Abstract
We have constructed a number of abbreviated dihydrofolate reductase (DHFR) genes by using cloned mouse genomic and cDNA sequences. These genes contain 1.0 kilobase of 5' flanking genomic sequence and varying portions of the 3' non-coding region. Two of the genes contain the first two introns of the DHFR gene; the other three lack introns. Transfection of DHFR-deficient Chinese hamster ovary cells with any of these constructed genes results in cells with the DHFR+ phenotype. Treatment of the transfectants with methotrexate, a folate antagonist, leads to the emergence of methotrexate-resistant colonies which have amplified the transfected genes.
Full text
PDF




Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Alt F. W., Kellems R. E., Bertino J. R., Schimke R. T. Selective multiplication of dihydrofolate reductase genes in methotrexate-resistant variants of cultured murine cells. J Biol Chem. 1978 Mar 10;253(5):1357–1370. [PubMed] [Google Scholar]
- Brinster R. L., Chen H. Y., Warren R., Sarthy A., Palmiter R. D. Regulation of metallothionein--thymidine kinase fusion plasmids injected into mouse eggs. Nature. 1982 Mar 4;296(5852):39–42. doi: 10.1038/296039a0. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
- Gruss P., Khoury G. Rescue of a splicing defective mutant by insertion of an heterologous intron. Nature. 1980 Aug 7;286(5773):634–637. doi: 10.1038/286634a0. [DOI] [PubMed] [Google Scholar]
- Haber D. A., Beverley S. M., Kiely M. L., Schimke R. T. Properties of an altered dihydrofolate reductase encoded by amplified genes in cultured mouse fibroblasts. J Biol Chem. 1981 Sep 25;256(18):9501–9510. [PubMed] [Google Scholar]
- Hamer D. H., Leder P. Splicing and the formation of stable RNA. Cell. 1979 Dec;18(4):1299–1302. doi: 10.1016/0092-8674(79)90240-x. [DOI] [PubMed] [Google Scholar]
- Kafatos F. C., Jones C. W., Efstratiadis A. Determination of nucleic acid sequence homologies and relative concentrations by a dot hybridization procedure. Nucleic Acids Res. 1979 Nov 24;7(6):1541–1552. doi: 10.1093/nar/7.6.1541. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kurtz D. T. Hormonal inducibility of rat alpha 2u globulin genes in transfected mouse cells. Nature. 1981 Jun 25;291(5817):629–631. doi: 10.1038/291629a0. [DOI] [PubMed] [Google Scholar]
- Lai C. J., Khoury G. Deletion mutants of simian virus 40 defective in biosynthesis of late viral mRNA. Proc Natl Acad Sci U S A. 1979 Jan;76(1):71–75. doi: 10.1073/pnas.76.1.71. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lee F., Mulligan R., Berg P., Ringold G. Glucocorticoids regulate expression of dihydrofolate reductase cDNA in mouse mammary tumour virus chimaeric plasmids. Nature. 1981 Nov 19;294(5838):228–232. doi: 10.1038/294228a0. [DOI] [PubMed] [Google Scholar]
- Mariani B. D., Slate D. L., Schimke R. T. S phase-specific synthesis of dihydrofolate reductase in Chinese hamster ovary cells. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4985–4989. doi: 10.1073/pnas.78.8.4985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McGrogan M., Raskas H. J. Species identification and genome mapping of cytoplasmic adenovirus type 2 RNAs synthesized late in infection. J Virol. 1977 Aug;23(2):240–249. doi: 10.1128/jvi.23.2.240-249.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Palmiter R. D., Chen H. Y., Brinster R. L. Differential regulation of metallothionein-thymidine kinase fusion genes in transgenic mice and their offspring. Cell. 1982 Jun;29(2):701–710. doi: 10.1016/0092-8674(82)90186-6. [DOI] [PubMed] [Google Scholar]
- Perucho M., Hanahan D., Wigler M. Genetic and physical linkage of exogenous sequences in transformed cells. Cell. 1980 Nov;22(1 Pt 1):309–317. doi: 10.1016/0092-8674(80)90178-6. [DOI] [PubMed] [Google Scholar]
- Robins D. M., Ripley S., Henderson A. S., Axel R. Transforming DNA integrates into the host chromosome. Cell. 1981 Jan;23(1):29–39. doi: 10.1016/0092-8674(81)90267-1. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Subramani S., Mulligan R., Berg P. Expression of the mouse dihydrofolate reductase complementary deoxyribonucleic acid in simian virus 40 vectors. Mol Cell Biol. 1981 Sep;1(9):854–864. doi: 10.1128/mcb.1.9.854. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Wiedemann L. M., Johnson L. F. Regulation of dihydrofolate reductase synthesis in an overproducing 3T6 cell line during transition from resting to growing state. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2818–2822. doi: 10.1073/pnas.76.6.2818. [DOI] [PMC free article] [PubMed] [Google Scholar]