Abstract
Adh distal factor-1 (Adf-1) is a sequence-specific DNA-binding activity originally identified in Drosophila tissue culture cells and embryos. Adf-1 binds to upstream recognition elements in each of the two promoters of the Drosophila alcohol dehydrogenase gene (Adh), and binding of Adf-1 to the Adh distal promoter site activates transcription. We have carried out a mutational analysis of the Adh distal promoter using both an in vitro transcription assay and a transient transfection assay in Drosophila tissue culture cells, and in both cases find that deletion of sequences required for Adf-1 binding leads to a 3-4-fold drop in transcription. We have purified Adf-1 and demonstrate by a sodium dodecyl sulfate-gel renaturation assay that it is a 34-kDa protein. Purified Adf-1 activates Adh distal promoter transcription in vitro in a binding site-dependent manner. DNase I footprint analysis shows that the purified protein binds not only to the two previously characterized sites in Adh but also to transcriptional regulatory elements in the dopa decarboxylase (Ddc) and Antennapedia (Antp) P1 promoters. Thus, it appears that Adf-1 may play an important role not only in the regulation of Adh expression but also in the transcription of other Drosophila genes as well.
Highlights
alcohol dehydrogenase gene (Adh) distal factor-1(Adf-1) is a sequence-specific DNA-binding activityoriginally identified in Drosophila tissue culture cells and embryos
In addition to binding to the A d h promoter sites and stimulating distal promotetrranscription, we have found that purified Adf-1 binds in a similar manner to several other developmentally regulated Drosophila
T o verify that the observeddifferences inCAT enzyme activity are due to diffelreevnetls of Adh-chloramphenicol acetyltransferase (CAT)
Summary
T o verify that the observeddifferences inCAT enzyme activity are due to diffelreevnetls of Adh-CAT. In Viuo and in Vitro Promoter Deletion Analysis-Previous RNAinitiating from thecorrectstartsites, we havealso DNase I footprint studies showed that Adf-1 protects Adh analyzed poly(A+) RNA from the transfected cells by primer promotersequences from -85 to -47 relative to the RNA extension analysis and find that the relative levels of accustartsite,andthis Adf-1-binding siteis required for high rately initiated Adh-CAT RNA parallel the levels of CAT levels of transcription in vitro (Heberlein et al, 1985; Heber- activity observed (data not shown). 1).The distal promoterdeletions were fused to the CATgene structedadditional 5’ promoterdeletionswithendpoints and tested by measuring CAT enzyme activity produced fol- between those of the previouslyanalyzed -33 and -86 lowing transient transfection into Schneider l2inceells.
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