Analysis in Cos-1 cells of processing and polyadenylation signals by using derivatives of the herpes simplex virus type 1 thymidine kinase gene
Analysis in Cos-1 cells of processing and polyadenylation signals by using derivatives of the herpes simplex virus type 1 thymidine kinase gene
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DOI：
10.1128/mcb.3.2.267-279.1983
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发表时间：
1983-02
影响因子：
5.3
通讯作者：
C. Cole;G. Santangelo
中科院分区：
文献类型：
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作者：
C. Cole;G. Santangelo
使用 Bal31 核酸酶从 3' 末端切除单纯疱疹病毒 1 型胸苷激酶 (tk) 基因，并分离出质粒 pTK206，该质粒缺乏通常在该基因 3' 末端发现的加工和聚腺苷酸化信号。将野生型基因 pTK2 和 pTK206 分别转移到 pSV010，这是一种含有猿猴病毒 40 (SV40) DNA 复制起点的质粒，允许在 Cos-1 细胞中复制和分析转录模式。将含有来自 SV40 和多瘤病毒的加工和聚腺苷酸化信号的 DNA 片段插入到切除的 tk 基因 pTK206 的 3' 端。我们发现tk基因表达需要加工和多腺苷酸化信号，来自SV40和多瘤病毒的信号可以替代单纯疱疹病毒tk信号，并且在被这些基因构建体转染的Cos-1细胞中tk mRNA水平存在相当大的差异。此外，在插入SV40早期区域中部的88个碱基对片段后，tk基因表达恢复到较低水平。 SV40 中该片段附近不会发生加工和聚腺苷酸化，即使它包含六核苷酸 5'-AAUAAA-3'。
Bal31 nuclease was used to resect the herpes simplex virus type 1 thymidine kinase (tk) gene from its 3' end, and a plasmid, pTK206, was isolated that lacked the processing and polyadenylation signals normally found at the 3' end of the gene. The wild-type gene, pTK2, and pTK206 were each transferred to pSV010, a plasmid containing the simian virus 40 (SV40) origin of DNA replication, allowing replication and analysis of the patterns of transcription in Cos-1 cells. Fragments of DNA containing processing and polyadenylation signals from SV40 and polyoma virus were inserted into the 3' end of the resected tk gene, pTK206. We found that tk gene expression requires a processing and polyadenylation signal, that signals from SV40 and polyoma virus could substitute for the herpes simplex virus tk signal, and that considerable differences in the levels of tk mRNA were present in Cos-1 cells transfected by these gene constructs. In addition, tk gene expression was restored to a low level after the insertion of an 88-base-pair fragment from the middle of the SV40 early region. Processing and polyadenylation do not occur in the vicinity of this fragment in SV40, even though it contains the hexanucleotide 5'-AAUAAA-3'.