Molecular cloning of a gene encoding an ARS binding factor from the yeast Saccharomyces cerevisiae.

We report the isolation of the gene for origin binding factor 1 (OBF1) from the yeast Saccharomyces cerevisiae by screening a yeast genomic DNA library in lambda gt11 with an ARS-specific oligonucleotide probe. One recombinant encoded a fusion protein of approximately 180 kDa that bound ARS-specific oligonucleotide probes in vitro. The restriction map of this gene was determined after isolation of the complete gene by screening a yeast genomic DNA library in YEp24. Characterization of the gene for OBF1 by pulsed-field gel electrophoresis and Northern and Southern blot analyses demonstrated that (i) the gene is located in chromosome IV, (ii) the gene is a single-copy gene, (iii) the mRNA is approximately 3.8 kilobases, which could code for an approximately 130-kDa polypeptide, consistent with the reported size of OBF1. An antibody, affinity-purified using the lysogen-encoded fusion protein, specifically detected an approximately 130-kDa polypeptide in yeast extract. The isolation of the gene for OBF1 should allow further analysis of the mechanism of action of this protein in vitro and in vivo. IMAGES: