Astigiano S, Barkai U, Abarzua P, Tan S C, Harper M I, Sherman M I
Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110.
Differentiation. 1991 Feb;46(1):61-7. doi: 10.1111/j.1432-0436.1991.tb00866.x.
cDNA libraries have been generated from Nulli-SCCl murine embryonal carcinoma (EC) cells untreated or treated for 24 h with all-trans retinoic acid (RA) or hexamethylenebisacetamide (HMBA), two chemically unrelated inducers of differentiation of EC cells. The libraries were screened for gene sequences whose expression was differentially regulated by one or both compounds. Of 20,000 cDNA clones screened, only 12 showed reproducible quantitative differences. One of the latter clones (pH 34) has been studied in detail. pH 34 cDNA hybridizes with a polyadenylated RNA (650 nucleotides) which is abundant in untreated Nulli-SCCl EC cells but whose steady-state levels decrease within 6 h of exposure to HMBA, reaching a minimum at 24 h. RA has a less-marked effect on this mRNA. Addition of inducers to the cells in fresh medium produces an early (15 min) transient increase in pH 34 mRNA levels. Nuclear run-on experiments are consistent with the view that the decrease in pH 34 mRNA is due to post-transcriptional events. Subclones of pH 34 in pGEM-4 were used to synthesize mRNA which could be translated in vitro into a 14-kDa protein. DNA sequencing of the pH 34 cDNA revealed that it is 607 bp in length with a single open reading frame capable of encoding a protein of 118 amino acids. Primer extension experiments revealed that the insert contains the full 5' sequence. Comparison with known sequences failed to reveal significant homology with previously sequenced proteins.
已从未经处理或用全反式维甲酸（RA）或六亚甲基双乙酰胺（HMBA）处理24小时的Nulli-SCC1小鼠胚胎癌细胞系构建了cDNA文库，RA和HMBA是两种化学结构不相关的胚胎癌细胞系分化诱导剂。筛选这些文库以寻找其表达受一种或两种化合物差异调节的基因序列。在筛选的20,000个cDNA克隆中，只有12个显示出可重复的定量差异。对其中一个克隆（pH 34）进行了详细研究。pH 34 cDNA与一种聚腺苷酸化RNA（650个核苷酸）杂交，该RNA在未经处理的Nulli-SCC1胚胎癌细胞中含量丰富，但其稳态水平在暴露于HMBA后6小时内下降，在24小时时达到最低。RA对该mRNA的影响较小。在新鲜培养基中向细胞添加诱导剂会导致pH 34 mRNA水平在早期（15分钟）出现短暂升高。细胞核连续转录实验与pH 34 mRNA的减少是由于转录后事件这一观点一致。pGEM-4中的pH 34亚克隆用于合成可在体外翻译成14 kDa蛋白质的mRNA。pH 34 cDNA的DNA测序显示其长度为607 bp，有一个单一的开放阅读框，能够编码一个118个氨基酸的蛋白质。引物延伸实验表明该插入片段包含完整的5'序列。与已知序列比较未发现与先前测序的蛋白质有显著同源性。
