Ruiz J C, Wahl G M
Mol Cell Biol. 1986 Sep;6(9):3050-8. doi: 10.1128/mcb.6.9.3050-3058.1986.
Eucaryotic expression vectors containing the Escherichia coli pyrB gene (pyrB encodes the catalytic subunit of aspartate transcarbamylase [ATCase]) and the Tn5 phosphotransferase gene (G418 resistance module) were transfected into a mutant Chinese hamster ovary cell line possessing a CAD multifunctional protein lacking ATCase activity. G418-resistant transformants were isolated and analyzed for ATCase activity, the ability to complement the CAD ATCase defect, and the ability to resist high concentrations of the ATCase inhibitor N-(phosphonacetyl)-L-aspartate (PALA) by amplifying the donated pyrB gene sequences. We report that bacterial ATCase is expressed in these lines, that it complements the CAD ATCase defect in trans, and that its amplification engenders PALA resistance. In addition, we derived rapid and sensitive assay conditions which enable the determination of bacterial ATCase enzyme activity in the presence of mammalian ATCase.
将含有大肠杆菌pyrB基因（pyrB编码天冬氨酸转氨甲酰酶[ATCase]的催化亚基）和Tn5磷酸转移酶基因（G418抗性模块）的真核表达载体转染到一种突变的中国仓鼠卵巢细胞系中，该细胞系的CAD多功能蛋白缺乏ATCase活性。分离出G418抗性转化体，并通过扩增捐赠的pyrB基因序列，分析其ATCase活性、弥补CAD ATCase缺陷的能力以及抵抗高浓度ATCase抑制剂N-（膦酰乙酰基）-L-天冬氨酸（PALA）的能力。我们报告称，细菌ATCase在这些细胞系中表达，它能反式弥补CAD ATCase缺陷，其扩增会产生PALA抗性。此外，我们得出了快速且灵敏的检测条件，能够在存在哺乳动物ATCase的情况下测定细菌ATCase酶活性。
