Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1985 Jul;5(7):1735–1742. doi: 10.1128/mcb.5.7.1735

Construction of a cDNA to the hamster CAD gene and its application toward defining the domain for aspartate transcarbamylase.

K Shigesada, G R Stark, J A Maley, L A Niswander, J N Davidson
PMCID: PMC367292  PMID: 2862577

Abstract

cDNA complementary to hamster mRNA encoding the CAD protein, a multifunctional protein which carries the first three enzymes of pyrimidine biosynthesis, was constructed. The longest of these recombinants (pCAD142) covers 82% of the 7.9-kilobase mRNA. Portions of the cDNA were excised and replaced by a lac promoter-operator-initiation codon segment. The resultant plasmids were transfected into an Escherichia coli mutant defective in aspartate transcarbamylase, the second enzyme of the pathway. Complementation of the bacterial defect was observed with as little as 2.2 kilobases of cDNA sequence, corresponding to the 3' region of the mRNA. DNA sequencing in this region of the hamster cDNA reveals stretches which are highly homologous to the E. coli gene for the catalytic subunit of aspartate transcarbamylase; other stretches show no homology. The highly conserved regions probably reflect areas of protein structure critical to catalysis, while the nonconserved regions may reflect differences between the quaternary structures of E. coli and mammalian aspartate transcarbamylases, one such difference being that the bacterial enzyme in its native form is allosterically regulated and the mammalian enzyme is not.

Full text

PDF
1735

Images in this article

1736Image
on p.1736
1738Image
on p.1738

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., Stark G. R. Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5350–5354. doi: 10.1073/pnas.74.12.5350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anderson S., Gait M. J., Mayol L., Young I. G. A short primer for sequencing DNA cloned in the single-stranded phage vector M13mp2. Nucleic Acids Res. 1980 Apr 25;8(8):1731–1743. doi: 10.1093/nar/8.8.1731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Aviv H., Leder P. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1408–1412. doi: 10.1073/pnas.69.6.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bailey J. M., Davidson N. Methylmercury as a reversible denaturing agent for agarose gel electrophoresis. Anal Biochem. 1976 Jan;70(1):75–85. doi: 10.1016/s0003-2697(76)80049-8. [DOI] [PubMed] [Google Scholar]
  5. Biggin M. D., Gibson T. J., Hong G. F. Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3963–3965. doi: 10.1073/pnas.80.13.3963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brothers V. M., Tsubota S. I., Germeraad S. E., Fristrom J. W. Rudimentary locus of Drosophila melanogaster: partial purification of a carbamylphosphate synthase--aspartate transcarbamylase--dihydroorotase complex. Biochem Genet. 1978 Apr;16(3-4):321–332. doi: 10.1007/BF00484088. [DOI] [PubMed] [Google Scholar]
  7. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  8. 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]
  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. Dretzen G., Bellard M., Sassone-Corsi P., Chambon P. A reliable method for the recovery of DNA fragments from agarose and acrylamide gels. Anal Biochem. 1981 Apr;112(2):295–298. doi: 10.1016/0003-2697(81)90296-7. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Grayson D. R., Evans D. R. The isolation and characterization of the aspartate transcarbamylase domain of the multifunctional protein, CAD. J Biol Chem. 1983 Apr 10;258(7):4123–4129. [PubMed] [Google Scholar]
  14. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Heidecker G., Messing J., Gronenborn B. A versatile primer for DNA sequencing in the M13mp2 cloning system. Gene. 1980 Jun;10(1):69–73. doi: 10.1016/0378-1119(80)90145-6. [DOI] [PubMed] [Google Scholar]
  16. Hoover T. A., Roof W. D., Foltermann K. F., O'Donovan G. A., Bencini D. A., Wild J. R. Nucleotide sequence of the structural gene (pyrB) that encodes the catalytic polypeptide of aspartate transcarbamoylase of Escherichia coli. Proc Natl Acad Sci U S A. 1983 May;80(9):2462–2466. doi: 10.1073/pnas.80.9.2462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jones M. E. Pyrimidine nucleotide biosynthesis in animals: genes, enzymes, and regulation of UMP biosynthesis. Annu Rev Biochem. 1980;49:253–279. doi: 10.1146/annurev.bi.49.070180.001345. [DOI] [PubMed] [Google Scholar]
  18. Kacian D. L., Myers J. C. Synthesis of extensive, possibly complete, DNA copies of poliovirus RNA in high yields and at high specific activities. Proc Natl Acad Sci U S A. 1976 Jul;73(7):2191–2195. doi: 10.1073/pnas.73.7.2191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Land H., Grez M., Hauser H., Lindenmaier W., Schütz G. 5'-Terminal sequences of eucaryotic mRNA can be cloned with high efficiency. Nucleic Acids Res. 1981 May 25;9(10):2251–2266. doi: 10.1093/nar/9.10.2251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lue P. F., Kaplan J. G. Heat-induced disaggregation of a multifunctional enzyme complex catalyzing the first steps in pyrimidine biosynthesis in bakers' yeast. Can J Biochem. 1970 Feb;48(2):155–159. doi: 10.1139/o70-024. [DOI] [PubMed] [Google Scholar]
  21. Makoff A. J., Radford A. Genetics and biochemistry of carbamoyl phosphate biosynthesis and its utilization in the pyrimidine biosynthetic pathway. Microbiol Rev. 1978 Jun;42(2):307–328. doi: 10.1128/mr.42.2.307-328.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. McDonell M. W., Simon M. N., Studier F. W. Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J Mol Biol. 1977 Feb 15;110(1):119–146. doi: 10.1016/s0022-2836(77)80102-2. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Myers J. C., Spiegelman S. Sodium pyrophosphate inhibition of RNA.DNA hybrid degradation by reverse transcriptase. Proc Natl Acad Sci U S A. 1978 Nov;75(11):5329–5333. doi: 10.1073/pnas.75.11.5329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Okayama H., Berg P. A cDNA cloning vector that permits expression of cDNA inserts in mammalian cells. Mol Cell Biol. 1983 Feb;3(2):280–289. doi: 10.1128/mcb.3.2.280. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. Padgett R. A., Wahl G. M., Stark G. R. Structure of the gene for CAD, the multifunctional protein that initiates UMP synthesis in Syrian hamster cells. Mol Cell Biol. 1982 Mar;2(3):293–301. doi: 10.1128/mcb.2.3.293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  31. Robey E. A., Schachman H. K. Site-specific mutagenesis of aspartate transcarbamoylase. Replacement of tyrosine 165 in the catalytic chain by serine reduces enzymatic activity. J Biol Chem. 1984 Sep 25;259(18):11180–11183. [PubMed] [Google Scholar]
  32. Rothenberg E., Baltimore D. Increased length of DNA made by virions of murine leukemia virus at limiting magnesium ion concentration. J Virol. 1977 Jan;21(1):168–178. doi: 10.1128/jvi.21.1.168-178.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rumsby P. C., Campbell P. C., Niswander L. A., Davidson J. N. Organization of a multifunctional protein in pyrimidine biosynthesis. A domain hypersensitive to proteolysis. Biochem J. 1984 Jan 15;217(2):435–440. doi: 10.1042/bj2170435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sanger F., Coulson A. R., Barrell B. G., Smith A. J., Roe B. A. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol. 1980 Oct 25;143(2):161–178. doi: 10.1016/0022-2836(80)90196-5. [DOI] [PubMed] [Google Scholar]
  35. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Segraves W. A., Louis C., Tsubota S., Schedl P., Rawls J. M., Jarry B. P. The rudimentary locus of Drosophila melanogaster. J Mol Biol. 1984 May 5;175(1):1–17. doi: 10.1016/0022-2836(84)90441-8. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Takanami M. RNA polymerase nascent product analysis. Methods Enzymol. 1980;65(1):497–499. doi: 10.1016/s0076-6879(80)65058-7. [DOI] [PubMed] [Google Scholar]
  39. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Williams L. G., Bernhardt S., Davis R. H. Copurification of pyrimidine-specific carbamyl phosphate synthetase and aspartate transcarbamylase of Neurospora crassa. Biochemistry. 1970 Oct 27;9(22):4329–4335. doi: 10.1021/bi00824a013. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES