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The effect of the human O(6)-alkylguanine-DNA alkyltransferase on the mutational specificity of bis-chloroethylnitrosourea in the Chinese hamster ovary cell line, D422

Minnick, Dana Thorne
http://rave.ohiolink.edu/etdc/view?acc_num=case1056573098

Abstract Details

1992, Doctor of Philosophy, Case Western Reserve University, Environmental Health Science.
The mutational specificity of the alkylating agent bis-chloroethylnitrosourea (BCNU) was analyzed at the endogenous hemizygous adenine phosphoribosyl transferase (aprt) gene of the O6-alkyguanine-DNA alkyltransferase-deficient Chinese hamster ovary cell line, D422. A one hour treatment of the CHO cells with 50 uM BCNU resulted in a toxicity level of 62% and induced mutation at this target with a frequency of 32.1 mutants/106 survivors (six-fold above background). Analysis of 49 BCNU-induced mutants at the DNA sequence level revealed that BCNU induced primarily base substitutions. The predominant BCNU-induced mutations were G:C → T:A transversions, which comprised 51% (25/49) of the mutations; while, G:C → A:T transitions, expected from miscoding of O6-alkylguanine for thymine, were the second most frequent mutation recovered representing 16.3% (8/49) of the mutations. Furthermore, two "hotspots" for BCNU-induced G:C → T:A transversions were observed, which involved different surrounding DNA sequences but identical helix parameters when analyzed by an application of Calladine's Rules. In an effort to address the importance of the O6-alkylguanine mediated lesions in BCNU-induced mutation in CH O D422, and to determine the probable role of these adducts as promutational lesions in the generation of the G:C → T:A and G:C → A:T base substitutions, the human O6-alkylguanine-DNA alkyltransferase cDNA (MGMT) was transfected into CHO D422. The alkyltransferase-transformed cell line exhibited 100-fold protection against BCNU-induced toxicity and a maximal four-fold reduction in the overall mutation frequency at the aprt gene target in comparison to the parental cell line. When the mutational specificity of BCNU in the alkyltransferase-transformed cell line was compared to that of the parental cell line, the frequence of G:C → T:A transversions was reduced 25-fold in the alkyltransferase-transformed cell line. Furthermore, G:C → A:T transitions were reduced 6-fold. These findings suggest that both the G:C → T:A transversions and G:C→A:T transitions were O6-alkylguanine mediated mutations. In the presence of proficient repair of the BCNU-induced O6-alkylguanine lesions, T:A → G:C transversions emerged as the predominant BCNU-induced mutation, comprising 45% (23/51) of the recovered mutations. In addition, these mutations occurred as multiple isolates at several sites suggesting that the focus of BCNU induced mutations at T:A base pairs was DNA sequence dependent.
David Sedwick (Advisor)
201 p.
Environmental Sciences
effect human O(6) alkylguanine DNA alkyltransferase mutational specificity bis-chloroethylnitrosourea Chinese hamster ovary cell line D422

Recommended Citations

Citations

  • Minnick, D. T. (1992). The effect of the human O(6)-alkylguanine-DNA alkyltransferase on the mutational specificity of bis-chloroethylnitrosourea in the Chinese hamster ovary cell line, D422 [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1056573098

    APA Style (7th edition)

  • Minnick, Dana. The effect of the human O(6)-alkylguanine-DNA alkyltransferase on the mutational specificity of bis-chloroethylnitrosourea in the Chinese hamster ovary cell line, D422. 1992. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1056573098.

    MLA Style (8th edition)

  • Minnick, Dana. "The effect of the human O(6)-alkylguanine-DNA alkyltransferase on the mutational specificity of bis-chloroethylnitrosourea in the Chinese hamster ovary cell line, D422." Doctoral dissertation, Case Western Reserve University, 1992. http://rave.ohiolink.edu/etdc/view?acc_num=case1056573098

    Chicago Manual of Style (17th edition)

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© 1992, all rights reserved.
This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.