The Adh gene promoters of Drosophila melanogaster and Drosophila orena are functionally conserved and share features of sequence structure and nuclease-protected sites.

Abstract

The sibling species Drosophila melanogaster and D. orena show similar patterns of alcohol dehydrogenase expression, both spatially and temporally. These two species diverged from a common ancestor 6 million to 15 million years ago, and the DNA sequences of the promoter regions of their Adh genes show a mosaic pattern of conservation and change. By interspecific transformation of D. orena sequences into D. melanogaster, we demonstrate a functional equivalence between these sequences. Using both D. melanogaster embryo extracts and purified transcription factor Adf-1, we compare the protection of these promoter sequences from nuclease, demonstrating considerable conservation.The transcription of genes by RNA polymerase II is usually regulated by the binding of specific proteins to contiguous DNA sequences. The availability of these transcription factors may determine both the temporal and spatial specificity of transcription of particular genes (for reviews, see references 12, 24, and 27). Different transcription factors may affect the same promoter, affording a mechanism for complex control. This has been seen for several genes in Drosophila melanogaster, including the yolk protein genes (15,29,30), fushi tarazu (19), Hsp26 (10), Ubx (4), and Dopa decarboxylase (7,25). Alternatively, a single gene may possess more than one promoter, different transcripts then coding for the same or different protein products (for a review, see reference 35). The Drosophila Adh (3), actin SC (8), and hunchback (40) genes are examples of genes that have different promoters but whose alternative transcripts code for identical polypeptides. In all of these cases there are both temporal and spatial differences in the expression of the alternative mRNAs. The significance of the complexity in control of these genes is not understood.The two promoters, distal (PD) and proximal (Pp), of the Adh gene of D. melanogaster are about 700 base pairs (bp) apart and encode mRNAs that differ only in their 5' untranslated ends (3, 18; for a review, see reference 36). In D. melanogaster the distal transcript initiates 707 bp 5' to the proximal transcript. The distal primary transcript is spliced to remove a 653-nucleotide intron that includes the sequence of the proximal promoter. Experiments with probes that are specific for either the distal or proximal mRNA have shown that these two RNAs differ in their temporal and spatial expression (23a, 33, 34). Although the unfertilized egg of D. melanogaster inherits from its mother both distal and proximal Adh mRNAs, these have decayed by 3 to 4 h after fertilization. Zygotic expression of Adh begins at about 8 h after fertilization and involves both promoters. The expression-from PD is transient at this stage, and by 12 to 16 h only the proximal mRNA can be detected. This RNA accumulates throughout larval life and is most abundant in the fat body and midgut. A few hours before puparium formation, proximal transcripts disappear; at the same time, a transient * Corresponding author. expression of the distal t...

Conserved enhancer and silencer elements responsible for differential Adh transcription in Drosophila cell lines.

“…8); 60 to 70 bp separate these two motifs. The conservation of these upstream elements in such distantly related species and the fact that they have been shown to be protein-binding sites (35) suggest that they are important for regulating ADH expression in the organism and may represent binding sites of important regulatory proteins.…”

“…AP-1-binding activity has been detected in organisms as diverse as Saccharomyces cerevisiae and Drosophila melanogaster (yAP-1 [35], dAP-1 [36]). The Drosophila AP-1 protein is also composed of a Jun-like and a Fos-like protein (37).…”

“…Moses et al (35) compared DNase I footprints on the Adh promoters of two closely related Drosophila species by using partially fractionated embryonic nuclear extracts. They showed that several blocks of highly conserved sequences are sites of protein binding.…”

“…Three of these footprints are within the enhancer-silencer region identified in this report. They are positioned over the octamer-like (-576 to -551), DTF-1-like (-538 to -516), and AP-1-like (-491 to -473) elements (35 It has recently been reported that theAdh larval enhancer (-660 to -5000) contains several repeats of two sequence motifs which are also present in theAdh adult enhancer (14), namely the octamer-like and DTF-1-like elements. Although no evidence of function or protein binding has been ascribed to these larval (proximal) enhancer sequences, it is curious that they are also present within the 61-bp distal-specific silencer element identified in this study.…”

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.

“…They also proposed that transcription from the distal promoter interferes with the proximal promoter, resulting in the inactivation of proximal transcription (11). Stage (68), the binding site of the rat transcription factor NGFI-B (69), three reported consensus ecdysone response elements (10,14,37), the binding site of CF1 from the Drosophila chorion sl5 gene, and the consensus FTZ-F1 site (65 (26,27,40) (56), whose recognition site also resembles the binding sites of DEP1, DEP2, and FTZ-F1 (Fig. 10b).…”