γ射线诱发大鼠胚胎细胞转化与N－ras的激活

Activation of N-ras Gene is Associated With γ-Radiation-Induced Transformation of Rat Em-bryo Cells.

以γ射线诱发转化的大鼠胚胎细胞（ＲＥＣ：ｍｙｃ：γ３３）的ＤＮＡ构建粘粒基因库，用总基因库ＤＮＡ转染ＮＩＨ／３Ｔ３细胞，产生转化灶的ＤＮＡ作二轮转染，二轮转化的ＮＩＨ／３Ｔ３细胞内有大鼠ＲＥＣ：ｍｙｃ：γ３３ＤＮＡ中具转化活性的Ｎ－ｒａｓ基因，用不对称ＰＣＲ和ＤＮＡ序列分析法证明，ＲＥＣ：ｍｙｃ：γ３３细胞中鼠Ｎ－ｒａｓ的活化是由于第６１位密码子的Ａ→Ｇ点突变。ＮＩＨ／３Ｔ３转化灶中鼠Ｎ－ｒａｓ也有同样点突变，但ＮＩＨ／３Ｔ３细胞的内源性Ｎ－ｒａｓ基因则无此突变。

cosmid library,constructed from DNA ofthe yradiation-transformed REC: myc cellline, designated REC:myc:γ33.was trans-fected into NIH/3T3 cells.yielding foci.An-other round transfection of DNA from the firstround focus into fresh NIH/3T3 cells producedsecond round foci.An active N-ras gene whichoriginated from rat REC.myc.γ33 cells wasdetected in the NIH/3T3 secondary transfor-mants.With PCR and direct DNA sequencingtechniques,rat N-ras gene was found activatedin the REC:myc.γ33 cells by CAA→CGApoint mutation at codon 61,but not in theREC myc cells.Also rat N-ras gene was iden-tified as a point mutated gene in the NIH/3T3transformants. and the endogenous N-ras genein the NIH/3T3 recipient cells remains nor-mal.What was found to be more interesting isthat five out of six γ-radiation transformedREC.myc cell lines bear the same point muta-tion (CAA→CGA) indicating association of γ-radiation-induced transformation with pointmutation in the N-ras gene.