The use of in vitro transcription to probe regulatory functions of viral protein domains.-猫眼课题宝

使用体外转录来探测病毒蛋白结构域的调节功能。

DOI：

10.1007/978-1-59745-277-9_2

发表时间：

2007

期刊：

Methods in molecular medicine

影响因子：

通讯作者：

中科院分区：

文献类型：

作者：

Loewenstein,PaulM;Song,Chao-Zhong;Green,Maurice

与其他DNA肿瘤病毒一样，腺病毒（Ads）也进化出特异性调控基因，通过控制其他病毒基因以及关键细胞基因的转录来促进病毒复制。在这方面，E1A转录单位含有多个蛋白质结构域，其可以转录激活或抑制参与细胞增殖和细胞分化调节的细胞基因。使用体外转录的研究提供了一个基础，从分子上理解病毒调控蛋白与细胞转录机制的相互作用，并继续告知我们的理解转录调控。本章提供了使用体外转录分析转录激活和转录抑制纯化，重组Ad E1A蛋白结构域和单个氨基酸取代突变体，以及使用蛋白质亲和层析，以确定参与病毒转录调控的宿主细胞转录因子的例子。详细描述了制备核转录提取物、制备生物活性蛋白质结构域、制备亲和力耗尽的转录提取物以及通过引物延伸和通过使用裸DNA模板的径流测定来分析转录的方法。

Adenoviruses (Ads), like other DNA tumor viruses, have evolved specific regulatory genes that facilitate virus replication by controlling the transcription of other viral genes as well as that of key cellular genes. In this regard, the E1A transcription unit contains multiple protein domains that can transcriptionally activate or repress cellular genes involved in the regulation of cell proliferation and cell differentiation. Studies using in vitro transcription have provided a basis for a molecular understanding of the interaction of viral regulatory proteins with the transcriptional machinery of the cell and continue to inform our understanding of transcription regulation. This chapter provides examples of the use of in vitro transcription to analyze transcriptional activation and transcriptional repression by purified, recombinant Ad E1A protein domains and single amino acid substitution mutants as well as the use of protein-affinity chromatography to identify host cell transcription factors involved in viral transcriptional regulation. A detailed description is provided of the methodology to prepare nuclear transcription extract, to prepare biologically active protein domains, to prepare affinity depleted transcription extracts, and to analyze transcription by primer extension and by run-off assay using naked DNA templates.