Transformation of intact yeast cells treated with alkali cations

Journal of Bacteriology - Tập 153 Số 1 - Trang 163-168 - 1983
Hisao Ito, Yasuki Fukuda, Kousaku Murata, Akira Kimura

Tóm tắt

Intact yeast cells treated with alkali cations took up plasmid DNA. Li+, Cs+, Rb+, K+, and Na+ were effective in inducing competence. Conditions for the transformation of Saccharomyces cerevisiae D13-1A with plasmid YRp7 were studied in detail with CsCl. The optimum incubation time was 1 h, and the optimum cell concentration was 5 x 10(7) cells per ml. The optimum concentration of Cs+ was 1.0 M. Transformation efficiency increased with increasing concentrations of plasmid DNA. Polyethylene glycol was absolutely required. Heat pulse and various polyamines or basic proteins stimulated the uptake of plasmid DNA. Besides circular DNA, linear plasmid DNA was also taken up by Cs+-treated yeast cells, although the uptake efficiency was considerably reduced. The transformation efficiency with Cs+ or Li+ was comparable with that of conventional protoplast methods for a plasmid containing ars1, although not for plasmids containing a 2 microns origin replication.

Từ khóa


Tài liệu tham khảo

Beach , D. , and P. Nurse . 1981 . High-frequency transformation of the fission yeast Schizosaccharomyces pombe . Nature (London) 290 : 140 - 142 .

Cameron , J. R. , P. Phillppsen , and R. W. Davis . 1977 . Analysis of chromosomal integration and deletions of yeast plasmids . Nucleic Acids Res. 4 : 1429 - 1448 .

Ferenczy , L. , and A. Maraz . 1977 . Transfer of mitochondria by protoplast fusion in Saccharomyces cerevisiae . Nature (London) 268 : 524 - 525 .

Grhdus , L. 1980 . Nucleic acid transport driven by ion gradient across cell membrane . FEBS Lett. 113 : 1 - 10 .

HelUng , R. B. , H. M. Goodman , and H. W. Boyer . 1974 . Analysis of endonuclease R * EcoRI fragments of DNA from lambdoid bacteriophages and other viruses by agarose-gel electrophoresis . J. Virol. 14 : 1235 - 1244 .

Hlnnen , A. , J. B. Hicks , and G. R. Flnk . 1978 . Transformation of yeast . Proc. Natl. Acad. Sci. U.S.A. 75 : 1929 - 1933 .

Kao , K. N. , and M. R. Mlchayluk . 1974 . A method for high frequency intergeneric fusion of plant protoplasts . Planta 115 : 355 - 367 .

Kimura , A. , K. Arima , and K. Murata . 1981 . Biofunctional change in yeast cell surface on treatment with Triton X-100 . Agric. Biol. Chem. 45 : 2627 - 2629 .

Kimura , A. , and M. Morita . 1975 . Fermentative formation of CDP-choline by intact cells of yeast: Saccharomyces carlsbergen.sis (IFO 0641) treated with a detergent, Triton X-100 . Agric. Biol. Chem. 39 : 1469 - 1474 .

Mandel , M. , and A. Hig . 1970 . Calcium-dependent bacteriophage DNA infection . J. Mol. Biol. 53 : 159 - 162 .

Meyers , J. A. , D. Sanchez , L. P. Elwell , ad S. Falkow . 1976 . Simple agarose gel electrophoretic method for the identification and characterization of plasmid deoxyribonucleic acid . J. Bacteriol. 127 : 1529 - 1537 .

Naharl K. 1979. DNA kumikae gijutsu p. 162-163. In K. Sakaguchi and M. Okanishi (ed.) Bunshi ikushu to oyo biseibtsu. Kodansha Tokyo. (In Japanese.)

Struhl ,d, D.T. Dhdecmb, S. Scherer , and R. W. Davis . 1979 . High-frequwecy transformation of yeast: autonomous replication 'f hybrid DNA molecules . Proc. Natl. Acad. Sci. U.S.A. 75 : 1929 - 1933 .

Sinkl M. ad I. Tanabe. 1976. Uptake of single-strand bacteriophage DNA by isolated tobacco protoplasts. Z. PManzenphysiol. 7&21-433.

va Sollngen ,- P. , aig L. B. van der Plaat . 1977 . Fusion of yeast spheroplasts . J. Bacteriol. 130 : 946 - 947 .

Yanofsky , C. , V. Horn , M. Bonner , and S. Ttasiowskl . 1971 . Polrity and enzyme functions in mutants of the first three genes of the tryptophan operon of Escherichia coli . Genetics 69 : 409 - 433 .