Marshall M S, Gibbs J B, Scolnick E M, Sigal I S
Mol Cell Biol. 1987 Jul;7(7):2309-15. doi: 10.1128/mcb.7.7.2309-2315.1987.
Activating mutations (valine 19 or leucine 68) were introduced into the Saccharomyces cerevisiae RAS1 and RAS2 genes. In addition, a deletion was introduced into the wild-type gene and into an activated RAS2 gene, removing the segment of the coding region for the unique C-terminal domain that lies between the N-terminal 174 residues and the penultimate 8-residue membrane attachment site. At low levels of expression, a dominant activated phenotype, characterized by low glycogen levels and poor sporulation efficiency, was observed for both full-length RAS1 and RAS2 variants having impaired GTP hydrolytic activity. Lethal CDC25 mutations were bypassed by the expression of mutant RAS1 or RAS2 proteins with activating amino acid substitutions, by expression of RAS2 proteins lacking the C-terminal domain, or by normal and oncogenic mammalian Harvey ras proteins. Biochemical measurements of adenylate cyclase in membrane preparations showed that the expression of RAS2 proteins lacking the C-terminal domain can restore adenylate cyclase activity to cdc25 membranes.
将激活突变（缬氨酸19或亮氨酸68）引入酿酒酵母的RAS1和RAS2基因中。此外，在野生型基因和激活的RAS2基因中引入缺失，去除了位于N端174个残基和倒数第二个8个残基的膜附着位点之间的独特C端结构域的编码区片段。在低表达水平下，对于具有受损GTP水解活性的全长RAS1和RAS2变体，均观察到以低糖原水平和差的孢子形成效率为特征的显性激活表型。致死性CDC25突变可通过具有激活氨基酸取代的突变RAS1或RAS2蛋白的表达、缺乏C端结构域的RAS2蛋白的表达或正常和致癌性哺乳动物哈维ras蛋白的表达而被绕过。膜制剂中腺苷酸环化酶的生化测量表明，缺乏C端结构域的RAS2蛋白的表达可将腺苷酸环化酶活性恢复到cdc25膜中。
