Isolation of the gene encoding yeast single-stranded nucleic acid binding protein 1

Abstract

A yeast gene encoding SSB-1, a single-stranded nucleic acid binding protein, has been isolated by screening a lambda gt11 genomic DNA library. The gene is located on a 1.84-kilobase chromosomal Bgl II-BamHI fragment. Yeast strains carrying the high-copy-number vector YEp24 with an SSB1 gene insert overproduce SSB-1 3-fold and SSB-1 mRNA 10-fold. A typical haploid cell contains about 20,000 molecules of SSB-1; thus, the cells can tolerate up to 60,000 copies. Yeast SSB-1 was expressed in Escherichia coli cells by using a phage T7 expression system. Spores containing the gene disrupted at a point within the coding sequence germinate and grow normally; thus, the gene is not essential. Protein blots show that no SSB-1 or novel immunologically related species that might retain SSB-1 activity are present in cells containing the disrupted SSB1 genes. Southern analysis and protein blots suggest the presence in yeast of a second, related, but nonidentical gene and two immunologically related proteins of 55 kDa and 75 kDa.