Department of Microbiology and Immunology, Center for Molecular and Tumor Virology, Louisiana State University Health Sciences Center, Shreveport, LA 71130-3932, United States.
J Virol Methods. 2012 Jun;182(1-2):18-26. doi: 10.1016/j.jviromet.2012.02.010. Epub 2012 Mar 8.
Bacterial artificial chromosome (BAC) recombineering using galK selection allows DNA cloned in Escherichia coli to be modified without introducing an unwanted selectable marker at the modification site. Genomes of some herpesviruses have a pair of inverted repeat sequences that makes it very difficult to introduce mutations into diploid (duplicate) genes using the galK selection method. To mutate diploid genes, we developed a galK-UTR BAC recombineering procedure that blocks one copy of the target diploid gene by insertion of a galK untranslated region (UTR), which enables the simple mutation of the other copy. The blocked copy can then be replaced with an UTR-specific primer pair. The IR2 gene of equine herpesvirus 1 (EHV-1) maps within both the internal (IR) and terminal repeat (TR) of the genomic short region and is expressed at low levels because its promoter is TATA-less. Both IR2 promoters in EHV-1 BAC were replaced with a mutant IR2 promoter containing three Sp1-binding motifs and a consensus TATA box by galK-UTR BAC recombineering. The expression level of the IR2 protein controlled by the modified promoter increased approximately 4-fold as compared to that of wild-type EHV-1. The galK-UTR method will provide a useful tool in studies of herpesviruses.
细菌人工染色体（BAC）重组使用 galK 选择允许在大肠杆菌中克隆的 DNA 进行修饰，而不会在修饰部位引入不必要的选择标记。一些疱疹病毒的基因组有一对反向重复序列，这使得使用 galK 选择方法很难在二倍体（重复）基因中引入突变。为了突变二倍体基因，我们开发了一种 galK-UTR BAC 重组方法，通过插入 galK 非翻译区（UTR）来阻断靶二倍体基因的一个拷贝，从而能够简单地突变另一个拷贝。然后可以用 UTR 特异性引物对替换被阻断的拷贝。马疱疹病毒 1（EHV-1）的 IR2 基因位于基因组短区的内部（IR）和末端重复（TR）内，由于其启动子无 TATA 框，因此表达水平较低。通过 galK-UTR BAC 重组，用含有三个 Sp1 结合基序和一个共识 TATA 框的突变 IR2 启动子替换了 EHV-1 BAC 中的两个 IR2 启动子。由修饰启动子控制的 IR2 蛋白的表达水平与野生型 EHV-1 相比增加了约 4 倍。galK-UTR 方法将为疱疹病毒的研究提供一种有用的工具。
