Skip to main content
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1988 Jan;85(2):524–528. doi: 10.1073/pnas.85.2.524

Transformation of yeast with synthetic oligonucleotides.

R P Moerschell 1, S Tsunasawa 1, F Sherman 1
PMCID: PMC279583  PMID: 2829192

Abstract

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.

Full text

PDF
524

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baim S. B., Pietras D. F., Eustice D. C., Sherman F. A mutation allowing an mRNA secondary structure diminishes translation of Saccharomyces cerevisiae iso-1-cytochrome c. Mol Cell Biol. 1985 Aug;5(8):1839–1846. doi: 10.1128/mcb.5.8.1839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beggs J. D. Transformation of yeast by a replicating hybrid plasmid. Nature. 1978 Sep 14;275(5676):104–109. doi: 10.1038/275104a0. [DOI] [PubMed] [Google Scholar]
  3. Burke D. T., Olson M. V. Oligodeoxynucleotide-directed mutagenesis of Escherichia coli and yeast by simple cotransformation of the primer and template. DNA. 1986 Aug;5(4):325–332. doi: 10.1089/dna.1986.5.325. [DOI] [PubMed] [Google Scholar]
  4. Dente L., Cesareni G., Cortese R. pEMBL: a new family of single stranded plasmids. Nucleic Acids Res. 1983 Mar 25;11(6):1645–1655. doi: 10.1093/nar/11.6.1645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Downie J. A., Stewart J. W., Brockman N., Schweingruber A. M., Sherman F. Structural gene for yeast iso-2-cytochrome c. J Mol Biol. 1977 Jun 25;113(2):369–384. doi: 10.1016/0022-2836(77)90147-4. [DOI] [PubMed] [Google Scholar]
  6. Downie J. A., Stewart J. W., Sherman F. Yeast mutants defective in iso-2-cytochrome c. J Mol Biol. 1977 Dec 5;117(2):369–386. doi: 10.1016/0022-2836(77)90133-4. [DOI] [PubMed] [Google Scholar]
  7. Driessen H. P., de Jong W. W., Tesser G. I., Bloemendal H. The mechanism of N-terminal acetylation of proteins. CRC Crit Rev Biochem. 1985;18(4):281–325. doi: 10.3109/10409238509086784. [DOI] [PubMed] [Google Scholar]
  8. Hampsey D. M., Das G., Sherman F. Amino acid replacements in yeast iso-1-cytochrome c. Comparison with the phylogenetic series and the tertiary structure of related cytochromes c. J Biol Chem. 1986 Mar 5;261(7):3259–3271. [PubMed] [Google Scholar]
  9. Hewick R. M., Hunkapiller M. W., Hood L. E., Dreyer W. J. A gas-liquid solid phase peptide and protein sequenator. J Biol Chem. 1981 Aug 10;256(15):7990–7997. [PubMed] [Google Scholar]
  10. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Mandecki W. Oligonucleotide-directed double-strand break repair in plasmids of Escherichia coli: a method for site-specific mutagenesis. Proc Natl Acad Sci U S A. 1986 Oct;83(19):7177–7181. doi: 10.1073/pnas.83.19.7177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Schold M., Colombero A., Reyes A. A., Wallace R. B. Oligonucleotide-directed mutagenesis using plasmid DNA templates and two primers. DNA. 1984 Dec;3(6):469–477. doi: 10.1089/dna.1.1984.3.469. [DOI] [PubMed] [Google Scholar]
  14. Sherman F., Stewart J. W., Parker J. H., Inhaber E., Shipman N. A., Putterman G. J., Gardisky R. L., Margoliash E. The mutational alteration of the primary structure of yeast iso-1-cytochrome c. J Biol Chem. 1968 Oct 25;243(20):5446–5456. [PubMed] [Google Scholar]
  15. Sherman F., Stewart J. W., Tsunasawa S. Methionine or not methionine at the beginning of a protein. Bioessays. 1985 Jul;3(1):27–31. doi: 10.1002/bies.950030108. [DOI] [PubMed] [Google Scholar]
  16. Simon J. R., Moore P. D. Homologous recombination between single-stranded DNA and chromosomal genes in Saccharomyces cerevisiae. Mol Cell Biol. 1987 Jul;7(7):2329–2334. doi: 10.1128/mcb.7.7.2329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Stiles J. I., Friedman L. R., Helms C., Consaul S., Sherman F. Transposition of the gene cluster CYC1-OSM1-RAD7 in yeast. J Mol Biol. 1981 Jun 5;148(4):331–346. doi: 10.1016/0022-2836(81)90179-0. [DOI] [PubMed] [Google Scholar]
  18. Tsunasawa S., Kondo J., Sakiyama F. Isocratic separation of PTH-amino acids at picomole level by reverse-phase HPLC in the presence of sodium dodecylsulfate. J Biochem. 1985 Feb;97(2):701–704. doi: 10.1093/oxfordjournals.jbchem.a135108. [DOI] [PubMed] [Google Scholar]
  19. Tsunasawa S., Narita K. Micro-identification of amino-terminal acetylamino acids in proteins. J Biochem. 1982 Sep;92(3):607–613. doi: 10.1093/oxfordjournals.jbchem.a133971. [DOI] [PubMed] [Google Scholar]
  20. Tsunasawa S., Stewart J. W., Sherman F. Amino-terminal processing of mutant forms of yeast iso-1-cytochrome c. The specificities of methionine aminopeptidase and acetyltransferase. J Biol Chem. 1985 May 10;260(9):5382–5391. [PubMed] [Google Scholar]
  21. Walder R. Y., Walder J. A. Oligodeoxynucleotide-directed mutagenesis using the yeast transformation system. Gene. 1986;42(2):133–139. doi: 10.1016/0378-1119(86)90289-1. [DOI] [PubMed] [Google Scholar]
  22. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 1983;100:468–500. doi: 10.1016/0076-6879(83)00074-9. [DOI] [PubMed] [Google Scholar]
  23. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. DNA. 1984 Dec;3(6):479–488. doi: 10.1089/dna.1.1984.3.479. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES