Development of a Yeast Stop Codon Assay Readily and Generally Applicable to Human Genes
作者: Akihiko KataokaMitsuhiro TadaMasahiro YanoKeiji FuruuchiSantoso CornainJun-ichi HamadaGaku SuzukiHidehisa YamadaSatoru TodoTetsuya Moriuchi
作者单位: 1First Department of Surgery, Hokkaido University School of Medicine, Sapporo
2Third Department of Internal Medicine, Hokkaido University School of Medicine, Sapporo
3Divisions of Cancer-Related Genes and Gene Therapy Development, Hokkaido University, Sapporo, Japan
4Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan
刊名: The American Journal of Pathology, 2001, Vol.159 (4), pp.1239-1245
来源数据库: Elsevier Journal
DOI: 10.1016/S0002-9440(10)62510-2
原始语种摘要: We established a yeast-based method to screen chain-terminating mutations that is readily applicable to any gene of interest. Based on the finding that 18- to 24-base-long homologous sequences are sufficient for gap repair in vivo in yeast, we used a strategy to amplify a test-gene fragment with addition of 24-bp sequences homologous to both cut-ends of a yeast expression vector, pMT18. After co-transformation with the amplified fragment and the linearized pMT18, each yeast ( Saccharomyces cerevisiae ) cell automatically forms a single-copy circular plasmid (because of CEN/ARS), which expresses a test-gene::ADE2 chimera protein. When the reading frame of the test-gene contains a nonsense or frameshift mutation, truncation of the chimera protein results in lack of ADE2 activity, leading to...
全文获取路径: Elsevier  (合作)
影响因子:4.522 (2012)