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The lacI gene has been used extensively for the recovery and analysis of mutations in bacteria with various DNA repair backgrounds and after exposure to a wide variety of mutagens. This has resulted in a large database of information on mutational mechanisms and specificity of many mutagens, as well as the effect of DNA repair background on mutagenicity. Most importantly, knowledge about the mutational sensitivity of the lacI gene is now available, yielding information about mutable nucleotides. This popularity and available knowledge resulted in the use of the lacI gene in transgenic rodents for the study of mutagenesis in mammals, where it resides in [approximately]40 repeated copies. As the number of sequenced mutations recovered from these animals increases, we are able to analyze the sites at which mutations have been recovered in great detail and to compare the recovered sites between bacteria and transgenic animals. The nucleotides that code for the DNA-binding domain are nearly saturated with base substitutions. Even after determining the sequences of [approximately]10,000 mutations recovered from the animals, however, new sites and new changes are still being recovered. In addition, we compare the nature of deletion mutations between bacteria and animals. based on the nature of deletions in the animals, we conclude that each deletion occurs in a single copy of the gene.

A study was conducted to examine the mutational specificity of 1-nitroso-8-nitropyrene (1,8-NONP) in the lacI gene of Escherichia coli strains. The strains varied across nucleotide excision repair and MucA/B-mediated error-prone translesion synthesis. Varied mutation classes were recovered whose conditions were caused by 1,8NONP treatment. Results show that DNA sequence context of the adduct and DNA cell repair background condition 1,8NONP mutational specificity.

The study of mutational frequency (Mf) and specificity in aging Big Blue lacI transgenic mice provides a unique opportunity to determine mutation rates (MR) in vivo in different tissues. We found that MR are not static, but rather, vary with the age or developmental stage of the tissue. Although Mf increase more rapidly early in life, MR are actually lower in younger animals than in older animals. For example, we estimate that the changes in Mf are 4.9 x [10.sup.-8] and 1.1 x [10.sup.-8] mutations/base pair/month in the livers of younger mice ([is less than] 1.5 months old) and older mice ([is greater than or equal to] 1.5 months old), respectively (a 4-fold decrease), and that the MR are 3.9 x [10.sup.-9] and 1.3 x [10.sup.-7] mutations/base pair/cell division, respectively (~30-fold increase). These data also permit art estimate of the MR of GC [right arrow] AT transitions occurring at 5'-CpG-3' (CpG) dinucleotide sequences. Subsequently, the contribution of these transitions to age-related demethylation of genomic DNA can be evaluated. Finally, to better understand the origin of observed Mf, we consider the contribution of various factors, including DNA damage and repair, by constructing a descriptive mutational model. We then apply this model to estimate the efficiency of repair of deaminated 5-methylcytosine nucleosides occurring at CpG dinucleotide sequences, as well as the influence of the [Msh2.sup.-/-] DNA repair defect on overall DNA repair efficiency in Big Blue mice. We conclude that even slight changes in DNA repair efficiency could lead to significant increases in mutation frequencies, potentially contributing significantly to human pathogenesis, including cancer.

Mutation frequency and specificity were determined as a function of age in nuclear DNA from liver, bladder, and brain of Big Blue lad transgenic mice aged 1.5-25 months. Mutations accumulated with age in liver and accumulated more rapidly in bladder. In the brain a small initial increase in mutation frequency was observed in young animals; however, no further increase was observed in adult mice. To investigate the origin of mutations, the mutational spectra for each tissue and age were determined. DNA sequence analysis of mutant lacI transgenes revealed no significant changes in mutational specificity in any tissue at any age. The spectra of mutations found in aging animals were identical to those in younger animals, suggesting that they originated from a common set of DNA lesions manifested during DNA replication. The data also indicated that there were no significant age-related mutational changes due to oxidative damage, or errors resulting from either changes in the fidelity of DNA polymerase or the efficiency of DNA repair. Hence, no evidence was found to support hypotheses that predict that oxidative damage or accumulation of errors in nuclear DNA contributes significantly to the aging process, at least in these three somatic tissues.