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.     

Correct developmental expression of a cloned alcohol dehydrogenase gene transduced into the Drosophila germ line

We have used P-element-mediated transformation to introduce a cloned Drosophila alcohol dehydrogenase (Adh) gene into the germ line of ADH null flies. Six independent transformants expressing ADH were identified by their acquired resistance to ethanol. Each transformant carries a single copy of the cloned Adh gene in a different chromosomal location. Four of the six transformant lines exhibit normal Adh expression by the following criteria: quantitative levels of ADH enzyme activity in larvae and adults; qualitative tissue specificity; the size of stable Adh mRNA; and the characteristic developmental switch in utilization of two different Adh promoters. The remaining two transformants express ADH enzyme activity with the correct tissue specificity, but at a lower level than wild type. These results demonstrate that an 11.8 kb chromosomal fragment containing the Adh gene includes the cis-acting sequences necessary for its correct developmental expression, and that a variety of chromosomal sites permit proper Adh gene function.     

Drosophila Alcohol Dehydrogenase Polymorphism and Carbon-13 Fluxes: Opportunities for Epistasis and Natural Selection

The influence of genetic variations in Drosophila alcohol dehydrogenase (ADH) on steady-state metabolic fluxes was studied by means of (13)C NMR spectroscopy. Four pathways were found to be operative during 8 hr of ethanol degradation in third instar larvae of Drosophila. Seven strains differed by 18-25% in the ratio between two major pathway fluxes, i.e., into glutamate-glutamine-proline vs. lactate-alanine-trehalose. In general, Adh genotypes with higher ADH activity exhibit a twofold difference in relative carbon flux from malate into lactate and alanine vs. α,α-trehalose compared to low ADH activity genotypes. Trehalose was degraded by the pentose-phosphate shunt. The pentose-phosphate shunt and malic enzyme could supply NADPH necessary for lipid synthesis from ethanol. Lactate and/or proline synthesis may maintain the NADH/NAD(+) balance during ethanol degradation. After 24 hr the flux into trehalose is increased, while the flux into lipids declines in Adh(F) larvae. In Adh(S) larvae the flux into lipids remains high. This co-ordinated nature of metabolism and the genotype-dependent differences in metabolic fluxes may form the basis for various epistatic interactions and ultimately for variations in organismal fitness.     

Molecular analysis of the Adh region of the genome of Drosophila melanogaster

A small region of the genome of Drosophila melanogaster has been cloned in a series of overlapping phage. A length of 165 X 10(3) base-pairs of contiguous DNA that spans polytene chromosome region 35A4 to 35B1 and includes the structural gene for alcohol dehydrogenase (Adh) as well as at least two other genes, outspread (osp) and no-ocelli (noc), has been characterized by mapping chromosome aberrations to the DNA. The relationship between osp and Adh is surprising: of nine osp alleles associated with chromosome breakpoints, five map distal (i.e. 5') to Adh and four map proximal (i.e. 3') to this gene. None affects the expression of Adh. As defined by these and other breakpoints, the osp gene spans at least 52 X 10(3) base-pairs and overlaps the Adh gene. The noc gene, as defined by the mapping of nearly 30 breakpoints, is at least 50 X 10(3) base-pairs in size. Alleles of noc and noc- deletions show either of two kinds of interaction with the recessive lethality of l(2)br29ScoR+1, a lethal that maps immediately distal to noc. One class of noc allele is viable when heterozygous with ScoR+1, while the other class is lethal or semi-lethal. Both classes, however, are homozygous or hemizygous viable. The locations of these two classes of noc allele on the DNA fall into two clusters, with those that are viable with ScoR+1 located proximal to those that are not. The physical boundary between these classes lies at a site just distal to that of the breakpoint of the inversion associated with ScoR+1 itself.     

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.