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.
Biswas
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EE
Division of Endocrinology, University of Maryland School of Medicine, Baltimore 21201.
Stefanec
M J
MJ
Biswas
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SB
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