Parameters affecting the frequencies of transformation and co-transformation with synthetic oligonucleotides in yeast.
Factors influencing the direct transformation of the yeast Saccharomyces cerevisiae with synthetic oligonucleotides were investigated by selecting for cyc1 transformants that contained at least partially functional iso-1-cytochrome c. Approximately 3 x 10(4) transformants, constituting 0.1% of the cells, were obtained by using 1 mg of oligonucleotide in the reaction mixture. Carrier, such as heterogeneous oligonucleotides, enhanced transformation frequencies. Transformation frequencies were dramatically reduced if the oligonucleotides had a large number of mismatches or had terminally located mismatches. Transformation with oligonucleotides, but not with linearized double-strand plasmid, was efficient in a rad52- strain, suggesting that the pathway for transformation with oligonucleotides is different from that with linearized double-strand plasmid. We describe a procedure of co-transformation with two oligonucleotides, one correcting the cyc1 defect of the target allele in the host strain, and the other producing a desired amino acid alteration elsewhere in the iso-1-cytochrome c molecule; approximately 20% of the transformants obtained by co-transformation contained these desired second alterations.
Yamamoto
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T
Department of Biochemistry, University of Rochester School of Medicine and Dentistry, New York 14642.
Moerschell
R P
RP
Wakem
L P
LP
Ferguson
D
D
Sherman
F
F
D000595:Amino Acid Sequence; D001483:Base Sequence; D003574:Cytochrome c Group / Q000235:genetics; D045304:Cytochromes c; D004247:DNA / Q000494:pharmacology; D004260:DNA Repair / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000235:genetics*; D012343:RNA, Transfer / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D014170:Transformation, Genetic / Q000187:drug effects
C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D009838:Oligodeoxyribonucleotides; D029701:Saccharomyces cerevisiae Proteins; D045304:Cytochromes c; D004247:DNA; D012343:RNA, Transfer