Cloning of a yeast gene coding for arginine-specific carbamoyl-phosphate synthetase.

Abstract

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.