Mellon P, Parker V, Gluzman Y, Maniatis T
Cell. 1981 Dec;27(2 Pt 1):279-88. doi: 10.1016/0092-8674(81)90411-6.
We have developed a rapid and simple method for studying the transcription of cloned eucaryotic genes, which involves transfecting SV40-transformed monkey cell lines (COS cells) with derivatives of the plasmid pBR322 that contain the SV40 viral replication origin but lack regions necessary for viral transcription (SV-ORI vectors). Because COS cells produce SV40 T antigen and are permissive for SV40 viral replication, transfected SV-ORI plasmids replicate to a high copy number. SV-ORI plasmids carrying a human alpha-globin gene are also replicated in COS cells. Moreover, the alpha-globin gene is faithfully transcribed to produce high levels of RNA, which is accurately processed to produce authentic alpha-globin mRNA. We have used this transcription system to demonstrate that a sequence located between 55 and 87 base pairs upstream from the mRNA capping site is required for efficient transcription of the alpha-globin gene in COS cells.
我们已经开发出一种快速简便的方法来研究克隆的真核基因转录，该方法包括用质粒pBR322的衍生物转染SV40转化的猴细胞系（COS细胞），这些衍生物含有SV40病毒复制起点，但缺乏病毒转录所需的区域（SV-ORI载体）。由于COS细胞产生SV40 T抗原且允许SV40病毒复制，转染的SV-ORI质粒可复制至高拷贝数。携带人α-珠蛋白基因的SV-ORI质粒也能在COS细胞中复制。此外，α-珠蛋白基因能被如实地转录以产生高水平的RNA，这些RNA能被准确加工以产生真实的α-珠蛋白mRNA。我们利用这个转录系统证明，在COS细胞中，α-珠蛋白基因高效转录需要位于mRNA帽位点上游55至87个碱基对之间的序列。
