Moerschell R P, Tsunasawa S, Sherman F
Department of Biophysics, University of Rochester School of Medicine and Dentistry, NY 14642.
Proc Natl Acad Sci U S A. 1988 Jan;85(2):524-8. doi: 10.1073/pnas.85.2.524.
Genomic DNA of the yeast, Saccharomyces cerevisiae, can be conveniently and specifically altered by transforming spheroplasts or lithium acetate-treated cells directly with synthetic oligonucleotides. Altered forms of iso-1-cytochrome c were generated by transforming a cyc1 mutant with oligonucleotides and selecting for at least partially functional revertants; the oligonucleotides contained a sequence that corrected the cyc1 mutation and produced additional alterations at nearby sites. Transformation has been accomplished with oligonucleotides as short as 20 nucleotides and with amounts as low as 100 micrograms. This method of site-directed mutagenesis in vivo has been used to produce alterations in the NH2-terminal region of iso-1-cytochrome c in which the NH2-terminal methionine is excised and the penultimate residue is acetylated.
通过直接用合成寡核苷酸转化酵母（酿酒酵母）的原生质体或经醋酸锂处理的细胞，可方便且特异性地改变其基因组DNA。通过用寡核苷酸转化cyc1突变体并选择至少部分功能回复体，产生了异-1-细胞色素c的改变形式；这些寡核苷酸包含一个校正cyc1突变并在附近位点产生额外改变的序列。已使用短至20个核苷酸、低至100微克的寡核苷酸完成了转化。这种体内定点诱变方法已用于在异-1-细胞色素c的NH2末端区域产生改变，其中NH2末端的甲硫氨酸被切除，倒数第二个残基被乙酰化。
