Analysis of cell transformation by small molecular weight RNA.

本研究旨在利用从小鼠胸腺瘤细胞中提取的小分子量RNA进行体外细胞转化。负责转化活性的RNA被命名为转化RNA。用异硫氰酸胍（GTC）-热酚提取法从化学诱导的小鼠淋巴瘤细胞中获得细胞总RNA。通过20%天然聚丙烯酰胺凝胶电泳两个循环或通过10%变性聚丙烯酰胺凝胶电泳三个循环来纯化转化RNA。将RNA转染大鼠成纤维细胞系3 Y1细胞。当用RNA转染的3 Y1细胞保持不连续传代时，转化细胞灶在43至53天的潜伏期出现。通过在软琼脂上形成细胞集落来确认3 Y1细胞的转化表型，为了确认由此分离的转化RNA的纯度，通过使用[3 ′-32 P]RNA的直接化学方法对RNA进行测序。读取RNA的部分110个碱基序列。结果表明，转化RNA序列与核内小RNA U 5 RNA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.