By complementation of the cyri-) mutation in Saccharomyces cerevisiae, we have isolated yeast genomic DNA containing the structural gene that encodes the catalytic unit of adenylate cyclase (EC 4.6.1.1). The isolated DNA restored adenylate cyclase activity to cyri-) mutants and directed integration at the CYR] locus. Wild-type strains transformed with CYR] DNA on the high copy number vector YEp24 contained 4-to 6-fold more adenylate cyclase activity than strains carrying the plasmid with no insert. This result suggests that expression of the CYRI gene product, rather than that of other polypeptide components of the adenylate cyclase system, limits total adenylate cyclase activity in S. cerevisiae. CYR)-containing plasmids also complemented the temperature-sensitive growth defect of the cell division cycle mutation cdc35-1, which confers a phenotype under restrictive conditions similar to that of cyri-) and maps to the same locus. Further, cdc35-1 cam mutants, which contain mutations that enable them to take up cAMP from the medium, grew at the restrictive temperature in the presence of exogenous cAMP. These observations support the view that CDC35 and CYR] are allelic and confirm the hypothesis that cAMP synthesis is required for cells to pass through the "start" position of the cell division cycle.Adenosine 3',5'-monophosphate (cAMP) is thought to play at least two important regulatory roles in the yeast Saccharomyces cerevisiae: As a negative regulator of sporulation in a/a diploids (1) and as a positive regulator at the "start" position of the cell division cycle (2). The
MATERIALS AND METHODSStrains, Plasmids, and Media. Table 1 shows the yeast strains used. Strain GC26-7B was derived from a cross between HR125-5Da and AM18-5C, followed by two backcrosses of cyrl-1 segregants to HR125-5Da or 1369. NW23-6D and NW23-9C were similarly obtained from a cross between GC26-7B and 1369 and two additional backcrosses of cyrl-J segregants to HR125-5Da or 1369. NW56-8A was obtained as an Ade-Cyr' cam segregant from a cross between AM3-4B and NW23-9C with three subsequent backcrosses of such segregants to NW23-6D or NW23-9C. NW33-1 was a ura3-52 cdc35-1 segregant from a cross between BR314-4A and 1369. The Escherichia coli strain was HB101.Plasmids used were YEp24 [pBR322 containing the yeast URA3 gene and a portion of yeast 2-,um DNA (10)] and YIp5 [pBR322 containing the yeast URA3 gene (11)].Rich medium for cultivation of yeast was YEPD (1% yeast extract/2% Bacto-peptone/2% glucose). Defined medium was 6.7 g of yeast nitrogen base without amino acids per liter and 2% glucose, supplemented with appropriate factors to support growth of auxotrophic strains. Solid medium contained 2% Bacto-agar. Media for growth of cyri-l mutants were supplemented with 2 mM cAMP.Preparation of Yeast Particulate Extracts. Strains to be tested for adenylate cyclase activity were grown in defined medium lacking uracil to maintain the URA3 plasmids. Cells (5 x 108) were harvested at a density of 1-2 x 107 per ml, washed once with d...
Developmental activation of the Drosophila alcohol dehydrogenase (Adh) distal promoter is controlled by the Adh adult enhancer (AAE). Within this 150 bp, complex enhancer is a small (12 bp) positive cis-acting element that is required for high levels of distal transcription in adult flies and ADH-expressing tissue culture cells. We previously reported that the steroid receptor superfamily member FTZ-F1 binds to this site. We have identified a second steroid receptor superfamily member, DHR39, which also binds to this site. DHR39 is expressed throughout development in transcripts of several sizes. In situ hybridization to embryos has shown that DHR39 RNA is found primarily in the central nervous system, and not in embryonic tissues that express ADH. FTZ-F1 RNA, however, shows temporal-specific patterns similar to those of the distal promoter. FTZ-F1 and DHR39 have identical amino acids in the 'P-box' of the DNA binding domain, suggesting that they have identical DNA recognition characteristics. By electrophoretic mobility shift analysis we show that a DHR39 fusion protein binds specifically to two FTZ-F1 binding sites. By over expressing the full length DHR39 protein in a transient co-transfection assay we have shown that it represses distal Adh expression in a dosage- and binding site-dependent manner. Over expression of an alternative DHR39 open reading frame that lacks part of the putative ligand binding domain does not alter Adh expression. In contrast, over expression of FTZ-F1 specifically activates distal Adh expression.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.