Alkylating agents (e.g., methylating and ethylating nitrosamines) are likely to be important carcinogens. The relatively reactive ethylating agents (e.g., ENU) give an unexpectedly high fraction of AT->TA mutations compared to other methylating and ethylating agents in a variety of eukaryotic systems including mouse models. There are arguments to suggest that these mutations might be due to alk2T (or possible alk3T) lesions. The principal investigator wishes to investigate mutagenesis by e2T and e3T adducts using site-specific techniques. The principal investigator has developed a workable EBV-based shuttle plasmid system to investigate the lesions of interests in human (293) cells in culture. The background MF is low, and methods to isolate mutants, while slightly laborious, are workable. The principal investigator has synthesized and characterized appropriate oligonucleotides containing both e2T and e3T. In studies with other lesions (m6G and several from ethylene oxide) the principal investigator has demonstrated his ability to build such oligonucleotides into shuttle plasmids and study lesions in cells. The principal investigator has studied e2T and e3T in vitro using both Kf and T7 DNA polymerase and observed dTTP incorporation opposite both lesions. If this were true in cells, then these lesions would be good candidates for AT->TA mutations.