Lau L F, Nathans D
EMBO J. 1985 Dec 1;4(12):3145-51. doi: 10.1002/j.1460-2075.1985.tb04057.x.
To identify previously undetected genes that may be involved in the transition from a resting state (G0) to a proliferative state (G1) of mammalian cells, we set out to isolate cDNA clones derived from mRNAs that appear in serum-stimulated cells in the absence of protein synthesis. A lambda cDNA library was prepared using poly(A)+ RNA from BALB/c 3T3 cells that had been brought to quiescence and subsequently stimulated with serum in the presence of cycloheximide. Approximately 50 000 recombinant phage plaques were screened, and 357 clones were isolated that hybridized to probes derived from stimulated-cell RNA but not to probes from resting-cell RNA. Cross hybridization analysis showed that four RNA sequence families account for approximately 90% of these clones. One of the clones hybridized to an actin probe; none hybridized to any of 13 oncogene probes tested. Five different RNAs that appear to be previously uncharacterized have been further analyzed. These RNAs accumulate and decay rapidly following stimulation by serum or purified growth factors, or by a tumor promoter, and they are superinduced by serum in the presence of cycloheximide. Three of the RNAs could be enriched by hybridization to cDNAs and translated in vitro, yielding proteins of approximately 43, 40 and 35 kd, respectively.
为了鉴定可能参与哺乳动物细胞从静止状态（G0）转变为增殖状态（G1）的先前未被检测到的基因，我们着手分离源自血清刺激细胞中出现的、在无蛋白质合成情况下的mRNA的cDNA克隆。使用来自已进入静止状态并随后在放线菌酮存在下用血清刺激的BALB/c 3T3细胞的聚腺苷酸加尾RNA制备λ cDNA文库。筛选了约50000个重组噬菌体噬菌斑，分离出357个与源自刺激细胞RNA的探针杂交但不与静止细胞RNA探针杂交的克隆。交叉杂交分析表明，四个RNA序列家族约占这些克隆的90%。其中一个克隆与肌动蛋白探针杂交；没有一个与所测试的13种癌基因探针中的任何一种杂交。对五个似乎先前未被表征的不同RNA进行了进一步分析。这些RNA在血清或纯化生长因子或肿瘤启动子刺激后迅速积累和降解，并且在放线菌酮存在下被血清超诱导。其中三个RNA可通过与cDNA杂交富集并在体外翻译，分别产生约43、40和35kd的蛋白质。
