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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 4.9x10(-8) and 1.1 x 10(-8) mutations/base pair/month in the livers of younger mice (<1. 5 months old) and older mice (> or =1.5 months old), respectively (a 4-fold decrease), and that the MR are 3.9 x 10(-9) and 1.3 x 10(-7) mutations/base pair/cell division, respectively ( approximately 30-fold increase). These data also permit an estimate of the MR of GC --> 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(-/-) 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 lacI 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.

Examination of the literature for hprt mutant frequencies from peripheral T cells yielded data from 1194 human subjects. Relationships between mutant frequency, age, sex, and smoking were examined, and the kinetics were described. Mutant frequency increases rapidly with age until about age 15. Afterward, the rate of increase falls such that after age 53, the hprt mutant frequency is largely stabilized. Sex had no effect on mutant frequency. Cigarette smoking increased mean mutant frequency compared to nonsmokers, but did not alter age vs. mutant frequency relationships. An hprt in vivo mutant database containing 795 human hprt mutants from 342 individuals was prepared. No difference in mutational spectra was observed comparing smokers to nonsmokers, confirming previous reports. Sex affected the frequency of deletions (>1 bp) that are recovered more than twice as frequently in females (P = 0. 008) compared to males. There is no indication of a significant shift in mutational spectra with age for individuals older than 19 yr, with the exception of A:T --> C:G transversions. These events are recovered more frequently in older individuals.

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.

The role of genomic alterations in mutagenesis induced by ionizing radiation has been the subject of considerable speculation. By Southern blotting analysis we show here that 9 of 55 (approximately 1/6) gamma-ray-induced mutants at the adenine phosphoribosyl transferase (aprt) locus of Chinese hamster ovary (CHO) cells have a detectable genomic rearrangement. These fall into two classes: intragenic deletions and chromosomal rearrangements. In contrast, no major genomic alterations were detected among 67 spontaneous mutants, although two restriction site loss events were observed. Three gamma-ray-induced mutants were found to be intragenic deletions; all may have identical break-points. The remaining six gamma-ray-induced mutants demonstrating a genomic alteration appear to be the result of chromosomal rearrangements, possibly translocation or inversion events. None of the remaining gamma-ray-induced mutants showed any observable alteration in blotting pattern indicating a substantial role for point mutation in gamma-ray-induced mutagenesis at the aprt locus.

The accuracy of replication and transfer of a lacI gene on an F' plasmid was measured. Following conjugal transfer of the F', a small but reproducible increase (1.8-fold) in the frequency of lacI- mutations was detected. Among these, however, the frequency of nonsense mutations was 15-fold higher than in the absence of transfer. This corresponds to a 300-fold increase in the rate of base substitutions per round of replication compared with normal vegetative DNA replication. The amber mutational spectra revealed that, following conjugal transfer, mutation frequencies were increased markedly at all sites detected. In addition, an increase in G:C leads to A:T transitions was noted and was due almost entirely to an enhanced proportion of mutants recovered at the spontaneous hotspots (amber sites 6, 15 and 34). recA-dependent processes were not responsible for the increase in mutation, since similar results were observed with various recA- donor and recipient combinations. These results demonstrate that the fidelity of conjugal DNA replication is considerably lower than that of vegetative DNA replication.

We have employed conjugal transfer of an F' lac episome to examine targeted and untargeted mutagenesis in the lacI gene of Escherichia coli and to determine the relative importance of pyrimidine dimers as premutational UV lesions compared to (6-4) photoproducts that also may have a mutational role. This conjugal system allowed us to assess the premutagenic role of UV lesions independently from any role as inducers of SOS functions. F' DNA was transferred to an SOS-induced recipient strain from: unirradiated donor cells, UV-treated donor cells or donor cells that were irradiated and then exposed to photoreactivating light. The results indicate that SOS-related, untargeted events may account for as much as one-third of the nonsense mutations (i.e., base substitutions) recovered after undamaged F' DNA is transferred to UV-irradiated recipients. When the donor strain also is irradiated, in excess of 90% of the mutations detected following conjugation appear to be targeted. Photoreactivation of the UV-treated donors cells, prior to F' transfer to the SOS-induced recipient strain, demonstrated that in this experimental system virtually all recovered UV-induced mutations are targeted by photoreactivable lesions. We presume that these lesions are pyrimidine dimers because (6-4) photoproducts are not photoreactivable.

EMS-induced mutations within a 180 base pair region of the lacI gene of E. coli were cloned and sequenced. In total, 105 and 79 EMS-induced mutations from a Uvr+ and a UvrB- strain, respectively, were sequenced. The specificity of EMS-induced mutagenesis was very similar in the two strains; G:C----A:T transitions accounted for all but three of the mutants. The overall frequency of induced mutation was fivefold higher in the UvrB- strain compared to the Uvr+ strain. This demonstrates, at the DNA sequence level, that the presumed premutagenic lesion, O6-ethylguanine, is subject to repair by the uvrABC excision repair system of E. coli. An analysis of mutation frequencies with respect to neighboring base sequence, in the two strains, shows that O6-ethylguanine lesions adjacent to A:T base pairs present better targets for the excision repair machinery than those not adjacent to A:T base pairs.

The mechanism responsible for somatic mutation in the variable genes of antibodies is unknown and may differ from previously described mechanisms that produce mutation in DNA. We have analyzed 421 somatic mutations from the rearranged immunoglobulin variable genes of mice to determine if the nucleotide substitutions differ from those generated during meiosis and if the presence of nearby direct and inverted repeated sequences could template mutations around the variable gene. The results reveal a difference in the pattern of substitutions obtained from somatic mutations vs. meiotic mutations. An increased frequency of T:A to C:G transitions and a decreased frequency of mutations involving a G in the somatic mutants compared to the meiotic mutants is indicated. This suggests that the mutational processes responsible for somatic mutations in antibody genes differs from that responsible for mutation during meiosis. An analysis of the local DNA sequences revealed many direct repeats and palindromic sequences that were capable of templating some of the known mutations. Although additional factors may be involved in targeting mutations to the variable gene, mistemplating by nearby repeats may provide a mechanism for the enhancement of somatic mutation.