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  • Title: Parameters affecting the frequencies of transformation and co-transformation with synthetic oligonucleotides in yeast.
    Author: Yamamoto T, Moerschell RP, Wakem LP, Ferguson D, Sherman F.
    Journal: Yeast; 1992 Nov; 8(11):935-48. PubMed ID: 1336288.
    Abstract:
    Factors influencing the direct transformation of the yeast Saccharomyces cerevisiae with synthetic oligonucleotides were investigated by selecting for cyc1 transformants that contained at least partially functional iso-1-cytochrome c. Approximately 3 x 10(4) transformants, constituting 0.1% of the cells, were obtained by using 1 mg of oligonucleotide in the reaction mixture. Carrier, such as heterogeneous 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, suggesting 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 amino 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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