Construction of transgenic mice with tissue-specific acceleration of mitochondrial DNA mutagenesis

Transgenic mice having rapid accumulation of mitochondrial DNA (mtDNA) mutations specifically in the heart were created. These mice contained a transgene encoding a proofreading-deficient, mouse mitochondrial DNA polymerase (pol gamma) driven by the promoter for the cardiac-specific alpha-myosin heavy chain. Starting shortly after birth greater than 95% of all pol gamma mRNA in the heart was transgene derived; expression in other tissues was low or absent. Mutations in cardiac mtDNA began to accumulate by 7 days after birth. At 1 month of age the frequency of point mutations was 0.014% as determined by DNA sequencing of cloned mtDNA. By long-extension PCR multiple different deletion mutations that had removed several thousand basepairs of genomic sequence were also detected. Sequencing of two deletion molecules showed that one was flanked at the breakpoint by direct repeat sequences. The expression of proofreading-deficient pol gamma had no apparent deleterious effect on mitochondrial DNA and protein content, gene expression, or respiratory function. However, associated with the rise in mtDNA mutation levels was the development of cardiomyopathy as evidenced by enlarged hearts in the transgenic mice. These mice may prove to be useful models to study the pathogenic effects of elevated levels of mitochondrial DNA mutations in specific tissues.     

Inhibition of human cytochrome P450 1B1, 1A1 and 1A2 by antigenotoxic compounds,purpurin and alizarin

Expanded simple tandem repeat (ESTR) loci include some of the most unstable DNA in the mouse genome and have been extensively used in pedigree studies of germline mutation. We now show that repeat DNA instability at the mouse ESTR locus Ms6-hm can also be monitored by single molecule PCR analysis of genomic DNA. Unlike unstable human minisatellites which mutate almost exclusively in the germline by a meiotic recombination-based process, mouse Ms6-hm shows repeat instability both in germinal (sperm) DNA and in somatic (spleen, brain) DNA. There is no significant variation in mutation frequency between mice of the same inbred strain. However, significant variation occurs between tissues, with mice showing the highest mutation frequency in sperm. The size spectra of somatic and sperm mutants are indistinguishable and heavily biased towards gains and losses of only a few repeat units, suggesting repeat turnover by a mitotic replication slippage process operating both in the soma and in the germline. Analysis of male mice following acute pre-meiotic exposure to X-rays showed a significant increase in sperm but not somatic mutation frequency, though no change in the size spectrum of mutants. The level of radiation-induced mutation at Ms6-hm was indistinguishable from that established by conventional pedigree analysis following paternal irradiation. This confirms that mouse ESTR loci are very sensitive to ionizing radiation and establishes that induced germline mutation results from radiation-induced mutant alleles being present in sperm, rather than from unrepaired sperm DNA lesions that subsequently lead to the appearance of mutants in the early embryo. This single molecule monitoring system has the potential to substantially reduce the number of mice needed for germline mutation monitoring, and can be used to study not only germline mutation but also somatic mutation in vivo and in cell culture.     

Genomic instability induced by mutations in Saccharomyces cerevisiae POL1

Mutations of chromosome replication genes can be one of the early events that promote genomic instability. Among genes that are involved in chromosomal replication, DNA polymerase alpha is essential for initiation of replication and lagging-strand synthesis. Here we examined the effect of two mutations in S. cerevisiae POL1, pol1-1 and pol1-17, on a microsatellite (GT)(16) tract. The pol1-17 mutation elevated the mutation rate 13-fold by altering sequences both inside and downstream of the (GT)(16) tract, whereas the pol1-1 mutation increased the mutation rate 54-fold by predominantly altering sequences downstream of the (GT)(16) tract in a RAD52-dependent manner. In a rad52 null mutant background pol1-1 and pol1-17 also exhibited different plasmid and chromosome loss phenotypes. Deletions of mismatch repair (MMR) genes induce a differential synergistic increase in the mutation rates of pol1-1 and pol1-17. These findings suggest that perturbations of DNA replication in these two pol1 mutants are caused by different mechanisms, resulting in various types of mutations. Thus, mutations of POL1 can induce a variety of mutator phenotypes and can be a source of genomic instability in cells.     

Mouse models of mitochondrial disease, oxidative stress, and senescence.

During the course of normal respiration, reactive oxygen species are produced which are particularly detrimental to mitochondrial function. This is shown by recent studies with a mouse that lacks the mitochondrial form of superoxide dismutase (Sod2). Tissues that are heavily dependent on mitochondrial function such as the brain and heart are most severely affected in the Sod2 mutant mouse. Recent work with a mouse mutant for the heart/muscle specific isoform of the mitochondrial adenine nuclear translocator (Ant1) demonstrates a potential link between mitochondrial oxidative stress and mitochondrial DNA mutations. These mutations can be detected by Long-extension PCR, a method for detecting a wide variety of mutations of the mitochondrial genome. Such mutations have also been observed in the mitochondrial genome with senescence regardless of the mean or maximal lifespan of the organism being studied. Mutations have been detected with age in Caenorhabditis elegans, mice, chimpanzees, and humans. This implies that a causal relationship may exist between mitochondrial reactive oxygen species production, and the senescence specific occurrence of mitochondrial DNA mutations.     

Age-associated increase of spontaneous mutant frequency and molecular nature of mutation in newborn and old lacZ-transgenic mouse

Accumulation of mutation has long been hypothesized to be a cause of aging and contribute to many of the degenerative diseases, which appear in the senescent phase of life. To test this hypothesis, age-associated changes in spontaneous mutation in different tissues of the body as well as the molecular nature of such changes should be examined. This kind of approach has become feasible only lately with a development of new transgenic mice suitable for mutation assay. Here, using one of these transgenic mice harboring lacZ gene, we have shown that the age-associated increase in spontaneous mutant frequency is common to all tissues examined; spleen, liver, heart, brain, skin and testis, while the rates of increase in mutant frequency differed among the tissues. DNA sequencing of the 496 lacZ mutants recovered from the tissues of newborn and old mice has revealed that spectra of mutations are similar at the two age points with G:C to A:T transition at CpG site being a predominant type of mutation. Furthermore, some mutations in old tissues are complex type and not found in tissues of newborn mice. These results suggest that similar mechanisms may be operating for mutation induction in fetal and postnatal aging process. In addition, the appearance of complex types of mutations in the old tissues suggests a unique cause for these mutations in aging tissues.     

Mutation frequency and specificity with age in liver, bladder and brain of lacI transgenic mice

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.     

Enhanced mutability at the tk locus in the radiosensitive double-strand break repair mutant xrs5

The thymidine kinase locus (tk) has been utilised as the target locus to measure the induced mutation frequency following X-irradiation in the X-ray-sensitive xrs5 mutant and its parent CHO K1 line of Chinese hamster cells. Mutations to tk- cells were measured by plating cells in selective medium containing trifluorothymidine after a post-irradiation expression time of 4 days. Our results show that the mutation frequency was 3-4 times higher in the xrs5 mutant than in the CHO K1 cell line. This enhanced mutation frequency in xrs5 is though to result from the deficiency in DNA double-strand break repair in this cell line which also results in the enhanced cell killing and higher frequencies of chromosomal aberrations in response to X-irradiation. The findings of the present study suggest that DNA double-strand break is a critical lesion leading to mutations in irradiated cells.     

Mechanisms of interference with simian virus 40 (SV40) DNA replication by trans-dominant mutants of SV40 large T antigen.

Mutations at multiple sites within the simian virus 40 (SV40) early region yield large T antigens which interfere trans dominantly with the replicative activities of wild-type T antigen. A series of experiments were conducted to study possible mechanisms of interference with SV40 DNA replication caused by these mutant T antigens. First, the levels of wild-type T antigen expression in cells cotransfected with wild-type and mutant SV40 DNAs were examined; approximately equal levels of wild-type T antigen were seen, regardless of whether the cotransfected mutant was trans dominant or not. Second, double mutants that contained the mutation of inA2827, a strong trans-dominant mutation with a 12-bp linker inserted at the position encoding amino acid 520, and various mutations in other parts of the large-T-antigen coding region were constructed. The trans-dominant interference of inA2827 was not affected by second mutations within the p105Rb binding site or the amino or carboxy terminus of large T antigen. Mutation of the nuclear localization signal partially reduced the trans dominance of inA2827. The large T antigen of mutant inA2815 contains an insertion of 4 amino acids at position 168 of large T; this T antigen fails to bind SV40 DNA but is not trans dominant for DNA replication. The double mutant containing the mutations of both inA2815 and in A2827 was not trans dominant. The large T antigen of dlA2433 lacks amino acids 587 to 589, was unstable, and failed to bind p53. Combining the dlA2433 mutation with the inA2827 mutation also reversed the trans dominance completely, but the effect of the dlA2433 mutation on trans dominance can be explained by the instability of this double mutant protein. In addition, we examined several mutants with conservative point mutations in the DNA binding domain and found that most of them were not trans dominant. The implications of the results of these experiments on possible mechanisms of trans dominance are discussed.     

Mitochondria harbouring mutant mtDNA--a cuckoo in the nest?

Mutations of the mitochondrial DNA (mtDNA) are associated with a number of human diseases. To become relevant in terms of pathology, a mutation must generally affect at least 50-70% of mtDNA molecules in a tissue. One way to reach this level is by inheritance. Mitotic segregation of mtDNA in the female germline can result in large increases in the percentage of mutant mtDNA between generations. A different explanation is required if a particular mtDNA mutation accumulates over time in somatic cells. We discuss the possibility that mutant mtDNA, by causing deficient oxidative phosphorylation, may become preferentially replicated and may thus thrive in the cell like a cuckoo in the nest. However, despite preferential replication, a de novo mtDNA mutation will be confined to that particular cell or a small clone of daughter cells. Significant accumulation can only occur if the cell harbouring the mutant mtDNA undergoes malignant transformation and therefore starts proliferating continuously. This type of amplification of mutant mtDNA has recently been demonstrated in certain bone marrow disorders (myelodysplastic syndromes) and in colon cancer cell lines. Finally, in postmitotic tissues, an inherited mutation which is present in virtually all cells of the tissue, may accumulate through replicative advantage. This may contribute to the development of degenerative diseases.     

C1 inhibitor gene sequence facilitates frameshift mutations.

Mutations disrupting the function or production of C1 inhibitor cause the disease hereditary angioneurotic edema. Patient mutations identified an imperfect inverted repeat sequence that was postulated to play a mechanistic role in the mutations. To test this hypothesis, the inverted repeat was cloned into the chloramphenicol acetyltransferase gene in pBR325 and its mutation rate was studied in four bacterial strains. These strains were selected to assay the effects of recombination and superhelical tension on mutation frequency. Mutations that revert bacteria to chloramphenicol resistance (Cmr) were scored. Both pairs of isogenic strains had reversion frequencies of approximately 10(-8). These rare reversion events in bacteria were most often a frameshift that involved the imperfect inverted repeat with a deletion or a tandem duplication, an event very similar to the human mutations. Increased DNA superhelical tension, which would be expected to enhance cruciform extrusion, did not accentuate mutagenesis. This finding suggests that the imperfect inverted repeat may form a stem-loop structure in the single-stranded DNA created by the duplex DNA melting prior to replication. Models explaining the slippage can be drawn using the lagging strand of the replication fork. In this model, the formation of a stem-loop structure is responsible for bringing the end of the deletion or duplication into close proximity.     

(A)symmetric stem cell replication and cancer

Most tissues in metazoans undergo continuous turnover due to cell death or epithelial shedding. Since cellular replication is associated with an inherent risk of mutagenesis, tissues are maintained by a small group of stem cells (SCs) that replicate slowly to maintain their own population and that give rise to differentiated cells. There is increasing evidence that many tumors are also maintained by a small population of cancer stem cells that may arise by mutations from normal SCs. SC replication can be either symmetric or asymmetric. The former can lead to expansion of the SC pool. We describe a simple model to evaluate the impact of (a)symmetric SC replication on the expansion of mutant SCs and to show that mutations that increase the probability of asymmetric replication can lead to rapid mutant SC expansion in the absence of a selective fitness advantage. Mutations in several genes can lead to this process and may be at the root of the carcinogenic process.     

A method for mutagenesis of mouse mtDNA and a resource of mouse mtDNA mutations for modeling human pathological conditions

Mutations in human mitochondrial DNA (mtDNA) can cause mitochondrial disease and have been associated with neurodegenerative disorders, cancer, diabetes and aging. Yet our progress toward delineating the precise contributions of mtDNA mutations to these conditions is impeded by the limited availability of faithful transmitochondrial animal models. Here, we report a method for the isolation of mutations in mouse mtDNA and its implementation for the generation of a collection of over 150 cell lines suitable for the production of transmitochondrial mice. This method is based on the limited mutagenesis of mtDNA by proofreading-deficient DNA-polymerase γ followed by segregation of the resulting highly heteroplasmic mtDNA population by means of intracellular cloning. Among generated cell lines, we identify nine which carry mutations affecting the same amino acid or nucleotide positions as in human disease, including a mutation in the ND4 gene responsible for 70% of Leber Hereditary Optic Neuropathies (LHON). Similar to their human counterparts, cybrids carrying the homoplasmic mouse LHON mutation demonstrated reduced respiration, reduced ATP content and elevated production of mitochondrial reactive oxygen species (ROS). The generated resource of mouse mtDNA mutants will be useful both in modeling human mitochondrial disease and in understanding the mechanisms of ROS production mediated by mutations in mtDNA.     

Evolution meets disease: penetrance and functional epistasis of mitochondrial tRNA mutations

About half of the mitochondrial DNA (mtDNA) mutations causing diseases in humans occur in tRNA genes. Particularly intriguing are those pathogenic tRNA mutations than can reach homoplasmy and yet show very different penetrance among patients. These mutations are scarce and, in addition to their obvious interest for understanding human pathology, they can be excellent experimental examples to model evolution and fixation of mitochondrial tRNA mutations. To date, the only source of this type of mutations is human patients. We report here the generation and characterization of the first mitochondrial tRNA pathological mutation in mouse cells, an m.3739G>A transition in the mitochondrial mt-Ti gene. This mutation recapitulates the molecular hallmarks of a disease-causing mutation described in humans, an m.4290T>C transition affecting also the human mt-Ti gene. We could determine that the pathogenic molecular mechanism, induced by both the mouse and the human mutations, is a high frequency of abnormal folding of the tRNA(Ile) that cannot be charged with isoleucine. We demonstrate that the cells harboring the mouse or human mutant tRNA have exacerbated mitochondrial biogenesis triggered by an increase in mitochondrial ROS production as a compensatory response. We propose that both the nature of the pathogenic mechanism combined with the existence of a compensatory mechanism can explain the penetrance pattern of this mutation. This particular behavior can allow a scenario for the evolution of mitochondrial tRNAs in which the fixation of two alleles that are individually deleterious can proceed in two steps and not require the simultaneous mutation of both.     

Detection of mutations in mitochondrial DNA by droplet digital PCR

Mutations in mitochondrial DNA (mtDNA) may result in various pathological processes. Detection of mutant mtDNAs is a problem for diagnostic practice that is complicated by heteroplasmy – a phenomenon of the inferring presence of at least two allelic variants of the mitochondrial genome. Also, the level of heteroplasmy largely determines the profile and severity of clinical manifestations. Here we discuss detection of mutations in heteroplasmic mtDNA using up-todate methods that have not yet been introduced as routine clinical assays. These methods can be used for detecting mutations in mtDNA to verify diagnosis of “mitochondrial disease”, studying dynamics of mutant mtDNA in body tissues of patients, as well as investigating structural features of mtDNAs. Original data on allele-specific discrimination of m.11778G>A mutation by droplet digital PCR are presented, which demonstrate an opportunity for simultaneous detection and quantitative assessment of mutations in mtDNAs.     

Induction of lacZ mutation by 7,12-dimethylbenz[a]anthracene in various tissues of transgenic mice.

The induction of gene mutations was examined in MutaMouse after an intraperitoneal injection of 7, 8-dimethylbenz[a]anthracene (DMBA) at 20 mg/kg in a collaborative study participated by four laboratories. Although the DMBA dose used was lower than the level that has been reported to induce micronucleated erythrocytes maximally in several mouse strains, a killing effect appeared after day 9 of the post-treatment interval. Mutations in lacZ transgene were detected by the positive selection assay following in vitro packaging of phage lambda from the genomic DNA of the transgenic animals that survived. The mutant induction was evaluated in the bone marrow, liver, skin, colon, kidney, thymus, and testis 7 to 28 days after the treatment. In the bone marrow, the mutant frequency reached a maximum, approximately a 30-fold increase, 14 days after the treatment and the increased frequency persisted at least up to day 28 of the post-treatment. Induction of mutants was detected in the liver, colon, thymus, and skin to lesser extents. Marginal responses were obtained in the kidney and testis. The slight increases in the mutant frequencies in the kidney and testis observed in some laboratories were within laboratory-to-laboratory or animal-to-animal variations. In contrast to the gene mutation induction in the bone marrow, the frequency of micronucleated reticulocytes increased transiently 3 days after the treatment and returned to a control level before day 8 of the post-treatment. It was suggested that DMBA induced gene mutation is fixed in stem cells depending on cell proliferation while DNA damages responsible for chromosome breakage are not transmitted to progeny cells.     

Molecular mechanisms of substitution mutagenesis. An experimental test of the Watson-Crick and topal-fresco models of base mispairings

The proteins coded by bacteriophate T4 replication genes 32, 41, 43, 44, 45, 61, and 62 together can replicate phi X174 DNA templates very efficiently. The fidelity of this in vitro replication reaction has been measured using an infectivity assay. The product molecules have the same specific infectivity as the template DNA. When an amber mutant DNA template is used, no increase in the frequency of revertants is seen even after more than 60 duplications in vitro. By using imbalances in the concentrations of deoxynucleotide substrates, the error rate during DNA replication in vitro can be greatly increased. Control experiments indicate that the increased mutagenesis is not due to the presence of dITP or dUTP as contaminants in the deoxynucleotide substrates used. The increase in the frequency of revertants is linearly related to the ratio of the correct and the incorrect deoxynucleotides. Determination of the DNA sequence of the revertants induced shows that a change in DNA sequence of the amber site predicted from the nucleotide bias occurs. DNA synthesis in vitro resembles in vivo replication in that the error rate depends not only upon the base change required for reversion but also upon the neighboring DNA sequences. The error rate is estimated to be 5 X 10(-6) at am3 site, 6.4 X 10(-7) at am86 site, and less than 2.9 X 10(-7) at am9 site. Comparison of the frequency of G-T and A-C mispairs reveals that most AT leads to GC transition mutations occur through G-T mispairs. Measurement of the frequency of the mispairs required to induce transversion mutations reveals that these occur primarily through purine-purine mispairs. Transition mutations are more frequent than transversion mutations at both the am3 and the am86 sites. These observations support the models for base pairing errors proposed by Watson and Crick ((1953) Nature 171, 964-967) and Topal and Fresco ((1976) Nature 263, 285-289).     

Estimation of in-vivo miscoding rates

The replication of premutagenic DNA lesions generates mutant progeny in patterns that distinguish lesions that rarely produce a mutation per DNA replication from those that frequently do so. The quantitative aspects of this distinction were tested in studies of heat-mutagenized bacteriophage T4. Previous T4 studies had demonstrated that transition mutations produced at G.C base-pairs depended upon heat-induced DNA lesions distinct from those responsible for transversions at G.C pairs. In this study the transversion mutations are shown to arise in patterns predicted for mutations produced from lesions that miscode rarely (fewer than 10% per replication). In contrast, the transition mutations arise in patterns predicted for mutations produced from lesions that miscode at about 20 to 60% per replication. The fact that the two classes of DNA lesions are distinguishable as predicted by the quantitative model suggests that such studies may in general be useful in quantifying the behavior of mutation-generating DNA lesions. The method employed also estimates the frequency of premutagenic lesions in DNA.