Abstract
The yeast gene CPA1 coding for the small subunit of arginine-specific carbamyl phosphate synthetase has been cloned by complementation of a cpa 1 mutant with a plasmid library of total yeast chromosomal DNA. Two of the plasmids, pJL113/ST4 and pJL113/ST15, contain DNA inserts in opposite orientations with overlapping sequences of 2.6 kilobases. The nucleotide sequence of a 2.2-kilobase region of the DNA insert carrying the CPA1 gene has been determined. The CPA1 gene has been identified to be 1233 nucleotides long and to code for a polypeptide of 411 amino acids with a calculated molecular weight of 45,358. The amino acid sequence encoded in CPA1 is homologous to the recently determined sequence of the small subunit of Escherichia coli carbamyl phosphate synthetase (Piette, J., Nyunoya, H., Lusty, C.J., Cunin, R., Weyens, G., Crabeel, M., Charlier, D., Glandsdorff, N., and Pierard, A. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 4134-4138) over the entire length of the polypeptide chain. Comparison of the amino acid sequences of the small subunits of yeast and E. coli carbamyl phosphate synthetases to the sequences of Component II of anthranilate and p-aminobenzoate synthases suggests that these amidotransferases are evolutionarily related. The most highly conserved region of the yeast and E. coli enzymes includes a cysteine residue previously found to be at the active site of Pseudomonas putida anthranilate synthase Component II (Kawamura, M., Keim, P.S., Goto, Y., Zalkin, H., and Heinrikson, R.L. (1978) J. Biol. Chem. 253, 4659-4668). Based on the observed homologies in the primary sequences of the other amidotransferases examined, we propose a 13-amino acid long sequence to be part of the catalytic domain of this class of enzymes.
Highlights
From the Molecular Genetics Laboratory,The Public Health Research Institute of The City of New York, Inc., New York, New York 10016
NucleotideSequence of CPAl-The yeast gene CPAl coding for the small subunit of arginine-specific carbamyl phosphate synthetase (4)was isolated as described under "Materialsand Methods." The two recombinant plasmids pJL113/ST4 and pJL113/ST15 with the CPAl gene have 3.1- and 2.6-kb yeast DNA inserts, respectively, in the YEpl[3] shuttle vector (24) (Fig. 1).The presence of the CPAl gene in the recombinant clones was confirmed by in uiuo complementation of yeast strains with mutations in the structural genes of the small subunit of arginine-specific carbamyl phosphate synthetase and in the pyrimidine-specific carbamyl phosphate synthetase
In previous studies (12),we reported that thelarge subunits of yeast arginine-specificcarbamyl phosphate synthetase and E. coli carbamyl phosphate synthetase are structurarlelylated
Summary
Nitrogen to a large subunit (-130 kDa) that catalyzes carba- Yeast and Bacterial Strains-The yeast strain S. cerevisiae JL113 myl phosphate formation from NH3,HCOT, and ATP (3). Cloning of Yeast CPAI-Yeast CPAl was isolated by a previously described protocol (11) from a recombinant plasmid pool of total yeast nuclear DNA ligated to the BarnHI site of the hybrid vector YEpl[3] (24). JL113 was transformed with 10 pgof the recombinant plasmid pool, and 28 independent clones (Leu+Cps') were obtained by selection for growth on minimal medium (2% glucose, 0.67% yeast nitrogen base without amino acids (Difco)/l.2. Plasmid DNA digested with Hind[11] plus SphI produced two fragments of 10.2 (vector) and 3.6 kb in the case of pJL113/ST4 or 3.2 kb with pJL113/ST15 (cf Fig. 1).The smaller fragments containing the yeast DNA inserts were separated on preparative agarose gels and isolated. RNA was transferred to nitrocellulose filters and hybridized as described by Thomas (29) with a radiolabeled probe prepared by nick-translation (30) of a 1088-nucleotidelong BstEII-Ban fragment containing almost all the coding sequence of the CPAl gene
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