title: Parameters affecting the frequencies of transformation and co-transformation with synthetic oligonucleotides in yeast.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Rochester School of Medicine and Dentistry, New York 14642.
authors.forename: T
authors.initials: T
authors.lastname: Yamamoto
authors.forename: R P
authors.initials: RP
authors.lastname: Moerschell
authors.forename: L P
authors.initials: LP
authors.lastname: Wakem
authors.forename: D
authors.initials: D
authors.lastname: Ferguson
authors.forename: F
authors.initials: F
authors.lastname: Sherman
mesh_terms: 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
chemical_list: C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D009838:Oligodeoxyribonucleotides; D029701:Saccharomyces cerevisiae Proteins; D045304:Cytochromes c; D004247:DNA; D012343:RNA, Transfer