Isolation and characterization of the genomic region from Drosophila kuntzei containing the Adh and Adhr genes

The nucleotide sequences of the Adh and Adhr genes of Drosophila kuntzei were derived from combined overlapping sequences of clones isolated from a genomic library and from cloned PCR and inverse-PCR fragments. Only a proximal promoter was detected upstream of the Adh gene, indicating that D. kuntzei Adh is regulated by a one-promoter system. Further upstream of the Adh structural gene, an adult enhancer region (AAE) was found that contains most of the regulatory sequences described for AAEs of other Drosophila species. Analysis of the ADH protein showed an amino acid change from valine to threonine in the active site at position 189 which is also found in D. funebris but is otherwise unique among DROSOPHILA: This difference alone may be responsible for the very low ADH activity found in this species and may cause a difference in substrate usage pattern. Codon bias in Adh and Adhr was comparable and found to be very low compared with other species. Phylogenetic analysis showed that D. kuntzei is closest related to D. funebris and D. immigrans. The time of divergence between D. kuntzei and D. funebris was estimated to be 14.2-20.2 Myr and that between D. kuntzei-D. funebris and D. immigrans to be 30.8-44.0 Myr. An analysis of the genetic variation in the Adh gene and upstream sequences of four European strains showed that this gene was highly variable. Overall nucleotide diversity (pi) was 0.0139, which is two times higher than that in D. melanogaster.     

DEAF-1, a novel protein that binds an essential region in a Deformed response element.

A 120 bp homeotic response element that is regulated specifically by Deformed in Drosophila embryos contains a single binding site for Deformed protein. However, a 24 bp sub-element containing this site does not constitute a Deformed response element. Specific activation requires a second region in the 120 bp element, which presumably contains one or more binding sites for Deformed cofactors. We have isolated a novel protein from Drosophila nuclear extracts which binds specifically to a site in this second region. This protein, which we call DEAF-1 (Deformed epidermal autoregulatory factor-1), contains three conserved domains. One of these includes a cysteine repeat motif that is similar to a motif found in Drosophila Nervy and the human MTG8 proto-oncoprotein, and another matches a region of Drosophila Trithorax. Mutations in the response element designed to improve binding to DEAF-1 in vitro resulted in increased embryonic expression. Conversely, small mutations designed to diminish binding to DEAF-1 resulted in reduced expression of the element. Thus, DEAF-1 is likely to contribute to the functional activity, and perhaps to the homeotic specificity, of this response element. Consistent with this hypothesis, we have discovered DEAF-1 binding sites in other Deformed response elements.     

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.     

Direct repression of splicing by transformer-2

The Drosophila melanogaster sex determination factor Tra2 positively regulates the splicing of both doublesex (dsx) and fruitless (fru) pre-mRNAs but negatively affects the splicing of the M1 intron in tra2 pre-mRNA. Retention of the M1 intron is known to be part of a negative-feedback mechanism wherein the Tra2 protein limits its own synthesis, but the mechanism responsible for accumulation of M1-containing RNA is unknown. Here we show that the recombinant Tra2 protein specifically represses M1 splicing in Drosophila nuclear extracts. We find that the Tra2 protein binds directly to several sites in and near the M1 intron and that, when Tra2 binding is competed with other RNAs, the splicing of M1 is restored. Mapping the RNA sequences functionally required for M1 repression identified both a 34-nucleotide (nt) A/C-rich sequence immediately upstream of the M1 5' splice site and a region within the intron itself. The AC-rich sequence is largely composed of a repeated 4-nt sequence that also forms a subrepeat within the repeated 13-nt splicing enhancer elements of fru and dsx RNAs. Although required for repression, the element also enhances M1 splicing in the absence of Tra2. We propose that Tra2 represses M1 splicing by interacting with multiple sequences in the pre-mRNA and interfering with enhancer function.     

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.     

A tissue-specific transcription enhancer from the Drosophila yolk protein 1 gene

The yolk protein 1 gene (yp1) of Drosophila melanogaster is expressed only in the ovarian follicle cells and the fat bodies of adult females. We have previously shown that a different cis-acting DNA region is required for each of these tissue specificities. In this paper we use germ line transformation to localize and characterized one of these tissue-specific regulatory regions. We demonstrate that a 125 bp segment of DNA located 196 bp upstream of the yp1 cap site is sufficient to determine the sex-, stage-, and fat body-specific expression of the yp1 gene. We also find that this region can confer yp1-specific expression on a heterologous Drosophila promoter. This specificity is retained when the region is in different orientations and at different distances from the heterologous promoter. Thus a small regulatory region acts in vivo as a positive enhancer to determine the fat body expression pattern of yp1.     

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.