Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1982 Apr;79(7):2240–2244. doi: 10.1073/pnas.79.7.2240

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

C J Lusty, J Lu
PMCID: PMC346167  PMID: 6285375

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.

Full text

PDF
2240

Images in this article

2242Image
on p.2242
2242Image
on p.2242

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alwine J. C., Kemp D. J., Parker B. A., Reiser J., Renart J., Stark G. R., Wahl G. M. Detection of specific RNAs or specific fragments of DNA by fractionation in gels and transfer to diazobenzyloxymethyl paper. Methods Enzymol. 1979;68:220–242. doi: 10.1016/0076-6879(79)68017-5. [DOI] [PubMed] [Google Scholar]
  2. Beggs J. D. Transformation of yeast by a replicating hybrid plasmid. Nature. 1978 Sep 14;275(5676):104–109. doi: 10.1038/275104a0. [DOI] [PubMed] [Google Scholar]
  3. Bernhardt S. A., Davis R. H. Carbamoyl phosphate compartmentation in Neurospora: histochemical localization of aspartate and ornithine transcarbamoylases. Proc Natl Acad Sci U S A. 1972 Jul;69(7):1868–1872. doi: 10.1073/pnas.69.7.1868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Broach J. R., Strathern J. N., Hicks J. B. Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene. Gene. 1979 Dec;8(1):121–133. doi: 10.1016/0378-1119(79)90012-x. [DOI] [PubMed] [Google Scholar]
  6. Coleman P. F., Suttle D. P., Stark G. R. Purification from hamster cells of the multifunctional protein that initiates de novo synthesis of pyrimidine nucleotides. J Biol Chem. 1977 Sep 25;252(18):6379–6385. [PubMed] [Google Scholar]
  7. Davidson J. N., Patterson D. Alteration in structure of multifunctional protein from Chinese hamster ovary cells defective in pyrimidine biosynthesis. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1731–1735. doi: 10.1073/pnas.76.4.1731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Davis R. H. Metabolite distribution in cells. Science. 1972 Nov 24;178(4063):835–840. doi: 10.1126/science.178.4063.835. [DOI] [PubMed] [Google Scholar]
  9. Davis R. H., Ristow J. L., Hanson B. A. Carbamyl phosphate synthetase A of Neurospora crassa. J Bacteriol. 1980 Jan;141(1):144–155. doi: 10.1128/jb.141.1.144-155.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Davis R. W., Thomas M., Cameron J., St John T. P., Scherer S., Padgett R. A. Rapid DNA isolations for enzymatic and hybridization analysis. Methods Enzymol. 1980;65(1):404–411. doi: 10.1016/s0076-6879(80)65051-4. [DOI] [PubMed] [Google Scholar]
  11. Hinnen A., Hicks J. B., Fink G. R. Transformation of yeast. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1929–1933. doi: 10.1073/pnas.75.4.1929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jeffreys A. J., Flavell R. A. A physical map of the DNA regions flanking the rabbit beta-globin gene. Cell. 1977 Oct;12(2):429–439. doi: 10.1016/0092-8674(77)90119-2. [DOI] [PubMed] [Google Scholar]
  13. Lacroute F., Piérard A., Grenson M., Wiame J. M. The biosynthesis of carbamoyl phosphate in Saccharomyces cerevisiae. J Gen Microbiol. 1965 Jul;40(1):127–142. doi: 10.1099/00221287-40-1-127. [DOI] [PubMed] [Google Scholar]
  14. Lusty C. J. Carbamoylphosphate synthetase I of rat-liver mitochondria. Purification, properties, and polypeptide molecular weight. Eur J Biochem. 1978 Apr 17;85(2):373–383. doi: 10.1111/j.1432-1033.1978.tb12249.x. [DOI] [PubMed] [Google Scholar]
  15. Mergeay M., Gigot D., Beckmann J., Glansdorff N., Piérard A. Physiology and genetics of carbamoylphosphate synthesis in Escherichia coli K12. Mol Gen Genet. 1974;133(4):299–316. doi: 10.1007/BF00332706. [DOI] [PubMed] [Google Scholar]
  16. Nagy M., Laporte J., Penverne B., Hervé G. Nuclear localization of aspartate transcabamoylase in Saccharomyces cerevisiae. J Cell Biol. 1982 Mar;92(3):790–794. doi: 10.1083/jcb.92.3.790. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Nasmyth K. A., Reed S. I. Isolation of genes by complementation in yeast: molecular cloning of a cell-cycle gene. Proc Natl Acad Sci U S A. 1980 Apr;77(4):2119–2123. doi: 10.1073/pnas.77.4.2119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Peacock A. C., Dingman C. W. Molecular weight estimation and separation of ribonucleic acid by electrophoresis in agarose-acrylamide composite gels. Biochemistry. 1968 Feb;7(2):668–674. doi: 10.1021/bi00842a023. [DOI] [PubMed] [Google Scholar]
  19. Petes T. D., Broach J. R., Wensink P. C., Hereford L. M., Fink G. R., Botstein D. Isolation and analysis of recombinant DNA molecules containing yeast DNA. Gene. 1978 Sep;4(1):37–49. doi: 10.1016/0378-1119(78)90013-6. [DOI] [PubMed] [Google Scholar]
  20. Piérard A., Schröter B. Structure-function relationships in the arginine pathway carbamoylphosphate synthase of Saccharomyces cerevisiae. J Bacteriol. 1978 Apr;134(1):167–176. doi: 10.1128/jb.134.1.167-176.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Potvin B., Gooder H. Carbamyl phosphate synthesis in Bacillus subtilis. Biochem Genet. 1975 Feb;13(1-2):125–143. doi: 10.1007/BF00486011. [DOI] [PubMed] [Google Scholar]
  22. Thuriaux P., Ramos F., Piérard A., Grenson M., Wiame J. M. Regulation of the carbamoylphosphate synthetase belonging to the arginine biosynthetic pathway of Saccharomyces cerevisiae. J Mol Biol. 1972 Jun 20;67(2):277–287. doi: 10.1016/0022-2836(72)90241-0. [DOI] [PubMed] [Google Scholar]
  23. Urrestarazu L. A., Vissers S., Wiame J. M. Change in location of ornithine carbamoyltransferase and carbamoylphosphate synthetase among yeasts in relation to the arginase/ornithine carbamoyltransferase regulatory complex and the energy status of the cells. Eur J Biochem. 1977 Oct 3;79(2):473–481. doi: 10.1111/j.1432-1033.1977.tb11830.x. [DOI] [PubMed] [Google Scholar]
  24. Weiss R. L., Davis R. H. Intracellular localization of enzymes of arginine metabolism in Neurospora. J Biol Chem. 1973 Aug 10;248(15):5403–5408. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES