Mutants of cultured chinese hamster cells deficient in adenine phosphoribosyl transferase

Jones, Gary E.; Sargent, P. A.

doi:10.1016/0092-8674(74)90007-5

Cited by 122 publications

(40 citation statements)

References 25 publications

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“…After 20 hr of growth, the cells were spun out and the cell-free supernatants were tested for levels of secreted myeloma protein idiotype by using a radioimmunoassay as described in (13). We have previously shown that a small decrease in the amounts of tumor-specific idiotypic surface antigens can make myeloma (35,36). It is clear from our study that changes in other surface markers such as gp7l and histocompatibility antigens need not be correlated with changes in immunoglobulin expression.…”

Section: Resultsmentioning

confidence: 52%

Clonal evolution of myeloma cells leads to quantitative changes in immunoglobulin secretion and surface antigen expression.

Leibson¹,

Loken²,

Panem³

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

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Section: Resultsmentioning

confidence: 52%

Clonal evolution of myeloma cells leads to quantitative changes in immunoglobulin secretion and surface antigen expression.

Leibson¹,

Loken²,

Panem³

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

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“…We ensured that the mutants selected were truly independent by treating parallel cultures in which there were no preexisting mutants and then selecting only one AAr mutant from each independently maintained culture. Of the mutants tested for APRT activity (6,9), two thirds had an undetectable level (<1%) and reversion frequencies below 10-6, and the remainder had some residual activity (2 to 20%).…”

mentioning

confidence: 99%

Alterations of Gene Structure in Ethyl Methane Sulfonate-Induced Mutants of Mammalian Cells

Meuth

Arrand

1982

Mol. Cell. Biol.

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To determine the types of alterations in gene structure induced by DNA-alkylating agents, we analyzed the restriction enzyme cleavage patterns of adenine phosphoribosyltransferase gene sequences in mutant strains of Chinese hamster ovary cells deficient in this enzyme. Base pair changes as detected by loss of restriction enzyme sites were found, but no major internal gene rearrangements could be detected.

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“…Consequently, all classes of mutational events can in principle be expected. Simple single-step selections for both forward and reverse mutations have been well characterized (19,20). Advantages of the CHO APRT locus for molecular analyses include the availability of hemizygous strains (6, 21), the apparent absence of pseudogenes (8, 9), and the small [2.6 kilobases (kb)] size of the functional CHO APRT gene, as determined by DNA sequencing analysis and DNA transfection studies (unpublished results).…”

mentioning

confidence: 99%

Spectrum of spontaneous mutation at the APRT locus of Chinese hamster ovary cells: an analysis at the DNA sequence level.

Jong

Grosovsky

Glickman

1988

Proc. Natl. Acad. Sci. U.S.A.

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The spectrum of spontaneous mutation of an endogenous mammalian cell gene has been determined at the DNA sequence level. Thirty independent spontaneous APRTmutations were cloned and subsequently completely sequenced. Twenty-seven contained single base substitutions. Of these, 22 were G-C to A-T transitions, suggesting a major role for the deamination of cytosine in spontaneous mutagenesis of Chinese hamster ovary cells. The remaining mutants included a tandem double substitution, a -1 frameshift, and a 17-base-pair deletion flanked by a 2-base-pair direct repeat. Many of the independently recovered mutants were clustered at sites of multiple occurrence (hot spots). One site accounted for >25% of all independently recovered events. Mutations were generally located within the coding sequence, although two mutations occurred within the consensus sequence for a 3' splice site.A fundamental understanding of mutagenesis in mammalian cells ultimately requires specific knowledge of the DNA sequence alterations involved. A complete analysis would describe the spectrum of mutational alterations for a random collection of independent mutants at a given locus. Such analyses have recently been reported in bacteria (1-3), but logistical difficulties have prevented such an analysis of an endogenous mammalian cell gene. Other endpoints have, by necessity, been used to construct a preliminary view of mutation in mammalian cells. These include comparative mutation induction at several selectable loci (4, 5), twodimensional gel electrophoresis analysis of mutant proteins (6), and Southern blot analysis of restriction site polymorphisms (7-10). Recently, recombinant shuttle vectors, which can be propagated in both prokaryotic and eukaryotic cells, have been developed for the analysis of mutant DNA sequences in mammalian cells (reviewed in ref. 11). Although the use of shuttle vectors facilitates the recovery and sequence analysis of mutations, this experimental approach has several drawbacks, often including an extraordinarily high mutation frequency and a high percentage of deletions and rearrangements among the recovered plasmids (12-16). This is true even under conditions in which the shuttle vector has been incorporated into the chromosome, although some inserts appear to approach the stability expected for natural genes (17, 18). These technical problems and the artificial nature of such constructs make it questionable whether these systems accurately represent mutagenesis at endogenous cellular genes.Our efforts have therefore concentrated on the development of a practical approach to the cloning and sequencing of mutant alleles of an endogenous gene. Our system involves the analysis of mutants at the adenine phosphoribosyltransferase (APRT) locus of Chinese hamster ovary (CHO) cells. This locus is well suited for mutational studies. It encodes an enzyme in the nucleotide salvage pathway and is thus nonessential for cell survival in ordinary growth medium. Consequently, all classes of mutational events can in principl...

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Mutants of cultured chinese hamster cells deficient in adenine phosphoribosyl transferase

Cited by 122 publications

References 25 publications

Clonal evolution of myeloma cells leads to quantitative changes in immunoglobulin secretion and surface antigen expression.

Clonal evolution of myeloma cells leads to quantitative changes in immunoglobulin secretion and surface antigen expression.

Alterations of Gene Structure in Ethyl Methane Sulfonate-Induced Mutants of Mammalian Cells

Spectrum of spontaneous mutation at the APRT locus of Chinese hamster ovary cells: an analysis at the DNA sequence level.

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