern of product inhibition of ADH-71k suggests a rapid equilibrium random mechanism for ethanol oxidation. Thus, although the ADH variants only differ by a few amino acids, these differences exert a far larger impact on their intrinsic properties than previously thought. How differences in product inhibition may be of significance in the evolution of the ADHs is discussed.     

Physiological Significance of the Alcohol Dehydrogenase Polymorphism in Larvae of Drosophila

This study deals with biochemical and metabolic-physiological aspects of the relationship between variation in in vivo alcohol dehydrogenase activity and fitness in larvae homozygous for the alleles Adh71k, AdhF, AdhS, of Drosophila melanogaster, and for the common Adh allele of Drosophila simulans. The Adh genotypes differ in the maximum oxidation rates of propan-2-ol into acetone in vivo. There are smaller differences between the Adh genotypes in rates of ethanol elimination. Rates of accumulation of ethanol in vivo are negatively associated with larval-to-adult survival of the Adh genotypes. The rank order of the maximum rates of the ADHs in elimination of propan-2-ol, as well as ethanol, is ADH-71k greater than ADH-F greater than ADH-S greater than simulans-ADH. The ratio of this maximum rate to ADH quantity reveals the rank order of ADH-S greater than ADH-F greater than ADH-71k greater than simulans-ADH, suggesting a compensation for allozymic efficiency by the ADH quantity in D. melanogaster.Our findings show that natural selection may act on the Adh polymorphism in larvae via differences in rates of alcohol metabolism.     

Extensive amino acid polymorphism at the pgm locus is consistent with adaptive protein evolution in Drosophila melanogaster

PGM plays a central role in the glycolytic pathway at the branch point leading to glycogen metabolism and is highly polymorphic in allozyme studies of many species. We have characterized the nucleotide diversity across the Pgm gene in Drosophila melanogaster and D. simulans to investigate the role that protein polymorphism plays at this crucial metabolic branch point shared with several other enzymes. Although D. melanogaster and D. simulans share common allozyme mobility alleles, we find these allozymes are the result of many different amino acid changes at the nucleotide level. In addition, specific allozyme classes within species contain several amino acid changes, which may explain the absence of latitudinal clines for PGM allozyme alleles, the lack of association of PGM allozymes with the cosmopolitan In(3L)P inversion, and the failure to detect differences between PGM allozymes in functional studies. We find a significant excess of amino acid polymorphisms within D. melanogaster when compared to the complete absence of fixed replacements with D. simulans. There is also strong linkage disequilibrium across the 2354 bp of the Pgm locus, which may be explained by a specific amino acid haplotype that is high in frequency yet contains an excess of singleton polymorphisms. Like G6pd, Pgm shows strong evidence for a branch point enzyme that exhibits adaptive protein evolution.     

Nucleotide variation at the hypervariable esterase 6 isozyme locus of Drosophila simulans

Esterase 6 (Est-6/EST6) is polymorphic in both Drosophila melanogaster and D. simulans for two common allozyme forms, as well as for several other less common variants. Parallel latitudinal clines in the frequencies of the common EST6-F and EST6-S allozymes in these species have previously been interpreted in terms of a shared amino acid polymorphism that distinguishes the two variants and is subject to selection. Here we compare the sequences of four D. simulans Est-6 isolates and show that overall estimates of nucleotide heterozygosity in both coding and 5' flanking regions are more than threefold higher than those obtained previously for this gene in D. melanogaster. Nevertheless, the ratio of replacement to exon silent-site polymorphism in D. simulans is less than the ratio of replacement to silent divergence between D. simulans and D. melanogaster, which could be the result of increased efficiency of selection against replacement polymorphisms in D. simulans or to divergent selection between the two species. We also find that the amino acid polymorphisms separating EST6- F and EST6-S in D. simulans are not the same as those that separate these allozymes in D. melanogaster, implying that the shared clines do not reflect shared molecular targets for selection. All comparisons within and between the two species reveal a remarkable paucity of variation in a stretch of nearly 400 bp immediately 5' of the gene, indicative of strong selective constraint to retain essential aspects of Est-6 promoter function.      

Evidence for a multiple function of the alcohol dehydrogenase allozyme ADH71k of Drosophila melanogaster

Alcohol dehydrogenase of Drosophila melanogaster catalyzes the oxidation of many primary and secondary alcohols. We show that sarcosine, choline and dihydroorotate are substrates of ADH in vitro. The first two substrates are regular substrates of the choline shunt, and the latter of the de novo pyrimidine synthesis. Differences in oxidative ability towards sarcosine and dihydroorotate between two ADH allozymes, ADH71k and ADHF, are observed. The catalytic activity of ADH71k towards sarcosine and dihydroorotate might be responsible for its allelic fixation in Notch8 mutant stocks, in which Notch females have a decreased level of the regular enzymes for these substrates. Their oxidation by ADH71k might act as a bypass, which restores at least part of the decreased activity of enzymes encoded by the Notch locus.     

Dehydrogenase-dependent ethanol metabolism in deer mice (Peromyscus maniculatus) lacking cytosolic alcohol dehydrogenase. Reversibility and isotope effects in vivo and in subcellular fractions

Elimination of [2H]ethanol in vivo as studied by gas chromatography/mass spectrometry occurred at about half the rate in deer mice reported to lack alcohol dehydrogenase (ADH-) compared with ADH+ deer mice and exhibited kinetic isotope effects on Vmax and Km (D(V/K] of 2.2 +/- 0.1 and 3.2 +/- 0.8 in the two strains, respectively. To an equal extent in both strains, ethanol elimination was accompanied by an ethanol-acetaldehyde exchange with an intermolecular transfer of hydrogen atoms, indicating the occurrence of dehydrogenase activity. This exchange was also observed in perfused deer mouse livers. Based on calculations it was estimated that at least 50% of ethanol elimination in ADH- deer mice was caused by the action of dehydrogenase systems. NADPH-supported cytochrome P-450-dependent ethanol oxidation in liver microsomes from ADH+ and ADH- deer mice was not stereoselective and occurred with a D(V/K) of 3.6. The D(V/K) value of catalase-dependent oxidation was 1.8, whereas a kinetic isotope effect of cytosolic ADH in the ADH+ strain was 3.2. Mitochondria from both ADH+ and ADH- deer mice catalyzed NAD+-dependent ethanol oxidation and NADH-dependent acetaldehyde reduction. The kinetic isotope effects of NAD+-dependent ethanol oxidation in the mitochondrial fraction from ADH+ and ADH- deer mice were 2.0 +/- 0.1 and 2.3 +/- 0.3, respectively. The results indicate only a minor contribution by cytochrome P-450 to ethanol elimination, whereas the isotope effects are consistent with ethanol oxidation by the catalase-H2O2 system in ADH- deer mice in addition to the dehydrogenase systems.     

Analysis of the gene encoding the multifunctional alcohol dehydrogenase allozyme ADH-71k of Drosophila melanogaster

The nucleotide sequence of the alcohol dehydrogenase gene Adh71k has been determined. The Adh71k allele encodes the thermostable and multifunctional ADH-71k allozyme of Drosophila melanogaster. Comparison with the sequences of AdhS, AdhF, and AdhFChD reveals differences in the coding and noncoding regions of the gene. Conceptual translation of the Adh71k sequence indicates that ADH-71k shares with ADH-F and ADH-FCHD an amino acid replacement at residue 192 and with ADH-FCHD an additional replacement of serine for proline at residue 214. Three unique differences were found in the nontranslated regions. It is proposed that a nucleotide deletion in the adult intron is related to the difference in expression level of the Adh71k allele, relative to the other alleles. An insertion of five nucleotides, additional to a single base deletion at that site, was detected in one of the larval enhancer regions in the 5' flanking region of the Adh71k allele, creating a palindromic structure in that area.     

Isolation of Protease-Free Alcohol Dehydrogenase (ADH) from Drosophila simulans and Several Homozygous and Heterozygous Drosophila melanogaster Variants

The enzyme alcohol dehydrogenase (ADH) fromseveral naturally occurring ADH variants ofDrosophila melanogaster and Drosophilasimulans was isolated. Affinity chromatography withthe ligand Cibacron Blue and elution with NAD⁺ showed similarbehavior for D. melanogaster ADH-FF, ADH-71k,and D. simulans ADH. Introduction of a secondCibacron Blue affinity chromatography step, withgradient elution with NAD⁺, resulted in pure and stable enzymes. D.melanogaster ADH-SS cannot be eluted from theaffinity chromatography column at a high concentrationof NAD⁺ and required a pH gradient for itspurification, preceded by a wash step with a high concentration ofNAD⁺. Hybrid Drosophila melanogasteralcohol dehydrogenase FS has been isolated fromheterozygous flies, using affinity chromatography withfirst elution at a high concentration NAD⁺, directlyfollowed by affinity chromatography elution with a pHgradient. Incubation of equal amounts of pure homodimersof Drosophila melanogaster ADH-FF and ADH-SS,in the presence of 3 M urea at pH 8.6, for 30 min at roomtemperature, followed by reassociation yielded activeDrosophila melanogaster ADH-FS heterodimers. Noproteolytic degradation was found after incubation ofpurified enzyme preparations in the absence or presenceof SDS, except for some degradation of ADH-SS after verylong incubation times. The thermostabilities of D.melanogaster ADH-71k and ADH-SS were almostidentical and were higher than those of D.melanogaster ADH-FF and D. simulans ADH. Thethermostability of D. melanogaster ADH-FS waslower than those of D. melanogaster ADH-FF andADH-SS. D. melanogaster ADH-FF and ADH-71k have identical inhibition constantswith the ligand Cibacron Blue at pH 8.6, which are twotimes higher at pH 9.5. The K_i values forD. simulans ADH are three times lower at bothpH values. D. melanogaster ADH-SS and ADH-FS havesimilar K_i values, which are lower than thosefor D. melanogaster ADH-FF at pH 8.6. But at pH9.5 the K_i value for ADH-FS is the same as atpH 8.6, while that of ADH-SS is seven times higher. Kinetic parameters ofDrosophila melanogaster ADH-FF, ADH-SS, andADH-71k and Drosophila simulans ADH, at pH 8.6and 9.5, showed little or no variation inK_m ^eth values. TheK_m ^NAD values measured at pH 9.5for Drosophila alcohol dehydrogenases are alllower than those measured at pH 8.6. The rate constants(k_cat) determined for all fourDrosophila alcohol dehydrogenases are higher at pH 9.5 than at pH 8.6. D.melanogaster ADH-FS showed nonlinear kinetics.     

Structure-function relationships in Drosophila melanogaster alcohol dehydrogenase allozymes ADH(S), ADH(F) and ADH(UF), and distantly related forms.

Drosophila melanogaster alcohol dehydrogenase (ADH), a paradigm for gene-enzyme molecular evolution and natural selection studies, presents three main alleloforms (ADHS, ADHF and ADHUF) differing by one or two substitutions that render different biochemical properties to the allelozymes. A three-dimensional molecular model of the three allozymes was built by homology modeling using as a template the available crystal structure of the orthologous D. lebanonensis ADH, which shares a sequence identity of 82.2%. Comparison between D. lebanonensis and D. melanogaster structures showed that there is almost no amino-acid change near the substrate or coenzyme binding sites and that the hydrophobic active site cavity is strictly conserved. Nevertheless, substitutions are not distributed at random in nonconstricted positions, or located in external loops, but they appear clustered mainly in secondary structure elements. From comparisons between D. melanogaster allozymes and with D. simulans, a very closely related species, a model based on changes in the electrostatic potential distribution is presented to explain their differential behavior. The depth of knowledge on Drosophila ADH genetics and kinetics, together with the recently obtained structural information, could provide a better understanding of the mechanisms underlying molecular evolution and population genetics.     

Historical Selection, Amino Acid Polymorphism and Lineage-Specific Divergence at the G6pd Locus in Drosophila Melanogaster and D. Simulans

The nucleotide diversity across 1705 bp of the G6pd gene is studied in 50 Drosophila melanogaster and 12 D. simulans lines. Our earlier report contrasted intraspecific polymorphism and interspecific differences at silent and replacement sites in these species. This report expands the number of European and African lines and examines the pattern of polymorphism with respect to the common A/B allozymes. In D. melanogaster the silent nucleotide diversity varies 2.8-fold across localities. The B allele sequences are two- to fourfold more variable than the derived A allele, and differences between allozymes are twice as among B alleles. There is strong linkage disequilibrium across the G6pd region. In both species the level of silent polymorphism increases from the 5' to 3' ends, while there is no comparable pattern in level of silent site divergence or fixation. The neutral model is not rejected in either species. Using D. yakuba as an outgroup, the D. melanogaster lineage shows a twofold greater rate of silent fixation, but less than half the rate of amino acid replacement. Lineage-specific differences in mutation fixation are inconsistent with neutral expectations and suggest the interaction of species-specific population size differences with both weakly advantageous and deleterious selection.     

Nucleotide variation in the triosephosphate isomerase (Tpi) locus of Drosophila melanogaster and Drosophila simulans

DNA sequence variation in a 1.1-kb region including the coding portion of the Tpi locus was examined in 25 homozygous third-chromosome lines of Drosophila melanogaster, nine lines of Drosophila simulans, and one line of Drosophila yakuba. Our data show that the widespread allozyme polymorphism observed in cosmopolitan D. melanogaster is due to a glutamic acid substitution occurring in a phylogenetically conserved lysine that has been identified as part of the "hinged-lid" active site of the enzyme. This observation suggests that the replacement polymorphism may have important functional consequences. One replacement polymorphism was also observed in D. simulans, although its functional relevance is more difficult to assess, since it affects a site that is not strongly conserved. This amino acid change in D. simulans is associated with a single lineage possessing seven unique silent substitutions, which may be indicative of balancing selection or population subdivision. The absence of fixed amino acid differences between D. melanogaster and D. simulans and only a single difference with D. yakuba suggests that triose phosphate isomerase is under strong functional constraint. Silent variation is slightly higher for D. melanogaster than for D. simulans. Finally, we outline the general lack of evidence for old balanced polymorphisms at allozyme loci in D. melanogaster.      

Identification of P-450ALC in microsomes from alcohol dehydrogenase-deficient deermice: contribution to ethanol elimination in vivo

Isozyme 3a of rabbit hepatic cytochrome P-450, also termed P-450ALC, was previously isolated and characterized and was shown to be induced 3- to 5-fold by exposure to ethanol. In the present study, antibody against rabbit P-450ALC was used to identify a homologous protein in alcohol dehydrogenase-negative (ADH-) and -positive (ADH+) deermice, Peromyscus maniculatus. The antibody reacts with a single protein having an apparent molecular weight of 52,000 on immunoblots of hepatic microsomes from untreated and ethanol-treated deermice from both strains. The level of the homologous protein was about 2-fold greater in microsomes from naive ADH- than from naive ADH+ animals. Ethanol treatment induced the protein about 3-fold in the ADH+ strain and about 4-fold in the ADH- strain. The antibody to rabbit P-450ALC inhibited the microsomal metabolism of ethanol and aniline. The homologous protein, termed deermouse P-450ALC, catalyzed from 70 to 80% of the oxidation of ethanol and about 90% of the hydroxylation of aniline by microsomes from both strains after ethanol treatment. The antibody-inhibited portion of the microsomal activities, which are attributable to the P-450ALC homolog, increased about 3-fold upon ethanol treatment in the ADH+ strain and about 4-fold in the ADH- strain, in excellent agreement with the results from immunoblots. The total microsomal P-450 content and the rate of ethanol oxidation were induced 1.4-fold and 2.2-fold, respectively, by ethanol in the ADH+ strain and 1.9-fold and 3.3-fold, respectively, in the ADH- strain. Thus, the total microsomal P-450 content and ethanol oxidation underestimate the induction of the P-450ALC homolog in both strains. A comparison of the rates of microsomal ethanol oxidation in vitro with rates of ethanol elimination in vivo indicates that deermouse P-450ALC could account optimally for 3 and 8% of total ethanol elimination in naive ADH+ and ADH- strains, respectively. After chronic ethanol treatment, P-450ALC could account maximally for 8% of the total ethanol elimination in the ADH+ strain and 22% in the ADH- strain. Further, cytochrome P-450ALC appears to be responsible for about one-half of the increase in the rate of ethanol elimination in vivo after chronic treatment with ethanol. These results indicate that the contribution of P-450ALC to ethanol oxidation in the deermouse is relatively small. Desferrioxamine had no effect on rates of ethanol uptake by perfused livers from ADH-negative deermice, indicating that ethanol oxidation by a hydroxyl radical-mediated mechanism was not involved in ethanol metabolism in this mutant.(ABSTRACT TRUNCATED AT 400 WORDS)     

Selective expansion of the newly evolved genomic variants of retrotransposon 1731 in the Drosophila genomes

The structural variants of the regulatory and coding regions of the LTR-retrotransposon 1731 are described. Two classes of genomic copies of retrotransposon 1731, with and without frameshifting strategy to express Gag and Pol proteins, were earlier revealed in the D. melanogaster genome. Copies without frameshifting are shown to be evolved from an ancient variant with frameshifting and are widespread in the genomes of the melanogaster complex species. Position of a rare codon responsible for ribosome pausing and efficient frameshifting is identified. Two structural variants of 1731 LTRs were detected in the melanogaster complex species: the predominant structural variant A1A2 of 1731 LTR in the D. melanogaster, D. simulans, and D. sechellia genomes contains duplicated and diverged copies of 28 bp in the U3 region, whereas A1 variant lacking this duplication is expanded in the D. mauritiana genome. Selective expansion of the A1A2 variant was detected in the independently established D. melanogaster cell cultures. A1A2 variant is expressed in embryos, cell culture, and testes, whereas A1 is expressed only in testes of D. melanogaster. Relief of expression of the A1A2 but not A1 variant in the ovaries as a result of mutation in the RNA interference (RNAi) spn-E gene is shown. Thus, expansion of the recently evolved genomic variants of the LTR retrotransposon 1731 possessing a new translation strategy, duplication in the U3 region, and extended profile of expression is revealed.     

Ethanol-metabolizing pathways in deermice. Estimation of flux calculated from isotope effects

The apparent deuterium isotope effects on Vmax/Km (D(V/K] of ethanol oxidation in two deermouse strains (one having and one lacking hepatic alcohol dehydrogenase (ADH] were used to calculate flux through the ADH, microsomal ethanol-oxidizing system (MEOS), and catalase pathways. In vitro, D(V/K) values were 3.22 for ADH, 1.13 for MEOS, and 1.83 for catalase under physiological conditions of pH, temperature, and ionic strength. In vivo, in deermice lacking ADH (ADH-), D(V/K) was 1.20 +/- 0.09 (mean +/- S.E.) at 7.0 +/- 0.5 mM blood ethanol and 1.08 +/- 0.10 at 57.8 +/- 10.2 mM blood ethanol, consistent with ethanol oxidation principally by MEOS. Pretreatment of ADH- animals with the catalase inhibitor 3-amino-1,2,4-triazole did not significantly change D(V/K). ADH+ deermice exhibited D(V/K) values of 1.87 +/- 0.06 (untreated), 1.71 +/- 0.13 (pretreated with 3-amino-1,2,4-triazole), and 1.24 +/- 0.13 (after the ADH inhibitor, 4-methylpyrazole) at 5-7 mM blood ethanol levels. At elevated blood ethanol concentrations (58.1 +/- 2.4 mM), a D(V/K) of 1.37 +/- 0.21 was measured in the ADH+ strain. For measured D(V/K) values to accurately reflect pathway contributions, initial reaction conditions are essential. These were shown to exist by the following criteria: negligible fractional conversion of substrate to product and no measurable back reaction in deermice having a reversible enzyme (ADH). Thus, calculations from D(V/K) indicate that, even when ADH is present, non-ADH pathways (mostly MEOS) participate significantly in ethanol metabolism at all concentrations tested and play a major role at high levels.     

Substrate and inhibitor specificities of the thermostable alcohol dehydrogenase allozymes ADH-71k and ADH-FCh.D. ofDrosophila melanogaster

Purified thermostable alcohol dehydrogenase allozymes ADH-71k and ADH-FCh.D. ofDrosophila melanogaster have been compared with the two common enzyme forms ADH-F and ADH-S. Enzyme kinetic parameters for various primary and secondary alcohols were determined under standard conditions used previously. Both ADH-71k and ADH-FCh.D. show ADH-S-like reaction kinetics andK _m values, due to retrograde evolution at site 214, Pro Ser. Inhibition studies with alcohol dehydrogenase inhibitors pyrazole, 4-methylpyrazole, and cibacron blue 3GA were also performed. Activity measurements on crude extracts of larvae and flies from isogenic lines of ADH-FCh.D. revealed a consistently higher activity than in ADH-71k-containing strains, in contrast to the original strains.K.Th.E is indebted to the Royal Norwegian Council for Technological and Scientific Research for their postdoctoral fellowship. Prof. J. S. McKinley-McKee gave me the opportunity to work in his laboratory. I thank Dr. Knut Sletten of the Biochemical Institute for the kind gift of 2-methoxyethanol and amino acid analysis of some samples. The Biological Institute, Oslo, Section of General Genetics, is gratefully acknowledged for enabling me to use their fly-breeding facilities. Dr. John B. Gibson provided us with a sample of FCh.D. flies for the construction of isogenic lines in which Dr. Johan Hageman participated, owing to Postdoctoral Grant 436-931-P from the Foundation of Biological Research (BION), which is subsidized by the Netherlands Organization for Scientific Research (NWO). J. H. and Paula Truyens were involved in the measurements on the crude extracts. Work at Victoria University was supported by the VUW Internal Grant Committee.     

Polymorphism and divergence at the prune locus in Drosophila melanogaster and D. simulans

The prune locus of Drosophila melanogaster lies at the tip of the X chromosome, in a region of reduced recombination in which nearby loci show reduced variation relative to evolutionary divergence from D. simulans. DNA sequencing of prune alleles from D. melanogaster and D. simulans reveals extremely low variation in D. melanogaster but greater variation in D. simulans. Divergence between the two species is not reduced. This pattern may be explained by either positive selection leading to hitchhiking of neutral variation or background selection against deleterious mutations. The pattern of silent versus replacement polymorphism and divergence at prune is consistent with either a model of weakly deleterious selection against amino acid substitutions or balancing selection.      

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.     

Synthesis of Drosophila melanogaster alcohol dehydrogenase in yeast

Expression systems for the heterologous expression of Drosophila melanogaster alcohol dehydrogenase (ADH) in Saccharomyces cerevisiae have been designed, analyzed and compared. Four different yeast/Escherichia coli shuttle vectors were constructed and used to transform four different yeast strains. Expression was detectable in ADH- yeast strains, from either a constitutive promoter, yeast ADH1 promoter (ADCp), or a regulated promoter, yeast GALp. The highest amount of D. melanogaster ADH was obtained from a multicopy plasmid with the D. melanogaster Adh gene expressed constitutively under the control of yeast ADCp promoter. The D. melanogaster enzyme was produced in cell extracts, as assessed by Coomassie blue staining and Western blotting after polyacrylamide-gel electrophoresis and it was fully active and able to complement the yeast ADH deficiency. Results show that D. melanogaster ADH subunits synthesized in yeast are able to assemble into functional dimeric forms. The synthesized D. melanogaster ADH represents up to 3.5% of the total extracted yeast protein.     

Wolbachia Infections in Drosophila Melanogaster and D. Simulans: Polymorphism and Levels of Cytoplasmic Incompatibility

Wolbachia are endosymbiotic bacteria, widespread in terrestrial Arthropods. They are mainly transmitted vertically, from mothers to offspring and induce various alterations of their hosts' sexuality and reproduction, the most commonly reported phenomenon being Cytoplasmic Incompatibility (CI), observed in Drosophila melanogaster and D. simulans. Basically, CI results in a more or less intense embryonic mortality, occurring in crosses between males infected by Wolbachia and uninfected females. In D. simulans, Wolbachia and CI were observed in 1986. Since then, this host species has become a model system for investigating the polymorphism of Wolbachia infections and CI. In this review we describe the different Wolbachia infections currently known to occur in D. melanogaster and D. simulans. The two species are highly contrasting with regard to symbiotic diversity: while five Wolbachia variants have been described in D. simulans natural populations, D. melanogaster seems to harbor one Wolbachia variant only. Another marked difference between these two Drosophila species is their permissiveness with regard to CI, which seems to be fully expressed in D. simulans but partially or totally repressed in D. melanogaster, demonstrating the involvement of host factors in the control of CI levels. The potential of the two host species regarding the understanding of CI and its evolution is also discussed.     

Comparative analysis of transposable elements in the melanogaster subgroup sequenced genomes

Transposable elements (TEs) are indwelling components of genomes, and their dynamics have been a driving force in genome evolution. Although we now have more information concerning their amounts and characteristics in various organisms, we still have little data from overall comparisons of their sequences in very closely-related species. While the Drosophila melanogaster genome has been extensively studied, we have only limited knowledge regarding the precise TE sequences in the genomes of the related species Drosophila simulans, Drosophila sechellia and Drosophila yakuba. In this study we analyzed the number and structure of TE copies in the sequenced genomes of these four species. Our findings show that, unexpectedly, the number of TE insertions in D. simulans is greater than that in D. melanogaster, but that most of the copies in D. simulans are degraded and in small fragments, as in D. sechellia and D. yakuba. This suggests that all three species were invaded by numerous TEs a long time ago, but have since regulated their activity, as the present TE copies are degraded, with very few full-length elements. In contrast, in D. melanogaster, a recent activation of TEs has resulted in a large number of almost-identical TE copies. We have detected variants of some TEs in D. simulans and D. sechellia, that are almost identical to the reference TE sequences in D. melanogaster, suggesting that D. melanogaster has recently been invaded by active TE variants from the other species. Our results indicate that the three species D. simulans, D. sechellia, and D. yakuba seem to be at a different stage of their TE life cycle when compared to D. melanogaster. Moreover, we show that D. melanogaster has been invaded by active TE variants for several TE families likely to come from D. simulans or the ancestor of D. simulans and D. sechellia. The numerous horizontal transfer events implied to explain these results could indicate introgression events between these species.