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Isolation of transformation-suppressor genes by using a cDNA-expression library having sense and anti-sense inserts.

使用具有正义和反义插入片段的 cDNA 表达文库分离转化抑制基因。

基本信息

批准号：
02454145
负责人：
KATAOKA Tohru
金额：
$ 4.42万
依托单位：
Kobe University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-02454145/
关键词：
ras oncogene RNA helicase cDNA-expression library reversion リバ-ジョン RNAhelicase 癌抑制遺伝子

项目摘要

1. We used artificial overexpression of cDNAs synthesized from NIH3T3 cell mRNA to induce reversion of NIH3T3 cells transformed by the activated Harvey-ras oncogene. The revertants were screened by observation under microscope for "flat" appearance. Among about 100 revertant cell lines obtained, we have chosen one revertant, Rev6-4, which showed marked decrease in its ability to form colonies in soft agar and to form tumors in nude mice, and isolated an incorporatcd cDNA from its genomic DNA by molecular cloning after PCR (polymerase chain reaction) amplification.2. Analysis of the CDNA isolated from Rev6-4 showed that the CDNA encoded P68, which had been shown to have ATP-dependent RNA helicase activity, and that the cDNA appeared to be over-expressed in an anti-sense manner in Rev6-4 cells. Concominantly, a decrease in the amount of endogenous P68 mRNA was observed. We have confirmed that the cloned Rev6-4 CDNA could induce decrease in colony formation in soft agar, in tumor formation in nude mice and in saturation density when overexpressed in the ras-transformed NIH3T3 cells in the anti-sense manner.3. We have isolated a full-length cDNA encoding P68. When the cDNA was over-expressed in NIH3T3 cells in a sense direction, formation of foci which appeared morphologically-transformed was observed.However, cells in the foci could not form tumors when injected into nude mice. Presently, we do not know if the P68 gene is an oncogene whose product is acting in downstream of ras. We are currently analysing a physiological function of P68 by using antibodies against it.4. We have isolated cDNAs from two other revertants and are currently analysing their structure and examining for their ability to induce reversion.

1.我们利用人工过表达由NIH 3 T3细胞mRNA合成的cDNA来诱导由活化的Harvey-ras癌基因转化的NIH 3 T3细胞的逆转。通过在显微镜下观察“扁平”外观筛选回复突变体。从获得的100株回复突变株中，筛选出软琼脂集落形成能力和裸鼠成瘤能力明显下降的回复突变株Rev 6 -4，并通过PCR扩增后的分子克隆，从其基因组DNA中分离得到了一个特异性的cDNA.从Rev 6 -4分离的cDNA的分析表明，该cDNA编码P68，其已被证明具有ATP依赖性RNA解旋酶活性，并且该cDNA似乎在Rev 6 -4细胞中以反义方式过表达。与此同时，内源性P68 mRNA的量减少。我们已经证实，克隆的Rev 6 -4 cDNA以反义方式在ras转化的NIH 3 T3细胞中过表达时，可以诱导软琼脂集落形成、裸鼠成瘤以及饱和密度的降低.我们已经分离出了编码P68的全长cDNA。当该cDNA在NIH 3 T3细胞中以正义方向过表达时，观察到形成了形态学转化的病灶，但病灶中的细胞注射到裸鼠体内时不能形成肿瘤。目前尚不清楚P68基因是否是一种癌基因，其产物是否作用于ras下游。我们目前正在分析P68的生理功能，通过使用抗体。我们已经从另外两个回复突变体中分离出cDNA，目前正在分析它们的结构，并研究它们诱导回复突变的能力。