Yamamoto T, Moerschell R P, Wakem L P, Ferguson D, Sherman F
Department of Biochemistry, University of Rochester School of Medicine and Dentistry, New York 14642.
Yeast. 1992 Nov;8(11):935-48. doi: 10.1002/yea.320081104.
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.
通过选择含有至少部分功能性同工 -1- 细胞色素 c 的 cyc1 转化体，研究了影响酿酒酵母用合成寡核苷酸直接转化的因素。在反应混合物中使用 1 mg 寡核苷酸，获得了约 3×10⁴ 个转化体，占细胞总数的 0.1%。载体，如异源寡核苷酸，可提高转化频率。如果寡核苷酸有大量错配或末端有错配，转化频率会显著降低。在 rad52⁻ 菌株中，用寡核苷酸转化有效，而用线性化双链质粒转化则无效，这表明用寡核苷酸转化的途径与用线性化双链质粒转化的途径不同。我们描述了一种用两种寡核苷酸共转化的方法，一种纠正宿主菌株中靶等位基因的 cyc1 缺陷，另一种在同工 -1- 细胞色素 c 分子的其他位置产生所需的氨基酸改变；通过共转化获得的转化体中约 20% 含有这些所需的二次改变。
