Detection of deletion mutations in pSV2gpt-transformed cells.

Tindall, Kenneth R.; Stankowski, Leon F.; Machanoff, Richard; Hsie, Abraham W.

doi:10.1128/mcb.4.7.1411

Cited by 128 publications

(74 citation statements)

References 19 publications

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“…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)(13)(14)(15)(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.…”

mentioning

confidence: 98%

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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mentioning

confidence: 98%

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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“…For instance, a promising system based on the bacterial gpt gene embedded into the chromosomes of hamster cells has been devised in which mutations are analyzed by Southern blotting (31). On the other hand, with this methodology mutation selection and analysis are relatively time consuming and molecular genetics cannot be used to further characterize the mutations.…”

Section: Cactccagt Total 39mentioning

confidence: 99%

The lacI shuttle: rapid analysis of the mutagenic specificity of ultraviolet light in human cells.

Lebkowski¹,

Clancy²,

Miller³

et al. 1985

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

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A system has been devised that allows the effect of mutagens acting in human cells to be readily analyzed at the DNA sequence level. The bacterial gene WIcI, carried on a shuttle vector, is introduced into human tissue culture cells by transfection and allowed to replicate in the cell nucleus.Twenty-four to 48 hr after transfection, the cells are exposed to a mutagen. After 1-2 days of further replication, vector DNA is purified and transfected back intoEscherichia coli for scoring and analysis of mutations in laW. The (1)(2)(3)(4). In these experiments, the target gene is carried on a shuttle vector, which can replicate in both mammalian and bacterial cells. Thus, the gene can be mutated in mammalian cells and then transferred to bacteria for analysis of mutations. The advantage of this scheme is that in bacteria mutations can be rapidly and unambiguously scored. Furthermore, if lad is used as the mutational target, sophisticated bacterial molecular genetics can be applied to characterize the mutations (5, 6). In lad, an extensive deletion map allows precise localization of mutations by using genetic techniques. Moreover, accurate DNA sequence assignments can be made for nonsense mutations by using simple genetic techniques.The use of shuttle vectors for determination of mutagenic specificity has been limited by a high spontaneous mutation frequency associated with transfection (1-4) and by an inability of the vectors to replicate efficiently in human cells. Recently, both of these problems were solved by the discovery that the human cell line 293 (7) replicates simian virus 40 (SV40)-based shuttle vectors extremely well with a relatively low spontaneous mutation frequency (4). When 293 cells containing shuttle vectors are exposed to mutagens, an increase in mutation frequency above the spontaneous background is readily obtained. Therefore, induced mutations can be rapidly isolated and characterized.We demonstrate here the operation ofthe lacd shuttle using UV light as the mutagen. The DNA sequence changes for 53 UV light-induced point mutations, as well as for 32 point mutations from unmutagenized cells, are reported. The mutagenic specificity of UV light thereby obtained in human cells has close parallels with that obtained previously for UV light treatment of Escherichia coli. This result suggests that human and bacterial cells may react to UV light damage in similar ways. It also argues that the lacd shuttle system is indeed detecting true UV light-induced mutations. Thus, it appears that this experimental system can rapidly elucidate the outcome of a mutagen's interaction with DNA in human cells at the most fundamental genetic level, the DNA sequence change. MATERIALS AND METHODSVector DNA. The previously described (4) plasmid pJYMib was used as the shuttle vector. pJYMib contains all of SV40, pML (a pBR322 derivative), the entire lacI gene, and the amino-terminal portion of lacZ. Plasmid DNA was prepared by the alkaline lysis procedure and purified on cesium chloride gradients as described (8)....

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“…The historical spontaneous mutation frequency of AS52 cells in our laboratory is 10.5 F 2.7 TG r mutants/10 6 clonable cells. As a positive control, AS52 cells were treated with ethylmethanesulfonate (EMS) as described previously (Tindall et al, 1984;Ariza and Williams, 1996). The mutation frequency under these conditions was 346.7 F 22.4 TG r mutants/10 6 clonable cells.…”

Section: Mutagenesismentioning

confidence: 99%

“…Since the gpt gene is not essential for growth or survival of AS52 cells, it is possible to isolate mutants containing a variety of mutations, including: point, interchromosomal deletions, mitotic recombinations, gene -chromosomal conversions and multilocus deletions. We, as well as others, have demonstrated that AS52 cells can be used to determine molecular and mechanistic features associated with mutagenesis in mammalian cells (Tindall et al, 1984(Tindall et al, , 1986(Tindall et al, , 1987Tindall and Stankowski, 1989;Hsie et al, 1990;Ariza and Williams, 1996;Ariza et al, 1998).…”

Section: Introductionmentioning

confidence: 99%

“…For these studies, we used the Chinese hamster ovary cell line, AS52. AS52 cells are a transgenic cell line that lack the normal X-linked mammalian hypoxanthine guanine phosphoribosyltransferase (hprt) gene, but that contain a single functional copy of the Escherichia coli xanthine-guanine phosphoribosyltransferase (gpt) gene stably integrated into the CHO genome (Tindall et al, 1984(Tindall et al, , 1986(Tindall et al, , 1987Tindall and Stankowski, 1989;Hart and Tindall, 1997). Since the gpt gene is not essential for growth or survival of AS52 cells, it is possible to isolate mutants containing a variety of mutations, including: point, interchromosomal deletions, mitotic recombinations, gene -chromosomal conversions and multilocus deletions.…”

Section: Introductionmentioning

confidence: 99%

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