Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1982 Mar;2(3):293–301. doi: 10.1128/mcb.2.3.293

Structure of the gene for CAD, the multifunctional protein that initiates UMP synthesis in Syrian hamster cells.

R A Padgett, G M Wahl, G R Stark
PMCID: PMC369788  PMID: 6125880

Abstract

Two adjacent fragments of genomic DNA spanning the gene for CAD, which encodes the first three enzymes of UMP biosynthesis, were cloned from a mutant Syrian hamster cell line containing multiple copies of this gene. The mutant was selected for resistance to N-(phosphonacetyl)-L-aspartate, a potent and specific inhibitor of aspartate transcarbamylase, the second enzyme in the pathway. The sizes and positions of about 37 intervening sequences within the 25-kilobase CAD gene were mapped by electron microscopy, and the locations of the 5' and 3' ends of the 7.9-kilobase CAD mRNA were established by electron microscopy and by other hybridization methods. The coding sequences are small (100 to 400 bases), as are most of the intervening sequences (50 to 300 bases). However, there are also several large intervening sequences of up to 5,000 bases each. Two small cytoplasmic polyadenylated RNAs are transcribed from a region just beyond the 5' end of the CAD gene, and their abundance reflects the degree of gene amplification.

Full text

PDF
293

Images in this article

296Image
on p.296
298Image
on p.298
298Image
on p.298
299Image
on p.299
300Image
on p.300
301Image
on p.301

Selected References

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

  1. Benton W. D., Davis R. W. Screening lambdagt recombinant clones by hybridization to single plaques in situ. Science. 1977 Apr 8;196(4286):180–182. doi: 10.1126/science.322279. [DOI] [PubMed] [Google Scholar]
  2. Blattner F. R., Williams B. G., Blechl A. E., Denniston-Thompson K., Faber H. E., Furlong L., Grunwald D. J., Kiefer D. O., Moore D. D., Schumm J. W. Charon phages: safer derivatives of bacteriophage lambda for DNA cloning. Science. 1977 Apr 8;196(4286):161–169. doi: 10.1126/science.847462. [DOI] [PubMed] [Google Scholar]
  3. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Enquist L., Sternberg N. In vitro packaging of lambda Dam vectors and their use in cloning DNA fragments. Methods Enzymol. 1979;68:281–298. doi: 10.1016/0076-6879(79)68020-5. [DOI] [PubMed] [Google Scholar]
  7. Gilbert W. Why genes in pieces? Nature. 1978 Feb 9;271(5645):501–501. doi: 10.1038/271501a0. [DOI] [PubMed] [Google Scholar]
  8. Jarry B. Isolation of a multifunctional complex containing the first three enzymes of pyrimidine biosynthesis in drosophila melanogaster. FEBS Lett. 1976 Nov;70(1):71–75. doi: 10.1016/0014-5793(76)80728-4. [DOI] [PubMed] [Google Scholar]
  9. Kempe T. D., Swyryd E. A., Bruist M., Stark G. R. Stable mutants of mammalian cells that overproduce the first three enzymes of pyrimidine nucleotide biosynthesis. Cell. 1976 Dec;9(4 Pt 1):541–550. doi: 10.1016/0092-8674(76)90036-2. [DOI] [PubMed] [Google Scholar]
  10. McEntee K. Protein X is the product of the recA gene of Escherichia coli. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5275–5279. doi: 10.1073/pnas.74.12.5275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Padgett R. A., Wahl G. M., Stark G. R. Properties of dispersed, highly repeated DNA within and near the hamster CAD gene. Mol Cell Biol. 1982 Mar;2(3):302–307. doi: 10.1128/mcb.2.3.302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  16. Thomas M., Davis R. W. Studies on the cleavage of bacteriophage lambda DNA with EcoRI Restriction endonuclease. J Mol Biol. 1975 Jan 25;91(3):315–328. doi: 10.1016/0022-2836(75)90383-6. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Wahl G. M., Stern M., Stark G. R. Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3683–3687. doi: 10.1073/pnas.76.8.3683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Wahl G. M., Vitto L., Padgett R. A., Stark G. R. Single-copy and amplified CAD genes in Syrian hamster chromosomes localized by a highly sensitive method for in situ hybridization. Mol Cell Biol. 1982 Mar;2(3):308–319. doi: 10.1128/mcb.2.3.308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Wahli W., Dawid I. B., Wyler T., Weber R., Ryffel G. U. Comparative analysis of the structural organization of two closely related vitellogenin genes in X. laevis. Cell. 1980 May;20(1):107–117. doi: 10.1016/0092-8674(80)90239-1. [DOI] [PubMed] [Google Scholar]
  21. Williams L. G., Bernhardt S. A., Davis R. H. Evidence for two discrete carbamyl phosphate pools in Neurospora. J Biol Chem. 1971 Feb 25;246(4):973–978. [PubMed] [Google Scholar]
  22. de Saint Vincent B. R., Delbrück S., Eckhart W., Meinkoth J., Vitto L., Wahl G. The cloning and reintroduction into animal cells of a functional CAD gene, a dominant amplifiable genetic marker. Cell. 1981 Dec;27(2 Pt 1):267–277. doi: 10.1016/0092-8674(81)90410-4. [DOI] [PubMed] [Google Scholar]

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

RESOURCES