Studies were made of 2 mum based chimaeric plasmid copy number in Saccharomyces cerevisiae. A plasmid (pAYE56) containing three selectable genes in yeast (yeast LEU2, bacterial CAT and HSV-1 - TK) was constructed to reflect changes in copy number. Yeast transformants could be grown under three selection regimes and plasmid copy number estimated. During selective growth for the LEU2 gene there are about 20 plasmids per cell. This increases to about 100 during selective growth for the TK gene and furthermore the copy number can be controlled by the stringency of selection. Simultaneous selection for the TK and CAT genes may lead to a further increase (160 copies). Two models are proposed to account for these increases. The amplification model proposes plasmid replication without cell growth whilst the selection model suggests that plasmid copy number varies greatly in a population of transformants and cells with a high copy number are selected for growth under the TK/CAT selection conditions. Whilst the mechanism of copy number increase is unclear, an attempt was made to relate the expression of a heterologous gene (Human alpha2-IFN) to gene dosage using the promotion and secretion signals of the alpha-factor gene. Production of intracellular alpha2-IFN was unaffected by copy number whilst secreted material showed a 100 fold increase over a ten fold increase in gene dosage. Attempts were made to isolate plasmid copy number mutants. After mutagenesis (of cells or plasmid) transformants were selected under conditions for simultaneous over-expression of the TK and CAT genes. Mutants capable of growth under these conditions were obtained. In one group the mutant phenotype was lost upon curing but did not return upon retransformation. In a second group a chromosomal mutation was isolated. Plasmid copy number estimates indicated that this was unchanged however. Alternative strategies are discussed for the isolation of mutants.
|Date of Award||1985|