Spontaneous mutation spectrum at the lambda cII locus in liver, lung, and spleen tissue of Big Blue® transgenic mice

Big Blue® mice harbor a recoverable transgene in a lambda/LIZ shuttle vector. In the standard assay, in vivo mutations are measured in the bacterial lacI gene using a labor‐intensive color plaque assay. Applying a simpler assay [Jakubczak et al. (1996): Proc Natl Acad Sci USA 93:9073–9078], we measured mutations in the lambda cII gene portion of the transgene. Spontaneous clear plaque mutants were analyzed from liver, lung, and spleen of five untreated mice. Of 314 mutants, 182 (58%) had independent mutations, 74 (23.5%) appeared clonal, and 58 (18.5%) showed no cII mutations. Of 182 independent cII mutations, 156 (85.7%) were base substitutions, 20 (10.9%) were frameshifts, and 6 (3.2%) were multiple substitutions and one deletion. G:C → A:T transitions were the predominant base substitution (78% of these at CpG sites). The major mutation hotspot, a six G run and its 3′ flanking T at bases 179 to 185, comprised 18.7% of the independent mutations. Other hotspots were positions 103, 196, and 212. The in vivo cII spectrum had a significantly higher proportion of G → A and G → T mutations and fewer frameshifts than reported in vitro. The cII and published lacI spectra are similar, though G → A transitions and deletions were fewer in the cII gene. The cI gene was sequenced in 48 mutants with no cII mutations and most had cI mutations: 81.3% base substitutions and 18.7% frameshifts. We conclude that the cII/cI system is insensitive to deletion events, but is useful for detecting point mutations. Environ. Mol. Mutagen. 33:132–143, 1999 © 1999 Wiley‐Liss, Inc.

[1]  T. Skopek,et al.  A comparative study of in vivo mutation assays: analysis of hprt, lacI, cII/cI and as mutational targets for N-nitroso-N-methylurea and benzo[a]pyrene in Big Blue mice. , 1998, Mutation research.

[2]  K. Tindall,et al.  Spontaneous and ENU-induced mutation spectra at the cII locus in Big Blue Rat2 embryonic fibroblasts. , 1998, Mutagenesis.

[3]  B. Glickman,et al.  The lacI gene as a target for mutation in transgenic rodents and Escherichia coli. , 1998, Genetics.

[4]  K. Tindall,et al.  Spontaneous mutation in lacI transgenic mice: a comparison of tissues. , 1998, Mutagenesis.

[5]  J. Shaddock,et al.  Molecular analysis of lacI mutations in Rat2 cells exposed to 7,12-dimethylbenz[a]anthracene: evidence for DNA sequence and DNA strand biases for mutation. , 1996, Mutation research.

[6]  J. French,et al.  Analysis of genetic instability during mammary tumor progression using a novel selection-based assay for in vivo mutations in a bacteriophage lambda transgene target. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[7]  Samuel H. Wilson,et al.  Enzyme-DNA Interactions Required for Efficient Nucleotide Incorporation and Discrimination in Human DNA Polymerase β(*) , 1996, The Journal of Biological Chemistry.

[8]  G. Douglas,et al.  Sequence spectra of spontaneous lacZ gene mutations in transgenic mouse somatic and germline tissues. , 1994, Mutagenesis.

[9]  J. Vijg,et al.  DNA sequence analysis of spontaneous mutations at a LacZ transgene integrated on the mouse X chromosome. , 1993, Mutagenesis.

[10]  W. Miller,et al.  A time-efficient, linear-space local similarity algorithm , 1991 .

[11]  J. Short,et al.  The use of transgenic mice for short-term, in vivo mutagenicity testing. , 1990, Genetic analysis, techniques and applications.

[12]  G. Coetzee,et al.  5-Methylcytosine as an endogenous mutagen in the human LDL receptor and p53 genes. , 1990, Science.

[13]  J. Short,et al.  Development of a short-term, in vivo mutagenesis assay: the effects of methylation on the recovery of a lambda phage shuttle vector from transgenic mice. , 1990, Nucleic acids research.

[14]  M. Rosenberg,et al.  Identification of the DNA binding domain of the phage lambda cII transcriptional activator and the direct correlation of cII protein stability with its oligomeric forms. , 1988, Genes & development.

[15]  Adams Wt,et al.  Statistical test for the comparison of samples from mutational spectra , 1987 .

[16]  I. Herskowitz,et al.  hflB, a new Escherichia coli locus regulating lysogeny and the level of bacteriophage lambda cII protein. , 1986, Journal of molecular biology.

[17]  D. Court,et al.  Promoter for the establishment of repressor synthesis in bacteriophage lambda. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[18]  P. Harbach,et al.  Effect of plating medium and phage storage on mutant frequency and titer in the lambda cII transgenic mutation assay , 1998, Environmental and molecular mutagenesis.

[19]  K. Tindall,et al.  Spectrum of spontaneous mutations in liver tissue of lacI transgenic mice , 1997, Environmental and molecular mutagenesis.

[20]  P. Harbach,et al.  System issue: Spontaneous and ethylnitrosourea‐induced mutation fixation and molecular spectra at the lacl transgene in the Big Blue® Rat‐2 embryo cell line , 1996 .

[21]  D. Schaid,et al.  System issues: Spontaneous mutation in Big Blue® transgenic mice: Analysis of age, gender, and tissue type , 1996 .

[22]  M. Rosenberg,et al.  Mutational analysis of a regulatory region in bacteriophage lambda that has overlapping signals for the initiation of transcription and translation. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[23]  M. Rosenberg,et al.  Establishment of Repressor Synthesis , 1983 .

[24]  I. Herskowitz,et al.  The lysis-lysogeny decision of phage lambda: explicit programming and responsiveness. , 1980, Annual review of genetics.