Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1983 Apr 11;11(7):1997–2012. doi: 10.1093/nar/11.7.1997

Structure of the dihydrofolate reductase gene in Chinese hamster ovary cells.

A M Carothers, G Urlaub, N Ellis, L A Chasin
PMCID: PMC325857  PMID: 6300788

Abstract

Overlapping recombinant lambda 1059 phages carrying regions of the dhfr locus from the amplified Chinese hamster ovary (CHO) cell clone MK42 have been isolated. In addition, dhfr cDNAs from this cell line have been cloned into plasmid pBR322. Restriction analysis of these recombinant molecules has led to a map of the Chinese hamster dhfr gene. This gene has a minimum size of 26 kb and contains six exons as defined by hybridization to a combination of mouse and CHO cDNA probes. The latter probes reveal 3' exonic sequences that are not present in mouse cDNA. The CHO dhfr gene thus extends about 700 bp further 3' than in the mouse, consistent with the larger size of the hamster mRNA. At least five intervening sequences are present, of approximate sizes: 0.3, 2.5, 8.6, 2.6 and 9.4 kb. Four sequences from highly repeated families are situated in introns within the dhfr gene. The overall structure of this gene is strikingly similar to that of the mouse. Evolutionary conservation of interrupted gene structure among mammals thus extends to genes that code for household enzymes as well as specialized or structural proteins.

Full text

PDF
1997

Images in this article

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. 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]
  3. Baralle F. E., Shoulders C. C., Goodbourn S., Jeffreys A., Proudfoot N. J. The 5' flanking region of human epsilon-globin gene. Nucleic Acids Res. 1980 Oct 10;8(19):4393–4404. doi: 10.1093/nar/8.19.4393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bennetzen J. L., Hall B. D. The primary structure of the Saccharomyces cerevisiae gene for alcohol dehydrogenase. J Biol Chem. 1982 Mar 25;257(6):3018–3025. [PubMed] [Google Scholar]
  5. 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]
  6. Bolivar F. Construction and characterization of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique Eco RI sites for selection of Eco RI generated recombinant DNA molecules. Gene. 1978 Oct;4(2):121–136. doi: 10.1016/0378-1119(78)90025-2. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Breathnach R., Chambon P. Organization and expression of eucaryotic split genes coding for proteins. Annu Rev Biochem. 1981;50:349–383. doi: 10.1146/annurev.bi.50.070181.002025. [DOI] [PubMed] [Google Scholar]
  9. Buell G. N., Wickens M. P., Payvar F., Schimke R. T. Synthesis of full length cDNAs from four partially purified oviduct mRNAs. J Biol Chem. 1978 Apr 10;253(7):2471–2482. [PubMed] [Google Scholar]
  10. 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]
  11. Crouse G. F., Simonsen C. C., McEwan R. N., Schimke R. T. Structure of amplified normal and variant dihydrofolate reductase genes in mouse sarcoma S180 cells. J Biol Chem. 1982 Jul 10;257(13):7887–7897. [PubMed] [Google Scholar]
  12. Dolnick B. J., Berenson R. J., Bertino J. R., Kaufman R. J., Nunberg J. H., Schimke R. T. Correlation of dihydrofolate reductase elevation with gene amplification in a homogeneously staining chromosomal region in L5178Y cells. J Cell Biol. 1979 Nov;83(2 Pt 1):394–402. doi: 10.1083/jcb.83.2.394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dugaiczyk A., Boyer H. W., Goodman H. M. Ligation of EcoRI endonuclease-generated DNA fragments into linear and circular structures. J Mol Biol. 1975 Jul 25;96(1):171–184. doi: 10.1016/0022-2836(75)90189-8. [DOI] [PubMed] [Google Scholar]
  14. Eckhardt T. A rapid method for the identification of plasmid desoxyribonucleic acid in bacteria. Plasmid. 1978 Sep;1(4):584–588. doi: 10.1016/0147-619x(78)90016-1. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Gasser C. S., Simonsen C. C., Schilling J. W., Schimke R. T. Expression of abbreviated mouse dihydrofolate reductase genes in cultured hamster cells. Proc Natl Acad Sci U S A. 1982 Nov;79(21):6522–6526. doi: 10.1073/pnas.79.21.6522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Graf L. H., Jr, Chasin L. A. Direct demonstration of genetic alterations at the dihydrofolate reductase locus after gamma irradiation. Mol Cell Biol. 1982 Jan;2(1):93–96. doi: 10.1128/mcb.2.1.93. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Grosveld F. G., Dahl H. H., de Boer E., Flavell R. A. Isolation of beta-globin-related genes from a human cosmid library. Gene. 1981 Apr;13(3):227–237. doi: 10.1016/0378-1119(81)90028-7. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Hanahan D., Meselson M. Plasmid screening at high colony density. Gene. 1980 Jun;10(1):63–67. doi: 10.1016/0378-1119(80)90144-4. [DOI] [PubMed] [Google Scholar]
  21. Jelinek W. R., Toomey T. P., Leinwand L., Duncan C. H., Biro P. A., Choudary P. V., Weissman S. M., Rubin C. M., Houck C. M., Deininger P. L. Ubiquitous, interspersed repeated sequences in mammalian genomes. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1398–1402. doi: 10.1073/pnas.77.3.1398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kapuściński J., Skoczylas B. Simple and rapid fluorimetric method for DNA microassay. Anal Biochem. 1977 Nov;83(1):252–257. doi: 10.1016/0003-2697(77)90533-4. [DOI] [PubMed] [Google Scholar]
  23. Karn J., Brenner S., Barnett L., Cesareni G. Novel bacteriophage lambda cloning vector. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5172–5176. doi: 10.1073/pnas.77.9.5172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kaufman R. J., Sharp P. A. Construction of a modular dihydrofolate reductase cDNA gene: analysis of signals utilized for efficient expression. Mol Cell Biol. 1982 Nov;2(11):1304–1319. doi: 10.1128/mcb.2.11.1304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Konecki D. S., Brennand J., Fuscoe J. C., Caskey C. T., Chinault A. C. Hypoxanthine-guanine phosphoribosyltransferase genes of mouse and Chinese hamster: construction and sequence analysis of cDNA recombinants. Nucleic Acids Res. 1982 Nov 11;10(21):6763–6775. doi: 10.1093/nar/10.21.6763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lewis J. A., Davide J. P., Melera P. W. Selective amplification of polymorphic dihydrofolate reductase gene loci in Chinese hamster lung cells. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6961–6965. doi: 10.1073/pnas.79.22.6961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lewis J. A., Kurtz D. T., Melera P. W. Molecular cloning of Chinese hamster dihydrofolate reductase-specific cDNA and the identification of multiple dihydrofolate reductase mRNAs in antifolate-resistant Chinese hamster lung fibroblasts. Nucleic Acids Res. 1981 Mar 25;9(6):1311–1322. doi: 10.1093/nar/9.6.1311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Liu C. P., Tucker P. W., Mushinski J. F., Blattner F. R. Mapping of heavy chain genes for mouse immunoglobulins M and D. Science. 1980 Sep 19;209(4463):1348–1353. doi: 10.1126/science.6774414. [DOI] [PubMed] [Google Scholar]
  29. Maniatis T., Hardison R. C., Lacy E., Lauer J., O'Connell C., Quon D., Sim G. K., Efstratiadis A. The isolation of structural genes from libraries of eucaryotic DNA. Cell. 1978 Oct;15(2):687–701. doi: 10.1016/0092-8674(78)90036-3. [DOI] [PubMed] [Google Scholar]
  30. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  31. McMaster G. K., Carmichael G. G. Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4835–4838. doi: 10.1073/pnas.74.11.4835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Melera P. W., Hession C. A., Davide J. P., Scotto K. W., Biedler J. L., Meyérs M. B., Shanske S. Antifolate-resistant Chinese Hamster Cells. mRNA directed overproduction of multiple dihydrofolate reductases from a series of independently derived sublines containing amplified dihydrofolate reductase genes. J Biol Chem. 1982 Nov 10;257(21):12939–12949. [PubMed] [Google Scholar]
  33. Nunberg J. H., Kaufman R. J., Chang A. C., Cohen S. N., Schimke R. T. Structure and genomic organization of the mouse dihydrofolate reductase gene. Cell. 1980 Feb;19(2):355–364. doi: 10.1016/0092-8674(80)90510-3. [DOI] [PubMed] [Google Scholar]
  34. Nunberg J. H., Kaufman R. J., Schimke R. T., Urlaub G., Chasin L. A. Amplified dihydrofolate reductase genes are localized to a homogeneously staining region of a single chromosome in a methotrexate-resistant Chinese hamster ovary cell line. Proc Natl Acad Sci U S A. 1978 Nov;75(11):5553–5556. doi: 10.1073/pnas.75.11.5553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Pellicer A., Wigler M., Axel R., Silverstein S. The transfer and stable integration of the HSV thymidine kinase gene into mouse cells. Cell. 1978 May;14(1):133–141. doi: 10.1016/0092-8674(78)90308-2. [DOI] [PubMed] [Google Scholar]
  36. Prem veer Reddy G., Pardee A. B. Multienzyme complex for metabolic channeling in mammalian DNA replication. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3312–3316. doi: 10.1073/pnas.77.6.3312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. Sanger F., Air G. M., Barrell B. G., Brown N. L., Coulson A. R., Fiddes C. A., Hutchison C. A., Slocombe P. M., Smith M. Nucleotide sequence of bacteriophage phi X174 DNA. Nature. 1977 Feb 24;265(5596):687–695. doi: 10.1038/265687a0. [DOI] [PubMed] [Google Scholar]
  39. Schoner R. G., Littlefield J. W. The organization of the dihydrofolate reductase gene of baby hamster kidney fibroblasts. Nucleic Acids Res. 1981 Dec 11;9(23):6601–6614. doi: 10.1093/nar/9.23.6601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Setzer D. R., McGrogan M., Nunberg J. H., Schimke R. T. Size heterogeneity in the 3' end of dihydrofolate reductase messenger RNAs in mouse cells. Cell. 1980 Nov;22(2 Pt 2):361–370. doi: 10.1016/0092-8674(80)90346-3. [DOI] [PubMed] [Google Scholar]
  41. Setzer D. R., McGrogan M., Schimke R. T. Nucleotide sequence surrounding multiple polyadenylation sites in the mouse dihydrofolate reductase gene. J Biol Chem. 1982 May 10;257(9):5143–5147. [PubMed] [Google Scholar]
  42. 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]
  43. 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]
  44. Urlaub G., Chasin L. A. Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4216–4220. doi: 10.1073/pnas.77.7.4216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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]
  46. Wickens M. P., Buell G. N., Schimke R. T. Synthesis of double-stranded DNA complementary to lysozyme, ovomucoid, and ovalbumin mRNAs. Optimization for full length second strand synthesis by Escherichia coli DNA polymerase I. J Biol Chem. 1978 Apr 10;253(7):2483–2495. [PubMed] [Google Scholar]
  47. veer Reddy G. P., Pardee A. B. Coupled ribonucleoside diphosphate reduction, channeling, and incorporation into DNA of mammalian cells. J Biol Chem. 1982 Nov 10;257(21):12526–12531. [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES