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Journal article
Organization of a Chinese hamster ovary dihydrofolate reductase gene identified by phenotypic rescue
Molecular and cellular biology, v 3(7), pp 1266-1273
01 Jul 1983
PMID: 6310370
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Abstract
We have constructed a genomic DNA library from a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) in the cosmid vector pHC79. By utilizing a murine dihydrofolate reductase (DHFR) cDNA clone, we have identified 66 DHFR+ clones among the 11,000 colonies screened by colony hybridization. To isolate a recombinant cosmid containing the entire DHFR gene, we have tested these colonies for their ability to rescue a DHFR- Chinese hamster ovary cell line, using the spheroplast fusion method of gene transfer developed by W. Schaffner (Proc. Natl. Acad. Sci. U.S.A. 77:2163-2167, 1980). One clone (cH1) was able to transform DHFR- cells to the DHFR+ phenotype and was shown in hybridization studies to contain all of the gene except a small portion of the 3' untranslated region. We have mapped cosmid cH1 and several overlapping cosmids with a variety of restriction enzymes and have determined the approximate positions of the five (and possibly six) exons within the DHFR gene. Differences between the sizes of homologous genes in hamster cells (24.5 kilobases [kb]) and in mouse cells (31.5 kb) are shown to reside primarily in the length of the 3' intron, which is 8 kb in the hamster gene and 16 kb in length in the mouse gene. Our studies confirm the utility of cosmid libraries for the isolation of large genes, as previously shown by R. de Saint Vincent et al. (Cell 27:267-277, 1981). In addition, a cosmid that contains a functional DHFR gene will be a useful vector for the co-amplification and subsequent overexpression of other cloned genes.
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Details
- Title
- Organization of a Chinese hamster ovary dihydrofolate reductase gene identified by phenotypic rescue
- Creators
- J D MilbrandtJ C AzizkhanK S GreisenJ L Hamlin
- Publication Details
- Molecular and cellular biology, v 3(7), pp 1266-1273
- Publisher
- American Society for Microbiology (ASM)
- Grant note
- AM22125 / NIADDK NIH HHS GM26108 / NIGMS NIH HHS
- Resource Type
- Journal article
- Language
- English
- Academic Unit
- Biochemistry and Molecular Biology
- Web of Science ID
- WOS:A1983QW22200012
- Scopus ID
- 2-s2.0-0020556270
- Other Identifier
- 991020200754304721
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- Web of Science research areas
- Biochemistry & Molecular Biology
- Cell Biology