Transformation of yeast with synthetic oligonucleotides.
Genomic DNA of the yeast, Saccharomyces cerevisiae, can be conveniently and specifically altered by transforming spheroplasts or lithium acetate-treated cells directly with synthetic oligonucleotides. Altered forms of iso-1-cytochrome c were generated by transforming a cyc1 mutant with oligonucleotides and selecting for at least partially functional revertants; the oligonucleotides contained a sequence that corrected the cyc1 mutation and produced additional alterations at nearby sites. Transformation has been accomplished with oligonucleotides as short as 20 nucleotides and with amounts as low as 100 micrograms. This method of site-directed mutagenesis in vivo has been used to produce alterations in the NH2-terminal region of iso-1-cytochrome c in which the NH2-terminal methionine is excised and the penultimate residue is acetylated.
Moerschell
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RP
Department of Biophysics, University of Rochester School of Medicine and Dentistry, NY 14642.
Tsunasawa
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S
Sherman
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F
D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D003574:Cytochrome c Group / Q000235:genetics; D045304:Cytochromes c; D005796:Genes; D005814:Genes, Viral; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D014170:Transformation, Genetic
C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D009838:Oligodeoxyribonucleotides; D029701:Saccharomyces cerevisiae Proteins; D045304:Cytochromes c