Drosophila Adh Research Articles

MIA: Mutual Information Analyzer, a graphic user interface program that calculates entropy, vertical and horizontal mutual information of molecular sequence sets.

BackgroundShort and long range correlations in biological sequences are central in genomic studies of covariation. These correlations can be studied using mutual information because it measures the amount of information one random variable contains about the other. Here we present MIA (Mutual Information Analyzer) a user friendly graphic interface pipeline that calculates spectra of vertical entropy (VH), vertical mutual information (VMI) and horizontal mutual information (HMI), since currently there is no user friendly integrated platform that in a single package perform all these calculations. MIA also calculates Jensen-Shannon Divergence (JSD) between pair of different species spectra, herein called informational distances. Thus, the resulting distance matrices can be presented by distance histograms and informational dendrograms, giving support to discrimination of closely related species.ResultsIn order to test MIA we analyzed sequences from Drosophila Adh locus, because the taxonomy and evolutionary patterns of different Drosophila species are well established and the gene Adh is extensively studied. The search retrieved 959 sequences of 291 species. From the total, 450 sequences of 17 species were selected. With this dataset MIA performed all tasks in less than three hours: gathering, storing and aligning fasta files; calculating VH, VMI and HMI spectra; and calculating JSD between pair of different species spectra. For each task MIA saved tables and graphics in the local disk, easily accessible for future analysis.ConclusionsOur tests revealed that the “informational model free” spectra may represent species signatures. Since JSD applied to Horizontal Mutual Information spectra resulted in statistically significant distances between species, we could calculate respective hierarchical clusters, herein called Informational Dendrograms (ID). When compared to phylogenetic trees all Informational Dendrograms presented similar taxonomy and species clusterization.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-015-0837-0) contains supplementary material, which is available to authorized users.

Experimentally Increased Codon Bias in the DrosophilaAdhGene Leads to an Increase in Larval, But Not Adult, Alcohol Dehydrogenase Activity

Although most amino acids can be encoded by more than one codon, the synonymous codons are not used with equal frequency. This phenomenon is known as codon bias and appears to be a universal feature of genomes. The translational selection hypothesis posits that the use of optimal codons, which match the most abundant species of isoaccepting tRNAs, results in increased translational efficiency and accuracy. Previous work demonstrated that the experimental reduction of codon bias in the Drosophila alcohol dehydrogenase (Adh) gene led to a significant decrease in ADH protein expression. In this study we performed the converse experiment: we replaced seven suboptimal leucine codons that occur naturally in the Drosophila melanogaster Adh gene with the optimal codon. We then compared the in vivo ADH activities imparted by the wild-type and mutant alleles. The introduction of optimal leucine codons led to an increase in ADH activity in third-instar larvae. In adult flies, however, the introduction of optimal codons led to a decrease in ADH activity. There is no evidence that other selectively constrained features of the Adh gene, or its rate of transcription, were altered by the synonymous replacements. These results are consistent with translational selection for codon bias being stronger in the larval stage and suggest that there may be a selective conflict over optimal codon usage between different developmental stages.

Degradation of hsp70 and Other mRNAs in Drosophila via the 5′–3′ Pathway and Its Regulation by Heat Shock

Two general pathways of mRNA decay have been characterized in yeast. Both start with deadenylation. The major pathway then proceeds via cap hydrolysis and 5'-exonucleolytic degradation whereas the minor pathway consists of 3'-exonucleolytic decay followed by hydrolysis of the remaining cap structure. In higher eukaryotes, these pathways of mRNA decay are believed to be conserved but have not been well characterized. We have investigated the decay of the hsp70 mRNA in Drosophila Schneider cells. As shown by the use of reporter constructs, rapid deadenylation of this mRNA is directed by its 3'-untranslated region. The main deadenylase is the CCR4.NOT complex; the PAN nuclease makes a lesser contribution. Heat shock prevents deadenylation not only of the hsp70 but also of bulk mRNA. A completely deadenylated capped hsp70 mRNA decay intermediate accumulates transiently and is degraded via cap hydrolysis and 5'-decay. Thus, decapping is a slow step in the degradation pathway. Cap hydrolysis is also inhibited during heat shock. Degradation of reporter RNAs from the 3'-end became detectable only upon inhibition of 5'-decay and thus represents a minor decay pathway. Because two reporter RNAs and at least two endogenous mRNAs were degraded primarily from the 5'-end with cap hydrolysis as a slow step, this pathway appears to be of general importance for mRNA decay in Drosophila.

Partial Characterization and Evolution of Adh-Adhr in Drosophila dunni

We sequenced 2123 bp of the Adh-Adhr genomic region of Drosophila dunni of the cardini group from two cloned DNA PCR fragments and from two cDNA clones of an Adh transcript. This comprises the Adh coding region and introns, 3' UTR, intergenic sequence, and most of Adhr, which is 260 bp downstream of Adh. Both genes have the typical Drosophila melanogaster Adh structure of three exons and two introns, except for changes in the putative 8 bp sequence involved in downregulation within the 3' UTR of Adh. Two amino acid substitutions could explain the low activity previously reported for this enzyme in D. dunni: Thr --> Lys at position 191 and Val --> Thr at position 189. D. dunni's Adh has the lowest codon bias reported so far for Drosophila species, and based on analysis of the nucleotide substitution rate, it is less conserved than Adhr.

Drosophila Alcohol Dehydrogenase: Acetate–Enzyme Interactions and Novel Insights into the Effects of Electrostatics on Catalysis

Drosophila alcohol dehydrogenase (DADH) is an NAD+-dependent enzyme that catalyzes the oxidation of alcohols to aldehydes/ketones and that is also able to further oxidize aldehydes to their corresponding carboxylic acids. The structure of the ternary enzyme-NADH-acetate complex of the slow alleloform of Drosophila melanogaster ADH (DmADH-S) was solved at 1.6 A resolution by X-ray crystallography. The coenzyme stereochemistry of the aldehyde dismutation reaction showed that the obtained enzyme-NADH-acetate complex reflects a productive ternary complex although no enzymatic reaction occurs. The stereochemistry of the acetate binding in the bifurcated substrate-binding site, along with previous stereochemical studies of aldehyde reduction and alcohol oxidation shows that the methyl group of the aldehyde in the reduction reaction binds to the R1 and in the oxidation reaction to the R2 sub-site. NMR studies along with previous kinetic studies show that the formed acetaldehyde intermediate in the oxidation of ethanol to acetate leaves the substrate site prior to the reduced coenzyme, and then binds to the newly formed enzyme-NAD+ complex. Here, we compare the three-dimensional structure of D.melanogaster ADH-S and a previous theoretically built model, evaluate the differences with the crystal structures of five Drosophila lebanonensis ADHs in numerous complexed forms that explain the substrate specificity as well as subtle kinetic differences between these two enzymes based on their crystal structures. We also re-examine the electrostatic influence of charged residues on the surface of the protein on the catalytic efficiency of the enzyme.

Pleiotropic effect of disrupting a conserved sequence involved in a long-range compensatory interaction in the Drosophila Adh gene.

Recent advances in experimental analyses of the evolution of RNA secondary structures suggest a more complex scenario than that typically considered by Kimura's classical model of compensatory evolution. In this study, we examine one such case in more detail. Previous experimental analysis of long-range compensatory interactions between the two ends of Drosophila Adh mRNA failed to fit the classical model of compensatory evolution. To further investigate and verify long-range pairing in Drosophila Adh with respect to models of compensatory evolution and its potential functional role, we introduced site-directed mutations in the Drosophila melanogaster Adh gene. We explore two alternative hypotheses for why previous analysis of long-range compensatory interactions failed to fit the classical model. Specifically, we investigate whether the disruption of a conserved short-range pairing within Adh exon 2 has an effect on Adh expression or if there is a dual functional role of a conserved sequence in the 3'-UTR in both long-range pairing and the negative regulation of Adh expression. We find that a classical result was not observed due to the pleiotropic effect of changing a nucleotide involved in both long-range base pairing and the negative regulation of gene expression.

A UAS site substitution approach to the in vivo dissection of promoters: interplay between the GATAb activator and the AEF-1 repressor at a Drosophila ecdysone response unit.

An ecdysone response unit (EcRU) directs the expression of the Fat body protein 1 (Fbp1) gene in the third instar larval Drosophila fat body. The tissue-specific activity of this regulatory element necessitates the binding of both the ligand-activated EcR/USP ecdysone receptor and GATAb. To analyze the role played by GATAb in the regulation of the Fbp1 EcRU activity, we have replaced the GATA-binding sites GBS1, GBS2 and GBS3 in the Fbp1 EcRU with UAS sites for the yeast GAL4 activator and tested the activity of the mutagenized Fbp1 EcRUs in transgenic lines, either in the presence or absence of ubiquitously expressed GAL4. Our results reveal that GATAb plays two distinguishable roles at the Fbp1 EcRU that contribute to the tissue-specific activity of this regulatory element. On the one hand, GATAb mediates a fat body-specific transcriptional activation. On the other hand, it antagonizes specifically in the fat body a ubiquitous repressor that maintains the Fbp1 EcRU in an inactive state, refractory to activation by GAL4. We identified this repressor as AEF-1, a factor previously shown to be involved in the regulation of the Drosophila Adh and yp1-yp2 genes. These results show that, for a functional dissection of complex promoter-dependent regulatory pathways, the replacement of specific regulatory target sites by UAS GAL4 binding sites is a powerful alternative to the widely used disruption approach.

A highly conserved sequence in the 3'-untranslated region of the drosophila Adh gene plays a functional role in Adh expression.

Phylogenetic analysis identified a highly conserved eight-base sequence (AAGGCTGA) within the 3'-untranslated region (UTR) of the Drosophila alcohol dehydrogenase gene, Adh. To examine the functional significance of this conserved motif, we performed in vitro deletion mutagenesis on the D. melanogaster Adh gene followed by P-element-mediated germline transformation. Deletion of all or part of the eight-base sequence leads to a twofold increase in in vivo ADH enzymatic activity. The increase in activity is temporally and spatially general and is the result of an underlying increase in Adh transcript. These results indicate that the conserved 3'-UTR motif plays a functional role in the negative regulation of Adh gene expression. The evolutionary significance of our results may be understood in the context of the amino acid change that produces the ADH-F allele and also leads to a twofold increase in ADH activity. While there is compelling evidence that the amino acid replacement has been a target of positive selection, the conservation of the 3'-UTR sequence suggests that it is under strong purifying selection. The selective difference between these two sequence changes, which have similar effects on ADH activity, may be explained by different metabolic costs associated with the increase in activity.

RNA secondary structure and compensatory evolution.

The classic concept of epistatic fitness interactions between genes has been extended to study interactions within gene regions, especially between nucleotides that are important in maintaining pre-mRNA/mRNA secondary structures. It is shown that the majority of linkage disequilibria found within the Drosophila Adh gene are likely to be caused by epistatic selection operating on RNA secondary structures. A recently proposed method of RNA secondary structure prediction based on DNA sequence comparisons is reviewed and applied to several types of RNAs, including tRNA, rRNA, and mRNA. The patterns of covariation in these RNAs are analyzed based on Kimura's compensatory evolution model. The results suggest that this model describes the substitution process in the pairing regions (helices) of RNA secondary structures well when the helices are evolutionarily conserved and thermodynamically stable, but fails in some other cases. Epistatic selection maintaining pre-mRNA/mRNA secondary structures is compared to weak selective forces that determine features such as base composition and synonymous codon usage. The relationships among these forces and their relative strengths are addressed. Finally, our mutagenesis experiments using the Drosophila Adh locus are reviewed. These experiments analyze long-range compensatory interactions between the 5' and 3' ends of Adh mRNA, the different constraints on secondary structures in introns and exons, and the possible role of secondary structures in RNA splicing.

Regulation of Drosophila Adh promoter switching by an initiator-targeted repression mechanism.

The stage-specific expression of the Drosophila alcohol dehydrogenase (Adh) gene is achieved through the alternate activation of two tandem promoters. The proximal promoter is active primarily during late embryonic development and early larval stages, while the distal promoter is active in late third instar larvae and adults. Here, we provide evidence that this Adh promoter switch is regulated by a zinc finger repressor protein (AEF-1) that is expressed predominantly in adult flies and targets the initiator region of the proximal promoter. We propose that AEF-1 plays a critical role in Adh promoter switching by blocking interactions between a component of the general transcription machinery and the initiator region of the proximal promoter.

Drosophila melanogaster alcohol dehydrogenase: mechanism of aldehyde oxidation and dismutation.

Drosophila alcohol dehydrogenase (Adh) catalyses the oxidation of both alcohols and aldehydes. In the latter case, the oxidation is followed by a reduction of the aldehyde, i.e. a dismutation reaction. At high pH, dismutation is accompanied by a small release of NADH, which is not observed at neutral pH. Previously it has been emphasized that kinetic coefficients obtained by measuring the increase in A340, i.e. the release of NADH at high pH is not a direct measure of the aldehyde oxidation reaction and these values cannot be compared with those for alcohol dehydrogenation. In this article we demonstrate that this is not entirely true, and that the coefficients phiB and phiAB, where B is the aldehyde and A is NAD+, are the same for a dismutation reaction and a simple aldehyde dehydrogenase reaction. Thus the substrate specificity of the aldehyde oxidation reaction can be determined by simply measuring the NADH release. The coefficients for oxidation and dehydrogenation reactions (phi0d and phiAd respectively) are complex and involve the constants for the dismutation reaction. However, dead-end inhibitors can be used to determine the quantitative contribution of the kinetic constants for the aldehyde oxidation and reduction pathways to the phi0d and phiAd coefficients. The combination of dead-end and product inhibitors can be used to determine the reaction mechanism for the aldehyde oxidation pathway. Previously, we showed that with Drosophila Adh, the interconversion between alcohols and aldehydes followed a strictly compulsory ordered pathway, although aldehydes and ketones formed binary complexes with the enzyme. This raised the question regarding the reaction mechanism for the oxidation of aldehydes, i.e. whether a random ordered pathway was followed. In the present work, the mechanism for the oxidation of different aldehydes and the accompanying dismutation reaction with the slow alleloenzyme (AdhS) from Drosophila melanogaster has been studied. To obtain reliable results for the liberation of NADH during the initial-rate phase, the reaction was measured with a sensitive recording filter fluorimeter, and the complexes formed with the different dead-end and product inhibitors have been interpreted on the basis of a full dismutation reaction. The results are only consistent with a compulsory ordered reaction mechanism, with the formation of a dead-end binary enzyme-aldehyde complex. Under initial-velocity conditions, the rate of acetate release was calculated to be larger than 2.5 s-1, which is more than ten times that of NADH. The substrate specificity constant (kcat/Km or 1/phiB) with respect to the oxidation of substrates was propan-2-ol>ethanol>acetaldehyde>trimethylacetaldehyde.

Cis-acting sequences controlling the adult-specific transcription pattern of theDrosophila affinidisjuncta Adh gene

The cis-acting sequences required for the adult-specific expression pattern of the alcohol dehydrogenase (Adh) gene of the Hawaiian picture-winged fruit fly, Drosophila affinidisjuncta were analyzed by germline transformation. Normally this gene produces two developmentally regulated transcripts. The upstream (distal) promoter produces a distal transcript, which makes up about 80% of the total in adults, while the downstream (proximal) promoter produces a corresponding proximal transcript, which accounts for the remainder. Previously constructed genes lacking regions corresponding to regulatory elements within the Drosophila melanogaster Adh gene or regions known to be required for full expression of the D. affinidisjuncta Adh gene in larvae were analyzed by introduction into the germline of D. melanogaster followed by RNase-protection analysis of RNA levels. In addition, to test a model of preferential promoter utilization by which transcription at the proximal promoter is inhibited by transcription initiated at the upstream distal promoter, a construction lacking the distal promoter was analyzed. Sequences homologous to the adult enhancer of the Adh gene of D. melanogaster appear to play a similar role in the D. affinidisjuncta gene. In contrast to what has been reported for other Drosophila Adh genes, this and some other regulatory elements are shared by the two promoters of the D. affinidisjuncta gene. Taken together, the results favor a model of stage-specific switching between the two promoters of the D. affinidisjuncta gene that involves competition for limiting components stimulating transcription, rather than interference by read-through from the upstream promoter.

A transcriptional role for conserved footprinting sequences within the larval promoter of a Drosophila alcohol dehydrogenase gene

All Drosophila alcohol dehydrogenase (Adh) genes that are expressed in larvae display strong transcription in the larval fat body. To identify and characterize elements needed for Adh promoter function, footprinting analysis of the Drosophila affinidisjuncta Adh gene was performed with stage-specific nuclear proteins from embryos and larvae. Multiple sites upstream of the larval promoter were protected from deoxyribonuclease digestion by both embryonic and larval extracts. Comparison with foot-printing results for Adh genes from other Drosophila species revealed only one nuclease-protected region that is conserved in both sequence and position. Clustered point mutations in this sequence were analyzed by footprinting analysis, transient transformation and in vitro transcription. Two separate sequences in this footprinting region exerted positive effects on transcription from the Adh proximal promoter in the larval fat body. The effects of these sequences on gene expression were synergistic. One of these sequences, TGATAA, bound in vitro to Drosophila melanogaster box A binding factor protein, as shown by gel mobility shift assays. This is the first direct demonstration of specific protein-DNA interactions influencing transcription of a Drosophila Adh gene in the larval fat body.

Drosophila melanogaster alcohol dehydrogenase: product-inhibition studies

The Drosophila melanogaster alleloenzymes AdhS and AdhF have been studied with respect to product inhibition by using the two substrate couples propan-2-ol/acetone and ethanol/acetaldehyde together with the coenzyme couple NAD+/NADH. With both substrate couples the reaction was consistent with an ordered Bi Bi mechanism. The substrates added to the enzyme in a compulsory order, with coenzyme as the leading substrate, to give two interconverting ternary complexes. The second ternary complex broke down with release of products in an obligatory order, with the aldehyde/ketone leaving first. Both the acetaldehyde and acetone products formed binary complexes with the enzyme that affected NAD+ binding. However, only an enzyme-acetone complex seemed to affect NADH binding and hence the reverse reaction. The inhibitory pattern with acetaldehyde as product was also affected by the formation of a ternary enzyme-NAD(+)-acetaldehyde complex, which broke down to acetic acid and NADH. The product-inhibition pattern shown in the present work is different from that published for Drosophila Adh previously and this discrepancy can not be explained by the use of different variants of Drosophila Adh.

Isolation and partial characterization of two alcohol dehydrogenase isozymes from the medfly Ceratitis capitata

Two alcohol dehydrogenase isozymes, namely ADH-1 and ADH-2 from Ceratitis capitata were purified to homogeneity and further characterized. After ammonium sulphate precipitation from an extract of whole third instar larvae, the two isozymes were separated by ion exchange chromatography on Q-Sepharose. A combination of affinity chromatography, gel filtration and ion exchange chromatography was then used to purify each isozyme (50 and 57 times with 53 and 58% yields, for ADH-1 and -2 respectively). A crucial step for obtaining homogeneous enzyme preparations was affinity chromatography on Cibacron Blue Sepharose coupled with specific elution with NAD. Each of the isozymes is a dimer with subunit molecular weight of ∼27 kDa. Both isozymes show a pH optimum of 9.6. ADH-1 proved to be immunochemically similar to ADH-2 when tested by Western blot analysis using polyclonal antibodies raised against ADH-1. While crude extracts of Dacus oleae ADH cross-react with these antibodies, no cross reactivity was observed with Drosophila melanogaster extracts. The sequence of a 22-residue peptide from ADH-1 was determined and showed 36% identity with residues 26–47 of the Drosophila melanogaster ADH sequence. Both the sizes of the purified proteins and the observed sequence similarity between ADH-1 and Drosophila ADH strongly suggest that the medfly ADH isozymes belong to the family of short chain dehydrogenases.

On the detection of nonrandom associations between DNA polymorphisms in natural populations of Drosophila.

The capacity to detect nonrandom associations between restriction-map variants was examined in eight gene regions of Drosophila melanogaster (yellow-achaetescute, white, Zw, Adh, Est6, and rosy) and D. pseudoobscura (Adh and Xdh), on the basis of published population data. The statistical power from individual pairwise tests was both heterogeneous and generally low across gene regions. Sample sizes larger than those currently being used are needed to ensure any power to detect disequilibrium by individual tests. It is found that the heterogeneity in power is mostly explained by large differences in the intensity of sample disequilibrium among regions. The yellow-achaete-scute, Zw, and Adh loci of D. melanogaster displayed both the highest mean power (approximately 0.4) and a very great disequilibrium (mean absolute values of D' were 0.8-1). By contrast, all the other gene regions exhibited lower mean power (approximately 0.2) and moderate levels of disequilibrium (0.4-0.6). Although the proportion of significant pairwise associations, especially for white, Est6, and rosy in D. melanogaster and for Adh and Xdh in D. pseudoobscura, is more or less close to the type I error, simultaneous-inference significance tests show that gametic disequilibrium is occurring at the eight DNA regions examined.

Effect of site-directed mutagenesis on conserved positions of Drosophila alcohol dehydrogenase

Tyr 152 and Lys 156 may be functionally important residues in Drosophila ADH as they are conserved in the genus and in all short-chain dehydrogenases. In addition, unaltered Gly positions could have a crucial role in the building of the structural framework. We have modified Drosophila ADH and expressed the mutant forms in E. coli. Mutation of Tyr 152 to Glu or Gin, Lys 156 to Ile, Gly 184 to Leu, and the double mutant Gly 130 to Cys and Gly 133 to Ile, all rendered, with different substrates and at different pHs, an inactive enzyme. Results suggest that Tyr 152 and Lys 156are involved in catalysis and that Gly 130, Gly 133 and Gly 184 contribute substantially to the structure of the active form.

Colorimetric assay to determine alcohol dehydrogenase activity

A colorimetric assay has been developed to quantify alcohol dehydrogenase (ADH) activity. The advantage of this method over conventional spectrophotometric assays is that many samples can be processed at once in a 96-well plate, monitoring the absorbance at 590 nm with a microtiter reader. ADH inhibitors have been used to show the specificity of this method. The assay offers an easy and reliable test for monitoring routine measurements of ADH activity and could be a valuable tool for those involved in the biomedical research of alcoholism, as well as in gene expression methodology in which Drosophila Adh has been widely used as reporter gene in transfection assays.

DNA-histone interactions are sufficient to position a single nucleosome juxtaposing Drosophila Adh adult enhancer and distal promoter.

The alcohol dehydrogenase gene (Adh) of Drosophila melanogaster is transcribed from two tandem promoters in distinct developmental and tissue-specific patterns. Both promoters are regulated by separate upstream enhancer regions. In its wild-type context the adult enhancer specifically stimulates only the distal promoter, approximately 400 bp downstream, and not the proximal promoter, which is approximately 700 bp further downstream. Genomic footprinting and micrococcal nuclease analyses have revealed a specifically positioned nucleosome between the distal promoter and adult enhancer. In vitro reconstitution of this nucleosome demonstrated that DNA-core histone interactions alone are sufficient to position the nucleosome. Based on this observation and sequence periodicities in the underlying DNA, the mechanism of positioning appears to involve specific DNA structural features (ie flexibility or curvature). We have observed this nucleosome positioned early during development, before tissue differentiation, and before non-histone protein-DNA interactions are established at the distal promoter or adult enhancer. This nucleosome positioning element in the Adh regulatory region could be involved in establishing a specific tertiary nucleoprotein structure that facilitates specific cis-element accessibility and/or distal promoter-adult enhancer interactions.

Drosophila transcriptional repressor protein that binds specifically to negative control elements in fat body enhancers.

Expression of the Drosophila melanogaster Adh gene in adults requires a fat body-specific enhancer called the Adh adult enhancer (AAE). We have identified a protein in Drosophila nuclear extracts that binds specifically to a site within the AAE (adult enhancer factor 1 [AEF-1]). In addition, we have shown that AEF-1 binds specifically to two other Drosophila fat body enhancers. Base substitutions in the AEF-1 binding site that disrupt AEF-1 binding in vitro result in a significant increase in the level of Adh expression in vivo. Thus, the AEF-1 binding site is a negative regulatory element within the AAE. A cDNA encoding the AEF-1 protein was isolated and shown to act as a repressor of the AAE in cotransfection studies. The AEF-1 protein contains four zinc fingers and an alanine-rich sequence. The latter motif is found in other eukaryotic proteins known to be transcriptional repressors.