Stone J C, Vass W C, Willumsen B M, Lowy D R
Jackson Laboratory, Bar Harbor, Maine 04609.
Mol Cell Biol. 1988 Aug;8(8):3565-9. doi: 10.1128/mcb.8.8.3565-3569.1988.
A series of mutations encoding single-amino-acid substitutions within the v-rasH effector domain were constructed, and the ability of the mutants to induce focal transformation of NIH 3T3 cells was studied. The mutations, which spanned codons 32 to 40, were made by a "cassette" mutagenesis technique that involved replacing this portion of the v-rasH effector domain with a linker carrying two BspMI sites in opposite orientations. Since BspMI cleaves outside its recognition sequence, BspMI digestion of the plasmid completely removed the linker, creating a double-stranded gap whose missing ras sequences were reconstructed as an oligonucleotide cassette. Based upon the ability of the mutants to induce focal transformation of NIH 3T3 cells, a range of phenotypes from virtually full activity to none (null mutants) was seen. Three classes of codons were present in this segment: one which could not be altered, even conservatively, without a loss of function (codons 32 and 35); one which retained detectable biologic activity with conservative changes but which lost function with more drastic substitutions (codons 36 and 40); and one which retained function even with a nonconservative substitution (codon 39).
构建了一系列编码v-rasH效应结构域内单氨基酸取代的突变体，并研究了这些突变体诱导NIH 3T3细胞灶性转化的能力。这些跨越密码子32至40的突变是通过“盒式”诱变技术产生的，该技术涉及用携带两个相反方向BspMI位点的接头替换v-rasH效应结构域的这一部分。由于BspMI在其识别序列之外切割，对质粒进行BspMI消化可完全去除接头，产生一个双链缺口，其缺失的ras序列作为寡核苷酸盒进行重建。根据突变体诱导NIH 3T3细胞灶性转化的能力，观察到一系列从几乎完全活性到无活性（无效突变体）的表型。该片段中存在三类密码子：一类即使保守改变也不能改变而不丧失功能（密码子32和35）；一类保守改变时保留可检测的生物学活性，但更剧烈的取代时丧失功能（密码子36和40）；一类即使非保守取代也保留功能（密码子39）。
