Mandal N C, Silverman P M
Proc Natl Acad Sci U S A. 1977 Apr;74(4):1657-61. doi: 10.1073/pnas.74.4.1657.
We report the isolation of E. coli mutant capable of supporting replication of bacteriophage Qbeta at 33 degrees, but not at 40 degrees. Coliphages f2, R23, fd, and yamma formed plaques on mutant cells at both temperatures. Temperature-shift experiments showed that bacteriophage Q beta replication was blocked in the mutant within the first 20-30 min of infection. The defect did not prevent translation of the Qbeta polymerase gene or assembly of catalytically active Qbeta replicase molecules. In fact, mutant cells infected at 40 degrees hyperinduced replicase active both in vivo and in vitro. However, zone sedimentation of the in vivo RNA product showed it to consist of partially double-stranded material sedimenting at 9 S, with little or no viral 32S RNA. The 9S RNA was also found, along with a predominant peak of 32S RNA in parental cells infected at 40 degrees, but not in cells infected at 33 degrees. It thus appears that the temperature-sensitive component is required for viral RNA replication, but not for other RNA synthesis catalyzed by the replicase. Uninfected mutant cells grew normally at 40 degrees in nutrient broth, but not in glucose- or glycerol-minimal media. Revertants selected for their abillity to grow in minimal medium at 40 degrees also supported bacteriophage Qbeta replication at 40 degrees.
我们报告了一种大肠杆菌突变体的分离情况，该突变体能够在33℃支持噬菌体Qβ的复制，但在40℃则不能。大肠杆菌噬菌体f2、R23、fd和γ在两个温度下均可在突变体细胞上形成噬菌斑。温度转换实验表明，噬菌体Qβ的复制在感染后的最初20 - 30分钟内在突变体中被阻断。该缺陷并不妨碍Qβ聚合酶基因的翻译或催化活性Qβ复制酶分子的组装。事实上，在40℃感染的突变体细胞在体内和体外均超诱导了复制酶活性。然而，体内RNA产物的区带沉降显示其由沉降系数为9S的部分双链物质组成，几乎没有或没有病毒32S RNA。在40℃感染的亲代细胞中，除了主要的32S RNA峰外也发现了9S RNA，但在33℃感染的细胞中未发现。因此，温度敏感成分似乎是病毒RNA复制所必需的，但不是复制酶催化的其他RNA合成所必需的。未感染的突变体细胞在营养肉汤中于40℃正常生长，但在葡萄糖或甘油基本培养基中则不能。为其在基本培养基中于40℃生长的能力而筛选出的回复突变体也能在40℃支持噬菌体Qβ的复制。
