Srivastava A K, Schlessinger D
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110.
Gene. 1991 Jul 15;103(1):53-9. doi: 10.1016/0378-1119(91)90390-w.
To facilitate studies of gene expression and homologous recombination, plasmids have been developed which permit the insertion of neomycin resistance-encoding gene (NmR) into either the human DNA insert or the vector arm of a yeast artificial chromosome (YAC). To integrate into the YAC arm, the plasmid pRV1 contains a LYS2 (encoding alpha-aminoadipate reductase) gene for selection in the yeast host, and a NmR gene for subsequent selection after transfection of mammalian cells. These two sequences are bracketed by fragments of the URA3 gene (encoding orotidine-5'-phosphate decarboxylase) that can disrupt the URA3 gene in the YAC arm by homologous recombination in yeast. To integrate a selectable marker into the insert, the plasmid pRV2 contains a NmR gene and an intact copy of the URA3 gene, bracketed by segments of an L1 (LINEs) repetitive element. In this case, the vector has been designed for use with YACs that have already been fitted in the vector arm with a different marker (i.e., TK) that has disrupted the URA3 gene in the vector arm. Selection is for the restoration of URA3 gene activity attendant on recombination into an L1 element in the YAC insert. Use of the vectors is illustrated with a YAC clone containing ribosomal DNA.
为便于进行基因表达和同源重组研究，已开发出质粒，可将新霉素抗性编码基因（NmR）插入人类DNA插入片段或酵母人工染色体（YAC）的载体臂中。为了整合到YAC臂中，质粒pRV1含有用于在酵母宿主中进行选择的LYS2（编码α-氨基己二酸还原酶）基因，以及用于在转染哺乳动物细胞后进行后续选择的NmR基因。这两个序列由URA3基因（编码乳清苷-5'-磷酸脱羧酶）的片段包围，这些片段可通过酵母中的同源重组破坏YAC臂中的URA3基因。为了将选择标记整合到插入片段中，质粒pRV2含有一个NmR基因和URA3基因的完整拷贝，由L1（长散在核元件）重复元件的片段包围。在这种情况下，该载体设计用于与已经在载体臂中装配有不同标记（即TK）的YAC一起使用，该标记破坏了载体臂中的URA3基因。选择是为了恢复伴随重组到YAC插入片段中的L1元件而产生的URA3基因活性。使用包含核糖体DNA的YAC克隆说明了这些载体的用途。
