title: When a glycolytic gene on a yeast 2 mu ORI-STB plasmid is made essential for growth its expression level is a major determinant of plasmid copy number.
abstract: This study demonstrates how varying the promoter strength of an essential gene on a yeast 2 mu ORI-STB YEp multicopy vector can influence vector copy levels. A phosphoglycerate kinase gene (PGK) on this plasmid was made essential for fermentative growth by transformation into a pgk- yeast strain. When in these PGK+ transformants the requirement for PGK expression was the sole selective criterion for plasmid maintenance, PGK promoter activity was inversely related to vector copy levels. Plasmids with an efficiently-transcribed PGK gene were maintained at approximately one copy per cell, whereas those lacking the UAS that normally directs high basal PGK transcription levels were present at up to 10-15 copies. All cultures of these PGK+ transformants contained only a low proportion of pgk- cells. Since mitotic loss of the plasmid arrests growth through loss of a functional PGK allele, PGK confers high stability to the YEp vector in such a pgk- genetic background. In this system YEp vector levels are probably influenced by PGK transcription because high expression of PGK is needed in rapid fermentative growth. Remarkably, low plasmid PGK promoter activity caused PGK mRNA levels slightly higher than those found in yeast with normal PGK regulation. A higher plasmid copy number is therefore not the only factor counteracting the effects of low PGK transcription, and it is possible that PGK mRNA becomes more stable in response to inefficient PGK transcription.
authors.affiliation: Department of Biochemistry, University College London, UK.
authors.forename: P W
authors.initials: PW
authors.lastname: Piper
authors.forename: B P
authors.initials: BP
authors.lastname: Curran
mesh_terms: D005285:Fermentation; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D006019:Glycolysis; D010735:Phosphoglycerate Kinase / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D014158:Transcription, Genetic; D014170:Transformation, Genetic
chemical_list: D010735:Phosphoglycerate Kinase