The objective of this proposal is the development of a mutagenesis assay system in human cell line that will provide, ultimately, the capability to analyze mutational events at the level of the DNA sequence. Prokaryotic systems measuring the fidelity of DNA replication on synthetic polymers, or single stranded plasmids/phage, containing chemically-altered nucleosides or quantitating specific locus mutagenesis in plasmid/phage/bacterial genomes have successfully predicted which chemicals will be mutagenic in mammalian cells. However, the prokaryotic systems are of unknown value for predicting the mechanisms by which mammalian cells replicate a segment of DNA containing a site of chemical, the actual mutational event. The studies proposed will validate a new mutational assay with the lambda immunoglobulin gene in a human lymphoblastoid cell as the target DNA. The lambda gene was evaluated as a potential target for a mutational assay because the gene product has a neutral survival value for the cell and because clones containing mutational events in the lambda gene can be selected by an isoelectric focusing analysis (IEF). These two criteria assure there are no inherent biases in the selection of the widest possible diversity of mutagenic events. The initial analysis of clones from chemically-exposed populations identifies three classes of mutational events; isoelectric variants (point mutations within the coding region of the gene), clones with the specific mRNA but lacking an immunologically detectable gene product (point mutations affecting translation), or clones lacking the mRNA (terminal deletion). Evaluation of over 3000 control clones has not identified a single phenotypic mutant. In contrast, a mutation rate (combination of isoelectric variants plus those not producing the lambda protein) of approximately 2% was observed in clones from populations exposed to MNNG. This assay rapidly selects a wide diversity of mutational events and provides the mutant clones for subsequent analysis at the DNA sequence level pending the development of these analytical protocols. In addition, a portion of these clones will represent a panel of mutants deficient in the individual steps of protein synthesis, a powerful group of probes for studying protein synthesis in mammalian cells.
