ADH enzyme activity and Adh gene expression in Drosophila melanogaster lines differentially selected for increased alcohol tolerance

In Drosophila melanogaster, alcohol dehydrogenase (ADH) activity is essential for ethanol tolerance, but its role may not be restricted to alcohol metabolism alone. Here we describe ADH activity and Adh expression level upon selection for increased alcohol tolerance in different life-stages of D. melanogaster lines with two distinct Adh genotypes: Adh(FF) and Adh(SS). We demonstrate a positive within genotype response for increased alcohol tolerance. Life-stage dependent selection was observed in larvae only. A slight constitutive increase in adult ADH activity for all selection regimes and genotypes was observed, that was not paralleled by Adh expression. Larval Adh expression showed a constitutive increase, that was not reflected in ADH activity. Upon exposure to environmental ethanol, sex, selection regime life stage and genotype appear to have differential effects. Increased ADH activity accompanies increased ethanol tolerance in D. melanogaster but this increase is not paralleled by expression of the Adh gene.     

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.     

Regulatory elements involved in Drosophila Adh gene expression are conserved in divergent species and separate elements mediate expression in different tissues.

Alcohol dehydrogenase (ADH) is expressed in a complex temporal and spatial pattern from tandem promoters (proximal and distal) in Drosophila melanogaster, and from two closely linked genes (Adh-1 and Adh-2) in D. mulleri. The expression patterns of Adh-1 and the proximal promoter, and Adh-2 and the distal promoter are similar, but not identical. We show that the mulleri Adh genes are appropriately expressed when introduced into the melanogaster genome, indicating that the cis- and trans-acting elements which regulate the corresponding promoters are functionally equivalent in the two species. By analyzing the expression of in vitro generated mutants of the mulleri Adh locus, we define at least three regulatory regions of the mulleri Adh genes and show that different control elements mediate the expression of Adh in different tissues.     

Mutations that induce the heat shock response of Drosophila

We have isolated a number of mutations in D. melanogaster that result in the constitutive expression of the heat shock response in a tissue-specific manner. These mutations induce alcohol dehydrogenase (ADH) when the ADH structural gene is fused to the promoter for the 70 kd heat shock protein (hsp70) gene. Flies carrying these mutations, the hsp70-Adh fusion, and a deletion in their endogenous Adh genes are ethanol tolerant and exhibit elevated ADH levels. Several of the tissue-specific mutations have also been shown to induce an hsp26-Adh fusion gene in trans. The mutation Act88FKM75, a G----A transition in the indirect flight muscle-specific actin gene, also exhibits this phenotype. Comparisons with the Act88FKM75 mutation suggest that the tissue-specific mutations induce the heat shock response by disrupting the physiology of the cells in which the variant gene product is expressed.     

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.     

Developmental profile and tissue distribution of Drosophila alcohol dehydrogenase: an immunochemical analysis with monoclonal antibodies.

To analyze Drosophila alcohol dehydrogenase gene (Adh) expression and tissue distribution at various developmental stages, we devised several immunochemical techniques making use of monoclonal antibodies against Drosophila alcohol dehydrogenase (ADH), which had been obtained previously. We here report their application to analyze the expression of Adh in a wild-type strain of D. melanogaster. s-ELISA tests were performed to evaluate fluctuations in ADH content and specific activity during development in individual organs as well as in whole individuals. In all cases, ADH specific activity appeared to be quite constant, which implies that variations in enzyme activity reflect differences in protein content. Immunoblottings of crude homogenates revealed immunoreactive low relative molecular mass peptides in addition to the 27 KD monomeric band, showing a conserved banding pattern in different organs and developmental stages. Immunohistochemical assays on whole organs were used to analyze the general pattern of ADH distribution. Immunoperoxidase staining of cryosections proved to be of crucial relevance, as it yielded full details of the tissue localization of ADH within the ADH-positive organs. We have shown not only that ADH displays a specific distribution in some organs but also that the enzyme is restricted to certain cell types.     

Increased Variation in Adh Enzyme Activity in Drosophila Mutation-Accumulation Experiment Is Not Due to Transposable Elements at the Adh Structural Gene

We present here a molecular analysis of the region surrounding the structural gene encoding alcohol dehydrogenase (Adh) in 47 lines of Drosophila melanogaster that have each accumulated mutations for 300 generations. While these lines show a significant increase in variation of alcohol dehydrogenase enzyme activity compared to control lines, we found no restriction map variation in a 13-kb region including the complete Adh structural gene and roughly 5 kb of both 5' and 3' sequences. Thus, the rapid accumulation of ADH activity variation after 28,200 allele generations does not appear to have been due to the mobilization of transposable elements into or out of the Adh structural gene region.     

Distinct retinoid metabolic functions for alcohol dehydrogenase genes Adh1 and Adh4 in protection against vitamin A toxicity or deficiency revealed in double null mutant mice

The ability of class I alcohol dehydrogenase (ADH1) and class IV alcohol dehydrogenase (ADH4) to metabolize retinol to retinoic acid is supported by genetic studies in mice carrying Adh1 or Adh4 gene disruptions. To differentiate the physiological roles of ADH1 and ADH4 in retinoid metabolism we report here the generation of an Adh1/4 double null mutant mouse and its comparison to single null mutants. We demonstrate that loss of both ADH1 and ADH4 does not have additive effects, either for production of retinoic acid needed for development or for retinol turnover to minimize toxicity. During gestational vitamin A deficiency Adh4 and Adh1/4 mutants exhibit completely penetrant postnatal lethality by day 15 and day 24, respectively, while 60% of Adh1 mutants survive to adulthood similar to wild-type. Following administration of a 50-mg/kg dose of retinol to examine retinol turnover, Adh1 and Adh1/4 mutants exhibit similar 10-fold decreases in retinoic acid production, whereas Adh4 mutants have only a slight decrease. LD(50) studies indicate a large increase in acute retinol toxicity for Adh1 mutants, a small increase for Adh4 mutants, and an intermediate increase for Adh1/4 mutants. Chronic retinol supplementation during gestation resulted in 65% postnatal lethality in Adh1 mutants, whereas only approximately 5% for Adh1/4 and Adh4 mutants. These studies indicate that ADH1 provides considerable protection against vitamin A toxicity, whereas ADH4 promotes survival during vitamin A deficiency, thus demonstrating largely non-overlapping functions for these enzymes in retinoid metabolism.     

Variation between electrophoretically identical alleles at the alcohol dehydrogenase locus in Drosophila melanogaster

A new variant of alcohol dehydrogenase (ADH 7lk) was found in a laboratory stock of Drosophila melanogaster. ADH in this stock had the same electrophoretic mobility as the F variant both on acrylamide and on agar. Activity levels were similar to the levels in F flies at temperature between 15 and 25 C. But while ADH F enzyme is inactivated rapidly at 40 C, ADH 7lk is still active. Also, ADH S is not inactivated at this temperature, but has a far lower activity per fly than ADH 7lk. Genetic analysis showed that the new variant is an allele of the Adh locus.     

Evolution of the Adh locus in the Drosophila willistoni group: the loss of an intron, and shift in codon usage

We report here the DNA sequence of the alcohol dehydrogenase gene (Adh) cloned from Drosophila willistoni. The three major findings are as follows: (1) Relative to all other Adh genes known from Drosophila, D. willistoni Adh has the last intron precisely deleted; PCR directly from total genomic DNA indicates that the deletion exists in all members of the willistoni group but not in any other group, including the closely related saltans group. Otherwise the structure and predicted protein are very similar to those of other species. (2) There is a significant shift in codon usage, especially compared with that in D. melanogaster Adh. The most striking shift is from C to U in the wobble position (both third and first position). Unlike the codon-usage-bias pattern typical of highly biased genes in D. melanogaster, including Adh, D. willistoni has nearly 50% G + C in the third position. (3) The phylogenetic information provided by this new sequence is in agreement with almost all other molecular and morphological data, in placing the obscura group closer to the melanogaster group, with the willistoni group farther distant but still clearly within the subgenus Sophophora.      

An association between ADH protein levels and polymorphic nucleotide variation in the Adh gene of Drosophila melanogaster.

Southern analysis of the Adh region of 212 Drosophila melanogaster lines collected from the Tahbilk winery revealed linkage disequilibrium between a 37-bp insertion [designated delta 2 by Kreitman (1983)] and the fast electrophoretic variant of alcohol dehydrogenase (ADH-F). Among these lines 34% contained the insert and encoded ADH-F, 33.5% encoded ADH-F and did not have the insert, and 32.5% encoded the slow electrophoretic variant of alcohol dehydrogenase (ADH-S). Strong linkage association between this insert and ADH-F is evident worldwide. Twenty-nine of the second chromosome lines were characterized for ADH protein quantity by using radial immunodiffusion. ADH quantity was estimated in both larvae and adult males raised in the presence and absence of alcohol supplement to each of two different food media. Analyses of variance indicated higher levels of ADH protein in larvae from lines with the insert (all ADH-F), compared with those without (both ADH-F and ADH-S), independent of either dietary alcohol or media type. No such difference in ADH quantity between insert- and noninsert-containing ADH-F lines was detected in adults, although the expected higher levels occurred in ADH-F lines compared with ADH-S lines. Given the high levels of linkage disequilibrium in the Adh region, these data suggest that either polymorphic nucleotide-site variants positively associated with delta 2 on the second chromosome or delta 2 itself increases larval levels of ADH protein.     

Organization and evolution of the alcohol dehydrogenase gene in Drosophila

The alcohol dehydrogenase (Adh) gene was isolated from Drosophila simulans and D. mauritiana, and the DNA sequence of a 4.6-kb region, containing the structural gene and flanking sequence, was determined for each. These sequences were compared with the Adh region of D. melanogaster to characterize changes that occur in the Drosophila genome during evolution and to identify conserved sequences of functional importance. Drosophila simulans and D. mauritiana Adh are organized in a manner similar to that of D. melanogaster Adh, including the presence of two promoters for the single Adh gene. This study identified conserved flanking elements that, in conjunction with other studies, suggest regions that may be involved in the control of Adh expression. Inter- and intraspecies comparisons revealed differences in the kinds of sequence changes that have accumulated. Sequence divergence in and around the Adh gene was used to assess inter- and intraspecies evolutionary relationships. Finally, there appears to be an unrelated structural gene located directly 3' of the Adh transcribed region.      

Differential regulation of duplicate alcohol dehydrogenase genes in Drosophila mojavensis

These studies report the existence of multiple forms of alcohol dehydrogenase in extracts of Drosophila mojavensis. The existence of these forms can be best explained by the hypothesis of a duplication for the Adh locus in D. mojavensis. Electrophoretic variants at each locus have been identified and crosses between individuals carrying alternative alleles at each locus result in F1 progeny with six bands of ADH. This pattern is consistent with these individuals being heterozygous at two loci. The loci have been named Adh-1 and Adh-2. Examination of the isozyme content during development shows that the two Adh genes are not coordinately controlled but have separate developmental programs. In embryos and first and second instar larvae only Adh-1 is expressed. At about the time of the second molt Adh-2 expression commences in some of the same cells that previously expressed and continue to express Adh-1. This is evidenced by the existence of an interlocus heterodimer in third instar larvae. Both genes are expressed throughout pupation. Shortly after emergence Adh-1 expression declines. In mature males only ADH-2 is present. In mature females both Adh-1 and Adh-2 are expressed but not in the same cells since the interlocus heterodimer is absent. Examination of specific tissues reveals that most of the larval ADH is found in fat body cells and as in most tissues of third instar larvae both Adh-1 and Adh-2 are expressed. The single exception appears to be larval gut which contains ADH-1 but little if any ADH-2. In mature males and female flies all ADH containing tissues have only ADH-2. However, mature ovaries contain substantial quantities of ADH-1 which is apparently deposited into eggs. Given the extensive amount of available information on the Adh gene-enzyme system of D. melanogaster and the tools that can be applied to the analysis of homologous systems, the ADH duplication of D. mojavensis, and its regulation may be a useful one for studying differential gene regulation in specific cell types.     

Tissue-Specific Expression Phenotypes of Hawaiian Drosophila Adh Genes in Drosophila Melanogaster Transformants

Interspecific differences in the tissue-specific patterns of expression displayed by the alcohol dehydrogenase (Adh) genes within the Hawaiian picture-winged Drosophila represent a rich source of evolutionary variation in gene regulation. Study of the cis-acting elements responsible for regulatory differences between Adh genes from various species is greatly facilitated by analyzing the behavior of the different Adh genes in a homogeneous background. Accordingly, the Adh gene from Drosophila grimshawi was introduced into the germ line of Drosophila melanogaster by means of P element-mediated transformation, and transformants carrying this gene were compared to transformants carrying the Adh genes from Drosophila affinidisjuncta and Drosophila hawaiiensis. The results indicate that the D. affinidisjuncta and D. grimshawi genes have relatively higher levels of expression and broader tissue distribution of expression than the D. hawaiiensis gene in larvae. All three genes are expressed at similar overall levels in adults, with differences in tissue distribution of enzyme activity corresponding to the pattern in the donor species. However, certain systematic differences between Adh gene expression in transformants and in the Hawaiian Drosophila are noted along with tissue-specific position effects in some cases. The implications of these findings for the understanding of evolved regulatory variation are discussed.