Doctoral Dissertations
Quantitative and molecular analyses of mutation in pSV2g̲p̲ṯ transformed Chinese hamster ovary cells
Date of Award
6-1984
Degree Type
Dissertation
Degree Name
Doctor of Philosophy
Major
Biomedical Sciences
Major Professor
Abraham W. Hsie
Committee Members
Stephen J. Kennel, Peter A. Lalley, Frank W. Larimer, Julian Preston
Abstract
A pSV2gpt transformed Chinese hamster ovary (CHO) cell line has been used to study mutation at the molecular level. This cell line, designated AS52, was constructed from a hypoxanthine-guanine phosphoribosyl transferase (HPRT) deficient CHO cell line, and has been previously shown to contain a single, functional copy of the E. coli xanthine-guanine phosphoribosyl transferase (XPRT) gene (gpt) stably integrated into the Chinese hamster genome. In this study, conditions for its use in the study of mammalian cell mutagenesis have been strigently defined. The rate of spontaneous mutation at the gpt locus (2 x 10-6 /cell division), and phenotypic expression time of XPRT mutants (seven days), compare favorably with that of the hprt locus in, and expression time of HPRT mutants derived from, the wild type CH0-K1-BH4 cells.
AS52 and CH0-K1-BH4 cells exhibit similar cytotoxic responses to the four agents that have been examined. However, statistically significant (p< .005) differences occur in mutation induction by three mutagens. Ratios of XPRT- to HPRT- mutants induced per unit dose of ethyl methanesulfonate (EMS), UV-light and ICR 191 are 0.70, 1.4 and 1.6, respectively. This ratio is also considerably larger than 1.0 for X-irradiation (6.1-19, depending upon dose), but exact comparisons cannot be made due to a difference in the shapes of the dose-response curves.
A large number of HPRT and XPRT mutant lines which arose spontaneously or following treatment with these agents were analyzed by Southern blot hybridization. All HPRT mutant cell lines which arose following treatment with EMS (21/21), UV-light (23/23), or ICR 191 (22/22) exhibited no alterations of the hprt locus detectable by this technique. Similar observations were made for the gpt locus in EMS- (20/22), UV-light- (21/26) and ICR 191-induced (20/24) XPRT mutant cell lines. X-ray-induced mutants, however, contained a large proportiori of deletion mutations affecting both the hprt (15/21) and gpt (26/26) loci. In contrast, a disparity was found between spontaneous HPRT and XPRT mutants. Most spontaneous hprt mutants (18/23) exhibited no detectable alterations, while most gpt mutants (14/23) contained deletions. These results indicate that, for the most part, each of these agents induces similar lesions at both loci.
The technique of plasmid rescue has been used to recover pSV2 gpt sequences from the AS52 cell line. Transformation of ^ coli with DNA prepared from AS52 cells resulted in the recovery of ampicillin resistant (Apr) colonies which also exhibited the gpt+ phenotype. In r similar experiments, selection for the Apr marker presumably allowed recovery, in plasmid form, of adjacent mutant gpt loci from all three XPRT derivatives of AS52 tested. While none of the plasmids recovered are useful in their present form, further refinements in cloning techniques should allow the recovery of mutant gpt sequences from the mammalian host cell line on a routine basis. The combined results of this study indicate that the AS52 cell promises to be particularly useful for future study of mutation in mammalian cells at the DNA sequence level.
Recommended Citation
Stankowski, Leon F., "Quantitative and molecular analyses of mutation in pSV2g̲p̲ṯ transformed Chinese hamster ovary cells. " PhD diss., University of Tennessee, 1984.
https://trace.tennessee.edu/utk_graddiss/12964