Cole C N, Santangelo G M
Mol Cell Biol. 1983 Feb;3(2):267-79. doi: 10.1128/mcb.3.2.267-279.1983.
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'.
使用Bal31核酸酶从其3'端切除单纯疱疹病毒1型胸苷激酶（tk）基因，并分离出一种质粒pTK206，该质粒缺乏通常在基因3'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pSV010，一种含有猿猴病毒40（SV40）DNA复制起点的质粒，从而允许在Cos-1细胞中复制并分析转录模式。将含有来自SV40和多瘤病毒的加工和聚腺苷酸化信号的DNA片段插入切除的tk基因pTK206的3'端。我们发现tk基因表达需要一个加工和聚腺苷酸化信号，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，在插入来自SV40早期区域中间的一个88碱基对片段后，tk基因表达恢复到低水平。尽管该片段含有六核苷酸5'-AAUAAA-3'，但在SV40中该片段附近不会发生加工和聚腺苷酸化。
