Mutation Research/DNAging最新文献

Using either the colony formation assay or flow cytometry, it is feasible to measure the frequency of rare mutant lymphocytes or erythrocytcs in human peripheral blood. Accordingly, we have investigated the mutant cell frequencies of the hypoxanthine-guanine phosphoribosyltransferase and T-cell receptor genes in T lymphocytes and of the glycophorin A gene in erythrocytes of several hundred persons aged 0–96 years. The mutant frequency of every one of these genes increased significantly with age. A simple accumulation of mutations in hematopoietic stem cells over time may explain the age-dependent increase in the frequency of glycophorin A mutants. In contrast, a balance between mutant cell generation and loss should be taken into account for the mechanism of the increase of T-cell mutations.

Theories on the causes of aging, based on the accumulation of somatic mutations in tissues of an organism, were formulated decades ago, but remain insufficiently tested. Transgenic animals, equipped with integrated bacterial reporter genes that can be efficiently rescued from total genomic DNA of all tissues and organs, represent ideal tools for investigating the types and frequencies of spontaneous mutants accumulating during aging. The first of such systems, based on the transgenic integration of bacteriophage lambda shuttle vectors that contain the bacterial lacZ gene as mutational target, was constructed in our laboratory and is now routinely used. Results obtained with this and the related LacI system that are relevant for the somatic mutation theory of aging will be discussed. One conclusion is that, due to the nature of the transgene. lambda-based systems have the disadvantage that deletion type mutations are underrepresented in comparison to point mutations. To overcome those limitations, we constructed a new transgenic mouse model carrying a pUR288 plasmid shuttle vector with the lacZ reporter gene. Some preliminary data obtained with this model serve to illustrate its potential use to extensively test the somatic mutation theory of aging.

The primary focus of this review is on correlations found between DNA damage, repair, and aging. New techniques for the measurement of DNA damage and repair at the level of individual genes, in individual DNA strands and in individual nucleotides will allow us to gain information regarding the nature of these correlations. Fine structure studies of DNA damage and repair in specific regions, including active genes, telomeres, and mitochondria have begun. Considerable intragenomic DNA repair heterogeneity has been found, and there have been indications of relationships between aging and repair in specific regions. More studies are necessary, however, particularly studies of the repair of endogenous damage. It is emphasized that the information obtained must be viewed from a perspective that takes into account the total responses of the cell to damaging events and the inter-relationships that exist between DNA repair and transcription.

Deletions and point mutations of mitochondrial DNA (mtDNA), which are characteristic of various human mitochondrial diseases, have been identified mainly in postmitotic tissues like brain, heart and skeletal muscle of healthy humans of advanced age but not in young people. An exponential increase with age was described for deletions of mtDNA. This paper reviews the molecular basis and experimental results on mutations of mtDNA in patients with mitochondrial diseases and in aged individuals. In addition new data on the exponential increase of point mutations of mtDNA, characteristic for MERRF and MELAS disease, in extraocular muscle from elderly humans are shown. Finally the ‘mitochondrial hypothesis on aging’ based on stochastic somatic mutations of mtDNA is presented.

Analysis of two human populations for dependence of somatic mutation on age has revealed both similarities and differences. The studies performed employed peripheral blood lymphocytes and measured the efficiency with which these cells form clones in vitro (cloning efficiency, CE) and the frequency of cells with inactivating mutations of the hypoxanthine phosphoribosyltransferase gene (mutant frequency, MF). The people studied were between 19 and 64 years of age. In one population, composed of 78 never smokers and 140 current smokers from the United States (US), both CE and MF were dependent on age: CE declined with age (p = 0.005); MF increased 0.15 per 10⁶ cells per year of age for nonsmokers (p < 0.001) and at 1.3 times that rate for smokers (p = 0.01). In the second population, 80 people of unknown smoking status from Russia, the increase in MF per year was even greater, 2.5 times that of the US nonsmokers (p = 0.001) but the dependence of CE on age was the same as for the US population (p = 0.043). Because the increase of MF of the Russians with age is 2-fold greater than that of the US smokers, the intensity of smoking and/or other environmental exposures, or the susceptibility to these exposures, must account for the difference in age dependent MF increase, not the proportion of Russians that are smokers. Differences in the lymphocyte subpopulations that survived the longer transit from Russia may have contributed to the observed differences in MF. However, overall, the mutant frequency results suggest that the Russians were chronically exposed to higher levels of agents that induce somatic mutation and that, on an age adjusted basis, the Russia population studied is at increased risk for health consequences from accumulated genetic damage.

Evidence is presented in support of the hypothesis that P DNA element movement in somatic cells of Drosophila melanogaster induces genetic damage that significantly reduces lifespan. The lifespan of D. melanogaster males was significantly reduced by the somatic movement of a single P element in the presence of P[ry⁺ Δ2–3](99B) transposase. In addition, the P[ry⁺ SalI](89D) repressor of P[ry⁺ Δ2–3](99B) somatic transposasc was observed to reduce the effect of P element movement on lifespan. Finally, the frequency of somatic-cell chromosome breakage was significantly increased in neuroblasts of males with somatically active P elements.

These results show that lifespan in D. melanogaster is decreased with increased somatic genetic damage from DNA-element movement. Although this conclusion does not confirm that transposable element movement is a cause of natural senescence, this conclusion is clear evidence in support of a close relationship between somatic genetic damage and aging.

The spontaneous frequencies of micronucleated reticulocytes (MNRETs) were examined monthly over the life spans of animals belonging to nine mouse strains for the 7th collaborative study organized by the CSGMT/JEMS MMS. Both sexes of the BDF1 strain and females of the A/J strain showed a statistically significant increase in mean spontaneous MNRET frequency in their last month of life, suggesting the possibility of strain-specific, age-dependent chromosomal instability. SAMP6/Tan, an accelerated senescence-prone strain, showed the same tendency, although it was not statistically significant. The other strains studied, ddY, CD-1, B6C3F1, SAMR1, and MS/Ac, did not show significant age-related differences in mean of MNRET frequencies. More extensive statistical analyses are underway, and the outcomes will be reported separately.

Spontaneous mutant frequency of lacZ gene in spleen of transgenic Muta^™ mouse was examined at different ages. It was (3.2 ± 1.3 (SD)) × 10⁻⁵ at newborn and increased almost linearly with age up to (8.3 ± 1.8) × 10⁻⁵ at one year. Since the mutation of the gene is not likely to be subject to selection in vivo, the data support the idea that spontaneous mutation takes place throughout aging process and accumulates with age if not selected out by cell death.

The factors that influence the spontaneous mutant frequencies in mammalian tissues have been ranked on the basis of data from our laboratory together with published data. Some of the data come from the endogenous hprt and Dlb-1 loci, but most come from transgenic mice carrying the bacterial lad and lacI genes in recoverable lambda phage vectors. Since there is evidence that these bacterial loci are selectively neutral, the mutant frequency observed is the integral of the mutation rates from the formation of the zygote. The factors that affect the inferred mutation rate, in decreasing order of importance are: site of integration of the transgene, age, tissue, and strain. Insufficient data exist to determine the influence of gender (probably small) and inter-laboratory variables (probably at least as important as age). The two most surprising results arc (1) that about half of all mutations arise during development (and half of these in utero) and (2) that most somatic tissues, whether queiscent or actively proliferating, have similar mutant frequencies and similar increases during adult life.

There are many methods of inference in common use in biology that are based on population sampling, including such diverse areas as sampling organisms to determine the population structure of an ecosystem, sampling a set of DNA sequences to infer evolutionary history, sampling genetic loci to build a genetic map, sampling differentially expressed genes to find phenotypic markers, and many others. Recently developed PCR-based methods for nucleic acid fingerprinting can be used as sampling tools with general applicability in molecular biology, evolution and genetics. These methods include arbitrarily primed PCR (AP-PCR; Welsh and McClelland, 1990) and random amplified polymorphic DNA (RAPD; Williams et al., 1990) for the fingerprinting of DNA, and RNA arbitrarily primed PCR (RAP-PCR; Welsh et al., 1992a) and differential display (DD; Liang and Pardee, 1992) for the fingerprinting of RNA. Novel ways of looking at genetic control are facilitated by the high data-acquisition capabilities of the fingerprinting methods. In this article, we review some of the applications of DNA fingerprinting to the study of mutagenesis, and of RNA fingerprinting to the study of normal and abnormal signal transduction. We propose that these fingerprinting approaches may also have applications in the study of senescence and aging.