The copy number of 2 mu DNA-derived plasmids in CIR+ Saccharomyces cerevisiae transformants is determined by its selective marker and is usually much lower than that of the endogenous plasmid. Only plasmids containing the leu2 allele of pJDB219, designated as leu2-d, under selective conditions displayed a higher copy number than did endogenous 2 mu DNA and by displacement generated cured cells. Spontaneous loss of 2 mu DNA occurred with a frequency of about 0.02% per generation. Curing plasmids, like pMP78, have copy numbers of 35; noncuring plasmids, like pDB248 or YEp6, have copy numbers of 4 to 8. The 2 mu DNA copy number in strains AH22 and YNN27 were determined to be 40 and 100, respectively. The high copy number of leu2-d-containing plasmids can be explained by its weak expression of less than 5% that of the wild-type LEU2 gene. The leu2-d allele has a deletion of the 5'-end sequence starting from 29 base pairs before the ATG initiation codon, but surprisingly, its expression is still regulated. On YRp7, which contains the chromosomal autonomic replication sequence ARS1, the defective leu2-d allele could not complement a leu2 host strain. This suggests a more stringent control of replication of ARS1-containing plasmids than of 2 mu-containing plasmids.
A general procedure for the curing of 2-μm in Saccharomyces cerevisiae is described. The method is based on the displacement of endogenous 2-μm DNA by the recombinant plasmid pMP78-1, which carries the yeast leu2 gene and the 2 -μm DNA replicon, but cannot be maintained stably in a yeast cell without endogenous 2-μm DNA. After transformation with pMP78-1 cells are grown selectively to displace 2-μm DNA. During the non-selective growth which follows, plasmid pMP78-1 is lost and up to 100% of the cells completely lack plasmids. In conjunction with a kanamycin resistance marker, as present in plasmid pMP81, this method should be applicable to cure any wild-type yeast strain. The stability of recombinant plasmids in cir (+) and cir (0) strains has been compared.
Wild or industrial yeast strains cannot be transformed by most selective vectors due to a lack of auxotrophic mutations. To enable identification of transformants of such yeast species, we have developed a 2-µm DNA vector with an indicator gene that can be used without any additional marker. The Escherichia coli gene for β-lactamase (bla) was placed under the control of the yeast promoter for the structural gene encoding ADHI. This increased the amount of β-lactamase produced in Saccharomyces cerevisiae 100-fold giving an enzyme activity in transformant colonies which is high enough to be detected directly on indicator plates. Non-selectively, the transformation frequency is even higher than under selective conditions indicating that selection does not assist the establishment of new plasmids. Transformants isolated non-selectively were found to retain the endogenous 2-µm DNA. Under control of appropriate promoters, the bacterial bla gene may also provide a convenient marker for other eukaryotic transformation systems.
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