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
. 1983 Nov;80(22):6897–6901. doi: 10.1073/pnas.80.22.6897

Partial cDNA sequence to a hamster gene corrects defect in Escherichia coli pyrB mutant.

J N Davidson, L A Niswander
PMCID: PMC390093  PMID: 6139812

Abstract

The first three enzymes of pyrimidine biosynthesis (carbamoyl-phosphate synthetase, aspartate carbamoyl-transferase, and dihydro-orotase) are carried on a multifunctional protein in mammalian cells and are on separate proteins in bacteria. A plasmid containing a cDNA sequence corresponding to 80% of a hamster mRNA for this protein was transformed into Escherichia coli mutants lacking aspartate carbamoyltransferase (pyrB) or dihydro-orotase (pyrC). Only pyrB transformants were able to grow in the absence of uracil. Plasmid recovered from primary transformants was similar in size to the original plasmid and could yield prototrophs after secondary transformation of E. coli pyrB mutants. When cell extracts were prepared from pyrB transformants, high levels of aspartate carbamoyltransferase activity were found, and the enzyme had properties identical to the mammalian enzyme, including lack of allosteric regulation, precipitation by antiserum specific to the hamster multifunctional protein, and presence of a strong aggregation center. These results demonstrate that (i) a partial hamster protein can complement E. coli defective in pyrimidine biosynthesis, (ii) the order of the enzyme domains of the multifunctional protein is likely to be NH2-dihydro-orotase-carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-COOH, and (iii) the enzyme domains appear to be self-contained at the DNA and protein levels. The protocol described here may be a general means for studying the domains of multifunctional proteins and for isolating other mammalian genes for which bacterial mutants have been prepared. It also permits study of the structure and function of the same gene in both prokaryotic and eukaryotic cells and may provide new insight into the evolution of complex genes.

Full text

PDF
6897

Selected References

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

  1. Aitken D. M., Bhatti A. R., Kaplan J. G. Characterization of the aspartate carbamoyltransferase subunit obtained from a multienzyme aggregate in the pyrimidine pathway of yeast. Activity and physical properties. Biochim Biophys Acta. 1973 May 5;309(1):50–57. doi: 10.1016/0005-2744(73)90316-1. [DOI] [PubMed] [Google Scholar]
  2. Bachmann B. J., Low K. B. Linkage map of Escherichia coli K-12, edition 6. Microbiol Rev. 1980 Mar;44(1):1–56. doi: 10.1128/mr.44.1.1-56.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bethell M. R., Smith K. E., White J. S., Jones M. E. Carbamyl phosphate: an allosteric substrate for aspartate transcarbamylase of Escherichia coli. Proc Natl Acad Sci U S A. 1968 Aug;60(4):1442–1449. doi: 10.1073/pnas.60.4.1442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. Chang A. C., Nunberg J. H., Kaufman R. J., Erlich H. A., Schimke R. T., Cohen S. N. Phenotypic expression in E. coli of a DNA sequence coding for mouse dihydrofolate reductase. Nature. 1978 Oct 19;275(5681):617–624. doi: 10.1038/275617a0. [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. Dagert M., Ehrlich S. D. Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene. 1979 May;6(1):23–28. doi: 10.1016/0378-1119(79)90082-9. [DOI] [PubMed] [Google Scholar]
  8. Davidson J. N., Carnright D. V., Patterson D. Biochemical genetic analysis of pyrimidine biosynthesis in mammalian cells: III. Association of carbamyl phosphate synthetase, aspartate transcarbamylase, and dihydroorotase in mutants of cultured Chinese hamster cells. Somatic Cell Genet. 1979 Mar;5(2):175–191. doi: 10.1007/BF01539159. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Davidson J. N., Rumsby P. C., Tamaren J. Organization of a multifunctional protein in pyrimidine biosynthesis. Analyses of active, tryptic fragments. J Biol Chem. 1981 May 25;256(10):5220–5225. [PubMed] [Google Scholar]
  11. Denis-Duphil M., Kaplan J. G. Fine structure of the URA2 locus in Saccharomyces cerevisiae. II. Meiotic and mitotic mapping studies. Mol Gen Genet. 1976 Jun 15;145(3):259–271. doi: 10.1007/BF00325822. [DOI] [PubMed] [Google Scholar]
  12. Exinger F., Lacroute F. Genetic evidence for the creation of a reinitiation site by mutation inside the yeast ura 2 gene. Mol Gen Genet. 1979 May 23;173(1):109–113. doi: 10.1007/BF00267696. [DOI] [PubMed] [Google Scholar]
  13. Fausto-Sterling A. Studies on the female sterile mutant rudimentary of Drosophila melanogaster. II. An analysis of aspartate transcarbamylase and dihydroorotase activities in wild-type and rudimentary strains. Biochem Genet. 1977 Aug;15(7-8):803–815. doi: 10.1007/BF00484105. [DOI] [PubMed] [Google Scholar]
  14. GERHART J. C., PARDEE A. B. The enzymology of control by feedback inhibition. J Biol Chem. 1962 Mar;237:891–896. [PubMed] [Google Scholar]
  15. Jarry B., Falk D. Functional diversity within the rudimentary locus of Drosophila melanogaster. Mol Gen Genet. 1974;135(2):113–122. doi: 10.1007/BF00264779. [DOI] [PubMed] [Google Scholar]
  16. Kantrowitz E. R., Foote J., Reed H. W., Vensel L. A. Isolation and preliminary characterization of single amino acid substitution mutants of aspartate carbamoyltransferase. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3249–3253. doi: 10.1073/pnas.77.6.3249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Makoff A. J., Buxton F. P., Radford A. A possible model for the structure of the Neurospora carbamoyl phosphate synthase-aspartate carbamoyl transferase complex enzyme. Mol Gen Genet. 1978 May 31;161(3):297–304. doi: 10.1007/BF00331004. [DOI] [PubMed] [Google Scholar]
  18. Mally M. I., Grayson D. R., Evans D. R. Controlled proteolysis of the multifunctional protein that initiates pyrimidine biosynthesis in mammalian cells: evidence for discrete structural domains. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6647–6651. doi: 10.1073/pnas.78.11.6647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Meagher R. B., Tait R. C., Betlach M., Boyer H. W. Protein expression in E. coli minicells by recombinant plasmids. Cell. 1977 Mar;10(3):521–536. doi: 10.1016/0092-8674(77)90039-3. [DOI] [PubMed] [Google Scholar]
  20. Mori M., Tatibana M. Multi-enzyme complex of glutamine-dependent carbamoyl-phosphate synthetase with aspartate carbamoyltransferase and dihydroorotase from rat ascites-hepatoma cells. Purification, molecular properties and limited proteolysis. Eur J Biochem. 1978 May 16;86(2):381–388. doi: 10.1111/j.1432-1033.1978.tb12320.x. [DOI] [PubMed] [Google Scholar]
  21. Mori M., Tatibana M. Purification of homogeneous glutamine-dependent carbamyl phosphate synthetase from ascites hepatoma cells as a complex with aspartate transcarbamylase and dihydroorotase. J Biochem. 1975 Jul;78(1):239–242. [PubMed] [Google Scholar]
  22. Norgard M. V., Keem K., Monahan J. J. Factors affecting the transformation of Escherichia coli strain chi1776 by pBR322 plasmid DNA. Gene. 1978 Jul;3(4):279–292. doi: 10.1016/0378-1119(78)90038-0. [DOI] [PubMed] [Google Scholar]
  23. Okayama H., Berg P. High-efficiency cloning of full-length cDNA. Mol Cell Biol. 1982 Feb;2(2):161–170. doi: 10.1128/mcb.2.2.161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Padgett R. A., Wahl G. M., Coleman P. F., Stark G. R. N-(Phosphonacetyl)-L-aspartate-resistant hamster cells overaccumulate a single mRNA coding for the multifunctional protein that catalyzes the first steps of UMP synthesis. J Biol Chem. 1979 Feb 10;254(3):974–980. [PubMed] [Google Scholar]
  25. Patterson D., Carnright D. V. Biochemical genetic analysis of pyrimidine biosynthesis in mammalian cells: I. Isolation of a mutant defective in the early steps of de novo pyrimidine synthesis. Somatic Cell Genet. 1977 Sep;3(5):483–495. doi: 10.1007/BF01539120. [DOI] [PubMed] [Google Scholar]
  26. Rawls J. M., Fristrom J. W. A complex genetic locus that controls of the first three steps of pyrimidine biosynthesis in Drosophila. Nature. 1975 Jun 26;255(5511):738–740. doi: 10.1038/255738a0. [DOI] [PubMed] [Google Scholar]
  27. Read S. M., Northcote D. H. Minimization of variation in the response to different proteins of the Coomassie blue G dye-binding assay for protein. Anal Biochem. 1981 Sep 1;116(1):53–64. doi: 10.1016/0003-2697(81)90321-3. [DOI] [PubMed] [Google Scholar]
  28. Shoaf W. T., Jones M. E. Uridylic acid synthesis in Ehrlich ascites carcinoma. Properties, subcellular distribution, and nature of enzyme complexes of the six biosynthetic enzymes. Biochemistry. 1973 Oct 9;12(21):4039–4051. doi: 10.1021/bi00745a004. [DOI] [PubMed] [Google Scholar]
  29. Souciet J. L., Hubert J. C., Lacroute F. Cloning and restriction mapping of the yeast URA2 gene coding for the carbamyl phosphate synthetase aspartate-transcarbamylase complex. Mol Gen Genet. 1982;186(3):385–390. doi: 10.1007/BF00729458. [DOI] [PubMed] [Google Scholar]
  30. Spector T. Refinement of the coomassie blue method of protein quantitation. A simple and linear spectrophotometric assay for less than or equal to 0.5 to 50 microgram of protein. Anal Biochem. 1978 May;86(1):142–146. doi: 10.1016/0003-2697(78)90327-5. [DOI] [PubMed] [Google Scholar]
  31. Sutcliffe J. G. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):77–90. doi: 10.1101/sqb.1979.043.01.013. [DOI] [PubMed] [Google Scholar]
  32. Wahl G. M., Padgett R. A., Stark G. R. Gene amplification causes overproduction of the first three enzymes of UMP synthesis in N-(phosphonacetyl)-L-aspartate-resistant hamster cells. J Biol Chem. 1979 Sep 10;254(17):8679–8689. [PubMed] [Google Scholar]
  33. Weber K. New structural model of E. coli aspartate transcarbamylase and the amino-acid sequence of the regulatory polypeptide chain. Nature. 1968 Jun 22;218(5147):1116–1119. doi: 10.1038/2181116a0. [DOI] [PubMed] [Google Scholar]
  34. Wiley D. C., Lipscomb W. N. Crystallographic determination of symmetry of aspartate transcarbamylase. Nature. 1968 Jun 22;218(5147):1119–1121. doi: 10.1038/2181119a0. [DOI] [PubMed] [Google Scholar]
  35. Woods D., Crampton J., Clarke B., Williamson R. The construction of a recombinant cDNA library representative of the poly(A)+ mRNA population from normal human lymphocytes. Nucleic Acids Res. 1980 Nov 25;8(22):5157–5168. doi: 10.1093/nar/8.22.5157. [DOI] [PMC free article] [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