Lusty C J, Lu J
Proc Natl Acad Sci U S A. 1982 Apr;79(7):2240-4. doi: 10.1073/pnas.79.7.2240.
Several recombinant plasmids containing cpaII, the gene that encodes the large subunit of yeast arginine-specific carbamoyl-phosphate synthetase [carbamoyl-phosphate synthetase (glutamine-hydrolyzing), carbon-dioxide: L-glutamine amido-ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.3.5], have been isolated. The plasmids were selected by transformation of a yeast strain with a mutation in the structural gene of the large subunit of carbamoyl-phosphate synthetase. By using a recombinant pool with inserts of yeast nuclear DNA of 5-20 kilobase pairs, we obtained 13 transformants. Of five transformants studied, three have been found to have stable plasmid inserts. These plasmids could be amplified in Escherichia coli and transferred back into the yeast carbamoyl-phosphate synthetase-deficient strains with concomitant complementation of the nuclear mutation. Plasmids pJL2/T1 and pJL2/T5 contain identical nuclear DNA inserts of 5.9 kilobase pairs. Although the insert of plasmid pJL2/T3 is also 5.9 kilobase pairs long, the sequence overlap with pJL2/T1 and pJL2/T5 is only 4.5 kilobase pairs long. The T3 insert has an orientation in the vector opposite to that of the T1 and T5 inserts. The recombinant plasmids with the yeast cpaII gene fail to cross-hybridize with a cloned fragment of E. coli DNA containing the carA and carB genes for the bacterial carbamoyl-phosphate synthetase.
已分离出几种含有cpaII的重组质粒，cpaII是编码酵母精氨酸特异性氨甲酰磷酸合成酶大亚基[氨甲酰磷酸合成酶（谷氨酰胺水解），二氧化碳：L-谷氨酰胺酰胺连接酶（ADP形成，氨基甲酸酯磷酸化），EC 6.3.3.5]的基因。这些质粒是通过用氨甲酰磷酸合成酶大亚基结构基因突变的酵母菌株进行转化而筛选出来的。利用插入有5 - 20千碱基对酵母核DNA的重组文库，我们获得了13个转化体。在所研究的5个转化体中，发现有3个具有稳定的质粒插入片段。这些质粒可在大肠杆菌中扩增，并转回酵母氨甲酰磷酸合成酶缺陷型菌株，同时使核突变得到互补。质粒pJL2/T1和pJL2/T5含有5.9千碱基对的相同核DNA插入片段。虽然质粒pJL2/T3的插入片段也是5.9千碱基对长，但与pJL2/T1和pJL2/T5的序列重叠部分只有4.5千碱基对长。T3插入片段在载体中的方向与T1和T5插入片段相反。含有酵母cpaII基因的重组质粒不能与含有细菌氨甲酰磷酸合成酶carA和carB基因的大肠杆菌DNA克隆片段杂交。
