Parameters affecting the frequencies of transformation and co‐transfromation with synthetic oligonucleotides in yeast

Yeast - Tập 8 Số 11 - Trang 935-948 - 1992
Tetsuro Yamamoto1, R P Moerschell2, Paul Wakem1, David J. Conway1, Fred Sherman1,2
1Depatment of Biochemistry, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, U.S.A.
2Depatment of Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, U.S.A.

Tóm tắt

AbstractFactors influencing the direct transformation of the yeast Saccharomyces cerevisiae with synthetic oligonucleotides were investigated by selecting for cyc1 transformants that contained at least partially fuctional iso‐1‐cytochrome c. Aproximately 3 × 104 transformanrs, constituting 0·1% of the cells, were obtained by using 1 mg of oligonucleotide in the reaction mixture. Carrier, such as heterogenous oligonucleotides, enhanced transformation frequencies. Transformation frequencies were dramatically reduced if the oligonucleotides had a large number of mismatches or had terminally located mismatches. Transformation with oligonucleotides, but not with linearized double‐strand plasmid, was efficient in a rad52− strain, ssuggesting that the pathway for transformation with oligonucleotides is different from that with linearized double‐strand plasmid. We describe a procedure of co‐transformation with two oligonucleotides, one correcting the cyc1 defect of the target allele in the host strain, and the other producing a desired amono acid alteration elsewhere in the iso‐1‐cytochrome c molecule; approximately 20% of the transformants obtained by co‐transformation contained these desired second alterations.

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Yeast

Tập 8 Số 11

935-948

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