Yeast 2 microm plasmid copy number is elevated by a mutation in the nuclear gene UBC4.
The copy number of the Saccharomyces cerevisiae endogenous 2 microm plasmid is under strict control to ensure efficient propagation to the daughter cell without significantly reducing the growth rate of the mother or the daughter cell. A recessive mutation has been identified that resulted in an elevated but stable 2 microm plasmid copy number, which could be complemented by a genomic DNA clone containing the UBC4 gene, encoding an E2 ubiquitin-conjugating enzyme. A ubc4::URA3 deletion resulted in the same elevated 2 microm plasmid copy number. An analysis of the endogenous 2 microm transcripts revealed that the steady-state abundance of REP1, REP2, FLP and RAF were all increased 4-5-fold in the mutant. Analysis of the mutant ubc4 allele identified a single base pair mutation within the UBC4 coding region, which would generate a glutamic acid to lysine amino acid substitution within a region of conserved tertiary structure located within the first alpha-helix of Ubc4p. These investigations represent the first molecular characterization of a mutation within a Saccharomyces cerevisiae nuclear gene shown to affect 2 microm steady-state plasmid copy number and implicate the ubiquitin-dependent proteolytic pathway in host control of 2 microm plasmid copy number.
Delta Biotechnology Ltd, Castle Court, 59 Castle Boulevard, Nottingham NG7 1FD, UK. darrell.sleep@aventis.com
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D015139:Blotting, Southern; D004254:DNA Nucleotidyltransferases / Q000096:biosynthesis / Q000235:genetics; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D018628:Gene Dosage; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D005816:Genetic Complementation Test; D008025:Ligases / Q000737:chemistry / Q000235:genetics*; D010957:Plasmids / Q000737:chemistry / Q000235:genetics*; D017354:Point Mutation / Q000235:genetics*; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics; D019908:Proto-Oncogene Proteins c-raf / Q000096:biosynthesis / Q000235:genetics; D012331:RNA, Fungal / Q000737:chemistry; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators / Q000096:biosynthesis / Q000235:genetics; D044763:Ubiquitin-Conjugating Enzymes
D004268:DNA-Binding Proteins; D011498:Protein Precursors; C491137:REP1 protein, S cerevisiae; C490689:REP2 protein, S cerevisiae; D012331:RNA, Fungal; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D044763:Ubiquitin-Conjugating Enzymes; C063109:ubiquitin-conjugating enzyme UBC4; D019908:Proto-Oncogene Proteins c-raf; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D008025:Ligases