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. 1984 Apr;4(4):771–778. doi: 10.1128/mcb.4.4.771

Transformation of protoplasted yeast cells is directly associated with cell fusion.

S Harashima, A Takagi, Y Oshima
PMCID: PMC368796  PMID: 6371497

Abstract

The frequency of cell fusion during transformation of yeast protoplasts with various yeast plasmids with a chromosome replicon (YRp or YCp) or 2 mu DNA (YEp) was estimated by two methods. In one method, a mixture of protoplasts of two haploid strains with identical mating type and complementary auxotrophic nuclear markers with or without cytoplasmic markers was transformed. When the number of various phenotypic classes of transformants for the nuclear markers was analyzed by equations derived from binominal distribution theory, the frequency of nuclear fusion among the transformants was 42 to 100% in transformations with the YRp or YCp plasmids and 28 to 39% with the YEp plasmids. In another method, a haploid bearing the sir mutation, which allows a diploid (or polyploid) homozygous for the MAT (mating type) locus to sporulate by the expression of the silent mating-type loci HML and HMR, was transformed with the plasmids. Sporulation ability was found in 43 to 95% of the transformants with the YRp or YCp plasmids, and 26 to 31% of the YEp transformants. When cytoplasmic mixing was included with the nuclear fusion, 96 to 100% of the transformants were found to be cell fusants. Based upon these observations, we concluded that transformation of yeast protoplasts is directly associated with cell fusion.

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Selected References

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  1. Anagnostopoulos C., Spizizen J. REQUIREMENTS FOR TRANSFORMATION IN BACILLUS SUBTILIS. J Bacteriol. 1961 May;81(5):741–746. doi: 10.1128/jb.81.5.741-746.1961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arima K., Takano I. Multiple fusion of protoplasts in Saccharomyces yeasts. Mol Gen Genet. 1979 Jun 20;173(3):271–277. doi: 10.1007/BF00268637. [DOI] [PubMed] [Google Scholar]
  3. Bachmann B. J. Linkage map of Escherichia coli K-12, edition 7. Microbiol Rev. 1983 Jun;47(2):180–230. doi: 10.1128/mr.47.2.180-230.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Bolivar F., Rodriguez R. L., Betlach M. C., Boyer H. W. Construction and characterization of new cloning vehicles. I. Ampicillin-resistant derivatives of the plasmid pMB9. Gene. 1977;2(2):75–93. doi: 10.1016/0378-1119(77)90074-9. [DOI] [PubMed] [Google Scholar]
  6. Botstein D., Falco S. C., Stewart S. E., Brennan M., Scherer S., Stinchcomb D. T., Struhl K., Davis R. W. Sterile host yeasts (SHY): a eukaryotic system of biological containment for recombinant DNA experiments. Gene. 1979 Dec;8(1):17–24. doi: 10.1016/0378-1119(79)90004-0. [DOI] [PubMed] [Google Scholar]
  7. Broach J. R., Strathern J. N., Hicks J. B. Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene. Gene. 1979 Dec;8(1):121–133. doi: 10.1016/0378-1119(79)90012-x. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Clewell D. B., Helinski D. R. Properties of a supercoiled deoxyribonucleic acid-protein relaxation complex and strand specificity of the relaxation event. Biochemistry. 1970 Oct 27;9(22):4428–4440. doi: 10.1021/bi00824a026. [DOI] [PubMed] [Google Scholar]
  10. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  11. Haber J. E., George J. P. A mutation that permits the expression of normally silent copies of mating-type information in Saccharomyces cerevisiae. Genetics. 1979 Sep;93(1):13–35. doi: 10.1093/genetics/93.1.13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Harashima S., Sidhu R. S., Toh-e A., Oshima Y. Cloning of the HIS5 gene of Saccharomyces cerevisiae by yeast transformation. Gene. 1981 Dec;16(1-3):335–341. doi: 10.1016/0378-1119(81)90091-3. [DOI] [PubMed] [Google Scholar]
  13. Hicks J. B., Hinnen A., Fink G. R. Properties of yeast transformation. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 2):1305–1313. doi: 10.1101/sqb.1979.043.01.149. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Johnston J., Hilger F., Mortimer R. Variation in frequency of transformation by plasmid YRp7 in Saccharomyces cerevisiae. Gene. 1981 Dec;16(1-3):325–329. doi: 10.1016/0378-1119(81)90089-5. [DOI] [PubMed] [Google Scholar]
  17. Klar A. J., Fogel S., Macleod K. MAR1-a Regulator of the HMa and HMalpha Loci in SACCHAROMYCES CEREVISIAE. Genetics. 1979 Sep;93(1):37–50. doi: 10.1093/genetics/93.1.37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lawson J. W., Gooder H. Growth and development of competence in the group H streptococci. J Bacteriol. 1970 Jun;102(3):820–825. doi: 10.1128/jb.102.3.820-825.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  20. Morrison D. A. Transformation in Escherichia coli: cryogenic preservation of competent cells. J Bacteriol. 1977 Oct;132(1):349–351. doi: 10.1128/jb.132.1.349-351.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Rine J., Strathern J. N., Hicks J. B., Herskowitz I. A suppressor of mating-type locus mutations in Saccharomyces cerevisiae: evidence for and identification of cryptic mating-type loci. Genetics. 1979 Dec;93(4):877–901. doi: 10.1093/genetics/93.4.877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Spencer H. T., Herriott R. M. Development of competence of Haemophilus influenzae. J Bacteriol. 1965 Oct;90(4):911–920. doi: 10.1128/jb.90.4.911-920.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Stinchcomb D. T., Mann C., Davis R. W. Centromeric DNA from Saccharomyces cerevisiae. J Mol Biol. 1982 Jun 25;158(2):157–190. doi: 10.1016/0022-2836(82)90427-2. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Toh-e A., Tada S., Oshima Y. 2-micrometers DNA-like plasmids in the osmophilic haploid yeast Saccharomyces rouxii. J Bacteriol. 1982 Sep;151(3):1380–1390. doi: 10.1128/jb.151.3.1380-1390.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Toh-e A., Wickner R. B. Curing of the 2 mu DNA plasmid from Saccharomyces cerevisiae. J Bacteriol. 1981 Mar;145(3):1421–1424. doi: 10.1128/jb.145.3.1421-1424.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Tschumper G., Carbon J. Sequence of a yeast DNA fragment containing a chromosomal replicator and the TRP1 gene. Gene. 1980 Jul;10(2):157–166. doi: 10.1016/0378-1119(80)90133-x. [DOI] [PubMed] [Google Scholar]
  28. Uchimiya H., Murashige T. Quantitative analysis of the fate of exogenous DNA in Nicotiana protoplasts. Plant Physiol. 1977 Feb;59(2):301–308. doi: 10.1104/pp.59.2.301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Weston A., Brown M. G., Perkins H. R., Saunders J. R., Humphreys G. O. Transformation of Escherichia coli with plasmid deoxyribonucleic acid: calcium-induced binding of deoxyribonucleic acid to whole cells and to isolated membrane fractions. J Bacteriol. 1981 Feb;145(2):780–787. doi: 10.1128/jb.145.2.780-787.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Zakian V. A., Scott J. F. Construction, replication, and chromatin structure of TRP1 RI circle, a multiple-copy synthetic plasmid derived from Saccharomyces cerevisiae chromosomal DNA. Mol Cell Biol. 1982 Mar;2(3):221–232. doi: 10.1128/mcb.2.3.221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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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