Drosophila Adh Research Articles

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.

The Binding Site of a Steroid Hormone Receptor-Like Protein within the Drosophila Adh Adult Enhancer Is Required for High Levels of Tissue-Specific Alcohol Dehydrogenase Expression

Developmental and tissue-specific transcription from the Adh distal promoter is regulated in part by the Adh adult enhancer, located 450 to 600 bp upstream from the distal RNA start site. We have characterized four proteins (DEP1 to DEP4), present in Drosophila tissue culture cell nuclear extracts, which bind to this enhancer. DEP1 and DEP2 bind to a positive cis-acting element (-492 to -481) and share nucleotide contacts. A small linker replacement deletion mutation, which disrupts the overlapping DEP1- and DEP2-binding sites, reduces Adh distal transcription in an alcohol dehydrogenase (ADH)-expressing cultured cell line, in the adult fat body (the major tissue of ADH expression), as well as in some but not all adult tissues where ADH is normally expressed. This enhancer element contains an imperfect palindromic sequence similar to steroid hormone receptor superfamily response elements. Binding-site screening of a lambda gt11 expression library has identified the steroid receptor superfamily member fushi tarazu factor 1 (FTZ-F1) as a protein that binds to this site. Anti-FTZ-F1 antibodies have identified DEP1 as FTZ-F1. DEP2 also binds to the FTZ-F1 site from the fushi tarazu zebra element, suggesting that DEP2 may also be a steroid receptor superfamily member. Our results raise the possibility that Adh regulation in certain adult tissues involves a hormone-mediated pathway. Because DEP1 (FTZ-F1) and DEP2 contact some of the same nucleotides within the positive cis element, it is unlikely that they can bind simultaneously. Such alternative binding may play a role in the tissue-specific and developmental transcription of Adh.

The binding site of a steroid hormone receptor-like protein within the Drosophila Adh adult enhancer is required for high levels of tissue-specific alcohol dehydrogenase expression.

Developmental and tissue-specific transcription from the Adh distal promoter is regulated in part by the Adh adult enhancer, located 450 to 600 bp upstream from the distal RNA start site. We have characterized four proteins (DEP1 to DEP4), present in Drosophila tissue culture cell nuclear extracts, which bind to this enhancer. DEP1 and DEP2 bind to a positive cis-acting element (-492 to -481) and share nucleotide contacts. A small linker replacement deletion mutation, which disrupts the overlapping DEP1- and DEP2-binding sites, reduces Adh distal transcription in an alcohol dehydrogenase (ADH)-expressing cultured cell line, in the adult fat body (the major tissue of ADH expression), as well as in some but not all adult tissues where ADH is normally expressed. This enhancer element contains an imperfect palindromic sequence similar to steroid hormone receptor superfamily response elements. Binding-site screening of a lambda gt11 expression library has identified the steroid receptor superfamily member fushi tarazu factor 1 (FTZ-F1) as a protein that binds to this site. Anti-FTZ-F1 antibodies have identified DEP1 as FTZ-F1. DEP2 also binds to the FTZ-F1 site from the fushi tarazu zebra element, suggesting that DEP2 may also be a steroid receptor superfamily member. Our results raise the possibility that Adh regulation in certain adult tissues involves a hormone-mediated pathway. Because DEP1 (FTZ-F1) and DEP2 contact some of the same nucleotides within the positive cis element, it is unlikely that they can bind simultaneously. Such alternative binding may play a role in the tissue-specific and developmental transcription of Adh.

Drosophila fat body protein P6 and alcohol dehydrogenase are derived from a common ancestral protein

Drosophila melanogaster alcohol dehydrogenase is an example of convergent evolution: it is not related to the ADHs of other organisms, but to short-chain dehydrogenases, which until now have been found only in bacteria and in mammalian steroid hormone metabolism. We present evidence that the Drosophila ADH is phylogenetically more closely related to P6, another highly expressed protein from the fat body of Drosophila, than it is to the short-chain dehydrogenases. The polypeptide sequence of P6 was inferred from DNA sequence analysis. Both ADH and P6 polypeptides have retained a high structural similarity with respect to the Chou-Fasman prediction of secondary structure and hydropathy. P6 is also homologous to the 25-kd protein from the fat body of Sarcophaga peregrina, whose sequence we have reexamined. The evolution of the P6-ADH family of proteins is characterized by a dramatic increase in the methionine content of P6. Methionine accounts for 20% of P6 amino acids. This is in contrast with the absence of this amino acid in mature ADH. There is evidence that P6 and the 25-kd protein have undergone a parallel and independent enrichment in methionine. When corrected for this, the rate of amino acid replacement shows that the P6-25-kd lineage diverged from insect ADH shortly before the divergence of the ADH gene (Adh) from its 3'-duplication (Adh-dup).

Introduction of single-stranded ADH genes into drosophila results in tissue-specific expression

We injected single-stranded circular DNA containing a Drosophila Adh gene into ADH-negative embryos of Drosophila melanogaster and performed ADH histochemical staining on third instar larvae of the injected generation. Introduction of either the coding or non-coding strand resulted in correct tissue-specific expression of the Adh gene in larvae. Southern blotting revealed that the bulk of the injected DNA became double-stranded shortly after injection and was not integrated into the genome.

ADrosophila Adhgene can be activated intransby an enhancer

The ability of a segment of the Drosophila Adh gene to produce ADH activity in larvae is dependent upon the presence of a 53 bp sequence (called NS1) located between 289 and 341 bp upstream of the larval transcription start site. This sequence behaves like an enhancer in that it can stimulate gene activity when it is placed at various distances from, or on either side of, an Adh gene. Like a typical enhancer, NS1 does not ordinarily function in trans. However, when an Adh gene lacking NS1 is placed on one plasmid, and a second gene carrying NS1 is placed on another, and the two plasmids are interlocked in a catenane, both genes are active. This finding supports the mechanism of loop-mediated enhancer action.

Tissue specific expression of the Drosophila Adh gene: a comparison of in situ hybridization and immunocytochemistry

The tissue specific patterns for Drosophila melanogaster alcohol dehydrogenase (Adh) mRNA and protein expression were determined using in situ hybridization and immunocytochemical techniques. Alcohol dehydrogenase mRNAs were localized in thin sections of frozen tissue using the hybridization of single stranded RNA probes. Alcohol dehydrogenase protein was identified in frozen tissue samples using ADH antisera, a biotinylated secondary antibody, and streptavidin conjugated to horseradish peroxidase. In tissues such as fat body, gastric caeca, and adult cardiac valve, the patterns of expression for ADH protein and mRNA were identical. Other tissues such as oocytes, nurse cells, imaginal disks, and brain show levels of Adh mRNA that are lower than or equivalent to those observed in the previously mentioned tissues, but they exhibit little or no ADH protein. The lack of concordance between Adh mRNA and ADH protein expression in oocytes and nurse cells may reflect the packaging of maternal mRNAs (but not ADH protein) for use in early development. The reason(s) for the other discrepancies in protein and mRNA expression are not known at this time but may be due to post-transcriptional regulation in these specific tissues.

Analysis of formaldehyde-inducedAdh mutations inDrosophila by RNA structure mapping and direct sequencing of PCR-amplified genomic DNA

Two formaldehyde-induced mutations at the Drosophila Adh locus (Adhfn45 and Adhfn46) were analyzed by determining RNA structures at different developmental stages, polymerase chain reaction (PCR) amplification of the affected genomic regions, and direct sequencing of the resulting double-stranded DNA fragments. Adhfn46 adults and larvae accumulate abundant ADH-like distal (adult) and proximal (larval) transcripts that are shorter than transcripts in wild-type flies by a lesion located in the second ADH protein-coding exon. Direct sequencing of the amplified DNA region showed that Adhfn46 contains a 69-bp in-frame deletion that removes 23 amino acids near one border of the second exon. Consistent with these findings, we observed a shorter ADHfn46 protein present at only 3% of wild-type levels. In contrast, Adhfn45 adults and larvae accumulate much smaller amounts of ADH-like distal and proximal transcripts. Both RNAs have an identical aberration in RNA splicing of the 65-base intron sequence. Direct sequencing of the amplified mutated DNA region showed that Adhfn45 contains a 21-bp deletion that removed and rearranged DNA at the 5' splice junction of the 65-bp intron. No ADH cross-reacting material is detected in Adhfn45 flies. Direct-repeat sequences (3-11 bp) are present flanking and within the mutated DNA regions. The patterns of DNA deletion and deletion accompanied by sequence addition at the mutant sites suggest a slipped mispairing mechanism during DNA replication or repair that involves local DNA homology.

Alternative DNA-protein interactions in variable-length internucleosomal regions associated withDrosophila Adhdistal promoter expression

Chromatin at the Drosophila Adh distal promoter displays an ordered but different conformation in different cell types as detected by a modified exonuclease protection assay and accessibility to endonucleases. In cells not transcribing Adh (ADH-) sequences between -40 to +30 of the distal RNA initiation site exist as a DNA linker between positioned nucleosomes, and appear to interact with a specific DNA-binding protein. In contrast, a longer linker DNA, from -140 to +30, is bound in a multi-protein transcription initiation complex in cells that specifically transcribe the distal (adult) ADH RNA (ADH+A). These DNA-protein interactions can account for a localized open chromatin structure at the distal promoter in ADH+A cells. The observed mutually exclusive patterns of DNA-protein interactions in the linkers of different ADH cell types between -40 to +30 suggest a model for organizing alternative chromatin structure associated with gene regulation. Two DNA binding proteins, one being a TATA box binding factor, compete for overlapping sites to allow either assembly of a transcription initiation complex and transcription, or positioning of nucleosomes for stable repression.

The role of specific enhancer-promoter interactions in the Drosophila Adh promoter switch.

The Drosophila melanogaster alcohol dehydrogenase (Adh) gene is transcribed from two promoters active at different developmental stages. In this paper we show that the promoters are differentially stimulated by two enhancers, the Adh larval enhancer and the Adh adult enhancer. In early larval stages, the larval enhancer stimulates transcription from the proximal promoter; in late larval stages, the two enhancers act synergistically to stimulate transcription from the distal promoter; and in adults, the adult enhancer stimulates transcription from the distal promoter. To determine the basis for these enhancer-promoter interactions, we examined the effect of each enhancer on three different promoters. We found that the adult enhancer is stage specific and stimulates transcription from all three promoters. In contrast, the larval enhancer is potentially active in all stages and stimulates transcription from only two of the three promoters. These observations suggest that normal temporal expression of Adh depends on the stage-specific activity of the adult enhancer and the differential response of the proximal and distal promoters to the larval enhancer.

On the oxidation of aldehydes by alcohol dehydrogenase of Drosophila melanogaster: Evidence for the gem-diol as the reacting substrate

Purified alcohol dehydrogenase from Drosophila melanogaster is able to catalyze the oxidation of aldehydes in addition to primary and secondary alcohols. Acetaldehyde, methoxy-acetaldehyde, chloro-acetaldehyde, and trichloro-acetaldehyde (chloral hydrate) are used here to show that the gem-diol structure is the reacting form of the aldehydes. The reaction rates depend primarily on the degree of hydration of the aldehydes and to a lesser extent on the resulting electronic state of the hydrated molecules. These results resemble the chemical oxidation of aldehydes. The V K m values for the oxidation of acetaldehyde hydrate and chloro-acetaldehyde hydrate are very similar to that for ethanol. The very high oxidation rates of secondary alcohols compared to primary alcohols by Drosophila ADH are explained as the result of the structural resemblance to hydrated aldehydes and a more favorable electronic state. This behavior is in contrast to mammalian and yeast ADH, which have a low affinity for secondary alcohols and aldehydes. The oxidation of aldehydes by ADH apparently enables D. melanogaster to live in alcoholic environments.

Drosophila Adh: A promoter element expands the tissue specificity of an enhancer

Drosophila alcohol dehydrogenase ( Adh) genes are expressed in the fat body and in species-specific sets of other tissues during larval and adult development. The Drosophila mulleri Adh-1 gene is expressed in the larval fat body and in three other larval tissues. In this paper, we show that Adh-1 expression in multiple cell types is the result of synergistic interactions between fat body-specific enhancers and a specific Adh-1 promoter element.

Molecular consequences of two formaldehyde-induced mutations in the alcohol dehydrogenase gene ofDrosophila melanogaster

Adhfn23 and Adhfn24 are two formaldehyde-induced, homozygous-viable, alcohol dehydrogenase-null mutants that bear lesions in the gene that codes for the alcohol dehydrogenase (ADH; EC 1.1.1.1) of Drosophila melanogaster. Adhfn23 contains a 34-base pair deletion in the C-terminal coding region of the alcohol dehydrogenase structural gene. By immunological and molecular analysis, we show that the deletion shifts the translation reading frame and results in a prematurely truncated polypeptide product (10 amino acids shorter than wild type) that cross-reacts with antibody raised against ADH. The steady-state level of alcohol dehydrogenase mRNA present in this mutant is close (97%) to that in the wild type, but the steady-state level of alcohol dehydrogenase-like protein is 50% lower. Moreover, the rate of alcohol dehydrogenase synthesis in Adhfn23 flies is reduced to 60% of that found in the wild type. Hence both the rate of synthesis and the rate of degradation of alcohol dehydrogenase are affected. In contrast, Adhfn24 which contains an 11-base pair deletion in the N-terminal coding region of the ADH gene, synthesizes no immunodetectable protein, and the amount of alcohol dehydrogenase mRNA is less than half that of wild-type flies. As with Adhfn23, the deletion in Adhfn24 results in a change in the reading frame. Unlike Adhfn23, however, nucleic acid sequence data indicate that polypeptide chain elongation can proceed for a considerable distance (over 130 amino acids) beyond the deletion. Based upon antigenic binding-site predictions, the resultant aberrant protein (projected 195 amino acids in length) would share few antigenic sites with the alcohol dehydrogenase from the wild type, which may account for the lack of immunoprecipitable material in this mutant. The contrasting effects these two deletions have on the Drosophila ADH mRNA levels and ADH protein levels are discussed.

Developmental regulation by an enhancer from the Sgs-4 gene of Drosophila

A cis acting regulatory region has previously been identified 300-500 bp upstream of the Drosophila glue protein gene, Sgs-4. The functional capabilities of this region have now been examined by fusing it to the Drosophila Adh gene and determining the pattern of expression from the fused construct after transformation. The results show that the Sgs-4 sequences between -150 and -568 are able to direct Adh expression in late third-instar salivary glands, the appropriate tissue and timing for Sgs-4 expression. In addition, the Sgs-4 sequence elevates Adh expression in the anterior midgut and fat body, despite the fact that Sgs-4 is not normally expressed there. All three regulatory activities, tissue specificity, timing and enhancement, show the positional flexibility of enhancer elements. In addition, the Sgs-4 and Adh regulatory elements combine to direct expression in novel spatial/temporal combinations in which neither would normally be expressed.

Temporal variation of Drosophila melanogaster Adh allele frequencies, inversion freqencies, and population sizes

The frequencies of cosmopolitan inversions and of Adh alleles in D. melanogaster, and population sizes of D. melanogaster and D. simulans have been monitored for two years in seven south-eastern Queensland sites. D. melanogaster population sizes showed greater ‘summer’ increases and ‘winter’ decreases than those of D. simulans. Adh allele frequencies showed no seasonal or yearly variation, and no significant correlations with environmental variables after accounting for linkage disequilibrium with In(2L)t. Some inversion frequencies showed regular ‘summer’ to ‘winter’ declines, yearly variation, and significant correlations with population size. Thus, the temporal variation of some inversion frequencies, unlike that for Adh allele frequencies, paralleled previously reported frequency variation over latitude.

The effect of temperature on biochemical and molecular properties of Drosophila alcohol dehydrogenase.

The gene products of the two major alleles of alcohol dehydrogenase (ADH-F and ADH-S) have been subjected to kinetic and biochemical analyses over a range of temperatures. Although temperature was found to have a significant effect on both kinetic and biochemical properties of Drosophila ADH, no significant differential effect was observed between the major ADH allozymes. The results are discussed within the context of the selective maintenance of Adh polymorphism in natural populations.

Copiais transcriptionally responsive to environmental stress

Adult Drosophila subjected to a variety of environmental stresses that induce classic Drosophila heat shock response simultaneously exhibit a rapid and significant rise in copia homologous transcripts. Levels of Drosophila Adh (alcohol dehydrogenase gene) and 18s ribosomal RNA were unaffected by environmental stress. Copia's ability to be induced by stress is correlated with the presence of sequences homologous to the heat shock promoter consensus sequence which appear to be appropriately positioned within the element's long terminal repeat (LTR). Although the copia-like element 297 also contains homologous sequences within its LTR, they are atypically positioned relative to the element's transcription start site and are non-functional in that the 297 element was not stress inducible. Hence, the position of the consensus sequence relative to a gene's transcription start site may be a factor in stress inducibility.

Replication, integration and expression of exogenous DNA injected into fertilized eggs of Xenopus laevis

We have analyzed the fate of circular and linear DNA molecules following microinjection into the cytoplasm of fertilized eggs of Xenopus laevis. Recombinant plasmids containing sea urchin histone genes (pSp 102), Drosophila ADH genes (SAC-1), and SV40 (SV2 CAT) replicate during the development of the injected frog embryo. In contrast, pBR322 either as monomers or multimers does not appear to replicate as efficiently. Generally, injected circular DNAs were not detectable by the gastrula stage of development, although there were several examples in which these molecules persisted until larval stages. In 90% of the cases, injected linear DNAs persisted as discrete molecules into early embryonic stages. A portion of the DNA sequences complementary to injected linear and circular molecules was detected comigrating with the high-molecular-weight cellular frog DNA (48 kb or larger) from mid-cleavage stages onward. Restriction enzyme analysis of DNA from injected embryos suggested some copies of the injected DNAs were integrated into the frog genome. This occurred in about 10%–30% of the cases of injected circular DNA and approximately 60%–70% of the cases of injected linear DNA. We were able to rescue circular plasmids from the injected blastulae by retransforming Escherichia coli. Restriction enzyme analysis of this DNA suggested that the majority of injected circular DNAs were not modified following replication in the frog embryo. The DNA of Xenopus embryos was highly methylated. On the other hand, injected DNA sequences were not methylated de novo even after many replication cycles in the frog embryo. Ribonucleic acid (RNA) transcripts from the injected DNAs were detectable by the late blastula stage of development.

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.

Alcohol dehydrogenase gene of Drosophila melanogaster: relationship of intervening sequences to functional domains in the protein.

The gene that codes for Drosophila alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase EC 1.1.1.1) was identified in a bacteriophage lambda library of genomic Drosophila DNA by using ADH cDNA cloned DNA as a probe. The DNA sequence of the protein encoding region was shown to be in agreement with the amino acid sequence of the ADH. Two intervening DNA sequences (introns) were identified within the protein encoding region: one was 65 nucleotides and located between the codons for amino acid residues 32 and 33, and one was 70 nucleotides and located between the codons for amino acid residues 167 and 168. Both contained the 5' G-T and 3' A-G dinucleotides characteristic of intron boundaries of eukaryotic genes. On the basis of secondary structure predictions, the first 140 amino acid residues of Drosophila ADH are in an alternating beta-sheet/alpha-helix arrangement which is characteristic of the coenzyme binding domain of dehydrogenases. The smaller of the two introns interrupts the domain predicted to bind the adenine portion of the coenzyme.