Factors governing the expression of a bacterial gene in mammalian cells.
Factors governing the expression of a bacterial gene in mammalian cells.
复制标题
控制哺乳动物细胞中细菌基因表达的因素。
DOI：
10.1128/mcb.1.5.449-459.1981
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发表时间：
1981
影响因子：
5.3
通讯作者：
Berg,P
中科院分区：
文献类型：
--
作者：
Mulligan,RC;Berg,P
用猴病毒40（SV 40）-pBR 322衍生的脱氧核糖核酸（DNA）载体转染培养的猴肾细胞，该载体含有编码黄嘌呤-鸟嘌呤磷酸核糖基转移酶（XGPRT）的大肠杆菌基因（Ecogpt，orgpt），可合成细菌酶。本文描述了gpt表达过程中形成的信使核糖核酸（mRNA）的结构和gptDNA片段中核苷酸序列的一个意想不到的特征。对CV1细胞感染过程中产生的gpt特异性mRNA的分析表明，除了根据已知的猿猴病毒40 RNA剪接模式预期的mRNA外，还存在一种新的SV 40-gpthybrid mRNA。这种新的mRNA含有一个SV 40前导片段，该片段与从细菌DNA片段转录的RNA序列拼接。gptDNA片段5′端345个核苷酸的序列表明，唯一的开放翻译期始于距gptDNA末端约200个核苷酸的AUG。两个额外的AUG以及翻译终止子密码子在所有三个阶段之前的XGPRT起始密码子。缺失推定起始密码子上游的两个增加了转染细胞中XGPRT的产生水平;缺失含有所提出的XGPRT起始子AUG的序列消除了酶的产生。基于XGPRT编码序列在重组体中的位置和mRNA的结构，我们推断该细菌酶可以从距mRNA 5′端400至800个核苷酸的起始AUG翻译，并且之前有2至6个AUG三联体。
Cultured monkey kidney cells transfected with simian virus 40 (SV40)-pBR322–derived deoxyribonucleic acid (DNA) vectors containing theEscherichia coligene (Ecogpt, orgpt) coding for the enzyme xanthine-guanine phosphoribosyltransferase (XGPRT) synthesize the bacterial enzyme. This paper describes the structure of the messenger ribonucleic acids (mRNA's) formed during the expression ofgptand an unexpected feature of the nucleotide sequence in thegptDNA segment. Analyses of thegpt-specific mRNA's produced during infection of CV1 cells indicate that in addition to the mRNA's expected on the basis of known simian virus 40 RNA splicing patterns, there is a novel SV40-gpthybrid mRNA. The novel mRNA contains an SV40 leader segment spliced to RNA sequences transcribed from the bacterial DNA segment. The sequence of the 5′-proximal 345 nucleotides of thegptDNA segment indicates that the only open translation phase begins with an AUG about 200 nucleotides from the end of thegptDNA. Two additional AUGs as well as translation terminator codons in all three phases precede the XGPRT initiator codon. Deletion of the two that are upstream of the putative start codon increases the level of XGPRT production in transfected cells; deletion of sequences that contain the proposed XGPRT initiator AUG abolishes enzyme production. Based on the location of the XGPRT coding sequence in the recombinants and the structure of the mRNA's, we infer that the bacterial enzyme can be translated from an initiator AUG that is 400 to 800 nucleotides from the 5′ terminus of the mRNA and preceded by two to six AUG triplets.