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Sample records for mutation research dna

  1. Minisequencing mitochondrial DNA pathogenic mutations

    Directory of Open Access Journals (Sweden)

    Carracedo Ángel

    2008-04-01

    Full Text Available Abstract Background There are a number of well-known mutations responsible of common mitochondrial DNA (mtDNA diseases. In order to overcome technical problems related to the analysis of complete mtDNA genomes, a variety of different techniques have been proposed that allow the screening of coding region pathogenic mutations. Methods We here propose a minisequencing assay for the analysis of mtDNA mutations. In a single reaction, we interrogate a total of 25 pathogenic mutations distributed all around the whole mtDNA genome in a sample of patients suspected for mtDNA disease. Results We have detected 11 causal homoplasmic mutations in patients suspected for Leber disease, which were further confirmed by standard automatic sequencing. Mutations m.11778G>A and m.14484T>C occur at higher frequency than expected by change in the Galician (northwest Spain patients carrying haplogroup J lineages (Fisher's Exact test, P-value Conclusion We here developed a minisequencing genotyping method for the screening of the most common pathogenic mtDNA mutations which is simple, fast, and low-cost. The technique is robust and reproducible and can easily be implemented in standard clinical laboratories.

  2. Recent research in DNA repair, mutation and recombination: a report of the DNA Repair Network meeting, held at City University, London on 18 December 1995.

    Science.gov (United States)

    Jones, N J; Strike, P

    1996-09-02

    The now traditional one day Christmas DNA Repair meeting was held at City University, London for the third year in succession. With over 130 participants and a programme consisting of a total of 24 pre-offered presentations the meeting reached record dimensions. Attendees were from 24 institutions throughout the United Kingdom, and with several distinct research groups contained within the large contingents from the ICRF Clare Hall Laboratories and the MRC Cell Mutation Unit in Brighton, this indicates the increasing interest and depth of UK research in DNA repair. One slight disappointment of the meeting was the fall in the numbers of non-UK participants. Although the meeting in 1994 (Strike, 1995) saw an increase in presentations from Continental Europe (six countries including France, Germany. The Netherlands and Switzerland), the trend did not continue this year, with only Denmark being represented. The 24 contributors consisted of approximately equal numbers of postgraduate students, postdoctoral researchers and more "established' scientists reflecting the continuing policy of encouraging younger members of the repair community to present their work. The mix of presenters was particularly well illustrated by two excellent and consecutive talks by Professor Bryn Bridges (MRC Cell Mutation Unit) and Alison Mitchell, a postgraduate student in Stephen West's laboratory (ICRF, Clare Hall). The organisms under study were as equally disparate and included Archaebacteria, Escherichia coli. Saccharomyces cerevisiae, Schizosaccharomyces pombe, Aspergillus, mice and men. The range of topics was also varied and included bacterial mutagenesis, NMR studies of Ada protein, preferential DNA repair, cell cycle checkpoint genes, reconstitution of nucleotide excision repair and V(D)J recombination in vitro, creation of repair deficient transgenic mice and mismatch defects in human cells. The result was a very successful meeting which was characterized by the consistently high

  3. Application of DNA based marker mutations for improvement of cereals and other sexually reproduced crop plants. Proceedings of a final research co-ordination meeting

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1998-03-01

    The Co-ordinated Research Programme (CRP) on the Application of DNA Based Marker Mutations for Improvement of Cereals and Other Sexually Reproduced Crop Plants represents the first of three CRPs dealing with the application of molecular markers to mutations and plant breeding and was implemented between 1992 and 1996. A second companion CRP entitled Use of Novel DNA Fingerprinting Techniques for the Detection and Characterization of Genetic Variation in Vegetatively Propagated Crops devoted to the application of molecular markers in vegetatively propagated crops species was implemented between 1993 and 1997. One positive consequence of these two CRPs has been the implementation of a third CRP entitled Radioactively Labeled DNA Probes for Crop Improvement, which began in 1995 and aims to provide enabling technologies, in the form of probes and primers, to laboratories in developing countries. The rapid development of molecular marker technologies has also resulted in a dramatic increase in request from developing Member States for technical co-operation projects utilizing molecular markers to improve local varieties for biotic and abiotic stresses and other traits of relevance. With the intensified use of induced mutations in genetic studies, it will be important to continue the important work of understanding induced mutations at the molecular level. Evidence of the progress made in implementing molecular marker technologies in laboratories around the world is presented in this publication, which contains the results presented by the participants at the fourth and final Research Co-ordination Meeting of the CRP held in Vienna, 4-8 November 1996. The FAO and IAEA wish to express their sincere appreciation to the participants of the meeting for their work during the project period resulting in the summary and scientific reports presented in this publication. Refs, figs, tabs.

  4. Application of DNA based marker mutations for improvement of cereals and other sexually reproduced crop plants. Proceedings of a final research co-ordination meeting

    International Nuclear Information System (INIS)

    1998-03-01

    The Co-ordinated Research Programme (CRP) on the Application of DNA Based Marker Mutations for Improvement of Cereals and Other Sexually Reproduced Crop Plants represents the first of three CRPs dealing with the application of molecular markers to mutations and plant breeding and was implemented between 1992 and 1996. A second companion CRP entitled Use of Novel DNA Fingerprinting Techniques for the Detection and Characterization of Genetic Variation in Vegetatively Propagated Crops devoted to the application of molecular markers in vegetatively propagated crops species was implemented between 1993 and 1997. One positive consequence of these two CRPs has been the implementation of a third CRP entitled Radioactively Labeled DNA Probes for Crop Improvement, which began in 1995 and aims to provide enabling technologies, in the form of probes and primers, to laboratories in developing countries. The rapid development of molecular marker technologies has also resulted in a dramatic increase in request from developing Member States for technical co-operation projects utilizing molecular markers to improve local varieties for biotic and abiotic stresses and other traits of relevance. With the intensified use of induced mutations in genetic studies, it will be important to continue the important work of understanding induced mutations at the molecular level. Evidence of the progress made in implementing molecular marker technologies in laboratories around the world is presented in this publication, which contains the results presented by the participants at the fourth and final Research Co-ordination Meeting of the CRP held in Vienna, 4-8 November 1996. The FAO and IAEA wish to express their sincere appreciation to the participants of the meeting for their work during the project period resulting in the summary and scientific reports presented in this publication

  5. Mitochondrial DNA mutations in human tumor cells

    OpenAIRE

    LI, HUI; HONG, ZE-HUI

    2012-01-01

    Mitochondria play significant roles in cellular energy metabolism, free radical generation and apoptosis. The dysfunction of mitochondria is correlated with the origin and progression of tumors; thus, mutations in the mitochondrial genome that affect mitochondrial function may be one of the causal factors of tumorigenesis. Although the role of mitochondrial DNA (mtDNA) mutations in carcinogenesis has been investigated extensively by various approaches, the conclusions remain controversial to ...

  6. Chimeric proteins for detection and quantitation of DNA mutations, DNA sequence variations, DNA damage and DNA mismatches

    Science.gov (United States)

    McCutchen-Maloney, Sandra L.

    2002-01-01

    Chimeric proteins having both DNA mutation binding activity and nuclease activity are synthesized by recombinant technology. The proteins are of the general formula A-L-B and B-L-A where A is a peptide having DNA mutation binding activity, L is a linker and B is a peptide having nuclease activity. The chimeric proteins are useful for detection and identification of DNA sequence variations including DNA mutations (including DNA damage and mismatches) by binding to the DNA mutation and cutting the DNA once the DNA mutation is detected.

  7. Roles of Mitochondrial DNA Mutations in Stem Cell Ageing

    Directory of Open Access Journals (Sweden)

    Tianhong Su

    2018-03-01

    Full Text Available Mitochondrial DNA (mtDNA mutations accumulate in somatic stem cells during ageing and cause mitochondrial dysfunction. In this review, we summarize the studies that link mtDNA mutations to stem cell ageing. We discuss the age-related behaviours of the somatic mtDNA mutations in stem cell populations and how they potentially contribute to stem cell ageing by altering mitochondrial properties in humans and in mtDNA-mutator mice. We also draw attention to the diverse fates of the mtDNA mutations with different origins during ageing, with potential selective pressures on the germline inherited but not the somatic mtDNA mutations.

  8. Spontaneous mutation by mutagenic repair of spontaneous lesions in DNA

    International Nuclear Information System (INIS)

    Hastings, P.J.; Quah, S.-K.; Borstel, R.C. von

    1976-01-01

    It is stated that strains of yeast carrying mutations in many of the steps in pathways repairing radiation-induced damage to DNA have enhanced spontaneous mutation rates. Most strains isolated because they have enhanced spontaneous mutation carry mutations in DNA repair systems. This suggests that much spontaneous mutation arises by mutagenic repair of spontaneous lesions. (author)

  9. Replicative DNA polymerase mutations in cancer☆

    Science.gov (United States)

    Heitzer, Ellen; Tomlinson, Ian

    2014-01-01

    Three DNA polymerases — Pol α, Pol δ and Pol ɛ — are essential for DNA replication. After initiation of DNA synthesis by Pol α, Pol δ or Pol ɛ take over on the lagging and leading strand respectively. Pol δ and Pol ɛ perform the bulk of replication with very high fidelity, which is ensured by Watson–Crick base pairing and 3′exonuclease (proofreading) activity. Yeast models have shown that mutations in the exonuclease domain of Pol δ and Pol ɛ homologues can cause a mutator phenotype. Recently, we identified germline exonuclease domain mutations (EDMs) in human POLD1 and POLE that predispose to ‘polymerase proofreading associated polyposis’ (PPAP), a disease characterised by multiple colorectal adenomas and carcinoma, with high penetrance and dominant inheritance. Moreover, somatic EDMs in POLE have also been found in sporadic colorectal and endometrial cancers. Tumors with EDMs are microsatellite stable and show an ‘ultramutator’ phenotype, with a dramatic increase in base substitutions. PMID:24583393

  10. Replicative DNA polymerase mutations in cancer.

    Science.gov (United States)

    Heitzer, Ellen; Tomlinson, Ian

    2014-02-01

    Three DNA polymerases - Pol α, Pol δ and Pol ɛ - are essential for DNA replication. After initiation of DNA synthesis by Pol α, Pol δ or Pol ɛ take over on the lagging and leading strand respectively. Pol δ and Pol ɛ perform the bulk of replication with very high fidelity, which is ensured by Watson-Crick base pairing and 3'exonuclease (proofreading) activity. Yeast models have shown that mutations in the exonuclease domain of Pol δ and Pol ɛ homologues can cause a mutator phenotype. Recently, we identified germline exonuclease domain mutations (EDMs) in human POLD1 and POLE that predispose to 'polymerase proofreading associated polyposis' (PPAP), a disease characterised by multiple colorectal adenomas and carcinoma, with high penetrance and dominant inheritance. Moreover, somatic EDMs in POLE have also been found in sporadic colorectal and endometrial cancers. Tumors with EDMs are microsatellite stable and show an 'ultramutator' phenotype, with a dramatic increase in base substitutions. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  11. Tumor‐associated DNA mutation detection in individuals undergoing colonoscopy

    OpenAIRE

    Fleshner, Phillip; Braunstein, Glenn D.; Ovsepyan, Gayane; Tonozzi, Theresa R.; Kammesheidt, Anja

    2017-01-01

    Abstract The majority of colorectal cancers (CRC) harbor somatic mutations and epigenetic modifications in the tumor tissue, and some of these mutations can be detected in plasma as circulating tumor DNA (ctDNA). Precancerous colorectal lesions also contain many of these same mutations. This study examined plasma for ctDNA from patients undergoing a screening or diagnostic colonoscopy to determine the sensitivity and specificity of the ctDNA panel for detecting CRC and precancerous lesions. T...

  12. Compound mitochondrial DNA mutations in a neurological patient ...

    Indian Academy of Sciences (India)

    Compound mitochondrial DNA mutations in a neurological patient with ataxia, myoclonus and deafness. Ji Hoon Park, Bo Ram Yoon, Hye Jin Kim, Phil Hyu Lee, Byung-Ok Choi and Ki Wha Chung. J. Genet. 93, 173–177. Table 1. Variations from the whole mtDNA sequence in the AMDF patient. Mutation. Report. Locus/ ...

  13. Presence of a consensus DNA motif at nearby DNA sequence of the mutation susceptible CG nucleotides.

    Science.gov (United States)

    Chowdhury, Kaushik; Kumar, Suresh; Sharma, Tanu; Sharma, Ankit; Bhagat, Meenakshi; Kamai, Asangla; Ford, Bridget M; Asthana, Shailendra; Mandal, Chandi C

    2018-01-10

    Complexity in tissues affected by cancer arises from somatic mutations and epigenetic modifications in the genome. The mutation susceptible hotspots present within the genome indicate a non-random nature and/or a position specific selection of mutation. An association exists between the occurrence of mutations and epigenetic DNA methylation. This study is primarily aimed at determining mutation status, and identifying a signature for predicting mutation prone zones of tumor suppressor (TS) genes. Nearby sequences from the top five positions having a higher mutation frequency in each gene of 42 TS genes were selected from a cosmic database and were considered as mutation prone zones. The conserved motifs present in the mutation prone DNA fragments were identified. Molecular docking studies were done to determine putative interactions between the identified conserved motifs and enzyme methyltransferase DNMT1. Collective analysis of 42 TS genes found GC as the most commonly replaced and AT as the most commonly formed residues after mutation. Analysis of the top 5 mutated positions of each gene (210 DNA segments for 42 TS genes) identified that CG nucleotides of the amino acid codons (e.g., Arginine) are most susceptible to mutation, and found a consensus DNA "T/AGC/GAGGA/TG" sequence present in these mutation prone DNA segments. Similar to TS genes, analysis of 54 oncogenes not only found CG nucleotides of the amino acid Arg as the most susceptible to mutation, but also identified the presence of similar consensus DNA motifs in the mutation prone DNA fragments (270 DNA segments for 54 oncogenes) of oncogenes. Docking studies depicted that, upon binding of DNMT1 methylates to this consensus DNA motif (C residues of CpG islands), mutation was likely to occur. Thus, this study proposes that DNMT1 mediated methylation in chromosomal DNA may decrease if a foreign DNA segment containing this consensus sequence along with CG nucleotides is exogenously introduced to dividing

  14. Mutated genes as research tool

    International Nuclear Information System (INIS)

    1981-01-01

    Green plants are the ultimate source of all resources required for man's life, his food, his clothes, and almost all his energy requirements. Primitive prehistoric man could live from the abundance of nature surrounding him. Man today, dominating nature in terms of numbers and exploiting its limited resources, cannot exist without employing his intelligence to direct natural evolution. Plant sciences, therefore, are not a matter of curiosity but an essential requirement. From such considerations, the IAEA and FAO jointly organized a symposium to assess the value of mutation research for various kinds of plant science, which directly or indirectly might contribute to sustaining and improving crop production. The benefit through developing better cultivars that plant breeders can derive from using the additional genetic resources resulting from mutation induction has been assessed before at other FAO/IAEA meetings (Rome 1964, Pullman 1969, Ban 1974, Ibadan 1978) and is also monitored in the Mutation Breeding Newsletter, published by IAEA twice a year. Several hundred plant cultivars which carry economically important characters because their genes have been altered by ionizing radiation or other mutagens, are grown by farmers and horticulturists in many parts of the world. But the benefit derived from such mutant varieties is without any doubt surpassed by the contribution which mutation research has made towards the advancement of genetics. For this reason, a major part of the papers and discussions at the symposium dealt with the role induced-mutation research played in providing insight into gene action and gene interaction, the organization of genes in plant chromosomes in view of homology and homoeology, the evolutionary role of gene duplication and polyploidy, the relevance of gene blocks, the possibilities for chromosome engineering, the functioning of cytroplasmic inheritance and the genetic dynamics of populations. In discussing the evolutionary role of

  15. RADIA: RNA and DNA integrated analysis for somatic mutation detection.

    Directory of Open Access Journals (Sweden)

    Amie J Radenbaugh

    Full Text Available The detection of somatic single nucleotide variants is a crucial component to the characterization of the cancer genome. Mutation calling algorithms thus far have focused on comparing the normal and tumor genomes from the same individual. In recent years, it has become routine for projects like The Cancer Genome Atlas (TCGA to also sequence the tumor RNA. Here we present RADIA (RNA and DNA Integrated Analysis, a novel computational method combining the patient-matched normal and tumor DNA with the tumor RNA to detect somatic mutations. The inclusion of the RNA increases the power to detect somatic mutations, especially at low DNA allelic frequencies. By integrating an individual's DNA and RNA, we are able to detect mutations that would otherwise be missed by traditional algorithms that examine only the DNA. We demonstrate high sensitivity (84% and very high precision (98% and 99% for RADIA in patient data from endometrial carcinoma and lung adenocarcinoma from TCGA. Mutations with both high DNA and RNA read support have the highest validation rate of over 99%. We also introduce a simulation package that spikes in artificial mutations to patient data, rather than simulating sequencing data from a reference genome. We evaluate sensitivity on the simulation data and demonstrate our ability to rescue back mutations at low DNA allelic frequencies by including the RNA. Finally, we highlight mutations in important cancer genes that were rescued due to the incorporation of the RNA.

  16. DNA mutation motifs in the genes associated with inherited diseases.

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    Michal Růžička

    Full Text Available Mutations in human genes can be responsible for inherited genetic disorders and cancer. Mutations can arise due to environmental factors or spontaneously. It has been shown that certain DNA sequences are more prone to mutate. These sites are termed hotspots and exhibit a higher mutation frequency than expected by chance. In contrast, DNA sequences with lower mutation frequencies than expected by chance are termed coldspots. Mutation hotspots are usually derived from a mutation spectrum, which reflects particular population where an effect of a common ancestor plays a role. To detect coldspots/hotspots unaffected by population bias, we analysed the presence of germline mutations obtained from HGMD database in the 5-nucleotide segments repeatedly occurring in genes associated with common inherited disorders, in particular, the PAH, LDLR, CFTR, F8, and F9 genes. Statistically significant sequences (mutational motifs rarely associated with mutations (coldspots and frequently associated with mutations (hotspots exhibited characteristic sequence patterns, e.g. coldspots contained purine tract while hotspots showed alternating purine-pyrimidine bases, often with the presence of CpG dinucleotide. Using molecular dynamics simulations and free energy calculations, we analysed the global bending properties of two selected coldspots and two hotspots with a G/T mismatch. We observed that the coldspots were inherently more flexible than the hotspots. We assume that this property might be critical for effective mismatch repair as DNA with a mutation recognized by MutSα protein is noticeably bent.

  17. Mitochondrial DNA mutations in mutator mice confer respiration defects and B-cell lymphoma development.

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    Takayuki Mito

    Full Text Available Mitochondrial DNA (mtDNA mutator mice are proposed to express premature aging phenotypes including kyphosis and hair loss (alopecia due to their carrying a nuclear-encoded mtDNA polymerase with a defective proofreading function, which causes accelerated accumulation of random mutations in mtDNA, resulting in expression of respiration defects. On the contrary, transmitochondrial mito-miceΔ carrying mtDNA with a large-scale deletion mutation (ΔmtDNA also express respiration defects, but not express premature aging phenotypes. Here, we resolved this discrepancy by generating mtDNA mutator mice sharing the same C57BL/6J (B6J nuclear background with that of mito-miceΔ. Expression patterns of premature aging phenotypes are very close, when we compared between homozygous mtDNA mutator mice carrying a B6J nuclear background and selected mito-miceΔ only carrying predominant amounts of ΔmtDNA, in their expression of significant respiration defects, kyphosis, and a short lifespan, but not the alopecia. Therefore, the apparent discrepancy in the presence and absence of premature aging phenotypes in mtDNA mutator mice and mito-miceΔ, respectively, is partly the result of differences in the nuclear background of mtDNA mutator mice and of the broad range of ΔmtDNA proportions of mito-miceΔ used in previous studies. We also provided direct evidence that mtDNA abnormalities in homozygous mtDNA mutator mice are responsible for respiration defects by demonstrating the co-transfer of mtDNA and respiration defects from mtDNA mutator mice into mtDNA-less (ρ(0 mouse cells. Moreover, heterozygous mtDNA mutator mice had a normal lifespan, but frequently developed B-cell lymphoma, suggesting that the mtDNA abnormalities in heterozygous mutator mice are not sufficient to induce a short lifespan and aging phenotypes, but are able to contribute to the B-cell lymphoma development during their prolonged lifespan.

  18. Polymorphism and mutation analysis of genomic DNA on cancer

    International Nuclear Information System (INIS)

    Ohta, Tsutomu

    2003-01-01

    DNA repair is a universal process in living cells that maintains the structural integrity of chromosomal DNA molecules in face of damage. A deficiency in DNA damage repair is associated with an increased cancer risk by increasing a mutation frequency of cancer-related genes. Variation in DNA repair capacity may be genetically determined. Therefore, we searched single-nucleotide polymorphisms (SNPs) in major DNA repair genes. This led to the finding of 600 SNPs and mutations including many novel SNPs in Japanese population. Case-control studies to explore the contribution of the SNPs in DNA repair genes to the risk of lung cancer revealed that five SNPs are associated with lung carcinogenesis. One of these SNPs is found in RAD54L gene, which is involved in double-strand DNA repair. We analyzed and reported activities of Rad54L protein with SNP and mutations. (authors)

  19. Performance of mitochondrial DNA mutations detecting early stage cancer

    International Nuclear Information System (INIS)

    Jakupciak, John P; Srivastava, Sudhir; Sidransky, David; O'Connell, Catherine D; Maragh, Samantha; Markowitz, Maura E; Greenberg, Alissa K; Hoque, Mohammad O; Maitra, Anirban; Barker, Peter E; Wagner, Paul D; Rom, William N

    2008-01-01

    Mutations in the mitochondrial genome (mtgenome) have been associated with cancer and many other disorders. These mutations can be point mutations or deletions, or admixtures (heteroplasmy). The detection of mtDNA mutations in body fluids using resequencing microarrays, which are more sensitive than other sequencing methods, could provide a strategy to measure mutation loads in remote anatomical sites. We determined the mtDNA mutation load in the entire mitochondrial genome of 26 individuals with different early stage cancers (lung, bladder, kidney) and 12 heavy smokers without cancer. MtDNA was sequenced from three matched specimens (blood, tumor and body fluid) from each cancer patient and two matched specimens (blood and sputum) from smokers without cancer. The inherited wildtype sequence in the blood was compared to the sequences present in the tumor and body fluid, detected using the Affymetrix Genechip ® Human Mitochondrial Resequencing Array 1.0 and supplemented by capillary sequencing for noncoding region. Using this high-throughput method, 75% of the tumors were found to contain mtDNA mutations, higher than in our previous studies, and 36% of the body fluids from these cancer patients contained mtDNA mutations. Most of the mutations detected were heteroplasmic. A statistically significantly higher heteroplasmy rate occurred in tumor specimens when compared to both body fluid of cancer patients and sputum of controls, and in patient blood compared to blood of controls. Only 2 of the 12 sputum specimens from heavy smokers without cancer (17%) contained mtDNA mutations. Although patient mutations were spread throughout the mtDNA genome in the lung, bladder and kidney series, a statistically significant elevation of tRNA and ND complex mutations was detected in tumors. Our findings indicate comprehensive mtDNA resequencing can be a high-throughput tool for detecting mutations in clinical samples with potential applications for cancer detection, but it is

  20. Self-cytoplasmic DNA upregulates the mutator enzyme APOBEC3A leading to chromosomal DNA damage.

    Science.gov (United States)

    Suspène, Rodolphe; Mussil, Bianka; Laude, Hélène; Caval, Vincent; Berry, Noémie; Bouzidi, Mohamed S; Thiers, Valérie; Wain-Hobson, Simon; Vartanian, Jean-Pierre

    2017-04-07

    Foreign and self-cytoplasmic DNA are recognized by numerous DNA sensor molecules leading to the production of type I interferons. Such DNA agonists should be degraded otherwise cells would be chronically stressed. Most human APOBEC3 cytidine deaminases can initiate catabolism of cytoplasmic mitochondrial DNA. Using the human myeloid cell line THP-1 with an interferon inducible APOBEC3A gene, we show that cytoplasmic DNA triggers interferon α and β production through the RNA polymerase III transcription/RIG-I pathway leading to massive upregulation of APOBEC3A. By catalyzing C→U editing in single stranded DNA fragments, the enzyme prevents them from re-annealing so attenuating the danger signal. The price to pay is chromosomal DNA damage in the form of CG→TA mutations and double stranded DNA breaks which, in the context of chronic inflammation, could drive cells down the path toward cancer. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  1. Impact of Emergent Circulating Tumor DNA RAS Mutation in Panitumumab-Treated Chemoresistant Metastatic Colorectal Cancer.

    Science.gov (United States)

    Kim, Tae Won; Peeters, Marc; Thomas, Anne L; Gibbs, Peter; Hool, Kristina; Zhang, Jianqi; Ang, Agnes; Bach, Bruce Allen; Price, Timothy

    2018-06-13

    The accumulation of emergent RAS mutations during anti-epidermal growth factor receptor (EGFR) therapy is of interest as a mechanism for acquired resistance to anti-EGFR treatment. Plasma analysis of circulating tumor (ct) DNA is a minimally invasive and highly sensitive method to determine RAS mutational status. This biomarker analysis of the global phase III ASPECCT study used next-generation sequencing to detect expanded RAS ctDNA mutations in panitumumab-treated patients. Plasma samples collected at baseline and posttreatment were analyzed categorically for the presence of RAS mutations by the Plasma Select -R™ 64-gene panel at 0.1% sensitivity. Among panitumumab-treated patients with evaluable plasma samples at baseline (n = 238), 188 (79%) were wild-type (WT) RAS, and 50 (21%) were mutant RAS Of the 188 patients with baseline ctDNA WT RAS status, 164 had evaluable posttreatment results with a 32% rate of emergent RAS mutations. The median overall survival (OS) for WT and RAS mutant status by ctDNA at baseline was 13.7 (95% confidence interval: 11.5-15.4) and 7.9 months (6.4-9.6), respectively ( P < 0.0001). Clinical outcomes were not significantly different between patients with and without emergent ctDNA RAS mutations. Although patients with baseline ctDNA RAS mutations had worse outcomes than patients who were WT RAS before initiating treatment, emergent ctDNA RAS mutations were not associated with less favorable patient outcomes in panitumumab-treated patients. Further research is needed to determine a clinically relevant threshold for baseline and emergent ctDNA RAS mutations. Copyright ©2018, American Association for Cancer Research.

  2. Current study on ionizing radiation-induced mitochondial DNA damage and mutations

    International Nuclear Information System (INIS)

    Zhou Xin; Wang Zhenhua; Zhang Hong

    2012-01-01

    Current advance in ionizing radiation-induced mitochondrial DNA damage and mutations is reviewed, in addition with the essential differences between mtDNA and nDNA damage and mutations. To extent the knowledge about radiation induced mitochondrial alterations, the researchers in Institute of Modern Physics, Chinese Academy of Sciences developed some technics such as real-time PCR, long-PCR for accurate quantification of radiation induced damage and mutations, and in-depth investigation about the functional changes of mitochondria based on mtDNA damage and mutations were also carried out. In conclusion, the important role of mitochondrial study in radiation biology is underlined, and further study on mitochondrial study associated with late effect and metabolism changes in radiation biology is pointed out. (authors)

  3. A comprehensive characterization of mitochondrial DNA mutations in glioblastoma multiforme.

    Science.gov (United States)

    Vidone, Michele; Clima, Rosanna; Santorsola, Mariangela; Calabrese, Claudia; Girolimetti, Giulia; Kurelac, Ivana; Amato, Laura Benedetta; Iommarini, Luisa; Trevisan, Elisa; Leone, Marco; Soffietti, Riccardo; Morra, Isabella; Faccani, Giuliano; Attimonelli, Marcella; Porcelli, Anna Maria; Gasparre, Giuseppe

    2015-06-01

    Glioblastoma multiforme (GBM) is the most malignant brain cancer in adults, with a poor prognosis, whose molecular stratification still represents a challenge in pathology and clinics. On the other hand, mitochondrial DNA (mtDNA) mutations have been found in most tumors as modifiers of the bioenergetics state, albeit in GBM a characterization of the mtDNA status is lacking to date. Here, a characterization of the burden of mtDNA mutations in GBM samples was performed. First, investigation of tumor-specific vs. non tumor-specific mutations was carried out with the MToolBox bioinformatics pipeline by analyzing 45 matched tumor/blood samples, from whole genome or whole exome sequencing datasets obtained from The Cancer Genome Atlas (TCGA) consortium. Additionally, the entire mtDNA sequence was obtained in a dataset of 104 fresh-frozen GBM samples. Mitochondrial mutations with potential pathogenic interest were prioritized based on heteroplasmic fraction, nucleotide variability, and in silico prediction of pathogenicity. A preliminary biochemical analysis of the activity of mitochondrial respiratory complexes was also performed on fresh-frozen GBM samples. Although a high number of mutations was detected, we report that the large majority of them does not pass the prioritization filters. Therefore, a relatively limited burden of pathogenic mutations is indeed carried by GBM, which did not appear to determine a general impairment of the respiratory chain. This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Exploring the common molecular basis for the universal DNA mutation bias: Revival of Loewdin mutation model

    International Nuclear Information System (INIS)

    Fu, Liang-Yu; Wang, Guang-Zhong; Ma, Bin-Guang; Zhang, Hong-Yu

    2011-01-01

    Highlights: → There exists a universal G:C → A:T mutation bias in three domains of life. → This universal mutation bias has not been sufficiently explained. → A DNA mutation model proposed by Loewdin 40 years ago offers a common explanation. -- Abstract: Recently, numerous genome analyses revealed the existence of a universal G:C → A:T mutation bias in bacteria, fungi, plants and animals. To explore the molecular basis for this mutation bias, we examined the three well-known DNA mutation models, i.e., oxidative damage model, UV-radiation damage model and CpG hypermutation model. It was revealed that these models cannot provide a sufficient explanation to the universal mutation bias. Therefore, we resorted to a DNA mutation model proposed by Loewdin 40 years ago, which was based on inter-base double proton transfers (DPT). Since DPT is a fundamental and spontaneous chemical process and occurs much more frequently within GC pairs than AT pairs, Loewdin model offers a common explanation for the observed universal mutation bias and thus has broad biological implications.

  5. Human Genome Research: Decoding DNA

    Science.gov (United States)

    dropdown arrow Site Map A-Z Index Menu Synopsis Human Genome Research: Decoding DNA Resources with of the DNA double helix during April 2003. James D. Watson, Francis Crick, and Maurice Wilkins were company Celera announced the completion of a "working draft" reference DNA sequence of the human

  6. Mitochondrial DNA Mutation Associated with Leber's Hereditary Optic Neuropathy

    Science.gov (United States)

    Wallace, Douglas C.; Singh, Gurparkash; Lott, Marie T.; Hodge, Judy A.; Schurr, Theodore G.; Lezza, Angela M. S.; Elsas, Louis J.; Nikoskelainen, Eeva K.

    1988-12-01

    Leber's hereditary optic neuropathy is a maternally inherited disease resulting in optic nerve degeneration and cardiac dysrhythmia. A mitochondrial DNA replacement mutation was identified that correlated with this disease in multiple families. This mutation converted a highly conserved arginine to a histidine at codon 340 in the NADH dehydrogenase subunit 4 gene and eliminated an Sfa NI site, thus providing a simple diagnostic test. This finding demonstrated that a nucleotide change in a mitochondrial DNA energy production gene can result in a neurological disease.

  7. Prospects for DNA methods to measure human heritable mutation rates

    International Nuclear Information System (INIS)

    Mendelsohn, M.L.

    1985-01-01

    A workshop cosponsored by ICPEMC and the US Department of Energy was held in Alta, Utah, December 9-13, 1984 to examine the extent to which DNA-oriented methods might provide new approaches to the important but intractable problem of measuring mutation rates in control and exposed human populations. The workshop identified and analyzed six DNA methods for detection of human heritable mutation, including several created at the meeting, and concluded that none of the methods combine sufficient feasibility and efficiency to be recommended for general application. 8 refs

  8. Mitochondrial DNA mutation load in a family with the m.8344A>G point mutation and lipomas

    DEFF Research Database (Denmark)

    Jeppesen, Tina Dysgaard; Al-Hashimi, Noor; Duno, Morten

    2017-01-01

    Studies have shown that difference in mtDNA mutation load among tissues is a result of postnatal modification. We present five family members with the m.8344A>G with variable phenotypes but uniform intrapersonal distribution of mutation load, indicating that there is no postnatal modification of mt......DNA mutation load in this genotype....

  9. Review: Clinical aspects of hereditary DNA Mismatch repair gene mutations

    NARCIS (Netherlands)

    Sijmons, Rolf H.; Hofstra, Robert M. W.

    Inherited mutations of the DNA Mismatch repair genes MLH1, MSH2, MSH6 and PMS2 can result in two hereditary tumor syndromes: the adult-onset autosomal dominant Lynch syndrome, previously referred to as Hereditary Non-Polyposis Colorectal Cancer (HNPCC) and the childhood-onset autosomal recessive

  10. Mitochondrial DNA Mutations in Epithelial Ovarian Tumor Progression

    Science.gov (United States)

    2007-12-01

    Panici PL, Fazio VM: Mutations of D310 mitochondrial mononu- cleotide repeat in primary tumors and cytological speci- mens . Cancer Lett 2003, 190:73...BR: Detection of LOH and mitochondrial DNA alter- ations in ductal lavage and nipple aspirate fluids from high- risk patients. Breast Cancer Res

  11. Mutation analysis with random DNA identifiers (MARDI) catalogs Pig-a mutations in heterogeneous pools of CD48-deficient T cells derived from DMBA-treated rats.

    Science.gov (United States)

    Revollo, Javier R; Crabtree, Nathaniel M; Pearce, Mason G; Pacheco-Martinez, M Monserrat; Dobrovolsky, Vasily N

    2016-03-01

    Identification of mutations induced by xenotoxins is a common task in the field of genetic toxicology. Mutations are often detected by clonally expanding potential mutant cells and genotyping each viable clone by Sanger sequencing. Such a "clone-by-clone" approach requires significant time and effort, and sometimes is even impossible to implement. Alternative techniques for efficient mutation identification would greatly benefit both basic and regulatory genetic toxicology research. Here, we report the development of Mutation Analysis with Random DNA Identifiers (MARDI), a novel high-fidelity Next Generation Sequencing (NGS) approach that circumvents clonal expansion and directly catalogs mutations in pools of mutant cells. MARDI uses oligonucleotides carrying Random DNA Identifiers (RDIs) to tag progenitor DNA molecules before PCR amplification, enabling clustering of descendant DNA molecules and eliminating NGS- and PCR-induced sequencing artifacts. When applied to the Pig-a cDNA analysis of heterogeneous pools of CD48-deficient T cells derived from DMBA-treated rats, MARDI detected nearly all Pig-a mutations that were previously identified by conventional clone-by-clone analysis and discovered many additional ones consistent with DMBA exposure: mostly A to T transversions, with the mutated A located on the non-transcribed DNA strand. © 2015 Wiley Periodicals, Inc.

  12. Optimal control of gene mutation in DNA replication.

    Science.gov (United States)

    Yu, Juanyi; Li, Jr-Shin; Tarn, Tzyh-Jong

    2012-01-01

    We propose a molecular-level control system view of the gene mutations in DNA replication from the finite field concept. By treating DNA sequences as state variables, chemical mutagens and radiation as control inputs, one cell cycle as a step increment, and the measurements of the resulting DNA sequence as outputs, we derive system equations for both deterministic and stochastic discrete-time, finite-state systems of different scales. Defining the cost function as a summation of the costs of applying mutagens and the off-trajectory penalty, we solve the deterministic and stochastic optimal control problems by dynamic programming algorithm. In addition, given that the system is completely controllable, we find that the global optimum of both base-to-base and codon-to-codon deterministic mutations can always be achieved within a finite number of steps.

  13. Fidelity and mutational spectrum of Pfu DNA polymerase on a human mitochondrial DNA sequence.

    Science.gov (United States)

    André, P; Kim, A; Khrapko, K; Thilly, W G

    1997-08-01

    The study of rare genetic changes in human tissues requires specialized techniques. Point mutations at fractions at or below 10(-6) must be observed to discover even the most prominent features of the point mutational spectrum. PCR permits the increase in number of mutant copies but does so at the expense of creating many additional mutations or "PCR noise". Thus, each DNA sequence studied must be characterized with regard to the DNA polymerase and conditions used to avoid interpreting a PCR-generated mutation as one arising in human tissue. The thermostable DNA polymerase derived from Pyrococcus furiosus designated Pfu has the highest fidelity of any DNA thermostable polymerase studied to date, and this property recommends it for analyses of tissue mutational spectra. Here, we apply constant denaturant capillary electrophoresis (CDCE) to separate and isolate the products of DNA amplification. This new strategy permitted direct enumeration and identification of point mutations created by Pfu DNA polymerase in a 96-bp low melting domain of a human mitochondrial sequence despite the very low mutant fractions generated in the PCR process. This sequence, containing part of the tRNA glycine and NADH dehydrogenase subunit 3 genes, is the target of our studies of mitochondrial mutagenesis in human cells and tissues. Incorrectly synthesized sequences were separated from the wild type as mutant/wild-type heteroduplexes by sequential enrichment on CDCE. An artificially constructed mutant was used as an internal standard to permit calculation of the mutant fraction. Our study found that the average error rate (mutations per base pair duplication) of Pfu was 6.5 x 10(-7), and five of its more frequent mutations (hot spots) consisted of three transversions (GC-->TA, AT-->TA, and AT-->CG), one transition (AT-->GC), and one 1-bp deletion (in an AAAAAA sequence). To achieve an even higher sensitivity, the amount of Pfu-induced mutants must be reduced.

  14. 21 CFR 864.7280 - Factor V Leiden DNA mutation detection systems.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Factor V Leiden DNA mutation detection systems....7280 Factor V Leiden DNA mutation detection systems. (a) Identification. Factor V Leiden deoxyribonucleic acid (DNA) mutation detection systems are devices that consist of different reagents and...

  15. Forty years of mutation breeding in Japan. Research and fruits

    International Nuclear Information System (INIS)

    Yamaguchi, Isao

    2003-01-01

    The radiation source used for breeding in the early years was mainly X rays. After the 2nd World War, gamma ray sources such as 60 Co and 137 Cs came to take a leading role in radiation breeding. The institute of Radiation Breeding (IRB) of the Ministry of Agriculture, Forestry and Fisheries (MAFF) was established on April 16, 1960. A gamma field with 2000Ci of a 60 Co source, the main irradiation facility of the IRB, was installed to study the genetically responses of crop plants to chronic exposures of ionizing radiation and their practical application to plant breeding. This paper consisted of 'forty years of research on radiobiology and mutation breeding in Japan', 'topics of mutation breeding research in IRB', 'outline of released varieties by mutation breeding' and 'future of mutation breeding'. The number of varieties released by the direct use of induced mutation in Japan amounts to 163 as of November 2001. Crops in which mutant varieties have been released range widely: rice and other cereals, industrial crops, forage crops, vegetables, ornamentals, mushrooms and fruit trees, the number of which reaches 48. The number of mutant varieties is highest (31) in chrysanthemum, followed by 22 in rice and 13 in soybean. By the indirect use of mutants, a total of 15 varieties of wheat, barley, soybean, mat rush and tomato have been registered by MAFF. Recent advances in biotechnological techniques have made it possible to determine DNA sequences of mutant genes. Accumulating information of DNA sequences and other molecular aspects of many mutant genes will throw light on the mechanisms of mutation induction and develop a new field of mutation breeding. (S.Y.)

  16. Aging of hematopoietic stem cells: DNA damage and mutations?

    Science.gov (United States)

    Moehrle, Bettina M; Geiger, Hartmut

    2016-10-01

    Aging in the hematopoietic system and the stem cell niche contributes to aging-associated phenotypes of hematopoietic stem cells (HSCs), including leukemia and aging-associated immune remodeling. Among others, the DNA damage theory of aging of HSCs is well established, based on the detection of a significantly larger amount of γH2AX foci and a higher tail moment in the comet assay, both initially thought to be associated with DNA damage in aged HSCs compared with young cells, and bone marrow failure in animals devoid of DNA repair factors. Novel data on the increase in and nature of DNA mutations in the hematopoietic system with age, the quality of the DNA damage response in aged HSCs, and the nature of γH2AX foci question a direct link between DNA damage and the DNA damage response and aging of HSCs, and rather favor changes in epigenetics, splicing-factors or three-dimensional architecture of the cell as major cell intrinsic factors of HSCs aging. Aging of HSCs is also driven by a strong contribution of aging of the niche. This review discusses the DNA damage theory of HSC aging in the light of these novel mechanisms of aging of HSCs. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  17. The influence of DNA repair inhibitors on the mutation rate

    International Nuclear Information System (INIS)

    Auzinger, Th.; Hruby, R.

    1980-12-01

    The simultaneous influence of gamma-radiation and DNA-repair inhibiting substances on the mutation frequency of mice was investigated in vivo with the micronucleus test. The detergens Tween 80, vitamin A, and the antiphlogisticum phenylbutazone were used as DNA-repair inhibiting substances. Using the same irradiation doses, a statistic significant increase of mutagenicity respectively micronucleus frequency was found in high concentrations of Tween 80 and in all used dosages of vitamin A, but not in phenylbutazone and in low concentrations of tween. (auth.)

  18. R248Q mutation--Beyond p53-DNA binding.

    Science.gov (United States)

    Ng, Jeremy W K; Lama, Dilraj; Lukman, Suryani; Lane, David P; Verma, Chandra S; Sim, Adelene Y L

    2015-12-01

    R248 in the DNA binding domain (DBD) of p53 interacts directly with the minor groove of DNA. Earlier nuclear magnetic resonance (NMR) studies indicated that the R248Q mutation resulted in conformation changes in parts of DBD far from the mutation site. However, how information propagates from the mutation site to the rest of the DBD is still not well understood. We performed a series of all-atom molecular dynamics (MD) simulations to dissect sterics and charge effects of R248 on p53-DBD conformation: (i) wild-type p53 DBD; (ii) p53 DBD with an electrically neutral arginine side-chain; (iii) p53 DBD with R248A; (iv) p53 DBD with R248W; and (v) p53 DBD with R248Q. Our results agree well with experimental observations of global conformational changes induced by the R248Q mutation. Our simulations suggest that both charge- and sterics are important in the dynamics of the loop (L3) where the mutation resides. We show that helix 2 (H2) dynamics is altered as a result of a change in the hydrogen bonding partner of D281. In turn, neighboring L1 dynamics is altered: in mutants, L1 predominantly adopts the recessed conformation and is unable to interact with the major groove of DNA. We focused our attention the R248Q mutant that is commonly found in a wide range of cancer and observed changes at the zinc-binding pocket that might account for the dominant negative effects of R248Q. Furthermore, in our simulations, the S6/S7 turn was more frequently solvent exposed in R248Q, suggesting that there is a greater tendency of R248Q to partially unfold and possibly lead to an increased aggregation propensity. Finally, based on the observations made in our simulations, we propose strategies for the rescue of R248Q mutants. © 2015 Wiley Periodicals, Inc.

  19. Mitochondrial DNA deletion mutations in adult mouse cardiac side population cells

    International Nuclear Information System (INIS)

    Lushaj, Entela B.; Lozonschi, Lucian; Barnes, Maria; Anstadt, Emily; Kohmoto, Takushi

    2012-01-01

    We investigated the presence and potential role of mitochondrial DNA (mtDNA) deletion mutations in adult cardiac stem cells. Cardiac side population (SP) cells were isolated from 12-week-old mice. Standard polymerase chain reaction (PCR) was used to screen for the presence of mtDNA deletion mutations in (a) freshly isolated SP cells and (b) SP cells cultured to passage 10. When present, the abundance of mtDNA deletion mutation was analyzed in single cell colonies. The effect of different levels of deletion mutations on SP cell growth and differentiation was determined. MtDNA deletion mutations were found in both freshly isolated and cultured cells from 12-week-old mice. While there was no significant difference in the number of single cell colonies with mtDNA deletion mutations from any of the groups mentioned above, the abundance of mtDNA deletion mutations was significantly higher in the cultured cells, as determined by quantitative PCR. Within a single clonal cell population, the detectable mtDNA deletion mutations were the same in all cells and unique when compared to deletions of other colonies. We also found that cells harboring high levels of mtDNA deletion mutations (i.e. where deleted mtDNA comprised more than 60% of total mtDNA) had slower proliferation rates and decreased differentiation capacities. Screening cultured adult stem cells for mtDNA deletion mutations as a routine assessment will benefit the biomedical application of adult stem cells.

  20. cDNA sequencing improves the detection of P53 missense mutations in colorectal cancer

    International Nuclear Information System (INIS)

    Szybka, Malgorzata; Kordek, Radzislaw; Zakrzewska, Magdalena; Rieske, Piotr; Pasz-Walczak, Grazyna; Kulczycka-Wojdala, Dominika; Zawlik, Izabela; Stawski, Robert; Jesionek-Kupnicka, Dorota; Liberski, Pawel P

    2009-01-01

    Recently published data showed discrepancies beteween P53 cDNA and DNA sequencing in glioblastomas. We hypothesised that similar discrepancies may be observed in other human cancers. To this end, we analyzed 23 colorectal cancers for P53 mutations and gene expression using both DNA and cDNA sequencing, real-time PCR and immunohistochemistry. We found P53 gene mutations in 16 cases (15 missense and 1 nonsense). Two of the 15 cases with missense mutations showed alterations based only on cDNA, and not DNA sequencing. Moreover, in 6 of the 15 cases with a cDNA mutation those mutations were difficult to detect in the DNA sequencing, so the results of DNA analysis alone could be misinterpreted if the cDNA sequencing results had not also been available. In all those 15 cases, we observed a higher ratio of the mutated to the wild type template by cDNA analysis, but not by the DNA analysis. Interestingly, a similar overexpression of P53 mRNA was present in samples with and without P53 mutations. In terms of colorectal cancer, those discrepancies might be explained under three conditions: 1, overexpression of mutated P53 mRNA in cancer cells as compared with normal cells; 2, a higher content of cells without P53 mutation (normal cells and cells showing K-RAS and/or APC but not P53 mutation) in samples presenting P53 mutation; 3, heterozygous or hemizygous mutations of P53 gene. Additionally, for heterozygous mutations unknown mechanism(s) causing selective overproduction of mutated allele should also be considered. Our data offer new clues for studying discrepancy in P53 cDNA and DNA sequencing analysis

  1. DNA methyltransferase 1 mutations and mitochondrial pathology: is mtDNA methylated?

    Directory of Open Access Journals (Sweden)

    Alessandra eMaresca

    2015-03-01

    Full Text Available Autosomal dominant cerebellar ataxia, deafness and narcolepsy (ADCA-DN and Hereditary sensory neuropathy with dementia and hearing loss (HSN1E are two rare, overlapping neurodegenerative syndromes that have been recently linked to allelic dominant pathogenic mutations in the DNMT1 gene, coding for DNA (cytosine-5-methyltransferase 1. DNMT1 is the enzyme responsible for maintaining the nuclear genome methylation patterns during the DNA replication and repair, thus regulating gene expression. The mutations responsible for ADCA-DN and HSN1E affect the replication foci targeting sequence domain, which regulates DNMT1 binding to chromatin. DNMT1 dysfunction is anticipated to lead to a global alteration of the DNA methylation pattern with predictable downstream consequences on gene expression. Interestingly, ADCA-DN and HSN1E phenotypes share some clinical features typical of mitochondrial diseases, such as optic atrophy, peripheral neuropathy and deafness, and some biochemical evidence of mitochondrial dysfunction. The recent discovery of a mitochondrial isoform of DNMT1 and its proposed role in methylating mitochondrial DNA (mtDNA suggests that DNMT1 mutations may directly affect mtDNA and mitochondrial physiology. On the basis of this latter finding the link between DNMT1 abnormal activity and mitochondrial dysfunction in ADCA-DN and HSN1E appears intuitive, however mtDNA methylation remains highly debated. In the last years several groups demonstrated the presence of 5-methylcytosine in mtDNA by different approaches, but, on the other end, the opposite evidence that mtDNA is not methylated has also been published. Since over 1500 mitochondrial proteins are encoded by the nuclear genome, the altered methylation of these genes may well have a critical role in leading to the mitochondrial impairment observed in ADCA-DN and HSN1E. Thus, many open questions still remain unanswered, such as why mtDNA should be methylated, and how this process is

  2. Infantile presentation of the mtDNA A3243G tRNA(Leu (UUR)) mutation.

    NARCIS (Netherlands)

    Okhuijsen-Kroes, E.J.; Trijbels, J.M.F.; Sengers, R.C.A.; Mariman, E.C.M.; Heuvel, L.P.W.J. van den; Wendel, U.A.H.; Koch, G.; Smeitink, J.A.M.

    2001-01-01

    Mitochondrial DNA (mtDNA) disorders are clinically very heterogeneous, ranging from single organ involvement to severe multisystem disease. One of the most frequently observed mtDNA mutations is the A-to-G transition at position 3243 of the tRNA(Leu (UUR)) gene. This mutation is often related to

  3. Role of DNA deletion length in mutation and cell survival

    International Nuclear Information System (INIS)

    Braby, L.A.; Morgan, T.L.

    1992-01-01

    A model is presented which is based on the assumption that malignant transformation, mutation, chromosome aberration, and reproductive death of cells are all manifestations of radiation induced deletions in the DNA of the cell, and that the size of the deletion in relation to the spacing of essential genes determines the consequences of that deletion. It is assumed that two independent types of potentially lethal lesions can result in DNA deletions, and that the relative numbers of these types of damage is dependent on radiation quality. The repair of the damage reduces the length of a deletion, but does not always eliminate it. The predictions of this model are in good agreement with a wide variety of experimental evidence. (author)

  4. The role of DNA polymerase {iota} in UV mutational spectra

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Jun-Hyuk [Division of Biology, Beckman Research Institute, City of Hope, Duarte, CA 91010 (United States); Besaratinia, Ahmad [Division of Biology, Beckman Research Institute, City of Hope, Duarte, CA 91010 (United States); Lee, Dong-Hyun [Division of Biology, Beckman Research Institute, City of Hope, Duarte, CA 91010 (United States); Lee, Chong-Soon [Department of Biochemistry, College of Natural Sciences, Yeungnam University, Gyongsan 712-749 (Korea, Republic of); Pfeifer, Gerd P. [Division of Biology, Beckman Research Institute, City of Hope, Duarte, CA 91010 (United States)]. E-mail: gpfeifer@coh.org

    2006-07-25

    UVB (280-320 nm) and UVC (200-280 nm) irradiation generate predominantly cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts in DNA. CPDs are thought to be responsible for most of the UV-induced mutations. Thymine-thymine CPDs, and probably also CPDs containing cytosine, are replicated in vivo in a largely accurate manner by a DNA polymerase {eta} (Pol {eta}) dependent process. Pol {eta} is a DNA damage-tolerant and error-prone DNA polymerase encoded by the POLH (XPV) gene in humans. Another member of the Y family of error-prone DNA polymerases is POLI encoding DNA polymerase iota (Pol {iota}). In order to clarify the specific role of Pol {iota} in UV mutagenesis, we have used an siRNA knockdown approach in combination with a supF shuttle vector which replicates in mammalian cells, similar as we have previously done for Pol {eta}. Synthetic RNA duplexes were used to efficiently inhibit Pol {iota} expression in 293T cells. The supF shuttle vector was irradiated with 254 nm UVC and replicated in 293T cells in presence of anti-Pol {iota} siRNA. Surprisingly, there was a consistent reduction of recovered plasmid from cells with Pol {iota} knockdown and this was independent of UV irradiation of the plasmid. The supF mutant frequency was unchanged in the siRNA knockdown cells relative to control cells confirming that Pol {iota} does not play an important role in UV mutagenesis. UV-induced supF mutants were sequenced from siRNA-treated cells and controls. Neither the type of mutations nor their distribution along the supF gene were significantly different between controls and siRNA knockdown cells and were predominantly C to T and CC to TT transitions at dipyrimidine sites. These results show that Pol {iota} has no significant role in UV lesion bypass and mutagenesis in vivo and provides some initial data suggesting that this polymerase may be involved in replication of extrachromosomal DNA.

  5. Ultra-deep sequencing of mouse mitochondrial DNA: mutational patterns and their origins.

    Directory of Open Access Journals (Sweden)

    Adam Ameur

    2011-03-01

    Full Text Available Somatic mutations of mtDNA are implicated in the aging process, but there is no universally accepted method for their accurate quantification. We have used ultra-deep sequencing to study genome-wide mtDNA mutation load in the liver of normally- and prematurely-aging mice. Mice that are homozygous for an allele expressing a proof-reading-deficient mtDNA polymerase (mtDNA mutator mice have 10-times-higher point mutation loads than their wildtype siblings. In addition, the mtDNA mutator mice have increased levels of a truncated linear mtDNA molecule, resulting in decreased sequence coverage in the deleted region. In contrast, circular mtDNA molecules with large deletions occur at extremely low frequencies in mtDNA mutator mice and can therefore not drive the premature aging phenotype. Sequence analysis shows that the main proportion of the mutation load in heterozygous mtDNA mutator mice and their wildtype siblings is inherited from their heterozygous mothers consistent with germline transmission. We found no increase in levels of point mutations or deletions in wildtype C57Bl/6N mice with increasing age, thus questioning the causative role of these changes in aging. In addition, there was no increased frequency of transversion mutations with time in any of the studied genotypes, arguing against oxidative damage as a major cause of mtDNA mutations. Our results from studies of mice thus indicate that most somatic mtDNA mutations occur as replication errors during development and do not result from damage accumulation in adult life.

  6. Role of Mitochondrial DNA Mutations in Cellular Vulnerability to Mitochondria-Specific Environmental Toxins

    National Research Council Canada - National Science Library

    Hirsch, Etienne C

    2005-01-01

    In recent years, growing evidence has shown that mutations of mitochondrial DNA (mtDNA) are an important cause of mitochondrial disorders in humans, and have been associated with common neurodegenerative disorders, aging and cancers...

  7. Genotoxicity of formaldehyde: Molecular basis of DNA damage and mutation

    Directory of Open Access Journals (Sweden)

    Masanobu eKawanishi

    2014-09-01

    Full Text Available Formaldehyde is commonly used in the chemical industry and is present in the environment, such as vehicle emissions, some building materials, food and tobacco smoke. It also occurs as a natural product in most organisms, the sources of which include a number of metabolic processes. It causes various acute and chronic adverse effects in humans if they inhale its fumes. Among the chronic effects on human health, we summarize data on genotoxicity and carcinogenicity in this review, and we particularly focus on the molecular mechanisms involved in the formaldehyde mutagenesis. Formaldehyde mainly induces N-hydroxymethyl mono-adducts on guanine, adenine and cytosine, and N-methylene crosslinks between adjacent purines in DNA. These crosslinks are types of DNA damage potentially fatal for cell survival if they are not removed by the nucleotide excision repair pathway. In the previous studies, we showed evidence that formaldehyde causes intra-strand crosslinks between purines in DNA using a unique method (Matsuda et al. Nucleic Acids Res. 26, 1769-1774,1998. Using shuttle vector plasmids, we also showed that formaldehyde as well as acetaldehyde induces tandem base substitutions, mainly at 5’-GG and 5’-GA sequences, which would arise from the intra-strand crosslinks. These mutation features are different from those of other aldehydes such as crotonaldehyde, acrolein, glyoxal and methylglyoxal. These findings provide molecular clues to improve our understanding of the genotoxicity and carcinogenicity of formaldehyde.

  8. Droplet digital PCR-based EGFR mutation detection with an internal quality control index to determine the quality of DNA.

    Science.gov (United States)

    Kim, Sung-Su; Choi, Hyun-Jeung; Kim, Jin Ju; Kim, M Sun; Lee, In-Seon; Byun, Bohyun; Jia, Lina; Oh, Myung Ryurl; Moon, Youngho; Park, Sarah; Choi, Joon-Seok; Chae, Seoung Wan; Nam, Byung-Ho; Kim, Jin-Soo; Kim, Jihun; Min, Byung Soh; Lee, Jae Seok; Won, Jae-Kyung; Cho, Soo Youn; Choi, Yoon-La; Shin, Young Kee

    2018-01-11

    In clinical translational research and molecular in vitro diagnostics, a major challenge in the detection of genetic mutations is overcoming artefactual results caused by the low-quality of formalin-fixed paraffin-embedded tissue (FFPET)-derived DNA (FFPET-DNA). Here, we propose the use of an 'internal quality control (iQC) index' as a criterion for judging the minimum quality of DNA for PCR-based analyses. In a pre-clinical study comparing the results from droplet digital PCR-based EGFR mutation test (ddEGFR test) and qPCR-based EGFR mutation test (cobas EGFR test), iQC index ≥ 0.5 (iQC copies ≥ 500, using 3.3 ng of FFPET-DNA [1,000 genome equivalents]) was established, indicating that more than half of the input DNA was amplifiable. Using this criterion, we conducted a retrospective comparative clinical study of the ddEGFR and cobas EGFR tests for the detection of EGFR mutations in non-small cell lung cancer (NSCLC) FFPET-DNA samples. Compared with the cobas EGFR test, the ddEGFR test exhibited superior analytical performance and equivalent or higher clinical performance. Furthermore, iQC index is a reliable indicator of the quality of FFPET-DNA and could be used to prevent incorrect diagnoses arising from low-quality samples.

  9. Significance of somatic mutations and content alteration of mitochondrial DNA in esophageal cancer

    Directory of Open Access Journals (Sweden)

    Wang Yu-Fen

    2006-04-01

    Full Text Available Abstract Background The roles of mitochondria in energy metabolism, the generation of ROS, aging, and the initiation of apoptosis have implicated their importance in tumorigenesis. In this study we aim to establish the mutation spectrum and to understand the role of somatic mtDNA mutations in esophageal cancer. Methods The entire mitochondrial genome was screened for somatic mutations in 20 pairs (18 esophageal squamous cell carcinomas, one adenosquamous carcinoma and one adenocarcinoma of tumor/surrounding normal tissue of esophageal cancers, using temporal temperature gradient gel electrophoresis (TTGE, followed by direct DNA sequencing to identify the mutations. Results Fourteen somatic mtDNA mutations were identified in 55% (11/20 of tumors analyzed, including 2 novel missense mutations and a frameshift mutation in ND4L, ATP6 subunit, and ND4 genes respectively. Nine mutations (64% were in the D-loop region. Numerous germline variations were found, at least 10 of them were novel and five were missense mutations, some of them occurred in evolutionarily conserved domains. Using real-time quantitative PCR analysis, the mtDNA content was found to increase in some tumors and decrease in others. Analysis of molecular and other clinicopathological findings does not reveal significant correlation between somatic mtDNA mutations and mtDNA content, or between mtDNA content and metastatic status. Conclusion Our results demonstrate that somatic mtDNA mutations in esophageal cancers are frequent. Some missense and frameshift mutations may play an important role in the tumorigenesis of esophageal carcinoma. More extensive biochemical and molecular studies will be necessary to determine the pathological significance of these somatic mutations.

  10. Enzyme-Free Detection of Mutations in Cancer DNA Using Synthetic Oligonucleotide Probes and Fluorescence Microscopy

    DEFF Research Database (Denmark)

    Miotke, Laura; Maity, Arindam; Ji, Hanlee

    2015-01-01

    BACKGROUND: Rapid reliable diagnostics of DNA mutations are highly desirable in research and clinical assays. Current development in this field goes simultaneously in two directions: 1) high-throughput methods, and 2) portable assays. Non-enzymatic approaches are attractive for both types...... 1000-fold above the potential detection limit. CONCLUSION: Overall, the novel assay we describe could become a new approach to rapid, reliable and enzyme-free diagnostics of cancer or other associated DNA targets. Importantly, stoichiometry of wild type and mutant targets is conserved in our assay...... of methods since they would allow rapid and relatively inexpensive detection of nucleic acids. Modern fluorescence microscopy is having a huge impact on detection of biomolecules at previously unachievable resolution. However, no straightforward methods to detect DNA in a non-enzymatic way using fluorescence...

  11. Whole-exome sequencing of muscle-invasive bladder cancer identifies recurrent mutations of UNC5C and prognostic importance of DNA repair gene mutations on survival.

    Science.gov (United States)

    Yap, Kai Lee; Kiyotani, Kazuma; Tamura, Kenji; Antic, Tatjana; Jang, Miran; Montoya, Magdeline; Campanile, Alexa; Yew, Poh Yin; Ganshert, Cory; Fujioka, Tomoaki; Steinberg, Gary D; O'Donnell, Peter H; Nakamura, Yusuke

    2014-12-15

    Because of suboptimal outcomes in muscle-invasive bladder cancer even with multimodality therapy, determination of potential genetic drivers offers the possibility of improving therapeutic approaches and discovering novel prognostic indicators. Using pTN staging, we case-matched 81 patients with resected ≥pT2 bladder cancers for whom perioperative chemotherapy use and disease recurrence status were known. Whole-exome sequencing was conducted in 43 cases to identify recurrent somatic mutations and targeted sequencing of 10 genes selected from the initial screening in an additional 38 cases was completed. Mutational profiles along with clinicopathologic information were correlated with recurrence-free survival (RFS) in the patients. We identified recurrent novel somatic mutations in the gene UNC5C (9.9%), in addition to TP53 (40.7%), KDM6A (21.0%), and TSC1 (12.3%). Patients who were carriers of somatic mutations in DNA repair genes (one or more of ATM, ERCC2, FANCD2, PALB2, BRCA1, or BRCA2) had a higher overall number of somatic mutations (P = 0.011). Importantly, after a median follow-up of 40.4 months, carriers of somatic mutations (n = 25) in any of these six DNA repair genes had significantly enhanced RFS compared with noncarriers [median, 32.4 vs. 14.8 months; hazard ratio of 0.46, 95% confidence interval (CI), 0.22-0.98; P = 0.0435], after adjustment for pathologic pTN staging and independent of adjuvant chemotherapy usage. Better prognostic outcomes of individuals carrying somatic mutations in DNA repair genes suggest these mutations as favorable prognostic events in muscle-invasive bladder cancer. Additional mechanistic investigation into the previously undiscovered role of UNC5C in bladder cancer is warranted. ©2014 American Association for Cancer Research.

  12. Study in mutation of alfalfa genome DNA due to low energy N+ implantation using RAPD

    International Nuclear Information System (INIS)

    Chen Roulei; Song Daojun; Yu Zengliang; Li Yufeng; Liang Yunzhang

    2001-01-01

    After implanted by various dosage N + beams, germination rate of alfalfa seeds appears to be saddle line with dosage increasing. The authors have studied in mutation of genome DNA due to low energy N + implantation, and concluded that 30 differential DNA fragments have been amplified by 8 primers (S 41 , S 42 , S 45 , S 46 , S 50 , S 52 , S 56 , S 58 ) in 100 primers, moreover, number of differential DNA fragments between CK and treatments increases with dosage. Consequently, low energy ion implantation can cause mutation of alfalfa genome DNA. The more dosage it is, the more mutation alfalfa will be

  13. Analysis of morphology, DNA and isozyme of leaf mutation in Brassica napus L

    International Nuclear Information System (INIS)

    Luo Zhen; Hu Dongwei; Li Xiaobai

    2008-01-01

    This paper aims to study the rule of irradiating effects, provide the effective way of analyzing mutant, and discuss the production application of mutant. By irradiating the 040B of Brassica napus L with . 0Co γ- ray, an obvious leaf mutation (ML) with large leaf area was found. The ML which has been inherited stably after three generations was compared with wide-type (CK) on the morphologic, DNA and isozymic levels. Results showed that S 4 and S17 from RAPD were two molecular markers which can express good polymorphism and have close relationships with leaf mutation sites. And in the analysis of EST and POD between ML and CK, the polymorphisms also proved that many discrepancies exist between ML and CK on the protein level. In addition, the research results in question can be applied to the breeding and genetic research of Brassica napus L

  14. Mitochondrial DNA mutation screening of male patients with obstructive sleep apnea-hypopnea syndrome.

    Science.gov (United States)

    Huang, Xiao-Ying; Li, Hong; Xu, Xiao-Mei; Wang, Liang-Xing

    2014-08-01

    The aim of the present study was to analyze the differences between the genes of the mitochondrial DNA (mtDNA) displacement loop (D-loop) region and the Cambridge Reference sequence, in order to screen the mutation sites and investigate the correlation between mutations, clinical parameters and complications associated with obstructive sleep apnea-hypopnea syndrome (OSAHS). mtDNA was obtained from male patients with OSAHS in the Zhejiang Province. In total, 60 male patients with OSAHS and 102 healthy adults were assessed to determine the levels of fasting blood glucose, total cholesterol, triglyceride (TG) and high-density and low-density lipoproteins (LDL). Furthermore, peripheral mtDNA was extracted and bidirectional sequencing was conducted to enable mutation screening. In the mtDNA D-loop region, 178 mutation sites were identified, of which 115 sites were present in the two groups. The number of non-common sites in the OSAHS group was significantly higher compared with the control group (P0.05). A total of 21 cases in the severe OSAHS group exhibited mutation rates of >10%. In the control group, there were 24 cases where the np73A-G and np263A-G mutations were predominant. The np303-np315 region was identified to be the highly variable region and various mutation forms were observed. Statistically significant differences were observed in the neck perimeter, TG and LDL levels among the OSAHS-no-mutation subgroups (P<0.05) and LDL was shown to be associated with an mtDNA mutation in the OSAHS group. Numerous polymorphic mutation sites were identified in the mtDNA D-loop region of the OSAHS group. Therefore, mtDNA mutation sites may be closely associated with the clinical manifestations and complications of OSAHS.

  15. mtDNA mutation C1494T, haplogroup A, and hearing loss in Chinese

    International Nuclear Information System (INIS)

    Wang Chengye; Kong Qingpeng; Yao Yonggang; Zhang Yaping

    2006-01-01

    Mutation C1494T in mitochondrial 12S rRNA gene was recently reported in two large Chinese families with aminoglycoside-induced and nonsyndromic hearing loss (AINHL) and was claimed to be pathogenic. This mutation, however, was first reported in a sample from central China in our previous study that was aimed to reconstruct East Asian mtDNA phylogeny. All these three mtDNAs formed a subclade defined by mutation C1494T in mtDNA haplogroup A. It thus seems that mutation C1494T is a haplogroup A-associated mutation and this matrilineal background may contribute a high risk for the penetrance of mutation C1494T in Chinese with AINHL. To test this hypothesis, we first genotyped mutation C1494T in 553 unrelated individuals from three regional Chinese populations and performed an extensive search for published complete or near-complete mtDNA data sets (>3000 mtDNAs), we then screened the C1494T mutation in 111 mtDNAs with haplogroup A status that were identified from 1823 subjects across China. The search for published mtDNA data sets revealed no other mtDNA besides the above-mentioned three carrying mutation C1494T. None of the 553 randomly selected individuals and the 111 haplogroup A mtDNAs was found to bear this mutation. Therefore, our results suggest that C1494T is a very rare event. The mtDNA haplogroup A background in general is unlikely to play an active role in the penetrance of mutation C1494T in AINHL

  16. Random mtDNA mutations modulate proliferation capacity in mouse embryonic fibroblasts

    International Nuclear Information System (INIS)

    Kukat, Alexandra; Edgar, Daniel; Bratic, Ivana; Maiti, Priyanka; Trifunovic, Aleksandra

    2011-01-01

    Highlights: → Increased mtDNA mutations in MEFs lead to high level of spontaneous immortalization. → This process is independent of endogenous ROS production. → Aerobic glycolysis significantly contributes to spontaneous immortalization of MEFs. -- Abstract: An increase in mtDNA mutation load leads to a loss of critical cells in different tissues thereby contributing to the physiological process of organismal ageing. Additionally, the accumulation of senescent cells that display changes in metabolic function might act in an active way to further disrupt the normal tissue function. We believe that this could be the important link missing in our understanding of the molecular mechanisms of premature ageing in the mtDNA mutator mice. We tested proliferation capacity of mtDNA mutator cells in vitro. When cultured in physiological levels of oxygen (3%) their proliferation capacity is somewhat lower than wild-type cells. Surprisingly, in conditions of increased oxidative stress (20% O 2 ) mtDNA mutator mouse embryonic fibroblasts exhibit continuous proliferation due to spontaneous immortalization, whereas the same conditions promote senescence in wild-type cells. We believe that an increase in aerobic glycolysis observed in mtDNA mutator mice is a major mechanism behind this process. We propose that glycolysis promotes proliferation and allows a fast turnover of metabolites, but also leads to energy crisis due to lower ATP production rate. This could lead to compromised replication and/or repair and therefore, in rare cases, might lead to mutations in tumor suppressor genes and spontaneous immortalization.

  17. Application of DNA chips in the analysis of gene mutation in HBV

    International Nuclear Information System (INIS)

    Wang Yongzhong; Ruan Lihua; Zhou Guoping; Wu Guoxiang; Chen Min

    2005-01-01

    Objective: To investigate the clinical applicability of DNA chips for analysis of gene mutation in HBV. Methods: Serum HBV DNA from 47 patients with viral hepatitis type B was amplified with PCR. Possible gene mutations were searched for in site 1896 of pre-C section, sites 1762,1764 of BCP section and sites 528, 552 of P section with DNA chip method based upon membrane coloration. Results: In the 32 patients without lamivudine treatment, the results were as follows: (1) 6 specimens with HBsAg + , HBeAg + , HBeAb - , no mutations observed. (2) 13 specimens with HBsAg + , HBeAg - , HBeAb + , mutations at site 1896, pre- C 4 cases, mutations at sites 1762,1764, BCP 11 cases. (3) 13 specimens with HBsAg + , HBeAg + , HBeAb + , mutations at site 1896 pre -C 4 cases, mutations at sites 1762,1764 BCP 13 cases. In the 15 patients after 48 weeks treatment with lamivudine but remained HBV DNA positive, mutations were observed at: site 1896 pre-C, 5 cases, sites 1762,1764 BCP, 6 cases, site 528 P section, 2 cases, site 552 P section, YVDD 4 cases, YIDD 7 cases. Conclusion: Mutations at sites 1896, 1762,1764 were more frequent in patients with HBeAb + and were related to the negative expression of HBeAg, Mutations at 1762,1764 BCP were closely related to the changes of HBeAg/HBeAb. P section mutations were only observed after lamivadine treatment and were related to resistance against the drug. DNA chip method based upon membrane coloration for detection of gene mutation was expedient and specific and worth popularization. (authors)

  18. DNA sequence analysis of X-ray induced Adh null mutations in Drosophila melanogaster

    International Nuclear Information System (INIS)

    Mahmoud, J.; Fossett, N.G.; Arbour-Reily, P.; McDaniel, M.; Tucker, A.; Chang, S.H.; Lee, W.R.

    1991-01-01

    The mutational spectrum for 28 X-ray induced mutations and 2 spontaneous mutations, previously determined by genetic and cytogenetic methods, consisted of 20 multilocus deficiencies (19 induced and 1 spontaneous) and 10 intragenic mutations (9 induced and 1 spontaneous). One of the X-ray induced intragenic mutations was lost, and another was determined to be a recombinant with the allele used in the recovery scheme. The DNA sequence of two X-ray induced intragenic mutations has been published. This paper reports the results of DNA sequence analysis of the remaining intragenic mutations and a summary of the X-ray induced mutational spectrum. The combination of DNA sequence analysis with genetic complementation analysis shows a continuous distribution in size of deletions rather than two different types of mutations consisting of deletions and 'point mutations'. Sequencing is shown to be essential for detecting intragenic deletions. Of particular importance for future studies is the observation that all of the intragenic deletions consist of a direct repeat adjacent to the breakpoint with one of the repeats deleted

  19. A novel approach to detect KRAS/BRAF mutation for colon cancer: Highly sensitive simultaneous detection of mutations and simple pre-treatment without DNA extraction.

    Science.gov (United States)

    Suzuki, Shun-Ichi; Matsusaka, Satoshi; Hirai, Mitsuharu; Shibata, Harumi; Takagi, Koichi; Mizunuma, Nobuyuki; Hatake, Kiyohiko

    2015-07-01

    It has been reported that colon cancer patients with KRAS and BRAF mutations that lie downstream of epidermal growth factor receptor (EGFR) acquire resistance against therapy with anti‑EGFR antibodies, cetuximab and panitumumab. On the other hand, some reports say KRAS codon 13 mutation (p.G13D) has lower resistance against anti-EGFR antibodies, thus there is a substantial need for detection of specific KRAS mutations. We have established a state-of-the-art measurement system using QProbe (QP) method that allows simultaneous measurement of KRAS codon 12/13, p.G13D and BRAF mutation, and compared this method against Direct Sequencing (DS) using 182 specimens from colon cancer patients. In addition, 32 biopsy specimens were processed with a novel pre-treatment method without DNA purification in order to detect KRAS/BRAF. As a result of KRAS mutation measurement, concordance rate between the QP method and DS method was 81.4% (144/177) except for the 5 specimens that were undeterminable. Among them, 29 specimens became positive with QP method and negative with DS method. BRAF was measured with QP method only, and the mutation detection rate was 3.9% (6/153). KRAS measurement using a simple new pre-treatment method without DNA extraction resulted in 31 good results out of 32, all of them matching with the DS method. We have established a simple but highly sensitive simultaneous detection system for KRAS/BRAF. Moreover, introduction of the novel pre-treatment technology eliminated the inconvenient DNA extraction process. From this research achievement, we not only anticipate quick and accurate results returned in the clinical field but also contribution in improving the test quality and work efficiency.

  20. Role of Mitochondrial DNA Mutations in Cellular Vulnerability to Mitochondria-Specific Environmental Toxins

    National Research Council Canada - National Science Library

    Hirsch, Etienne C

    2005-01-01

    .... To test such a hypothesis in Parkinson's disease we proposed to: 1) develop an animal model with accumulated mtDNA mutations in catecholaminergic neurons by creating a transgenic mouse containing a tyrosine hydroxylase (TH...

  1. Simultaneous Profiling of DNA Mutation and Methylation by Melting Analysis Using Magnetoresistive Biosensor Array

    DEFF Research Database (Denmark)

    Rizzi, Giovanni; Lee, Jung-Rok; Dahl, Christina

    2017-01-01

    specificity. Genomic (mutation) or bisulphite-treated (methylation) DNA is amplified using nondiscriminatory primers, and the amplicons are then hybridized to a giant magnetoresistive (GMR) biosensor array followed by melting curve measurements. The GMR biosensor platform offers scalable multiplexed detection...

  2. Validation of high-resolution DNA melting analysis for mutation scanning of the CDKL5 gene: identification of novel mutations.

    Science.gov (United States)

    Raymond, Laure; Diebold, Bertrand; Leroux, Céline; Maurey, Hélène; Drouin-Garraud, Valérie; Delahaye, Andre; Dulac, Olivier; Metreau, Julia; Melikishvili, Gia; Toutain, Annick; Rivier, François; Bahi-Buisson, Nadia; Bienvenu, Thierry

    2013-01-01

    Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) have been predominantly described in epileptic encephalopathies of female, including infantile spasms with Rett-like features. Up to now, detection of mutations in this gene was made by laborious, expensive and/or time consuming methods. Here, we decided to validate high-resolution melting analysis (HRMA) for mutation scanning of the CDKL5 gene. Firstly, using a large DNA bank consisting to 34 samples carrying different mutations and polymorphisms, we validated our analytical conditions to analyse the different exons and flanking intronic sequences of the CDKL5 gene by HRMA. Secondly, we screened CDKL5 by both HRMA and denaturing high performance liquid chromatography (dHPLC) in a cohort of 135 patients with early-onset seizures. Our results showed that point mutations and small insertions and deletions can be reliably detected by HRMA. Compared to dHPLC, HRMA profiles are more discriminated, thereby decreasing unnecessary sequencing. In this study, we identified eleven novel sequence variations including four pathogenic mutations (2.96% prevalence). HRMA appears cost-effective, easy to set up, highly sensitive, non-toxic and rapid for mutation screening, ideally suited for large genes with heterogeneous mutations located along the whole coding sequence, such as the CDKL5 gene. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. Determining the Location of DNA Modification and Mutation Caused by UVB Light in Skin Cancer

    Science.gov (United States)

    2015-09-01

    Award Number: W81XWH-12-1-0333 TITLE: Determining the Location of DNA Modification and Mutation Caused by UVB Light in Skin Cancer PRINCIPAL...COVERED 15 Aug 2012 – 14 Aug 2015 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER W81XWH-12-1-0333 Determining the Location of DNA Modification and Mutation ...sequencing libraries generated for both yeast and human cells show pyrimidine bias on the 5’ end, indicating that we are sequencing the dimers

  4. Induced mutations - a tool in plant research

    International Nuclear Information System (INIS)

    1981-01-01

    These proceedings include 34 papers and 18 brief descriptions of poster presentations in the following areas as they are affected by induced mutations: advancement of genetics, plant evolution, plant physiology, plant parasites, plant symbioses, in vitro culture, gene ecology and plant breeding. Only a relatively small number of papers are of direct nuclear interest essentially in view of the mutations being induced by ionizing radiations. The papers of nuclear interest have been entered as separate and individual items of input

  5. DNA damage by reactive species: Mechanisms, mutation and repair

    Indian Academy of Sciences (India)

    DNA is continuously attacked by reactive species that can affect its structure and function severely. Structural modifications to DNA mainly arise from modifications in its bases that primarily occur due to their exposure to different reactive species. Apart from this, DNA strand break, inter- and intra-strand crosslinks and ...

  6. Effects of TET2 mutations on DNA methylation in chronic myelomonocytic leukemia

    Science.gov (United States)

    TET2 enzymatically converts 5-methyl-cytosine to 5-hydroxymethyl-cytosine, possibly leading to loss of DNA methylation. TET2 mutations are common in myeloid leukemia and were proposed to contribute to leukemogenesis through DNA methylation. To expand on this concept, we studied chronic myelomonocyti...

  7. Mutations of mtDNA polymerase-γ and hyperlactataemia in the HIV ...

    African Journals Online (AJOL)

    Mutations of mtDNA polymerase-γ and hyperlactataemia in the HIV-infected Zulu population of South Africa. ... D B A Ojwach, C Aldous, P Kocheleff, B Sartorius ... of their capacity to impede human mitochondrial DNA polymerase-γ (POLG), ...

  8. Mutation of Haemophilus influenzae transforming DNA in vitro with near-ultraviolet radiation: action spectrum

    Energy Technology Data Exchange (ETDEWEB)

    Cabrera-Juarez, E; Setlow, J K [Escuela Nacional de Ciencias Biologicas, Mexico City. Dept. de Bioquimica; Oak Ridge National Lab., Tenn. (USA). Biology Div.)

    1976-05-01

    Mutations were produced in purified transforming DNA from Haemophilus influenzae by near UV radiation and were assayed as mutants among cells transformed with irradiated DNA. The maximum efficiency of mutation induction was at around 334 nm, and the efficiency dropped off steeply at lower and higher wavelengths. The difference between the action spectrum for mutation and that for the oxygen-independent inactivation of transforming DNA, which had a shoulder at 365 nm, indicates that there are different lesions involved in the inactivating and mutagenic effects of near-UV. The presence of histidine during irradiation enhanced the mutagenic effect at 334 and 365 nm, although it protected against inactivation at 365 nm. The effective near-UV wavelengths for in vitro mutation are to some extent the same as the effective wavelengths for mutation in vivo reported previously. These findings indicate that mutations are produced in vivo by near-UV with DNA as the primary target molecule rather than by a secondary non-photochemical reaction between DNA and some other cell component.

  9. Mutations affecting RNA polymerase I-stimulated exchange and rDNA recombination in yeast

    International Nuclear Information System (INIS)

    Lin, Y.H.; Keil, R.L.

    1991-01-01

    HOT1 is a cis-acting recombination-stimulatory sequence isolated from the rDNA repeat unit of yeast. The ability of HOT1 to stimulate mitotic exchange appears to depend on its ability to promote high levels of RNA polymerase I transcription. A qualitative colony color sectoring assay was developed to screen for trans-acting mutations that alter the activity of HOT1. Both hypo-recombination and hyper-recombination mutants were isolated. Genetic analysis of seven HOT1 recombination mutants (hrm) that decrease HOT1 activity shows that they behave as recessive nuclear mutations and belong to five linkage groups. Three of these mutations, hrm1, hrm2, and hrm3, also decrease rDNA exchange but do not alter recombination in the absence of HOT1. Another mutation, hrm4, decreases HOT1-stimulated recombination but does not affect rDNA recombination or exchange in the absence of HOT1. Two new alleles of RAD52 were also isolated using this screen. With regard to HOT1 activity, rad52 is epistatic to all four hrm mutations indicating that the products of the HRM genes and of RAD52 mediate steps in the same recombination pathway. Finding mutations that decrease both the activity of HOT1 and exchange in the rDNA supports the hypothesis that HOT1 plays a role in rDNA recombination

  10. RAPD analysis of alfalfa DNA mutation via N+ implantation

    International Nuclear Information System (INIS)

    Li Yufeng; Huang Qunce; Yu Zengliang; Liang Yunzhang

    2003-01-01

    Germination capacity of alfalfa seeds under low energy N + implantation manifests oscillations going down with dose strength. From analyzing alfalfa genome DNA under low energy N + implantation by RAPD (Random Amplified Polymorphous DNA), it is recommended that 30 polymorphic DNA fragments be amplified with 8 primers in total 100 primers, and fluorescence intensity of the identical DNA fragment amplified by RAPD is different between CK and treatments. Number of different polymorphic DNA fragments between treatment and CK via N + implantation manifests going up with dose strength

  11. Myopathic mtDNA Depletion Syndrome Due to Mutation in TK2 Gene.

    Science.gov (United States)

    Martín-Hernández, Elena; García-Silva, María Teresa; Quijada-Fraile, Pilar; Rodríguez-García, María Elena; Rivera, Henry; Hernández-Laín, Aurelio; Coca-Robinot, David; Fernández-Toral, Joaquín; Arenas, Joaquín; Martín, Miguel A; Martínez-Azorín, Francisco

    2017-01-01

    Whole-exome sequencing was used to identify the disease gene(s) in a Spanish girl with failure to thrive, muscle weakness, mild facial weakness, elevated creatine kinase, deficiency of mitochondrial complex III and depletion of mtDNA. With whole-exome sequencing data, it was possible to get the whole mtDNA sequencing and discard any pathogenic variant in this genome. The analysis of whole exome uncovered a homozygous pathogenic mutation in thymidine kinase 2 gene ( TK2; NM_004614.4:c.323 C>T, p.T108M). TK2 mutations have been identified mainly in patients with the myopathic form of mtDNA depletion syndromes. This patient presents an atypical TK2-related myopathic form of mtDNA depletion syndromes, because despite having a very low content of mtDNA (TK2 gene in mtDNA depletion syndromes and expanded the phenotypic spectrum.

  12. MELAS syndrome, cardiomyopathy, rhabdomyolysis, and autism associated with the A3260G mitochondrial DNA mutation.

    Science.gov (United States)

    Connolly, Barbara S; Feigenbaum, Annette S J; Robinson, Brian H; Dipchand, Anne I; Simon, David K; Tarnopolsky, Mark A

    2010-11-12

    The A to G transition mutation at position 3260 of the mitochondrial genome is usually associated with cardiomyopathy and myopathy. One Japanese kindred reported the phenotype of mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS syndrome) in association with the A3260G mtDNA mutation. We describe the first Caucasian cases of MELAS syndrome associated with the A3260G mutation. Furthermore, this mutation was associated with exercise-induced rhabdomyolysis, hearing loss, seizures, cardiomyopathy, and autism in the large kindred. We conclude that the A3260G mtDNA mutation is associated with wide phenotypic heterogeneity with MELAS and other "classical" mitochondrial phenotypes being manifestations. Copyright © 2010 Elsevier Inc. All rights reserved.

  13. Hair Dye–DNA Interaction: Plausible Cause of Mutation

    Directory of Open Access Journals (Sweden)

    Swati Maiti

    2015-09-01

    Full Text Available Hair dye is one of the most popular cosmetic products which are used more widely and frequently to improve an individual’s appearance. Although the genotoxic effects of dye ingredients are widely reported, hair dye in its usable form is not reported extensively. In this contribution, we report the possible mode of interaction of hair dye with DNA which leads to genotoxicity. The effect of dye DNA interaction was studied on the most popular and globally used hair dye with Calf Thymus DNA and plasmid DNA. This interaction of dye DNA was studied by spectroscopic analyses and gel electrophoresis. The result had shown positive interaction of dye with DNA. Gel electrophoresis study confirms the binding of dye with DNA which results in linearization and fragmentation of the plasmid DNA. Dye–DNA interaction causes fragmentation and oxidation of DNA in absence of any catalyst, implies high toxicity of commercial hair dyes. Thus, it can be deduced from the present studies that hair dye in its usable form may lead to its penetration through skin affecting genomic DNA possesses genotoxic property and can be treated as one of the most common mutagen.

  14. Clinical differences in patients with mitochondriocytopathies due to nuclear versus mitochondrial DNA mutations.

    Science.gov (United States)

    Rubio-Gozalbo, M E; Dijkman, K P; van den Heuvel, L P; Sengers, R C; Wendel, U; Smeitink, J A

    2000-01-01

    Defects in oxidative phosphorylation (OXPHOS) are genetically unique because the different components involved in this process, respiratory chain enzyme complexes (I, III, and IV) and complex V, are encoded by nuclear and mitochondrial genome. The objective of the study was to assess whether there are clinical differences in patients suffering from OXPHOS defects caused by nuclear or mitochondrial DNA (mtDNA) mutations. We studied 16 families with > or = two siblings with a genetically established OXPHOS deficiency, four due to a nuclear gene mutation and 12 due to a mtDNA mutation. Siblings with a nuclear gene mutation showed very similar clinical pictures that became manifest in the first years (ranging from first months to early childhood). There was a severe progressive course. Seven of the eight children died in their first decade. Conversely, siblings with a mtDNA mutation had clinical pictures that varied from almost alike to very distinct. They became symptomatic at an older age (ranging from childhood to adulthood), with the exception of defects associated with Leigh or Leigh-like phenotype. The clinical course was more gradual and relatively less severe; four of the 26 patients died, one in his second year, another in her second decade and two in their sixth decade. There are differences in age at onset, severity of clinical course, outcome, and intrafamilial variability in patients affected of an OXPHOS defect due to nuclear or mtDNA mutations. Patients with nuclear mutations become symptomatic at a young age, and have a severe clinical course. Patients with mtDNA mutations show a wider clinical spectrum of age at onset and severity. These differences may be of importance regarding the choice of which genome to study in affected patients as well as with respect to genetic counseling. Copyright 2000 Wiley-Liss, Inc.

  15. Optimised Pre-Analytical Methods Improve KRAS Mutation Detection in Circulating Tumour DNA (ctDNA) from Patients with Non-Small Cell Lung Cancer (NSCLC)

    Science.gov (United States)

    Sherwood, James L.; Corcoran, Claire; Brown, Helen; Sharpe, Alan D.; Musilova, Milena; Kohlmann, Alexander

    2016-01-01

    Introduction Non-invasive mutation testing using circulating tumour DNA (ctDNA) is an attractive premise. This could enable patients without available tumour sample to access more treatment options. Materials & Methods Peripheral blood and matched tumours were analysed from 45 NSCLC patients. We investigated the impact of pre-analytical variables on DNA yield and/or KRAS mutation detection: sample collection tube type, incubation time, centrifugation steps, plasma input volume and DNA extraction kits. Results 2 hr incubation time and double plasma centrifugation (2000 x g) reduced overall DNA yield resulting in lowered levels of contaminating genomic DNA (gDNA). Reduced “contamination” and increased KRAS mutation detection was observed using cell-free DNA Blood Collection Tubes (cfDNA BCT) (Streck), after 72 hrs following blood draw compared to EDTA tubes. Plasma input volume and use of different DNA extraction kits impacted DNA yield. Conclusion This study demonstrated that successful ctDNA recovery for mutation detection in NSCLC is dependent on pre-analytical steps. Development of standardised methods for the detection of KRAS mutations from ctDNA specimens is recommended to minimise the impact of pre-analytical steps on mutation detection rates. Where rapid sample processing is not possible the use of cfDNA BCT tubes would be advantageous. PMID:26918901

  16. Optimised Pre-Analytical Methods Improve KRAS Mutation Detection in Circulating Tumour DNA (ctDNA from Patients with Non-Small Cell Lung Cancer (NSCLC.

    Directory of Open Access Journals (Sweden)

    James L Sherwood

    Full Text Available Non-invasive mutation testing using circulating tumour DNA (ctDNA is an attractive premise. This could enable patients without available tumour sample to access more treatment options.Peripheral blood and matched tumours were analysed from 45 NSCLC patients. We investigated the impact of pre-analytical variables on DNA yield and/or KRAS mutation detection: sample collection tube type, incubation time, centrifugation steps, plasma input volume and DNA extraction kits.2 hr incubation time and double plasma centrifugation (2000 x g reduced overall DNA yield resulting in lowered levels of contaminating genomic DNA (gDNA. Reduced "contamination" and increased KRAS mutation detection was observed using cell-free DNA Blood Collection Tubes (cfDNA BCT (Streck, after 72 hrs following blood draw compared to EDTA tubes. Plasma input volume and use of different DNA extraction kits impacted DNA yield.This study demonstrated that successful ctDNA recovery for mutation detection in NSCLC is dependent on pre-analytical steps. Development of standardised methods for the detection of KRAS mutations from ctDNA specimens is recommended to minimise the impact of pre-analytical steps on mutation detection rates. Where rapid sample processing is not possible the use of cfDNA BCT tubes would be advantageous.

  17. Blocking DNA Repair in Advanced BRCA-Mutated Cancer

    Science.gov (United States)

    In this trial, patients with relapsed or refractory advanced cancer and confirmed BRCA mutations who have not previously been treated with a PARP inhibitor will be given BMN 673 by mouth once a day in 28-day cycles.

  18. Somatic mutations in stilbene estrogen-induced Syrian hamster kidney tumors identified by DNA fingerprinting

    Directory of Open Access Journals (Sweden)

    Roy Deodutta

    2004-01-01

    Full Text Available Abstract Kidney tumors from stilbene estrogen (diethylstilbestrol-treated Syrian hamsters were screened for somatic genetic alterations by Random Amplified Polymorphic DNA-polymerase chain-reaction (RAPD-PCR fingerprinting. Fingerprints from tumor tissue were generated by single arbitrary primers and compared with fingerprints for normal tissue from the same animal, as well as normal and tumor tissues from different animals. Sixty one of the arbitrary primers amplified 365 loci that contain approximately 476 kbp of the hamster genome. Among these amplified DNA fragments, 44 loci exhibited either qualitative or quantitative differences between the tumor tissues and normal kidney tissues. RAPD-PCR loci showing decreased and increased intensities in tumor tissue DNA relative to control DNA indicate that loci have undergone allelic losses and gains, respectively, in the stilbene estrogen-induced tumor cell genome. The presence or absence of the amplified DNA fragments indicate homozygous insertions or deletions in the kidney tumor DNA compared to the age-matched normal kidney tissue DNA. Seven of 44 mutated loci also were present in the kidney tissues adjacent to tumors (free of macroscopic tumors. The presence of mutated loci in uninvolved (non-tumor surrounding tissue adjacent to tumors from stilbene estrogen-treated hamsters suggests that these mutations occurred in the early stages of carcinogenesis. The cloning and sequencing of RAPD amplified loci revealed that one mutated locus had significant sequence similarity with the hamster Cyp1A1 gene. The results show the ability of RAPD-PCR to detect and isolate, in a single step, DNA sequences representing genetic alterations in stilbene estrogen-induced cancer cells, including losses of heterozygosity, and homozygous deletion and insertion mutations. RAPD-PCR provides an alternative molecular approach for studying cancer cytogenetics in stilbene estrogen-induced tumors in humans and experimental

  19. Frequent occurrence of mitochondrial DNA mutations in Barrett's metaplasia without the presence of dysplasia.

    Directory of Open Access Journals (Sweden)

    Soong Lee

    Full Text Available BACKGROUND: Barrett's esophagus (BE is one of the most common premalignant lesions and can progress to esophageal adenocarcinoma (EA. The numerous molecular events may play a role in the neoplastic transformation of Barrett's mucosa such as the change of DNA ploidy, p53 mutation and alteration of adhesion molecules. However, the molecular mechanism of the progression of BE to EA remains unclear and most studies of mitochondrial DNA (mtDNA mutations in BE have performed on BE with the presence of dysplasia. METHODS/FINDINGS: Thus, the current study is to investigate new molecular events (Barrett's esophageal tissue-specific-mtDNA alterations/instabilities in mitochondrial genome and causative factors for their alterations using the corresponding adjacent normal mucosal tissue (NT and tissue (BT from 34 patients having Barrett's metaplasia without the presence of dysplasia. Eighteen patients (53% exhibited mtDNA mutations which were not found in adjacent NT. mtDNA copy number was about 3 times higher in BT than in adjacent NT. The activity of the mitochondrial respiratory chain enzyme complexes in tissues from Barrett's metaplasia without the presence of dysplasia was impaired. Reactive oxygen species (ROS level in BT was significantly higher than those in corresponding samples. CONCLUSION/SIGNIFICANCE: High ROS level in BT may contribute to the development of mtDNA mutations, which may play a crucial role in disease progression and tumorigenesis in BE.

  20. DNA methylation patterns of candidate genes regulated by thymine DNA glycosylase in patients with TP53 germline mutations

    Energy Technology Data Exchange (ETDEWEB)

    Fortes, F.P. [CIPE, Laboratrio de Oncogentica Molecular, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Kuasne, H. [CIPE, Laboratrio NeoGene, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Departamento de Urologia, Faculdade de Medicina, Universidade Estadual Paulista, Botucatu, SP (Brazil); Marchi, F.A. [CIPE, Laboratrio NeoGene, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Programa Inter-Institucional em Bioinformtica, Instituto de Matemtica e Estatstica, Universidade So Paulo, So Paulo, SP (Brazil); Miranda, P.M. [CIPE, Laboratrio NeoGene, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Rogatto, S.R. [CIPE, Laboratrio NeoGene, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Departamento de Urologia, Faculdade de Medicina, Universidade Estadual Paulista, Botucatu, SP (Brazil); Achatz, M.I. [CIPE, Laboratrio de Oncogentica Molecular, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Departamento de Oncogentica, A.C. Camargo Cancer Center, So Paulo, SP (Brazil)

    2015-04-28

    Li-Fraumeni syndrome (LFS) is a rare, autosomal dominant, hereditary cancer predisposition disorder. In Brazil, the p.R337H TP53 founder mutation causes the variant form of LFS, Li-Fraumeni-like syndrome. The occurrence of cancer and age of disease onset are known to vary, even in patients carrying the same mutation, and several mechanisms such as genetic and epigenetic alterations may be involved in this variability. However, the extent of involvement of such events has not been clarified. It is well established that p53 regulates several pathways, including the thymine DNA glycosylase (TDG) pathway, which regulates the DNA methylation of several genes. This study aimed to identify the DNA methylation pattern of genes potentially related to the TDG pathway (CDKN2A, FOXA1, HOXD8, OCT4, SOX2, and SOX17) in 30 patients with germline TP53mutations, 10 patients with wild-type TP53, and 10 healthy individuals. We also evaluated TDG expression in patients with adrenocortical tumors (ADR) with and without the p.R337H TP53 mutation. Gene methylation patterns of peripheral blood DNA samples assessed by pyrosequencing revealed no significant differences between the three groups. However, increased TDG expression was observed by quantitative reverse transcription PCR in p.R337H carriers with ADR. Considering the rarity of this phenotype and the relevance of these findings, further studies using a larger sample set are necessary to confirm our results.

  1. Preimplantation genetic diagnosis for mitochondrial DNA mutations: analysis of one blastomere suffices.

    Science.gov (United States)

    Sallevelt, Suzanne C E H; Dreesen, Joseph C F M; Coonen, Edith; Paulussen, Aimee D C; Hellebrekers, Debby M E I; de Die-Smulders, Christine E M; Smeets, Hubert J M; Lindsey, Patrick

    2017-10-01

    Preimplantation genetic diagnosis (PGD) is a reproductive strategy for mitochondrial DNA (mtDNA) mutation carriers, strongly reducing their risk of affected offspring. Embryos either without the mutation or with mutation load below the phenotypic threshold are transferred to the uterus. Because of incidental heteroplasmy deviations in single blastomere and the relatively limited data available, we so far preferred relying on two blastomeres rather than one. Considering the negative effect of a two-blastomere biopsy protocol compared with a single-blastomere biopsy protocol on live birth delivery rate, we re-evaluated the error rate in our current dataset. For the m.3243A>G mutation, sufficient embryos/blastomeres were available for a powerful analysis. The diagnostic error rate, defined as a potential false-negative result, based on a threshold of 15%, was determined in 294 single blastomeres analysed in 73 embryos of 9 female m.3243A>G mutation carriers. Only one out of 294 single blastomeres (0.34%) would have resulted in a false-negative diagnosis. False-positive diagnoses were not detected. Our findings support a single-blastomere biopsy PGD protocol for the m.3243A>G mutation as the diagnostic error rate is very low. As in the early preimplantation embryo no mtDNA replication seems to occur and the mtDNA is divided randomly among the daughter cells, we conclude this result to be independent of the specific mutation and therefore applicable to all mtDNA mutations. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  2. Electrostatic study of Alanine mutational effects on transcription: application to GATA-3:DNA interaction complex.

    Science.gov (United States)

    El-Assaad, Atlal; Dawy, Zaher; Nemer, Georges

    2015-01-01

    Protein-DNA interaction is of fundamental importance in molecular biology, playing roles in functions as diverse as DNA transcription, DNA structure formation, and DNA repair. Protein-DNA association is also important in medicine; understanding Protein-DNA binding kinetics can assist in identifying disease root causes which can contribute to drug development. In this perspective, this work focuses on the transcription process by the GATA Transcription Factor (TF). GATA TF binds to DNA promoter region represented by `G,A,T,A' nucleotides sequence, and initiates transcription of target genes. When proper regulation fails due to some mutations on the GATA TF protein sequence or on the DNA promoter sequence (weak promoter), deregulation of the target genes might lead to various disorders. In this study, we aim to understand the electrostatic mechanism behind GATA TF and DNA promoter interactions, in order to predict Protein-DNA binding in the presence of mutations, while elaborating on non-covalent binding kinetics. To generate a family of mutants for the GATA:DNA complex, we replaced every charged amino acid, one at a time, with a neutral amino acid like Alanine (Ala). We then applied Poisson-Boltzmann electrostatic calculations feeding into free energy calculations, for each mutation. These calculations delineate the contribution to binding from each Ala-replaced amino acid in the GATA:DNA interaction. After analyzing the obtained data in view of a two-step model, we are able to identify potential key amino acids in binding. Finally, we applied the model to GATA-3:DNA (crystal structure with PDB-ID: 3DFV) binding complex and validated it against experimental results from the literature.

  3. A Constant Rate of Spontaneous Mutation in DNA-Based Microbes

    Science.gov (United States)

    Drake, John W.

    1991-08-01

    In terms of evolution and fitness, the most significant spontaneous mutation rate is likely to be that for the entire genome (or its nonfrivolous fraction). Information is now available to calculate this rate for several DNA-based haploid microbes, including bacteriophages with single- or double-stranded DNA, a bacterium, a yeast, and a filamentous fungus. Their genome sizes vary by ≈6500-fold. Their average mutation rates per base pair vary by ≈16,000-fold, whereas their mutation rates per genome vary by only ≈2.5-fold, apparently randomly, around a mean value of 0.0033 per DNA replication. The average mutation rate per base pair is inversely proportional to genome size. Therefore, a nearly invariant microbial mutation rate appears to have evolved. Because this rate is uniform in such diverse organisms, it is likely to be determined by deep general forces, perhaps by a balance between the usually deleterious effects of mutation and the physiological costs of further reducing mutation rates.

  4. DNA Damage Repair System in Plants: A Worldwide Research Update.

    Science.gov (United States)

    Gimenez, Estela; Manzano-Agugliaro, Francisco

    2017-10-30

    Living organisms are usually exposed to various DNA damaging agents so the mechanisms to detect and repair diverse DNA lesions have developed in all organisms with the result of maintaining genome integrity. Defects in DNA repair machinery contribute to cancer, certain diseases, and aging. Therefore, conserving the genomic sequence in organisms is key for the perpetuation of life. The machinery of DNA damage repair (DDR) in prokaryotes and eukaryotes is similar. Plants also share mechanisms for DNA repair with animals, although they differ in other important details. Plants have, surprisingly, been less investigated than other living organisms in this context, despite the fact that numerous lethal mutations in animals are viable in plants. In this manuscript, a worldwide bibliometric analysis of DDR systems and DDR research in plants was made. A comparison between both subjects was accomplished. The bibliometric analyses prove that the first study about DDR systems in plants (1987) was published thirteen years later than that for other living organisms (1975). Despite the increase in the number of papers about DDR mechanisms in plants in recent decades, nowadays the number of articles published each year about DDR systems in plants only represents 10% of the total number of articles about DDR. The DDR research field was done by 74 countries while the number of countries involved in the DDR & Plant field is 44. This indicates the great influence that DDR research in the plant field currently has, worldwide. As expected, the percentage of studies published about DDR systems in plants has increased in the subject area of agricultural and biological sciences and has diminished in medicine with respect to DDR studies in other living organisms. In short, bibliometric results highlight the current interest in DDR research in plants among DDR studies and can open new perspectives in the research field of DNA damage repair.

  5. TRAIP promotes DNA damage response during genome replication and is mutated in primordial dwarfism.

    Science.gov (United States)

    Harley, Margaret E; Murina, Olga; Leitch, Andrea; Higgs, Martin R; Bicknell, Louise S; Yigit, Gökhan; Blackford, Andrew N; Zlatanou, Anastasia; Mackenzie, Karen J; Reddy, Kaalak; Halachev, Mihail; McGlasson, Sarah; Reijns, Martin A M; Fluteau, Adeline; Martin, Carol-Anne; Sabbioneda, Simone; Elcioglu, Nursel H; Altmüller, Janine; Thiele, Holger; Greenhalgh, Lynn; Chessa, Luciana; Maghnie, Mohamad; Salim, Mahmoud; Bober, Michael B; Nürnberg, Peter; Jackson, Stephen P; Hurles, Matthew E; Wollnik, Bernd; Stewart, Grant S; Jackson, Andrew P

    2016-01-01

    DNA lesions encountered by replicative polymerases threaten genome stability and cell cycle progression. Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism. We establish that TRAIP relocalizes to sites of DNA damage, where it is required for optimal phosphorylation of H2AX and RPA2 during S-phase in response to ultraviolet (UV) irradiation, as well as fork progression through UV-induced DNA lesions. TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes. Human genetics thus identifies TRAIP as a component of the DNA damage response to replication-blocking DNA lesions.

  6. In utero DNA damage from environmental pollution is associated with somatic gene mutation in newborns

    Energy Technology Data Exchange (ETDEWEB)

    Perera, F.; Hemminki, K.; Jedrychowski, W.; Whyatt, R.; Campbell, U.; Hsu, Y.Z.; Santella, R.; Albertini, R.; O' Neill, J.P. [Columbia University, New York, NY (United States). School of Public Health

    2002-10-01

    Transplacental exposure to carcinogenic air pollutants from the combustion of fossil fuels is a growing health concern, given evidence of the heightened susceptibility of the fetus. These mutagenic/carcinogenic pollutants include aromatic compounds such as polycyclic aromatic hydrocarbons that bind to DNA, forming chemical-DNA adducts. The genotoxic effects of transplacental exposure in humans has been investigated by analyzing aromatic-DNA adducts and the frequency of gene mutations at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus in umbilical cord and maternal blood samples. Here the authors show, in a cross-sectional study of 67 mothers and 64 newborns from the Krakow Region of Poland, that aromatic-DNA adducts measured by P-32-postlabeling are positively associated with HPRT mutant frequency in the newborns (beta = 0.56, P = 0.03) after controlling for exposure to tobacco smoke, diet, and socioeconomic status. In contrast to the fetus, HPRT mutations and DNA adducts do not reflect similar exposure periods in the mother, and the maternal biomarkers were not correlated. Adducts were higher in the newborn than the mother, indicating differential susceptibility of the fetus to DNA damage; but HPRT mutation frequency was 4-fold lower, consistent with the long lifetime of the biomarker. These results provide the first demonstration of a molecular link between somatic mutation in the newborn and transplacental exposure to common air pollutants, a finding that is relevant to cancer risk assessment.

  7. Prevalence of migraine in persons with the 3243A>G mutation in mitochondrial DNA

    DEFF Research Database (Denmark)

    Guo, S.; Esserlind, A-L; Andersson, Z

    2016-01-01

    % vs. 6%; P persons with the mDNA 3243A>G mutation was found. This finding suggests a clinical association between a monogenetically inherited disorder......BACKGROUND AND PURPOSE: Over the last three decades mitochondrial dysfunction has been postulated to be a potential mechanism in migraine pathogenesis. The lifetime prevalence of migraine in persons carrying the 3243A>G mutation in mitochondrial DNA was investigated. METHODS: In this cross......-sectional study, 57 mDNA 3243A>G mutation carriers between May 2012 and October 2014 were included. As a control group, a population-based cohort from our epidemiological studies on migraine in Danes was used. History of headache and migraine was obtained by telephone interview, based on a validated semi...

  8. Chemical cleavage reactions of DNA on solid support: application in mutation detection

    Directory of Open Access Journals (Sweden)

    Cotton Richard GH

    2003-05-01

    Full Text Available Abstract Background The conventional solution-phase Chemical Cleavage of Mismatch (CCM method is time-consuming, as the protocol requires purification of DNA after each reaction step. This paper describes a new version of CCM to overcome this problem by immobilizing DNA on silica solid supports. Results DNA test samples were loaded on to silica beads and the DNA bound to the solid supports underwent chemical modification reactions with KMnO4 (potassium permanganate and hydroxylamine in 3M TEAC (tetraethylammonium chloride solution. The resulting modified DNA was then simultaneously cleaved by piperidine and removed from the solid supports to afford DNA fragments without the requirement of DNA purification between reaction steps. Conclusions The new solid-phase version of CCM is a fast, cost-effective and sensitive method for detection of mismatches and mutations.

  9. Assessing the contribution of the herpes simplex virus DNA polymerase to spontaneous mutations

    Directory of Open Access Journals (Sweden)

    Leary Jeffry J

    2002-05-01

    Full Text Available Abstract Background The thymidine kinase (tk mutagenesis assay is often utilized to determine the frequency of herpes simplex virus (HSV replication-mediated mutations. Using this assay, clinical and laboratory HSV-2 isolates were shown to have a 10- to 80-fold higher frequency of spontaneous mutations compared to HSV-1. Methods A panel of HSV-1 and HSV-2, along with polymerase-recombinant viruses expressing type 2 polymerase (Pol within a type 1 genome, were evaluated using the tk and non-HSV DNA mutagenesis assays to measure HSV replication-dependent errors and determine whether the higher mutation frequency of HSV-2 is a distinct property of type 2 polymerases. Results Although HSV-2 have mutation frequencies higher than HSV-1 in the tk assay, these errors are assay-specific. In fact, wild type HSV-1 and the antimutator HSV-1 PAAr5 exhibited a 2–4 fold higher frequency than HSV-2 in the non-HSV DNA mutatagenesis assay. Furthermore, regardless of assay, HSV-1 recombinants expressing HSV-2 Pol had error rates similar to HSV-1, whereas the high mutator virus, HSV-2 6757, consistently showed signficant errors. Additionally, plasmid DNA containing the HSV-2 tk gene, but not type 1 tk or LacZ DNA, was shown to form an anisomorphic DNA stucture. Conclusions This study suggests that the Pol is not solely responsible for the virus-type specific differences in mutation frequency. Accordingly, it is possible that (a mutations may be modulated by other viral polypeptides cooperating with Pol, and (b the localized secondary structure of the viral genome may partially account for the apparently enhanced error frequency of HSV-2.

  10. A comparative study on low-energy ion beam and neutralized beam modifications of naked DNA and biological effect on mutation

    Science.gov (United States)

    Sarapirom, S.; Thongkumkoon, P.; Prakrajang, K.; Anuntalabhochai, S.; Yu, L. D.

    2012-02-01

    DNA conformation change or damage induced by low-energy ion irradiation has been of great interest owing to research developments in ion beam biotechnology and ion beam application in biomedicine. Mechanisms involved in the induction of DNA damage may account for effect from implanting ion charge. In order to check this effect, we used both ion beam and neutralized beam at keV energy to bombard naked DNA. Argon or nitrogen ion beam was generated and extracted from a radiofrequency (RF) ion source and neutralized by microwave-driven plasma in the beam path. Plasmid DNA pGFP samples were irradiated with the ion or neutralized beam in vacuum, followed by gel electrophoresis to observe changes in the DNA conformations. It was revealed that the ion charge played a certain role in inducing DNA conformation change. The subsequent DNA transfer into bacteria Escherichia coli ( E. coli) for mutation analysis indicated that the charged ion beam induced DNA change had high potential in mutation induction while neutralized beam did not. The intrinsic reason was attributed to additional DNA deformation and contortion caused by ion charge exchange effect so that the ion beam induced DNA damage could hardly be completely repaired, whereas the neutralized beam induced DNA change could be more easily recoverable owing to absence of the additional DNA deformation and contortion.

  11. A comparative study on low-energy ion beam and neutralized beam modifications of naked DNA and biological effect on mutation

    International Nuclear Information System (INIS)

    Sarapirom, S.; Thongkumkoon, P.; Prakrajang, K.; Anuntalabhochai, S.; Yu, L.D.

    2012-01-01

    DNA conformation change or damage induced by low-energy ion irradiation has been of great interest owing to research developments in ion beam biotechnology and ion beam application in biomedicine. Mechanisms involved in the induction of DNA damage may account for effect from implanting ion charge. In order to check this effect, we used both ion beam and neutralized beam at keV energy to bombard naked DNA. Argon or nitrogen ion beam was generated and extracted from a radiofrequency (RF) ion source and neutralized by microwave-driven plasma in the beam path. Plasmid DNA pGFP samples were irradiated with the ion or neutralized beam in vacuum, followed by gel electrophoresis to observe changes in the DNA conformations. It was revealed that the ion charge played a certain role in inducing DNA conformation change. The subsequent DNA transfer into bacteria Escherichia coli (E. coli) for mutation analysis indicated that the charged ion beam induced DNA change had high potential in mutation induction while neutralized beam did not. The intrinsic reason was attributed to additional DNA deformation and contortion caused by ion charge exchange effect so that the ion beam induced DNA damage could hardly be completely repaired, whereas the neutralized beam induced DNA change could be more easily recoverable owing to absence of the additional DNA deformation and contortion.

  12. Episodic weakness due to mitochondrial DNA MT-ATP6/8 mutations.

    Science.gov (United States)

    Auré, Karine; Dubourg, Odile; Jardel, Claude; Clarysse, Lucie; Sternberg, Damien; Fournier, Emmanuel; Laforêt, Pascal; Streichenberger, Nathalie; Petiot, Philippe; Gervais-Bernard, Hélène; Vial, Christophe; Bedat-Millet, Anne-Laure; Drouin-Garraud, Valérie; Bouillaud, Frédéric; Vandier, Christophe; Fontaine, Bertrand; Lombès, Anne

    2013-11-19

    To report that homoplasmic deleterious mutations in the mitochondrial DNA MT-ATP6/8 genes may be responsible for acute episodes of limb weakness mimicking periodic paralysis due to channelopathies and dramatically responding to acetazolamide. Mitochondrial DNA sequencing and restriction PCR, oxidative phosphorylation functional assays, reactive oxygen species metabolism, and patch-clamp technique in cultured skin fibroblasts. Occurrence of a typical MELAS (mitochondrial encephalopathy with lactic acidosis and stroke-like episodes) syndrome in a single member of a large pedigree with episodic weakness associated with a later-onset distal motor neuropathy led to the disclosure of 2 deleterious mitochondrial DNA mutations. The MT-ATP6 m.9185T>C p.Leu220Pro mutation, previously associated with Leigh syndrome, was present in all family members, while the MT-TL1 m.3271T>C mutation, a known cause of MELAS syndrome, was observed in the sole patient with MELAS presentation. Significant defect of complexes V and I as well as oxidative stress were observed in both primary fibroblasts and cybrid cells with 100% m.9185T>C mutation. Permanent plasma membrane depolarization and altered permeability to K(+) in fibroblasts provided a link with the paralysis episodes. Screening of 9 patients, based on their clinical phenotype, identified 4 patients with similar deleterious MT-ATP6 mutations (twice m.9185T>C and once m.9176T>C or m.8893T>C). A fifth patient presented with an original potentially deleterious MT-ATP8 mutation (m.8403T>C). All mutations were associated with almost-normal complex V activity but significant oxidative stress and permanent plasma membrane depolarization. Homoplasmic mutations in the MT-ATP6/8 genes may cause episodic weakness responding to acetazolamide treatment.

  13. DNA polymerase η mutational signatures are found in a variety of different types of cancer.

    Science.gov (United States)

    Rogozin, Igor B; Goncearenco, Alexander; Lada, Artem G; De, Subhajyoti; Yurchenko, Vyacheslav; Nudelman, German; Panchenko, Anna R; Cooper, David N; Pavlov, Youri I

    2018-01-01

    DNA polymerase (pol) η is a specialized error-prone polymerase with at least two quite different and contrasting cellular roles: to mitigate the genetic consequences of solar UV irradiation, and promote somatic hypermutation in the variable regions of immunoglobulin genes. Misregulation and mistargeting of pol η can compromise genome integrity. We explored whether the mutational signature of pol η could be found in datasets of human somatic mutations derived from normal and cancer cells. A substantial excess of single and tandem somatic mutations within known pol η mutable motifs was noted in skin cancer as well as in many other types of human cancer, suggesting that somatic mutations in A:T bases generated by DNA polymerase η are a common feature of tumorigenesis. Another peculiarity of pol ηmutational signatures, mutations in YCG motifs, led us to speculate that error-prone DNA synthesis opposite methylated CpG dinucleotides by misregulated pol η in tumors might constitute an additional mechanism of cytosine demethylation in this hypermutable dinucleotide.

  14. Clustering of Caucasian Leber hereditary optic neuropathy patients containing the 11778 or 14484 mutations on an mtDNA lineage

    Energy Technology Data Exchange (ETDEWEB)

    Brown, M.D.; Sun, F.; Wallace, D.C. [Emory Univ. School of Medicine, Atlanta, GA (United States)

    1997-02-01

    Leber hereditary optic neuropathy (LHON) is a type of blindness caused by mtDNA mutations. Three LHON mtDNA mutations at nucleotide positions 3460, 11778, and 14484 are specific for LHON and account for 90% of worldwide cases and are thus designated as {open_quotes}primary{close_quotes} LHON mutations. Fifteen other {open_quotes}secondary{close_quotes} LHON mtDNA mutations have been identified, but their pathogenicity is unclear. mtDNA haplotype and phylogenetic analysis of the primary LHON mutations in North American Caucasian patients and controls has shown that, unlike the 3460 and 11778 mutations, which are distributed throughout the European-derived (Caucasian) mtDNA phylogeny, patients containing the 14484 mutation tended to be associated with European mtDNA haplotype J. To investigate this apparent clustering, we performed {chi}{sup 2}-based statistical analyses to compare the distribution of LHON patients on the Caucasian phylogenetic tree. Our results indicate that, unlike the 3460 and 11778 mutations, the 14484 mutation was not distributed on the phylogeny in proportion to the frequencies of the major Caucasian mtDNA haplogroups found in North America. The 14484 mutation was next shown to occur on the haplogroup J background more frequently that expected, consistent with the observation that {approximately}75% of worldwide 14484-positive LHON patients occur in association with haplogroup J. The 11778 mutation also exhibited a moderate clustering on haplogroup J. These observations were supported by statistical analysis using all available mutation frequencies reported in the literature. This paper thus illustrates the potential importance of genetic background in certain mtDNA-based diseases, speculates on a pathogenic role for a subset of LHON secondary mutations and their interaction with primary mutations, and provides support for a polygenic model for LHON expression in some cases. 18 refs., 3 tabs.

  15. Evolutionary constraints and the neutral theory. [mutation-caused nucleotide substitutions in DNA

    Science.gov (United States)

    Jukes, T. H.; Kimura, M.

    1984-01-01

    The neutral theory of molecular evolution postulates that nucleotide substitutions inherently take place in DNA as a result of point mutations followed by random genetic drift. In the absence of selective constraints, the substitution rate reaches the maximum value set by the mutation rate. The rate in globin pseudogenes is about 5 x 10 to the -9th substitutions per site per year in mammals. Rates slower than this indicate the presence of constraints imposed by negative (natural) selection, which rejects and discards deleterious mutations.

  16. Deficiency of the DNA repair protein nibrin increases the basal but not the radiation induced mutation frequency in vivo

    International Nuclear Information System (INIS)

    Wessendorf, Petra; Vijg, Jan; Nussenzweig, André; Digweed, Martin

    2014-01-01

    Highlights: • lacZ mutant frequencies measured in vivo in mouse models of radiosensitive Nijmegen Breakage Syndrome. • Spontaneous mutation frequencies are increased in lymphatic tissue due to Nbn mutation. • Single base transitions, not deletions, dominate the mutation spectrum. • Radiation induced mutation frequencies are not increased due to Nbn mutation. - Abstract: Nibrin (NBN) is a member of a DNA repair complex together with MRE11 and RAD50. The complex is associated particularly with the repair of DNA double strand breaks and with the regulation of cell cycle check points. Hypomorphic mutation of components of the complex leads to human disorders characterised by radiosensitivity and increased tumour occurrence, particularly of the lymphatic system. We have examined here the relationship between DNA damage, mutation frequency and mutation spectrum in vitro and in vivo in mouse models carrying NBN mutations and a lacZ reporter plasmid. We find that NBN mutation leads to increased spontaneous DNA damage in fibroblasts in vitro and high basal mutation rates in lymphatic tissue of mice in vivo. The characteristic mutation spectrum is dominated by single base transitions rather than the deletions and complex rearrangements expected after abortive repair of DNA double strand breaks. We conclude that in the absence of wild type nibrin, the repair of spontaneous errors, presumably arising during DNA replication, makes a major contribution to the basal mutation rate. This applies also to cells heterozygous for an NBN null mutation. Mutation frequencies after irradiation in vivo were not increased in mice with nibrin mutations as might have been expected considering the radiosensitivity of NBS patient cells in vitro. Evidently apoptosis is efficient, even in the absence of wild type nibrin

  17. Deficiency of the DNA repair protein nibrin increases the basal but not the radiation induced mutation frequency in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Wessendorf, Petra [Institute of Medical and Human Genetics, Charité – Universitätsmedizin Berlin, Augustenburger Platz 1, D-13353 Berlin (Germany); Vijg, Jan [Albert Einstein College of Medicine, Michael F. Price Center, 1301 Morris Park Avenue, Bronx, NY 10461 (United States); Nussenzweig, André [Laboratory of Genome Integrity, National Cancer Institute, National Institute of Health, 37 Convent Drive, Room 1106, Bethesda, MD 20892 (United States); Digweed, Martin, E-mail: martin.digweed@charite.de [Institute of Medical and Human Genetics, Charité – Universitätsmedizin Berlin, Augustenburger Platz 1, D-13353 Berlin (Germany)

    2014-11-15

    Highlights: • lacZ mutant frequencies measured in vivo in mouse models of radiosensitive Nijmegen Breakage Syndrome. • Spontaneous mutation frequencies are increased in lymphatic tissue due to Nbn mutation. • Single base transitions, not deletions, dominate the mutation spectrum. • Radiation induced mutation frequencies are not increased due to Nbn mutation. - Abstract: Nibrin (NBN) is a member of a DNA repair complex together with MRE11 and RAD50. The complex is associated particularly with the repair of DNA double strand breaks and with the regulation of cell cycle check points. Hypomorphic mutation of components of the complex leads to human disorders characterised by radiosensitivity and increased tumour occurrence, particularly of the lymphatic system. We have examined here the relationship between DNA damage, mutation frequency and mutation spectrum in vitro and in vivo in mouse models carrying NBN mutations and a lacZ reporter plasmid. We find that NBN mutation leads to increased spontaneous DNA damage in fibroblasts in vitro and high basal mutation rates in lymphatic tissue of mice in vivo. The characteristic mutation spectrum is dominated by single base transitions rather than the deletions and complex rearrangements expected after abortive repair of DNA double strand breaks. We conclude that in the absence of wild type nibrin, the repair of spontaneous errors, presumably arising during DNA replication, makes a major contribution to the basal mutation rate. This applies also to cells heterozygous for an NBN null mutation. Mutation frequencies after irradiation in vivo were not increased in mice with nibrin mutations as might have been expected considering the radiosensitivity of NBS patient cells in vitro. Evidently apoptosis is efficient, even in the absence of wild type nibrin.

  18. Plasma circulating tumor DNA as an alternative to metastatic biopsies for mutational analysis in breast cancer.

    Science.gov (United States)

    Rothé, F; Laes, J-F; Lambrechts, D; Smeets, D; Vincent, D; Maetens, M; Fumagalli, D; Michiels, S; Drisis, S; Moerman, C; Detiffe, J-P; Larsimont, D; Awada, A; Piccart, M; Sotiriou, C; Ignatiadis, M

    2014-10-01

    Molecular screening programs use next-generation sequencing (NGS) of cancer gene panels to analyze metastatic biopsies. We interrogated whether plasma could be used as an alternative to metastatic biopsies. The Ion AmpliSeq™ Cancer Hotspot Panel v2 (Ion Torrent), covering 2800 COSMIC mutations from 50 cancer genes was used to analyze 69 tumor (primary/metastases) and 31 plasma samples from 17 metastatic breast cancer patients. The targeted coverage for tumor DNA was ×1000 and for plasma cell-free DNA ×25 000. Whole blood normal DNA was used to exclude germline variants. The Illumina technology was used to confirm observed mutations. Evaluable NGS results were obtained for 60 tumor and 31 plasma samples from 17 patients. When tumor samples were analyzed, 12 of 17 (71%, 95% confidence interval (CI) 44% to 90%) patients had ≥1 mutation (median 1 mutation per patient, range 0-2 mutations) in either p53, PIK3CA, PTEN, AKT1 or IDH2 gene. When plasma samples were analyzed, 12 of 17 (71%, 95% CI: 44-90%) patients had ≥1 mutation (median 1 mutation per patient, range 0-2 mutations) in either p53, PIK3CA, PTEN, AKT1, IDH2 and SMAD4. All mutations were confirmed. When we focused on tumor and plasma samples collected at the same time-point, we observed that, in four patients, no mutation was identified in either tumor or plasma; in nine patients, the same mutations was identified in tumor and plasma; in two patients, a mutation was identified in tumor but not in plasma; in two patients, a mutation was identified in plasma but not in tumor. Thus, in 13 of 17 (76%, 95% CI 50% to 93%) patients, tumor and plasma provided concordant results whereas in 4 of 17 (24%, 95% CI 7% to 50%) patients, the results were discordant, providing complementary information. Plasma can be prospectively tested as an alternative to metastatic biopsies in molecular screening programs. © The Author 2014. Published by Oxford University Press on behalf of the European Society for Medical Oncology

  19. Determining the Location of DNA Modification and Mutation Caused by UVB Light in Skin Cancer

    Science.gov (United States)

    2013-09-01

    reads were then processed to determine the dinucleotide composition on the 5’ end by separating the Watson and Crick strands, and the dinucleotide...AD_________________ Award Number: W81XWH-12-1-0333 TITLE: Determining the Location of DNA ...COVERED 15 August 2012 – 14 August 2013 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Determining the Location of DNA Modification and Mutation Caused

  20. Does aerobic exercises induce mtDNA mutation in human blood ...

    African Journals Online (AJOL)

    The aim of this study was to determine the effect of eight weeks aerobic training on mitochondrial DNA (mtDNA) mutation in human blood leucocytes. Twenty untrained healthy students (training group: n =10, age = 20.7±1.5 yrs, weight = 67.7±10 kg, BF% = 17.5±7.35 & control group: n =10, age = 21±1.3 yrs, weight ...

  1. Somatic mitochondrial DNA mutations in cancer escape purifying selection and high pathogenicity mutations lead to the oncocytic phenotype: pathogenicity analysis of reported somatic mtDNA mutations in tumors

    International Nuclear Information System (INIS)

    Pereira, Luísa; Soares, Pedro; Máximo, Valdemar; Samuels, David C

    2012-01-01

    The presence of somatic mitochondrial DNA (mtDNA) mutations in cancer cells has been interpreted in controversial ways, ranging from random neutral accumulation of mutations, to positive selection for high pathogenicity, or conversely to purifying selection against high pathogenicity variants as occurs at the population level. Here we evaluated the predicted pathogenicity of somatic mtDNA mutations described in cancer and compare these to the distribution of variations observed in the global human population and all possible protein variations that could occur in human mtDNA. We focus on oncocytic tumors, which are clearly associated with mitochondrial dysfunction. The protein variant pathogenicity was predicted using two computational methods, MutPred and SNPs&GO. The pathogenicity score of the somatic mtDNA variants were significantly higher in oncocytic tumors compared to non-oncocytic tumors. Variations in subunits of Complex I of the electron transfer chain were significantly more common in tumors with the oncocytic phenotype, while variations in Complex V subunits were significantly more common in non-oncocytic tumors. Our results show that the somatic mtDNA mutations reported over all tumors are indistinguishable from a random selection from the set of all possible amino acid variations, and have therefore escaped the effects of purifying selection that act strongly at the population level. We show that the pathogenicity of somatic mtDNA mutations is a determining factor for the oncocytic phenotype. The opposite associations of the Complex I and Complex V variants with the oncocytic and non-oncocytic tumors implies that low mitochondrial membrane potential may play an important role in determining the oncocytic phenotype

  2. Effect of cold atmospheric pressure He-plasma jet on DNA change and mutation

    Science.gov (United States)

    Yaopromsiri, C.; Yu, L. D.; Sarapirom, S.; Thopan, P.; Boonyawan, D.

    2015-12-01

    Cold atmospheric pressure plasma jet (CAPPJ) effect on DNA change was studied for assessment of its safety. The experiment utilized a home-developed CAPPJ using 100% helium to directly treat naked DNA plasmid pGFP (plasmid green fluorescent protein). A traversal electric field was applied to separate the plasma components and both dry and wet sample conditions were adopted to investigate various factor roles in changing DNA. Plasma species were measured by using optical emission spectroscopy. DNA topological form change was analyzed by gel electrophoresis. The plasma jet treated DNA was transferred into bacterial Escherichia coli cells for observing mutation. The results show that the He-CAPPJ could break DNA strands due to actions from charge, radicals and neutrals and potentially cause genetic modification of living cells.

  3. Effect of cold atmospheric pressure He-plasma jet on DNA change and mutation

    Energy Technology Data Exchange (ETDEWEB)

    Yaopromsiri, C. [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Yu, L.D., E-mail: yuld@thep-center.org [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand); Sarapirom, S. [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Faculty of Science, Maejo University, Bang Khen, Chiang Mai 50290 (Thailand); Thopan, P.; Boonyawan, D. [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand)

    2015-12-15

    Cold atmospheric pressure plasma jet (CAPPJ) effect on DNA change was studied for assessment of its safety. The experiment utilized a home-developed CAPPJ using 100% helium to directly treat naked DNA plasmid pGFP (plasmid green fluorescent protein). A traversal electric field was applied to separate the plasma components and both dry and wet sample conditions were adopted to investigate various factor roles in changing DNA. Plasma species were measured by using optical emission spectroscopy. DNA topological form change was analyzed by gel electrophoresis. The plasma jet treated DNA was transferred into bacterial Escherichia coli cells for observing mutation. The results show that the He-CAPPJ could break DNA strands due to actions from charge, radicals and neutrals and potentially cause genetic modification of living cells.

  4. DNA damage by reactive species: Mechanisms, mutation and repair

    Indian Academy of Sciences (India)

    2012-06-25

    Jun 25, 2012 ... crosslinks can also affect the structure of DNA significantly. ... H2O2 by converting it into water, reaction of H2O2 with ..... Damaged nucleotide flipping by (a) AGT due to intercalation of an amino acid (Arg128) (pdb 1t38) and ...

  5. Progress on clustered DNA damage in radiation research

    International Nuclear Information System (INIS)

    Yang Li'na; Zhang Hong; Di Cuixia; Zhang Qiuning; Wang Xiaohu

    2012-01-01

    Clustered DNA damage which caused by high LET heavy ion radiation can lead to mutation, tumorigenesis and apoptosis. Promoting apoptosis of cancer cells is always the basis of cancer treatment. Clustered DNA damage has been the hot topic in radiobiology. The detect method is diversity, but there is not a detail and complete protocol to analyze clustered DNA damage. In order to provide reference for clustered DNA damage in the radiotherapy study, the clustered DNA damage characteristics, the latest progresses on clustered DNA damage and the detecting methods are reviewed and discussed in detail in this paper. (authors)

  6. Running on empty: does mitochondrial DNA mutation limit replicative lifespan in yeast?: Mutations that increase the division rate of cells lacking mitochondrial DNA also extend replicative lifespan in Saccharomyces cerevisiae.

    Science.gov (United States)

    Dunn, Cory D

    2011-10-01

    Mitochondrial DNA (mtDNA) mutations escalate with increasing age in higher organisms. However, it has so far been difficult to experimentally determine whether mtDNA mutation merely correlates with age or directly limits lifespan. A recent study shows that budding yeast can also lose functional mtDNA late in life. Interestingly, independent studies of replicative lifespan (RLS) and of mtDNA-deficient cells show that the same mutations can increase both RLS and the division rate of yeast lacking the mitochondrial genome. These exciting, parallel findings imply a potential causal relationship between mtDNA mutation and replicative senescence. Furthermore, these results suggest more efficient methods for discovering genes that determine lifespan. Copyright © 2011 WILEY Periodicals, Inc.

  7. Mitochondrial DNA depletion syndrome due to mutations in the RRM2B gene.

    Science.gov (United States)

    Bornstein, Belén; Area, Estela; Flanigan, Kevin M; Ganesh, Jaya; Jayakar, Parul; Swoboda, Kathryn J; Coku, Jorida; Naini, Ali; Shanske, Sara; Tanji, Kurenai; Hirano, Michio; DiMauro, Salvatore

    2008-06-01

    Mitochondrial DNA depletion syndrome (MDS) is characterized by a reduction in mtDNA copy number and has been associated with mutations in eight nuclear genes, including enzymes involved in mitochondrial nucleotide metabolism (POLG, TK2, DGUOK, SUCLA2, SUCLG1, PEO1) and MPV17. Recently, mutations in the RRM2B gene, encoding the p53-controlled ribonucleotide reductase subunit, have been described in seven infants from four families, who presented with various combinations of hypotonia, tubulopathy, seizures, respiratory distress, diarrhea, and lactic acidosis. All children died before 4 months of age. We sequenced the RRM2B gene in three unrelated cases with unexplained severe mtDNA depletion. The first patient developed intractable diarrhea, profound weakness, respiratory distress, and died at 3 months. The other two unrelated patients had a much milder phenotype and are still alive at ages 27 and 36 months. All three patients had lactic acidosis and severe depletion of mtDNA in muscle. Muscle histochemistry showed RRF and COX deficiency. Sequencing the RRM2B gene revealed three missense mutations and two single nucleotide deletions in exons 6, 8, and 9, confirming that RRM2B mutations are important causes of MDS and that the clinical phenotype is heterogeneous and not invariably fatal in infancy.

  8. Somatic mtDNA mutation spectra in the aging human putamen.

    Directory of Open Access Journals (Sweden)

    Siôn L Williams

    Full Text Available The accumulation of heteroplasmic mitochondrial DNA (mtDNA deletions and single nucleotide variants (SNVs is a well-accepted facet of the biology of aging, yet comprehensive mutation spectra have not been described. To address this, we have used next generation sequencing of mtDNA-enriched libraries (Mito-Seq to investigate mtDNA mutation spectra of putamen from young and aged donors. Frequencies of the "common" deletion and other "major arc" deletions were significantly increased in the aged cohort with the fold increase in the frequency of the common deletion exceeding that of major arc deletions. SNVs also increased with age with the highest rate of accumulation in the non-coding control region which contains elements necessary for translation and replication. Examination of predicted amino acid changes revealed a skew towards pathogenic SNVs in the coding region driven by mutation bias. Levels of the pathogenic m.3243A>G tRNA mutation were also found to increase with age. Novel multimeric tandem duplications that resemble murine control region multimers and yeast ρ(- mtDNAs, were identified in both young and aged specimens. Clonal ∼50 bp deletions in the control region were found at high frequencies in aged specimens. Our results reveal the complex manner in which the mitochondrial genome alters with age and provides a foundation for studies of other tissues and disease states.

  9. CSF studies facilitate DNA diagnosis in familial Alzheimer's disease due to a presenilin-1 mutation

    NARCIS (Netherlands)

    de Bot, Susanne T; Kremer, H P H; Dooijes, Dennis; Verbeek, Marcel M

    2009-01-01

    In sporadic Alzheimer's disease (AD), cerebrospinal fluid (CSF) analysis is becoming increasingly relevant to establish an early diagnosis. We present a case of familial AD due to a presenilin-1 mutation in which CSF studies suggested appropriate DNA diagnostics. A 38 year old Dutch man presented

  10. Fidelity and Mutational Spectrum of Pfu DNA Polymerase on a Human Mitochondrial DNA Sequence

    Science.gov (United States)

    André, Paulo; Kim, Andrea; Khrapko, Konstantin; Thilly, William G.

    1997-01-01

    The study of rare genetic changes in human tissues requires specialized techniques. Point mutations at fractions at or below 10−6 must be observed to discover even the most prominent features of the point mutational spectrum. PCR permits the increase in number of mutant copies but does so at the expense of creating many additional mutations or “PCR noise”. Thus, each DNA sequence studied must be characterized with regard to the DNA polymerase and conditions used to avoid interpreting a PCR-generated mutation as one arising in human tissue. The thermostable DNA polymerase derived from Pyrococcus furiosus designated Pfu has the highest fidelity of any DNA thermostable polymerase studied to date, and this property recommends it for analyses of tissue mutational spectra. Here, we apply constant denaturant capillary electrophoresis (CDCE) to separate and isolate the products of DNA amplification. This new strategy permitted direct enumeration and identification of point mutations created by Pfu DNA polymerase in a 96-bp low melting domain of a human mitochondrial sequence despite the very low mutant fractions generated in the PCR process. This sequence, containing part of the tRNA glycine and NADH dehydrogenase subunit 3 genes, is the target of our studies of mitochondrial mutagenesis in human cells and tissues. Incorrectly synthesized sequences were separated from the wild type as mutant/wild-type heteroduplexes by sequential enrichment on CDCE. An artificially constructed mutant was used as an internal standard to permit calculation of the mutant fraction. Our study found that the average error rate (mutations per base pair duplication) of Pfu was 6.5 × 10−7, and five of its more frequent mutations (hot spots) consisted of three transversions (GC → TA, AT → TA, and AT → CG), one transition (AT → GC), and one 1-bp deletion (in an AAAAAA sequence). To achieve an even higher sensitivity, the amount of Pfu-induced mutants must be

  11. Pigmentary retinopathy associated with the mitochondrial DNA 3243 point mutation.

    Science.gov (United States)

    Sue, C M; Mitchell, P; Crimmins, D S; Moshegov, C; Byrne, E; Morris, J G

    1997-10-01

    Fourteen patients from four unrelated families were studied to determine the prevalence of retinal pigmentary abnormalities associated with the MELAS A to G 3243 point mutation. Neurologic and ophthalmic examinations, retinal photography, pattern shift visual evoked potentials, and electroretinography were performed in all patients. Eight of the 14 patients had retinal pigmentary abnormalities characterized by symmetric areas of depigmentation involving predominantly the posterior pole and midperipheral retina. None of the patients had optic atrophy and only one patient with pigmentary retinal abnormalities had impaired visual acuity. None of the diabetic subjects (n = 6) had signs of diabetic retinopathy. Fluorescein angiography demonstrated mottled hyper- and hypofluorescent areas indicating multiple window defects in the retinal pigmentary epithelium. Visual evoked potentials showed delayed P100 responses in four of the eight patients with retinal pigmentary abnormalities. We conclude that there is a high prevalence of retinal pigmentary abnormalities in patients with MELAS A to G 3243 point mutation. These abnormalities are usually asymptomatic and best detected by retinal photography.

  12. Detection of somatic mutations by high-resolution DNA melting (HRM) analysis in multiple cancers.

    Science.gov (United States)

    Gonzalez-Bosquet, Jesus; Calcei, Jacob; Wei, Jun S; Garcia-Closas, Montserrat; Sherman, Mark E; Hewitt, Stephen; Vockley, Joseph; Lissowska, Jolanta; Yang, Hannah P; Khan, Javed; Chanock, Stephen

    2011-01-17

    Identification of somatic mutations in cancer is a major goal for understanding and monitoring the events related to cancer initiation and progression. High resolution melting (HRM) curve analysis represents a fast, post-PCR high-throughput method for scanning somatic sequence alterations in target genes. The aim of this study was to assess the sensitivity and specificity of HRM analysis for tumor mutation screening in a range of tumor samples, which included 216 frozen pediatric small rounded blue-cell tumors as well as 180 paraffin-embedded tumors from breast, endometrial and ovarian cancers (60 of each). HRM analysis was performed in exons of the following candidate genes known to harbor established commonly observed mutations: PIK3CA, ERBB2, KRAS, TP53, EGFR, BRAF, GATA3, and FGFR3. Bi-directional sequencing analysis was used to determine the accuracy of the HRM analysis. For the 39 mutations observed in frozen samples, the sensitivity and specificity of HRM analysis were 97% and 87%, respectively. There were 67 mutation/variants in the paraffin-embedded samples, and the sensitivity and specificity for the HRM analysis were 88% and 80%, respectively. Paraffin-embedded samples require higher quantity of purified DNA for high performance. In summary, HRM analysis is a promising moderate-throughput screening test for mutations among known candidate genomic regions. Although the overall accuracy appears to be better in frozen specimens, somatic alterations were detected in DNA extracted from paraffin-embedded samples.

  13. Detection of somatic mutations by high-resolution DNA melting (HRM analysis in multiple cancers.

    Directory of Open Access Journals (Sweden)

    Jesus Gonzalez-Bosquet

    Full Text Available Identification of somatic mutations in cancer is a major goal for understanding and monitoring the events related to cancer initiation and progression. High resolution melting (HRM curve analysis represents a fast, post-PCR high-throughput method for scanning somatic sequence alterations in target genes. The aim of this study was to assess the sensitivity and specificity of HRM analysis for tumor mutation screening in a range of tumor samples, which included 216 frozen pediatric small rounded blue-cell tumors as well as 180 paraffin-embedded tumors from breast, endometrial and ovarian cancers (60 of each. HRM analysis was performed in exons of the following candidate genes known to harbor established commonly observed mutations: PIK3CA, ERBB2, KRAS, TP53, EGFR, BRAF, GATA3, and FGFR3. Bi-directional sequencing analysis was used to determine the accuracy of the HRM analysis. For the 39 mutations observed in frozen samples, the sensitivity and specificity of HRM analysis were 97% and 87%, respectively. There were 67 mutation/variants in the paraffin-embedded samples, and the sensitivity and specificity for the HRM analysis were 88% and 80%, respectively. Paraffin-embedded samples require higher quantity of purified DNA for high performance. In summary, HRM analysis is a promising moderate-throughput screening test for mutations among known candidate genomic regions. Although the overall accuracy appears to be better in frozen specimens, somatic alterations were detected in DNA extracted from paraffin-embedded samples.

  14. Mutations in the DNA methyltransferase gene DNMT3A cause an overgrowth syndrome with intellectual disability.

    Science.gov (United States)

    Tatton-Brown, Katrina; Seal, Sheila; Ruark, Elise; Harmer, Jenny; Ramsay, Emma; Del Vecchio Duarte, Silvana; Zachariou, Anna; Hanks, Sandra; O'Brien, Eleanor; Aksglaede, Lise; Baralle, Diana; Dabir, Tabib; Gener, Blanca; Goudie, David; Homfray, Tessa; Kumar, Ajith; Pilz, Daniela T; Selicorni, Angelo; Temple, I Karen; Van Maldergem, Lionel; Yachelevich, Naomi; van Montfort, Robert; Rahman, Nazneen

    2014-04-01

    Overgrowth disorders are a heterogeneous group of conditions characterized by increased growth parameters and other variable clinical features such as intellectual disability and facial dysmorphism. To identify new causes of human overgrowth, we performed exome sequencing in ten proband-parent trios and detected two de novo DNMT3A mutations. We identified 11 additional de novo mutations by sequencing DNMT3A in a further 142 individuals with overgrowth. The mutations alter residues in functional DNMT3A domains, and protein modeling suggests that they interfere with domain-domain interactions and histone binding. Similar mutations were not present in 1,000 UK population controls (13/152 cases versus 0/1,000 controls; P < 0.0001). Mutation carriers had a distinctive facial appearance, intellectual disability and greater height. DNMT3A encodes a DNA methyltransferase essential for establishing methylation during embryogenesis and is commonly somatically mutated in acute myeloid leukemia. Thus, DNMT3A joins an emerging group of epigenetic DNA- and histone-modifying genes associated with both developmental growth disorders and hematological malignancies.

  15. The prognostic value of KRAS mutated plasma DNA in advanced non-small cell lung cancer

    DEFF Research Database (Denmark)

    Nygaard, Anneli Dowler; Garm Spindler, Karen-Lise; Pallisgaard, Niels

    2013-01-01

    BACKGROUND: Lung cancer is one of the most common malignant diseases worldwide and associated with considerable morbidity and mortality. New agents targeting the epidermal growth factor system are emerging, but only a subgroup of the patients will benefit from the therapy. Cell free DNA (cf......DNA) in the blood allows for tumour specific analyses, including KRAS-mutations, and the aim of the study was to investigate the possible prognostic value of plasma mutated KRAS (pmKRAS) in patients with non-small cell lung cancer (NSCLC). MATERIAL AND METHODS: Patients with newly diagnosed, advanced NSCLC eligible....... RESULTS: The study included 246 patients receiving a minimum of 1 treatment cycle, and all but four were evaluable for response according to RECIST. Forty-three patients (17.5%) presented with a KRAS mutation. OS was 8.9 months and PFS by intention to treat 5.4 months. Patients with a detectable plasma...

  16. Mutations in BALB mitochondrial DNA induce CCL20 up-regulation promoting tumorigenic phenotypes

    Energy Technology Data Exchange (ETDEWEB)

    Sligh, James [Department of Medicine—Dermatology Division, University of Arizona, Tucson, AZ 857 24 (United States); University of Arizona Cancer Center, Tucson, AZ 85724 (United States); Janda, Jaroslav [University of Arizona Cancer Center, Tucson, AZ 85724 (United States); Jandova, Jana, E-mail: jjandova@email.arizona.edu [Department of Medicine—Dermatology Division, University of Arizona, Tucson, AZ 857 24 (United States); University of Arizona Cancer Center, Tucson, AZ 85724 (United States)

    2014-11-15

    Highlights: • Alterations in mitochondrial DNA are commonly found in various human cancers. • Mutations in BALB mitochondrial DNA induce up-regulation of chemokine CCL20. • Increased growth and motility of mtBALB cells is associated with CCL20 levels. • mtDNA changes in BALB induce in vivo tumor growth through CCL20 up-regulation. • Mutations in mitochondrial DNA play important roles in keratinocyte neoplasia. - Abstract: mtDNA mutations are common in human cancers and are thought to contribute to the process of neoplasia. We examined the role of mtDNA mutations in skin cancer by generating fibroblast cybrids harboring a mutation in the gene encoding the mitochondrial tRNA for arginine. This somatic mutation (9821insA) was previously reported in UV-induced hyperkeratotic skin tumors in hairless mice and confers specific tumorigenic phenotypes to mutant cybrids. Microarray analysis revealed and RT-PCR along with Western blot analysis confirmed the up-regulation of CCL20 and its receptor CCR6 in mtBALB haplotype containing the mt-Tr 9821insA allele compared to wild type mtB6 haplotype. Based on reported role of CCL20 in cancer progression we examined whether the hyper-proliferation and enhanced motility of mtBALB haplotype would be associated with CCL20 levels. Treatment of both genotypes with recombinant CCL20 (rmCCL20) resulted in enhanced growth and motility of mtB6 cybrids. Furthermore, the acquired somatic alteration increased the in vivo tumor growth of mtBALB cybrids through the up-regulation of CCL20 since neutralizing antibody significantly decreased in vivo tumor growth of these cells; and tumors from anti-CCL20 treated mice injected with mtBALB cybrids showed significantly decreased CCL20 levels. When rmCCL20 or mtBALB cybrids were used as chemotactic stimuli, mtB6 cybrids showed increased motility while anti-CCL20 antibody decreased the migration and in vivo tumor growth of mtBALB cybrids. Moreover, the inhibitors of MAPK signaling and NF

  17. Deep sequencing shows that oocytes are not prone to accumulate mtDNA heteroplasmic mutations during ovarian ageing.

    Science.gov (United States)

    Boucret, L; Bris, C; Seegers, V; Goudenège, D; Desquiret-Dumas, V; Domin-Bernhard, M; Ferré-L'Hotellier, V; Bouet, P E; Descamps, P; Reynier, P; Procaccio, V; May-Panloup, P

    2017-10-01

    Does ovarian ageing increase the number of heteroplasmic mitochondrial DNA (mtDNA) point mutations in oocytes? Our results suggest that oocytes are not subject to the accumulation of mtDNA point mutations during ovarian ageing. Ageing is associated with the alteration of mtDNA integrity in various tissues. Primary oocytes, present in the ovary since embryonic life, may accumulate mtDNA mutations during the process of ovarian ageing. This was an observational study of 53 immature oocyte-cumulus complexes retrieved from 35 women undergoing IVF at the University Hospital of Angers, France, from March 2013 to March 2014. The women were classified in two groups, one including 19 women showing signs of ovarian ageing objectified by a diminished ovarian reserve (DOR), and the other, including 16 women with a normal ovarian reserve (NOR), which served as a control group. mtDNA was extracted from isolated oocytes, and from their corresponding cumulus cells (CCs) considered as a somatic cell compartment. The average mtDNA content of each sample was assessed by using a quantitative real-time PCR technique. Deep sequencing was performed using the Ion Torrent Proton for Next-Generation Sequencing. Signal processing and base calling were done by the embedded pre-processing pipeline and the variants were analyzed using an in-house workflow. The distribution of the different variants between DOR and NOR patients, on one hand, and oocyte and CCs, on the other, was analyzed with the generalized mixed linear model to take into account the cluster of cells belonging to a given mother. There were no significant differences between the numbers of mtDNA variants between the DOR and the NOR patients, either in the oocytes (P = 0.867) or in the surrounding CCs (P = 0.154). There were also no differences in terms of variants with potential functional consequences. De-novo mtDNA variants were found in 28% of the oocytes and in 66% of the CCs with the mean number of variants being

  18. Mitochondrial DNA triplication and punctual mutations in patients with mitochondrial neuromuscular disorders

    Energy Technology Data Exchange (ETDEWEB)

    Mkaouar-Rebai, Emna, E-mail: emna.mkaouar@gmail.com [Département des Sciences de la Vie, Faculté des Sciences de Sfax, Université de Sfax (Tunisia); Felhi, Rahma; Tabebi, Mouna [Laboratoire de Génétique Moléculaire Humaine, Faculté de Médecine de Sfax, Université de Sfax (Tunisia); Alila-Fersi, Olfa; Chamkha, Imen [Département des Sciences de la Vie, Faculté des Sciences de Sfax, Université de Sfax (Tunisia); Maalej, Marwa; Ammar, Marwa [Laboratoire de Génétique Moléculaire Humaine, Faculté de Médecine de Sfax, Université de Sfax (Tunisia); Kammoun, Fatma [Service de pédiatrie, C.H.U. Hedi Chaker de Sfax (Tunisia); Keskes, Leila [Laboratoire de Génétique Moléculaire Humaine, Faculté de Médecine de Sfax, Université de Sfax (Tunisia); Hachicha, Mongia [Service de pédiatrie, C.H.U. Hedi Chaker de Sfax (Tunisia); Fakhfakh, Faiza, E-mail: faiza.fakhfakh02@gmail.com [Département des Sciences de la Vie, Faculté des Sciences de Sfax, Université de Sfax (Tunisia)

    2016-04-29

    Mitochondrial diseases are a heterogeneous group of disorders caused by the impairment of the mitochondrial oxidative phosphorylation system which have been associated with various mutations of the mitochondrial DNA (mtDNA) and nuclear gene mutations. The clinical phenotypes are very diverse and the spectrum is still expanding. As brain and muscle are highly dependent on OXPHOS, consequently, neurological disorders and myopathy are common features of mtDNA mutations. Mutations in mtDNA can be classified into three categories: large-scale rearrangements, point mutations in tRNA or rRNA genes and point mutations in protein coding genes. In the present report, we screened mitochondrial genes of complex I, III, IV and V in 2 patients with mitochondrial neuromuscular disorders. The results showed the presence the pathogenic heteroplasmic m.9157G>A variation (A211T) in the MT-ATP6 gene in the first patient. We also reported the first case of triplication of 9 bp in the mitochondrial NC7 region in Africa and Tunisia, in association with the novel m.14924T>C in the MT-CYB gene in the second patient with mitochondrial neuromuscular disorder. - Highlights: • We reported 2 patients with mitochondrial neuromuscular disorders. • The heteroplasmic MT-ATP6 9157G>A variation was reported. • A triplication of 9 bp in the mitochondrial NC7 region was detected. • The m.14924T>C transition (S60P) in the MT-CYB gene was found.

  19. Endurance exercise rescues progeroid aging and induces systemic mitochondrial rejuvenation in mtDNA mutator mice

    Science.gov (United States)

    Safdar, Adeel; Bourgeois, Jacqueline M.; Ogborn, Daniel I.; Little, Jonathan P.; Hettinga, Bart P.; Akhtar, Mahmood; Thompson, James E.; Melov, Simon; Mocellin, Nicholas J.; Kujoth, Gregory C.; Prolla, Tomas A.; Tarnopolsky, Mark A.

    2011-01-01

    A causal role for mitochondrial DNA (mtDNA) mutagenesis in mammalian aging is supported by recent studies demonstrating that the mtDNA mutator mouse, harboring a defect in the proofreading-exonuclease activity of mitochondrial polymerase gamma, exhibits accelerated aging phenotypes characteristic of human aging, systemic mitochondrial dysfunction, multisystem pathology, and reduced lifespan. Epidemiologic studies in humans have demonstrated that endurance training reduces the risk of chronic diseases and extends life expectancy. Whether endurance exercise can attenuate the cumulative systemic decline observed in aging remains elusive. Here we show that 5 mo of endurance exercise induced systemic mitochondrial biogenesis, prevented mtDNA depletion and mutations, increased mitochondrial oxidative capacity and respiratory chain assembly, restored mitochondrial morphology, and blunted pathological levels of apoptosis in multiple tissues of mtDNA mutator mice. These adaptations conferred complete phenotypic protection, reduced multisystem pathology, and prevented premature mortality in these mice. The systemic mitochondrial rejuvenation through endurance exercise promises to be an effective therapeutic approach to mitigating mitochondrial dysfunction in aging and related comorbidities. PMID:21368114

  20. Study of hepatitis B virus gene mutations with enzymatic colorimetry-based DNA microarray.

    Science.gov (United States)

    Mao, Hailei; Wang, Huimin; Zhang, Donglei; Mao, Hongju; Zhao, Jianlong; Shi, Jian; Cui, Zhichu

    2006-01-01

    To establish a modified microarray method for detecting HBV gene mutations in the clinic. Site-specific oligonucleotide probes were immobilized to microarray slides and hybridized to biotin-labeled HBV gene fragments amplified from two-step PCR. Hybridized targets were transferred to nitrocellulose membranes, followed by intensity measurement using BCIP/NBT colorimetry. HBV genes from 99 Hepatitis B patients and 40 healthy blood donors were analyzed. Mutation frequencies of HBV pre-core/core and basic core promoter (BCP) regions were found to be significantly higher in the patient group (42%, 40% versus 2.5%, 5%, P colorimetry method exhibited the same level of sensitivity and reproducibility. An enzymatic colorimetry-based DNA microarray assay was successfully established to monitor HBV mutations. Pre-core/core and BCP mutations of HBV genes could be major causes of HBV infection in HBeAg-negative patients and could also be relevant to chronicity and aggravation of hepatitis B.

  1. DNA microarray analysis of fim mutations in Escherichia coli

    DEFF Research Database (Denmark)

    Schembri, Mark; Ussery, David; Workman, Christopher

    2002-01-01

    Bacterial adhesion is often mediated by complex polymeric surface structures referred to as fimbriae. Type I fimbriae of Escherichia coli represent the archetypical and best characterised fimbrial system. These adhesive organelles mediate binding to D-mannose and are directly associated...... we have used DNA microarray analysis to examine the molecular events involved in response to fimbrial gene expression in E. coli K-12. Observed differential expression levels of the fim genes were in good agreement with our current knowledge of the stoichiometry of type I fimbriae. Changes in fim...

  2. DNA nanotechnology and its applications in biomedical research.

    Science.gov (United States)

    Sun, Lifan; Yu, Lu; Shen, Wanqiu

    2014-09-01

    DNA nanotechnology, which uses DNA as a material to self-assemble designed nanostructures, including DNA 2D arrays, 3D nanostructures, DNA nanotubes and DNA nanomechanical devices, has showed great promise in biomedical applications. Various DNA nanostructures have been used for protein characterization, enzyme assembly, biosensing, drug delivery and biomimetic assemblies. In this review, we will present recent advances of DNA nanotechnology and its applications in biomedical research field.

  3. Mutation Processes in 293-Based Clones Overexpressing the DNA Cytosine Deaminase APOBEC3B.

    Directory of Open Access Journals (Sweden)

    Monica K Akre

    Full Text Available Molecular, cellular, and clinical studies have combined to demonstrate a contribution from the DNA cytosine deaminase APOBEC3B (A3B to the overall mutation load in breast, head/neck, lung, bladder, cervical, ovarian, and other cancer types. However, the complete landscape of mutations attributable to this enzyme has yet to be determined in a controlled human cell system. We report a conditional and isogenic system for A3B induction, genomic DNA deamination, and mutagenesis. Human 293-derived cells were engineered to express doxycycline-inducible A3B-eGFP or eGFP constructs. Cells were subjected to 10 rounds of A3B-eGFP exposure that each caused 80-90% cell death. Control pools were subjected to parallel rounds of non-toxic eGFP exposure, and dilutions were done each round to mimic A3B-eGFP induced population fluctuations. Targeted sequencing of portions of TP53 and MYC demonstrated greater mutation accumulation in the A3B-eGFP exposed pools. Clones were generated and microarray analyses were used to identify those with the greatest number of SNP alterations for whole genome sequencing. A3B-eGFP exposed clones showed global increases in C-to-T transition mutations, enrichments for cytosine mutations within A3B-preferred trinucleotide motifs, and more copy number aberrations. Surprisingly, both control and A3B-eGFP clones also elicited strong mutator phenotypes characteristic of defective mismatch repair. Despite this additional mutational process, the 293-based system characterized here still yielded a genome-wide view of A3B-catalyzed mutagenesis in human cells and a system for additional studies on the compounded effects of simultaneous mutation mechanisms in cancer cells.

  4. SNPase-ARMS qPCR: Ultrasensitive Mutation-Based Detection of Cell-Free Tumor DNA in Melanoma Patients.

    Directory of Open Access Journals (Sweden)

    Julia Stadler

    Full Text Available Cell-free circulating tumor DNA in the plasma of cancer patients has become a common point of interest as indicator of therapy options and treatment response in clinical cancer research. Especially patient- and tumor-specific single nucleotide variants that accurately distinguish tumor DNA from wild type DNA are promising targets. The reliable detection and quantification of these single-base DNA variants is technically challenging. Currently, a variety of techniques is applied, with no apparent "gold standard". Here we present a novel qPCR protocol that meets the conditions of extreme sensitivity and specificity that are required for detection and quantification of tumor DNA. By consecutive application of two polymerases, one of them designed for extreme base-specificity, the method reaches unprecedented sensitivity and specificity. Three qPCR assays were tested with spike-in experiments, specific for point mutations BRAF V600E, PTEN T167A and NRAS Q61L of melanoma cell lines. It was possible to detect down to one copy of tumor DNA per reaction (Poisson distribution, at a background of up to 200 000 wild type DNAs. To prove its clinical applicability, the method was successfully tested on a small cohort of BRAF V600E positive melanoma patients.

  5. DNA adduct formation and mutation induction by aristolochic acid in rat kidney and liver

    International Nuclear Information System (INIS)

    Mei, Nan; Arlt, Volker M.; Phillips, David H.; Heflich, Robert H.; Chen, Tao

    2006-01-01

    Aristolochic acid (AA) is a potent nephrotoxin and carcinogen and is the causative factor for Chinese herb nephropathy. AA has been associated with the development of urothelial cancer in humans, and kidney and forestomach tumors in rodents. To investigate the molecular mechanisms responsible for the tumorigenicity of AA, we determined the DNA adduct formation and mutagenicity of AA in the liver (nontarget tissue) and kidney (target tissue) of Big Blue rats. Groups of six male rats were gavaged with 0, 0.1, 1.0 and 10.0 mg AA/kg body weight five times/week for 3 months. The rats were sacrificed 1 day after the final treatment, and the livers and kidneys were isolated. DNA adduct formation was analyzed by 32 P-postlabeling and mutant frequency (MF) was determined using the λ Select-cII Mutation Detection System. Three major adducts (7-[deoxyadenosin-N 6 -yl]-aristolactam I, 7-[deoxyadenosin-N 6 -yl]-aristolactam II and 7-[deoxyguanosin-N 2 -yl]-aristolactam I) were identified. There were strong linear dose-responses for AA-induced DNA adducts in treated rats, ranging from 25 to 1967 adducts/10 8 nucleotides in liver and 95-4598 adducts/10 8 nucleotides in kidney. A similar trend of dose-responses for mutation induction also was found, the MFs ranging from 37 to 666 x 10 -6 in liver compared with the MFs of 78-1319 x 10 -6 that we previously reported for the kidneys of AA-treated rats. Overall, kidneys had at least two-fold higher levels of DNA adducts and MF than livers. Sequence analysis of the cII mutants revealed that there was a statistically significant difference between the mutation spectra in both kidney and liver of AA-treated and control rats, but there was no significant difference between the mutation spectra in AA-treated livers and kidneys. A:T → T:A transversion was the predominant mutation in AA-treated rats; whereas G:C → A:T transition was the main type of mutation in control rats. These results indicate that the AA treatment that eventually

  6. DNA adduct formation and mutation induction by aristolochic acid in rat kidney and liver

    Energy Technology Data Exchange (ETDEWEB)

    Mei, Nan [Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, FDA, Jefferson, AR 72079 (United States)]. E-mail: nan.mei@fda.hhs.gov; Arlt, Volker M. [Section of Molecular Carcinogenesis, Institute of Cancer Research, Cotswold Road, Sutton, Surrey SM2 5NG (United Kingdom); Phillips, David H. [Section of Molecular Carcinogenesis, Institute of Cancer Research, Cotswold Road, Sutton, Surrey SM2 5NG (United Kingdom); Heflich, Robert H. [Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, FDA, Jefferson, AR 72079 (United States); Chen, Tao [Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, FDA, Jefferson, AR 72079 (United States)

    2006-12-01

    Aristolochic acid (AA) is a potent nephrotoxin and carcinogen and is the causative factor for Chinese herb nephropathy. AA has been associated with the development of urothelial cancer in humans, and kidney and forestomach tumors in rodents. To investigate the molecular mechanisms responsible for the tumorigenicity of AA, we determined the DNA adduct formation and mutagenicity of AA in the liver (nontarget tissue) and kidney (target tissue) of Big Blue rats. Groups of six male rats were gavaged with 0, 0.1, 1.0 and 10.0 mg AA/kg body weight five times/week for 3 months. The rats were sacrificed 1 day after the final treatment, and the livers and kidneys were isolated. DNA adduct formation was analyzed by {sup 32}P-postlabeling and mutant frequency (MF) was determined using the {lambda} Select-cII Mutation Detection System. Three major adducts (7-[deoxyadenosin-N {sup 6}-yl]-aristolactam I, 7-[deoxyadenosin-N {sup 6}-yl]-aristolactam II and 7-[deoxyguanosin-N {sup 2}-yl]-aristolactam I) were identified. There were strong linear dose-responses for AA-induced DNA adducts in treated rats, ranging from 25 to 1967 adducts/10{sup 8} nucleotides in liver and 95-4598 adducts/10{sup 8} nucleotides in kidney. A similar trend of dose-responses for mutation induction also was found, the MFs ranging from 37 to 666 x 10{sup -6} in liver compared with the MFs of 78-1319 x 10{sup -6} that we previously reported for the kidneys of AA-treated rats. Overall, kidneys had at least two-fold higher levels of DNA adducts and MF than livers. Sequence analysis of the cII mutants revealed that there was a statistically significant difference between the mutation spectra in both kidney and liver of AA-treated and control rats, but there was no significant difference between the mutation spectra in AA-treated livers and kidneys. A:T {sup {yields}} T:A transversion was the predominant mutation in AA-treated rats; whereas G:C {sup {yields}} A:T transition was the main type of mutation in control

  7. Germline mutation rates at tandem repeat loci in DNA-repair deficient mice

    International Nuclear Information System (INIS)

    Barber, Ruth C.; Miccoli, Laurent; Buul, Paul P.W. van; Burr, Karen L.-A.; Duyn-Goedhart, Annemarie van; Angulo, Jaime F.; Dubrova, Yuri E.

    2004-01-01

    Mutation rates at two expanded simple tandem repeat (ESTR) loci were studied in the germline of non-exposed and irradiated severe combined immunodeficient (scid) and poly(ADP-ribose) polymerase (PARP-1 -/- ) deficient male mice. Non-exposed scid and PARP -/- male mice showed considerably elevated ESTR mutation rates, far higher than those in wild-type isogenic mice and other inbred strains. The irradiated scid and PARP-1 -/- male mice did not show any detectable increases in their mutation rate, whereas significant ESTR mutation induction was observed in the irradiated wild-type isogenic males. ESTR mutation spectra in the scid and PARP-1 -/- strains did not differ from those in the isogenic wild-type strains. Considering these data and the results of previous studies, we propose that a delay in repair of DNA damage in scid and PARP-1 -/- mice could result in replication fork pausing which, in turn, may affect ESTR mutation rate in the non-irradiated males. The lack of mutation induction in irradiated scid and PARP-1 -/- can be explained by the high cell killing effects of irradiation on the germline of deficient mice

  8. Transcription Restores DNA Repair to Heterochromatin, Determining Regional Mutation Rates in Cancer Genomes

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    Christina L. Zheng

    2014-11-01

    Full Text Available Somatic mutations in cancer are more frequent in heterochromatic and late-replicating regions of the genome. We report that regional disparities in mutation density are virtually abolished within transcriptionally silent genomic regions of cutaneous squamous cell carcinomas (cSCCs arising in an XPC−/− background. XPC−/− cells lack global genome nucleotide excision repair (GG-NER, thus establishing differential access of DNA repair machinery within chromatin-rich regions of the genome as the primary cause for the regional disparity. Strikingly, we find that increasing levels of transcription reduce mutation prevalence on both strands of gene bodies embedded within H3K9me3-dense regions, and only to those levels observed in H3K9me3-sparse regions, also in an XPC-dependent manner. Therefore, transcription appears to reduce mutation prevalence specifically by relieving the constraints imposed by chromatin structure on DNA repair. We model this relationship among transcription, chromatin state, and DNA repair, revealing a new, personalized determinant of cancer risk.

  9. Inspecting Targeted Deep Sequencing of Whole Genome Amplified DNA Versus Fresh DNA for Somatic Mutation Detection: A Genetic Study in Myelodysplastic Syndrome Patients.

    Science.gov (United States)

    Palomo, Laura; Fuster-Tormo, Francisco; Alvira, Daniel; Ademà, Vera; Armengol, María Pilar; Gómez-Marzo, Paula; de Haro, Nuri; Mallo, Mar; Xicoy, Blanca; Zamora, Lurdes; Solé, Francesc

    2017-08-01

    Whole genome amplification (WGA) has become an invaluable method for preserving limited samples of precious stock material and has been used during the past years as an alternative tool to increase the amount of DNA before library preparation for next-generation sequencing. Myelodysplastic syndromes (MDS) are a group of clonal hematopoietic stem cell disorders characterized by presenting somatic mutations in several myeloid-related genes. In this work, targeted deep sequencing has been performed on four paired fresh DNA and WGA DNA samples from bone marrow of MDS patients, to assess the feasibility of using WGA DNA for detecting somatic mutations. The results of this study highlighted that, in general, the sequencing and alignment statistics of fresh DNA and WGA DNA samples were similar. However, after variant calling and when considering variants detected at all frequencies, there was a high level of discordance between fresh DNA and WGA DNA (overall, a higher number of variants was detected in WGA DNA). After proper filtering, a total of three somatic mutations were detected in the cohort. All somatic mutations detected in fresh DNA were also identified in WGA DNA and validated by whole exome sequencing.

  10. Detection of Hepatitis B Virus M204I Mutation by Quantum Dot-Labeled DNA Probe

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    Cheng Zhang

    2017-04-01

    Full Text Available Quantum dots (QDs are semiconductor nanoparticles with a diameter of less than 10 nm, which have been widely used as fluorescent probes in biochemical analysis and vivo imaging because of their excellent optical properties. Sensitive and convenient detection of hepatitis B virus (HBV gene mutations is important in clinical diagnosis. Therefore, we developed a sensitive, low-cost and convenient QDs-mediated fluorescent method for the detection of HBV gene mutations in real serum samples from chronic hepatitis B (CHB patients who had received lamivudine or telbivudine antiviral therapy. We also evaluated the efficiency of this method for the detection of drug-resistant mutations compared with direct sequencing. In CHB, HBV DNA from the serum samples of patients with poor response or virological breakthrough can be hybridized to probes containing the M204I mutation to visualize fluorescence under fluorescence microscopy, where fluorescence intensity is related to the virus load, in our method. At present, the limits of the method used to detect HBV genetic variations by fluorescence quantum dots is 103 IU/mL. These results show that QDs can be used as fluorescent probes to detect viral HBV DNA polymerase gene variation, and is a simple readout system without complex and expensive instruments, which provides an attractive platform for the detection of HBV M204I mutation.

  11. Quantitative evaluation of DNA damage and mutation rate by atmospheric and room-temperature plasma (ARTP) and conventional mutagenesis.

    Science.gov (United States)

    Zhang, Xue; Zhang, Chong; Zhou, Qian-Qian; Zhang, Xiao-Fei; Wang, Li-Yan; Chang, Hai-Bo; Li, He-Ping; Oda, Yoshimitsu; Xing, Xin-Hui

    2015-07-01

    DNA damage is the dominant source of mutation, which is the driving force of evolution. Therefore, it is important to quantitatively analyze the DNA damage caused by different mutagenesis methods, the subsequent mutation rates, and their relationship. Atmospheric and room temperature plasma (ARTP) mutagenesis has been used for the mutation breeding of more than 40 microorganisms. However, ARTP mutagenesis has not been quantitatively compared with conventional mutation methods. In this study, the umu test using a flow-cytometric analysis was developed to quantify the DNA damage in individual viable cells using Salmonella typhimurium NM2009 as the model strain and to determine the mutation rate. The newly developed method was used to evaluate four different mutagenesis systems: a new ARTP tool, ultraviolet radiation, 4-nitroquinoline-1-oxide (4-NQO), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis. The mutation rate was proportional to the corresponding SOS response induced by DNA damage. ARTP caused greater DNA damage to individual living cells than the other conventional mutagenesis methods, and the mutation rate was also higher. By quantitatively comparing the DNA damage and consequent mutation rate after different types of mutagenesis, we have shown that ARTP is a potentially powerful mutagenesis tool with which to improve the characteristics of microbial cell factories.

  12. Human aging and somatic point mutations in mtDNA: a comparative study of generational differences (grandparents and grandchildren

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    Anderson Nonato do Rosário Marinho

    2011-01-01

    Full Text Available The accumulation of somatic mutations in mtDNA is correlated with aging. In this work, we sought to identify somatic mutations in the HVS-1 region (D-loop of mtDNA that might be associated with aging. For this, we compared 31 grandmothers (mean age: 63 ± 2.3 years and their 62 grandchildren (mean age: 15 ± 4.1 years, the offspring of their daughters. Direct DNA sequencing showed that mutations absent in the grandchildren were detected in a presumably homoplasmic state in three grandmothers and in a heteroplasmic state in an additional 13 grandmothers; no mutations were detected in the remaining 15 grandmothers. However, cloning followed by DNA sequencing in 12 grandmothers confirmed homoplasia in only one of the three mutations previously considered to be homoplasmic and did not confirm heteroplasmy in three out of nine grandmothers found to be heteroplasmic by direct sequencing. Thus, of 12 grandmothers in whom mtDNA was analyzed by cloning, eight were heteroplasmic for mutations not detected in their grandchildren. In this study, the use of genetically related subjects allowed us to demonstrate the occurrence of age-related (> 60 years old mutations (homoplasia and heteroplasmy. It is possible that both of these situations (homoplasia and heteroplasmy were a long-term consequence of mitochondrial oxidative phosphorylation that can lead to the accumulation of mtDNA mutations throughout life.

  13. Gamma radiation-induced heritable mutations at repetitive DNA loci in out-bred mice

    International Nuclear Information System (INIS)

    Somers, C.M.; Sharma, R.; Quinn, J.S.; Boreham, D.R.

    2004-01-01

    Recent studies have shown that expanded-simple-tandem-repeat (ESTR) DNA loci are efficient genetic markers for detecting radiation-induced germ line mutations in mice. Dose responses following irradiation, however, have only been characterized in a small number of inbred mouse strains, and no studies have applied Esters to examine potential modifiers of radiation risk, such as adaptive response. We gamma-irradiated groups of male out-bred Swiss-Webster mice with single acute doses of 0.5 and 1.0 Gy, and compared germ line mutation rates at ESTR loci to a sham-irradiated control. To test for evidence of adaptive response we treated a third group with a total dose of 1.1 Gy that was fractionated into a 0.1 Gy adapting dose, followed by a challenge dose of 1.0 Gy 24 h later. Paternal mutation rates were significantly elevated above the control in the 0.5 Gy (2.8-fold) and 1.0 Gy (3.0-fold) groups, but were similar to each other despite the difference in radiation dose. The doubling dose for paternal mutation induction was 0.26 Gy (95% CI = 0.14-0.51 Gy). Males adapted with a 0.1 Gy dose prior to a 1.0 Gy challenge dose had mutation rates that were not significantly elevated above the control, and were 43% reduced compared to those receiving single doses. We conclude that pre-meiotic male germ cells in out-bred Swiss-Webster mice are sensitive to ESTR mutations induced by acute doses of ionizing radiation, but mutation induction may become saturated at a lower dose than in some strains of inbred mice. Reduced mutation rates in the adapted group provide intriguing evidence for suppression of ESTR mutations in the male germline through adaptive response. Repetitive DNA markers may be useful tools for exploration of biological factors affecting the probability of heritable mutations caused by low-dose ionizing radiation exposure. The biological significance of ESTR mutations in terms of radiation risk assessment, however, is still undetermined

  14. Genotype-Phenotype Correlation of Maternally Inherited Disorders due to Mutations in Mitochondrial DNA

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    Peterus Thajeb

    2006-09-01

    Full Text Available Mitochondrial disorders are heterogeneous systemic ailments that are most often caused by maternal inheritance of a variety of mutations of the mitochondrial (mt DNA. Paternal inheritance and somatic mutation are rare. The disorders are well recognized not only for the genotypic heterogeneity, but also the phenotypic variation among the affected members of a single family. The genotype-phenotype correlation of the diversity of the syndromic and non-syndromic features of mitochondrial disorders are discussed. Some aspects of the molecular mechanisms of this heterogeneity, and the histopathologic findings are highlighted.

  15. Absence of correlation between serum CRP levels and mitochondrial D-loop DNA mutations in gastro-oesophageal adenocarcinoma

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    Benjamin H. L. Tan

    2014-01-01

    Full Text Available Introduction: Both inflammation and mitochondrial DNA (mtDNA mutation are thought to play a role in the many human cancers. The aim of this study was to evaluate the relationship between inflammation and accumulation of mitochondrial DNA (mtDNA mutations in the D-loop region in carcinogenesis of gastro-oesophageal adenocarcinomas. Materials and Methods: Blood samples of 20 patients with gastro-oesophageal adenocarcinoma were taken for measurement of serum C-reactive protein (CRP concentration. Direct sequencing of mtDNA in the D-loop region was done in the 20 adenocarcinoma samples and their corresponding surrounding non-cancerous tissue. Sequences were compared with existing mtDNA databases to identify mutations. Results: mtDNA mutations in the D-loop region occur commonly with almost identical frequency in both non-cancerous tissue (3.0 ΁ 1.6 and adenocarcinoma (3.1 ΁ 1.9 (P = 0.916, paired t-test. CRP levels are not predictive of the number of D-loop mutations in both adenocarcinoma (β: -0.131; 95% CI: -2.354-1.364; P = 0.583 and non-cancerous tissue samples (β: 0.130; 95% CI: -1.125-1.933; P = 0.586. Five new mutations were identified that were not recorded previously in mtDNA databases. Conclusion: D-loop mtDNA mutations are common in both gastro-oesophageal adenocarcinoma and surrounding non-cancerous tissue. However, the accumulation of such mutations appears to occur independent of systemic inflammation. The frequency of D-loop mutations is likely not useful as a marker for carcinogenesis in gastro-oesophageal adenocarcinoma.

  16. DNA repair in Haemophilus influenzae: isolation and characterization of an ultraviolet sensitive mutator mutant

    International Nuclear Information System (INIS)

    Walter, R.B.

    1985-01-01

    DNA repair in Haemophilus influenzae appears to be quite different from that seen in Escherichia coli in that H. influenzae shows neither SOS nor adaptation phenomena. Repair of DNA lesions in H. influenzae has been seen to occur via recombinational, excision, and mismatch repair pathways acting independently of one another. The author has isolated an ultraviolet (UV)-sensitive mutator mutant (mutB1) of H. influenzae Rd which shows deficiencies in both recombinational and mismatch repair pathways. This mutant is sensitive to a variety of DNA damaging agents as well as being hypermutable by alkylating agents and base analogues. MutB1 cells do not show post-UV DNA breakdown but do begin excision after UV irradiation. Genetic transformation with UV-irradiated DNA on mut B1 recipients shows that high (HE) and low (LE) efficiency markers are transformed at a ratio of 1.0 as in the mismatch repair deficient hex 1 mutant; however, kinetics of UV-inactivation experiments indicate that HE markers are sensitized and act as LE markers do on wild type recipients. Thus, the mutB gene product appears to play a role in both DNA repair and genetic transformation. A model is outlined which presents a role for a DNA helicase in both DNA repair and genetic transformation of H. influenzae

  17. Mutations in the DNA methyltransferase gene, DNMT3A, cause an overgrowth syndrome with intellectual disability

    Science.gov (United States)

    Tatton-Brown, Katrina; Seal, Sheila; Ruark, Elise; Harmer, Jenny; Ramsay, Emma; del Vecchio Duarte, Silvana; Zachariou, Anna; Hanks, Sandra; O’Brien, Eleanor; Aksglaede, Lise; Baralle, Diana; Dabir, Tabib; Gener, Blanca; Goudie, David; Homfray, Tessa; Kumar, Ajith; Pilz, Daniela T; Selicorni, Angelo; Temple, I Karen; Van Maldergem, Lionel; Yachelevich, Naomi; van Montfort, Robert; Rahman, Nazneen

    2014-01-01

    Overgrowth disorders are a heterogeneous group of conditions characterised by increased growth parameters and variable other clinical features, such as intellectual disability and facial dysmorphism1. To identify novel causes of human overgrowth we performed exome sequencing in 10 proband-parent trios and detected two de novo DNMT3A mutations. We identified 11 additional de novo mutations through DNMT3A sequencing of a further 142 individuals with overgrowth. The mutations were all located in functional DNMT3A domains and protein modelling suggests they interfere with domain-domain interactions and histone binding. No similar mutations were present in 1000 UK population controls (13/152 vs 0/1000; P<0.0001). Mutation carriers had a distinctive facial appearance, intellectual disability and increased height. DNMT3A encodes a key methyltransferase essential for establishing the methylation imprint in embryogenesis and is commonly somatically mutated in acute myeloid leukaemia2-4. Thus DNMT3A joins an emerging group of epigenetic DNA and histone modifying genes associated with both developmental growth disorders and haematological malignancies5. PMID:24614070

  18. Mutation and Methylation Analysis of the Chromodomain-Helicase-DNA Binding 5 Gene in Ovarian Cancer

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    Kylie L. Gorringe

    2008-11-01

    Full Text Available Chromodomain, helicase, DNA binding 5 (CHD5 is a member of a subclass of the chromatin remodeling Swi/Snf proteins and has recently been proposed as a tumor suppressor in a diverse range of human cancers. We analyzed all 41 coding exons of CHD5 for somatic mutations in 123 primary ovarian cancers as well as 60 primary breast cancers using high-resolution melt analysis. We also examined methylation of the CHD5 promoter in 48 ovarian cancer samples by methylation-specific single-stranded conformation polymorphism and bisulfite sequencing. In contrast to previous studies, no mutations were identified in the breast cancers, but somatic heterozygous missense mutations were identified in 3 of 123 ovarian cancers. We identified promoter methylation in 3 of 45 samples with normal CHD5 and in 2 of 3 samples with CHD5 mutation, suggesting these tumors may have biallelic inactivation of CHD5. Hemizygous copy number loss at CHD5 occurred in 6 of 85 samples as assessed by single nucleotide polymorphism array. Tumors with CHD5 mutation or methylation were more likely to have mutation of KRAS or BRAF (P = .04. The aggregate frequency of CHD5 haploinsufficiency or inactivation is 16.2% in ovarian cancer. Thus, CHD5 may play a role as a tumor suppressor gene in ovarian cancer; however, it is likely that there is another target of the frequent copy number neutral loss of heterozygosity observed at 1p36.

  19. Identification of unique repeated patterns, location of mutation in DNA finger printing using artificial intelligence technique.

    Science.gov (United States)

    Mukunthan, B; Nagaveni, N

    2014-01-01

    In genetic engineering, conventional techniques and algorithms employed by forensic scientists to assist in identification of individuals on the basis of their respective DNA profiles involves more complex computational steps and mathematical formulae, also the identification of location of mutation in a genomic sequence in laboratories is still an exigent task. This novel approach provides ability to solve the problems that do not have an algorithmic solution and the available solutions are also too complex to be found. The perfect blend made of bioinformatics and neural networks technique results in efficient DNA pattern analysis algorithm with utmost prediction accuracy.

  20. Detection of DNA oligonucleotides with base mutations by terahertz spectroscopy and microstructures.

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    Mingjie Tang

    Full Text Available DNA oligonucleotides with a 5-base mutation at the 3'-terminus were investigated by terahertz (THz spectroscopy in a marker-free manner. The four single-stranded oligonucleotides with 17nt have been detected with specificity on a microfluidic chip, and corroborated by spectral measurements with split-ring resonators. The number of hydrogen bonds formed between the oligonucleotide and its surrounding water molecules, deemed a key contribution to the THz absorption of biological solutions, was explored by molecular dynamics simulations to explain the experimental findings. Our work underlies the feasibility of THz spectroscopy combined with microstructures for marker-free detection of DNA, which may form the basis of a prospective diagnostic tool for studying genic mutation.

  1. A Phenotype-Driven Approach to Generate Mouse Models with Pathogenic mtDNA Mutations Causing Mitochondrial Disease

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    Johanna H.K. Kauppila

    2016-09-01

    Full Text Available Mutations of mtDNA are an important cause of human disease, but few animal models exist. Because mammalian mitochondria cannot be transfected, the development of mice with pathogenic mtDNA mutations has been challenging, and the main strategy has therefore been to introduce mutations found in cell lines into mouse embryos. Here, we describe a phenotype-driven strategy that is based on detecting clonal expansion of pathogenic mtDNA mutations in colonic crypts of founder mice derived from heterozygous mtDNA mutator mice. As proof of concept, we report the generation of a mouse line transmitting a heteroplasmic pathogenic mutation in the alanine tRNA gene of mtDNA displaying typical characteristics of classic mitochondrial disease. In summary, we describe a straightforward and technically simple strategy based on mouse breeding and histology to generate animal models of mtDNA-mutation disease, which will be of great importance for studies of disease pathophysiology and preclinical treatment trials.

  2. dNTP pool levels modulate mutator phenotypes of error-prone DNA polymerase ε variants.

    Science.gov (United States)

    Williams, Lindsey N; Marjavaara, Lisette; Knowels, Gary M; Schultz, Eric M; Fox, Edward J; Chabes, Andrei; Herr, Alan J

    2015-05-12

    Mutator phenotypes create genetic diversity that fuels tumor evolution. DNA polymerase (Pol) ε mediates leading strand DNA replication. Proofreading defects in this enzyme drive a number of human malignancies. Here, using budding yeast, we show that mutator variants of Pol ε depend on damage uninducible (Dun)1, an S-phase checkpoint kinase that maintains dNTP levels during a normal cell cycle and up-regulates dNTP synthesis upon checkpoint activation. Deletion of DUN1 (dun1Δ) suppresses the mutator phenotype of pol2-4 (encoding Pol ε proofreading deficiency) and is synthetically lethal with pol2-M644G (encoding altered Pol ε base selectivity). Although pol2-4 cells cycle normally, pol2-M644G cells progress slowly through S-phase. The pol2-M644G cells tolerate deletions of mediator of the replication checkpoint (MRC) 1 (mrc1Δ) and radiation sensitive (Rad) 9 (rad9Δ), which encode mediators of checkpoint responses to replication stress and DNA damage, respectively. The pol2-M644G mutator phenotype is partially suppressed by mrc1Δ but not rad9Δ; neither deletion suppresses the pol2-4 mutator phenotype. Thus, checkpoint activation augments the Dun1 effect on replication fidelity but is not required for it. Deletions of genes encoding key Dun1 targets that negatively regulate dNTP synthesis, suppress the dun1Δ pol2-M644G synthetic lethality and restore the mutator phenotype of pol2-4 in dun1Δ cells. DUN1 pol2-M644G cells have constitutively high dNTP levels, consistent with checkpoint activation. In contrast, pol2-4 and POL2 cells have similar dNTP levels, which decline in the absence of Dun1 and rise in the absence of the negative regulators of dNTP synthesis. Thus, dNTP pool levels correlate with Pol ε mutator severity, suggesting that treatments targeting dNTP pools could modulate mutator phenotypes for therapy.

  3. Sensitive detection of point mutation by electrochemiluminescence and DNA ligase-based assay

    Science.gov (United States)

    Zhou, Huijuan; Wu, Baoyan

    2008-12-01

    The technology of single-base mutation detection plays an increasingly important role in diagnosis and prognosis of genetic-based diseases. Here we reported a new method for the analysis of point mutations in genomic DNA through the integration of allele-specific oligonucleotide ligation assay (OLA) with magnetic beads-based electrochemiluminescence (ECL) detection scheme. In this assay the tris(bipyridine) ruthenium (TBR) labeled probe and the biotinylated probe are designed to perfectly complementary to the mutant target, thus a ligation can be generated between those two probes by Taq DNA Ligase in the presence of mutant target. If there is an allele mismatch, the ligation does not take place. The ligation products are then captured onto streptavidin-coated paramagnetic beads, and detected by measuring the ECL signal of the TBR label. Results showed that the new method held a low detection limit down to 10 fmol and was successfully applied in the identification of point mutations from ASTC-α-1, PANC-1 and normal cell lines in codon 273 of TP53 oncogene. In summary, this method provides a sensitive, cost-effective and easy operation approach for point mutation detection.

  4. Increasing Nucleosome Occupancy Is Correlated with an Increasing Mutation Rate so Long as DNA Repair Machinery Is Intact

    Science.gov (United States)

    Taylor, Jared F.; Khattab, Omar S.; Chen, Yu-Han; Chen, Yumay; Jacobsen, Steven E.; Wang, Ping H.

    2015-01-01

    Deciphering the multitude of epigenomic and genomic factors that influence the mutation rate is an area of great interest in modern biology. Recently, chromatin has been shown to play a part in this process. To elucidate this relationship further, we integrated our own ultra-deep sequenced human nucleosomal DNA data set with a host of published human genomic and cancer genomic data sets. Our results revealed, that differences in nucleosome occupancy are associated with changes in base-specific mutation rates. Increasing nucleosome occupancy is associated with an increasing transition to transversion ratio and an increased germline mutation rate within the human genome. Additionally, cancer single nucleotide variants and microindels are enriched within nucleosomes and both the coding and non-coding cancer mutation rate increases with increasing nucleosome occupancy. There is an enrichment of cancer indels at the theoretical start (74 bp) and end (115 bp) of linker DNA between two nucleosomes. We then hypothesized that increasing nucleosome occupancy decreases access to DNA by DNA repair machinery and could account for the increasing mutation rate. Such a relationship should not exist in DNA repair knockouts, and we thus repeated our analysis in DNA repair machinery knockouts to test our hypothesis. Indeed, our results revealed no correlation between increasing nucleosome occupancy and increasing mutation rate in DNA repair knockouts. Our findings emphasize the linkage of the genome and epigenome through the nucleosome whose properties can affect genome evolution and genetic aberrations such as cancer. PMID:26308346

  5. Increasing Nucleosome Occupancy Is Correlated with an Increasing Mutation Rate so Long as DNA Repair Machinery Is Intact.

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    Puya G Yazdi

    Full Text Available Deciphering the multitude of epigenomic and genomic factors that influence the mutation rate is an area of great interest in modern biology. Recently, chromatin has been shown to play a part in this process. To elucidate this relationship further, we integrated our own ultra-deep sequenced human nucleosomal DNA data set with a host of published human genomic and cancer genomic data sets. Our results revealed, that differences in nucleosome occupancy are associated with changes in base-specific mutation rates. Increasing nucleosome occupancy is associated with an increasing transition to transversion ratio and an increased germline mutation rate within the human genome. Additionally, cancer single nucleotide variants and microindels are enriched within nucleosomes and both the coding and non-coding cancer mutation rate increases with increasing nucleosome occupancy. There is an enrichment of cancer indels at the theoretical start (74 bp and end (115 bp of linker DNA between two nucleosomes. We then hypothesized that increasing nucleosome occupancy decreases access to DNA by DNA repair machinery and could account for the increasing mutation rate. Such a relationship should not exist in DNA repair knockouts, and we thus repeated our analysis in DNA repair machinery knockouts to test our hypothesis. Indeed, our results revealed no correlation between increasing nucleosome occupancy and increasing mutation rate in DNA repair knockouts. Our findings emphasize the linkage of the genome and epigenome through the nucleosome whose properties can affect genome evolution and genetic aberrations such as cancer.

  6. Thermodynamic framework to assess low abundance DNA mutation detection by hybridization

    Science.gov (United States)

    Willems, Hanny; Jacobs, An; Hadiwikarta, Wahyu Wijaya; Venken, Tom; Valkenborg, Dirk; Van Roy, Nadine; Vandesompele, Jo; Hooyberghs, Jef

    2017-01-01

    The knowledge of genomic DNA variations in patient samples has a high and increasing value for human diagnostics in its broadest sense. Although many methods and sensors to detect or quantify these variations are available or under development, the number of underlying physico-chemical detection principles is limited. One of these principles is the hybridization of sample target DNA versus nucleic acid probes. We introduce a novel thermodynamics approach and develop a framework to exploit the specific detection capabilities of nucleic acid hybridization, using generic principles applicable to any platform. As a case study, we detect point mutations in the KRAS oncogene on a microarray platform. For the given platform and hybridization conditions, we demonstrate the multiplex detection capability of hybridization and assess the detection limit using thermodynamic considerations; DNA containing point mutations in a background of wild type sequences can be identified down to at least 1% relative concentration. In order to show the clinical relevance, the detection capabilities are confirmed on challenging formalin-fixed paraffin-embedded clinical tumor samples. This enzyme-free detection framework contains the accuracy and efficiency to screen for hundreds of mutations in a single run with many potential applications in molecular diagnostics and the field of personalised medicine. PMID:28542229

  7. Tyr120Asp mutation alters domain flexibility and dynamics of MeCP2 DNA binding domain leading to impaired DNA interaction: Atomistic characterization of a Rett syndrome causing mutation.

    Science.gov (United States)

    D'Annessa, Ilda; Gandaglia, Anna; Brivio, Elena; Stefanelli, Gilda; Frasca, Angelisa; Landsberger, Nicoletta; Di Marino, Daniele

    2018-05-01

    Mutations in the X-linked MECP2 gene represent the main origin of Rett syndrome, causing a profound intellectual disability in females. MeCP2 is an epigenetic transcriptional regulator containing two main functional domains: a methyl-CpG binding domain (MBD) and a transcription repression domain (TRD). Over 600 pathogenic mutations were reported to affect the whole protein; almost half of missense mutations affect the MBD. Understanding the impact of these mutations on the MBD structure and interaction with DNA will foster the comprehension of their pathogenicity and possibly genotype/phenotype correlation studies. Herein, we use molecular dynamics simulations to obtain a detailed view of the dynamics of WT and mutated MBD in the presence and absence of DNA. The pathogenic mutation Y120D is used as paradigm for our studies. Further, since the Y120 residue was previously found to be a phosphorylation site, we characterize the dynamic profile of the MBD also in the presence of Y120 phosphorylation (pY120). We found that addition of a phosphate group to Y120 or mutation in aspartic acid affect domain mobility that samples an alternative conformational space with respect to the WT, leading to impaired ability to interact with DNA. Experimental assays showing a significant reduction in the binding affinity between the mutated MBD and the DNA confirmed our predictions. Copyright © 2018 Elsevier B.V. All rights reserved.

  8. Ataxia telangiectasia mutated (ATM) interacts with p400 ATPase for an efficient DNA damage response.

    Science.gov (United States)

    Smith, Rebecca J; Savoian, Matthew S; Weber, Lauren E; Park, Jeong Hyeon

    2016-11-04

    Ataxia telangiectasia mutated (ATM) and TRRAP proteins belong to the phosphatidylinositol 3-kinase-related kinase family and are involved in DNA damage repair and chromatin remodeling. ATM is a checkpoint kinase that is recruited to sites of DNA double-strand breaks where it phosphorylates a diverse range of proteins that are part of the chromatin and DNA repair machinery. As an integral subunit of the TRRAP-TIP60 complexes, p400 ATPase is a chromatin remodeler that is also targeted to DNA double-strand break sites. While it is understood that DNA binding transcriptional activators recruit p400 ATPase into a regulatory region of the promoter, how p400 recognises and moves to DNA double-strand break sites is far less clear. Here we investigate a possibility whether ATM serves as a shuttle to deliver p400 to break sites. Our data indicate that p400 co-immunoprecipitates with ATM independently of DNA damage state and that the N-terminal domain of p400 is vital for this interaction. Heterologous expression studies using Sf9 cells revealed that the ATM-p400 complex can be reconstituted without other mammalian bridging proteins. Overexpression of ATM-interacting p400 regions in U2OS cells induced dominant negative effects including the inhibition of both DNA damage repair and cell proliferation. Consistent with the dominant negative effect, the stable expression of an N-terminal p400 fragment showed a decrease in the association of p400 with ATM, but did not alter the association of p400 with TRRAP. Taken together, our findings suggest that a protein-protein interaction between ATM and p400 ATPase occurs independently of DNA damage and contributes to efficient DNA damage response and repair.

  9. The influence of calf thymus DNA and deoxyribonucleosides on the induction of different mutation types in Drosophila

    International Nuclear Information System (INIS)

    Ondrej, M.

    1975-01-01

    The influence of an exogenous DNA on the induction of mutations by X rays was compared with the influence of an equimolar mixture of four deoxyribonucleosides. Pre-treatment and post-treatment with the calf thymus DNA did not influence mutation frequency in the specific loci dp, b, cn and bw as well as Minute mutations induced in the Drosophila sperm by X radiation. Pre-treatment with the equimolar mixture of four deoxyribonucleosides increased the frequency of the Minutes but did not affect mutation frequency in the loci dp, b, cn, bw. The equimolar mixture of nucleosides alone induced a low frequency of Minute mutations in the Drosophila sperm. DNA alone induced a low frequency of recessive lethals. These lethals arose as mosaics of small sectors of the gonads of the F 1 females and were revealed as late as in the F 3 generation. (author)

  10. Molecular targets, DNA breakage, DNA repair: Their roles in mutation induction in mammalian germ cells

    International Nuclear Information System (INIS)

    Sega, G.A.

    1989-01-01

    Variability in genetic sensitivity among different germ-cell stages in the mammal to various mutagens could be the result of how much chemical reaches the different stages, what molecular targets may be affected in the different stages and whether or not repair of lesions occurs. Several chemicals have been found to bind very strongly to protamine in late-spermatid and early-spermatozoa stages in the mouse. The chemicals also produce their greatest genetic damage in these same germ-cell stages. While chemical binding to DNA has not been correlated with the level of induced genetic damage, DNA breakage in the sensitive stages has been shown to increase. This DNA breakage is believed to indirectly result from chemical binding to sulfhydryl groups in protamine which prevents normal chromatin condensation within the sperm nucleus. 22 refs., 5 figs

  11. DNA and cancer biology: role in radiation and drug sensitivity, carcinogenesis and mutations

    International Nuclear Information System (INIS)

    Yielding, K.L.

    1974-01-01

    The DNA excision repair mechanism is an important factor in the resistance exhibited by tumor cells toward both x rays and alkylating agents as demonstrated by the fact that the chemical alterations to cellular DNA caused by these agents are substrates for the repair enzymes. Furthermore, experiments performed in our laboratory demonstrate that: (a) tumor sensitivity to alkylating agents and x-ray can be increased by inhibition of the repair process, and (b) there is a suggestion that this sensitization can be achieved with some degree of selectivity, thereby improving the balance of sensitivites between tumor and normal tissue. Other work from this laboratory has shown that cocarcinogens probably act by preventing repair of carcinogenic damage to the DNA genome. The possibility has also been raised that mistakes made during repair synthesis might be responsible for some genetic diversity and for the mutations which arise in resting cells. (U.S.)

  12. Radiation-induced germ-line mutations detected by a direct comparison of parents and children DNA sequences containing SNPs

    International Nuclear Information System (INIS)

    Morimyo, M.; Hongo, E.; Higashi, T.; Wu, J.; Matsumoto, I.; Okamoto, M.; Kawano, A.; Tsuji, S.

    2003-01-01

    Full text: Germ-line mutation is detected in mice but not in humans. To estimate genetic risk of humans, a new approach to extrapolate from animal data to humans or to directly detect radiation-induced mutations in man is expected. We have developed a new method to detect germ-line mutations by directly comparing DNA sequences of parents and children. The nucleotide sequences among mouse strains are almost identical except SNP markers that are detected at 1/1000 frequency. When gamma-irradiated male mice are mated with female mice, heterogeneous nucleotide sequences induced in children DNA are a candidate of mutation, whose assignment can be done by SNP analysis. This system can easily detect all types of mutations such as transition, transversion, frameshift and deletion induced by radiation and can be applied to humans having genetically heterogeneous nucleotide sequences and many SNP markers. C3H male mice of 8 weeks of gestation were irradiated with gamma rays of 3 and 1 Gy and after 3 weeks, they were mated with the same aged C57BL female mice. After 3 weeks breeding, DNA was extracted from parents and children mice. The nucleotide sequences of 150 STS markers containing 300-900 bp and SNPs of parents and children DNA were determined by a direct sequencing; amplification of STS markers by Taq DNA polymerase, purification of PCR products, and DNA sequencing with a dye-terminator method. At each radiation dose, a total amount of 5 Mb DNA sequences were examined to detect radiation-induced mutations. We could find 6 deletions in 3 Gy irradiated mice but not in 1 Gy and control mice. The mutation frequency was about 4.0 x 10 -7 /bp/ Gy or 1.6 x 10 -4 /locus/Gy, and suggested the non-linear increase of mutation rate with dose

  13. PMS2 gene mutational analysis: direct cDNA sequencing to circumvent pseudogene interference.

    Science.gov (United States)

    Wimmer, Katharina; Wernstedt, Annekatrin

    2014-01-01

    The presence of highly homologous pseudocopies can compromise the mutation analysis of a gene of interest. In particular, when using PCR-based strategies, pseudogene co-amplification has to be effectively prevented. This is often achieved by using primers designed to be parental gene specific according to the reference sequence and by applying stringent PCR conditions. However, there are cases in which this approach is of limited utility. For example, it has been shown that the PMS2 gene exchanges sequences with one of its pseudogenes, named PMS2CL. This results in functional PMS2 alleles containing pseudogene-derived sequences at their 3'-end and in nonfunctional PMS2CL pseudogene alleles that contain gene-derived sequences. Hence, the paralogues cannot be distinguished according to the reference sequence. This shortcoming can be effectively circumvented by using direct cDNA sequencing. This approach is based on the selective amplification of PMS2 transcripts in two overlapping 1.6-kb RT-PCR products. In addition to avoiding pseudogene co-amplification and allele dropout, this method has also the advantage that it allows to effectively identify deletions, splice mutations, and de novo retrotransposon insertions that escape the detection of most DNA-based mutation analysis protocols.

  14. Mechanisms of mtDNA segregation and mitochondrial signalling in cells with the pathogenic A3243G mutation

    NARCIS (Netherlands)

    Jahangir Tafrechi, Roshan Sakineh

    2008-01-01

    Using newly developed single cell A3243G mutation load assays a novel mechanism of mtDNA segregation was identified in which the multi-copy mtDNA nucleoid takes a central position. Furthermore, likely due to low level changes in gene expression, no genes or gene sets could be identified with gene

  15. DNA mutations mediate microevolution between host-adapted forms of the pathogenic fungus Cryptococcus neoformans.

    Directory of Open Access Journals (Sweden)

    Denise A Magditch

    Full Text Available The disease cryptococcosis, caused by the fungus Cryptococcus neoformans, is acquired directly from environmental exposure rather than transmitted person-to-person. One explanation for the pathogenicity of this species is that interactions with environmental predators select for virulence. However, co-incubation of C. neoformans with amoeba can cause a "switch" from the normal yeast morphology to a pseudohyphal form, enabling fungi to survive exposure to amoeba, yet conversely reducing virulence in mammalian models of cryptococcosis. Like other human pathogenic fungi, C. neoformans is capable of microevolutionary changes that influence the biology of the organism and outcome of the host-pathogen interaction. A yeast-pseudohyphal phenotypic switch also happens under in vitro conditions. Here, we demonstrate that this morphological switch, rather than being under epigenetic control, is controlled by DNA mutation since all pseudohyphal strains bear mutations within genes encoding components of the RAM pathway. High rates of isolation of pseudohyphal strains can be explained by the physical size of RAM pathway genes and a hypermutator phenotype of the strain used in phenotypic switching studies. Reversion to wild type yeast morphology in vitro or within a mammalian host can occur through different mechanisms, with one being counter-acting mutations. Infection of mice with RAM mutants reveals several outcomes: clearance of the infection, asymptomatic maintenance of the strains, or reversion to wild type forms and progression of disease. These findings demonstrate a key role of mutation events in microevolution to modulate the ability of a fungal pathogen to cause disease.

  16. SOLAR RADIATION AND INDUCTION OF DNA DAMAGE, MUTATIONS AND SKIN CANCERS.

    Energy Technology Data Exchange (ETDEWEB)

    SETLOW,R.B.

    2007-05-10

    An understanding of the effects of sunlight on human skin begins with the effects on DNA and extends to cells, animals and humans. The major DNA photoproducts arising from UVB (280-320 nm) exposures are cyclobutane pyrimidine dimers. If unrepaired, they may kill or mutate cells and result in basal and squamous cell carcinomas. Although UVA (320-400 nm) and visible wavelengths are poorly absorbed by DNA, the existing data indicate clearly that exposures to these wavelengths are responsible, in an animal model, for {approx}95 % of the incidence of cutaneous malignant melanoma (CMM). Six lines of evidence, to be discussed in detail, support the photosensitizing role of melanin in the induction of this cancer. They are: (1) Melanomas induced in backcross hybrids of small tropical fish of the genus Xiphophorus, exposed to wavelengths from 302-547 nm, indicate that {approx}95% of the cancers induced by exposure to sunlight would arise from UVA + visible wavelengths; (2) The action spectrum for inducing melanin-photosensitized oxidant production is very similar to the spectrum for inducing melanoma; (3) Albino whites and blacks, although very sensitive to sunburn and the sunlight induction of non-CMM, have very low incidences of CMM; (4) The incidence of CMM as a function of latitude is very similar to that of UVA, but not UVB; (5) Use of UVA-exposing sun-tanning parlors by the young increases the incidence rate of CMM and (6) Major mutations observed in CMM are not UVB-induced.

  17. Senataxin Mutation Reveals How R-Loops Promote Transcription by Blocking DNA Methylation at Gene Promoters.

    Science.gov (United States)

    Grunseich, Christopher; Wang, Isabel X; Watts, Jason A; Burdick, Joshua T; Guber, Robert D; Zhu, Zhengwei; Bruzel, Alan; Lanman, Tyler; Chen, Kelian; Schindler, Alice B; Edwards, Nancy; Ray-Chaudhury, Abhik; Yao, Jianhua; Lehky, Tanya; Piszczek, Grzegorz; Crain, Barbara; Fischbeck, Kenneth H; Cheung, Vivian G

    2018-02-01

    R-loops are three-stranded nucleic acid structures found abundantly and yet often viewed as by-products of transcription. Studying cells from patients with a motor neuron disease (amyotrophic lateral sclerosis 4 [ALS4]) caused by a mutation in senataxin, we uncovered how R-loops promote transcription. In ALS4 patients, the senataxin mutation depletes R-loops with a consequent effect on gene expression. With fewer R-loops in ALS4 cells, the expression of BAMBI, a negative regulator of transforming growth factor β (TGF-β), is reduced; that then leads to the activation of the TGF-β pathway. We uncovered that genome-wide R-loops influence promoter methylation of over 1,200 human genes. DNA methyl-transferase 1 favors binding to double-stranded DNA over R-loops. Thus, in forming R-loops, nascent RNA blocks DNA methylation and promotes further transcription. Hence, our results show that nucleic acid structures, in addition to sequences, influence the binding and activity of regulatory proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Electrical signatures of single-stranded DNA with single base mutations in a nanopore capacitor

    International Nuclear Information System (INIS)

    Gracheva, Maria E; Aksimentiev, Aleksei; Leburton, Jean-Pierre

    2006-01-01

    In this paper, we evaluate the magnitude of the electrical signals produced by DNA translocation through a 1 nm diameter nanopore in a capacitor membrane with a numerical multi-scale approach, and assess the possibility of resolving individual nucleotides as well as their types in the absence of conformational disorder. We show that the maximum recorded voltage caused by the DNA translocation is about 35 mV, while the maximum voltage signal due to the DNA backbone is about 30 mV, and the maximum voltage of a DNA base is about 8 mV. Signals from individual nucleotides can be identified in the recorded voltage traces, suggesting a 1 nm diameter pore in a capacitor can be used to accurately count the number of nucleotides in a DNA strand. Furthermore, we study the effect of a single base substitution on the voltage trace, and calculate the differences among the voltage traces due to a single base mutation for the sequences C 3 AC 7 , C 3 CC 7 , C 3 GC 7 and C 3 TC 7 . The calculated voltage differences are in the 5-10 mV range. The calculated maximum voltage caused by the translocation of individual bases varies from 2 to 9 mV, which is experimentally detectable

  19. Arsenic trioxide promotes mitochondrial DNA mutation and cell apoptosis in primary APL cells and NB4 cell line.

    Science.gov (United States)

    Meng, Ran; Zhou, Jin; Sui, Meng; Li, ZhiYong; Feng, GuoSheng; Yang, BaoFeng

    2010-01-01

    This study aimed to investigate the effects of arsenic trioxide (As(2)O(3)) on the mitochondrial DNA (mtDNA) of acute promyelocytic leukemia (APL) cells. The NB4 cell line was treated with 2.0 micromol/L As(2)O(3) in vitro, and the primary APL cells were treated with 2.0 micromol/L As(2)O(3) in vitro and 0.16 mg kg(-1) d(-1) As(2)O(3) in vivo. The mitochondrial DNA of all the cells above was amplified by PCR, directly sequenced and analyzed by Sequence Navigatore and Factura software. The apoptosis rates were assayed by flow cytometry. Mitochondrial DNA mutation in the D-loop region was found in NB4 and APL cells before As(2)O(3) use, but the mutation spots were remarkably increased after As(2)O(3) treatment, which was positively correlated to the rates of cellular apoptosis, the correlation coefficient: r (NB4-As2O3)=0.973818, and r (APL-As2O3)=0.934703. The mutation types include transition, transversion, codon insertion or deletion, and the mutation spots in all samples were not constant and regular. It is revealed that As(2)O(3) aggravates mtDNA mutation in the D-loop region of acute promyelocytic leukemia cells both in vitro and in vivo. Mitochondrial DNA might be one of the targets of As(2)O(3) in APL treatment.

  20. Cadmium induces DNA damage in tobacco roots, but no DNA damage, somatic mutations or homologous recombination in tobacco leaves

    Czech Academy of Sciences Publication Activity Database

    Gichner, Tomáš; Patková, Zdeňka; Száková, J.; Demnerová, K.

    2004-01-01

    Roč. 559, 1/2 (2004), s. 49-57 ISSN 1383-5718 R&D Projects: GA ČR GA526/02/0293; GA ČR GA521/02/0400; GA MŠk LN00B030 Institutional research plan: CEZ:AV0Z5038910 Keywords : beta-Glucuronidase * Chlorophyll mutations * Comet assay Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.020, year: 2004

  1. Reconstruction of DNA sequences using genetic algorithms and cellular automata: towards mutation prediction?

    Science.gov (United States)

    Mizas, Ch; Sirakoulis, G Ch; Mardiris, V; Karafyllidis, I; Glykos, N; Sandaltzopoulos, R

    2008-04-01

    Change of DNA sequence that fuels evolution is, to a certain extent, a deterministic process because mutagenesis does not occur in an absolutely random manner. So far, it has not been possible to decipher the rules that govern DNA sequence evolution due to the extreme complexity of the entire process. In our attempt to approach this issue we focus solely on the mechanisms of mutagenesis and deliberately disregard the role of natural selection. Hence, in this analysis, evolution refers to the accumulation of genetic alterations that originate from mutations and are transmitted through generations without being subjected to natural selection. We have developed a software tool that allows modelling of a DNA sequence as a one-dimensional cellular automaton (CA) with four states per cell which correspond to the four DNA bases, i.e. A, C, T and G. The four states are represented by numbers of the quaternary number system. Moreover, we have developed genetic algorithms (GAs) in order to determine the rules of CA evolution that simulate the DNA evolution process. Linear evolution rules were considered and square matrices were used to represent them. If DNA sequences of different evolution steps are available, our approach allows the determination of the underlying evolution rule(s). Conversely, once the evolution rules are deciphered, our tool may reconstruct the DNA sequence in any previous evolution step for which the exact sequence information was unknown. The developed tool may be used to test various parameters that could influence evolution. We describe a paradigm relying on the assumption that mutagenesis is governed by a near-neighbour-dependent mechanism. Based on the satisfactory performance of our system in the deliberately simplified example, we propose that our approach could offer a starting point for future attempts to understand the mechanisms that govern evolution. The developed software is open-source and has a user-friendly graphical input interface.

  2. Mitochondrial DNA depletion, mitochondrial mutations and high TFAM expression in hepatocellular carcinoma

    OpenAIRE

    Qiao, Lihua; Ru, Guoqing; Mao, Zhuochao; Wang, Chenghui; Nie, Zhipeng; Li, Qiang; Huang-yang, Yiyi; Zhu, Ling; Liang, Xiaoyang; Yu, Jialing; Jiang, Pingping

    2017-01-01

    We investigated the role of mitochondrial genetic alterations in hepatocellular carcinoma by directly comparing the mitochondrial genomes of 86 matched pairs of HCC and non-tumor liver samples. Substitutions in 637 mtDNA sites were detected, comprising 89.80% transitions and 6.60% transversions. Forty-six somatic variants, including 15 novel mutations, were identified in 40.70% of tumor tissues. Of those, 21 were located in the non-coding region and 25 in the protein-coding region. Twenty-two...

  3. Risk of colorectal cancer for people with a mutation in both a MUTYH and a DNA mismatch repair gene

    Science.gov (United States)

    Win, Aung Ko; Reece, Jeanette C.; Buchanan, Daniel D.; Clendenning, Mark; Young, Joanne P.; Cleary, Sean P.; Kim, Hyeja; Cotterchio, Michelle; Dowty, James G.; MacInnis, Robert J.; Tucker, Katherine M.; Winship, Ingrid M.; Macrae, Finlay A.; Burnett, Terrilea; Le Marchand, Loïc; Casey, Graham; Haile, Robert W.; Newcomb, Polly A.; Thibodeau, Stephen N.; Lindor, Noralane M.; Hopper, John L.; Gallinger, Steven; Jenkins, Mark A.

    2015-01-01

    The base excision repair protein, MUTYH, functionally interacts with the DNA mismatch repair (MMR) system. As genetic testing moves from testing one gene at a time, to gene panel and whole exome next generation sequencing approaches, understanding the risk associated with co-existence of germline mutations in these genes will be important for clinical interpretation and management. From the Colon Cancer Family Registry, we identified 10 carriers who had both a MUTYH mutation (6 with c.1187G>A p.(Gly396Asp), 3 with c.821G>A p.(Arg274Gln), and 1 with c.536A>G p.(Tyr179Cys)) and a MMR gene mutation (3 in MLH1, 6 in MSH2, and 1 in PMS2), 375 carriers of a single (monoallelic) MUTYH mutation alone, and 469 carriers of a MMR gene mutation alone. Of the 10 carriers of both gene mutations, 8 were diagnosed with colorectal cancer. Using a weighted cohort analysis, we estimated that risk of colorectal cancer for carriers of both a MUTYH and a MMR gene mutation was substantially higher than that for carriers of a MUTYH mutation alone [hazard ratio (HR) 21.5, 95 % confidence interval (CI) 9.19–50.1; p colorectal cancer for carriers of a MMR gene mutation alone. Our finding suggests MUTYH mutation testing in MMR gene mutation carriers is not clinically informative. PMID:26202870

  4. A hotspot in the glucocorticoid receptor DNA-binding domain susceptible to loss of function mutation

    Science.gov (United States)

    Banuelos, Jesus; Shin, Soon Cheon; Lu, Nick Z.

    2015-01-01

    Glucocorticoids (GCs) are used to treat a variety of inflammatory disorders and certain cancers. However, GC resistance occurs in subsets of patients. We found that EL4 cells, a GC-resistant mouse thymoma cell line, harbored a point mutation in their GC receptor (GR) gene, resulting in the substitution of arginine 493 by a cysteine in the second zinc finger of the DNA-binding domain. Allelic discrimination analyses revealed that the R493C mutation occurred on both alleles. In the absence of GCs, the GR in EL4 cells localized predominantly in the cytoplasm and upon dexamethasone treatment underwent nuclear translocation, suggesting the ligand binding ability of the GR in EL4 cells was intact. In transient transfection assays, the R493C mutant could not transactivate the MMTV-luciferase reporter. Site-directed mutagenesis to revert the R493C mutation restored the transactivation activity. Cotransfection experiments showed that the R493C mutant did not inhibit the transcriptional activities of the wild-type GR. In addition, the R493C mutant did not repress either the AP-1 or NF-κB reporters as effectively as WT GR. Furthermore, stable expression of the WT GR in the EL4 cells enabled GC-mediated gene regulation, specifically upregulation of IκBα and downregulation of interferon γ and interleukin 17A. Arginine 493 is conserved among multiple species and all human nuclear receptors and its mutation has also been found in the human GR, androgen receptor, and mineralocorticoid receptor. Thus, R493 is necessary for the transcriptional activity of the GR and a hotspot for mutations that result in GC resistance. PMID:25676786

  5. Differences in K-ras and mitochondrial DNA mutations and microsatellite instability between colorectal cancers of Vietnamese and Japanese patients.

    Science.gov (United States)

    Miwata, Tomohiro; Hiyama, Toru; Quach, Duc Trong; Le, Huy Minh; Hua, Ha Ngoc Thi; Oka, Shiro; Tanaka, Shinji; Arihiro, Koji; Chayama, Kazuaki

    2014-11-30

    The incidence of early-onset (under 50 years of age) colorectal cancer (CRC) in the Vietnamese has been reported to be quite higher than that in the Japanese. To clarify the differences in genetic alterations between Vietnamese and Japanese CRCs, we investigated mutations in K-ras and mitochondrial DNA (mtDNA) and high-frequency microsatellite instability (MSI-H) in the CRCs of Vietnamese and Japanese patients. We enrolled 60 Vietnamese and 233 Japanese patients with invasive CRCs. DNA was extracted from formalin-fixed, paraffin-embedded tissue sections. K-ras mutations were examined with PCR-single-strand conformation polymorphism analysis. mtDNA mutations and MSI-H were examined with microsatellite analysis using D310 and BAT-26, respectively. K-ras mutations were examined in 60 Vietnamese and 45 Japanese CRCs. The frequency of the mutations in the Vietnamese CRCs was significantly higher than that in the Japanese CRCs (8 of 24 [33%] vs 5 of 45 [11%], p =0.048). MSI-H was examined in 60 Vietnamese and 130 Japanese CRCs. The frequency of MSI-H in the Vietnamese CRCs was also significantly higher than that in the Japanese CRCs (6 of 27 [22%] vs 10 of 130 [8%], p =0.030). mtDNA mutations were examined in 60 Vietnamese and 138 Japanese CRCs. The frequency of mtDNA mutations in the Vietnamese CRCs was significantly higher than that in the Japanese CRCs (19 of 44 [43%] vs 11 of 133 [9%], p Vietnamese and Japanese patients. These results indicate that the developmental pathways of CRCs in the Vietnamese may differ from those of CRCs in the Japanese.

  6. Analysis of mutation/rearrangement frequencies and methylation patterns at a given DNA locus using restriction fragment length polymorphism.

    Science.gov (United States)

    Boyko, Alex; Kovalchuk, Igor

    2010-01-01

    Restriction fragment length polymorphism (RFLP) is a difference in DNA sequences of organisms belonging to the same species. RFLPs are typically detected as DNA fragments of different lengths after digestion with various restriction endonucleases. The comparison of RFLPs allows investigators to analyze the frequency of occurrence of mutations, such as point mutations, deletions, insertions, and gross chromosomal rearrangements, in the progeny of stressed plants. The assay involves restriction enzyme digestion of DNA followed by hybridization of digested DNA using a radioactively or enzymatically labeled probe. Since DNA can be digested with methylation sensitive enzymes, the assay can also be used to analyze a methylation pattern of a particular locus. Here, we describe RFLP analysis using methylation-insensitive and methylation-sensitive enzymes.

  7. rDNA Copy Number Variants Are Frequent Passenger Mutations in Saccharomyces cerevisiae Deletion Collections and de Novo Transformants

    Directory of Open Access Journals (Sweden)

    Elizabeth X. Kwan

    2016-09-01

    Full Text Available The Saccharomyces cerevisiae ribosomal DNA (rDNA locus is known to exhibit greater instability relative to the rest of the genome. However, wild-type cells preferentially maintain a stable number of rDNA copies, suggesting underlying genetic control of the size of this locus. We performed a screen of a subset of the Yeast Knock-Out (YKO single gene deletion collection to identify genetic regulators of this locus and to determine if rDNA copy number correlates with yeast replicative lifespan. While we found no correlation between replicative lifespan and rDNA size, we identified 64 candidate strains with significant rDNA copy number differences. However, in the process of validating candidate rDNA variants, we observed that independent isolates of our de novo gene deletion strains had unsolicited but significant changes in rDNA copy number. Moreover, we were not able to recapitulate rDNA phenotypes from the YKO yeast deletion collection. Instead, we found that the standard lithium acetate transformation protocol is a significant source of rDNA copy number variation, with lithium acetate exposure being the treatment causing variable rDNA copy number events after transformation. As the effects of variable rDNA copy number are being increasingly reported, our finding that rDNA is affected by lithium acetate exposure suggested that rDNA copy number variants may be influential passenger mutations in standard strain construction in S. cerevisiae.

  8. rDNA Copy Number Variants Are Frequent Passenger Mutations in Saccharomyces cerevisiae Deletion Collections and de Novo Transformants.

    Science.gov (United States)

    Kwan, Elizabeth X; Wang, Xiaobin S; Amemiya, Haley M; Brewer, Bonita J; Raghuraman, M K

    2016-09-08

    The Saccharomyces cerevisiae ribosomal DNA (rDNA) locus is known to exhibit greater instability relative to the rest of the genome. However, wild-type cells preferentially maintain a stable number of rDNA copies, suggesting underlying genetic control of the size of this locus. We performed a screen of a subset of the Yeast Knock-Out (YKO) single gene deletion collection to identify genetic regulators of this locus and to determine if rDNA copy number correlates with yeast replicative lifespan. While we found no correlation between replicative lifespan and rDNA size, we identified 64 candidate strains with significant rDNA copy number differences. However, in the process of validating candidate rDNA variants, we observed that independent isolates of our de novo gene deletion strains had unsolicited but significant changes in rDNA copy number. Moreover, we were not able to recapitulate rDNA phenotypes from the YKO yeast deletion collection. Instead, we found that the standard lithium acetate transformation protocol is a significant source of rDNA copy number variation, with lithium acetate exposure being the treatment causing variable rDNA copy number events after transformation. As the effects of variable rDNA copy number are being increasingly reported, our finding that rDNA is affected by lithium acetate exposure suggested that rDNA copy number variants may be influential passenger mutations in standard strain construction in S. cerevisiae. Copyright © 2016 Kwan et al.

  9. High-fidelity target sequencing of individual molecules identified using barcode sequences: de novo detection and absolute quantitation of mutations in plasma cell-free DNA from cancer patients.

    Science.gov (United States)

    Kukita, Yoji; Matoba, Ryo; Uchida, Junji; Hamakawa, Takuya; Doki, Yuichiro; Imamura, Fumio; Kato, Kikuya

    2015-08-01

    Circulating tumour DNA (ctDNA) is an emerging field of cancer research. However, current ctDNA analysis is usually restricted to one or a few mutation sites due to technical limitations. In the case of massively parallel DNA sequencers, the number of false positives caused by a high read error rate is a major problem. In addition, the final sequence reads do not represent the original DNA population due to the global amplification step during the template preparation. We established a high-fidelity target sequencing system of individual molecules identified in plasma cell-free DNA using barcode sequences; this system consists of the following two steps. (i) A novel target sequencing method that adds barcode sequences by adaptor ligation. This method uses linear amplification to eliminate the errors introduced during the early cycles of polymerase chain reaction. (ii) The monitoring and removal of erroneous barcode tags. This process involves the identification of individual molecules that have been sequenced and for which the number of mutations have been absolute quantitated. Using plasma cell-free DNA from patients with gastric or lung cancer, we demonstrated that the system achieved near complete elimination of false positives and enabled de novo detection and absolute quantitation of mutations in plasma cell-free DNA. © The Author 2015. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

  10. Targeted mutations induced by a single acetylaminofluorene DNA adduct in mammalian cells and bacteria

    International Nuclear Information System (INIS)

    Moryia, M.; Takeshita, M.; Johnson, F.; Peden, K.; Will, S.; Grollman, A.P.

    1988-01-01

    Mutagenic specificity of 2-acetylaminofluorene (AAF) has been established in mammalian cells and several strains of bacteria by using a shuttle plasmid vector containing a single N-(deoxyguanosin-8-yl)acetylaminofluorene (C8-dG-AAF) adduct. The nucleotide sequence of the gene conferring tetracycline resistance was modified by conservative codon replacement so as to accommodate the sequence d(CCTTCGCTAC) flanked by two restriction sites, Bsm I and Xho I. The corresponding synthetic oligodeoxynucleotide underwent reaction with 2-(N-acetoxy-N-acetylamino)-fluorene (AAAF), forming a single dG-AAF adduct. This modified oligodeoxynucleotide was hybridized to its complementary strand and ligated between the Bsm I and Xho I sites of the vector. Plasmids containing the C8-dG-AAF adduct were used to transfect simian virus 40-transformed simian kidney (COS-1) cells and to transform several AB strains of Escherichia coli. Colonies containing mutant plasmides were detected by hybridization to 32 P-labeled oligodeoxynucleotides. Presence of the single DNA adduct increased the mutation frequency by 8-fold in both COS cells and E. coli. Over 80% of mutations detected in both systems were targeted and represented G x C → C x G or G x C → T x A transversions or single nucleotide deletions. The authors conclude that modification of a deoxyguanosine residue with AAF preferentially induces mutations targeted at this site when a plasmid containing a single C8-dG-AAF adduct is introduced into mammalian cells or bacteria

  11. NSD1 mutations generate a genome-wide DNA methylation signature.

    LENUS (Irish Health Repository)

    Choufani, S

    2015-12-22

    Sotos syndrome (SS) represents an important human model system for the study of epigenetic regulation; it is an overgrowth\\/intellectual disability syndrome caused by mutations in a histone methyltransferase, NSD1. As layered epigenetic modifications are often interdependent, we propose that pathogenic NSD1 mutations have a genome-wide impact on the most stable epigenetic mark, DNA methylation (DNAm). By interrogating DNAm in SS patients, we identify a genome-wide, highly significant NSD1(+\\/-)-specific signature that differentiates pathogenic NSD1 mutations from controls, benign NSD1 variants and the clinically overlapping Weaver syndrome. Validation studies of independent cohorts of SS and controls assigned 100% of these samples correctly. This highly specific and sensitive NSD1(+\\/-) signature encompasses genes that function in cellular morphogenesis and neuronal differentiation, reflecting cardinal features of the SS phenotype. The identification of SS-specific genome-wide DNAm alterations will facilitate both the elucidation of the molecular pathophysiology of SS and the development of improved diagnostic testing.

  12. Exome sequencing identifies rare deleterious mutations in DNA repair genes FANCC and BLM as potential breast cancer susceptibility alleles.

    Directory of Open Access Journals (Sweden)

    Ella R Thompson

    2012-09-01

    Full Text Available Despite intensive efforts using linkage and candidate gene approaches, the genetic etiology for the majority of families with a multi-generational breast cancer predisposition is unknown. In this study, we used whole-exome sequencing of thirty-three individuals from 15 breast cancer families to identify potential predisposing genes. Our analysis identified families with heterozygous, deleterious mutations in the DNA repair genes FANCC and BLM, which are responsible for the autosomal recessive disorders Fanconi Anemia and Bloom syndrome. In total, screening of all exons in these genes in 438 breast cancer families identified three with truncating mutations in FANCC and two with truncating mutations in BLM. Additional screening of FANCC mutation hotspot exons identified one pathogenic mutation among an additional 957 breast cancer families. Importantly, none of the deleterious mutations were identified among 464 healthy controls and are not reported in the 1,000 Genomes data. Given the rarity of Fanconi Anemia and Bloom syndrome disorders among Caucasian populations, the finding of multiple deleterious mutations in these critical DNA repair genes among high-risk breast cancer families is intriguing and suggestive of a predisposing role. Our data demonstrate the utility of intra-family exome-sequencing approaches to uncover cancer predisposition genes, but highlight the major challenge of definitively validating candidates where the incidence of sporadic disease is high, germline mutations are not fully penetrant, and individual predisposition genes may only account for a tiny proportion of breast cancer families.

  13. PMS2 gene mutation results in DNA mismatch repair system failure in a case of adult granulosa cell tumor

    OpenAIRE

    Wang, Wen-Chung; Lee, Ya-Ting; Lai, Yen-Chein

    2017-01-01

    Background Granulosa cell tumors are rare ovarian malignancies. Their characteristics include unpredictable indolent growth with malignant potential and late recurrence. Approximately 95% are of adult type. Recent molecular studies have characterized the FOXL2 402C?>?G mutation in adult granulosa cell tumor. Our previous case report showed that unique FOXL2 402C?>?G mutation and defective DNA mismatch repair system are associated with the development of adult granulosa cell tumor. Findings In...

  14. Effects of the ssb-1 and ssb-113 mutations on survival and DNA repair in UV-irradiated delta uvrB strains of Escherichia coli K-12.

    OpenAIRE

    Wang, T C; Smith, K C

    1982-01-01

    The molecular defect in DNA repair caused by ssb mutations (single-strand binding protein) was studied by analyzing DNA synthesis and DNA double-strand break production in UV-irradiated Escherichia coli delta uvrB strains. The presence of the ssb-113 mutation produced a large inhibition of DNA synthesis and led to the formation of double-strand breaks, whereas the ssb-1 mutation produced much less inhibition of DNA synthesis and fewer double-strand breaks. We suggest that the single-strand bi...

  15. Mutation of mtDNA ND1 Gene in 20 Type 2 Diabetes Mellitus Patients of Gorontalonese and Javanese Ethnicity

    Directory of Open Access Journals (Sweden)

    AMIEN RAMADHAN ISHAK

    2014-12-01

    Full Text Available Mitochondrial gene mutation plays a role in the development of type two diabetes mellitus (T2DM. A point mutation in the mitochondrial gene Nicotinamide adenine dinucleotide dehydrogenase 1 (mtDNA ND1 gene mainly reported as the most common mutation related to T2DM. However, several studies have identified another SNP (single-nucleotide polymorphisms in the RNA region of mtDNA from patients from specific ethnic populations in Indonesia. Building on those findings, this study aimed to use PCR and DNA sequencing technology to identify nucleotides in RNA and ND1 fragment from 20 Gorontalonese and 20 Javanese T2DM patients, that may trigger T2DM expression. The results showed successful amplification of RNA along 294 bp for all samples. From these samples, we found two types of point mutation in Javanese patients in the G3316A and T3200C points of the rRNA and ND1 gene. In samples taken from Gorontalonese patients, no mutation were found in the RNA or ND1 region. We conclude that T2DM was triggered differently in our two populations. While genetic mutation is implicated for the 20 Javanese patients, T2DM pathogenesis in the Gorontalonese patients must be traced to other genetic, environmental, or behavioral factors.

  16. DNA impedance biosensor for detection of cancer, TP53 gene mutation, based on gold nanoparticles/aligned carbon nanotubes modified electrode.

    Science.gov (United States)

    Fayazfar, H; Afshar, A; Dolati, M; Dolati, A

    2014-07-11

    For the first time, a new platform based on electrochemical growth of Au nanoparticles on aligned multi-walled carbon nanotubes (A-MWCNT) was developed for sensitive lable-free DNA detection of the TP53 gene mutation, one of the most popular genes in cancer research. Electrochemical impedance spectroscopy (EIS) was used to monitor the sequence-specific DNA hybridization events related to TP53 gene. Compared to the bare Ta or MWCNT/Ta electrodes, the synergistic interactions of vertically aligned MWCNT array and gold nanoparticles at modified electrode could improve the density of the probe DNA attachment and resulting the sensitivity of the DNA sensor greatly. Using EIS, over the extended DNA concentration range, the change of charge transfer resistance was found to have a linear relationship in respect to the logarithm of the complementary oligonucleotides sequence concentrations in the wide range of 1.0×10(-15)-1.0×10(-7)M, with a detection limit of 1.0×10(-17)M (S/N=3). The prepared sensor also showed good stability (14 days), reproducibility (RSD=2.1%) and could be conveniently regenerated via dehybridization in hot water. The significant improvement in sensitivity illustrates that combining gold nanoparticles with the on-site fabricated aligned MWCNT array represents a promising platform for achieving sensitive biosensor for fast mutation screening related to most human cancer types. Copyright © 2014. Published by Elsevier B.V.

  17. The effects of radiation on p53-mutated glioma cells using cDNA microarray technique

    International Nuclear Information System (INIS)

    Ngo, F.Q.H.; Hsiao, Y.-Y.H.

    2003-01-01

    Full text: In this study, we investigated the effects of 10-Gy irradiation on cell-cycle arrest, apoptosis and clonogenic death in the p53-mutated human U138MG (malignant glioblastoma) cell line. In order to evaluate time-dependent events in cellular responses to radiation, we did a time course study by incubating cells ranging from 0.5 to 48 hours after irradiation. Cell-cycle distribution and apoptosis were evaluated by flow cytometry using propidium iodide (PI) and annexin-V plus PI staining. Cell viability and proliferative capacity were studied by colony formation assay. Dual fluorescence cDNA microarray technique was used to examine the differential expression patterns of the irradiated cells. The cDNA microarray chips used contained DNA sequences corresponding to 12,814 human genes. From the flow cytometry data, it can be observed that radiation induced G2/M phase arrest and that late apoptosis was more evident following G2/M arrest. After 36 hours, some cells underwent senescence and the remains continued on with the cell cycle. Microarray analyses revealed changes in the expression of a small number of cell-cycle-related genes (p21, cyclin B1, etc.) and cell-death genes (tumor necrosis factors, DDB2, etc.) suggesting their involvement in radiation-induced cell-cycle arrest and apoptosis. In silico interpretations of the molecular mechanisms responsible for these radiation effects are in progress

  18. The Decrease in Mitochondrial DNA Mutation Load Parallels Visual Recovery in a Leber Hereditary Optic Neuropathy Patient

    Directory of Open Access Journals (Sweden)

    Sonia Emperador

    2018-02-01

    Full Text Available The onset of Leber hereditary optic neuropathy is relatively rare in childhood and, interestingly, the rate of spontaneous visual recovery is very high in this group of patients. Here, we report a child harboring a rare pathological mitochondrial DNA mutation, present in heteroplasmy, associated with the disease. A patient follow-up showed a rapid recovery of the vision accompanied by a decrease of the percentage of mutated mtDNA. A retrospective study on the age of recovery of all childhood-onset Leber hereditary optic neuropathy patients reported in the literature suggested that this process was probably related with pubertal changes.

  19. DNA replication in necessary for fixing induced mutations to streptomycin-resistance in UV-irradiated Escherichia coli cells

    Energy Technology Data Exchange (ETDEWEB)

    Dubinin, N P; Filippov, V D

    1986-01-01

    A suspension of E.coli cells has been subjected to UV radiation, then it has been incubated in the growth medium for 15 min. After that one of the portions was incubated with nalidixic acid (NA), and the other one without it in the presence of an antibiotic. Frequency of mutations depending on or irrespective of photoactivation, has been determined. Dependence of Str mutation fixing, induced by low UV radiation doses, on DNA synthesis is determined. Results indicate that both photoreactivation of mutations and its senstivity to mfd system are simultaneously lost.

  20. Preliminary studies on DNA retardation by MutS applied to the detection of point mutations in clinical samples

    International Nuclear Information System (INIS)

    Stanislawska-Sachadyn, Anna; Paszko, Zygmunt; Kluska, Anna; Skasko, Elzibieta; Sromek, Maria; Balabas, Aneta; Janiec-Jankowska, Aneta; Wisniewska, Alicja; Kur, Jozef; Sachadyn, Pawel

    2005-01-01

    MutS ability to bind DNA mismatches was applied to the detection of point mutations in PCR products. MutS recognized mismatches from single up to five nucleotides and retarded the electrophoretic migration of mismatched DNA. The electrophoretic detection of insertions/deletions above three nucleotides is also possible without MutS, thanks to the DNA mobility shift caused by the presence of large insertion/deletion loops in the heteroduplex DNA. Thus, the method enables the search for a broad range of mutations: from single up to several nucleotides. The mobility shift assays were carried out in polyacrylamide gels stained with SYBR-Gold. One assay required 50-200 ng of PCR product and 1-3 μg of Thermus thermophilus his 6 -MutS protein. The advantages of this approach are: the small amounts of DNA required for the examination, simple and fast staining, no demand for PCR product purification, no labelling and radioisotopes required. The method was tested in the detection of cancer predisposing mutations in RET, hMSH2, hMLH1, BRCA1, BRCA2 and NBS1 genes. The approach appears to be promising in screening for unknown point mutations

  1. The SRS2 suppressor of rad6 mutations of Saccharomyces cerevisiae acts by channeling DNA lesions into the RAD52 DNA repair pathway

    International Nuclear Information System (INIS)

    Schiestl, R.H.; Prakash, S.; Prakash, L.

    1990-01-01

    rad6 mutants of Saccharomyces cerevisiae are defective in the repair of damaged DNA, DNA damage induced mutagenesis, and sporulation. In order to identify genes that can substitute for RAD6 function, the authors have isolated genomic suppressors of the UV sensitivity of rad6 deletion (rad6Δ) mutations and show that they also suppress the γ-ray sensitivity but not the UV mutagenesis or sporulation defects of rad6. The suppressors show semidominance for suppression of UV sensitivity and dominance for suppression of γ-ray sensitivity. The six suppressor mutations they isolated are all alleles of the same locus and are also allelic to a previously described suppressor of the rad6-1 nonsense mutation, SRS2. They show that suppression of rad6Δ is dependent on the RAD52 recombinational repair pathway since suppression is not observed in the rad6Δ SRS2 strain containing an additional mutation in either the RAD51, RAD52, RAD54, RAD55 or RAD57 genes. Possible mechanisms by which SRS2 may channel unrepaired DNA lesions into the RAD52 DNA repair pathway are discussed

  2. Investigating the effects of dietary folic acid on sperm count, DNA damage and mutation in Balb/c mice

    International Nuclear Information System (INIS)

    Swayne, Breanne G.; Kawata, Alice; Behan, Nathalie A.; Williams, Andrew; Wade, Mike G.; MacFarlane, Amanda J.; Yauk, Carole L.

    2012-01-01

    To date, fewer than 50 mutagens have been studied for their ability to cause heritable mutations. The majority of those studied are classical mutagens like radiation and anti-cancer drugs. Very little is known about the dietary variables influencing germline mutation rates. Folate is essential for DNA synthesis and methylation and can impact chromatin structure. We therefore determined the effects of folic acid-deficient (0 mg/kg), control (2 mg/kg) and supplemented (6 mg/kg) diets in early development and during lactation or post-weaning on mutation rates and chromatin quality in sperm of adult male Balb/c mice. The sperm chromatin structure assay and mutation frequencies at expanded simple tandem repeats (ESTRs) were used to evaluate germline DNA integrity. Treatment of a subset of mice fed the control diet with the mutagen ethylnitrosourea (ENU) at 8 weeks of age was included as a positive control. ENU treated mice exhibited decreased cauda sperm counts, increased DNA fragmentation and increased ESTR mutation frequencies relative to non-ENU treated mice fed the control diet. Male mice weaned to the folic acid deficient diet had decreased cauda sperm numbers, increased DNA fragmentation index, and increased ESTR mutation frequency. Folic acid deficiency in early development did not lead to changes in sperm counts or chromatin integrity in adult mice. Folic acid supplementation in early development or post-weaning did not affect germ cell measures. Therefore, adequate folic acid intake in adulthood is important for preventing chromatin damage and mutation in the male germline. Folic acid supplementation at the level achieved in this study does not improve nor is it detrimental to male germline chromatin integrity.

  3. Investigating the effects of dietary folic acid on sperm count, DNA damage and mutation in Balb/c mice

    Energy Technology Data Exchange (ETDEWEB)

    Swayne, Breanne G.; Kawata, Alice [Environmental Health Science and Research Bureau, Health Canada, Ottawa, Ontario, K1A 0K9 (Canada); Behan, Nathalie A. [Nutrition Research Division, Food Directorate, Health Products and Food Branch, Health Canada, Ottawa, Ontario, K1A 0K9 (Canada); Williams, Andrew; Wade, Mike G. [Environmental Health Science and Research Bureau, Health Canada, Ottawa, Ontario, K1A 0K9 (Canada); MacFarlane, Amanda J. [Nutrition Research Division, Food Directorate, Health Products and Food Branch, Health Canada, Ottawa, Ontario, K1A 0K9 (Canada); Yauk, Carole L., E-mail: carole.yauk@hc-sc.ga.ca [Environmental Health Science and Research Bureau, Health Canada, Ottawa, Ontario, K1A 0K9 (Canada)

    2012-09-01

    To date, fewer than 50 mutagens have been studied for their ability to cause heritable mutations. The majority of those studied are classical mutagens like radiation and anti-cancer drugs. Very little is known about the dietary variables influencing germline mutation rates. Folate is essential for DNA synthesis and methylation and can impact chromatin structure. We therefore determined the effects of folic acid-deficient (0 mg/kg), control (2 mg/kg) and supplemented (6 mg/kg) diets in early development and during lactation or post-weaning on mutation rates and chromatin quality in sperm of adult male Balb/c mice. The sperm chromatin structure assay and mutation frequencies at expanded simple tandem repeats (ESTRs) were used to evaluate germline DNA integrity. Treatment of a subset of mice fed the control diet with the mutagen ethylnitrosourea (ENU) at 8 weeks of age was included as a positive control. ENU treated mice exhibited decreased cauda sperm counts, increased DNA fragmentation and increased ESTR mutation frequencies relative to non-ENU treated mice fed the control diet. Male mice weaned to the folic acid deficient diet had decreased cauda sperm numbers, increased DNA fragmentation index, and increased ESTR mutation frequency. Folic acid deficiency in early development did not lead to changes in sperm counts or chromatin integrity in adult mice. Folic acid supplementation in early development or post-weaning did not affect germ cell measures. Therefore, adequate folic acid intake in adulthood is important for preventing chromatin damage and mutation in the male germline. Folic acid supplementation at the level achieved in this study does not improve nor is it detrimental to male germline chromatin integrity.

  4. Integrated genomic classification of melanocytic tumors of the central nervous system using mutation analysis, copy number alterations and DNA methylation profiling.

    Science.gov (United States)

    Griewank, Klaus; Koelsche, Christian; van de Nes, Johannes A P; Schrimpf, Daniel; Gessi, Marco; Möller, Inga; Sucker, Antje; Scolyer, Richard A; Buckland, Michael E; Murali, Rajmohan; Pietsch, Torsten; von Deimling, Andreas; Schadendorf, Dirk

    2018-06-11

    In the central nervous system, distinguishing primary leptomeningeal melanocytic tumors from melanoma metastases and predicting their biological behavior solely using histopathologic criteria can be challenging. We aimed to assess the diagnostic and prognostic value of integrated molecular analysis. Targeted next-generation-sequencing, array-based genome-wide methylation analysis and BAP1 immunohistochemistry was performed on the largest cohort of central nervous system melanocytic tumors analyzed to date, incl. 47 primary tumors of the central nervous system, 16 uveal melanomas. 13 cutaneous melanoma metastasis and 2 blue nevus-like melanomas. Gene mutation, DNA-methylation and copy-number profiles were correlated with clinicopathological features. Combining mutation, copy-number and DNA-methylation profiles clearly distinguished cutaneous melanoma metastases from other melanocytic tumors. Primary leptomeningeal melanocytic tumors, uveal melanomas and blue nevus-like melanoma showed common DNA-methylation, copy-number alteration and gene mutation signatures. Notably, tumors demonstrating chromosome 3 monosomy and BAP1 alterations formed a homogeneous subset within this group. Integrated molecular profiling aids in distinguishing primary from metastatic melanocytic tumors of the central nervous system. Primary leptomeningeal melanocytic tumors, uveal melanoma and blue nevus-like melanoma share molecular similarity with chromosome 3 and BAP1 alterations markers of poor prognosis. Copyright ©2018, American Association for Cancer Research.

  5. Comparing the DNA hypermethylome with gene mutations in human colorectal cancer.

    Directory of Open Access Journals (Sweden)

    Kornel E Schuebel

    2007-09-01

    Full Text Available We have developed a transcriptome-wide approach to identify genes affected by promoter CpG island DNA hypermethylation and transcriptional silencing in colorectal cancer. By screening cell lines and validating tumor-specific hypermethylation in a panel of primary human colorectal cancer samples, we estimate that nearly 5% or more of all known genes may be promoter methylated in an individual tumor. When directly compared to gene mutations, we find larger numbers of genes hypermethylated in individual tumors, and a higher frequency of hypermethylation within individual genes harboring either genetic or epigenetic changes. Thus, to enumerate the full spectrum of alterations in the human cancer genome, and to facilitate the most efficacious grouping of tumors to identify cancer biomarkers and tailor therapeutic approaches, both genetic and epigenetic screens should be undertaken.

  6. Comparison of the quantification of KRAS mutations by digital PCR and E-ice-COLD-PCR in circulating-cell-free DNA from metastatic colorectal cancer patients.

    Science.gov (United States)

    Sefrioui, David; Mauger, Florence; Leclere, Laurence; Beaussire, Ludivine; Di Fiore, Frédéric; Deleuze, Jean-François; Sarafan-Vasseur, Nasrin; Tost, Jörg

    2017-02-01

    Circulating cell-free DNA (ccfDNA) bears great promise as biomarker for personalized medicine, but ccfDNA is present only at low levels in the plasma or serum of cancer patients. E-ice-COLD-PCR is a recently developed enrichment method to detect and identify mutations present at low-abundance in clinical samples. However, recent studies have shown the importance to accurately quantify low-abundance mutations as clinically important decisions will depend on certain mutation thresholds. The possibility for an enrichment method to accurately quantify the mutation levels remains a point of concern and might limit its clinical applicability. In the present study, we compared the quantification of KRAS mutations in ccfDNA from metastatic colorectal cancer patients by E-ice-COLD-PCR with two digital PCR approaches. For the quantification of mutations by E-ice-COLD-PCR, cell lines with known mutations diluted into WT genomic DNA were used for calibration. E-ice-COLD-PCR and the two digital PCR approaches showed the same range of the mutation level and were concordant for mutation levels below the clinical relevant threshold. E-ice-COLD-PCR can accurately detect and quantify low-abundant mutations in ccfDNA and has a shorter time to results making it compatible with the requirements of analyses in a clinical setting without the loss of quantitative accuracy. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  7. Structural DNA Nanotechnology: Artificial Nanostructures for Biomedical Research.

    Science.gov (United States)

    Ke, Yonggang; Castro, Carlos; Choi, Jong Hyun

    2018-04-04

    Structural DNA nanotechnology utilizes synthetic or biologic DNA as designer molecules for the self-assembly of artificial nanostructures. The field is founded upon the specific interactions between DNA molecules, known as Watson-Crick base pairing. After decades of active pursuit, DNA has demonstrated unprecedented versatility in constructing artificial nanostructures with significant complexity and programmability. The nanostructures could be either static, with well-controlled physicochemical properties, or dynamic, with the ability to reconfigure upon external stimuli. Researchers have devoted considerable effort to exploring the usability of DNA nanostructures in biomedical research. We review the basic design methods for fabricating both static and dynamic DNA nanostructures, along with their biomedical applications in fields such as biosensing, bioimaging, and drug delivery. Expected final online publication date for the Annual Review of Biomedical Engineering Volume 20 is June 4, 2018. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

  8. Sensitive electrochemical determination of unlabeled MutS protein and detection of point mutations in DNA

    Czech Academy of Sciences Publication Activity Database

    Paleček, Emil; Masařík, Michal; Kizek, René; Kuhlmeier, D.; Hassmann, J.; Schülein, J.

    2004-01-01

    Roč. 76, č. 19 (2004), s. 5930-5936 ISSN 0003-2700 R&D Projects: GA AV ČR IBS5004355; GA AV ČR KSK4055109 Institutional research plan: CEZ:AV0Z5004920 Keywords : MutS protein * DNA repair * mercury electrodes Subject RIV: BO - Biophysics Impact factor: 5.450, year: 2004

  9. Somatic point mutations in mtDNA control region are influenced by genetic background and associated with healthy aging: a GEHA study

    DEFF Research Database (Denmark)

    Rose, Giuseppina; Romeo, Giuseppe; Dato, Serena

    2010-01-01

    and of mortality risk in the elderly. Our study provides new evidence on the relevance of mtDNA somatic mutations in aging and longevity and confirms that the occurrence of specific point mutations in the mtDNA control region may represent a strategy for the age-related remodelling of organismal functions....

  10. High prevalence of impaired glucose homeostasis and myopathy in asymptomatic and oligosymptomatic 3243A>G mitochondrial DNA mutation-positive subjects

    DEFF Research Database (Denmark)

    Frederiksen, A.L.; Jeppesen, T.D.; Vissing, J.

    2009-01-01

    combinations. Consequently, it is difficult to predict the "phenotypic risk profile" of 3243A>G mutation-positive subjects. The 3243A>G mutation coexists in cells with wild-type mtDNA, a phenomenon called heteroplasmy. The marked variability in mutation loads in different tissues is the main explanation...

  11. PMS2 gene mutation results in DNA mismatch repair system failure in a case of adult granulosa cell tumor.

    Science.gov (United States)

    Wang, Wen-Chung; Lee, Ya-Ting; Lai, Yen-Chein

    2017-03-27

    Granulosa cell tumors are rare ovarian malignancies. Their characteristics include unpredictable indolent growth with malignant potential and late recurrence. Approximately 95% are of adult type. Recent molecular studies have characterized the FOXL2 402C > G mutation in adult granulosa cell tumor. Our previous case report showed that unique FOXL2 402C > G mutation and defective DNA mismatch repair system are associated with the development of adult granulosa cell tumor. In this study, the DNA sequences of four genes, MSH2, MLH1, MSH6, and PMS2, in the DNA mismatch repair system were determined via direct sequencing to elucidate the exact mechanism for the development of this granulosa cell tumor. The results showed that two missense germline mutations, T485K and N775L, inactivate the PMS2 gene. The results of this case study indicated that although FOXL2 402C > G mutation determines the development of granulosa cell tumor, PMS2 mutation may be the initial driver of carcinogenesis. Immunohistochemistry-based tumor testing for mismatch repair gene expression may be necessary for granulosa cell tumors to determine their malignant potential or if they are part of Lynch syndrome.

  12. Ultraviolet-irradiated simian virus 40 activates a mutator function in rat cells under conditions preventing viral DNA replication

    Energy Technology Data Exchange (ETDEWEB)

    Cornelis, J.; Su, Z.Z.; Dinsart, C.; Rommelaere, J. (Universite libre de Bruxelles, Rhode St Genese (Belgium))

    The UV-irradiated temperature-sensitive early SV40 mutant tsA209 is able to activate at the nonpermissive temperature the expression of mutator and recovery functions in rat cells. Unirradiated SV40 activates these functions only to a low extent. The expression of these mutator and recovery functions in SV40-infected cells was detected using the single-stranded DNA parvovirus H-1 as a probe. Because early SV40 mutants are defective in the initiation of viral DNA synthesis at the nonpermissive temperature, these results suggest that replication of UV-damaged DNA is not a prerequisite for the activation of mutator and recovery functions in mammalian cells. The expression of the mutator function is dose-dependent, i.e., the absolute number of UV-irradiated SV40 virions introduced per cell determines its level. Implications for the interpretation of mutation induction curves in the progeny of UV-irradiated SV40 in permissive host cells are discussed.

  13. Frequent mutations in EGFR, KRAS and TP53 genes in human lung cancer tumors detected by ion torrent DNA sequencing.

    Directory of Open Access Journals (Sweden)

    Xin Cai

    Full Text Available Lung cancer is the most common malignancy and the leading cause of cancer deaths worldwide. While smoking is by far the leading cause of lung cancer, other environmental and genetic factors influence the development and progression of the cancer. Since unique mutations patterns have been observed in individual cancer samples, identification and characterization of the distinctive lung cancer molecular profile is essential for developing more effective, tailored therapies. Until recently, personalized DNA sequencing to identify genetic mutations in cancer was impractical and expensive. The recent technological advancements in next-generation DNA sequencing, such as the semiconductor-based Ion Torrent sequencing platform, has made DNA sequencing cost and time effective with more reliable results. Using the Ion Torrent Ampliseq Cancer Panel, we sequenced 737 loci from 45 cancer-related genes to identify genetic mutations in 76 human lung cancer samples. The sequencing analysis revealed missense mutations in KRAS, EGFR, and TP53 genes in the breast cancer samples of various histologic types. Thus, this study demonstrates the necessity of sequencing individual human cancers in order to develop personalized drugs or combination therapies to effectively target individual, breast cancer-specific mutations.

  14. Current status and research of plant space mutation breeding

    International Nuclear Information System (INIS)

    Qiu Xinmian

    2011-01-01

    Plant space mutation breeding and discussed themechanism of plant space mutagenesis. The variations of organisms were induced by the comprehensive effects of high vacuum, microgravity,incense radiat ion and so on. The application of space mutation breeding and inheritance in specially good grmplasm material in China were well summarized. The prospects of space mutat ion breeding was described. The space mutagenesis will provided a new way for the future breeding. (author)

  15. Mitochondrial DNA mutations in preneoplastic lesions of the gastrointestinal tract: A biomarker for the early detection of cancer

    Directory of Open Access Journals (Sweden)

    Montgomery Elizabeth A

    2006-12-01

    Full Text Available Abstract Background Somatic mutations of mitochondrial DNA (mtDNA are common in many human cancers. We have described an oligonucleotide microarray ("MitoChip" for rapid sequencing of the entire mitochondrial genome (Zhou et al, J Mol Diagn 2006, facilitating the analysis of mtDNA mutations in preneoplastic lesions. We examined 14 precancerous lesions, including seven Barrett esophagus biopsies, with or without associated dysplasia; four colorectal adenomas; and three inflammatory colitis-associated dysplasia specimens. In all cases, matched normal tissues from the corresponding site were obtained as germline control. MitoChip analysis was performed on DNA obtained from cryostat-embedded specimens. Results A total of 513,639 bases of mtDNA were sequenced in the 14 samples, with 490,224 bases (95.4% bases assigned by the automated genotyping software. All preneoplastic lesions examined demonstrated at least one somatic mtDNA sequence alteration. Of the 100 somatic mtDNA alterations observed in the 14 cases, 27 were non-synonymous coding region mutations (i.e., resulting in an amino acid change, 36 were synonymous, and 37 involved non-coding mtDNA. Overall, somatic alterations most commonly involved the COI, ND4 and ND5 genes. Notably, somatic mtDNA alterations were observed in preneoplastic lesions of the gastrointestinal tract even in the absence of histopathologic evidence of dysplasia, suggesting that the mitochondrial genome is susceptible at the earliest stages of multistep cancer progression. Conclusion Our findings further substantiate the rationale for exploring the mitochondrial genome as a biomarker for the early diagnosis of cancer, and confirm the utility of a high-throughput array-based platform for this purpose from a clinical applicability standpoint.

  16. Mutation of Mitochondrial DNA G13513A Presenting with Leigh Syndrome, Wolff-Parkinson-White Syndrome and Cardiomyopathy

    Directory of Open Access Journals (Sweden)

    Shi-Bing Wang

    2008-08-01

    Full Text Available Mutation of mitochondrial DNA (mtDNA G13513A, encoding the ND5 subunit of respiratory chain complex I, can cause mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS and Leigh syndrome. Wolff-Parkinson-White (WPW syndrome and optic atrophy were reported in a high proportion of patients with this mutation. We report an 18-month-old girl, with an 11-month history of psychomotor regression who was diagnosed with WPW syndrome and hypertrophic cardiomyopathy, in association with Leigh syndrome. Supplementation with coenzyme Q10, thiamine and carnitine prevented further regression in gross motor function but the patient's heart function deteriorated and dilated cardiomyopathy developed 11 months later. She was found to have a mutation of mtDNA G13513A. We suggest that mtDNA G13513A mutation is an important factor in patients with Leigh syndrome associated with WPW syndrome and/or optic atrophy, and serial heart function monitoring by echocardiography is recommended in this group of patients.

  17. Mutation and DNA replication in Escherichia coli treated with low concentrations of N-methyl-N'-nitro-N-nitrosoguanidine

    International Nuclear Information System (INIS)

    Jimenez-Sanchez, A.; Cerda-Olmedo, E.

    1975-01-01

    N-Methyl-N'-nitro-N-nitrosoguanidine (nitrosoguanidine) causes an unexpectedly high frequency of closely linked double mutants because of its specificity for chromosome regions in replication. Low nitrosoguanidine concentrations (I μg/ml) in liquid cultures allow replication at the normal rate and are mutagenic. It was expected that mutations would be spread over the chromosome as it replicated, but a high frequency of closely linked double mutants was found. If a thymine auxotroph is grown in the presence of 5-bromodeoxyuridine (BUdR) and nitrosoguanidine and then exposed to 313-nm radiation (which destroys BUdR-substituted DNA), the mutation frequency is much higher among survivors than among non-irradiated cells. It is concluded that nitrosoguanidine inhibits DNA replication in a small fraction of the population and that mutations are induced in that same fraction. Nitrosoguanidine treatment leads to a high frequency of closely linked double mutants under all known conditions

  18. Creation of chimeric human/rabbit APOBEC1 with HIV-1 restriction and DNA mutation activities

    Science.gov (United States)

    Ikeda, Terumasa; Ong, Eugene Boon Beng; Watanabe, Nobumoto; Sakaguchi, Nobuo; Maeda, Kazuhiko; Koito, Atsushi

    2016-01-01

    APOBEC1 (A1) proteins from lagomorphs and rodents have deaminase-dependent restriction activity against HIV-1, whereas human A1 exerts a negligible effect. To investigate these differences in the restriction of HIV-1 by A1 proteins, a series of chimeric proteins combining rabbit and human A1s was constructed. Homology models of the A1s indicated that their activities derive from functional domains that likely act in tandem through a dimeric interface. The C-terminal region containing the leucine-rich motif and the dimerization domains of rabbit A1 is important for its anti-HIV-1 activity. The A1 chimeras with strong anti-HIV-1 activity were incorporated into virions more efficiently than those without anti-HIV-1 activity, and exhibited potent DNA-mutator activity. Therefore, the C-terminal region of rabbit A1 is involved in both its packaging into the HIV-1 virion and its deamination activity against both viral cDNA and genomic RNA. This study identifies the novel molecular mechanism underlying the target specificity of A1.

  19. Concordance of mutation detection in circulating tumor DNA in early clinical trials using different blood collection protocols

    DEFF Research Database (Denmark)

    Ahlborn, Lise B.; Madsen, Mette; Jonson, Lars

    2017-01-01

    in a clinical setting. Here we investigate the concordance between standard blood collection for molecular analysis using immediate separation of plasma, compared to the use of collection tubes allowing for delayed processing. Methods: In this study, we measured the fractional abundance of tumor specific...... patients with advanced solid cancers enrolled in early clinical trials. Results: Concordance in the fractional abundance of mutations in ctDNA isolated from blood collected in either K3EDTA or BCT tubes from patients with different solid cancers was observed. Conclusions: This study indicates that BCT...... mutations (BRAF p.V600E and PIK3CA p.H1047R) in ctDNA isolated from blood samples collected in either cell-stabilizing Cell-Free DNA BCT tubes (delayed processing within 72 hours) or standard K3EDTA tubes (immediate processing within 15 minutes). Twenty-five blood sample pairs (EDTA/BCT) were collected from...

  20. MELAS syndrome associated with both A3243G-tRNALeu mutation and multiple mitochondrial DNA deletions.

    Science.gov (United States)

    Aharoni, Sharon; Traves, Teres A; Melamed, Eldad; Cohen, Sarit; Silver, Esther Leshinsky

    2010-09-15

    The syndrome of mitochondrial encephalopathy, lactic acidosis, and stroke-like episode (MELAS) is characterized clinically by recurrent focal neurological deficits, epilepsy, and short stature. The phenotypic spectrum is extremely diverse, with multisystemic organ involvement leading to isolated diabetes, deafness, renal tubulopathy, hypertrophic cardiomyopathy, and retinitis pigmentosa. In 80% of cases, the syndrome is associated with an AG transmission mutation (A3243G) in the tRNALeu gene of the mitochondrial DNA (mtDNA). We describe a woman with a unique combination of the MELAS A3243G mutation and multiple mtDNA deletions with normal POLG sequence. The patient presented with diabetes mellitus, sensorineural deafness, short stature, and mental disorientation. All her three children died in early adolescence. 2010 Elsevier B.V. All rights reserved.

  1. Influence of inhibitors of poly(ADP-ribose) polymerase on DNA repair, chromosomal alterations, and mutations

    Energy Technology Data Exchange (ETDEWEB)

    Natarajan, A.T.; van Zeeland, A.A.; Zwanenburg, T.S.

    1983-01-01

    The influence of inhibitors of poly(ADP-ribose) polymerase such as 3-aminobenzamide (3AB) and benzamide (B) on the spontaneously occurring as well as mutagen induced chromosomal aberrations, sister chromatid exchanges (SCEs) and point mutations has been studied. In addition, the influence of 3AB on DNA repair was measured following treatment with physical and chemical mutagens. Post treatment of X-irradiated mammalian cells with 3AB increases the frequencies of induced chromosomal aberrations by a factor of 2 to 3. 3AB, when present in the medium containing bromodeoxyuridine(BrdUrd) during two cell cycles, increases the frequencies of SCEs in Chinese hamster ovary cells (CHO) in a concentration dependent manner leading to about a 10-fold increase at 10 mM concentration. The extent of increase in the frequencies of SCEs due to 1 mM 3AB in several human cell lines has been studied, including those derived from patients suffering from genetic diseases such as ataxia telangiectasia (A-T), Fanconi's anemia (FA), and Huntington's chorea. None of these syndromes showed any increased response when compared to normal cells. 3AB, however, increased the frequencies of spontaneously occurring chromosomal aberrations in A-T and FA cells. 3AB does not influence the frequencies of SCEs induced by UV or mitomycin C (MMC) in CHO cells. However, it increases the frequencies of SCEs induced by ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS). Under the conditions in which 3AB increases the frequencies of spontaneously occurring as well as induced SCEs, it does not increase the frequencies of point mutations in hypoxanthine-guanine phosphoribosyltransferase (HGPRT) locus. 3AB does not influence the amount of repair replication following dimethylsulphate (DMS) treatment of human fibroblasts, or UV irradiated human lymphocytes.

  2. DCB - DNA and Chromosome Aberrations Research

    Science.gov (United States)

    Part of NCI's Division of Cancer Biology's research portfolio, this research area is focused on making clear the genetic and epigenetic mechanisms of tumorigenesis and mechanisms of chemical and physical carcinogenesis.

  3. DNA synthesis time in germinating rice and pattern of diethylsulphate induced mutations in pre-soaked seeds

    International Nuclear Information System (INIS)

    Narahari, P.

    1978-01-01

    DNA synthesis pattern in germinating rice seeds, pre-soaked in water for varying periods upto 48 hr, was determined by following the pulse incorporation of 3 H-thymidine into the TCA-insoluble nucleoprotein. Synthesis of DNA commenced at 24 hr, progressively increased to a first peak at about 38 hr, thereafter showed a 1/3rd drop and subsequently increased to a 2nd and still higher peak at 46 to 48 hr of pre-soaking. Treatments of diethylsulphate (dES) at a low concentration (0.2%-2hr) administered at various progressing stages of DNA synthesis resulted in decrease in seedling height and survival, and increase in mutation frequency at 45 hr. pre-soaking, maximum mutation frequencies of 20, 10 and 2% on M 1 plants, M 1 spikes and M 2 seedling bases, respectively were observed. Higher dES concentration (0.3%-2hr) given at later periods of pre-soaking showed near lethal effects and consequently decreased mutation frequencies. Treatments of sodium fluoride given singly or in combination with dES did not show any substantially different results as compared to those of the respective controls. Mutation spectra observed after dES treatments to germinating seeds, at different pre-soaking periods, were quite dissimilar. Specific mutations of economic importance like semi-dwarf mutants were isolated from the treatment of germinating seeds pre-soaked for 37.5 hr or more when shoot apex cells were undergoing DNA synthesis. (author)

  4. Mutation breeding research of wheat (T. aestivum) in China

    International Nuclear Information System (INIS)

    Wang, L.; Zhang, B.; Shi, J.; Tao, S.; Fan, Q.

    1990-01-01

    Full text: 78 cultivars and various valuable strains have been obtained through induced genic mutation and chromosome translocation. Irradiation of hybrid seeds, gametes, zygotes and in vitro cultured cells, gave increased mutation frequency and expanded spectrum. Various physical agents were examined either singly or in combination with chemical agents. Combined use of γ-irradiation by low dose and in vitro culture proved effective in raising the percentage of seed-set in wide-crosses. (author)

  5. MethylMeter(®): bisulfite-free quantitative and sensitive DNA methylation profiling and mutation detection in FFPE samples.

    Science.gov (United States)

    McCarthy, David; Pulverer, Walter; Weinhaeusel, Andreas; Diago, Oscar R; Hogan, Daniel J; Ostertag, Derek; Hanna, Michelle M

    2016-06-01

    Development of a sensitive method for DNA methylation profiling and associated mutation detection in clinical samples. Formalin-fixed and paraffin-embedded tumors received by clinical laboratories often contain insufficient DNA for analysis with bisulfite or methylation sensitive restriction enzymes-based methods. To increase sensitivity, methyl-CpG DNA capture and Coupled Abscription PCR Signaling detection were combined in a new assay, MethylMeter(®). Gliomas were analyzed for MGMT methylation, glioma CpG island methylator phenotype and IDH1 R132H. MethylMeter had 100% assay success rate measuring all five biomarkers in formalin-fixed and paraffin-embedded tissue. MGMT methylation results were supported by survival and mRNA expression data. MethylMeter is a sensitive and quantitative method for multitarget DNA methylation profiling and associated mutation detection. The MethylMeter-based GliomaSTRAT assay measures methylation of four targets and one mutation to simultaneously grade gliomas and predict their response to temozolomide. This information is clinically valuable in management of gliomas.

  6. The mitochondrial DNA 10197 G > A mutation causes MELAS/Leigh overlap syndrome presenting with acute auditory agnosia.

    Science.gov (United States)

    Leng, Yinglin; Liu, Yuhe; Fang, Xiaojing; Li, Yao; Yu, Lei; Yuan, Yun; Wang, Zhaoxia

    2015-04-01

    Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes/Leigh (MELAS/LS) overlap syndrome is a mitochondrial disorder subtype with clinical and magnetic resonance imaging (MRI) features that are characteristic of both MELAS and Leigh syndrome (LS). Here, we report an MELAS/LS case presenting with cortical deafness and seizures. Cranial MRI revealed multiple lesions involving bilateral temporal lobes, the basal ganglia and the brainstem, which conformed to neuroimaging features of both MELAS and LS. Whole mitochondrial DNA (mtDNA) sequencing and PCR-RFLP revealed a de novo heteroplasmic m.10197 G > A mutation in the NADH dehydrogenase subunit 3 gene (ND3), which was predicted to cause an alanine to threonine substitution at amino acid 47. Although the mtDNA m.10197 G > A mutation has been reported in association with LS, Leber hereditary optic neuropathy and dystonia, it has never been linked with MELAS/LS overlap syndrome. Our patient therefore expands the phenotypic spectrum of the mtDNA m.10197 G > A mutation.

  7. Quantitative cell-free DNA, KRAS, and BRAF mutations in plasma from patients with metastatic colorectal cancer during treatment with cetuximab and irinotecan

    DEFF Research Database (Denmark)

    Spindler, Karen-Lise Garm; Pallisgaard, Niels; Vogelius, Ivan Storgaard

    2012-01-01

    The present study investigated the levels of circulating cell-free DNA (cfDNA) in plasma from patients with metastatic colorectal cancer (mCRC) in relation to third-line treatment with cetuximab and irinotecan and the quantitative relationship of cfDNA with tumor-specific mutations in plasma....

  8. Finding cancer driver mutations in the era of big data research.

    Science.gov (United States)

    Poulos, Rebecca C; Wong, Jason W H

    2018-04-02

    In the last decade, the costs of genome sequencing have decreased considerably. The commencement of large-scale cancer sequencing projects has enabled cancer genomics to join the big data revolution. One of the challenges still facing cancer genomics research is determining which are the driver mutations in an individual cancer, as these contribute only a small subset of the overall mutation profile of a tumour. Focusing primarily on somatic single nucleotide mutations in this review, we consider both coding and non-coding driver mutations, and discuss how such mutations might be identified from cancer sequencing datasets. We describe some of the tools and database that are available for the annotation of somatic variants and the identification of cancer driver genes. We also address the use of genome-wide variation in mutation load to establish background mutation rates from which to identify driver mutations under positive selection. Finally, we describe the ways in which mutational signatures can act as clues for the identification of cancer drivers, as these mutations may cause, or arise from, certain mutational processes. By defining the molecular changes responsible for driving cancer development, new cancer treatment strategies may be developed or novel preventative measures proposed.

  9. Increasing global participation in genetics research through DNA barcoding.

    Science.gov (United States)

    Adamowicz, Sarah J; Steinke, Dirk

    2015-12-01

    DNA barcoding--the sequencing of short, standardized DNA regions for specimen identification and species discovery--has promised to facilitate rapid access to biodiversity knowledge by diverse users. Here, we advance our opinion that increased global participation in genetics research is beneficial, both to scientists and for science, and explore the premise that DNA barcoding can help to democratize participation in genetics research. We examine publication patterns (2003-2014) in the DNA barcoding literature and compare trends with those in the broader, related domain of genomics. While genomics is the older and much larger field, the number of nations contributing to the published literature is similar between disciplines. Meanwhile, DNA barcoding exhibits a higher pace of growth in the number of publications as well as greater evenness among nations in their proportional contribution to total authorships. This exploration revealed DNA barcoding to be a highly international discipline, with growing participation by researchers in especially biodiverse nations. We briefly consider several of the challenges that may hinder further participation in genetics research, including access to training and molecular facilities as well as policy relating to the movement of genetic resources.

  10. Better plants through mutations

    International Nuclear Information System (INIS)

    1988-01-01

    This is a public relations film describing problems associated with the genetic improvement of crop plants through induced mutations. Mutations are the ultimate source of genetic variation in plants. Mutation induction is now established as a practical tool in plant breeding. The Joint FAO/IAEA Division and the IAEA's laboratory at Seibersdorf have supported research and practical implementation of mutation breeding of both seed propagated and vegetatively propagated plants. Plant biotechnology based on in vitro culture and recombinant DNA technology will make a further significant contribution to plant breeding

  11. Cold Spring Harbor symposia on quantitative biology. Volume XLVII, Part 1. Structures of DNA

    International Nuclear Information System (INIS)

    Anon.

    1983-01-01

    The proceedings for the 47th Annual Cold Spring Harbor Symposia on Quantitative Biology are presented. This symposium focused on the Structure of DNA. Topics presented covered research in the handedness of DNA, conformational analysis, chemically modified DNA, chemical synthesis of DNA, DNA-protein interactions, DNA within nucleosomes, DNA methylation, DNA replication, gyrases and topoisomerases, recombining and mutating DNA, transcription of DNA and its regulation, the organization of genes along DNA, repetitive DNA and pseudogenes, and origins of replication, centromeres, and teleomeres

  12. Simultaneous DNA and RNA mapping of somatic mitochondrial mutations across diverse human cancers

    DEFF Research Database (Denmark)

    Stewart, James B.; Alaei-Mahabadi, Babak; Radhakrishnan, Sabarinathan

    2015-01-01

    of evidence from both genomic and transcriptomic sequencing. We find that there is selective pressure against deleterious coding mutations, supporting that functional mitochondria are required in tumor cells, and also observe a strong mutational strand bias, compatible with endogenous replication...

  13. Highly sensitive detection of ESR1 mutations in cell-free DNA from patients with metastatic breast cancer using molecular barcode sequencing.

    Science.gov (United States)

    Masunaga, Nanae; Kagara, Naofumi; Motooka, Daisuke; Nakamura, Shota; Miyake, Tomohiro; Tanei, Tomonori; Naoi, Yasuto; Shimoda, Masafumi; Shimazu, Kenzo; Kim, Seung Jin; Noguchi, Shinzaburo

    2018-01-01

    We aimed to develop a highly sensitive method to detect ESR1 mutations in cell-free DNA (cfDNA) using next-generation sequencing with molecular barcode (MB-NGS) targeting the hotspot segment (c.1600-1713). The sensitivity of MB-NGS was tested using serially diluted ESR1 mutant DNA and then cfDNA samples from 34 patients with metastatic breast cancer were analyzed with MB-NGS. The results of MB-NGS were validated in comparison with conventional NGS and droplet digital PCR (ddPCR). MB-NGS showed a higher sensitivity (0.1%) than NGS without barcode (1%) by reducing background errors. Of the cfDNA samples from 34 patients with metastatic breast cancer, NGS without barcode revealed seven mutations in six patients (17.6%) and MB-NGS revealed six additional mutations including three mutations not reported in the COSMIC database of breast cancer, resulting in total 13 ESR1 mutations in ten patients (29.4%). Regarding the three hotspot mutations, all the patients with mutations detected by MB-NGS had identical mutations detected by droplet digital PCR (ddPCR), and mutant allele frequency correlated very well between both (r = 0.850, p < 0.01). Moreover, all the patients without these mutations by MB-NGS were found to have no mutations by ddPCR. In conclusion, MB-NGS could successfully detect ESR1 mutations in cfDNA with a higher sensitivity of 0.1% than conventional NGS and was considered as clinically useful as ddPCR.

  14. A combinatorial role for MutY and Fpg DNA glycosylases in mutation avoidance in Mycobacterium smegmatis

    International Nuclear Information System (INIS)

    Hassim, Farzanah; Papadopoulos, Andrea O.; Kana, Bavesh D.; Gordhan, Bhavna G.

    2015-01-01

    Highlights: • We studied the combined role of MutY and Fpg DNA glycosylases in M. smegmatis. • Loss of MutY showed increased sensitivity to oxidative damage. • Loss of MutY together with the Fpg glycosylases showed increased mutation rates. • Our data indicate interplay between these enzymes to control mutagenesis. - Abstract: Hydroxyl radical (·OH) among reactive oxygen species cause damage to nucleobases with thymine being the most susceptible, whilst in contrast, the singlet oxygen ( 1 0 2 ) targets only guanine bases. The high GC content of mycobacterial genomes predisposes these organisms to oxidative damage of guanine. The exposure of cellular DNA to ·OH and one-electron oxidants results in the formation of two main degradation products, the pro-mutagenic 8-oxo-7,8-dihydroguanine (8-oxoGua) and the cytotoxic 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua). These lesions are repaired through the base excision repair (BER) pathway and we previously, demonstrated a combinatorial role for the mycobacterial Endonuclease III (Nth) and the Nei family of DNA glycosylases in mutagenesis. In addition, the formamidopyrimidine (Fpg/MutM) and MutY DNA glycosylases have also been implicated in mutation avoidance and BER in mycobacteria. In this study, we further investigate the combined role of MutY and the Fpg/Nei DNA glycosylases in Mycobacterium smegmatis and demonstrate that deletion of mutY resulted in enhanced sensitivity to oxidative stress, an effect which was not exacerbated in Δfpg1 Δfpg2 or Δnei1 Δnei2 double mutant backgrounds. However, combinatorial loss of the mutY, fpg1 and fpg2 genes resulted in a significant increase in mutation rates suggesting interplay between these enzymes. Consistent with this, there was a significant increase in C → A mutations with a corresponding change in cell morphology of rifampicin resistant mutants in the Δfpg1 Δfpg2 ΔmutY deletion mutant. In contrast, deletion of mutY together with the nei homologues

  15. The erratic mitochondrial clock: variations of mutation rate, not population size, affect mtDNA diversity across birds and mammals

    Directory of Open Access Journals (Sweden)

    Galtier Nicolas

    2009-03-01

    Full Text Available Abstract Background During the last ten years, major advances have been made in characterizing and understanding the evolution of mitochondrial DNA, the most popular marker of molecular biodiversity. Several important results were recently reported using mammals as model organisms, including (i the absence of relationship between mitochondrial DNA diversity and life-history or ecological variables, (ii the absence of prominent adaptive selection, contrary to what was found in invertebrates, and (iii the unexpectedly large variation in neutral substitution rate among lineages, revealing a possible link with species maximal longevity. We propose to challenge these results thanks to the bird/mammal comparison. Direct estimates of population size are available in birds, and this group presents striking life-history trait differences with mammals (higher mass-specific metabolic rate and longevity. These properties make birds the ideal model to directly test for population size effects, and to discriminate between competing hypotheses about the causes of substitution rate variation. Results A phylogenetic analysis of cytochrome b third-codon position confirms that the mitochondrial DNA mutation rate is quite variable in birds, passerines being the fastest evolving order. On average, mitochondrial DNA evolves slower in birds than in mammals of similar body size. This result is in agreement with the longevity hypothesis, and contradicts the hypothesis of a metabolic rate-dependent mutation rate. Birds show no footprint of adaptive selection on cytochrome b evolutionary patterns, but no link between direct estimates of population size and cytochrome b diversity. The mutation rate is the best predictor we have of within-species mitochondrial diversity in birds. It partly explains the differences in mitochondrial DNA diversity patterns observed between mammals and birds, previously interpreted as reflecting Hill-Robertson interferences with the W

  16. A combinatorial role for MutY and Fpg DNA glycosylases in mutation avoidance in Mycobacterium smegmatis

    Energy Technology Data Exchange (ETDEWEB)

    Hassim, Farzanah; Papadopoulos, Andrea O.; Kana, Bavesh D.; Gordhan, Bhavna G., E-mail: bhavna.gordhan@nhls.ac.za

    2015-09-15

    Highlights: • We studied the combined role of MutY and Fpg DNA glycosylases in M. smegmatis. • Loss of MutY showed increased sensitivity to oxidative damage. • Loss of MutY together with the Fpg glycosylases showed increased mutation rates. • Our data indicate interplay between these enzymes to control mutagenesis. - Abstract: Hydroxyl radical (·OH) among reactive oxygen species cause damage to nucleobases with thymine being the most susceptible, whilst in contrast, the singlet oxygen ({sup 1}0{sub 2}) targets only guanine bases. The high GC content of mycobacterial genomes predisposes these organisms to oxidative damage of guanine. The exposure of cellular DNA to ·OH and one-electron oxidants results in the formation of two main degradation products, the pro-mutagenic 8-oxo-7,8-dihydroguanine (8-oxoGua) and the cytotoxic 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua). These lesions are repaired through the base excision repair (BER) pathway and we previously, demonstrated a combinatorial role for the mycobacterial Endonuclease III (Nth) and the Nei family of DNA glycosylases in mutagenesis. In addition, the formamidopyrimidine (Fpg/MutM) and MutY DNA glycosylases have also been implicated in mutation avoidance and BER in mycobacteria. In this study, we further investigate the combined role of MutY and the Fpg/Nei DNA glycosylases in Mycobacterium smegmatis and demonstrate that deletion of mutY resulted in enhanced sensitivity to oxidative stress, an effect which was not exacerbated in Δfpg1 Δfpg2 or Δnei1 Δnei2 double mutant backgrounds. However, combinatorial loss of the mutY, fpg1 and fpg2 genes resulted in a significant increase in mutation rates suggesting interplay between these enzymes. Consistent with this, there was a significant increase in C → A mutations with a corresponding change in cell morphology of rifampicin resistant mutants in the Δfpg1 Δfpg2 ΔmutY deletion mutant. In contrast, deletion of mutY together with the nei

  17. Introduction to mutation breeding and genetic research of soybean in China

    International Nuclear Information System (INIS)

    Zhan Mingkui; Zhao Jingrong

    1988-01-01

    This paper summarized the achievements and developments in mutation breeding and genetic research of soybean. The optimal irradiation dosage was determined for 22 varieties of soybean which have been released and popularized so far. Analyses of mutants, mutant characters and mutation frequency in the generations of M 1 , M 2 and M 3 of soybean were carried out and a procedure of mutation breeding was described. Discussion of the effect of different radiant agents, the selection of progeny induced by radiation, the breeding method by combining mutation with hybridization and resistant varieties with good quality ones have been conducted

  18. Pathophysiological consequences and benefits of HFE mutations: 20 years of research

    Science.gov (United States)

    Hollerer, Ina; Bachmann, André; Muckenthaler, Martina U.

    2017-01-01

    Mutations in the HFE (hemochromatosis) gene cause hereditary hemochromatosis, an iron overload disorder that is hallmarked by excessive accumulation of iron in parenchymal organs. The HFE mutation p.Cys282Tyr is pathologically most relevant and occurs in the Caucasian population with a carrier frequency of up to 1 in 8 in specific European regions. Despite this high prevalence, the mutation causes a clinically relevant phenotype only in a minority of cases. In this review, we summarize historical facts and recent research findings about hereditary hemochromatosis, and outline the pathological consequences of the associated gene defects. In addition, we discuss potential advantages of HFE mutations in asymptomatic carriers. PMID:28280078

  19. Identification of DNA polymerase molecules repairing DNA irradiated damage and molecular biological study on modified factors of mutation rate

    Energy Technology Data Exchange (ETDEWEB)

    Yamada, Koichi; Inoue, Shuji [National Inst. of Healthand Nutrition, Tokyo (Japan)

    1999-02-01

    DNA repairing polymerase has not been identified in human culture cells because the specificities of enzyme inhibitors used in previous studies were not so high. In this study, anti-sense oligonucleotides were transfected into human fibroblast cells by electroporation and several clones selected by geneticin treatment were found to express the RNA of the incorporated DNA. However, the expression was not significant and its reproducibility was poor. Then, a study on repairing mechanism was made using XP30 RO and XP 115 LO cells which are variant cells of xeroderma pigmentosum, a human hereditary disease aiming to identify the DNA polymerase related to the disease. There were abnormalities in DNA polymerase subunit {delta} or {epsilon} which consists DNA replication complex. Thus, it was suggested that the DNA replication of these mutant cells might terminate at the site containing such abnormality. (M.N.)

  20. RESEARCH ARTICLE Fitness-compensatory mutations facilitate the ...

    Indian Academy of Sciences (India)

    Navya

    2016-12-23

    Dec 23, 2016 ... and Cellular Biology, Division of Molecular Biology and Human Genetics, Faculty of .... macrophage cell line (ATCC TIB-202) was propagated in RPMI-1640 ..... tolerance to anti-TB drugs and is thought to occur as a result of metabolic ... Mutations in genes related to intermediary respiration and metabolism.

  1. Ultraviolet light induction of diphtheria toxin-resistant mutations in normal and DNA repair-deficient human and Chinese hamster fibroblasts

    International Nuclear Information System (INIS)

    Trosko, J.E.; Schultz, R.S.; Chang, C.C.; Glover, T.

    1980-01-01

    The role on unrepaired DNA lesions in the production of mutations is suspected of contributing to the initiation phase of carcinogenesis. Since the molecular basis of mutagenesis is not understood in eukaryotic cells, development of new genetic markers for quantitative in vitro measurement of mutations for mammalian cells is needed. Furthermore, mammalian cells, genetically deficient for various DNA repair enzymes, will be needed to study the role of unrepaired DNA lesions in mutagenesis. The results in this report relate to preliminary attempts to characterize the diphtheria toxin resistance marker as a useful quantitative genetic marker in human cells and to isolate and characterize various DNA repair-deficient Chinese hamster cells

  2. High prevalence of impaired glucose homeostasis and myopathy in asymptomatic and oligosymptomatic 3243A>G mitochondrial DNA mutation-positive subjects

    DEFF Research Database (Denmark)

    Frederiksen, A.L.; Jeppesen, T.D.; Vissing, J.

    2009-01-01

    controls were subjected to an oral glucose tolerance test. Twenty-six adult 3243A>G carriers with unknown myopathy status and 17 healthy controls had a maximal cycle test and a muscle biopsy performed. The mutation loads were quantified in blood and muscle biopsies and correlated to the clinical......INTRODUCTION: The point mutation of 3243A>G mtDNA is the most frequent cause of mitochondrial diabetes, often presenting as the syndrome maternally inherited diabetes and deafness (MIDD). The mutation may also cause myopathy, ataxia, strokes, ophthalmoplegia, epilepsy, and cardiomyopathy in various...... combinations. Consequently, it is difficult to predict the "phenotypic risk profile" of 3243A>G mutation-positive subjects. The 3243A>G mutation coexists in cells with wild-type mtDNA, a phenomenon called heteroplasmy. The marked variability in mutation loads in different tissues is the main explanation...

  3. Retrospective assessment of the most common mitochondrial DNA mutations in a large Hungarian cohort of suspect mitochondrial cases.

    Science.gov (United States)

    Remenyi, Viktoria; Inczedy-Farkas, Gabriella; Komlosi, Katalin; Horvath, Rita; Maasz, Anita; Janicsek, Ingrid; Pentelenyi, Klara; Gal, Aniko; Karcagi, Veronika; Melegh, Bela; Molnar, Maria Judit

    2015-08-01

    Prevalence estimations for mitochondrial disorders still vary widely and only few epidemiologic studies have been carried out so far. With the present work we aim to give a comprehensive overview about frequencies of the most common mitochondrial mutations in Hungarian patients. A total of 1328 patients were tested between 1999 and 2012. Among them, 882 were screened for the m.3243A > G, m.8344A > G, m.8993T > C/G mutations and deletions, 446 for LHON primary mutations. The mutation frequency in our cohort was 2.61% for the m.3243A > G, 1.47% for the m.8344A > G, 17.94% for Leber's Hereditary Optic Neuropathy (m.3460G > A, m.11778G > A, m.14484T > C) and 0.45% for the m.8993T > C/G substitutions. Single mtDNA deletions were detected in 14.97%, while multiple deletions in 6.01% of the cases. The mutation frequency in Hungarian patients suggestive of mitochondrial disease was similar to other Caucasian populations. Further retrospective studies of different populations are needed in order to accurately assess the importance of mitochondrial diseases and manage these patients.

  4. Research progress on the space-flight mutation breeding of woodyplant

    International Nuclear Information System (INIS)

    Cui Binbin; Sun Yuhan; Li Yun

    2013-01-01

    The space-flight mutation breeding conception, characteristics, mutagenic effects, research progress at home and abroad in woody plant were reviewed in this paper. Compared with crops, although the research of the woody plants space-flight mutation breeding in China started later, but it has developed rapidly and has gotten certain achievement. Now the satellite and high-altitude balloon experiment were conducted with over 20 tree species such as Populus ussuriensis and 50 flower species such as Paeonia suffruticosa. The above work will has profound significance for space-flight breeding technology application on woody plants. In the end, this thesis analyzes the prospect in the future from four aspects such as using woody plants asexual reproduction characteristic, strengthening the space mutation mechanism study, enhancing new space mutation varieties screen and strengthening ornamental specific types selection. This thesis also thinks that the space mutation breeding is expected to become an effective way in woody plant genetic breeding. (authors)

  5. Induced mutation research in plant breeding; Recherche sur les mutations radioinduites en phytogenetique

    Energy Technology Data Exchange (ETDEWEB)

    Briggs, R. W. [Biology Department, Brookhaven National Laboratory, Upton, NY (United States)

    1970-01-15

    The improvement of plants is of great importance to the developing countries. The author briefly describes, with references, recent work on mutation breeding by means of ionizing radiations. The aim of this work is to increase the quantity and quality of plants, e.g. by increasing yield, by developing disease- or insect-resistant varieties, by increasing nutritive value, by improving taste, storage life and appearance. (author) [French] L'amélioration des plantes présente une grande importance pour les pays en voie de développement. L’auteur décrit succinctement, en s'appuyant sur des exemples, certaines recherches récentes en phytogénétique faisant intervenir des mutations radioinduites. L'objet du travail exposé dans le mémoire est d'améliorer quantitativement et qualitativement les plantes, par exemple en augmentant les rendements, en créant des variétés résistant aux maladies et aux insectes, en augmentant la valeur nutritive des produits, ou en améliorant leur goût, leur durée de conservation et leur aspect. (author)

  6. Homoplasmy of the G7444A mtDNA and heterozygosity of the GJB2 c.35delG mutations in a family with hearing loss

    DEFF Research Database (Denmark)

    Kokotas, Haris; Grigoriadou, Maria; Yang, Li

    2011-01-01

    Mitochondrial mutations have been shown to be responsible for syndromic as well as non-syndromic hearing loss. The G7444A mitochondrial DNA mutation affects COI/the precursor of tRNA(Ser(UCN)), encoding the first subunit of cytochrome oxidase. Here we report on the first Greek family with the G74...

  7. High mutation detection rate in the COL4A5 collagen gene in suspected Alport syndrome using PCR and direct DNA sequencing

    DEFF Research Database (Denmark)

    Martin, P; Heiskari, N; Zhou, J

    1998-01-01

    -amplified and sequenced from DNA of 50 randomly chosen patients with suspected Alport syndrome. Mutations were found in 41 patients, giving a mutation detection rate of 82%. Retrospective analysis of clinical data revealed that two of the cases might be autosomal. Although it could not be determined whether the remaining...

  8. Research for structure and function of protein promoting mutation induction

    International Nuclear Information System (INIS)

    Nohmi, Takehiko; Gruz, P.; Shimizu, M.

    2004-01-01

    The nature of Y family DNA polymerase, which takes a damaged deoxynucleotide 3 phosphate (dNTP) into the template, was discussed in detail. Bacterial endoenzyme DNA pol Y1 took in the oxidized dNTP uniquely. The pol Y1 showed following singularity: (1) taking 2-OH-dGTP in opposite side of template A, (2) taking 2-OH-dATP in opposite side of template G and T, (3) not only taking in both of the oxidized dNTP, but also carrying out an elongation reaction of the dNTP, (4) after taking in the oxidized nucleotide continuously, the elongation reaction stopped. Human Y family DNA polymerase, hpol η showed the same nature of the preceding item (1) and (2), also. Above mentioned natures indicated possibility of common nature of Y family polymerase. (M. Suetake)

  9. Molecular nature of X-ray-induced mutations compared with that of spontaneous ones in human c-hprt gene integrated into mammalian chromosomal DNA

    International Nuclear Information System (INIS)

    Kimura, Hiroshi; Kato, Takesi.

    1992-01-01

    X-ray-induced mutations were analysed at molecular levels in comparison with spontaneous mutations. Altered sequences were determined tentatively of 30 independent X-ray-induced mutations in a cDNA of the human hprt gene which was integrated into mammalian chromosome as a part of a shuttle vector. Mutations consisted of base substitutions (37 %), frameshifts (27 %), deletions (27 %) and others (10 %). All these mutational events were distributed randomly over the gene without there being hot spots. The spectrum and distribution of X-ray-induced mutations resembled those of spontaneous mutations. Among base substitutions, transversions were predominant and base substitution mutations occurred more at A:T sites than at G:C sites, which is also the case in spontaneous mutations. Most of the frameshift and deletion mutations induced by X-rays, as well as those spontaneously arising, were characterized by the existence of short direct repeats of several identical bases in a row at the sites of the mutations. A slippage misalignment mechanism in replication well accounts for the generation of these classes of mutations. Judging from the data accumulated so far, it can be concluded that X-ray-induced mutations at molecular levels are similar to those spontaneously occurring. (author)

  10. Emergence of Tetracycline Resistance in Helicobacter pylori: Multiple Mutational Changes in 16S Ribosomal DNA and Other Genetic Loci

    Science.gov (United States)

    Dailidiene, Daiva; Bertoli, M. Teresita; Miciuleviciene, Jolanta; Mukhopadhyay, Asish K.; Dailide, Giedrius; Pascasio, Mario Alberto; Kupcinskas, Limas; Berg, Douglas E.

    2002-01-01

    Tetracycline is useful in combination therapies against the gastric pathogen Helicobacter pylori. We found 6 tetracycline-resistant (Tetr) strains among 159 clinical isolates (from El Salvador, Lithuania, and India) and obtained the following four results: (i) 5 of 6 Tetr isolates contained one or two nucleotide substitutions in one part of the primary tetracycline binding site in 16S rRNA (AGA965-967 [Escherichia coli coordinates] changed to gGA, AGc, guA, or gGc [lowercase letters are used to represent the base changes]), whereas the sixth (isolate Ind75) retained AGA965-967; (ii) PCR products containing mutant 16S ribosomal DNA (rDNA) alleles transformed recipient strains to Tetr phenotypes, but transformants containing alleles with single substitutions (gGA and AGc) were less resistant than their Tetr parents; (iii) each of 10 Tetr mutants of reference strain 26695 (in which mutations were induced with metronidazole, a mutagenic anti-H. pylori agent) contained the normal AGA965-967 sequence; and (iv) transformant derivatives of Ind75 and of one of the Tetr 26695 mutants that had acquired mutant rDNA alleles were resistant to tetracycline at levels higher than those to which either parent strain was resistant. Thus, tetracycline resistance in H. pylori results from an accumulation of changes that may affect tetracycline-ribosome affinity and/or other functions (perhaps porins or efflux pumps). We suggest that the rarity of tetracycline resistance among clinical isolates reflects this need for multiple mutations and perhaps also the deleterious effects of such mutations on fitness. Formally equivalent mutations with small but additive effects are postulated to contribute importantly to traits such as host specificity and virulence and to H. pylori's great genetic diversity. PMID:12435699

  11. Analysis of UV-induced mutation spectra in Escherichia coli by DNA polymerase {eta} from Arabidopsis thaliana

    Energy Technology Data Exchange (ETDEWEB)

    Santiago, Maria Jesus [Departamento de Genetica, Facultad de Ciencias, Edificio Gregor Mendel, Campus Rabanales, Universidad de Cordoba (Spain); Alejandre-Duran, Encarna [Departamento de Genetica, Facultad de Ciencias, Edificio Gregor Mendel, Campus Rabanales, Universidad de Cordoba (Spain); Ruiz-Rubio, Manuel [Departamento de Genetica, Facultad de Ciencias, Edificio Gregor Mendel, Campus Rabanales, Universidad de Cordoba (Spain)]. E-mail: ge1rurum@uco.es

    2006-10-10

    DNA polymerase {eta} belongs to the Y-family of DNA polymerases, enzymes that are able to synthesize past template lesions that block replication fork progression. This polymerase accurately bypasses UV-associated cis-syn cyclobutane thymine dimers in vitro and therefore may contributes to resistance against sunlight in vivo, both ameliorating survival and decreasing the level of mutagenesis. We cloned and sequenced a cDNA from Arabidopsis thaliana which encodes a protein containing several sequence motifs characteristics of Pol{eta} homologues, including a highly conserved sequence reported to be present in the active site of the Y-family DNA polymerases. The gene, named AtPOLH, contains 14 exons and 13 introns and is expressed in different plant tissues. A strain from Saccharomyces cerevisiae, deficient in Pol{eta} activity, was transformed with a yeast expression plasmid containing the AtPOLH cDNA. The rate of survival to UV irradiation in the transformed mutant increased to similar values of the wild type yeast strain, showing that AtPOLH encodes a functional protein. In addition, when AtPOLH is expressed in Escherichia coli, a change in the mutational spectra is detected when bacteria are irradiated with UV light. This observation might indicate that AtPOLH could compete with DNA polymerase V and then bypass cyclobutane pyrimidine dimers incorporating two adenylates.

  12. scid mutation in mice confers hypersensitivity to ionizing radiation and a deficiency in DNA double-strand break repair

    International Nuclear Information System (INIS)

    Biedermann, K.A.; Sun, J.R.; Giaccia, A.J.; Tosto, L.M.; Brown, J.M.

    1991-01-01

    C.B-17 severe combined immunodeficient (scid) mice carry the scid mutation and are severely deficient in both T cell- and B cell-mediated immunity, apparently as a result of defective V(D)J joining of the immunoglobulin and T-cell receptor gene elements. In the present studies, we have defined the tissue, cellular, and molecular basis of another characteristic of these mice: their hypersensitivity to ionizing radiation. Bone marrow stem cells, intestinal crypt cells, and epithelial skin cells from scid mice are 2- to 3-fold more sensitive when irradiated in situ than are congenic BALB/c or C.B-17 controls. Two independently isolated embryo fibroblastic scid mouse cell lines display similar hypersensitivities to gamma-rays. In addition, these cell lines are sensitive to cell killing by bleomycin, which also produces DNA strand breaks, but not by the DNA crosslinking agent mitomycin C or UV irradiation. Measurement of the rejoining of gamma-ray-induced DNA double-strand breaks by pulsed-field gel electrophoresis indicates that these animals are defective in this repair system. This suggests that the gamma-ray sensitivity of the scid mouse fibroblasts could be the result of reduced repair of DNA double-strand breaks. Therefore, a common factor may participate in both the repair of DNA double-strand breaks as well as V(D)J rejoining during lymphocyte development. This murine autosomal recessive mutation should prove extremely useful in fundamental studies of radiation-induced DNA damage and repair

  13. Improvement of some ornamental plants by induced somatic mutations at National Botanical Research Institute

    International Nuclear Information System (INIS)

    Gupta, M.N.

    1980-01-01

    Research work on improvement of some ornamental plants by induced somatic mutations has been in progress at the National Botanical Research Institute, Lucknow, since 1964. The methods of treatments with gamma rays, detection, isolation and multiplication of induced somatic mutations have been given for Bougainvillea, Chrysanthemum, perennial Portulaca, rose and tuberose. During the last 15 years, a total of 38 new cultivars of different ornamentals evolved by gamna induced somatic mutations have been released. They include Bougainvillea 1; Chrysanthemum 28; perennial portulaca 6; rose 1 and tuberose 2. Descriptions of the original cultivars and their gamma induced mutants are given along with other pertinent details. (author)

  14. Prevalence of Germline Mutations in Genes Engaged in DNA Damage Repair by Homologous Recombination in Patients with Triple-Negative and Hereditary Non-Triple-Negative Breast Cancers.

    Directory of Open Access Journals (Sweden)

    Pawel Domagala

    Full Text Available This study sought to assess the prevalence of common germline mutations in several genes engaged in the repair of DNA double-strand break by homologous recombination in patients with triple-negative breast cancers and hereditary non-triple-negative breast cancers. Tumors deficient in this type of DNA damage repair are known to be especially sensitive to DNA cross-linking agents (e.g., platinum drugs and to poly(ADP-ribose polymerase (PARP inhibitors.Genetic testing was performed for 36 common germline mutations in genes engaged in the repair of DNA by homologous recombination, i.e., BRCA1, BRCA2, CHEK2, NBN, ATM, PALB2, BARD1, and RAD51D, in 202 consecutive patients with triple-negative breast cancers and hereditary non-triple-negative breast cancers.Thirty five (22.2% of 158 patients in the triple-negative group carried mutations in genes involved in DNA repair by homologous recombination, while 10 (22.7% of the 44 patients in the hereditary non-triple-negative group carried such mutations. Mutations in BRCA1 were most frequent in patients with triple-negative breast cancer (18.4%, and mutations in CHEK2 were most frequent in patients with hereditary non-triple-negative breast cancers (15.9%. In addition, in the triple-negative group, mutations in CHEK2, NBN, and ATM (3.8% combined were found, while mutations in BRCA1, NBN, and PALB2 (6.8% combined were identified in the hereditary non-triple-negative group.Identifying mutations in genes engaged in DNA damage repair by homologous recombination other than BRCA1/2 can substantially increase the proportion of patients with triple-negative breast cancer and hereditary non-triple-negative breast cancer who may be eligible for therapy using PARP inhibitors and platinum drugs.

  15. Rats with a missense mutation in Atm display neuroinflammation and neurodegeneration subsequent to accumulation of cytosolic DNA following unrepaired DNA damage.

    Science.gov (United States)

    Quek, Hazel; Luff, John; Cheung, KaGeen; Kozlov, Sergei; Gatei, Magtouf; Lee, C Soon; Bellingham, Mark C; Noakes, Peter G; Lim, Yi Chieh; Barnett, Nigel L; Dingwall, Steven; Wolvetang, Ernst; Mashimo, Tomoji; Roberts, Tara L; Lavin, Martin F

    2017-04-01

    Mutations in the ataxia-telangiectasia (A-T)-mutated ( ATM ) gene give rise to the human genetic disorder A-T, characterized by immunodeficiency, cancer predisposition, and neurodegeneration. Whereas a series of animal models recapitulate much of the A-T phenotype, they fail to present with ataxia or neurodegeneration. We describe here the generation of an Atm missense mutant [amino acid change of leucine (L) to proline (P) at position 2262 (L2262P)] rat by intracytoplasmic injection (ICSI) of mutant sperm into oocytes. Atm -mutant rats ( Atm L2262P/L2262P ) expressed low levels of ATM protein, suggesting a destabilizing effect of the mutation, and had a significantly reduced lifespan compared with Atm +/+ Whereas these rats did not show cerebellar atrophy, they succumbed to hind-limb paralysis (45%), and the remainder developed tumors. Closer examination revealed the presence of both dsDNA and ssDNA in the cytoplasm of cells in the hippocampus, cerebellum, and spinal cord of Atm L2262P/L2262P rats. Significantly increased levels of IFN-β and IL-1β in all 3 tissues were indicative of DNA damage induction of the type 1 IFN response. This was further supported by NF-κB activation, as evidenced by p65 phosphorylation (P65) and translocation to the nucleus in the spinal cord and parahippocampus. Other evidence of neuroinflammation in the brain and spinal cord was the loss of motor neurons and the presence of increased activation of microglia. These data provide support for a proinflammatory phenotype that is manifested in the Atm mutant rat as hind-limb paralysis. This mutant represents a useful model to investigate the importance of neuroinflammation in A-T. © Society for Leukocyte Biology.

  16. Development and applications of Bacillus subtilis test systems for mutagens, involving DNA-repair deficiency and suppressible auxotrophic mutations

    International Nuclear Information System (INIS)

    Tanooka, H.

    1977-01-01

    A mutagen-tester of Bacillus subtilis was constructed and tested with known carcinogens. The parental strain HA101 of Okubo and Yanagida carrying suppressible nonsense mutations in his and met genes was transformed to carry an excision-repair deficiency mutation. The constructed strain TKJ5211 showed a 20-30-fold higher sensitivity for His + reversion than the parental strain when treated with UV and UV-mimetic chemicals but unchanged mutation frequency with X-rays and methyl methanesulfonate. The tester strain was used in a spot test of 30 selected chemicals and also for testing with liver homogenate activation. The results showed an almost equivalent but somewhat broader detection spectrum than the Salmonella typhimurium TA100 system. Another test method used a pair of B. subtilis strains differing in their DNA-repair capacity, i.e. the most UV-sensitive mutant HJ-15 and a wild-type strain, to detect repair-dependent DNA damage produced by chemicals. Spores could be used in either test

  17. Association between mutation spectra and stable and unstable DNA adduct profiles in Salmonella for benzo[a]pyrene and dibenzo[a,l]pyrene

    Energy Technology Data Exchange (ETDEWEB)

    DeMarini, David M., E-mail: demarini.david@epa.gov [Integrated Systems Toxicology Division, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711 (United States); Hanley, Nancy M.; Warren, Sarah H.; Adams, Linda D.; King, Leon C. [Integrated Systems Toxicology Division, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711 (United States)

    2011-09-01

    Highlights: {yields} Benzo[a]pyrene (BP) induces stable DNA adducts and mutations primarily at guanine. {yields} Dibenzo[a,l]pyrene (DBP) induces them primarily at adenine. {yields} BP induces abasic sites, but DBP does not in the Salmonella mutagenicity assay. {yields} Stable DNA adducts alone appear to account for the mutation spectrum of DBP. {yields} Stable DNA adducts and possibly abasic sites account for the mutation spectrum of BP. - Abstract: Benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP) are two polycyclic aromatic hydrocarbons (PAHs) that exhibit distinctly different mutagenicity and carcinogenicity profiles. Although some studies show that these PAHs produce unstable DNA adducts, conflicting data and arguments have been presented regarding the relative roles of these unstable adducts versus stable adducts, as well as oxidative damage, in the mutagenesis and tumor-mutation spectra of these PAHs. However, no study has determined the mutation spectra along with the stable and unstable DNA adducts in the same system with both PAHs. Thus, we determined the mutagenic potencies and mutation spectra of BP and DBP in strains TA98, TA100 and TA104 of Salmonella, and we also measured the levels of abasic sites (aldehydic-site assay) and characterized the stable DNA adducts ({sup 32}P-postlabeling/HPLC) induced by these PAHs in TA104. Our results for the mutation spectra and site specificity of stable adducts were consistent with those from other systems, showing that DBP was more mutagenic than BP in TA98 and TA100. The mutation spectra of DBP and BP were significantly different in TA98 and TA104, with 24% of the mutations induced by BP in TA98 being complex frameshifts, whereas DBP produced hardly any of these mutations. In TA104, BP produced primarily GC to TA transversions, whereas DBP produced primarily AT to TA transversions. The majority (96%) of stable adducts induced by BP were at guanine, whereas the majority (80%) induced by DBP were at adenine

  18. Research progress of IDH1 and IDH2 mutations in gliomas

    Directory of Open Access Journals (Sweden)

    Shan-shan ZHANG

    2015-11-01

    Full Text Available The gene mutations of isocitrate dehydrogenase 1 and 2 (IDH1/2 mainly occur in astrocytoma, anaplastic astrocytoma, oligodendroglioma, anaplastic oligodendroglioma, oligoastrocytoma, anaplastic oligoastrocytoma and secondary glioblastoma. The IDH1/2 gene mutation can alter proteinase function, consume α-ketoglutarate and nicotinamide adenine dinucleotide phosphate-reduced (NADPH and thus produce carcinogenic metabolite, 2-hydroxyglutarate. The intracellular accumulation of 2-hydroxyglutarate will induce a series of downstream effects which may result in the development of gliomas mentioned above. Both IDH1/2 mutations and other concomitant hereditary variations are biomarkers for differential diagnosis and IDH1/2 mutations are also independent factors for the prognosis of gliomas. The molecular targeting therapy for IDH1/2 mutations has become the research focus of glioma treatment. This review summarizes the recent progress of this field. DOI: 10.3969/j.issn.1672-6731.2015.11.017

  19. A combinatorial role for MutY and Fpg DNA glycosylases in mutation avoidance in Mycobacterium smegmatis.

    Science.gov (United States)

    Hassim, Farzanah; Papadopoulos, Andrea O; Kana, Bavesh D; Gordhan, Bhavna G

    2015-09-01

    Hydroxyl radical (OH) among reactive oxygen species cause damage to nucleobases with thymine being the most susceptible, whilst in contrast, the singlet oxygen ((1)02) targets only guanine bases. The high GC content of mycobacterial genomes predisposes these organisms to oxidative damage of guanine. The exposure of cellular DNA to OH and one-electron oxidants results in the formation of two main degradation products, the pro-mutagenic 8-oxo-7,8-dihydroguanine (8-oxoGua) and the cytotoxic 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua). These lesions are repaired through the base excision repair (BER) pathway and we previously, demonstrated a combinatorial role for the mycobacterial Endonuclease III (Nth) and the Nei family of DNA glycosylases in mutagenesis. In addition, the formamidopyrimidine (Fpg/MutM) and MutY DNA glycosylases have also been implicated in mutation avoidance and BER in mycobacteria. In this study, we further investigate the combined role of MutY and the Fpg/Nei DNA glycosylases in Mycobacterium smegmatis and demonstrate that deletion of mutY resulted in enhanced sensitivity to oxidative stress, an effect which was not exacerbated in Δfpg1 Δfpg2 or Δnei1 Δnei2 double mutant backgrounds. However, combinatorial loss of the mutY, fpg1 and fpg2 genes resulted in a significant increase in mutation rates suggesting interplay between these enzymes. Consistent with this, there was a significant increase in C → A mutations with a corresponding change in cell morphology of rifampicin resistant mutants in the Δfpg1 Δfpg2 ΔmutY deletion mutant. In contrast, deletion of mutY together with the nei homologues did not result in any growth/survival defects or changes in mutation rates. Taken together these data indicate that the mycobacterial mutY, in combination with the Fpg DNA N-glycosylases, plays an important role in controlling mutagenesis under oxidative stress. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. A novel class of mutations that affect DNA replication in E. coli

    DEFF Research Database (Denmark)

    Nordman, Jared; Skovgaard, Ole; Wright, Andrew

    2007-01-01

    Over-initiation of DNA replication in cells containing the cold-sensitive dnaA(cos) allele has been shown to lead to extensive DNA damage, potentially due to head-to-tail replication fork collisions that ultimately lead to replication fork collapse, growth stasis and/or cell death. Based on the a...

  1. DNA repair and its relation to recombination-deficient and other mutations in Bacillus subtilis

    International Nuclear Information System (INIS)

    Ganesan, A.T.

    1975-01-01

    DNA repair processes operating in Bacillus subtilis are similar to other transformable bacterial systems. Radiation-sensitive, recombination-deficient mutants are blocked in distinct steps leading to recombination. DNA polymerase I is essential for the repair of x-ray-induced damage to DNA but not for recombination

  2. Specific mutations in the C-terminus domain of HBV surface antigen significantly correlate with low level of serum HBV-DNA in patients with chronic HBV infection

    NARCIS (Netherlands)

    Mirabelli, Carmen; Surdo, Matteo; van Hemert, Formijn; Lian, Zhichao; Salpini, Romina; Cento, Valeria; Cortese, Maria Francesca; Aragri, Marianna; Pollicita, Michela; Alteri, Claudia; Bertoli, Ada; Berkhout, Ben; Micheli, Valeria; Gubertini, Guido; Santoro, Maria Mercedes; Romano, Sara; Visca, Michela; Bernassola, Martina; Longo, Roberta; de Sanctis, Giuseppe Maria; Trimoulet, Pascal; Fleury, Hervè; Marino, Nicoletta; Mazzotta, Francesco; Cappiello, Giuseppina; Spanò, Alberto; Sarrecchia, Cesare; Zhang, Jing Maria; Andreoni, Massimo; Angelico, Mario; Verheyen, Jens; Perno, Carlo Federico; Svicher, Valentina

    2015-01-01

    Background: To define HBsAg-mutations correlated with different serum HBV-DNA levels in HBV chronically-infected drug-naive patients. Methods: This study included 187 patients stratified into the following ranges of serum HBV-DNA: 12-2000 IU/ml, 2000-100,000 IU/ml, and > 100,000 IU/ml.

  3. Enzymic colorimetry-based DNA chip: a rapid and accurate assay for detecting mutations for clarithromycin resistance in the 23S rRNA gene of Helicobacter pylori.

    Science.gov (United States)

    Xuan, Shi-Hai; Zhou, Yu-Gui; Shao, Bo; Cui, Ya-Lin; Li, Jian; Yin, Hong-Bo; Song, Xiao-Ping; Cong, Hui; Jing, Feng-Xiang; Jin, Qing-Hui; Wang, Hui-Min; Zhou, Jie

    2009-11-01

    Macrolide drugs, such as clarithromycin (CAM), are a key component of many combination therapies used to eradicate Helicobacter pylori. However, resistance to CAM is increasing in H. pylori and is becoming a serious problem in H. pylori eradication therapy. CAM resistance in H. pylori is mostly due to point mutations (A2142G/C, A2143G) in the peptidyltransferase-encoding region of the 23S rRNA gene. In this study an enzymic colorimetry-based DNA chip was developed to analyse single-nucleotide polymorphisms of the 23S rRNA gene to determine the prevalence of mutations in CAM-related resistance in H. pylori-positive patients. The results of the colorimetric DNA chip were confirmed by direct DNA sequencing. In 63 samples, the incidence of the A2143G mutation was 17.46 % (11/63). The results of the colorimetric DNA chip were concordant with DNA sequencing in 96.83 % of results (61/63). The colorimetric DNA chip could detect wild-type and mutant signals at every site, even at a DNA concentration of 1.53 x 10(2) copies microl(-1). Thus, the colorimetric DNA chip is a reliable assay for rapid and accurate detection of mutations in the 23S rRNA gene of H. pylori that lead to CAM-related resistance, directly from gastric tissues.

  4. CRISPR/Cas9 DNA cleavage at SNP-derived PAM enables both in vitro and in vivo KRT12 mutation-specific targeting.

    Science.gov (United States)

    Courtney, D G; Moore, J E; Atkinson, S D; Maurizi, E; Allen, E H A; Pedrioli, D M L; McLean, W H I; Nesbit, M A; Moore, C B T

    2016-01-01

    CRISPR/Cas9-based therapeutics hold the possibility for permanent treatment of genetic disease. The potency and specificity of this system has been used to target dominantly inherited conditions caused by heterozygous missense mutations through inclusion of the mutated base in the short-guide RNA (sgRNA) sequence. This research evaluates a novel approach for targeting heterozygous single-nucleotide polymorphisms (SNPs) using CRISPR/Cas9. We determined that a mutation within KRT12, which causes Meesmann's epithelial corneal dystrophy (MECD), leads to the occurrence of a novel protospacer adjacent motif (PAM). We designed an sgRNA complementary to the sequence adjacent to this SNP-derived PAM and evaluated its potency and allele specificity both in vitro and in vivo. This sgRNA was found to be highly effective at reducing the expression of mutant KRT12 mRNA and protein in vitro. To assess its activity in vivo we injected a combined Cas9/sgRNA expression construct into the corneal stroma of a humanized MECD mouse model. Sequence analysis of corneal genomic DNA revealed non-homologous end-joining repair resulting in frame-shifting deletions within the mutant KRT12 allele. This study is the first to demonstrate in vivo gene editing of a heterozygous disease-causing SNP that results in a novel PAM, further highlighting the potential for CRISPR/Cas9-based therapeutics.

  5. Role of specific DNA mutations in the peripheral blood of colorectal cancer patients for the assessment of tumor stage and residual disease following tumor resection

    Science.gov (United States)

    Norcic, Gregor; Jelenc, Franc; Cerkovnik, Petra; Stegel, Vida; Novakovic, Srdjan

    2016-01-01

    In the present study, the detection of tumor-specific KRAS proto-oncogene, GTPase (KRAS) and B-Raf proto-oncogene, serine/threonine kinase (BRAF) mutations in the peripheral blood of colorectal cancer (CRC) patients at all stages and adenomas was used for the estimation of disease stage prior to surgery and for residual disease following surgery. A total of 65 CRC patients were enrolled. The primary tumor tested positive for the specific mutations (KRAS mutations in codons 12, 13, 61, 117 or 146 and BRAF mutations in codon 600) in 35 patients. In all these patients, the specimen of normal bowel resected with the tumor was also tested for the presence of the same mutations in order to exclude the germ-line mutations. Only patients who tested positive for the specific mutation in the primary tumor were included in further analysis for the presence of tumor-specific mutation in the peripheral blood. No statistically significant differences were found between the detection rates of tumor mutations in the blood and different tumor stages (P=0.491). However, statistically significant differences in the proportions of patients with detected tumor-specific DNA mutations in the peripheral blood were found when comparing the groups of patients with R0 and R2 resections (P=0.038). Tumor-specific DNA mutations in the peripheral blood were more frequently detected in the patients with an incomplete surgical clearance of the tumor due to macroscopic residual disease (R2 resections). Therefore, the study concludes that the follow-up of somatic KRAS- and BRAF-mutated DNA in the peripheral blood of CRC patients may be useful in assessing the surgical clearance of the disease. PMID:27900004

  6. DNA sequence analysis of the mutational specificity of u.v. light in the SUP4-o gene of yeast

    International Nuclear Information System (INIS)

    Kunz, B.A.; Mis, J.R.A.; Pierce, M.K.; Giroux, C.N.

    1987-01-01

    Mutations induced in the SUP4-o gene of Saccharomyces cerevisiae by u.v. irradiation have been characterized. DNA sequence analysis of 120 mutants revealed that u.v. induced all types of base substitutions, although transitions, in particular G:C → A:T events predominated. In addition, a small number of single base pair deletions and double mutations, occurring in tandem or separated by a few base pairs, were recovered. The base pair substitutions were not distributed randomly in the SUP4-o gene and, with one exception, were all located at sites of adjacent pyrimidines, suggesting they were targeted by u.v. photolesions. A substantial fraction of the mutations were detected at hotspots for u.v. mutagenesis. The majority of changes occurred at the 3' base of dipyrimidine sequences where both cyclobutane dimers and [6-4]-photoproducts could form. Approximately one-third of the induced base substitutions were found at potential pyrimidine dimer sites where [6-4]-photoproducts would be expected to occur rarely. Possible origins of the induced mutations and the role of cyclobutane dimers as premutational u.v. lesions in yeast are considered. (author)

  7. Structure-guided Mutational Analysis of the Nucleotidyltransferase Domain of Escherichia coli DNA Ligase (LigA).

    Science.gov (United States)

    Wang, Li Kai; Zhu, Hui; Shuman, Stewart

    2009-03-27

    NAD(+)-dependent DNA ligases (LigA) are ubiquitous in bacteria, where they are essential for growth and present attractive targets for antimicrobial drug discovery. LigA has a distinctive modular structure in which a nucleotidyltransferase catalytic domain is flanked by an upstream NMN-binding module and by downstream OB-fold, zinc finger, helix-hairpin-helix, and BRCT domains. Here we conducted a structure-function analysis of the nucleotidyltransferase domain of Escherichia coli LigA, guided by the crystal structure of the LigA-DNA-adenylate intermediate. We tested the effects of 29 alanine and conservative mutations at 15 amino acids on ligase activity in vitro and in vivo. We thereby identified essential functional groups that coordinate the reactive phosphates (Arg(136)), contact the AMP adenine (Lys(290)), engage the phosphodiester backbone flanking the nick (Arg(218), Arg(308), Arg(97) plus Arg(101)), or stabilize the active domain fold (Arg(171)). Finer analysis of the mutational effects revealed step-specific functions for Arg(136), which is essential for the reaction of LigA with NAD(+) to form the covalent ligase-AMP intermediate (step 1) and for the transfer of AMP to the nick 5'-PO(4) to form the DNA-adenylate intermediate (step 2) but is dispensable for phosphodiester formation at a preadenylylated nick (step 3).

  8. Sequence-specific DNA alkylation targeting for Kras codon 13 mutation by pyrrole-imidazole polyamide seco-CBI conjugates.

    Science.gov (United States)

    Taylor, Rhys Dylan; Asamitsu, Sefan; Takenaka, Tomohiro; Yamamoto, Makoto; Hashiya, Kaori; Kawamoto, Yusuke; Bando, Toshikazu; Nagase, Hiroki; Sugiyama, Hiroshi

    2014-01-27

    Hairpin N-methylpyrrole-N-methylimidazole polyamide seco-CBI conjugates 2-6 were designed for synthesis by Fmoc solid-phase synthesis, and their DNA-alkylating activities against the Kras codon 13 mutation were compared by high-resolution denaturing gel electrophoresis with 225 base pair (bp) DNA fragments. Conjugate 5 had high reactivity towards the Kras codon 13 mutation site, with alkylation occurring at the A of the sequence 5'-ACGTCACCA-3' (site 2), including minor 1 bp-mismatch alkylation against wild type 5'-ACGCCACCA-3' (site 3). Conjugate 6, which differs from conjugate 5 by exchanging one Py unit with a β unit, showed high selectivity but only weakly alkylated the A of 5'-ACGTCACCA-3' (site 2). The hairpin polyamide seco-CBI conjugate 5 thus alkylates according to Dervan's pairing rule with the pairing recognition which β/β pair targets T-A and A-T pairs. SPR and a computer-minimized model suggest that 5 binds to the target sequence with high affinity in a hairpin conformation, allowing for efficient DNA alkylation. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Mutations on the DNA binding surface of TBP discriminate between yeast TATA and TATA-less gene transcription.

    Science.gov (United States)

    Kamenova, Ivanka; Warfield, Linda; Hahn, Steven

    2014-08-01

    Most RNA polymerase (Pol) II promoters lack a TATA element, yet nearly all Pol II transcription requires TATA binding protein (TBP). While the TBP-TATA interaction is critical for transcription at TATA-containing promoters, it has been unclear whether TBP sequence-specific DNA contacts are required for transcription at TATA-less genes. Transcription factor IID (TFIID), the TBP-containing coactivator that functions at most TATA-less genes, recognizes short sequence-specific promoter elements in metazoans, but analogous promoter elements have not been identified in Saccharomyces cerevisiae. We generated a set of mutations in the yeast TBP DNA binding surface and found that most support growth of yeast. Both in vivo and in vitro, many of these mutations are specifically defective for transcription of two TATA-containing genes with only minor defects in transcription of two TATA-less, TFIID-dependent genes. TBP binds several TATA-less promoters with apparent high affinity, but our results suggest that this binding is not important for transcription activity. Our results are consistent with the model that sequence-specific TBP-DNA contacts are not important at yeast TATA-less genes and suggest that other general transcription factors or coactivator subunits are responsible for recognition of TATA-less promoters. Our results also explain why yeast TBP derivatives defective for TATA binding appear defective in activated transcription. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  10. Impact of Somatic Mutations in the D-Loop of Mitochondrial DNA on the Survival of Oral Squamous Cell Carcinoma Patients

    Science.gov (United States)

    Lin, Jin-Ching; Wang, Chen-Chi; Jiang, Rong-San; Wang, Wen-Yi; Liu, Shih-An

    2015-01-01

    Objectives The aim of this study was to investigate somatic mutations in the D-loop of mitochondrial DNA (mtDNA) and their impact on survival in oral squamous cell carcinoma patients. Materials and Methods Surgical specimen confirmed by pathological examination and corresponding non-cancerous tissues were collected from 120 oral squamous cell carcinoma patients. The sequence in the D-loop of mtDNA from non-cancerous tissues was compared with that from paired cancer samples and any sequence differences were recognized as somatic mutations. Results Somatic mutations in the D-loop of mtDNA were identified in 75 (62.5%) oral squamous cell carcinoma patients and most of them occurred in the poly-C tract. Although there were no significant differences in demographic and tumor-related features between participants with and without somatic mutation, the mutation group had a better survival rate (5 year disease-specific survival rate: 64.0% vs. 43.0%, P = 0.0266). Conclusion Somatic mutation in D-loop of mtDNA was associated with a better survival in oral squamous cell carcinoma patients. PMID:25906372

  11. Mutations in DNA repair genes are associated with the Haarlem lineage of Mycobacterium tuberculosis independently of their antibiotic resistance.

    Science.gov (United States)

    Olano, Juanita; López, Beatriz; Reyes, Alejandro; Lemos, María del Pilar; Correa, Nidia; Del Portillo, Patricia; Barrera, Lucia; Robledo, Jaime; Ritacco, Viviana; Zambrano, María Mercedes

    2007-11-01

    The analysis of the DNA repair genes ogt and ung was carried out in 117 Mycobacterium tuberculosis clinical isolates from Argentina and Colombia in order to explore correlation between mutations in these genes and multi-drug resistance. With the exception of two Beijing family isolates, the rest of the strains harbored either two wild-type or two mutant alleles with identical single nucleotide polymorphisms (SNPs) in each gene (ogt44 and ung501). These ogt44 and ung501 mutations were not associated with multi-drug resistance and occurred simultaneously in circulating Haarlem genotype M. tuberculosis strains. We therefore propose the use of these markers as tools in phylogenetic and epidemiologic studies.

  12. Structural and mutational analysis of Escherichia coli AlkB provides insight into substrate specificity and DNA damage searching.

    Directory of Open Access Journals (Sweden)

    Paul J Holland

    Full Text Available BACKGROUND: In Escherichia coli, cytotoxic DNA methyl lesions on the N1 position of purines and N3 position of pyrimidines are primarily repaired by the 2-oxoglutarate (2-OG iron(II dependent dioxygenase, AlkB. AlkB repairs 1-methyladenine (1-meA and 3-methylcytosine (3-meC lesions, but it also repairs 1-methylguanine (1-meG and 3-methylthymine (3-meT at a much less efficient rate. How the AlkB enzyme is able to locate and identify methylated bases in ssDNA has remained an open question. METHODOLOGY/PRINCIPAL FINDINGS: We determined the crystal structures of the E. coli AlkB protein holoenzyme and the AlkB-ssDNA complex containing a 1-meG lesion. We coupled this to site-directed mutagenesis of amino acids in and around the active site, and tested the effects of these mutations on the ability of the protein to bind both damaged and undamaged DNA, as well as catalyze repair of a methylated substrate. CONCLUSIONS/SIGNIFICANCE: A comparison of our substrate-bound AlkB-ssDNA complex with our unliganded holoenzyme reveals conformational changes of residues within the active site that are important for binding damaged bases. Site-directed mutagenesis of these residues reveals novel insight into their roles in DNA damage recognition and repair. Our data support a model that the AlkB protein utilizes at least two distinct conformations in searching and binding methylated bases within DNA: a "searching" mode and "repair" mode. Moreover, we are able to functionally separate these modes through mutagenesis of residues that affect one or the other binding state. Finally, our mutagenesis experiments show that amino acid D135 of AlkB participates in both substrate specificity and catalysis.

  13. Mutational analysis of an archaeal minichromosome maintenance protein exterior hairpin reveals critical residues for helicase activity and DNA binding

    Directory of Open Access Journals (Sweden)

    Brewster Aaron S

    2010-08-01

    Full Text Available Abstract Background The mini-chromosome maintenance protein (MCM complex is an essential replicative helicase for DNA replication in Archaea and Eukaryotes. While the eukaryotic complex consists of six homologous proteins (MCM2-7, the archaeon Sulfolobus solfataricus has only one MCM protein (ssoMCM, six subunits of which form a homohexamer. We have recently reported a 4.35Å crystal structure of the near full-length ssoMCM. The structure reveals a total of four β-hairpins per subunit, three of which are located within the main channel or side channels of the ssoMCM hexamer model generated based on the symmetry of the N-terminal Methanothermobacter thermautotrophicus (mtMCM structure. The fourth β-hairpin, however, is located on the exterior of the hexamer, near the exit of the putative side channels and next to the ATP binding pocket. Results In order to better understand this hairpin's role in DNA binding and helicase activity, we performed a detailed mutational and biochemical analysis of nine residues on this exterior β-hairpin (EXT-hp. We examined the activities of the mutants related to their helicase function, including hexamerization, ATPase, DNA binding and helicase activities. The assays showed that some of the residues on this EXT-hp play a role for DNA binding as well as for helicase activity. Conclusions These results implicate several current theories regarding helicase activity by this critical hexameric enzyme. As the data suggest that EXT-hp is involved in DNA binding, the results reported here imply that the EXT-hp located near the exterior exit of the side channels may play a role in contacting DNA substrate in a manner that affects DNA unwinding.

  14. MELAS and Kearns–Sayre overlap syndrome due to the mtDNA m. A3243G mutation and large-scale mtDNA deletions

    Directory of Open Access Journals (Sweden)

    Nian Yu

    2016-09-01

    Full Text Available This paper reported an unusual manifestation of a 19-year-old Chinese male patient presented with a complex phenotype of mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS syndrome and Kearns–Sayre syndrome (KSS. He was admitted to our hospital with the chief complaint of “acute fever, headache and slow reaction for 21 days”. He was initially misdiagnosed as “viral encephalitis”. This Chinese man with significant past medical history of intolerating fatigue presented paroxysmal neurobehavioral attacks that started about 10 years ago. During this span, 3 or 4 attack clusters were described during which several attacks occurred over a few days. The further examination found that the hallmark signs of this patient included progressive myoclonus epilepsy, cerebellar ataxia, hearing loss, myopathic weakness, ophthalmoparesis, pigmentary retinopathy and bifascicular heart block (Wolff–Parkinson–White syndrome. By young age the disease progression is characterized by the addition of migraine, vomiting, and stroke-like episodes, symptoms of MELAS expression, which indicated completion of the MELAS/KSS overlap syndrome. The m. A3243G mitochondrial DNA mutation and single large-scale mtDNA deletions were found in this patient. This mutation has been reported with MELAS, KSS, myopathy, deafness and mental disorder with cognitive impairment. This is the first description with a MELAS/KSS syndrome in Chinese.

  15. Novel primer specific false terminations during DNA sequencing reactions: danger of inaccuracy of mutation analysis in molecular diagnostics

    Science.gov (United States)

    Anwar, R; Booth, A; Churchill, A J; Markham, A F

    1996-01-01

    The determination of nucleotide sequence is fundamental to the identification and molecular analysis of genes. Direct sequencing of PCR products is now becoming a commonplace procedure for haplotype analysis, and for defining mutations and polymorphism within genes, particularly for diagnostic purposes. A previously unrecognised phenomenon, primer related variability, observed in sequence data generated using Taq cycle sequencing and T7 Sequenase sequencing, is reported. This suggests that caution is necessary when interpreting DNA sequence data. This is particularly important in situations where treatment may be dependent on the accuracy of the molecular diagnosis. Images PMID:16696096

  16. Transfection with extracellularly UV-damaged DNA induces human and rat cells to express a mutator phenotype towards parvovirus H-1

    International Nuclear Information System (INIS)

    Dinsart, C.; Cornelis, J.J.; Klein, B.; van der Eb, A.J.; Rommelaere, J.

    1984-01-01

    Human and rat cells transfected with UV-irradiated linear double-stranded DNA from calf thymus displayed a mutator activity. This phenotype was identified by growing a lytic thermosensitive single-stranded DNA virus (parvovirus H-1) in those cells and determining viral reversion frequencies. Likewise, exogenous UV-irradiated closed circular DNAs, either double-stranded (simian virus 40) or single-stranded (phi X174), enhanced the ability of recipient cells to mutate parvovirus H-1. The magnitude of mutator activity expression increased along with the number of UV lesions present in the inoculated DNA up to a saturation level. Unirradiated DNA displayed little inducing capacity, irrespective of whether it was single or double stranded. Deprivation of a functional replication origin did not impede UV-irradiated simian virus 40 DNA from providing rat and human cells with a mutator function. Our data suggest that in mammalian cells a trans-acting mutagenic signal might be generated from UV-irradiated DNA without the necessity for damaged DNA to replicate

  17. DNA Variations in Oculocutaneous Albinism: An Updated Mutation List and Current Outstanding Issues in Molecular Diagnostics

    Science.gov (United States)

    Simeonov, Dimitre R.; Wang, Xinjing; Wang, Chen; Sergeev, Yuri; Dolinska, Monika; Bower, Matthew; Fischer, Roxanne; Winer, David; Dubrovsky, Genia; Balog, Joan Z.; Huizing, Marjan; Hart, Rachel; Zein, Wadih M.; Gahl, William A.; Brooks, Brian P.; Adams, David R.

    2014-01-01

    Oculocutaneous albinism (OCA) is a rare genetic disorder of melanin synthesis that results in hypopigmented hair, skin, and eyes. There are four types of OCA, caused by mutations in TYR (OCA-1), OCA2 (OCA-2), TYRP1 (OCA-3), or SLC45A2 (OCA-4). Here we report 22 novel mutations; 14 from a cohort of 61 patients seen as part of the NIH OCA Natural History Study and 8 from a prior study at the University of Minnesota. We also include a comprehensive list of almost 600 previously reported OCA mutations, along with ethnicity information, carrier frequencies, and in silico pathogenicity predictions. In addition to discussing the clinical and molecular features of OCA, we address the cases of apparent missing heritability. In our cohort, 25% of patients did not have two mutations in a single OCA gene. We demonstrate the utility of multiple detection methods to reveal mutations missed by Sanger sequencing. Finally, we review the TYR p.R402Q temperature sensitive variant and confirm its association with cases of albinism with only one identifiable TYR mutation. PMID:23504663

  18. A Comparative Study for Detection of EGFR Mutations in Plasma Cell-Free DNA in Korean Clinical Diagnostic Laboratories

    Directory of Open Access Journals (Sweden)

    Yoonjung Kim

    2018-01-01

    Full Text Available Liquid biopsies to genotype the epidermal growth factor receptor (EGFR for targeted therapy have been implemented in clinical decision-making in the field of lung cancer, but harmonization of detection methods is still scarce among clinical laboratories. We performed a pilot external quality assurance (EQA scheme to harmonize circulating tumor DNA testing among laboratories. For EQA, we created materials containing different levels of spiked cell-free DNA (cfDNA in normal plasma. The limit of detection (LOD of the cobas® EGFR Mutation Test v2 (Roche Molecular Systems was also evaluated. From November 2016 to June 2017, seven clinical diagnostic laboratories participated in the EQA program. The majority (98.94% of results obtained using the cobas assay and next-generation sequencing (NGS were acceptable. Quantitative results from the cobas assay were positively correlated with allele frequencies derived from digital droplet PCR measurements and showed good reproducibility among laboratories. The LOD of the cobas assay was 5~27 copies/mL for p.E746_A750del (exon 19 deletion, 35~70 copies/mL for p.L858R, 18~36 copies/mL for p.T790M, and 15~31 copies/mL for p.A767_V769dup (exon 20 insertion. Deep sequencing of materials (>100,000X depth of coverage resulted in detection of low-level targets present at frequencies of 0.06~0.13%. Our results indicate that the cobas assay is a reliable and rapid method for detecting EGFR mutations in plasma cfDNA. Careful interpretation is particularly important for p.T790M detection in the setting of relapse. Individual laboratories should optimize NGS performance to maximize clinical utility.

  19. Prime-boost therapeutic vaccination in mice with DNA/DNA or DNA/Fowlpox virus recombinants expressing the Human Papilloma Virus type 16 E6 and E7 mutated proteins fused to the coat protein of Potato virus X.

    Science.gov (United States)

    Illiano, Elena; Bissa, Massimiliano; Paolini, Francesca; Zanotto, Carlo; De Giuli Morghen, Carlo; Franconi, Rosella; Radaelli, Antonia; Venuti, Aldo

    2016-10-02

    The therapeutic antitumor potency of a prime-boost vaccination strategy was explored, based on the mutated, nontransforming forms of the E6 (E6 F47R ) and E7 (E7 GGG ) oncogenes of Human Papilloma Virus type 16 (HPV16), fused to the Potato virus X (PVX) coat protein (CP) sequence. Previous data showed that CP fusion improves the immunogenicity of tumor-associated antigens and may thus increase their efficacy. After verifying the correct expression of E6 F47R CP and E7 GGG CP inserted into DNA and Fowlpox virus recombinants by Western blotting and immunofluorescence, their combined use was evaluated for therapy in a pre-clinical mouse model of HPV16-related tumorigenicity. Immunization protocols were applied using homologous (DNA/DNA) or heterologous (DNA/Fowlpox) prime-boost vaccine regimens. The humoral immune responses were determined by ELISA, and the therapeutic efficacy evaluated by the delay in tumor appearance and reduced tumor volume after inoculation of syngeneic TC-1* tumor cells. Homologous DNA/DNA genetic vaccines were able to better delay tumor appearance and inhibit tumor growth when DNAE6 F47R CP and DNAE7 GGG CP were administered in combination. However, the heterologous DNA/Fowlpox vaccination strategy was able to delay tumor appearance in a higher number of animals when E6 F47R CP and in particular E7 GGG CP were administered alone. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Strikingly different penetrance of LHON in two Chinese families with primary mutation G11778A is independent of mtDNA haplogroup background and secondary mutation G13708A

    International Nuclear Information System (INIS)

    Wang Huawei; Jia Xiaoyun; Ji Yanli; Kong Qingpeng; Zhang Qingjiong; Yao Yonggang; Zhang Yaping

    2008-01-01

    The penetrance of Leber's hereditary optic neuropathy (LHON) in families with primary mitochondrial DNA (mtDNA) mutations is very complex. Matrilineal and nuclear genetic background, as well as environmental factors, have been reported to be involved in different affected pedigrees. Here we describe two large Chinese families that show a striking difference in the penetrance of LHON, in which 53.3% and 15.0% of members were affected (P < 0.02), respectively. Analysis of the complete mtDNA genome of the two families revealed the presence of the primary mutation G11778A and several other variants suggesting the same haplogroup status G2a. The family with higher penetrance contained a previously described secondary mutation G13708A, which presents a polymorphism in normal Chinese samples and does not affect in vivo mitochondrial oxidative metabolism as described in a previous study. Evolutionary analysis failed to indicate any putatively pathogenic mutation that cosegregated with G11778A in these two pedigrees. Our results suggest that the variable penetrance of LHON in the two Chinese families is independent of both their mtDNA haplotype background and a secondary mutation G13708A. As a result, it is likely that unknown nuclear gene involvement and/or other factors contribute to the strikingly different penetrance of LHON

  1. Strikingly different penetrance of LHON in two Chinese families with primary mutation G11778A is independent of mtDNA haplogroup background and secondary mutation G13708A

    Energy Technology Data Exchange (ETDEWEB)

    Wang Huawei [Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223 (China)]|[Laboratory for Conservation and Utilization of Bio-resource, Yunnan University, Kunming 650091 (China); Jia Xiaoyun; Ji Yanli [State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060 (China); Kong Qingpeng [State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223 (China); Zhang Qingjiong [State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060 (China)], E-mail: qingjiongzhang@yahoo.com; Yao Yonggang [Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223 (China)]|[State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223 (China)], E-mail: ygyaozh@yahoo.com; Zhang Yaping [Laboratory for Conservation and Utilization of Bio-resource, Yunnan University, Kunming 650091 (China)]|[State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223 (China)

    2008-08-25

    The penetrance of Leber's hereditary optic neuropathy (LHON) in families with primary mitochondrial DNA (mtDNA) mutations is very complex. Matrilineal and nuclear genetic background, as well as environmental factors, have been reported to be involved in different affected pedigrees. Here we describe two large Chinese families that show a striking difference in the penetrance of LHON, in which 53.3% and 15.0% of members were affected (P < 0.02), respectively. Analysis of the complete mtDNA genome of the two families revealed the presence of the primary mutation G11778A and several other variants suggesting the same haplogroup status G2a. The family with higher penetrance contained a previously described secondary mutation G13708A, which presents a polymorphism in normal Chinese samples and does not affect in vivo mitochondrial oxidative metabolism as described in a previous study. Evolutionary analysis failed to indicate any putatively pathogenic mutation that cosegregated with G11778A in these two pedigrees. Our results suggest that the variable penetrance of LHON in the two Chinese families is independent of both their mtDNA haplotype background and a secondary mutation G13708A. As a result, it is likely that unknown nuclear gene involvement and/or other factors contribute to the strikingly different penetrance of LHON.

  2. Oncogenetic tree model of somatic mutations and DNA methylation in colon tumors.

    Science.gov (United States)

    Sweeney, Carol; Boucher, Kenneth M; Samowitz, Wade S; Wolff, Roger K; Albertsen, Hans; Curtin, Karen; Caan, Bette J; Slattery, Martha L

    2009-01-01

    Our understanding of somatic alterations in colon cancer has evolved from a concept of a series of events taking place in a single sequence to a recognition of multiple pathways. An oncogenetic tree is a model intended to describe the pathways and sequence of somatic alterations in carcinogenesis without assuming that tumors will fall in mutually exclusive categories. We applied this model to data on colon tumor somatic alterations. An oncogenetic tree model was built using data on mutations of TP53, KRAS2, APC, and BRAF genes, methylation at CpG sites of MLH1 and TP16 genes, methylation in tumor (MINT) markers, and microsatellite instability (MSI) for 971 colon tumors from a population-based series. Oncogenetic tree analysis resulted in a reproducible tree with three branches. The model represents methylation of MINT markers as initiating a branch and predisposing to MSI, methylation of MHL1 and TP16, and BRAF mutation. APC mutation is the first alteration in an independent branch and is followed by TP53 mutation. KRAS2 mutation was placed a third independent branch, implying that it neither depends on, nor predisposes to, the other alterations. Individual tumors were observed to have alteration patterns representing every combination of one, two, or all three branches. The oncogenetic tree model assumptions are appropriate for the observed heterogeneity of colon tumors, and the model produces a useful visual schematic of the sequence of events in pathways of colon carcinogenesis.

  3. Genetic mutation analysis of HBV covalently closed circular DNA in peripheral blood mononuclear cells from chronic hepatitis B patients with nucleos(tide analog-resistant mutations in serum virions

    Directory of Open Access Journals (Sweden)

    Zhong-bin LI

    2012-06-01

    Full Text Available Objective  To analyze the characteristics of genetic mutations in reverse-transcriptase (RT domain of HBV covalently closed circular DNA (cccDNA in peripheral blood mononuclear cells (PBMCs obtained from chronic hepatitis B (CHB patients with drug-resistant mutations in serum virions during nucleoside/nucleotide analog (NA therapy. Methods  A total of 30 CHB patients admitted to 302 Hospital of PLA from July 2010 to August 2011 were included in this study. All the patients were confirmed to harbor the drug-resistant mutations in serum virions during an NA therapy longer than 6 months. Total DNA was extracted from PBMCs isolated from 30 whole blood samples at the same time point as that of serum analysis. Plasmid-safe ATP-dependent DNase (PSAD digestion in combination with rolling circle amplification and gap-spanning semi-nested PCR were used to amplify the RT region of HBV cccDNA. NA-resistant-associated mutations were analyzed at nine sites. Results  HBV cccDNA was efficiently amplified in 16 out of 30 (53.3% PBMC samples, and the detection rate was not correlated with HBeAg-positive rate, serum ALT level or HBV DNA load. Five of 16 (31.3% patients were sustained to have genotype B HBV infection, and 11 of 16 (68.8% were of genotype C HBV infection, and the result was consistent with the genotyping results using serum HBV. Different from drug-resistant mutations detected in the serum virions, the viruses detected in HBV cccDNA of 16 PBMC samples were all wild-type viruses without NA-resistant-associated mutations in RT region. Conclusions  During NA antiviral treatment, if drug-resistant mutations occur in serum HBV DNA of CHB patients, the dominant species of HBV cccDNA in PBMCs from the same patient is still the original wild-type strains. It is speculated that PBMCs might be the potential "repository" of HBV wild-type strain in vivo.

  4. Genome-wide DNA methylation analysis of transient neonatal diabetes type 1 patients with mutations in ZFP57.

    Science.gov (United States)

    Bak, Mads; Boonen, Susanne E; Dahl, Christina; Hahnemann, Johanne M D; Mackay, Deborah J D G; Tümer, Zeynep; Grønskov, Karen; Temple, I Karen; Guldberg, Per; Tommerup, Niels

    2016-04-14

    Transient neonatal diabetes mellitus 1 (TNDM1) is a rare imprinting disorder characterized by intrautering growth retardation and diabetes mellitus usually presenting within the first six weeks of life and resolves by the age of 18 months. However, patients have an increased risk of developing diabetes mellitus type 2 later in life. Transient neonatal diabetes mellitus 1 is caused by overexpression of the maternally imprinted genes PLAGL1 and HYMAI on chromosome 6q24. One of the mechanisms leading to overexpression of the locus is hypomethylation of the maternal allele of PLAGL1 and HYMAI. A subset of patients with maternal hypomethylation at PLAGL1 have hypomethylation at additional imprinted loci throughout the genome, including GRB10, ZIM2 (PEG3), MEST (PEG1), KCNQ1OT1 and NESPAS (GNAS-AS1). About half of the TNDM1 patients carry mutations in ZFP57, a transcription factor involved in establishment and maintenance of methylation of imprinted loci. Our objective was to investigate whether additional regions are aberrantly methylated in ZFP57 mutation carriers. Genome-wide DNA methylation analysis was performed on four individuals with homozygous or compound heterozygous ZFP57 mutations, three relatives with heterozygous ZFP57 mutations and five controls. Methylation status of selected regions showing aberrant methylation in the patients was verified using bisulfite-sequencing. We found large variability among the patients concerning the number and identity of the differentially methylated regions, but more than 60 regions were aberrantly methylated in two or more patients and a novel region within PPP1R13L was found to be hypomethylated in all the patients. The hypomethylated regions in common between the patients are enriched for the ZFP57 DNA binding motif. We have expanded the epimutational spectrum of TNDM1 associated with ZFP57 mutations and found one novel region within PPP1R13L which is hypomethylated in all TNDM1 patients included in this study. Functional

  5. Impact of HBV genotype and mutations on HBV DNA and qHBsAg levels in patients with HBeAg-negative chronic HBV infection.

    Science.gov (United States)

    Kuhnhenn, L; Jiang, B; Kubesch, A; Vermehren, J; Knop, V; Susser, S; Dietz, J; Carra, G; Finkelmeier, F; Grammatikos, G; Zeuzem, S; Sarrazin, C; Hildt, E; Peiffer, K-H

    2018-04-10

    HBV DNA and quantitative (q)HBsAg levels as prognostic markers for HBV-related disease are mostly validated in Asia and their significance in Western populations is uncertain. To analyse the impact of the HBV genotype and frequent mutations in precore (PC), basal core promoter (BCP) and preS on HBV DNA and qHBsAg levels. HBV DNA and qHBsAg serum levels of 465 patients with HBeAg-negative chronic HBV infection were correlated with the HBV genotype and mutations in PC, BCP and preS. For a detailed analysis of the molecular virology, genotype A2 genomes harbouring these mutations were analysed for replication efficacy and HBsAg release in cell culture. While no impact of the HBV genotype on HBV DNA levels was observed, qHBsAg levels differed up to 1.4 log among the genotypes (P HBV DNA levels (P HBV genome harbouring a preS deletion. In contrast, a perinuclear HBsAg accumulation was detected for the PC and BCP-variants, reflecting an impaired HBsAg release. qHBsAg serum levels depend on the HBV genotype and together with HBV DNA levels on frequent mutations in PC, BCP and preS in HBeAg-negative patients. qHBsAg cut-offs when used as prognostic markers require genotype-dependent validation. © 2018 John Wiley & Sons Ltd.

  6. Development, distribution and application of DNA markers for cereal research

    International Nuclear Information System (INIS)

    Qi, X.; Stephenson, P.; Devos, K.M.; Gale, M.D.

    2001-01-01

    DNA probes and primers are important resources for molecular genetic research and molecular breeding. Presently, more than 2500 wheat probes, 400 barley probes, 800 foxtail, pearl millet and finger millet probes, and approximately 150 wheat microsatellite (SSR) primer pairs have been developed and maintained in our DNA Resource Centre at the John Innes Centre (JIC). To accelerate probe and primer distribution, an 'anchor set' and a 'supplementary anchor set', containing 73 and 31 wheat RFLP probes, respectively, and a standard set of 42 primer pairs for wheat SSR markers were selected. Similarly, a set of 52 pearl millet probes has been selected for distribution. More than 8000 wheat RFLP probes, 2000 wheat SSR primer pairs, 700 millet probes and 200 barley probes have been distributed to more than 250 research groups in 40 countries. Our wheat and millet probes and other grass cDNA probes have been used for comparative genetic studies. The revealed conservation of gene content and gene order has been used to construct maps of many grass species and to predict the locations of key genes from one crop species to another. Developed SSR and AFLP markers in wheat, barley and millet are particularly suited for genetic diversity analyses and map construction. (author)

  7. Jeremy Rifkin challenges recombinant DNA research: A rhetoric of heresy

    Energy Technology Data Exchange (ETDEWEB)

    Futrell, W.M.

    1992-01-01

    One significant issue to come before the public in recent years is recombinant DNA research or genetic engineering and its applications. An important spokesman on this issue is Jeremy Rifkin. Rifkin is of rhetorical interest because of his strategies to sustain the dialogue and define the parameters in which it occurs. This dissertation analyzes a broad range of Rifkin's rhetorical artifacts and those of scientists engaged in recombinant DNA research. They are examined against criteria developed to identify and understand heresy. The five areas of analysis are: the nearness/remoteness phenomenon, the social construction of heresy, the social consequences of heresy, the doctrinal consequences of heresy, and the heresy-hunt ritual. The first two criteria focus on the rhetorical strategies of the heretic. The last three concentrate on the rhetorical strategies of the defenders of the institutional orthodoxy. This dissertation examines the rhetorical strategies of a heretical challenge to the scientific establishment and the consequences of that challenge. This dissertation also analyzes the rhetorical strategies employed by the defenders of the scientific orthodoxy. Although an understanding of the rhetorical strategies employed on both sides of this conflict is important, the implications for the role of rhetoric in highly controversial issues such as recombinant DNA are even more critical.

  8. Clinical heterogeneity within xeroderma pigmentosum associated with mutations in the DNA repair and transcription gene ERCC3

    Energy Technology Data Exchange (ETDEWEB)

    Vermeulen, W.; Kleijer, W.J.; Bootsma, D.; Hoeijmakers, J.H.J.; Weeda, G. (Erasmus Univ., Rotterdam (Netherlands)); Scott, R.J.; Rodgers, S.; Mueller, H.J. (Univ. Hospital, Basel (Switzerland)); Cole, J.; Arlett, C.F. (Univ. of Sussex, Brighton (United Kingdom))

    1994-02-01

    The human DNA excision repair gene ERCC3 specifically corrects the nucleotide excision repair (NER) defect of xeroderma pigmentosum (XP) complementation group B. In addition to its function in NER, the ERCC3 DNA helicase was recently identified as one of the components of the human BTF2/TFIIH transcription factor complex, which is required for initiation of transcription of class II genes. To date, a single patient (XP11BE) has been assigned to this XP group B (XP-B), with the remarkable conjunction of two autosomal recessive DNA repair deficiency disorders: XP and Cockayne syndrome (CS). The intriguing involvement of the ERCC3 protein in the vital process of transcription may provide an explanation for the rarity, severity, and wide spectrum of clinical features in this complementation group. Here the authors report the identification of two new XP-B patients: XPCS1BA and XPCS2BA (siblings), by microneedle injection of the cloned ERCC3 repair gene as well as by cell hybridization. Molecular analysis of the ERCC3 gene in both patients revealed a single base substitution causing a missense mutation in a region that is completely conserved in yeast, Drosophila, mouse, and human ERCC3. As in patient XP11BE, the expression of only one allele (paternal) is detected. The mutation causes a virtually complete inactivation of the NER function of the protein. Despite this severe NER defect, both patients display a late onset of neurologic impairment, mild cutaneous symptoms, and a striking absence of skin tumors even at an age of >40 years. Analysis of the frequency of hprt[sup [minus

  9. Mutations of mtDNA polymerase-γ and hyperlactataemia in the HIV-infected Zulu population of South Africa.

    Science.gov (United States)

    Ojwach, D B A; Aldous, C; Kochleff, P; Sartorius, B

    2016-12-01

    Mitochondrial toxicity, particularly symptomatic hyperlactataemia or lactic acidosis (SHL/LA), has been attributed to the use of nucleoside reverse transcriptase inhibitors (NRTIs), possibly because of their capacity to impede human mitochondrial DNA polymerase-γ (POLG), which is responsible for the replication of mitochondrial DNA. To determine whether known monogenic POLG1 polymorphisms could be linked with the unexpectedly high incidence of SHL/LA observed in HIV-infected Zulu-speaking patients exposed to the NRTIs stavudine or zidovudine in their antiretroviral therapy. One hundred and sixteen patients from Edendale Hospital, Pietermaritzburg, South Africa, participated in the study between March and August 2014. Fifty-nine symptomatic cases were compared with 57 non-symptomatic controls on stavudine for ≥24 months. Among the symptomatic patients, 13 had SHL with measured lactate between 3.0 and 4.99 mmol/L, and 46 had LA with a lactate level ≥5 mmol/L. Genomic DNA from 113 samples was used for subsequent allelic discrimination polymerase chain reaction screening for the R964C and E1143G single-nucleotide polymorphisms of POLG1. Sequencing was performed for 40/113 randomly selected samples for confirmation of the genotyping results. Neither of the two known POLG1 mutations was observed. The cases presented with SHL/LA between 4 and 18 months on stavudine. Females (70.4%) were significantly (p<0.001) more likely to be cases (adjusted odds ratio 24.24, 95% CI 5.14 - 114.25) compared with males. This study has shown that our sample of the Zulu-speaking population does not exhibit a genetic predisposition to SHL/LA associated with known monogenic POLG1 mutations, indicating another possible predisposing factor for increased risk of SHL/LA.

  10. Promoter hypermethylation of the DNA repair gene O(6)-methylguanine-DNA methyltransferase is associated with the presence of G:C to A:T transition mutations in p53 in human colorectal tumorigenesis.

    Science.gov (United States)

    Esteller, M; Risques, R A; Toyota, M; Capella, G; Moreno, V; Peinado, M A; Baylin, S B; Herman, J G

    2001-06-15

    Defects in DNA repair may be responsible for the genesis of mutations in key genes in cancer cells. The tumor suppressor gene p53 is commonly mutated in human cancer by missense point mutations, most of them G:C to A:T transitions. A recognized cause for this type of change is spontaneous deamination of the methylcytosine. However, the persistence of a premutagenic O(6)-methylguanine can also be invoked. This last lesion is removed in the normal cell by the DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT). In many tumor types, epigenetic silencing of MGMT by promoter hypermethylation has been demonstrated and linked to the appearance of G to A mutations in the K-ras oncogene in colorectal tumors. To study the relevance of defective MGMT function by aberrant methylation in relation to the presence of p53 mutations, we studied 314 colorectal tumors for MGMT promoter hypermethylation and p53 mutational spectrum. Inactivation of MGMT by aberrant methylation was associated with the appearance of G:C to A:T transition mutations at p53 (Fischer's exact test, two-tailed; P = 0.01). Overall, MGMT methylated tumors displayed p53 transition mutations in 43 of 126 (34%) cases, whereas MGMT unmethylated tumors only showed G:C to A:T changes in 37 of 188 (19%) tumors. A more striking association was found in G:C to A:T transitions in non-CpG dinucleotides; 71% (12 of 17) of the total non-CpG transition mutations in p53 were observed in MGMT aberrantly methylated tumors (Fischer's exact test, two-tailed; P = 0.008). Our data suggest that epigenetic silencing of MGMT by promoter hypermethylation may lead to G:C to A:T transition mutations in p53.

  11. The effect of DNA replication on mutation of the Saccharomyces cerevisiae CDC8 gene.

    Science.gov (United States)

    Zaborowska, D; Zuk, J

    1990-04-01

    Incubation in YPD medium under permissive conditions when DNA replication is going on, strongly stimulates the induction of cdc+ colonies of UV-irradiated cells of yeast strains HB23 (cdc8-1/cdc8-3), HB26 (cdc8-3/cdc8-3) and HB7 (cdc8-1/cdc8-1). Inhibition of DNA replication by hydroxyurea, araCMP, cycloheximide or caffeine or else by incubation in phosphate buffer pH 7.0, abolishes this stimulation. Thus the replication of DNA is strongly correlated with the high induction of cdc+ colonies by UV irradiation. It is postulated that these UV-induced cdc+ colonies arise as the result infidelity in DNA replication.

  12. Rapid detection of ERG11 gene mutations in clinical Candida albicans isolates with reduced susceptibility to fluconazole by rolling circle amplification and DNA sequencing

    OpenAIRE

    Wang, Huiping; Kong, Fanrong; Sorrell, Tania C; Wang, Bin; McNicholas, Paul; Pantarat, Namfon; Ellis, David; Xiao, Meng; Widmer, Fred; Chen, Sharon CA

    2009-01-01

    Abstract Background Amino acid substitutions in the target enzyme Erg11p of azole antifungals contribute to clinically-relevant azole resistance in Candida albicans. A simple molecular method for rapid detection of ERG11 gene mutations would be an advantage as a screening tool to identify potentially-resistant strains and to track their movement. To complement DNA sequencing, we developed a padlock probe and rolling circle amplification (RCA)-based method to detect a series of mutations in th...

  13. Heart tissue of harlequin (hq)/Big Blue mice has elevated reactive oxygen species without significant impact on the frequency and nature of point mutations in nuclear DNA

    Energy Technology Data Exchange (ETDEWEB)

    Crabbe, Rory A. [Department of Biology, University of Western Ontario, London, Ontario, N6A 5B7 (Canada); Hill, Kathleen A., E-mail: khill22@uwo.ca [Department of Biology, University of Western Ontario, London, Ontario, N6A 5B7 (Canada)

    2010-09-10

    Age is a major risk factor for heart disease, and cardiac aging is characterized by elevated mitochondrial reactive oxygen species (ROS) with compromised mitochondrial and nuclear DNA integrity. To assess links between increased ROS levels and mutations, we examined in situ levels of ROS and cII mutation frequency, pattern and spectrum in the heart of harlequin (hq)/Big Blue mice. The hq mouse is a model of premature aging with mitochondrial dysfunction and increased risk of oxidative stress-induced heart disease with the means for in vivo mutation detection. The hq mutation produces a significant downregulation in the X-linked apoptosis-inducing factor gene (Aif) impairing both the antioxidant and oxidative phosphorylation functions of AIF. Brain and skin of hq disease mice have elevated frequencies of point mutations in nuclear DNA and histopathology characterized by cell loss. Reports of associated elevations in ROS in brain and skin have mixed results. Herein, heart in situ ROS levels were elevated in hq disease compared to AIF-proficient mice (p < 0.0001) yet, mutation frequency and pattern were similar in hq disease, hq carrier and AIF-proficient mice. Heart cII mutations were also assessed 15 days following an acute exposure to an exogenous ROS inducer (10 mg paraquat/kg). Acute paraquat exposure with a short mutant manifestation period was insufficient to elevate mutation frequency or alter mutation pattern in the post-mitotic heart tissue of AIF-proficient mice. Paraquat induction of ROS requires mitochondrial complex I and thus is likely compromised in hq mice. Results of this preliminary survey and the context of recent literature suggest that determining causal links between AIF deficiency and the premature aging phenotypes of specific tissues is better addressed with assay of mitochondrial ROS and large-scale changes in mitochondrial DNA in specific cell types.

  14. Heart tissue of harlequin (hq)/Big Blue mice has elevated reactive oxygen species without significant impact on the frequency and nature of point mutations in nuclear DNA

    International Nuclear Information System (INIS)

    Crabbe, Rory A.; Hill, Kathleen A.

    2010-01-01

    Age is a major risk factor for heart disease, and cardiac aging is characterized by elevated mitochondrial reactive oxygen species (ROS) with compromised mitochondrial and nuclear DNA integrity. To assess links between increased ROS levels and mutations, we examined in situ levels of ROS and cII mutation frequency, pattern and spectrum in the heart of harlequin (hq)/Big Blue mice. The hq mouse is a model of premature aging with mitochondrial dysfunction and increased risk of oxidative stress-induced heart disease with the means for in vivo mutation detection. The hq mutation produces a significant downregulation in the X-linked apoptosis-inducing factor gene (Aif) impairing both the antioxidant and oxidative phosphorylation functions of AIF. Brain and skin of hq disease mice have elevated frequencies of point mutations in nuclear DNA and histopathology characterized by cell loss. Reports of associated elevations in ROS in brain and skin have mixed results. Herein, heart in situ ROS levels were elevated in hq disease compared to AIF-proficient mice (p < 0.0001) yet, mutation frequency and pattern were similar in hq disease, hq carrier and AIF-proficient mice. Heart cII mutations were also assessed 15 days following an acute exposure to an exogenous ROS inducer (10 mg paraquat/kg). Acute paraquat exposure with a short mutant manifestation period was insufficient to elevate mutation frequency or alter mutation pattern in the post-mitotic heart tissue of AIF-proficient mice. Paraquat induction of ROS requires mitochondrial complex I and thus is likely compromised in hq mice. Results of this preliminary survey and the context of recent literature suggest that determining causal links between AIF deficiency and the premature aging phenotypes of specific tissues is better addressed with assay of mitochondrial ROS and large-scale changes in mitochondrial DNA in specific cell types.

  15. Synergistic effects of UdgB and Ung in mutation prevention and protection against commonly encountered DNA damaging agents in Mycobacterium smegmatis.

    Science.gov (United States)

    Malshetty, Vidyasagar S; Jain, Ruchi; Srinath, Thiruneelakantan; Kurthkoti, Krishna; Varshney, Umesh

    2010-03-01

    The incorporation of dUMP during replication or the deamination of cytosine in DNA results in the occurrence of uracils in genomes. To maintain genomic integrity, uracil DNA glycosylases (UDGs) excise uracil from DNA and initiate the base-excision repair pathway. Here, we cloned, purified and biochemically characterized a family 5 UDG, UdgB, from Mycobacterium smegmatis to allow us to use it as a model organism to investigate the physiological significance of the novel enzyme. Studies with knockout strains showed that compared with the wild-type parent, the mutation rate of the udgB( -) strain was approximately twofold higher, whereas the mutation rate of a strain deficient in the family 1 UDG (ung(- )) was found to be approximately 8.4-fold higher. Interestingly, the mutation rate of the double-knockout (ung(-)/ udgB(-)) strain was remarkably high, at approximately 19.6-fold. While CG to TA mutations predominated in the ung(-) and ung(-)/udgB(-) strains, AT to GC mutations were enhanced in the udgB(-) strain. The ung(-)/udgB(-) strain was notably more sensitive to acidified nitrite and hydrogen peroxide stresses compared with the single knockouts (ung(-) or udgB(-)). These observations reveal a synergistic effect of UdgB and Ung in DNA repair, and could have implications for the generation of attenuated strains of Mycobacterium tuberculosis.

  16. Rapid detection of ERG11 gene mutations in clinical Candida albicans isolates with reduced susceptibility to fluconazole by rolling circle amplification and DNA sequencing

    Directory of Open Access Journals (Sweden)

    Ellis David

    2009-08-01

    Full Text Available Abstract Background Amino acid substitutions in the target enzyme Erg11p of azole antifungals contribute to clinically-relevant azole resistance in Candida albicans. A simple molecular method for rapid detection of ERG11 gene mutations would be an advantage as a screening tool to identify potentially-resistant strains and to track their movement. To complement DNA sequencing, we developed a padlock probe and rolling circle amplification (RCA-based method to detect a series of mutations in the C. albicans ERG11 gene using "reference" azole-resistant isolates with known mutations. The method was then used to estimate the frequency of ERG11 mutations and their type in 25 Australian clinical C. albicans isolates with reduced susceptibility to fluconazole and in 23 fluconazole-susceptible isolates. RCA results were compared DNA sequencing. Results The RCA assay correctly identified all ERG11 mutations in eight "reference" C. albicans isolates. When applied to 48 test strains, the RCA method showed 100% agreement with DNA sequencing where an ERG11 mutation-specific probe was used. Of 20 different missense mutations detected by sequencing in 24 of 25 (96% isolates with reduced fluconazole susceptibility, 16 were detected by RCA. Five missense mutations were detected by both methods in 18 of 23 (78% fluconazole-susceptible strains. DNA sequencing revealed that mutations in non-susceptible isolates were all due to homozygous nucleotide changes. With the exception of the mutations leading to amino acid substitution E266D, those in fluconazole-susceptible strains were heterozygous. Amino acid substitutions common to both sets of isolates were D116E, E266D, K128T, V437I and V488I. Substitutions unique to isolates with reduced fluconazole susceptibility were G464 S (n = 4 isolates, G448E (n = 3, G307S (n = 3, K143R (n = 3 and Y123H, S405F and R467K (each n = 1. DNA sequencing revealed a novel substitution, G450V, in one isolate. Conclusion The sensitive RCA

  17. Mutations in Cytosine-5 tRNA Methyltransferases Impact Mobile Element Expression and Genome Stability at Specific DNA Repeats

    Directory of Open Access Journals (Sweden)

    Bianca Genenncher

    2018-02-01

    Full Text Available The maintenance of eukaryotic genome stability is ensured by the interplay of transcriptional as well as post-transcriptional mechanisms that control recombination of repeat regions and the expression and mobility of transposable elements. We report here that mutations in two (cytosine-5 RNA methyltransferases, Dnmt2 and NSun2, impact the accumulation of mobile element-derived sequences and DNA repeat integrity in Drosophila. Loss of Dnmt2 function caused moderate effects under standard conditions, while heat shock exacerbated these effects. In contrast, NSun2 function affected mobile element expression and genome integrity in a heat shock-independent fashion. Reduced tRNA stability in both RCMT mutants indicated that tRNA-dependent processes affected mobile element expression and DNA repeat stability. Importantly, further experiments indicated that complex formation with RNA could also contribute to the impact of RCMT function on gene expression control. These results thus uncover a link between tRNA modification enzymes, the expression of repeat DNA, and genomic integrity.

  18. Genetic signatures from amplification profiles characterize DNA mutation in somatic and radiation-induced sports of chrysanthemum

    International Nuclear Information System (INIS)

    Trigiano, R.N.; Scott, M.C.; Caetano-Anolles, G.

    1998-01-01

    The chrysanthemum (Dendranthema grandiflora Tzvelev.) cultivars 'Dark Charm', 'Salmon Charm', 'Coral Charm' and 'Dark Bronze Charm' are either radiation-induced mutants or spontaneous sports of 'Charm' and constitute a family or series of plants that primarily differ in flower color. These cultivars, which were difficult to differentiate genetically by DNA amplification fingerprinting (DAF), were easily identified by using arbitrary signatures from amplification profiles (ASAP). Genomic DNA was first amplified with three standard octamer arbitrary primers, all of which produced monomorphic profiles. Products from each of these DNA fingerprints were subsequently reamplified using four minihairpin decamer primers. The 12 primer combinations produced signatures containing approximately 37% polymorphic character loci, which were used to estimate genetic relationships between cultivars. Forty-six (32%) unique amplification products were associated with individual cultivars. The number of ASAP polymorphisms detected provided an estimate of the mutation rate in the mutant cultivars, ranging from 0.03% to 1.6% of nucleotide changes within an average of 18 kb of arbitrary amplified DAF sequence. The ASAP technique permits the clear genetic identification of somatic mutants and radiation-induced sports that are genetically highly homogeneous and should facilitate marker assisted breeding and protection of plant breeders rights of varieties or cultivars

  19. DNA Microarray Technologies: A Novel Approach to Geonomic Research

    Energy Technology Data Exchange (ETDEWEB)

    Hinman, R.; Thrall, B.; Wong, K,

    2002-01-01

    A cDNA microarray allows biologists to examine the expression of thousands of genes simultaneously. Researchers may analyze the complete transcriptional program of an organism in response to specific physiological or developmental conditions. By design, a cDNA microarray is an experiment with many variables and few controls. One question that inevitably arises when working with a cDNA microarray is data reproducibility. How easy is it to confirm mRNA expression patterns? In this paper, a case study involving the treatment of a murine macrophage RAW 264.7 cell line with tumor necrosis factor alpha (TNF) was used to obtain a rough estimate of data reproducibility. Two trials were examined and a list of genes displaying either a > 2-fold or > 4-fold increase in gene expression was compiled. Variations in signal mean ratios between the two slides were observed. We can assume that erring in reproducibility may be compensated by greater inductive levels of similar genes. Steps taken to obtain results included serum starvation of cells before treatment, tests of mRNA for quality/consistency, and data normalization.

  20. An innovative strategy to clone positive modifier genes of defects caused by mtDNA mutations: MRPS18C as suppressor gene of m.3946G>A mutation in MT-ND1 gene.

    Science.gov (United States)

    Rodríguez-García, María Elena; Cotrina-Vinagre, Francisco Javier; Carnicero-Rodríguez, Patricia; Martínez-Azorín, Francisco

    2017-07-01

    We have developed a new functional complementation approach to clone modifier genes which overexpression is able to suppress the biochemical defects caused by mtDNA mutations (suppressor genes). This strategy consists in transferring human genes into respiratory chain-deficient fibroblasts, followed by a metabolic selection in a highly selective medium. We used a normalized expression cDNA library in an episomal vector (pREP4) to transfect the fibroblasts, and a medium with glutamine and devoid of any carbohydrate source to select metabolically. Growing the patient's fibroblasts in this selective medium, the deficient cells rapidly disappear unless they are rescued by the cDNA of a suppressor gene. The use of an episomal vector allows us to carry out several rounds of transfection/selection (cyclical phenotypic rescue) to enrich the rescue with true clones of suppressor genes. Using fibroblasts from a patient with epileptic encephalopathy with the m.3946G>A (p.E214K) mutation in the MT-ND1 gene, several candidate genes were identified and one of them was characterized functionally. Thus, overexpression of MRPS18C gene (that encode for bS18m protein) suppressed the molecular defects produced by this mtDNA mutation, recovering the complex I activity and reducing the ROS produced by this complex to normal levels. We suggest that modulation of bS18m expression may be an effective therapeutic strategy for the patients with this mutation.

  1. Research highlights on: the use of induced mutations for plant improvement in Malaysia

    International Nuclear Information System (INIS)

    1998-01-01

    Nuclear techniques play an increasingly valuable role in agricultural research and development. The collaborative work of IAEA and FAO has been instrumental to the progress. The nuclear techniques are now used in a wide range of applications including crop improvement. In the initial years, many plant breeders had difficulty in believing that induced mutations through radiation had any relevance to their conventional procedures. But attitudes have greatly changed, in great part due to the Joint Division's programmes. The result was a high number of improved new varieties bred to date with the help of induced mutations, including some in this country. This publication is intended primarily to gather and put in order all information on the research and work on the use of induced mutations for plant breeding in the country. Its main purpose is to see if ensuing research efforts could be better coordinated, focused and enhanced in order to supplement the plant improvement programmes in the country. The task of collating the relevant information was not without difficulty since many of the work had been carried out long time ago; their objectives generally were quite broad; and the results essentially not published, with some exceptions. Section I begins with thefntroduction, giving a brief account of the developments of induced mutations in Malaysia, the facilities available in various institutions and the role played by the National Committee on the Use of Mutations in Plant Breeding. The collaborative efforts of IAEA and IFNCC are also briefly described here, together with all the activates which they had supported in the past. Section 11 briefly describes the induced mutations and how they are produced and utilised in the plant improvement programmes, taking into consideration the safety precautions required, and the requirements of different crop species. Section III describes in greater details all the research that had been carried out in the country. The

  2. Use of spontaneously mutated human DNA as competitive internal standard for nucleic acid quantification by reverse transcription-polymerase chain reaction (RT-PCR)

    International Nuclear Information System (INIS)

    Rudnicka, L.; Diaz, A.; Varga, J.; Jimenez, S.A.; Christiano, A.; Uitto, J.

    1995-01-01

    Quantification of gene expression is of increasing interest in many medical sciences. Methods based on reverse transcription-polymerase chain reactions (RT-PCRs) are timesaving and require only very small amounts of RNA. A limiting factor, however, is the significant fluctuation in the efficacy of reverse transcription as well in the polymerase chain reactions. Various external and internal standards have been suggested for correcting these fluctuations. We describe a novel way of creating an internal standard for assessing the expression of type VII collagen in human cells. The total RNA of a patient with hereditary 'epidermilysis bulosa dystrophica' associated with a homozygous T to A point mutation in type VII collagen gene was reverse transcribed and a 382bp fragment of type VII collagen cDNA containing the mutation was amplified. The mutated cDNA, unlike normal type VII collagen cDNA could be cleaved by 'Ear I' endonuclease into 244bp and 138bp fragments. Semiquantitative PCR was performed with the mutated cDNA as internal standard and the studied cDNA sample in the same tube in the presence of α 32 P-labelled dCTP. The reaction was followed by 'Ear I' digestion, electrophoresis on a polyacrylamide gel and exposure to a X-ray film. In conclusion, we describe a timesaving method for creating internal standards for semiquantitative RT-PCR. (author). 12 refs, 3 figs

  3. Molecular and clinical characterization of the myopathic form of mitochondrial DNA depletion syndrome caused by mutations in the thymidine kinase (TK2) gene.

    Science.gov (United States)

    Chanprasert, Sirisak; Wang, Jing; Weng, Shao-Wen; Enns, Gregory M; Boué, Daniel R; Wong, Brenda L; Mendell, Jerry R; Perry, Deborah A; Sahenk, Zarife; Craigen, William J; Alcala, Francisco J Climent; Pascual, Juan M; Melancon, Serge; Zhang, Victor Wei; Scaglia, Fernando; Wong, Lee-Jun C

    2013-01-01

    Mitochondrial DNA (mtDNA) depletion syndromes (MDSs) are a clinically and molecularly heterogeneous group of mitochondrial cytopathies characterized by severe mtDNA copy number reduction in affected tissues. Clinically, MDSs are mainly categorized as myopathic, encephalomyopathic, hepatocerebral, or multi-systemic forms. To date, the myopathic form of MDS is mainly caused by mutations in the TK2 gene, which encodes thymidine kinase 2, the first and rate limiting step enzyme in the phosphorylation of pyrimidine nucleosides. We analyzed 9 unrelated families with 11 affected subjects exhibiting the myopathic form of MDS, by sequencing the TK2 gene. Twelve mutations including 4 novel mutations were detected in 9 families. Skeletal muscle specimens were available from 7 out of 11 subjects. Respiratory chain enzymatic activities in skeletal muscle were measured in 6 subjects, and enzymatic activities were reduced in 3 subjects. Quantitative analysis of mtDNA content in skeletal muscle was performed in 5 subjects, and marked mtDNA content reduction was observed in each. In addition, we outline the molecular and clinical characteristics of this syndrome in a total of 52 patients including those previously reported, and a total of 36 TK2 mutations are summarized. Clinically, hypotonia and proximal muscle weakness are the major phenotypes present in all subjects. In summary, our study expands the molecular and clinical spectrum associated with TK2 deficiency. © 2013.

  4. Heterozygous SSBP1 start loss mutation co-segregates with hearing loss and the m.1555A>G mtDNA variant in a large multigenerational family.

    Science.gov (United States)

    Kullar, Peter J; Gomez-Duran, Aurora; Gammage, Payam A; Garone, Caterina; Minczuk, Michal; Golder, Zoe; Wilson, Janet; Montoya, Julio; Häkli, Sanna; Kärppä, Mikko; Horvath, Rita; Majamaa, Kari; Chinnery, Patrick F

    2018-01-01

    The m.1555A>G mtDNA variant causes maternally inherited deafness, but the reasons for the highly variable clinical penetrance are not known. Exome sequencing identified a heterozygous start loss mutation in SSBP1, encoding the single stranded binding protein 1 (SSBP1), segregating with hearing loss in a multi-generational family transmitting m.1555A>G, associated with mtDNA depletion and multiple deletions in skeletal muscle. The SSBP1 mutation reduced steady state SSBP1 levels leading to a perturbation of mtDNA metabolism, likely compounding the intra-mitochondrial translation defect due to m.1555A>G in a tissue-specific manner. This family demonstrates the importance of rare trans-acting genetic nuclear modifiers in the clinical expression of mtDNA disease. © The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain.

  5. [DNA mutations associated to rifampicin or isoniazid resistance in M. tuberculosis clinical isolates from Sonora, Mexico].

    Science.gov (United States)

    Bolado-Martínez, Enrique; Pérez-Mendoza, Ansix; Alegría-Morquecho, Francisco Monserrat; Candia-Plata, María del Carmen; Aguayo-Verdugo, María del Rosario; Alvarez-Hernández, Gerardo

    2012-01-01

    To perform the analysis of specific regions of the major genes associated with resistance to isoniazid or rifampin. Twenty two M. tuberculosis strains, isolated from human samples obtained in Sonora, Mexico. Specific primers for hotspots of the rpoB, katG, inhA genes and the ahpC-oxyR intergenic region were used. The purified PCR products were sequenced. Mutations in the promoter of inhA, the ahpC-oxyR region, and codon 315 of katG and in 451 or 456 codons of rpoB, were identified. Detection of mutations not previously reported requires further genotypic analysis of Mycobacterium tuberculosis isolates in Sonora.

  6. Specific UV-induced mutation spectrum in the p53 gene of skin tumors from DNA-repair-deficient xeroderma pigmentosum patients

    International Nuclear Information System (INIS)

    Dumaz, N.; Drougard, C.; Sarasin, A.; Daya-Grosjean, L.

    1993-01-01

    The UV component of sunlight is the major carcinogen involved in the etiology of skin cancers. The authors have studied the rare, hereditary syndrome xeroderma pigmentosum (XP), which is characterized by a very high incidence of cutaneous tumors on exposed skin at an early age, probably due to a deficiency in excision repair of UV-induced lesions. It is interesting to determine the UV mutation spectrum in XP skin tumors in order to correlate the absence of repair of specific DNA lesions and the initiation of skin tumors. The p53 gene is frequently mutated in human cancers and represents a good target for studying mutation spectra since there are >100 potential sites for phenotypic mutations. Using reverse transcription-PCR and single-strand conformation polymorphism to analyze >40 XP skin tumors (mainly basal and squamous cell carcinomas), the authors have found that 40% (17 out of 43) contained at least one point mutation on the p53 gene. All the mutations were located at dipyrimidine sites, essentially at CC sequences, which are hot spots for UV-induced DNA lesions. Sixty-one percent of these mutations were tandem CC → TT mutations considered to be unique to UV-induced lesions; these mutations are not observed in internal human tumors. All the mutations, except two, must be due to translesion synthesis of unrepaired dipyrimidine lesions left on the nontranscribed strand. These results show the existence of preferential repair of UV lesions [either pyrimidine dimers or pyrimidine-pyrimidone (6-4) photoproducts] on the transcribed strand in human tissues

  7. Protective action of DNA preparations on the survival of cells and yield of 8-azaguanine resistant mutations in X-irradiated cell culture of chinese hamsters

    International Nuclear Information System (INIS)

    Kuznetsova, N.N.; Feoktistova, T.P.

    1976-01-01

    A DNA preparation (molecular weight 19.6-21.0x1O 6 daltons) administered to cell culture of Chinese hamsters in concentrations of 100 to 122 μg/ml 60 minutes before and in the course of 3 days after X-irradiation (600 R) decreased the lethality of irradiated cells and reduced induction of 8-azaguanine resistant genic mutations. DNA preparations with the concentrations under study had no toxic action on cells and were not mutagenous

  8. [The mutations of the D-loop hypervariable region II and hypervariable region III of mitochondrial DNA in oral squamous cell carcinoma].

    Science.gov (United States)

    Wang, Yao-Zhong; Jia, Mu-Yun; Yuan, Rong-Tao; Han, Guo-Dong; Bu, Ling-Xue

    2010-06-01

    To investigate the frequency of mitochondrial DNA (mtDNA) D-loop hypervariable region II (HVR II) and hypervariable region III (HVR III) mutations in oral squamous cell carcinoma (OSCC) and their correlation to provide the new targets for the prevention and treatment of OSCC. The D-loop HVR II and HVR III regions of mtDNA in seven cases with OSCC tissues, matched with paracancerous tissues and normal mucosa tissues from the same case, were amplified by polymerase chain raction (PCR), then were detected by direct sequencing to find the mutantsites after the comparison of all sequencing results with the mtDNA Cambridge sequence in the GenBank database. 82 (56 species) nucleotide changes, with 51(26 species) nucleotide polymorphism, were found after the comparison of all sequencing results with the mtDNA Cambridge sequence in the GenBank database. 31(30 species) mutations, with 21 located within the HVR II and HVR III regions, were found in 3 tumor tissue samples, their paracancerous and normal mucosa tissue were found more polymorphic changes but no mutation. The mtDNA D-loop HVR II and HVR III regions mutation rate was 42.9% (3/7) in OSCC. The mtDNA D-loop HVR II and HVR III regions were highly polymorphic and mutable regions in OSCC. It suggested that the D-loop HVR II and HVR III regions of mtDNA might play a significant role in the tumorigenesis of OSCC. It may become new targets for the gene therapy of OSCC by regulating the above indexes.

  9. Microsatellites in the Eukaryotic DNA Mismatch Repair Genes as Modulators of Evolutionary Mutation Rate

    Science.gov (United States)

    Chang, Dong Kyung; Metzgar, David; Wills, Christopher; Boland, C. Richard

    2003-01-01

    All "minor" components of the human DNA mismatch repair (MMR) system-MSH3, MSH6, PMS2, and the recently discovered MLH3-contain mononucleotide microsatellites in their coding sequences. This intriguing finding contrasts with the situation found in the major components of the DNA MMR system-MSH2 and MLH1-and, in fact, most human genes. Although eukaryotic genomes are rich in microsatellites, non-triplet microsatellites are rare in coding regions. The recurring presence of exonal mononucleotide repeat sequences within a single family of human genes would therefore be considered exceptional.

  10. Base-pairing preferences, physicochemical properties and mutational behaviour of the DNA lesion 8-nitroguanine †

    OpenAIRE

    Bhamra, Inder; Compagnone-Post, Patricia; O’Neil, Ian A.; Iwanejko, Lesley A.; Bates, Andrew D.; Cosstick, Richard

    2012-01-01

    8-Nitro-2′-deoxyguanosine (8-nitrodG) is a relatively unstable, mutagenic lesion of DNA that is increasingly believed to be associated with tissue inflammation. Due to the lability of the glycosidic bond, 8-nitrodG cannot be incorporated into oligodeoxynucleotides (ODNs) by chemical DNA synthesis and thus very little is known about its physicochemical properties and base-pairing preferences. Here we describe the synthesis of 8-nitro-2′-O-methylguanosine, a ribonucleoside analogue of this lesi...

  11. Structure-guided mutational analysis of the OB, HhH, and BRCT domains of Escherichia coli DNA ligase.

    Science.gov (United States)

    Wang, Li Kai; Nair, Pravin A; Shuman, Stewart

    2008-08-22

    NAD(+)-dependent DNA ligases (LigAs) are ubiquitous in bacteria and essential for growth. LigA enzymes have a modular structure in which a central catalytic core composed of nucleotidyltransferase and oligonucleotide-binding (OB) domains is linked via a tetracysteine zinc finger to distal helix-hairpin-helix (HhH) and BRCT (BRCA1-like C-terminal) domains. The OB and HhH domains contribute prominently to the protein clamp formed by LigA around nicked duplex DNA. Here we conducted a structure-function analysis of the OB and HhH domains of Escherichia coli LigA by alanine scanning and conservative substitutions, entailing 43 mutations at 22 amino acids. We thereby identified essential functional groups in the OB domain that engage the DNA phosphodiester backbone flanking the nick (Arg(333)); penetrate the minor grove and distort the nick (Val(383) and Ile(384)); or stabilize the OB fold (Arg(379)). The essential constituents of the HhH domain include: four glycines (Gly(455), Gly(489), Gly(521), Gly(553)), which bind the phosphate backbone across the minor groove at the outer margins of the LigA-DNA interface; Arg(487), which penetrates the minor groove at the outer margin on the 3 (R)-OH side of the nick; and Arg(446), which promotes protein clamp formation via contacts to the nucleotidyltransferase domain. We find that the BRCT domain is required in its entirety for effective nick sealing and AMP-dependent supercoil relaxation.

  12. Mitochondrial DNA polymerase editing mutation, PolgD257A, reduces the diabetic phenotype of Akita male mice by suppressing appetite.

    Science.gov (United States)

    Fox, Raymond; Kim, Hyung-Suk; Reddick, Robert L; Kujoth, Gregory C; Prolla, Tomas A; Tsutsumi, Shuichi; Wada, Youichiro; Smithies, Oliver; Maeda, Nobuyo

    2011-05-24

    Diabetes and the development of its complications have been associated with mitochondrial DNA (mtDNA) dysfunction, but causal relationships remain undetermined. With the objective of testing whether increased mtDNA mutations exacerbate the diabetic phenotype, we have compared mice heterozygous for the Akita diabetogenic mutation (Akita) with mice homozygous for the D257A mutation in mitochondrial DNA polymerase gamma (Polg) or with mice having both mutations (Polg-Akita). The Polg-D257A protein is defective in proofreading and increases mtDNA mutations. At 3 mo of age, the Polg-Akita and Akita male mice were equally hyperglycemic. Unexpectedly, as the Polg-Akita males aged to 9 mo, their diabetic symptoms decreased. Thus, their hyperglycemia, hyperphagia and urine output declined significantly. The decrease in their food intake was accompanied by increased plasma leptin and decreased plasma ghrelin, while hypothalamic expression of the orexic gene, neuropeptide Y, was lower and expression of the anorexic gene, proopiomelanocortin, was higher. Testis function progressively worsened with age in the double mutants, and plasma testosterone levels in 9-mo-old Polg-Akita males were significantly reduced compared with Akita males. The hyperglycemia and hyperphagia returned in aged Polg-Akita males after testosterone administration. Hyperglycemia-associated distal tubular damage in the kidney also returned, and Polg-D257A-associated proximal tubular damage was enhanced. The mild diabetes of female Akita mice was not affected by the Polg-D257A mutation. We conclude that reduced diabetic symptoms of aging Polg-Akita males results from appetite suppression triggered by decreased testosterone associated with damage to the Leydig cells of the testis.

  13. Malignant chondroblastoma presenting as a recurrent pelvic tumor with DNA aneuploidy and p53 mutation as supportive evidence of malignancy

    Energy Technology Data Exchange (ETDEWEB)

    Ostrowski, M.L. [Department of Pathology and Laboratory Medicine, Baylor College of Medicine, The Methodist Hospital and Texas Children' s Hospital, Houston, Texas (United States); Department of Pathology and Laboratory Medicine, Houston, TX (United States). Methodist Hospital; Johnson, M.E. [Department of Orthopedic Surgery, Baylor College of Medicine, The Methodist Hospital and Texas Children' s Hospital, Houston, Texas (United States); Truong, L.D.; Hicks, M.J.; Spjut, H.J. [Department of Pathology and Laboratory Medicine, Baylor College of Medicine, The Methodist Hospital and Texas Children' s Hospital, Houston, Texas (United States); Smith, F.E. [Department of Oncology, Baylor College of Medicine, The Methodist Hospital and Texas Children' s Hospital, Houston, Texas (United States)

    1999-11-01

    We report a rare case of malignant chondroblastoma, which presented in a 47-year-old man as a recurrent tumor, 18 years following wide excision of a typical pelvic chondroblastoma. Radiologic studies of the recurrent tumor showed a large, lytic, destructive lesion of the right pelvic bones and femur, with a pathologic fracture of the latter, a large pelvic soft tissue mass, and multiple pulmonary metastases. Biopsy tissue showed typical features of chondroblastoma, but also increased nuclear atypia, hyperchromasia, and pleomorphism, compared to the original tumor, and, most significantly, abnormal mitotic figures. Immunohistochemical studies of the recurrent tumor revealed p53 mutation and extensive proliferative activity, and flow cytometric studies showed DNA aneuploidy, none of which was present in the original tumor. The patient received chemotherapy and radiation, but died of disease eight months after presentation. We also review chondroblastoma in general, to assign this unusual lesion to a tumor subtype. (orig.)

  14. Genetic and epigenetic mutations affect the DNA binding capability of human ZFP57 in transient neonatal diabetes type 1.

    Science.gov (United States)

    Baglivo, Ilaria; Esposito, Sabrina; De Cesare, Lucia; Sparago, Angela; Anvar, Zahra; Riso, Vincenzo; Cammisa, Marco; Fattorusso, Roberto; Grimaldi, Giovanna; Riccio, Andrea; Pedone, Paolo V

    2013-05-21

    In the mouse, ZFP57 contains three classical Cys2His2 zinc finger domains (ZF) and recognizes the methylated TGC(met)CGC target sequence using the first and the second ZFs. In this study, we demonstrate that the human ZFP57 (hZFP57) containing six Cys2His2 ZFs, binds the same methylated sequence through the third and the fourth ZFs, and identify the aminoacids critical for DNA interaction. In addition, we present evidences indicating that hZFP57 mutations and hypomethylation of the TNDM1 ICR both associated with Transient Neonatal Diabetes Mellitus type 1 result in loss of hZFP57 binding to the TNDM1 locus, likely causing PLAGL1 activation. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  15. DNA detection and single nucleotide mutation identification using SERS for molecular diagnostics and global health

    Science.gov (United States)

    Ngo, Hoan T.; Gandra, Naveen; Fales, Andrew M.; Taylor, Steve M.; Vo-Dinh, Tuan

    2017-02-01

    Nucleic acid-based molecular diagnostics at the point-of-care (POC) and in resource-limited settings is still a challenge. We present a sensitive yet simple DNA detection method with single nucleotide polymorphism (SNP) identification capability. The detection scheme involves sandwich hybridization of magnetic beads conjugated with capture probes, target sequences, and ultrabright surface-enhanced Raman Scattering (SERS) nanorattles conjugated with reporter probes. Upon hybridization, the sandwich probes are concentrated at the detection focus controlled by a magnetic system for SERS measurements. The ultrabright SERS nanorattles, consisting of a core and a shell with resonance Raman reporters loaded in the gap space between the core and the shell, serve as SERS tags for ultrasensitive signal detection. Specific DNA sequences of the malaria parasite Plasmodium falciparum and dengue virus 1 (DENV1) were used as the model marker system. Detection limit of approximately 100 attomoles was achieved. Single nucleotide polymorphism (SNP) discrimination of wild type malaria DNA and mutant malaria DNA, which confers resistance to artemisinin drugs, was also demonstrated. The results demonstrate the molecular diagnostic potential of the nanorattle-based method to both detect and genotype infectious pathogens. The method's simplicity makes it a suitable candidate for molecular diagnosis at the POC and in resource-limited settings.

  16. Desmin common mutation is associated with multi-systemic disease manifestations and depletion of mitochondria and mitochondrial DNA

    Directory of Open Access Journals (Sweden)

    Elizabeth eMcCormick

    2015-06-01

    Full Text Available Desmin (DES is a major muscle scaffolding protein that also functions to anchor mitochondria. Pathogenic DES mutations, however, have not previously been recognized as a cause of multi-systemic mitochondrial disease. Here, we describe a 45-year-old man who presented to The Children’s Hospital of Philadelphia Mitochondrial-Genetics Diagnostic Clinic for evaluation of progressive cardiac, neuromuscular, gastrointestinal, and mood disorders. Muscle biopsy at age 45 was remarkable for cytoplasmic bodies, as well as ragged red fibers and SDH positive/COX negative fibers that were suggestive of a mitochondrial myopathy. Muscle also showed significant reductions in mitochondrial content (16% of control mean for citrate synthase activity and mitochondrial DNA (35% of control mean. His family history was significant for cardiac conduction defects and myopathy in multiple maternal relatives. Multiple single gene and panel-based sequencing studies were unrevealing. Whole exome sequencing identified a known pathogenic p.S13F mutation in DES that had previously been associated with desmin-related myopathy. Desmin-related myopathy is an autosomal dominant disorder characterized by right ventricular hypertrophic cardiomyopathy, myopathy, and arrhythmias. However, neuropathy, gastrointestinal dysfunction, and depletion of both mitochondria and mitochondrial DNA have not previously been widely recognized in this disorder. Recognition that mitochondrial dysfunction occurs in desmin-related myopathy clarifies the basis for the multi-systemic manifestations, as are typical of primary mitochondrial disorders. Understanding the mitochondrial pathophysiology of desmin-related myopathy highlights the possibility of new therapies for the otherwise untreatable and often fatal class of disease. We postulate that drug treatments aimed at improving mitochondrial biogenesis or reducing oxidative stress may be effective therapies to ameliorate the effects of desmin

  17. Creation of an open-access, mutation-defined fibroblast resource for neurological disease research.

    Directory of Open Access Journals (Sweden)

    Selina Wray

    Full Text Available Our understanding of the molecular mechanisms of many neurological disorders has been greatly enhanced by the discovery of mutations in genes linked to familial forms of these diseases. These have facilitated the generation of cell and animal models that can be used to understand the underlying molecular pathology. Recently, there has been a surge of interest in the use of patient-derived cells, due to the development of induced pluripotent stem cells and their subsequent differentiation into neurons and glia. Access to patient cell lines carrying the relevant mutations is a limiting factor for many centres wishing to pursue this research. We have therefore generated an open-access collection of fibroblast lines from patients carrying mutations linked to neurological disease. These cell lines have been deposited in the National Institute for Neurological Disorders and Stroke (NINDS Repository at the Coriell Institute for Medical Research and can be requested by any research group for use in in vitro disease modelling. There are currently 71 mutation-defined cell lines available for request from a wide range of neurological disorders and this collection will be continually expanded. This represents a significant resource that will advance the use of patient cells as disease models by the scientific community.

  18. The resistance of Micrococcus radiodurans to killing and mutation by agents which damage DNA

    International Nuclear Information System (INIS)

    Sweet, D.M.; Moseley, B.E.B.

    1976-01-01

    The resistance of Micrococcus radiodurans to the lethal and mutagenic action of ultraviolet (UV) light, ionising (γ) radiation, mitomycin C (MTC), nitrous acid (NA), hydroxylamine (HA), N-methyl-N'-nitro-N-nitrosoguanidine (NG), ethylmethanesulphonate (EMS) and β-propiolactone (βPL) has been compared with that of Escherichia coli B/r. M. radiodurans was much more resistant than E. coli B/r to the lethal effects of UV light (by a factor of 33), γ-radiation (55), NG (15) and NA (62), showed intermediate resistance to MTC (4) and HA (7), but was sensitive to EMS (1) and βPL (2). M. radiodurans was very resistant to mutagens producing damage which can be repaired by a recombination system, indicating that it possesses an extremely efficient recombination repair mechanism. Both species were equally sensitive to mutation to trimethoprim resistance by NG, but M. radiodurans was more resistant than E. coli B/r to the other mutagens tested, being non-mutable by UV light, γ-radiation, MTC and HA, and only slightly sensitive to mutation by NA, EMS, and βPL. The resistance of M. radiodurans to mutation by UV light, γ-radiation and MTC is consistent with an hypothesis that recombination repair in M. radiodurans is accurate since these mutagens may depend on an 'error-prone' recombination system for their mutagenic effect in E. coli B/r. However, because M. radiodurans is also resistant to mutagens such as HA and EMS, which are mutagenic in E. coli in the absence of an 'error-prone' system, we propose that all the mutagens tested may have a common mode of action in E. coli B/r, but that this mutagenic pathway is missing in M. radiodurans

  19. Resistance of Micrococcus radiodurans to killing and mutation by agents which damage DNA

    Energy Technology Data Exchange (ETDEWEB)

    Sweet, D M; Moseley, B E.B. [Edinburgh Univ. (UK). School of Agriculture

    1976-02-01

    The resistance of Micrococcus radiodurans to the lethal and mutagenic action of ultraviolet (UV) light, ionising (..gamma..) radiation, mitomycin C (MTC), nitrous acid (NA), hydroxylamine (HA), N-methyl-N'-nitro-N-nitrosoguanidine (NG), ethylmethanesulphonate (EMS) and ..beta..-propiolactone (..beta..PL) has been compared with that of Escherichia coli B/r. M. radiodurans was much more resistant than E. coli B/r to the lethal effects of UV light (by a factor of 33), ..gamma..-radiation (55), NG (15) and NA (62), showed intermediate resistance to MTC (4) and HA (7), but was sensitive to EMS (1) and ..beta..PL (2). M. radiodurans was very resistant to mutagens producing damage which can be repaired by a recombination system, indicating that it possesses an extremely efficient recombination repair mechanism. Both species were equally sensitive to mutation to trimethoprim resistance by NG, but M. radiodurans was more resistant than E. coli B/r to the other mutagens tested, being non-mutable by UV light, ..gamma..-radiation, MTC and HA, and only slightly sensitive to mutation by NA, EMS, and ..beta..PL. The resistance of M. radiodurans to mutation by UV light, ..gamma.. radiation and MTC is consistent with an hypothesis that recombination repair in M. radiodurans is accurate since these mutagens may depend on an 'error-prone' recombination system for their mutagenic effect in E. coli B/r. However, because M. radiodurans is also resistant to mutagens such as HA and EMS, which are mutagenic in E. coli in the absence of an 'error-prone' system, we propose that all the mutagens tested may have a common mode of action in E. coli B/r, but that this mutagenic pathway is missing in M. radiodurans.

  20. Missense mutations located in structural p53 DNA-binding motifs are associated with extremely poor survival in chronic lymphocytic leukemia.

    Science.gov (United States)

    Trbusek, Martin; Smardova, Jana; Malcikova, Jitka; Sebejova, Ludmila; Dobes, Petr; Svitakova, Miluse; Vranova, Vladimira; Mraz, Marek; Francova, Hana Skuhrova; Doubek, Michael; Brychtova, Yvona; Kuglik, Petr; Pospisilova, Sarka; Mayer, Jiri

    2011-07-01

    There is a distinct connection between TP53 defects and poor prognosis in chronic lymphocytic leukemia (CLL). It remains unclear whether patients harboring TP53 mutations represent a homogenous prognostic group. We evaluated the survival of patients with CLL and p53 defects identified at our institution by p53 yeast functional assay and complementary interphase fluorescence in situ hybridization analysis detecting del(17p) from 2003 to 2010. A defect of the TP53 gene was identified in 100 of 550 patients. p53 mutations were strongly associated with the deletion of 17p and the unmutated IgVH locus (both P DBMs), structurally well-defined parts of the DNA-binding domain, manifested a clearly shorter median survival (12 months) compared with patients having missense mutations outside DBMs (41 months; P = .002) or nonmissense alterations (36 months; P = .005). The difference in survival was similar in the analysis limited to patients harboring mutation accompanied by del(17p) and was also confirmed in a subgroup harboring TP53 defect at diagnosis. The patients with p53 DBMs mutation (at diagnosis) also manifested a short median time to first therapy (TTFT; 1 month). The substantially worse survival and the short TTFT suggest a strong mutated p53 gain-of-function phenotype in patients with CLL with DBMs mutations. The impact of p53 DBMs mutations on prognosis and response to therapy should be analyzed in investigative clinical trials.

  1. The effect of defective DNA double-strand break repair on mutations and chromosome aberrations in the Chinese hamster cell mutant XR-V15B

    International Nuclear Information System (INIS)

    Helbig, R.; Speit, G.; Zdzienicka, M.Z.

    1995-01-01

    The radiosensitive Chinese hamster cell line XR-V15B was used to study the effect of decreased rejoining of DNA double-strand breaks (DSBs) on gene mutations and chromosome aberrations. XR-V15B cells are hypersensitive to the cytotoxic effects of neocarzinostatin (NCS) and methyl methanesulfonate (MMS). Both mutagens induced more chromosome aberrations in XR-V15B cells than in the parental cell strain. The clastogenic action of NCS was characterized by the induction of predominantly chromosome-type aberrations in cells of both strains, whereas MMS induced mainly chromatid aberrations. The frequency of induced gene mutations at the hprt locus was not increased compared to the parental V79 cells when considering the same survival level. Molecular analysis by multiplex polymerase chain reaction (PCR) of mutants induced by NCS revealed a high frequency of deletions in cells of both cell lines. Methyl methane-sulfonate induced mainly mutations without visible change in the PCR pattern, which probably represent point mutations. Our findings suggest a link between a defect in DNA DSB repair and increased cytotoxic and clastogenic effects. However, a decreased ability to rejoin DNA DSBs does not seem to influence the incidence and types of gene mutations at the hprt locus induced by NCS and MMS. 28 refs., 4 figs., 3 tabs

  2. Structural Effects of Some Relevant Missense Mutations on the MECP2-DNA Binding: A MD Study Analyzed by Rescore+, a Versatile Rescoring Tool of the VEGA ZZ Program.

    Science.gov (United States)

    Pedretti, Alessandro; Granito, Cinzia; Mazzolari, Angelica; Vistoli, Giulio

    2016-09-01

    DNA methylation plays key roles in mammalian cells and is modulated by a set of proteins which recognize symmetrically methylated nucleotides. Among them, the protein MECP2 shows multifunctional roles repressing and/or activating genes by binding to both methylated and unmethylated regions of the genome. The interest for this protein markedly increased from the observation that its mutations are the primary cause of Rett syndrome, a neurodevelopmental disorder which causes mental retardation in young females. Thus, the present study is aimed to investigate the effects of some of these known pathogenic missense mutations (i.e. R106Q, R106W, R111G, R133C and R133H) on the MECP2 folding and DNA binding by molecular dynamics simulations. The effects of the simulated mutations are also parameterized by using a here proposed new tool, named Rescore+, implemented in the VEGA ZZ suite of programs, which calculates a set of scoring functions on all frames of a trajectory or on all complexes contained in a database thus allowing an easy rescoring of results coming from MD or docking simulations. The obtained results revealed that the reported loss of the MECP2 function induced by the simulated mutations can be ascribed to both stabilizing and destabilizing effect on DNA binding. The study confirms that MD simulations are particularly useful to rationalize and predict the mutation effects offering insightful information for diagnostics and drug design. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Novel Point Mutations and A8027G Polymorphism in Mitochondrial-DNA-Encoded Cytochrome c Oxidase II Gene in Mexican Patients with Probable Alzheimer Disease

    Directory of Open Access Journals (Sweden)

    Verónica Loera-Castañeda

    2014-01-01

    Full Text Available Mitochondrial dysfunction has been thought to contribute to Alzheimer disease (AD pathogenesis through the accumulation of mitochondrial DNA mutations and net production of reactive oxygen species (ROS. Mitochondrial cytochrome c-oxidase plays a key role in the regulation of aerobic production of energy and is composed of 13 subunits. The 3 largest subunits (I, II, and III forming the catalytic core are encoded by mitochondrial DNA. The aim of this work was to look for mutations in mitochondrial cytochrome c-oxidase gene II (MTCO II in blood samples from probable AD Mexican patients. MTCO II gene was sequenced in 33 patients with diagnosis of probable AD. Four patients (12% harbored the A8027G polymorphism and three of them were early onset (EO AD cases with familial history of the disease. In addition, other four patients with EOAD had only one of the following point mutations: A8003C, T8082C, C8201T, or G7603A. Neither of the point mutations found in this work has been described previously for AD patients, and the A8027G polymorphism has been described previously; however, it hasn’t been related to AD. We will need further investigation to demonstrate the role of the point mutations of mitochondrial DNA in the pathogenesis of AD.

  4. Research progress in mutational effects of aerospace on crop and ground simulation on aerospace environment factors

    International Nuclear Information System (INIS)

    Liu Luxiang; Wang Jing; Zhao Linshu; Guo Huijun; Zhao Shirong; Zheng Qicheng; Yang Juncheng

    2004-01-01

    In this paper, the current status of aerospace botany research in aboard was briefly introduced. The research progress of mutational effects of aerospace on crop seed and its application in germplasm enhancement and new variety development by using recoverable satellite techniques in China has been reviewed. The approaches and its experimental advances of ground simulation on aerospace environmental factors were analyzed at different angles of particle biology, physical field biology and gravity biology

  5. DNA Glycosylases Involved in Base Excision Repair May Be Associated with Cancer Risk in BRCA1 and BRCA2 Mutation Carriers

    DEFF Research Database (Denmark)

    Osorio, Ana; Milne, Roger L; Kuchenbaecker, Karoline

    2014-01-01

    Single Nucleotide Polymorphisms (SNPs) in genes involved in the DNA Base Excision Repair (BER) pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between one of th...

  6. DNA Glycosylases Involved in Base Excision Repair May Be Associated with Cancer Risk in BRCA1 and BRCA2 Mutation Carriers

    NARCIS (Netherlands)

    A. Osorio (Ana); R.L. Milne (Roger); K.B. Kuchenbaecker (Karoline); T. Vaclová (Tereza); G. Pita (Guillermo); R. Alonso (Rosario); P. Peterlongo (Paolo); I. Blanco (Ignacio); M. de La Hoya (Miguel); M. Durán (Mercedes); O. Díez (Orland); T. Ramon Y Cajal; I. Konstantopoulou (I.); C. Martínez-Bouzas (Cristina); R. Andrés Conejero (Raquel); P. Soucy (Penny); L. McGuffog (Lesley); D. Barrowdale (Daniel); A. Lee (Andrew); B. Arver (Brita Wasteson); J. Rantala (Johanna); N. Loman (Niklas); H. Ehrencrona (Hans); O.I. Olopade (Olofunmilayo); M.S. Beattie (Mary); S.M. Domchek (Susan); K.L. Nathanson (Katherine); R. Rebbeck (Timothy); B.K. Arun (Banu); B.Y. Karlan (Beth); C.S. Walsh (Christine); K.J. Lester (Kathryn); E.M. John (Esther); A.S. Whittemore (Alice); M.B. Daly (Mary); M.C. Southey (Melissa); J.L. Hopper (John); M.-B. Terry (Mary-Beth); S.S. Buys (Saundra); R. Janavicius (Ramunas); C.M. Dorfling (Cecilia); E.J. van Rensburg (Elizabeth); L. Steele (Linda); S.L. Neuhausen (Susan); Y.C. Ding (Yuan); T.V.O. Hansen (Thomas); L. Jønson (Lars); B. Ejlertsen (Bent); A-M. Gerdes (Anne-Marie); J. Infante (Jon); B. Herráez (Belén); L.T. Moreno (Leticia Thais); J.N. Weitzel (Jeffrey); J. Herzog (Josef); K. Weeman (Kisa); S. Manoukian (Siranoush); B. Peissel (Bernard); D. Zaffaroni (D.); G. Scuvera (Giulietta); B. Bonnani (Bernardo); F. Mariette (F.); S. Volorio (Sara); A. Viel (Alessandra); L. Varesco (Liliana); L. Papi (Laura); L. Ottini (Laura); M.G. Tibiletti (Maria Grazia); P. Radice (Paolo); D. Yannoukakos (Drakoulis); J. Garber; S.D. Ellis (Steve); D. Frost (Debra); R. Platte (Radka); E. Fineberg (Elena); D.G. Evans (Gareth); F. Lalloo (Fiona); L. Izatt (Louise); R. Eeles (Rosalind); J.W. Adlard (Julian); R. Davidson (Rosemarie); T.J. Cole (Trevor); D. Eccles (Diana); J. Cook (Jackie); S.V. Hodgson (Shirley); C. Brewer (Carole); M. Tischkowitz (Marc); F. Douglas (Fiona); M.E. Porteous (Mary); L. Side (Lucy); L.J. Walker (Lisa); P.J. Morrison (Patrick); A. Donaldson (Alan); J. Kennedy (John); C. Foo (Claire); A.K. Godwin (Andrew); R.K. Schmutzler (Rita); B. Wapenschmidt (Barbara); K. Rhiem (Kerstin); C.W. Engel (Christoph); A. Meindl (Alfons); N. Ditsch (Nina); N. Arnold (Norbert); H. Plendl (Hansjoerg); D. Niederacher (Dieter); C. Sutter (Christian); S. Wang-Gohrke (Shan); D. Steinemann (Doris); S. Preisler-Adams (Sabine); K. Kast (Karin); R. Varon-Mateeva (Raymonda); P.A. Gehrig (Paola A.); D. Stoppa-Lyonnet (Dominique); O. Sinilnikova (Olga); S. Mazoyer (Sylvie); F. Damiola (Francesca); B. Poppe (Bruce); K. Claes (Kathleen); M. Piedmonte (Marion); K. Tucker (Kathryn); F.J. Backes (Floor); P.M. Rodríguez; W. Brewster (Wendy); K. Wakeley (Katie); T. Rutherford (Thomas); T. Caldes (Trinidad); H. Nevanlinna (Heli); K. Aittomäki (Kristiina); M.A. Rookus (Matti); T.A.M. van Os (Theo); L. van der Kolk (Lizet); J.L. de Lange (J.); E.J. Meijers-Heijboer (Hanne); A.H. van der Hout (Annemarie); C.J. van Asperen (Christi); E.B. Gómez García (Encarna); N. Hoogerbrugge (Nicoline); J.M. Collée (Margriet); C.H.M. van Deurzen (Carolien); R.B. van der Luijt (Rob); P. Devilee (Peter); E. Olah (Edith); C. Lazaro (Conxi); A. Teulé (A.); M. Menéndez (Mireia); A. Jakubowska (Anna); C. Cybulski (Cezary); J. Gronwald (Jacek); J. Lubinski (Jan); K. Durda (Katarzyna); K. Jaworska-Bieniek (Katarzyna); O.T. Johannson (Oskar); C. Maugard; M. Montagna (Marco); S. Tognazzo (Silvia); P.J. Teixeira; S. Healey (Sue); C. Olswold (Curtis); L. Guidugli (Lucia); N.M. Lindor (Noralane); S. Slager (Susan); C. Szabo (Csilla); J. Vijai (Joseph); M. Robson (Mark); N. Kauff (Noah); L. Zhang (Lingling); R. Rau-Murthy (Rohini); A. Fink-Retter (Anneliese); C.F. Singer (Christian); C. Rappaport (Christine); D. Geschwantler Kaulich (Daphne); G. Pfeiler (Georg); M.-K. Tea; A. Berger (Annemarie); C. Phelan (Catherine); M.H. Greene (Mark); P.L. Mai (Phuong); F. Lejbkowicz (Flavio); I.L. Andrulis (Irene); A.M. Mulligan (Anna Marie); G. Glendon (Gord); A.E. Toland (Amanda); S.E. Bojesen (Stig); I.S. Pedersen (Inge Sokilde); L. Sunde (Lone); M. Thomassen (Mads); T.A. Kruse (Torben); U.B. Jensen; E. Friedman (Eitan); Y. Laitman (Yael); S.P. Shimon (Shani Paluch); J. Simard (Jacques); D.F. Easton (Douglas); K. Offit (Kenneth); F.J. Couch (Fergus); G. Chenevix-Trench (Georgia); A.C. Antoniou (Antonis); J. Benítez (Javier)

    2014-01-01

    textabstractSingle Nucleotide Polymorphisms (SNPs) in genes involved in the DNA Base Excision Repair (BER) pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between

  7. DNA glycosylases involved in base excision repair may be associated with cancer risk in BRCA1 and BRCA2 mutation carriers

    NARCIS (Netherlands)

    Osorio, A.; Milne, R.L.; Kuchenbaecker, K.; Vaclova, T.; Pita, G.; Alonso, R.; Peterlongo, P.; Blanco, I.; Hoya, M. de la; Duran, M.; Diez, O.; Ramon, Y.C.T.; Konstantopoulou, I.; Martinez-Bouzas, C.; Conejero, R. Andres; Soucy, P.; McGuffog, L.; Barrowdale, D.; Lee, A.; Swe, B.; Arver, B.; Rantala, J.; Loman, N.; Ehrencrona, H.; Olopade, O.I.; Beattie, M.S.; Domchek, S.M.; Nathanson, K.; Rebbeck, T.R.; Arun, B.K.; Karlan, B.Y.; Walsh, C.; Lester, J.; John, E.M.; Whittemore, A.S.; Daly, M.B.; Southey, M.; Hopper, J.; Terry, M.B.; Buys, S.S.; Janavicius, R.; Dorfling, C.M.; Rensburg, E.J. van; Steele, L.; Neuhausen, S.L.; Ding, Y.C.; Hansen, T.V.; Jonson, L.; Ejlertsen, B.; Gerdes, A.M.; Infante, M.; Herraez, B.; Moreno, L.T.; Weitzel, J.N.; Herzog, J.; Weeman, K.; Manoukian, S.; Peissel, B.; Zaffaroni, D.; Scuvera, G.; Bonanni, B.; Mariette, F.; Volorio, S.; Viel, A.; Varesco, L.; Papi, L.; Ottini, L.; Tibiletti, M.G.; Radice, P.; Yannoukakos, D.; Garber, J.; Ellis, S.; Frost, D.; Platte, R.; Fineberg, E.; Evans, G.; Lalloo, F.; Izatt, L.; Eeles, R.; Adlard, J.; Davidson, R.; Cole, T.; Eccles, D.; Cook, J; Hodgson, S.; Brewer, C.; Tischkowitz, M.; Douglas, F.; Porteous, M.; Side, L.; Walker, L.; Morrison, P.; Donaldson, A.; Kennedy, J.; Foo, C.; Godwin, A.K.; Schmutzler, R.K.; Wappenschmidt, B.; Rhiem, K.; Engel, C.; Hoogerbrugge-van der Linden, N.; et al.,

    2014-01-01

    Single Nucleotide Polymorphisms (SNPs) in genes involved in the DNA Base Excision Repair (BER) pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between one of the

  8. The mitochondrial DNA mutation ND6*14,484C associated with leber hereditary optic neuropathy, leads to deficiency of complex I of the respiratory chain

    NARCIS (Netherlands)

    Oostra, R. J.; van Galen, M. J.; Bolhuis, P. A.; Bleeker-Wagemakers, E. M.; van den Bogert, C.

    1995-01-01

    The electron transfer activity of Complex I of the respiratory chain and Complex I-linked ATP synthesis were investigated in leukocytes of four males affected by Leber hereditary optic neuropathy and a mutation in the ND6 gene at nucleotide position 14,484 of mtDNA. The electron transfer activity in

  9. INDUCTION OF DNA ADDUCTS, TUMORS, AND KI-RAS ONCOGENE MUTATIONS IN STRAIN A/J MOUSE LUNG BY IP. ADMINISTRATION OF DIBENZ[A,H]ANTHRACENE

    Science.gov (United States)

    Induction of DNA adducts, tumors, and Ki-ras oncogene mutations in strain AlJ mouse lung by ip. administration of dibenz[a,h]anthracene Previous studies of polycyclic aromatic hydrocarbon (P AH) induced lung tumors in the strain NJ mouse model system have demonstrated qua...

  10. A plastome mutation affects processing of both chloroplast and nuclear DNA-encoded plastid proteins.

    Science.gov (United States)

    Johnson, E M; Schnabelrauch, L S; Sears, B B

    1991-01-01

    Immunoblotting of a chloroplast mutant (pm7) of Oenothera showed that three proteins, cytochrome f and the 23 kDa and 16 kDa subunits of the oxygen-evolving subcomplex of photosystem II, were larger than the corresponding mature proteins of the wild type and, thus, appear to be improperly processed in pm7. The mutant is also chlorotic and has little or no internal membrane development in the plastids. The improperly processed proteins, and other proteins that are completely missing, represent products of both the plastid and nuclear genomes. To test for linkage of these defects, a green revertant of pm7 was isolated from cultures in which the mutant plastids were maintained in a nuclear background homozygous for the plastome mutator (pm) gene. In this revertant, all proteins analyzed co-reverted to the wild-type condition, indicating that a single mutation in a plastome gene is responsible for the complex phenotype of pm7. These results suggest that the defect in pm7 lies in a gene that affects a processing protease encoded in the chloroplast genome.

  11. A novel RUNX2 missense mutation predicted to disrupt DNA binding causes cleidocranial dysplasia in a large Chinese family with hyperplastic nails

    Directory of Open Access Journals (Sweden)

    Wang Xiaoqin

    2007-12-01

    Full Text Available Abstract Background Cleidocranial dysplasia (CCD is a dominantly inherited disease characterized by hypoplastic or absent clavicles, large fontanels, dental dysplasia, and delayed skeletal development. The purpose of this study is to investigate the genetic basis of Chinese family with CCD. Methods Here, a large Chinese family with CCD and hyperplastic nails was recruited. The clinical features displayed a significant intrafamilial variation. We sequenced the coding region of the RUNX2 gene for the mutation and phenotype analysis. Results The family carries a c.T407C (p.L136P mutation in the DNA- and CBFβ-binding Runt domain of RUNX2. Based on the crystal structure, we predict this novel missense mutation is likely to disrupt DNA binding by RUNX2, and at least locally affect the Runt domain structure. Conclusion A novel missense mutation was identified in a large Chinese family with CCD with hyperplastic nails. This report further extends the mutation spectrum and clinical features of CCD. The identification of this mutation will facilitate prenatal diagnosis and preimplantation genetic diagnosis.

  12. A switch from high-fidelity to error-prone DNA double-strand break repair underlies stress-induced mutation.

    Science.gov (United States)

    Ponder, Rebecca G; Fonville, Natalie C; Rosenberg, Susan M

    2005-09-16

    Special mechanisms of mutation are induced in microbes under growth-limiting stress causing genetic instability, including occasional adaptive mutations that may speed evolution. Both the mutation mechanisms and their control by stress have remained elusive. We provide evidence that the molecular basis for stress-induced mutagenesis in an E. coli model is error-prone DNA double-strand break repair (DSBR). I-SceI-endonuclease-induced DSBs strongly activate stress-induced mutations near the DSB, but not globally. The same proteins are required as for cells without induced DSBs: DSBR proteins, DinB-error-prone polymerase, and the RpoS starvation-stress-response regulator. Mutation is promoted by homology between cut and uncut DNA molecules, supporting a homology-mediated DSBR mechanism. DSBs also promote gene amplification. Finally, DSBs activate mutation only during stationary phase/starvation but will during exponential growth if RpoS is expressed. Our findings reveal an RpoS-controlled switch from high-fidelity to mutagenic DSBR under stress. This limits genetic instability both in time and to localized genome regions, potentially important evolutionary strategies.

  13. Mutation of Breast Cancer Cell Genomic DNA by APOBEC3B

    Science.gov (United States)

    2013-09-01

    Future work is needed to understand A3B regulation and the potential interaction with other oncogenes and tumour suppressors . For example, although...Limited. All rights reserved©2013 METHODS SUMMARY Flash -frozen tissues were obtained from the University of Minnesota Tissue Procurement Facility...used for RNA isolation, cDNA synthesis and qPCR as described14. Tissue RNA was from 100mg flash -frozen tissue disrupted by a 2-h water bath sonication in

  14. cDNA analyses of CAPN3 enhance mutation detection and reveal a low prevalence of LGMD2A patients in Denmark

    DEFF Research Database (Denmark)

    Duno, M.; Sveen, M.L.; Schwartz, M.

    2008-01-01

    Calpainopathy or limb-girdle muscular dystrophy type 2A (LGMD2A) is generally recognized as the most prevalent form of recessive LGMD and is caused by mutations in the CAPN3 gene. Out of a cohort of 119 patients fulfilling clinical criteria for LGMD2, referred to our neuromuscular clinic, 46 were....... In three other, only one heterozygous mutation could be identified on the genomic level; however, CAPN3 cDNA analyses demonstrated homozygosity for the mutant allele, indicating the presence of an unidentified allele that somehow compromise correct CAPN3 RNA processing. In the three remaining patients...... origin, indicating a five- to sixfold lower prevalence in Denmark compared to other European countries. A total of 16 different CAPN3 mutations were identified, of which 5 were novel. The present study demonstrates the value of cDNA analysis for CAPN3 in LGMD2A patients and indicates that calpainopathy...

  15. A novel mutation in the mitochondrial DNA cytochrome b gene (MTCYB) in a patient with mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes syndrome.

    Science.gov (United States)

    Emmanuele, Valentina; Sotiriou, Evangelia; Rios, Purificación Gutierrez; Ganesh, Jaya; Ichord, Rebecca; Foley, A Reghan; Akman, H Orhan; Dimauro, Salvatore

    2013-02-01

    Mutations in the mitochondrial DNA cytochrome b gene (MTCYB) have been commonly associated with isolated mitochondrial myopathy and exercise intolerance, rarely with multisystem disorders, and only once with a parkinsonism/mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS) overlap syndrome. Here, we describe a novel mutation (m.14864 T>C) in MTCYB in a 15-year-old girl with a clinical history of migraines, epilepsy, sensorimotor neuropathy, and strokelike episodes, a clinical picture reminiscent of MELAS.  The mutation, which changes a highly conserved cysteine to arginine at amino acid position 40 of cytochrome b, was heteroplasmic in muscle, blood, fibroblasts, and urinary sediment from the patient but absent in accessible tissues from her asymptomatic mother. This case demonstrates that MTCYB must be included in the already long list of mitochondrial DNA genes that have been associated with the MELAS phenotype.

  16. The mutation studies of mutagen-sensitive and DNA repair mutants of Chinese hamster fibroblasts

    International Nuclear Information System (INIS)

    Schultz, R.A.; Chang, C.C.; Trosko, J.E.

    1981-01-01

    We have previously reported the isolation and partial characterization of DNA repair and/or mutagen-sensitive mutant Chinese hamster cell strains. Here we present the results of a detailed study of the ultraviolet light (UV)-induced mutability of one of these strains, UVs-7, and provide preliminary mutability data on two additional lines, UVr-23 and UVs-40. UVs-7 in extremely deficient in unscheduled DNA synthesis (UDS) but only slightly more sensitive to UV than the parental line. When examined for the UV-inducibility of mutants resistant to ouabain, 6-thioguanine, or diphtheria toxin, UVs-7 was found to be hypermutable at all three loci as compared to the parental line. The degree of hypermutability was not the same for any two loci. UVs-40, a highly UV-sensitive strain, was also found to be hypermutable at the ouabain-resistant (ouar) locus. UVr-23, which is UV-resistant and more proficient at UDS than the parental line, appeared to exhibit a tendency toward hypomutability at both the ouabain(ouar) and 6-thioguanine--resistant (6TGr) loci. Further characterization of all these lines should aid in delineating mammalian mechanisms of DNA repair and mutagenesis

  17. Base-pairing preferences, physicochemical properties and mutational behaviour of the DNA lesion 8-nitroguanine.

    Science.gov (United States)

    Bhamra, Inder; Compagnone-Post, Patricia; O'Neil, Ian A; Iwanejko, Lesley A; Bates, Andrew D; Cosstick, Richard

    2012-11-01

    8-Nitro-2'-deoxyguanosine (8-nitrodG) is a relatively unstable, mutagenic lesion of DNA that is increasingly believed to be associated with tissue inflammation. Due to the lability of the glycosidic bond, 8-nitrodG cannot be incorporated into oligodeoxynucleotides (ODNs) by chemical DNA synthesis and thus very little is known about its physicochemical properties and base-pairing preferences. Here we describe the synthesis of 8-nitro-2'-O-methylguanosine, a ribonucleoside analogue of this lesion, which is sufficiently stable to be incorporated into ODNs. Physicochemical studies demonstrated that 8-nitro-2'-O-methylguanosine adopts a syn conformation about the glycosidic bond; thermal melting studies and molecular modelling suggest a relatively stable syn-8-nitroG·anti-G base pair. Interestingly, when this lesion analogue was placed in a primer-template system, extension of the primer by either avian myeloblastosis virus reverse transcriptase (AMV-RT) or human DNA polymerase β (pol β), was significantly impaired, but where incorporation opposite 8-nitroguanine did occur, pol β showed a 2:1 preference to insert dA over dC, while AMV-RT incorporated predominantly dC. The fact that no 8-nitroG·G base pairing is seen in the primer extension products suggests that the polymerases may discriminate against this pairing system on the basis of its poor geometric match to a Watson-Crick pair.

  18. Base-pairing preferences, physicochemical properties and mutational behaviour of the DNA lesion 8-nitroguanine†

    Science.gov (United States)

    Bhamra, Inder; Compagnone-Post, Patricia; O’Neil, Ian A.; Iwanejko, Lesley A.; Bates, Andrew D.; Cosstick, Richard

    2012-01-01

    8-Nitro-2′-deoxyguanosine (8-nitrodG) is a relatively unstable, mutagenic lesion of DNA that is increasingly believed to be associated with tissue inflammation. Due to the lability of the glycosidic bond, 8-nitrodG cannot be incorporated into oligodeoxynucleotides (ODNs) by chemical DNA synthesis and thus very little is known about its physicochemical properties and base-pairing preferences. Here we describe the synthesis of 8-nitro-2′-O-methylguanosine, a ribonucleoside analogue of this lesion, which is sufficiently stable to be incorporated into ODNs. Physicochemical studies demonstrated that 8-nitro-2′-O-methylguanosine adopts a syn conformation about the glycosidic bond; thermal melting studies and molecular modelling suggest a relatively stable syn-8-nitroG·anti-G base pair. Interestingly, when this lesion analogue was placed in a primer-template system, extension of the primer by either avian myeloblastosis virus reverse transcriptase (AMV-RT) or human DNA polymerase β (pol β), was significantly impaired, but where incorporation opposite 8-nitroguanine did occur, pol β showed a 2:1 preference to insert dA over dC, while AMV-RT incorporated predominantly dC. The fact that no 8-nitroG·G base pairing is seen in the primer extension products suggests that the polymerases may discriminate against this pairing system on the basis of its poor geometric match to a Watson–Crick pair. PMID:22965127

  19. Mutation Breeding Newsletter. No. 39

    International Nuclear Information System (INIS)

    1992-01-01

    This newsletter contains brief articles on the use of radiation to induce mutations in plants; radiation-induced mutants in Chrysanthemum; disrupting the association between oil and protein content in soybean seeds; mutation studies on bougainvillea; a new pepper cultivar; and the use of mutation induction to improve the quality of yam beans. A short review of the seminar on the use of mutation and related biotechnology for crop improvement in the Middle East and Mediterranean regions, and a description of a Co-ordinated Research Programme on the application of DNA-based marker mutations for the improvement of cereals and other sexually reproduced crop species are also included. Two tables are given: these are based on the ''FAO/IAEA Mutant Varieties Database'' and show the number of mutated varieties and the number of officially released mutant varieties in particular crops/species. Refs and tabs

  20. Structure-guided mutational analysis of the nucleotidyltransferase domain of Escherichia coli NAD+-dependent DNA ligase (LigA).

    Science.gov (United States)

    Zhu, Hui; Shuman, Stewart

    2005-04-01

    NAD+-dependent DNA ligase (LigA) is essential for bacterial growth and a potential target for antimicrobial drug discovery. Here we queried the role of 14 conserved amino acids of Escherichia coli LigA by alanine scanning and thereby identified five new residues within the nucleotidyltransferase domain as being essential for LigA function in vitro and in vivo. Structure activity relationships were determined by conservative mutagenesis for the Glu-173, Arg-200, Arg-208, and Arg-277 side chains, as well as four other essential side chains that had been identified previously (Lys-115, Asp-117, Asp-285, and Lys-314). In addition, we identified Lys-290 as important for LigA activity. Reference to the structure of Enterococcus faecalis LigA allowed us to discriminate three classes of essential/important side chains that: (i) contact NAD+ directly (Lys-115, Glu-173, Lys-290, and Lys-314); (ii) comprise the interface between the NMN-binding domain (domain Ia) and the nucleotidyltransferase domain or comprise part of a nick-binding site on the surface of the nucleotidyltransferase domain (Arg-200 and Arg-208); or (iii) stabilize the active site fold of the nucleotidyltransferase domain (Arg-277). Analysis of mutational effects on the isolated ligase adenylylation and phosphodiester formation reactions revealed different functions for essential side chains at different steps of the DNA ligase pathway, consistent with the proposal that the active site is serially remodeled as the reaction proceeds.

  1. A common mutation in the 5,10-methylenetetrahydrofolate reductase gene affects genomic DNA methylation through an interaction with folate status

    Science.gov (United States)

    Friso, Simonetta; Choi, Sang-Woon; Girelli, Domenico; Mason, Joel B.; Dolnikowski, Gregory G.; Bagley, Pamela J.; Olivieri, Oliviero; Jacques, Paul F.; Rosenberg, Irwin H.; Corrocher, Roberto; Selhub, Jacob

    2002-01-01

    DNA methylation, an essential epigenetic feature of DNA that modulates gene expression and genomic integrity, is catalyzed by methyltransferases that use the universal methyl donor S-adenosyl-l-methionine. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the synthesis of 5-methyltetrahydrofolate (5-methylTHF), the methyl donor for synthesis of methionine from homocysteine and precursor of S-adenosyl-l-methionine. In the present study we sought to determine the effect of folate status on genomic DNA methylation with an emphasis on the interaction with the common C677T mutation in the MTHFR gene. A liquid chromatography/MS method for the analysis of nucleotide bases was used to assess genomic DNA methylation in peripheral blood mononuclear cell DNA from 105 subjects homozygous for this mutation (T/T) and 187 homozygous for the wild-type (C/C) MTHFR genotype. The results show that genomic DNA methylation directly correlates with folate status and inversely with plasma homocysteine (tHcy) levels (P < 0.01). T/T genotypes had a diminished level of DNA methylation compared with those with the C/C wild-type (32.23 vs.62.24 ng 5-methylcytosine/μg DNA, P < 0.0001). When analyzed according to folate status, however, only the T/T subjects with low levels of folate accounted for the diminished DNA methylation (P < 0.0001). Moreover, in T/T subjects DNA methylation status correlated with the methylated proportion of red blood cell folate and was inversely related to the formylated proportion of red blood cell folates (P < 0.03) that is known to be solely represented in those individuals. These results indicate that the MTHFR C677T polymorphism influences DNA methylation status through an interaction with folate status. PMID:11929966

  2. Development of the adverse outcome pathway "alkylation of DNA in male premeiotic germ cells leading to heritable mutations" using the OECD's users' handbook supplement.

    Science.gov (United States)

    Yauk, Carole L; Lambert, Iain B; Meek, M E Bette; Douglas, George R; Marchetti, Francesco

    2015-12-01

    The Organisation for Economic Cooperation and Development's (OECD) Adverse Outcome Pathway (AOP) programme aims to develop a knowledgebase of all known pathways of toxicity that lead to adverse effects in humans and ecosystems. A Users' Handbook was recently released to provide supplementary guidance on AOP development. This article describes one AOP-alkylation of DNA in male premeiotic germ cells leading to heritable mutations. This outcome is an important regulatory endpoint. The AOP describes the biological plausibility and empirical evidence supporting that compounds capable of alkylating DNA cause germ cell mutations and subsequent mutations in the offspring of exposed males. Alkyl adducts are subject to DNA repair; however, at high doses the repair machinery becomes saturated. Lack of repair leads to replication of alkylated DNA and ensuing mutations in male premeiotic germ cells. Mutations that do not impair spermatogenesis persist and eventually are present in mature sperm. Thus, the mutations are transmitted to the offspring. Although there are some gaps in empirical support and evidence for essentiality of the key events for certain aspects of this AOP, the overall AOP is generally accepted as dogma and applies broadly to any species that produces sperm. The AOP was developed and used in an iterative process to test and refine the Users' Handbook, and is one of the first publicly available AOPs. It is our hope that this AOP will be leveraged to develop other AOPs in this field to advance method development, computational models to predict germ cell effects, and integrated testing strategies. © 2015 Her Majesty the Queen in Right of Canada.

  3. Investigating the structural impacts of I64T and P311S mutations in APE1-DNA complex: a molecular dynamics approach.

    Directory of Open Access Journals (Sweden)

    C George Priya Doss

    Full Text Available Elucidating the molecular dynamic behavior of Protein-DNA complex upon mutation is crucial in current genomics. Molecular dynamics approach reveals the changes on incorporation of variants that dictate the structure and function of Protein-DNA complexes. Deleterious mutations in APE1 protein modify the physicochemical property of amino acids that affect the protein stability and dynamic behavior. Further, these mutations disrupt the binding sites and prohibit the protein to form complexes with its interacting DNA.In this study, we developed a rapid and cost-effective method to analyze variants in APE1 gene that are associated with disease susceptibility and evaluated their impacts on APE1-DNA complex dynamic behavior. Initially, two different in silico approaches were used to identify deleterious variants in APE1 gene. Deleterious scores that overlap in these approaches were taken in concern and based on it, two nsSNPs with IDs rs61730854 (I64T and rs1803120 (P311S were taken further for structural analysis.Different parameters such as RMSD, RMSF, salt bridge, H-bonds and SASA applied in Molecular dynamic study reveals that predicted deleterious variants I64T and P311S alters the structure as well as affect the stability of APE1-DNA interacting functions. This study addresses such new methods for validating functional polymorphisms of human APE1 which is critically involved in causing deficit in repair capacity, which in turn leads to genetic instability and carcinogenesis.

  4. Influence of anoxia on the induction of mutations by phenylalanine radicals during gamma-irradiation of plasmid DNA in aqueous solution.

    Science.gov (United States)

    Kuipers, Gitta K; Slotman, Ben J; Reitsma-Wijker, Carola A; van Andel, Rob J; Poldervaart, Hester A; Lafleur, M Vincent M

    2004-12-21

    When DNA is irradiated in aqueous solution, most of the damage is inflicted by water-derived radicals. This is called the indirect effect of ionizing radiation. However in whole cells not only the primary formed water radicals play a role, because some cellular compounds form secondary radicals which can also damage DNA. It is known that the amino acid phenylalanine is able to react with water radicals, resulting in the production of secondary phenylalanine radicals which can damage and inactivate DNA. In a previous study the influence of the presence of phenylalanine during gamma-irradiation of DNA in aqueous solution under oxic conditions was studied. Under anoxic irradiation conditions different amounts and types of reactive water-derived radicals are formed compared to oxic conditions and also different phenylalanine radicals are formed. Therefore, this study examines the influence of the presence of phenylalanine under anoxic conditions on the gamma-radiation-induced mutation spectrum. The results indicate that phenylalanine radicals are damaging to DNA, but less effective compared to primary water radicals. On the mutational level, in the presence of phenylalanine radicals under anoxic conditions, the amount of mutations on G:C base pairs was significantly decreased as compared to oxic conditions. Furthermore, the results of this study indicate that nucleotide excision repair is involved in repair of both inactivating and mutagenic damage induced by phenylalanine radicals under anoxic conditions.

  5. Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is associated with G to A mutations in K-ras in colorectal tumorigenesis.

    Science.gov (United States)

    Esteller, M; Toyota, M; Sanchez-Cespedes, M; Capella, G; Peinado, M A; Watkins, D N; Issa, J P; Sidransky, D; Baylin, S B; Herman, J G

    2000-05-01

    O6-methylguanine DNA methyltransferase (MGMT) is a DNA repair protein that removes mutagenic and cytotoxic adducts from the O6 position of guanine. O6-methylguanine mispairs with thymine during replication, and if the adduct is not removed, this results in conversion from a guanine-cytosine pair to an adenine-thymine pair. In vitro assays show that MGMT expression avoids G to A mutations and MGMT transgenic mice are protected against G to A transitions at ras genes. We have recently demonstrated that the MGMT gene is silenced by promoter methylation in many human tumors, including colorectal carcinomas. To study the relevance of defective MGMT function by aberrant methylation in relation to the presence of K-ras mutations, we studied 244 colorectal tumor samples for MGMT promoter hypermethylation and K-ras mutational status. Our results show a clear association between the inactivation of MGMT by promoter hypermethylation and the appearance of G to A mutations at K-ras: 71% (36 of 51) of the tumors displaying this particular type of mutation had abnormal MGMT methylation, whereas only 32% (12 of 37) of those with other K-ras mutations not involving G to A transitions and 35% (55 of 156) of the tumors without K-ras mutations demonstrated MGMT methylation (P = 0.002). In addition, MGMT loss associated with hypermethylation was observed in the small adenomas, including those that do not yet contain K-ras mutations. Hypermethylation of other genes such as p16INK4a and p14ARF was not associated with either MGMT hypermethylation or K-ras mutation. Our data suggest that epigenetic silencing of MGMT by promoter hypermethylation may lead to a particular genetic change in human cancer, specifically G to A transitions in the K-ras oncogene.

  6. Hearing loss in a patient with the myopathic form of mitochondrial DNA depletion syndrome and a novel mutation in the TK2 gene.

    Science.gov (United States)

    Martí, Ramon; Nascimento, Andrés; Colomer, Jaume; Lara, Mari C; López-Gallardo, Ester; Ruiz-Pesini, Eduardo; Montoya, Julio; Andreu, Antoni L; Briones, Paz; Pineda, Mercè

    2010-08-01

    Mitochondrial DNA (mtDNA) depletion syndrome (MDS) is a devastating disorder of infancy caused by a significant reduction of the number of copies of mitochondrial DNA in one or more tissues. We report a Spanish patient with the myopathic form of MDS, harboring two mutations in the thymidine kinase 2 gene (TK2): a previously reported deletion (p.K244del) and a novel nucleotide duplication in the exon 2, generating a frameshift and premature stop codon. Sensorineural hearing loss was a predominant symptom in the patient and a novel feature of MDS due to TK2 mutations. The patient survived up to the age of 8.5 y, which confirms that survival above the age of 5 y is not infrequent in patients with MDS due to TK2 deficiency.

  7. Use of DNA markers in forest tree improvement research

    Science.gov (United States)

    D.B. Neale; M.E. Devey; K.D. Jermstad; M.R. Ahuja; M.C. Alosi; K.A. Marshall

    1992-01-01

    DNA markers are rapidly being developed for forest trees. The most important markers are restriction fragment length polymorphisms (RFLPs), polymerase chain reaction- (PCR) based markers such as random amplified polymorphic DNA (RAPD), and fingerprinting markers. DNA markers can supplement isozyme markers for monitoring tree improvement activities such as; estimating...

  8. Mutational specificity of alkylating agents and the influence of DNA repair

    Energy Technology Data Exchange (ETDEWEB)

    Horsfall, M.J.; Gordon, A.J.; Burns, P.A.; Zielenska, M.; van der Vliet, G.M.; Glickman, B.W. (York Univ., Toronto, Ontario (Canada))

    1990-01-01

    Alkylating treatments predominantly induce G:C = greater than A:T transitions, consistent with the predicted significance of the miscoding potential of the O6-alG lesion. However, the frequency and distribution of these events induced by any one compound may be diagnostic. SN1 agents that act via an alkyldiazonium cation, such as the N-nitroso compounds, preferentially generate G:C = greater than A:T transitions at 5'-RG-3' sites, while the more SN2 alkylsulfates and alkylalkane-sulfonates do not. The precise nature of this site bias and the possibility of strand bias are target dependent. The extent of this site bias and the contribution of other base substitutions are substituent size dependent. A similar 5'-RT-3' effect is seen for A:T = greater than G:C transitions, presumably directed by O4-alT lesions. The 5'-RG-3' effect, at least, likely reflects a deposition specificity arising from some aspect of helix geometry, although it may be further exaggerated by alkylation-specific repair. Excision repair appears to preferentially reduce the occurrence of ethylation-induced G:C = greater than A:T and A:T = greater than G:C transitions at sites flanked by A:T base pairs. This may be due to an enhancement of the helical distortion imposed by damage at such positions. A similar effect is not seen for methylation-induced mutations and in the case of propyl adducts, the influence of excision repair on the ultimate distribution of mutation cannot be as easily defined with respect to neighbouring sequence. 199 references.

  9. Germline PMS2 and somatic POLE exonuclease mutations cause hypermutability of the leading DNA strand in biallelic mismatch repair deficiency syndrome brain tumours.

    Science.gov (United States)

    Andrianova, Maria A; Chetan, Ghati Kasturirangan; Sibin, Madathan Kandi; Mckee, Thomas; Merkler, Doron; Narasinga, Rao Kvl; Ribaux, Pascale; Blouin, Jean-Louis; Makrythanasis, Periklis; Seplyarskiy, Vladimir B; Antonarakis, Stylianos E; Nikolaev, Sergey I

    2017-11-01

    Biallelic mismatch repair deficiency (bMMRD) in tumours is frequently associated with somatic mutations in the exonuclease domains of DNA polymerases POLE or POLD1, and results in a characteristic mutational profile. In this article, we describe the genetic basis of ultramutated high-grade brain tumours in the context of bMMRD. We performed exome sequencing of two second-cousin patients from a large consanguineous family of Indian origin with early onset of high-grade glioblastoma and astrocytoma. We identified a germline homozygous nonsense variant, p.R802*, in the PMS2 gene. Additionally, by genome sequencing of these tumours, we found extremely high somatic mutation rates (237/Mb and 123/Mb), as well as somatic mutations in the proofreading domain of POLE polymerase (p.P436H and p.L424V), which replicates the leading DNA strand. Most interestingly, we found, in both cancers, that the vast majority of mutations were consistent with the signature of POLE exo - , i.e. an abundance of C>A and C>T mutations, particularly in special contexts, on the leading strand. We showed that the fraction of mutations under positive selection among mutations in tumour suppressor genes is more than two-fold lower in ultramutated tumours than in other glioblastomas. Genetic analyses enabled the diagnosis of the two consanguineous childhood brain tumours as being due to a combination of PMS2 germline and POLE somatic variants, and confirmed them as bMMRD/POLE exo - disorders. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  10. Molecular insight into mitochondrial DNA depletion syndrome in two patients with novel mutations in the deoxyguanosine kinase and thymidine kinase 2 genes.

    Science.gov (United States)

    Wang, Liya; Limongelli, Anna; Vila, Maya R; Carrara, Franco; Zeviani, Massimo; Eriksson, Staffan

    2005-01-01

    Thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) are the two key enzymes in mitochondrial DNA (mtDNA) precursor synthesis. Deficiencies in TK2 or dGK activity, due to genetic alteration, have been shown to cause tissue-specific depletion of mtDNA. In the case of TK2 deficiency, affected individuals suffer severe myopathy and, in the case of dGK deficiency, devastating liver or multi-systemic disease. Here, we report clinical and biochemical findings from two patients with mtDNA depletion syndrome. Patient A was a compound heterozygote carrying the previously reported T77M mutation and a novel mutation (R161K) in the TK2 gene. Patient B carried a novel mutation (L250S) in the dGK gene. The clinical symptoms of patient A included muscular weakness and exercise intolerance due to a severe mitochondrial myopathy associated with a 92% reduction in mtDNA. There was minimal involvement of other organs. Patient B suffered from rapidly progressive, early onset fatal liver failure associated with profoundly decreased mtDNA levels in liver and, to a lesser extent, in skeletal muscle. Site-directed mutagenesis was used to introduce the mutations detected in patients A and B into the TK2 and dGK cDNAs, respectively. We then characterized each of these recombinant enzymes. Catalytic activities of the three mutant enzymes were reduced to about 2-4% for TK2 and 0.5% for dGK as compared to the wild-type enzymes. Altered competition between dCyd and dThd was observed for the T77M mutant. The residual activities of the two mitochondrial enzymes correlated directly with disease development.

  11. Determining the role of missense mutations in the POU domain of HNF1A that reduce the DNA-binding affinity: A computational approach.

    Directory of Open Access Journals (Sweden)

    Sneha P

    Full Text Available Maturity-onset diabetes of the young type 3 (MODY3 is a non-ketotic form of diabetes associated with poor insulin secretion. Over the past years, several studies have reported the association of missense mutations in the Hepatocyte Nuclear Factor 1 Alpha (HNF1A with MODY3. Missense mutations in the POU homeodomain (POUH of HNF1A hinder binding to the DNA, thereby leading to a dysfunctional protein. Missense mutations of the HNF1A were retrieved from public databases and subjected to a three-step computational mutational analysis to identify the underlying mechanism. First, the pathogenicity and stability of the mutations were analyzed to determine whether they alter protein structure and function. Second, the sequence conservation and DNA-binding sites of the mutant positions were assessed; as HNF1A protein is a transcription factor. Finally, the biochemical properties of the biological system were validated using molecular dynamic simulations in Gromacs 4.6.3 package. Two arginine residues (131 and 203 in the HNF1A protein are highly conserved residues and contribute to the function of the protein. Furthermore, the R131W, R131Q, and R203C mutations were predicted to be highly deleterious by in silico tools and showed lower binding affinity with DNA when compared to the native protein using the molecular docking analysis. Triplicate runs of molecular dynamic (MD simulations (50ns revealed smaller changes in patterns of deviation, fluctuation, and compactness, in complexes containing the R131Q and R131W mutations, compared to complexes containing the R203C mutant complex. We observed reduction in the number of intermolecular hydrogen bonds, compactness, and electrostatic potential, as well as the loss of salt bridges, in the R203C mutant complex. Substitution of arginine with cysteine at position 203 decreases the affinity of the protein for DNA, thereby destabilizing the protein. Based on our current findings, the MD approach is an important

  12. A novel mutation in homeobox DNA binding domain of HOXC13 gene underlies pure hair and nail ectodermal dysplasia (ECTD9) in a Pakistani family.

    Science.gov (United States)

    Khan, Anwar Kamal; Muhammad, Noor; Aziz, Abdul; Khan, Sher Alam; Shah, Khadim; Nasir, Abdul; Khan, Muzammil Ahmad; Khan, Saadullah

    2017-04-12

    Pure hair and nail ectodermal dysplasia (PHNED) is a congenital disorder of hair abnormalities and nail dysplasia. Both autosomal recessive and dominant inheritance fashion of PHNED occurs. In literature, to date, five different forms of PHNED have been reported at molecular level, having three genes known and two loci with no gene yet. In this study, a four generations consanguineous family of Pakistani origin with autosomal recessive PHNED was investigated. Affected members exhibited PHNED phenotypes with involvement of complete hair loss and nail dysplasia. To screen for mutation in the genes (HOXC13, KRT74, KRT85), its coding exons and exons-intron boundaries were sequenced. The 3D models of normal and mutated HOXC13 were predicted by using homology modeling. Through investigating the family to known loci, the family was mapped to ectodermal dysplasia 9 (ECTD9) loci with genetic address of 12q13.13. Mutation screening revealed a novel missense mutation (c.929A > C; p.Asn310Thr) in homeobox DNA binding domain of HOXC13 gene in affected members of the family. Due to mutation, loss of hydrogen bonding and difference in potential energy occurs, which may resulting in alteration of protein function. This is the first mutation reported in homeodomain, while 5 th mutation reported in HOXC13 gene causing PHNED.

  13. Germline Mutations in PALB2, BRCA1, and RAD51C, Which Regulate DNA Recombination Repair, in Patients with Gastric Cancer

    Science.gov (United States)

    Sahasrabudhe, Ruta; Lott, Paul; Bohorquez, Mabel; Toal, Ted; Estrada, Ana P.; Suarez, John J.; Brea-Fernández, Alejandro; Cameselle-Teijeiro, José; Pinto, Carla; Ramos, Irma; Mantilla, Alejandra; Prieto, Rodrigo; Corvalan, Alejandro; Norero, Enrique; Alvarez, Carolina; Tapia, Teresa; Carvallo, Pilar; Gonzalez, Luz M.; Cock-Rada, Alicia; Solano, Angela; Neffa, Florencia; Valle, Adriana Della; Yau, Chris; Soares, Gabriela; Borowsky, Alexander; Hu, Nan; He, Li-Ji; Han, Xiao-You; Taylor, Philip R.; Goldstein, Alisa M.; Torres, Javier; Echeverry, Magdalena; Ruiz-Ponte, Clara; Teixeira, Manuel R.; Carvajal Carmona, Luis G.

    2016-01-01

    Up to 10% of cases of gastric cancer are familial, but so far, only mutations in CDH1 have been associated with gastric cancer risk. To identify genetic variants that affect risk for gastric cancer, we collected blood samples from 28 patients with hereditary diffuse gastric cancer (HDGC) not associated with mutations in CDH1 and performed whole-exome sequence analysis. We then analyzed sequences of candidate genes in 333 independent HDGC and non-HDGC cases. We identified 11 cases with mutations in PALB2, BRCA1, or RAD51C genes, which regulate homologous DNA recombination. We found these mutations in 2 of 31 patients with HDGC (6.5%) and 9 of 331 patients with sporadic gastric cancer (2.8%). Most of these mutations had been previously associated with other types of tumors and partially co-segregated with gastric cancer in our study. Tumors that developed in patients with these mutations had a mutation signature associated with somatic homologous recombination deficiency. Our findings indicate that defects in homologous recombination increase risk for gastric cancer. PMID:28024868

  14. Rapid point-of-care testing for epidermal growth factor receptor gene mutations in patients with lung cancer using cell-free DNA from cytology specimen supernatants.

    Science.gov (United States)

    Asaka, Shiho; Yoshizawa, Akihiko; Saito, Kazusa; Kobayashi, Yukihiro; Yamamoto, Hiroshi; Negishi, Tatsuya; Nakata, Rie; Matsuda, Kazuyuki; Yamaguchi, Akemi; Honda, Takayuki

    2018-06-01

    Epidermal growth factor receptor (EGFR) mutations are associated with responses to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in non-small-cell lung cancer (NSCLC). Our previous study revealed a rapid point-of-care system for detecting EGFR mutations. This system analyzes cell pellets from cytology specimens using droplet-polymerase chain reaction (d-PCR), and has a reaction time of 10 min. The present study aimed to validate the performance of the EGFR d-PCR assay using cell-free DNA (cfDNA) from supernatants obtained from cytology specimens. Assay results from cfDNA supernatant analyses were compared with those from cell pellets for 90 patients who were clinically diagnosed with, or suspected of having, lung cancer (80 bronchial lavage fluid samples, nine pleural effusion samples and one spinal fluid sample). EGFR mutations were identified in 12 and 15 cases using cfDNA supernatants and cell pellets, respectively. The concordance rates between cfDNA-supernatant and cell‑pellet assay results were 96.7% [kappa coefficient (K)=0.87], 98.9% (K=0.94), 98.9% (K=0.79) and 98.9% (K=0.79) for total EGFR mutations, L858R, E746_A750del and T790M, respectively. All 15 patients with EGFR mutation-positive results, as determined by EGFR d-PCR assay using cfDNA supernatants or cell pellets, also displayed positive results by conventional EGFR assays using tumor tissue or cytology specimens. Notably, EGFR mutations were even detected in five cfDNA supernatants for which the cytological diagnoses of the corresponding cell pellets were 'suspicious for malignancy', 'atypical' or 'negative for malignancy.' In conclusion, this rapid point-of-care system may be considered a promising novel screening method that may enable patients with NSCLC to receive EGFR-TKI therapy more rapidly, whilst also reserving cell pellets for additional morphological and molecular analyses.

  15. Was Muller's 1946 Nobel Prize research for radiation-induced gene mutations peer-reviewed?

    Science.gov (United States)

    Calabrese, Edward J

    2018-06-06

    This historical analysis indicates that it is highly unlikely that the Nobel Prize winning research of Hermann J. Muller was peer-reviewed. The published paper of Muller lacked a research methods section, cited no references, and failed to acknowledge and discuss the work of Gager and Blakeslee (PNAS 13:75-79, 1927) that claimed to have induced gene mutation via ionizing radiation six months prior to Muller's non-data Science paper (Muller, Science 66(1699):84-87, 1927a). Despite being well acclimated into the scientific world of peer-review, Muller choose to avoid the peer-review process on his most significant publication. It appears that Muller's actions were strongly influenced by his desire to claim primacy for the discovery of gene mutation. The actions of Muller have important ethical lessons and implications today, when self-interest trumps one's obligations to society and the scientific culture that supports the quest for new knowledge and discovery.

  16. Research progress on the mutation of diploid microspore in Ginkgo biloba L

    International Nuclear Information System (INIS)

    Sun Yuhan; Li Yun; Yang Nina; Cheng Jinxin; Hu Junyan; Wang Yaru

    2011-01-01

    With the gradual development of Ginkgo's comprehensive utilization in recent years, the requirements of leaf yield and growth rate are increased year by year. And the results show that the triploid variety is predominant in these sides, so the ploidy breeding research has important promote significance to variety improvement in Ginkgo. The paper briefly introduced the prophase research of Ginkgo's ploidy breeding, and made a comprehensive discussion of the mutation research and influence factors in the diploid microspore of Ginkgo. (authors)

  17. Evaluation of the metabolic fate of munitions material (TNT & RDX) in plant systems. Initial assessment of plant DNA mutation spectra as a biomarker

    Energy Technology Data Exchange (ETDEWEB)

    Leung, F.; Cataldo, D.A.; Fellows, R.J.; Jarrell, A.E.; Harvey, S.D.

    1995-09-01

    Munitions material can enter the environment as a result of manufacturing activities and field usage. Predictor methodologies, or biomarkers would enhance evaluation of environmental impacts. The goal of this exploratory study deoxyribonucleic acid (DNA) mutation frequency as a biomarker for munitions exposure. The approach e resolution of an effective repetitive sequence probe for the identification of characteristic mutations, and (2) the development of a testing media [a clonal cell line of carrot (Daucus carota) spension cells]. Commercially available probes demonstrated marginal resolution therefore a low-C{sub o}t library was then constructed. Three colonies from the low-C{sub o}t DNA library were screened and the DNA isolates sequenced. A suspension culture of carrot (Daucus carota) was developed. A mutation spectra experiment was initiated at a 10-mg TNT/L exposure concentration with the attempt to clone over 1500 single TNT-exposed cells. Over the following six months greater than 98% of the initially isolated cells were unable to survive and produce micro calluses. The remaining calli were too few to be statistically significant and the experiment was terminated. The biomarker concept itself remains to be disproved, but the need for large numbers of uniform clones to differentiate true mutations suggest that more direct techniques using whole tissues need to be developed.

  18. Pms2 and uracil-DNA glycosylases act jointly in the mismatch repair pathway to generate Ig gene mutations at A-T base pairs.

    Science.gov (United States)

    Girelli Zubani, Giulia; Zivojnovic, Marija; De Smet, Annie; Albagli-Curiel, Olivier; Huetz, François; Weill, Jean-Claude; Reynaud, Claude-Agnès; Storck, Sébastien

    2017-04-03

    During somatic hypermutation (SHM) of immunoglobulin genes, uracils introduced by activation-induced cytidine deaminase are processed by uracil-DNA glycosylase (UNG) and mismatch repair (MMR) pathways to generate mutations at G-C and A-T base pairs, respectively. Paradoxically, the MMR-nicking complex Pms2/Mlh1 is apparently dispensable for A-T mutagenesis. Thus, how detection of U:G mismatches is translated into the single-strand nick required for error-prone synthesis is an open question. One model proposed that UNG could cooperate with MMR by excising a second uracil in the vicinity of the U:G mismatch, but it failed to explain the low impact of UNG inactivation on A-T mutagenesis. In this study, we show that uracils generated in the G1 phase in B cells can generate equal proportions of A-T and G-C mutations, which suggests that UNG and MMR can operate within the same time frame during SHM. Furthermore, we show that Ung -/- Pms2 -/- mice display a 50% reduction in mutations at A-T base pairs and that most remaining mutations at A-T bases depend on two additional uracil glycosylases, thymine-DNA glycosylase and SMUG1. These results demonstrate that Pms2/Mlh1 and multiple uracil glycosylases act jointly, each one with a distinct strand bias, to enlarge the immunoglobulin gene mutation spectrum from G-C to A-T bases. © 2017 Girelli Zubani et al.

  19. Parkinson's disease brain mitochondria have impaired respirasome assembly, age-related increases in distribution of oxidative damage to mtDNA and no differences in heteroplasmic mtDNA mutation abundance

    Directory of Open Access Journals (Sweden)

    Keeney Paula M

    2009-09-01

    Full Text Available Abstract Background Sporadic Parkinson's disease (sPD is a nervous system-wide disease that presents with a bradykinetic movement disorder and is frequently complicated by depression and cognitive impairment. sPD likely has multiple interacting causes that include increased oxidative stress damage to mitochondrial components and reduced mitochondrial bioenergetic capacity. We analyzed mitochondria from postmortem sPD and CTL brains for evidence of oxidative damage to mitochondrial DNA (mtDNA, heteroplasmic mtDNA point mutations and levels of electron transport chain proteins. We sought to determine if sPD brains possess any mtDNA genotype-respiratory phenotype relationships. Results Treatment of sPD brain mtDNA with the mitochondrial base-excision repair enzyme 8-oxyguanosine glycosylase-1 (hOGG1 inhibited, in an age-dependent manner, qPCR amplification of overlapping ~2 kbase products; amplification of CTL brain mtDNA showed moderate sensitivity to hOGG1 not dependent on donor age. hOGG1 mRNA expression was not different between sPD and CTL brains. Heteroplasmy analysis of brain mtDNA using Surveyor nuclease® showed asymmetric distributions and levels of heteroplasmic mutations across mtDNA but no patterns that statistically distinguished sPD from CTL. sPD brain mitochondria displayed reductions of nine respirasome proteins (respiratory complexes I-V. Reduced levels of sPD brain mitochondrial complex II, III and V, but not complex I or IV proteins, correlated closely with rates of NADH-driven electron flow. mtDNA levels and PGC-1α expression did not differ between sPD and CTL brains. Conclusion PD brain mitochondria have reduced mitochondrial respiratory protein levels in complexes I-V, implying a generalized defect in respirasome assembly. These deficiencies do not appear to arise from altered point mutational burden in mtDNA or reduction of nuclear signaling for mitochondrial biogenesis, implying downstream etiologies. The origin of age

  20. DNA glycosylases involved in base excision repair may be associated with cancer risk in BRCA1 and BRCA2 mutation carriers.

    Directory of Open Access Journals (Sweden)

    Ana Osorio

    2014-04-01

    Full Text Available Single Nucleotide Polymorphisms (SNPs in genes involved in the DNA Base Excision Repair (BER pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between one of the components of the BER pathway, PARP1 (poly ADP ribose polymerase, and both BRCA1 and BRCA2. In the present study, we have performed a comprehensive analysis of 18 genes involved in BER using a tagging SNP approach in a large series of BRCA1 and BRCA2 mutation carriers. 144 SNPs were analyzed in a two stage study involving 23,463 carriers from the CIMBA consortium (the Consortium of Investigators of Modifiers of BRCA1 and BRCA2. Eleven SNPs showed evidence of association with breast and/or ovarian cancer at p<0.05 in the combined analysis. Four of the five genes for which strongest evidence of association was observed were DNA glycosylases. The strongest evidence was for rs1466785 in the NEIL2 (endonuclease VIII-like 2 gene (HR: 1.09, 95% CI (1.03-1.16, p = 2.7 × 10(-3 for association with breast cancer risk in BRCA2 mutation carriers, and rs2304277 in the OGG1 (8-guanine DNA glycosylase gene, with ovarian cancer risk in BRCA1 mutation carriers (HR: 1.12 95%CI: 1.03-1.21, p = 4.8 × 10(-3. DNA glycosylases involved in the first steps of the BER pathway may be associated with cancer risk in BRCA1/2 mutation carriers and should be more comprehensively studied.

  1. Mutations in the DNA-binding domain of NR2E3 affect in vivo dimerization and interaction with CRX.

    Directory of Open Access Journals (Sweden)

    Raphael Roduit

    Full Text Available BACKGROUND: NR2E3 (PNR is an orphan nuclear receptor essential for proper photoreceptor determination and differentiation. In humans, mutations in NR2E3 have been associated with the recessively inherited enhanced short wavelength sensitive (S- cone syndrome (ESCS and, more recently, with autosomal dominant retinitis pigmentosa (adRP. NR2E3 acts as a suppressor of the cone generation program in late mitotic retinal progenitor cells. In adult rod photoreceptors, NR2E3 represses cone-specific gene expression and acts in concert with the transcription factors CRX and NRL to activate rod-specific genes. NR2E3 and CRX have been shown to physically interact in vitro through their respective DNA-binding domains (DBD. The DBD also contributes to homo- and heterodimerization of nuclear receptors. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed NR2E3 homodimerization and NR2E3/CRX complex formation in an in vivo situation by Bioluminescence Resonance Energy Transfer (BRET(2. NR2E3 wild-type protein formed homodimers in transiently transfected HEK293T cells. NR2E3 homodimerization was impaired in presence of disease-causing mutations in the DBD, except for the p.R76Q and p.R104W mutant proteins. Strikingly, the adRP-linked p.G56R mutant protein interacted with CRX with a similar efficiency to that of NR2E3 wild-type and p.R311Q proteins. In contrast, all other NR2E3 DBD-mutant proteins did not interact with CRX. The p.G56R mutant protein was also more effective in abolishing the potentiation of rhodospin gene transactivation by the NR2E3 wild-type protein. In addition, the p.G56R mutant enhanced the transrepression of the M- and S-opsin promoter, while all other NR2E3 DBD-mutants did not. CONCLUSIONS/SIGNIFICANCE: These results suggest different disease mechanisms in adRP- and ESCS-patients carrying NR2E3 mutations. Titration of CRX by the p.G56R mutant protein acting as a repressor in trans may account for the severe clinical phenotype in adRP patients.

  2. Mutation Detection in Patients With Advanced Cancer by Universal Sequencing of Cancer-Related Genes in Tumor and Normal DNA vs Guideline-Based Germline Testing.

    Science.gov (United States)

    Mandelker, Diana; Zhang, Liying; Kemel, Yelena; Stadler, Zsofia K; Joseph, Vijai; Zehir, Ahmet; Pradhan, Nisha; Arnold, Angela; Walsh, Michael F; Li, Yirong; Balakrishnan, Anoop R; Syed, Aijazuddin; Prasad, Meera; Nafa, Khedoudja; Carlo, Maria I; Cadoo, Karen A; Sheehan, Meg; Fleischut, Megan H; Salo-Mullen, Erin; Trottier, Magan; Lipkin, Steven M; Lincoln, Anne; Mukherjee, Semanti; Ravichandran, Vignesh; Cambria, Roy; Galle, Jesse; Abida, Wassim; Arcila, Marcia E; Benayed, Ryma; Shah, Ronak; Yu, Kenneth; Bajorin, Dean F; Coleman, Jonathan A; Leach, Steven D; Lowery, Maeve A; Garcia-Aguilar, Julio; Kantoff, Philip W; Sawyers, Charles L; Dickler, Maura N; Saltz, Leonard; Motzer, Robert J; O'Reilly, Eileen M; Scher, Howard I; Baselga, Jose; Klimstra, David S; Solit, David B; Hyman, David M; Berger, Michael F; Ladanyi, Marc; Robson, Mark E; Offit, Kenneth

    2017-09-05

    Guidelines for cancer genetic testing based on family history may miss clinically actionable genetic changes with established implications for cancer screening or prevention. To determine the proportion and potential clinical implications of inherited variants detected using simultaneous sequencing of the tumor and normal tissue ("tumor-normal sequencing") compared with genetic test results based on current guidelines. From January 2014 until May 2016 at Memorial Sloan Kettering Cancer Center, 10 336 patients consented to tumor DNA sequencing. Since May 2015, 1040 of these patients with advanced cancer were referred by their oncologists for germline analysis of 76 cancer predisposition genes. Patients with clinically actionable inherited mutations whose genetic test results would not have been predicted by published decision rules were identified. Follow-up for potential clinical implications of mutation detection was through May 2017. Tumor and germline sequencing compared with the predicted yield of targeted germline sequencing based on clinical guidelines. Proportion of clinically actionable germline mutations detected by universal tumor-normal sequencing that would not have been detected by guideline-directed testing. Of 1040 patients, the median age was 58 years (interquartile range, 50.5-66 years), 65.3% were male, and 81.3% had stage IV disease at the time of genomic analysis, with prostate, renal, pancreatic, breast, and colon cancer as the most common diagnoses. Of the 1040 patients, 182 (17.5%; 95% CI, 15.3%-19.9%) had clinically actionable mutations conferring cancer susceptibility, including 149 with moderate- to high-penetrance mutations; 101 patients tested (9.7%; 95% CI, 8.1%-11.7%) would not have had these mutations detected using clinical guidelines, including 65 with moderate- to high-penetrance mutations. Frequency of inherited mutations was related to case mix, stage, and founder mutations. Germline findings led to discussion or initiation of

  3. Translesion DNA synthesis and mutation induced in a plasmid with a single adduct of the environmental contaminant 3-nitrobenzanthrone in SOS-induced Escherichia coli

    International Nuclear Information System (INIS)

    Kawanishi, M.; Kanno, T.; Yagi, T.; Enya-Takamura, T.; Fuchs, R.P.

    2003-01-01

    Full text: 3-Nitrobenzanthrone (NBA) is a powerfully mutagenic nitrated aromatic hydrocarbon found in diesel exhaust and in airborne particulate matters. NBA forms an unusual DNA adduct in vitro that has a C-C bond between the C-8 position of deoxyguanosine and the C-2 position of NBA. We previously found that this adduct is also present in the human cells treated with NBA, and induces mutations in supF shuttle vector system. In this study, we analyzed translesion DNA synthesis (TLS) over a single adduct in lacZ' gene in a plasmid in uvrAmutS Escherichia coli. The result showed that the adduct blocked DNA replication and an observed TLS frequency was 5.4% in non-SOS-induced E. coli. All progenies after the TLS had no mutation. On the other hand, TLS increased to 11.3%, and 4.8% of them had mostly G to T mutations in SOS-induced E. coli. These results suggest that this unusual adduct would be one of causes of lung cancer that is increasing in the urban areas polluted with diesel exhaust. It must be interesting to reveal which DNA polymerase is involved in this TLS

  4. Sequence analysis of mitochondrial DNA hypervariable region III of ...

    African Journals Online (AJOL)

    Aghomotsegin

    2015-07-01

    Jul 1, 2015 ... population genetics research, studies based on mitochondrial DNA (mtDNA) and Y-chromosome DNA are an excellent way of illustrating population structure .... avoid landing investigators into serious situations of medical genetic privacy and ethnics, especially for. mtDNA coding area whose mutation often ...

  5. Frameshift mutations in infectious cDNA clones of Citrus tristeza virus: a strategy to minimize the toxicity of viral sequences to Escherichia coli

    International Nuclear Information System (INIS)

    Satyanarayana, Tatineni; Gowda, Siddarame; Ayllon, Maria A.; Dawson, William O.

    2003-01-01

    The advent of reverse genetics revolutionized the study of positive-stranded RNA viruses that were amenable for cloning as cDNAs into high-copy-number plasmids of Escherichia coli. However, some viruses are inherently refractory to cloning in high-copy-number plasmids due to toxicity of viral sequences to E. coli. We report a strategy that is a compromise between infectivity of the RNA transcripts and toxicity to E. coli effected by introducing frameshift mutations into 'slippery sequences' near the viral 'toxicity sequences' in the viral cDNA. Citrus tristeza virus (CTV) has cDNA sequences that are toxic to E. coli. The original full-length infectious cDNA of CTV and a derivative replicon, CTV-ΔCla, cloned into pUC119, resulted in unusually limited E. coli growth. However, upon sequencing of these cDNAs, an additional uridinylate (U) was found in a stretch of U's between nts 3726 and 3731 that resulted in a change to a reading frame with a stop codon at nt 3734. Yet, in vitro produced RNA transcripts from these clones infected protoplasts, and the resulting progeny virus was repaired. Correction of the frameshift mutation in the CTV cDNA constructs resulted in increased infectivity of in vitro produced RNA transcripts, but also caused a substantial increase of toxicity to E. coli, now requiring 3 days to develop visible colonies. Frameshift mutations created in sequences not suspected to facilitate reading frame shifting and silent mutations introduced into oligo(U) regions resulted in complete loss of infectivity, suggesting that the oligo(U) region facilitated the repair of the frameshift mutation. Additional frameshift mutations introduced into other oligo(U) regions also resulted in transcripts with reduced infectivity similarly to the original clones with the +1 insertion. However, only the frameshift mutations introduced into oligo(U) regions that were near and before the toxicity region improved growth and stability in E. coli. These data demonstrate that

  6. Effects of space flight on DNA mutation and secondary metabolites of licorice (Glycyrrhiza uralensis Fisch.)

    Institute of Scientific and Technical Information of China (English)

    GAO WenYuan; LI KeFeng; YAN Shuo; GAO XiuMei; HU LiMin

    2009-01-01

    Licorice (Glycyrrhiza uralensis Fisch.) seeds were flown on a recoverable satellite for 18 days(the average radiation dose in the flight recovery module was 0.102 mGy/d, the distance from flight apogee to earth was 350 km, gravity 10~(-6)). After returning to earth, the seeds were germinated and grown to maturity. The parallel ground-based seeds were also planted under the same conditions. The leaves of licorice were used for inter-simple sequence repeat (ISSR) analysis and the two main secondary metabolites in one-year-old roots were analyzed by high performance liquid chromatography (HPLC).Among 22 random primers used in this experiment, 6 primers generated different DNA band types. Analysis of HPLC showed that the content of glycyrrhizic acid (GA) and liquiritin (LQ) in the roots from seeds flown in space was respectively 2.19, 1.18 times higher than that of the control group. The results demonstrated that the extraterrestrial environment induced mutagenic effects on licorice and affected its secondary metabolites. These changes indicated that extraterrestrial orbit is possible means of breeding of licorice so as to preserve this endangered medicinal plant.

  7. Effects of space flight on DNA mutation and secondary metabolites of licorice (Glycyrrhiza uralensis Fisch.)

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Licorice (Glycyrrhiza uralensis Fisch.) seeds were flown on a recoverable satellite for 18 days(the average radiation dose in the flight recovery module was 0.102 mGy/d, the distance from flight apogee to earth was 350 km, gravity 10-6). After returning to earth, the seeds were germinated and grown to maturity. The parallel ground-based seeds were also planted under the same conditions. The leaves of licorice were used for inter-simple sequence repeat (ISSR) analysis and the two main secondary me-tabolites in one-year-old roots were analyzed by high performance liquid chromatography (HPLC). Among 22 random primers used in this experiment, 6 primers generated different DNA band types. Analysis of HPLC showed that the content of glycyrrhizic acid (GA) and liquiritin (LQ) in the roots from seeds flown in space was respectively 2.19, 1.18 times higher than that of the control group. The results demonstrated that the extraterrestrial environment induced mutagenic effects on licorice and affected its secondary metabolites. These changes indicated that extraterrestrial orbit is possible means of breeding of licorice so as to preserve this endangered medicinal plant.

  8. Induced mutations in connection with biotechnology for crop improvement in Latin America. Proceedings of a final research co-ordination meeting

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2001-05-01

    This publication results from the second Co-ordinated Research Project (CRP) on Plant Breeding and Genetics organized on a regional basis in Latin America. The present CRP and the previous one were initiated and implemented in response to the pressing need to enhance the productivity of economic plants, viz. food crops, fruits and ornamentals. Improvement of crop production has become the highest priority in most countries of Latin America, as in other regions. Breeding superior varieties is often the only feasible solution where inputs are limited; well adapted varieties are required to meet specific agro-environmental conditions. Such varieties provide yield stability on an economically required level. The most important and common factors limiting crop production are abiotic, e.g. cold, salinity, soil aluminium toxicity and drought; as well as biotic, e.g. diseases and pests. Modern biotechnology and induced mutations offer new means and significant potential to breed desired varieties in a relatively short time. Additionally, both approaches facilitate the breeding of some vegetatively propagated crops which until now were improved mainly through selection of rare spontaneous mutants in natural or cultivated populations. Using some of these techniques it recently became possible to produce, in some crops, true-to-type mutated lines or clones within a few months. Biotechnology can also facilitate selection, description and molecular characterization of promising mutants. Currently used DNA markers, such as restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) as well as other polymerase chain reaction (PCR)-based techniques, were included in this CRP to benefit the important crops of this region. Also included in this CRP were doubled haploids (DH), which are obtained from anther or microspore cultures and are very suitable biotechnology methods. In connection with radiation-induced mutations, they can speed up conventional

  9. Induced mutations in connection with biotechnology for crop improvement in Latin America. Proceedings of a final research co-ordination meeting

    International Nuclear Information System (INIS)

    2001-05-01

    This publication results from the second Co-ordinated Research Project (CRP) on Plant Breeding and Genetics organized on a regional basis in Latin America. The present CRP and the previous one were initiated and implemented in response to the pressing need to enhance the productivity of economic plants, viz. food crops, fruits and ornamentals. Improvement of crop production has become the highest priority in most countries of Latin America, as in other regions. Breeding superior varieties is often the only feasible solution where inputs are limited; well adapted varieties are required to meet specific agro-environmental conditions. Such varieties provide yield stability on an economically required level. The most important and common factors limiting crop production are abiotic, e.g. cold, salinity, soil aluminium toxicity and drought; as well as biotic, e.g. diseases and pests. Modern biotechnology and induced mutations offer new means and significant potential to breed desired varieties in a relatively short time. Additionally, both approaches facilitate the breeding of some vegetatively propagated crops which until now were improved mainly through selection of rare spontaneous mutants in natural or cultivated populations. Using some of these techniques it recently became possible to produce, in some crops, true-to-type mutated lines or clones within a few months. Biotechnology can also facilitate selection, description and molecular characterization of promising mutants. Currently used DNA markers, such as restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) as well as other polymerase chain reaction (PCR)-based techniques, were included in this CRP to benefit the important crops of this region. Also included in this CRP were doubled haploids (DH), which are obtained from anther or microspore cultures and are very suitable biotechnology methods. In connection with radiation-induced mutations, they can speed up conventional

  10. Characterization of Ofloxacin Interaction with Mutated (A91V) Quinolone Resistance Determining Region of DNA Gyrase in Mycobacterium Leprae through Computational Simulation.

    Science.gov (United States)

    Nisha, J; Shanthi, V

    2018-06-01

    Mycobacterium leprae, the causal agent of leprosy is non-cultivable in vitro. Thus, the assessment of antibiotic activity against Mycobacterium leprae depends primarily upon the time-consuming mouse footpad system. The GyrA protein of Mycobacterium leprae is the target of the antimycobacterial drug, Ofloxacin. In recent times, the GyrA mutation (A91V) has been found to be resistant to Ofloxacin. This phenomenon has necessitated the development of new, long-acting antimycobacterial compounds. The underlying mechanism of drug resistance is not completely known. Currently, experimentally crystallized GyrA-DNA-OFLX models are not available for highlighting the binding and mechanism of Ofloxacin resistance. Hence, we employed computational approaches to characterize the Ofloxacin interaction with both the native and mutant forms of GyrA complexed with DNA. Binding energy measurements obtained from molecular docking studies highlights hydrogen bond-mediated efficient binding of Ofloxacin to Asp47 in the native GyrA-DNA complex in comparison with that of the mutant GyrA-DNA complex. Further, molecular dynamics studies highlighted the stable binding of Ofloxacin with native GyrA-DNA complex than with the mutant GyrA-DNA complex. This mechanism provided a plausible reason for the reported, reduced effect of Ofloxacin to control leprosy in individuals with the A91V mutation. Our report is the first of its kind wherein the basis for the Ofloxacin drug resistance mechanism has been explored with the help of ternary Mycobacterium leprae complex, GyrA-DNA-OFLX. These structural insights will provide useful information for designing new drugs to target the Ofloxacin-resistant DNA gyrase.

  11. UVA activation of N-dialkylnitrosamines releasing nitric oxide, producing strand breaks as well as oxidative damages in DNA, and inducing mutations in the Ames test

    International Nuclear Information System (INIS)

    Arimoto-Kobayashi, Sakae; Sano, Kayoko; Machida, Masaki; Kaji, Keiko; Yakushi, Keiko

    2010-01-01

    We investigated the photo-mutagenicity and photo-genotoxicity of N-dialkylnitrosamines and its mechanisms of UVA activation. With simultaneous irradiation of UVA, photo-mutagenicity of seven N-dialkylnitrosamines was observed in Ames bacteria (Salmonella typhimurium TA1535) in the absence of metabolic activation. Mutagenicity of pre-irradiated N-dialkylnitrosamines was also observed with S. typhimurium hisG46, TA100, TA102 and YG7108 in the absence of metabolic activation. UVA-mediated mutation with N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) decreased by adding either the NO or OH radical scavenger. When superhelical DNA was irradiated with N-dialkylnitrosamines, nicked circular DNA appeared. Ten N-dialkylnitrosamines examined produced strand breaks in the treated DNA in the presence of UVA. The level of single-strand breaks in φX174 DNA mediated by N-nitrosomorpholine (NMOR) and UVA decreased by adding either a radical scavenger or superoxide dismutase. When calf thymus DNA was treated with N-dialkylnitrosamines (NDMA, NDEA, NMOR, N-nitrosopyrrolidine (NPYR) and N-nitrosopiperidine (NPIP)) and UVA, the ratio of 8-oxodG/dG in the DNA increased. Action spectra were obtained to determine if nitrosamine acts as a sensitizer of UVA. Both mutation frequency and NO formation were highest at the absorption maximum of nitrosamines, approximately 340 nm. The plots of NO formation and mutation frequency align with the absorption curve of NPYR, NMOR and NDMA. A significant linear correlation between the optical density of N-dialkynitrosamines at 340 nm and NO formation in each irradiated solution was revealed by ANOVA. We would like to propose the hypothesis that the N-nitroso moiety of N-dialkylnitrosamines absorbs UVA photons, UVA-photolysis of N-dialkylnitrosamines brings release of nitric oxide, and subsequent production of alkyl radical cations and active oxygen species follow as secondary events, which cause DNA strand breaks, oxidative and

  12. UVA activation of N-dialkylnitrosamines releasing nitric oxide, producing strand breaks as well as oxidative damages in DNA, and inducing mutations in the Ames test

    Energy Technology Data Exchange (ETDEWEB)

    Arimoto-Kobayashi, Sakae [Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima, Okayama 700-8530 (Japan); Sano, Kayoko; Machida, Masaki; Kaji, Keiko; Yakushi, Keiko [Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima, Okayama 700-8530 (Japan)

    2010-09-10

    We investigated the photo-mutagenicity and photo-genotoxicity of N-dialkylnitrosamines and its mechanisms of UVA activation. With simultaneous irradiation of UVA, photo-mutagenicity of seven N-dialkylnitrosamines was observed in Ames bacteria (Salmonella typhimurium TA1535) in the absence of metabolic activation. Mutagenicity of pre-irradiated N-dialkylnitrosamines was also observed with S. typhimurium hisG46, TA100, TA102 and YG7108 in the absence of metabolic activation. UVA-mediated mutation with N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) decreased by adding either the NO or OH radical scavenger. When superhelical DNA was irradiated with N-dialkylnitrosamines, nicked circular DNA appeared. Ten N-dialkylnitrosamines examined produced strand breaks in the treated DNA in the presence of UVA. The level of single-strand breaks in {phi}X174 DNA mediated by N-nitrosomorpholine (NMOR) and UVA decreased by adding either a radical scavenger or superoxide dismutase. When calf thymus DNA was treated with N-dialkylnitrosamines (NDMA, NDEA, NMOR, N-nitrosopyrrolidine (NPYR) and N-nitrosopiperidine (NPIP)) and UVA, the ratio of 8-oxodG/dG in the DNA increased. Action spectra were obtained to determine if nitrosamine acts as a sensitizer of UVA. Both mutation frequency and NO formation were highest at the absorption maximum of nitrosamines, approximately 340 nm. The plots of NO formation and mutation frequency align with the absorption curve of NPYR, NMOR and NDMA. A significant linear correlation between the optical density of N-dialkynitrosamines at 340 nm and NO formation in each irradiated solution was revealed by ANOVA. We would like to propose the hypothesis that the N-nitroso moiety of N-dialkylnitrosamines absorbs UVA photons, UVA-photolysis of N-dialkylnitrosamines brings release of nitric oxide, and subsequent production of alkyl radical cations and active oxygen species follow as secondary events, which cause DNA strand breaks, oxidative and

  13. Population data and mutation rates of 19 STR loci in seven provinces from China based on Goldeneye™ DNA ID System 20A.

    Science.gov (United States)

    Liu, Qiu-Ling; Chen, Ye-Fei; Huang, Xiao-Ling; Liu, Kai-Yan; Zhao, Hu; Lu, De-Jian

    2017-05-01

    Short tandem repeat (STR) analysis is a primary tool in forensic casework. Population data and mutation rates of STRs are very important for paternity testing and forensic genetics. However, the population data and mutation rates of STRs in Han nationality based on large samples have still not been fully described in China. In this study, the allelic frequencies, forensic parameters, and mutation rate of 19 STR loci (D19S433, D5S818, D21S11, D18S51, D6S1043, D3S1358, D13S317, D7S820, D16S539, CSFIPO, PentaD, vWA, D8S1179, TPOX, Penta E, TH01, D12S391, D2S1338, and FGA) based on the Goldeneye™ DNA ID System 20A in Southern China Han nationality among seven provinces were investigated. Furthermore, population stratification of Southern China Han nationality among seven provinces was established. The multidimensional scaling (MDS) plot based on genetic distances (Fst) showed that the studied populations can be clustered into two major groups. However, relationships among populations were weak (Fst < 0.0043). A total of 376 cases of mutation were detected from the 19 selected loci in 15,396 meioses. The average mutation rate for the 19 loci was estimated to be 1.3 × 10 -3 per meiosis. The mutation was mainly single step; the paternal mutation rate was higher than the maternal; and paternal mutation rate increases with paternal age.

  14. Clinical and Molecular Characteristics in 100 Chinese Pediatric Patients with m.3243A>G Mutation in Mitochondrial DNA

    Directory of Open Access Journals (Sweden)

    Chang-Yu Xia

    2016-01-01

    Conclusions: Our study showed that half of Chinese pediatric patients with m.3243A>G mutation presented seizures, short stature, abnormal MRI/CT changes, elevated plasma lactate, vomiting, and headache. Pediatric patients with these recurrent symptoms should be considered for screening m.3243A>G mutation. Clinical manifestations and laboratory abnormalities should be carefully monitored in patients with this point mutation.

  15. Small-Molecule Inhibitors Targeting DNA Repair and DNA Repair Deficiency in Research and Cancer Therapy.

    Science.gov (United States)

    Hengel, Sarah R; Spies, M Ashley; Spies, Maria

    2017-09-21

    To maintain stable genomes and to avoid cancer and aging, cells need to repair a multitude of deleterious DNA lesions, which arise constantly in every cell. Processes that support genome integrity in normal cells, however, allow cancer cells to develop resistance to radiation and DNA-damaging chemotherapeutics. Chemical inhibition of the key DNA repair proteins and pharmacologically induced synthetic lethality have become instrumental in both dissecting the complex DNA repair networks and as promising anticancer agents. The difficulty in capitalizing on synthetically lethal interactions in cancer cells is that many potential targets do not possess well-defined small-molecule binding determinates. In this review, we discuss several successful campaigns to identify and leverage small-molecule inhibitors of the DNA repair proteins, from PARP1, a paradigm case for clinically successful small-molecule inhibitors, to coveted new targets, such as RAD51 recombinase, RAD52 DNA repair protein, MRE11 nuclease, and WRN DNA helicase. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Seventy five years of research on induced mutations with special reference to crop improvement in India

    International Nuclear Information System (INIS)

    Kharkwal, M.C.; Pawar, S.E.; Pandey, R.N.

    2001-01-01

    Mutation research programmes for crop improvement were initiated about seven decades ago, immediately after the discovery of mutagenic effects X-rays on Drosophila by Muller 1927 and barley and maize by Stadler in 1929. During fifties and sixties, several countries including China, India, the Netherlands, USA and Japan took up the task of crop improvement through mutation breeding approaches. By the end of the 20 th century about 2252 mutant varieties of crop plants including cereals, oil seeds, pulses, vegetables, fruits, fibres and ornamentals have been developed and released for cultivation worldwide. More than 60% of the varieties were released after 1985. While 1585 varieties were released as direct mutants, 667 varieties were developed by using mutants in cross breeding. Physical mutagens (X-rays, gamma rays, thermal and fast neutrons) account for the development of about 89% of the total varieties. Gamma rays alone were used to develop about 60% of the mutant varieties. India has made an appreciable contribution with the development of 301 mutant varieties (> 13% ) of forty four crop species. The mutant varieties have been improved for plant architecture, yield, quality and tolerance to biotic and abiotic stresses. Mutation breeding has made a significant contribution to the national economy of the countries like China, India, Japan, Pakistan and USA. Induced mutagenesis is gaining importance in plant molecular biology as a tool to identify and isolate gene and to study their structure and function. (author)

  17. Neratinib Efficacy and Circulating Tumor DNA Detection of HER2 Mutations in HER2 Nonamplified Metastatic Breast Cancer.

    Science.gov (United States)

    Ma, Cynthia X; Bose, Ron; Gao, Feng; Freedman, Rachel A; Telli, Melinda L; Kimmick, Gretchen; Winer, Eric; Naughton, Michael; Goetz, Matthew P; Russell, Christy; Tripathy, Debu; Cobleigh, Melody; Forero, Andres; Pluard, Timothy J; Anders, Carey; Niravath, Polly Ann; Thomas, Shana; Anderson, Jill; Bumb, Caroline; Banks, Kimberly C; Lanman, Richard B; Bryce, Richard; Lalani, Alshad S; Pfeifer, John; Hayes, Daniel F; Pegram, Mark; Blackwell, Kimberly; Bedard, Philippe L; Al-Kateb, Hussam; Ellis, Matthew J C

    2017-10-01

    Purpose: Based on promising preclinical data, we conducted a single-arm phase II trial to assess the clinical benefit rate (CBR) of neratinib, defined as complete/partial response (CR/PR) or stable disease (SD) ≥24 weeks, in HER2 mut nonamplified metastatic breast cancer (MBC). Secondary endpoints included progression-free survival (PFS), toxicity, and circulating tumor DNA (ctDNA) HER2 mut detection. Experimental Design: Tumor tissue positive for HER2 mut was required for eligibility. Neratinib was administered 240 mg daily with prophylactic loperamide. ctDNA sequencing was performed retrospectively for 54 patients (14 positive and 40 negative for tumor HER2 mut ). Results: Nine of 381 tumors (2.4%) sequenced centrally harbored HER2 mut (lobular 7.8% vs. ductal 1.6%; P = 0.026). Thirteen additional HER2 mut cases were identified locally. Twenty-one of these 22 HER2 mut cases were estrogen receptor positive. Sixteen patients [median age 58 (31-74) years and three (2-10) prior metastatic regimens] received neratinib. The CBR was 31% [90% confidence interval (CI), 13%-55%], including one CR, one PR, and three SD ≥24 weeks. Median PFS was 16 (90% CI, 8-31) weeks. Diarrhea (grade 2, 44%; grade 3, 25%) was the most common adverse event. Baseline ctDNA sequencing identified the same HER2 mut in 11 of 14 tumor-positive cases (sensitivity, 79%; 90% CI, 53%-94%) and correctly assigned 32 of 32 informative negative cases (specificity, 100%; 90% CI, 91%-100%). In addition, ctDNA HER2 mut variant allele frequency decreased in nine of 11 paired samples at week 4, followed by an increase upon progression. Conclusions: Neratinib is active in HER2 mut , nonamplified MBC. ctDNA sequencing offers a noninvasive strategy to identify patients with HER2 mut cancers for clinical trial participation. Clin Cancer Res; 23(19); 5687-95. ©2017 AACR . ©2017 American Association for Cancer Research.

  18. Differential radiosensitivity phenotypes of DNA-PKcs mutations affecting NHEJ and HRR systems following irradiation with gamma-rays or very low fluences of alpha particles.

    Science.gov (United States)

    Lin, Yu-Fen; Nagasawa, Hatsumi; Little, John B; Kato, Takamitsu A; Shih, Hung-Ying; Xie, Xian-Jin; Wilson, Paul F; Brogan, John R; Kurimasa, Akihiro; Chen, David J; Bedford, Joel S; Chen, Benjamin P C

    2014-01-01

    We have examined cell-cycle dependence of chromosomal aberration induction and cell killing after high or low dose-rate γ irradiation in cells bearing DNA-PKcs mutations in the S2056 cluster, the T2609 cluster, or the kinase domain. We also compared sister chromatid exchanges (SCE) production by very low fluences of α-particles in DNA-PKcs mutant cells, and in homologous recombination repair (HRR) mutant cells including Rad51C, Rad51D, and Fancg/xrcc9. Generally, chromosomal aberrations and cell killing by γ-rays were similarly affected by mutations in DNA-PKcs, and these mutant cells were more sensitive in G1 than in S/G2 phase. In G1-irradiated DNA-PKcs mutant cells, both chromosome- and chromatid-type breaks and exchanges were in excess than wild-type cells. For cells irradiated in late S/G2 phase, mutant cells showed very high yields of chromatid breaks compared to wild-type cells. Few exchanges were seen in DNA-PKcs-null, Ku80-null, or DNA-PKcs kinase dead mutants, but exchanges in excess were detected in the S2506 or T2609 cluster mutants. SCE induction by very low doses of α-particles is resulted from bystander effects in cells not traversed by α-particles. SCE seen in wild-type cells was completely abolished in Rad51C- or Rad51D-deficient cells, but near normal in Fancg/xrcc9 cells. In marked contrast, very high levels of SCEs were observed in DNA-PKcs-null, DNA-PKcs kinase-dead and Ku80-null mutants. SCE induction was also abolished in T2609 cluster mutant cells, but was only slightly reduced in the S2056 cluster mutant cells. Since both non-homologous end-joining (NHEJ) and HRR systems utilize initial DNA lesions as a substrate, these results suggest the possibility of a competitive interference phenomenon operating between NHEJ and at least the Rad51C/D components of HRR; the level of interaction between damaged DNA and a particular DNA-PK component may determine the level of interaction of such DNA with a relevant HRR component.

  19. Mutation-Induced Population Shift in the MexR Conformational Ensemble Disengages DNA Binding: A Novel Mechanism for MarR Family Derepression.

    Science.gov (United States)

    Anandapadamanaban, Madhanagopal; Pilstål, Robert; Andresen, Cecilia; Trewhella, Jill; Moche, Martin; Wallner, Björn; Sunnerhagen, Maria

    2016-08-02

    MexR is a repressor of the MexAB-OprM multidrug efflux pump operon of Pseudomonas aeruginosa, where DNA-binding impairing mutations lead to multidrug resistance (MDR). Surprisingly, the crystal structure of an MDR-conferring MexR mutant R21W (2.19 Å) presented here is closely similar to wild-type MexR. However, our extended analysis, by molecular dynamics and small-angle X-ray scattering, reveals that the mutation stabilizes a ground state that is deficient of DNA binding and is shared by both mutant and wild-type MexR, whereas the DNA-binding state is only transiently reached by the more flexible wild-type MexR. This population shift in the conformational ensemble is effected by mutation-induced allosteric coupling of contact networks that are independent in the wild-type protein. We propose that the MexR-R21W mutant mimics derepression by small-molecule binding to MarR proteins, and that the described allosteric model based on population shifts may also apply to other MarR family members. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. A point mutation in the DNA-binding domain of HPV-2 E2 protein increases its DNA-binding capacity and reverses its transcriptional regulatory activity on the viral early promoter

    Directory of Open Access Journals (Sweden)

    Gao Chen

    2012-02-01

    Full Text Available Abstract Background The human papillomavirus (HPV E2 protein is a multifunctional DNA-binding protein. The transcriptional activity of HPV E2 is mediated by binding to its specific binding sites in the upstream regulatory region of the HPV genomes. Previously we reported a HPV-2 variant from a verrucae vulgaris patient with huge extensive clustered cutaneous, which have five point mutations in its E2 ORF, L118S, S235P, Y287H, S293R and A338V. Under the control of HPV-2 LCR, co-expression of the mutated HPV E2 induced an increased activity on the viral early promoter. In the present study, a series of mammalian expression plasmids encoding E2 proteins with one to five amino acid (aa substitutions for these mutations were constructed and transfected into HeLa, C33A and SiHa cells. Results CAT expression assays indicated that the enhanced promoter activity was due to the co-expressions of the E2 constructs containing A338V mutation within the DNA-binding domain. Western blots analysis demonstrated that the transiently transfected E2 expressing plasmids, regardless of prototype or the A338V mutant, were continuously expressed in the cells. To study the effect of E2 mutations on its DNA-binding activity, a serial of recombinant E2 proteins with various lengths were expressed and purified. Electrophoresis mobility shift assays (EMSA showed that the binding affinity of E2 protein with A338V mutation to both an artificial probe with two E2 binding sites or HPV-2 and HPV-16 promoter-proximal LCR sequences were significantly stronger than that of the HPV-2 prototype E2. Furthermore, co-expression of the construct containing A338V mutant exhibited increased activities on heterologous HPV-16 early promoter P97 than that of prototype E2. Conclusions These results suggest that the mutation from Ala to Val at aa 338 is critical for E2 DNA-binding and its transcriptional regulation.

  1. First description of a novel mitochondrial mutation in the MT-TI gene associated with multiple mitochondrial DNA deletion and depletion in family with severe dilated mitochondrial cardiomyopathy.

    Science.gov (United States)

    Alila-Fersi, Olfa; Tabebi, Mouna; Maalej, Marwa; Belguith, Neila; Keskes, Leila; Mkaouar-Rebai, Emna; Fakhfakh, Faiza

    2018-03-18

    Mitochondria are essential for early cardiac development and impaired mitochondrial function was described associated with heart diseases such as hypertrophic or dilated mitochondrial cardiomyopathy. In this study, we report a family including two individuals with severe dilated mitochondrial cardiomyopathy. The whole mitochondrial genome screening showed the presence of several variations and a novel homoplasmic mutation m.4318-4322delC in the MT-TI gene shared by the two patients and their mother and leading to a disruption of the tRNA Ile secondary structure. In addition, a mitochondrial depletion was present in blood leucocyte of the two affected brother whereas a de novo heteroplasmic multiple deletion in the major arc of mtDNA was present in blood leucocyte and mucosa of only one of them. These deletions in the major arc of the mtDNA resulted to the loss of several protein-encoding genes and also some tRNA genes. The mtDNA deletion and depletion could result to an impairment of the oxidative phosphorylation and energy metabolism in the respiratory chain in the studied patients. Our report is the first description of a family with severe lethal dilated mitochondrial cardiomyopathy and presenting several mtDNA abnormalities including punctual mutation, deletion and depletion. Copyright © 2018 Elsevier Inc. All rights reserved.

  2. Effect of the uvr D3 mutation on ultraviolet radiation-induced DNA-repair replication in Escherichia coli K12

    International Nuclear Information System (INIS)

    Carlson, K.M.; Smith, K.C.

    1981-01-01

    Ultraviolet-radiation-induced DNA-repair replication was measured in wild-type, polA1, uvrD3, and polA1 uvrD3 strains of Escherichia coli K 12. A large stimulation of repair replication was observed in the uvrD3 strain, compared to the wild-type and polA1 strains. This enhanced repair replication was reduced in the polA1 uvrD3 strain. Therefore, a uvrD3 mutation appears to affect the amount of repair replication performed by DNA polymerase I. In the polA1 strain, there also appears to be an effect of the uvrD3 mutation on the amount of repair replication performed by DNA polymerase III (and/or II). The enhanced repair replication observed for the uvrD3 strains appears to be in response to the enhanced DNA degradation observed for these strains. (orig.)

  3. Detection of mutations using microarrays of poly(C)10-poly(T)10 modified DNA probes immobilized on agarose films

    DEFF Research Database (Denmark)

    Dufva, Hans Martin; Petersen, Jesper; Stoltenborg, M.

    2006-01-01

    Allele-specific hybridization to a DNA microarray call be a useful method for genotyping patient DNA. In this article, we demonstrate that 13- to 17-base oligonucleotides tagged with a poly(T)10-poly(C)10 tail (TC tag), but otherwise unmodified, can be crosslinked by UV light irradiation to an ag......Allele-specific hybridization to a DNA microarray call be a useful method for genotyping patient DNA. In this article, we demonstrate that 13- to 17-base oligonucleotides tagged with a poly(T)10-poly(C)10 tail (TC tag), but otherwise unmodified, can be crosslinked by UV light irradiation...... to an agarose film grafted onto unmodified glass. Microarrays of TC-tagged probes immobilized on the agarose film can be used to diagnose Mutations in the human P-globin gene, which encodes the beta-chains in hemoglobin. Although the probes differed widely regarding inciting point temperature (similar to 20...... degrees C), a single stringency wash still gave sufficiently high discrimination signals between perfect match and mismatch probes to allow robust mutation detection. In all, 270 genotypings were performed on patient materials, and no genotype was incorrectly classified. Quality control experiments...

  4. Deletion mutations of bacteriophage

    International Nuclear Information System (INIS)

    Ryo, Yeikou

    1975-01-01

    Resolution of mutation mechanism with structural changes of DNA was discussed through the studies using bacteriophage lambda. One of deletion mutations inductions of phage lambda is the irradiation of ultraviolet ray. It is not clear if the inductions are caused by errors in reparation of ultraviolet-induced damage or by the activation of int gene. Because the effective site of int gene lies within the regions unnecessary for existing, it is considered that int gene is connected to deletion mutations induction. A certain system using prophage complementarity enables to detect deletion mutations at essential hereditary sites and to solve the relations of deletion mutations with other recombination system, DNA reproduction and repairment system. Duplication and multiplication of hereditary elements were discussed. If lambda deletion mutations of the system, which can control recombination, reproduction and repairment of added DNA, are constructed, mutations mechanism with great changes of DNA structure can be solved by phage lambda. (Ichikawa, K.)

  5. Repair Mechanism of UV-damaged DNA in Xeroderma Pigmentosum | Center for Cancer Research

    Science.gov (United States)

    Xeroderma pigmentosum (XP) is a rare, inherited disorder characterized by extreme skin sensitivity to ultraviolet (UV) rays from sunlight. XP is caused by mutations in genes involved in nucleotide excision repair (NER) of damaged DNA. Normal cells are usually able to fix this damage before it leads to problems; however, the DNA damage is not repaired normally in patients with XP. As more abnormalities form in DNA, cells malfunction and eventually become cancerous or die. XP patients have more than a 10,000-fold increased risk of developing skin cancer. Kenneth Kraemer, M.D., in CCR’s Dermatology Branch, has been studying XP patients at the Clinical Center for more than 40 years.

  6. Public involvement in pharmacogenomics research: a national survey on public attitudes towards pharmacogenomics research and the willingness to donate DNA samples to a DNA bank in Japan.

    Science.gov (United States)

    Kobayashi, Eriko; Satoh, Nobunori

    2009-11-01

    To assess the attitudes of the Japanese general public towards pharmacogenomics research and a DNA bank for identifying genomic markers associated with ADRs and their willingness to donate DNA samples, we conducted a national survey for 1,103 Japanese adults from the general public, not a patient population. The response rate was 36.8%. The majority of the respondents showed a positive attitude towards pharmacogenomics research (81.0%) and a DNA bank (70.4%). Considering fictitious clinical situations such as taking medications and experiencing ADRs, the willingness to donate DNA samples when experiencing ADRs (61.7%) was higher than when taking medications (45.3%). Older generations were significantly associated with a decreased willingness to donate (OR = 0.45, CI 0.28-0.72 in 50s. OR = 0.49, CI: 0.31-0.77 in 60s). Positive attitudes towards pharmacogenomics research, a DNA bank, blood/bone marrow/organ donation were significantly associated with an increased willingness. However, the respondents had the following concerns regarding a DNA bank: the confidentiality of their personal information, the manner by which research results were utilized and simply the use of their own DNA for research. In order to attain public understanding to overcome these concerns, a process of public awareness should be put into place to emphasize the beneficial aspects of identifying genomic markers associated with ADRs and to address these concerns raised in our study. Further study is needed to assess the willingness of actual patients taking medications in real situations, since the respondents in our study were from the general public, not a patient population, and their willingness was assessed on the condition of assuming that they were patients taking medications.

  7. Synchrotron radiation. 4. Analyses of biological samples using synchrotron radiation. 3. Research on radiation damage to DNA using synchrotron radiation

    International Nuclear Information System (INIS)

    Takakura, Kaoru

    1998-01-01

    This review described how the synchrotron radiation (SR) is used to solve problems unknown hitherto in radiation biology. Historically, the target substance of UV light in bacterial death was suggested to be nucleic acid in 1930. Researches on the radiation damage to DNA were begun at around 1960 and have mainly used UV light, X-ray and γray. Soft X-ray and vacuum UV whose energy covering from several eV to scores of keV have not been used since UV and X-ray lack the energy of this range. This is one of reasons why detailed process leading to radiation-induced death, carcinogenicity and mutation has not been known hitherto. RS possesses wide range of energy, i.e., from UV to hard X-ray, of high intensity, which is helpful for studying the unknown problems. The RS studies were begun in nineteen-seventies. Those include the action spectrum studies and atomic target studies. In the former, the course of the effect, e.g., the mechanism of DNA double strand breakage, can be elucidated. In the latter, photon of known energy can be irradiated to the specified atom like phosphorus in DNA which elucidating the precise physicochemical process of the breakage. Use of RS in these studies is thought still meaningful in future. (K.H.) 62 refs

  8. A comparative investigation of DNA strand breaks, sister chromatid exchanges and K-ras gene mutations induced by cadmium salts in cultured human cells

    International Nuclear Information System (INIS)

    Mouron, Silvana Andrea; Grillo, Claudia Alejandra; Dulout, Fernando Noel; Golijow, Carlos Daniel

    2004-01-01

    Cadmium (Cd) is a toxic heavy metal of continuing occupational and environmental concern with a wide variety of adverse effects. Several studies have shown that cadmium produces DNA strand breaks, DNA-protein cross-links, oxidative DNA damage, chromosomal aberrations, dysregulation of gene expression resulting in enhanced proliferation, depressed apoptosis and/or altered DNA repair. This study was undertaken to investigate the ability of cadmium chloride (CdCl 2 ) and cadmium sulphate (CdSO 4 ) to induce point mutations in codon 12 of the K-ras protooncogene assessed by polymerase chain reaction-single strand conformation polymorphisms (PCR-SSCP) and RFLP-enriched PCR methods. Also their genotoxic effects were analyzed by the comet assay and sister chromatid exchanges test. The human lung fibroblast cell line MRC-5 was used for the experiments. Sister chromatid exchanges assay (SCEs) frequencies were significantly increased in cells exposed to cadmium salts in relation to controls (p < 0.001). Despite the slow increment observed in the three comet parameters considered when cells were treated with cadmium chloride, significant differences between groups were only found in the variable comet moment (CM) (p < 0.005). On the other hand, when cells were exposed to cadmium sulphate, the Kruskal-Wallis test showed highly significant differences between groups for migration, tail moment and comet moment parameters (p < 0.001). Nevertheless, a null or weak point mutation induction in K-ras protooncogene was detected using polymerase chain reaction-low ionic strength-single strand conformation polymorphisms (PCR-LIS-SSCP) and RFLP-enriched PCR methods when cells were treated with cadmium salts. Thus, inorganic cadmium produces genotoxicity in human lung fibroblast MRC-5 cells, in the absence of significant point mutation of the K-ras gene

  9. Traceless splicing enabled by substrate-induced activation of the Nostoc punctiforme Npu DnaE intein after mutation of a catalytic cysteine to serine.

    Science.gov (United States)

    Cheriyan, Manoj; Chan, Siu-Hong; Perler, Francine

    2014-12-12

    Inteins self-catalytically cleave out of precursor proteins while ligating the surrounding extein fragments with a native peptide bond. Much attention has been lavished on these molecular marvels with the hope of understanding and harnessing their chemistry for novel biochemical transformations including coupling peptides from synthetic or biological origins and controlling protein function. Despite an abundance of powerful applications, the use of inteins is still hampered by limitations in our understanding of their specificity (defined as flanking sequences that permit splicing) and the challenge of inserting inteins into target proteins. We examined the frequently used Nostoc punctiforme Npu DnaE intein after the C-extein cysteine nucleophile (Cys+1) was mutated to serine or threonine. Previous studies demonstrated reduced rates and/or splicing yields with the Npu DnaE intein after mutation of Cys+1 to Ser+1. In this study, genetic selection identified extein sequences with Ser+1 that enabled the Npu DnaE intein to splice with only a 5-fold reduction in rate compared to the wild-type Cys+1 intein and without mutation of the intein itself to activate Ser+1 as a nucleophile. Three different proteins spliced efficiently after insertion of the intein flanked by the selected sequences. We then used this selected specificity to achieve traceless splicing in a targeted enzyme at a location predicted by primary sequence similarity to only the selected C-extein sequence. This study highlights the latent catalytic potential of the Npu DnaE intein to splice with an alternative nucleophile and enables broader intein utility by increasing insertion site choices. Copyright © 2014. Published by Elsevier Ltd.

  10. Toehold-mediated strand displacement reaction triggered isothermal DNA amplification for highly sensitive and selective fluorescent detection of single-base mutation.

    Science.gov (United States)

    Zhu, Jing; Ding, Yongshun; Liu, Xingti; Wang, Lei; Jiang, Wei

    2014-09-15

    Highly sensitive and selective detection strategy for single-base mutations is essential for risk assessment of malignancy and disease prognosis. In this work, a fluorescent detection method for single-base mutation was proposed based on high selectivity of toehold-mediated strand displacement reaction (TSDR) and powerful signal amplification capability of isothermal DNA amplification. A discrimination probe was specially designed with a stem-loop structure and an overhanging toehold domain. Hybridization between the toehold domain and the perfect matched target initiated the TSDR along with the unfolding of the discrimination probe. Subsequently, the target sequence acted as a primer to initiate the polymerization and nicking reactions, which released a great abundant of short sequences. Finally, the released strands were annealed with the reporter probe, launching another polymerization and nicking reaction to produce lots of G-quadruplex DNA, which could bind the N-methyl mesoporphyrin IX to yield an enhanced fluorescence response. However, when there was even a single base mismatch in the target DNA, the TSDR was suppressed and so subsequent isothermal DNA amplification and fluorescence response process could not occur. The proposed approach has been successfully implemented for the identification of the single-base mutant sequences in the human KRAS gene with a detection limit of 1.8 pM. Furthermore, a recovery of 90% was obtained when detecting the target sequence in spiked HeLa cells lysate, demonstrating the feasibility of this detection strategy for single-base mutations in biological samples. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Preselection of EGFR mutations in non-small-cell lung cancer patients by immunohistochemistry: comparison with DNA-sequencing, EGFR wild-type expression, gene copy number gain and clinicopathological data.

    Science.gov (United States)

    Gaber, Rania; Watermann, Iris; Kugler, Christian; Vollmer, Ekkehard; Perner, Sven; Reck, Martin; Goldmann, Torsten

    2017-01-01

    Targeting epidermal growth factor receptor (EGFR) in patients with non-small-cell lung cancer (NSCLC) having EGFR mutations is associated with an improved overall survival. The aim of this study is to verify, if EGFR mutations detected by immunohistochemistry (IHC) is a convincing way to preselect patients for DNA-sequencing and to figure out, the statistical association between EGFR mutation, wild-type EGFR overexpression, gene copy number gain, which are the main factors inducing EGFR tumorigenic activity and the clinicopathological data. Two hundred sixteen tumor tissue samples of primarily chemotherapeutic naïve NSCLC patients were analyzed for EGFR mutations E746-A750del and L858R and correlated with DNA-sequencing. Two hundred six of which were assessed by IHC, using 6B6 and 43B2 specific antibodies followed by DNA-sequencing of positive cases and 10 already genotyped tumor tissues were also included to investigate debugging accuracy of IHC. In addition, EGFR wild-type overexpression was IHC evaluated and EGFR gene copy number determination was performed by fluorescence in situ hybridization (FISH). Forty-one÷206 (19.9%) cases were positive for mutated EGFR by IHC. Eight of them had EGFR mutations of exons 18-21 by DNA-sequencing. Hit rate of 10 already genotyped NSCLC mutated cases was 90% by IHC. Positive association was found between EGFR mutations determined by IHC and both EGFR overexpression and increased gene copy number (p=0.002 and p<0.001, respectively). Additionally, positive association was detected between EGFR mutations, high tumor grade and clinical stage (p<0.001). IHC staining with mutation specific antibodies was demonstrated as a possible useful screening test to preselect patients for DNA-sequencing.

  12. Radioactively labelled DNA probes for crop improvement. Proceedings of a final research co-ordination meeting

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2001-11-01

    With the advent of DNA molecular marker technology in the 1980s plant breeding had a new and powerful tool with which to increase its efficacy. Such markers are abundant and directly reveal information about the genotype and therefore are more useful than simple phenotypic markers. In plant breeding applications, molecular markers reveal information about variability and genetic relationships, and enable genetic mapping, which greatly assists the breeder in selection of parents and progeny, as well as in management of breeding strategies. Furthermore, molecular markers linked to phenotypic traits permit very early selection of superior progenies from breeding populations, therefore significantly reducing the need for field testing and greatly increasing efficiency of plant breeding programmes. For this to occur the oligonucleotide probes for labelling genetic markers and/or the primers for polymerase chain reactions to amplify genetic markers needed to be also accessible to scientists in developing Member States. In addition, technical information, training and troubleshooting were needed to support the utilization of DNA markers. In the early 1990s there was a dramatic increase in requests for access to this technology. This co-ordinated research project (CRP) facilitated the transfer of molecular marker technology, in terms of both material and information, from advanced laboratories to assist breeding programmes in developing countries. Two other CRPs were conducted concurrently in order to assist developing Member States to utilise molecular markers - Application of DNA Based Marker Mutations for Improvement of Cereals and other Sexually Reproduced Crop Plants, and Use of Novel DNA Fingerprinting Techniques for the Detection and Characterisation of Genetic Variation in Vegetatively Propagated Crops (IAEA-TECDOC-1010 and IAEA-TECDOC-1047, respectively). The present CRP built upon the success of the former projects by ensuring the availability of probes

  13. Radioactively labelled DNA probes for crop improvement. Proceedings of a final research co-ordination meeting

    International Nuclear Information System (INIS)

    2001-11-01

    With the advent of DNA molecular marker technology in the 1980s plant breeding had a new and powerful tool with which to increase its efficacy. Such markers are abundant and directly reveal information about the genotype and therefore are more useful than simple phenotypic markers. In plant breeding applications, molecular markers reveal information about variability and genetic relationships, and enable genetic mapping, which greatly assists the breeder in selection of parents and progeny, as well as in management of breeding strategies. Furthermore, molecular markers linked to phenotypic traits permit very early selection of superior progenies from breeding populations, therefore significantly reducing the need for field testing and greatly increasing efficiency of plant breeding programmes. For this to occur the oligonucleotide probes for labelling genetic markers and/or the primers for polymerase chain reactions to amplify genetic markers needed to be also accessible to scientists in developing Member States. In addition, technical information, training and troubleshooting were needed to support the utilization of DNA markers. In the early 1990s there was a dramatic increase in requests for access to this technology. This co-ordinated research project (CRP) facilitated the transfer of molecular marker technology, in terms of both material and information, from advanced laboratories to assist breeding programmes in developing countries. Two other CRPs were conducted concurrently in order to assist developing Member States to utilise molecular markers - Application of DNA Based Marker Mutations for Improvement of Cereals and other Sexually Reproduced Crop Plants, and Use of Novel DNA Fingerprinting Techniques for the Detection and Characterisation of Genetic Variation in Vegetatively Propagated Crops (IAEA-TECDOC-1010 and IAEA-TECDOC-1047, respectively). The present CRP built upon the success of the former projects by ensuring the availability of probes

  14. Development of a human live attenuated West Nile infectious DNA vaccine: Identification of a minimal mutation set conferring the attenuation level acceptable for a human vaccine

    Energy Technology Data Exchange (ETDEWEB)

    Yamshchikov, Vladimir, E-mail: yaximik@gmail.com; Manuvakhova, Marina; Rodriguez, Efrain; Hébert, Charles

    2017-01-15

    ABSTRACT: For the development of a human West Nile (WN) infectious DNA (iDNA) vaccine, we created highly attenuated chimeric virus W1806 with the serological identity of highly virulent WN-NY99. Earlier, we attempted to utilize mutations found in the E protein of the SA14-14-2 vaccine to bring safety of W1806 to the level acceptable for human use (). Here, we analyzed effects of the SA14-14-2 changes on growth properties and neurovirulence of W1806. A set including the E138K, K279M, K439R and G447D changes was identified as the perspective subset for satisfying the target safety profile without compromising immunogenicity of the vaccine candidate. The genetic stability of the attenuated phenotype was found to be unsatisfactory being dependent on a subset of attenuating changes incorporated in W1806. Elucidation of underlying mechanisms influencing selection of pathways for restoration of the envelope protein functionality will facilitate resolution of the emerged genetic stability issue. - Highlights: •Effect of mutations in E on properties of WN1806 is determined. •A subset of attenuating mutations suitable for a human vaccine is defined. •Mechanism of attenuation is proposed and illustrated. •Underlying mechanisms of neurovirulence reversion are suggested.

  15. No evidence of association between optic neuritis and secondary LHON mtDNA mutations in patients with multiple sclerosis

    DEFF Research Database (Denmark)

    Andalib, Sasan; Talebi, Mahnaz; Sakhinia, Ebrahim

    2017-01-01

    Leber's Hereditary Optic Neuropathy (LHON) shares features with Multiple Sclerosis (MS). Both diseases develop optic lesions. Frequent secondary LHON mutations in MS patients may explain the optic damage. Here, we tested the hypothesis that secondary LHON mutations are associated with optic...

  16. The impact of SF3B1 mutations in CLL on the DNA-damage response

    DEFF Research Database (Denmark)

    Te Raa, G D; Derks, I A M; Navrkalova, V

    2015-01-01

    Mutations or deletions in TP53 or ATM are well-known determinants of poor prognosis in chronic lymphocytic leukemia (CLL), but only account for approximately 40% of chemo-resistant patients. Genome-wide sequencing has uncovered novel mutations in the splicing factor sf3b1, that were in part assoc...

  17. Screening individuals with intellectual disability, autism and Tourette's syndrome for KCNK9 mutations and aberrant DNA methylation within the 8q24 imprinted cluster.

    Science.gov (United States)

    Sánchez Delgado, Marta; Camprubí, Cristina; Tümer, Zeynep; Martínez, Francisco; Milà, Montserrat; Monk, David

    2014-09-01

    The phenotype overlap between autism spectrum disorders (ASD) & intellectual disabilities (ID) is mirrored at the genetic level, with common genes being reported mutated in variety of developmental disabilities. However despite widespread genetic screening for mutations, in approximately 40-60% of childhood developmental disorders the genetic cause remains unknown. Several genome-wide linkage screens in ASD have identified a locus mapping to distal 8q. We have recently identified a novel brain-specific imprinted cluster at this location, which contains the reciprocally expressed maternal KCNK9 and paternally expressed non-coding PEG13 transcripts, the latter located within an intron of TRAPPC9. Interestingly, mutations of KCNK9 and TRAPPC9 have been reported in Birk-Barel mental retardation and non-syndromic familial forms of ID, respectively. Here, we report a genetic screen for KCNK9 coding mutations and potential epigenetic aberrations that could result in deregulated imprinting in a cohort of 120 ID, 86 ASD and 86 Tourette syndrome patients. Fifteen of the ID patients had clinical characteristics overlapping with Birk-Barel syndrome. Sequencing of the two coding exons of KCNK9 failed to identify pathologic mutations, with only one variant, rs2615374, being present with allele frequencies similar to those described in dbSNP database. DNA methylation profiling of the KCNK9 and TRAPPC9 promoters, the maternally methylated PEG13 DMR and a long-range enhancer region were normal in all patients. Our findings suggest that mutations of KCNK9 or epigenetic disturbances within the PEG13 imprinted cluster do not significantly contribute to the cause of the developmental disabilities tested in this study. © 2014 Wiley Periodicals, Inc.

  18. Hereditary 1,25-dihydroxyvitamin D-resistant rickets with alopecia resulting from a novel missense mutation in the DNA-binding domain of the vitamin D receptor

    Science.gov (United States)

    Malloy, Peter J.; Wang, Jining; Srivastava, Tarak; Feldman, David

    2009-01-01

    The rare genetic recessive disease, hereditary vitamin D resistant rickets (HVDRR), is caused by mutations in the vitamin D receptor (VDR) that result in resistance to the active hormone 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3 or calcitriol). In this study, we examined the VDR from a young boy with clinical features of HVDRR including severe rickets, hypocalcemia, hypophosphatemia and partial alopecia. The pattern of alopecia was very unusual with areas of total baldness, adjacent to normal hair and regions of scant hair. The child failed to improve on oral calcium and vitamin D therapy but his abnormal chemistries and his bone x-rays normalized with intravenous calcium therapy. We found that the child was homozygous for a unique missense mutation in the VDR gene that converted valine to methionine at amino acid 26 (V26M) in the VDR DNA-binding domain (DBD). The mutant VDR was studied in the patient’s cultured skin fibroblasts and found to exhibit normal [3H]1,25-(OH)2D3 binding and protein expression. However, the fibroblasts were unresponsive to treatment with high concentrations of 1,25(OH)2D3 as demonstrated by their failure to induce CYP24A1 gene expression, a marker of 1,25(OH)2D3 responsiveness. We recreated the V26M mutation in the WT VDR and showed that in transfected COS-7 cells the mutation abolished 1,25(OH)2D3-mediated transactivation. The mutant VDR exhibited normal ligand-induced binding to RXRα and to the coactivator DRIP205. However, the V26M mutation inhibited VDR binding to a consensus vitamin D response element (VDRE). In summary, we have identified a novel V26M mutation in the VDR DBD as the molecular defect in a patient with HVDRR and an unusual pattern of alopecia. PMID:19815438

  19. DNA-repair and mutations in immuncompetent cells from patients with rheumatic diseases and corresponding animal models

    International Nuclear Information System (INIS)

    Altmann, H.

    1977-01-01

    Unscheduled DNA synthesis was investigated in lymphocytes of patients with different inflammatory rheumatic diseases. After γ-irradiation H 3 -thymidin incorporation in DNA and DNA rejoining was reduced. After UV-irradiation the first step (90 min) of unscheduled DNA synthesis was above the controls. Some animal models for human diseases showed the same trend. An infectious ethiology was discussed for some of these diseases. (author)

  20. One-by-one single-molecule detection of mutated nucleobases by monitoring tunneling current using a DNA tip.

    Science.gov (United States)

    Bui, Phuc Tan; Nishino, Tomoaki; Shiigi, Hiroshi; Nagaoka, Tsutomu

    2015-01-31

    A DNA molecule was utilized as a probe tip to achieve single-molecule genetic diagnoses. Hybridization of the probe and target DNAs resulted in electron tunneling along the emergent double-stranded DNA. Simple stationary monitoring of the tunneling current leads to single-molecule DNA detection and discovery of base mismatches and methylation.

  1. Site-directed mutational analysis of structural interactions of low molecule compounds binding to the N-terminal 8 kDa domain of DNA polymerase β

    International Nuclear Information System (INIS)

    Murakami, Shizuka; Kamisuki, Shinji; Takata, Kei-ichi; Kasai, Nobuyuki; Kimura, Seisuke; Mizushina, Yoshiyuki; Ohta, Keisuke; Sugawara, Fumio; Sakaguchi, Kengo

    2006-01-01

    We previously reported the mode of inhibition of DNA polymerase β (pol. β) by long chain fatty acids and a bile acid, involving binding analyses to the N-terminal 8-kDa DNA binding domain. Here we describe a site-directed mutational analysis in which the key amino acids (L11, K35, H51, K60, L77, and T79), which are direct interaction sites in the domain, were substituted with K, A, A, A, K, and A, respectively. And their pol. β interactions with a C24-long chain fatty acid, nervonic acid (NA), and a bile acid, lithocholic acid (LCA), were investigated by gel mobility shift assay and NMR spectroscopy. In the case of K35A, there was complete loss of DNA binding activity while K60A hardly has any activity. In contrast the other mutations had no appreciable effects. Thus, K35 and K60 are key amino acid sites for binding to template DNA. The DNA binding activities of L11K, H51A, and T79A as well as the wild type were inhibited by NA to the same extent. T79A demonstrated a disturbed interaction with LCA. 1 H- 15 N HSQC NMR analysis indicated that despite their many similarities, the wild-type and the mutant proteins displayed some significant chemical shift differences. Not only were the substituted amino acid residues three-dimensionally shifted, but some amino acids which are positioned far distant from the key amino acids showed a shift. These results suggest that the interaction surface was significantly distorted with the result that LCA could not bind to the domain. These findings confirm our previous biochemical and 3D structural proposals concerning inhibition by NA and LCA

  2. Spontaneous HBsAg loss in Korean patients: relevance of viral genotypes, S gene mutations, and covalently closed circular DNA copy numbers

    Directory of Open Access Journals (Sweden)

    Kyun-Hwan Kim

    2014-09-01

    Full Text Available Background/AimsOccult HBV infection can persist following HBsAg loss and be transmitted, but the virological features are not well defined.MethodsHere we investigated 25 Korean patients who lost HBsAg during follow up, either spontaneously or subsequent to therapy.ResultsWhereas subtype adr (genotype C was found in 96% of HBsAg positive patients, 75 % of patients who lost HBsAg spontaneously were seemed to be infected with the ayw subtype with sequence similar to genotype D. Mutations in the major hydrophilic region (MHR of HBsAg were found in 7 patients who lost HBsAg spontaneously. The mutations include T123S, M125I/N, C139R, D144E, V177A, L192F, and W196L, some of which have not been reported before. Functional analysis via transfection experiments indicate that the C139R and D144E mutations drastically reduced HBsAg antigenicity, while the Y225del mutation found in one interferon-treated patient impaired HBsAg secretion.ConclusionsLack of detectable HBsAg in patient serum could be explained by low level of ccc DNA in liver tissue, low antigenicity of the surface protein, or its secretion defect.

  3. Alzheimer neuropathology without frontotemporal lobar degeneration hallmarks (TAR DNA-binding protein 43 inclusions) in missense progranulin mutation Cys139Arg.

    Science.gov (United States)

    Redaelli, Veronica; Rossi, Giacomina; Maderna, Emanuela; Kovacs, Gabor G; Piccoli, Elena; Caroppo, Paola; Cacciatore, Francesca; Spinello, Sonia; Grisoli, Marina; Sozzi, Giuliano; Salmaggi, Andrea; Tagliavini, Fabrizio; Giaccone, Giorgio

    2018-01-01

    Null mutations in progranulin gene (GRN) reduce the progranulin production resulting in haploinsufficiency and are tightly associated with tau-negative frontotemporal lobar degeneration with TAR DNA-binding protein 43-positive inclusions (FTLD-TDP). Missense mutations of GRN were also identified, but their effects are not completely clear, in particular unanswered is the question of what neuropathology they elicit, also considering that their occurrence has been reported in patients with typical clinical features of Alzheimer disease. They describe two fraternal twins carrying the missense GRN Cys139Arg mutation affected by late-onset dementia and we report the neuropathological study of one of them. Both patients were examined by neuroimaging, neuropsychological assessment and genetic analysis of GRN and other genes associated with dementia. The brain of one was obtained at autopsy and examined neuropathologically. One sister presented clinical and MRI features leading to the diagnosis of Alzheimer disease. The other underwent autopsy and the brain showed neuropathological hallmarks of Alzheimer disease with abundant Aβ-amyloid deposition and Braak stage V of neurofibrillary pathology, in the absence of the hallmark lesions of FTLD-TDP. Their findings may contribute to better clarify the role of progranulin in neurodegenerative diseases indicating that some GRN mutations, in particular missense ones, may act as strong risk factor for Alzheimer disease rather than induce FTLD-TDP. © 2016 International Society of Neuropathology.

  4. Only male matrilineal relatives with Leber's hereditary optic neuropathy in a large Chinese family carrying the mitochondrial DNA G11778A mutation

    International Nuclear Information System (INIS)

    Qu Jia; Li Ronghua; Tong Yi; Hu Yongwu; Zhou Xiangtian; Qian Yaping; Lu Fan; Guan Minxin

    2005-01-01

    We report here the characterization of a five-generation large Chinese family with Leber's hereditary optic neuropathy (LHON). Very strikingly, six affected individuals of 38 matrilineal relatives (17 females/21 males) are exclusively males in this Chinese family. These matrilineal relatives in this family exhibited late-onset/progressive visual impairment with a wide range of severity, ranging from blindness to normal vision. The age of onset in visual impairment varies from 17 to 30 years. Sequence analysis of the complete mitochondrial genome in this pedigree revealed the presence of the G11778A mutation in ND4 gene and 29 other variants. This mitochondrial genome belongs to the Southern Chinese haplogroup B5b. We showed that the G11778A mutation is present at near homoplasmy in matrilineal relatives of this Chinese family but not in 164 Chinese controls. Incomplete penetrance of LHON in this family indicates the involvement of modulatory factors in the phenotypic expression of visual dysfunction associated with the G11778A mutation. However, none of other mtDNA variants are evolutionarily conserved and implicated to have significantly functional consequence. Thus, nuclear modifier gene(s) or environmental factor(s) seem to account for the penetrance and phenotypic variability of LHON in this Chinese family carrying the G11778A mutation

  5. Clinical and molecular features of an infant patient affected by Leigh Disease associated to m.14459G > A mitochondrial DNA mutation: a case report

    Directory of Open Access Journals (Sweden)

    Moggio Maurizio

    2011-07-01

    Full Text Available Abstract Background Leigh Syndrome (LS is a severe neurodegenerative disorder characterized by bilateral symmetrical necrotic lesions in the basal ganglia and brainstem. Onset is in early infancy and prognosis is poor. Causative mutations have been disclosed in mitochondrial DNA and nuclear genes affecting respiratory chain subunits and assembly factors. Case presentation Here we report the clinical and molecular features of a 15-month-old female LS patient. Direct sequencing of her muscle-derived mtDNA revealed the presence of two apparently homoplasmic variants: the novel m.14792C > G and the already known m.14459G > A resulting in p.His16Asp change in cytochrome b (MT-CYB and p.Ala72Val substitution in ND6 subunit, respectively. The m.14459G > A was heteroplasmic in the mother's blood-derived DNA. Conclusions The m.14459G > A might lead to LS, complicated LS or Leber Optic Hereditary Neuropathy. A comprehensive re-evaluation of previously described 14459G > A-mutated patients does not explain this large clinical heterogeneity.

  6. The mitochondrial ND1 m.3337G>A mutation associated to multiple mitochondrial DNA deletions in a patient with Wolfram syndrome and cardiomyopathy.

    Science.gov (United States)

    Mezghani, Najla; Mnif, Mouna; Mkaouar-Rebai, Emna; Kallel, Nozha; Salem, Ikhlass Haj; Charfi, Nadia; Abid, Mohamed; Fakhfakh, Faiza

    2011-07-29

    Wolfram syndrome (WFS) is a rare hereditary disorder also known as DIDMOAD (diabetes insipidus, diabetes mellitus, optic atrophy, and deafness). It is a heterogeneous disease and full characterization of all clinical and biological features of this disorder is difficult. The wide spectrum of clinical expression, affecting several organs and tissues, and the similarity in phenotype between patients with Wolfram syndrome and those with certain types of respiratory chain diseases suggests mitochondrial DNA (mtDNA) involvement in Wolfram syndrome patients. We report a Tunisian patient with clinical features of moderate Wolfram syndrome including diabetes, dilated cardiomyopathy and neurological complications. The results showed the presence of the mitochondrial ND1 m.3337G>A mutation in almost homoplasmic form in 3 tested tissues of the proband (blood leukocytes, buccal mucosa and skeletal muscle). In addition, the long-range PCR amplifications revealed the presence of multiple deletions of the mitochondrial DNA extracted from the patient's skeletal muscle removing several tRNA and protein-coding genes. Our study reported a Tunisian patient with clinical features of moderate Wolfram syndrome associated with cardiomyopathy, in whom we detected the ND1 m.3337G>A mutation with mitochondrial multiple deletions. Copyright © 2011 Elsevier Inc. All rights reserved.

  7. Juvenile Leigh syndrome, optic atrophy, ataxia, dystonia, and epilepsy due to T14487C mutation in the mtDNA-ND6 gene: a mitochondrial syndrome presenting from birth to adolescence.

    Science.gov (United States)

    Leshinsky-Silver, Esther; Shuvalov, Ruslan; Inbar, Shani; Cohen, Sarit; Lev, Dorit; Lerman-Sagie, Tally

    2011-04-01

    An increasing number of reports describe mutations in mitochondrial DNA coding regions, especially in mitochondrial DNA- encoded nicotinamide adenine dinucleotide dehydrogenase subunit genes of the respiratory chain complex I, as causing early-onset Leigh syndrome. The authors report the molecular findings in a 24-year-old patient with juvenile-onset Leigh syndrome presenting with optic atrophy, ataxia dystonia, and epilepsy. A brain magnetic resonance imaging revealed bilateral basal ganglia and thalamic hypointensities, and a magnetic resonance spectroscopy revealed an increased lactate peak. The authors identified a T14487C change causing M63V substitution in the mitochondrial ND6 gene. The mutation was heteroplasmic in muscle and blood samples, with different mutation loads, and was absent in the patient's mother's urine and blood samples. They suggest that the T14487C mtDNA mutation should be analyzed in Leigh syndrome, presenting with optic atrophy, ataxia, dystonia, and epilepsy, regardless of age.

  8. DNA Packaging by λ-Like Bacteriophages: Mutations Broadening the Packaging Specificity of Terminase, the λ-Packaging Enzyme

    OpenAIRE

    Feiss, Michael; Reynolds, Erin; Schrock, Morgan; Sippy, Jean

    2010-01-01

    The DNA-packaging specificities of phages λ and 21 depend on the specific DNA interactions of the small terminase subunits, which have support helix-turn-recognition helix-wing DNA-binding motifs. λ-Terminase with the recognition helix of 21 preferentially packages 21 DNA. This chimeric terminase's ability to package λDNA is reduced ∼20-fold. Phage λ with the chimeric terminase is unable to form plaques, but pseudorevertants are readily obtained. Some pseudorevertants have trans-acting suppre...

  9. Mutation of the mouse Syce1 gene disrupts synapsis and suggests a link between synaptonemal complex structural components and DNA repair.

    Directory of Open Access Journals (Sweden)

    Ewelina Bolcun-Filas

    2009-02-01

    Full Text Available In mammals, the synaptonemal complex is a structure required to complete crossover recombination. Although suggested by cytological work, in vivo links between the structural proteins of the synaptonemal complex and the proteins of the recombination process have not previously been made. The central element of the synaptonemal complex is traversed by DNA at sites of recombination and presents a logical place to look for interactions between these components. There are four known central element proteins, three of which have previously been mutated. Here, we complete the set by creating a null mutation in the Syce1 gene in mouse. The resulting disruption of synapsis in these animals has allowed us to demonstrate a biochemical interaction between the structural protein SYCE2 and the repair protein RAD51. In normal meiosis, this interaction may be responsible for promoting homologous synapsis from sites of recombination.

  10. X-ray-induced mutations in Escherichia coli K-12 strains with altered DNA polymerase I activities

    International Nuclear Information System (INIS)

    Nagata, Yuki; Kawata, Masakado; Komura, Jun-ichiro; Ono, Tetsuya; Yamamoto, Kazuo

    2003-01-01

    Spectra of ionizing radiation mutagenesis were determined by sequencing X-ray-induced endogenous tonB gene mutations in Escherichia coli polA strains. We used two polA alleles, the polA1 mutation, defective for Klenow domain, and the polA107 mutation, defective for flap domain. We demonstrated that irradiation of 75 and 50 Gy X-rays could induce 3.8- and 2.6-fold more of tonB mutation in polA1 and polA107 strains, respectively, than spontaneous level. The radiation induced spectrum of 51 tonB mutations in polA1 and 51 in polA107 indicated that minus frameshift, A:T→T:A transversion and G:C→T:A transversion were the types of mutations increased. Previously, we have reported essentially the same X-ray-induced tonB mutation spectra in the wild-type strain. These results indicate that (1) X-rays can induce minus frameshift, A:T→T:A transversion and G:C→T:A transversion in E. coli and (2) presence or absence of polymerase I (PolI) of E. coli does not have any effects on the process of X-ray mutagenesis

  11. Methyl DNA adducts, DNA repair, and hypoxanthine-guanine phosphoribosyl transferase mutations in peripheral white blood cells from patients with malignant melanoma treated with dacarbazine and hydroxyurea

    NARCIS (Netherlands)

    Philip, P.A.; Souliotis, V.L.; Harris, A.L.; Salisbury, A.; Tates, A.D.; Mitchell, K.; Delft, J.H.M. van; Ganesan, T.S.; Kyrtopoulos, S.A.

    1996-01-01

    Dacarbazine (DTIC) is a DNA-methylating drug used in the treatment of malignant melanoma. Among the DNA dducts induced by DTIC are N7-methylguanine (N7-meG) and O6-methylguamne (O6-meG). The latter adduct, in particular, may be important in the mutagenic as well as the cytotoxic activity of DTIC.

  12. Mitochondrial DNA polymerase editing mutation, PolgD257A, disturbs stem-progenitor cell cycling in the small intestine and restricts excess fat absorption.

    Science.gov (United States)

    Fox, Raymond G; Magness, Scott; Kujoth, Gregory C; Prolla, Tomas A; Maeda, Nobuyo

    2012-05-01

    Changes in intestinal absorption of nutrients are important aspects of the aging process. To address this issue, we investigated the impact of accelerated mitochondrial DNA mutations on the stem/progenitor cells in the crypts of Lieberkühn in mice homozygous for a mitochondrial DNA polymerase gamma mutation, Polg(D257A), that exhibit accelerated aging phenotype. As early as 3-7 mo of age, the small intestine was significantly enlarged in the PolgD257A mice. The crypts of the PolgD257A mice contained 20% more cells than those of their wild-type littermates and exhibited a 10-fold increase in cellular apoptosis primarily in the stem/progenitor cell zones. Actively dividing cells were proportionally increased, yet a significantly smaller proportion of cells was in the S phase of the cell cycle. Stem cell-derived organoids from PolgD257A mice failed to develop fully in culture and exhibited fewer crypt units, indicating an impact of the mutation on the intestinal epithelial stem/progenitor cell maintenance. In addition, epithelial cell migration along the crypt-villus axis was slowed and less organized, and the ATP content in the villi was significantly reduced. On a high-fat, high-carbohydrate diet, PolgD257A mice showed significantly restricted absorption of excess lipids accompanied by an increase in fecal steatocrits. We conclude that the PolgD257A mutation causes cell cycle dysregulation in the crypts leading to the age-associated changes in the morphology of the small intestine and contributes to the restricted absorption of dietary lipids.

  13. 75 FR 31795 - Office of Biotechnology Activities; Recombinant DNA Research: Amended Notice of Meeting

    Science.gov (United States)

    2010-06-04

    ... DEPARTMENT OF HEALTH AND HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities; Recombinant DNA Research: Amended Notice of Meeting ACTION: Notice of cancellation of... information. Dated: May 26, 2010. Jacqueline Corrigan-Curay, Acting Director, Office of Biotechnology...

  14. The mitochondrial ND1 m.3337G>A mutation associated to multiple mitochondrial DNA deletions in a patient with Wolfram syndrome and cardiomyopathy

    Energy Technology Data Exchange (ETDEWEB)

    Mezghani, Najla [Laboratoire de Genetique Moleculaire Humaine, Faculte de Medecine de Sfax, Universite de Sfax (Tunisia); Mnif, Mouna [Service d' endocrinologie, C.H.U. Habib Bourguiba de Sfax (Tunisia); Mkaouar-Rebai, Emna, E-mail: emna_mkaouar@mail2world.com [Laboratoire de Genetique Moleculaire Humaine, Faculte de Medecine de Sfax, Universite de Sfax (Tunisia); Kallel, Nozha [Service d' endocrinologie, C.H.U. Habib Bourguiba de Sfax (Tunisia); Salem, Ikhlass Haj [Laboratoire de Genetique Moleculaire Humaine, Faculte de Medecine de Sfax, Universite de Sfax (Tunisia); Charfi, Nadia; Abid, Mohamed [Service d' endocrinologie, C.H.U. Habib Bourguiba de Sfax (Tunisia); Fakhfakh, Faiza [Laboratoire de Genetique Moleculaire Humaine, Faculte de Medecine de Sfax, Universite de Sfax (Tunisia)

    2011-07-29

    Highlights: {yields} We reported a patient with Wolfram syndrome and dilated cardiomyopathy. {yields} We detected the ND1 mitochondrial m.3337G>A mutation in 3 tested tissues (blood leukocytes, buccal mucosa and skeletal muscle). {yields} Long-range PCR amplification revealed the presence of multiple mitochondrial deletions in the skeletal muscle. {yields} The deletions remove several tRNA and protein-coding genes. -- Abstract: Wolfram syndrome (WFS) is a rare hereditary disorder also known as DIDMOAD (diabetes insipidus, diabetes mellitus, optic atrophy, and deafness). It is a heterogeneous disease and full characterization of all clinical and biological features of this disorder is difficult. The wide spectrum of clinical expression, affecting several organs and tissues, and the similarity in phenotype between patients with Wolfram syndrome and those with certain types of respiratory chain diseases suggests mitochondrial DNA (mtDNA) involvement in Wolfram syndrome patients. We report a Tunisian patient with clinical features of moderate Wolfram syndrome including diabetes, dilated cardiomyopathy and neurological complications. The results showed the presence of the mitochondrial ND1 m.3337G>A mutation in almost homoplasmic form in 3 tested tissues of the proband (blood leukocytes, buccal mucosa and skeletal muscle). In addition, the long-range PCR amplifications revealed the presence of multiple deletions of the mitochondrial DNA extracted from the patient's skeletal muscle removing several tRNA and protein-coding genes. Our study reported a Tunisian patient with clinical features of moderate Wolfram syndrome associated with cardiomyopathy, in whom we detected the ND1 m.3337G>A mutation with mitochondrial multiple deletions.

  15. The mitochondrial ND1 m.3337G>A mutation associated to multiple mitochondrial DNA deletions in a patient with Wolfram syndrome and cardiomyopathy

    International Nuclear Information System (INIS)

    Mezghani, Najla; Mnif, Mouna; Mkaouar-Rebai, Emna; Kallel, Nozha; Salem, Ikhlass Haj; Charfi, Nadia; Abid, Mohamed; Fakhfakh, Faiza

    2011-01-01

    Highlights: → We reported a patient with Wolfram syndrome and dilated cardiomyopathy. → We detected the ND1 mitochondrial m.3337G>A mutation in 3 tested tissues (blood leukocytes, buccal mucosa and skeletal muscle). → Long-range PCR amplification revealed the presence of multiple mitochondrial deletions in the skeletal muscle. → The deletions remove several tRNA and protein-coding genes. -- Abstract: Wolfram syndrome (WFS) is a rare hereditary disorder also known as DIDMOAD (diabetes insipidus, diabetes mellitus, optic atrophy, and deafness). It is a heterogeneous disease and full characterization of all clinical and biological features of this disorder is difficult. The wide spectrum of clinical expression, affecting several organs and tissues, and the similarity in phenotype between patients with Wolfram syndrome and those with certain types of respiratory chain diseases suggests mitochondrial DNA (mtDNA) involvement in Wolfram syndrome patients. We report a Tunisian patient with clinical features of moderate Wolfram syndrome including diabetes, dilated cardiomyopathy and neurological complications. The results showed the presence of the mitochondrial ND1 m.3337G>A mutation in almost homoplasmic form in 3 tested tissues of the proband (blood leukocytes, buccal mucosa and skeletal muscle). In addition, the long-range PCR amplifications revealed the presence of multiple deletions of the mitochondrial DNA extracted from the patient's skeletal muscle removing several tRNA and protein-coding genes. Our study reported a Tunisian patient with clinical features of moderate Wolfram syndrome associated with cardiomyopathy, in whom we detected the ND1 m.3337G>A mutation with mitochondrial multiple deletions.

  16. Single-strand conformation polymorphism (SSCP)-based mutation scanning approaches to fingerprint sequence variation in ribosomal DNA of ascaridoid nematodes.

    Science.gov (United States)

    Zhu, X Q; Gasser, R B

    1998-06-01

    In this study, we assessed single-strand conformation polymorphism (SSCP)-based approaches for their capacity to fingerprint sequence variation in ribosomal DNA (rDNA) of ascaridoid nematodes of veterinary and/or human health significance. The second internal transcribed spacer region (ITS-2) of rDNA was utilised as the target region because it is known to provide species-specific markers for this group of parasites. ITS-2 was amplified by PCR from genomic DNA derived from individual parasites and subjected to analysis. Direct SSCP analysis of amplicons from seven taxa (Toxocara vitulorum, Toxocara cati, Toxocara canis, Toxascaris leonina, Baylisascaris procyonis, Ascaris suum and Parascaris equorum) showed that the single-strand (ss) ITS-2 patterns produced allowed their unequivocal identification to species. While no variation in SSCP patterns was detected in the ITS-2 within four species for which multiple samples were available, the method allowed the direct display of four distinct sequence types of ITS-2 among individual worms of T. cati. Comparison of SSCP/sequencing with the methods of dideoxy fingerprinting (ddF) and restriction endonuclease fingerprinting (REF) revealed that also ddF allowed the definition of the four sequence types, whereas REF displayed three of four. The findings indicate the usefulness of the SSCP-based approaches for the identification of ascaridoid nematodes to species, the direct display of sequence variation in rDNA and the detection of population variation. The ability to fingerprint microheterogeneity in ITS-2 rDNA using such approaches also has implications for studying fundamental aspects relating to mutational change in rDNA.

  17. Public involvement in pharmacogenomics research: a national survey on patients' attitudes towards pharmacogenomics research and the willingness to donate DNA samples to a DNA bank in Japan.

    Science.gov (United States)

    Kobayashi, Eriko; Sakurada, Tomoya; Ueda, Shiro; Satoh, Nobunori

    2011-05-01

    To assess the attitude of Japanese patients towards pharmacogenomics research and a DNA bank for identifying genomic markers associated with adverse drug reactions (ADRs) and their willingness to donate DNA samples, we conducted a survey of 550 male and female patients. The majority of the respondents showed a positive attitude towards pharmacogenomics research (87.6%) and a DNA bank (75.1%). The willingness to donate DNA samples when experiencing severe ADRs (55.8%) was higher than when taking medications (40.4%). Positive attitudes towards a DNA bank and organ donation were significantly associated with an increased willingness to donate. Though the level of positive attitude in the patient population was higher than that in the general public in our former study (81.0 and 70.4%, respectively), the level of the willingness of patients to donate was 40.4% when taking medications and 55.8% when experiencing severe ADRs which was lower than that of the general public in our former study (45.3 and 61.7%). The results suggested that the level of true willingness in the patient population was lower than that of the general public considering the fictitious situation presented to the public (to suppose that they were patients receiving medication). It is important to assess the willingness of patients who are true potential donors, not the general public.

  18. Genome-wide SNP scan of pooled DNA reveals nonsense mutation in FGF20 in the scaleless line of featherless chickens

    Directory of Open Access Journals (Sweden)

    Wells Kirsty L

    2012-06-01

    Full Text Available Abstract Background Scaleless (sc/sc chickens carry a single recessive mutation that causes a lack of almost all body feathers, as well as foot scales and spurs, due to a failure of skin patterning during embryogenesis. This spontaneous mutant line, first described in the 1950s, has been used extensively to explore the tissue interactions involved in ectodermal appendage formation in embryonic skin. Moreover, the trait is potentially useful in tropical agriculture due to the ability of featherless chickens to tolerate heat, which is at present a major constraint to efficient poultry meat production in hot climates. In the interests of enhancing our understanding of feather placode development, and to provide the poultry industry with a strategy to breed heat-tolerant meat-type chickens (broilers, we mapped and identified the sc mutation. Results Through a cost-effective and labour-efficient SNP array mapping approach using DNA from sc/sc and sc/+ blood sample pools, we map the sc trait to chromosome 4 and show that a nonsense mutation in FGF20 is completely associated with the sc/sc phenotype. This mutation, common to all sc/sc individuals and absent from wild type, is predicted to lead to loss of a highly conserved region of the FGF20 protein important for FGF signalling. In situ hybridisation and quantitative RT-PCR studies reveal that FGF20 is epidermally expressed during the early stages of feather placode patterning. In addition, we describe a dCAPS genotyping assay based on the mutation, developed to facilitate discrimination between wild type and sc alleles. Conclusions This work represents the first loss of function genetic evidence supporting a role for FGF ligand signalling in feather development, and suggests FGF20 as a novel central player in the development of vertebrate skin appendages, including hair follicles and exocrine glands. In addition, this is to our knowledge the first report describing the use of the chicken SNP array to

  19. Replicating animal mitochondrial DNA

    Directory of Open Access Journals (Sweden)

    Emily A. McKinney

    2013-01-01

    Full Text Available The field of mitochondrial DNA (mtDNA replication has been experiencing incredible progress in recent years, and yet little is certain about the mechanism(s used by animal cells to replicate this plasmid-like genome. The long-standing strand-displacement model of mammalian mtDNA replication (for which single-stranded DNA intermediates are a hallmark has been intensively challenged by a new set of data, which suggests that replication proceeds via coupled leading-and lagging-strand synthesis (resembling bacterial genome replication and/or via long stretches of RNA intermediates laid on the mtDNA lagging-strand (the so called RITOLS. The set of proteins required for mtDNA replication is small and includes the catalytic and accessory subunits of DNA polymerase y, the mtDNA helicase Twinkle, the mitochondrial single-stranded DNA-binding protein, and the mitochondrial RNA polymerase (which most likely functions as the mtDNA primase. Mutations in the genes coding for the first three proteins are associated with human diseases and premature aging, justifying the research interest in the genetic, biochemical and structural properties of the mtDNA replication machinery. Here we summarize these properties and discuss the current models of mtDNA replication in animal cells.

  20. Identification of DNA Fragments that Showed Linkage to the Radiation-induced Yellow Vein Mosaic Disease Resistance Mutation in Okra

    International Nuclear Information System (INIS)

    Boonsirichai, Kanokporn; Phadvibulya, Valailak; Adthalungrong, Amnuai; Srithongchai, Wanphen; Puripunyavanich, Vichai

    2007-08-01

    Full text: The yellow vein mosaic disease resistant mutant of okra was crossed to Pichit 03, a susceptible variety. Their progeny showed prolonged resistance when compared with Pichit 03. DNA fingerprints of F2 and BC1F1 individuals from the cross indicated that most DNA bands did not segregate with either the resistance or the susceptible characteristics. Nonetheless, polymorphic DNA bands could be identified between the mutant and Okura, the parental variety

  1. Evolutionary analyses of entire genomes do not support the association of mtDNA mutations with Ras/MAPK pathway syndromes.

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    Alberto Gómez-Carballa

    Full Text Available BACKGROUND: There are several known autosomal genes responsible for Ras/MAPK pathway syndromes, including Noonan syndrome (NS and related disorders (such as LEOPARD, neurofibromatosis type 1, although mutations of these genes do not explain all cases. Due to the important role played by the mitochondrion in the energetic metabolism of cardiac muscle, it was recently proposed that variation in the mitochondrial DNA (mtDNA genome could be a risk factor in the Noonan phenotype and in hypertrophic cardiomyopathy (HCM, which is a common clinical feature in Ras/MAPK pathway syndromes. In order to test these hypotheses, we sequenced entire mtDNA genomes in the largest series of patients suffering from Ras/MAPK pathway syndromes analyzed to date (n = 45, most of them classified as NS patients (n = 42. METHODS/PRINCIPAL FINDINGS: The results indicate that the observed mtDNA lineages were mostly of European ancestry, reproducing in a nutshell the expected haplogroup (hg patterns of a typical Iberian dataset (including hgs H, T, J, and U. Three new branches of the mtDNA phylogeny (H1j1, U5b1e, and L2a5 are described for the first time, but none of these are likely to be related to NS or Ras/MAPK pathway syndromes when observed under an evolutionary perspective. Patterns of variation in tRNA and protein genes, as well as redundant, private and heteroplasmic variants, in the mtDNA genomes of patients were as expected when compared with the patterns inferred from a worldwide mtDNA phylogeny based on more than 8700 entire genomes. Moreover, most of the mtDNA variants found in patients had already been reported in healthy individuals and constitute common polymorphisms in human population groups. CONCLUSIONS/SIGNIFICANCE: As a whole, the observed mtDNA genome variation in the NS patients was difficult to reconcile with previous findings that indicated a pathogenic role of mtDNA variants in NS.

  2. Evolutionary Analyses of Entire Genomes Do Not Support the Association of mtDNA Mutations with Ras/MAPK Pathway Syndromes

    Science.gov (United States)

    Cerezo, María; Balboa, Emilia; Heredia, Claudia; Castro-Feijóo, Lidia; Rica, Itxaso; Barreiro, Jesús; Eirís, Jesús; Cabanas, Paloma; Martínez-Soto, Isabel; Fernández-Toral, Joaquín; Castro-Gago, Manuel; Pombo, Manuel; Carracedo, Ángel; Barros, Francisco

    2011-01-01

    Background There are several known autosomal genes responsible for Ras/MAPK pathway syndromes, including Noonan syndrome (NS) and related disorders (such as LEOPARD, neurofibromatosis type 1), although mutations of these genes do not explain all cases. Due to the important role played by the mitochondrion in the energetic metabolism of cardiac muscle, it was recently proposed that variation in the mitochondrial DNA (mtDNA) genome could be a risk factor in the Noonan phenotype and in hypertrophic cardiomyopathy (HCM), which is a common clinical feature in Ras/MAPK pathway syndromes. In order to test these hypotheses, we sequenced entire mtDNA genomes in the largest series of patients suffering from Ras/MAPK pathway syndromes analyzed to date (n = 45), most of them classified as NS patients (n = 42). Methods/Principal Findings The results indicate that the observed mtDNA lineages were mostly of European ancestry, reproducing in a nutshell the expected haplogroup (hg) patterns of a typical Iberian dataset (including hgs H, T, J, and U). Three new branches of the mtDNA phylogeny (H1j1, U5b1e, and L2a5) are described for the first time, but none of these are likely to be related to NS or Ras/MAPK pathway syndromes when observed under an evolutionary perspective. Patterns of variation in tRNA and protein genes, as well as redundant, private and heteroplasmic variants, in the mtDNA genomes of patients were as expected when compared with the patterns inferred from a worldwide mtDNA phylogeny based on more than 8700 entire genomes. Moreover, most of the mtDNA variants found in patients had already been reported in healthy individuals and constitute common polymorphisms in human population groups. Conclusions/Significance As a whole, the observed mtDNA genome variation in the NS patients was difficult to reconcile with previous findings that indicated a pathogenic role of mtDNA variants in NS. PMID:21526175

  3. A new sieving matrix for DNA sequencing, genotyping and mutation detection and high-throughput genotyping with a 96-capillary array system

    Energy Technology Data Exchange (ETDEWEB)

    Gao, David [Iowa State Univ., Ames, IA (United States)

    1999-11-08

    Capillary electrophoresis has been widely accepted as a fast separation technique in DNA analysis. In this dissertation, a new sieving matrix is described for DNA analysis, especially DNA sequencing, genetic typing and mutation detection. A high-throughput 96 capillary array electrophoresis system was also demonstrated for simultaneous multiple genotyping. The authors first evaluated the influence of different capillary coatings on the performance of DNA sequencing. A bare capillary was compared with a DB-wax, an FC-coated and a polyvinylpyrrolidone dynamically coated capillary with PEO as sieving matrix. It was found that covalently-coated capillaries had no better performance than bare capillaries while PVP coating provided excellent and reproducible results. The authors also developed a new sieving Matrix for DNA separation based on commercially available poly(vinylpyrrolidone) (PVP). This sieving matrix has a very low viscosity and an excellent self-coating effect. Successful separations were achieved in uncoated capillaries. Sequencing of M13mp18 showed good resolution up to 500 bases in treated PVP solution. Temperature gradient capillary electrophoresis and PVP solution was applied to mutation detection. A heteroduplex sample and a homoduplex reference were injected during a pair of continuous runs. A temperature gradient of 10 C with a ramp of 0.7 C/min was swept throughout the capillary. Detection was accomplished by laser induced fluorescence detection. Mutation detection was performed by comparing the pattern changes between the homoduplex and the heteroduplex samples. High throughput, high detection rate and easy operation were achieved in this system. They further demonstrated fast and reliable genotyping based on CTTv STR system by multiple-capillary array electrophoresis. The PCR products from individuals were mixed with pooled allelic ladder as an absolute standard and coinjected with a 96-vial tray. Simultaneous one-color laser-induced fluorescence

  4. Persistence of Repair Proteins at Unrepaired DNA Damage Distinguishes Diseases with ERCC2 (XPD) Mutations: Cancer-Prone Xeroderma Pigmentosum vs. Non-Cancer-Prone Trichothiodystrophy

    Science.gov (United States)

    Boyle, Jennifer; Ueda, Takahiro; Oh, Kyu-Seon; Imoto, Kyoko; Tamura, Deborah; Jagdeo, Jared; Khan, Sikandar G.; Nadem, Carine; DiGiovanna, John J.; Kraemer, Kenneth H.

    2012-01-01

    Patients with xeroderma pigmentosum (XP) have a 1,000-fold increase in ultraviolet (UV)-induced skin cancers while trichothiodystrophy (TTD) patients, despite mutations in the same genes, ERCC2 (XPD) or ERCC3 (XPB), are cancer-free. Unlike XP cells, TTD cells have a nearly normal rate of removal of UV-induced 6-4 photoproducts (6-4PP) in their DNA and low levels of the basal transcription factor, TFIIH. We examined seven XP, TTD, and XP/TTD complex patients and identified mutations in the XPD gene. We discovered large differences in nucleotide excision repair (NER) protein recruitment to sites of localized UV damage in TTD cells compared to XP or normal cells. XPC protein was rapidly localized in all cells. XPC was redistributed in TTD, and normal cells by 3 hr postirradiation, but remained localized in XP cells at 24-hr postirradiation. In XP cells recruitment of other NER proteins (XPB, XPD, XPG, XPA, and XPF) was also delayed and persisted at 24 hr (p < 0.001). In TTD cells with defects in the XPD, XPB, or GTF2H5 (TTDA) genes, in contrast, recruitment of these NER proteins was reduced compared to normals at early time points (p < 0.001) and remained low at 24 hr postirradiation. These data indicate that in XP persistence of NER proteins at sites of unrepaired DNA damage is associated with greatly increased skin cancer risk possibly by blockage of translesion DNA synthesis. In contrast, in TTD, low levels of unstable TFIIH proteins do not accumulate at sites of unrepaired photoproducts and may permit normal translesion DNA synthesis without increased skin cancer. PMID:18470933

  5. Amelogenesis Imperfecta and Screening of Mutation in Amelogenin Gene

    Directory of Open Access Journals (Sweden)

    Fernanda Veronese Oliveira

    2014-01-01

    Full Text Available The aim of this study was to report the clinical findings and the screening of mutations of amelogenin gene of a 7-year-old boy with amelogenesis imperfecta (AI. The genomic DNA was extracted from saliva of patient and his family, followed by PCR and direct DNA sequencing. The c.261C>T mutation was found in samples of mother, father, and brother, but the mutation was not found in the sequence of the patient. This mutation is a silent mutation and a single-nucleotide polymorphism (rs2106416. Thus, it is suggested that the mutation found was not related to the clinical presence of AI. Further research is necessary to examine larger number of patients and genes related to AI.

  6. The retinitis pigmentosa-mutated RP2 protein exhibits exonuclease activity and translocates to the nucleus in response to DNA damage

    International Nuclear Information System (INIS)

    Yoon, Jung-Hoon; Qiu Junzhuan; Cai Sheng; Chen Yuan; Cheetham, Michael E.; Shen Binghui; Pfeifer, Gerd P.

    2006-01-01

    Retinitis pigmentosa (RP) is a genetically heterogeneous disease characterized by degeneration of the retina. Mutations in the RP2 gene are linked to the second most frequent form of X-linked retinitis pigmentosa. RP2 is a plasma membrane-associated protein of unknown function. The N-terminal domain of RP2 shares amino acid sequence similarity to the tubulin-specific chaperone protein co-factor C. The C-terminus consists of a domain with similarity to nucleoside diphosphate kinases (NDKs). Human NDK1, in addition to its role in providing nucleoside triphosphates, has recently been described as a 3' to 5' exonuclease. Here, we show that RP2 is a DNA-binding protein that exhibits exonuclease activity, with a preference for single-stranded or nicked DNA substrates that occur as intermediates of base excision repair pathways. Furthermore, we show that RP2 undergoes re-localization into the nucleus upon treatment of cells with DNA damaging agents inducing oxidative stress, most notably solar simulated light and UVA radiation. The data suggest that RP2 may have previously unrecognized roles as a DNA damage response factor and 3' to 5' exonuclease

  7. Label-free detection of DNA hybridization and single point mutations in a nano-gap biosensor

    International Nuclear Information System (INIS)

    Zaffino, R L; Mir, M; Samitier, J

    2014-01-01

    We describe a conductance-based biosensor that exploits DNA-mediated long-range electron transport for the label-free and direct electrical detection of DNA hybridization. This biosensor platform comprises an array of vertical nano-gap biosensors made of gold and fabricated through standard photolithography combined with focused ion beam lithography. The nano-gap walls are covalently modified with short, anti-symmetric thiolated DNA probes, which are terminated by 19 bases complementary to both the ends of a target DNA strand. The nano-gaps are separated by a distance of 50nm, which was adjusted to fit the length of the DNA target plus the DNA probes. The hybridization of the target DNA closes the gap circuit in a switch on/off fashion, in such a way that it is readily detected by an increase in the current after nano-gap closure. The nano-biosensor shows high specificity in the discrimination of base-pair mismatching and does not require signal indicators or enhancing molecules. The design of the biosensor platform is applicable for multiplexed detection in a straightforward manner. The platform is well-suited to mass production, point-of-care diagnostics, and wide-scale DNA analysis applications. (paper)

  8. Effect of vitamin E on cytotoxicity, DNA single strand breaks, chromosomal aberrations, and mutation in Chinese hamster V-79 cells exposed to ultraviolet-B light

    International Nuclear Information System (INIS)

    Sugiyama, M.; Tsuzuki, K.; Matsumoto, K.; Ogura, R.

    1992-01-01

    The effect of pretreatment with vitamin E on cytotoxicity, DNA single strand breaks, and chromosomal aberrations as well as on mutation induced by ultraviolet-B light (UV-B) was investigated in Chinese hamster V-79 cells. Cellular pretreatment with non-toxic levels of 25 μM α-tocopherol succinate (vitamin E) for 24h prior to exposure resulted in a 10-fold increase in cellular levels of α-tocopherol. Using a colony-forming assay, this pretreatment decreased the cytotoxicity of UV-B light. However, alkaline elution assays demonstrated that pretreatment with vitamin E did not affect the number of DNA single strand breaks caused by UV-B light. UV-B exposure produced a dose-dependent induction of chromosomal aberrations and mutations at the HGPRT locus, and neither of these actions of UV-B was influenced by pretreatment with the vitamin. These results suggest that vitamin E protects cells from UV-B-induced cytotoxicity, possibly through its ability to scavenge free radicals. The genotoxicity induced by UV-B light may not correlate directly with the cytotoxic action of this wavelength region in sunlight. (author)

  9. Resistance mutations and CTL epitopes in archived HIV-1 DNA of patients on antiviral treatment: toward a new concept of vaccine.

    Directory of Open Access Journals (Sweden)

    Jennifer Papuchon

    Full Text Available Eleven patients responding successfully to first-line antiretroviral therapy (ART were investigated for proviral drug resistance mutations (DRMs in RT by ultra-deep pyrosequencing (UDPS. After molecular typing of the class I alleles A and B, the CTL epitopes in the Gag, Nef and Pol regions of the provirus were sequenced and compared to the reference HXB2 HIV-1 epitopes. They were then matched with the HLA alleles with determination of theoretical affinity (TA. For 3 patients, the results could be compared with an RNA sample of the circulating virus at initiation of therapy. Five out of 11 patients exhibited DRMs by UDPS. The issue is whether a therapeutic switch is relevant in these patients by taking into account the identity of the archived resistance mutations. When the archived CTL epitopes were determined on the basis of the HLA alleles, different patterns were observed. Some epitopes were identical to those reported for the reference with the same TA, while others were mutated with a decrease in TA. In 2 cases, an epitope was observed as a combination of subpopulations at entry and was retrieved as a single population with lower TA at success. With regard to immunological stimulation and given the variability of the archived CTL epitopes, we propose a new concept of curative vaccine based on identification of HIV-1 CTL epitopes after prior sequencing of proviral DNA and matching with HLA class I alleles.

  10. Polysomnographic and neurometabolic features may mark preclinical autosomal dominant cerebellar ataxia, deafness, and narcolepsy due to a mutation in the DNA (cytosine-5-)-methyltransferase gene, DNMT1.

    Science.gov (United States)

    Moghadam, Keivan Kaveh; Pizza, Fabio; Tonon, Caterina; Lodi, Raffaele; Carelli, Valerio; Poli, Francesca; Franceschini, Christian; Barboni, Piero; Seri, Marco; Ferrari, Simona; La Morgia, Chiara; Testa, Claudia; Cornelio, Ferdinando; Liguori, Rocco; Winkelmann, Juliane; Lin, Ling; Mignot, Emmanuel; Plazzi, Giuseppe

    2014-05-01

    We aimed to report the clinical picture of two asymptomatic daughters of a patient with autosomal dominant cerebellar ataxia, deafness, and narcolepsy (ADCA-DN) due to a mutation in the DNA (cytosine-5-)-methyltransferase gene, DNMT1. Clinical assessment based on history, neurologic examination, sleep recordings, neurophysiologic neuroimaging, and genetic tests was performed. History and neurologic examination in both subjects were unremarkable. Genetic analysis disclosed in both the paternally-inherited heterozygous point mutation in the DNMT1 gene. Sleep recordings found sleep-onset rapid eye movement periods (SOREMPs) and proton magnetic resonance spectroscopy (MRS) revealed increased cerebellar myoinositol (mI) in both subjects. Auditory and ophthalmologic investigations as well as structural brain magnetic resonance imaging (MRI) scans revealed no abnormalities. The two asymptomatic carriers of the heterozygous DNMT1 mutation for ADCA-DN, a late-onset neurodegenerative disease, presented with SOREMPs associated with an increase of mI in the brain, a marker of glial cell activity and density characteristic of early stages of neurodegenerative diseases. Therefore, SOREMPs may precede the clinical picture of ADCA-DN as an early polysomnographic marker of central nervous system involvement detected by MRS. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Analysis of mutations in DNA gyrase and topoisomerase IV of Ureaplasma urealyticum and Ureaplasma parvum serovars resistant to fluoroquinolones.

    Science.gov (United States)

    Piccinelli, Giorgio; Gargiulo, Franco; Biscaro, Valeria; Caccuri, Francesca; Caruso, Arnaldo; De Francesco, Maria Antonia

    2017-01-01

    This study aims to determine the prevalence of fluoroquinolone resistance of Ureaplasma biovars and serovars isolated from urogenital clinical samples and determine the underlying molecular mechanism for quinolone resistance for all resistant isolates. Of 105 samples confirmed as positive for U. urealyticum/U. parvum, 85 were resistant to quinolones by the Mycoplasma-IST2 kit. However, only 43 out of 85 quinolone resistant isolates had amino acid substitutions in GyrA, GyrB, ParC and ParE proteins underlining that this assay have mis-identified as fluoroquinolone resistant 42 isolates. The known ParC E87K and ParC S83L mutations were found in 1 and 10 isolates, respectively. An original mutation of ureaplasmal ParC (E87Q, 1 isolate) was found. Furthermore, we found a ParE R448K mutation in one isolate, already described. Among the additional alterations detected, the most prevalent mutation found was L176F in GyrA protein in 18 isolates with single infection and in 3 isolates with mixed ureaplasma infections. Mutations in GyrB (E502Q, 4 isolates), ParE (Q412K, Q412P, Q412T, 3 independent isolates), whose role is unknown, were also found. Other sporadic mutations in the four genes were identified. This investigation is the result of monitoring the data for molecular fluoroquinone resistance in Ureaplasma spp. in Italy. Resulting that this acquired resistance is high and that continued local epidemiological studies are essential to monitor and document their antimicrobial resistance trends. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Generation of KCL025 research grade human embryonic stem cell line carrying a mutation in NF1 gene

    Directory of Open Access Journals (Sweden)

    Heema Hewitson

    2016-03-01

    Full Text Available The KCL025 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation in the NF1 gene encoding neurofibromin (c.3739–3742 ΔTTTG. Mutations in this gene have been linked to neurofibromatosis type 1, juvenile myelomonocytic leukemia and Watson syndrome. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays.

  13. Gene editing by co-transformation of TALEN and chimeric RNA/DNA oligonucleotides on the rice OsEPSPS gene and the inheritance of mutations.

    Directory of Open Access Journals (Sweden)

    Mugui Wang

    Full Text Available Although several site-specific nucleases (SSNs, such as zinc-finger nucleases (ZFNs, transcription activator-like effector nucleases (TALENs, and the clustered regularly interspaced short palindromic repeat (CRISPR/Cas, have emerged as powerful tools for targeted gene editing in many organisms, to date, gene targeting (GT in plants remains a formidable challenge. In the present study, we attempted to substitute a single base in situ on the rice OsEPSPS gene by co-transformation of TALEN with chimeric RNA/DNA oligonucleotides (COs, including different strand composition such as RNA/DNA (C1 or DNA/RNA (C2 but contained the same target base to be substituted. In contrast to zero GT event obtained by the co-transformation of TALEN with homologous recombination plasmid (HRP, we obtained one mutant showing target base substitution although accompanied by undesired deletion of 12 bases downstream the target site from the co-transformation of TALEN and C1. In addition to this typical event, we also obtained 16 mutants with different length of base deletions around the target site among 105 calli lines derived from transformation of TALEN alone (4/19 as well as co-transformation of TELAN with either HRP (5/30 or C1 (2/25 or C2 (5/31. Further analysis demonstrated that the homozygous gene-edited mutants without foreign gene insertion could be obtained in one generation. The induced mutations in transgenic generation were also capable to pass to the next generation stably. However, the genotypes of mutants did not segregate normally in T1 population, probably due to lethal mutations. Phenotypic assessments in T1 generation showed that the heterozygous plants with either one or three bases deletion on target sequence, called d1 and d3, were more sensitive to glyphosate and the heterozygous d1 plants had significantly lower seed-setting rate than wild-type.

  14. Development of techniques using DNA analysis method for detection/analysis of radiation-induced mutation. Development of an useful probe/primer and improvement of detection efficacy

    International Nuclear Information System (INIS)

    Maekawa, Hideaki; Tsuchida, Kozo; Hashido, Kazuo; Takada, Naoko; Kameoka, Yosuke; Hirata, Makoto

    1999-01-01

    Previously, it was demonstrated that detection of centromere became easy and reliable through fluorescent staining by FISH method using a probe of the sequence preserved in α-satelite DNA. Since it was, however, found inappropriate to detect dicentrics based on the relative amount of DNA probe on each chromosome. A prove which allows homogeneous detection of α-satelite DNA for each chromosome was constructed. A presumed sequence specific to kinetochore, CENP-B box was amplified by PCR method and the product DNA was used as a probe. However, the variation in amounts of probe DNA among chromosomes was decreased by only about 20%. Then, a program for image processing of the results obtained from FISH using α-satelite DNA was constructed to use as a marker for centromere. When compared with detection of abnormal chromosomes stained by the conventional method, calculation efficacy for only detection of centromere was improved by the use of this program. Calculation to discriminate the normal or not was still complicated and the detection efficacy was little improved. Chromosomal abnormalities in lymphocytes were used to detect the effects of radiation. In this method, it is needed to shift the phase of cells into metaphase. The mutation induced by radiation might be often repaired during shifting. To exclude this possibility, DNA extraction was conducted at a low temperature and immediately after exposure to 137 Cs, and a rapid genome detection method was established using the genome DNA. As the model genomes, the following three were used: 1) long chain repeated sequences widely dispersed over chromosome, 2) cluster genes, 3) single copy genes. The effects of radiation were detectable at 1-2 Gy for the long repeated sequences and at 7 Gy for the cluster genes, respectively, whereas no significant effects were observed at any Gy tested for the single copy genes. Amplification was marked in the cells exposed at 1-10 Gy (peak at 4 Gy), suggesting that these regions had

  15. Mutation and crystallization of the first KH domain of human polycytosine-binding protein 1 (PCBP1) in complex with DNA

    International Nuclear Information System (INIS)

    Yoga, Yano M. K.; Traore, Daouda A. K.; Wilce, Jacqueline A.; Wilce, Matthew C. J.

    2011-01-01

    The successful preparation of a mutant KH domain representing the first KH domain of PCBP1 and its crystallization in complex with a C-rich DNA are reported. This structure is anticipated to provide high-resolution information that will allow better understanding of the basis of cytosine specificity by PCBPs. Polycytosine-binding proteins (PCBPs) are triple KH-domain proteins that play an important role in the regulation of translation of eukaryotic mRNA. They are also utilized by viral RNA and have been shown to interact with ssDNA. Underlying their function is the specific recognition of C-rich nucleotides by their KH domains. However, the structural basis of this recognition is only partially understood. Here, the preparation of a His-tagged KH domain is described, representing the first domain of PCBP1 that incorporates a C54S mutation as well as the addition of a C-terminal tryptophan. This construct has facilitated the preparation of highly diffracting crystals in complex with C-rich DNA (sequence ACCCCA). Crystals of the KH1–DNA complex were grown using the hanging-drop vapour-diffusion method in 0.1 M phosphate–citrate pH 4.2, 40%(v/v) PEG 300. X-ray diffraction data were collected to 1.77 Å resolution and the diffraction was consistent with space group P2 1 , with unit-cell parameters a = 38.59, b = 111.88, c = 43.42 Å, α = γ = 90.0, β = 93.37°. The structure of the KH1–DNA complex will further our insight into the basis of cytosine specificity by PCBPs

  16. Two novel mutations in thymidine kinase-2 cause early onset fatal encephalomyopathy and severe mtDNA depletion.

    Science.gov (United States)

    Lesko, Nicole; Naess, Karin; Wibom, Rolf; Solaroli, Nicola; Nennesmo, Inger; von Döbeln, Ulrika; Karlsson, Anna; Larsson, Nils-Göran

    2010-03-01

    Deficiency of thymidine kinase-2 (TK2) has been described in children with early onset fatal skeletal myopathy. TK2 is a mitochondrial deoxyribonucleoside kinase required for the phosphorylation of deoxycytidine and deoxythymidine and hence is vital for the maintenance of a balanced mitochondrial dNTP pool in post-mitotic tissues. We describe a patient with two novel TK2 mutations, which caused disease onset shortly after birth and death at the age of three months. One mutation (219insCG) generated an early stop codon, thus preventing the synthesis of a functional protein. The second mutation (R130W) resulted in an amino acid substitution, which caused a severe reduction (TK2 enzyme activity. These two novel TK2 mutations cause an extremely severe phenotype with overwhelming central nervous system symptoms not commonly seen in patients with TK2-deficiency. We conclude that the severe clinical presentation in this patient was due to a virtual lack of mitochondrial TK2 activity. Copyright 2009 Elsevier B.V. All rights reserved.

  17. Yeast Studies Lead to a New DNA-Based Model for Research on Development | Poster

    Science.gov (United States)

    A paper from Amar J. S. Klar, Ph.D., with the RNA Biology Laboratory in NCI’s Center for Cancer Research, has identified a model for DNA research that explains the congenital disorder of mirror hand movements in humans. A mirror movement is when an intentional movement on one side of the body is mirrored by an involuntary movement on the other.

  18. DNA adducts, mutant frequencies and mutation spectra in λlacZ transgenic mice treated with N-nitrosodimethylamine

    NARCIS (Netherlands)

    Souliotis, V.L.; Delft, J.H.M. van; Steenwinkel, M.-J.S.T.; Baan, R.A.; Kyrtopoulos, S.A.

    1998-01-01

    Groups of λlacZ transgenic mice were treated i.p. with N-nitrosodimethylamine (NDMA) as single doses of 5 mg/kg or 10 mg/kg or as 10 daily doses of 1 mg/kg and changes in DNA N7- or O6-methylguanine or the repair enzyme O6-alkylguanine-DNA alkyltransferase (AGT) were followed for up to 14 days in

  19. Study on detection of mutation DNA fragment in gastric cancer by restriction endonuclease fingerprinting with capillary electrophoresis.

    Science.gov (United States)

    Wang, Rong; Xie, Hua; Xu, Yue-Bing; Jia, Zheng-Ping; Meng, Xian-Dong; Zhang, Juan-Hong; Ma, Jun; Wang, Juan; Wang, Xian-Hua

    2012-03-01

    The DNA fragment detection focusing technique has further enhanced the sensitivity and information of DNA targets. The DNA fragment detection method was established by capillary electrophoresis with laser-induced fluorescence detection and restriction endonuclease chromatographic fingerprinting (CE-LIF-REF) in our experiment. The silica capillary column was coated with short linear polyarclarylamide (SLPA) using nongel sieving technology. The excision product of various restricted enzymes of DNA fragments was obtained by REF with the molecular biology software Primer Premier 5. The PBR322/BsuRI DNA marker was used to establish the optimization method. The markers were focused electrophoretically and detected by CE-LIF. The results demonstrate that the CE-LIF-REF with SLPA can improve separation, sensitivity and speed of analysis. This technique may be applied to analysis of the excision product of various restricted enzymes of prokaryotic plasmid (pIRES2), eukaryote plasmid (pcDNA3.1) and the PCR product of codon 248 region of gastric cancer tissue. The results suggest that this method could very sensitively separate the excision products of various restricted enzymes at a much better resolution than the traditional agarose electrophoresis. Copyright © 2011 John Wiley & Sons, Ltd.

  20. A molecular biological study on the identification of the molecular species of DNA polymerases for repairing radiation-damaged DNA and the factors modifying the mutation rate

    Energy Technology Data Exchange (ETDEWEB)

    Yamada, Koichi; Inoue, Shuji [National Inst. of Health and Nutrition, Tokyo (Japan)

    1997-02-01

    Aiming at prevention and treatment of radiation damages, the authors have been investigating DNA damages by X-ray and its repairing mechanism, however, the molecular species of DNA polymerase which mediate the repairing could not been identified by biochemical methods using various inhibitors because of their low specificity. Therefore, in this study, anti-sense oligonucleotides for DNA polymerase {alpha}, {delta} and {epsilon} were obtained by chemical synthesis and transduced into human fibroblast cell, NB1RGB by three methods; endocytotic method, electroporation method and lipofection method. For the first method, the addition of those peptides into the cell culture at 5 {mu}M inhibited the polymerase activity by up to 30% and it was economically difficult to use at higher concentrations than it. For the electroporation method, different conditions were tested in the respects of initial potential, time constant and buffer, but the uptake of thimidine was scarcely decreased in the surviving cells, suggesting that the surviving rate would be short in the cells electroporated with those anti-sense peptides. For the lipofection method, among several cationic lipids tested, lipofectamine significantly enlarged the decrease of thymidine uptake by anti-sense {delta}, however it was considered that its application to DNA repairing is difficult because lipofectamine is strongly cytotoxic. Therefore, construction of a vector which allows to express anti-sense RNA in those cells is undertaken. (M.N.)

  1. A molecular biological study on the identification of the molecular species of DNA polymerases for repairing radiation-damaged DNA and the factors modifying the mutation rate

    International Nuclear Information System (INIS)

    Yamada, Koichi; Inoue, Shuji

    1997-01-01

    Aiming at prevention and treatment of radiation damages, the authors have been investigating DNA damages by X-ray and its repairing mechanism, however, the molecular species of DNA polymerase which mediate the repairing could not been identified by biochemical methods using various inhibitors because of their low specificity. Therefore, in this study, anti-sense oligonucleotides for DNA polymerase α, δ and ε were obtained by chemical synthesis and transduced into human fibroblast cell, NB1RGB by three methods; endocytotic method, electroporation method and lipofection method. For the first method, the addition of those peptides into the cell culture at 5 μM inhibited the polymerase activity by up to 30% and it was economically difficult to use at higher concentrations than it. For the electroporation method, different conditions were tested in the respects of initial potential, time constant and buffer, but the uptake of thimidine was scarcely decreased in the surviving cells, suggesting that the surviving rate would be short in the cells electroporated with those anti-sense peptides. For the lipofection method, among several cationic lipids tested, lipofectamine significantly enlarged the decrease of thymidine uptake by anti-sense δ, however it was considered that its application to DNA repairing is difficult because lipofectamine is strongly cytotoxic. Therefore, construction of a vector which allows to express anti-sense RNA in those cells is undertaken. (M.N.)

  2. Long span DNA paired-end-tag (DNA-PET sequencing strategy for the interrogation of genomic structural mutations and fusion-point-guided reconstruction of amplicons.

    Directory of Open Access Journals (Sweden)

    Fei Yao

    Full Text Available Structural variations (SVs contribute significantly to the variability of the human genome and extensive genomic rearrangements are a hallmark of cancer. While genomic DNA paired-end-tag (DNA-PET sequencing is an attractive approach to identify genomic SVs, the current application of PET sequencing with short insert size DNA can be insufficient for the comprehensive mapping of SVs in low complexity and repeat-rich genomic regions. We employed a recently developed procedure to generate PET sequencing data using large DNA inserts of 10-20 kb and compared their characteristics with short insert (1 kb libraries for their ability to identify SVs. Our results suggest that although short insert libraries bear an advantage in identifying small deletions, they do not provide significantly better breakpoint resolution. In contrast, large inserts are superior to short inserts in providing higher physical genome coverage for the same sequencing cost and achieve greater sensitivity, in practice, for the identification of several classes of SVs, such as copy number neutral and complex events. Furthermore, our results confirm that large insert libraries allow for the identification of SVs within repetitive sequences, which cannot be spanned by short inserts. This provides a key advantage in studying rearrangements in cancer, and we show how it can be used in a fusion-point-guided-concatenation algorithm to study focally amplified regions in cancer.

  3. Illustrative cases for monitoring by quantitative analysis of BRAF/NRAS ctDNA mutations in liquid biopsies of metastatic melanoma patients who gained clinical benefits from anti-PD1 antibody therapy.

    Science.gov (United States)

    Seremet, Teofila; Planken, Simon; Schreuer, Max; Jansen, Yanina; Delaunoy, Mélanie; El Housni, Hakim; Lienard, Danielle; Del Marmol, Véronique; Heimann, Pierre; Neyns, Bart

    2018-02-01

    Anti-programmed death 1 (PD-1) monoclonal antibodies improve the survival of metastatic melanoma patients. Predictive or monitoring biomarkers for response to this therapy could improve the clinical management of these patients. To date, no established biomarkers are available for monitoring the response to immunotherapy. Tumor- specific mutations in circulating tumor DNA (ctDNA) such as BRAF and NRAS mutations for melanoma patients have been proposed for monitoring of immunotherapy response. We present seven illustrative cases for the use of ctDNA BRAF and NRAS mutations' monitoring in plasma. The cases described exemplify four distinct clinical benefit patterns: rapid and durable complete response (CR), early progression, followed by CR, CR followed by early progression after interrupting treatment and long-term disease stabilization. These representative cases suggest that comprehensive BRAF/NRAS ctDNA monitoring during anti-PD1 therapy is informative and can be of added value for the monitoring of melanoma patients gaining clinical benefit on anti-PD1 treatment. An important advantage of our approach is that using the cartridge system on the Idylla platform for mutation analysis, the results become available the same day 2 h after plasma collection. Therefore, in the future, the ctDNA level can be an element in the clinical management of the patients.

  4. High prevalence of HIV-1 transmitted drug-resistance mutations from proviral DNA massively parallel sequencing data of therapy-naïve chronically infected Brazilian blood donors.

    Directory of Open Access Journals (Sweden)

    Rodrigo Pessôa

    Full Text Available An improved understanding of the prevalence of low-abundance transmitted drug-resistance mutations (TDRM in therapy-naïve HIV-1-infected patients may help determine which patients are the best candidates for therapy. In this study, we aimed to obtain a comprehensive picture of the evolving HIV-1 TDRM across the massive parallel sequences (MPS of the viral entire proviral genome in a well-characterized Brazilian blood donor naïve to antiretroviral drugs.The MPS data from 128 samples used in the analysis were sourced from Brazilian blood donors and were previously classified by less-sensitive (LS or "detuned" enzyme immunoassay as non-recent or longstanding HIV-1 infections. The Stanford HIV Resistance Database (HIVDBv 6.2 and IAS-USA mutation lists were used to interpret the pattern of drug resistance. The minority variants with TDRM were identified using a threshold of ≥ 1.0% and ≤ 20% of the reads sequenced. The rate of TDRM in the MPS data of the proviral genome were compared with the corresponding published consensus sequences of their plasma viruses.No TDRM were detected in the integrase or envelope regions. The overall prevalence of TDRM in the protease (PR and reverse transcriptase (RT regions of the HIV-1 pol gene was 44.5% (57/128, including any mutations to the nucleoside analogue reverse transcriptase inhibitors (NRTI and non-nucleoside analogue reverse transcriptase inhibitors (NNRTI. Of the 57 subjects, 43 (75.4% harbored a minority variant containing at least one clinically relevant TDRM. Among the 43 subjects, 33 (76.7% had detectable minority resistant variants to NRTIs, 6 (13.9% to NNRTIs, and 16 (37.2% to PR inhibitors. The comparison of viral sequences in both sources, plasma and cells, would have detected 48 DNA provirus disclosed TDRM by MPS previously missed by plasma bulk analysis.Our findings revealed a high prevalence of TDRM found in this group, as the use of MPS drastically increased the detection of these

  5. Research progress in plant mutation by combining ion beam irradiations and tissue culture

    International Nuclear Information System (INIS)

    Zhou Linbin; Li Wenjian; Qu Ying; Li Ping

    2007-01-01

    About a new mutation breeding method which combines plant tissue culture technique with heavy ion beam irradiations were discussed in this paper with the principles, operation steps, molecular mechanisms, etc. The mutation method developed a few advantages coming from plant tissue culture, which can produce offspring by asexual ways. Meanwhile, using this method, the study of biological effects of high energy particles with different linear energy transfer values on plant tissues or cells can be explored and optimized in theory or practice. (authors)

  6. Cancer spectrum in DNA mismatch repair gene mutation carriers: results from a hospital based Lynch syndrome registry.

    Science.gov (United States)

    Pande, Mala; Wei, Chongjuan; Chen, Jinyun; Amos, Christopher I; Lynch, Patrick M; Lu, Karen H; Lucio, Laura A; Boyd-Rogers, Stephanie G; Bannon, Sarah A; Mork, Maureen E; Frazier, Marsha L

    2012-09-01

    The spectrum of cancers seen in a hospital based Lynch syndrome registry of mismatch repair gene mutation carriers was examined to determine the distribution of cancers and examine excess cancer risk. Overall there were 504 cancers recorded in 368 mutation carriers from 176 families. These included 236 (46.8 %) colorectal and 268 (53.2 %) extracolonic cancers. MLH1 mutation carriers had a higher frequency of colorectal cancers whereas MSH2, MSH6 and PMS2 mutation carriers had more extracolonic cancers although these differences were not statistically significant. Men had fewer extracolonic cancers than colorectal (45.3 vs. 54.7 %), whereas women had more extracolonic than colorectal cancers (59.0 vs. 41.0 %). The mean age at diagnosis overall for extracolonic cancers was older than for colorectal, 49.1 versus 44.8 years (P ≤ 0.001). As expected, the index cancer was colorectal in 58.1 % of patients and among the extracolonic index cancers, endometrial was the most common (13.8 %). A significant number of non-Lynch syndrome index cancers were recorded including breast (n = 5) prostate (n = 3), thyroid (n = 3), cervix (n = 3), melanoma (n = 3), and 1 case each of thymoma, sinus cavity, and adenocarcinoma of the lung. However, standardized incidence ratios calculated to assess excess cancer risk showed that only those cancers known to be associated with Lynch syndrome were significant in our sample. We found that Lynch syndrome patients can often present with cancers that are not considered part of Lynch syndrome. This has clinical relevance both for diagnosis of Lynch syndrome and surveillance for cancers of different sites during follow-up of these patients.

  7. Molecular barcoding, DNA from snake venom, and toxinological research: Considerations and concerns.

    Science.gov (United States)

    Powell, Randy L; Reyes, Steven R; Lannutti, Dominic I

    2006-12-15

    The problem of species identification in toxinological research and solutions such as molecular barcoding and DNA extraction from venom samples are addressed. Molecular barcoding is controversial with both perceived advantages and inherent problems. A method of species identification utilizing mitochondrial DNA from venom has been identified. This method could result in deemphasizing the importance of obtaining detailed information on the venom source prior to analysis. Additional concerns include; a cost prohibitive factor, intraspecific venom variation, and venom processing issues. As researchers demand more stringent records and verification, venom suppliers may be prompted to implement improved methods and controls.

  8. DNA methylation changes in genes frequently mutated in sporadic colorectal cancer and in the DNA repair and Wnt/β-catenin signaling pathway genes

    Czech Academy of Sciences Publication Activity Database

    Farkas, S. A.; Vymetálková, Veronika; Vodičková, Ludmila; Vodička, Pavel; Torbjörn, K. N.

    2014-01-01

    Roč. 6, č. 2 (2014), s. 179-191 ISSN 1750-1911 R&D Projects: GA ČR GPP304/11/P715; GA ČR(CZ) GAP304/12/1585; GA MZd NT14329 Institutional support: RVO:68378041 Keywords : CpG * DNA repair genes * sporadic colorectal cancer Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.649, year: 2014

  9. Factors influencing parents' decision to donate their healthy infant's DNA for minimal-risk genetic research.

    Science.gov (United States)

    Hatfield, Linda A; Pearce, Margaret M

    2014-11-01

    To examine factors that influence a parent's decision to donate their healthy infant's DNA for minimal-risk genetic research. Grounded theory, using semi-structured interviews conducted with 35 postpartum mother or mother-father dyads in an urban teaching hospital. Data were collected from July 2011 to January 2012. Audiorecorded semistructured interviews were conducted in private rooms with mothers or mother-father dyads 24 to 48 hr after the birth of their healthy, full-term infant. Data-driven content analysis using selected principles of grounded theory was performed. Parents' willingness to donate their healthy infant's DNA for minimal-risk pediatric genetic research emerged as a process involving three interacting components: the parents, the scientist, and the comfort of the child embedded within the context of benefit to the child. The purpose of the study and parents' perception of their commitment of time and resources determined their willingness to participate. The scientist's ability to communicate trust in the research process influenced parents' decisions. Physical discomfort of the child shaped parents' decision to donate DNA. Parental perception of a direct benefit to their child affected their willingness to discuss genetic research and its outcomes. Significant gaps and misunderstandings in parental knowledge of pediatric genetic research may affect parental willingness to donate their healthy child's DNA. Nurses knowledgeable about the decision-making process parents utilize to donate their healthy infant's DNA for minimal-risk genetic research and the factors influencing that decision are well positioned to educate parents about the role of genetics in health and illness and reassure potential research participants of the value and safeguards in pediatric genetic research. © 2014 Sigma Theta Tau International.

  10. The development of ideas about the effect of DNA repair on the induction of gene mutations and chromosomal aberrations by radiation and by chemicals

    International Nuclear Information System (INIS)

    Kimball, R.F.

    1987-01-01

    An historical overview is given of the development of ideas about chromosomal and DNA repair as they relate to the induction of mutations, chromosomal aberrations, and sister-chromatid exchanges by radiations and chemicals. The genetic and molecular bases of the various repair pathways are reviewed whenever possible. Work on both prokaryotes and eukaryotes is included. Mention is made, when deemed appropriate, of major developments in other areas that served as essential background for the repair work, but no attempt is made to cover these background developments in any detail. Near the end, a brief review is given of factors affecting polymerase fidelity. The history is subdivided into approximately 10-year intervals. For the most part, references are to reviews and symposia in which the ideas of the time were brought together. The implications of these findings for some practical problems in genetic toxicology and for our understanding of the maintenance of the genome are discussed at the end. 147 refs

  11. Correlation of the UV-induced mutational spectra and the DNA damage distribution of the human HPRT gene: Automating the analysis

    International Nuclear Information System (INIS)

    Kotturi, G.; Erfle, H.; Koop, B.F.; Boer, J.G. de; Glickman, B.W.

    1994-01-01

    Automated DNA sequencers can be readily adapted for various types of sequence-based nucleic acid analysis: more recently it was determined the distribution of UV photoproducts in the E. coli laci gene using techniques developed for automated fluorescence-based analysis. We have been working to improve the automated approach of damage distribution. Our current method is more rigorous. We have new software that integrates the area under the individual peaks, rather than measuring the height of the curve. In addition, we now employ an internal standard. The analysis can also be partially automated. Detection limits for both major types of UV-photoproducts (cyclobutane dimers and pyrimidine (6-4) pyrimidone photoproducts) are reported. The UV-induced damage distribution in the hprt gene is compared to the mutational spectra in human and rodents cells

  12. Characterization of vanadate-dependent NADH oxidation activity and isolation of yeast DNA which complements a class 1 vanadate resistance mutation

    International Nuclear Information System (INIS)

    Minasi, L.E.

    1989-01-01

    A vanadate-dependent NADH oxidation activity has been characterized in plasma membranes from the yeast S cerevisiae. NADH oxidation activity was maximally stimulated at pH 5.0 in phosphate buffer. NADH oxidation was not dependent on the concentration of plasma membranes. The vanadate-dependent NADH oxidation activity was abolished under anaerobic conditions and the concomitant uptake of oxygen occurred during NADH oxidation. The activity was inhibited by superoxide dismutase and stimulated by the presence of paraquat. These results indicate that the vanadate stimulation of NADH oxidation in yeast plasma membranes occurs as a result of the vanadate-dependent oxidation of NADH by superoxide, generated by a plasma membrane NADH oxidase. 51 V-NMR results indicated that a phosphate-vanadate anhydride was the stimulatory species in pH 5.0 and pH 7.0 phosphate buffer. Yeast DNA has been isolated which complements a class 1 vanadate resistance mutation

  13. Development of ideas about the effect of DNA repair on the induction of gene mutations and chromosomal aberrations by radiation and by chemicals

    Energy Technology Data Exchange (ETDEWEB)

    Kimball, R F

    1987-07-01

    An historical overview is given of the development of ideas about chromosomal and DNA repair as they relate to the induction of mutations, chromosomal aberrations, and sister-chromatid exchanges by radiations and chemicals. The genetic and molecular bases of the various repair pathways are reviewed whenever possible. Work on both prokaryotes and eukaryotes is included. Mention is made, when deemed appropriate, of major developments in other areas that served as essential background for the repair work, but no attempt is made to cover these background developments in any detail. Near the end, a brief review is given of factors affecting polymerase fidelity. The history is subdivided into approximately 10-year intervals. For the most part, references are to reviews and symposia in which the ideas of the time were brought together. The implications of these findings for some practical problems in genetic toxicology and for our understanding of the maintenance of the genome are discussed at the end. 147 refs.

  14. A nonsense mutation causing decreased levels of insulin receptor mRNA: Detection by a simplified technique for direct sequencing of genomic DNA amplified by the polymerase chain reaction

    International Nuclear Information System (INIS)

    Kadowaki, T.; Kadowaki, H.; Taylor, S.I.

    1990-01-01

    Mutations in the insulin receptor gene can render the cell resistant to the biological action of insulin. The authors have studied a patient with leprechaunism (leprechaun/Minn-1), a genetic syndrome associated with intrauterine growth retardation and extreme insulin resistance. Genomic DNA from the patient was amplified by the polymerase chain reaction catalyzed by Thermus aquaticus (Taq) DNA polymerase, and the amplified DNA was directly sequenced. A nonsense mutations was identified at codon 897 in exon 14 in the paternal allele of the patient's insulin receptor gene. Levels of insulin receptor mRNA are decreased to <10% of normal in Epstein-Barr virus-transformed lymphoblasts and cultured skin fibroblasts from this patient. Thus, this nonsense mutation appears to cause a decrease in the levels of insulin receptor mRNA. In addition, they have obtained indirect evidence that the patient's maternal allele of the insulin receptor gene contains a cis-acting dominant mutation that also decreases the level of mRNA, but by a different mechanism. The nucleotide sequence of the entire protein-coding domain and the sequences of the intron-exon boundaries for all 22 exons of the maternal allele were normal. Presumably, the mutation in the maternal allele maps elsewhere in the insulin receptor gene. Thus, they conclude that the patient is a compound heterozygote for two cis-acting dominant mutations in the insulin receptor gene: (i) a nonsense mutation in the paternal allel that reduces the level of insulin receptor mRNA and (ii) an as yet unidentified mutation in the maternal allele that either decreases the rate of transcription or decreases the stability of the mRNA

  15. [Research progress of mutational spectrum and pathophysiology of WFS1 gene in Wolfram syndrome and nonsyndromic low frequency sensorineural hearing loss].

    Science.gov (United States)

    Shi, S M; Han, Y H; Wang, H B

    2016-09-07

    Compound homozygous or heterozygous mutations in WFS 1 can lead to autosomal recessive Wolfram syndrome (WS), and heterozygous mutations in WFS 1 can lead to autosomal dominant non-syndromic low frequency sensorineural hearing loss (LFSNHL). In addition, mutations in the WFS region has relationship with diabetes and psychiatric diseases. In this paper, we provide an overview of genetic research with different phenotypes, including WS and LFSNHL.

  16. 4-Aminobiphenyl (4-ABP) - DNA Damage in Breast Tissue and Relationship to p53 Mutations and Polymorphisms of Metabolizing Genes

    National Research Council Canada - National Science Library

    Niguidula, Nancy

    2000-01-01

    .... The analysis of the CYP1A2 gene is currently in progress. Due to the difficulty in obtaining large fragments of DNA from the tumor tissue sections required for PCR-RFLP, a new method is under development for genotyping NAT2...

  17. 1999 Gordon Research Conference on Mammalian DNA Repair. Final Progress Report

    International Nuclear Information System (INIS)

    NONE

    1999-01-01

    This Conference will examine DNA repair as the key component in genomic surveillance that is so crucial to the overall integrity and function of mammalian cells. Recent discoveries have catapulted the field of DNA repair into a pivotal position for fundamental investigations into oncology, aging, environmental health, and developmental biology. We hope to highlight the most promising and exciting avenues of research in robust discussions at this conference. This Mammalian DNA Repair Gordon Conference differs from the past conferences in this series, in which the programs were broader in scope, with respect to topics and biological systems covered. A conference sponsored by the Genetics Society in April 1998 emphasized recombinational mechanisms for double-strand break repair and the role of mismatch repair deficiency in colorectal cancer. These topics will therefore receive somewhat less emphasis in the upcoming Conference. In view of the recent mechanistic advances in mammalian DNA repair, an upcoming comprehensive DNA repair meeting next autumn at Hilton Head; and the limited enrollment for Gordon Conferences we have decided to focus session-by-session on particular areas of controversy and/or new developments specifically in mammalian systems. Thus, the principal presentations will draw upon results from other cellular systems only to the extent that they impact our understanding of mammalian DNA repair

  18. 1999 Gordon Research Conference on Mammalian DNA Repair. Final Progress Report

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1999-02-12

    This Conference will examine DNA repair as the key component in genomic surveillance that is so crucial to the overall integrity and function of mammalian cells. Recent discoveries have catapulted the field of DNA repair into a pivotal position for fundamental investigations into oncology, aging, environmental health, and developmental biology. We hope to highlight the most promising and exciting avenues of research in robust discussions at this conference. This Mammalian DNA Repair Gordon Conference differs from the past conferences in this series, in which the programs were broader in scope, with respect to topics and biological systems covered. A conference sponsored by the Genetics Society in April 1998 emphasized recombinational mechanisms for double-strand break repair and the role of mismatch repair deficiency in colorectal cancer. These topics will therefore receive somewhat less emphasis in the upcoming Conference. In view of the recent mechanistic advances in mammalian DNA repair, an upcoming comprehensive DNA repair meeting next autumn at Hilton Head; and the limited enrollment for Gordon Conferences we have decided to focus session-by-session on particular areas of controversy and/or new developments specifically in mammalian systems. Thus, the principal presentations will draw upon results from other cellular systems only to the extent that they impact our understanding of mammalian DNA repair.

  19. Mutations reducing replication from R-loops suppress the defects of growth, chromosome segregation and DNA supercoiling in cells lacking topoisomerase I and RNase HI activity.

    Science.gov (United States)

    Usongo, Valentine; Martel, Makisha; Balleydier, Aurélien; Drolet, Marc

    2016-04-01

    R-loop formation occurs when the nascent RNA hybridizes with the template DNA strand behind the RNA polymerase. R-loops affect a wide range of cellular processes and their use as origins of replication was the first function attributed to them. In Escherichia coli, R-loop formation is promoted by the ATP-dependent negative supercoiling activity of gyrase (gyrA and gyrB) and is inhibited by topoisomerase (topo) I (topA) relaxing transcription-induced negative supercoiling. RNase HI (rnhA) degrades the RNA moiety of R-loops. The depletion of RNase HI activity in topA null mutants was previously shown to lead to extensive DNA relaxation, due to DNA gyrase inhibition, and to severe growth and chromosome segregation defects that were partially corrected by overproducing topo III (topB). Here, DNA gyrase assays in crude cell extracts showed that the ATP-dependent activity (supercoiling) of gyrase but not its ATP-independent activity (relaxation) was inhibited in topA null cells lacking RNase HI. To characterize the cellular event(s) triggered by the absence of RNase HI, we performed a genetic screen for suppressors of the growth defect of topA rnhA null cells. Suppressors affecting genes in replication (holC2::aph and dnaT18::aph) nucleotide metabolism (dcd49::aph), RNA degradation (rne59::aph) and fimbriae synthesis (fimD22::aph) were found to reduce replication from R-loops and to restore supercoiling, thus pointing to a correlation between R-loop-dependent replication in topA rnhA mutants and the inhibition of gyrase activity and growth. Interestingly, the position of fimD on the E. coli chromosome corresponds to the site of one of the five main putative origins of replication from R-loops in rnhA null cells recently identified by next-generation sequencing, thus suggesting that the fimD22::aph mutation inactivated one of these origins. Furthermore, we show that topo III overproduction is unable to complement the growth defect of topA rnhA null mutants at low

  20. TP53 mutations, human papilloma virus DNA and inflammation markers in esophageal squamous cell carcinoma from the Rift Valley, a high-incidence area in Kenya

    Directory of Open Access Journals (Sweden)

    Martel-Planche Ghislaine

    2011-10-01

    Full Text Available Abstract Background Squamous Cell Carcinoma of Esophagus is one of the most common malignancies in both men and women in eastern and south-eastern Africa. In Kenya, clinical observations suggest that this cancer is frequent in the Rift Valley area. However, so far, there has been no report on the molecular characteristics of esophageal squamous cell carcinoma (ESCC in this area. Results We have analyzed TP53 mutations, the presence of human papilloma virus (HPV DNA and expression of inflammation markers Cyclooxygenase 2 (Cox-2 and Nitrotyrosine (NTyR in 28 cases (13 males and 15 females of archived ESCC tissues collected at the Moi Teaching and Referral Hospital in Eldoret, Kenya. Eleven mutations were detected in TP53 exons 5 to 8 (39%. All ESCC samples were negative for HPV 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73 and 82. Immunohistochemical analysis of Cox-2 and NTyR showed a low proportion of positive cases (17.4% and 39.1%, respectively. No association between the above markers and suspected risk factors (alcohol or tobacco use, hot tea drinking, use of charcoal for cooking was found. Conclusion Our findings suggest that mechanisms of esophageal carcinogenesis in eastern Africa might be different from other parts of the world. Low prevalence of TP53 mutation compared with other intermediate or high incidence areas of the world highlights this hypothesis. Our data did not support a possible ole of HPV in this series of cases. Further studies are needed to assess and compare the molecular patterns of ESCC from Kenya with those of high-incidence areas such as China or Central Asia.

  1. Mutator activity in Schizophyllum commune

    Energy Technology Data Exchange (ETDEWEB)

    Shneyour, Y.; Koltin, Y. (Tel Aviv Univ. (Israel). Dept. of Microbiology)

    1983-01-01

    A strain with an elevated level of spontaneous mutations and an especially high rate of reversion at a specific locus (pab/sup -/) was identified. The mutator trait is recessive. UV sensitivity and the absence of a UV-specific endonucleolytic activity were associated with the enhancement of the mutation rate in mutator strains. The endonuclease associated with the regulation of the mutation rate also acted on single-stranded DNA. The molecular weight of this enzyme is about 38,000 daltons.

  2. Reduction of spontaneous somatic mutation frequency by a low-dose X irradiation of Drosophila larvae and possible involvement of DNA single-strand damage repair.

    Science.gov (United States)

    Koana, Takao; Takahashi, Takashi; Tsujimura, Hidenobu

    2012-03-01

    The third instar larvae of Drosophila were irradiated with X rays, and the somatic mutation frequency in their wings was measured after their eclosion. In the flies with normal DNA repair and apoptosis functions, 0.2 Gy irradiation at 0.05 Gy/min reduced the frequency of the so-called small spot (mutant cell clone with reduced reproductive activity) compared with that in the sham-irradiated flies. When apoptosis was suppressed using the baculovirus p35 gene, the small spot frequency increased four times in the sham-irradiated control group, but the reduction by the 0.2-Gy irradiation was still evident. In a non-homologous end joining-deficient mutant, the small spot frequency was also reduced by 0.2 Gy radiation. In a mutant deficient in single-strand break repair, no reduction in the small spot frequency by 0.2 Gy radiation was observed, and the small spot frequency increased with the radiation dose. Large spot (mutant cell clone with normal reproductive activity) frequency was not affected by suppression of apoptosis and increased monotonically with radiation dose in wild-type larvae and in mutants for single- or double-strand break repair. It is hypothesized that some of the small spots resulted from single-strand damage and, in wild-type larvae, 0.2 Gy radiation activated the normal single-strand break repair gene, which reduced the background somatic mutation frequency.

  3. Are There Mutator Polymerases?

    Directory of Open Access Journals (Sweden)

    Miguel Garcia-Diaz

    2003-01-01

    Full Text Available DNA polymerases are involved in different cellular events, including genome replication and DNA repair. In the last few years, a large number of novel DNA polymerases have been discovered, and the biochemical analysis of their properties has revealed a long list of intriguing features. Some of these polymerases have a very low fidelity and have been suggested to play mutator roles in different processes, like translesion synthesis or somatic hypermutation. The current view of these processes is reviewed, and the current understanding of DNA polymerases and their role as mutator enzymes is discussed.

  4. Antibiotic resistance in Pseudomonas aeruginosa strains with increased mutation frequency due to inactivation of the DNA oxidative repair system

    DEFF Research Database (Denmark)

    Mandsberg, Lotte F; Ciofu, Oana; Kirkby, N

    2009-01-01

    ,8-dihydro-8-oxodeoxyguanosine) than PAO1 after exposure to PMNs, and they developed resistance to antibiotics more frequently. The mechanisms of resistance were increased β-lactamase production and overexpression of the MexCD-OprJ efflux-pump. Mutations in either the mutT or the mutY gene were found...... in resistant HP clinical isolates from patients with CF, and complementation with wild-type genes reverted the phenotype. In conclusion, oxidative stress might be involved in the development of resistance to antibiotics. We therefore suggest the possible use of antioxidants for CF patients to prevent...

  5. Generation of KCL035 research grade human embryonic stem cell line carrying a mutation in HBB gene

    Directory of Open Access Journals (Sweden)

    Heema Hewitson

    2016-03-01

    Full Text Available The KCL035 human embryonic stem cell line was derived from an embryo donated for research that carried a mutation in the HBB gene, which is linked to the β-thalassemia syndrome. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays.

  6. [beta]-hexosaminidase isozymes from cells cotransfected with [alpha] and [beta] cDNA constructs: Analysis of the [alpha]-subunit missense mutation associated with the adult form of Tay-Sachs disease

    Energy Technology Data Exchange (ETDEWEB)

    Brown, C.A.; Mahuran, D.J. (Univ. of Toronto (Canada))

    1993-08-01

    In vitro mutagenesis and transient expression in COS cells has been used to associate a missense mutation with a clinical or biochemical phenotype. Mutations affecting the [alpha]-subunit of [beta]-hexosaminidase A ([alpha][beta]) (E.C.3.2.1.52) result in Tay-Sachs disease. Because hexosaminidase A is heterodimeric, analysis of [alpha]-chain mutations is not straightforward. The authors examine three approaches utilizing previously identified mutations affecting [alpha]-chain folding. These involve transfection of (1) the [alpha] cDNA alone; (2) a [beta] cDNA construct encoding a [beta]-subunit substituted at a position homologous to that of the [alpha]-subunit, and (3) both [alpha] and [beta] cDNAs. The latter two procedures amplified residual activity levels over that of patient samples, an effect not previously found with mutations affecting an [open quotes]active[close quotes] [alpha]Arg residue. This effect may help to discriminate between protein-folding and active-site mutations. The authors conclude that, with proper controls, the latter method of cotransfection can be used to evaluate the effects and perhaps to predict the clinical course of some [alpha]-chain mutations. Using this technique, they demonstrate that the adult-onset Tay-Sachs mutation, [alpha]Gly[yields]Ser[sup 269], does not directly affect [alpha][beta] dimerization but exerts an indirect effect on the dimer through destabilizing the folded [alpha]-subunit at physiological temperatures. Two other [alpha] mutations linked to more severe phenotypes appear to inhibit the initial folding of the subunit. 36 refs., 2 figs., 5 tabs.

  7. EGFR T790M mutation testing of non-small cell lung cancer tissue and blood samples artificially spiked with circulating cell-free tumor DNA: results of a round robin trial.

    Science.gov (United States)

    Fassunke, Jana; Ihle, Michaela Angelika; Lenze, Dido; Lehmann, Annika; Hummel, Michael; Vollbrecht, Claudia; Penzel, Roland; Volckmar, Anna-Lena; Stenzinger, Albrecht; Endris, Volker; Jung, Andreas; Lehmann, Ulrich; Zeugner, Silke; Baretton, Gustavo; Kreipe, Hans; Schirmacher, Peter; Kirchner, Thomas; Dietel, Manfred; Büttner, Reinhard; Merkelbach-Bruse, Sabine

    2017-10-01

    The European Commision (EC) recently approved osimertinib for the treatment of adult patients with locally advanced or metastatic non-small-cell lung cancer (NSCLC) harboring EGFR T790M mutations. Besides tissue-based testing, blood samples containing cell-free circulating tumor DNA (ctDNA) can be used to interrogate T790M status. Herein, we describe the conditions and results of a round robin trial (RRT) for T790M mutation testing in NSCLC tissue specimens and peripheral blood samples spiked with cell line DNA mimicking tumor-derived ctDNA. The underlying objectives of this two-staged external quality assessment (EQA) approach were (a) to evaluate the accuracy of T790M mutations testing across multiple centers and (b) to investigate if a liquid biopsy-based testing for T790M mutations in spiked blood samples is feasible in routine diagnostic. Based on a successfully completed internal phase I RRT, an open RRT for EGFR T790M mutation testing in tumor tissue and blood samples was initiated. In total, 48 pathology centers participated in the EQA. Of these, 47 (97.9%) centers submitted their analyses within the pre-defined time frame and 44 (tissue), respectively, 40 (plasma) successfully passed the test. The overall success rates in the RRT phase II were 91.7% (tissue) and 83.3% (blood), respectively. Thirty-eight out of 48 participants (79.2%) successfully passed both parts of the RRT. The RRT for blood-based EGFR testing initiated in Germany is, to the best of our knowledge, the first of his kind in Europe. In summary, our results demonstrate that blood-based genotyping for EGFR resistance mutations can be successfully integrated in routine molecular diagnostics complementing the array of molecular methods already available at pathology centers in Germany.

  8. Genome-wide DNA methylation analysis of transient neonatal diabetes type 1 patients with mutations in ZFP57

    DEFF Research Database (Denmark)

    Bak, Mads; Boonen, Susanne E; Dahl, Christina

    2016-01-01

    of developing diabetes mellitus type 2 later in life. Transient neonatal diabetes mellitus 1 is caused by overexpression of the maternally imprinted genes PLAGL1 and HYMAI on chromosome 6q24. One of the mechanisms leading to overexpression of the locus is hypomethylation of the maternal allele of PLAGL1......BACKGROUND: Transient neonatal diabetes mellitus 1 (TNDM1) is a rare imprinting disorder characterized by intrautering growth retardation and diabetes mellitus usually presenting within the first six weeks of life and resolves by the age of 18 months. However, patients have an increased risk...... and HYMAI. A subset of patients with maternal hypomethylation at PLAGL1 have hypomethylation at additional imprinted loci throughout the genome, including GRB10, ZIM2 (PEG3), MEST (PEG1), KCNQ1OT1 and NESPAS (GNAS-AS1). About half of the TNDM1 patients carry mutations in ZFP57, a transcription factor...

  9. Sesame improvement by induced mutations: Results of the co-ordinated research project and recommendation for future studies

    International Nuclear Information System (INIS)

    Zanten, L. van

    2001-01-01

    The FAO/IAEA Co-ordinated Research Project has brought together sesame breeders from 11 countries. They, together with pathologists, agronomists and physiologists, have made considerable effort to advance the genetic improvement in sesame. The results and conclusions from this project cover the mutation techniques used for the genetic improvement of various aspects of sesame. These recommendations do not only deal with the application of mutation induction, but also with the wider plant breeding related objectives and methods to be considered for this semi-domesticated crop. It is clear that more advanced techniques can and should be incorporated in the process which would enhance the genetic improvement. Although five years is a relatively limited time in a plant breeding programme, the participants have been able to produce and make available a considerable pool of agronomically interesting mutant sesame germplasm. The participants in the CRP considered that, together with other specialists, plant breeders can gain fuller benefit from the mutations induced by radiation or chemicals. Work on these mutants must continue in co-operation/consultation with plant physiologists and pathologists, and with biotechnologists who may in the future be able to provide in the future methods for introducing beneficial traits from other crops into sesame. The sesame programme should include scientists from the Member States where sesame grows and scientists from developed countries who may have greater access to physiological and molecular research facilities. (author)

  10. In vitro mutation breeding of bananas and plantains. Final reports of an FAO/IAEA co-ordinated research programme from 1988 to 1993

    International Nuclear Information System (INIS)

    1995-06-01

    This document contains 9 final reports of the participants at the FAO/IAEA co-ordinated research programme on 'In vitro mutation breeding of bananas and plantains'. A separate abstract was prepared for each report. Refs, figs and tabs

  11. In vitro mutation breeding of bananas and plantains. Final reports of an FAO/IAEA co-ordinated research programme from 1988 to 1993

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1995-06-01

    This document contains 9 final reports of the participants at the FAO/IAEA co-ordinated research programme on `In vitro mutation breeding of bananas and plantains`. A separate abstract was prepared for each report. Refs, figs and tabs.

  12. Mutagenic DNA repair in Escherichia coli. Pt. 2. Factors affecting loss of photoreversibility of UV induced mutations

    Energy Technology Data Exchange (ETDEWEB)

    Doubleday, O P; Bridges, B A; Green, M H.L. [Medical Research Council, Brighton (UK). Cell Mutation Unit

    1975-01-01

    The photoreversibility of UV-induced mutations to Trp/sup +/ in strain Escherichia coli WP2 uvr A trp (unable to excise pyrimidine dimers) was lost at different rates during incubation in different media. In Casamino acids medium after a short initial lag, photoreversibility was lost over about one generation time; in minimal medium with tryptophan, photoreversibility persisted for more than two generations; in Casamino acids medium with pantoyl lactone photoreversibility was lost extremely slowly. The rate of loss of photoreversibility was unaffected by UV dose in either Casamino acids medium or in minimal medium. The same eventual number of induced mutants was obtained when cells were incubated for two generations in any of the three media before being transferred to selective plates supplemented with Casamino acids. Thus in each the proportion of cells capable of giving rise to a mutant was the same and only the rate at which these cells did so during post-irradiation growth varied, suggesting that there might be a specific fraction of pyrimidine dimers at a given site capable of initiating a mutagenic repair event, and that the size of this fraction is dose dependent. Segregation experiments have shown that error-prone repair appears to occur once only and is not repeated in subsequent replication cycles, in contrast to (presumed error-free) recombination repair. The results are discussed in the light of current models of UV mutagenesis.

  13. Proton nuclear magnetic resonance studies on bulge-containing DNA oligonucleotides from a mutational hot-spot sequence

    International Nuclear Information System (INIS)

    Woodson, S.A.; Crothers, D.M.

    1987-01-01

    A series of bulge-containing and normal double-helical synthetic oligodeoxyribonucleotides, of sequence corresponding to a frame-shift mutational hot spot in the λ C/sub I/ gene, are compared by proton magnetic resonance spectroscopy at 500 MHz. The imino proton resonances are assigned by one-dimensional nuclear Overhauser effect spectroscopy. Nonselective T 1 inversion-recovery experiments are used to determine exchangeable proton lifetimes and to compare helix stability and dynamics of the three duplexes. An extra adenosine flanking the internal G-C base pairs has a strongly localized effect on helix stability, but the destabilizing effect of an extra cytidine in a C tract is delocalized over the entire G-C run. These data lead to the conclusion that the position of the bulge migrates along the run in the fast-exchange limit on the NMR time scale. Rapid migration of the bulge defect in homopolymeric sequences may help rationalize both frame-shift mutagenesis and translational frame shifting. The authors estimate that the unfavorable free energy of a localized bulge defect is 2.9-3.2 kcal/mol, in good agreement with earlier estimates for RNA helices

  14. Mutation avoidance and DNA repair proficiency in Ustilago maydis are differentially lost with progressive truncation of the REC1 gene product

    Energy Technology Data Exchange (ETDEWEB)

    Onel, K.; Thelen, M.P.; Ferguson, D.O.; Bennett, R.L.; Holloman, W.K. [Cornell Univ. Medical College, NY, NY (United States)

    1995-10-01

    The REC1 gene of Ustilago maydis has an uninterrupted open reading frame, predicted from the genomic sequence to encode a protein of 522 amino acid residues. Nevertheless, an intron is present, and functional activity of the gene in mitotic cells requires an RNA processing event to remove the intron. This results in a change in reading frame and production of a protein of 463 amino acid residues. The 3{prime}{r_arrow}5{prime} exonuclease activity of proteins derived form the REC1 genomic open reading frame, the intronless open reading frame, and several mutants was investigated. The mutants included a series of deletions constructed by removing restriction fragments at the 3{prime} end of the cloned REC1 gene and a set of mutant alleles previously isolated in screens for radiation sensitivity. The results indicated that elimination of the C-terminal third of the protein did not result in a serious reduction in 3{prime}{r_arrow}5{prime} exonuclease activity, but deletion into the midsection caused a severe loss of activity. The biological activity of the rec1-1 allele, which encodes a truncated polypeptide with full 3{prime}{r_arrow}5{prime} exonuclease activity, and the rec1-5 allele, which encodes a more severely truncated polypeptide with no exonuclease activity, was investigated. The two mutants were equally sensitive to the lethal effect of UV light, but the spontaneous mutation rate was elevated 10-fold over the wild-type rate in the rec1-1 mutant and 100-fold in the rec1-5 mutant. The elevated spontaneous mutation rate correlated with the ablation of exonuclease activity, but the radiation sensitivity did not. These results indicate that the C-terminal portion of the Rec1 protein is not essential for exonuclease activity but is crucial in the role of REC1 in DNA damage repair. 49 refs., 3 figs., 1 tab.

  15. Achievements, Challenges, and Opportunities in DNA-Encoded Library Research: An Academic Point of View.

    Science.gov (United States)

    Yuen, Lik Hang; Franzini, Raphael M

    2017-05-04

    DNA-encoded chemical libraries (DECLs) are pools of DNA-tagged small molecules that enable facile screening and identification of bio-macromolecule binders. The successful development of DECLs has led to their increasingly important role in drug development, and screening hits have entered clinical trials. In this review, we summarize the development and currently active research areas of DECLs with a focus on contributions from groups at academic institutes. We further look at opportunities and future directions of DECL research in medicinal chemistry and chemical biology based on the symbiotic relationship between academia and industry. Challenges associated with the application of DECLs in academic drug discovery are further discussed. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Research in Undergraduate Instruction: A Biotech Lab Project for Recombinant DNA Protein Expression in Bacteria

    Science.gov (United States)

    Brockman, Mark; Ordman, Alfred B.; Campbell, A. Malcolm

    1996-06-01

    In the sophomore-level Molecular Biology and Biotechnology course at Beloit College, students learn basic methods in molecular biology in the context of pursuing a semester-long original research project. We are exploring how DNA sequence affects expression levels of proteins. A DNA fragment encoding all or part of the guanylate monokinase (gmk) sequence is cloned into pSP73 and expressed in E. coli. A monoclonal antibody is made to gmk. The expression level of gmk is determined by SDS gel elctrophoresis, a Western blot, and an ELISA assay. Over four years, an increase in enrollment in the course from 9 to 34 students, the 85% of majors pursuing advanced degrees, and course evaluations all support the conclusion that involving students in research during undergraduate courses encourages them to pursue careers in science.

  17. Rice breeding with induced mutations II. Report of an FAO/IAEA research co-ordination meeting

    Energy Technology Data Exchange