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Retrotransposons are a class of transposable elements capable of self-replication and insertion into new genomic locations. Across species, the mobilization of retrotransposons in somatic cells has been suggested to contribute to the cell and tissue functional decline that occurs during aging. Retrotransposons are broadly expressed across cell types, and de novo insertions have been observed to correlate with tumorigenesis. However, the extent to which new retrotransposon insertions occur during normal aging and their effect on cellular and animal function remains understudied. Here, we use a single nucleus whole genome sequencing approach in Drosophila to directly test whether transposon insertions increase with age in somatic cells. Analyses of nuclei from thoraces and indirect flight muscles using a newly developed pipeline, Retrofind, revealed no significant increase in the number of transposon insertions with age. Despite this, reducing the expression of two different retrotransposons, 412 and Roo, extended lifespan, but did not alter indicators of health such as stress resistance. This suggests a key role for transposon expression and not insertion in regulating longevity. Transcriptomic analyses revealed similar changes to gene expression in 412 and Roo knockdown flies and highlighted changes to genes involved in proteolysis and immune function as potential contributors to the observed changes in longevity. Combined, our data show a clear link between retrotransposon expression and aging., Competing Interests: Conflicts of interest J.V. is a co-founder of Singulomics Inc., and MutaGentech Inc. The other authors declare no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

De novo mutations, a consequence of errors in DNA repair or replication, have been reported to accumulate with age in normal tissues of humans and model organisms. This accumulation during development and aging has been implicated as a causal factor in aging and age-related pathology, including but not limited to cancer. Due to their generally very low abundance mutations have been difficult to detect in normal tissues. Only with recent advances in DNA sequencing of single-cells, clonal lineages or ultra-high-depth sequencing of small tissue biopsies, somatic mutation frequencies and spectra have been unveiled in several tissue types. The rapid accumulation of such data prompted us to develop a platform called SomaMutDB (https://vijglab.einsteinmed.org/SomaMutDB) to catalog the 2.42 million single nucleotide variations (SNVs) and 0.12 million small insertions and deletions (INDELs) thus far identified using these advanced methods in nineteen human tissues or cell types as a function of age or environmental stress conditions. SomaMutDB employs a user-friendly interface to display and query somatic mutations with their functional annotations. Moreover, the database provides six powerful tools for analyzing mutational signatures associated with the data. We believe such an integrated resource will prove valuable for understanding somatic mutations and their possible role in human aging and age-related diseases., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Stochastic epigenetic changes drive biological processes, such as development, aging and disease. Yet, epigenetic information is typically collected from millions of cells, thereby precluding a more precise understanding of cell-to-cell variability and the pathogenic history of epimutations. Here we present a novel procedure for directly detecting epimutations in DNA methylation patterns using single-cell, locus-specific bisulfite sequencing (SLBS). We show that within gene promoter regions of mouse hepatocytes the epimutation rate is two orders of magnitude higher than the mutation rate., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Next generation sequencing has become a powerful tool in dissecting and identifying mutations and genomic structural variants that accompany tumourigenesis. Sequence analysis of glioblastoma multiforme (GBM) illustrates the ability to rapidly identify mutations that may affect phenotype. Approximately 50% of human GBMs overexpress epidermal growth factor receptor (EGFR) which renders the EGFR protein a compelling therapeutic target. In brain tumours, attempts to target EGFR as a cancer therapeutic, however, have achieved little or no benefit. The mechanisms that drive therapeutic resistance to EGFR inhibitors in brain tumours are not well defined, and drug resistance contributes to the deadly and aggressive nature of the disease. Whole genome sequencing of four primary GBMs revealed multiple pathways by which EGFR protein abundance becomes deregulated in these tumours and will guide the development of new strategies for treating EGFR overexpressing tumours. Each of the four tumours displayed a different mechanism leading to increased EGFR protein levels. One mechanism is mediated by gene amplification and tandem duplication of the kinase domain. A second involves an intragenic deletion that generates a constitutively active form of the protein. A third combines the loss of a gene which encodes a protein that regulates EGFR abundance as well as an miRNA that modulates EGFR expression. A fourth mechanism entails loss of an ubiquitin ligase docking site in the C-terminal part of the protein whose absence inhibits turnover of the receptor., (© The Author 2014. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Unlabelled: Here we present the open-source R/Bioconductor software package BEAT (BS-Seq Epimutation Analysis Toolkit). It implements all bioinformatics steps required for the quantitative high-resolution analysis of DNA methylation patterns from bisulfite sequencing data, including the detection of regional epimutation events, i.e. loss or gain of DNA methylation at CG positions relative to a reference. Using a binomial mixture model, the BEAT package aggregates methylation counts per genomic position, thereby compensating for low coverage, incomplete conversion and sequencing errors., Availability and Implementation: BEAT is freely available as part of Bioconductor at www.bioconductor.org/packages/devel/bioc/html/BEAT.html. The package is distributed under the GNU Lesser General Public License 3.0., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

DNA mutations are the inevitable consequences of errors that arise during replication and repair of DNA damage. Because of their random and infrequent occurrence, quantification and characterization of DNA mutations in the genome of somatic cells has been difficult. Random, low-abundance mutations are currently inaccessible by standard high-throughput sequencing approaches because they cannot be distinguished from sequencing errors. One way to circumvent this problem and simultaneously account for the mutational heterogeneity within tissues is whole genome sequencing of a representative number of single cells. Here, we show elevated mutation levels in single cells from Drosophila melanogaster S2 and mouse embryonic fibroblast populations after treatment with the powerful mutagen N-ethyl-N-nitrosourea. This method can be applied as a direct measure of exposure to mutagenic agents and for assessing genotypic heterogeneity within tissues or cell populations.

Noncanonical DNA structures are postulated to be responsible for some breakpoint hotspots that occur frequently in cancers. We developed a novel mouse model system using the naturally occurring H-DNA structure that deviate from the familiar right-handed helical B form found at the breakage hotspot in the human c-MYC promoter and a Z-DNA-forming CG repeat to test this idea directly. Large-scale chromosomal deletions and/or translocations occurred in 5 (7.7%, 95% confidence interval [CI] = 3.7% to 12.8%) of the 65 mice carrying the H-DNA-forming sequences and in 7 (6.6%, 95% CI = 3.8% to 11.6%) of the 106 mice carrying the Z-DNA-forming sequences, but in 0 of the 63 control mice (P = .042 and P = .035, respectively, two-sided test). Thus, the DNA structure itself can introduce instability in a mammalian genome.

Background: Hydrogen peroxide (H(2)O(2)) has been shown to induce vascular smooth muscle cell contraction in vitro. In this study, the effect of endogenously produced H(2)O(2) on blood pressure (BP) was examined using a transgenic mouse model (hCatTg(+/0)) in which catalase is overexpressed., Methods: The hCatTg(+/0) and wild-type mice received a bolus injection of norepinephrine (NE; 1 microg/g) or angiotensin II (Ang II; 0.5 microg/g), or an osmotic minipump infusion of NE (2.5 microg/g/day) or Ang II (0.5 microg/g/day) for 7 days. Systolic BP (SBP) was measured using a tail-cuff apparatus. H(2)O(2) release from mouse aortas was measured using an H(2)O(2) assay kit., Results: The hCatTg(+/0) and wild-type mice showed similar basal levels of systolic BP (SBP) and H(2)O(2) release from the aorta. A bolus injection of NE or Ang II increased SBP 31 +/- 5 and 37 +/- 6 mm Hg, respectively, in wild-type mice. In contrast, same doses of NE and Ang II increased SBP only 15 +/- 3 and 17 +/- 4 mm Hg, respectively, in hCatTg(+/0) mice. Osmotic minipump infusion of NE or Ang II increased SBP by approximately 30 mm Hg in wild-type mice, but only by about 10 mm Hg in hCatTg(+/0) mice. The addition of NE or Ang II to the incubation media significantly increased H(2)O(2) release from the aortic segment of wild-type mice but did not alter H(2)O(2) release from the aortic segment of hCatTg(+/0) mice., Conclusion: Overexpression of catalase diminishes the pressor response to NE and Ang II by reducing H(2)O(2) production in the arterial wall.

Using a lacZ plasmid transgenic mouse model, spectra of spontaneous point mutations were determined in brain, heart, liver, spleen and small intestine in young and old mice. While similar at a young age, the mutation spectra among these organs were significantly different in old age. In brain and heart G:C-->A:T transitions at CpG sites were the predominant mutation, suggesting that oxidative damage is not a major mutagenic event in these tissues. Other base changes, especially those affecting A:T base pairs, positively correlated with increasing proliferative activity of the different tissues. A relatively high percentage of base changes at A:T base pairs and compound mutants were found in both spleen and spontaneous lymphoma, suggesting a possible role of the hypermutation process in splenocytes in carcinogenesis. The similar mutant spectra observed at a young age may reflect a common mutation mechanism for all tissues that could be driven by the rapid cell division that takes place during development. However, the spectra of the young tissues did not resemble that of the most proliferative aged tissue, implying that replicative history per se is not the underlying causal factor of age-related organ-specific differences in mutation spectra. Rather, differences in organ function, possibly in association with replicative history, may explain the divergence in mutation spectra during aging.

Quantitative information on the cell-to-cell distribution of all possible mitochondrial DNA (mtDNA) mutations in young and aged tissues is needed to assess the relevance of these mutations to the aging process. In the present study, we used PCR amplification of full-length mitochondrial genomes from single cells to scan human cardiomyocytes for all possible large deletions in mtDNA. Analysis of more than 350 individual cells that were derived from three middle-aged and four centenarian donors demonstrates that while most of the cells contain no deletions, in certain cardiomyocytes a significant portion of the mtDNA molecules carried one particular deletion. Different affected cells contained different deletions. Although similar numbers of cells were screened for each donor, these deletion-rich cells were found only in the hearts of old donors, where they occurred at a frequency of up to one in seven cells. These initial observations demonstrate the efficiency of the method and indicate that mitochondrial mutations have the potential to play an important role in human myocardial aging.

The plasmid-based transgenic mouse model, which uses the lacZ gene as the target for mutation, is sensitive to a wide range of in vivo mutations, ranging from point mutations to insertions and deletions extending far into the mouse genome. In this study, the nature of subtle lacZ mutations, which do not completely abolish beta-galactosidase activity, as detected by positive selection, was investigated. These subtle mutants are called 'color mutants' due to their light blue staining on X-gal medium. Replating of color mutants and retransformation of plasmid DNA, purified from individual color mutants, resulted in the same phenotype as the original color mutant. The p-gal positive selection system tolerates approximately 10% of wild-type activity as indicated by spectrophotometric determination of beta-galactosidase activity of individual color mutants. Restriction digestion and size separation of plasmid DNA revealed no visible change in the size of the plasmid in color mutants. Sequence analysis confirmed the presence of a point mutation in each lacZ gene of nine different color mutants. The results indicate that color mutants are caused neither by the presence of a mixture of wild-type and mutated lacZ plasmids within the same host cell nor by a mixture of cells within the original mutant colony which carry either wild-type or mutated lacZ plasmids. In addition, it was discovered that the mouse line studied harbors four polymorphic base changes among the integrated plasmid copies.

With the current rapid pace at which human disease genes are identified there is a need for practical, cost-efficient genetic screening tests. Two-dimensional electrophoretic separation of PCR-amplified gene fragments on the basis of size and base pair sequence, in non-denaturing and denaturing gradient polyacrylamide gels respectively, provides a rapid parallel approach to gene mutational scanning. Accuracy of the denaturing gradient gel electrophoresis (DGGE) component of this system strongly depends on the design of the PCR primers and the melting characteristics of the fragments they encompass. We have developed a fully automated generally applicable procedure to generate optimal two-dimensional test designs at a minimum amount of time and effort. Designs were generated for the RB1 , TP53 , MLH1 and BRCA1 genes that can be readily implemented in research and clinical laboratories as low cost genetic screening tests.

To study in vivo somatic mutations a C57BL/6 transgenic mouse model was constructed harboring multiple chromosomally integrated copies of the plasmid pUR288, which carried the lacZ reporter gene as the mutational target. We previously demonstrated that lacZ-containing plasmids could be rescued from their integrated state efficient enough to detect mutations in lacZ by positive selection. The smaller size of the plasmid vector, as compared with our earlier transgenic mouse model based on bacteriophage lambda vectors, should offer considerable advantages in terms of rescue efficiency and sensitivity to large size alterations in the lacZ gene. To evaluate the plasmid-based mouse model for its suitability to detect in vivo mutations, we determined mutant frequencies in different organs of untreated and ethyl nitrosourea (ENU)-treated animals using a new, improved protocol. The rescue efficiencies obtained were as high as 200,000/micrograms genomic DNA; millions of transformants could be obtained in one single experiment. The average spontaneous mutant frequency in four different organs of 4- to 8-week-old mice ranged from 4.41 to 6.82 x 10(-5), compared with a mutant frequency of the same plasmid grown in Escherichia coli of approximately 1 x 10(-5) or less. Single treatments with 100 and 250 mg ENU/kg body wt resulted in a 7- and 14-fold increase, respectively, in spleen mutant frequency at 14 days after i.p. administration of the alkylating agent. Restriction enzyme analysis showed that a considerable portion of spontaneous mutants were size changes varying from approximately 100 to 3000 bp. Some mutant plasmids contained mouse genomic sequences, which is indicative of large genetic rearrangement events involving the 3' flanking regions of the transgene cluster. Among the ENU-induced mutants, size changes comprised only a minor fraction of the total, which is in keeping with the known ENU mutation spectra in vitro and in vivo. The high rescue efficiency of this plasmid-based model, in combination with its sensitivity to a broad spectrum of mutations, including large deletions, makes it very suitable as a general in vivo mutagenicity test system.

Transgenic mice with integrated shuttle vectors containing the LacZ mutational target gene were used to study spontaneous mutational events in vivo. The transgenic mouse strain used carries the LacZ transgene on the X chromosome and was previously found to be characterized by approximately 25-fold higher spontaneous mutation frequency in liver and brain compared with at least three other transgenic mouse strains. To determine the nature of in vivo spontaneous mutational events, 35 mutant LacZ genes isolated from liver and brain of mice from strain 35.5 were analyzed at the DNA sequence level. The results obtained indicate that single base-pair changes were predominant in both liver and brain. However, in liver the majority of mutations were transitions whereas in brain transversions were predominantly observed. Six mutants appeared to contain multiple dispersed mutations, separated by as much as 44 bp. Mutations were generally located within a 500 bp region encoding the active site of the beta-galactosidase protein. Our results indicate that spontaneous mutations at the LacZ transgene are tissue specific and dependent on the chromosomal position of the LacZ transgene.

Using the alkaline filter elution technique we determined the induction and disappearance of single-strand breaks (SSB) in freshly isolated peripheral blood lymphocytes (PBL) from 43 Alzheimer disease (AD) patients and from 48 healthy, age- and sex-matched control subjects following in vitro exposure to N-ethyl-N-nitrosourea (ENU), methyl methanesulphonate (MMS), or gamma (gamma)-radiation. No differences in SSB disappearance between AD patients and controls were observed after treatment of PBL with MMS or gamma-rays. After treatment with ENU, however, the amount of SSB disappearance was significantly lower in PBL from familial, but not in PBL from sporadic AD patients. ENU repair in PBL from neurological controls was comparable to that found in normal age-matched controls, indicating that the lower amount of ENU repair in familial AD patients is not a consequence of neuronal degeneration. These tentative findings are discussed in relation to the aetiology of AD.

A transgenic mouse strain with a high copy number of rescuable lacZ sequences was evaluated for its effectiveness in detecting lacZ- mutations in selected tissues. Procarbazine, cyclophosphamide, ethylnitrosourea, 7,12-dimethylbenz[a]anthracene (DMBA), acrylamide and chlorambucil were tested following either single or repeated dosing regimens. Bone marrow, liver, skin and testis tissues were selected to assess as target sites for mutation. Bone marrow, liver and testis tissues were examined for mutation following exposures to ethylnitrosourea and chlorambucil. Increased mutant frequencies were found for both chemicals in all three tissues. Bone marrow tissue was examined for mutation following procarbazine, cyclophosphamide and acrylamide exposures, and skin was examined for mutation following dermal application of DMBA. Mutation induction was observed in all cases. The results obtained from this investigation demonstrate the applicability of this transgenic mouse as an effective model to detect and analyze gene mutation in selected organs including germinal tissues. Studies of organotrophic chemical mutagens and carcinogens are possible with this model as are studies of the susceptibility of germinal tissues to mutagen exposures.

Many species have been shown to contain tandemly repeated short sequence DNA known as minisatellites and simple sequence motifs. Due to allelic variation in the copy number of the repeat unit these loci are usually highly polymorphic. Here we demonstrate the presence of sequences in the genome of the nematode Caenorhabditis elegans which are homologous to two sets of short sequence DNA. However, when two independent strains were compared no polymorphism for these sequences could be detected.