Nakamichi N, Rhoads D D, Roufa D J
J Biol Chem. 1983 Nov 10;258(21):13236-42.
We used an intersecting pool strategy to recognize chimeric plasmids containing Chinese hamster ribosomal protein cDNAs. The screening procedure involved hybridization-selection of messenger RNAs, cell-free translation of selected mRNAs, and electrophoresis of polypeptide products on one- and two-dimensional polyacrylamide gels. The protocol was designed to recognize ribosomal protein S14 cDNAs specifically. Of 500 chimeric plasmids screened, two possessed cDNAs complementary to S14 mRNA and 18 contained sequences complementary to other ribosomal protein messages. Previously we demonstrated that mutations affecting Chinese hamster ovary cell ribosomal protein S14 are responsible for genetic resistance to the translational inhibitor emetine (emt b). Because emetine-resistant mutant and wild type Chinese hamster ovary cells elaborate mRNAs that encode electrophoretically distinguishable forms of S14 protein, we were able to identify S14 cDNA clones unambiguously. The data described here indicate that: 1) clone pCS14-1 contains most, if not all, of the S14 coding sequence as a cDNA; 2) S14 mRNA is approximately 0.01% of a Chinese hamster cell's polyadenylated messenger RNA; and 3) genomic DNA-encoding ribosomal protein S14 is a low, perhaps single, copy sequence with a complex structure, including several, long intervening sequences.
我们采用交叉文库策略来识别含有中国仓鼠核糖体蛋白cDNA的嵌合质粒。筛选过程包括信使RNA的杂交选择、所选mRNA的无细胞翻译以及多肽产物在一维和二维聚丙烯酰胺凝胶上的电泳。该方案旨在特异性识别核糖体蛋白S14 cDNA。在筛选的500个嵌合质粒中，有两个含有与S14 mRNA互补的cDNA，18个含有与其他核糖体蛋白信使互补的序列。此前我们证明，影响中国仓鼠卵巢细胞核糖体蛋白S14的突变是对翻译抑制剂依米丁（emt b）产生遗传抗性的原因。由于依米丁抗性突变体和野生型中国仓鼠卵巢细胞产生的mRNA编码电泳上可区分的S14蛋白形式，我们能够明确鉴定出S14 cDNA克隆。此处描述的数据表明：1）克隆pCS14 - 1包含作为cDNA的大部分（如果不是全部）S14编码序列；2）S14 mRNA约占中国仓鼠细胞多聚腺苷酸化信使RNA的0.01%；3）编码核糖体蛋白S14的基因组DNA是一种低拷贝（可能是单拷贝）序列，结构复杂，包括几个长的间隔序列。
