Analysis of mutation in DNA transformed Chinese hamster ovary cells

Author

Kenneth Raymond Tindall

Date of Award

12-1982

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

A. W. Hsie

Committee Members

Howard Adler, Jim Epler, Frank Kenney, Peter Lalley

Abstract

DNA-mediated gene transfer was used to study mutation at the xanthine-guanine phosphoribosyl transferase (gpt) locus in hypoxanthine-guanine phosphoribosyl transferase (HGPRT)-deficient Chinese hamster ovary (CHO) cells transformed by the pSV2gpt plasmid vector. The pSV2gpt vector carries the E. coli gpt gene in an SV40-pBR322 recombinant plasmid and, through the use of the SV40 early promoter, expresses the gpt gene product, XGPRT, in pSV2gpt transformed mammalian cells. E. coli XGPRT is the bacterial equivalent of the mammalian purine salvage pathway enzyme, HGPRT. Selection protocols utilizing the purine analog, 6-thioguanine (TG), that have been developed for the isolation of mutants at the hgprt locus in CHO cells were shown to be applicable for the isolation of mutants at the gpt locus in several pSV2gpt transformed CHO cell lines.

The HGPRT-deficient CHO transformation host has been characterized and determined to carry a stable, nonrevertible mutation at the hgprt locus. Analyses of several isolated pSV2gpt transformed CHO clones have demonstrated E. coli XGPRT activity in cell free extracts of each line assayed. In addition, genomic DNA derived from several of these pSV2gpt transformed cell lines has been analyzed by Southern blot hybridization establishing the presence of one to several copies of the gpt gene integrated in the high molecular weight DNA. These data indicate the functional expression of the gpt gene in these transformed CHO cell lines.

One pSV2gpt transformant has a single copy of the gpt gene integrated in the CHO genomic DNA. Induction of TG-resistant (TG^r) mutants of this cell line is quantitative following irradiation with ultraviolet light or X-rays. Both the spontaneous and induced TG^r mutants lack XGPRT activity. Furthermore, Southern blot hybridization analyses established specific deletions through gpt structural gene sequences in two of the TG^r mutants analyzed. These data indicate the utility of this gpt transformed cell line as an easily manipulated system for quantitative and molecular analyses of gene mutation in CHO cells.

Recommended Citation

Tindall, Kenneth Raymond, "Analysis of mutation in DNA transformed Chinese hamster ovary cells. " PhD diss., University of Tennessee, 1982.
https://trace.tennessee.edu/utk_graddiss/13336