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.
Division of Endocrinology, University of Maryland School of Medicine, Baltimore 21201.
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D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D015723:Gene Library; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D011061:Poly A / Q000235:genetics; D012313:RNA / Q000235:genetics; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors
C061886:ABF1 protein, S cerevisiae; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D011061:Poly A; D012313:RNA