Methods to detect and analyze somatic mutations in higher organisms are critically important in view of their causal role in cancer, heritable diseases, and, possibly, aging. Here, we describe ...detailed protocols for the use of a mutational reporter system based on lacZ-containing plasmids integrated in the germline of Mus musculus and Drosophila melanogaster. Plasmids containing the bacterial lacZ gene integrated at one or more chromosomal sites can be excised, purified and recovered in suitable Escherichia coli hosts allowing the positive selection of mutant lacZ genes and their further molecular characterization. This system is capable of detecting a broad range of mutational events, varying from small mutations in the lacZ reporter gene to large genome rearrangements with one breakpoint in lacZ and the other breakpoint elsewhere in the genome.
Accurate detection of gene mutations is important in many areas of biology and medicine. In fundamental studies of mutagenesis it is often necessary to assess all possible mutations, either ...spontaneous or induced by genotoxic agents, in a particular gene or gene sequence to explain a given cellular or physiological endpoint. In molecular medicine comprehensive detection of all possible mutations in a disease gene is required before clinical genetic testing becomes feasible. Of the many mutation detection methods currently available none is capable of scanning for all possible mutations in a cost-effective manner. Here we show that by two-dimensional DNA electrophoretic separation, on the basis of both size and base pair sequence, in principle all mutations in a given gene can be detected. This is illustrated by some data on 2-D electrophoresis of 10 exons of the cystic fibrosis gene.
To study spontaneous and induced mutagenesis in vivo we recently constructed a series of transgenic mice harboring different numbers of bacteriophage lambda shuttle vectors, provided with a LacZ ...mutational target gene, integrated in their genome. The transgenic mice enabled analysis of spontaneous and induced mutation frequencies in postmitotic tissues like liver and brain. The obtained data indicated spontaneous mutation frequencies in the order of 10(-5)-10(-6). Here we report a 25-100 times higher spontaneous mutation frequency in liver and brain DNA of mice from strain 35.5, with the lambda-gt10LacZ concatemer integrated on the X-chromosome. These results indicate the presence of a mutational 'hot spot' in the mammalian somatic genome in vivo.
An attempt was made to assign mouse lifespan-associated interstrain differences in DNA repair to a specific chromosomal region using a set of congenic mice. The sensitive 32P-postlabeling assay was ...employed to measure the removal of benzoapyrene-induced DNA adducts in liver DNA of three different chromosome 4 congenic mouse strains: B6.C-H-15c, B6.C-H-16c, and B6.C-H-26c and the two parental strains, C57B1/6 and BALB/c. The removal of the one main adduct detected, trans-(7R)-N2-10-(7 beta,8 alpha,9 alpha-trihydroxy)-7,8,9,10- tetrahydrobenzo(a)-pyrene-yl-deoxyguanosine (BPDE-N2-dG), in liver DNA of C57Bl/6 and BALB/c mice between one and three days after treatment, was approximately 86% and 57%, respectively. The percentage removal of BPDE-N2-dG in two of the three congenic mouse strains, B6.C-H-16c and B6.C-H-26c, resembled that found in BALB/c, whereas the third strain, B6.C-H-15c, removed about the same amount as C57B1/6, i.e., approximately 88% of BPDE-N2-dG between one and three days after treatment. The usefulness of congenic mouse strains for identifying genes putatively involved in aging and/or disease susceptibility is discussed.
