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.     

Structural and functional conservation of the Drosophila doublesex splicing enhancer repeat elements.

We have compared the RNA sequences and secondary structures of the Drosophila melanogaster and Drosophila virilis doublesex (dsx) splicing enhancers. The sequences of the two splicing enhancers are highly divergent except for the presence of nearly identical 13-nt repeat elements (six in D. melanogaster and four in D. virilis) and a stretch of nucleotides at the 5' and 3' ends of the enhancers. In vitro RNA structure probing of the two enhancers revealed that the 13-nt repeats are predominantly single-stranded. Thus, both the primary sequences and single-stranded nature of the repeats are conserved between the two species. The significance of the primary sequence conservation was demonstrated by showing that the two enhancers are functionally interchangeable in Tra-/Tra2-dependent in vitro splicing. In addition, inhibition of splicing enhancer activity by antisense oligonucleotides complementary to the repeats demonstrated the importance of the conserved single-stranded structure of the repeats. In vitro binding studies revealed that Tra2 interacts with each of the D. melanogaster repeat elements, except for repeat 2, with affinities that are indistinguishable, whereas Tra binds nonspecifically to the enhancer. Taken together, these observations indicate that the organization of sequences within the dsx splicing enhancers of D. melanogaster and D. virilis results in a structure in which each of the repeat elements is single-stranded and therefore accessible for specific recognition by the RNA-binding domain of Tra2.     

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.     

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.     

Redundant cis-acting elements control expression of the Drosophila affinidisjuncta Adh gene in the larval fat body.

The alcohol dehydrogenase (Adh) gene in the Hawaiian species of fruit fly, Drosophila affinidisjuncta, like the Adh genes from all Drosophila species analyzed, is expressed at high levels in the larval fat body via a larval-specific promoter. To identify the cis-acting elements involved in this highly conserved aspect of Adh gene expression, deleted D. affinidisjuncta genes were introduced into D. melanogaster by somatic transformation. Unlike previously described methods, this transformation system allows analysis of Adh gene expression specifically in the larval fat body. The arrangement of sequences influencing expression of the proximal promoter of this gene in the larval fat body differs markedly from that described for the Adh gene from the distant relative, D. melanogaster. Multiple redundant elements dispersed 5' and 3' to the gene, only some of which map to regions carrying evolutionarily conserved sequences, affect expression in the fat body. D. affinidisjuncta employs a novel mode of Adh gene regulation in which the proximal promoter is influenced by sequences having roles in expression of the distal promoter. This gene is also unique in that far upstream sequences can compensate for loss of sequences within 200 bp of the proximal RNA start site. Furthermore, expression is influenced in an unusual, context-dependent manner by a naturally-occurring 3' duplication of the proximal promoter--a feature found only in Hawaiian species.     

Dynamics of heart differentiation, visualized utilizing heart enhancer elements of the Drosophila melanogaster bHLH transcription factor Hand

Drosophila melanogaster has become one of the important model systems to investigate the development and differentiation of the heart. After 24h after egg deposition (h AED), a simple tube-like organ is formed, consisting of essentially only two cell types, the contractile cardioblasts and non-myogenic pericardial cells. In contrast to the detailed knowledge of heart formation during embryogenesis, only a few studies deal with later changes in heart morphology and/or function. This is mainly due to the difficulties to carry out whole mount stainings in later stages without complicated dissections or treatments of the cuticle and puparium. In this paper we describe the identification of a hand genomic region, which is fully sufficient to drive GFP expression in heart cells of embryos, larvae, and adults. This serves as an initial step to understand the position of hand in the early regulatory network in heart development. Furthermore, we demonstrate that our newly created GFP reporter line is extremely useful to study postembryonic heart differentiation. For the first time we document heart differentiation in living animals throughout all developmental stages of Drosophila melanogaster, including embryogenesis, all three larval stages, metamorphosis, and the adult life with respect to pericardial cells and cardiomyocytes.     

Synergistic effect of Adh alleles in Drosophila melanogaster.

In laboratory cultures of Drosophila melanogaster derived from an African population, the quantities of six out of seven enzymes (G6PD, IDH, GPDH, ME, MDH, PGM and ADH) were higher in Adh-FF homozygotes than they were in Adh-SS. In crosses between Adh-FF and Adh-SS flies, the differences segregated as co-dominant alleles of a single Mendelian gene closely linked, or identical, to the Adh locus. The generality of these associations was suggested by the study of a French population with a very different history and genetic background. The possibility that the associations were caused by artefacts of the immunodiffusion techniques, or to a linked inversion (In(2L)t), was excluded. Possible ways by which the Adh locus may affect the quantities of other enzymes are discussed.