The
Transgenic
and
In Vivo Mutagenesis
Interest Group

Minutes, Transgenic and In Vivo Mutagenesis Interest Group, Pittsburgh, PA, October 2004.

TIVM Interest Group Meeting

Dr. Carrie Valentine, chair, described the activity of the interest group newsletter to circulate reviews of scientific articles of interest to the group through the electronic newsletter. These reviews are also posted on the EMS web site under the TIVM interest group.

The officers of the interest group were re-elected for another year: Chair: Dr. Carrie Valentine of the National Center for Toxicological Research, FDA, Jefferson AR ; Secretary: Dr. Kathleen Hill , now of the U. Western Ontario, London ON . Kathleen will be taking over the EMS newsletter this next year; therefore, next year the interest group will need a new secretary.

Scientific Report #1.

Dr. Carrie Valentine reported on work in progress on determining the mutant frequency in spleen lymphocytes of FX174 mice treated with ENU at 40 mg/kg for the purpose of comparing the sensitivity of single burst analysis of the FX174 transgene to the lacI transgene. She explained the work of Dohet et al., 1985, PNAS USA 82:503-505, in which it was demonstrated that mismatches in the cII gene of lambda, when not methylated by the E. coli dam protein at adenines in GACT sequences, is promptly repaired by mismatch repair in E. coli. Without proper methylation the repair is relatively random and would effectively fix a mutation in E. coli (or accurately repair it to wild type). Therefore, she argued that the mosaic plaque assay, which purports to identify mutations fixed in vitro by the mosaicism of the original blue plaque, would not be expected to identify mutations fixed in E. coli from mismatches. The FX174 genome does not contain a GATC sequence for mismatch repair and, therefore, is not subject to mismatch repair in E. coli.

Mismatches may be recognized by the FX174 forward assay by virtue of their producing half-sized bursts. Single burst analysis evaluates the number of mutant plaque-forming units produced after replication in a single bacterial cell, the first one electroporated. If a mutation was fixed in vivo, a full burst of mutant PFUs will be produced. Currently, only those plaques from aliquots with greater than 150 PFUs are considered as derived from in vivo bursts. Carrie presented preliminary results from 6 control animals and 5 ENU-treated animals (out of a total of 8 controls and 6 treated) by single burst analysis. The mutant frequency for controls was 0.037 x 10^-5 and the mutant frequency for ENU-treated samples was 0.83 x 10^-5, for a fold increase of 22. The same dose of ENU produced a 7.8-fold increase in the lacI transgene (Skopek et al., 1995, EMM 26:9-15). The cII/cI transgene produces an 8.5-fold increase at a dose 2.5 times higher (100 mk/kg; Zimmer et al., 1999, EMM 33:249-256) and the lacZ transgene 2.4-fold at 50 mg/kg (van Delft et al., 1998, Mut. Res. 415:85-96).

Dr. David Josephy pointed out that these comparisons are based on finding only one in vivo burst so far among control animals. Dr. Steve Sommer commented that in his experience selective assays are more prone to repair of adducts in E. coli. Dr. Don Ennis remarked that there he saw no reason why selective assays should have more of a problem with ex vivo fixation than non-selective assays. Dr. Ennis is currently developing a selective mutational assay for the lacI transgene.

Scientific Report #2.

Tanya Day reported for Dr Pamela J. Sykes of Flinders University and Medical Centre, Adelaide , South Australia who was ill and unable to attend the EMS meeting. Tanya Day presented data from experiments assessing the effect of treatment of low dose X-rays on chromosomal inversion in the pKZ1 transgenic mouse recombination mutagenesis assay.

The pKZ1 mouse assay (Science 251:81-86, 1991) enables quantification of somatic intrachromosomal recombination (SICR) in vivo. The pKZ1 transgene contains an E. coli LacZ gene in inverse orientation with respect to a chicken beta-actin enhancer promoter complex. The LacZ gene is flanked by mouse immunoglobulin gene recombination signal sequences which can facilitate the formation of a stem-and-loop structure, followed by inversion of the intervening DNA segment, enabling LacZ gene expression. Cells expressing the LacZ gene product are visualised in pKZ1 tissues using a chromogenic substrate (X-gal), and quantified using light microscopy to determine mutation frequency.

Recombination in the pKZ1 assay is thought to be mediated by non-homologous end-joining, a highly conserved DNA repair process which is similar to V(D)J recombination. Recombination in the pKZ1 assay is not thought to be directly related to Rag1 and Rag2 mediated V(D)J recombination because inversions have been observed in a variety of non-lymphoid mouse tissues, and Rag1 and Rag2 expression is thought to be confined to lymphoid tissues.

An increase in inversion frequency in pKZ1 spleen has been observed 3 days after treatment with high doses of mitomycin C, etoposide, methylene chloride and X-rays (Mutation Research 427:1-9, 1999). The pKZ1 assay is more than 1000X more sensitive for the detection of a mutagenic effect after treatment with cyclophosphamide than BigBlue. Interestingly, when pKZ1 mice were treated with low doses of etoposide, the inversion frequency decreased below the endogenous inversion frequency, indicating a J-shaped dose response (Mutation Research 500: 117-124, 2002). The spontaneous mutation frequency in the pKZ1 assay is approximately 1.5 x 10^-4, which allows the detection of a reduction in inversion frequency below endogenous frequency without the need to screen a prohibitive number of cells.

The pKZ1 assay was employed to characterise the mutagenic effect of low doses of X-rays. Mice were treated with various doses of X-rays, ranging from 2 Gy to 1 mGy, and three days later spleens were removed and screened to quantify inversion frequency. The doses were divided into three dose ranges, high dose (³ 100 mGy), low dose (0.1 – 10 mGy) and ultra-low dose (1 mGy – 20 mGy). An induction in inversions was observed at high and ultra-low doses of radiation exposure and a reduction below endogenous inversion frequency was observed at low doses (Radiation Research 162: 447-452, 2004). This was an unusual response in the low and ultra-low dose range. These results have been reproduced in a pKZ1 hybridoma cell line for the high and low dose radiation exposures, although the dose-response curve is shifted to the left by a factor of 10. Further experiments are required to determine if an induction in inversion frequency is also observed at ultra-low doses in vitro. Present studies in the laboratory are aimed at understanding the mechanism behind these responses.

Scientific Report #3. Dr. Kathleen Hill .

Kathleen reviewed analyses of spontaneous mutation frequency and pattern in individual tissues over the lifespan of the Big Blue mouse. This work was carried out in the laboratory of Steve Sommer over the past eight years. An initial study by this research group repor ted a plateau in mutation frequency in young adulthood to middle adulthood (3 to 10 months of age) and a mutation pattern unchanged with the threefold increase in age and different tissue types (Nishino et al. 1996 Envion Mol Mutagen 28:229). Last year, the group repor ted observations of tissue specific profiles of mutation frequency and no significant change in mutation pattern with aging ( Hill et al. 2004 Environ Mol Mutagen 43:110). Adipose tissue and liver showed eleva ted mutation frequency in late adulthood (25 compared with 10 months of age) while neurons and germ cells showed constant mutation frequency in middle to late adulthood (10 and 25 months). To better define the time course of spontaneous mutation frequency in middle to late adulthood of the mouse, measurements were made at 10, 14, 17, 23, 25 and 30 months of age, in samples of adipose tissue, liver, cerebellum (90% neurons) and the male germline (95% germ cells). The findings confirm (i) the previously observed occurrence of at least two tissue-specific profiles of spontaneous mutation frequency (constancy in neurons and male germ cells and elevation with age in liver and adipose tissue), (ii) a low mutation frequency in the male germline, and (iii) a constancy of mutation pattern with age within a tissue. These findings appear to extend to old age (30 mo).

Additional findings include subtle, but highly significant differences in the mutation pattern between some tissues (P = 0.0002), consistent with a minor effect of tissue-specific metabolism and larger inter-animal variation in spontaneous mutation frequency in liver and adipose tissues. The presumptive unaltered balance of DNA damage and repair with age in the germline has evolutionary consequences, is unexpec ted through old age, and is of particular interest given the controversy over whether or not increasing germline mutation frequency with paternal age is the cause of reports associating older fathers with a higher incidence of some types of genetic disease.