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Nucleic Acids Research, 1992, Vol. 20, No. 6 1349-1354
© 1992

MOLECULAR BIOLOGY

Non-phenotypic selection of N-methyl-N-nitrosourea-induced mutations in human cells

F. Palombo, M. Bignami and E. Dogliotti*

Laboratory of Comparative Toxicology and Ecotoxicology, Istituto Superiore di Sanita', Viale Regina Elena 299 00161 Rome, Italy

*To whom correspondence should be addressed

Received November 13, 1991. Revised February 13, 1992. Accepted February 13, 1992.

The distribution of mutations In a particular gene as detected by a selective mutation assay could be affected by the structural properties of the target protein. To investigate this, we have analysed N-methyl-N-nitrosourea (MNU)-induced mutations in two restriction recognition sequences of a target gene for mutation analysis and compared these data with what previously observed in a phenotypic mutation assay. DNA base changes in the Ncil and EcoRV sites of the gpt gene maintained In human cells by a shuttle vector system were measured by restriction fragment length polymorphism/polymerase chain reaction (RFLP/PCR) technique. After MNU-treatment of human cells, mutations were detected in the Ncil recognition sequence but not in the EcoRV site. DNA sequencing analysis revealed that all Neil-resistant mutations were GC to AT transitions located over four bases of the Neil recognition sequence. Only one of these mutations drastically affected the functionality of the GPT protein. The Neil-resistant mutations were randomly distributed in both DNA strands of the gpt gene and were preferentially targeted at guanine residues flanked 5' by a guanine. Our results indicate that the structure of the GPT protein is the main contributor to the strand-specificity of MNU-induced mutations previously reported by using a phenotypic mutation assay. The potential use of the RFLP/PCR technique as a general tool for mutation detection is also discussed.


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