Huang K N, Odinsky S A, Cross F R
The Rockefeller University, New York, New York 10021, USA.
Mol Cell Biol. 1997 Aug;17(8):4654-66. doi: 10.1128/MCB.17.8.4654.
We have generated 50 new alleles of the yeast CLN2 gene by using site-directed mutagenesis. With the recently obtained crystal structure of cyclin A as a guide, a peptide linker sequence was inserted at 13 sites within the cyclin box of Cln2 to determine if the architecture of Cln2 is similar to that of cyclin A. Linkers inserted in what are predicted to be helices 1, 2, 3, and 5 of the cyclin box resulted in nonfunctional Cln2 molecules. Linkers inserted between these putative helix sites and in the region believed to contain a fourth helix did not have significant effects upon Cln2 function. A series of deletions in the region between the third and fifth helices indicate that the putative fourth helix may lie at the C-terminal end of this region yet is not essential for function. Two residues that are predicted to form a buried salt bridge important for interaction of two helices of the cyclin box were also mutated, and an additional set of 31 mutant alleles was generated by clustered-charge-to-alanine scanning mutagenesis. All of the mutant CLN2 alleles made in this study were tested in a variety of genetic and functional assays previously demonstrated to differentiate specific cyclin functions. Some alleles demonstrated restricted patterns of defects, suggesting that these mutations may interfere with specific aspects of Cln2 function.
我们通过定点诱变产生了酵母CLN2基因的50个新等位基因。以最近获得的细胞周期蛋白A的晶体结构为指导，在Cln2细胞周期蛋白框内的13个位点插入了一个肽接头序列，以确定Cln2的结构是否与细胞周期蛋白A的结构相似。插入到细胞周期蛋白框预测的螺旋1、2、3和5中的接头导致Cln2分子无功能。插入这些假定螺旋位点之间以及被认为包含第四个螺旋的区域中的接头对Cln2功能没有显著影响。在第三和第五个螺旋之间区域的一系列缺失表明，假定的第四个螺旋可能位于该区域的C末端，但对功能并非必不可少。预测形成对细胞周期蛋白框的两个螺旋相互作用很重要的埋藏盐桥的两个残基也发生了突变，并且通过聚类电荷到丙氨酸扫描诱变产生了另外一组31个突变等位基因。本研究中产生的所有突变CLN2等位基因都在先前证明可区分特定细胞周期蛋白功能的各种遗传和功能试验中进行了测试。一些等位基因表现出受限的缺陷模式，表明这些突变可能会干扰Cln2功能的特定方面。
