Rhoads D D, Roufa D J
Division of Biology, Kansas State University, Manhattan 66506.
Mol Cell Biol. 1987 Oct;7(10):3767-74. doi: 10.1128/mcb.7.10.3767-3774.1987.
Cloned fragments of human ribosomal protein S14 DNA (RPS14) were transfected into cultured Chinese hamster (CHO) cells. Transient expression assays indicated that DNA with as little as 31 base pairs of upstream flanking sequence was transcribed into a polyadenylated, 650-base mRNA that is largely bound to the polyribosomes. In these respects the exogenous human S14 message appeared to function normally in CHO cells. Interestingly, transcription of human RPS14 did not require the TATA sequence located 26 base pairs upstream of exon 1. Stably transformed clones were selected from cultures of emetine-resistant CHO cells (Emr-2) after transfection with pSV2Neo-human RPS14 constructs. Human RPS14 complemented the mutationally based drug resistance of the Chinese hamster cells, demonstrating that the cloned human ribosomal protein gene is functional in rodent cells. Analysis of transformed cells with different amounts of integrated RPS14 indicated that human S14 mRNA levels are not tightly regulated by CHO cells. In contrast, the steady-state S14 level fluctuated only slightly, if at all, in transformed clones whose S14 message contents differed by more than 30-fold. These data support the conclusion that expression of human RPS14 is regulated, at least partially, posttranscriptionally.
将人核糖体蛋白S14 DNA（RPS14）的克隆片段转染到培养的中国仓鼠（CHO）细胞中。瞬时表达分析表明，带有仅31个碱基对上游侧翼序列的DNA被转录成一种多聚腺苷酸化的650个碱基的mRNA，该mRNA大部分与多核糖体结合。在这些方面，外源性人S14信使RNA在CHO细胞中似乎正常发挥功能。有趣的是，人RPS14的转录不需要位于外显子1上游26个碱基对处的TATA序列。在用pSV2Neo-人RPS14构建体转染后，从耐依米丁的CHO细胞（Emr-2）培养物中筛选出稳定转化的克隆。人RPS14补充了中国仓鼠细胞基于突变的耐药性，表明克隆的人核糖体蛋白基因在啮齿动物细胞中具有功能。对具有不同整合量RPS14的转化细胞的分析表明，人S14 mRNA水平不受CHO细胞的严格调控。相比之下，在S14信使RNA含量相差超过30倍的转化克隆中，稳态S14水平即使有波动也非常轻微。这些数据支持这样的结论，即人RPS14的表达至少部分是在转录后受到调控的。
