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Analysis of N-Methyl-N-nitrosourea-induced Mutations in a Shuttle Vector Plasmid Propagated in Mouse O⁶-Methylguanine-DNA Methyltransferase-deficient Cells in Comparison with Proficient Cells¹

Departments of Experimental Radiology [S-i. M., T. Y., H. T.] and Dermatology [S-i. M., C. N., S. I.], Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan

A shuttle vector plasmid, pYZ289, was constructed from the pZ189 plasmid and polyoma virus DNA. The plasmid contains a supF gene as a marker of mutation and can replicate in both Escherichia coli and mouse cells. The pYZ289 plasmids treated with N-methyl-N-nitrosourea were passed through mouse cells originating from skin tumors, which are either proficient (HL18) or deficient (HL8) in O⁶-methylguanine-DNA methyltransferase activity, and mutations in the supF gene were analyzed. In the repair-deficient HL8 cells, N-methyl-N-nitrosourea-treated pYZ289 showed lower plasmid survival and higher mutation frequency than in the repair-proficient HL18 cells. DNA sequence analysis in the mutated supF gene revealed that most mutations occurred in G:C base pairs (86% for HL8, 76% for HL18), and the frequency of G:C A:T transition was higher in HL8 cells (69%) than in HL18 cells (35%). G:C T:A transversions occurred more frequently in HL18 cells (31%) than in HL8 cells (12%). Mutations occurred frequently at the base pair positions of 123 and 159 of supF gene in HL18 cells and at 169 in HL8 cells. Analysis of the bases neighboring the mutations appeared to be related to the mutability of the base pairs with the sequence of 5'-purine-G-G-3' being the most frequently mutated. These results show that the new pYZ289 plasmid is useful for the analysis of mutations and that a deficiency in O⁶-methylguanine-DNA methyltransferase enhances the N-methyl-N-nitrosourea-induced mutation with significant specificity.

¹ This work was supported by Grants-in-Aid from the Ministry of Education, Science and Culture, Japan.

² To whom requests for reprints should be addressed, at Department of Experimental Radiology, Faculty of Medicine, Kyoto University, Yoshidakonoecho, Sakyo-ku, Kyoto 606, Japan.

Received 4/11/91. Accepted 9/19/91.