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. 1979 Aug;76(8):3829–3833. doi: 10.1073/pnas.76.8.3829

High-frequency transformation of yeast by plasmids containing the cloned yeast ARG4 gene

Chu-Lai Hsiao 1, John Carbon 1
PMCID: PMC383928  PMID: 386351

Abstract

Hybrid ColE1 plasmids, containing cloned DNA from the yeast ARG4 region [e.g., pYe(arg4)1], transform yeast arg4 mutants to ARG4+ with a frequency of 10-4 (about 103 transformants per μg of plasmid DNA) and can replicate autonomously without integrating into the yeast genome. The yeast transformants are genetically unstable when grown on nonselective medium, but can be readily grown and maintained on minimal medium lacking arginine. The existence of unintegrated replicating plasmid DNA in the yeast transformants was demonstrated by Southern gel hybridization and by transformation of Escherichia coli argH mutants with DNA preparations from yeast transformants and subsequent recovery of intact plasmid DNA from the bacterial transformants. Plasmid DNAs recovered from the E. coli-yeast-E. coli “shuttle” remain essentially unchanged, as judged by DNA restriction fragment patterns. Some plasmid mutations leading to increased efficiency of expression of the ARG4 gene in E. coli do not appear to affect expression of the cloned ARG4 gene in yeast. Appropriate derivatives of these ARG4 plasmids are of potential usefulness as vectors for cloning genes in yeast and for studying the mechanism of yeast DNA replication.

Keywords: recombinant DNA, yeast replicon, yeast plasmid, argininosuccinate lyase

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Selected References

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

  1. 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]
  2. Chinault A. C., Carbon J. Overlap hybridization screening: isolation and characterization of overlapping DNA fragments surrounding the leu2 gene on yeast chromosome III. Gene. 1979 Feb;5(2):111–126. doi: 10.1016/0378-1119(79)90097-0. [DOI] [PubMed] [Google Scholar]
  3. Clarke L., Carbon J. Biochemical construction and selection of hybrid plasmids containing specific segments of the Escherichia coli genome. Proc Natl Acad Sci U S A. 1975 Nov;72(11):4361–4365. doi: 10.1073/pnas.72.11.4361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Clarke L., Carbon J. Functional expression of cloned yeast DNA in Escherichia coli: specific complementation of argininosuccinate lyase (argH) mutations. J Mol Biol. 1978 Apr 25;120(4):517–532. doi: 10.1016/0022-2836(78)90351-0. [DOI] [PubMed] [Google Scholar]
  5. Clewell D. B. Nature of Col E 1 plasmid replication in Escherichia coli in the presence of the chloramphenicol. J Bacteriol. 1972 May;110(2):667–676. doi: 10.1128/jb.110.2.667-676.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hilger F., Culot M., Minet M., Pierard A., Grenson M., Wiame J. M. Studies on the kinetics of the enzyme sequence mediating arginine synthesis in Saccharomyces cerevisiae. J Gen Microbiol. 1973 Mar;75(1):33–41. doi: 10.1099/00221287-75-1-33. [DOI] [PubMed] [Google Scholar]
  7. Hinnen A., Hicks J. B., Fink G. R. Transformation of yeast. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1929–1933. doi: 10.1073/pnas.75.4.1929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. MAAS W. K. Studies on repression of arginine biosynthesis in Escherichia coli. Cold Spring Harb Symp Quant Biol. 1961;26:183–191. doi: 10.1101/sqb.1961.026.01.023. [DOI] [PubMed] [Google Scholar]
  9. Mortimer R. K., Hawthorne D. C. Genetic mapping in Saccharomyces. Genetics. 1966 Jan;53(1):165–173. doi: 10.1093/genetics/53.1.165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Petes T. D., Williamson D. H. Fiber autoradiography of replicating yeast DNA. Exp Cell Res. 1975 Oct 1;95(1):103–110. doi: 10.1016/0014-4827(75)90614-x. [DOI] [PubMed] [Google Scholar]
  11. Ratzkin B., Carbon J. Functional expression of cloned yeast DNA in Escherichia coli. Proc Natl Acad Sci U S A. 1977 Feb;74(2):487–491. doi: 10.1073/pnas.74.2.487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  13. Sharp P. A., Sugden B., Sambrook J. Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose--ethidium bromide electrophoresis. Biochemistry. 1973 Jul 31;12(16):3055–3063. doi: 10.1021/bi00740a018. [DOI] [PubMed] [Google Scholar]
  14. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  15. Struhl K., Stinchcomb D. T., Scherer S., Davis R. W. High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1035–1039. doi: 10.1073/pnas.76.3.1035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. van Solingen P., van der Plaat J. B. Fusion of yeast spheroplasts. J Bacteriol. 1977 May;130(2):946–947. doi: 10.1128/jb.130.2.946-947.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]

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