Imbert J, Clertant P, de Bovis B, Planche J, Birg F
J Virol. 1983 Sep;47(3):442-51. doi: 10.1128/JVI.47.3.442-451.1983.
The stabilities of in vivo [35S]methionine-labeled large T and small t proteins, synthesized in temperature-sensitive (type N) and temperature-insensitive (type A) FR 3T3 rat cells transformed by an early temperature-sensitive mutant of simian virus 40 (SV40), tsA30, were analyzed at the permissive and restrictive temperatures. The two polypeptides, detected in greatly reduced amounts in cells of the N type at the restrictive temperature, were also unstable at the permissive temperature. However, both were made in similar amounts and were apparently stable in cells of the A type, irrespective of the temperature. The structures of the viral RNAs present at the permissive temperature were analyzed for transformants representative of each type, and containing a single integration of viral DNA. The two cell lines synthesized transcripts identical to the large T and small t mRNAs identified in SV40-infected monkey cells. Similar amounts of viral RNA were found in A and N transformants in active growth at the permissive and restrictive temperatures, which argued against a control at a transcriptional level. Assay of a defined function of the protein, namely, the binding of nucleotide detected by affinity labeling with periodate-oxidized [alpha-32P]ATP, clearly showed that the large T proteins from both types of transformants exhibited, at least for that particular biochemical function, the same in vitro temperature sensitivity. In transformants of the A type only could a reduced binding activity be detected in extracts from cells grown at the restrictive temperature. Thus, the temperature-independent behavior of the A transformants may result from an in vivo partial stabilization of the newly synthesized large T protein, probably through interaction with a cellular component(s).
在允许温度和限制温度下，分析了在由猿猴病毒40（SV40）的早期温度敏感突变体tsA30转化的温度敏感型（N型）和温度不敏感型（A型）FR 3T3大鼠细胞中合成的体内[35S]甲硫氨酸标记的大T蛋白和小t蛋白的稳定性。在限制温度下，N型细胞中检测到的这两种多肽数量大幅减少，在允许温度下它们也不稳定。然而，在A型细胞中，无论温度如何，这两种多肽的合成量相似且显然稳定。分析了允许温度下存在的病毒RNA的结构，这些病毒RNA来自代表每种类型且含有单个病毒DNA整合的转化体。这两种细胞系合成的转录本与在SV40感染的猴细胞中鉴定出的大T和小t mRNA相同。在允许温度和限制温度下处于活跃生长状态的A和N转化体中发现了相似数量的病毒RNA，这表明转录水平上不存在调控。通过用高碘酸盐氧化的[α-32P]ATP进行亲和标记检测蛋白质的特定功能，即核苷酸结合，结果清楚地表明，至少对于该特定生化功能而言，两种类型转化体中的大T蛋白在体外表现出相同的温度敏感性。仅在A型转化体中，在限制温度下生长的细胞提取物中可检测到结合活性降低。因此，A型转化体的温度不依赖行为可能是由于新合成的大T蛋白在体内部分稳定化，可能是通过与一种或多种细胞成分相互作用实现的。
