UV-exposure,endogenous DNA damage,and DNA replication errors shape the spectra of genome changes in human skin

Human skin is continuously exposed to environmental DNA damage leading to the accumulation of somatic mutations over the lifetime of an individual. Mutagenesis in human skin cells can be also caused by endogenous DNA damage and by DNA replication errors. The contributions of these processes to the somatic mutation load in the skin of healthy humans has so far not been accurately assessed because the low numbers of mutations from current sequencing methodologies preclude the distinction between sequencing errors and true somatic genome changes. In this work, we sequenced genomes of single cell-derived clonal lineages obtained from primary skin cells of a large cohort of healthy individuals across a wide range of ages. We report here the range of mutation load and a comprehensive view of the various somatic genome changes that accumulate in skin cells. We demonstrate that UV-induced base substitutions, insertions and deletions are prominent even in sun-shielded skin. In addition, we detect accumulation of mutations due to spontaneous deamination of methylated cytosines as well as insertions and deletions characteristic of DNA replication errors in these cells. The endogenously induced somatic mutations and indels also demonstrate a linear increase with age, while UV-induced mutation load is age-independent. Finally, we show that DNA replication stalling at common fragile sites are potent sources of gross chromosomal rearrangements in human cells. Thus, somatic mutations in skin of healthy individuals reflect the interplay of environmental and endogenous factors in facilitating genome instability and carcinogenesis.     

Identification of three populations ofOphiostoma ulmi (aggressive subgroup) by mitochondrial DNA restriction-site mapping and nuclear DNA fingerprinting

Three genetically distinct populations of the Dutch elm pathogenOphistoma ulmi within the aggressive subgroup were defined by the hybridization of a human minisatellite DNA sequence (HVR 33.6) to polymorphic dispersed DNA sequences within theO. ulmi nuclear genomes. For the 10 isolates examined there was a close correlation between nuclear DNA fingerprints and mitochondrial (mt) DNA restriction patterns. A restriction-site map was constructed for the mitochondrial genomes for each of these populations. The three mt DNA maps corresponded to genome sizes of 49.1 (Type I), 49.9 (Type II), and 53.9 (Type III) kilobase pairs (kbp) of DNA. The Type I and Type II mt genomes differed from the Type III mt genome by discrete length mutations of 4.8 and 4.0 kbp, respectively. It is unknown whether these length mutations resulted from insertions into or deletions from a progenitor mitochondrial genome. There was no correlation between the mitochondrial or nuclear genotypes and the geographical source of the isolates.     

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.     

Genetic study of saccharomycete yeast sensitivity to the lethal and mutagenic action of nitrous acid. III. Relation between mitochondrial genome variability and the unstable sensitivity of yeast cells to inactivation by nitrous acid

The sensitivity of the yeast Saccharomyces cerevisiae to nitrous acid (NA) is significantly influenced by various spontaneous mutations of the mitochondrial (mt) genome as well as by the nuclear mutation mmg 1 leading to a decrease in the spontaneous mutability of the mt genome. The mmg 1 locus and the mt genome most probably interact and this nucleo-cytoplasmic interaction plays a role in determining the NA sensitivity of yeast cells. A significant subclonal variation of the NA sensitivity has already been reported for the strains under study. Here we show this variability to decrease significantly when the cells are devoid of the mt DNA or carry the mmg 1 mutation. These data suggest a direct relation between the unstable NA sensitivity and the variability of the mt genome.     

A study of mitochondrial DNA mutations in peripheral lymphocytes in an aging cohort

Somatic mutation in the mitochondrial genome occurs much more rapidly than in the nuclear genome and is a feature, possibly contributory, of the aging of cells and tissues. Identifying mitochondrial sequence changes in blood DNA of elderly subjects may provide a maker for the epigenetic changes of mitochondrial DNA known to occur in tissues with lower cellular turnover, and would also have implications for immunosenescence. No large-scale epidemiological studies have been reported previously. In this study we have established long-PCR banks of the mitochondrial genome from peripheral lymphocytes for an elderly cohort of 716 individuals with a range of measured aging phenotypes, and we have established assays for three widely reported mutations: the 4977 bp and 8048 bp deletions and point mutation A3243G. No individuals were identified with detectable heteroplasmy for these changes. Implications for tissue and population prevalence are discussed. The mitochondrial long-PCR DNA banks established will be useful for a wide range of studies of somatic mutation and of germline haplotypes in relation to aging.     

Temporal and tissue specific regulation of RP-associated splicing factor genes PRPF3, PRPF31 and PRPC8--implications in the pathogenesis of RP

Background

Genetic mutations in several ubiquitously expressed RNA splicing genes such as PRPF3, PRP31 and PRPC8, have been found to cause retina-specific diseases in humans. To understand this intriguing phenomenon, most studies have been focused on testing two major hypotheses. One hypothesis assumes that these mutations interrupt retina-specific interactions that are important for RNA splicing, implying that there are specific components in the retina interacting with these splicing factors. The second hypothesis suggests that these mutations have only a mild effect on the protein function and thus affect only the metabolically highly active cells such as retinal photoreceptors.

Methodology/Principal Findings

We examined the second hypothesis using the PRPF3 gene as an example. We analyzed the spatial and temporal expression of the PRPF3 gene in mice and found that it is highly expressed in retinal cells relative to other tissues and its expression is developmentally regulated. In addition, we also found that PRP31 and PRPC8 as well as snRNAs are highly expressed in retinal cells.

Conclusions/Significance

Our data suggest that the retina requires a relatively high level of RNA splicing activity for optimal tissue-specific physiological function. Because the RP18 mutation has neither a debilitating nor acute effect on protein function, we suggest that retinal degeneration is the accumulative effect of decades of suboptimal RNA splicing due to the mildly impaired protein.     

Localization of retina/pineal-expressed sequences: identification of novel candidate genes for inherited retinal disorders.

More than 100 genes causing inherited retinal diseases have been mapped to chromosomal locations, but less than half of these genes have been cloned. Mutations in many retina/pineal-specific genes are known to cause inherited retinal diseases. Examples include mutations in arrestin, rhodopsin kinase, and the cone-rod homeobox gene, CRX. To identify additional candidate genes for inherited retinal disorders, novel retina/pineal-expressed EST clusters were identified from the TIGR Human Gene Index database and mapped to specific chromosomal sites. After known human gene sequences were excluded, and repeat sequences were masked, 26 novel retina and pineal gland cDNA clusters were identified. The retinal expression of each novel EST cluster was confirmed by PCR assay of a retinal cDNA library, and each cluster was localized in the genome using the GeneBridge 4.0 radiation hybrid panel. In silico expression data from the TIGR database suggest that these EST clusters are retina/pineal-specific or predominantly expressed in these tissues. This combination of database analysis and laboratory investigation has localized several EST clusters that are potential candidates for genes causing inherited retinopathy.     

Mutation analyses of integrated HBV genome in hepatitis B patients

Little has been learnt in the last 30 years about detection of HBV genome as well as its mutation analysis between hepatitis B fathers （HBF） and their children. In this study, we used nest polymerase chain reaction （PCR）, fluorescence in situ hybridization （FISH）, and DNA sequencing analysis, to examine the integrated HBV genome in paraffin-embedded testis tissues, which were taken as samples from HBE and in peripheral blood mononuclear cells （PBMC） from 74 cases of HBFs and their children who were born after their fathers＇ HBV infection （caHBF）. We found that HBV DNA existed in testis tissues, mainly in the basilar parts of the seminiferous tubules, and also in PBMC of HBE It was also documented that there were point mutations of poly-loci, insertions and deletions of nucleotides in integrated HBV genomes, and the types of gene mutations in the HBFs were similar to those in caHBE This study addresses the major types of gene mutations in integrated HBV genome in human patients and also presents reliable evidence of possible genetic transmission of hepatitis B.     

A nuclear mutation decreasing rho- production modifies the unstable nitrous-acid sensitivity of yeast

The nuclear mmgl mutation, which reduces rho- mutability in Saccharomyces cerevisiae, renders the rho+ cells less sensitive to inactivation by nitrous acid (NA) but has little or no effect on the NA sensitivity of the rho0 cells devoid of mitochondrial (mt) DNA. Therefore the cells' NA sensitivity seems to be influenced by an interaction of the mmgl mutation and the mt genome rather than the mmgl mutation itself. The clonal variation of NA sensitivity is high in MMG+ yeast and significantly reduced in rho0 mutants and mmgl cells. The results presented suggest that frequent spontaneous heritable changes of the mt genome occur in MMG+ cells, which, (i) unlike rho- mutations, do not damage the respiratory capacity, and (ii) manifest themselves in a high clonal variation of NA sensitivity.     

Ultraviolet radiation exposure accelerates the accumulation of the aging-dependent T414G mitochondrial DNA mutation in human skin

The accumulation of mitochondrial DNA (mtDNA) mutations has been proposed as an underlying cause of the aging process. Such mutations are thought to be generated principally through mechanisms involving oxidative stress. Skin is frequently exposed to a potent mutagen in the form of ultraviolet (UV) radiation and mtDNA deletion mutations have previously been shown to accumulate with photoaging. Here we report that the age-related T414G point mutation originally identified in skin fibroblasts from donors over 65 years also accumulates with age in skin tissue. Moreover, there is a significantly greater incidence of this mutation in skin from sun-exposed sites (chi(2)= 6.8, P < 0.01). Identification and quantification of the T414G mutation in dermal skin tissue from 108 donors ranging from 8 to 97 years demonstrated both increased occurrence with photoaging as well as an increase in the proportion of molecules affected. In addition, we have discovered frequent genetic linkage between a common photoaging-associated mtDNA deletion and the T414G mutation. This linkage indicates that mtDNA mutations such as these are unlikely to be distributed equally across the mtDNA population within the skin tissue, increasing their likelihood of exerting focal effects at the cellular level. Taken together, these data significantly contribute to our understanding of the DNA damaging effects of UV exposure and how resultant mutations may ultimately contribute towards premature aging.     

The perplexed and confused mutations affect distinct stages during the transition from proliferating to post-mitotic cells within the zebrafish retina

To identify and study genes essential for vertebrate retinal development, we are screening zebrafish embryos for mutations that disrupt retinal histogenesis. Key steps in retinogenesis include withdrawal from mitosis by multipotent neuroepithelial cells, specification to particular cell types, migration to the appropriate laminar positions, and molecular and morphological differentiation. In this study, we have identified two recessive mutations that affect the transition of proliferating neuroepithelial cells to postmitotic retinal cells. Both the perplexed and confused mutant phenotypes were initially detectable when the first retinal neuroepithelial cells began to leave the cell cycle. At this time, each mutant retina showed increased cell death and a lack of morphological differentiation. Cell death was found to be apoptotic in both perplexed and confused retinas based on TUNEL analysis and activation of caspase-3. TUNEL-phosphoRb-BrdU colocalization studies indicated that the perplexed mutation caused death in cells transitioning from a proliferative to postmitotic state. For the confused mutation, TUNEL-phosphoRb-BrdU analysis revealed that only a subset of postmitotic cells were induced to activate apoptosis. Mosaic analysis demonstrated that within the retina the perplexed mutation functions noncell-autonomously. Furthermore, whole lens or eye cup transplantations indicated that the retinal defect was intrinsic to the retina. Mosaic analysis with confused embryos showed this mutation acts cell-autonomously. From these studies, we conclude that the perplexed and confused genes are essential at distinct stages during the transition from proliferating to postmitotic cells within the zebrafish retina.     

Mitochondrial DNA polymorphism in mitochondrial myopathy

Summary In order to test the hypothesis that mitochondrial myopathy may be caused by mutation of the mitochondrial (mt) genome, restriction fragment length polymorphism in leucocyte mt DNA has been studied in 38 patients with mitochondrial myopathy, 44 of their unaffected matrilineal relatives, and 35 normal control subjects. Previously unreported mt DNA polymorphisms were identified in both patients and controls. No differences in restriction fragment patterns were observed between affected and unaffected individuals in the same maternal line, and there was no evidence of major deletion of mt DNA in patients. This study provides no positive evidence of mitochondrial inheritance in mitochondrial myopathy, but this has not been excluded.     

Mutations in mitochondrial DNA accumulate differentially in three different human tissues during ageing.

In 60 human tissue samples (encompassing skeletal muscle, heart and kidney) obtained from subjects aged from under 1 to 90 years, we used quantitative PCR procedures to quantify mitochondrial DNA (mtDNA) molecules carrying the 4977 bp deletion (mtDNA4977) and 3243 A-->G base substitution. In addition, the prevalence of multiple mtDNA deletions was assessed in a semi-quantitative manner. For all three tissues, the correlations between the accumulation of the particular mtDNA mutations and age of the subject are highly significant. However, differential extents of accumulation of the two specific mutations in the various tissues were observed. Thus, the mean abundance (percentage of mutant mtDNA out of total mtDNA) of mtDNA4977in a subset of age-matched adults is substantially higher in skeletal muscle than in heart and kidney. However, the mean abundance of the 3243 A-->G mutation in skeletal muscle was found to be lower than that in heart and kidney. Visualisation of arrays of PCR products arising from multiple mtDNA deletions in DNA extracted from adult skeletal muscle, was readily made after 30 cycles of PCR. By contrast, in DNA extracted from adult heart or kidney, amplification for 35 cycles of PCR was required to detect multiple mtDNA deletions. Although such multiple deletions are less abundant in heart and kidney than in skeletal muscle, in all tissue extracts there are unique patterns of bands, even from different tissues of the same subject. The differential accumulation of mtDNA4977, other mtDNA deletions and the 3243 A-->G mutation in the three tissues analysed presumably reflects different metabolic and senescence characteristics of these various tissues.     

Conservative and compensatory evolution in oxidative phosphorylation complexes of angiosperms with highly divergent rates of mitochondrial genome evolution

Interactions between nuclear and mitochondrial gene products are critical for eukaryotic cell function. Nuclear genes encoding mitochondrial‐targeted proteins (N‐mt genes) experience elevated rates of evolution, which has often been interpreted as evidence of nuclear compensation in response to elevated mitochondrial mutation rates. However, N‐mt genes may be under relaxed functional constraints, which could also explain observed increases in their evolutionary rate. To disentangle these hypotheses, we examined patterns of sequence and structural evolution in nuclear‐ and mitochondrial‐encoded oxidative phosphorylation proteins from species in the angiosperm genus Silene with vastly different mitochondrial mutation rates. We found correlated increases in N‐mt gene evolution in species with fast‐evolving mitochondrial DNA. Structural modeling revealed an overrepresentation of N‐mt substitutions at positions that directly contact mutated residues in mitochondrial‐encoded proteins, despite overall patterns of conservative structural evolution. These findings support the hypothesis that selection for compensatory changes in response to mitochondrial mutations contributes to the elevated rate of evolution in N‐mt genes. We discuss these results in light of theories implicating mitochondrial mutation rates and mitonuclear coevolution as drivers of speciation and suggest comparative and experimental approaches that could take advantage of heterogeneity in rates of mtDNA evolution across eukaryotes to evaluate such theories.     

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.     

Analysis of the Zebrafish perplexed mutation reveals tissue-specific roles for de novo pyrimidine synthesis during development

The zebrafish perplexed mutation disrupts cell proliferation and differentiation during retinal development. In addition, growth and morphogenesis of the tectum, jaw, and pectoral fins are also affected. Positional cloning was used to identify a mutation in the carbamoyl-phosphate synthetase2-aspartate transcarbamylase-dihydroorotase (cad) gene as possibly causative of the perplexed mutation and this was confirmed by gene knockdown and pyrimidine rescue experiments. CAD is required for de novo biosynthesis of pyrimidines that are required for DNA, RNA, and UDP-dependent protein glycosylation. Developmental studies of several vertebrate species showed high levels of cad expression in tissues where mutant phenotypes were observed. Confocal time-lapse analysis of perplexed retinal cells in vivo showed a near doubling of the cell cycle period length. We also compared the perplexed mutation with mutations that affect either DNA synthesis or UDP-dependent protein glycosylation. Cumulatively, our results suggest an essential role for CAD in facilitating proliferation and differentiation events in a tissue-specific manner during vertebrate development. Both de novo DNA synthesis and UDP-dependent protein glycosylation are important for the perplexed phenotypes.     

On the origin of unusual mutations recovered from the lacI transgenic assay

Amongst approximately 25,000 mutants recovered from tissues of the lacI mouse and rat transgenic mutation assay, we identified seven mutants that carry changes that are unlike the majority of mutations that are normally recovered in these systems. The recovered mutants feature replacements and insertions of sequences that originate in the animal's genome, in the bacteriophage lambda construct that harbors the lacI gene, and in the genome of the E. coli plating host. These mutants demonstrate that mutations resulting from diverse mechanisms, in addition to the normal point mutations, can be recovered. In addition, the data indicate that such mutations may often not be of animal origin.     

Latent mitochondrial DNA deletion mutations drive muscle fiber loss at old age

With age, somatically derived mitochondrial DNA (mtDNA) deletion mutations arise in many tissues and species. In skeletal muscle, deletion mutations clonally accumulate along the length of individual fibers. At high intrafiber abundances, these mutations disrupt individual cell respiration and are linked to the activation of apoptosis, intrafiber atrophy, breakage, and necrosis, contributing to fiber loss. This sequence of molecular and cellular events suggests a putative mechanism for the permanent loss of muscle fibers with age. To test whether mtDNA deletion mutation accumulation is a significant contributor to the fiber loss observed in aging muscle, we pharmacologically induced deletion mutation accumulation. We observed a 1200% increase in mtDNA deletion mutation‐containing electron transport chain‐deficient muscle fibers, an 18% decrease in muscle fiber number and 22% worsening of muscle mass loss. These data affirm the hypothesized role for mtDNA deletion mutation in the etiology of muscle fiber loss at old age.     

Variegated expression and delayed retinal pigmentation during development in transgenic mice with a deletion in the locus control region of the tyrosinase gene

Deletion of the tyrosinase locus control region (LCR) in transgenic mice results in variegated expression in the skin. Here we investigate the pigmentation pattern of other tissues that express tyrosinase: iris, choroid, and retina in the same animals. A mosaic distribution of pigmentation appears in the iris and choroid. Interestingly, a markedly different mosaic pattern is found in the retina, where central areas contain little or no melanin while pigmentation rises to normal levels towards periphery. Further, there is a temporal delay in the initiation and accumulation of pigment in retinal pigmented epithelium (RPE) cells during development, and patterns of adult retinal melanisation in these mice appear arrested at a stage found in early embryogenesis in wild-type mice. These results demonstrate that the tyrosinase LCR is needed for the correct establishment and maintenance of this expression domain throughout development, but particularly during the later stages of retinal melanisation.     

The complete mitochondrial genome of yarrowia lipolytica

We here report the complete nucleotide sequence of the 47.9 kb mitochondrial (mt) genome from the obligate aerobic yeast Yarrowia lipolytica. It encodes, all on the same strand, seven subunits of NADH: ubiquinone oxidoreductase (ND1-6, ND4L), apocytochrome b (COB), three subunits of cytochrome oxidase (COX1, 2, 3), three subunits of ATP synthetase (ATP6, 8 and 9), small and large ribosomal RNAs and an incomplete set of tRNAs. The Y. lipolytica mt genome is very similar to the Hansenula wingei mt genome, as judged from blocks of conserved gene order and from sequence homology. The extra DNA in the Y. lipolytica mt genome consists of 17 group 1 introns and stretches of A+Trich sequence, interspersed with potentially transposable GC clusters. The usual mould mt genetic code is used. Interestingly, there is no tRNA able to read CGN (arginine) codons. CGN codons could not be found in exonic open reading frames, whereas they do occur in intronic open reading frames. However, several of the intronic open reading frames have accumulated mutations and must be regarded as pseudogenes. We propose that this may have been triggered by the presence of untranslatable CGN codons. This sequence is available under EMBL Accession No. AJ307410.