Transformation of Yeast with Synthetic Oligonucleotides

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 85; no. 2; pp. 524 - 528
• Main Authors: Moerschell, Richard P., Tsunasawa, Susumu, Sherman, Fred
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 01.01.1988; National Acad Sciences
• Subjects: Alleles; Amino Acid Sequence; Amino acids; Base Sequence; Biological and medical sciences; Cytochrome c Group - genetics; Cytochromes c; DNA; Fundamental and applied biological sciences. Psychology; Genes; Genes, Viral; Genetic mutation; Lithium; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Mutagenesis. Repair; Nucleotides; Oligodeoxyribonucleotides - chemical synthesis; Oligonucleotides; Plasmids; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae Proteins; Spheroplasts; Transformation, Genetic; Yeasts; Fungi; In vivo; Yeast; Synthetic product; Genetic transformation; Site directed mutagenesis; Ascomycetes; Oligonucleotide; Saccharomyces cerevisiae; Thallophyta
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.85.2.524

Genomic DNA of the yeast, Saccharomyces cerevisiae, can be conveniently and specifically altered by transforming spheroplasts of 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 μ g. 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.