Curing of Saccharomyces cerevisiae 2-μm DNA by transformation

SummaryA 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 cir0 strains has been compared.

[1]  J. Beggs.  Transformation of yeast by a replicating hybrid plasmid , 1978, Nature.

[2]  J. Hartley,et al.  Nucleotide sequence of the yeast plasmid , 1980, Nature.

[3]  D. M. Livingston.  Inheritance of the 2 micrometer m DNA plasmid from Saccharomyces. , 1977, Genetics.

[4]  G. Fink,et al.  Transformation of yeast. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[5]  M. Guérineau,et al.  2 μm plasmid copy number in different yeast strains and repartition of endogenous and 2 μm chimeric plasmids in transformed strains , 1980, Current Genetics.

[6]  P. Slonimski,et al.  Characterization of a new class of circular DNA molecules in yeast. , 1971, Biochemical and biophysical research communications.

[7]  C. Hollenberg,et al.  Mitochondrial DNA: V. A 25-μ closed circular duplex DNA molecule in wild-type yeast mitochondria. Structure and genetic complexity , 1970 .

[8]  T. Maniatis,et al.  Nucleotide sequence of the rightward operator of phage lambda. , 1975, Proceedings of the National Academy of Sciences of the United States of America.

[9]  C. Hollenberg,et al.  Inheritance of multiple drug resistance in Saccharomyces cerevisiae: Linkage to leu1 and analyses of 2 μm DNA in partial revertants , 1979, Molecular and General Genetics MGG.

[10]  M. Aigle,et al.  Qualitative detection of penicillinase produced by yeast strains carrying chimeric yeast‐coli plasmids , 1979, FEBS letters.

[11]  D. Hogness,et al.  Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. , 1975, Proceedings of the National Academy of Sciences of the United States of America.

[12]  F. Bolivar.  Construction and characterization of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique Eco RI sites for selection of Eco RI generated recombinant DNA molecules. , 1978, Gene.

[13]  H. Tabak.  Absence of 2 μm DNA sequences in Saccharomyces cerevisiae Y 379‐5D , 1977 .

[14]  M. Dobson,et al.  Loss of 2 um DNA from Saccharomyces cerevisiae transformed with the chimaeric plasmid pJDB219 , 1980, Current Genetics.

[15]  R. W. Davis,et al.  High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[16]  M. Guérineau,et al.  Stable yeast transformation with chimeric plasmids using a 2 μm-circular DNA-less strain as a recipient , 1979, Molecular and General Genetics MGG.

[17]  E. Southern.  Detection of specific sequences among DNA fragments separated by gel electrophoresis. , 1975, Journal of molecular biology.

[18]  A. Jiménez,et al.  Expression of a transposable antibiotic resistance element in Saccharomyces , 1980, Nature.

[19]  C. Hollenberg.  Mapping of regions on cloned saccharomyces cerevisiae 2-μm DNA coding for polypeptides synthesized in Escherichia coli minicells , 1978, Molecular and General Genetics MGG.