Mizunaga T, Tkacz J S, Rodriguez L, Hackel R A, Lampen J O
Waksman Institute of Microbiology, Rutgers University, Piscataway, New Jersey 08854.
Mol Cell Biol. 1981 May;1(5):460-8. doi: 10.1128/mcb.1.5.460-468.1981.
Mutagenesis of the sucrose-fermenting (SUC1) Saccharomyces cerevisiae strain 4059-358D yielded an invertase-negative mutant (D10). Subsequent mutagenic treatment of D10 gave a sucrose-fermenting revertant (D10-ER1) that contained the same amount of large (mannoprotein) invertase as strain 4059-358D but only trace amounts of the smaller intracellular nonglycosylated enzyme. Limited genetic evidence indicated that the mutations in D10 and D10-ER1 are allelic to the SUC1 gene. The large invertases from D10-ER1 and 4059-358D were purified and compared. The two enzymes have similar specific activity and Km for sucrose, cross-react immunologically, and show the same subunit molecular weight after removal of the carbohydrate with endo-beta-N-acetylglucosaminidae H. They differ in that the large enzyme from the revertant is rapidly inactivated at 55 degrees C, whereas that from the parent is relatively stable at 65 degrees C. The small invertase in extracts of D10-ER1 is also heat sensitive as compared to the small enzyme from the original parent strain. The low level of small invertase in mutant D10-ER1 may reflect increased intracellular degradation of this heat-labile form. In several crosses of D10-ER1 with strains carrying the SUC1 or SUC3 genes, the temperature sensitivity of the large and small invertases and the low cellular level of small invertase appeared to cosegregate. These findings are evidence that SUC1 is a structural gene for invertase and that both large and small forms are encoded by a single gene. A detailed genetic analysis is presented in a companion paper.
对蔗糖发酵型（SUC1）酿酒酵母菌株4059 - 358D进行诱变处理，得到了一种转化酶阴性突变体（D10）。随后对D10进行诱变处理，获得了一个蔗糖发酵回复突变体（D10 - ER1），该突变体所含的大分子量（甘露糖蛋白）转化酶的量与菌株4059 - 358D相同，但仅含有微量较小的细胞内非糖基化酶。有限的遗传学证据表明，D10和D10 - ER1中的突变与SUC1基因等位。对来自D10 - ER1和4059 - 358D的大分子量转化酶进行了纯化和比较。这两种酶对蔗糖具有相似的比活性和Km值，能发生免疫交叉反应，并且在用内切β - N - 乙酰葡糖胺糖苷酶H去除碳水化合物后，显示出相同的亚基分子量。它们的不同之处在于，回复突变体中的大分子量酶在55℃时迅速失活，而亲本菌株中的大分子量酶在65℃时相对稳定。与原始亲本菌株中的小分子量转化酶相比，D10 - ER1提取物中的小分子量转化酶也对热敏感。突变体D10 - ER1中小分子量转化酶的低水平可能反映了这种热不稳定形式在细胞内的降解增加。在D10 - ER1与携带SUC1或SUC3基因的菌株的几次杂交中，大、小分子量转化酶的温度敏感性以及小分子量转化酶的低细胞水平似乎是共分离的。这些发现证明SUC1是转化酶的结构基因，并且大、小两种形式均由单个基因编码。在一篇配套论文中给出了详细的遗传分析。
