Analysis of cell transformation by small molecular weight RNA.

本研究从小鼠胸腺瘤细胞中提取小分子量RNA进行体外细胞转化。负责转化活性的RNA被指定为转化RNA。采用异硫氰酸胍-热酚萃取法提取化学诱导小鼠淋巴瘤细胞总RNA。转化RNA通过20%天然聚丙烯酰胺凝胶电泳2次或10%变性聚丙烯酰胺凝胶电泳3次得到纯化。将RNA转染大鼠成纤维细胞系3Y1细胞。转染RNA的3Y1细胞不连续传代时，潜伏期43 ~ 53 d出现转化细胞灶。通过软琼脂中细胞的集落形成证实了3Y1细胞的转化表型。为了确认所分离的转化RNA的纯度，采用[3'-32P]RNA直接化学法对其进行测序。读取RNA的部分110个碱基序列。结果表明，转化RNA序列与其中一种小核RNA U5具有显著的同源性。

This study is focused on in vitro cell transformation with small molecular weight RNA extracted from mouse thymoma cells. The RNA responsible for transforming activity was designated as transforming RNA. Total cellular RNA was obtained from chemically-induced lymphoma cells in mice by Guanidinium isothiocyanate (GTC)-hot phenol extraction. Transforming RNA was purified by the two cycles of electrophoresis through 20% native polyacrylamide gels or three cycles of electrophoresis through 10% denaturing polyacrylamide gels. The RNA was transfected into rat fibroblastic cell line, 3Y1 cells. When 3Y1 cells transfected with the RNA was maintained without serial passage, transformed cell foci appeared at the latency of 43 to 53 days. The transformed phenotype of 3Y1 cells was confirmed by colony formation of the cells in soft agar.In order to confirm the purity of transforming RNA thus isolated, the RNA was sequenced by the direct chemical method using [3'-32P]RNA. The partial 110 bases sequence of the RNA was read. It was revealed that transforming RNA sequence had remarkable homology to one of the small nuclear RNA, namely U5 RNA.