Summary
By using two chimeric plasmids containing yeast URA3 gene as a selection marker and 2 μm yeast DNA linked to the bacterial plasmid pCR1, a yeast strain devoid of any 2 μm DNA sequence was transformed. Recovery in E. coli of plasmids from yeast transformants showed that the 2 μm-less strain was able to maintain the chimeric plasmids as autonomous replicons, with very infrequent plasmid recombination. Hybridization experiments gave no evidence for integration of the URA3 DNA sequence in the chromosomal DNA. The transformed clones showed a high stability of the ura+ character during vegetative multiplication, even in the absence of selective pressure. The specific activity of orotidine 5′ monophosphate decarboxylase (coded by the URA3 gene) was 5 to 10 fold higher than in the wild type.
These features should offer new possibilities for cloning with yeast.
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Bach, M.L., Lacroute, F., Botstein, D.: Evidence for transcriptional regulation of OMP-decarboxylase in yeast by hybridization of mRNA to the yeast structural gene cloned in E. coli. Proc. Natl. Acad. Sci. U.S.A. 76, 386–390 (1979)
Becwith, J.R., Pardee, A.B., Austrian, R., Jacob, F.: Coordination of the synthesis of the enzymes in the pyrimidine pathway of E. coli. J. Mol. Biol. 5, 618–634 (1962)
Beggs, J.D.B.: Transformation of yeast by a replicating hybrid plasmid. Nature 275, 104–109 (1978)
Blanc, H., Dujon, B., Guerineau, M., Slonimski, P.P.: Detection of specific DNA sequences in yeast by colony hybridization. Mol. Gen. Genet. 161, 311–315 (1978)
Burgess, R.R., Travers, A.A.: DNA-dependant RNA polymerase. In: Procedures in nucleic acid research, Cantoni, G.I. and Davies, D.R. (eds.), Vol. 2, pp. 851–863. New-York: Harper and Row 1971
Cohen, S.N., Chang, A.C.Y., Hsu, L.: Non chromosomal antibiotic resistance in bacteria: Genetic transformation of Escherichia coli by R-factor DNA. Proc. Natl. Acad. Sci. U.S.A. 69, 2110–2114 (1972)
Gerbaud, C., Fournier, P., Blanc, H., Aigle, M., Heslot, H., and Guérineau, M.: High frequency of yeast transformation by plasmids carrying part or entire 2-μm yeast plasmid. Gene 5, 233–253 (1979)
Guérineau, M.: Expression of a yeast episome: RNA-DNA hybridizations studies. FEBS Lett. 80, 426–428 (1977)
Guérineau, M.: Plasmid DNA in yeast. In: Viruses and plasmids in fungi, Lemke, P.A. (ed.), pp. 539–593. New York: Marcel Dekker 1979
Guérineau, M., Slonimski, P.P., Avner, P.R.: Yeast episome: oligomycin resistance associated with a small covalently closed non mitochondrial circular DNA. Biochem. Biophys. Res. Commun. 61, 462–469 (1974)
Hinnen, A., Hicks, J.B., Fink, G.R.: Transformation of yeast. Proc. Natl. Acad. Sci U.S.A. 75, 1929–1933 (1978)
Hollenberg, C.P., Degelmann, A., Kustermann-Kuhn, B., Royer, H.D.: Characterization of 2 μm DNA of S. cerevisiae by restriction fragment analysis and integration in an E. coli plasmid. Proc. Natl. Acad. Sci. U.S.A. 73, 2072–2076 (1976)
Korch, C.T., Lacroute, F., Exinger, F.: A regulatory interaction between pyrimidine and purine biosynthesis via ureidosuccinic acid. Mol. Gen. Genet. 133, 63–75 (1974)
Lacroute, F.: Regulation of pyrimidine biosynthesis in Saccharomyces cerevisiae. J. Bacteriol. 95, 824–832 (1968)
Rigby, P.W.J., Dieckmann, M., Rhodes, C., Berg, P.: Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113, 237–251 (1977)
Stickland, L.H.: The determination of small quantities of bacteria by means of the biuret reaction. J. Gen. Microbiol. 5, 698–703 (1951)
Struhl, K., Stinchcomb, D.T, Scherrer, S., Davis, R.W.: High frequency transformation of yeast: Autonomous replication of hybrid DNA molecules. Proc. Natl. Acad. Sci. U.S.A. 75, 1035–1039 (1979)
Thuring, R.W.J., Sanders, J.P.M., Borst, P.: A freeze-squeeze method for recovering long DNA from agarose gels. Anal. Biochem. 66, 213–220 (1975)
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Communicated by W. Gajewski
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Blanc, H., Gerbaud, C., Piotr Slonimski, P. et al. Stable yeast transformation with chimeric plasmids using a 2 μm-circular DNA-less strain as a recipient. Molec. gen. Genet. 176, 335–342 (1979). https://doi.org/10.1007/BF00333095
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DOI: https://doi.org/10.1007/BF00333095