High Efficiency Transformation of Intact Yeast Cells Using Single Stranded Nucleic Acids As a Carrier

Overview

Journal Curr Genet

Specialty Genetics

Date 1989 Dec 1

PMID 2692852

Citations 914

Authors

R H Schiestl

R D Gietz

Affiliations

Soon will be listed here.

Abstract

A method, using LiAc to yield competent cells, is described that increased the efficiency of genetic transformation of intact cells of Saccharomyces cerevisiae to more than 1 X 10(5) transformants per microgram of vector DNA and to 1.5% transformants per viable cell. The use of single stranded, or heat denaturated double stranded, nucleic acids as carrier resulted in about a 100 fold higher frequency of transformation with plasmids containing the 2 microns origin of replication. Single stranded DNA seems to be responsible for the effect since M13 single stranded DNA, as well as RNA, was effective. Boiled carrier DNA did not yield any increased transformation efficiency using spheroplast formation to induce DNA uptake, indicating a difference in the mechanism of transformation with the two methods.

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References

Struhl K, Stinchcomb D, Scherer S, Davis R . High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci U S A. 1979; 76(3):1035-9. PMC: 383183. DOI: 10.1073/pnas.76.3.1035. View

Hanahan D . Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983; 166(4):557-80. DOI: 10.1016/s0022-2836(83)80284-8. View

Keszenman-Pereyra D, Hieda K . A colony procedure for transformation of Saccharomyces cerevisiae. Curr Genet. 1988; 13(1):21-3. DOI: 10.1007/BF00365751. View

Hoffman C, Winston F . A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene. 1987; 57(2-3):267-72. DOI: 10.1016/0378-1119(87)90131-4. View

Bruschi C, Comer A, Howe G . Specificity of DNA uptake during whole cell transformation of S. cerevisiae. Yeast. 1987; 3(2):131-7. DOI: 10.1002/yea.320030209. View

Aviv H, Leder P . Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972; 69(6):1408-12. PMC: 426713. DOI: 10.1073/pnas.69.6.1408. View

Fasullo M, Davis R . Recombinational substrates designed to study recombination between unique and repetitive sequences in vivo. Proc Natl Acad Sci U S A. 1987; 84(17):6215-9. PMC: 299041. DOI: 10.1073/pnas.84.17.6215. View

Brzobohaty B, Kovac L . Factors enhancing genetic transformation of intact yeast cells modify cell wall porosity. J Gen Microbiol. 1986; 132(11):3089-93. DOI: 10.1099/00221287-132-11-3089. View

Kunkel T, Roberts J, Zakour R . Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987; 154:367-82. DOI: 10.1016/0076-6879(87)54085-x. View

10.

Schiestl R, Reynolds P, Prakash S, Prakash L . Cloning and sequence analysis of the Saccharomyces cerevisiae RAD9 gene and further evidence that its product is required for cell cycle arrest induced by DNA damage. Mol Cell Biol. 1989; 9(5):1882-96. PMC: 362979. DOI: 10.1128/mcb.9.5.1882-1896.1989. View

11.

Klebe R, Harriss J, Sharp Z, DOUGLAS M . A general method for polyethylene-glycol-induced genetic transformation of bacteria and yeast. Gene. 1983; 25(2-3):333-41. DOI: 10.1016/0378-1119(83)90238-x. View

12.

Jensen R, Sprague Jr G, Herskowitz I . Regulation of yeast mating-type interconversion: feedback control of HO gene expression by the mating-type locus. Proc Natl Acad Sci U S A. 1983; 80(10):3035-9. PMC: 393968. DOI: 10.1073/pnas.80.10.3035. View

13.

Burke D, Carle G, Olson M . Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science. 1987; 236(4803):806-12. DOI: 10.1126/science.3033825. View

14.

Holm C, FANGMAN W, Botstein D . A rapid, efficient method for isolating DNA from yeast. Gene. 1986; 42(2):169-73. DOI: 10.1016/0378-1119(86)90293-3. View

15.

Harashima S, Takagi A, Oshima Y . Transformation of protoplasted yeast cells is directly associated with cell fusion. Mol Cell Biol. 1984; 4(4):771-8. PMC: 368796. DOI: 10.1128/mcb.4.4.771-778.1984. View

16.

Schiestl R, Wintersberger U . X-ray enhances mating type switching in heterothallic strains of Saccharomyces cerevisiae. Mol Gen Genet. 1982; 186(4):512-7. DOI: 10.1007/BF00337958. View

17.

Schiestl R, Prakash S . RAD1, an excision repair gene of Saccharomyces cerevisiae, is also involved in recombination. Mol Cell Biol. 1988; 8(9):3619-26. PMC: 365417. DOI: 10.1128/mcb.8.9.3619-3626.1988. View

18.

Rothstein R . One-step gene disruption in yeast. Methods Enzymol. 1983; 101:202-11. DOI: 10.1016/0076-6879(83)01015-0. View

19.

Beggs J . Transformation of yeast by a replicating hybrid plasmid. Nature. 1978; 275(5676):104-9. DOI: 10.1038/275104a0. View

20.

Johnston J, Hilger F, Mortimer R . Variation in frequency of transformation by plasmid YRp7 in Saccharomyces cerevisiae. Gene. 1981; 16(1-3):325-9. DOI: 10.1016/0378-1119(81)90089-5. View