The presence of a defective LEU2 gene on 2 mu DNA recombinant plasmids of Saccharomyces cerevisiae is responsible for curing and high copy number.

摘要: The copy number of 2 mu DNA-derived plasmids in CIR+ Saccharomyces cerevisiae transformants is determined by its selective marker and usually much lower than that the endogenous plasmid. Only containing leu2 allele pJDB219, designated as leu2-d, under conditions displayed a higher did DNA displacement generated cured cells. Spontaneous loss occurred with frequency about 0.02% per generation. Curing plasmids, like pMP78, have numbers 35; noncuring pDB248 or YEp6, 4 to 8. strains AH22 YNN27 were be 40 100, respectively. high leu2-d-containing can explained weak expression less 5% wild-type LEU2 gene. leu2-d has deletion 5'-end sequence starting from 29 base pairs before ATG initiation codon, but surprisingly, still regulated. On YRp7, which contains chromosomal autonomic replication ARS1, defective could not complement host strain. This suggests more stringent control ARS1-containing mu-containing plasmids.