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The GAGA transcription factor of Drosophila melanogaster is ubiquitous and plays multiple roles. Characterization of cDNA clones and detection by domain- specific antibodies has revealed that the 70-90 kDa major GAGA species are encoded by two open reading frames producing GAGA factor proteins of 519 amino acids (GAGA-519) and 581 amino acids (GAGA-581), which share a common N-terminal region that is linked to two different glutamine-rich C-termini. Purified recombinant GAGA-519 and GAGA-581 proteins can form homomeric complexes that bind specifically to a single GAGA sequence in vitro. The two GAGA isoforms also function similarly in transient transactivation assays in tissue culture cells and in chromatin remodeling experiments in vitro . Only GAGA-519 protein accumulates during the first 6 h of embryogenesis. Thereafter, both GAGA proteins are present in nearly equal amounts throughout development; in larval salivary gland nuclei they colocalize completely to specific regions along the euchromatic arms of the polytene chromosomes. Coimmunoprecipitation of GAGA-519 and GAGA-581 from crude nuclear extracts and from mixtures of purified recombinant proteins, indicates direct interactions. We suggest that homomeric complexes of GAGA-519 may function during early embryogenesis; both homomeric and heteromeric complexes of GAGA-519 and GAGA-581 may function later.

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The distal promoter ofAdh is differentially expressed in Drosophila tissue culture cell lines. After transfection with an exogenous Adh gene, there was a specific increase in distal alcohol dehydrogenase (ADH) transcripts in ADH-expressing (ADH+) cells above the levels observed in transfected ADH-nonexpressing (ADH-) cells. We used deletion mutations and a comparative transient-expression assay to identify the cis-acting elements responsible for enhanced Adh distal transcription in ADH+ cells. DNA sequences controlling high levels of distal transcription were localized to a 15-base-pair (bp) region nearly 500 bp upstream of the distal RNA start site. In addition, a 61-bp negative cis-acting element was found upstream from and adjacent to the enhancer. When this silencer element was deleted, distal transcription increased only in the ADH+ cell line. These distant upstream elements must interact with the promoter elements, the Adf-l-binding site and the TATA box, as they only influenced transcription when at least one of these two positive distal promoter elements was present. Internal deletions targeted to the Adf-l-binding site or the TATA box reduced transcription in both cell types but did not affect the transcription initiation site. Distal transcription in transfected ADH-cells appears to be controlled primarily through these promoter elements and does not involve the upstream regulatory elements. Evolutionary conservation in distantly related Drosophila species suggests the importance of these upstream elements in correct developmental and tissue-specffic expression of ADH.The alcohol dehydrogenase gene (Adh) of Drosophila melanogaster is transcribed from two promoters, each of which has separate developmental and tissue-specific patterns of expression (6, 44; reviewed in references 11 and 51). Because differential gene expression in eucaryotes can be controlled at the level of transcription initiation by interactions between cis-acting elements and sequence-specific trans-acting factors (16, 33), we have begun to characterize the cis-and trans-acting elements responsible for differential ADH expression.Our model system for studying Adh gene regulation has been a collection of Drosophila cell lines (48) that show large (ADH+) or barely detectable (ADH-) expression of the Adh gene (4). Analysis of the mRNA in these cell lines indicated differing levels of ADH mRNA specific to the distal promoter, whereas the proximal transcript appeared to be entirely absent (4). Detailed characterization of the Adh locus in these cell lines has shown that the gene is intact (4, 16a; also unpublished observations). We have hypothesized that the observed levels of distal transcripts reflect differential distal promoter transcriptional activity in different cell lines, which may in turn result from differences in transacting factors present in these cells.To test this hypothesis in vivo and to define the DNA sequences responsible for the differential pattern of expression in Drosophila cell lines, we used a transient-expressio...