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Sample records for canine genomic dna

  1. Genomic profiling reveals extensive heterogeneity in somatic DNA copy number aberrations of canine hemangiosarcoma.

    Science.gov (United States)

    Thomas, Rachael; Borst, Luke; Rotroff, Daniel; Motsinger-Reif, Alison; Lindblad-Toh, Kerstin; Modiano, Jaime F; Breen, Matthew

    2014-09-01

    Canine hemangiosarcoma is a highly aggressive vascular neoplasm associated with extensive clinical and anatomical heterogeneity and a grave prognosis. Comprehensive molecular characterization of hemangiosarcoma may identify novel therapeutic targets and advanced clinical management strategies, but there are no published reports of tumor-associated genome instability and disrupted gene dosage in this cancer. We performed genome-wide microarray-based somatic DNA copy number profiling of 75 primary intra-abdominal hemangiosarcomas from five popular dog breeds that are highly predisposed to this disease. The cohort exhibited limited global genomic instability, compared to other canine sarcomas studied to date, and DNA copy number aberrations (CNAs) were predominantly of low amplitude. Recurrent imbalances of several key cancer-associated genes were evident; however, the global penetrance of any single CNA was low and no distinct hallmark aberrations were evident. Copy number gains of dog chromosomes 13, 24, and 31, and loss of chromosome 16, were the most recurrent CNAs involving large chromosome regions, but their relative distribution within and between cases suggests they most likely represent passenger aberrations. CNAs involving CDKN2A, VEGFA, and the SKI oncogene were identified as potential driver aberrations of hemangiosarcoma development, highlighting potential targets for therapeutic modulation. CNA profiles were broadly conserved between the five breeds, although subregional variation was evident, including a near twofold lower incidence of VEGFA gain in Golden Retrievers versus other breeds (22 versus 40 %). These observations support prior transcriptional studies suggesting that the clinical heterogeneity of this cancer may reflect the existence of multiple, molecularly distinct subtypes of canine hemangiosarcoma.

  2. Polymerase spiral reaction (PSR): a novel, visual isothermal amplification method for detection of canine parvovirus 2 genomic DNA.

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    Gupta, Vikas; Chakravarti, Soumendu; Chander, Vishal; Majumder, Saurabh; Bhat, Shabir Ahmad; Gupta, Vivek Kumar; Nandi, Sukdeb

    2017-07-01

    Canine parvovirus-2 (CPV-2), which is ubiquitously distributed worldwide, causes severe and often fatal gastroenteritis in dogs. Accurate, differential and rapid diagnosis of canine parvoviral enteritis remains a challenge for clinicians. A recently developed isothermal amplification technique, polymerase spiral reaction (PSR), was optimized for the first time for a viral pathogen with reference recombinant plasmid standards from different CPV-2 antigenic variants (CPV-2, CPV-2a, CPV-2b and CPV-2c) and subsequently validated using clinical samples. Addition of chromogenic substrate SYBR Green I after the completion of the reaction resulted in bright green fluorescence in positive samples, while negative samples and a no-template control remained orange. These results were further substantiated through visualization of a laddering pattern of the PSR-amplified product in an agarose gel in positive cases and the absence of this pattern in no-template control and negative samples. The PSR assay was found to be highly specific, as it did not react with other putative canine pathogens (canine adenovirus 1 and canine distemper virus). The sensitivity of the newly developed PSR technique was compared with that of conventional PCR, real-time PCR and LAMP, using a serial tenfold dilution of canine parvovirus DNA. The detection limit of PSR was found to be at the femtogram level, which is comparable with that of real-time PCR and LAMP, which are ten times more sensitive than conventional PCR. The assay was validated using 90 clinical samples, of which 54 were found positive, while only 45 samples were positive in conventional PCR. This novel assay, which is fully compliant with the 'ASSURED' concept for disease diagnosis, provides a simple, rapid, specific, sensitive and cost-effective method for diagnosis of canine parvoviral enteritis in veterinary clinics.

  3. The canine genome.

    Science.gov (United States)

    Ostrander, Elaine A; Wayne, Robert K

    2005-12-01

    The dog has emerged as a premier species for the study of morphology, behavior, and disease. The recent availability of a high-quality draft sequence lifts the dog system to a new threshold. We provide a primer to use the dog genome by first focusing on its evolutionary history. We overview the relationship of dogs to wild canids and discuss their origin and domestication. Dogs clearly originated from a substantial number of gray wolves and dog breeds define distinct genetic units that can be divided into at least four hierarchical groupings. We review evidence showing that dogs have high levels of linkage disequilibrium. Consequently, given that dog breeds express specific phenotypic traits and vary in behavior and the incidence of genetic disease, genomic-wide scans for linkage disequilibrium may allow the discovery of genes influencing breed-specific characteristics. Finally, we review studies that have utilized the dog to understand the genetic underpinning of several traits, and we summarize genomic resources that can be used to advance such studies. We suggest that given these resources and the unique characteristics of breeds, that the dog is a uniquely valuable resource for studying the genetic basis of complex traits.

  4. Complete Genome Sequence of Canine Papillomavirus Type 10

    Science.gov (United States)

    Luff, Jennifer; Moore, Peter; Zhou, Dan; Wang, Jingang; Usuda, Yukari; Affolter, Verena; Schlegel, Richard

    2012-01-01

    Papillomaviruses are epitheliotropic, nonenveloped, circular, double-stranded DNA viruses within the family Papillomaviridae that are associated with benign and malignant tumors in humans and animals. We report the complete genome sequence of canine papillomavirus type 10 identified from a pigmented plaque located on the head of a mixed-breed bloodhound. PMID:22997424

  5. Forensic Analysis of Canine DNA Samples in the Undergraduate Biochemistry Laboratory

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    Carson, Tobin M.; Bradley, Sharonda Q.; Fekete, Brenda L.; Millard, Julie T.; LaRiviere, Frederick J.

    2009-01-01

    Recent advances in canine genomics have allowed the development of highly distinguishing methods of analysis for both nuclear and mitochondrial DNA. We describe a laboratory exercise suitable for an undergraduate biochemistry course in which the polymerase chain reaction is used to amplify hypervariable regions of DNA from dog hair and saliva…

  6. Genome Sequence of Canine Adenovirus Type 1 Isolated from a Wolf (Canis lupus) in Southern Italy.

    Science.gov (United States)

    Pizzurro, Federica; Marcacci, Maurilia; Zaccaria, Guendalina; Orsini, Massimiliano; Cito, Francesca; Rosamilia, Alfonso; Di Renzo, Ludovica; Malatesta, Daniela; Di Sabatino, Daria; Lorusso, Alessio

    2017-04-20

    Canine adenovirus type 1 (CAdV-1), a DNA virus of the family Adenoviridae, causes infectious canine hepatitis, a highly contagious disease primarily affecting canids. In this report, we describe the isolation and whole-genome sequence of a CAdV-1 isolate from the liver of a free-ranging wolf (Canis lupus). Copyright © 2017 Pizzurro et al.

  7. Genomic prediction of traits related to canine hip dysplasia

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    Enrique eSanchez-Molano

    2015-03-01

    Full Text Available Increased concern for the welfare of pedigree dogs has led to development of selection programs against inherited diseases. An example is canine hip dysplasia (CHD, which has a moderate heritability and a high prevalence in some large-sized breeds. To date, selection using phenotypes has led to only modest improvement, and alternative strategies such as genomic selection may prove more effective. The primary aims of this study were to compare the performance of pedigree- and genomic-based breeding against CHD in the UK Labrador retriever population and to evaluate the performance of different genomic selection methods. A sample of 1179 Labrador Retrievers evaluated for CHD according to the UK scoring method (hip score, HS was genotyped with the Illumina CanineHD BeadChip. Twelve functions of HS and its component traits were analyzed using different statistical methods (GBLUP, Bayes C and Single-Step methods, and results were compared with a pedigree-based approach (BLUP using cross-validation. Genomic methods resulted in similar or higher accuracies than pedigree-based methods with training sets of 944 individuals for all but the untransformed HS, suggesting that genomic selection is an effective strategy. GBLUP and Bayes C gave similar prediction accuracies for HS and related traits, indicating a polygenic architecture. This conclusion was also supported by the low accuracies obtained in additional GBLUP analyses performed using only the SNPs with highest test statistics, also indicating that marker-assisted selection would not be as effective as genomic selection. A Single-Step method that combines genomic and pedigree information also showed higher accuracy than GBLUP and Bayes C for the log-transformed HS, which is currently used for pedigree based evaluations in UK. In conclusion, genomic selection is a promising alternative to pedigree-based selection against CHD, requiring more phenotypes with genomic data to improve further the accuracy

  8. Canine adenovirus type 2 vector generation via I-Sce1-mediated intracellular genome release.

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    Sandy Ibanes

    Full Text Available When canine adenovirus type 2 (CAdV-2, or also commonly referred to as CAV-2 vectors are injected into the brain parenchyma they preferentially transduce neurons, are capable of efficient axonal transport to afferent regions, and allow transgene expression for at last >1 yr. Yet, translating these data into a user-friendly vector platform has been limited because CAV-2 vector generation is challenging. Generation of E1-deleted adenovirus vectors often requires transfection of linear DNA fragments of >30 kb containing the vector genome into an E1-transcomplementing cell line. In contrast to human adenovirus type 5 vector generation, CAV-2 vector generation is less efficient due, in part, to a reduced ability to initiate replication and poor transfectibility of canine cells with large, linear DNA fragments. To improve CAV-2 vector generation, we generated an E1-transcomplementing cell line expressing the estrogen receptor (ER fused to I-SceI, a yeast meganuclease, and plasmids containing the I-SceI recognition sites flanking the CAV-2 vector genome. Using transfection of supercoiled plasmid and intracellular genome release via 4-OH-tamoxifen-induced nuclear translocation of I-SceI, we improved CAV-2 vector titers 1,000 fold, and in turn increased the efficacy of CAV-2 vector generation.

  9. Canine COL4A3 and COL4A4: sequencing, mapping and genomic organization.

    Science.gov (United States)

    Wiersma, Anje C; Millon, Lee V; Hestand, Matthew S; Van Oost, Bernard A; Bannasch, Danika L

    2005-08-01

    Canine alpha3 and alpha4 chains of collagen type IV genes (COL4A3 and COL4A4) are expressed in the renal glomerular basement membrane, where they provide a critical structural and functional matrix for other basement membrane components. These genes are candidates for hereditary nephritis (Alport syndrome) in several dog breeds (e.g. English Cocker Spaniel and Bull Terrier). Using RACE and PCR, the cDNA of both genes was cloned and sequenced. Both COL4A3 and COL4A4, as well as canine NPPC (Natriuretic Peptide Precursor C), were mapped to CFA25 using an RH panel. Conservation of the tight linkage of COL4A3 and COL4A4 as seen in human and mouse was verified in the dog. Intron-exon boundaries in both genes were determined by BLAST analysis of the Canis Familiaris Trace Archive. The elucidation of the cDNA sequences, genomic organization and the open reading frames of canine COL4A3 and COL4A4 provide the groundwork for screening these genes for mutations in hereditary nephritis in dogs.

  10. Invariants of DNA genomic signals

    Science.gov (United States)

    Cristea, Paul Dan A.

    2005-02-01

    For large scale analysis purposes, the conversion of genomic sequences into digital signals opens the possibility to use powerful signal processing methods for handling genomic information. The study of complex genomic signals reveals large scale features, maintained over the scale of whole chromosomes, that would be difficult to find by using only the symbolic representation. Based on genomic signal methods and on statistical techniques, the paper defines parameters of DNA sequences which are invariant to transformations induced by SNPs, splicing or crossover. Re-orienting concatenated coding regions in the same direction, regularities shared by the genomic material in all exons are revealed, pointing towards the hypothesis of a regular ancestral structure from which the current chromosome structures have evolved. This property is not found in non-nuclear genomic material, e.g., plasmids.

  11. A web resource on DNA tests for canine and feline hereditary diseases.

    Science.gov (United States)

    Slutsky, Jeffrey; Raj, Karthik; Yuhnke, Scott; Bell, Jerold; Fretwell, Neale; Hedhammar, Ake; Wade, Claire; Giger, Urs

    2013-08-01

    Following the first identification of a disease-causing mutation in dogs in 1989 and the more recent completion of canine and feline genome sequences, much progress has been made in the molecular characterization of hereditary diseases in dogs and cats. To increase access to information on diagnosing hereditary diseases in dogs and cats, a web application has been developed to collect, organize and display information on available DNA tests and other supporting information, including gene and chromosomal locations, mutations, primary research citations and disease descriptions. The DNA testing information can be accessed at the URL: http://research.vet.upenn.edu/WSAVA-LabSearch. There are currently 131 molecular genetic tests available for hereditary diseases in dogs and cats offered by 43 laboratories worldwide. This tool should provide clinicians, researchers, breeders and companion animal owners with a single comprehensive, up-to-date and readily searchable webpage for information on hereditary disease testing. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. An Improved microRNA Annotation of the Canine Genome.

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    Penso-Dolfin, Luca; Swofford, Ross; Johnson, Jeremy; Alföldi, Jessica; Lindblad-Toh, Kerstin; Swarbreck, David; Moxon, Simon; Di Palma, Federica

    2016-01-01

    The domestic dog, Canis familiaris, is a valuable model for studying human diseases. The publication of the latest Canine genome build and annotation, CanFam3.1 provides an opportunity to enhance our understanding of gene regulation across tissues in the dog model system. In this study, we used the latest dog genome assembly and small RNA sequencing data from 9 different dog tissues to predict novel miRNAs in the dog genome, as well as to annotate conserved miRNAs from the miRBase database that were missing from the current dog annotation. We used both miRCat and miRDeep2 algorithms to computationally predict miRNA loci. The resulting, putative hairpin sequences were analysed in order to discard false positives, based on predicted secondary structures and patterns of small RNA read alignments. Results were further divided into high and low confidence miRNAs, using the same criteria. We generated tissue specific expression profiles for the resulting set of 811 loci: 720 conserved miRNAs, (207 of which had not been previously annotated in the dog genome) and 91 novel miRNA loci. Comparative analyses revealed 8 putative homologues of some novel miRNA in ferret, and one in microbat. All miRNAs were also classified into the genic and intergenic categories, based on the Ensembl RefSeq gene annotation for CanFam3.1. This additionally allowed us to identify four previously undescribed MiRtrons among our total set of miRNAs. We additionally annotated piRNAs, using proTRAC on the same input data. We thus identified 263 putative clusters, most of which (211 clusters) were found to be expressed in testis. Our results represent an important improvement of the dog genome annotation, paving the way to further research on the evolution of gene regulation, as well as on the contribution of post-transcriptional regulation to pathological conditions.

  13. DNA integrity of fresh and frozen canine epididymal spermatozoa.

    Science.gov (United States)

    Varesi, Sara; Vernocchi, Valentina; Morselli, Maria Giorgia; Luvoni, Gaia Cecilia

    2014-12-01

    The aims of this study were to evaluate: (1) the effect of cryopreservation on DNA fragmentation of canine epididymal spermatozoa, and (2) the potential protective effect of melatonin on post-thaw sperm quality (motility, morphology, acrosomal and DNA integrity). Epididymal spermatozoa were collected after orchiectomy of ten dogs. Sperm samples were frozen in the presence or absence of melatonin (1mM). DNA fragmentation index (percentage of spermatozoa with fragmented DNA) was similar in fresh samples (3.3±3.6) and samples frozen with (4.2±3.8) or without (3.6±3.7) melatonin. Sperm motility was significantly (psperm motility. Proportions of spermatozoa with normal morphology were similar in fresh and frozen samples, irrespective of the presence of melatonin in the extender. Acrosome integrity was significantly decreased (pepididymal spermatozoa was not affected by the freezing procedure, and the presence of melatonin did not preserve motility and acrosome integrity which were adversely affected by cryopreservation. The evaluation of DNA status of thawed gametes is particularly relevant for epididymal spermatozoa since these spermatozoa are usually stored and used in assisted reproductive techniques. Copyright © 2014 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  14. Brief Guide to Genomics: DNA, Genes and Genomes

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    Skip to main content A Brief Guide to Genomics Enter Search Term(s): Español Research Funding An Overview ... Breve guía de genómica A Brief Guide to Genomics DNA, Genes and Genomes Deoxyribonucleic acid (DNA) is ...

  15. An Improved microRNA Annotation of the Canine Genome.

    Directory of Open Access Journals (Sweden)

    Luca Penso-Dolfin

    Full Text Available The domestic dog, Canis familiaris, is a valuable model for studying human diseases. The publication of the latest Canine genome build and annotation, CanFam3.1 provides an opportunity to enhance our understanding of gene regulation across tissues in the dog model system. In this study, we used the latest dog genome assembly and small RNA sequencing data from 9 different dog tissues to predict novel miRNAs in the dog genome, as well as to annotate conserved miRNAs from the miRBase database that were missing from the current dog annotation. We used both miRCat and miRDeep2 algorithms to computationally predict miRNA loci. The resulting, putative hairpin sequences were analysed in order to discard false positives, based on predicted secondary structures and patterns of small RNA read alignments. Results were further divided into high and low confidence miRNAs, using the same criteria. We generated tissue specific expression profiles for the resulting set of 811 loci: 720 conserved miRNAs, (207 of which had not been previously annotated in the dog genome and 91 novel miRNA loci. Comparative analyses revealed 8 putative homologues of some novel miRNA in ferret, and one in microbat. All miRNAs were also classified into the genic and intergenic categories, based on the Ensembl RefSeq gene annotation for CanFam3.1. This additionally allowed us to identify four previously undescribed MiRtrons among our total set of miRNAs. We additionally annotated piRNAs, using proTRAC on the same input data. We thus identified 263 putative clusters, most of which (211 clusters were found to be expressed in testis. Our results represent an important improvement of the dog genome annotation, paving the way to further research on the evolution of gene regulation, as well as on the contribution of post-transcriptional regulation to pathological conditions.

  16. Genome-wide association study identifies a novel canine glaucoma locus.

    Science.gov (United States)

    Ahonen, Saija J; Pietilä, Elina; Mellersh, Cathryn S; Tiira, Katriina; Hansen, Liz; Johnson, Gary S; Lohi, Hannes

    2013-01-01

    Glaucoma is an optic neuropathy and one of the leading causes of blindness. Its hereditary forms are classified into primary closed-angle (PCAG), primary open-angle (POAG) and primary congenital glaucoma (PCG). Although many loci have been mapped in human, only a few genes have been identified that are associated with the development of glaucoma and the genetic basis of the disease remains poorly understood. Glaucoma has also been described in many dog breeds, including Dandie Dinmont Terriers (DDT) in which it is a late-onset (>7 years) disease. We designed clinical and genetic studies to better define the clinical features of glaucoma in the DDT and to identify the genetic cause. Clinical diagnosis was based on ophthalmic examinations of the affected dogs and 18 additionally investigated unaffected DDTs. We collected DNA from over 400 DTTs and a genome wide association study was performed in a cohort of 23 affected and 23 controls, followed by a fine mapping, a replication study and candidate gene sequencing. The clinical study suggested that ocular abnormalities including abnormal iridocorneal angles and pectinate ligament dysplasia are common (50% and 72%, respectively) in the breed and the disease resembles human PCAG. The genetic study identified a novel 9.5 Mb locus on canine chromosome 8 including the 1.6 Mb best associated region (p = 1.63 × 10(-10), OR = 32 for homozygosity). Mutation screening in five candidate genes did not reveal any causative variants. This study indicates that although ocular abnormalities are common in DDTs, the genetic risk for glaucoma is conferred by a novel locus on CFA8. The canine locus shares synteny to a region in human chromosome 14q, which harbors several loci associated with POAG and PCG. Our study reveals a new locus for canine glaucoma and ongoing molecular studies will likely help to understand the genetic etiology of the disease.

  17. Genome-wide association study identifies a novel canine glaucoma locus.

    Directory of Open Access Journals (Sweden)

    Saija J Ahonen

    Full Text Available Glaucoma is an optic neuropathy and one of the leading causes of blindness. Its hereditary forms are classified into primary closed-angle (PCAG, primary open-angle (POAG and primary congenital glaucoma (PCG. Although many loci have been mapped in human, only a few genes have been identified that are associated with the development of glaucoma and the genetic basis of the disease remains poorly understood. Glaucoma has also been described in many dog breeds, including Dandie Dinmont Terriers (DDT in which it is a late-onset (>7 years disease. We designed clinical and genetic studies to better define the clinical features of glaucoma in the DDT and to identify the genetic cause. Clinical diagnosis was based on ophthalmic examinations of the affected dogs and 18 additionally investigated unaffected DDTs. We collected DNA from over 400 DTTs and a genome wide association study was performed in a cohort of 23 affected and 23 controls, followed by a fine mapping, a replication study and candidate gene sequencing. The clinical study suggested that ocular abnormalities including abnormal iridocorneal angles and pectinate ligament dysplasia are common (50% and 72%, respectively in the breed and the disease resembles human PCAG. The genetic study identified a novel 9.5 Mb locus on canine chromosome 8 including the 1.6 Mb best associated region (p = 1.63 × 10(-10, OR = 32 for homozygosity. Mutation screening in five candidate genes did not reveal any causative variants. This study indicates that although ocular abnormalities are common in DDTs, the genetic risk for glaucoma is conferred by a novel locus on CFA8. The canine locus shares synteny to a region in human chromosome 14q, which harbors several loci associated with POAG and PCG. Our study reveals a new locus for canine glaucoma and ongoing molecular studies will likely help to understand the genetic etiology of the disease.

  18. First complete genome sequence of canine bocavirus 2 in mainland China

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    S.-L. Zhai

    2017-07-01

    Full Text Available We obtained the first full-length genome sequence of canine bocavirus 2 (CBoV2 from the faeces of a healthy dog in Guangzhou city, Guangdong province, mainland China. The genome of GZHD15 consisted of 5059 nucleotides. Sequence analysis suggested that GZHD15 was close to a previously circulated Hong Kong isolate.

  19. Direct detection of methylation in genomic DNA

    NARCIS (Netherlands)

    Bart, A.; van Passel, M. W. J.; van Amsterdam, K.; van der Ende, A.

    2005-01-01

    The identification of methylated sites on bacterial genomic DNA would be a useful tool to study the major roles of DNA methylation in prokaryotes: distinction of self and nonself DNA, direction of post-replicative mismatch repair, control of DNA replication and cell cycle, and regulation of gene

  20. Chromosomal mapping of canine-derived BAC clones to the red fox and American mink genomes.

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    Kukekova, Anna V; Vorobieva, Nadegda V; Beklemisheva, Violetta R; Johnson, Jennifer L; Temnykh, Svetlana V; Yudkin, Dmitry V; Trut, Lyudmila N; Andre, Catherine; Galibert, Francis; Aguirre, Gustavo D; Acland, Gregory M; Graphodatsky, Alexander S

    2009-01-01

    High-quality sequencing of the dog (Canis lupus familiaris) genome has enabled enormous progress in genetic mapping of canine phenotypic variation. The red fox (Vulpes vulpes), another canid species, also exhibits a wide range of variation in coat color, morphology, and behavior. Although the fox genome has not yet been sequenced, canine genomic resources have been used to construct a meiotic linkage map of the red fox genome and begin genetic mapping in foxes. However, a more detailed gene-specific comparative map between the dog and fox genomes is required to establish gene order within homologous regions of dog and fox chromosomes and to refine breakpoints between homologous chromosomes of the 2 species. In the current study, we tested whether canine-derived gene-containing bacterial artificial chromosome (BAC) clones can be routinely used to build a gene-specific map of the red fox genome. Forty canine BAC clones were mapped to the red fox genome by fluorescence in situ hybridization (FISH). Each clone was uniquely assigned to a single fox chromosome, and the locations of 38 clones agreed with cytogenetic predictions. These results clearly demonstrate the utility of FISH mapping for construction of a whole-genome gene-specific map of the red fox. The further possibility of using canine BAC clones to map genes in the American mink (Mustela vison) genome was also explored. Much lower success was obtained for this more distantly related farm-bred species, although a few BAC clones were mapped to the predicted chromosomal locations.

  1. Cloning, localization and focus formation at DNA damage sites of canine XRCC4.

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    Koike, Manabu; Yutoku, Yasutomo; Koike, Aki

    2017-01-10

    Various chemotherapies and radiation therapies are useful for killing cancer cells mainly by inducing DNA double-strand breaks (DSBs). Uncovering the molecular mechanisms of DSB repair processes is crucial for developing next-generation radiotherapies and chemotherapeutics for human and animal cancers. XRCC4 plays a critical role in Ku-dependent nonhomologous DNA-end joining (NHEJ) in human cells, and is one of the core NHEJ factors. The localization of core NHEJ factors, such as human Ku70 and Ku80, might play a crucial role in regulating NHEJ activity. Recently, companion animals, such as canines, have been proposed to be a good model in many aspects of cancer research. However, the localization and regulation mechanisms of core NHEJ factors in canine cells have not been elucidated. Here, we show that the expression and subcellular localization of canine XRCC4 changes dynamically during the cell cycle. Furthermore, EYFP-canine XRCC4 accumulates quickly at laser-microirradiated DSB sites. The structure of a putative human XRCC4 nuclear localization signal (NLS) is highly conserved in canine, chimpanzee and mouse XRCC4. However, the amino acid residue corresponding to the human XRCC4 K210, thought to be important for nuclear localization, is not conserved in canine XRCC4. Our findings might be useful for the study of the molecular mechanisms of Ku-dependent NHEJ in canine cells and the development of new radiosensitizers that target XRCC4.

  2. Canine DNA Profiling in Forensic Casework: The Tail Wagging the Dog.

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    Berger, C; Berger, B; Parson, W

    2009-01-01

    The popularity of dogs as faithful human companions instigates forensically relevant issues on a regular basis. Domestic dogs take an active role as the causes of accidents and as perpetrators of attacks; even more frequently, dogs act as links between victims and suspects in crime cases due to the fact that dog owners/keepers live in an environment rich with canine material. Mitochondrial and nuclear DNA analyses of canine cells have been applied successfully in individual cases. However, a review of published casework amply demonstrates great interlaboratory variability not only in methodological but also in general terms. We screened the literature for application of canine DNA analysis in the forensic context and found 12 publications presented by 10 different laboratories. In almost no case did employed DNA markers widely overlap between these studies. Even worse is the situation with respect to allele nomenclature, where a plethora of variants has been reported by the different groups. Despite great technological achievements in the recent past, it seems that in forensic canine DNA analysis the cart was put before the horse. The canine forensic field faces the urgent need for general standardization and harmonization activities such as those that have taken place in the human forensic field in the past years. In particular, the nature and selection of DNA markers to be tested, the implementation of a generally compatible allele nomenclature, and a settlement on standardized statistical calculation methods adopted for the specific genetic peculiarities of dog populations need thorough consideration. Copyright © 2009 Central Police University.

  3. Genomic approaches to DNA repair and mutagenesis.

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    Wyrick, John J; Roberts, Steven A

    2015-12-01

    DNA damage is a constant threat to cells, causing cytotoxicity as well as inducing genetic alterations. The steady-state abundance of DNA lesions in a cell is minimized by a variety of DNA repair mechanisms, including DNA strand break repair, mismatch repair, nucleotide excision repair, base excision repair, and ribonucleotide excision repair. The efficiencies and mechanisms by which these pathways remove damage from chromosomes have been primarily characterized by investigating the processing of lesions at defined genomic loci, among bulk genomic DNA, on episomal DNA constructs, or using in vitro substrates. However, the structure of a chromosome is heterogeneous, consisting of heavily protein-bound heterochromatic regions, open regulatory regions, actively transcribed genes, and even areas of transient single stranded DNA. Consequently, DNA repair pathways function in a much more diverse set of chromosomal contexts than can be readily assessed using previous methods. Recent efforts to develop whole genome maps of DNA damage, repair processes, and even mutations promise to greatly expand our understanding of DNA repair and mutagenesis. Here we review the current efforts to utilize whole genome maps of DNA damage and mutation to understand how different chromosomal contexts affect DNA excision repair pathways. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Genome analysis of canine astroviruses reveals genetic heterogeneity and suggests possible inter-species transmission.

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    Mihalov-Kovács, Eszter; Martella, Vito; Lanave, Gianvito; Bodnar, Livia; Fehér, Enikő; Marton, Szilvia; Kemenesi, Gábor; Jakab, Ferenc; Bányai, Krisztián

    2017-03-15

    Canine astrovirus RNA was detected in the stools of 17/63 (26.9%) samples, using either a broadly reactive consensus RT-PCR for astroviruses or random RT-PCR coupled with massive deep sequencing. The complete or nearly complete genome sequence of five canine astroviruses was reconstructed that allowed mapping the genome organization and to investigate the genetic diversity of these viruses. The genome was about 6.6kb in length and contained three open reading frames (ORFs) flanked by a 5' UTR, and a 3' UTR plus a poly-A tail. ORF1a and ORF1b overlapped by 43 nucleotides while the ORF2 overlapped by 8 nucleotides with the 3' end of ORF1b. Upon genome comparison, four strains (HUN/2012/2, HUN/2012/6, HUN/2012/115, and HUN/2012/135) were more related genetically to each other and to UK canine astroviruses (88-96% nt identity), whilst strain HUN/2012/126 was more divergent (75-76% nt identity). In the ORF1b and ORF2, strains HUN/2012/2, HUN/2012/6, and HUN/2012/135 were related genetically to other canine astroviruses identified formerly in Europe and China, whereas strain HUN/2012/126 was related genetically to a divergent canine astrovirus strain, ITA/2010/Zoid. For one canine astrovirus, HUN/2012/8, only a 3.2kb portion of the genome, at the 3' end, could be determined. Interestingly, this strain possessed unique genetic signatures (including a longer ORF1b/ORF2 overlap and a longer 3'UTR) and it was divergent in both ORF1b and ORF2 from all other canine astroviruses, with the highest nucleotide sequence identity (68% and 63%, respectively) to a mink astrovirus, thus suggesting a possible event of interspecies transmission. The genetic heterogeneity of canine astroviruses may pose a challenge for the diagnostics and for future prophylaxis strategies. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Genome instabilities arising from ribonucleotides in DNA.

    Science.gov (United States)

    Klein, Hannah L

    2017-08-01

    Genomic DNA is transiently contaminated with ribonucleotide residues during the process of DNA replication through misincorporation by the replicative DNA polymerases α, δ and ε, and by the normal replication process on the lagging strand, which uses RNA primers. These ribonucleotides are efficiently removed during replication by RNase H enzymes and the lagging strand synthesis machinery. However, when ribonucleotides remain in DNA they can distort the DNA helix, affect machineries for DNA replication, transcription and repair, and can stimulate genomic instabilities which are manifest as increased mutation, recombination and chromosome alterations. The genomic instabilities associated with embedded ribonucleotides are considered here, along with a discussion of the origin of the lesions that stimulate particular classes of instabilities. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Cloning, localization and focus formation at DNA damage sites of canine XLF.

    Science.gov (United States)

    Koike, Manabu; Yutoku, Yasutomo; Koike, Aki

    2017-01-20

    Understanding the molecular mechanisms of DNA double-strand break (DSB) repair processes, especially nonhomologous DNA-end joining (NHEJ), is critical for developing next-generation radiotherapies and chemotherapeutics for human and animal cancers. The localization, protein-protein interactions and post-translational modifications of core NHEJ factors, such as human Ku70 and Ku80, might play critical roles in controlling NHEJ activity. XRCC4-like factor (XLF) is a core NHEJ factor and plays a key role in the Ku-dependent NHEJ repair process in human cells. Recently, companion animals, such as canines, have been proposed to be a good model for many aspects of cancer research, including the development of chemotherapeutics. However, the localization and regulation of core NHEJ factors in canine cells have not been elucidated. Here, we show that the localization of canine XLF changes dynamically during the cell cycle. EYFP-canine XLF localizes in the nuclei of interphase cells and accumulates immediately at microirradiated DSB sites. The structure of a putative human XLF nuclear localization signal (NLS) and a putative 14-3-3 binding motif are evolutionarily conserved in canine, chimpanzee and mouse XLF. However, the putative β-TRCP-recognizable degron of human XLF is not conserved in canine and mouse. Additionally, some vital human XLF phosphorylation sites, including the ATM major phosphorylation site (S251), are not conserved in canine XLF. Our findings might be useful for the study of the molecular mechanisms of NHEJ in canine cells and for the development of new radiosensitizers that target XLF.

  7. Sequencing intractable DNA to close microbial genomes.

    Directory of Open Access Journals (Sweden)

    Richard A Hurt

    Full Text Available Advancement in high throughput DNA sequencing technologies has supported a rapid proliferation of microbial genome sequencing projects, providing the genetic blueprint for in-depth studies. Oftentimes, difficult to sequence regions in microbial genomes are ruled "intractable" resulting in a growing number of genomes with sequence gaps deposited in databases. A procedure was developed to sequence such problematic regions in the "non-contiguous finished" Desulfovibrio desulfuricans ND132 genome (6 intractable gaps and the Desulfovibrio africanus genome (1 intractable gap. The polynucleotides surrounding each gap formed GC rich secondary structures making the regions refractory to amplification and sequencing. Strand-displacing DNA polymerases used in concert with a novel ramped PCR extension cycle supported amplification and closure of all gap regions in both genomes. The developed procedures support accurate gene annotation, and provide a step-wise method that reduces the effort required for genome finishing.

  8. DNA Microarrays in Comparative Genomics and Transcriptomics

    DEFF Research Database (Denmark)

    Willenbrock, Hanni

    2007-01-01

    of each method’s ability to analyze DNA copy number data. Moreover, our study shows that analysis methods developed for cancer research may also successfully be applied to DNA copy number profiles from bacterial genomes. However, here the purpose is to characterize variations in the gene content...... to verify predictions of highly expressed genes. Moreover, the codon bias of microbial genomes was found to constitute an environmental signature. For example, soil bacteria have very similar codon bias....

  9. Detection of Non-Amplified Genomic DNA

    CERN Document Server

    Corradini, Roberto

    2012-01-01

    This book offers a state-of-the-art overview on non amplified DNA detection methods and provides chemists, biochemists, biotechnologists and material scientists with an introduction to these methods. In fact all these fields have dedicated resources to the problem of nucleic acid detection, each contributing with their own specific methods and concepts. This book will explain the basic principles of the different non amplified DNA detection methods available, highlighting their respective advantages and limitations. The importance of non-amplified DNA sequencing technologies will be also discussed. Non-amplified DNA detection can be achieved by adopting different techniques. Such techniques have allowed the commercialization of innovative platforms for DNA detection that are expected to break into the DNA diagnostics market. The enhanced sensitivity required for the detection of non amplified genomic DNA has prompted new strategies that can achieve ultrasensitivity by combining specific materials with specifi...

  10. Demographic history, selection and functional diversity of the canine genome.

    Science.gov (United States)

    Ostrander, Elaine A; Wayne, Robert K; Freedman, Adam H; Davis, Brian W

    2017-12-01

    The domestic dog represents one of the most dramatic long-term evolutionary experiments undertaken by humans. From a large wolf-like progenitor, unparalleled diversity in phenotype and behaviour has developed in dogs, providing a model for understanding the developmental and genomic mechanisms of diversification. We discuss pattern and process in domestication, beginning with general findings about early domestication and problems in documenting selection at the genomic level. Furthermore, we summarize genotype-phenotype studies based first on single nucleotide polymorphism (SNP) genotyping and then with whole-genome data and show how an understanding of evolution informs topics as different as human history, adaptive and deleterious variation, morphological development, ageing, cancer and behaviour.

  11. An integrated 4249 marker FISH/RH map of the canine genome

    Directory of Open Access Journals (Sweden)

    Mahairas Gregory G

    2004-09-01

    Full Text Available Abstract Background The 156 breeds of dog recognized by the American Kennel Club offer a unique opportunity to map genes important in genetic variation. Each breed features a defining constellation of morphological and behavioral traits, often generated by deliberate crossing of closely related individuals, leading to a high rate of genetic disease in many breeds. Understanding the genetic basis of both phenotypic variation and disease susceptibility in the dog provides new ways in which to dissect the genetics of human health and biology. Results To facilitate both genetic mapping and cloning efforts, we have constructed an integrated canine genome map that is both dense and accurate. The resulting resource encompasses 4249 markers, and was constructed using the RHDF5000-2 whole genome radiation hybrid panel. The radiation hybrid (RH map features a density of one marker every 900 Kb and contains 1760 bacterial artificial chromosome clones (BACs localized to 1423 unique positions, 851 of which have also been mapped by fluorescence in situ hybridization (FISH. The two data sets show excellent concordance. Excluding the Y chromosome, the map features an RH/FISH mapped BAC every 3.5 Mb and an RH mapped BAC-end, on average, every 2 Mb. For 2233 markers, the orthologous human genes have been established, allowing the identification of 79 conserved segments (CS between the dog and human genomes, dramatically extending the length of most previously described CS. Conclusions These results provide a necessary resource for the canine genome mapping community to undertake positional cloning experiments and provide new insights into the comparative canine-human genome maps.

  12. An improved canine genome and a comprehensive catalogue of coding genes and non-coding transcripts.

    Directory of Open Access Journals (Sweden)

    Marc P Hoeppner

    Full Text Available The domestic dog, Canis familiaris, is a well-established model system for mapping trait and disease loci. While the original draft sequence was of good quality, gaps were abundant particularly in promoter regions of the genome, negatively impacting the annotation and study of candidate genes. Here, we present an improved genome build, canFam3.1, which includes 85 MB of novel sequence and now covers 99.8% of the euchromatic portion of the genome. We also present multiple RNA-Sequencing data sets from 10 different canine tissues to catalog ∼175,000 expressed loci. While about 90% of the coding genes previously annotated by EnsEMBL have measurable expression in at least one sample, the number of transcript isoforms detected by our data expands the EnsEMBL annotations by a factor of four. Syntenic comparison with the human genome revealed an additional ∼3,000 loci that are characterized as protein coding in human and were also expressed in the dog, suggesting that those were previously not annotated in the EnsEMBL canine gene set. In addition to ∼20,700 high-confidence protein coding loci, we found ∼4,600 antisense transcripts overlapping exons of protein coding genes, ∼7,200 intergenic multi-exon transcripts without coding potential, likely candidates for long intergenic non-coding RNAs (lincRNAs and ∼11,000 transcripts were reported by two different library construction methods but did not fit any of the above categories. Of the lincRNAs, about 6,000 have no annotated orthologs in human or mouse. Functional analysis of two novel transcripts with shRNA in a mouse kidney cell line altered cell morphology and motility. All in all, we provide a much-improved annotation of the canine genome and suggest regulatory functions for several of the novel non-coding transcripts.

  13. Genomic DNA extraction method from Annona senegalensis Pers ...

    African Journals Online (AJOL)

    aghomotsegin

    2014-02-05

    Feb 5, 2014 ... DNA fruit. 4 μl DNA was loaded per lane. (b) Restriction enzymes digestion of Annona senegalensis genomic. DNA. Lanes 1-3: DNA digested with Sau3A. Figure 2. Amplification of purified DNA with SSR-PCR. DNA was purified using the method described. The purified DNA was amplified using SSR-.

  14. Genomic signal processing for DNA sequence clustering.

    Science.gov (United States)

    Mendizabal-Ruiz, Gerardo; Román-Godínez, Israel; Torres-Ramos, Sulema; Salido-Ruiz, Ricardo A; Vélez-Pérez, Hugo; Morales, J Alejandro

    2018-01-01

    Genomic signal processing (GSP) methods which convert DNA data to numerical values have recently been proposed, which would offer the opportunity of employing existing digital signal processing methods for genomic data. One of the most used methods for exploring data is cluster analysis which refers to the unsupervised classification of patterns in data. In this paper, we propose a novel approach for performing cluster analysis of DNA sequences that is based on the use of GSP methods and the K-means algorithm. We also propose a visualization method that facilitates the easy inspection and analysis of the results and possible hidden behaviors. Our results support the feasibility of employing the proposed method to find and easily visualize interesting features of sets of DNA data.

  15. The DNA replication, virogenesis and infection of canine minute virus in non-permissive and permissive cells.

    Science.gov (United States)

    Li, Fang; Zhang, Qian; Yao, Qing; Chen, Long; Li, Jianning; Qiu, Jianming; Sun, Yuning

    2014-01-22

    Canine minute virus (CnMV), a kind of autonomous parvovirus, is a member of genus bocavirus in parvovirdae family. In our previous study, we constructed and obtained infectious clones of CnMV, analyzed genome characteristics, RNA transcription profile, and revealed some molecular mechanisms of cytopathic effect of target cells. The purpose of this study was to investigate DNA replication, virogenesis and infectious tropism of CnMV in non-permissive and permissive cells. We demonstrated that the genomic DNA of CnMV, besides WRD cells, could replicate significantly in some non-permissive cells (CrFK, EBtR and COS-7) following transfection with infectious clone of CnMV, pI-MVC. Moreover, by using Western blotting and immunofluorescence, we found that the NS1 protein of CnMV was obviously expressed in both 293, CrFK, EBtR and COS-7 cells transfected with pI-MVC. Meanwhile, two-rounds of reinfection on WRD cells (blind passage) of the transfected cell lysates in CrFK, EBtR and COS-7 cells tranfected with pI-MVC showed that pI-MVC could produce infectious virions in these types of non-permissive cells. Furthermore, it is confirmed that CnMV only infected WRD cells (permissive cells for CnMV), could not infect any non-permissive cells including CrFK, EBtR, COS-7, HK293, A549 and A9 cells. Taken together, for the first time, we have demonstrated that bocavirus CnMV DNA could replicate and form infectious progeny virus in some non-permissive cells. And what is more, unlike other parvoviruses, CnMV did not infect some non-permissive cells, although the DNA replication of CnMV occurred in these cells. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Plant genome size variation: bloating and purging DNA

    National Research Council Canada - National Science Library

    Michael, Todd P

    2014-01-01

    Plant genome size variation is a dynamic process of bloating and purging DNA. While it was thought plants were on a path to obesity through continual DNA bloating, recent research supports that most plants activity purge DNA...

  17. Immunogenicity of a DNA-launched replicon-based canine parvovirus DNA vaccine expressing VP2 antigen in dogs.

    Science.gov (United States)

    Dahiya, Shyam S; Saini, Mohini; Kumar, Pankaj; Gupta, Praveen K

    2012-10-01

    A replicon-based DNA vaccine encoding VP2 gene of canine parvovirus (CPV) was developed by cloning CPV-VP2 gene into a replicon-based DNA vaccine vector (pAlpha). The characteristics of a replicon-based DNA vaccine like, self-amplification of transcripts and induction of apoptosis were analyzed in transfected mammalian cells. When the pAlpha-CPV-VP2 was injected intradermal as DNA-launched replicon-based DNA vaccine in dogs, it induced CPV-specific humoral and cell mediated immune responses. The virus neutralization antibody and lymphocyte proliferative responses were higher than conventional CPV DNA vaccine and commercial CPV vaccine. These results indicated that DNA-launched replicon-based CPV DNA vaccine was effective in inducing both CPV-specific humoral and cellular immune responses and can be considered as effective alternative to conventional CPV DNA vaccine and commercial CPV vaccine. Crown Copyright © 2012. Published by Elsevier India Pvt Ltd. All rights reserved.

  18. Chromatin Dynamics in Genome Stability: Roles in Suppressing Endogenous DNA Damage and Facilitating DNA Repair

    Directory of Open Access Journals (Sweden)

    Nidhi Nair

    2017-07-01

    Full Text Available Genomic DNA is compacted into chromatin through packaging with histone and non-histone proteins. Importantly, DNA accessibility is dynamically regulated to ensure genome stability. This is exemplified in the response to DNA damage where chromatin relaxation near genomic lesions serves to promote access of relevant enzymes to specific DNA regions for signaling and repair. Furthermore, recent data highlight genome maintenance roles of chromatin through the regulation of endogenous DNA-templated processes including transcription and replication. Here, we review research that shows the importance of chromatin structure regulation in maintaining genome integrity by multiple mechanisms including facilitating DNA repair and directly suppressing endogenous DNA damage.

  19. Analysis of genomic mutation and immunohistochemistry of platelet-derived growth factor receptors in canine vascular tumours.

    Science.gov (United States)

    Abou Asa, S; Mori, T; Maruo, K; Khater, A; El-Sawak, A; Abd el-Aziz, E; Yanai, T; Sakai, H

    2015-09-01

    We examined whether mutation of the platelet-derived growth factor receptor protein tyrosine kinase (PDGFR)-α and PDGFR-β genes contributes to their overexpression in canine vascular tumours. Genomic sequences of trans- or juxtamembrane regions of PDGFR-α and PDGFR-β were analysed with immunohistochemical staining and polymerase chain reaction-direct sequencing using DNA from paraffin-embedded neoplastic tissues of 27 hemangiosarcomas (HSAs) and 20 hemangiomas (HAs). Immunohistochemically, 75% of the HA cases were positive for PDGFR-α and almost most of the HA cases were negative for PDGFR-β. Of the HSA cases, 55.6% were negative for PDGFR-α and 63% were strongly positive for PDGFR-β. Among the HA cases, 1 missense mutation was detected in PDGFR-α exon 18 and 1 in PDGFR-β exon 17. Two HSA cases had missense mutations in exon 14 and 1 in exon 17 of PDGFR-β. Thus, genomic mutation of trans- or juxtamembrane regions of PDGFRs was not the main mechanism driving the activation of receptors in HSA and HA. © 2013 Blackwell Publishing Ltd.

  20. DNA damage in canine transmissible venereal tumor cells

    OpenAIRE

    Amaral,Anne Santos do; Ferreira, Isabelle [UNESP; Colodel, Márcia Moleta; Salvadori, Daisy Maria Favero [UNESP; Rocha, Noeme Sousa [UNESP

    2011-01-01

    The Transmissible Venereal Tumor (TVT) has been classified according to the predominant cell type as follows: lymphocytoid, plasmocytoid, and mixed. Various degrees of aggressiveness with large array of biological behaviour have been described according to the TVT cell lineages, the present study was designed to investigate the level of DNA damage in the three TVT cell types aiming a better understanding of mechanisms related to the aggressiveness of this neoplasia. A total of 35 dogs were ev...

  1. Nuclear DNA-Content in Mesenchymal Lesions in Dogs: Its Value as Marker of Malignancy and Extent of Genomic Instability

    Energy Technology Data Exchange (ETDEWEB)

    Boerkamp, Kim M., E-mail: K.M.Boerkamp@uu.nl; Rutteman, Gerard R. [Department of Clinical Science of Companion Animals, Faculty of Veterinary Medicine, UU, Yalelaan 104, 3584 CM, Utrecht (Netherlands); Kik, Marja J. L. [Department of Pathobiology, Faculty of Veterinary Medicine, UU, Yalelaan 1, 3508 TD, Utrecht (Netherlands); Kirpensteijn, Jolle [Department of Clinical Science of Companion Animals, Faculty of Veterinary Medicine, UU, Yalelaan 104, 3584 CM, Utrecht (Netherlands); Schulze, Christoph; Grinwis, Guy C. M. [Department of Pathobiology, Faculty of Veterinary Medicine, UU, Yalelaan 1, 3508 TD, Utrecht (Netherlands)

    2012-12-03

    DNA-aneuploidy may reflect the malignant nature of mesenchymal proliferations and herald gross genomic instability as a mechanistic factor in tumor genesis. DNA-ploidy and -index were determined by flow cytometry in canine inflammatory or neoplastic mesenchymal tissues and related to clinico-pathological features, biological behavior and p53 gene mutational status. Half of all sarcomas were aneuploid. Benign mesenchymal neoplasms were rarely aneuploid and inflammatory lesions not at all. The aneuploidy rate was comparable to that reported for human sarcomas with significant variation amongst subtypes. DNA-ploidy status in canines lacked a relation with histological grade of malignancy, in contrast to human sarcomas. While aneuploidy was related to the development of metastases in soft tissue sarcomas it was not in osteosarcomas. No relation amongst sarcomas was found between ploidy status and presence of P53 gene mutations. Heterogeneity of the DNA index between primary and metastatic sarcoma sites was present in half of the cases examined. Hypoploidy is more common in canine sarcomas and hyperploid cases have less deviation of the DNA index than human sarcomas. The variation in the presence and extent of aneuploidy amongst sarcoma subtypes indicates variation in genomic instability. This study strengthens the concept of interspecies variation in the evolution of gross chromosomal aberrations during cancer development.

  2. Nuclear DNA-Content in Mesenchymal Lesions in Dogs: Its Value as Marker of Malignancy and Extent of Genomic Instability

    Directory of Open Access Journals (Sweden)

    Christoph Schulze

    2012-12-01

    Full Text Available DNA-aneuploidy may reflect the malignant nature of mesenchymal proliferations and herald gross genomic instability as a mechanistic factor in tumor genesis. DNA-ploidy and -index were determined by flow cytometry in canine inflammatory or neoplastic mesenchymal tissues and related to clinico-pathological features, biological behavior and p53 gene mutational status. Half of all sarcomas were aneuploid. Benign mesenchymal neoplasms were rarely aneuploid and inflammatory lesions not at all. The aneuploidy rate was comparable to that reported for human sarcomas with significant variation amongst subtypes. DNA-ploidy status in canines lacked a relation with histological grade of malignancy, in contrast to human sarcomas. While aneuploidy was related to the development of metastases in soft tissue sarcomas it was not in osteosarcomas. No relation amongst sarcomas was found between ploidy status and presence of P53 gene mutations. Heterogeneity of the DNA index between primary and metastatic sarcoma sites was present in half of the cases examined. Hypoploidy is more common in canine sarcomas and hyperploid cases have less deviation of the DNA index than human sarcomas. The variation in the presence and extent of aneuploidy amongst sarcoma subtypes indicates variation in genomic instability. This study strengthens the concept of interspecies variation in the evolution of gross chromosomal aberrations during cancer development.

  3. Artificial DNA cutters for DNA manipulation and genome engineering.

    Science.gov (United States)

    Aiba, Yuichiro; Sumaoka, Jun; Komiyama, Makoto

    2011-12-01

    This tutorial review provides recent developments in artificial cutters for site-selective scission of DNA with the focus on chemistry-based DNA cutters. They are useful tools for molecular biology and biotechnology, since their site-selectivity of scission is much higher than that of naturally occurring restriction enzymes and also their scission site is freely chosen. In order to prepare these cutters, a DNA-cutting molecule is combined with a sequence-recognizing molecule in a covalent or non-covalent way. At targeted sites in single-stranded and double-stranded DNAs, the scission occurs via either oxidative cleavage of nucleotides or hydrolysis of phosphodiester linkages. Among many successful examples, an artificial restriction DNA cutter, prepared from Ce(iv)/EDTA and pseudo-complementary peptide nucleic acid, hydrolyzed double-stranded DNA at the target site. The scission site and scission specificity are determined simply in terms of the Watson-Crick rule so that even the whole genome of human beings was selectively cut at one predetermined site. Consistently, homologous recombination in human cells was successfully promoted by this tool. For the purpose of comparison, protein-based DNA cutters (e.g., zinc finger nucleases) are also briefly described. The potential applications of these cutters and their future aspects are discussed.

  4. Genome-Wide Analysis Identifies Germ-Line Risk Factors Associated with Canine Mammary Tumours.

    Directory of Open Access Journals (Sweden)

    Malin Melin

    2016-05-01

    Full Text Available Canine mammary tumours (CMT are the most common neoplasia in unspayed female dogs. CMTs are suitable naturally occurring models for human breast cancer and share many characteristics, indicating that the genetic causes could also be shared. We have performed a genome-wide association study (GWAS in English Springer Spaniel dogs and identified a genome-wide significant locus on chromosome 11 (praw = 5.6x10-7, pperm = 0.019. The most associated haplotype spans a 446 kb region overlapping the CDK5RAP2 gene. The CDK5RAP2 protein has a function in cell cycle regulation and could potentially have an impact on response to chemotherapy treatment. Two additional loci, both on chromosome 27, were nominally associated (praw = 1.97x10-5 and praw = 8.30x10-6. The three loci explain 28.1±10.0% of the phenotypic variation seen in the cohort, whereas the top ten associated regions account for 38.2±10.8% of the risk. Furthermore, the ten GWAS loci and regions with reduced genetic variability are significantly enriched for snoRNAs and tumour-associated antigen genes, suggesting a role for these genes in CMT development. We have identified several candidate genes associated with canine mammary tumours, including CDK5RAP2. Our findings enable further comparative studies to investigate the genes and pathways in human breast cancer patients.

  5. A genetic predictive model for canine hip dysplasia: integration of Genome Wide Association Study (GWAS) and candidate gene approaches.

    Science.gov (United States)

    Bartolomé, Nerea; Segarra, Sergi; Artieda, Marta; Francino, Olga; Sánchez, Elisenda; Szczypiorska, Magdalena; Casellas, Joaquim; Tejedor, Diego; Cerdeira, Joaquín; Martínez, Antonio; Velasco, Alfonso; Sánchez, Armand

    2015-01-01

    Canine hip dysplasia is one of the most prevalent developmental orthopedic diseases in dogs worldwide. Unfortunately, the success of eradication programs against this disease based on radiographic diagnosis is low. Adding the use of diagnostic genetic tools to the current phenotype-based approach might be beneficial. The aim of this study was to develop a genetic prognostic test for early diagnosis of hip dysplasia in Labrador Retrievers. To develop our DNA test, 775 Labrador Retrievers were recruited. For each dog, a blood sample and a ventrodorsal hip radiograph were taken. Dogs were divided into two groups according to their FCI hip score: control (A/B) and case (D/E). C dogs were not included in the sample. Genetic characterization combining a GWAS and a candidate gene strategy using SNPs allowed a case-control population association study. A mathematical model which included 7 SNPs was developed using logistic regression. The model showed a good accuracy (Area under the ROC curve = 0.85) and was validated in an independent population of 114 dogs. This prognostic genetic test represents a useful tool for choosing the most appropriate therapeutic approach once genetic predisposition to hip dysplasia is known. Therefore, it allows a more individualized management of the disease. It is also applicable during genetic selection processes, since breeders can benefit from the information given by this test as soon as a blood sample can be collected, and act accordingly. In the authors' opinion, a shift towards genomic screening might importantly contribute to reducing canine hip dysplasia in the future. In conclusion, based on genetic and radiographic information from Labrador Retrievers with hip dysplasia, we developed an accurate predictive genetic test for early diagnosis of hip dysplasia in Labrador Retrievers. However, further research is warranted in order to evaluate the validity of this genetic test in other dog breeds.

  6. A genetic predictive model for canine hip dysplasia: integration of Genome Wide Association Study (GWAS and candidate gene approaches.

    Directory of Open Access Journals (Sweden)

    Nerea Bartolomé

    Full Text Available Canine hip dysplasia is one of the most prevalent developmental orthopedic diseases in dogs worldwide. Unfortunately, the success of eradication programs against this disease based on radiographic diagnosis is low. Adding the use of diagnostic genetic tools to the current phenotype-based approach might be beneficial. The aim of this study was to develop a genetic prognostic test for early diagnosis of hip dysplasia in Labrador Retrievers. To develop our DNA test, 775 Labrador Retrievers were recruited. For each dog, a blood sample and a ventrodorsal hip radiograph were taken. Dogs were divided into two groups according to their FCI hip score: control (A/B and case (D/E. C dogs were not included in the sample. Genetic characterization combining a GWAS and a candidate gene strategy using SNPs allowed a case-control population association study. A mathematical model which included 7 SNPs was developed using logistic regression. The model showed a good accuracy (Area under the ROC curve = 0.85 and was validated in an independent population of 114 dogs. This prognostic genetic test represents a useful tool for choosing the most appropriate therapeutic approach once genetic predisposition to hip dysplasia is known. Therefore, it allows a more individualized management of the disease. It is also applicable during genetic selection processes, since breeders can benefit from the information given by this test as soon as a blood sample can be collected, and act accordingly. In the authors' opinion, a shift towards genomic screening might importantly contribute to reducing canine hip dysplasia in the future. In conclusion, based on genetic and radiographic information from Labrador Retrievers with hip dysplasia, we developed an accurate predictive genetic test for early diagnosis of hip dysplasia in Labrador Retrievers. However, further research is warranted in order to evaluate the validity of this genetic test in other dog breeds.

  7. Effects of dietary selenium supplementation on DNA damage and apoptosis in canine prostate.

    Science.gov (United States)

    Waters, David J; Shen, Shuren; Cooley, Dawn M; Bostwick, David G; Qian, Junqi; Combs, Gerald F; Glickman, Lawrence T; Oteham, Carol; Schlittler, Deborah; Morris, J Steven

    2003-02-05

    The trace mineral selenium inhibits cancer development in a variety of experimental animal models. We used an in vivo canine model to evaluate the effects of dietary selenium supplementation on DNA damage in prostate tissue and on apoptosis in prostate epithelial cells. Sexually intact elderly male beagle dogs were randomly assigned to receive an unsupplemented diet (control group) or diets that were supplemented with selenium (treatment group), either as selenomethionine or as high-selenium yeast at 3 micro g/kg or 6 micro g/kg body weight per day for 7 months. The extent of DNA damage in prostate cells and in peripheral blood lymphocytes, as determined by the alkaline comet assay, was lower among the selenium-supplemented dogs than among the control dogs (prostate Pdogs and 1.7 (interquartile range = 0.2-2.8) for the control dogs ( P =.04, Mann-Whitney U test). These data suggest that dietary selenium supplementation decreases DNA damage and increases epithelial cell apoptosis within the aging canine prostate.

  8. Combinatorial treatment of DNA and chromatin-modifying drugs cause cell death in human and canine osteosarcoma cell lines.

    Directory of Open Access Journals (Sweden)

    Venugopal Thayanithy

    Full Text Available Downregulation of microRNAs (miRNAs at the 14q32 locus stabilizes the expression of cMYC, thus significantly contributing to osteosarcoma (OS pathobiology. Here, we show that downregulation of 14q32 miRNAs is epigenetically regulated. The predicted promoter regions of miRNA clusters at 14q32 locus showed no recurrent patterns of differential methylation, but Saos2 cells showed elevated histone deacetylase (HDAC activity. Treatment with 4-phenylbutyrate increased acetylation of histones associated with 14q32 miRNAs, but interestingly, robust restoration of 14q32 miRNA expression, attenuation of cMYC expression, and induction of apoptosis required concomitant treatment with 5-Azacytidine, an inhibitor of DNA methylation. These events were associated with genome-wide gene expression changes including induction of pro-apoptotic genes and downregulation of cell cycle genes. Comparable effects were achieved in human and canine OS cells using the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA/Vorinostat and the DNA methylation inhibitor Zebularine (Zeb, with significantly more pronounced cytotoxicity in cells whose molecular phenotypes were indicative of aggressive biological behavior. These results suggested that the combination of these chromatin-modifying drugs may be a useful adjuvant in the treatment of rapidly progressive OS.

  9. Genome wide application of DNA melting analysis.

    Science.gov (United States)

    Jost, Daniel; Everaers, Ralf

    2009-01-21

    Correspondences between functional and thermodynamic melting properties in a genome are being increasingly employed for ab initio gene finding and for the interpretation of the evolution of genomes. Here we present the first systematic genome wide comparison between biologically coding domains and thermodynamically stable regions. In particular, we develop statistical methods to estimate the reliability of the resulting predictions. Not surprisingly, we find that the success of the approach depends on the difference in GC content between the coding and the non-coding parts of the genome and on the percentage of coding base-pairs in the sequence. These prerequisites vary strongly between species, where we observe no systematic differences between eukaryotes and prokaryotes. We find a number of organisms in which the strong correlation of coding domains and thermodynamically stable regions allows us to identify putative exons or genes to complement existing approaches. In contrast to previous investigations along these lines we have not employed the Poland-Scheraga (PS) model of DNA melting but use the earlier Zimm-Bragg (ZB) model. The Ising-like form of the ZB model can be viewed as an approximation to the PS model, with averaged loop entropies included into the cooperative factor [Formula: see text]. This results in a speed-up by a factor of 20-100 compared to the Fixman-Freire algorithm for the solution of the PS model. We show that for genomic sequences the resulting systematic errors are negligible compared to the parameterization uncertainty of the models. We argue that for limited computing resources, available CPU power is better invested in broadening the statistical base for genomic investigations than in marginal improvements of the description of the physical melting behavior.

  10. LUPA: a European initiative taking advantage of the canine genome architecture for unravelling complex disorders in both human and dogs.

    Science.gov (United States)

    Lequarré, Anne-Sophie; Andersson, Leif; André, Catherine; Fredholm, Merete; Hitte, Christophe; Leeb, Tosso; Lohi, Hannes; Lindblad-Toh, Kerstin; Georges, Michel

    2011-08-01

    The domestic dog offers a unique opportunity to explore the genetic basis of disease, morphology and behaviour. Humans share many diseases with our canine companions, making dogs an ideal model organism for comparative disease genetics. Using newly developed resources, genome-wide association studies in dog breeds are proving to be exceptionally powerful. Towards this aim, veterinarians and geneticists from 12 European countries are collaborating to collect and analyse the DNA from large cohorts of dogs suffering from a range of carefully defined diseases of relevance to human health. This project, named LUPA, has already delivered considerable results. The consortium has collaborated to develop a new high density single nucleotide polymorphism (SNP) array. Mutations for four monogenic diseases have been identified and the information has been utilised to find mutations in human patients. Several complex diseases have been mapped and fine mapping is underway. These findings should ultimately lead to a better understanding of the molecular mechanisms underlying complex diseases in both humans and their best friend. Copyright © 2011 Elsevier Ltd. All rights reserved.

  11. Less is More--Optimization of DNA Extraction from Canine Feces.

    Science.gov (United States)

    Lindquist, Christina D; Wictum, Elizabeth J

    2016-01-01

    Although most DNA crime laboratories may not encounter fecal samples often, they are a familiar sample type in non-human forensic laboratories due to their prevalence in the environment. Fecal matter can be challenging due to low numbers of nucleated cells and the presence of inhibitors that impede amplification success. Sampling location (internal vs. external), sampling quantity (10-200 mg), and various extraction protocols (silica matrix, bead beating, and clean-up column) were evaluated to maximize DNA yield. The greatest yield of intact DNA was obtained using a modified silica matrix extraction protocol (VGL-Fecal) on 30-50 mg of fecal matter collected from the external surface of a stool that had been dried for 24 h. This optimized sampling and extraction protocol was applied to a pilot study where DNA yield and genotyping success were evaluated. By optimizing our collection, sampling, and extraction procedures, a reliable method for maximizing the yield of canine fecal DNA was developed. © 2015 American Academy of Forensic Sciences.

  12. Full-length genomic characterization and molecular evolution of canine parvovirus in China.

    Science.gov (United States)

    Zhou, Ling; Tang, Qinghai; Shi, Lijun; Kong, Miaomiao; Liang, Lin; Mao, Qianqian; Bu, Bin; Yao, Lunguang; Zhao, Kai; Cui, Shangjin; Leal, Élcio

    2016-06-01

    Canine parvovirus type 2 (CPV-2) can cause acute haemorrhagic enteritis in dogs and myocarditis in puppies. This disease has become one of the most serious infectious diseases of dogs. During 2014 in China, there were many cases of acute infectious diarrhoea in dogs. Some faecal samples were negative for the CPV-2 antigen based on a colloidal gold test strip but were positive based on PCR, and a viral strain was isolated from one such sample. The cytopathic effect on susceptible cells and the results of the immunoperoxidase monolayer assay, PCR, and sequencing indicated that the pathogen was CPV-2. The strain was named CPV-NY-14, and the full-length genome was sequenced and analysed. A maximum likelihood tree was constructed using the full-length genome and all available CPV-2 genomes. New strains have replaced the original strain in Taiwan and Italy, although the CPV-2a strain is still predominant there. However, CPV-2a still causes many cases of acute infectious diarrhoea in dogs in China.

  13. Analysis of phage Mu DNA transposition by whole-genome ...

    Indian Academy of Sciences (India)

    (Trilink Biotechnologies) were employed. Sample DNA. (ChIP or processed Mu DNA) was amplified with Cy5-9mer primer, and reference DNA (Input or whole genome DNA) with Cy3-9mer primer. The samples were loaded on microarray slides and subjected to standard hybridization procedures (NimbleGen Arrays User's ...

  14. Detection of DNA Methylation by Whole-Genome Bisulfite Sequencing.

    Science.gov (United States)

    Li, Qing; Hermanson, Peter J; Springer, Nathan M

    2018-01-01

    DNA methylation plays an important role in the regulation of the expression of transposons and genes. Various methods have been developed to assay DNA methylation levels. Bisulfite sequencing is considered to be the "gold standard" for single-base resolution measurement of DNA methylation levels. Coupled with next-generation sequencing, whole-genome bisulfite sequencing (WGBS) allows DNA methylation to be evaluated at a genome-wide scale. Here, we described a protocol for WGBS in plant species with large genomes. This protocol has been successfully applied to assay genome-wide DNA methylation levels in maize and barley. This protocol has also been successfully coupled with sequence capture technology to assay DNA methylation levels in a targeted set of genomic regions.

  15. Early life DNA vaccination with the H gene of Canine distemper virus induces robust protection against distemper

    DEFF Research Database (Denmark)

    Jensen, Trine Hammer; Nielsen, Line; Aasted, Bent

    2009-01-01

    Young mink kits (n = 8)were vaccinated withDNA plasmids encoding the viral haemagglutinin protein (H) of a vaccine strain of Canine distemper virus (CDV). Virus neutralising (VN) antibodieswere induced after 2 immunisations and after the third immunisation all kits had high VN antibody titres...

  16. Whole genome sequence analysis of circulating Bluetongue virus serotype 11 strains from the United States including two domestic canine isolates.

    Science.gov (United States)

    Gaudreault, Natasha N; Jasperson, Dane C; Dubovi, Edward J; Johnson, Donna J; Ostlund, Eileen N; Wilson, William C

    2015-07-01

    Bluetongue virus (BTV) is a vector-transmitted pathogen that typically infects and causes disease in domestic and wild ruminants. BTV is also known to infect domestic canines as discovered when dogs were vaccinated with a BTV-contaminated vaccine. Canine BTV infections have been documented through serological surveys, and natural infection by the Culicoides vector has been suggested. The report of isolation of BTV serotype 11 (BTV-11) from 2 separate domestic canine abortion cases in the states of Texas in 2011 and Kansas in 2012, were apparently unrelated to BTV-contaminated vaccination or consumption of BTV-contaminated raw meat as had been previously speculated. To elucidate the origin and relationship of these 2 domestic canine BTV-11 isolates, whole genome sequencing was performed. Six additional BTV-11 field isolates from Texas, Florida, and Washington, submitted for diagnostic investigation during 2011 and 2013, were also fully sequenced and analyzed. The phylogenetic analysis indicates that the BTV-11 domestic canine isolates are virtually identical, and both share high identity with 2 BTV-11 isolates identified from white-tailed deer in Texas in 2011. The results of the current study further support the hypothesis that a BTV-11 strain circulating in the Midwestern states could have been transmitted to the dogs by the infected Culicoides vector. Our study also expands the short list of available BTV-11 sequences, which may aid BTV surveillance and epidemiology. © 2015 The Author(s).

  17. Whole-genome methylation caller designed for methyl-DNA ...

    African Journals Online (AJOL)

    DNA methylation is an indispensable epigenetic modification required for regulating the expression of mammalian genomes. Continued efforts have been made to unravel the methylation states genome-wide, featuring the methyl-DNA immunoprecipitation (MeDIP) coupled with next-generation sequencing. Our method ...

  18. Rapid DNA extraction of bacterial genome using laundry detergents ...

    African Journals Online (AJOL)

    Genomic DNA extraction from bacterial cells involves processes normally performed in most biological laboratories. Therefore, various methods have been offered, manually and kit, but these methods may be time consuming and costly. In this paper, genomic DNA extraction of Pseudomonas aeruginosa was investigated ...

  19. Efficient method for the extraction of genomic DNA from wormwood ...

    African Journals Online (AJOL)

    Suitable method for isolation of genomic DNA is really important. The best method can make the best result for genetic study. About five differences methods were used amongst which were Sarkosyl Method, CTAB method, Kit Method, SDS Method and Phenol-Chloroform Method. Isolated genomic DNA showed high purity ...

  20. Activity of the DNA minor groove cross-linking agent SG2000 (SJG-136) against canine tumours.

    Science.gov (United States)

    Mellinas-Gomez, Maria; Spanswick, Victoria J; Paredes-Moscosso, Solange R; Robson, Matthew; Pedley, R Barbara; Thurston, David E; Baines, Stephen J; Stell, Anneliese; Hartley, John A

    2015-08-19

    Cancer is the leading cause of death in older dogs and its prevalence is increasing. There is clearly a need to develop more effective anti-cancer drugs in dogs. SG2000 (SJG-136) is a sequence selective DNA minor groove cross-linking agent. Based on its in vitro potency, the spectrum of in vivo and clinical activity against human tumours, and its tolerability in human patients, SG2000 has potential as a novel therapeutic against spontaneously occurring canine malignancies. In vitro cytotoxicity was assessed using SRB and MTT assays, and in vivo activity was assessed using canine tumour xenografts. DNA interstrand cross-linking (ICL) was determined using a modification of the single cell gel electrophoresis (comet) assay. Effects on cell cycle distribution were assessed by flow cytometry and measurement of γ-H2AX by immunofluorescence and immunohistochemistry. SG2000 had a multi-log differential cytotoxic profile against a panel of 12 canine tumour cell lines representing a range of common tumour types in dogs. In the CMeC-1 melanoma cell line, DNA ICLs increased linearly with dose following a 1 h treatment. Peak ICL was achieved within 1 h and no removal was observed over 48 h. A relationship between DNA ICL formation and cytotoxicity was observed across cell lines. The formation of γ-H2AX foci was slow, becoming evident after 4 h and reaching a peak at 24 h. SG2000 exhibited significant anti-tumour activity against two canine melanoma tumour models in vivo. Anti-tumour activity was observed at 0.15 and 0.3 mg/kg given i.v. either once, or weekly x 3. Dose-dependent DNA ICL was observed in tumours (and to a lower level in peripheral blood mononuclear cells) at 2 h and persisted at 24 h. ICL increased following the second and third doses in a repeated dose schedule. At 24 h, dose dependent γ-H2AX foci were more numerous than at 2 h, and greater in tumours than in peripheral blood mononuclear cells. SG2000-induced H2AX phosphorylation measured by

  1. Genomic DNA k-mer Spectra: Models and Modalities

    Science.gov (United States)

    Chor, Benny; Horn, David; Goldman, Nick; Levy, Yaron; Massingham, Tim

    Background: The empirical frequencies of DNA k-mers in whole genome sequences provide an interesting perspective on genomic complexity, and the availability of large segments of genomic sequence from many organisms means that analysis of k-mers with non-trivial lengths is now possible.

  2. Resurrection of DNA Function In Vivo from an Extinct Genome

    OpenAIRE

    Pask, Andrew J; Behringer, Richard R.; Renfree, Marilyn B

    2008-01-01

    There is a burgeoning repository of information available from ancient DNA that can be used to understand how genomes have evolved and to determine the genetic features that defined a particular species. To assess the functional consequences of changes to a genome, a variety of methods are needed to examine extinct DNA function. We isolated a transcriptional enhancer element from the genome of an extinct marsupial, the Tasmanian tiger (Thylacinus cynocephalus or thylacine), obtained from 100 ...

  3. [A method for preparation of genomic DNA from Grifola frondosa and construction of a genomic library].

    Science.gov (United States)

    Xu, Zhi-Xiang; Cheng, Du; Li, Bao-Jian

    2004-09-01

    Grifola frondosa, is a valuable medicinal fungus. High quality total genomic DNA is difficult to prepare due to its high polysaccharide content. A method for the preparation of Grifola frondosa total genomic DNA and construction of Grifola frondosa, genomic library is described. Genomic DNA prepared by this method is digested by Sau3A I restriction enzyme. Constructed genomic library give a titer of 2 x 10(5) transformants/50mg , with a average insert size of 14kb. This has paved way for the cloning of other Grifola frondosa genes and molecular biology studies.

  4. Plant genome size variation: bloating and purging DNA.

    Science.gov (United States)

    Michael, Todd P

    2014-07-01

    Plant genome size variation is a dynamic process of bloating and purging DNA. While it was thought plants were on a path to obesity through continual DNA bloating, recent research supports that most plants activity purge DNA. Plant genome size research has greatly benefited from the cataloguing of genome size estimates at the Kew Plant DNA C-values Database, and the recent availability of over 50 fully sequenced and published plant genomes. The emerging trend is that plant genomes bloat due to the copy-and-paste proliferation of a few long terminal repeat retrotransposons (LTRs) and aggressively purge these proliferating LTRs through several mechanisms including illegitimate and incomplete recombination, and double-strand break repair through non-homologous end joining. However, ultra-small genomes such as Utricularia gibba (Bladderwort), which is 82 megabases (Mb), purge excess DNA through genome fractionation and neofunctionalization during multiple rounds of whole genome duplication (WGD). In contrast, the largest published genome, Picea abies (Norway Spruce) at 19 800 Mb, has no detectable WGD but has bloated with diverse and diverged LTRs that either have evaded purging mechanisms or these purging mechanism are absent in gymnosperms. Finally, advances in DNA methylation studies suggest that smaller genomes have a more aggressive epigenomic surveillance system to purge young LTR retrotransposons, which is less active or missing in larger genomes like the bloated gymnosperms. While genome size may not reflect genome complexity, evidence is mounting that genome size may reflect evolutionary status. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  5. Developmental Validation of Short Tandem Repeat Reagent Kit for Forensic DNA Profiling of Canine Biological Materials

    Science.gov (United States)

    Dayton, Melody; Koskinen, Mikko T; Tom, Bradley K; Mattila, Anna-Maria; Johnston, Eric; Halverson, Joy; Fantin, Dennis; DeNise, Sue; Budowle, Bruce; Smith, David Glenn; Kanthaswamy, Sree

    2009-01-01

    Aim To develop a reagent kit that enables multiplex polymerase chain reaction (PCR) amplification of 18 short tandem repeats (STR) and the canine sex-determining Zinc Finger marker. Methods Validation studies to determine the robustness and reliability in forensic DNA typing of this multiplex assay included sensitivity testing, reproducibility studies, intra- and inter-locus color balance studies, annealing temperature and cycle number studies, peak height ratio determination, characterization of artifacts such as stutter percentages and dye blobs, mixture analyses, species-specificity, case type samples analyses and population studies. Results The kit robustly amplified domesticated dog samples and consistently generated full 19-locus profiles from as little as 125 pg of dog DNA. In addition, wolf DNA samples could be analyzed with the kit. Conclusion The kit, which produces robust, reliable, and reproducible results, will be made available for the forensic research community after modifications based on this study’s evaluation to comply with the quality standards expected for forensic casework. PMID:19480022

  6. Genome Sequence of Canine Parvovirus Strain SC02/2011, Isolated from a Puppy with Severe Diarrhea in South China

    Science.gov (United States)

    Cheng, Yi; Ji, Yikuan; Wang, Yu; Sun, Leilei; Huang, Jiaxin

    2012-01-01

    A widespread hemorrhagic gastroenteritis in young dogs occurred in South China. A virulent field canine parvovirus (CPV) strain, SC02/2011, was isolated from a puppy showing enteric signs in Guangdong, China. The genome of CPV strain SC02/2011 was sequenced and analyzed, which will promote a better understanding of the molecular epidemiology and genetic diversity of CPV field isolates in South China. PMID:23166228

  7. Comparison of canine parvovirus with mink enteritis virus by restriction site mapping.

    OpenAIRE

    McMaster, G K; Tratschin, J D; Siegl, G

    1981-01-01

    The genomes of canine parvovirus and mink enteritis virus were compared by restriction enzyme analysis of their replicative-form DNAs. Of 79 mapped sites, 68, or 86%, were found to be common for both types of DNA, indicating that canine parvovirus and mink enteritis virus are closely related viruses. Whether they evolved from a common precursor or whether canine parvovirus is derived from mink enteritis virus, however, cannot be deduced from our present data.

  8. A universal, rapid, and inexpensive method for genomic DNA ...

    Indian Academy of Sciences (India)

    MOHAMMED BAQUR SAHIB A. AL-SHUHAIB

    Abstract. There is no 'one' procedure for extracting DNA from the whole blood of both mammals and birds, since each species has a unique property that require different methods to release its own DNA. Therefore, to obtain genomic DNA, a universal, rapid, and noncostly method was developed. A very simple biological ...

  9. Isolating silkworm genomic DNA without liquid nitrogen suitable for ...

    African Journals Online (AJOL)

    Genomic DNA was isolated from posterior silk gland of silkworms, Antheraea assama. Absolute alcohol was used as tissue fixing solution instead of grinding in liquid nitrogen, which yielded high molecular weight DNA (>40 kb). Samples yielded similar amount of DNA when fixed in absolute alcohol (400 μmg/g of silk gland ...

  10. Leaf storage conditions and genomic DNA isolation efficiency in ...

    African Journals Online (AJOL)

    SERVER

    African Journal of Biotechnology Vol. 7 (5), pp. 557-564, 4 March, 2008 ... Storage of plant tissues for DNA is important to avoid degradation of DNA. Preliminary studies were conducted on Ocimum gratissimum L. in ..... A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acid Res.

  11. A universal, rapid, and inexpensive method for genomic DNA ...

    Indian Academy of Sciences (India)

    reagents for 'all in one' / ready to use tool to extract genomic. DNA (gDNA) from a very wide spectrum of blood samples. To meet these criteria, a universal and versatile DNA extrac- tion procedure should be developed with minimal chemicals and equipment. On the other hand, the interesting natural relation between RBCs ...

  12. Genomic DNA extraction from sapwood of Pinus roxburghii for ...

    African Journals Online (AJOL)

    A method for extraction of genomic DNA from sapwood tissues of mature tall trees of Pinus roxburghii, where collection of needle tissues is extremely difficult has been standardized. The extracted DNA was comparable to that obtained from the needle tissue in terms of yield and purity. The yield of extracted DNA ranged ...

  13. Validation of DESS as a DNA Preservation Method for the Detection of Strongyloides spp. in Canine Feces

    Directory of Open Access Journals (Sweden)

    Meruyert Beknazarova

    2017-06-01

    Full Text Available Strongyloides stercoralis is a gastrointestinal parasitic nematode with a life cycle that includes free-living and parasitic forms. For both clinical (diagnostic and environmental evaluation, it is important that we can detect Strongyloides spp. in both human and non-human fecal samples. Real-time PCR is the most feasible method for detecting the parasite in both clinical and environmental samples that have been preserved. However, one of the biggest challenges with PCR detection is DNA degradation during the postage time from rural and remote areas to the laboratory. This study included a laboratory assessment and field validation of DESS (dimethyl sulfoxide, disodium EDTA, and saturated NaCl preservation of Strongyloides spp. DNA in fecal samples. The laboratory study investigated the capacity of 1:1 and 1:3 sample to DESS ratios to preserve Strongyloides ratti in spike canine feces. It was found that both ratios of DESS significantly prevented DNA degradation compared to the untreated sample. This method was then validated by applying it to the field-collected canine feces and detecting Strongyloides DNA using PCR. A total of 37 canine feces samples were collected and preserved in the 1:3 ratio (sample: DESS and of these, 17 were positive for Strongyloides spp. The study shows that both 1:1 and 1:3 sample to DESS ratios were able to preserve the Strongyloides spp. DNA in canine feces samples stored at room temperature for up to 56 days. This DESS preservation method presents the most applicable and feasible method for the Strongyloides DNA preservation in field-collected feces.

  14. Validation of DESS as a DNA Preservation Method for the Detection of Strongyloides spp. in Canine Feces.

    Science.gov (United States)

    Beknazarova, Meruyert; Millsteed, Shelby; Robertson, Gemma; Whiley, Harriet; Ross, Kirstin

    2017-06-09

    Strongyloides stercoralis is a gastrointestinal parasitic nematode with a life cycle that includes free-living and parasitic forms. For both clinical (diagnostic) and environmental evaluation, it is important that we can detect Strongyloides spp. in both human and non-human fecal samples. Real-time PCR is the most feasible method for detecting the parasite in both clinical and environmental samples that have been preserved. However, one of the biggest challenges with PCR detection is DNA degradation during the postage time from rural and remote areas to the laboratory. This study included a laboratory assessment and field validation of DESS (dimethyl sulfoxide, disodium EDTA, and saturated NaCl) preservation of Strongyloides spp. DNA in fecal samples. The laboratory study investigated the capacity of 1:1 and 1:3 sample to DESS ratios to preserve Strongyloides ratti in spike canine feces. It was found that both ratios of DESS significantly prevented DNA degradation compared to the untreated sample. This method was then validated by applying it to the field-collected canine feces and detecting Strongyloides DNA using PCR. A total of 37 canine feces samples were collected and preserved in the 1:3 ratio (sample: DESS) and of these, 17 were positive for Strongyloides spp. The study shows that both 1:1 and 1:3 sample to DESS ratios were able to preserve the Strongyloides spp. DNA in canine feces samples stored at room temperature for up to 56 days. This DESS preservation method presents the most applicable and feasible method for the Strongyloides DNA preservation in field-collected feces.

  15. GBshape: a genome browser database for DNA shape annotations.

    Science.gov (United States)

    Chiu, Tsu-Pei; Yang, Lin; Zhou, Tianyin; Main, Bradley J; Parker, Stephen C J; Nuzhdin, Sergey V; Tullius, Thomas D; Rohs, Remo

    2015-01-01

    Many regulatory mechanisms require a high degree of specificity in protein-DNA binding. Nucleotide sequence does not provide an answer to the question of why a protein binds only to a small subset of the many putative binding sites in the genome that share the same core motif. Whereas higher-order effects, such as chromatin accessibility, cooperativity and cofactors, have been described, DNA shape recently gained attention as another feature that fine-tunes the DNA binding specificities of some transcription factor families. Our Genome Browser for DNA shape annotations (GBshape; freely available at http://rohslab.cmb.usc.edu/GBshape/) provides minor groove width, propeller twist, roll, helix twist and hydroxyl radical cleavage predictions for the entire genomes of 94 organisms. Additional genomes can easily be added using the GBshape framework. GBshape can be used to visualize DNA shape annotations qualitatively in a genome browser track format, and to download quantitative values of DNA shape features as a function of genomic position at nucleotide resolution. As biological applications, we illustrate the periodicity of DNA shape features that are present in nucleosome-occupied sequences from human, fly and worm, and we demonstrate structural similarities between transcription start sites in the genomes of four Drosophila species. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  16. A Genetic Predictive Model for Canine Hip Dysplasia : Integration of Genome Wide Association Study (GWAS) and Candidate Gene Approaches

    OpenAIRE

    Nerea Bartolomé; Sergi Segarra; Marta Artieda; Olga Francino; Elisenda Sánchez; Magdalena Szczypiorska; Joaquim Casellas; Diego Tejedor; Joaquín Cerdeira; Antonio Martínez; Alfonso Velasco; Armand Sánchez

    2015-01-01

    Canine hip dysplasia is one of the most prevalent developmental orthopedic diseases in dogs worldwide. Unfortunately, the success of eradication programs against this disease based on radiographic diagnosis is low. Adding the use of diagnostic genetic tools to the current phenotype-based approach might be beneficial. The aim of this study was to develop a genetic prognostic test for early diagnosis of hip dysplasia in Labrador Retrievers. To develop our DNA test, 775 Labrador Retrievers were ...

  17. GEAR: genomic enrichment analysis of regional DNA copy number changes.

    Science.gov (United States)

    Kim, Tae-Min; Jung, Yu-Chae; Rhyu, Mun-Gan; Jung, Myeong Ho; Chung, Yeun-Jun

    2008-02-01

    We developed an algorithm named GEAR (genomic enrichment analysis of regional DNA copy number changes) for functional interpretation of genome-wide DNA copy number changes identified by array-based comparative genomic hybridization. GEAR selects two types of chromosomal alterations with potential biological relevance, i.e. recurrent and phenotype-specific alterations. Then it performs functional enrichment analysis using a priori selected functional gene sets to identify primary and clinical genomic signatures. The genomic signatures identified by GEAR represent functionally coordinated genomic changes, which can provide clues on the underlying molecular mechanisms related to the phenotypes of interest. GEAR can help the identification of key molecular functions that are activated or repressed in the tumor genomes leading to the improved understanding on the tumor biology. GEAR software is available with online manual in the website, http://www.systemsbiology.co.kr/GEAR/.

  18. Genomic DNA extraction protocols from ovine hair

    Directory of Open Access Journals (Sweden)

    Jennifer Nonato da Silva Prate

    2013-12-01

    Full Text Available Genomic DNA extracted from animal cells can be used for several purposes, for example, to know genetic variability and genetic relationships between individuals, breeds and/or species, paternity tests, to describe the genetic profile for registration of the animal at association of breeders, detect genetic polymorphisms (SNP related to characteristics of commercial interest, disease diagnose, assess resistance or susceptibility to pathogens, etc. For such evaluations, in general, DNA is amplified by PCR (polymerase chain reaction, and then subjected to various techniques as RFLP (restriction fragments length polymorphism, SSCP (single strand conformation polymorphism, and sequencing. The DNA may be obtained from blood, buccal swabs, meat, cartilage or hair bulb. Among all, the last biological material has been preferred by farmers for its ease acquisition. Several methods for extracting DNA from hair bulb were reported without any consensus for its implementation. This study aimed to optimize a protocol for efficient DNA extraction for use in PCR-RFLP analysis of the Prion gene. For this study, were collected hair samples containing hair bulb from 131 Santa Inês sheep belonging to the Institute of Zootechny, Nova Odessa - SP. Two DNA extraction protocols were evaluated. The first, called phenol-chloroform-isoamyl alcohol (PCIA has long been used by Animal Genetic Laboratories, whose procedures are described below: in each microtube (1.5 mL containing 500 µL of TE-Tween solution (Tris-HCl 50 mM, EDTA 1 mM and 0.5% Tween 20 were added to approximately 30 hair bulb per animal which was incubated at 65°C with shaking at 170 rpm for 2 hours. Then was added 15 µL of proteinase K [10 mg mL-1] and incubated at 55°C at 170 rpm for 6-12 hours. At the end of digestion was added 1 volume of solution phenol-chloroform-isoamyl alcohol (25:24:1 followed by vigorous shaking for 10 seconds and centrifuged at 8000 rpm and 4°C for 10 minutes. The upper phase

  19. Whole-Genome Sequencing of a Canine Family Trio Reveals a FAM83G Variant Associated with Hereditary Footpad Hyperkeratosis

    Directory of Open Access Journals (Sweden)

    Shumaila Sayyab

    2016-03-01

    Full Text Available Over 250 Mendelian traits and disorders, caused by rare alleles have been mapped in the canine genome. Although each disease is rare in the dog as a species, they are collectively common and have major impact on canine health. With SNP-based genotyping arrays, genome-wide association studies (GWAS have proven to be a powerful method to map the genomic region of interest when 10–20 cases and 10–20 controls are available. However, to identify the genetic variant in associated regions, fine-mapping and targeted resequencing is required. Here we present a new approach using whole-genome sequencing (WGS of a family trio without prior GWAS. As a proof-of-concept, we chose an autosomal recessive disease known as hereditary footpad hyperkeratosis (HFH in Kromfohrländer dogs. To our knowledge, this is the first time this family trio WGS-approach has been used successfully to identify a genetic variant that perfectly segregates with a canine disorder. The sequencing of three Kromfohrländer dogs from a family trio (an affected offspring and both its healthy parents resulted in an average genome coverage of 9.2X per individual. After applying stringent filtering criteria for candidate causative coding variants, 527 single nucleotide variants (SNVs and 15 indels were found to be homozygous in the affected offspring and heterozygous in the parents. Using the computer software packages ANNOVAR and SIFT to functionally annotate coding sequence differences, and to predict their functional effect, resulted in seven candidate variants located in six different genes. Of these, only FAM83G:c155G > C (p.R52P was found to be concordant in eight additional cases, and 16 healthy Kromfohrländer dogs.

  20. Full-length genomic and molecular characterization of Canine parvovirus in dogs from North of Brazil.

    Science.gov (United States)

    Silva, S P; Silva, L N P P; Rodrigues, E D L; Cardoso, J F; Tavares, F N; Souza, W M; Santos, C M P; Martins, F M S; Jesus, I S; Brito, T C; Moura, T P C; Nunes, M R T; Casseb, L M N; Silva Filho, E; Casseb, A R

    2017-09-21

    With the objective of characterizing Canine parvovirus (CPV) from some suspected fecal samples of dogs collected from the Veterinarian Hospital in Belém city, five positive samples were found by PCR assay and an update molecular characterization was provided of the CPV-2 circulation in Belém. Through sequencing of the complete DNA sequences (NS1, NS2, VP1, and VP2 genes), the CPV-2 strain was identified as CPV-2b (Asn426Asp) circulating in Belém. The CPV-2b strain with a different change at the position Tyr324Leu was detected in all samples assessed and thus reported for the first time for the scientific community. Phylogenetic analysis indicated that Belém CPV-2b and CPV-2a strains would be related to a cluster with samples after the 1990s, suggesting that CPV-2b in Belém originated from CPV-2a circulating in Brazil after the 1990s. Potential recombination events were analyzed using RDP4 and SplitsTree4; therefore, results suggest that CPV-2 sequences here described were not potentially recombination events. Continuous monitoring and molecular characterization of CPV-2 samples are needed not only to identify possible genetic and antigenic changes that may interfere with the effectiveness of vaccines but also to bring a better understanding of the mechanisms that drive the evolution of CPV-2 in Brazil.

  1. Tracking genome engineering outcome at individual DNA breakpoints.

    Science.gov (United States)

    Certo, Michael T; Ryu, Byoung Y; Annis, James E; Garibov, Mikhail; Jarjour, Jordan; Rawlings, David J; Scharenberg, Andrew M

    2011-07-10

    Site-specific genome engineering technologies are increasingly important tools in the postgenomic era, where biotechnological objectives often require organisms with precisely modified genomes. Rare-cutting endonucleases, through their capacity to create a targeted DNA strand break, are one of the most promising of these technologies. However, realizing the full potential of nuclease-induced genome engineering requires a detailed understanding of the variables that influence resolution of nuclease-induced DNA breaks. Here we present a genome engineering reporter system, designated 'traffic light', that supports rapid flow-cytometric analysis of repair pathway choice at individual DNA breaks, quantitative tracking of nuclease expression and donor template delivery, and high-throughput screens for factors that bias the engineering outcome. We applied the traffic light system to evaluate the efficiency and outcome of nuclease-induced genome engineering in human cell lines and identified strategies to facilitate isolation of cells in which a desired engineering outcome has occurred.

  2. Concentrating Genomic Length DNA in a Microfabricated Array

    DEFF Research Database (Denmark)

    Chen, Yu; Abrams, Ezra S.; Boles, T. Christian

    2015-01-01

    We demonstrate that a microfabricated bump array can concentrate genomic-length DNA molecules efficiently at continuous, high flow velocities, up to 40 μm=s, if the single-molecule DNA globule has a sufficiently large shear modulus. Increase in the shear modulus is accomplished by compacting the ...... for next-generation DNA-sequencing libraries will be an important application of this development.......We demonstrate that a microfabricated bump array can concentrate genomic-length DNA molecules efficiently at continuous, high flow velocities, up to 40 μm=s, if the single-molecule DNA globule has a sufficiently large shear modulus. Increase in the shear modulus is accomplished by compacting...... the DNA molecules to minimal coil size using polyethylene glycol (PEG) derived depletion forces. We map out the sweet spot, where concentration occurs, as a function of PEG concentration and flow speed using a combination of theoretical analysis and experiment. Purification of DNA from enzymatic reactions...

  3. Current developments in canine genetics.

    Science.gov (United States)

    Marschall, Yvonne; Distl, Ottmar

    2010-01-01

    In recent years, canine genetics had made huge progress. In 1999 the first complete karyotype and ideogram of the dog was published. Several linkage and RH maps followed. Using these maps, sets of microsatellite markers for whole genome scans were compiled. In 2003 the sequencing of the DNA of a female Boxer began. Now the second version of the dog genome assembly has been put online, and recently, a microchip SNP array became available. Parallel to these developments, some causal mutations for different traits have been identified. Most of the identified mutations were responsible for monogenic canine hereditary diseases. With the tools available now, it is possible to use the advantages of the population structure of the various dog breeds to unravel complex genetic traits. Furthermore, the dog is a suitable model for the research of a large number of human hereditary diseases and particularly for cancer genetics, heart and neurodegenerative diseases. There are some examples where it was possible to benefit from the knowledge of canine genetics for human research. The search for quantitative trait loci (QTL), the testing of candidate genes and genome-wide association studies can now be performed in dogs. QTL for skeletal size variations and for canine hip dysplasia have been already identified and for these complex traits the responsible genes and their possible interactions can now be identified.

  4. Isolation and enrichment of Cryptosporidium DNA and verification of DNA purity for whole-genome sequencing.

    Science.gov (United States)

    Guo, Yaqiong; Li, Na; Lysén, Colleen; Frace, Michael; Tang, Kevin; Sammons, Scott; Roellig, Dawn M; Feng, Yaoyu; Xiao, Lihua

    2015-02-01

    Whole-genome sequencing of Cryptosporidium spp. is hampered by difficulties in obtaining sufficient, highly pure genomic DNA from clinical specimens. In this study, we developed procedures for the isolation and enrichment of Cryptosporidium genomic DNA from fecal specimens and verification of DNA purity for whole-genome sequencing. The isolation and enrichment of genomic DNA were achieved by a combination of three oocyst purification steps and whole-genome amplification (WGA) of DNA from purified oocysts. Quantitative PCR (qPCR) analysis of WGA products was used as an initial quality assessment of amplified genomic DNA. The purity of WGA products was assessed by Sanger sequencing of cloned products. Next-generation sequencing tools were used in final evaluations of genome coverage and of the extent of contamination. Altogether, 24 fecal specimens of Cryptosporidium parvum, C. hominis, C. andersoni, C. ubiquitum, C. tyzzeri, and Cryptosporidium chipmunk genotype I were processed with the procedures. As expected, WGA products with low (sequences in Sanger sequencing. The cloning-sequencing analysis, however, showed significant contamination in 5 WGA products (proportion of positive colonies derived from Cryptosporidium genomic DNA, ≤25%). Following this strategy, 20 WGA products from six Cryptosporidium species or genotypes with low (mostly sequencing, generating sequence data covering 94.5% to 99.7% of Cryptosporidium genomes, with mostly minor contamination from bacterial, fungal, and host DNA. These results suggest that the described strategy can be used effectively for the isolation and enrichment of Cryptosporidium DNA from fecal specimens for whole-genome sequencing. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  5. Rapid extraction and preservation of genomic DNA from human samples.

    Science.gov (United States)

    Kalyanasundaram, D; Kim, J-H; Yeo, W-H; Oh, K; Lee, K-H; Kim, M-H; Ryew, S-M; Ahn, S-G; Gao, D; Cangelosi, G A; Chung, J-H

    2013-02-01

    Simple and rapid extraction of human genomic DNA remains a bottleneck for genome analysis and disease diagnosis. Current methods using microfilters require cumbersome, multiple handling steps in part because salt conditions must be controlled for attraction and elution of DNA in porous silica. We report a novel extraction method of human genomic DNA from buccal swab and saliva samples. DNA is attracted onto a gold-coated microchip by an electric field and capillary action while the captured DNA is eluted by thermal heating at 70 °C. A prototype device was designed to handle four microchips, and a compatible protocol was developed. The extracted DNA using microchips was characterized by qPCR for different sample volumes, using different lengths of PCR amplicon, and nuclear and mitochondrial genes. In comparison with a commercial kit, an equivalent yield of DNA extraction was achieved with fewer steps. Room-temperature preservation for 1 month was demonstrated for captured DNA, facilitating straightforward collection, delivery, and handling of genomic DNA in an environment-friendly protocol.

  6. From barcodes to genomes: extending the concept of DNA barcoding.

    Science.gov (United States)

    Coissac, Eric; Hollingsworth, Peter M; Lavergne, Sébastien; Taberlet, Pierre

    2016-04-01

    DNA barcoding has had a major impact on biodiversity science. The elegant simplicity of establishing massive scale databases for a few barcode loci is continuing to change our understanding of species diversity patterns, and continues to enhance human abilities to distinguish among species. Capitalizing on the developments of next generation sequencing technologies and decreasing costs of genome sequencing, there is now the opportunity for the DNA barcoding concept to be extended to new kinds of genomic data. We illustrate the benefits and capacity to do this, and also note the constraints and barriers to overcome before it is truly scalable. We advocate a twin track approach: (i) continuation and acceleration of global efforts to build the DNA barcode reference library of life on earth using standard DNA barcodes and (ii) active development and application of extended DNA barcodes using genome skimming to augment the standard barcoding approach. © 2016 John Wiley & Sons Ltd.

  7. An automated annotation tool for genomic DNA sequences using ...

    Indian Academy of Sciences (India)

    Genomic sequence data are often available well before the annotated sequence is published. We present a method for analysis of genomic DNA to identify coding sequences using the GeneScan algorithm and characterize these resultant sequences by BLAST. The routines are used to develop a system for automated ...

  8. Prospective evaluation of a patented DNA test for canine hip dysplasia (CHD).

    Science.gov (United States)

    Manz, Eberhard; Tellhelm, Bernd; Krawczak, Michael

    2017-01-01

    Genetic testing has been propagated as a suitable means to specify individual risks for canine hip dysplasia (CHD). However, the current lack of validation of most genetic CHD tests has left dog owners and breeders in the dark about their practical utility. Therefore, the Society for German Shepherd Dogs (Verein für Deutsche Schäferhunde, SV) initiated a prospective study of 935 animals to assess independently the value of a genetic CHD test (European Patent Specification EP 2 123 777 B1) that was developed by Distl et al. (2009) on the basis of the SV animal stock. Dogs were followed-up for 3 years after birth, classified regarding their CHD phenotype using the scheme of the Fédération Cynologique Internationale, and genotyped for the 17 single nucleotide polymorphisms (SNPs) constituting the CHD test in question. Individual SNP genotypes were combined into animal-specific genomic breeding values (GBVs), calculated as the weighted sum of SNP-wise scores as laid down in the patent specification. Logistic regression analysis revealed that, unexpectedly, the odds ratio for CHD decreased, rather than increased, by a factor of 0.98 per unit increase of the GBV. Nevertheless, since this effect was not statistically significant (95% CI: 0.93-1.03), and the area-under-curve of the test was only 0.523, it must be concluded that the genetic test patented by Distl et al. (2009) is unsuitable for individual CHD risk assessment.

  9. Prospective evaluation of a patented DNA test for canine hip dysplasia (CHD.

    Directory of Open Access Journals (Sweden)

    Eberhard Manz

    Full Text Available Genetic testing has been propagated as a suitable means to specify individual risks for canine hip dysplasia (CHD. However, the current lack of validation of most genetic CHD tests has left dog owners and breeders in the dark about their practical utility. Therefore, the Society for German Shepherd Dogs (Verein für Deutsche Schäferhunde, SV initiated a prospective study of 935 animals to assess independently the value of a genetic CHD test (European Patent Specification EP 2 123 777 B1 that was developed by Distl et al. (2009 on the basis of the SV animal stock. Dogs were followed-up for 3 years after birth, classified regarding their CHD phenotype using the scheme of the Fédération Cynologique Internationale, and genotyped for the 17 single nucleotide polymorphisms (SNPs constituting the CHD test in question. Individual SNP genotypes were combined into animal-specific genomic breeding values (GBVs, calculated as the weighted sum of SNP-wise scores as laid down in the patent specification. Logistic regression analysis revealed that, unexpectedly, the odds ratio for CHD decreased, rather than increased, by a factor of 0.98 per unit increase of the GBV. Nevertheless, since this effect was not statistically significant (95% CI: 0.93-1.03, and the area-under-curve of the test was only 0.523, it must be concluded that the genetic test patented by Distl et al. (2009 is unsuitable for individual CHD risk assessment.

  10. Effects of nickel on DNA methyltransferase activity and genomic DNA methylation levels.

    Science.gov (United States)

    Lee, Y W; Broday, L; Costa, M

    1998-07-31

    Methylation of DNA plays an important role in organizing the genome into transcriptionally active and inactive zones. Nickel compounds cause chromatin condensation and DNA methylation in the transgenic gpt+ Chinese hamster cell line (G12). Here we show that nickel is an inhibitor of cytosine 5-methyltransferase activity in vivo and in vitro. In living cells, this inhibition is transient and following a recovery period after nickel treatment, Mtase activity slightly rebounds. Genomic DNA methylation levels are also somewhat decreased following nickel treatment, but with time, there is an elevation of total DNA methylation above basal levels and before any rebound of methyltransferase activity. These results suggest that nickel exposure can elevate total genomic DNA methylation levels even when DNA methyltransferase activity is depressed. These findings may explain the hypermethylation of senescence and tumor suppressor genes found during nickel carcinogenesis and support the model of a direct effect of Ni2+ on chromatin leading to de novo DNA methylation.

  11. Genomic Approaches to DNA repair and Mutagenesis

    OpenAIRE

    Wyrick, John J.; Roberts, Steven A.

    2015-01-01

    DNA damage is a constant threat to cells, causing cytotoxicity as well as inducing genetic alterations. The steady-state abundance of DNA lesions in a cell is minimized by a variety of DNA repair mechanisms, including DNA strand break repair, mismatch repair, nucleotide excision repair, base excision repair, and ribonucleotide excision repair. The efficiencies and mechanisms by which these pathways remove damage from chromosomes have been primarily characterized by investigating the processin...

  12. Short-sequence DNA repeats in prokaryotic genomes

    NARCIS (Netherlands)

    A.F. van Belkum (Alex); S. Scherer; L. van Alphen (Loek); H.A. Verbrugh (Henri)

    1998-01-01

    textabstractShort-sequence DNA repeat (SSR) loci can be identified in all eukaryotic and many prokaryotic genomes. These loci harbor short or long stretches of repeated nucleotide sequence motifs. DNA sequence motifs in a single locus can be identical and/or

  13. Rapid DNA extraction of bacterial genome using laundry detergents ...

    African Journals Online (AJOL)

    Yomi

    2012-01-03

    Jan 3, 2012 ... To evaluate the efficiency of the genomic DNA in the processes in which DNA is used as a template, the polymerase chain reaction ... purity and concentration is of common processes in molecular research and clinical ... these methods, using chemical compounds or physical methods to lysis the cell is the ...

  14. Genomic DNA extraction from sapwood of Pinus roxburghii for ...

    African Journals Online (AJOL)

    Ashish

    2013-02-22

    Feb 22, 2013 ... A method for extraction of genomic DNA from sapwood tissues of mature tall trees of Pinus roxburghii, where collection of needle tissues is extremely difficult has been standardized. The extracted DNA was comparable to that obtained from the needle tissue in terms of yield and purity. The yield of extracted.

  15. Genomic Stability: FANCJ-Dependent G4 DNA Repair

    OpenAIRE

    Maizels, Nancy

    2008-01-01

    G-rich regions have the potential to form G4 DNA upon replication, which can lead to genomic instability. FANCJ, a G4 DNA helicase, has been shown to be critical for the stability of regions that match the G4 signature motif by experiments analyzing its nematode homolog.

  16. A universal, rapid, and inexpensive method for genomic DNA ...

    Indian Academy of Sciences (India)

    ... of both mammals and birds, since each species has a unique property that require different methods to release its own DNA. Therefore, to obtain genomic DNA, a universal, rapid, and noncostly method was developed. A very simple biological basis is followed in this procedure, in which, when the bloodis placed in water, ...

  17. Genome-wide mapping of DNA strand breaks.

    Directory of Open Access Journals (Sweden)

    Frédéric Leduc

    Full Text Available Determination of cellular DNA damage has so far been limited to global assessment of genome integrity whereas nucleotide-level mapping has been restricted to specific loci by the use of specific primers. Therefore, only limited DNA sequences can be studied and novel regions of genomic instability can hardly be discovered. Using a well-characterized yeast model, we describe a straightforward strategy to map genome-wide DNA strand breaks without compromising nucleotide-level resolution. This technique, termed "damaged DNA immunoprecipitation" (dDIP, uses immunoprecipitation and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labeling (TUNEL to capture DNA at break sites. When used in combination with microarray or next-generation sequencing technologies, dDIP will allow researchers to map genome-wide DNA strand breaks as well as other types of DNA damage and to establish a clear profiling of altered genes and/or intergenic sequences in various experimental conditions. This mapping technique could find several applications for instance in the study of aging, genotoxic drug screening, cancer, meiosis, radiation and oxidative DNA damage.

  18. cDNA structure, genomic organization and expression patterns of ...

    African Journals Online (AJOL)

    Visfatin was a newly identified adipocytokine, which was involved in various physiologic and pathologic processes of organisms. The cDNA structure, genomic organization and expression patterns of silver Prussian carp visfatin were described in this report. The silver Prussian carp visfatin cDNA cloned from the liver was ...

  19. TALENs: customizable molecular DNA scissors for genome engineering of plants.

    Science.gov (United States)

    Chen, Kunling; Gao, Caixia

    2013-06-20

    Precise genome modification with engineered nucleases is a powerful tool for studying basic biology and applied biotechnology. Transcription activator-like effector nucleases (TALENs), consisting of an engineered specific (TALE) DNA binding domain and a Fok I cleavage domain, are newly developed versatile reagents for genome engineering in different organisms. Because of the simplicity of the DNA recognition code and their modular assembly, TALENs can act as customizable molecular DNA scissors inducing double-strand breaks (DSBs) at given genomic location. Thus, they provide a valuable approach to targeted genome modifications such as mutations, insertions, replacements or chromosome rearrangements. In this article, we review the development of TALENs, and summarize the principles and tools for TALEN-mediated gene targeting in plant cells, as well as current and potential strategies for use in plant research and crop improvement. Copyright © 2013. Published by Elsevier Ltd.

  20. Rapid isolation of yeast genomic DNA: Bust n' Grab

    Directory of Open Access Journals (Sweden)

    Peterson Kenneth R

    2004-04-01

    Full Text Available Abstract Background Mutagenesis of yeast artificial chromosomes (YACs often requires analysis of large numbers of yeast clones to obtain correctly targeted mutants. Conventional ways to isolate yeast genomic DNA utilize either glass beads or enzymatic digestion to disrupt yeast cell wall. Using small glass beads is messy, whereas enzymatic digestion of the cells is expensive when many samples need to be analyzed. We sought to develop an easier and faster protocol than the existing methods for obtaining yeast genomic DNA from liquid cultures or colonies on plates. Results Repeated freeze-thawing of cells in a lysis buffer was used to disrupt the cells and release genomic DNA. Cell lysis was followed by extraction with chloroform and ethanol precipitation of DNA. Two hundred ng – 3 μg of genomic DNA could be isolated from a 1.5 ml overnight liquid culture or from a large colony. Samples were either resuspended directly in a restriction enzyme/RNase coctail mixture for Southern blot hybridization or used for several PCR reactions. We demonstrated the utility of this method by showing an analysis of yeast clones containing a mutagenized human β-globin locus YAC. Conclusion An efficient, inexpensive method for obtaining yeast genomic DNA from liquid cultures or directly from colonies was developed. This protocol circumvents the use of enzymes or glass beads, and therefore is cheaper and easier to perform when processing large numbers of samples.

  1. Selective microbial genomic DNA isolation using restriction endonucleases.

    Science.gov (United States)

    Barnes, Helen E; Liu, Guohong; Weston, Christopher Q; King, Paula; Pham, Long K; Waltz, Shannon; Helzer, Kimberly T; Day, Laura; Sphar, Dan; Yamamoto, Robert T; Forsyth, R Allyn

    2014-01-01

    To improve the metagenomic analysis of complex microbiomes, we have repurposed restriction endonucleases as methyl specific DNA binding proteins. As an example, we use DpnI immobilized on magnetic beads. The ten minute extraction technique allows specific binding of genomes containing the DpnI Gm6ATC motif common in the genomic DNA of many bacteria including γ-proteobacteria. Using synthetic genome mixtures, we demonstrate 80% recovery of Escherichia coli genomic DNA even when only femtogram quantities are spiked into 10 µg of human DNA background. Binding is very specific with less than 0.5% of human DNA bound. Next Generation Sequencing of input and enriched synthetic mixtures results in over 100-fold enrichment of target genomes relative to human and plant DNA. We also show comparable enrichment when sequencing complex microbiomes such as those from creek water and human saliva. The technique can be broadened to other restriction enzymes allowing for the selective enrichment of trace and unculturable organisms from complex microbiomes and the stratification of organisms according to restriction enzyme enrichment.

  2. DNA Repair and Genome Maintenance in Bacillus subtilis

    Science.gov (United States)

    Lenhart, Justin S.; Schroeder, Jeremy W.; Walsh, Brian W.

    2012-01-01

    Summary: From microbes to multicellular eukaryotic organisms, all cells contain pathways responsible for genome maintenance. DNA replication allows for the faithful duplication of the genome, whereas DNA repair pathways preserve DNA integrity in response to damage originating from endogenous and exogenous sources. The basic pathways important for DNA replication and repair are often conserved throughout biology. In bacteria, high-fidelity repair is balanced with low-fidelity repair and mutagenesis. Such a balance is important for maintaining viability while providing an opportunity for the advantageous selection of mutations when faced with a changing environment. Over the last decade, studies of DNA repair pathways in bacteria have demonstrated considerable differences between Gram-positive and Gram-negative organisms. Here we review and discuss the DNA repair, genome maintenance, and DNA damage checkpoint pathways of the Gram-positive bacterium Bacillus subtilis. We present their molecular mechanisms and compare the functions and regulation of several pathways with known information on other organisms. We also discuss DNA repair during different growth phases and the developmental program of sporulation. In summary, we present a review of the function, regulation, and molecular mechanisms of DNA repair and mutagenesis in Gram-positive bacteria, with a strong emphasis on B. subtilis. PMID:22933559

  3. De novo assembly of the carrot mitochondrial genome using next generation sequencing of whole genomic DNA provides first evidence of DNA transfer into an angiosperm plastid genome

    Directory of Open Access Journals (Sweden)

    Iorizzo Massimo

    2012-05-01

    Full Text Available Abstract Background Sequence analysis of organelle genomes has revealed important aspects of plant cell evolution. The scope of this study was to develop an approach for de novo assembly of the carrot mitochondrial genome using next generation sequence data from total genomic DNA. Results Sequencing data from a carrot 454 whole genome library were used to develop a de novo assembly of the mitochondrial genome. Development of a new bioinformatic tool allowed visualizing contig connections and elucidation of the de novo assembly. Southern hybridization demonstrated recombination across two large repeats. Genome annotation allowed identification of 44 protein coding genes, three rRNA and 17 tRNA. Identification of the plastid genome sequence allowed organelle genome comparison. Mitochondrial intergenic sequence analysis allowed detection of a fragment of DNA specific to the carrot plastid genome. PCR amplification and sequence analysis across different Apiaceae species revealed consistent conservation of this fragment in the mitochondrial genomes and an insertion in Daucus plastid genomes, giving evidence of a mitochondrial to plastid transfer of DNA. Sequence similarity with a retrotransposon element suggests a possibility that a transposon-like event transferred this sequence into the plastid genome. Conclusions This study confirmed that whole genome sequencing is a practical approach for de novo assembly of higher plant mitochondrial genomes. In addition, a new aspect of intercompartmental genome interaction was reported providing the first evidence for DNA transfer into an angiosperm plastid genome. The approach used here could be used more broadly to sequence and assemble mitochondrial genomes of diverse species. This information will allow us to better understand intercompartmental interactions and cell evolution.

  4. Defining functional DNA elements in the human genome

    Science.gov (United States)

    Kellis, Manolis; Wold, Barbara; Snyder, Michael P.; Bernstein, Bradley E.; Kundaje, Anshul; Marinov, Georgi K.; Ward, Lucas D.; Birney, Ewan; Crawford, Gregory E.; Dekker, Job; Dunham, Ian; Elnitski, Laura L.; Farnham, Peggy J.; Feingold, Elise A.; Gerstein, Mark; Giddings, Morgan C.; Gilbert, David M.; Gingeras, Thomas R.; Green, Eric D.; Guigo, Roderic; Hubbard, Tim; Kent, Jim; Lieb, Jason D.; Myers, Richard M.; Pazin, Michael J.; Ren, Bing; Stamatoyannopoulos, John A.; Weng, Zhiping; White, Kevin P.; Hardison, Ross C.

    2014-01-01

    With the completion of the human genome sequence, attention turned to identifying and annotating its functional DNA elements. As a complement to genetic and comparative genomics approaches, the Encyclopedia of DNA Elements Project was launched to contribute maps of RNA transcripts, transcriptional regulator binding sites, and chromatin states in many cell types. The resulting genome-wide data reveal sites of biochemical activity with high positional resolution and cell type specificity that facilitate studies of gene regulation and interpretation of noncoding variants associated with human disease. However, the biochemically active regions cover a much larger fraction of the genome than do evolutionarily conserved regions, raising the question of whether nonconserved but biochemically active regions are truly functional. Here, we review the strengths and limitations of biochemical, evolutionary, and genetic approaches for defining functional DNA segments, potential sources for the observed differences in estimated genomic coverage, and the biological implications of these discrepancies. We also analyze the relationship between signal intensity, genomic coverage, and evolutionary conservation. Our results reinforce the principle that each approach provides complementary information and that we need to use combinations of all three to elucidate genome function in human biology and disease. PMID:24753594

  5. GAPDH Pseudogenes and the Quantification of Feline Genomic DNA Equivalents

    Directory of Open Access Journals (Sweden)

    A. Katrin Helfer-Hungerbuehler

    2013-01-01

    Full Text Available Quantitative real-time PCR (qPCR is broadly used to detect and quantify nucleic acid targets. In order to determine cell copy number and genome equivalents, a suitable reference gene that is present in a defined number in the genome is needed, preferably as a single copy gene. For most organisms, a variable number of glyceraldehyde-3-phosphate dehydrogenase (GAPDH pseudogenes have been reported. However, it has been suggested that a single-copy of the GAPDH pseudogene is present in the feline genome and that a GAPDH assay can therefore be used to quantify feline genomic DNA (gDNA. The aim of this study was to determine whether one or more GAPDH pseudogenes are present in the feline genome and to provide a suitable alternative qPCR system for the quantification of feline cell copy number and genome equivalents. Bioinformatics and sequencing results revealed that not just one but several closely related GAPDH-like sequences were present in the cat genome. We thus identified, developed, optimized, and validated an alternative reference gene assay using feline albumin (fALB. Our data emphasize the need for an alternative reference gene, apart from the GAPDH pseudogene, for the normalization of gDNA levels. We recommend using the fALB qPCR assay for future studies.

  6. Purification of genomic DNA with minimal contamination of proteins.

    Science.gov (United States)

    Hebron, Haroun R; Yang, Yu; Hang, Jun

    2009-12-01

    The purification is based on a set of solutions and a simple centrifugation procedure. Protocols are designed for an easy extraction and purification of genomic DNA from a wide range of samples, including whole blood, buffy coat, bone marrow, body fluids, buccal cells, tissues, mouse tails, etc. RBCs are lysed by dilution into a hypotonic solution. Tissues are broken down and digested by proteinase K in the presence of an anion detergent to release genomic DNA. After precipitation of the detergent and proteins, unique beads that bind proteins, lipids, and RNAs are added to achieve the supreme purity. Genomic DNA is then separated by alcohol precipitation. A proprietary nucleic acid precipitation reagent is used to enhance DNA recovery from low concentration samples. No DNA-binding beads or columns are used in the method, eliminating the problem of low yield and the risk of shearing of genomic DNA. The purified samples are free of proteins, lipids, salts, and RNA contamination. Purified samples are also stable for storage and suitable for all downstream applications.

  7. DNA Replicons for Plant Genome Engineering[W][OPEN

    Science.gov (United States)

    Baltes, Nicholas J.; Gil-Humanes, Javier; Cermak, Tomas; Atkins, Paul A.; Voytas, Daniel F.

    2014-01-01

    Sequence-specific nucleases enable facile editing of higher eukaryotic genomic DNA; however, targeted modification of plant genomes remains challenging due to ineffective methods for delivering reagents for genome engineering to plant cells. Here, we use geminivirus-based replicons for transient expression of sequence-specific nucleases (zinc-finger nucleases, transcription activator–like effector nucleases, and the clustered, regularly interspaced, short palindromic repeat/Cas system) and delivery of DNA repair templates. In tobacco (Nicotiana tabacum), replicons based on the bean yellow dwarf virus enhanced gene targeting frequencies one to two orders of magnitude over conventional Agrobacterium tumefaciens T-DNA. In addition to the nuclease-mediated DNA double-strand breaks, gene targeting was promoted by replication of the repair template and pleiotropic activity of the geminivirus replication initiator proteins. We demonstrate the feasibility of using geminivirus replicons to generate plants with a desired DNA sequence modification. By adopting a general plant transformation method, plantlets with a desired DNA change were regenerated in genome engineering. PMID:24443519

  8. Efficiency of methylated DNA immunoprecipitation bisulphite sequencing for whole-genome DNA methylation analysis.

    Science.gov (United States)

    Jeong, Hae Min; Lee, Sangseon; Chae, Heejoon; Kim, RyongNam; Kwon, Mi Jeong; Oh, Ensel; Choi, Yoon-La; Kim, Sun; Shin, Young Kee

    2016-08-01

    We compared four common methods for measuring DNA methylation levels and recommended the most efficient method in terms of cost and coverage. The DNA methylation status of liver and stomach tissues was profiled using four different methods, whole-genome bisulphite sequencing (WG-BS), targeted bisulphite sequencing (Targeted-BS), methylated DNA immunoprecipitation sequencing (MeDIP-seq) and methylated DNA immunoprecipitation bisulphite sequencing (MeDIP-BS). We calculated DNA methylation levels using each method and compared the results. MeDIP-BS yielded the most similar DNA methylation profile to WG-BS, with 20 times less data, suggesting remarkable cost savings and coverage efficiency compared with the other methods. MeDIP-BS is a practical cost-effective method for analyzing whole-genome DNA methylation that is highly accurate at base-pair resolution.

  9. Inconsistencies in Neanderthal genomic DNA sequences.

    Directory of Open Access Journals (Sweden)

    Jeffrey D Wall

    2007-10-01

    Full Text Available Two recently published papers describe nuclear DNA sequences that were obtained from the same Neanderthal fossil. Our reanalyses of the data from these studies show that they are not consistent with each other and point to serious problems with the data quality in one of the studies, possibly due to modern human DNA contaminants and/or a high rate of sequencing errors.

  10. Differential DNA Methylation Analysis without a Reference Genome.

    Science.gov (United States)

    Klughammer, Johanna; Datlinger, Paul; Printz, Dieter; Sheffield, Nathan C; Farlik, Matthias; Hadler, Johanna; Fritsch, Gerhard; Bock, Christoph

    2015-12-22

    Genome-wide DNA methylation mapping uncovers epigenetic changes associated with animal development, environmental adaptation, and species evolution. To address the lack of high-throughput methods for DNA methylation analysis in non-model organisms, we developed an integrated approach for studying DNA methylation differences independent of a reference genome. Experimentally, our method relies on an optimized 96-well protocol for reduced representation bisulfite sequencing (RRBS), which we have validated in nine species (human, mouse, rat, cow, dog, chicken, carp, sea bass, and zebrafish). Bioinformatically, we developed the RefFreeDMA software to deduce ad hoc genomes directly from RRBS reads and to pinpoint differentially methylated regions between samples or groups of individuals (http://RefFreeDMA.computational-epigenetics.org). The identified regions are interpreted using motif enrichment analysis and/or cross-mapping to annotated genomes. We validated our method by reference-free analysis of cell-type-specific DNA methylation in the blood of human, cow, and carp. In summary, we present a cost-effective method for epigenome analysis in ecology and evolution, which enables epigenome-wide association studies in natural populations and species without a reference genome. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  11. Differential DNA Methylation Analysis without a Reference Genome

    Directory of Open Access Journals (Sweden)

    Johanna Klughammer

    2015-12-01

    Full Text Available Genome-wide DNA methylation mapping uncovers epigenetic changes associated with animal development, environmental adaptation, and species evolution. To address the lack of high-throughput methods for DNA methylation analysis in non-model organisms, we developed an integrated approach for studying DNA methylation differences independent of a reference genome. Experimentally, our method relies on an optimized 96-well protocol for reduced representation bisulfite sequencing (RRBS, which we have validated in nine species (human, mouse, rat, cow, dog, chicken, carp, sea bass, and zebrafish. Bioinformatically, we developed the RefFreeDMA software to deduce ad hoc genomes directly from RRBS reads and to pinpoint differentially methylated regions between samples or groups of individuals (http://RefFreeDMA.computational-epigenetics.org. The identified regions are interpreted using motif enrichment analysis and/or cross-mapping to annotated genomes. We validated our method by reference-free analysis of cell-type-specific DNA methylation in the blood of human, cow, and carp. In summary, we present a cost-effective method for epigenome analysis in ecology and evolution, which enables epigenome-wide association studies in natural populations and species without a reference genome.

  12. Nucleosome Packaging and Nucleosome Positioning of Genomic DNA

    Science.gov (United States)

    Lowary, P. T.; Widom, J.

    1997-02-01

    The goals of this study were to assess the extent to which bulk genomic DNA sequences contribute to their own packaging in nucleosomes and to reveal the relationship between nucleosome packaging and positioning. Using a competitive nucleosome reconstitution assay, we found that at least 95% of bulk DNA sequences have an affinity for histone octamer in nucleosomes that is similar to that of randomly synthesized DNA; they contribute little to their own packaging at the level of individual nucleosomes. An equation was developed that relates the measured free energy to the fractional occupancy of specific nucleosome positions. Evidently, the bulk of eukaryotic genomic DNA is also not evolved or constrained for significant sequence-directed nucleosome positioning at the level of individual nucleosomes. Implications for gene regulation in vivo are discussed.

  13. Comparison of kDNA PCR-hybridization assay with three PCR methods for canines visceral Leishmaniasis diagnosis

    Energy Technology Data Exchange (ETDEWEB)

    Pilatti, Marcia M.; Andrade, Antero S.R. [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil)], e-mail: marciapilatti@yahoo.com.br, e-mail: antero@cdtn.br; Ferreira, Sidney A. [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Dept. de Parasitologia], e-mail: saninoalmeida@gmail.com

    2009-07-01

    The sensitivity of the kDNA PCR-Hybridization assay, which uses radioactive DNA probes (labeled with {sup 32}P), was compared with three conventional PCR methods used for canine visceral leishmaniasis diagnosis. All PCR methods had two steps: a first amplification followed by hybridization or by a new amplification (nested or semi nested). Two methods (kDNA PCR-Hybridization and kDNA snPCR) used primers addressed to kinetoplast minicircles and the other two methods to the coding (LnPCR) and intergenic noncoding regions (ITS-1 nPCR) of the ribosomal rRNA genes. The comparison was accomplished in two groups of 23 infected dogs using samples collected by the conjunctival swab procedure. In the Group 1 the DNA was extracted from cotton swabs by phenol-chloroform and in Group 2 by boiling. The most efficient PCR methods in the Group 1 were those based on kDNA targets. The kDNA PCR-Hybridization was able to detect parasites in 22/23 dogs (95.6%) and in 40/46 samples (86.9%). The kDNA snPCR was positive for 21/23 dogs (91.3%) and for 40/46 samples (86.9%). The positivities of the kDNA based methods were significantly higher than the positivities verified for the methods based on ribosomal rRNA genes (p<0.05). In the Group 2 the kDNA PCR- Hybridization showed a better performance detecting parasites in 18/23 dogs (78.3%) and in 31/46 samples (67.4%), significantly higher than the other three methods (p<0.05). The higher sensitivity of the minicircle kDNA based assays reported by others was confirmed in this study and kDNA PCR-Hybridization showed the best sensitivity among the assays evaluated. (author)

  14. Developmental validation of DogFiler, a novel multiplex for canine DNA profiling in forensic casework.

    Science.gov (United States)

    Wictum, Elizabeth; Kun, Teri; Lindquist, Christina; Malvick, Julia; Vankan, Dianne; Sacks, Benjamin

    2013-01-01

    While the analysis of human DNA has been the focus of large-scale collaborative endeavors, non-human forensic DNA analysis has not benefited from the same funding streams and coordination of effort. Consequently, the development of standard marker panels, allelic ladders and allele-specific sequence data comparable to those established for human forensic genetics has lagged. To meet that need for domestic dogs, we investigated sequence data provided by the published 7.6X dog genome for novel short tandem repeat markers that met our criteria for sensitivity, stability, robustness, polymorphic information content, and ease of scoring. Fifteen unlinked tetranucleotide repeat markers were selected from a pool of 3113 candidate markers and assembled with a sex-linked marker into a multiplex capable of generating a full profile with as little as 60pg of nuclear DNA. An accompanying allelic ladder was assembled and sequenced to obtain detailed repeat motif data. Validation was carried out according to SWGDAM guidelines, and the DogFiler panel has been integrated into forensic casework and accepted in courts across the U.S. Applying various formulae for calculating random match probabilities for inbred populations, estimates for this panel of markers have proven to be comparable to those obtained in human forensic genetics. The DogFiler panel and the associated allelic ladder represent the first published non-human profiling system to fully address all SWGDAM recommendations. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  15. DNA polymerase proofreading: Multiple roles maintain genome stability.

    Science.gov (United States)

    Reha-Krantz, Linda J

    2010-05-01

    DNA polymerase proofreading is a spell-checking activity that enables DNA polymerases to remove newly made nucleotide incorporation errors from the primer terminus before further primer extension and also prevents translesion synthesis. DNA polymerase proofreading improves replication fidelity approximately 100-fold, which is required by many organisms to prevent unacceptably high, life threatening mutation loads. DNA polymerase proofreading has been studied by geneticists and biochemists for >35 years. A historical perspective and the basic features of DNA polymerase proofreading are described here, but the goal of this review is to present recent advances in the elucidation of the proofreading pathway and to describe roles of DNA polymerase proofreading beyond mismatch correction that are also important for maintaining genome stability. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  16. A Genome-wide Linkage Scan in German Shepherd Dogs Localizes Canine Platelet Procoagulant Deficiency (Scott Syndrome) to Canine Chromosome 27

    Science.gov (United States)

    Brooks, Marjory; Etter, Krystal; Catalfamo, James; Brisbin, Abra; Bustamante, Carlos; Mezey, Jason

    2011-01-01

    Scott syndrome is a rare hereditary bleeding disorder associated with an inability of stimulated platelets to externalize the negatively charged phospholipid, phosphatidylserine (PS). Canine Scott syndrome (CSS) is the only naturally-occurring animal model of this defect and therefore represents a unique tool to discover a disease gene capable of producing this platelet phenotype. We undertook platelet function studies and linkage analyses in a pedigree of CSS-affected German shepherd dogs. Based on residual serum prothrombin and flow cytometric assays, CSS segregates as an autosomal recessive trait. An initial genome scan, performed by genotyping 48 dogs for 280 microsatellite markers, suggested linkage with markers on chromosome 27. Genotypes ultimately obtained for a total of 56 dogs at 11 markers on chromosome 27 revealed significant LOD scores for 2 markers near the centromere, with multipoint linkage indicating a CSS trait locus spanning approximately 14 cM. These results provide the basis for fine mapping studies to narrow the disease interval and target the evaluation of putative disease genes. PMID:19854246

  17. Effect of nickel chloride on Arabidopsis genomic DNA and methylation of 18S rDNA

    Directory of Open Access Journals (Sweden)

    Zhongai Li

    2015-01-01

    Conclusions: NiCl2 application caused variation of DNA methylation of the Arabidopsis genomic and offspring's. NiCl2 also resulted in nucleolar injury and deformity of root tip cells. The methylation rate of 18S rDNA also changed by adding NiCl2.

  18. Concentrating and labeling genomic DNA in a nanofluidic array

    DEFF Research Database (Denmark)

    Marie, Rodolphe; Pedersen, Jonas Nyvold; Mir, Kalim U.

    2018-01-01

    Nucleotide incorporation by DNA polymerase forms the basis of DNA sequencing-by-synthesis. In current platforms, either the single-stranded DNA or the enzyme is immobilized on a solid surface to locate the incorporation of individual nucleotides in space and/or time. Solid-phase reactions may, ho...... concentration can reach up to 100 μg mL-1, and the DNA is eluted from the array by increasing the flow rate. The device may be an important preparative module for carrying out enzymatic processing on DNA extracted from single-cells in a microfluidic chip.......Nucleotide incorporation by DNA polymerase forms the basis of DNA sequencing-by-synthesis. In current platforms, either the single-stranded DNA or the enzyme is immobilized on a solid surface to locate the incorporation of individual nucleotides in space and/or time. Solid-phase reactions may......, however, hinder the polymerase activity. We demonstrate a device and a protocol for the enzymatic labeling of genomic DNA arranged in a dense array of single molecules without attaching the enzyme or the DNA to a surface. DNA molecules accumulate in a dense array of pits embedded within a nanoslit due...

  19. Identifying 5-methylcytosine and related modifications in DNA genomes.

    OpenAIRE

    Rein, T.; DePamphilis, M L; Zorbas, H

    1998-01-01

    Intense interest in the biological roles of DNA methylation, particularly in eukaryotes, has produced at least eight different methods for identifying 5-methylcytosine and related modifications in DNA genomes. However, the utility of each method depends not only on its simplicity but on its specificity, resolution, sensitivity and potential artifacts. Since these parameters affect the interpretation of data, they should be considered in any application. Therefore, we have outlined the princip...

  20. Biased distribution of DNA uptake sequences towards genome maintenance genes

    DEFF Research Database (Denmark)

    Davidsen, T.; Rodland, E.A.; Lagesen, K.

    2004-01-01

    Repeated sequence signatures are characteristic features of all genomic DNA. We have made a rigorous search for repeat genomic sequences in the human pathogens Neisseria meningitidis, Neisseria gonorrhoeae and Haemophilus influenzae and found that by far the most frequent 9-10mers residing within...... in these organisms. Pasteurella multocida also displayed high frequencies of a putative DUS identical to that previously identified in H. influenzae and with a skewed distribution towards genome maintenance genes, indicating that this bacterium might be transformation competent under certain conditions....

  1. Diagnosis of canine Echinococcus multilocularis infections by copro-DNA tests: comparison of DNA extraction techniques and evaluation of diagnostic deworming.

    Science.gov (United States)

    Irie, Takao; Ito, Takuya; Kouguchi, Hirokazu; Yamano, Kimiaki; Uraguchi, Kohji; Yagi, Kinpei; Nonaka, Nariaki

    2017-08-01

    The use of copro-DNA detection methods for the diagnosis of canine Echinococcus multilocularis infection was evaluated with a focus on DNA extraction techniques: two commercial kits and a modified alkaline-sodium dodecyl sulfate (SDS) technique. Dog feces (0.2 g) mixed with a protoscolex or with 1 or 10 eggs of E. multilocularis were subjected to DNA detection following extraction by these methods. DNA was extracted from all protoscolex samples by all methods, but success for samples with eggs depended on extraction technique with the modified technique showing success on all samples. Following experimental infection of dogs, copro-DNA was successfully extracted from fecal samples (0.2 g) of dogs in the patent period by all methods. In the prepatent period, PCR testing of feces subsamples (0.2 g) extracted by each technique was positive at a rate of 79.6-94.4%. Extraction by the modified technique with fecal samples of over 1 g showed detection of copro-DNA in all samples in both the patent and prepatent periods, and it produced reproducible detection in the addition recovery test using feces from 72 different domestic dogs. As copro-DNA was detected for at least 1 day following deworming with administration of anthelmintic drugs in experimentally infected dogs, diagnostic deworming might be useful for clinical examination. Using the present detection method can provide quick and accurate diagnosis of canine E. multilocularis infection, which with prompt management and treatment of infected dogs can prevent pet owners from becoming infected and prevent echinococcosis from spreading into non-endemic areas.

  2. Concentrating and labeling genomic DNA in a nanofluidic array.

    Science.gov (United States)

    Marie, Rodolphe; Pedersen, Jonas N; Mir, Kalim U; Bilenberg, Brian; Kristensen, Anders

    2018-01-18

    Nucleotide incorporation by DNA polymerase forms the basis of DNA sequencing-by-synthesis. In current platforms, either the single-stranded DNA or the enzyme is immobilized on a solid surface to locate the incorporation of individual nucleotides in space and/or time. Solid-phase reactions may, however, hinder the polymerase activity. We demonstrate a device and a protocol for the enzymatic labeling of genomic DNA arranged in a dense array of single molecules without attaching the enzyme or the DNA to a surface. DNA molecules accumulate in a dense array of pits embedded within a nanoslit due to entropic trapping. We then perform ϕ29 polymerase extension from single-strand nicks created on the trapped molecules to incorporate fluorescent nucleotides into the DNA. The array of entropic traps can be loaded with λ-DNA molecules to more than 90% of capacity at a flow rate of 10 pL min-1. The final concentration can reach up to 100 μg mL-1, and the DNA is eluted from the array by increasing the flow rate. The device may be an important preparative module for carrying out enzymatic processing on DNA extracted from single-cells in a microfluidic chip.

  3. Identification of differential genes by suppression subtractive hybridization: I. Preparation of subtracted cDNA or genomic DNA library.

    Science.gov (United States)

    Rebrikov, Denis V

    2008-07-01

    INTRODUCTIONSuppression subtractive hybridization (SSH) is one of the most powerful and popular methods for generating subtracted cDNA or genomic DNA libraries. This technique can be used to compare two mRNA populations and obtain cDNAs representing genes that are either overexpressed or exclusively expressed in one population as compared to another. It can also be used for comparison of genomic DNA populations. This protocol describes the preparation of a subtracted cDNA or genomic DNA library, and includes methods for cDNA synthesis, tester and driver DNA digestion, and adapter ligation.

  4. Chromosomal assignment of canine THADA gene to CFA 10q25

    Directory of Open Access Journals (Sweden)

    Dolf Gaudenz

    2008-06-01

    Full Text Available Abstract Background Chromosomal translocations affecting the chromosome 2p21 cluster in a 450 kb breakpoint region are frequently observed in human benign thyroid adenomas. THADA (thyroid adenoma associated was identified as the affected gene within this breakpoint region. In contrast to man tumours of the thyroid gland of dogs (Canis lupus familiaris constitute mainly as follicular cell carcinomas, with malignant thyroid tumours being more frequent than benign thyroid adenomas. In order to elucidate if the THADA gene is also a target of chromosomal rearrangements in thyroid adenomas of the dog we have physically mapped the canine THADA gene to canine chromosome 10. A PCR was established to screen a canine genome library for a BAC clone containing the gene sequence of canine THADA. Further PCR reactions were done using the identified BAC clone as a template in order to verify the corresponding PCR product by sequencing. Canine whole blood was incubated with colcemid in order to arrest the cultured cells in metaphases. The verified BAC DNA was digoxigenin labeled and used as a probe in fluorescence in situ hybridization (FISH. Ten well spread metaphases were examined indicating a signal on canine chromosome 10 on both chromatids. A detailed fine mapping was performed indicating the canine THADA gene locus on the q-arm of chromosome 10. Results The canine THADA gene locus was mapped on chromosome 10q25. Our mapping results obtained in this study following the previously described nomenclature for the canine karyotype. Conclusion We analysed whether the THADA gene locus is a hotspot of canine chromosomal rearrangements in canine neoplastic lesions of the thyroid and in addition might play a role as a candidate gene for a possible malignant transformation of canine thyroid adenomas. Although the available cytogenetic data of canine thyroid adenomas are still insufficient the chromosomal region to which the canine THADA has been mapped seems to be no

  5. cDNA, genomic sequence cloning and overexpression of ribosomal ...

    African Journals Online (AJOL)

    RPS16 of eukaryote is a component of the 40S small ribosomal subunit encoded by RPS16 gene and is also a homolog of prokaryotic RPS9. The cDNA and genomic sequence of RPS16 was cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) using reverse transcription-polymerase chain ...

  6. cDNA, genomic sequence cloning and overexpression of ribosomal ...

    African Journals Online (AJOL)

    PRECIOUS

    2009-11-02

    Nov 2, 2009 ... RPS20 is a component of the 40S small ribosomal subunit encoded by RPS20 gene, which is conserved between eukaryotes, prokaryotes and archaebacteria. The cDNA and the genomic sequence of RPS20 were cloned successfully from the Giant Panda (Ailuropoda melanoleuca) using RT-PCR ...

  7. Genomic DNA isolation from Artemisia species grown in cold desert ...

    African Journals Online (AJOL)

    ... protocol to extract pure genomic DNA from different Artemisia species was tailored. The protocol was based on the CTAB method with slight modifications. In the study, 1.6 M NaCl, 2% cetyltrimethyl ammonium bromide (CTAB), 3% polyvinylpyrrolidone (PVP) and 0.5% β-mercaptoethanol was used in the extraction buffer.

  8. Chromosomal localization of rDNA genes and genomic organization ...

    Indian Academy of Sciences (India)

    Genetica 123, 227–233. Alves-Costa F. A., Wasko A. P., Oliveira C., Foresti F. and Martins. C. 2006 Genomic organization and evolution of the 5S ribosomal. DNA in Tilapiine fishes. Genetica 127, 243–252. Brinn A. M., Porto J. R. and Feldberg E. 2004 Karyological evi- dence for interspecific hybridization between Cichla ...

  9. cDNA, genomic sequence cloning and overexpression of ...

    African Journals Online (AJOL)

    Cytochrome c oxidase (COX) is a component of the mitochondria respiratory chain. COX6b1 is one of the COX small subunits encoded by nuclear genes. In currently study, the cDNA and the genomic sequence of COX6b1 were successfully cloned from the Ailuropoda melanoleuca with the RT-PCR technology and ...

  10. Viral and Cellular Genomes Activate Distinct DNA Damage Responses

    Science.gov (United States)

    Shah, Govind A.; O’Shea, Clodagh C.

    2015-01-01

    Summary In response to cellular genome breaks, MRE11/RAD50/NBS1 (MRN) activates a global ATM DNA damage response (DDR) that prevents cellular replication. Here we show that MRN-ATM also has critical functions in defending the cell against DNA viruses. We reveal temporally distinct responses to adenovirus genomes: a critical MRN-ATM DDR that must be inactivated by E1B-55K/E4-ORF3 viral oncoproteins and a global MRN independent ATM DDR to viral nuclear domains that does not impact viral replication. We show that MRN binds to adenovirus genomes and activates a localized ATM response that specifically prevents viral DNA replication. In contrast to chromosomal breaks, ATM activation is not amplified by H2AX across megabases of chromatin to induce global signaling and replicative arrest. Thus, γH2AX foci discriminate ‘self’ and ‘non-self’ genomes and determine if a localized anti-viral or global ATM response is appropriate. This provides an elegant mechanism to neutralize viral genomes without jeopardizing cellular viability. PMID:26317467

  11. Precision genome engineering with programmable DNA-nicking enzymes

    Science.gov (United States)

    Kim, Eunji; Kim, Sojung; Kim, Duk Hyoung; Choi, Beom-Soon; Choi, Ik-Young; Kim, Jin-Soo

    2012-01-01

    Zinc finger nucleases (ZFNs) are powerful tools of genome engineering but are limited by their inevitable reliance on error-prone nonhomologous end-joining (NHEJ) repair of DNA double-strand breaks (DSBs), which gives rise to randomly generated, unwanted small insertions or deletions (indels) at both on-target and off-target sites. Here, we present programmable DNA-nicking enzymes (nickases) that produce single-strand breaks (SSBs) or nicks, instead of DSBs, which are repaired by error-free homologous recombination (HR) rather than mutagenic NHEJ. Unlike their corresponding nucleases, zinc finger nickases allow site-specific genome modifications only at the on-target site, without the induction of unwanted indels. We propose that programmable nickases will be of broad utility in research, medicine, and biotechnology, enabling precision genome engineering in any cell or organism. PMID:22522391

  12. Resurrection of DNA function in vivo from an extinct genome.

    Science.gov (United States)

    Pask, Andrew J; Behringer, Richard R; Renfree, Marilyn B

    2008-05-21

    There is a burgeoning repository of information available from ancient DNA that can be used to understand how genomes have evolved and to determine the genetic features that defined a particular species. To assess the functional consequences of changes to a genome, a variety of methods are needed to examine extinct DNA function. We isolated a transcriptional enhancer element from the genome of an extinct marsupial, the Tasmanian tiger (Thylacinus cynocephalus or thylacine), obtained from 100 year-old ethanol-fixed tissues from museum collections. We then examined the function of the enhancer in vivo. Using a transgenic approach, it was possible to resurrect DNA function in transgenic mice. The results demonstrate that the thylacine Col2A1 enhancer directed chondrocyte-specific expression in this extinct mammalian species in the same way as its orthologue does in mice. While other studies have examined extinct coding DNA function in vitro, this is the first example of the restoration of extinct non-coding DNA and examination of its function in vivo. Our method using transgenesis can be used to explore the function of regulatory and protein-coding sequences obtained from any extinct species in an in vivo model system, providing important insights into gene evolution and diversity.

  13. Resurrection of DNA function in vivo from an extinct genome.

    Directory of Open Access Journals (Sweden)

    Andrew J Pask

    2008-05-01

    Full Text Available There is a burgeoning repository of information available from ancient DNA that can be used to understand how genomes have evolved and to determine the genetic features that defined a particular species. To assess the functional consequences of changes to a genome, a variety of methods are needed to examine extinct DNA function. We isolated a transcriptional enhancer element from the genome of an extinct marsupial, the Tasmanian tiger (Thylacinus cynocephalus or thylacine, obtained from 100 year-old ethanol-fixed tissues from museum collections. We then examined the function of the enhancer in vivo. Using a transgenic approach, it was possible to resurrect DNA function in transgenic mice. The results demonstrate that the thylacine Col2A1 enhancer directed chondrocyte-specific expression in this extinct mammalian species in the same way as its orthologue does in mice. While other studies have examined extinct coding DNA function in vitro, this is the first example of the restoration of extinct non-coding DNA and examination of its function in vivo. Our method using transgenesis can be used to explore the function of regulatory and protein-coding sequences obtained from any extinct species in an in vivo model system, providing important insights into gene evolution and diversity.

  14. Analysis of human accelerated DNA regions using archaic hominin genomes.

    Directory of Open Access Journals (Sweden)

    Hernán A Burbano

    Full Text Available Several previous comparisons of the human genome with other primate and vertebrate genomes identified genomic regions that are highly conserved in vertebrate evolution but fast-evolving on the human lineage. These human accelerated regions (HARs may be regions of past adaptive evolution in humans. Alternatively, they may be the result of non-adaptive processes, such as biased gene conversion. We captured and sequenced DNA from a collection of previously published HARs using DNA from an Iberian Neandertal. Combining these new data with shotgun sequence from the Neandertal and Denisova draft genomes, we determine at least one archaic hominin allele for 84% of all positions within HARs. We find that 8% of HAR substitutions are not observed in the archaic hominins and are thus recent in the sense that the derived allele had not come to fixation in the common ancestor of modern humans and archaic hominins. Further, we find that recent substitutions in HARs tend to have come to fixation faster than substitutions elsewhere in the genome and that substitutions in HARs tend to cluster in time, consistent with an episodic rather than a clock-like process underlying HAR evolution. Our catalog of sequence changes in HARs will help prioritize them for functional studies of genomic elements potentially responsible for modern human adaptations.

  15. Analysis of Human Accelerated DNA Regions Using Archaic Hominin Genomes

    Science.gov (United States)

    Burbano, Hernán A.; Green, Richard E.; Maricic, Tomislav; Lalueza-Fox, Carles; de la Rasilla, Marco; Rosas, Antonio; Kelso, Janet; Pollard, Katherine S.; Lachmann, Michael; Pääbo, Svante

    2012-01-01

    Several previous comparisons of the human genome with other primate and vertebrate genomes identified genomic regions that are highly conserved in vertebrate evolution but fast-evolving on the human lineage. These human accelerated regions (HARs) may be regions of past adaptive evolution in humans. Alternatively, they may be the result of non-adaptive processes, such as biased gene conversion. We captured and sequenced DNA from a collection of previously published HARs using DNA from an Iberian Neandertal. Combining these new data with shotgun sequence from the Neandertal and Denisova draft genomes, we determine at least one archaic hominin allele for 84% of all positions within HARs. We find that 8% of HAR substitutions are not observed in the archaic hominins and are thus recent in the sense that the derived allele had not come to fixation in the common ancestor of modern humans and archaic hominins. Further, we find that recent substitutions in HARs tend to have come to fixation faster than substitutions elsewhere in the genome and that substitutions in HARs tend to cluster in time, consistent with an episodic rather than a clock-like process underlying HAR evolution. Our catalog of sequence changes in HARs will help prioritize them for functional studies of genomic elements potentially responsible for modern human adaptations. PMID:22412940

  16. Multiplexed DNA sequence capture of mitochondrial genomes using PCR products.

    Directory of Open Access Journals (Sweden)

    Tomislav Maricic

    Full Text Available BACKGROUND: To utilize the power of high-throughput sequencers, target enrichment methods have been developed. The majority of these require reagents and equipment that are only available from commercial vendors and are not suitable for the targets that are a few kilobases in length. METHODOLOGY/PRINCIPAL FINDINGS: We describe a novel and economical method in which custom made long-range PCR products are used to capture complete human mitochondrial genomes from complex DNA mixtures. We use the method to capture 46 complete mitochondrial genomes in parallel and we sequence them on a single lane of an Illumina GA(II instrument. CONCLUSIONS/SIGNIFICANCE: This method is economical and simple and particularly suitable for targets that can be amplified by PCR and do not contain highly repetitive sequences such as mtDNA. It has applications in population genetics and forensics, as well as studies of ancient DNA.

  17. Criminal genomic pragmatism: prisoners' representations of DNA technology and biosecurity.

    Science.gov (United States)

    Machado, Helena; Silva, Susana

    2012-01-01

    Within the context of the use of DNA technology in crime investigation, biosecurity is perceived by different stakeholders according to their particular rationalities and interests. Very little is known about prisoners' perceptions and assessments of the uses of DNA technology in solving crime. To propose a conceptual model that serves to analyse and interpret prisoners' representations of DNA technology and biosecurity. A qualitative study using an interpretative approach based on 31 semi-structured tape-recorded interviews was carried out between May and September 2009, involving male inmates in three prisons located in the north of Portugal. The content analysis focused on the following topics: the meanings attributed to DNA and assessments of the risks and benefits of the uses of DNA technology and databasing in forensic applications. DNA was described as a record of identity, an exceptional material, and a powerful biometric identifier. The interviewees believed that DNA can be planted to incriminate suspects. Convicted offenders argued for the need to extend the criteria for the inclusion of DNA profiles in forensic databases and to restrict the removal of profiles. The conceptual model entitled criminal genomic pragmatism allows for an understanding of the views of prison inmates regarding DNA technology and biosecurity.

  18. Criminal Genomic Pragmatism: Prisoners' Representations of DNA Technology and Biosecurity

    Directory of Open Access Journals (Sweden)

    Helena Machado

    2012-01-01

    Full Text Available Background. Within the context of the use of DNA technology in crime investigation, biosecurity is perceived by different stakeholders according to their particular rationalities and interests. Very little is known about prisoners’ perceptions and assessments of the uses of DNA technology in solving crime. Aim. To propose a conceptual model that serves to analyse and interpret prisoners’ representations of DNA technology and biosecurity. Methods. A qualitative study using an interpretative approach based on 31 semi-structured tape-recorded interviews was carried out between May and September 2009, involving male inmates in three prisons located in the north of Portugal. The content analysis focused on the following topics: the meanings attributed to DNA and assessments of the risks and benefits of the uses of DNA technology and databasing in forensic applications. Results. DNA was described as a record of identity, an exceptional material, and a powerful biometric identifier. The interviewees believed that DNA can be planted to incriminate suspects. Convicted offenders argued for the need to extend the criteria for the inclusion of DNA profiles in forensic databases and to restrict the removal of profiles. Conclusions. The conceptual model entitled criminal genomic pragmatism allows for an understanding of the views of prison inmates regarding DNA technology and biosecurity.

  19. Base-Resolution Analysis of Cisplatin–DNA Adducts at the Genome Scale

    OpenAIRE

    Shu, Xiaoting; Xiong, Xushen; Song, Jinghui; He, Chuan; Yi, Chengqi

    2016-01-01

    Cisplatin, one of the most widely used anticancer drugs, crosslinks DNA and ultimately induces cell death. However, the genomic pattern of cisplatin–DNA adducts has remained unknown owing to the lack of a reliable and sensitive genome-wide method. Herein we present “cisplatin-seq” to identify genome-wide cisplatin crosslinking sites at base resolution. Cisplatin-seq reveals that mitochondrial DNA is a preferred target of cisplatin. For nuclear genomes, cisplatin–DNA adducts are enriched withi...

  20. Canine distemper virus DNA vaccination of mink can overcome interference by maternal antibodies

    DEFF Research Database (Denmark)

    Jensen, Trine Hammer; Nielsen, Line; Aasted, Bent

    2015-01-01

    Canine distemper virus (CDV) is highly contagious and can cause severe disease against which conventional live vaccines are ineffective in the presence of maternal antibodies. Vaccination in the presences of maternal antibodies was challenged by vaccination of 5 days old and 3 weeks old mink kits...... virus specific immune response in neonatal mink and protection against virulent CDV exposure later in life....

  1. Canine tumor cross-species genomics uncovers targets linked to osteosarcoma progression

    Directory of Open Access Journals (Sweden)

    Triche Timothy

    2009-12-01

    Full Text Available Abstract Background Pulmonary metastasis continues to be the most common cause of death in osteosarcoma. Indeed, the 5-year survival for newly diagnosed osteosarcoma patients has not significantly changed in over 20 years. Further understanding of the mechanisms of metastasis and resistance for this aggressive pediatric cancer is necessary. Pet dogs naturally develop osteosarcoma providing a novel opportunity to model metastasis development and progression. Given the accelerated biology of canine osteosarcoma, we hypothesized that a direct comparison of canine and pediatric osteosarcoma expression profiles may help identify novel metastasis-associated tumor targets that have been missed through the study of the human cancer alone. Results Using parallel oligonucleotide array platforms, shared orthologues between species were identified and normalized. The osteosarcoma expression signatures could not distinguish the canine and human diseases by hierarchical clustering. Cross-species target mining identified two genes, interleukin-8 (IL-8 and solute carrier family 1 (glial high affinity glutamate transporter, member 3 (SLC1A3, which were uniformly expressed in dog but not in all pediatric osteosarcoma patient samples. Expression of these genes in an independent population of pediatric osteosarcoma patients was associated with poor outcome (p = 0.020 and p = 0.026, respectively. Validation of IL-8 and SLC1A3 protein expression in pediatric osteosarcoma tissues further supported the potential value of these novel targets. Ongoing evaluation will validate the biological significance of these targets and their associated pathways. Conclusions Collectively, these data support the strong similarities between human and canine osteosarcoma and underline the opportunities provided by a comparative oncology approach as a means to improve our understanding of cancer biology and therapies.

  2. Design of different strategies of multivalent DNA-based vaccination against rabies and canine distemper in mice and dogs

    Directory of Open Access Journals (Sweden)

    Touihri Leila

    2012-12-01

    Full Text Available Abstract Background During the vaccination campaigns, puppies younger than 3 months old are not targeted and remain unvaccinated for at least the first year of their lives. Almost half of the reported rabid dogs are 6 months or younger. Hence, we should recommend the vaccination against rabies of young puppies. Unfortunately, owing to the exposure of puppies to infections with either canine parvovirus (CPV or distemper virus (CDV after the intervention of the vaccinators, owners are reluctant to vaccinate puppies against rabies. Therefore, it is necessary to include the CPV and CDV valences in the vaccine against rabies. Multivalent DNA-based vaccination in dogs, including rabies and distemper valences, could help in raising vaccine coverage. Methods We have designed monovalent and multivalent DNA-based vaccine candidates for in vitro and in vivo assays. These plasmids encode to the rabies virus glycoprotein and/or the canine distemper virus hemagglutinin. The first strategy of multivalent DNA-based vaccination is by mixing plasmids encoding to a single antigen each. The second is by simply fusing the genes of the antigens together. The third is by adding the foot and mouth disease virus (FMDV 2A oligopeptide gene into the antigen genes. The last strategy is by the design and use of a bicistronic plasmid with an “Internal Ribosome Entry Site” (IRES domain. Results The monovalent construct against canine distemper was efficiently validated by inducing higher humoral immune responses compared to cell-culture-derived vaccine both in mice and dogs. All multivalent plasmids efficiently expressed both valences after in vitro transfection of BHK-21 cells. In BALB/c mice, the bicistronic IRES-dependant construct was the most efficient inducer of virus-neutralizing antibodies against both valences. It was able to induce better humoral immune responses compared to the administration of either cell-culture-derived vaccines or monovalent plasmids. The

  3. Detection of Streptococcus mutans Genomic DNA in Human DNA Samples Extracted from Saliva and Blood

    Science.gov (United States)

    Vieira, Alexandre R.; Deeley, Kathleen B.; Callahan, Nicholas F.; Noel, Jacqueline B.; Anjomshoaa, Ida; Carricato, Wendy M.; Schulhof, Louise P.; DeSensi, Rebecca S.; Gandhi, Pooja; Resick, Judith M.; Brandon, Carla A.; Rozhon, Christopher; Patir, Asli; Yildirim, Mine; Poletta, Fernando A.; Mereb, Juan C.; Letra, Ariadne; Menezes, Renato; Wendell, Steven; Lopez-Camelo, Jorge S.; Castilla, Eduardo E.; Orioli, Iêda M.; Seymen, Figen; Weyant, Robert J.; Crout, Richard; McNeil, Daniel W.; Modesto, Adriana; Marazita, Mary L.

    2011-01-01

    Caries is a multifactorial disease, and studies aiming to unravel the factors modulating its etiology must consider all known predisposing factors. One major factor is bacterial colonization, and Streptococcus mutans is the main microorganism associated with the initiation of the disease. In our studies, we have access to DNA samples extracted from human saliva and blood. In this report, we tested a real-time PCR assay developed to detect copies of genomic DNA from Streptococcus mutans in 1,424 DNA samples from humans. Our results suggest that we can determine the presence of genomic DNA copies of Streptococcus mutans in both DNA samples from caries-free and caries-affected individuals. However, we were not able to detect the presence of genomic DNA copies of Streptococcus mutans in any DNA samples extracted from peripheral blood, which suggests the assay may not be sensitive enough for this goal. Values of the threshold cycle of the real-time PCR reaction correlate with higher levels of caries experience in children, but this correlation could not be detected for adults. PMID:21731912

  4. Ancient DNA: genomic amplification of Roman and medieval bovine bones

    Directory of Open Access Journals (Sweden)

    A. Valentini

    2010-04-01

    Full Text Available Cattle remains (bones and teeth of both roman and medieval age were collected in the archaeological site of Ferento (Viterbo, Italy with the aim of extracting and characterising nucleic acids. Procedures to minimize contamination with modern DNA and to help ancient DNA (aDNA preservation of the archaeological remains were adopted. Different techniques to extract aDNA (like Phenol/chloroform extraction from bovine bones were tested to identify the method that applies to the peculiar characteristics of the study site. Currently, aDNA investigation is mainly based on mtDNA, due to the ease of amplification of the small and high-copied genome and to its usefulness in evolutionary studies. Preliminary amplification of both mitochondrial and nuclear aDNA fragments from samples of Roman and medieval animals were performed and partial specific sequences of mitochondrial D-loop as well as of nuclear genes were obtained. The innovative amplification of nuclear aDNA could enable the analysis of genes involved in specific animal traits, giving insights of ancient economic and cultural uses, as well as providing information on the origin of modern livestock population.

  5. The contribution of co-transcriptional RNA:DNA hybrid structures to DNA damage and genome instability

    Science.gov (United States)

    Hamperl, Stephan; Cimprich, Karlene A.

    2014-01-01

    Accurate DNA replication and DNA repair are crucial for the maintenance of genome stability, and it is generally accepted that failure of these processes is a major source of DNA damage in cells. Intriguingly, recent evidence suggests that DNA damage is more likely to occur at genomic loci with high transcriptional activity. Furthermore, loss of certain RNA processing factors in eukaryotic cells is associated with increased formation of co-transcriptional RNA:DNA hybrid structures known as R-loops, resulting in double-strand breaks (DSBs) and DNA damage. However, the molecular mechanisms by which R-loop structures ultimately lead to DNA breaks and genome instability is not well understood. In this review, we summarize the current knowledge about the formation, recognition and processing of RNA:DNA hybrids, and discuss possible mechanisms by which these structures contribute to DNA damage and genome instability in the cell. PMID:24746923

  6. The contribution of co-transcriptional RNA:DNA hybrid structures to DNA damage and genome instability.

    Science.gov (United States)

    Hamperl, Stephan; Cimprich, Karlene A

    2014-07-01

    Accurate DNA replication and DNA repair are crucial for the maintenance of genome stability, and it is generally accepted that failure of these processes is a major source of DNA damage in cells. Intriguingly, recent evidence suggests that DNA damage is more likely to occur at genomic loci with high transcriptional activity. Furthermore, loss of certain RNA processing factors in eukaryotic cells is associated with increased formation of co-transcriptional RNA:DNA hybrid structures known as R-loops, resulting in double-strand breaks (DSBs) and DNA damage. However, the molecular mechanisms by which R-loop structures ultimately lead to DNA breaks and genome instability is not well understood. In this review, we summarize the current knowledge about the formation, recognition and processing of RNA:DNA hybrids, and discuss possible mechanisms by which these structures contribute to DNA damage and genome instability in the cell. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Rapid genomic DNA changes in allotetraploid fish hybrids.

    Science.gov (United States)

    Wang, J; Ye, L H; Liu, Q Z; Peng, L Y; Liu, W; Yi, X G; Wang, Y D; Xiao, J; Xu, K; Hu, F Z; Ren, L; Tao, M; Zhang, C; Liu, Y; Hong, Y H; Liu, S J

    2015-06-01

    Rapid genomic change has been demonstrated in several allopolyploid plant systems; however, few studies focused on animals. We addressed this issue using an allotetraploid lineage (4nAT) of freshwater fish originally derived from the interspecific hybridization of red crucian carp (Carassius auratus red var., ♀, 2n=100) × common carp (Cyprinus carpio L., ♂, 2n=100). We constructed a bacterial artificial chromosome (BAC) library from allotetraploid hybrids in the 20th generation (F20) and sequenced 14 BAC clones representing a total of 592.126 kb, identified 11 functional genes and estimated the guanine-cytosine content (37.10%) and the proportion of repetitive elements (17.46%). The analysis of intron evolution using nine orthologous genes across a number of selected fish species detected a gain of 39 introns and a loss of 30 introns in the 4nAT lineage. A comparative study based on seven functional genes among 4nAT, diploid F1 hybrids (2nF1) (first generation of hybrids) and their original parents revealed that both hybrid types (2nF1 and 4nAT) not only inherited genomic DNA from their parents, but also demonstrated rapid genomic DNA changes (homoeologous recombination, parental DNA fragments loss and formation of novel genes). However, 4nAT presented more genomic variations compared with their parents than 2nF1. Interestingly, novel gene fragments were found for the iqca1 gene in both hybrid types. This study provided a preliminary genomic characterization of allotetraploid F20 hybrids and revealed evolutionary and functional genomic significance of allopolyploid animals.

  8. A Novel Method of Genomic DNA Extraction for Cactaceae

    Directory of Open Access Journals (Sweden)

    Shannon D. Fehlberg

    2013-03-01

    Full Text Available Premise of the study: Genetic studies of Cactaceae can at times be impeded by difficult sampling logistics and/or high mucilage content in tissues. Simplifying sampling and DNA isolation through the use of cactus spines has not previously been investigated. Methods and Results: Several protocols for extracting DNA from spines were tested and modified to maximize yield, amplification, and sequencing. Sampling of and extraction from spines resulted in a simplified protocol overall and complete avoidance of mucilage as compared to typical tissue extractions. Sequences from one nuclear and three plastid regions were obtained across eight genera and 20 species of cacti using DNA extracted from spines. Conclusions: Genomic DNA useful for amplification and sequencing can be obtained from cactus spines. The protocols described here are valuable for any cactus species, but are particularly useful for investigators interested in sampling living collections, extensive field sampling, and/or conservation genetic studies.

  9. A novel method of genomic DNA extraction for Cactaceae1

    Science.gov (United States)

    Fehlberg, Shannon D.; Allen, Jessica M.; Church, Kathleen

    2013-01-01

    • Premise of the study: Genetic studies of Cactaceae can at times be impeded by difficult sampling logistics and/or high mucilage content in tissues. Simplifying sampling and DNA isolation through the use of cactus spines has not previously been investigated. • Methods and Results: Several protocols for extracting DNA from spines were tested and modified to maximize yield, amplification, and sequencing. Sampling of and extraction from spines resulted in a simplified protocol overall and complete avoidance of mucilage as compared to typical tissue extractions. Sequences from one nuclear and three plastid regions were obtained across eight genera and 20 species of cacti using DNA extracted from spines. • Conclusions: Genomic DNA useful for amplification and sequencing can be obtained from cactus spines. The protocols described here are valuable for any cactus species, but are particularly useful for investigators interested in sampling living collections, extensive field sampling, and/or conservation genetic studies. PMID:25202521

  10. Effect of nickel chloride on Arabidopsis genomic DNA and methylation of 18S rDNA

    OpenAIRE

    Li, Zhongai; Chen, Xin; Li, Suoping; Wang, Zicheng

    2015-01-01

    Background: In recent years, nickel (Ni) has been widely applied in industrial and agricultural production and has become a kind of environmental pollution. In this study, the effect of nickel chloride (NiCl2) with different concentrations on Arabidopsis genomic stability and DNA methylation has been demonstrated. The nucleolus variation and 18S rDNA methylation after NiCl2 treatment have been analyzed. Results: The results are as follows: (1) The NiCl2 could result in heritable genomic me...

  11. Characterization of noncoding regulatory DNA in the human genome.

    Science.gov (United States)

    Elkon, Ran; Agami, Reuven

    2017-08-08

    Genetic variants associated with common diseases are usually located in noncoding parts of the human genome. Delineation of the full repertoire of functional noncoding elements, together with efficient methods for probing their biological roles, is therefore of crucial importance. Over the past decade, DNA accessibility and various epigenetic modifications have been associated with regulatory functions. Mapping these features across the genome has enabled researchers to begin to document the full complement of putative regulatory elements. High-throughput reporter assays to probe the functions of regulatory regions have also been developed but these methods separate putative regulatory elements from the chromosome so that any effects of chromatin context and long-range regulatory interactions are lost. Definitive assignment of function(s) to putative cis-regulatory elements requires perturbation of these elements. Genome-editing technologies are now transforming our ability to perturb regulatory elements across entire genomes. Interpretation of high-throughput genetic screens that incorporate genome editors might enable the construction of an unbiased map of functional noncoding elements in the human genome.

  12. Studies on the effects of persistent RNA priming on DNA replication and genomic stability

    OpenAIRE

    Stuckey, Ruth

    2014-01-01

    [EN]: DNA replication and transcription take place on the same DNA template, and the correct interplay between these processes ensures faithful genome duplication. DNA replication must be highly coordinated with other cell cycle events, such as segregation of fully replicated DNA in order to maintain genomic integrity. Transcription generates RNA:DNA hybrids, transient intermediate structures that are degraded by the ribonuclease H (RNaseH) class of enzymes. RNA:DNA hybrids can form R-loops, ...

  13. Retrotransposon silencing by DNA methylation can drive mammalian genomic imprinting.

    Directory of Open Access Journals (Sweden)

    Shunsuke Suzuki

    2007-04-01

    Full Text Available Among mammals, only eutherians and marsupials are viviparous and have genomic imprinting that leads to parent-of-origin-specific differential gene expression. We used comparative analysis to investigate the origin of genomic imprinting in mammals. PEG10 (paternally expressed 10 is a retrotransposon-derived imprinted gene that has an essential role for the formation of the placenta of the mouse. Here, we show that an orthologue of PEG10 exists in another therian mammal, the marsupial tammar wallaby (Macropus eugenii, but not in a prototherian mammal, the egg-laying platypus (Ornithorhynchus anatinus, suggesting its close relationship to the origin of placentation in therian mammals. We have discovered a hitherto missing link of the imprinting mechanism between eutherians and marsupials because tammar PEG10 is the first example of a differentially methylated region (DMR associated with genomic imprinting in marsupials. Surprisingly, the marsupial DMR was strictly limited to the 5' region of PEG10, unlike the eutherian DMR, which covers the promoter regions of both PEG10 and the adjacent imprinted gene SGCE. These results not only demonstrate a common origin of the DMR-associated imprinting mechanism in therian mammals but provide the first demonstration that DMR-associated genomic imprinting in eutherians can originate from the repression of exogenous DNA sequences and/or retrotransposons by DNA methylation.

  14. Congruent evolution of different classes of non-coding DNA in prokaryotic genomes

    OpenAIRE

    Rogozin, Igor B.; Makarova, Kira S.; Natale, Darren A.; Spiridonov, Alexey N.; Tatusov, Roman L.; Wolf, Yuri I.; Yin, Jodie; Koonin, Eugene V.

    2002-01-01

    Prokaryotic genomes are considered to be ‘wall-to-wall’ genomes, which consist largely of genes for proteins and structural RNAs, with only a small fraction of the genomic DNA allotted to intergenic regions, which are thought to typically contain regulatory signals. The majority of bacterial and archaeal genomes contain 6–14% non-coding DNA. Significant positive correlations were detected between the fraction of non-coding DNA and inter- and intra-operonic distances, suggesting that different...

  15. Amplification of a Zygosaccharomyces bailii DNA segment in wine yeast genomes by extrachromosomal circular DNA formation.

    Directory of Open Access Journals (Sweden)

    Virginie Galeote

    Full Text Available We recently described the presence of large chromosomal segments resulting from independent horizontal gene transfer (HGT events in the genome of Saccharomyces cerevisiae strains, mostly of wine origin. We report here evidence for the amplification of one of these segments, a 17 kb DNA segment from Zygosaccharomyces bailii, in the genome of S. cerevisiae strains. The copy number, organization and location of this region differ considerably between strains, indicating that the insertions are independent and that they are post-HGT events. We identified eight different forms in 28 S. cerevisiae strains, mostly of wine origin, with up to four different copies in a single strain. The organization of these forms and the identification of an autonomously replicating sequence functional in S. cerevisiae, strongly suggest that an extrachromosomal circular DNA (eccDNA molecule serves as an intermediate in the amplification of the Z. bailii region in yeast genomes. We found little or no sequence similarity at the breakpoint regions, suggesting that the insertions may be mediated by nonhomologous recombination. The diversity between these regions in S. cerevisiae represents roughly one third the divergence among the genomes of wine strains, which confirms the recent origin of this event, posterior to the start of wine strain expansion. This is the first report of a circle-based mechanism for the expansion of a DNA segment, mediated by nonhomologous recombination, in natural yeast populations.

  16. Complete mitochondrial DNA genome of Pseudobagrus truncatus (Siluriformes: Bagridae).

    Science.gov (United States)

    Liang, Hong-wei; Meng, Yan; Li, Zhong; Zhang, Yan; Zou, Gui-wei

    2014-06-01

    In this study, the complete mitochondrial DNA (mtDNA) sequence of Pseudobagrus truncatus (Siluriformes: Bagridae) was determined. The complete mtDNA genome sequence of P. truncatus is 16,533 bp in size. It consists of 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and one non-coding control region. The gene order and genes were the same as that found in other previously reported catfishes. The overall-based composition was 31.6% A, 26.7% T, 14.9% G and 26.8% C, with a high A + T content (58.3%). This complete mitogenome of P. truncatus provides a basic data for studies on species identification, molecular systematics and conservation genetics.

  17. [Genomic profiling: from molecular cytogenetics to DNA arrays].

    Science.gov (United States)

    Theillet, C; Orsetti, B; Redon, R; Manoir, S D

    2001-03-01

    Genetic instability results, in a large majority of solid tumors, in deep chromosomal rearrangements. However, because chromosomal instability produces highly complex caryotypes, rarely showing stereotypic aberrations, it has not been possible to characterize solid cancers according to specific patterns of chromosomal rearrangements. This contrasts with the situation in hematological malignancies, where cytogenetics has allowed to lay out the basis of a renewed classification. New insights have been brought by the development of comparative genomic hybridization (CGH). This molecular cytogenetics approach was originally devised to detect regions in the genome of tumor cells undergoing quantitative changes, i.e. gains or losses of copy numbers. The large body of studies based on CGH show that solid tumors undergo frequent gains and losses and that every chromosomes show at least one region of anomaly. Furthermore, different tumor types present distinct CGH patterns of gains and losses. These observations favor the idea that it may be possible to type human solid cancers according to their patterns of genomic aberrations. However, despite the fact that a number of CGH based studies present data suggesting that different tumor types or cancers at different stages of evolution show distinct patterns of gains and losses, it has proven difficult to be conclusive. This can be mainly attributed to the lack of spatial resolution of CGH. Indeed, CGH uses metaphase chromosomes as hybridization targets and therefore its resolution is at the level of chromosomal banding. The recent adaptation of DNA array technology to CGH will allow to pass this limitation. In DNA array based CGH (array-CGH) metaphase chromosomes have been replaced by spots of cloned DNA. These DNA clones may either be genomic (BACs, YACs or cosmids) or coding (cDNAs). The resolution of array-CGH is therefore determined by the size of the cloned DNA insert (100 Kb for BACs, 1-2 kb for cDNAs). Data

  18. Effects of pretreatment on the denaturation and fragmentation of genomic DNA for DNA hybridization.

    Science.gov (United States)

    Wang, Xiaofang; Son, Ahjeong

    2013-12-01

    DNA hybridization is an important step for a number of bioassays such as fluorescence in situ hybridization, microarrays, as well as the NanoGene assay. Denaturation and fragmentation of genomic DNA are two critical pretreatments for DNA hybridization. However, no thorough and systematic characterization on denaturation and fragmentation has been carried out for the NanoGene assay so far. In this study, we investigated the denaturation and fragmentation of the bacterial gDNA with physical treatments (i.e., heating and sonication) and chemical treatments (i.e., dimethyl sulfoxide). First of all, a simple approach for indicating the denaturation fraction was developed based on the absorbance difference (i.e., hyperchromic effect) between the double-stranded DNA and single-stranded DNA fragments. Then the denaturation capabilities of the treatments to the gDNA were elucidated, followed by the examination of the possible renaturation over time. The fragmentation of the gDNA by each treatment was also investigated. Based on denaturation efficiency, minimum renaturation tendency, and fragmentation, the sonication method was found to be the best among the six methods. We further demonstrated that the sonication method produced the best result among the treatments examined for the DNA hybridization in the NanoGene assay.

  19. Beyond the dna: a prototype for functional genomics

    Energy Technology Data Exchange (ETDEWEB)

    Albala, J

    2000-03-02

    A prototype oligonucleotide ''functional chip'' has been developed to screen novel DNA repair proteins for their ability to bind or alter different forms of DNA. This chip has been developed as a functional genomics screen for analysis of protein-DNA interactions for novel proteins identified from the Human Genome Project The process of novel gene identification that has ensued as a consequence of available sequence information is remarkable. The challenge how lies in determining the function of newly identified gene products in a time-and cost-effective high-throughput manner. The functional chip is generated by the robotic application of DNA spotted in a microarray format onto a glass slide. Individual proteins are then analyzed against the different form of DNA bound to the slide. Several prototype functional chips were designed to contain various DNA fragments tethered to a glass slide for analysis of protein-DNA binding or enzymatic activity of known proteins. The technology has been developed to screen novel, putative DNA repair proteins for their ability to bind various types of DNA alone and in concert with protein partners. An additional scheme has been devised to screen putative repair enzymes for their ability to process different types of DNA molecules. Current methods to analyze gene expression primarily utilize either of two technologies. The oligonucleotide chip, pioneered by Fodor and co-workers and Affymetrix, Inc., consists of greater than 64,000 oligonucleotides attached in situ to a glass support. The oligonucleotide chip has been used primarily to identify specific mutations in a given gene by hybridization against a fluorescently-labeled substrate. The second method is the microarray, whereby DNA targets are systematically arranged on a glass slide and then hybridized with fluorescently-labeled complex targets for gene expression analysis (Jordan, 1998). By this technique, a large amount of information can be obtained

  20. Legume genomics: understanding biology through DNA and RNA sequencing

    Science.gov (United States)

    O'Rourke, Jamie A.; Bolon, Yung-Tsi; Bucciarelli, Bruna; Vance, Carroll P.

    2014-01-01

    Background The legume family (Leguminosae) consists of approx. 17 000 species. A few of these species, including, but not limited to, Phaseolus vulgaris, Cicer arietinum and Cajanus cajan, are important dietary components, providing protein for approx. 300 million people worldwide. Additional species, including soybean (Glycine max) and alfalfa (Medicago sativa), are important crops utilized mainly in animal feed. In addition, legumes are important contributors to biological nitrogen, forming symbiotic relationships with rhizobia to fix atmospheric N2 and providing up to 30 % of available nitrogen for the next season of crops. The application of high-throughput genomic technologies including genome sequencing projects, genome re-sequencing (DNA-seq) and transcriptome sequencing (RNA-seq) by the legume research community has provided major insights into genome evolution, genomic architecture and domestication. Scope and Conclusions This review presents an overview of the current state of legume genomics and explores the role that next-generation sequencing technologies play in advancing legume genomics. The adoption of next-generation sequencing and implementation of associated bioinformatic tools has allowed researchers to turn each species of interest into their own model organism. To illustrate the power of next-generation sequencing, an in-depth overview of the transcriptomes of both soybean and white lupin (Lupinus albus) is provided. The soybean transcriptome focuses on analysing seed development in two near-isogenic lines, examining the role of transporters, oil biosynthesis and nitrogen utilization. The white lupin transcriptome analysis examines how phosphate deficiency alters gene expression patterns, inducing the formation of cluster roots. Such studies illustrate the power of next-generation sequencing and bioinformatic analyses in elucidating the gene networks underlying biological processes. PMID:24769535

  1. Mobile small RNAs regulate genome-wide DNA methylation.

    Science.gov (United States)

    Lewsey, Mathew G; Hardcastle, Thomas J; Melnyk, Charles W; Molnar, Attila; Valli, Adrián; Urich, Mark A; Nery, Joseph R; Baulcombe, David C; Ecker, Joseph R

    2016-02-09

    RNA silencing at the transcriptional and posttranscriptional levels regulates endogenous gene expression, controls invading transposable elements (TEs), and protects the cell against viruses. Key components of the mechanism are small RNAs (sRNAs) of 21-24 nt that guide the silencing machinery to their nucleic acid targets in a nucleotide sequence-specific manner. Transcriptional gene silencing is associated with 24-nt sRNAs and RNA-directed DNA methylation (RdDM) at cytosine residues in three DNA sequence contexts (CG, CHG, and CHH). We previously demonstrated that 24-nt sRNAs are mobile from shoot to root in Arabidopsis thaliana and confirmed that they mediate DNA methylation at three sites in recipient cells. In this study, we extend this finding by demonstrating that RdDM of thousands of loci in root tissues is dependent upon mobile sRNAs from the shoot and that mobile sRNA-dependent DNA methylation occurs predominantly in non-CG contexts. Mobile sRNA-dependent non-CG methylation is largely dependent on the DOMAINS REARRANGED METHYLTRANSFERASES 1/2 (DRM1/DRM2) RdDM pathway but is independent of the CHROMOMETHYLASE (CMT)2/3 DNA methyltransferases. Specific superfamilies of TEs, including those typically found in gene-rich euchromatic regions, lose DNA methylation in a mutant lacking 22- to 24-nt sRNAs (dicer-like 2, 3, 4 triple mutant). Transcriptome analyses identified a small number of genes whose expression in roots is associated with mobile sRNAs and connected to DNA methylation directly or indirectly. Finally, we demonstrate that sRNAs from shoots of one accession move across a graft union and target DNA methylation de novo at normally unmethylated sites in the genomes of root cells from a different accession.

  2. The relationship between DNA replication and human genome organization.

    Science.gov (United States)

    Necsulea, Anamaria; Guillet, Claire; Cadoret, Jean-Charles; Prioleau, Marie-Noëlle; Duret, Laurent

    2009-04-01

    Assessment of the impact of DNA replication on genome architecture in Eukaryotes has long been hampered by the scarcity of experimental data. Recent work, relying on computational predictions of origins of replication, suggested that replication might be a major determinant of gene organization in human (Huvet et al. 2007. Human gene organization driven by the coordination of replication and transcription. Genome Res. 17:1278-1285). Here, we address this question by analyzing the first large-scale data set of experimentally determined origins of replication in human: 283 origins identified in HeLa cells, in 1% of the genome covered by ENCODE regions (Cadoret et al. 2008. Genome-wide studies highlight indirect links between human replication origins and gene regulation. Proc Natl Acad Sci USA. 105:15837-15842). We show that origins of replication are not randomly distributed as they display significant overlap with promoter regions and CpG islands. The hypothesis of a selective pressure to avoid frontal collisions between replication and transcription polymerases is not supported by experimental data as we find no evidence for gene orientation bias in the proximity of origins of replication. The lack of a significant orientation bias remains manifest even when considering only genes expressed at a high rate, or in a wide number of tissues, and is not affected by the regional replication timing. Gene expression breadth does not appear to be correlated with the distance from the origins of replication. We conclude that the impact of DNA replication on human genome organization is considerably weaker than previously proposed.

  3. Comparison of three genomic DNA extraction methods to obtain high DNA quality from maize.

    Science.gov (United States)

    Abdel-Latif, Amani; Osman, Gamal

    2017-01-01

    The world's top three cereals, based on their monetary value, are rice, wheat, and corn. In cereal crops, DNA extraction is difficult owing to rigid non-cellulose components in the cell wall of leaves and high starch and protein content in grains. The advanced techniques in molecular biology require pure and quick extraction of DNA. The majority of existing DNA extraction methods rely on long incubation and multiple precipitations or commercially available kits to produce contaminant-free high molecular weight DNA. In this study, we compared three different methods used for the isolation of high-quality genomic DNA from the grains of cereal crop, Zea mays, with minor modifications. The DNA from the grains of two maize hybrids, M10 and M321, was extracted using extraction methods DNeasy Qiagen Plant Mini Kit, CTAB-method (with/without 1% PVP) and modified Mericon extraction. Genes coding for 45S ribosomal RNA are organized in tandem arrays of up to several thousand copies and contain codes for 18S, 5.8S and 26S rRNA units separated by internal transcribed spacers ITS1 and ITS2. While the rRNA units are evolutionary conserved, ITS regions show high level of interspecific divergence and have been used frequently in genetic diversity and phylogenetic studies. In this study, the genomic DNA was then amplified with PCR using primers specific for ITS gene. PCR products were then visualized on agarose gel. The modified Mericon extraction method was found to be the most efficient DNA extraction method, capable to provide high DNA yields with better quality, affordable cost and less time.

  4. Whole-genome amplified DNA from stored dried blood spots is reliable in high resolution melting curve and sequencing analysis

    DEFF Research Database (Denmark)

    Winkel, Bo G; Hollegaard, Mads Vilhelm; Olesen, Morten S

    2011-01-01

    The use of dried blood spots (DBS) samples in genomic workup has been limited by the relative low amounts of genomic DNA (gDNA) they contain. It remains to be proven that whole genome amplified DNA (wgaDNA) from stored DBS samples, constitutes a reliable alternative to gDNA.We wanted to compare...

  5. Isolation and analysis of high quality nuclear DNA with reduced organellar DNA for plant genome sequencing and resequencing.

    Science.gov (United States)

    Lutz, Kerry A; Wang, Wenqin; Zdepski, Anna; Michael, Todd P

    2011-05-20

    High throughput sequencing (HTS) technologies have revolutionized the field of genomics by drastically reducing the cost of sequencing, making it feasible for individual labs to sequence or resequence plant genomes. Obtaining high quality, high molecular weight DNA from plants poses significant challenges due to the high copy number of chloroplast and mitochondrial DNA, as well as high levels of phenolic compounds and polysaccharides. Multiple methods have been used to isolate DNA from plants; the CTAB method is commonly used to isolate total cellular DNA from plants that contain nuclear DNA, as well as chloroplast and mitochondrial DNA. Alternatively, DNA can be isolated from nuclei to minimize chloroplast and mitochondrial DNA contamination. We describe optimized protocols for isolation of nuclear DNA from eight different plant species encompassing both monocot and eudicot species. These protocols use nuclei isolation to minimize chloroplast and mitochondrial DNA contamination. We also developed a protocol to determine the number of chloroplast and mitochondrial DNA copies relative to the nuclear DNA using quantitative real time PCR (qPCR). We compared DNA isolated from nuclei to total cellular DNA isolated with the CTAB method. As expected, DNA isolated from nuclei consistently yielded nuclear DNA with fewer chloroplast and mitochondrial DNA copies, as compared to the total cellular DNA prepared with the CTAB method. This protocol will allow for analysis of the quality and quantity of nuclear DNA before starting a plant whole genome sequencing or resequencing experiment. Extracting high quality, high molecular weight nuclear DNA in plants has the potential to be a bottleneck in the era of whole genome sequencing and resequencing. The methods that are described here provide a framework for researchers to extract and quantify nuclear DNA in multiple types of plants.

  6. Isolation and analysis of high quality nuclear DNA with reduced organellar DNA for plant genome sequencing and resequencing

    Directory of Open Access Journals (Sweden)

    Zdepski Anna

    2011-05-01

    Full Text Available Abstract Background High throughput sequencing (HTS technologies have revolutionized the field of genomics by drastically reducing the cost of sequencing, making it feasible for individual labs to sequence or resequence plant genomes. Obtaining high quality, high molecular weight DNA from plants poses significant challenges due to the high copy number of chloroplast and mitochondrial DNA, as well as high levels of phenolic compounds and polysaccharides. Multiple methods have been used to isolate DNA from plants; the CTAB method is commonly used to isolate total cellular DNA from plants that contain nuclear DNA, as well as chloroplast and mitochondrial DNA. Alternatively, DNA can be isolated from nuclei to minimize chloroplast and mitochondrial DNA contamination. Results We describe optimized protocols for isolation of nuclear DNA from eight different plant species encompassing both monocot and eudicot species. These protocols use nuclei isolation to minimize chloroplast and mitochondrial DNA contamination. We also developed a protocol to determine the number of chloroplast and mitochondrial DNA copies relative to the nuclear DNA using quantitative real time PCR (qPCR. We compared DNA isolated from nuclei to total cellular DNA isolated with the CTAB method. As expected, DNA isolated from nuclei consistently yielded nuclear DNA with fewer chloroplast and mitochondrial DNA copies, as compared to the total cellular DNA prepared with the CTAB method. This protocol will allow for analysis of the quality and quantity of nuclear DNA before starting a plant whole genome sequencing or resequencing experiment. Conclusions Extracting high quality, high molecular weight nuclear DNA in plants has the potential to be a bottleneck in the era of whole genome sequencing and resequencing. The methods that are described here provide a framework for researchers to extract and quantify nuclear DNA in multiple types of plants.

  7. Isolation and analysis of high quality nuclear DNA with reduced organellar DNA for plant genome sequencing and resequencing

    Science.gov (United States)

    2011-01-01

    Background High throughput sequencing (HTS) technologies have revolutionized the field of genomics by drastically reducing the cost of sequencing, making it feasible for individual labs to sequence or resequence plant genomes. Obtaining high quality, high molecular weight DNA from plants poses significant challenges due to the high copy number of chloroplast and mitochondrial DNA, as well as high levels of phenolic compounds and polysaccharides. Multiple methods have been used to isolate DNA from plants; the CTAB method is commonly used to isolate total cellular DNA from plants that contain nuclear DNA, as well as chloroplast and mitochondrial DNA. Alternatively, DNA can be isolated from nuclei to minimize chloroplast and mitochondrial DNA contamination. Results We describe optimized protocols for isolation of nuclear DNA from eight different plant species encompassing both monocot and eudicot species. These protocols use nuclei isolation to minimize chloroplast and mitochondrial DNA contamination. We also developed a protocol to determine the number of chloroplast and mitochondrial DNA copies relative to the nuclear DNA using quantitative real time PCR (qPCR). We compared DNA isolated from nuclei to total cellular DNA isolated with the CTAB method. As expected, DNA isolated from nuclei consistently yielded nuclear DNA with fewer chloroplast and mitochondrial DNA copies, as compared to the total cellular DNA prepared with the CTAB method. This protocol will allow for analysis of the quality and quantity of nuclear DNA before starting a plant whole genome sequencing or resequencing experiment. Conclusions Extracting high quality, high molecular weight nuclear DNA in plants has the potential to be a bottleneck in the era of whole genome sequencing and resequencing. The methods that are described here provide a framework for researchers to extract and quantify nuclear DNA in multiple types of plants. PMID:21599914

  8. Chromosomal Localization of DNA Amplifications in Neuroblastoma Tumors Using cDNA Microarray Comparative Genomic Hybridization

    Directory of Open Access Journals (Sweden)

    Ben Beheshti

    2003-01-01

    Full Text Available Conventional comparative genomic hybridization (CGH profiling of neuroblastomas has identified many genomic aberrations, although the limited resolution has precluded a precise localization of sequences of interest within amplicons. To map high copy number genomic gains in clinically matched stage IV neuroblastomas, CGH analysis using a 19,200-feature cDNA microarray was used. A dedicated (freely available algorithm was developed for rapid in silico determination of chromosomal localizations of microarray cDNA targets, and for generation of an ideogram-type profile of copy number changes. Using these methodologies, novel gene amplifications undetectable by chromosome CGH were identified, and larger MYCN amplicon sizes (in one tumor up to 6 Mb than those previously reported in neuroblastoma were identified. The genes HPCAL1, LPIN1/KIAA0188, NAG, and NSE1/LOC151354 were found to be coamplified with MYCN. To determine whether stage IV primary tumors could be further subclassified based on their genomic copy number profiles, hierarchical clustering was performed. Cluster analysis of microarray CGH data identified three groups: 1 no amplifications evident, 2 a small MYCN amplicon as the only detectable imbalance, and 3 a large MYCN amplicon with additional gene amplifications. Application of CGH to cDNA microarray targets will help to determine both the variation of amplicon size and help better define amplification-dependent and independent pathways of progression in neuroblastoma.

  9. Genomic DNA Interactions Mechanize Peptidotoxin-Mediated Anticancer Nanotherapy.

    Science.gov (United States)

    Misra, Santosh K; Schwartz-Duval, Aaron S; Pan, Dipanjan

    2017-07-03

    Host defense peptides (HDPs) are a class of evolutionarily conserved substances of the innate immune response that have been identified as major players in the defense system in many living organisms. Some of the HDPs are also referred to as peptidotoxins, which offer immense potential for anticancer therapy. However, their therapeutic potential is yet to be fully translated mainly due to their off-target toxicity. Here we show that their nanoenabled delivery may become beneficial in controlling their delivery in intracellular space. We introduced an amphiphilic polymer to synthesize a well-defined, self-assembled, rigid-cored polymeric nanoarchitecture for controlled delivery of three model peptidotoxins, i.e., melittin, TSAP-1, and a negative control peptide of synthetic origin. Interestingly, our results revealed strong interaction of peptidotoxins with duplex plasmid DNA. Extensive biophysical characterization (UV-vis spectroscopy, gel electrophoresis, MTT assay, and flow assisted cell sorting) experimentally verified that peptidotoxins were able to interact with genomic DNA in vitro and in turn influence the cancer cell growth. Thus, we unraveled that, through genomic DNA regulation, peptidotoxins can play a role in cell cycle regulation and exert their anticancer activities.

  10. The roles of adenoviral vectors and donor DNA structures on genome editing

    NARCIS (Netherlands)

    Holkers, Maarten

    2016-01-01

    Accurate and efficient genome editing is primarily dependent on the generation of a sequence-specific, genomic double-stranded DNA break (DSB) combined with the introduction of an exogenous DNA template into target cells. The exogenous template, called donor DNA, normally contains the foreign

  11. Noninvasive Prenatal DNA Testing: The Vanguard of Genomic Medicine.

    Science.gov (United States)

    Hui, Lisa; Bianchi, Diana W

    2017-01-14

    Noninvasive prenatal DNA testing is the vanguard of genomic medicine. In only four years, this screening test has revolutionized prenatal care globally and opened up new prospects for personalized medicine for the fetus. There are widespread implications for increasing the scope of human genetic variation that can be detected before birth, and for discovering more about maternofetal and placental biology. These include an urgent need to develop pretest education for all pregnant women and consistent post-test management recommendations for those with discordant test results. The reduction in invasive testing has had downstream effects on specialist training and caused many countries to re-examine their national approaches to prenatal screening. Finally, the accumulating datasets of genomic information on pregnant women and their fetuses raise ethical issues regarding consent for future data mining and intellectual property.

  12. Genome-Wide Prediction of DNA Methylation Using DNA Composition and Sequence Complexity in Human

    Directory of Open Access Journals (Sweden)

    Chengchao Wu

    2017-02-01

    Full Text Available DNA methylation plays a significant role in transcriptional regulation by repressing activity. Change of the DNA methylation level is an important factor affecting the expression of target genes and downstream phenotypes. Because current experimental technologies can only assay a small proportion of CpG sites in the human genome, it is urgent to develop reliable computational models for predicting genome-wide DNA methylation. Here, we proposed a novel algorithm that accurately extracted sequence complexity features (seven features and developed a support-vector-machine-based prediction model with integration of the reported DNA composition features (trinucleotide frequency and GC content, 65 features by utilizing the methylation profiles of embryonic stem cells in human. The prediction results from 22 human chromosomes with size-varied windows showed that the 600-bp window achieved the best average accuracy of 94.7%. Moreover, comparisons with two existing methods further showed the superiority of our model, and cross-species predictions on mouse data also demonstrated that our model has certain generalization ability. Finally, a statistical test of the experimental data and the predicted data on functional regions annotated by ChromHMM found that six out of 10 regions were consistent, which implies reliable prediction of unassayed CpG sites. Accordingly, we believe that our novel model will be useful and reliable in predicting DNA methylation.

  13. An Assessment of Whole Blood and Fractions by Nested PCR as a DNA Source for Diagnosing Canine Ehrlichiosis and Anaplasmosis

    Directory of Open Access Journals (Sweden)

    Tereza Emmanuelle de Farias Rotondano

    2012-01-01

    Full Text Available Ehrlichiosis and anaplasmosis are tick-borne diseases. Ehrlichia canis and Anaplasma platys infect mainly white cells and platelets, respectively. The main DNA source for PCR is peripheral blood, but the potential of blood cell fractions has not been extensively investigated. This study aims at assessment of whole blood (WB and blood fractions potential in nested PCR (nPCR to diagnose canine ehrlichiosis and anaplasmosis. The 16S rRNA gene was amplified in 71.4, 17.8, 31.57, and 30% of the WB, granulocyte (G, mononuclear cells (M, and buffy coat (BC samples. Compared to the WB, the sensitivity of the PCR was 42.86% for the M, and BC fractions, 21.43% for the G, and 33.33% for the blood clot (C. There was fair agreement between the WB and M, BC and C, and slight with the G. Fair agreement occurred between the nPCR and morulae in the blood smear. One animal was coinfected with A. platys and E. canis. This study provided the first evidence of A. platys infection in dogs in Paraíba, Brazil, and demonstrated that WB is a better DNA source than blood fractions to detect Ehrlichia and Anaplasma by nPCR, probably because of the plasma bacterial concentration following host cell lysis.

  14. Influence of chemotherapy for lymphoma in canine parvovirus DNA distribution and specific humoral immunity.

    Science.gov (United States)

    Elias, M A; Duarte, A; Nunes, T; Lourenço, A M; Braz, B S; Vicente, G; Henriques, J; Tavares, L

    2014-12-01

    In man, the combination of cancer and its treatment increases patients' susceptibility to opportunistic infections, due to immune system impairment. In veterinary medicine little information is available concerning this issue. In order to evaluate if a similar dysfunction is induced in small animals undergoing chemotherapy, we assessed the complete blood count, leukocytic, plasma and fecal canine parvovirus (CPV) viral load, and anti-CPV protective antibody titers, in dogs with lymphoma treated with CHOP (cyclophosphamide, doxorubicin, vincristine and prednisolone) protocol, before and during chemotherapy. There was no evidence of decreased immune response, either at admission or after two chemotherapy cycles, indicating that the previously established immunity against CPV was not significantly impaired, supporting the idea that immunosuppression as a result of hematopoietic neoplasms and their treatment in dogs requires further investigation and conclusions cannot be extrapolated from human literature. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Human adipogenesis is associated with genome-wide DNA methylation and gene-expression changes

    DEFF Research Database (Denmark)

    Broholm, Christa; Olsson, Anders Henrik; Perfilyev, Alexander

    2016-01-01

    AIM: To define the genomic distribution and function of DNA methylation changes during human adipogenesis. METHODS: We isolated adipocyte-derived stem cells from 13 individuals and analyzed genome-wide DNA methylation and gene expression in cultured adipocyte-derived stem cells and mature adipocy...... for cell cycle and adipokine signaling. CONCLUSION: Human adipogenesis is associated with significant DNA methylation changes across the entire genome and may impact regulation of cell cycle and adipokine signaling....

  16. Multi-scale coding of genomic information: From DNA sequence to genome structure and function

    Energy Technology Data Exchange (ETDEWEB)

    Arneodo, Alain, E-mail: alain.arneodo@ens-lyon.f [Universite de Lyon, F-69000 Lyon (France); Laboratoire Joliot-Curie and Laboratoire de Physique, CNRS, Ecole Normale Superieure de Lyon, F-69007 Lyon (France); Vaillant, Cedric, E-mail: cedric.vaillant@ens-lyon.f [Universite de Lyon, F-69000 Lyon (France); Laboratoire Joliot-Curie and Laboratoire de Physique, CNRS, Ecole Normale Superieure de Lyon, F-69007 Lyon (France); Audit, Benjamin, E-mail: benjamin.audit@ens-lyon.f [Universite de Lyon, F-69000 Lyon (France); Laboratoire Joliot-Curie and Laboratoire de Physique, CNRS, Ecole Normale Superieure de Lyon, F-69007 Lyon (France); Argoul, Francoise, E-mail: francoise.argoul@ens-lyon.f [Universite de Lyon, F-69000 Lyon (France); Laboratoire Joliot-Curie and Laboratoire de Physique, CNRS, Ecole Normale Superieure de Lyon, F-69007 Lyon (France); D' Aubenton-Carafa, Yves, E-mail: daubenton@cgm.cnrs-gif.f [Centre de Genetique Moleculaire, CNRS, Allee de la Terrasse, 91198 Gif-sur-Yvette (France); Thermes, Claude, E-mail: claude.thermes@cgm.cnrs-gif.f [Centre de Genetique Moleculaire, CNRS, Allee de la Terrasse, 91198 Gif-sur-Yvette (France)

    2011-02-15

    Understanding how chromatin is spatially and dynamically organized in the nucleus of eukaryotic cells and how this affects genome functions is one of the main challenges of cell biology. Since the different orders of packaging in the hierarchical organization of DNA condition the accessibility of DNA sequence elements to trans-acting factors that control the transcription and replication processes, there is actually a wealth of structural and dynamical information to learn in the primary DNA sequence. In this review, we show that when using concepts, methodologies, numerical and experimental techniques coming from statistical mechanics and nonlinear physics combined with wavelet-based multi-scale signal processing, we are able to decipher the multi-scale sequence encoding of chromatin condensation-decondensation mechanisms that play a fundamental role in regulating many molecular processes involved in nuclear functions.

  17. Genome and metagenome sequencing: Using the human methyl-binding domain to partition genomic DNA derived from plant tissues.

    Science.gov (United States)

    Yigit, Erbay; Hernandez, David I; Trujillo, Joshua T; Dimalanta, Eileen; Bailey, C Donovan

    2014-11-01

    Variation in the distribution of methylated CpG (methyl-CpG) in genomic DNA (gDNA) across the tree of life is biologically interesting and useful in genomic studies. We illustrate the use of human methyl-CpG-binding domain (MBD2) to fractionate angiosperm DNA into eukaryotic nuclear (methyl-CpG-rich) vs. organellar and prokaryotic (methyl-CpG-poor) elements for genomic and metagenomic sequencing projects. • MBD2 has been used to enrich prokaryotic DNA in animal systems. Using gDNA from five model angiosperm species, we apply a similar approach to identify whether MBD2 can fractionate plant gDNA into methyl-CpG-depleted vs. enriched methyl-CpG elements. For each sample, three gDNA libraries were sequenced: (1) untreated gDNA, (2) a methyl-CpG-depleted fraction, and (3) a methyl-CpG-enriched fraction. • Relative to untreated gDNA, the methyl-depleted libraries showed a 3.2-11.2-fold and 3.4-11.3-fold increase in chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA), respectively. Methyl-enriched fractions showed a 1.8-31.3-fold and 1.3-29.0-fold decrease in cpDNA and mtDNA, respectively. • The application of MBD2 enabled fractionation of plant gDNA. The effectiveness was particularly striking for monocot gDNA (Poaceae). When sufficiently effective on a sample, this approach can increase the cost efficiency of sequencing plant genomes as well as prokaryotes living in or on plant tissues.

  18. Genome and Metagenome Sequencing: Using the Human Methyl-Binding Domain to Partition Genomic DNA Derived from Plant Tissues

    Directory of Open Access Journals (Sweden)

    Erbay Yigit

    2014-11-01

    Full Text Available Premise of the study: Variation in the distribution of methylated CpG (methyl-CpG in genomic DNA (gDNA across the tree of life is biologically interesting and useful in genomic studies. We illustrate the use of human methyl-CpG-binding domain (MBD2 to fractionate angiosperm DNA into eukaryotic nuclear (methyl-CpG-rich vs. organellar and prokaryotic (methyl-CpG-poor elements for genomic and metagenomic sequencing projects. Methods: MBD2 has been used to enrich prokaryotic DNA in animal systems. Using gDNA from five model angiosperm species, we apply a similar approach to identify whether MBD2 can fractionate plant gDNA into methyl-CpG-depleted vs. enriched methyl-CpG elements. For each sample, three gDNA libraries were sequenced: (1 untreated gDNA, (2 a methyl-CpG-depleted fraction, and (3 a methyl-CpG-enriched fraction. Results: Relative to untreated gDNA, the methyl-depleted libraries showed a 3.2–11.2-fold and 3.4–11.3-fold increase in chloroplast DNA (cpDNA and mitochondrial DNA (mtDNA, respectively. Methyl-enriched fractions showed a 1.8–31.3-fold and 1.3–29.0-fold decrease in cpDNA and mtDNA, respectively. Discussion: The application of MBD2 enabled fractionation of plant gDNA. The effectiveness was particularly striking for monocot gDNA (Poaceae. When sufficiently effective on a sample, this approach can increase the cost efficiency of sequencing plant genomes as well as prokaryotes living in or on plant tissues.

  19. Highly sensitive polymerase chain reaction-free quantum dot-based quantification of forensic genomic DNA

    Energy Technology Data Exchange (ETDEWEB)

    Tak, Yu Kyung; Kim, Won Young; Kim, Min Jung; Han, Eunyoung [College of Pharmacy, Seoul National University, Seoul 151-742 (Korea, Republic of); Han, Myun Soo; Kim, Jong Jin [Forensic DNA Center, National Forensic Service, Seoul 158-707 (Korea, Republic of); Kim, Wook [Department of Biological Sciences, Dankook University, Cheonan 330-714 (Korea, Republic of); Lee, Jong Eun [DNA Link, VICHEN CITY Building 3F, Seoul 121-850 (Korea, Republic of); Song, Joon Myong, E-mail: jmsong@snu.ac.kr [College of Pharmacy, Seoul National University, Seoul 151-742 (Korea, Republic of)

    2012-04-06

    Highlights: Black-Right-Pointing-Pointer Genomic DNA quantification were performed using a quantum dot-labeled Alu sequence. Black-Right-Pointing-Pointer This probe provided PCR-free determination of human genomic DNA. Black-Right-Pointing-Pointer Qdot-labeled Alu probe-hybridized genomic DNAs had a 2.5-femtogram detection limit. Black-Right-Pointing-Pointer Qdot-labeled Alu sequence was used to assess DNA samples for human identification. - Abstract: Forensic DNA samples can degrade easily due to exposure to light and moisture at the crime scene. In addition, the amount of DNA acquired at a criminal site is inherently limited. This limited amount of human DNA has to be quantified accurately after the process of DNA extraction. The accurately quantified extracted genomic DNA is then used as a DNA template in polymerase chain reaction (PCR) amplification for short tandem repeat (STR) human identification. Accordingly, highly sensitive and human-specific quantification of forensic DNA samples is an essential issue in forensic study. In this work, a quantum dot (Qdot)-labeled Alu sequence was developed as a probe to simultaneously satisfy both the high sensitivity and human genome selectivity for quantification of forensic DNA samples. This probe provided PCR-free determination of human genomic DNA and had a 2.5-femtogram detection limit due to the strong emission and photostability of the Qdot. The Qdot-labeled Alu sequence has been used successfully to assess 18 different forensic DNA samples for STR human identification.

  20. The mitochondrial and plastid genomes of Volvox carteri: bloated molecules rich in repetitive DNA

    Directory of Open Access Journals (Sweden)

    Lee Robert W

    2009-03-01

    Full Text Available Abstract Background The magnitude of noncoding DNA in organelle genomes can vary significantly; it is argued that much of this variation is attributable to the dissemination of selfish DNA. The results of a previous study indicate that the mitochondrial DNA (mtDNA of the green alga Volvox carteri abounds with palindromic repeats, which appear to be selfish elements. We became interested in the evolution and distribution of these repeats when, during a cursory exploration of the V. carteri nuclear DNA (nucDNA and plastid DNA (ptDNA sequences, we found palindromic repeats with similar structural features to those of the mtDNA. Upon this discovery, we decided to investigate the diversity and evolutionary implications of these palindromic elements by sequencing and characterizing large portions of mtDNA and ptDNA and then comparing these data to the V. carteri draft nuclear genome sequence. Results We sequenced 30 and 420 kilobases (kb of the mitochondrial and plastid genomes of V. carteri, respectively – resulting in partial assemblies of these genomes. The mitochondrial genome is the most bloated green-algal mtDNA observed to date: ~61% of the sequence is noncoding, most of which is comprised of short palindromic repeats spread throughout the intergenic and intronic regions. The plastid genome is the largest (>420 kb and most expanded (>80% noncoding ptDNA sequence yet discovered, with a myriad of palindromic repeats in the noncoding regions, which have a similar size and secondary structure to those of the mtDNA. We found that 15 kb (~0.01% of the nuclear genome are homologous to the palindromic elements of the mtDNA, and 50 kb (~0.05% are homologous to those of the ptDNA. Conclusion Selfish elements in the form of short palindromic repeats have propagated in the V. carteri mtDNA and ptDNA, resulting in the distension of these genomes. Copies of these same repeats are also found in a small fraction of the nucDNA, but appear to be inert in this

  1. Solution-based targeted genomic enrichment for precious DNA samples

    Directory of Open Access Journals (Sweden)

    Shearer Aiden

    2012-05-01

    Full Text Available Abstract Background Solution-based targeted genomic enrichment (TGE protocols permit selective sequencing of genomic regions of interest on a massively parallel scale. These protocols could be improved by: 1 modifying or eliminating time consuming steps; 2 increasing yield to reduce input DNA and excessive PCR cycling; and 3 enhancing reproducible. Results We developed a solution-based TGE method for downstream Illumina sequencing in a non-automated workflow, adding standard Illumina barcode indexes during the post-hybridization amplification to allow for sample pooling prior to sequencing. The method utilizes Agilent SureSelect baits, primers and hybridization reagents for the capture, off-the-shelf reagents for the library preparation steps, and adaptor oligonucleotides for Illumina paired-end sequencing purchased directly from an oligonucleotide manufacturing company. Conclusions This solution-based TGE method for Illumina sequencing is optimized for small- or medium-sized laboratories and addresses the weaknesses of standard protocols by reducing the amount of input DNA required, increasing capture yield, optimizing efficiency, and improving reproducibility.

  2. Genome-Wide Discriminatory Information Patterns of Cytosine DNA Methylation

    Directory of Open Access Journals (Sweden)

    Robersy Sanchez

    2016-06-01

    Full Text Available Cytosine DNA methylation (CDM is a highly abundant, heritable but reversible chemical modification to the genome. Herein, a machine learning approach was applied to analyze the accumulation of epigenetic marks in methylomes of 152 ecotypes and 85 silencing mutants of Arabidopsis thaliana. In an information-thermodynamics framework, two measurements were used: (1 the amount of information gained/lost with the CDM changes I R and (2 the uncertainty of not observing a SNP L C R . We hypothesize that epigenetic marks are chromosomal footprints accounting for different ontogenetic and phylogenetic histories of individual populations. A machine learning approach is proposed to verify this hypothesis. Results support the hypothesis by the existence of discriminatory information (DI patterns of CDM able to discriminate between individuals and between individual subpopulations. The statistical analyses revealed a strong association between the topologies of the structured population of Arabidopsis ecotypes based on I R and on LCR, respectively. A statistical-physical relationship between I R and L C R was also found. Results to date imply that the genome-wide distribution of CDM changes is not only part of the biological signal created by the methylation regulatory machinery, but ensures the stability of the DNA molecule, preserving the integrity of the genetic message under continuous stress from thermal fluctuations in the cell environment.

  3. Comparative performance of two whole-genome capture methodologies on ancient DNA Illumina libraries

    OpenAIRE

    Ávila-Arcos María C; Sandoval-Velasco Marcela; Schroeder Hannes; Carpenter Meredith L.; Malaspinas Anna-Sapfo; Wales Nathan; Peñaloza Fernando; Bustamante Carlos D.; Gilbert M. Thomas P.

    2015-01-01

    Application of whole genome capture (WGC) methods to ancient DNA (aDNA) promises to increase efficiency of ancient genome sequencing. We compared the performance of two recent WGC methods in enriching human aDNA within Illumina libraries built using both double stranded and single stranded build protocols. Although both methods effectively enriched aDNA we observed consistent differences between the methods providing the opportunity to further explore parameters influencing WGC experiments. ...

  4. DNA bending facilitates the error-free DNA damage tolerance pathway and upholds genome integrity

    Science.gov (United States)

    Gonzalez-Huici, Victor; Szakal, Barnabas; Urulangodi, Madhusoodanan; Psakhye, Ivan; Castellucci, Federica; Menolfi, Demis; Rajakumara, Eerappa; Fumasoni, Marco; Bermejo, Rodrigo; Jentsch, Stefan; Branzei, Dana

    2014-01-01

    DNA replication is sensitive to damage in the template. To bypass lesions and complete replication, cells activate recombination-mediated (error-free) and translesion synthesis-mediated (error-prone) DNA damage tolerance pathways. Crucial for error-free DNA damage tolerance is template switching, which depends on the formation and resolution of damage-bypass intermediates consisting of sister chromatid junctions. Here we show that a chromatin architectural pathway involving the high mobility group box protein Hmo1 channels replication-associated lesions into the error-free DNA damage tolerance pathway mediated by Rad5 and PCNA polyubiquitylation, while preventing mutagenic bypass and toxic recombination. In the process of template switching, Hmo1 also promotes sister chromatid junction formation predominantly during replication. Its C-terminal tail, implicated in chromatin bending, facilitates the formation of catenations/hemicatenations and mediates the roles of Hmo1 in DNA damage tolerance pathway choice and sister chromatid junction formation. Together, the results suggest that replication-associated topological changes involving the molecular DNA bender, Hmo1, set the stage for dedicated repair reactions that limit errors during replication and impact on genome stability. PMID:24473148

  5. Gross genomic damage measured by DNA image cytometry independently predicts gastric cancer patient survival

    NARCIS (Netherlands)

    Belien, J.A.M.; Buffart, T.E.; Gill, A.; Broeckaert, M.A.M.; Quirke, P.; Meijer, G.A.; Grabsch, H.

    2009-01-01

    BACKGROUND: DNA aneuploidy reflects gross genomic changes. It can be measured by flow cytometry (FCM-DNA) or image cytometry (ICM-DNA). In gastric cancer, the prevalence of DNA aneuploidy has been reported to range from 27 to 100%, with conflicting associations with clinicopathological variables.

  6. A simple and efficient method for extraction of genomic DNA from ...

    African Journals Online (AJOL)

    SERVER

    2008-04-17

    Apr 17, 2008 ... DNA extraction in many plants is difficult because of metabolites that interfere with DNA isolation procedures and subsequent applications, such as DNA restriction, amplification and cloning. We have developed a reliable and efficient method for isolating genomic DNA free from polysaccharide,.

  7. A simple and efficient method for extraction of genomic DNA from ...

    African Journals Online (AJOL)

    DNA extraction in many plants is difficult because of metabolites that interfere with DNA isolation procedures and subsequent applications, such as DNA restriction, amplification and cloning. We have developed a reliable and efficient method for isolating genomic DNA free from polysaccharide, polyphenols and protein ...

  8. Genome-wide DNA methylation scan in major depressive disorder.

    Directory of Open Access Journals (Sweden)

    Sarven Sabunciyan

    Full Text Available While genome-wide association studies are ongoing to identify sequence variation influencing susceptibility to major depressive disorder (MDD, epigenetic marks, such as DNA methylation, which can be influenced by environment, might also play a role. Here we present the first genome-wide DNA methylation (DNAm scan in MDD. We compared 39 postmortem frontal cortex MDD samples to 26 controls. DNA was hybridized to our Comprehensive High-throughput Arrays for Relative Methylation (CHARM platform, covering 3.5 million CpGs. CHARM identified 224 candidate regions with DNAm differences >10%. These regions are highly enriched for neuronal growth and development genes. Ten of 17 regions for which validation was attempted showed true DNAm differences; the greatest were in PRIMA1, with 12-15% increased DNAm in MDD (p = 0.0002-0.0003, and a concomitant decrease in gene expression. These results must be considered pilot data, however, as we could only test replication in a small number of additional brain samples (n = 16, which showed no significant difference in PRIMA1. Because PRIMA1 anchors acetylcholinesterase in neuronal membranes, decreased expression could result in decreased enzyme function and increased cholinergic transmission, consistent with a role in MDD. We observed decreased immunoreactivity for acetylcholinesterase in MDD brain with increased PRIMA1 DNAm, non-significant at p = 0.08.While we cannot draw firm conclusions about PRIMA1 DNAm in MDD, the involvement of neuronal development genes across the set showing differential methylation suggests a role for epigenetics in the illness. Further studies using limbic system brain regions might shed additional light on this role.

  9. Simultaneous Whole Mitochondrial Genome Sequencing with Short Overlapping Amplicons Suitable for Degraded DNA Using the Ion Torrent Personal Genome Machine.

    Science.gov (United States)

    Chaitanya, Lakshmi; Ralf, Arwin; van Oven, Mannis; Kupiec, Tomasz; Chang, Joseph; Lagacé, Robert; Kayser, Manfred

    2015-12-01

    Whole mitochondrial (mt) genome analysis enables a considerable increase in analysis throughput, and improves the discriminatory power to the maximum possible phylogenetic resolution. Most established protocols on the different massively parallel sequencing (MPS) platforms, however, invariably involve the PCR amplification of large fragments, typically several kilobases in size, which may fail due to mtDNA fragmentation in the available degraded materials. We introduce a MPS tiling approach for simultaneous whole human mt genome sequencing using 161 short overlapping amplicons (average 200 bp) with the Ion Torrent Personal Genome Machine. We illustrate the performance of this new method by sequencing 20 DNA samples belonging to different worldwide mtDNA haplogroups. Additional quality control, particularly regarding the potential detection of nuclear insertions of mtDNA (NUMTs), was performed by comparative MPS analysis using the conventional long-range amplification method. Preliminary sensitivity testing revealed that detailed haplogroup inference was feasible with 100 pg genomic input DNA. Complete mt genome coverage was achieved from DNA samples experimentally degraded down to genomic fragment sizes of about 220 bp, and up to 90% coverage from naturally degraded samples. Overall, we introduce a new approach for whole mt genome MPS analysis from degraded and nondegraded materials relevant to resolve and infer maternal genetic ancestry at complete resolution in anthropological, evolutionary, medical, and forensic applications. © 2015 The Authors. **Human Mutation published by Wiley Periodicals, Inc.

  10. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes.

    Science.gov (United States)

    Versalovic, J; Koeuth, T; Lupski, J R

    1991-12-25

    Dispersed repetitive DNA sequences have been described recently in eubacteria. To assess the distribution and evolutionary conservation of two distinct prokaryotic repetitive elements, consensus oligonucleotides were used in polymerase chain reaction [PCR] amplification and slot blot hybridization experiments with genomic DNA from diverse eubacterial species. Oligonucleotides matching Repetitive Extragenic Palindromic [REP] elements and Enterobacterial Repetitive Intergenic Consensus [ERIC] sequences were synthesized and tested as opposing PCR primers in the amplification of eubacterial genomic DNA. REP and ERIC consensus oligonucleotides produced clearly resolvable bands by agarose gel electrophoresis following PCR amplification. These band patterns provided unambiguous DNA fingerprints of different eubacterial species and strains. Both REP and ERIC probes hybridized preferentially to genomic DNA from Gram-negative enteric bacteria and related species. Widespread distribution of these repetitive DNA elements in the genomes of various microorganisms should enable rapid identification of bacterial species and strains, and be useful for the analysis of prokaryotic genomes.

  11. Cloning, localization and focus formation at DNA damage sites of canine XLF

    OpenAIRE

    KOIKE, Manabu; YUTOKU, Yasutomo; KOIKE, Aki

    2016-01-01

    Understanding the molecular mechanisms of DNA double-strand break (DSB) repair processes, especially nonhomologous DNA-end joining (NHEJ), is critical for developing next-generation radiotherapies and chemotherapeutics for human and animal cancers. The localization, protein-protein interactions and post-translational modifications of core NHEJ factors, such as human Ku70 and Ku80, might play critical roles in controlling NHEJ activity. XRCC4-like factor (XLF) is a core NHEJ factor and plays a...

  12. Cloning, localization and focus formation at DNA damage sites of canine XRCC4

    OpenAIRE

    KOIKE, Manabu; YUTOKU, Yasutomo; KOIKE, Aki

    2016-01-01

    Various chemotherapies and radiation therapies are useful for killing cancer cells mainly by inducing DNA double-strand breaks (DSBs). Uncovering the molecular mechanisms of DSB repair processes is crucial for developing next-generation radiotherapies and chemotherapeutics for human and animal cancers. XRCC4 plays a critical role in Ku-dependent nonhomologous DNA-end joining (NHEJ) in human cells, and is one of the core NHEJ factors. The localization of core NHEJ factors, such as human Ku70 a...

  13. Volume visualization of multiple alignment of large genomicDNA

    Energy Technology Data Exchange (ETDEWEB)

    Shah, Nameeta; Dillard, Scott E.; Weber, Gunther H.; Hamann, Bernd

    2005-07-25

    Genomes of hundreds of species have been sequenced to date, and many more are being sequenced. As more and more sequence data sets become available, and as the challenge of comparing these massive ''billion basepair DNA sequences'' becomes substantial, so does the need for more powerful tools supporting the exploration of these data sets. Similarity score data used to compare aligned DNA sequences is inherently one-dimensional. One-dimensional (1D) representations of these data sets do not effectively utilize screen real estate. As a result, tools using 1D representations are incapable of providing informatory overview for extremely large data sets. We present a technique to arrange 1D data in 3D space to allow us to apply state-of-the-art interactive volume visualization techniques for data exploration. We demonstrate our technique using multi-millions-basepair-long aligned DNA sequence data and compare it with traditional 1D line plots. The results show that our technique is superior in providing an overview of entire data sets. Our technique, coupled with 1D line plots, results in effective multi-resolution visualization of very large aligned sequence data sets.

  14. Genome-Wide Analysis of DNA Methylation in Human Amnion

    Directory of Open Access Journals (Sweden)

    Jinsil Kim

    2013-01-01

    Full Text Available The amnion is a specialized tissue in contact with the amniotic fluid, which is in a constantly changing state. To investigate the importance of epigenetic events in this tissue in the physiology and pathophysiology of pregnancy, we performed genome-wide DNA methylation profiling of human amnion from term (with and without labor and preterm deliveries. Using the Illumina Infinium HumanMethylation27 BeadChip, we identified genes exhibiting differential methylation associated with normal labor and preterm birth. Functional analysis of the differentially methylated genes revealed biologically relevant enriched gene sets. Bisulfite sequencing analysis of the promoter region of the oxytocin receptor (OXTR gene detected two CpG dinucleotides showing significant methylation differences among the three groups of samples. Hypermethylation of the CpG island of the solute carrier family 30 member 3 (SLC30A3 gene in preterm amnion was confirmed by methylation-specific PCR. This work provides preliminary evidence that DNA methylation changes in the amnion may be at least partially involved in the physiological process of labor and the etiology of preterm birth and suggests that DNA methylation profiles, in combination with other biological data, may provide valuable insight into the mechanisms underlying normal and pathological pregnancies.

  15. An oral Sindbis virus replicon-based DNA vaccine containing VP2 gene of canine parvovirus delivered by Escherichia coli elicits immune responses in dogs.

    Science.gov (United States)

    Dahiya, S S; Saini, M; Kumar, P; Gupta, P K

    2011-01-01

    A Sindbis virus replicon-based DNA vaccine containing VP2 gene of canine parvovirus (CPV) was delivered by Escherichia coli to elicit immune responses. The orally immunized dogs developed CPV-specific serum IgG and virus neutralizing antibody responses. The cellular immune responses analyzed using lymphocyte proliferation test and flow cytometry indicated CPV-specific sensitization of both CD3+CD4+ and CD3+CD8+ lymphocytes. This study demonstrated that the oral CPV DNA vaccine delivered by E. coli can be considered as a promising approach for vaccination of dogs against CPV.

  16. Molecular characterization of transgenic shallots (Allium cepa L.) by adaptor ligation PCR (AL-PCR) and sequencing of genomic DNA flanking T-DNA borders

    NARCIS (Netherlands)

    Zheng, S.J.; Henken, G.; Sofiari, E.; Jacobsen, E.; Krens, F.A.

    2001-01-01

    Genomic DNA blot hybridization is traditionally used to demonstrate that, via genetic transformation, foreign genes are integrated into host genomes. However, in large genome species, such as Allium cepa L., the use of genomic DNA blot hybridization is pushed towards its limits, because a

  17. A protocol for large scale genomic DNA isolation for cacao genetics ...

    African Journals Online (AJOL)

    Advances in DNA technology, such as marker assisted selection, detection of quantitative trait loci and genomic selection also require the isolation of DNA from a large number of samples and the preservation of tissue samples for future use in cacao genome studies. The present study proposes a method for the ...

  18. Simplified extraction of good quality genomic DNA from a variety of ...

    African Journals Online (AJOL)

    Depending on the nature and complexity of plant material, proper method needs to be employed for extraction of genomic DNA, along with its performance evaluation by different molecular techniques. Here, we optimized and employed a simple genomic DNA isolation protocol suitable for a variety of plant materials ...

  19. DNA microarrays: from structural genomics to functional genomics. The applications of gene chips in dermatology and dermatopathology.

    Science.gov (United States)

    Sellheyer, Klaus; Belbin, Thomas J

    2004-11-01

    The human genome project was successful in sequencing the entire human genome and ended earlier than expected. The vast genetic information now available will have far-reaching consequences for medicine in the twenty-first century. The knowledge gained from the mapping and sequencing of human genes on a genome-wide scale--commonly referred to as structural genomics--is prerequisite for studies that focus on the functional aspects of genes. A recently invented technique, known as gene chip, or DNA microarray, technology, allows the study of the function of thousands of genes at once, thereby opening the door to the new field of functional genomics. At its core, the DNA microarray utilizes a unique feature of DNA known as complementary hybridization. As such, it is not different from Southern (DNA) blot or northern (RNA) blot hybridizations, or the polymerase chain reaction, with the exception that it allows expression profiling of the entire human genome in a single hybridization experiment. The article highlights the principles, technology, and applications of DNA microarrays as they pertain to the field of dermatology and dermatopathology. The most important applications are the gene expression profiling of skin cancer, especially of melanoma. Other potential applications include gene expression profiling of inflammatory skin diseases, the mutational analysis of genodermatoses, and polymorphism screening, as well as drug development and chemosensitivity prediction. cDNA microarrays will shape the diagnostic approach of the dermatology and the dermatopathology of the future and may lead to new therapeutic options.

  20. An analysis of the use of genomic DNA as a universal reference in two channel DNA microarrays

    Directory of Open Access Journals (Sweden)

    Kapur Vivek

    2005-05-01

    Full Text Available Abstract Background DNA microarray is an invaluable tool for gene expression explorations. In the two-dye microarray, fluorescence intensities of two samples, each labeled with a different dye, are compared after hybridization. To compare a large number of samples, the 'reference design' is widely used, in which all RNA samples are hybridized to a common reference. Genomic DNA is an attractive candidate for use as a universal reference, especially for bacterial systems with a low percentage of non-coding sequences. However, genomic DNA, comprising of both the sense and anti-sense strands, is unlike the single stranded cDNA usually used in microarray hybridizations. The presence of the antisense strand in the 'reference' leads to reactions between complementary labeled strands in solution and may cause the assay result to deviate from true values. Results We have developed a mathematical model to predict the validity of using genomic DNA as a reference in the microarray assay. The model predicts that the assay can accurately estimate relative concentrations for a wide range of initial cDNA concentrations. Experimental results of DNA microarray assay using genomic DNA as a reference correlated well to those obtained by a direct hybridization between two cDNA samples. The model predicts that the initial concentrations of labeled genomic DNA strands and immobilized strands, and the hybridization time do not significantly affect the assay performance. At low values of the rate constant for hybridization between immobilized and mobile strands, the assay performance varies with the hybridization time and initial cDNA concentrations. For the case where a microarray with immobilized single strands is used, results from hybridizations using genomic DNA as a reference will correspond to true ratios under all conditions. Conclusion Simulation using the mathematical model, and the experimental study presented here show the potential utility of microarray

  1. Genomic DNA extraction and barcoding of endophytic fungi.

    Science.gov (United States)

    Diaz, Patricia L; Hennell, James R; Sucher, Nikolaus J

    2012-01-01

    Endophytes live inter- and/or intracellularly inside healthy aboveground tissues of plants without causing disease. Endophytic fungi are found in virtually every vascular plant species examined. The origins of this symbiotic relationship between endophytes go back to the emergence of vascular plants. Endophytic fungi receive nutrition and protection from their hosts while the plants benefit from the production of fungal secondary metabolites, which enhance the host plants' resistance to herbivores, pathogens, and various abiotic stresses. Endophytic fungi have attracted increased interest as potential sources of secondary metabolites with agricultural, industrial, and medicinal use. This chapter provides detailed protocols for isolation of genomic DNA from fungal endophytes and its use in polymerase chain reaction-based amplification of the internal transcribed spacer region between the conserved flanking regions of the small and large subunit of ribosomal RNA for barcoding purposes.

  2. 78 FR 29698 - Availability of an Environmental Assessment for Field Testing a Canine Lymphoma Vaccine, DNA

    Science.gov (United States)

    2013-05-21

    ... the Virus-Serum-Toxin Act (21 U.S.C. 151 et seq.), a veterinary biological product must be shown to be..., Tennessee, and Texas. The above-mentioned product is a replication-incompetent DNA vaccine consisting of a...

  3. Pairagon: a highly accurate, HMM-based cDNA-to-genome aligner

    DEFF Research Database (Denmark)

    Lu, David V; Brown, Randall H; Arumugam, Manimozhiyan

    2009-01-01

    MOTIVATION: The most accurate way to determine the intron-exon structures in a genome is to align spliced cDNA sequences to the genome. Thus, cDNA-to-genome alignment programs are a key component of most annotation pipelines. The scoring system used to choose the best alignment is a primary...... heuristics. RESULTS: We present Pairagon, a pair hidden Markov model based cDNA-to-genome alignment program, as the most accurate aligner for sequences with high- and low-identity levels. We conducted a series of experiments testing alignment accuracy with varying sequence identity. We first created 'perfect......' simulated cDNA sequences by splicing the sequences of exons in the reference genome sequences of fly and human. The complete reference genome sequences were then mutated to various degrees using a realistic mutation simulator and the perfect cDNAs were aligned to them using Pairagon and 12 other aligners...

  4. Rapid and sensitive detection of canine parvovirus type 2 by recombinase polymerase amplification.

    Science.gov (United States)

    Wang, Jianchang; Liu, Libing; Li, Ruiwen; Wang, Jinfeng; Fu, Qi; Yuan, Wanzhe

    2016-04-01

    A novel recombinase polymerase amplification (RPA)-based method for detection of canine parvovirus type 2 (CPV-2) was developed. Sensitivity analysis showed that the detection limit of RPA was 10 copies of CPV-2 genomic DNA. RPA amplified both CPV-2a and -2b DNA but did not amplify the template of other important dog viruses (CCoV, PRV or CDV), demonstrating high specificity. The method was further validated with 57 canine fecal samples. An outstanding advantage of RPA is that it is an isothermal reaction and can be performed in a water bath, making RPA a potential alternative method for CPV-2 detection in resource-limited settings.

  5. Genome-wide survey of repetitive DNA elements in the button mushroom Agaricus bisporus

    NARCIS (Netherlands)

    Foulongne-Oriol, M.; Murat, C.; Castanera, R.; Ramírez, L.; Sonnenberg, A.S.M.

    2013-01-01

    Repetitive DNA elements are ubiquitous constituents of eukaryotic genomes. The biological roles of these repetitive elements, supposed to impact genome organization and evolution, are not completely elucidated yet. The availability of whole genome sequence offers the opportunity to draw a picture of

  6. Effects of DNA mass on multiple displacement whole genome amplification and genotyping performance

    Directory of Open Access Journals (Sweden)

    Haque Kashif A

    2005-09-01

    Full Text Available Abstract Background Whole genome amplification (WGA promises to eliminate practical molecular genetic analysis limitations associated with genomic DNA (gDNA quantity. We evaluated the performance of multiple displacement amplification (MDA WGA using gDNA extracted from lymphoblastoid cell lines (N = 27 with a range of starting gDNA input of 1–200 ng into the WGA reaction. Yield and composition analysis of whole genome amplified DNA (wgaDNA was performed using three DNA quantification methods (OD, PicoGreen® and RT-PCR. Two panels of N = 15 STR (using the AmpFlSTR® Identifiler® panel and N = 49 SNP (TaqMan® genotyping assays were performed on each gDNA and wgaDNA sample in duplicate. gDNA and wgaDNA masses of 1, 4 and 20 ng were used in the SNP assays to evaluate the effects of DNA mass on SNP genotyping assay performance. A total of N = 6,880 STR and N = 56,448 SNP genotype attempts provided adequate power to detect differences in STR and SNP genotyping performance between gDNA and wgaDNA, and among wgaDNA produced from a range of gDNA templates inputs. Results The proportion of double-stranded wgaDNA and human-specific PCR amplifiable wgaDNA increased with increased gDNA input into the WGA reaction. Increased amounts of gDNA input into the WGA reaction improved wgaDNA genotyping performance. Genotype completion or genotype concordance rates of wgaDNA produced from all gDNA input levels were observed to be reduced compared to gDNA, although the reduction was not always statistically significant. Reduced wgaDNA genotyping performance was primarily due to the increased variance of allelic amplification, resulting in loss of heterozygosity or increased undetermined genotypes. MDA WGA produces wgaDNA from no template control samples; such samples exhibited substantial false-positive genotyping rates. Conclusion The amount of gDNA input into the MDA WGA reaction is a critical determinant of genotyping performance of wgaDNA. At least 10 ng of

  7. Protection of the genome and central protein-coding sequences by non-coding DNA against DNA damage from radiation.

    Science.gov (United States)

    Qiu, Guo-Hua

    2015-01-01

    Non-coding DNA comprises a very large proportion of the total genomic content in higher organisms, but its function remains largely unclear. Non-coding DNA sequences constitute the majority of peripheral heterochromatin, which has been hypothesized to be the genome's 'bodyguard' against DNA damage from chemicals and radiation for almost four decades. The bodyguard protective function of peripheral heterochromatin in genome defense has been strengthened by the results from numerous recent studies, which are summarized in this review. These data have suggested that cells and/or organisms with a higher level of heterochromatin and more non-coding DNA sequences, including longer telomeric DNA and rDNAs, exhibit a lower frequency of DNA damage, higher radioresistance and longer lifespan after IR exposure. In addition, the majority of heterochromatin is peripherally located in the three-dimensional structure of genome organization. Therefore, the peripheral heterochromatin with non-coding DNA could play a protective role in genome defense against DNA damage from ionizing radiation by both absorbing the radicals from water radiolysis in the cytosol and reducing the energy of IR. However, the bodyguard protection by heterochromatin has been challenged by the observation that DNA damage is less frequently detected in peripheral heterochromatin than in euchromatin, which is inconsistent with the expectation and simulation results. Previous studies have also shown that the DNA damage in peripheral heterochromatin is rarely repaired and moves more quickly, broadly and outwardly to approach the nuclear pore complex (NPC). Additionally, it has been shown that extrachromosomal circular DNAs (eccDNAs) are formed in the nucleus, highly detectable in the cytoplasm (particularly under stress conditions) and shuttle between the nucleus and the cytoplasm. Based on these studies, this review speculates that the sites of DNA damage in peripheral heterochromatin could occur more

  8. Integrated Microarray-based Tools for Detection of Genomic DNA Damage and Repair Mechanisms.

    Science.gov (United States)

    van Eijk, Patrick; Teng, Yumin; Bennet, Mark R; Evans, Katie E; Powell, James R; Webster, Richard M; Reed, Simon H

    2018-01-01

    The genetic information contained within the DNA molecule is highly susceptible to chemical and physical insult, caused by both endogenous and exogenous sources that can generate in the order of thousands of lesions a day in each of our cells (Lindahl, Nature 362(6422):709-715, 1993). DNA damages interfere with DNA metabolic processes such as transcription and replication and can be potent inhibitors of cell division and gene expression. To combat these regular threats to genome stability, a host of DNA repair mechanisms have evolved. When DNA lesions are left unrepaired due to defects in the repair pathway, mutations can arise that may alter the genetic information of the cell. DNA repair is thus fundamental to genome stability and defects in all the major repair pathways can lead to cancer predisposition. Therefore, the ability to accurately measure DNA damage at a genomic scale and determine the level, position, and rates of removal by DNA repair can contribute greatly to our understanding of how DNA repair in chromatin is organized throughout the genome. For this reason, we developed the 3D-DIP-Chip protocol described in this chapter. Conducting such measurements has potential applications in a variety of other fields, such as genotoxicity testing and cancer treatment using DNA damage inducing chemotherapy. Being able to detect and measure genomic DNA damage and repair patterns in individuals following treatment with chemotherapy could enable personalized medicine by predicting response to therapy.

  9. Cloning and sequencing of cDNA and genomic DNA encoding PDM phosphatase of Fusarium moniliforme.

    Science.gov (United States)

    Yoshida, Hiroshi; Iizuka, Mari; Narita, Takao; Norioka, Naoko; Norioka, Shigemi

    2006-12-01

    PDM phosphatase was purified approximately 500-fold through six steps from the extract of dried powder of the culture filtrate of Fusarium moniliforme. The purified preparation appeared homogeneous on SDS-PAGE although the protein band was broad. Amino acid sequence information was collected on tryptic peptides from this preparation. cDNA cloning was carried out based on the information. A full-length cDNA was obtained and sequenced. The sequence had an open reading frame of 651 amino acid residues with a molecular mass of 69,988 Da. Cloning and sequencing of the genomic DNA corresponding to the cDNA was also conducted. The deduced amino acid sequence could account for many but not all of the tryptic peptides, suggesting presence of contaminant protein(s). SDS-PAGE analysis after chemical deglycosylation showed two proteins with molecular masses of 58 and 68 kDa. This implied that the 58 kDa protein had been copurified with PDM phosphatase. Homology search showed that PDM phosphatase belongs to the purple acid phosphatase family, which is widely distributed in the biosphere. Sequence data of fungal purple acid phosphatases were collected from the database. Processing of the data revealed presence of two types, whose evolutionary relationships were discussed.

  10. Chemically synthesized silver nanoparticles as cell lysis agent for bacterial genomic DNA isolation

    Science.gov (United States)

    Goswami, Gunajit; Boruah, Himangshu; Gautom, Trishnamoni; Jyoti Hazarika, Dibya; Barooah, Madhumita; Boro, Robin Chandra

    2017-12-01

    Silver nanoparticles (AgNPs) have seen a recent spurt of use in varied fields of science. In this paper, we showed a novel application of AgNP as a promising microbial cell-lysis agent for genomic DNA isolation. We utilized chemically synthesized AgNPs for lysing bacterial cells to isolate their genomic DNA. The AgNPs efficiently lysed bacterial cells to yield good quality DNA that could be subsequently used for several molecular biology works.

  11. A Comparison of Fresh Frozen vs. Formalin-Fixed, Paraffin-Embedded Specimens of Canine Mammary Tumors via Branched-DNA Assay

    Directory of Open Access Journals (Sweden)

    Florenza Lüder Ripoli

    2016-05-01

    Full Text Available Mammary neoplasms are the tumors most affecting female dogs and women. Formalin-fixed, paraffin-embedded (FFPE tissues are an invaluable source of archived biological material. Fresh frozen (FF tissue is considered ideal for gene expression analysis. However, strategies based on FFPE material offer several advantages. Branched-DNA assays permit a reliable and fast workflow when analyzing gene expression. The aim of this study was to assess the comparability of the branched-DNA assay when analyzing certain gene expression patterns between FF and FFPE samples in canine mammary tumors. RNA was isolated from 109 FFPE samples and from 93 FF samples of different canine mammary tissues. Sixteen (16 target genes (Tp53; Myc; HMGA1; Pik3ca; Mcl1; MAPK3; FOXO3; PTEN; GATA4; PFDN5; HMGB1; MAPK1; BRCA2; BRCA1; HMGA2; and Her2 were analyzed via branched-DNA assay (b-DNA. ACTB, GAPDH, and HPRT1 were used as data normalizers. Overall, the relative gene expression of the two different origins of samples showed an agreement of 63%. Still, care should be taken, as FFPE specimens showed lower expression of the analyzed targets when compared to FF samples. The fact that the gene expression in FFPE proved to be lower than in FF specimens is likely to have been caused by the effect of storage time. ACTB had the best performance as a data normalizer.

  12. The logic of DNA replication in double-stranded DNA viruses: insights from global analysis of viral genomes.

    Science.gov (United States)

    Kazlauskas, Darius; Krupovic, Mart; Venclovas, Česlovas

    2016-06-02

    Genomic DNA replication is a complex process that involves multiple proteins. Cellular DNA replication systems are broadly classified into only two types, bacterial and archaeo-eukaryotic. In contrast, double-stranded (ds) DNA viruses feature a much broader diversity of DNA replication machineries. Viruses differ greatly in both completeness and composition of their sets of DNA replication proteins. In this study, we explored whether there are common patterns underlying this extreme diversity. We identified and analyzed all major functional groups of DNA replication proteins in all available proteomes of dsDNA viruses. Our results show that some proteins are common to viruses infecting all domains of life and likely represent components of the ancestral core set. These include B-family polymerases, SF3 helicases, archaeo-eukaryotic primases, clamps and clamp loaders of the archaeo-eukaryotic type, RNase H and ATP-dependent DNA ligases. We also discovered a clear correlation between genome size and self-sufficiency of viral DNA replication, the unanticipated dominance of replicative helicases and pervasive functional associations among certain groups of DNA replication proteins. Altogether, our results provide a comprehensive view on the diversity and evolution of replication systems in the DNA virome and uncover fundamental principles underlying the orchestration of viral DNA replication. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Genomic and functional integrity of the hematopoietic system requires tolerance of oxidative DNA lesions

    NARCIS (Netherlands)

    Martín-Pardillos, Ana; Tsaalbi-Shtylik, Anastasia; Chen, Si; Lazare, Seka; van Os, Ronald P; Dethmers-Ausema, Albertina; Fakouri, Nima Borhan; Bosshard, Matthias; Aprigliano, Rossana; van Loon, Barbara; Salvatori, Daniela C F; Hashimoto, Keiji; Dingemanse-van der Spek, Celia; Moriya, Masaaki; Rasmussen, Lene Juel; de Haan, Gerald; Raaijmakers, Marc H G P; de Wind, Niels

    2017-01-01

    Endogenous DNA damage is causally associated with the functional decline and transformation of stem cells that characterize aging. DNA lesions that have escaped DNA repair can induce replication stress and genomic breaks that induce senescence and apoptosis. It is not clear how stem and

  14. De novo assembly and characterization of the carrot mitochondrial genome using next generation sequencing data from whole genomic DNA provides first evidence of DNA transfer into an angiosperm plastid genome

    Science.gov (United States)

    Sequence analysis of organelle genomes has revealed important aspects of plant cell evolution. The scope of this study was to develop an approach for de novo assembly of the carrot mitochondrial genome using next generation sequence data from total genomic DNA. Sequencing data from a carrot 454 whol...

  15. The Comet assay for detection of DNA damage in canine sperm.

    Science.gov (United States)

    Pereira, A F; Borges, P; Fontbonne, A; Cardoso, L; Gaivão, I; Martins-Bessa, A

    2017-12-01

    Sperm DNA integrity is a fundamental prerequisite in fertilization and embryo development. Among DNA integrity tests, the Comet assay is an accurate and sensitive test for the detection of sperm oxidative damage. The aim of this work was to evaluate sperm oxidative damage using the Comet assay and to study the correlation between Comet and routine assays for the evaluation of semen quality. Dogs were divided in two groups: group A (n = 6), comprising dogs with abnormal spermiogram, that is astheno-, terato- or oligoasthenoteratozoospermic (OAT); and group B (n = 8), comprising normospermic dogs. The distribution of sperm oxidative damage was significantly different between the two groups (p = .001): group A-median: 31.55%, interquartile range (IQR): 30.18-38.01; group B-median: 0.90%, IQR: 0.65-1.96. The correlation between oxidative damage and abnormal morphology was high (r = .846; p sperm quality, the Comet assay has ample potential for clinical and research purposes in dogs. © 2017 Blackwell Verlag GmbH.

  16. Immunological comparison of DNA vaccination using two delivery systems against canine leishmaniasis.

    Science.gov (United States)

    Shahbazi, Mehdi; Zahedifard, Farnaz; Saljoughian, Noushin; Doroud, Delaram; Jamshidi, Shahram; Mahdavi, Niousha; Shirian, Sadegh; Daneshbod, Yahya; Hamid Zarkesh-Esfahani, Sayyed; Papadopoulou, Barbara; Rafati, Sima

    2015-09-15

    Visceral leishmaniasis (VL) is a fatal disease caused by the intracellular protozoan parasite Leishmania infantum. Dogs are the primary reservoirs of this parasite, and vaccination of dogs could be an effective method to reduce its transfer to humans. In order to develop a vaccine against VL (apart from the choice of immunogenic candidate antigens), it is necessary to use an appropriate delivery system to promote a proper antigen-specific immune response. In this study, we compared two vaccine delivery systems, namely electroporation and cationic solid-lipid nanoparticle (cSLN) formulation, to administer a DNA vaccine containing the Leishmania donovani A2 antigen, and L. infantum cysteine proteinases of type I (CPA) and II (CPB) without its unusual C-terminal extension. The protective potencies of these two vaccine delivery systems were evaluated against L. infantum challenge in outbred dogs. Our results show that the administration of pcDNA-A2-CPA-CPB(-CTE)GFP vaccine as a prime-boost by either electroporation or cSLN formulation protects the dogs against L. infantum infection. Partial protection in vaccinated dogs is associated with significantly (pleishmaniasis. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Global mapping of protein-DNA interactions in vivo by digital genomic footprinting.

    Science.gov (United States)

    Hesselberth, Jay R; Chen, Xiaoyu; Zhang, Zhihong; Sabo, Peter J; Sandstrom, Richard; Reynolds, Alex P; Thurman, Robert E; Neph, Shane; Kuehn, Michael S; Noble, William S; Fields, Stanley; Stamatoyannopoulos, John A

    2009-04-01

    The orchestrated binding of transcriptional activators and repressors to specific DNA sequences in the context of chromatin defines the regulatory program of eukaryotic genomes. We developed a digital approach to assay regulatory protein occupancy on genomic DNA in vivo by dense mapping of individual DNase I cleavages from intact nuclei using massively parallel DNA sequencing. Analysis of >23 million cleavages across the Saccharomyces cerevisiae genome revealed thousands of protected regulatory protein footprints, enabling de novo derivation of factor binding motifs and the identification of hundreds of new binding sites for major regulators. We observed striking correspondence between single-nucleotide resolution DNase I cleavage patterns and protein-DNA interactions determined by crystallography. The data also yielded a detailed view of larger chromatin features including positioned nucleosomes flanking factor binding regions. Digital genomic footprinting should be a powerful approach to delineate the cis-regulatory framework of any organism with an available genome sequence.

  18. An Adenovirus DNA Replication Factor, but Not Incoming Genome Complexes, Targets PML Nuclear Bodies.

    Science.gov (United States)

    Komatsu, Tetsuro; Nagata, Kyosuke; Wodrich, Harald

    2015-11-25

    Promyelocytic leukemia protein nuclear bodies (PML-NBs) are subnuclear domains implicated in cellular antiviral responses. Despite the antiviral activity, several nuclear replicating DNA viruses use the domains as deposition sites for the incoming viral genomes and/or as sites for viral DNA replication, suggesting that PML-NBs are functionally relevant during early viral infection to establish productive replication. Although PML-NBs and their components have also been implicated in the adenoviral life cycle, it remains unclear whether incoming adenoviral genome complexes target PML-NBs. Here we show using immunofluorescence and live-cell imaging analyses that incoming adenovirus genome complexes neither localize at nor recruit components of PML-NBs during early phases of infection. We further show that the viral DNA binding protein (DBP), an early expressed viral gene and essential DNA replication factor, independently targets PML-NBs. We show that DBP oligomerization is required to selectively recruit the PML-NB components Sp100 and USP7. Depletion experiments suggest that the absence of one PML-NB component might not affect the recruitment of other components toward DBP oligomers. Thus, our findings suggest a model in which an adenoviral DNA replication factor, but not incoming viral genome complexes, targets and modulates PML-NBs to support a conducive state for viral DNA replication and argue against a generalized concept that PML-NBs target incoming viral genomes. The immediate fate upon nuclear delivery of genomes of incoming DNA viruses is largely unclear. Early reports suggested that incoming genomes of herpesviruses are targeted and repressed by PML-NBs immediately upon nuclear import. Genome localization and/or viral DNA replication has also been observed at PML-NBs for other DNA viruses. Thus, it was suggested that PML-NBs may immediately sense and target nuclear viral genomes and hence serve as sites for deposition of incoming viral genomes and

  19. The emerging role of nuclear architecture in DNA repair and genome maintenance.

    Science.gov (United States)

    Misteli, Tom; Soutoglou, Evi

    2009-04-01

    DNA repair and maintenance of genome stability are crucial to cellular and organismal function, and defects in these processes have been implicated in cancer and ageing. Detailed molecular, biochemical and genetic analyses have outlined the molecular framework involved in cellular DNA-repair pathways, but recent cell-biological approaches have revealed important roles for the spatial and temporal organization of the DNA-repair machinery during the recognition of DNA lesions and the assembly of repair complexes. It has also become clear that local higher-order chromatin structure, chromatin dynamics and non-random global genome organization are key factors in genome maintenance. These cell-biological features of DNA repair illustrate an emerging role for nuclear architecture in multiple aspects of genome maintenance.

  20. [Study on potential anti-caries DNA vaccine pcDNA3-gtfB integration into host cell genome].

    Science.gov (United States)

    Yang, Jinbo; Liu, Tianjia; Li, Jiyao

    2003-06-01

    Gene vaccine security is of concern because of the possibility of insertion mutagenesis resulting in inactivation of tumor suppressor or activation of oncogene. The purpose of this study was to examine the potential of anti-caries DNA vaccine pcDNA3-gtfB integrating into the host cell genome. Anti-caries DNA vaccine pcDNA3-gtfB was constructed by the previous study. The gtfB gene(904-4,578 bp, genebank M17361) was cloned from Streptococcus mutans GS-5. 36 Wistar rats were divided into 2 groups: submandibular gland-targeted injection(SGT) group and control group. Rats in SGT group were injected with 100 micrograms of plasmid pcDNA3-gtfB, rats in control group with PBS solution. Genomes from submandibular gland, kidney, heart, liver, lung, and brain tissues were isolated later in 12 weeks. Genomes from different tissues were purified by low-melting agarose electrophoresis. Using the purified genomes as template, plasmid integration were examined by PCR(upper primer: 5'-ATATGGTACCATGACCGAAGCGACATCTAAGCAAGA-3', lower primer: 5'-ACTACTCGAGTTAGAACCATTGACCCTG AGCATTGC-3'). The sensitivity level of PCR was determined by adding gradient plasmid copies into genomes in control group. The examination of 6 tissues failed in revealing any evidence of integration at the sensitivity level that could detect 1 copy integration in 10,000 nuclei. The potential frequency of plasmid pcDNA3-gtfB integration into host cell genome would not exceed that of the spontaneous mutation. It was indicated that pcDNA3-gtfB was genetically safe as a promising anti-carious DNA vaccine.

  1. Rapid and reliable extraction of genomic DNA from various wild-type and transgenic plants

    Directory of Open Access Journals (Sweden)

    Yang Moon-Sik

    2004-09-01

    Full Text Available Abstract Background DNA extraction methods for PCR-quality DNA from calluses and plants are not time efficient, since they require that the tissues be ground in liquid nitrogen, followed by precipitation of the DNA pellet in ethanol, washing and drying the pellet, etc. The need for a rapid and simple procedure is urgent, especially when hundreds of samples need to be analyzed. Here, we describe a simple and efficient method of isolating high-quality genomic DNA for PCR amplification and enzyme digestion from calluses, various wild-type and transgenic plants. Results We developed new rapid and reliable genomic DNA extraction method. With our developed method, plant genomic DNA extraction could be performed within 30 min. The method was as follows. Plant tissue was homogenized with salt DNA extraction buffer using hand-operated homogenizer and extracted by phenol:chloroform:isoamyl alcohol (25:24:1. After centrifugation, the supernatant was directly used for DNA template for PCR, resulting in successful amplification for RAPD from various sources of plants and specific foreign genes from transgenic plants. After precipitating the supernatant, the DNA was completely digested by restriction enzymes. Conclusion This DNA extraction procedure promises simplicity, speed, and efficiency, both in terms of time and the amount of plant sample required. In addition, this method does not require expensive facilities for plant genomic DNA extraction.

  2. Whole genome duplications and a 'function' for junk DNA? Facts and hypotheses.

    Directory of Open Access Journals (Sweden)

    Reiner A Veitia

    Full Text Available BACKGROUND: The lack of correlation between genome size and organismal complexity is understood in terms of the massive presence of repetitive and non-coding DNA. This non-coding subgenome has long been called "junk" DNA. However, it might have important functions. Generation of junk DNA depends on proliferation of selfish DNA elements and on local or global DNA duplication followed by genic non-functionalization. METHODOLOGY/PRINCIPAL FINDINGS: Evidence from genomic analyses and experimental data indicates that Whole Genome Duplications (WGD are often followed by a return to the diploid state, through DNA deletions and intra/interchromosomal rearrangements. We use simple theoretical models and simulations to explore how a WGD accompanied by sequence deletions might affect the dosage balance often required among several gene products involved in regulatory processes. We find that potential genomic deletions leading to changes in nuclear and cell volume might potentially perturb gene dosage balance. CONCLUSIONS/SIGNIFICANCE: The potentially negative impact of DNA deletions can be buffered if deleted genic DNA is, at least temporarily, replaced by repetitive DNA so that the nuclear/cell volume remains compatible with normal living. Thus, we speculate that retention of non-functionalized non-coding DNA, and replacement of deleted DNA through proliferation of selfish elements, might help avoid dosage imbalances in cycles of polyploidization and diploidization, which are particularly frequent in plants.

  3. Genome-Wide Association Mapping for Intelligence in Military Working Dogs: Canine Cohort, Canine Intelligence Assessment Regimen, Genome-Wide Single Nucleotide Polymorphism (SNP) Typing, and Unsupervised Classification Algorithm for Genome-Wide Association Data Analysis

    Science.gov (United States)

    2011-09-01

    Almasy, L, Blangero, J. (2009) Human QTL linkage mapping. Genetica 136:333-340. Amos, CI. (2007) Successful design and conduct of genome-wide...quantitative trait loci. Genetica 136:237-243. Skol AD, Scott LJ, Abecasis GR, Boehnke M. (2006) Joint analysis is more efficient than replication

  4. Molecular analysis and genomic organization of major DNA satellites in banana (Musa spp..

    Directory of Open Access Journals (Sweden)

    Jana Čížková

    Full Text Available Satellite DNA sequences consist of tandemly arranged repetitive units up to thousands nucleotides long in head-to-tail orientation. The evolutionary processes by which satellites arise and evolve include unequal crossing over, gene conversion, transposition and extra chromosomal circular DNA formation. Large blocks of satellite DNA are often observed in heterochromatic regions of chromosomes and are a typical component of centromeric and telomeric regions. Satellite-rich loci may show specific banding patterns and facilitate chromosome identification and analysis of structural chromosome changes. Unlike many other genomes, nuclear genomes of banana (Musa spp. are poor in satellite DNA and the information on this class of DNA remains limited. The banana cultivars are seed sterile clones originating mostly from natural intra-specific crosses within M. acuminata (A genome and inter-specific crosses between M. acuminata and M. balbisiana (B genome. Previous studies revealed the closely related nature of the A and B genomes, including similarities in repetitive DNA. In this study we focused on two main banana DNA satellites, which were previously identified in silico. Their genomic organization and molecular diversity was analyzed in a set of nineteen Musa accessions, including representatives of A, B and S (M. schizocarpa genomes and their inter-specific hybrids. The two DNA satellites showed a high level of sequence conservation within, and a high homology between Musa species. FISH with probes for the satellite DNA sequences, rRNA genes and a single-copy BAC clone 2G17 resulted in characteristic chromosome banding patterns in M. acuminata and M. balbisiana which may aid in determining genomic constitution in interspecific hybrids. In addition to improving the knowledge on Musa satellite DNA, our study increases the number of cytogenetic markers and the number of individual chromosomes, which can be identified in Musa.

  5. Molecular analysis and genomic organization of major DNA satellites in banana (Musa spp.).

    Science.gov (United States)

    Čížková, Jana; Hřibová, Eva; Humplíková, Lenka; Christelová, Pavla; Suchánková, Pavla; Doležel, Jaroslav

    2013-01-01

    Satellite DNA sequences consist of tandemly arranged repetitive units up to thousands nucleotides long in head-to-tail orientation. The evolutionary processes by which satellites arise and evolve include unequal crossing over, gene conversion, transposition and extra chromosomal circular DNA formation. Large blocks of satellite DNA are often observed in heterochromatic regions of chromosomes and are a typical component of centromeric and telomeric regions. Satellite-rich loci may show specific banding patterns and facilitate chromosome identification and analysis of structural chromosome changes. Unlike many other genomes, nuclear genomes of banana (Musa spp.) are poor in satellite DNA and the information on this class of DNA remains limited. The banana cultivars are seed sterile clones originating mostly from natural intra-specific crosses within M. acuminata (A genome) and inter-specific crosses between M. acuminata and M. balbisiana (B genome). Previous studies revealed the closely related nature of the A and B genomes, including similarities in repetitive DNA. In this study we focused on two main banana DNA satellites, which were previously identified in silico. Their genomic organization and molecular diversity was analyzed in a set of nineteen Musa accessions, including representatives of A, B and S (M. schizocarpa) genomes and their inter-specific hybrids. The two DNA satellites showed a high level of sequence conservation within, and a high homology between Musa species. FISH with probes for the satellite DNA sequences, rRNA genes and a single-copy BAC clone 2G17 resulted in characteristic chromosome banding patterns in M. acuminata and M. balbisiana which may aid in determining genomic constitution in interspecific hybrids. In addition to improving the knowledge on Musa satellite DNA, our study increases the number of cytogenetic markers and the number of individual chromosomes, which can be identified in Musa.

  6. Tax impairs DNA replication forks and increases DNA breaks in specific oncogenic genome regions.

    Science.gov (United States)

    Chaib-Mezrag, Hassiba; Lemaçon, Delphine; Fontaine, Hélène; Bellon, Marcia; Bai, Xue Tao; Drac, Marjorie; Coquelle, Arnaud; Nicot, Christophe

    2014-09-04

    Human T-cell leukemia virus type 1 (HTLV-I) is a human retrovirus associated with adult T-cell leukemia (ATL), an aggressive CD4 T-cell proliferative disease with dismal prognosis. The long latency preceding the development of the disease and the low incidence suggests that the virus itself is not sufficient for transformation and that genetic defects are required to create a permissive environment for leukemia. In fact, ATL cells are characterized by profound genetic modifications including structural and numerical chromosome alterations. In this study we used molecular combing techniques to study the effect of the oncoprotein Tax on DNA replication. We found that replication forks have difficulties replicating complex DNA, fork progression is slower, and they pause or stall more frequently in the presence of Tax expression. Our results also show that Tax-associated replication defects are partially compensated by an increase in the firing of back-up origins. Consistent with these effects of Tax on DNA replication, an increase in double strand DNA breaks (DDSB) was seen in Tax expressing cells. Tax-mediated increases in DDSBs were associated with the ability of Tax to activate NF-kB and to stimulate intracellular nitric oxide production. We also demonstrated a reduced expression of human translesion synthesis (TLS) DNA polymerases Pol-H and Pol-K in HTLV-I-transformed T cells and ATL cells. This was associated with an increase in DNA breaks induced by Tax at specific genome regions, such as the c-Myc and the Bcl-2 major breakpoints. Consistent with the notion that the non-homologous end joining (NHEJ) pathway is hyperactive in HTLV-I-transformed cells, we found that inhibition of the NHEJ pathway induces significant killing of HTLV-I transformed cells and patient-derived leukemic ATL cells. Our results suggest that, replication problems increase genetic instability in HTLV-I-transformed cells. As a result, abuse of NHEJ and a defective homologous repair (HR) DNA

  7. Epigenetic control of mobile DNA as an interface between experience and genome change

    Directory of Open Access Journals (Sweden)

    James A. Shapiro

    2014-04-01

    Full Text Available Mobile DNA in the genome is subject to RNA-targeted epigenetic control. This control regulates the activity of transposons, retrotransposons and genomic proviruses. Many different life history experiences alter the activities of mobile DNA and the expression of genetic loci regulated by nearby insertions. The same experiences induce alterations in epigenetic formatting and lead to trans-generational modifications of genome expression and stability. These observations lead to the hypothesis that epigenetic formatting directed by non-coding RNA provides a molecular interface between life history events and genome alteration.

  8. The emerging role of nuclear architecture in DNA repair and genome maintenance

    OpenAIRE

    Misteli, Tom; Soutoglou, Evi

    2009-01-01

    DNA repair and maintenance of genome stability are crucial to cellular and organismal function, and defects in these processes have been implicated in cancer and ageing. Detailed molecular, biochemical and genetic analyses have outlined the molecular framework involved in cellular DNA-repair pathways, but recent cell-biological approaches have revealed important roles for the spatial and temporal organization of the DNA-repair machinery during the recognition of DNA lesions and the assembly o...

  9. Pre-diagnostic leukocyte genomic DNA methylation and the risk of colorectal cancer in women.

    Directory of Open Access Journals (Sweden)

    Hongmei Nan

    Full Text Available Abnormal one-carbon metabolism may lead to general genomic (global hypomethylation, which may predispose an individual to the development of colorectal neoplasia.We evaluated the association between pre-diagnostic leukocyte genomic DNA methylation level and the risk of colorectal cancer in a nested case-control study of 358 colorectal cancer cases and 661 matched controls within the all-female cohort of the Nurses' Health Study (NHS. Among control subjects, we further examined major plasma components in the one-carbon metabolism pathway in relation to genomic DNA methylation level. Liquid chromatography/tandem mass spectrometry was used to examine leukocyte genomic DNA methylation level. We calculated odds ratios (ORs and 95% confidence intervals (95% CIs using logistic regression.Overall genomic DNA methylation level was not associated with the risk of colorectal cancer (p for trend, 0.45. Compared with women in the lowest quintile of methylation, the multivariate OR of colorectal cancer risk was 1.32 (95% CI, 0.82-2.13 for those in the highest quintile. We did not find significant associations between major plasma components of one-carbon metabolism or risk factors for colorectal cancer and genomic DNA methylation level (all p for trend >0.05. Also, neither one-carbon metabolism-related plasma components nor well-known risk factors for colorectal cancer modified the association between genomic DNA methylation level and the risk of colorectal cancer (all p for interaction >0.05.We found no evidence that hypomethylation of leukocyte genomic DNA increases risk of colorectal cancer among women. Additional studies are needed to investigate the association between pre-diagnostic genomic DNA methylation level and colorectal cancer risk among diverse populations.

  10. Genome DNA Sequence Variation, Evolution, and Function in Bacteria and Archaea.

    Science.gov (United States)

    Nishida, Hiromi

    2013-01-01

    Comparative genomics has revealed that variations in bacterial and archaeal genome DNA sequences cannot be explained by only neutral mutations. Virus resistance and plasmid distribution systems have resulted in changes in bacterial and archaeal genome sequences during evolution. The restriction-modification system, a virus resistance system, leads to avoidance of palindromic DNA sequences in genomes. Clustered, regularly interspaced, short palindromic repeats (CRISPRs) found in genomes represent yet another virus resistance system. Comparative genomics has shown that bacteria and archaea have failed to gain any DNA with GC content higher than the GC content of their chromosomes. Thus, horizontally transferred DNA regions have lower GC content than the host chromosomal DNA does. Some nucleoid-associated proteins bind DNA regions with low GC content and inhibit the expression of genes contained in those regions. This form of gene repression is another type of virus resistance system. On the other hand, bacteria and archaea have used plasmids to gain additional genes. Virus resistance systems influence plasmid distribution. Interestingly, the restriction-modification system and nucleoid-associated protein genes have been distributed via plasmids. Thus, GC content and genomic signatures do not reflect bacterial and archaeal evolutionary relationships.

  11. Phylogenetic relationships in three species of canine Demodex mite based on partial sequences of mitochondrial 16S rDNA.

    Science.gov (United States)

    Sastre, Natalia; Ravera, Ivan; Villanueva, Sergio; Altet, Laura; Bardagí, Mar; Sánchez, Armand; Francino, Olga; Ferrer, Lluís

    2012-12-01

    The historical classification of Demodex mites has been based on their hosts and morphological features. Genome sequencing has proved to be a very effective taxonomic tool in phylogenetic studies and has been applied in the classification of Demodex. Mitochondrial 16S rDNA has been demonstrated to be an especially useful marker to establish phylogenetic relationships. To amplify and sequence a segment of the mitochondrial 16S rDNA from Demodex canis and Demodex injai, as well as from the short-bodied mite called, unofficially, D. cornei and to determine their genetic proximity. Demodex mites were examined microscopically and classified as Demodex folliculorum (one sample), D. canis (four samples), D. injai (two samples) or the short-bodied species D. cornei (three samples). DNA was extracted, and a 338 bp fragment of the 16S rDNA was amplified and sequenced. The sequences of the four D. canis mites were identical and shared 99.6 and 97.3% identity with two D. canis sequences available at GenBank. The sequences of the D. cornei isolates were identical and showed 97.8, 98.2 and 99.6% identity with the D. canis isolates. The sequences of the two D. injai isolates were also identical and showed 76.6% identity with the D. canis sequence. Demodex canis and D. injai are two different species, with a genetic distance of 23.3%. It would seem that the short-bodied Demodex mite D. cornei is a morphological variant of D. canis. © 2012 The Authors. Veterinary Dermatology © 2012 ESVD and ACVD.

  12. Toxicological effects of benzo[a]pyrene on DNA methylation of whole genome in ICR mice.

    Science.gov (United States)

    Zhao, L; Zhang, S; An, X; Tan, W; Pang, D; Ouyang, H

    2015-10-30

    It has been well known that alterations in DNA methylation - an important regulator of gene transcription - lead to cancer. Therefore a change in the level of DNA methylation of whole genome has been considered as a biomarker of carcinogenesis. Previously, a large number of experimental results in genetic toxicology have showed that benzo[a]pyrene could cause DNA mutation and fragmentation. However, there was little to no studies on alterations in DNA methylation of genome directly result from exposure to benzo[a]pyrene. In this paper, possible mechanisms of alterations in whole genomic DNA methylation by benzo[a]pyrene were investigated using ICR mice after benzo[a]pyrene exposure. The blood, liver, pancreas, skin, lung and bladder of ICR mice were removed and checked after a fixed time interval (6 hours) of benzo[a]pyrene exposure, and whole genomic DNA methylation level was determined by high performance liquid chromatography (HPLC). The results exhibited tissue specificity, that is, the level of whole genomic DNA methylation decreases significantly in blood and liver, rather than pancreas, lung, skin and bladder of ICR mice. This study investigated the direct relationship between aberrant DNA methylation level and benzo[a]pyrene exposure, which might be helpful to clarify the toxicological mechanism of benzo[a]pyrene in epigenetic perspectives.

  13. Leaf storage conditions and genomic DNA isolation efficiency in ...

    African Journals Online (AJOL)

    Storage of plant tissues for DNA is important to avoid degradation of DNA. Preliminary studies were conducted on Ocimum gratissimum L. in order to establish the storage conditions for the collected samples before DNA extraction. Secondly, the aim was to determine the best protocol for the extraction of high quality DNA, ...

  14. Actionable mutations in canine hemangiosarcoma.

    Directory of Open Access Journals (Sweden)

    Guannan Wang

    Full Text Available Angiosarcomas (AS are rare in humans, but they are a deadly subtype of soft tissue sarcoma. Discovery sequencing in AS, especially the visceral form, is hampered by the rarity of cases. Most diagnostic material exists as archival formalin fixed, paraffin embedded tissue which serves as a poor source of high quality DNA for genome-wide sequencing. We approached this problem through comparative genomics. We hypothesized that exome sequencing a histologically similar tumor, hemangiosarcoma (HSA, that occurs in approximately 50,000 dogs per year, may lead to the identification of potential oncogenic drivers and druggable targets that could also occur in angiosarcoma.Splenic hemangiosarcomas are common in dogs, which allowed us to collect a cohort of archived matched tumor and normal tissue samples suitable for whole exome sequencing. Mapping of the reads to the latest canine reference genome (Canfam3 demonstrated that >99% of the targeted exomal regions were covered, with >80% at 20X coverage and >90% at 10X coverage.Sequence analysis of 20 samples identified somatic mutations in PIK3CA, TP53, PTEN, and PLCG1, all of which correspond to well-known tumor drivers in human cancer, in more than half of the cases. In one case, we identified a mutation in PLCG1 identical to a mutation observed previously in this gene in human visceral AS. Activating PIK3CA mutations present novel therapeutic targets, and clinical trials of targeted inhibitors are underway in human cancers. Our results lay a foundation for similar clinical trials in canine HSA, enabling a precision medicine approach to this disease.

  15. Actionable mutations in canine hemangiosarcoma.

    Science.gov (United States)

    Wang, Guannan; Wu, Ming; Maloneyhuss, Martha A; Wojcik, John; Durham, Amy C; Mason, Nicola J; Roth, David B

    2017-01-01

    Angiosarcomas (AS) are rare in humans, but they are a deadly subtype of soft tissue sarcoma. Discovery sequencing in AS, especially the visceral form, is hampered by the rarity of cases. Most diagnostic material exists as archival formalin fixed, paraffin embedded tissue which serves as a poor source of high quality DNA for genome-wide sequencing. We approached this problem through comparative genomics. We hypothesized that exome sequencing a histologically similar tumor, hemangiosarcoma (HSA), that occurs in approximately 50,000 dogs per year, may lead to the identification of potential oncogenic drivers and druggable targets that could also occur in angiosarcoma. Splenic hemangiosarcomas are common in dogs, which allowed us to collect a cohort of archived matched tumor and normal tissue samples suitable for whole exome sequencing. Mapping of the reads to the latest canine reference genome (Canfam3) demonstrated that >99% of the targeted exomal regions were covered, with >80% at 20X coverage and >90% at 10X coverage. Sequence analysis of 20 samples identified somatic mutations in PIK3CA, TP53, PTEN, and PLCG1, all of which correspond to well-known tumor drivers in human cancer, in more than half of the cases. In one case, we identified a mutation in PLCG1 identical to a mutation observed previously in this gene in human visceral AS. Activating PIK3CA mutations present novel therapeutic targets, and clinical trials of targeted inhibitors are underway in human cancers. Our results lay a foundation for similar clinical trials in canine HSA, enabling a precision medicine approach to this disease.

  16. Searching the Human Genome for Snail and Slug With DNA@Home

    OpenAIRE

    Zarns, Kristopher; Desell, Travis; Nechaev, Sergei; Dhasarathy, Archana

    2015-01-01

    DNA@Home is a volunteer computing project that aims to use Gibbs Sampling for the identification and location of DNA control signals on full genome-scale datasets. A fault tolerant and asynchronous implementation of Gibbs sampling using the Berkeley Open Infrastructure for Network Computing (BOINC) was used to identify the location of binding sites of the SNAI1 (Snail) and SNAI2 (Slug) transcription factors across the human genome. Genes regulated by Slug but not Snail, and genes regulated by...

  17. Substitutions of short heterologous DNA segments of intragenomic or extragenomic origins produce clustered genomic polymorphisms

    DEFF Research Database (Denmark)

    Harms, Klaus; Lunnan, Asbjørn; Hülter, Nils

    2016-01-01

    In a screen for unexplained mutation events we identified a previously unrecognized mechanism generating clustered DNA polymorphisms such as microindels and cumulative SNPs. The mechanism, short-patch double illegitimate recombination (SPDIR), facilitates short single-stranded DNA molecules to in...... gene transfer. The DNA replacements are nonreciprocal and locus independent. Bioinformatic approaches reveal occurrence of SPDIR events in the gram-positive human pathogen Streptococcus pneumoniae and in the human genome....... to invade and replace genomic DNA through two joint illegitimate recombination events. SPDIR is controlled by key components of the cellular genome maintenance machinery in the gram-negative bacterium Acinetobacter baylyi. The source DNA is primarily intragenomic but can also be acquired through horizontal...

  18. DNA Oncogenic Virus-Induced Oxidative Stress, Genomic Damage, and Aberrant Epigenetic Alterations

    Directory of Open Access Journals (Sweden)

    Mankgopo Magdeline Kgatle

    2017-01-01

    Full Text Available Approximately 20% of human cancers is attributable to DNA oncogenic viruses such as human papillomavirus (HPV, hepatitis B virus (HBV, and Epstein-Barr virus (EBV. Unrepaired DNA damage is the most common and overlapping feature of these DNA oncogenic viruses and a source of genomic instability and tumour development. Sustained DNA damage results from unceasing production of reactive oxygen species and activation of inflammasome cascades that trigger genomic changes and increased propensity of epigenetic alterations. Accumulation of epigenetic alterations may interfere with genome-wide cellular signalling machineries and promote malignant transformation leading to cancer development. Untangling and understanding the underlying mechanisms that promote these detrimental effects remain the major objectives for ongoing research and hope for effective virus-induced cancer therapy. Here, we review current literature with an emphasis on how DNA damage influences HPV, HVB, and EBV replication and epigenetic alterations that are associated with carcinogenesis.

  19. Targeted DNA methylation in pericentromeres with genome editing-based artificial DNA methyltransferase.

    Science.gov (United States)

    Yamazaki, Taiga; Hatano, Yu; Handa, Tetsuya; Kato, Sakiko; Hoida, Kensuke; Yamamura, Rui; Fukuyama, Takashi; Uematsu, Takayuki; Kobayashi, Noritada; Kimura, Hiroshi; Yamagata, Kazuo

    2017-01-01

    To study the impact of epigenetic changes on biological functions, the ability to manipulate the epigenetic status of certain genomic regions artificially could be an indispensable technology. "Epigenome editing" techniques have gradually emerged that apply TALE or CRISPR/Cas9 technologies with various effector domains isolated from epigenetic code writers or erasers such as DNA methyltransferase, 5-methylcytosine oxidase, and histone modification enzymes. Here we demonstrate that a TALE recognizing a major satellite, consisting of a repeated sequence in pericentromeres, could be fused with the bacterial CpG methyltransferase, SssI. ChIP-qPCR assays demonstrated that the fusion protein TALMaj-SssI preferentially bound to major chromosomal satellites in cultured cell lines. Then, TALMaj-SssI was expressed in fertilized mouse oocytes with hypomethylated major satellites (10-20% CpG islands). Bisulfite sequencing revealed that the DNA methylation status was increased specifically in major satellites (50-60%), but not in minor satellites or other repeat elements, such as Intracisternal A-particle (IAP) or long interspersed nuclear elements-1 (Line1) when the expression level of TALMaj-SssI is optimized in the cell. At a microscopic level, distal ends of chromosomes at the first mitotic stage were dramatically highlighted by the mCherry-tagged methyl CpG binding domain of human MBD1 (mCherry-MBD-NLS). Moreover, targeted DNA methylation to major satellites did not interfere with kinetochore function during early embryonic cleavages. Co-injection of dCas9 fused with SssI and guide RNA (gRNA) recognizing major satellite sequences enabled increment of the DNA methylation in the satellites, but a few off-target effects were also observed in minor satellites and retrotransposons. Although CRISPR can be applied instead of the TALE system, technical improvements to reduce off-target effects are required. We have demonstrated a new method of introducing DNA methylation without

  20. Targeted DNA methylation in pericentromeres with genome editing-based artificial DNA methyltransferase.

    Directory of Open Access Journals (Sweden)

    Taiga Yamazaki

    Full Text Available To study the impact of epigenetic changes on biological functions, the ability to manipulate the epigenetic status of certain genomic regions artificially could be an indispensable technology. "Epigenome editing" techniques have gradually emerged that apply TALE or CRISPR/Cas9 technologies with various effector domains isolated from epigenetic code writers or erasers such as DNA methyltransferase, 5-methylcytosine oxidase, and histone modification enzymes. Here we demonstrate that a TALE recognizing a major satellite, consisting of a repeated sequence in pericentromeres, could be fused with the bacterial CpG methyltransferase, SssI. ChIP-qPCR assays demonstrated that the fusion protein TALMaj-SssI preferentially bound to major chromosomal satellites in cultured cell lines. Then, TALMaj-SssI was expressed in fertilized mouse oocytes with hypomethylated major satellites (10-20% CpG islands. Bisulfite sequencing revealed that the DNA methylation status was increased specifically in major satellites (50-60%, but not in minor satellites or other repeat elements, such as Intracisternal A-particle (IAP or long interspersed nuclear elements-1 (Line1 when the expression level of TALMaj-SssI is optimized in the cell. At a microscopic level, distal ends of chromosomes at the first mitotic stage were dramatically highlighted by the mCherry-tagged methyl CpG binding domain of human MBD1 (mCherry-MBD-NLS. Moreover, targeted DNA methylation to major satellites did not interfere with kinetochore function during early embryonic cleavages. Co-injection of dCas9 fused with SssI and guide RNA (gRNA recognizing major satellite sequences enabled increment of the DNA methylation in the satellites, but a few off-target effects were also observed in minor satellites and retrotransposons. Although CRISPR can be applied instead of the TALE system, technical improvements to reduce off-target effects are required. We have demonstrated a new method of introducing DNA

  1. A systematic evaluation of whole genome amplification of bisulfite-modified DNA

    Directory of Open Access Journals (Sweden)

    Bundo Miki

    2012-11-01

    Full Text Available Abstract Background Studying DNA methylation profiles in detail should be the first step in epigenetic research. Although sodium bisulfite modification of genomic DNA is the gold standard method for DNA methylation analysis, this method results in the loss of the majority of the DNA material. Whole genome amplification (WGA of bisulfite-modified DNA is expected to provide a rich source of materials, but its validity has not been thoroughly evaluated. In this study, we evaluated the extent of biased amplification in the WGA of bisulfite-modified DNA and the reproducibility of independent WGA reactions. We performed the multiple displacement amplification-based WGA separately three times. Each experiment included two reactions using 10 or 50 ng of bisulfite-modified DNA as template. DNA methylation levels were compared between WGA products and original bisulfite-modified DNA at about 450,000 CpG sites. Results Using a sufficient amount of bisulfite-modified DNA for WGA was critical for downstream application. The considerable deviations from original bisulfite-modified DNA were found in the middle range of DNA methylation levels. Distribution of hyper- and hypomethylation were equal, which suggested that the deviation at each CpG site occurred randomly. Averaging the data from independently amplified WGA products dramatically improved the overall quality. Conclusions WGA of bisulfite-modified DNA could be a valuable tool for epigenetic research, but careful experimental design and data interpretation are required.

  2. Microsatellite DNA in genomic survey sequences and UniGenes of loblolly pine

    Science.gov (United States)

    Craig S Echt; Surya Saha; Dennis L Deemer; C Dana Nelson

    2011-01-01

    Genomic DNA sequence databases are a potential and growing resource for simple sequence repeat (SSR) marker development in loblolly pine (Pinus taeda L.). Loblolly pine also has many expressed sequence tags (ESTs) available for microsatellite (SSR) marker development. We compared loblolly pine SSR densities in genome survey sequences (GSSs) to those in non-redundant...

  3. Accurate DNA assembly and genome engineering with optimized uracil excision cloning

    DEFF Research Database (Denmark)

    Cavaleiro, Mafalda; Kim, Se Hyeuk; Seppala, Susanna

    2015-01-01

    that produces β-carotene to optimize assembly junctions and the uracil excision protocol. By combining uracil excision cloning with a genomic integration technology, we demonstrate that up to six DNA fragments can be assembled in a one-tube reaction for direct genome integration with high accuracy, greatly...... facilitating the advanced engineering of robust cell factories....

  4. Genome-Wide Motif Statistics are Shaped by DNA Binding Proteins over Evolutionary Time Scales

    Directory of Open Access Journals (Sweden)

    Long Qian

    2016-10-01

    Full Text Available The composition of a genome with respect to all possible short DNA motifs impacts the ability of DNA binding proteins to locate and bind their target sites. Since nonfunctional DNA binding can be detrimental to cellular functions and ultimately to organismal fitness, organisms could benefit from reducing the number of nonfunctional DNA binding sites genome wide. Using in vitro measurements of binding affinities for a large collection of DNA binding proteins, in multiple species, we detect a significant global avoidance of weak binding sites in genomes. We demonstrate that the underlying evolutionary process leaves a distinct genomic hallmark in that similar words have correlated frequencies, a signal that we detect in all species across domains of life. We consider the possibility that natural selection against weak binding sites contributes to this process, and using an evolutionary model we show that the strength of selection needed to maintain global word compositions is on the order of point mutation rates. Likewise, we show that evolutionary mechanisms based on interference of protein-DNA binding with replication and mutational repair processes could yield similar results and operate with similar rates. On the basis of these modeling and bioinformatic results, we conclude that genome-wide word compositions have been molded by DNA binding proteins acting through tiny evolutionary steps over time scales spanning millions of generations.

  5. A Clone ReArray System for DNA Finishing at the Joint Genome Institute

    OpenAIRE

    Pollard, Martin; Wilson, Steven; Roberts, Simon; Martinez, Michele

    2005-01-01

    A Clone ReArray System for DNA Finishing at the Joint Genome Institute, Martin Pollard, Steven Wilson, Simon Roberts, Michele Martinez, Department of Energy Joint Genome Institute. The Department of Energy Joint Genome Institute has designed and built a system to rearray clones from our sequencing libraries as part of our sequencing finishing process. The system consists of a Tecan Genesys 200 RSP and an automated refrigerator/freezer with a 1000 plate capacity. Library source plate and well ...

  6. Influence of pre-analytical procedures on genomic DNA integrity in blood samples: the SPIDIA experience.

    Science.gov (United States)

    Malentacchi, F; Ciniselli, C M; Pazzagli, M; Verderio, P; Barraud, L; Hartmann, C C; Pizzamiglio, S; Weisbuch, S; Wyrich, R; Gelmini, S

    2015-02-02

    DNA integrity is a critical part of the definition of genomic DNA (gDNA) quality and can influence downstream molecular applications. Pre-analytical variables as sample storage and DNA extraction methods can influence the quality and quantity of isolated DNA and affect molecular test performances. The aim of this paper is to investigate the role of blood sample storage and DNA extraction procedures on gDNA integrity and gDNA fragmentation impact on a molecular test. 157 DNA samples deriving from the Pan European 1st SPIDIA DNA External Quality Assessment (EQA), aimed to investigate the influence of blood storage on gDNA quality and quantity, have been analyzed by Pulsed Field Gel Electrophoresis and ImageJ imaging software. 157 DNA samples derived from the Pan European 1st SPIDIA DNA External Quality Assessment (EQA), which aimed to investigate the influence of blood storage on gDNA quality and quantity, have been analyzed by Pulsed Field Gel Electrophoresis and ImageJ imaging software. Our results demonstrate that blood sample storage and DNA extraction procedures influence gDNA integrity and that the same blood sample which underwent a long range multiplex PCR based analytical test can provide different results if the adopted pre-analytical procedures are not standardized. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Genome-Wide Negative Feedback Drives Transgenerational DNA Methylation Dynamics in Arabidopsis

    Science.gov (United States)

    Kassam, Mohamed; Duvernois-Berthet, Evelyne; Cortijo, Sandra; Takashima, Kazuya; Saze, Hidetoshi; Toyoda, Atsushi; Fujiyama, Asao; Colot, Vincent; Kakutani, Tetsuji

    2015-01-01

    Epigenetic variations of phenotypes, especially those associated with DNA methylation, are often inherited over multiple generations in plants. The active and inactive chromatin states are heritable and can be maintained or even be amplified by positive feedback in a transgenerational manner. However, mechanisms controlling the transgenerational DNA methylation dynamics are largely unknown. As an approach to understand the transgenerational dynamics, we examined long-term effect of impaired DNA methylation in Arabidopsis mutants of the chromatin remodeler gene DDM1 (Decrease in DNA Methylation 1) through whole genome DNA methylation sequencing. The ddm1 mutation induces a drastic decrease in DNA methylation of transposable elements (TEs) and repeats in the initial generation, while also inducing ectopic DNA methylation at hundreds of loci. Unexpectedly, this ectopic methylation can only be seen after repeated self-pollination. The ectopic cytosine methylation is found primarily in the non-CG context and starts from 3’ regions within transcription units and spreads upstream. Remarkably, when chromosomes with reduced DNA methylation were introduced from a ddm1 mutant into a DDM1 wild-type background, the ddm1-derived chromosomes also induced analogous de novo accumulation of DNA methylation in trans. These results lead us to propose a model to explain the transgenerational DNA methylation redistribution by genome-wide negative feedback. The global negative feedback, together with local positive feedback, would ensure robust and balanced differentiation of chromatin states within the genome. PMID:25902052

  8. Development of an efficient protocol for genomic DNA extraction from mango (Mangifera indica

    Directory of Open Access Journals (Sweden)

    MOHAMMAD AHSANUL KABIR

    2011-11-01

    Full Text Available Majumder DAN, Hassan L, Rahim MA, Kabir MA. 2011. Development of an efficient protocol for genomic DNA extraction from mango (Mangifera indica. Nusantara Bioscience 3: 105-111. A simple and efficient method for genomic DNA extraction from woody fruit crops containing high polysaccharide levels has been described here. In the present study, three kinds of plant DNA extraction protocols were studied and the target was to establish the water-saturated ether (WSE with 1.25 M NaCl method as the most efficient protocol for removing the highly concentrated polysaccharides from genomic DNA of woody fruit crops. This method involves the modified CTAB or SDS procedure employing a purification step to remove polysaccharides using the WSE method. Precipitation with an equal volume of isopropanol caused a DNA pellet to form. After being washed with 70% ethyl alcohol, the pellet became easily dissolved in TE buffer. Using these three methods, DNA was extracted from samples of 60 mango genotypes, including young, mature, old, frosted old and withered old leaves. Compared with the three studied DNA extraction protocols of mango, it was found that the WSE method with NaCl had the highest value of average percentage (85.44% in DNA content of the mango genotypes. The average yield of DNA ranged from 5.05 µg/µL to11.28 µg/µL. DNA was suitable for PCR and RAPD analyses and long-term storage for further use.

  9. Genome-wide negative feedback drives transgenerational DNA methylation dynamics in Arabidopsis.

    Science.gov (United States)

    Ito, Tasuku; Tarutani, Yoshiaki; To, Taiko Kim; Kassam, Mohamed; Duvernois-Berthet, Evelyne; Cortijo, Sandra; Takashima, Kazuya; Saze, Hidetoshi; Toyoda, Atsushi; Fujiyama, Asao; Colot, Vincent; Kakutani, Tetsuji

    2015-04-01

    Epigenetic variations of phenotypes, especially those associated with DNA methylation, are often inherited over multiple generations in plants. The active and inactive chromatin states are heritable and can be maintained or even be amplified by positive feedback in a transgenerational manner. However, mechanisms controlling the transgenerational DNA methylation dynamics are largely unknown. As an approach to understand the transgenerational dynamics, we examined long-term effect of impaired DNA methylation in Arabidopsis mutants of the chromatin remodeler gene DDM1 (Decrease in DNA Methylation 1) through whole genome DNA methylation sequencing. The ddm1 mutation induces a drastic decrease in DNA methylation of transposable elements (TEs) and repeats in the initial generation, while also inducing ectopic DNA methylation at hundreds of loci. Unexpectedly, this ectopic methylation can only be seen after repeated self-pollination. The ectopic cytosine methylation is found primarily in the non-CG context and starts from 3' regions within transcription units and spreads upstream. Remarkably, when chromosomes with reduced DNA methylation were introduced from a ddm1 mutant into a DDM1 wild-type background, the ddm1-derived chromosomes also induced analogous de novo accumulation of DNA methylation in trans. These results lead us to propose a model to explain the transgenerational DNA methylation redistribution by genome-wide negative feedback. The global negative feedback, together with local positive feedback, would ensure robust and balanced differentiation of chromatin states within the genome.

  10. Highly sensitive polymerase chain reaction-free quantum dot-based quantification of forensic genomic DNA.

    Science.gov (United States)

    Tak, Yu Kyung; Kim, Won Young; Kim, Min Jung; Han, Eunyoung; Han, Myun Soo; Kim, Jong Jin; Kim, Wook; Lee, Jong Eun; Song, Joon Myong

    2012-04-06

    Forensic DNA samples can degrade easily due to exposure to light and moisture at the crime scene. In addition, the amount of DNA acquired at a criminal site is inherently limited. This limited amount of human DNA has to be quantified accurately after the process of DNA extraction. The accurately quantified extracted genomic DNA is then used as a DNA template in polymerase chain reaction (PCR) amplification for short tandem repeat (STR) human identification. Accordingly, highly sensitive and human-specific quantification of forensic DNA samples is an essential issue in forensic study. In this work, a quantum dot (Qdot)-labeled Alu sequence was developed as a probe to simultaneously satisfy both the high sensitivity and human genome selectivity for quantification of forensic DNA samples. This probe provided PCR-free determination of human genomic DNA and had a 2.5-femtogram detection limit due to the strong emission and photostability of the Qdot. The Qdot-labeled Alu sequence has been used successfully to assess 18 different forensic DNA samples for STR human identification. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. Epigenetic changes of Arabidopsis genome associated with altered DNA methyltransferase and demethylase expressions after gamma irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Ji Eun; Cho, Eun Ju; Kim, Ji Hong; Chung, Byung Yeoup; Kim, Jin Hong [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2012-05-15

    DNA methylation at carbon 5 of cytosines is a hall mark of epigenetic inactivation and heterochromatin in both plants and mammals. In Arabidopsis, DNA methylation has two roles that protect the genome from selfish DNA elements and regulate gene expression. Plant genome has three types of DNA methyltransferase, METHYLTRANSFERASE 1 (MET1), DOMAINREARRANGED METHYLASE (DRM) and CHROMOMETHYLASE 3 (CMT3) that are capable of methylating CG, CHG (where H is A, T, or C) and CHH sites, respectively. MET1 is a maintenance DNA methyltransferase that controls CG methylation. Two members of the DRM family, DRM1 and DRM2, are responsible for de novo methylation of CG, CHG, and CHH sites but show a preference for CHH sites. Finally, CMT3 principally carries out CHG methylation and is involved in both de novo methylation and maintenance. Alternatively, active DNA demethylation may occur through the glycosylase activity by removing the methylcytosines from DNA. It may have essential roles in preventing transcriptional silencing of transgenes and endogenous genes and in activating the expression of imprinted genes. DNA demetylation in Arabidopsis is mediated by the DEMETER (DME) family of bifunctional DNA glycosylase. Three targets of DME are MEA (MEDEA), FWA (FLOWERING WAGENINGEN), and FIS2 (FERTILIZATION INDEPENDENT SEED 2). The DME family contains DEMETER-LIKE 2 (DML2), DML3, and REPRESSOR OF SILENING 1 (ROS1). DNA demetylation by ROS1, DML2, and DML3 protect the hypermethylation of specific genome loci. ROS1 is necessary to suppress the promoter methylation and the silencing of endogenous genes. In contrast, the function of DML2 and DML3 has not been reported. Several recent studies have suggested that epigenetic alterations such as change in DNA methylation and histone modification should be caused in plant genomes upon exposure to ionizing radiation. However, there is a lack of data exploring the underlying mechanisms. Therefore, the present study aims to characterize and

  12. [Isolation of genomic DNA from Cinnamomum cassia Presl].

    Science.gov (United States)

    Xu, Hong; Zhang, Jun; Zheng, Min; Yuan, Wenjie

    2004-05-01

    To extract high-quality DNA for RAPD and other molecular biology research from Chinese traditional medicine Cinnamomum cassia Presl.. Improved CTAB method, low pH medium with high salt method (LPHS) and urea method were used for DNA extract. The yield and quality of DNA was analysed by electrophoresis, ratios of A260/A280 and PCR amplification. The modified CTAB method and low pH medium with high salt method can produce high qualiy DNA for PCR amplification.

  13. DNA template strand sequencing of single-cells maps genomic rearrangements at high resolution

    NARCIS (Netherlands)

    Falconer, Ester; Hills, Mark; Naumann, Ulrike; Poon, Steven S. S.; Chavez, Elizabeth A.; Sanders, Ashley D.; Zhao, Yongjun; Hirst, Martin; Lansdorp, Peter M.

    2012-01-01

    DNA rearrangements such as sister chromatid exchanges (SCEs) are sensitive indicators of genomic stress and instability, but they are typically masked by single-cell sequencing techniques. We developed Strand-seq to independently sequence parental DNA template strands from single cells, making it

  14. Genome-wide DNA methylation levels and altered cortisol stress reactivity following childhood trauma in humans

    NARCIS (Netherlands)

    Houtepen, Lotte C|info:eu-repo/dai/nl/413662802; Vinkers, Christiaan H|info:eu-repo/dai/nl/304824755; Carrillo-Roa, Tania; Hiemstra, Marieke; van Lier, Pol A; Meeus, Wim; Branje, Susan; Heim, Christine M; Nemeroff, Charles B; Mill, Jonathan; Schalkwyk, Leonard C; Creyghton, Menno P|info:eu-repo/dai/nl/336269471; Kahn, René S|info:eu-repo/dai/nl/073778532; Joëls, Marian|info:eu-repo/dai/nl/070219249; Binder, Elisabeth B; Boks, Marco P M|info:eu-repo/dai/nl/286852071

    2016-01-01

    DNA methylation likely plays a role in the regulation of human stress reactivity. Here we show that in a genome-wide analysis of blood DNA methylation in 85 healthy individuals, a locus in the Kit ligand gene (KITLG; cg27512205) showed the strongest association with cortisol stress reactivity (P=5.8

  15. Complete Genomic DNA Sequence of the East Asian Spotted Fever Disease Agent Rickettsia japonica

    Science.gov (United States)

    Matsutani, Minenosuke; Ogawa, Motohiko; Takaoka, Naohisa; Hanaoka, Nozomu; Toh, Hidehiro; Yamashita, Atsushi; Oshima, Kenshiro; Hirakawa, Hideki; Kuhara, Satoru; Suzuki, Harumi; Hattori, Masahira; Kishimoto, Toshio; Ando, Shuji; Azuma, Yoshinao; Shirai, Mutsunori

    2013-01-01

    Rickettsia japonica is an obligate intracellular alphaproteobacteria that causes tick-borne Japanese spotted fever, which has spread throughout East Asia. We determined the complete genomic DNA sequence of R. japonica type strain YH (VR-1363), which consists of 1,283,087 base pairs (bp) and 971 protein-coding genes. Comparison of the genomic DNA sequence of R. japonica with other rickettsiae in the public databases showed that 2 regions (4,323 and 216 bp) were conserved in a very narrow range of Rickettsia species, and the shorter one was inserted in, and disrupted, a preexisting open reading frame (ORF). While it is unknown how the DNA sequences were acquired in R. japonica genomes, it may be a useful signature for the diagnosis of Rickettsia species. Instead of the species-specific inserted DNA sequences, rickettsial genomes contain Rickettsia-specific palindromic elements (RPEs), which are also capable of locating in preexisting ORFs. Precise alignments of protein and DNA sequences involving RPEs showed that when a gene contains an inserted DNA sequence, each rickettsial ortholog carried an inserted DNA sequence at the same locus. The sequence, ATGAC, was shown to be highly frequent and thus characteristic in certain RPEs (RPE-4, RPE-6, and RPE-7). This finding implies that RPE-4, RPE-6, and RPE-7 were derived from a common inserted DNA sequence. PMID:24039725

  16. Genome-wide DNA methylation analysis of the porcine hypothalamus-pituitary-ovary axis

    DEFF Research Database (Denmark)

    Yuan, Xiao Long; Zhang, Zhe; Li, Bin

    2017-01-01

    Previous studies have suggested that DNA methylation in both CpG and CpH (where H = C, T or A) contexts plays a critical role in biological functions of different tissues. However, the genome-wide DNA methylation patterns of porcine hypothalamus-pituitary-ovary (HPO) tissues remain virtually...

  17. TopBP1/Dpb11 binds DNA anaphase bridges to prevent genome instability

    DEFF Research Database (Denmark)

    Germann, Susanne M; Schramke, Vera; Pedersen, Rune Troelsgaard

    2014-01-01

    DNA anaphase bridges are a potential source of genome instability that may lead to chromosome breakage or nondisjunction during mitosis. Two classes of anaphase bridges can be distinguished: DAPI-positive chromatin bridges and DAPI-negative ultrafine DNA bridges (UFBs). Here, we establish budding...

  18. Elg1 forms an alternative RFC complex important for DNA replication and genome integrity

    NARCIS (Netherlands)

    Bellaoui, Mohammed; Chang, Michael; Ou, Jiongwen; Xu, Hong; Boone, Charles; Brown, Grant W

    2003-01-01

    Genome-wide synthetic genetic interaction screens with mutants in the mus81 and mms4 replication fork-processing genes identified a novel replication factor C (RFC) homolog, Elg1, which forms an alternative RFC complex with Rfc2-5. This complex is distinct from the DNA replication RFC, the DNA

  19. Mutagenic capacity of endogenous G4 DNA underlies genome instability in FANCJ-defective C. elegans.

    Science.gov (United States)

    Kruisselbrink, Evelien; Guryev, Victor; Brouwer, Karin; Pontier, Daphne B; Cuppen, Edwin; Tijsterman, Marcel

    2008-06-24

    To safeguard genetic integrity, cells have evolved an accurate but not failsafe mechanism of DNA replication. Not all DNA sequences tolerate DNA replication equally well [1]. Also, genomic regions that impose structural barriers to the DNA replication fork are a potential source of genetic instability [2, 3]. Here, we demonstrate that G4 DNA-a sequence motif that folds into quadruplex structures in vitro [4, 5]-is highly mutagenic in vivo and is removed from genomes that lack dog-1, the C. elegans ortholog of mammalian FANCJ [6, 7], which is mutated in Fanconi anemia patients [8-11]. We show that sequences that match the G4 DNA signature G3-5N1-3G3-5N1-3G3-5N1-3G3-5 are deleted in germ and somatic tissues of dog-1 animals. Unbiased aCGH analyses of dog-1 genomes that were allowed to accumulate mutations in >100 replication cycles indicate that deletions are found exclusively at G4 DNA; deletion frequencies can reach 4% per site per animal generation. We found that deletion sizes fall short of Okazaki fragment lengths [12], and no significant microhomology was observed at deletion junctions. The existence of 376,000 potentially mutagenic G4 DNA sites in the human genome could have major implications for the etiology of hereditary FancJ and nonhereditary cancers.

  20. Assessing the utility of whole genome amplified DNA for next-generation molecular ecology.

    Science.gov (United States)

    Blair, Christopher; Campbell, C Ryan; Yoder, Anne D

    2015-09-01

    DNA quantity can be a hindrance in ecological and evolutionary research programmes due to a range of factors including endangered status of target organisms, available tissue type, and the impact of field conditions on preservation methods. A potential solution to low-quantity DNA lies in whole genome amplification (WGA) techniques that can substantially increase DNA yield. To date, few studies have rigorously examined sequence bias that might result from WGA and next-generation sequencing of nonmodel taxa. To address this knowledge deficit, we use multiple displacement amplification (MDA) and double-digest RAD sequencing on the grey mouse lemur (Microcebus murinus) to quantify bias in genome coverage and SNP calls when compared to raw genomic DNA (gDNA). We focus our efforts in providing baseline estimates of potential bias by following manufacturer's recommendations for starting DNA quantities (>100 ng). Our results are strongly suggestive that MDA enrichment does not introduce systematic bias to genome characterization. SNP calling between samples when genotyping both de-novo and with a reference genome are highly congruent (>98%) when specifying a minimum threshold of 20X stack depth to call genotypes. Relative genome coverage is also similar between MDA and gDNA, and allelic dropout is not observed. SNP concordance varies based on coverage threshold, with 95% concordance reached at ~12X coverage genotyping de-novo and ~7X coverage genotyping with the reference genome. These results suggest that MDA may be a suitable solution for next-generation molecular ecological studies when DNA quantity would otherwise be a limiting factor. © 2015 John Wiley & Sons Ltd.

  1. DNA-DNA hybridization values and their relationship to whole-genome sequence similarities

    National Research Council Canada - National Science Library

    Goris, Johan; Konstantinidis, Konstantinos T; Klappenbach, Joel A; Coenye, Tom; Vandamme, Peter; Tiedje, James M

    2007-01-01

    .... Since the extent of hybridization between a pair of strains is ultimately governed by their respective genomic sequences, we examined the quantitative relationship between DDH values and genome...

  2. Genome Evolution and Meiotic Maps by Massively Parallel DNA Sequencing: Spotted Gar, an Outgroup for the Teleost Genome Duplication

    Science.gov (United States)

    Amores, Angel; Catchen, Julian; Ferrara, Allyse; Fontenot, Quenton; Postlethwait, John H.

    2011-01-01

    Genomic resources for hundreds of species of evolutionary, agricultural, economic, and medical importance are unavailable due to the expense of well-assembled genome sequences and difficulties with multigenerational studies. Teleost fish provide many models for human disease but possess anciently duplicated genomes that sometimes obfuscate connectivity. Genomic information representing a fish lineage that diverged before the teleost genome duplication (TGD) would provide an outgroup for exploring the mechanisms of evolution after whole-genome duplication. We exploited massively parallel DNA sequencing to develop meiotic maps with thrift and speed by genotyping F1 offspring of a single female and a single male spotted gar (Lepisosteus oculatus) collected directly from nature utilizing only polymorphisms existing in these two wild individuals. Using Stacks, software that automates the calling of genotypes from polymorphisms assayed by Illumina sequencing, we constructed a map containing 8406 markers. RNA-seq on two map-cross larvae provided a reference transcriptome that identified nearly 1000 mapped protein-coding markers and allowed genome-wide analysis of conserved synteny. Results showed that the gar lineage diverged from teleosts before the TGD and its genome is organized more similarly to that of humans than teleosts. Thus, spotted gar provides a critical link between medical models in teleost fish, to which gar is biologically similar, and humans, to which gar is genomically similar. Application of our F1 dense mapping strategy to species with no prior genome information promises to facilitate comparative genomics and provide a scaffold for ordering the numerous contigs arising from next generation genome sequencing. PMID:21828280

  3. Genomic DNA extraction method from pearl millet ( Pennisetum ...

    African Journals Online (AJOL)

    DNA extraction is difficult in a variety of plants because of the presence of metabolites that interfere with DNA isolation procedures and downstream applications such as DNA restriction, amplification, and cloning. Here we describe a modified procedure based on the hexadecyltrimethylammonium bromide (CTAB) method to ...

  4. Genomic DNA extraction method from Annona senegalensis Pers ...

    African Journals Online (AJOL)

    Extraction of DNA in many plants is difficult because of the presence of metabolites that interfere with DNA isolation procedures and downstream applications such as DNA restriction, replications, amplification, as well as cloning. Modified procedure based on the hexadecyltrimethyl ammoniumbromide (CTAB) method is ...

  5. Migration of mitochondrial DNA in the nuclear genome of colorectal adenocarcinoma.

    Science.gov (United States)

    Srinivasainagendra, Vinodh; Sandel, Michael W; Singh, Bhupendra; Sundaresan, Aishwarya; Mooga, Ved P; Bajpai, Prachi; Tiwari, Hemant K; Singh, Keshav K

    2017-03-29

    Colorectal adenocarcinomas are characterized by abnormal mitochondrial DNA (mtDNA) copy number and genomic instability, but a molecular interaction between mitochondrial and nuclear genome remains unknown. Here we report the discovery of increased copies of nuclear mtDNA (NUMT) in colorectal adenocarcinomas, which supports link between mtDNA and genomic instability in the nucleus. We name this phenomenon of nuclear occurrence of mitochondrial component as numtogenesis. We provide a description of NUMT abundance and distribution in tumor versus matched blood-derived normal genomes. Whole-genome sequence data were obtained for colon adenocarcinoma and rectum adenocarcinoma patients participating in The Cancer Genome Atlas, via the Cancer Genomics Hub, using the GeneTorrent file acquisition tool. Data were analyzed to determine NUMT proportion and distribution on a genome-wide scale. A NUMT suppressor gene was identified by comparing numtogenesis in other organisms. Our study reveals that colorectal adenocarcinoma genomes, on average, contains up to 4.2-fold more somatic NUMTs than matched normal genomes. Women colorectal tumors contained more NUMT than men. NUMT abundance in tumor predicted parallel abundance in blood. NUMT abundance positively correlated with GC content and gene density. Increased numtogenesis was observed with higher mortality. We identified YME1L1, a human homolog of yeast YME1 (yeast mitochondrial DNA escape 1) to be frequently mutated in colorectal tumors. YME1L1 was also mutated in tumors derived from other tissues. We show that inactivation of YME1L1 results in increased transfer of mtDNA in the nuclear genome. Our study demonstrates increased somatic transfer of mtDNA in colorectal tumors. Our study also reveals sex-based differences in frequency of NUMT occurrence and that NUMT in blood reflects NUMT in tumors, suggesting NUMT may be used as a biomarker for tumorigenesis. We identify YME1L1 as the first NUMT suppressor gene in human and

  6. Whole genome amplification strategy for forensic genetic analysis using single or few cell equivalents of genomic DNA.

    Science.gov (United States)

    Hanson, Erin K; Ballantyne, Jack

    2005-11-15

    Evidentiary items sometimes contain an insufficient quantity of DNA for routine forensic genetic analysis. These so-called low copy number DNA samples (limitations of routine DNA analysis methods. Theoretically, one way of making such intractable samples amenable to analysis would be to increase the number of starting genomes available for subsequent STR (short tandem repeat) analysis by a whole genome amplification strategy (WGA). Although numerous studies employing WGA have focused primarily on clinical applications, few in-depth studies have been conducted to evaluate the potential usefulness of these methods in forensic casework. After an initial evaluation of existing methods, a modified WGA strategy was developed that appears to have utility for low copy number forensic casework specimens. The method employs a slight, but important, modification of the "improved primer extension preamplification PCR" method (I-PEP-PCR), which we term mIPEP (modified-I-PEP-PCR). Complete autosomal STR and Y-STR (Y chromosome short tandem repeat) profiles were routinely obtained with 5 pg of template DNA, which is equivalent to 1-2 diploid cells. Remarkably, partial Y- and autosomal STR profiles were obtained from mIPEP-treated DNA recovered from bloodstains exposed to the outside environment for 1 year whereas non-mIPEP-treated samples did not produce profiles. STR profiles were obtained from contact DNA from single dermal ridge fingerprints when the DNA was subjected to prior mIPEP amplification but not when the mIPEP step was omitted.

  7. Aprataxin resolves adenylated RNA-DNA junctions to maintain genome integrity

    Science.gov (United States)

    Tumbale, Percy; Williams, Jessica S.; Schellenberg, Matthew J.; Kunkel, Thomas A.; Williams, R. Scott

    2014-01-01

    Faithful maintenance and propagation of eukaryotic genomes is ensured by three-step DNA ligation reactions employed by ATP-dependent DNA ligases1,2. Paradoxically, when DNA ligases encounter nicked DNA structures with abnormal DNA termini, DNA ligase catalytic activity can generate and/or exacerbate DNA damage through abortive ligation that produces chemically adducted, toxic 5′-adenylated (5′-AMP) DNA lesions3–6 (Fig. 1a). Aprataxin (Aptx) reverses DNA-adenylation but the context for deadenylation repair is unclear. Here we examine the importance of Aptx to RNaseH2-dependent excision repair (RER) of a lesion that is very frequently introduced into DNA, a ribonucleotide. We show that ligases generate adenylated 5′-ends containing a ribose characteristic of RNaseH2 incision. Aptx efficiently repairs adenylated RNA-DNA, and acting in an RNA-DNA damage response (RDDR), promotes cellular survival and prevents S-phase checkpoint activation in budding yeast undergoing RER. Structure-function studies of human Aptx/RNA-DNA/AMP/Zn complexes define a mechanism for detecting and reversing adenylation at RNA-DNA junctions. This involves A-form RNA-binding, proper protein folding and conformational changes, all of which are impacted by heritable APTX mutations in Ataxia with Oculomotor Apraxia 1 (AOA1). Together, these results suggest that accumulation of adenylated RNA-DNA may contribute to neurological disease. PMID:24362567

  8. High-Quality Exome Sequencing of Whole-Genome Amplified Neonatal Dried Blood Spot DNA

    DEFF Research Database (Denmark)

    Poulsen, Jesper Buchhave; Lescai, Francesco; Grove, Jakob

    2016-01-01

    be amplified to obtain micrograms of an otherwise limited resource, referred to as whole-genome amplified DNA (wgaDNA). Here we investigate the robustness of exome sequencing of wgaDNA of neonatal DBS samples. We conducted three pilot studies of seven, eight and seven subjects, respectively. For each subject...... from variant calls. No differences were observed substituting 2x3.2 with 2x1.6 mm discs, allowing for additional reduction of sample material in future projects....

  9. Recent advances in the genome-wide study of DNA replication origins in yeast

    OpenAIRE

    Peng, Chong; Luo, Hao; Zhang, Xi; Gao, Feng

    2015-01-01

    DNA replication, one of the central events in the cell cycle, is the basis of biological inheritance. In order to be duplicated, a DNA double helix must be opened at defined sites, which are called DNA replication origins (ORIs). Unlike in bacteria, where replication initiates from a single replication origin, multiple origins are utilized in the eukaryotic genomes. Among them, the ORIs in budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe have been best ch...

  10. Widespread Horizontal Gene Transfer from Circular Single-stranded DNA Viruses to Eukaryotic Genomes

    Directory of Open Access Journals (Sweden)

    Xie Jiatao

    2011-09-01

    Full Text Available Abstract Background In addition to vertical transmission, organisms can also acquire genes from other distantly related species or from their extra-chromosomal elements (plasmids and viruses via horizontal gene transfer (HGT. It has been suggested that phages represent substantial forces in prokaryotic evolution. In eukaryotes, retroviruses, which can integrate into host genome as an obligate step in their replication strategy, comprise approximately 8% of the human genome. Unlike retroviruses, few members of other virus families are known to transfer genes to host genomes. Results Here we performed a systematic search for sequences related to circular single-stranded DNA (ssDNA viruses in publicly available eukaryotic genome databases followed by comprehensive phylogenetic analysis. We conclude that the replication initiation protein (Rep-related sequences of geminiviruses, nanoviruses and circoviruses have been frequently transferred to a broad range of eukaryotic species, including plants, fungi, animals and protists. Some of the transferred viral genes were conserved and expressed, suggesting that these genes have been coopted to assume cellular functions in the host genomes. We also identified geminivirus-like and parvovirus-like transposable elements in genomes of fungi and lower animals, respectively, and thereby provide direct evidence that eukaryotic transposons could derive from ssDNA viruses. Conclusions Our discovery extends the host range of circular ssDNA viruses and sheds light on the origin and evolution of these viruses. It also suggests that ssDNA viruses act as an unforeseen source of genetic innovation in their hosts.

  11. Accurate DNA Assembly and Genome Engineering with Optimized Uracil Excision Cloning.

    Science.gov (United States)

    Cavaleiro, Ana Mafalda; Kim, Se Hyeuk; Seppälä, Susanna; Nielsen, Morten T; Nørholm, Morten H H

    2015-09-18

    Simple and reliable DNA editing by uracil excision (a.k.a. USER cloning) has been described by several research groups, but the optimal design of cohesive DNA ends for multigene assembly remains elusive. Here, we use two model constructs based on expression of gfp and a four-gene pathway that produces β-carotene to optimize assembly junctions and the uracil excision protocol. By combining uracil excision cloning with a genomic integration technology, we demonstrate that up to six DNA fragments can be assembled in a one-tube reaction for direct genome integration with high accuracy, greatly facilitating the advanced engineering of robust cell factories.

  12. The architecture of ArgR-DNA complexes at the genome-scale in Escherichia coli

    DEFF Research Database (Denmark)

    Cho, Suhyung; Cho, Yoo-Bok; Kang, Taek Jin

    2015-01-01

    DNA-binding motifs that are recognized by transcription factors (TFs) have been well studied; however, challenges remain in determining the in vivo architecture of TF-DNA complexes on a genome-scale. Here, we determined the in vivo architecture of Escherichia coli arginine repressor (Arg...... facilitate the non-specific contacts between ArgR subunits and the residual sequences. Additionally, our approach may also reveal other fundamental structural features of TF-DNA interactions that have implications for studying genome-scale transcriptional regulatory networks....

  13. Effect of Ar+ Implantation and Maize Genome DNA on Autotetraploid Rice

    Science.gov (United States)

    Li, Yufeng; Huang, Qunce; Yu, Zengliang

    2008-06-01

    The effect of Ar+ beam implantation and maize genome DNA on autotetraploid rice is studied. Better mutation types and higher mutation rates were discovered in M2 of T3 with ion implantation and immersion in maize genome DNA. In the five agronomic categories investigated, the mutation rate of the seed setting rate was 9.1%, and the total mutation rate was 14.8% in the T3. However, the total mutation rate was 2.1% with the treatment of only ion implantation and 1.3% with the treatment of only immersion in maize genome DNA. Mutant FA36(4) was discovered in M1 with ion beam implantation and immersion in maize genome DNA. Its RuBPCase activity, PEPCase activity and seed setting rate were 32%, 153%, and 36.79%, respectively, higher than its parent IR36(4). Rapid analysis of polymorphicDNA (RAPD) analysis of three M2 plants of FA36(4) (FM1, FM2, FM3) and two controls (purple maize and IR36(4)) were also conducted with 40 random primers. S5-3 was RAPD fragment amplified with a template of purple maize, FM2 and FM3 genome DNA using primer S5. There was no S5-3 in the RAPD pattern of IR36(4) or FM1.

  14. Global analysis of genomic instability caused by DNA replication stress in Saccharomyces cerevisiae.

    Science.gov (United States)

    Zheng, Dao-Qiong; Zhang, Ke; Wu, Xue-Chang; Mieczkowski, Piotr A; Petes, Thomas D

    2016-12-13

    DNA replication stress (DRS)-induced genomic instability is an important factor driving cancer development. To understand the mechanisms of DRS-associated genomic instability, we measured the rates of genomic alterations throughout the genome in a yeast strain with lowered expression of the replicative DNA polymerase δ. By a genetic test, we showed that most recombinogenic DNA lesions were introduced during S or G 2 phase, presumably as a consequence of broken replication forks. We observed a high rate of chromosome loss, likely reflecting a reduced capacity of the low-polymerase strains to repair double-stranded DNA breaks (DSBs). We also observed a high frequency of deletion events within tandemly repeated genes such as the ribosomal RNA genes. By whole-genome sequencing, we found that low levels of DNA polymerase δ elevated mutation rates, both single-base mutations and small insertions/deletions. Finally, we showed that cells with low levels of DNA polymerase δ tended to accumulate small promoter mutations that increased the expression of this polymerase. These deletions conferred a selective growth advantage to cells, demonstrating that DRS can be one factor driving phenotypic evolution.

  15. DNA-PKcs, ATM, and ATR Interplay Maintains Genome Integrity during Neurogenesis.

    Science.gov (United States)

    Enriquez-Rios, Vanessa; Dumitrache, Lavinia C; Downing, Susanna M; Li, Yang; Brown, Eric J; Russell, Helen R; McKinnon, Peter J

    2017-01-25

    The DNA damage response (DDR) orchestrates a network of cellular processes that integrates cell-cycle control and DNA repair or apoptosis, which serves to maintain genome stability. DNA-PKcs (the catalytic subunit of the DNA-dependent kinase, encoded by PRKDC), ATM (ataxia telangiectasia, mutated), and ATR (ATM and Rad3-related) are related PI3K-like protein kinases and central regulators of the DDR. Defects in these kinases have been linked to neurodegenerative or neurodevelopmental syndromes. In all cases, the key neuroprotective function of these kinases is uncertain. It also remains unclear how interactions between the three DNA damage-responsive kinases coordinate genome stability, particularly in a physiological context. Here, we used a genetic approach to identify the neural function of DNA-PKcs and the interplay between ATM and ATR during neurogenesis. We found that DNA-PKcs loss in the mouse sensitized neuronal progenitors to apoptosis after ionizing radiation because of excessive DNA damage. DNA-PKcs was also required to prevent endogenous DNA damage accumulation throughout the adult brain. In contrast, ATR coordinated the DDR during neurogenesis to direct apoptosis in cycling neural progenitors, whereas ATM regulated apoptosis in both proliferative and noncycling cells. We also found that ATR controls a DNA damage-induced G2/M checkpoint in cortical progenitors, independent of ATM and DNA-PKcs. These nonoverlapping roles were further confirmed via sustained murine embryonic or cortical development after all three kinases were simultaneously inactivated. Thus, our results illustrate how DNA-PKcs, ATM, and ATR have unique and essential roles during the DDR, collectively ensuring comprehensive genome maintenance in the nervous system. The DNA damage response (DDR) is essential for prevention of a broad spectrum of different human neurologic diseases. However, a detailed understanding of the DDR at a physiological level is lacking. In contrast to many in

  16. Artificial zinc finger DNA binding domains: versatile tools for genome engineering and modulation of gene expression.

    Science.gov (United States)

    Hossain, Mir A; Barrow, Joeva J; Shen, Yong; Haq, Md Imdadul; Bungert, Jörg

    2015-11-01

    Genome editing and alteration of gene expression by synthetic DNA binding activities gained a lot of momentum over the last decade. This is due to the development of new DNA binding molecules with enhanced binding specificity. The most commonly used DNA binding modules are zinc fingers (ZFs), TALE-domains, and the RNA component of the CRISPR/Cas9 system. These binding modules are fused or linked to either nucleases that cut the DNA and induce DNA repair processes, or to protein domains that activate or repress transcription of genes close to the targeted site in the genome. This review focuses on the structure, design, and applications of ZF DNA binding domains (ZFDBDs). ZFDBDs are relatively small and have been shown to penetrate the cell membrane without additional tags suggesting that they could be delivered to cells without a DNA or RNA intermediate. Advanced algorithms that are based on extensive knowledge of the mode of ZF/DNA interactions are used to design the amino acid composition of ZFDBDs so that they bind to unique sites in the genome. Off-target binding has been a concern for all synthetic DNA binding molecules. Thus, increasing the specificity and affinity of ZFDBDs will have a significant impact on their use in analytical or therapeutic settings. © 2015 Wiley Periodicals, Inc.

  17. Xenopus egg extract to study regulation of genome-wide and locus-specific DNA replication.

    Science.gov (United States)

    Raspelli, Erica; Falbo, Lucia; Costanzo, Vincenzo

    2017-01-01

    Faithful DNA replication, coupled with accurate repair of DNA damage, is essential to maintain genome stability and relies on different DNA metabolism genes. Many of these genes are involved in the assembly of replication origins, in the coordination of DNA repair to protect replication forks progression in the presence of DNA damage and in the replication of repetitive chromatin regions. Some DNA metabolism genes are essential in higher eukaryotes, suggesting the existence of specialized mechanisms of repair and replication in organisms with complex genomes. The impact on cell survival of many of these genes has so far precluded in depth molecular analysis of their function. The cell-free Xenopus laevis egg extract represents an ideal system to overcome survival issues and to facilitate the biochemical study of replication-associated functions of essential proteins in vertebrate organisms. Here, we will discuss how Xenopus egg extracts have been used to study cellular and molecular processes, such as DNA replication and DNA repair. In particular, we will focus on innovative imaging and proteomic-based experimental approaches to characterize the molecular function of a number of essential DNA metabolism factors involved in the duplication of complex vertebrate genomes. © 2017 Wiley Periodicals, Inc.

  18. Genomic and functional integrity of the hematopoietic system requires tolerance of oxidative DNA lesions

    DEFF Research Database (Denmark)

    Martín-Pardillos, Ana; Tsaalbi-Shtylik, Anastasia; Chen, Si

    2017-01-01

    Endogenous DNA damage is causally associated with the functional decline and transformation of stem cells that characterize aging. DNA lesions that have escaped DNA repair can induce replication stress and genomic breaks that induce senescence and apoptosis. It is not clear how stem......-distorting nucleotide lesions, resulted in the perinatal loss of hematopoietic stem cells, progressive loss of bone marrow, and fatal aplastic anemia between 3 and 4 months of age. This was associated with replication stress, genomic breaks, DNA damage signaling, senescence, and apoptosis in bone marrow. Surprisingly......), the postreplicative bypass of damaged nucleotides. Rev1 hematopoietic stem and progenitor cells displayed compromised proliferation, and replication stress that could be rescued with an antioxidant. The additional disruption of Xpc, essential for global-genome nucleotide excision repair (ggNER) of helix...

  19. High-Throughput Sequencing of Three Lemnoideae (Duckweeds) Chloroplast Genomes from Total DNA

    Science.gov (United States)

    Wang, Wenqin; Messing, Joachim

    2011-01-01

    Background Chloroplast genomes provide a wealth of information for evolutionary and population genetic studies. Chloroplasts play a particularly important role in the adaption for aquatic plants because they float on water and their major surface is exposed continuously to sunlight. The subfamily of Lemnoideae represents such a collection of aquatic species that because of photosynthesis represents one of the fastest growing plant species on earth. Methods We sequenced the chloroplast genomes from three different genera of Lemnoideae, Spirodela polyrhiza, Wolffiella lingulata and Wolffia australiana by high-throughput DNA sequencing of genomic DNA using the SOLiD platform. Unfractionated total DNA contains high copies of plastid DNA so that sequences from the nucleus and mitochondria can easily be filtered computationally. Remaining sequence reads were assembled into contiguous sequences (contigs) using SOLiD software tools. Contigs were mapped to a reference genome of Lemna minor and gaps, selected by PCR, were sequenced on the ABI3730xl platform. Conclusions This combinatorial approach yielded whole genomic contiguous sequences in a cost-effective manner. Over 1,000-time coverage of chloroplast from total DNA were reached by the SOLiD platform in a single spot on a quadrant slide without purification. Comparative analysis indicated that the chloroplast genome was conserved in gene number and organization with respect to the reference genome of L. minor. However, higher nucleotide substitution, abundant deletions and insertions occurred in non-coding regions of these genomes, indicating a greater genomic dynamics than expected from the comparison of other related species in the Pooideae. Noticeably, there was no transition bias over transversion in Lemnoideae. The data should have immediate applications in evolutionary biology and plant taxonomy with increased resolution and statistical power. PMID:21931804

  20. High-throughput sequencing of three Lemnoideae (duckweeds chloroplast genomes from total DNA.

    Directory of Open Access Journals (Sweden)

    Wenqin Wang

    Full Text Available BACKGROUND: Chloroplast genomes provide a wealth of information for evolutionary and population genetic studies. Chloroplasts play a particularly important role in the adaption for aquatic plants because they float on water and their major surface is exposed continuously to sunlight. The subfamily of Lemnoideae represents such a collection of aquatic species that because of photosynthesis represents one of the fastest growing plant species on earth. METHODS: We sequenced the chloroplast genomes from three different genera of Lemnoideae, Spirodela polyrhiza, Wolffiella lingulata and Wolffia australiana by high-throughput DNA sequencing of genomic DNA using the SOLiD platform. Unfractionated total DNA contains high copies of plastid DNA so that sequences from the nucleus and mitochondria can easily be filtered computationally. Remaining sequence reads were assembled into contiguous sequences (contigs using SOLiD software tools. Contigs were mapped to a reference genome of Lemna minor and gaps, selected by PCR, were sequenced on the ABI3730xl platform. CONCLUSIONS: This combinatorial approach yielded whole genomic contiguous sequences in a cost-effective manner. Over 1,000-time coverage of chloroplast from total DNA were reached by the SOLiD platform in a single spot on a quadrant slide without purification. Comparative analysis indicated that the chloroplast genome was conserved in gene number and organization with respect to the reference genome of L. minor. However, higher nucleotide substitution, abundant deletions and insertions occurred in non-coding regions of these genomes, indicating a greater genomic dynamics than expected from the comparison of other related species in the Pooideae. Noticeably, there was no transition bias over transversion in Lemnoideae. The data should have immediate applications in evolutionary biology and plant taxonomy with increased resolution and statistical power.

  1. High-throughput DNA Stretching in Continuous Elongational Flow for Genome Sequence Scanning

    Science.gov (United States)

    Meltzer, Robert; Griffis, Joshua; Safranovitch, Mikhail; Malkin, Gene; Cameron, Douglas

    2014-03-01

    Genome Sequence Scanning (GSS) identifies and compares bacterial genomes by stretching long (60 - 300 kb) genomic DNA restriction fragments and scanning for site-selective fluorescent probes. Practical application of GSS requires: 1) high throughput data acquisition, 2) efficient DNA stretching, 3) reproducible DNA elasticity in the presence of intercalating fluorescent dyes. GSS utilizes a pseudo-two-dimensional micron-scale funnel with convergent sheathing flows to stretch one molecule at a time in continuous elongational flow and center the DNA stream over diffraction-limited confocal laser excitation spots. Funnel geometry has been optimized to maximize throughput of DNA within the desired length range (>10 million nucleobases per second). A constant-strain detection channel maximizes stretching efficiency by applying a constant parabolic tension profile to each molecule, minimizing relaxation and flow-induced tumbling. The effect of intercalator on DNA elasticity is experimentally controlled by reacting one molecule of DNA at a time in convergent sheathing flows of the dye. Derivations of accelerating flow and non-linear tension distribution permit alignment of detected fluorescence traces to theoretical templates derived from whole-genome sequence data.

  2. Canine hip dysplasia is predictable by genotyping.

    Science.gov (United States)

    Guo, G; Zhou, Z; Wang, Y; Zhao, K; Zhu, L; Lust, G; Hunter, L; Friedenberg, S; Li, J; Zhang, Y; Harris, S; Jones, P; Sandler, J; Krotscheck, U; Todhunter, R; Zhang, Z

    2011-04-01

    To establish a predictive method using whole genome genotyping for early intervention in canine hip dysplasia (CHD) risk management, for the prevention of the progression of secondary osteoarthritis (OA), and for selective breeding. Two sets of dogs (six breeds) were genotyped with dense SNPs covering the entire canine genome. The first set contained 359 dogs upon which a predictive formula for genomic breeding value (GBV) was derived by using their estimated breeding value (EBV) of the Norberg angle (a measure of CHD) and their genotypes. To investigate how well the formula would work for an individual dog with genotype only (without using EBV), a cross validation was performed by masking the EBV of one dog at a time. The genomic data and the EBV of the remaining dogs were used to predict the GBV for the single dog that was left out. The second set of dogs included 38 new Labrador retriever dogs, which had no pedigree relationship to the dogs in the first set. The cross validation showed a strong correlation (R>0.7) between the EBV and the GBV. The independent validation showed a moderate correlation (R=0.5) between GBV for the Norberg angle and the observed Norberg angle (no EBV was available for the new 38 dogs). Sensitivity, specificity, positive and negative predictive values of the genomic data were all above 70%. Prediction of CHD from genomic data is feasible, and can be applied for risk management of CHD and early selection for genetic improvement to reduce the prevalence of CHD in breeding programs. The prediction can be implemented before maturity, at which age current radiographic screening programs are traditionally applied, and as soon as DNA is available. Copyright © 2010 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  3. Canine Hip Dysplasia is Predictable by Genotyping

    Science.gov (United States)

    Guo, Gang; Zhou, Zhengkui; Wang, Yachun; Zhao, Keyan; Zhu, Lan; Lust, George; Hunter, Linda; Friedenberg, Steven; Li, Junya; Zhang, Yuan; Harris, Stephen; Jones, Paul; Sandler, Jody; Krotscheck, Ursula; Todhunter, Rory; Zhang, Zhiwu

    2011-01-01

    Summary Objective To establish a predictive method using whole genome genotyping for early intervention in canine hip dysplasia (CHD) risk management, for the prevention of the progression of secondary osteoarthritis (OA), and for selective breeding. Design Two sets of dogs (6 breeds) were genotyped with dense SNPs covering the entire canine genome. The first set contained 359 dogs upon which a predictive formula for genomic breeding value (GBV) was derived by using their estimated breeding value (EBV) of the Norberg angle (a measure of CHD) and their genotypes. To investigate how well the formula would work for an individual dog with genotype only (without using EBV or phenotype), a cross validation was performed by masking the EBV of one dog at a time. The genomic data and the EBV of the remaining dogs were used to predict the GBV for the single dog that was left out. The second set of dogs included 38 new Labrador retriever dogs, which had no pedigree relationship to the dogs in the first set. Results The cross validation showed a strong correlation (r>0.7) between the EBV and the GBV. The independent validation showed a strong correlation (r=0.5) between GBV for the Norberg angle and the observed Norberg angle (no EBV was available for the new 38 dogs). Sensitivity, specificity, positive, and negative predictive value of the genomic data were all above 70%. Conclusions Prediction of CHD from genomic data is feasible, and can be applied for risk management of CHD and early selection for genetic improvement to reduce the prevalence of CHD in breeding programs. The prediction can be implemented before maturity, at which age current radiographic screening programs are traditionally applied, and as soon as DNA is available. PMID:21215318

  4. Aprataxin resolves adenylated RNA–DNA junctions to maintain genome integrity

    Energy Technology Data Exchange (ETDEWEB)

    Tumbale, Percy [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States). Lab. of Structural Biology; Williams, Jessica S. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States). Lab. of Structural Biology; Schellenberg, Matthew J. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States). Lab. of Structural Biology; Kunkel, Thomas A. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States). Lab. of Structural Biology and Lab. of Molecular Genetics; Williams, R. Scott [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States). Lab. of Structural Biology and Lab. Molecular Genetics

    2013-12-22

    Faithful maintenance and propagation of eukaryotic genomes is ensured by three-step DNA ligation reactions used by ATP-dependent DNA ligases. Paradoxically, when DNA ligases encounter nicked DNA structures with abnormal DNA termini, DNA ligase catalytic activity can generate and/or exacerbate DNA damage through abortive ligation that produces chemically adducted, toxic 5'-adenylated (5'-AMP) DNA lesions. Aprataxin (APTX) reverses DNA adenylation but the context for deadenylation repair is unclear. Here we examine the importance of APTX to RNase-H2-dependent excision repair (RER) of a lesion that is very frequently introduced into DNA, a ribonucleotide. We show that ligases generate adenylated 5' ends containing a ribose characteristic of RNase H2 incision. APTX efficiently repairs adenylated RNA–DNA, and acting in an RNA–DNA damage response (RDDR), promotes cellular survival and prevents S-phase checkpoint activation in budding yeast undergoing RER. Structure–function studies of human APTX–RNA–DNA–AMP–Zn complexes define a mechanism for detecting and reversing adenylation at RNA–DNA junctions. This involves A-form RNA binding, proper protein folding and conformational changes, all of which are affected by heritable APTX mutations in ataxia with oculomotor apraxia 1. Together, these results indicate that accumulation of adenylated RNA–DNA may contribute to neurological disease.

  5. Genome Partitioner: A web tool for multi-level partitioning of large-scale DNA constructs for synthetic biology applications.

    Directory of Open Access Journals (Sweden)

    Matthias Christen

    Full Text Available Recent advances in lower-cost DNA synthesis techniques have enabled new innovations in the field of synthetic biology. Still, efficient design and higher-order assembly of genome-scale DNA constructs remains a labor-intensive process. Given the complexity, computer assisted design tools that fragment large DNA sequences into fabricable DNA blocks are needed to pave the way towards streamlined assembly of biological systems. Here, we present the Genome Partitioner software implemented as a web-based interface that permits multi-level partitioning of genome-scale DNA designs. Without the need for specialized computing skills, biologists can submit their DNA designs to a fully automated pipeline that generates the optimal retrosynthetic route for higher-order DNA assembly. To test the algorithm, we partitioned a 783 kb Caulobacter crescentus genome design. We validated the partitioning strategy by assembling a 20 kb test segment encompassing a difficult to synthesize DNA sequence. Successful assembly from 1 kb subblocks into the 20 kb segment highlights the effectiveness of the Genome Partitioner for reducing synthesis costs and timelines for higher-order DNA assembly. The Genome Partitioner is broadly applicable to translate DNA designs into ready to order sequences that can be assembled with standardized protocols, thus offering new opportunities to harness the diversity of microbial genomes for synthetic biology applications. The Genome Partitioner web tool can be accessed at https://christenlab.ethz.ch/GenomePartitioner.

  6. Quantification of trace-level DNA by real-time whole genome amplification.

    Directory of Open Access Journals (Sweden)

    Min-Jung Kang

    Full Text Available Quantification of trace amounts of DNA is a challenge in analytical applications where the concentration of a target DNA is very low or only limited amounts of samples are available for analysis. PCR-based methods including real-time PCR are highly sensitive and widely used for quantification of low-level DNA samples. However, ordinary PCR methods require at least one copy of a specific gene sequence for amplification and may not work for a sub-genomic amount of DNA. We suggest a real-time whole genome amplification method adopting the degenerate oligonucleotide primed PCR (DOP-PCR for quantification of sub-genomic amounts of DNA. This approach enabled quantification of sub-picogram amounts of DNA independently of their sequences. When the method was applied to the human placental DNA of which amount was accurately determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES, an accurate and stable quantification capability for DNA samples ranging from 80 fg to 8 ng was obtained. In blind tests of laboratory-prepared DNA samples, measurement accuracies of 7.4%, -2.1%, and -13.9% with analytical precisions around 15% were achieved for 400-pg, 4-pg, and 400-fg DNA samples, respectively. A similar quantification capability was also observed for other DNA species from calf, E. coli, and lambda phage. Therefore, when provided with an appropriate standard DNA, the suggested real-time DOP-PCR method can be used as a universal method for quantification of trace amounts of DNA.

  7. Role of DNA Polymerases in Repeat-Mediated Genome Instability

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    Kartik A. Shah

    2012-11-01

    Full Text Available Expansions of simple DNA repeats cause numerous hereditary diseases in humans. We analyzed the role of DNA polymerases in the instability of Friedreich’s ataxia (GAAn repeats in a yeast experimental system. The elementary step of expansion corresponded to ∼160 bp in the wild-type strain, matching the size of Okazaki fragments in yeast. This step increased when DNA polymerase α was mutated, suggesting a link between the scale of expansions and Okazaki fragment size. Expandable repeats strongly elevated the rate of mutations at substantial distances around them, a phenomenon we call repeat-induced mutagenesis (RIM. Notably, defects in the replicative DNA polymerases δ and ∊ strongly increased rates for both repeat expansions and RIM. The increases in repeat-mediated instability observed in DNA polymerase δ mutants depended on translesion DNA polymerases. We conclude that repeat expansions and RIM are two sides of the same replicative mechanism.

  8. USE OF COMPETITIVE DNA HYBRIDIZATION TO IDENTIFY DIFFERENCES IN THE GENOMES OF TWO CLOSELY RELATED FECAL INDICATOR BACTERIA

    Science.gov (United States)

    Although recent technological advances in DNA sequencing and computational biology now allow scientists to compare entire microbial genomes, comparisons of closely related bacterial species and individual isolates by whole-genome sequencing approaches remains prohibitively expens...

  9. The Dunaliella salina organelle genomes: large sequences, inflated with intronic and intergenic DNA

    Energy Technology Data Exchange (ETDEWEB)

    Smith, David R.; Lee, Robert W.; Cushman, John C.; Magnuson, Jon K.; Tran, Duc; Polle, Juergen E.

    2010-05-07

    Abstract Background: Dunaliella salina Teodoresco, a unicellular, halophilic green alga belonging to the Chlorophyceae, is among the most industrially important microalgae. This is because D. salina can produce massive amounts of β-carotene, which can be collected for commercial purposes, and because of its potential as a feedstock for biofuels production. Although the biochemistry and physiology of D. salina have been studied in great detail, virtually nothing is known about the genomes it carries, especially those within its mitochondrion and plastid. This study presents the complete mitochondrial and plastid genome sequences of D. salina and compares them with those of the model green algae Chlamydomonas reinhardtii and Volvox carteri. Results: The D. salina organelle genomes are large, circular-mapping molecules with ~60% noncoding DNA, placing them among the most inflated organelle DNAs sampled from the Chlorophyta. In fact, the D. salina plastid genome, at 269 kb, is the largest complete plastid DNA (ptDNA) sequence currently deposited in GenBank, and both the mitochondrial and plastid genomes have unprecedentedly high intron densities for organelle DNA: ~1.5 and ~0.4 introns per gene, respectively. Moreover, what appear to be the relics of genes, introns, and intronic open reading frames are found scattered throughout the intergenic ptDNA regions -- a trait without parallel in other characterized organelle genomes and one that gives insight into the mechanisms and modes of expansion of the D. salina ptDNA. Conclusions: These findings confirm the notion that chlamydomonadalean algae have some of the most extreme organelle genomes of all eukaryotes. They also suggest that the events giving rise to the expanded ptDNA architecture of D. salina and other Chlamydomonadales may have occurred early in the evolution of this lineage. Although interesting from a genome evolution standpoint, the D. salina organelle DNA sequences will aid in the development of a viable

  10. The Dunaliella salina organelle genomes: large sequences, inflated with intronic and intergenic DNA.

    Science.gov (United States)

    Smith, David Roy; Lee, Robert W; Cushman, John C; Magnuson, Jon K; Tran, Duc; Polle, Jürgen E W

    2010-05-07

    Dunaliella salina Teodoresco, a unicellular, halophilic green alga belonging to the Chlorophyceae, is among the most industrially important microalgae. This is because D. salina can produce massive amounts of beta-carotene, which can be collected for commercial purposes, and because of its potential as a feedstock for biofuels production. Although the biochemistry and physiology of D. salina have been studied in great detail, virtually nothing is known about the genomes it carries, especially those within its mitochondrion and plastid. This study presents the complete mitochondrial and plastid genome sequences of D. salina and compares them with those of the model green algae Chlamydomonas reinhardtii and Volvox carteri. The D. salina organelle genomes are large, circular-mapping molecules with approximately 60% noncoding DNA, placing them among the most inflated organelle DNAs sampled from the Chlorophyta. In fact, the D. salina plastid genome, at 269 kb, is the largest complete plastid DNA (ptDNA) sequence currently deposited in GenBank, and both the mitochondrial and plastid genomes have unprecedentedly high intron densities for organelle DNA: approximately 1.5 and approximately 0.4 introns per gene, respectively. Moreover, what appear to be the relics of genes, introns, and intronic open reading frames are found scattered throughout the intergenic ptDNA regions -- a trait without parallel in other characterized organelle genomes and one that gives insight into the mechanisms and modes of expansion of the D. salina ptDNA. These findings confirm the notion that chlamydomonadalean algae have some of the most extreme organelle genomes of all eukaryotes. They also suggest that the events giving rise to the expanded ptDNA architecture of D. salina and other Chlamydomonadales may have occurred early in the evolution of this lineage. Although interesting from a genome evolution standpoint, the D. salina organelle DNA sequences will aid in the development of a viable

  11. The Dunaliella salina organelle genomes: large sequences, inflated with intronic and intergenic DNA

    Directory of Open Access Journals (Sweden)

    Tran Duc

    2010-05-01

    Full Text Available Abstract Background Dunaliella salina Teodoresco, a unicellular, halophilic green alga belonging to the Chlorophyceae, is among the most industrially important microalgae. This is because D. salina can produce massive amounts of β-carotene, which can be collected for commercial purposes, and because of its potential as a feedstock for biofuels production. Although the biochemistry and physiology of D. salina have been studied in great detail, virtually nothing is known about the genomes it carries, especially those within its mitochondrion and plastid. This study presents the complete mitochondrial and plastid genome sequences of D. salina and compares them with those of the model green algae Chlamydomonas reinhardtii and Volvox carteri. Results The D. salina organelle genomes are large, circular-mapping molecules with ~60% noncoding DNA, placing them among the most inflated organelle DNAs sampled from the Chlorophyta. In fact, the D. salina plastid genome, at 269 kb, is the largest complete plastid DNA (ptDNA sequence currently deposited in GenBank, and both the mitochondrial and plastid genomes have unprecedentedly high intron densities for organelle DNA: ~1.5 and ~0.4 introns per gene, respectively. Moreover, what appear to be the relics of genes, introns, and intronic open reading frames are found scattered throughout the intergenic ptDNA regions -- a trait without parallel in other characterized organelle genomes and one that gives insight into the mechanisms and modes of expansion of the D. salina ptDNA. Conclusions These findings confirm the notion that chlamydomonadalean algae have some of the most extreme organelle genomes of all eukaryotes. They also suggest that the events giving rise to the expanded ptDNA architecture of D. salina and other Chlamydomonadales may have occurred early in the evolution of this lineage. Although interesting from a genome evolution standpoint, the D. salina organelle DNA sequences will aid in the

  12. Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

    DEFF Research Database (Denmark)

    Møller, Henrik D.; Bojsen, Rasmus Kenneth; Tachibana, Chris

    2016-01-01

    Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods...... for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA...... circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types....

  13. Studies on the Interaction between Zinc-Hydroxybenzoite Complex and Genomic DNA

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    Hacali Necefoglu

    2006-04-01

    Full Text Available Zinc-Hydroxybenzoite ([Zn (H206] (p-HO-C6H4COO22H20 complex which wassynthesized and characterized by instrumental methods and the DNA samples which hadbeen isolated from cattle were allowed to interact at 37 oC for different time periods. Theinteraction of genomic DNA with this complex has been followed by agarose gelelectrophoresis at 50 V for 2 h. When DNA samples were allowed to interact with this metalcomplex, it was found that band intensities changed with the concentrations of the complex.In the result of interaction between this complex and genomic DNA samples, it wasdetermined that the intensities of bands were changed at the different concentrations of thecomplex. The brightness of the bands was increased and mobility of the bands wasdecreased, indicating the occurrence of increased covalent binding of the metal complexwith DNA. In this study it was concluded that the damage effect of ascorbate was reducedby Zinc-Hydroxybenzoite.

  14. Ectopic expression of cancer/testis antigen SSX2 induces DNA damage and promotes genomic instability

    DEFF Research Database (Denmark)

    Greve, Katrine Buch Vidén; Lindgreen, Jonas; Terp, Mikkel Green

    2015-01-01

    of senescence (i.e. an irregular and enlarged cell shape, enhanced β-galactosidase activity and DNA double-strand breaks). Since replication defects, DNA damage and senescence are interconnected and well-documented effects of oncogene expression, we tested the oncogenic potential of SSX2. Importantly, knockdown...... in an increased DNA content and enlargement of cell nuclei, suggestive of replication aberrations. The cells further displayed signs of DNA damage and genomic instability, associated with p53-mediated G1 cell cycle arrest and a late apoptotic response. These results suggest a model wherein SSX2-mediated...... replication stress translates into mitotic defects and genomic instability. Arrest of cell growth and induction of DNA double-strand breaks was also observed in MCF7 breast cancer cells in response to SSX2 expression. Additionally, MCF7 cells with ectopic SSX2 expression demonstrated typical signs...

  15. Database of Periodic DNA Regions in Major Genomes

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    Felix E. Frenkel

    2017-01-01

    Full Text Available Summary. We analyzed several prokaryotic and eukaryotic genomes looking for the periodicity sequences availability and employing a new mathematical method. The method envisaged using the random position weight matrices and dynamic programming. Insertions and deletions were allowed inside periodicities, thus adding a novelty to the results we obtained. A periodicity length, one of the key periodicity features, varied from 2 to 50 nt. Totally over 60,000 periodicity sequences were found in 15 genomes including some chromosomes of the H. sapiens (partial, C. elegans, D. melanogaster, and A. thaliana genomes.

  16. Patterns of genomic integration of nuclear chloroplast DNA fragments in plant species.

    Science.gov (United States)

    Yoshida, Takanori; Furihata, Hazuka Y; Kawabe, Akira

    2014-01-01

    The transfer of organelle DNA fragments to the nuclear genome is frequently observed in eukaryotes. These transfers are thought to play an important role in gene and genome evolution of eukaryotes. In plants, such transfers occur from plastid to nuclear [nuclear plastid DNAs (NUPTs)] and mitochondrial to nuclear (nuclear mitochondrial DNAs) genomes. The amount and genomic organization of organelle DNA fragments have been studied in model plant species, such as Arabidopsis thaliana and rice. At present, publicly available genomic data can be used to conduct such studies in non-model plants. In this study, we analysed the amount and genomic organization of NUPTs in 17 plant species for which genome sequences are available. The amount and distribution of NUPTs varied among the species. We also estimated the distribution of NUPTs according to the time of integration (relative age) by conducting sequence similarity analysis between NUPTs and the plastid genome. The age distributions suggested that the present genomic constitutions of NUPTs could be explained by the combination of the rapidly eliminated deleterious parts and few but constantly existing less deleterious parts.

  17. Genome-wide DNA methylation patterns and transcription analysis in sheep muscle.

    Directory of Open Access Journals (Sweden)

    Christine Couldrey

    Full Text Available DNA methylation plays a central role in regulating many aspects of growth and development in mammals through regulating gene expression. The development of next generation sequencing technologies have paved the way for genome-wide, high resolution analysis of DNA methylation landscapes using methodology known as reduced representation bisulfite sequencing (RRBS. While RRBS has proven to be effective in understanding DNA methylation landscapes in humans, mice, and rats, to date, few studies have utilised this powerful method for investigating DNA methylation in agricultural animals. Here we describe the utilisation of RRBS to investigate DNA methylation in sheep Longissimus dorsi muscles. RRBS analysis of ∼1% of the genome from Longissimus dorsi muscles provided data of suitably high precision and accuracy for DNA methylation analysis, at all levels of resolution from genome-wide to individual nucleotides. Combining RRBS data with mRNAseq data allowed the sheep Longissimus dorsi muscle methylome to be compared with methylomes from other species. While some species differences were identified, many similarities were observed between DNA methylation patterns in sheep and other more commonly studied species. The RRBS data presented here highlights the complexity of epigenetic regulation of genes. However, the similarities observed across species are promising, in that knowledge gained from epigenetic studies in human and mice may be applied, with caution, to agricultural species. The ability to accurately measure DNA methylation in agricultural animals will contribute an additional layer of information to the genetic analyses currently being used to maximise production gains in these species.

  18. Flow cytometric analysis of oil palm: a preliminary analysis for cultivars and genomic DNA alteration

    Directory of Open Access Journals (Sweden)

    Warawut Chuthammathat

    2005-12-01

    Full Text Available DNA contents of oil palm (Elaeis guineensis Jacq. cultivars were analyzed by flow cytometry using different external reference plant species. Analysis using corn (Zea mays line CE-777 as a reference plant gave the highest DNA content of oil palm (4.72±0.23 pg 2C-1 whereas the DNA content was found to be lower when using soybean (Glycine max cv. Polanka (3.77±0.09 pg 2C-1 or tomato (Lycopersicon esculentum cv. Stupicke (4.25±0.09 pg 2C-1 as a reference. The nuclear DNA contents of Dura (D109, Pisifera (P168 and Tenera (T38 cultivars were 3.46±0.04, 3.24±0.03 and 3.76±0.04 pg 2C-1 nuclei, respectively, using soybean as a reference. One haploid genome of oil palm therefore ranged from 1.56 to 1.81±109 base pairs. DNA contents from one-year-old calli and cell suspension of oil palm were found to be significantly different from those of seedlings. It thus should be noted that genomic DNA alteration occurred in these cultured tissues. We therefore confirm that flow cytometric analysis could verify cultivars, DNA content and genomic DNA alteration of oil palm using soybean as an external reference standard.

  19. Survey of protein–DNA interactions in Aspergillus oryzae on a genomic scale

    Science.gov (United States)

    Wang, Chao; Lv, Yangyong; Wang, Bin; Yin, Chao; Lin, Ying; Pan, Li

    2015-01-01

    The genome-scale delineation of in vivo protein–DNA interactions is key to understanding genome function. Only ∼5% of transcription factors (TFs) in the Aspergillus genus have been identified using traditional methods. Although the Aspergillus oryzae genome contains >600 TFs, knowledge of the in vivo genome-wide TF-binding sites (TFBSs) in aspergilli remains limited because of the lack of high-quality antibodies. We investigated the landscape of in vivo protein–DNA interactions across the A. oryzae genome through coupling the DNase I digestion of intact nuclei with massively parallel sequencing and the analysis of cleavage patterns in protein–DNA interactions at single-nucleotide resolution. The resulting map identified overrepresented de novo TF-binding motifs from genomic footprints, and provided the detailed chromatin remodeling patterns and the distribution of digital footprints near transcription start sites. The TFBSs of 19 known Aspergillus TFs were also identified based on DNase I digestion data surrounding potential binding sites in conjunction with TF binding specificity information. We observed that the cleavage patterns of TFBSs were dependent on the orientation of TF motifs and independent of strand orientation, consistent with the DNA shape features of binding motifs with flanking sequences. PMID:25883143

  20. Evolutionary consequences of DNA methylation on the GC content in vertebrate genomes.

    Science.gov (United States)

    Mugal, Carina F; Arndt, Peter F; Holm, Lena; Ellegren, Hans

    2015-01-15

    The genomes of many vertebrates show a characteristic variation in GC content. To explain its origin and evolution, mainly three mechanisms have been proposed: selection for GC content, mutation bias, and GC-biased gene conversion. At present, the mechanism of GC-biased gene conversion, i.e., short-scale, unidirectional exchanges between homologous chromosomes in the neighborhood of recombination-initiating double-strand breaks in favor for GC nucleotides, is the most widely accepted hypothesis. We here suggest that DNA methylation also plays an important role in the evolution of GC content in vertebrate genomes. To test this hypothesis, we investigated one mammalian (human) and one avian (chicken) genome. We used bisulfite sequencing to generate a whole-genome methylation map of chicken sperm and made use of a publicly available whole-genome methylation map of human sperm. Inclusion of these methylation maps into a model of GC content evolution provided significant support for the impact of DNA methylation on the local equilibrium GC content. Moreover, two different estimates of equilibrium GC content, one that neglects and one that incorporates the impact of DNA methylation and the concomitant CpG hypermutability, give estimates that differ by approximately 15% in both genomes, arguing for a strong impact of DNA methylation on the evolution of GC content. Thus, our results put forward that previous estimates of equilibrium GC content, which neglect the hypermutability of CpG dinucleotides, need to be reevaluated. Copyright © 2015 Mugal et al.

  1. Black African immigrant community leaders' views on participation in genomics research and DNA biobanking.

    Science.gov (United States)

    Buseh, Aaron G; Underwood, Sandra M; Stevens, Patricia E; Townsend, Leolia; Kelber, Sheryl T

    2013-01-01

    The emergence of DNA biobanks and the power they lend to genomics research promise substantial advances in disease prevention and treatment. Greater participation of racial/ethnic minority populations is necessary to assure a future of personalized medicine for all. The purpose of this study was to explore perspectives on genomics research and DNA biobanking among black African immigrants, an often overlooked US subpopulation. As part of a larger staged study using community-based participatory research (CBPR) methods, we conducted four focus groups with 27 leaders in the black African immigrant community, exploring perceptions about genomics, barriers, and facilitators to participation in DNA biobanks and ethical ways to engage communities. Prominent in their views on genomics research was the legacy of colonial mistreatment and exploitation by Western researchers in their home countries in sub-Saharan Africa. The central dilemma for participants was balancing responsibilities to protect their people from harm and to find solutions for African generations to come. They insisted that nothing short of a transformation of research practice would elicit the full and sustained participation of African immigrants in the genomics enterprise. To better align practice and policy in the field of genomics research and DNA biobanking with values expressed by African immigrant leaders, it is recommended that the field adopt a CBPR model for research and a benefit-sharing model for policy. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Sequencing of chloroplast genome using whole cellular DNA and Solexa sequencing technology

    Directory of Open Access Journals (Sweden)

    Jian eWu

    2012-11-01

    Full Text Available Sequencing of the chloroplast genome using traditional sequencing methods has been difficult because of its size (>120 kb and the complicated procedures required to prepare templates. To explore the feasibility of sequencing the chloroplast genome using DNA extracted from whole cells and Solexa sequencing technology, we sequenced whole cellular DNA isolated from leaves of three Brassica rapa accessions with one lane per accession. In total, 246 Mb, 362Mb, 361 Mb sequence data were generated for the three accessions Chiifu-401-42, Z16 and FT, respectively. Microreads were assembled by reference-guided assembly using the cpDNA sequences of B. rapa, Arabidopsis thaliana, and Nicotiana tabacum. We achieved coverage of more than 99.96% of the cp genome in the three tested accessions using the B. rapa sequence as the reference. When A. thaliana or N. tabacum sequences were used as references, 99.7–99.8% or 95.5–99.7% of the B. rapa chloroplast genome was covered, respectively. These results demonstrated that sequencing of whole cellular DNA isolated from young leaves using the Illumina Genome Analyzer is an efficient method for high-throughput sequencing of chloroplast genome.

  3. Ultra-barcoding in cacao (Theobroma spp.; Malvaceae) using whole chloroplast genomes and nuclear ribosomal DNA.

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    Kane, Nolan; Sveinsson, Saemundur; Dempewolf, Hannes; Yang, Ji Yong; Zhang, Dapeng; Engels, Johannes M M; Cronk, Quentin

    2012-02-01

    To reliably identify lineages below the species level such as subspecies or varieties, we propose an extension to DNA-barcoding using next-generation sequencing to produce whole organellar genomes and substantial nuclear ribosomal sequence. Because this method uses much longer versions of the traditional DNA-barcoding loci in the plastid and ribosomal DNA, we call our approach ultra-barcoding (UBC). We used high-throughput next-generation sequencing to scan the genome and generate reliable sequence of high copy number regions. Using this method, we examined whole plastid genomes as well as nearly 6000 bases of nuclear ribosomal DNA sequences for nine genotypes of Theobroma cacao and an individual of the related species T. grandiflorum, as well as an additional publicly available whole plastid genome of T. cacao. All individuals of T. cacao examined were uniquely distinguished, and evidence of reticulation and gene flow was observed. Sequence variation was observed in some of the canonical barcoding regions between species, but other regions of the chloroplast were more variable both within species and between species, as were ribosomal spacers. Furthermore, no single region provides the level of data available using the complete plastid genome and rDNA. Our data demonstrate that UBC is a viable, increasingly cost-effective approach for reliably distinguishing varieties and even individual genotypes of T. cacao. This approach shows great promise for applications where very closely related or interbreeding taxa must be distinguished.

  4. RNA-dependent DNA endonuclease Cas9 of the CRISPR system: Holy Grail of genome editing?

    Science.gov (United States)

    Gasiunas, Giedrius; Siksnys, Virginijus

    2013-11-01

    Tailor-made nucleases for precise genome modification, such as zinc finger or TALE nucleases, currently represent the state-of-the-art for genome editing. These nucleases combine a programmable protein module which guides the enzyme to the target site with a nuclease domain which cuts DNA at the addressed site. Reprogramming of these nucleases to cut genomes at specific locations requires major protein engineering efforts. RNA-guided DNA endonuclease Cas9 of the type II (clustered regularly interspaced short palindromic repeat) CRISPR-Cas system uses CRISPR RNA (crRNA) as a guide to locate the DNA target and the Cas9 protein to cut DNA. Easy programmability of the Cas9 endonuclease using customizable RNAs brings unprecedented flexibility and versatility for targeted genome modification. We highlight the potential of the Cas9 RNA-guided DNA endonuclease as a novel tool for genome surgery, and discuss possible constraints and future prospects. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. Genome dynamics of short oligonucleotides: the example of bacterial DNA uptake enhancing sequences.

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    Mohammed Bakkali

    Full Text Available Among the many bacteria naturally competent for transformation by DNA uptake-a phenomenon with significant clinical and financial implications- Pasteurellaceae and Neisseriaceae species preferentially take up DNA containing specific short sequences. The genomic overrepresentation of these DNA uptake enhancing sequences (DUES causes preferential uptake of conspecific DNA, but the function(s behind this overrepresentation and its evolution are still a matter for discovery. Here I analyze DUES genome dynamics and evolution and test the validity of the results to other selectively constrained oligonucleotides. I use statistical methods and computer simulations to examine DUESs accumulation in Haemophilus influenzae and Neisseria gonorrhoeae genomes. I analyze DUESs sequence and nucleotide frequencies, as well as those of all their mismatched forms, and prove the dependence of DUESs genomic overrepresentation on their preferential uptake by quantifying and correlating both characteristics. I then argue that mutation, uptake bias, and weak selection against DUESs in less constrained parts of the genome combined are sufficient enough to cause DUESs accumulation in susceptible parts of the genome with no need for other DUES function. The distribution of overrepresentation values across sequences with different mismatch loads compared to the DUES suggests a gradual yet not linear molecular drive of DNA sequences depending on their similarity to the DUES. Other genomically overrepresented sequences, both pro- and eukaryotic, show similar distribution of frequencies suggesting that the molecular drive reported above applies to other frequent oligonucleotides. Rare oligonucleotides, however, seem to be gradually drawn to genomic underrepresentation, thus, suggesting a molecular drag. To my knowledge this work provides the first clear evidence of the gradual evolution of selectively constrained oligonucleotides, including repeated, palindromic and protein

  6. Metabolism, Genomics, and DNA Repair in the Mouse Aging Liver

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    Michel Lebel

    2011-01-01

    Full Text Available The liver plays a pivotal role in the metabolism of nutrients, drugs, hormones, and metabolic waste products, thereby maintaining body homeostasis. The liver undergoes substantial changes in structure and function within old age. Such changes are associated with significant impairment of many hepatic metabolic and detoxification activities, with implications for systemic aging and age-related disease. It has become clear, using rodent models as biological tools, that genetic instability in the form of gross DNA rearrangements or point mutations accumulate in the liver with age. DNA lesions, such as oxidized bases or persistent breaks, increase with age and correlate well with the presence of senescent hepatocytes. The level of DNA damage and/or mutation can be affected by changes in carcinogen activation, decreased ability to repair DNA, or a combination of these factors. This paper covers some of the DNA repair pathways affecting liver homeostasis with age using rodents as model systems.

  7. The genome of the brown alga Ectocarpus siliculosus contains a series of viral DNA pieces, suggesting an ancient association with large dsDNA viruses

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    Boland Wilhelm

    2008-04-01

    Full Text Available Abstract Background Ectocarpus siliculosus virus-1 (EsV-1 is a lysogenic dsDNA virus belonging to the super family of nucleocytoplasmic large DNA viruses (NCLDV that infect Ectocarpus siliculosus, a marine filamentous brown alga. Previous studies indicated that the viral genome is integrated into the host DNA. In order to find the integration sites of the viral genome, a genomic library from EsV-1-infected algae was screened using labelled EsV-1 DNA. Several fragments were isolated and some of them were sequenced and analyzed in detail. Results Analysis revealed that the algal genome is split by a copy of viral sequences that have a high identity to EsV-1 DNA sequences. These fragments are interspersed with DNA repeats, pseudogenes and genes coding for products involved in DNA replication, integration and transposition. Some of these gene products are not encoded by EsV-1 but are present in the genome of other members of the NCLDV family. Further analysis suggests that the Ectocarpus algal genome contains traces of the integration of a large dsDNA viral genome; this genome could be the ancestor of the extant NCLDV genomes. Furthermore, several lines of evidence indicate that the EsV-1 genome might have originated in these viral DNA pieces, implying the existence of a complex integration and recombination system. A protein similar to a new class of tyrosine recombinases might be a key enzyme of this system. Conclusion Our results support the hypothesis that some dsDNA viruses are monophyletic and evolved principally through genome reduction. Moreover, we hypothesize that phaeoviruses have probably developed an original replication system.

  8. Capacitive DNA sensor for rapid and sensitive detection of whole genome human herpesvirus-1 dsDNA in serum.

    Science.gov (United States)

    Cheng, Cheng; Oueslati, Rania; Wu, Jayne; Chen, Jiangang; Eda, Shigetoshi

    2017-06-01

    This work presents a rapid, highly sensitive, low-cost, and specific capacitive DNA sensor for detection of whole genome human herpesvirus-1 DNA. This sensor is capable of direct DNA detection with a response time of 30 s, and it can be used to test standard buffer or serum samples. The sensing approach for DNA detection is based on alternating current (AC) electrokinetics. By applying an inhomogeneous AC electric field on sensor electrodes, positive dielectrophoresis is induced to accelerate DNA hybridization. The same applied AC signal also directly measures the hybridization of target with the probe on the sensor surface. Experiments are conducted to optimize the AC signal, as well as the buffers for probe immobilization and target DNA hybridization. The assay is highly sensitive and specific, with no response to human herpesvirus-2 DNA at 5 ng/mL and a LOD of 1.0 pg/mL (6.5 copies/μL or 10.7 aM) in standard buffer. When testing the double stranded (ds) DNA spiked in human serum samples, the sensor yields a LOD of 20.0 pg/mL (129.5 copies/μL or 0.21 femtomolar (fM)) in neat serum. In this work, the target is whole genome dsDNA, consequently the test can be performed without the use of enzyme or amplification, which considerably simplifies the sensor operation and is highly suitable for point of care disease diagnosis. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Enhancing Targeted Genomic DNA Editing in Chicken Cells Using the CRISPR/Cas9 System

    Science.gov (United States)

    Wang, Ling; Yang, Likai; Guo, Yijie; Du, Weili; Yin, Yajun; Zhang, Tao; Lu, Hongzhao

    2017-01-01

    The CRISPR/Cas9 system has enabled highly efficient genome targeted editing for various organisms. However, few studies have focused on CRISPR/Cas9 nuclease-mediated chicken genome editing compared with mammalian genomes. The current study combined CRISPR with yeast Rad52 (yRad52) to enhance targeted genomic DNA editing in chicken DF-1 cells. The efficiency of CRISPR/Cas9 nuclease-induced targeted mutations in the chicken genome was increased to 41.9% via the enrichment of the dual-reporter surrogate system. In addition, the combined effect of CRISPR nuclease and yRad52 dramatically increased the efficiency of the targeted substitution in the myostatin gene using 50-mer oligodeoxynucleotides (ssODN) as the donor DNA, resulting in a 36.7% editing efficiency after puromycin selection. Furthermore, based on the effect of yRad52, the frequency of exogenous gene integration in the chicken genome was more than 3-fold higher than that without yRad52. Collectively, these results suggest that ssODN is an ideal donor DNA for targeted substitution and that CRISPR/Cas9 combined with yRad52 significantly enhances chicken genome editing. These findings could be extensively applied in other organisms. PMID:28068387

  10. A genomic schism in birds revealed by phylogenetic analysis of DNA strings.

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    Edwards, Scott V; Fertil, Bernard; Giron, Alain; Deschavanne, Patrick J

    2002-08-01

    The molecular systematics of vertebrates has been based entirely on alignments of primary structures of macromolecules; however, higher order features of DNA sequences not used in traditional studies also contain valuable phylogenetic information. Recent molecular data sets conflict over the phylogenetic placement of flightless birds (ratites - paleognaths), but placement of this clade critically influences interpretation of character change in birds. To help resolve this issue, we applied a new bioinformatics approach to the largest molecular data set currently available. We distilled nearly one megabase (1 million base pairs) of heterogeneous avian genomic DNA from 20 birds and an alligator into genomic signatures, defined as the complete set of frequencies of short sequence motifs (strings), thereby providing a way to directly compare higher order features of nonhomologous DNA sequences. Phylogenetic analysis and principal component analysis of the signatures strongly support the traditional hypothesis of basal ratites and monophyly of the nonratite birds (neognaths) and imply that ratite genomes are linguistically primitive within birds, despite their base compositional similarity to neognath genomes. Our analyses show further that the phylogenetic signal of genomic signatures are strongest among deep splits within vertebrates. Despite clear problems with phylogenetic analysis of genomic signatures, our study raises intriguing issues about the biological and genomic differences that fundamentally differentiate paleognaths and neognaths.

  11. FA-SAT Is an Old Satellite DNA Frozen in Several Bilateria Genomes

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    Ferreira, Daniela; Mendes-da-Silva, Ana; Meles, Susana; Adega, Filomena

    2017-01-01

    Abstract In recent years, a growing body of evidence has recognized the tandem repeat sequences, and specifically satellite DNA, as a functional class of sequences in the genomic “dark matter.” Using an original, complementary, and thus an eclectic experimental design, we show that the cat archetypal satellite DNA sequence, FA-SAT, is “frozen” conservatively in several Bilateria genomes. We found different genomic FA-SAT architectures, and the interspersion pattern was conserved. In Carnivora genomes, the FA-SAT-related sequences are also amplified, with the predominance of a specific FA-SAT variant, at the heterochromatic regions. We inspected the cat genome project to locate FA-SAT array flanking regions and revealed an intensive intermingling with transposable elements. Our results also show that FA-SAT-related sequences are transcribed and that the most abundant FA-SAT variant is not always the most transcribed. We thus conclude that the DNA sequences of FA-SAT and their transcripts are “frozen” in these genomes. Future work is needed to disclose any putative function that these sequences may play in these genomes.

  12. Repair-mediated duplication by capture of proximal chromosomal DNA has shaped vertebrate genome evolution.

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    John K Pace

    2009-05-01

    Full Text Available DNA double-strand breaks (DSBs are a common form of cellular damage that can lead to cell death if not repaired promptly. Experimental systems have shown that DSB repair in eukaryotic cells is often imperfect and may result in the insertion of extra chromosomal DNA or the duplication of existing DNA at the breakpoint. These events are thought to be a source of genomic instability and human diseases, but it is unclear whether they have contributed significantly to genome evolution. Here we developed an innovative computational pipeline that takes advantage of the repetitive structure of genomes to detect repair-mediated duplication events (RDs that occurred in the germline and created insertions of at least 50 bp of genomic DNA. Using this pipeline we identified over 1,000 probable RDs in the human genome. Of these, 824 were intra-chromosomal, closely linked duplications of up to 619 bp bearing the hallmarks of the synthesis-dependent strand-annealing repair pathway. This mechanism has duplicated hundreds of sequences predicted to be functional in the human genome, including exons, UTRs, intron splice sites and transcription factor binding sites. Dating of the duplication events using comparative genomics and experimental validation revealed that the mechanism has operated continuously but with decreasing intensity throughout primate evolution. The mechanism has produced species-specific duplications in all primate species surveyed and is contributing to genomic variation among humans. Finally, we show that RDs have also occurred, albeit at a lower frequency, in non-primate mammals and other vertebrates, indicating that this mechanism has been an important force shaping vertebrate genome evolution.

  13. Local chromatin structure of heterochromatin regulates repeated DNA stability, nucleolus structure, and genome integrity

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    Peng, Jamy C. [Univ. of California, Berkeley, CA (United States)

    2007-01-01

    Heterochromatin constitutes a significant portion of the genome in higher eukaryotes; approximately 30% in Drosophila and human. Heterochromatin contains a high repeat DNA content and a low density of protein-encoding genes. In contrast, euchromatin is composed mostly of unique sequences and contains the majority of single-copy genes. Genetic and cytological studies demonstrated that heterochromatin exhibits regulatory roles in chromosome organization, centromere function and telomere protection. As an epigenetically regulated structure, heterochromatin formation is not defined by any DNA sequence consensus. Heterochromatin is characterized by its association with nucleosomes containing methylated-lysine 9 of histone H3 (H3K9me), heterochromatin protein 1 (HP1) that binds H3K9me, and Su(var)3-9, which methylates H3K9 and binds HP1. Heterochromatin formation and functions are influenced by HP1, Su(var)3-9, and the RNA interference (RNAi) pathway. My thesis project investigates how heterochromatin formation and function impact nuclear architecture, repeated DNA organization, and genome stability in Drosophila melanogaster. H3K9me-based chromatin reduces extrachromosomal DNA formation; most likely by restricting the access of repair machineries to repeated DNAs. Reducing extrachromosomal ribosomal DNA stabilizes rDNA repeats and the nucleolus structure. H3K9me-based chromatin also inhibits DNA damage in heterochromatin. Cells with compromised heterochromatin structure, due to Su(var)3-9 or dcr-2 (a component of the RNAi pathway) mutations, display severe DNA damage in heterochromatin compared to wild type. In these mutant cells, accumulated DNA damage leads to chromosomal defects such as translocations, defective DNA repair response, and activation of the G2-M DNA repair and mitotic checkpoints that ensure cellular and animal viability. My thesis research suggests that DNA replication, repair, and recombination mechanisms in heterochromatin differ from those in

  14. Genome wide survey of microsatellites in ssDNA viruses infecting vertebrates.

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    Jain, Ankit; Mittal, Nikhil; Sharma, Prakash C

    2014-12-01

    Microsatellites or Simple Sequence Repeats (SSRs) are tandem iterations of one to six base pairs, non-randomly distributed throughout prokaryotic and eukaryotic genomes. Limited knowledge is available about distribution of microsatellites in single stranded DNA (ssDNA) viruses, particularly vertebrate infecting viruses. We studied microsatellite distribution in 118 ssDNA virus genomes belonging to three families of vertebrate infecting viruses namely Circoviridae, Parvoviridae, and Anelloviridae, and found that microsatellites constitute an important component of these virus genomes. Mononucleotide repeats were predominant followed by dinucleotide and trinucleotide repeats. A strong positive relationship existed between number of mononucleotide repeats and genome size among all the three virus families. A similar relationship existed for the occurrence of DTTPH (di-, tri-, tetra-, penta- and hexa-nucleotide) repeats in the families Anelloviridae and Parvoviridae only. Relative abundance and relative density of mononucleotide repeats showed a strong positive relationship with genome size in Circoviridae and Parvoviridae. However, in the case of DTTPH repeats, these features showed a strong relationship with genome size in Circoviridae only. On the other hand, relative microsatellite abundance and relative density of mononucleotide repeats were negatively correlated with GC content (%) in Parvoviridae genomes. On the basis of available annotations, our analysis revealed maximum occurrence of mononucleotide as well as DTTPH repeats in the coding regions of these virus genomes. Interestingly, after normalizing the length of the coding and non-coding regions of each virus genome, we found relative density of microsatellites much higher in the non-coding regions. We understand that the present study will help in the better characterization of the stability, genome organization and evolution of these virus classes and may provide useful leads to decipher the

  15. Genome-wide mRNA sequencing of a single canine cerebellar cortical degeneration case leads to the identification of a disease associated SPTBN2 mutation

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    Forman Oliver P

    2012-07-01

    Full Text Available Abstract Background Neonatal cerebellar cortical degeneration is a neurodegenerative disease described in several canine breeds including the Beagle. Affected Beagles are unable to ambulate normally from the onset of walking and the main pathological findings include Purkinje cell loss with swollen dendritic processes. Previous reports suggest an autosomal recessive mode of inheritance. The development of massively parallel sequencing techniques has presented the opportunity to investigate individual clinical cases using genome-wide sequencing approaches. We used genome-wide mRNA sequencing (mRNA-seq of cerebellum tissue from a single Beagle with neonatal cerebellar cortical degeneration as a method of candidate gene sequencing, with the aim of identifying the causal mutation. Results A four-week old Beagle dog presented with progressive signs of cerebellar ataxia and the owner elected euthanasia. Histopathology revealed findings consistent with cerebellar cortical degeneration. Genome-wide mRNA sequencing (mRNA-seq of RNA from cerebellum tissue was used as a method of candidate gene sequencing. After analysis of the canine orthologues of human spinocerebellar ataxia associated genes, we identified a homozygous 8 bp deletion in the β-III spectrin gene, SPTBN2, associated with spinocerebellar type 5 in humans. Genotype analysis of the sire, dam, ten clinically unaffected siblings, and an affected sibling from a previous litter, showed the mutation to fully segregate with the disorder. Previous studies have shown that β-III spectrin is critical for Purkinje cell development, and the absence of this protein can lead to cell damage through excitotoxicity, consistent with the observed Purkinje cell loss, degeneration of dendritic processes and associated neurological dysfunction in this Beagle. Conclusions An 8 bp deletion in the SPTBN2 gene encoding β-III spectrin is associated with neonatal cerebellar cortical degeneration in Beagle dogs

  16. Sensitive and specific KRAS somatic mutation analysis on whole-genome amplified DNA from archival tissues.

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    van Eijk, Ronald; van Puijenbroek, Marjo; Chhatta, Amiet R; Gupta, Nisha; Vossen, Rolf H A M; Lips, Esther H; Cleton-Jansen, Anne-Marie; Morreau, Hans; van Wezel, Tom

    2010-01-01

    Kirsten RAS (KRAS) is a small GTPase that plays a key role in Ras/mitogen-activated protein kinase signaling; somatic mutations in KRAS are frequently found in many cancers. The most common KRAS mutations result in a constitutively active protein. Accurate detection of KRAS mutations is pivotal to the molecular diagnosis of cancer and may guide proper treatment selection. Here, we describe a two-step KRAS mutation screening protocol that combines whole-genome amplification (WGA), high-resolution melting analysis (HRM) as a prescreen method for mutation carrying samples, and direct Sanger sequencing of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue, from which limited amounts of DNA are available. We developed target-specific primers, thereby avoiding amplification of homologous KRAS sequences. The addition of herring sperm DNA facilitated WGA in DNA samples isolated from as few as 100 cells. KRAS mutation screening using high-resolution melting analysis on wgaDNA from formalin-fixed, paraffin-embedded tissue is highly sensitive and specific; additionally, this method is feasible for screening of clinical specimens, as illustrated by our analysis of pancreatic cancers. Furthermore, PCR on wgaDNA does not introduce genotypic changes, as opposed to unamplified genomic DNA. This method can, after validation, be applied to virtually any potentially mutated region in the genome.

  17. Extensive variation in the density and distribution of DNA polymorphism in sorghum genomes.

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    Joseph Evans

    Full Text Available Sorghum genotypes currently used for grain production in the United States were developed from African landraces that were imported starting in the mid-to-late 19(th century. Farmers and plant breeders selected genotypes for grain production with reduced plant height, early flowering, increased grain yield, adaptation to drought, and improved resistance to lodging, diseases and pests. DNA polymorphisms that distinguish three historically important grain sorghum genotypes, BTx623, BTx642 and Tx7000, were characterized by genome sequencing, genotyping by sequencing, genetic mapping, and pedigree-based haplotype analysis. The distribution and density of DNA polymorphisms in the sequenced genomes varied widely, in part because the lines were derived through breeding and selection from diverse Kafir, Durra, and Caudatum race accessions. Genomic DNA spanning dw1 (SBI-09 and dw3 (SBI-07 had identical haplotypes due to selection for reduced height. Lower SNP density in genes located in pericentromeric regions compared with genes located in euchromatic regions is consistent with background selection in these regions of low recombination. SNP density was higher in euchromatic DNA and varied >100-fold in contiguous intervals that spanned up to 300 Kbp. The localized variation in DNA polymorphism density occurred throughout euchromatic regions where recombination is elevated, however, polymorphism density was not correlated with gene density or DNA methylation. Overall, sorghum chromosomes contain distal euchromatic regions characterized by extensive, localized variation in DNA polymorphism density, and large pericentromeric regions of low gene density, diversity, and recombination.

  18. Genomic DNA Copy Number Aberrations, Histological Diagnosis, Oral Subsite and Aneuploidy in OPMDs/OSCCs

    Science.gov (United States)

    Monticone, Massimiliano; Malacarne, Davide; Cirmena, Gabriella; Brown, David; Aiello, Cinzia; Maffei, Massimo; Marino, Roberto; Giaretti, Walter; Pentenero, Monica

    2015-01-01

    Oral potentially malignant disorders (OPMDs) characterized by the presence of dysplasia and DNA copy number aberrations (CNAs), may reflect chromosomal instability (CIN) and predispose to oral squamous cell carcinoma (OSCC). Early detection of OPMDs with such characteristics may play a crucial role in OSCC prevention. The aim of this study was to explore the relationship between CNAs, histological diagnosis, oral subsite and aneuploidy in OPMDs/OSCCs. Samples from OPMDs and OSCCs were processed by high-resolution DNA flow cytometry (hr DNA-FCM) to determine the relative nuclear DNA content. Additionally, CNAs were obtained for a subset of these samples by genome-wide array comparative genomic hybridization (aCGH) using DNA extracted from either diploid or aneuploid nuclei suspension sorted by FCM. Our study shows that: i) aneuploidy, global genomic imbalance (measured as the total number of CNAs) and specific focal CNAs occur early in the development of oral cancer and become more frequent at later stages; ii) OPMDs limited to tongue (TNG) mucosa display a higher frequency of aneuploidy compared to OPMDs confined to buccal mucosa (BM) as measured by DNA-FCM; iii) TNG OPMDs/OSCCs show peculiar features of CIN compared to BM OPMDs/OSCCs given the preferential association with total broad and specific focal CNA gains. Follow-up studies are warranted to establish whether the presence of DNA aneuploidy and specific focal or broad CNAs may predict cancer development in non-dysplastic OPMDs. PMID:26540282

  19. Digital Droplet Multiple Displacement Amplification (ddMDA for Whole Genome Sequencing of Limited DNA Samples.

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    Minsoung Rhee

    Full Text Available Multiple displacement amplification (MDA is a widely used technique for amplification of DNA from samples containing limited amounts of DNA (e.g., uncultivable microbes or clinical samples before whole genome sequencing. Despite its advantages of high yield and fidelity, it suffers from high amplification bias and non-specific amplification when amplifying sub-nanogram of template DNA. Here, we present a microfluidic digital droplet MDA (ddMDA technique where partitioning of the template DNA into thousands of sub-nanoliter droplets, each containing a small number of DNA fragments, greatly reduces the competition among DNA fragments for primers and polymerase thereby greatly reducing amplification bias. Consequently, the ddMDA approach enabled a more uniform coverage of amplification over the entire length of the genome, with significantly lower bias and non-specific amplification than conventional MDA. For a sample containing 0.1 pg/μL of E. coli DNA (equivalent of ~3/1000 of an E. coli genome per droplet, ddMDA achieves a 65-fold increase in coverage in de novo assembly, and more than 20-fold increase in specificity (percentage of reads mapping to E. coli compared to the conventional tube MDA. ddMDA offers a powerful method useful for many applications including medical diagnostics, forensics, and environmental microbiology.

  20. A network of multi-tasking proteins at the DNA replication fork preserves genome stability.

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    Martin E Budd

    2005-12-01

    Full Text Available To elucidate the network that maintains high fidelity genome replication, we have introduced two conditional mutant alleles of DNA2, an essential DNA replication gene, into each of the approximately 4,700 viable yeast deletion mutants and determined the fitness of the double mutants. Fifty-six DNA2-interacting genes were identified. Clustering analysis of genomic synthetic lethality profiles of each of 43 of the DNA2-interacting genes defines a network (consisting of 322 genes and 876 interactions whose topology provides clues as to how replication proteins coordinate regulation and repair to protect genome integrity. The results also shed new light on the functions of the query gene DNA2, which, despite many years of study, remain controversial, especially its proposed role in Okazaki fragment processing and the nature of its in vivo substrates. Because of the multifunctional nature of virtually all proteins at the replication fork, the meaning of any single genetic interaction is inherently ambiguous. The multiplexing nature of the current studies, however, combined with follow-up supporting experiments, reveals most if not all of the unique pathways requiring Dna2p. These include not only Okazaki fragment processing and DNA repair but also chromatin dynamics.

  1. Evolution of DNA methylation patterns in the Brassicaceae is driven by differences in genome organization.

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    Danelle K Seymour

    2014-11-01

    Full Text Available DNA methylation is an ancient molecular modification found in most eukaryotes. In plants, DNA methylation is not only critical for transcriptionally silencing transposons, but can also affect phenotype by altering expression of protein coding genes. The extent of its contribution to phenotypic diversity over evolutionary time is, however, unclear, because of limited stability of epialleles that are not linked to DNA mutations. To dissect the relative contribution of DNA methylation to transposon surveillance and host gene regulation, we leveraged information from three species in the Brassicaceae that vary in genome architecture, Capsella rubella, Arabidopsis lyrata, and Arabidopsis thaliana. We found that the lineage-specific expansion and contraction of transposon and repeat sequences is the main driver of interspecific differences in DNA methylation. The most heavily methylated portions of the genome are thus not conserved at the sequence level. Outside of repeat-associated methylation, there is a surprising degree of conservation in methylation at single nucleotides located in gene bodies. Finally, dynamic DNA methylation is affected more by tissue type than by environmental differences in all species, but these responses are not conserved. The majority of DNA methylation variation between species resides in hypervariable genomic regions, and thus, in the context of macroevolution, is of limited phenotypic consequence.

  2. A Network of Multi-Tasking Proteins at the DNA Replication Fork Preserves Genome Stability.

    Directory of Open Access Journals (Sweden)

    2005-12-01

    Full Text Available To elucidate the network that maintains high fidelity genome replication, we have introduced two conditional mutant alleles of DNA2, an essential DNA replication gene, into each of the approximately 4,700 viable yeast deletion mutants and determined the fitness of the double mutants. Fifty-six DNA2-interacting genes were identified. Clustering analysis of genomic synthetic lethality profiles of each of 43 of the DNA2-interacting genes defines a network (consisting of 322 genes and 876 interactions whose topology provides clues as to how replication proteins coordinate regulation and repair to protect genome integrity. The results also shed new light on the functions of the query gene DNA2, which, despite many years of study, remain controversial, especially its proposed role in Okazaki fragment processing and the nature of its in vivo substrates. Because of the multifunctional nature of virtually all proteins at the replication fork, the meaning of any single genetic interaction is inherently ambiguous. The multiplexing nature of the current studies, however, combined with follow-up supporting experiments, reveals most if not all of the unique pathways requiring Dna2p. These include not only Okazaki fragment processing and DNA repair but also chromatin dynamics.

  3. Large-scale chromosome folding versus genomic DNA sequences: A discrete double Fourier transform technique.

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    Chechetkin, V R; Lobzin, V V

    2017-08-07

    Using state-of-the-art techniques combining imaging methods and high-throughput genomic mapping tools leaded to the significant progress in detailing chromosome architecture of various organisms. However, a gap still remains between the rapidly growing structural data on the chromosome folding and the large-scale genome organization. Could a part of information on the chromosome folding be obtained directly from underlying genomic DNA sequences abundantly stored in the databanks? To answer this question, we developed an original discrete double Fourier transform (DDFT). DDFT serves for the detection of large-scale genome regularities associated with domains/units at the different levels of hierarchical chromosome folding. The method is versatile and can be applied to both genomic DNA sequences and corresponding physico-chemical parameters such as base-pairing free energy. The latter characteristic is closely related to the replication and transcription and can also be used for the assessment of temperature or supercoiling effects on the chromosome folding. We tested the method on the genome of E. coli K-12 and found good correspondence with the annotated domains/units established experimentally. As a brief illustration of further abilities of DDFT, the study of large-scale genome organization for bacteriophage PHIX174 and bacterium Caulobacter crescentus was also added. The combined experimental, modeling, and bioinformatic DDFT analysis should yield more complete knowledge on the chromosome architecture and genome organization. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Nuclear DNA content in Sinningia (Gesneriaceae); intraspecific genome size variation and genome characterization in S. speciosa.

    Science.gov (United States)

    Zaitlin, David; Pierce, Andrew J

    2010-12-01

    The Gesneriaceae (Lamiales) is a family of flowering plants comprising >3000 species of mainly tropical origin, the most familiar of which is the cultivated African violet (Saintpaulia spp.). Species of Gesneriaceae are poorly represented in the lists of taxa sampled for genome size estimation; measurements are available for three species of Ramonda and one each of Haberlea, Saintpaulia, and Streptocarpus, all species of Old World origin. We report here nuclear genome size estimates for 10 species of Sinningia, a neotropical genus largely restricted to Brazil. Flow cytometry of leaf cell nuclei showed that holoploid genome size in Sinningia is very small (approximately two times the size of the Arabidopsis genome), and is small compared to the other six species of Gesneriaceae with genome size estimates. We also documented intraspecific genome size variation of 21%-26% within a group of wild Sinningia speciosa (Lodd.) Hiern collections. In addition, we analyzed 1210 genome survey sequences from S. speciosa to characterize basic features of the nuclear genome such as guanine-cytosine content, types of repetitive elements, numbers of protein-coding sequences, and sequences unique to S. speciosa. We included several other angiosperm species as genome size standards, one of which was the snapdragon (Antirrhinum majus L.; Veronicaceae, Lamiales). Multiple measurements on three accessions indicated that the genome size of A. majus is ~633 × 10⁶ base pairs, which is approximately 40% of the previously published estimate.

  5. Metabolism, genomics, and DNA repair in the mouse aging liver

    DEFF Research Database (Denmark)

    Lebel, Michel; de Souza-Pinto, Nadja C; Bohr, Vilhelm A

    2011-01-01

    The liver plays a pivotal role in the metabolism of nutrients, drugs, hormones, and metabolic waste products, thereby maintaining body homeostasis. The liver undergoes substantial changes in structure and function within old age. Such changes are associated with significant impairment of many......, such as oxidized bases or persistent breaks, increase with age and correlate well with the presence of senescent hepatocytes. The level of DNA damage and/or mutation can be affected by changes in carcinogen activation, decreased ability to repair DNA, or a combination of these factors. This paper covers some...... hepatic metabolic and detoxification activities, with implications for systemic aging and age-related disease. It has become clear, using rodent models as biological tools, that genetic instability in the form of gross DNA rearrangements or point mutations accumulate in the liver with age. DNA lesions...

  6. DNA repair, genome stability and cancer: a historical perspective.

    Science.gov (United States)

    Jeggo, Penny A; Pearl, Laurence H; Carr, Antony M

    2016-01-01

    The multistep process of cancer progresses over many years. The prevention of mutations by DNA repair pathways led to an early appreciation of a role for repair in cancer avoidance. However, the broader role of the DNA damage response (DDR) emerged more slowly. In this Timeline article, we reflect on how our understanding of the steps leading to cancer developed, focusing on the role of the DDR. We also consider how our current knowledge can be exploited for cancer therapy.

  7. Analysis of the mycoplasma genome by recombinant DNA technology

    DEFF Research Database (Denmark)

    Christiansen, C; Frydenberg, J; Christiansen, Gunna

    1984-01-01

    A library of DNA fragments from Mycoplasma sp. strain PG50 has been made in the vector pBR325. Analysis in Escherichia coli minicells of randomly picked clones from this library demonstrated that many plasmids can promote synthesis of mycoplasma protein in the E. coli genetic background. Screening....... The DNA sequence of 16S rRNA and the surrounding control regions has been determined....

  8. Optical Detection of Non-amplified Genomic DNA

    Science.gov (United States)

    Li, Di; Fan, Chunhai

    Nucleic acid sequences are unique to every living organisms including animals, plants and even bacteria and virus, which provide a practical molecular target for the identification and diagnosis of various diseases. DNA contains heterocyclic rings that has inherent optical absorbance at 260 nm, which is widely used to quantify single and double stranded DNA in biology. However, this simple quantification method could not differentiate sequences; therefore it is not suitable for sequence-specific analyte detection. In addition to a few exceptions such as chiral-related circular dichroism spectra, DNA hybridization does not produce significant changes in optical signals, thus an optical label is generally needed for sequence-specific DNA detection with optical means. During the last two decades, we have witnessed explosive progress in the area of optical DNA detection, especially with the help of simultaneously rapidly developed nanomaterials. In this chapter, we will summarize recent advances in optical DNA detection including colorimetric, fluorescent, luminescent, surface plasmon resonance (SPR) and Raman scattering assays. Challenges and problems remained to be addressed are also discussed.

  9. Genomic DNA Methylation Analyses Reveal the Distinct Profiles in Castor Bean Seeds with Persistent Endosperms1

    Science.gov (United States)

    Yang, Tianquan; Dong, Xue; Li, De-Zhu

    2016-01-01

    Investigations of genomic DNA methylation in seeds have been restricted to a few model plants. The endosperm genomic DNA hypomethylation has been identified in angiosperm, but it is difficult to dissect the mechanism of how this hypomethylation is established and maintained because endosperm is ephemeral and disappears with seed development in most dicots. Castor bean (Ricinus communis), unlike Arabidopsis (Arabidopsis thaliana), endosperm is persistent throughout seed development, providing an excellent model in which to dissect the mechanism of endosperm genomic hypomethylation in dicots. We characterized the DNA methylation-related genes encoding DNA methyltransferases and demethylases and analyzed their expression profiles in different tissues. We examined genomic methylation including CG, CHG, and CHH contexts in endosperm and embryo tissues using bisulfite sequencing and revealed that the CHH methylation extent in endosperm and embryo was, unexpectedly, substantially higher than in previously studied plants, irrespective of the CHH percentage in their genomes. In particular, we found that the endosperm exhibited a global reduction in CG and CHG methylation extents relative to the embryo, markedly switching global gene expression. However, CHH methylation occurring in endosperm did not exhibit a significant reduction. Combining with the expression of 24-nucleotide small interfering RNAs (siRNAs) mapped within transposable element (TE) regions and genes involved in the RNA-directed DNA methylation pathway, we demonstrate that the 24-nucleotide siRNAs played a critical role in maintaining CHH methylation and repressing the activation of TEs in persistent endosperm development. This study discovered a novel genomic DNA methylation pattern and proposes the potential mechanism occurring in dicot seeds with persistent endosperm. PMID:27208275

  10. Inter-genomic DNA Exchanges and Homeologous Gene Silencing Shaped the Nascent Allopolyploid Coffee Genome (Coffea arabica L.

    Directory of Open Access Journals (Sweden)

    Philippe Lashermes

    2016-09-01

    Full Text Available Allopolyploidization is a biological process that has played a major role in plant speciation and evolution. Genomic changes are common consequences of polyploidization, but their dynamics over time are still poorly understood. Coffea arabica, a recently formed allotetraploid, was chosen to study genetic changes that accompany allopolyploid formation. Both RNA-seq and DNA-seq data were generated from two genetically distant C. arabica accessions. Genomic structural variation was investigated using C. canephora, one of its diploid progenitors, as reference genome. The fate of 9047 duplicate homeologous genes was inferred and compared between the accessions. The pattern of SNP density along the reference genome was consistent with the allopolyploid structure. Large genomic duplications or deletions were not detected. Two homeologous copies were retained and expressed in 96% of the genes analyzed. Nevertheless, duplicated genes were found to be affected by various genomic changes leading to homeolog loss or silencing. Genetic and epigenetic changes were evidenced that could have played a major role in the stabilization of the unique ancestral allotetraploid and its subsequent diversification. While the early evolution of C. arabica mainly involved homeologous crossover exchanges, the later stage appears to have relied on more gradual evolution involving gene conversion and homeolog silencing.

  11. Complete mitochondrial DNA genome of Pseudobagrus medianalis (Siluriformes: Bagridae).

    Science.gov (United States)

    Liang, Hong-wei; Li, Zhong; Zou, Gui-wei; Shi, Ming-yan

    2016-01-01

    The complete mitochondrial genome was sequenced from the freshwater fish, Pseudobagrus medianalis (Siluriformes: Bagridae) in this study. The genome sequence was 16,647 bp in length, and the gene order and contents were identical with the bagridae fishes. The mitochondrial genome contains 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs and 2 non-coding regions (control region and origin of light-strand replication). All genes were encoded on the heavy strain except for ND6 and eight tRNA genes. The overall base composition is 25.7% A, 30.9% T, 28.0% G, 15.4% C, with an A+T bias of 56.6%. The complete mitogenome data provides useful genetic markers for the studies on the molecular identification, population genetics, phylogenetic analysis and conservation genetics.

  12. MultiPipMaker and supporting tools: alignments and analysis of multiple genomic DNA sequences

    OpenAIRE

    Schwartz, Scott; Elnitski, Laura; Li, Mei; Weirauch, Matt; Riemer, Cathy; Smit, Arian; Green, Eric D.; Hardison, Ross C.; Miller, Webb

    2003-01-01

    Analysis of multiple sequence alignments can generate important, testable hypotheses about the phylogenetic history and cellular function of genomic sequences. We describe the MultiPipMaker server, which aligns multiple, long genomic DNA sequences quickly and with good sensitivity (available at http://bio.cse.psu.edu/ since May 2001). Alignments are computed between a contiguous reference sequence and one or more secondary sequences, which can be finished or draft sequence. The outputs includ...

  13. Whole Genome mtDNA Sequencing on the Ion Torrent PGM

    OpenAIRE

    Powers, M.; Watkins, W; Potucek, Y.; Warner, Derek

    2012-01-01

    The mitochondrial genome is a 16.5 kb circular chromosome encoding 37 genes. Mutations in the mitochondrial genome have been linked to many diseases such as Autism, Kearns-Sayre syndrome (KSS), Leber's hereditary optic neuropathy (LHON), Leigh Syndrome, and myoclonic epilepsy with red ragged fibers (MERRF). In addition to clinical interest, there is strong interest in sequencing mtDNA for archeological and genealogical studies.

  14. cDNA2Genome: A tool for mapping and annotating cDNAs

    Directory of Open Access Journals (Sweden)

    Suhai Sandor

    2003-09-01

    Full Text Available Abstract Background In the last years several high-throughput cDNA sequencing projects have been funded worldwide with the aim of identifying and characterizing the structure of complete novel human transcripts. However some of these cDNAs are error prone due to frameshifts and stop codon errors caused by low sequence quality, or to cloning of truncated inserts, among other reasons. Therefore, accurate CDS prediction from these sequences first require the identification of potentially problematic cDNAs in order to speed up the posterior annotation process. Results cDNA2Genome is an application for the automatic high-throughput mapping and characterization of cDNAs. It utilizes current annotation data and the most up to date databases, especially in the case of ESTs and mRNAs in conjunction with a vast number of approaches to gene prediction in order to perform a comprehensive assessment of the cDNA exon-intron structure. The final result of cDNA2Genome is an XML file containing all relevant information obtained in the process. This XML output can easily be used for further analysis such us program pipelines, or the integration of results into databases. The web interface to cDNA2Genome also presents this data in HTML, where the annotation is additionally shown in a graphical form. cDNA2Genome has been implemented under the W3H task framework which allows the combination of bioinformatics tools in tailor-made analysis task flows as well as the sequential or parallel computation of many sequences for large-scale analysis. Conclusions cDNA2Genome represents a new versatile and easily extensible approach to the automated mapping and annotation of human cDNAs. The underlying approach allows sequential or parallel computation of sequences for high-throughput analysis of cDNAs.

  15. Deletion-bias in DNA double-strand break repair differentially contributes to plant genome shrinkage.

    Science.gov (United States)

    Vu, Giang T H; Cao, Hieu X; Reiss, Bernd; Schubert, Ingo

    2017-06-01

    In order to prevent genome instability, cells need to be protected by a number of repair mechanisms, including DNA double-strand break (DSB) repair. The extent to which DSB repair, biased towards deletions or insertions, contributes to evolutionary diversification of genome size is still under debate. We analyzed mutation spectra in Arabidopsis thaliana and in barley (Hordeum vulgare) by PacBio sequencing of three DSB-targeted loci each, uncovering repair via gene conversion, single strand annealing (SSA) or nonhomologous end-joining (NHEJ). Furthermore, phylogenomic comparisons between A. thaliana and two related species were used to detect naturally occurring deletions during Arabidopsis evolution. Arabidopsis thaliana revealed significantly more and larger deletions after DSB repair than barley, and barley displayed more and larger insertions. Arabidopsis displayed a clear net loss of DNA after DSB repair, mainly via SSA and NHEJ. Barley revealed a very weak net loss of DNA, apparently due to less active break-end resection and easier copying of template sequences into breaks. Comparative phylogenomics revealed several footprints of SSA in the A. thaliana genome. Quantitative assessment of DNA gain and loss through DSB repair processes suggests deletion-biased DSB repair causing ongoing genome shrinking in A. thaliana, whereas genome size in barley remains nearly constant. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  16. One-stop genomic DNA extraction by salicylic acid-coated magnetic nanoparticles.

    Science.gov (United States)

    Zhou, Zhongwu; Kadam, Ulhas S; Irudayaraj, Joseph

    2013-11-15

    Salicylic acid-coated magnetic nanoparticles were prepared via a modified one-step synthesis and used for a one-stop extraction of genomic DNA from mammalian cells. The synthesized magnetic particles were used for magnetic separation of cells from the media by nonspecific binding of the particles as well as extraction of genomic DNA from the lysate. The quantity and quality were confirmed by agarose gel electrophoresis and polymerase chain reaction. The entire process of extraction and isolation can be completed within 30 min. Compared with traditional methods based on centrifugation and filtration, the established method is fast, simple, reliable, and environmentally friendly. Copyright © 2013 Elsevier Inc. All rights reserved.

  17. Genomic signal processing methods for computation of alignment-free distances from DNA sequences.

    Science.gov (United States)

    Borrayo, Ernesto; Mendizabal-Ruiz, E Gerardo; Vélez-Pérez, Hugo; Romo-Vázquez, Rebeca; Mendizabal, Adriana P; Morales, J Alejandro

    2014-01-01

    Genomic signal processing (GSP) refers to the use of digital signal processing (DSP) tools for analyzing genomic data such as DNA sequences. A possible application of GSP that has not been fully explored is the computation of the distance between a pair of sequences. In this work we present GAFD, a novel GSP alignment-free distance computation method. We introduce a DNA sequence-to-signal mapping function based on the employment of doublet values, which increases the number of possible amplitude values for the generated signal. Additionally, we explore the use of three DSP distance metrics as descriptors for categorizing DNA signal fragments. Our results indicate the feasibility of employing GAFD for computing sequence distances and the use of descriptors for characterizing DNA fragments.

  18. [Bacterial infections as seen from the eukaryotic genome: DNA double strand breaks, inflammation and cancer].

    Science.gov (United States)

    Lemercier, Claudie

    2014-01-01

    An increasing number of studies report that infection by pathogenic bacteria alters the host genome, producing highly hazardous DNA double strand breaks for the eukaryotic cell. Even when DNA repair occurs, it often leaves "scars" on chromosomes that might generate genomic instability at the next cell division. Chronic intestinal inflammation promotes the expansion of genotoxic bacteria in the intestinal microbiote which in turn triggers tumor formation and colon carcinomas. Bacteria act at the level of the host DNA repair machinery. They also highjack the host cell cycle to allow themselves time for replication in an appropriate reservoir. However, except in the case of bacteria carrying the CDT nuclease, the molecular mechanisms responsible for DNA lesions are not well understood, even if reactive oxygen species released during infection make good candidates. © 2014 médecine/sciences – Inserm.

  19. Long-Range Correlations in Genomic DNA: A Signature of the Nucleosomal Structure

    Science.gov (United States)

    Audit, B.; Thermes, C.; Vaillant, C.; D'Aubenton-Carafa, Y.; Muzy, J. F.; Arneodo, A.

    2001-03-01

    We use the ``wavelet transform microscope'' to carry out a comparative statistical analysis of DNA bending profiles and of the corresponding DNA texts. In the three kingdoms, one reveals on both signals a characteristic scale of 100-200 bp that separates two different regimes of power-law correlations (PLC). In the small-scale regime, PLC are observed in eukaryotic, in double-strand DNA viral, and in archaeal genomes, which contrasts with their total absence in the genomes of eubacteria and their viruses. This strongly suggests that small-scale PLC are related to the mechanisms underlying the wrapping of DNA in the nucleosomal structure. We further speculate that the large scale PLC are the signature of the higher-order structure and dynamics of chromatin.

  20. The Role of N-α-acetyltransferase 10 Protein in DNA Methylation and Genomic Imprinting.

    Science.gov (United States)

    Lee, Chen-Cheng; Peng, Shih-Huan; Shen, Li; Lee, Chung-Fan; Du, Ting-Huei; Kang, Ming-Lun; Xu, Guo-Liang; Upadhyay, Anup K; Cheng, Xiaodong; Yan, Yu-Ting; Zhang, Yi; Juan, Li-Jung

    2017-10-05

    Genomic imprinting is an allelic gene expression phenomenon primarily controlled by allele-specific DNA methylation at the imprinting control region (ICR), but the underlying mechanism remains largely unclear. N-α-acetyltransferase 10 protein (Naa10p) catalyzes N-α-acetylation of nascent proteins, and mutation of human Naa10p is linked to severe developmental delays. Here we report that Naa10-null mice display partial embryonic lethality, growth retardation, brain disorders, and maternal effect lethality, phenotypes commonly observed in defective genomic imprinting. Genome-wide analyses further revealed global DNA hypomethylation and enriched dysregulation of imprinted genes in Naa10p-knockout embryos and embryonic stem cells. Mechanistically, Naa10p facilitates binding of DNA methyltransferase 1 (Dnmt1) to DNA substrates, including the ICRs of the imprinted allele during S phase. Moreover, the lethal Ogden syndrome-associated mutation of human Naa10p disrupts its binding to the ICR of H19 and Dnmt1 recruitment. Our study thus links Naa10p mutation-associated Ogden syndrome to defective DNA methylation and genomic imprinting. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Genome-wide analysis of DNA methylation dynamics during early human development.

    Science.gov (United States)

    Okae, Hiroaki; Chiba, Hatsune; Hiura, Hitoshi; Hamada, Hirotaka; Sato, Akiko; Utsunomiya, Takafumi; Kikuchi, Hiroyuki; Yoshida, Hiroaki; Tanaka, Atsushi; Suyama, Mikita; Arima, Takahiro

    2014-12-01

    DNA methylation is globally reprogrammed during mammalian preimplantation development, which is critical for normal development. Recent reduced representation bisulfite sequencing (RRBS) studies suggest that the methylome dynamics are essentially conserved between human and mouse early embryos. RRBS is known to cover 5-10% of all genomic CpGs, favoring those contained within CpG-rich regions. To obtain an unbiased and more complete representation of the methylome during early human development, we performed whole genome bisulfite sequencing of human gametes and blastocysts that covered>70% of all genomic CpGs. We found that the maternal genome was demethylated to a much lesser extent in human blastocysts than in mouse blastocysts, which could contribute to an increased number of imprinted differentially methylated regions in the human genome. Global demethylation of the paternal genome was confirmed, but SINE-VNTR-Alu elements and some other tandem repeat-containing regions were found to be specifically protected from this global demethylation. Furthermore, centromeric satellite repeats were hypermethylated in human oocytes but not in mouse oocytes, which might be explained by differential expression of de novo DNA methyltransferases. These data highlight both conserved and species-specific regulation of DNA methylation during early mammalian development. Our work provides further information critical for understanding the epigenetic processes underlying differentiation and pluripotency during early human development.

  2. BuD, a helix–loop–helix DNA-binding domain for genome modification

    Energy Technology Data Exchange (ETDEWEB)

    Stella, Stefano [Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029 Madrid (Spain); University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen (Denmark); Molina, Rafael; López-Méndez, Blanca [Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029 Madrid (Spain); Juillerat, Alexandre; Bertonati, Claudia; Daboussi, Fayza [Cellectis, 8 Rue de la Croix Jarry, 75013 Paris (France); Campos-Olivas, Ramon [Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029 Madrid (Spain); Duchateau, Phillippe [Cellectis, 8 Rue de la Croix Jarry, 75013 Paris (France); Montoya, Guillermo, E-mail: guillermo.montoya@cpr.ku.dk [Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029 Madrid (Spain); University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen (Denmark)

    2014-07-01

    Crystal structures of BurrH and the BurrH–DNA complex are reported. DNA editing offers new possibilities in synthetic biology and biomedicine for modulation or modification of cellular functions to organisms. However, inaccuracy in this process may lead to genome damage. To address this important problem, a strategy allowing specific gene modification has been achieved through the addition, removal or exchange of DNA sequences using customized proteins and the endogenous DNA-repair machinery. Therefore, the engineering of specific protein–DNA interactions in protein scaffolds is key to providing ‘toolkits’ for precise genome modification or regulation of gene expression. In a search for putative DNA-binding domains, BurrH, a protein that recognizes a 19 bp DNA target, was identified. Here, its apo and DNA-bound crystal structures are reported, revealing a central region containing 19 repeats of a helix–loop–helix modular domain (BurrH domain; BuD), which identifies the DNA target by a single residue-to-nucleotide code, thus facilitating its redesign for gene targeting. New DNA-binding specificities have been engineered in this template, showing that BuD-derived nucleases (BuDNs) induce high levels of gene targeting in a locus of the human haemoglobin β (HBB) gene close to mutations responsible for sickle-cell anaemia. Hence, the unique combination of high efficiency and specificity of the BuD arrays can push forward diverse genome-modification approaches for cell or organism redesign, opening new avenues for gene editing.

  3. Comprehensive analysis of genome-wide DNA methylation across human polycystic ovary syndrome ovary granulosa cell.

    Science.gov (United States)

    Xu, Jiawei; Bao, Xiao; Peng, Zhaofeng; Wang, Linlin; Du, Linqing; Niu, Wenbin; Sun, Yingpu

    2016-05-10

    Polycystic ovary syndrome (PCOS) affects approximately 7% of the reproductive-age women. A growing body of evidence indicated that epigenetic mechanisms contributed to the development of PCOS. The role of DNA modification in human PCOS ovary granulosa cell is still unknown in PCOS progression. Global DNA methylation and hydroxymethylation were detected between PCOS' and controls' granulosa cell. Genome-wide DNA methylation was profiled to investigate the putative function of DNA methylaiton. Selected genes expressions were analyzed between PCOS' and controls' granulosa cell. Our results showed that the granulosa cell global DNA methylation of PCOS patients was significant higher than the controls'. The global DNA hydroxymethylation showed low level and no statistical difference between PCOS and control. 6936 differentially methylated CpG sites were identified between control and PCOS-obesity. 12245 differential methylated CpG sites were detected between control and PCOS-nonobesity group. 5202 methylated CpG sites were significantly differential between PCOS-obesity and PCOS-nonobesity group. Our results showed that DNA methylation not hydroxymethylation altered genome-wide in PCOS granulosa cell. The different methylation genes were enriched in development protein, transcription factor activity, alternative splicing, sequence-specific DNA binding and embryonic morphogenesis. YWHAQ, NCF2, DHRS9 and SCNA were up-regulation in PCOS-obesity patients with no significance different between control and PCOS-nonobesity patients, which may be activated by lower DNA methylaiton. Global and genome-wide DNA methylation alteration may contribute to different genes expression and PCOS clinical pathology.

  4. Comparative assessment of genomic DNA extraction processes for Plasmodium: Identifying the appropriate method.

    Science.gov (United States)

    Mann, Riti; Sharma, Supriya; Mishra, Neelima; Valecha, Neena; Anvikar, Anupkumar R

    2015-12-01

    Plasmodium DNA, in addition to being used for molecular diagnosis of malaria, find utility in monitoring patient responses to antimalarial drugs, drug resistance studies, genotyping and sequencing purposes. Over the years, numerous protocols have been proposed for extracting Plasmodium DNA from a variety of sources. Given that DNA isolation is fundamental to successful molecular studies, here we review the most commonly used methods for Plasmodium genomic DNA isolation, emphasizing their pros and cons. A comparison of these existing methods has been made, to evaluate their appropriateness for use in different applications and identify the method suitable for a particular laboratory based study. Selection of a suitable and accessible DNA extraction method for Plasmodium requires consideration of many factors, the most important being sensitivity, cost-effectiveness and, purity and stability of isolated DNA. Need of the hour is to accentuate on the development of a method that upholds well on all these parameters.

  5. Preparation of genomic DNA for RAPD analysis from thick-walled dormant teliospores of Tilletia species.

    Science.gov (United States)

    Gang, D R; Weber, D J

    1995-07-01

    We describe a method for isolating genomic DNA from teliospores of Tilletia caries (DC) Tul., T. controversa Kuhn and T. foetida (T. laevis) (Wallr.) Liro. for random-amplified polymorphic DNA (RAPD) analysis. DNA analysis of teliospores of covered smut or bunt has been difficult because of the thick wall and the high lipid content of the spores. This method overcomes these problems and yields sufficient quantities of DNA from the three species' teliospores for RAPDs. DNA quality appears to be good with very little degradation. RAPD amplifications of the extracted DNAs are reproducible and produce numerous large molecular weight bands from each individual. This procedure should permit the use of DNA analysis techniques to study species and races of Tilletia as well as fungi with similar spore structure.

  6. Genome analysis of DNA repair genes in the alpha proteobacterium Caulobacter crescentus

    Directory of Open Access Journals (Sweden)

    Menck Carlos FM

    2007-03-01

    Full Text Available Abstract Background The integrity of DNA molecules is fundamental for maintaining life. The DNA repair proteins protect organisms against genetic damage, by removal of DNA lesions or helping to tolerate them. DNA repair genes are best known from the gamma-proteobacterium Escherichia coli, which is the most understood bacterial model. However, genome sequencing raises questions regarding uniformity and ubiquity of these DNA repair genes and pathways, reinforcing the need for identifying genes and proteins, which may respond to DNA damage in other bacteria. Results In this study, we employed a bioinformatic approach, to analyse and describe the open reading frames potentially related to DNA repair from the genome of the alpha-proteobacterium Caulobacter crescentus. This was performed by comparison with known DNA repair related genes found in public databases. As expected, although C. crescentus and E. coli bacteria belong to separate phylogenetic groups, many of their DNA repair genes are very similar. However, some important DNA repair genes are absent in the C. crescentus genome and other interesting functionally related gene duplications are present, which do not occur in E. coli. These include DNA ligases, exonuclease III (xthA, endonuclease III (nth, O6-methylguanine-DNA methyltransferase (ada gene, photolyase-like genes, and uracil-DNA-glycosylases. On the other hand, the genes imuA and imuB, which are involved in DNA damage induced mutagenesis, have recently been described in C. crescentus, but are absent in E. coli. Particularly interesting are the potential atypical phylogeny of one of the photolyase genes in alpha-proteobacteria, indicating an origin by horizontal transfer, and the duplication of the Ada orthologs, which have diverse structural configurations, including one that is still unique for C. crescentus. Conclusion The absence and the presence of certain genes are discussed and predictions are made considering the particular

  7. Reconstruction of a Bacterial Genome from DNA Cassettes

    Energy Technology Data Exchange (ETDEWEB)

    Christopher Dupont; John Glass; Laura Sheahan; Shibu Yooseph; Lisa Zeigler Allen; Mathangi Thiagarajan; Andrew Allen; Robert Friedman; J. Craig Venter

    2011-12-31

    This basic research program comprised two major areas: (1) acquisition and analysis of marine microbial metagenomic data and development of genomic analysis tools for broad, external community use; (2) development of a minimal bacterial genome. Our Marine Metagenomic Diversity effort generated and analyzed shotgun sequencing data from microbial communities sampled from over 250 sites around the world. About 40% of the 26 Gbp of sequence data has been made publicly available to date with a complete release anticipated in six months. Our results and those mining the deposited data have revealed a vast diversity of genes coding for critical metabolic processes whose phylogenetic and geographic distributions will enable a deeper understanding of carbon and nutrient cycling, microbial ecology, and rapid rate evolutionary processes such as horizontal gene transfer by viruses and plasmids. A global assembly of the generated dataset resulted in a massive set (5Gbp) of genome fragments that provide context to the majority of the generated data that originated from uncultivated organisms. Our Synthetic Biology team has made significant progress towards the goal of synthesizing a minimal mycoplasma genome that will have all of the machinery for independent life. This project, once completed, will provide fundamentally new knowledge about requirements for microbial life and help to lay a basic research foundation for developing microbiological approaches to bioenergy.

  8. An automated annotation tool for genomic DNA sequences using ...

    Indian Academy of Sciences (India)

    Unknown

    1999), which is one of the major causes of stomach ulcers. Proteins encoded by the genome con- tain double ... causes symptoms of disease. Third, the organization pro- duces two distinct types of cell ..... cated in mortality associated with cerebral malaria where infected erythrocytes bind to the endothelial layers in the brain ...

  9. Sugar Cane Genome Numbers Assumption by Ribosomal DNA FISH Techniques

    NARCIS (Netherlands)

    Thumjamras, S.; Jong, de H.; Iamtham, S.; Prammanee, S.

    2013-01-01

    Conventional cytological method is limited for polyploidy plant genome study, especially sugar cane chromosomes that show unstable numbers of each cultivar. Molecular cytogenetic as fluorescent in situ hybridization (FISH) techniques were used in this study. A basic chromosome number of sugar cane

  10. Targeted enrichment of genomic DNA regions for next generation sequencing

    NARCIS (Netherlands)

    Mertens, F.; El-Sharawy, A.; Sauer, S.; Van Helvoort, J.; Van der Zaag, P.J.; Franke, A.; Nilsson, M.; Lehrach. H.; Brookes, A.

    2011-01-01

    In this review we discuss the latest targeted enrichment methods, and aspects of their utilization along with second generation sequencing for complex genome analysis. In doing so we provide an overview of issues involved in detecting genetic variation, for which targeted enrichment has become a

  11. Whole-genome methylation caller designed for methyl- DNA ...

    African Journals Online (AJOL)

    etchie

    2013-02-20

    Feb 20, 2013 ... its response to environmental cues. Epigenetics provide stability and diversity to the cellular phenotype through chromatin marks that affect local transcriptional potential and are preserved or regenerated during cell division. Much of the human genome is CpG depleted with the exception of CpG islands ...

  12. Genomic DNA characterization of pork spleen by Raman spectroscopy

    Science.gov (United States)

    Guzmán-Embús, D. A.; Orrego Cardozo, M.; Vargas-Hernández, C.

    2013-11-01

    In this paper, the study of Raman signal enhancement due to interaction between ZnO rods and pork spleen DNA is reported. ZnO microstructures were synthesized by the Sol-Gel method and afterward combined with porcine spleen DNA extracted in the previous stages, following standardized cell lysis, deproteinization, and precipitation processes. Raman spectroscopy was used for the characterization of structures of ZnO and ZnO-DNA complex, and the results show the respective bands of ZnO wurtzite hexagonal phase for modes E2 (M), A1(TO), E2(High), E1(LO), and 2LO. Due to the SERS effect in the spectral range from 200 to 1800 cm,-1 Raman bands caused by vibrations of the deoxyribose C-O-C binding were also observed, producing deformation of the ring as shown in the 559 cm-1 peak. The broad band at 782 cm-1, together with the complex vibration of the string 5'-COPO-C3', is over a wide band of thymine (790 cm-1) or cytosine (780 cm-1). A prominent band near 1098 cm-1 assigned to symmetric stretching vibration phosphodioxy group (PO2-) DNA backbone is most favoured in intensity by the addition of ZnO particles originated by the SERS effect. This effect suggests a possible mechanism for enhancing the Raman signal due to the electromagnetic interaction between a DNA molecule and the flat surface of the ZnO rod.

  13. Human single-stranded DNA binding proteins are essential for maintaining genomic stability

    Science.gov (United States)

    2013-01-01

    The double-stranded conformation of cellular DNA is a central aspect of DNA stabilisation and protection. The helix preserves the genetic code against chemical and enzymatic degradation, metabolic activation, and formation of secondary structures. However, there are various instances where single-stranded DNA is exposed, such as during replication or transcription, in the synthesis of chromosome ends, and following DNA damage. In these instances, single-stranded DNA binding proteins are essential for the sequestration and processing of single-stranded DNA. In order to bind single-stranded DNA, these proteins utilise a characteristic and evolutionary conserved single-stranded DNA-binding domain, the oligonucleotide/oligosaccharide-binding (OB)-fold. In the current review we discuss a subset of these proteins involved in the direct maintenance of genomic stability, an important cellular process in the conservation of cellular viability and prevention of malignant transformation. We discuss the central roles of single-stranded DNA binding proteins from the OB-fold domain family in DNA replication, the restart of stalled replication forks, DNA damage repair, cell cycle-checkpoint activation, and telomere maintenance. PMID:23548139

  14. Automated genomic DNA purification options in agricultural applications using MagneSil paramagnetic particles

    Science.gov (United States)

    Bitner, Rex M.; Koller, Susan C.

    2002-06-01

    The automated high throughput purification of genomic DNA form plant materials can be performed using MagneSil paramagnetic particles on the Beckman-Coulter FX, BioMek 2000, and the Tecan Genesis robot. Similar automated methods are available for DNA purifications from animal blood. These methods eliminate organic extractions, lengthy incubations and cumbersome filter plates. The DNA is suitable for applications such as PCR and RAPD analysis. Methods are described for processing traditionally difficult samples such as those containing large amounts of polyphenolics or oils, while still maintaining a high level of DNA purity. The robotic protocols have ben optimized for agricultural applications such as marker assisted breeding, seed-quality testing, and SNP discovery and scoring. In addition to high yield purification of DNA from plant samples or animal blood, the use of Promega's DNA-IQ purification system is also described. This method allows for the purification of a narrow range of DNA regardless of the amount of additional DNA that is present in the initial sample. This simultaneous Isolation and Quantification of DNA allows the DNA to be used directly in applications such as PCR, SNP analysis, and RAPD, without the need for separate quantitation of the DNA.

  15. DNA Topoisomerase 1 Facilitates the Transcription and Replication of the Ebola Virus Genome

    OpenAIRE

    Takahashi, Kei; Halfmann, Peter; Oyama, Masaaki; Kozuka-Hata, Hiroko; Noda, Takeshi; Kawaoka, Yoshihiro

    2013-01-01

    Ebola virus (EBOV) protein L (EBOL) acts as a viral RNA-dependent RNA polymerase. To better understand the mechanisms underlying the transcription and replication of the EBOV genome, we sought to identify cellular factors involved in these processes via their coimmunoprecipitation with EBOL and by mass spectrometry. Of 65 candidate proteins identified, we focused on DNA topoisomerase 1 (TOP1), which localizes to the nucleus and unwinds helical DNA. We found that in the presence of EBOL, TOP1 ...

  16. Protocol for improved extraction and PCR amplification of genomic DNA from liverwort, Plagiochasma appendiculatum.

    Science.gov (United States)

    Soni, Arvind; Kumar, Anil

    2009-11-01

    A simplest method was followed for isolation of high quality genomic DNA form thallus of Plagiochasma appendiculatum that contained large quantities of polyphenols, terpenoids, tannins, contamination of high amount of RNA and polysaccharides. The method involved a modification of CTAB procedure using PVP (1%) and LiCl (4M) solution to remove polyphenols and RNA and some other binding proteins. The present protocol was found suitable for restriction enzyme digestion and random amplified polymorphic DNA (RAPD) analysis.

  17. The Genomic Impact of DNA CpG Methylation on Gene Expression; Relationships in Prostate Cancer

    Directory of Open Access Journals (Sweden)

    Mark D. Long

    2017-02-01

    Full Text Available The process of DNA CpG methylation has been extensively investigated for over 50 years and revealed associations between changing methylation status of CpG islands and gene expression. As a result, DNA CpG methylation is implicated in the control of gene expression in developmental and homeostasis processes, as well as being a cancer-driver mechanism. The development of genome-wide technologies and sophisticated statistical analytical approaches has ushered in an era of widespread analyses, for example in the cancer arena, of the relationships between altered DNA CpG methylation, gene expression, and tumor status. The remarkable increase in the volume of such genomic data, for example, through investigators from the Cancer Genome Atlas (TCGA, has allowed dissection of the relationships between DNA CpG methylation density and distribution, gene expression, and tumor outcome. In this manner, it is now possible to test that the genome-wide correlations are measurable between changes in DNA CpG methylation and gene expression. Perhaps surprisingly is that these associations can only be detected for hundreds, but not thousands, of genes, and the direction of the correlations are both positive and negative. This, perhaps, suggests that CpG methylation events in cancer systems can act as disease drivers but the effects are possibly more restricted than suspected. Additionally, the positive and negative correlations suggest direct and indirect events and an incomplete understanding. Within the prostate cancer TCGA cohort, we examined the relationships between expression of genes that control DNA methylation, known targets of DNA methylation and tumor status. This revealed that genes that control the synthesis of S-adenosyl-l-methionine (SAM associate with altered expression of DNA methylation targets in a subset of aggressive tumors.

  18. The pathological consequences of impaired genome integrity in humans; disorders of the DNA replication machinery.

    Science.gov (United States)

    O'Driscoll, Mark

    2017-01-01

    Accurate and efficient replication of the human genome occurs in the context of an array of constitutional barriers, including regional topological constraints imposed by chromatin architecture and processes such as transcription, catenation of the helical polymer and spontaneously generated DNA lesions, including base modifications and strand breaks. DNA replication is fundamentally important for tissue development and homeostasis; differentiation programmes are intimately linked with stem cell division. Unsurprisingly, impairments of the DNA replication machinery can have catastrophic consequences for genome stability and cell division. Functional impacts on DNA replication and genome stability have long been known to play roles in malignant transformation through a variety of complex mechanisms, and significant further insights have been gained from studying model organisms in this context. Congenital hypomorphic defects in components of the DNA replication machinery have been and continue to be identified in humans. These disorders present with a wide range of clinical features. Indeed, in some instances, different mutations in the same gene underlie different clinical presentations. Understanding the origin and molecular basis of these features opens a window onto the range of developmental impacts of suboptimal DNA replication and genome instability in humans. Here, I will briefly overview the basic steps involved in DNA replication and the key concepts that have emerged from this area of research, before switching emphasis to the pathological consequences of defects within the DNA replication network; the human disorders. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  19. Genome-wide DNA methylation is predictive of outcome in juvenile myelomonocytic leukemia

    OpenAIRE

    Stieglitz, Elliot; Mazor, Tali; Olshen, Adam B.; Geng, Huimin; Gelston, Laura C.; Akutagawa, Jon; Lipka, Daniel B.; Plass, Christoph; Flotho, Christian; Chehab, Farid F.; Braun, Benjamin S.; Costello, Joseph F.; Loh, Mignon L.

    2017-01-01

    Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative disorder of childhood caused by mutations in the Ras pathway. Outcomes in JMML vary markedly from spontaneous resolution to rapid relapse after hematopoietic stem cell transplantation. Here, we hypothesized that DNA methylation patterns would help predict disease outcome and therefore performed genome-wide DNA methylation profiling in a cohort of 39 patients. Unsupervised hierarchical clustering identifies three clusters of patie...

  20. Genomic DNA extraction from cells by electroporation on an integrated microfluidic platform.

    Science.gov (United States)

    Geng, Tao; Bao, Ning; Sriranganathanw, Nammalwar; Li, Liwu; Lu, Chang

    2012-11-06

    The vast majority of genetic analysis of cells involves chemical lysis for release of DNA molecules. However, chemical reagents required in the lysis interfere with downstream molecular biology and often require removal after the step. Electrical lysis based on irreversible electroporation is a promising technique to prepare samples for genetic analysis due to its purely physical nature, fast speed, and simple operation. However, there has been no experimental confirmation on whether electrical lysis extracts genomic DNA from cells in a reproducible and efficient fashion in comparison to chemical lysis, especially for eukaryotic cells that have most of the DNA enclosed in the nucleus. In this work, we construct an integrated microfluidic chip that physically traps a low number of cells, lyses the cells using electrical pulses rapidly, then purifies and concentrates genomic DNA. We demonstrate that electrical lysis offers high efficiency for DNA extraction from both eukaryotic cells (up to ∼36% for Chinese hamster ovary cells) and bacterial cells (up to ∼45% for Salmonella typhimurium) that is comparable to the widely used chemical lysis. The DNA extraction efficiency has dependence on both the electric parameters and relative amount of beads used for DNA adsorption. We envision that electroporation-based DNA extraction will find use in ultrasensitive assays that benefit from minimal dilution and simple procedures.

  1. Complete sequence and characterization of mitochondrial DNA genome of Channa asiatica (Perciformes: Channidae).

    Science.gov (United States)

    Meng, Yan; Zhang, Yan

    2016-01-01

    The complete nucleotide sequence of Channa asiatica mitochondrial (mtDNA) genome was determined in this study. The genome sequence (GenBank accession number KJ930190) was 16,550 base pairs in length, and the gene content and organization on the mitochondrial genome were similar to the other Channa fishes. The overall base composition of C. asiatica mitogenome is 29.4% A, 26.3% T, 15.3% G, 29.0% C, with a high A + T content of 55.7%. The mitochondrial sequence could provide useful genetic information for studying the molecular identification, population genetics, phylogenetic analysis and conservation genetics.

  2. Relationships between 16S-23S rRNA gene internal transcribed spacer DNA and genomic DNA similarities in the taxonomy of phototrophic bacteria

    Science.gov (United States)

    Okamura, K.; Hisada, T.; Takata, K.; Hiraishi, A.

    2013-04-01

    Rapid and accurate identification of microbial species is essential task in microbiology and biotechnology. In prokaryotic systematics, genomic DNA-DNA hybridization is the ultimate tool to determine genetic relationships among bacterial strains at the species level. However, a practical problem in this assay is that the experimental procedure is laborious and time-consuming. In recent years, information on the 16S-23S rRNA gene internal transcribed spacer (ITS) region has been used to classify bacterial strains at the species and intraspecies levels. It is unclear how much information on the ITS region can reflect the genome that contain it. In this study, therefore, we evaluate the quantitative relationship between ITS DNA and entire genomic DNA similarities. For this, we determined ITS sequences of several species of anoxygenic phototrophic bacteria belonging to the order Rhizobiales, and compared with DNA-DNA relatedness among these species. There was a high correlation between the two genetic markers. Based on the regression analysis of this relationship, 70% DNA-DNA relatedness corresponded to 92% ITS sequence similarity. This suggests the usefulness of the ITS sequence similarity as a criterion for determining the genospecies of the phototrophic bacteria. To avoid the effects of polymorphism bias of ITS on similarities, PCR products from all loci of ITS were used directly as genetic probes for comparison. The results of ITS DNA-DNA hybridization coincided well with those of genomic DNA-DNA relatedness. These collective data indicate that the whole ITS DNA-DNA similarity can be used as an alternative to genomic DNA-DNA similarity.

  3. Excavating Neandertal and Denisovan DNA from the genomes of Melanesian individuals.

    Science.gov (United States)

    Vernot, Benjamin; Tucci, Serena; Kelso, Janet; Schraiber, Joshua G; Wolf, Aaron B; Gittelman, Rachel M; Dannemann, Michael; Grote, Steffi; McCoy, Rajiv C; Norton, Heather; Scheinfeldt, Laura B; Merriwether, David A; Koki, George; Friedlaender, Jonathan S; Wakefield, Jon; Pääbo, Svante; Akey, Joshua M

    2016-04-08

    Although Neandertal sequences that persist in the genomes of modern humans have been identified in Eurasians, comparable studies in people whose ancestors hybridized with both Neandertals and Denisovans are lacking. We developed an approach to identify DNA inherited from multiple archaic hominin ancestors and applied it to whole-genome sequences from 1523 geographically diverse individuals, including 35 previously unknown Island Melanesian genomes. In aggregate, we recovered 1.34 gigabases and 303 megabases of the Neandertal and Denisovan genome, respectively. We use these maps of archaic sequences to show that Neandertal admixture occurred multiple times in different non-African populations, characterize genomic regions that are significantly depleted of archaic sequences, and identify signatures of adaptive introgression. Copyright © 2016, American Association for the Advancement of Science.

  4. Canine pyometra

    National Research Council Canada - National Science Library

    Melanie HAAS; Franz-Josef KAUP; Stephan NEUMANN

    2016-01-01

    .... Thus, we used naturally occurring canine pyometra as a model of inflammation, in order to examine the behavior of serum CXCL8 in relation to the disease intensity and commonly analyzed inflammatory mediators...

  5. A comparison of the reliability of two gene targets in loop-mediated isothermal amplification assays for detecting leptospiral DNA in canine urine.

    Science.gov (United States)

    Gentilini, Fabio; Zanoni, Renato Giulio; Zambon, Elisa; Turba, Maria Elena

    2017-01-01

    We compared 2 novel loop-mediated isothermal amplification (LAMP) assays that target either the 16S ribosomal RNA ( rrs) gene or the gene encoding a 32-kDa leptospiral lipoprotein ( lipL32) in order to assess the effect of the target on the accuracy of the LAMP assays. The most sensitive assay was the rrs assay with a limit of detection (LOD) of 1.2 × 101 genome equivalents per reaction. The novel lipL32 assay showed an LOD of 1.2 × 102 genome equivalents per reaction. Both assays showed adequate specificity when tested against a collection of bacteria commonly found in voided canine urine. However, when field samples were assayed, the rrs assays gave many false-positive results and a poor positive predictive value of 8.33%. In conclusion, even if the LAMP assay is used in low prevalence areas, the lipL32 assay would be preferable. Conversely, the higher analytical sensitivity of the rrs assay could be effectively used as a screening test in endemic areas with high disease prevalence, followed by confirmation of the positive results using the lipL32 assay.

  6. Extraction of ribosomal RNA and genomic DNA from soil for studying the diversity of the indigenous bacterial community

    NARCIS (Netherlands)

    Duarte, G.F.; Rosado, A.S.; Keijzer-Wolters, A.C.; Elsas, van J.D.

    1998-01-01

    A method for the indirect (cell extraction followed by nucleic acid extraction) isolation of bacterial ribosomal RNA (rRNA) and genomic DNA from soil was developed. The protocol allowed for the rapid parallel extraction of genomic DNA as well as small and large ribosomal subunit RNA from four soils

  7. Isolation of "Caenorhabditis elegans" Genomic DNA and Detection of Deletions in the "unc-93" Gene Using PCR

    Science.gov (United States)

    Lissemore, James L.; Lackner, Laura L.; Fedoriw, George D.; De Stasio, Elizabeth A.

    2005-01-01

    PCR, genomic DNA isolation, and agarose gel electrophoresis are common molecular biology techniques with a wide range of applications. Therefore, we have developed a series of exercises employing these techniques for an intermediate level undergraduate molecular biology laboratory course. In these exercises, students isolate genomic DNA from the…

  8. Assessing the utility of whole-genome amplified serum DNA for array-based high throughput genotyping.

    Science.gov (United States)

    Bucasas, Kristine L; Pandya, Gagan A; Pradhan, Sonal; Fleischmann, Robert D; Peterson, Scott N; Belmont, John W

    2009-12-18

    Whole genome amplification (WGA) offers new possibilities for genome-wide association studies where limited DNA samples have been collected. This study provides a realistic and high-precision assessment of WGA DNA genotyping performance from 20-year old archived serum samples using the Affymetrix Genome-Wide Human SNP Array 6.0 (SNP6.0) platform. Whole-genome amplified (WGA) DNA samples from 45 archived serum replicates and 5 fresh sera paired with non-amplified genomic DNA were genotyped in duplicate. All genotyped samples passed the imposed QC thresholds for quantity and quality. In general, WGA serum DNA samples produced low call rates (45.00 +/- 2.69%), although reproducibility for successfully called markers was favorable (concordance = 95.61 +/- 4.39%). Heterozygote dropouts explained the majority (>85% in technical replicates, 50% in paired genomic/serum samples) of discordant results. Genotyping performance on WGA serum DNA samples was improved by implementation of Corrected Robust Linear Model with Maximum Likelihood Classification (CRLMM) algorithm but at the loss of many samples which failed to pass its quality threshold. Poor genotype clustering was evident in the samples that failed the CRLMM confidence threshold. We conclude that while it is possible to extract genomic DNA and subsequently perform whole-genome amplification from archived serum samples, WGA serum DNA did not perform well and appeared unsuitable for high-resolution genotyping on these arrays.

  9. Hyper-expansion of large DNA segments in the genome of kuruma shrimp, Marsupenaeus japonicus

    Directory of Open Access Journals (Sweden)

    Kondo Hidehiro

    2010-02-01

    Full Text Available Abstract Background Higher crustaceans (class Malacostraca represent the most species-rich and morphologically diverse group of non-insect arthropods and many of its members are commercially important. Although the crustacean DNA sequence information is growing exponentially, little is known about the genome organization of Malacostraca. Here, we constructed a bacterial artificial chromosome (BAC library and performed BAC-end sequencing to provide genomic information for kuruma shrimp (Marsupenaeus japonicus, one of the most widely cultured species among crustaceans, and found the presence of a redundant sequence in the BAC library. We examined the BAC clone that includes the redundant sequence to further analyze its length, copy number and location in the kuruma shrimp genome. Results Mj024A04 BAC clone, which includes one redundant sequence, contained 27 putative genes and seemed to display a normal genomic DNA structure. Notably, of the putative genes, 3 genes encode homologous proteins to the inhibitor of apoptosis protein and 7 genes encode homologous proteins to white spot syndrome virus, a virulent pathogen known to affect crustaceans. Colony hybridization and PCR analysis of 381 BAC clones showed that almost half of the BAC clones maintain DNA segments whose sequences are homologous to the representative BAC clone Mj024A04. The Mj024A04 partial sequence was detected multiple times in the kuruma shrimp nuclear genome with a calculated copy number of at least 100. Microsatellites based BAC genotyping clearly showed that Mj024A04 homologous sequences were cloned from at least 48 different chromosomal loci. The absence of micro-syntenic relationships with the available genomic sequences of Daphnia and Drosophila suggests the uniqueness of these fragments in kuruma shrimp from current arthropod genome sequences. Conclusions Our results demonstrate that hyper-expansion of large DNA segments took place in the kuruma shrimp genome. Although we

  10. Hyper-expansion of large DNA segments in the genome of kuruma shrimp, Marsupenaeus japonicus.

    Science.gov (United States)

    Koyama, Takashi; Asakawa, Shuichi; Katagiri, Takayuki; Shimizu, Atsushi; Fagutao, Fernand F; Mavichak, Rapeepat; Santos, Mudjekeewis D; Fuji, Kanako; Sakamoto, Takashi; Kitakado, Toshihide; Kondo, Hidehiro; Shimizu, Nobuyoshi; Aoki, Takashi; Hirono, Ikuo

    2010-02-26

    Higher crustaceans (class Malacostraca) represent the most species-rich and morphologically diverse group of non-insect arthropods and many of its members are commercially important. Although the crustacean DNA sequence information is growing exponentially, little is known about the genome organization of Malacostraca. Here, we constructed a bacterial artificial chromosome (BAC) library and performed BAC-end sequencing to provide genomic information for kuruma shrimp (Marsupenaeus japonicus), one of the most widely cultured species among crustaceans, and found the presence of a redundant sequence in the BAC library. We examined the BAC clone that includes the redundant sequence to further analyze its length, copy number and location in the kuruma shrimp genome. Mj024A04 BAC clone, which includes one redundant sequence, contained 27 putative genes and seemed to display a normal genomic DNA structure. Notably, of the putative genes, 3 genes encode homologous proteins to the inhibitor of apoptosis protein and 7 genes encode homologous proteins to white spot syndrome virus, a virulent pathogen known to affect crustaceans. Colony hybridization and PCR analysis of 381 BAC clones showed that almost half of the BAC clones maintain DNA segments whose sequences are homologous to the representative BAC clone Mj024A04. The Mj024A04 partial sequence was detected multiple times in the kuruma shrimp nuclear genome with a calculated copy number of at least 100. Microsatellites based BAC genotyping clearly showed that Mj024A04 homologous sequences were cloned from at least 48 different chromosomal loci. The absence of micro-syntenic relationships with the available genomic sequences of Daphnia and Drosophila suggests the uniqueness of these fragments in kuruma shrimp from current arthropod genome sequences. Our results demonstrate that hyper-expansion of large DNA segments took place in the kuruma shrimp genome. Although we analyzed only a part of the duplicated DNA segments, our

  11. cDNA sequences reveal considerable gene prediction inaccuracy in the Plasmodium falciparum genome

    Directory of Open Access Journals (Sweden)

    Valenzuela Jesus G

    2007-07-01

    Full Text Available Abstract Background The completion of the Plasmodium falciparum genome represents a milestone in malaria research. The genome sequence allows for the development of genome-wide approaches such as microarray and proteomics that will greatly facilitate our understanding of the parasite biology and accelerate new drug and vaccine development. Designing and application of these genome-wide assays, however, requires accurate information on gene prediction and genome annotation. Unfortunately, the genes in the parasite genome databases were mostly identified using computer software that could make some erroneous predictions. Results We aimed to obtain cDNA sequences to examine the accuracy of gene prediction in silico. We constructed cDNA libraries from mixed blood stages of P. falciparum parasite using the SMART cDNA library construction technique and generated 17332 high-quality expressed sequence tags (EST, including 2198 from primer-walking experiments. Assembly of our sequence tags produced 2548 contigs and 2671 singletons versus 5220 contigs and 5910 singletons when our EST were assembled with EST in public databases. Comparison of all the assembled EST/contigs with predicted CDS and genomic sequences in the PlasmoDB database identified 356 genes with predicted coding sequences fully covered by EST, including 85 genes (23.6% with introns incorrectly predicted. Careful automatic software and manual alignments found an additional 308 genes that have introns different from those predicted, with 152 new introns discovered and 182 introns with sizes or locations different from those predicted. Alternative spliced and antisense transcripts were also detected. Matching cDNA to predicted genes also revealed silent chromosomal regions, mostly at subtelomere regions. Conclusion Our data indicated that approximately 24% of the genes in the current databases were predicted incorrectly, although some of these inaccuracies could represent alternatively

  12. Hormone Receptor Expression Analyses in Neoplastic and Non-Neoplastic Canine Mammary Tissue by a Bead Based Multiplex Branched DNA Assay: A Gene Expression Study in Fresh Frozen and Formalin-Fixed, Paraffin-Embedded Samples.

    Directory of Open Access Journals (Sweden)

    Annika Mohr

    Full Text Available Immunohistochemistry (IHC is currently considered the method of choice for steroid hormone receptor status evaluation in human breast cancer and, therefore, it is commonly utilized for assessing canine mammary tumors. In case of low hormone receptor expression, IHC is limited and thus is complemented by molecular analyses. In the present study, a multiplex bDNA assay was evaluated as a method for hormone receptor gene expression detection in canine mammary tissues. Estrogen receptor (ESR1, progesterone receptor (PGR, prolactin receptor (PRLR and growth hormone receptor (GHR gene expressions were evaluated in neoplastic and non-neoplastic canine mammary tissues. A set of 119 fresh frozen and 180 formalin-fixed, paraffin-embedded (FFPE was comparatively analyzed and used for assay evaluation. Furthermore, a possible association between the hormone receptor expression in different histological subtypes of canine malignant mammary tumors and the castration status, breed and invasive growth of the tumor were analyzed. The multiplex bDNA assay proved to be more sensitive for fresh frozen specimens. Hormone receptor expression found was significantly decreased in malignant mammary tumors in comparison to non-neoplastic tissue and benign mammary tumors. Among the histological subtypes the lowest gene expression levels of ESR1, PGR and PRLR were found in solid, anaplastic and ductal carcinomas. In summary, the evaluation showed that the measurement of hormone receptors with the multiplex bDNA assay represents a practicable method for obtaining detailed quantitative information about gene expression in canine mammary tissue for future studies. Still, comparison with IHC or quantitative real-time PCR is needed for further validation of the present method.

  13. Efficient method for the extraction of genomic DNA from wormwood ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-09-17

    Sep 17, 2008 ... The mixed were transferred to a test tube. (with cap), 2 ml phenol were added again and were centrifuged for. 5 min to separate phase. The upper aqueous phase was tran- sferred into new tube. Then, two volume of ice-cool 95% ethanol were added to the aqueous phase for ethanol precipitated DNA and.

  14. Extensive mapping of PPAR binding to genomic DNA

    DEFF Research Database (Denmark)

    Nielsen, Ronni; Pedersen, Thomas Åskov; Trindade, Luisa

    The peroxisome proliferator-activated receptor (PPAR) transcription factors a, d and g are members of the nuclear hormone receptor super family. The PPARs bind regulatory DNA elements (PPREs) as heterodimers with the retinoid X receptor (RXR) and thereby induce transcription in response to ligand...

  15. Chromosomal localization of rDNA genes and genomic organization ...

    Indian Academy of Sciences (India)

    In this study, classical and molecular cytogenetic analyses were performed in tilapia fishes, Oreochromis mossambicus (XX/XY sex determination system), O. urolepis ... Moreover, fluorescence in situ hybridization (FISH) with 5S rDNA probes showed six positive fluorescence signals on six chromosomes of all the analysed ...

  16. Ascaris phylogeny based on multiple whole mtDNA genomes

    DEFF Research Database (Denmark)

    Nejsum, Peter; Hawash, Mohamed B. F.; Betson, Martha

    2017-01-01

    Ascaris lumbricoides and A. suum are two parasitic nematodes infecting humans and pigs, respectively. There has been considerable debate as to whether Ascaris in the two hosts should be considered a single or two separate species. Previous studies identified at least three major clusters (A, B...... and C) of human and pig Ascaris based on partial cox1 sequences. In the present study, we selected major haplotypes from these different clusters to characterize their whole mitochondrial genomes for phylogenetic analysis. We also undertook coalescent simulations to investigate the evolutionary history...... of the different Ascaris haplotypes. The topology of the phylogenetic tree based on complete mitochondrial genomic sequences was found to be similar to partial cox1 sequencing, but the support at internal nodes was higher in the former. Coalescent simulations suggested the presence of at least two divergence...

  17. A comparative encyclopedia of DNA elements in the mouse genome.

    Science.gov (United States)

    Yue, Feng; Cheng, Yong; Breschi, Alessandra; Vierstra, Jeff; Wu, Weisheng; Ryba, Tyrone; Sandstrom, Richard; Ma, Zhihai; Davis, Carrie; Pope, Benjamin D; Shen, Yin; Pervouchine, Dmitri D; Djebali, Sarah; Thurman, Robert E; Kaul, Rajinder; Rynes, Eric; Kirilusha, Anthony; Marinov, Georgi K; Williams, Brian A; Trout, Diane; Amrhein, Henry; Fisher-Aylor, Katherine; Antoshechkin, Igor; DeSalvo, Gilberto; See, Lei-Hoon; Fastuca, Meagan; Drenkow, Jorg; Zaleski, Chris; Dobin, Alex; Prieto, Pablo; Lagarde, Julien; Bussotti, Giovanni; Tanzer, Andrea; Denas, Olgert; Li, Kanwei; Bender, M A; Zhang, Miaohua; Byron, Rachel; Groudine, Mark T; McCleary, David; Pham, Long; Ye, Zhen; Kuan, Samantha; Edsall, Lee; Wu, Yi-Chieh; Rasmussen, Matthew D; Bansal, Mukul S; Kellis, Manolis; Keller, Cheryl A; Morrissey, Christapher S; Mishra, Tejaswini; Jain, Deepti; Dogan, Nergiz; Harris, Robert S; Cayting, Philip; Kawli, Trupti; Boyle, Alan P; Euskirchen, Ghia; Kundaje, Anshul; Lin, Shin; Lin, Yiing; Jansen, Camden; Malladi, Venkat S; Cline, Melissa S; Erickson, Drew T; Kirkup, Vanessa M; Learned, Katrina; Sloan, Cricket A; Rosenbloom, Kate R; Lacerda de Sousa, Beatriz; Beal, Kathryn; Pignatelli, Miguel; Flicek, Paul; Lian, Jin; Kahveci, Tamer; Lee, Dongwon; Kent, W James; Ramalho Santos, Miguel; Herrero, Javier; Notredame, Cedric; Johnson, Audra; Vong, Shinny; Lee, Kristen; Bates, Daniel; Neri, Fidencio; Diegel, Morgan; Canfield, Theresa; Sabo, Peter J; Wilken, Matthew S; Reh, Thomas A; Giste, Erika; Shafer, Anthony; Kutyavin, Tanya; Haugen, Eric; Dunn, Douglas; Reynolds, Alex P; Neph, Shane; Humbert, Richard; Hansen, R Scott; De Bruijn, Marella; Selleri, Licia; Rudensky, Alexander; Josefowicz, Steven; Samstein, Robert; Eichler, Evan E; Orkin, Stuart H; Levasseur, Dana; Papayannopoulou, Thalia; Chang, Kai-Hsin; Skoultchi, Arthur; Gosh, Srikanta; Disteche, Christine; Treuting, Piper; Wang, Yanli; Weiss, Mitchell J; Blobel, Gerd A; Cao, Xiaoyi; Zhong, Sheng; Wang, Ting; Good, Peter J; Lowdon, Rebecca F; Adams, Leslie B; Zhou, Xiao-Qiao; Pazin, Michael J; Feingold, Elise A; Wold, Barbara; Taylor, James; Mortazavi, Ali; Weissman, Sherman M; Stamatoyannopoulos, John A; Snyder, Michael P; Guigo, Roderic; Gingeras, Thomas R; Gilbert, David M; Hardison, Ross C; Beer, Michael A; Ren, Bing

    2014-11-20

    The laboratory mouse shares the majority of its protein-coding genes with humans, making it the premier model organism in biomedical research, yet the two mammals differ in significant ways. To gain greater insights into both shared and species-specific transcriptional and cellular regulatory programs in the mouse, the Mouse ENCODE Consortium has mapped transcription, DNase I hypersensitivity, transcription factor binding, chromatin modifications and replication domains throughout the mouse genome in diverse cell and tissue types. By comparing with the human genome, we not only confirm substantial conservation in the newly annotated potential functional sequences, but also find a large degree of divergence of sequences involved in transcriptional regulation, chromatin state and higher order chromatin organization. Our results illuminate the wide range of evolutionary forces acting on genes and their regulatory regions, and provide a general resource for research into mammalian biology and mechanisms of human diseases.

  18. Draft genome sequence of an elite Dura palm and whole-genome patterns of DNA variation in oil palm.

    Science.gov (United States)

    Jin, Jingjing; Lee, May; Bai, Bin; Sun, Yanwei; Qu, Jing; Rahmadsyah; Alfiko, Yuzer; Lim, Chin Huat; Suwanto, Antonius; Sugiharti, Maria; Wong, Limsoon; Ye, Jian; Chua, Nam-Hai; Yue, Gen Hua

    2016-12-01

    Oil palm is the world's leading source of vegetable oil and fat. Dura, Pisifera and Tenera are three forms of oil palm. The genome sequence of Pisifera is available whereas the Dura form has not been sequenced yet. We sequenced the genome of one elite Dura palm, and re-sequenced 17 palm genomes. The assemble genome sequence of the elite Dura tree contained 10,971 scaffolds and was 1.701 Gb in length, covering 94.49% of the oil palm genome. 36,105 genes were predicted. Re-sequencing of 17 additional palm trees identified 18.1 million SNPs. We found high genetic variation among palms from different geographical regions, but lower variation among Southeast Asian Dura and Pisifera palms. We mapped 10,000 SNPs on the linkage map of oil palm. In addition, high linkage disequilibrium (LD) was detected in the oil palms used in breeding populations of Southeast Asia, suggesting that LD mapping is likely to be practical in this important oil crop. Our data provide a valuable resource for accelerating genetic improvement and studying the mechanism underlying phenotypic variations of important oil palm traits. © The Author 2016. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

  19. A DNA minor groove electronegative potential genome map based on photo-chemical probing

    DEFF Research Database (Denmark)

    Lindemose, Søren; Nielsen, Peter Eigil; Hansen, Morten

    2011-01-01

    The double-stranded DNA of the genome contains both sequence information directly relating to the protein and RNA coding as well as functional and structural information relating to protein recognition. Only recently is the importance of DNA shape in this recognition process being fully appreciated...... of any given sequence. We have validated this model on a series of protein-DNA binding sites known to involve minor groove electrostatic recognition as well as on stable nucleosome core complexes. The algorithm allows for the first time a full minor groove electrostatic description at the nucleotide...

  20. The Genomic Pattern of tDNA Operon Expression in E. coli.

    Directory of Open Access Journals (Sweden)

    2005-06-01

    Full Text Available In fast-growing microorganisms, a tRNA concentration profile enriched in major isoacceptors selects for the biased usage of cognate codons. This optimizes translational rate for the least mass invested in the translational apparatus. Such translational streamlining is thought to be growth-regulated, but its genetic basis is poorly understood. First, we found in reanalysis of the E. coli tRNA profile that the degree to which it is translationally streamlined is nearly invariant with growth rate. Then, using least squares multiple regression, we partitioned tRNA isoacceptor pools to predicted tDNA operons from the E. coli K12 genome. Co-expression of tDNAs in operons explains the tRNA profile significantly better than tDNA gene dosage alone. Also, operon expression increases significantly with proximity to the origin of replication, oriC, at all growth rates. Genome location explains about 15% of expression variation in a form, at a given growth rate, that is consistent with replication-dependent gene concentration effects. Yet the change in the tRNA profile with growth rate is less than would be expected from such effects. We estimated per-copy expression rates for all tDNA operons that were consistent with independent estimates for rDNA operons. We also found that tDNA operon location, and the location dependence of expression, were significantly different in the leading and lagging strands. The operonic organization and genomic location of tDNA operons are significant factors influencing their expression. Nonrandom patterns of location and strandedness shown by tDNA operons in E. coli suggest that their genomic architecture may be under selection to satisfy physiological demand for tRNA expression at high growth rates.

  1. Topological events in single molecules of long genomic DNA confined in nanochannels

    Science.gov (United States)

    Reifenberger, Jeffrey; Dorfman, Kevin; Cao, Han

    2014-03-01

    ct- We present a rapid genome-wide analysis method based on new NanoChannel Array technology (IrysTM System) that confines and linearizes extremely long DNA molecules (100 to 1,000 kilobases) for direct image analysis at tens to hundred of gigabases per run. Genomic DNA is stained with YOYO and labeled specifically at the `GCTCTTC' sequence with fluorescent dyes allowing each molecule to be uniquely patterned and mapped to its corresponding reference. This high-throughput platform automates the imaging of such barcoded patterns on genomic DNA to identify wide spread structural variations in a genome. Here we describe a method to rule out possible topologically altered molecules in linear confinement by identifying possible topological events through a T-test looking for spikes in the fluorescence of the YOYO stained DNA backbone. These events are confirmed through aligning the marked individual molecules to a standard reference and measuring a distance differential between labels surrounding the suspected topological event compared to the reference. Such events could be flagged to distinguish from true structural variations.

  2. Genomic DNA sequence and cytosine methylation changes of adult rice leaves after seeds space flight

    Science.gov (United States)

    Shi, Jinming

    In this study, cytosine methylation on CCGG site and genomic DNA sequence changes of adult leaves of rice after seeds space flight were detected by methylation-sensitive amplification polymorphism (MSAP) and Amplified fragment length polymorphism (AFLP) technique respectively. Rice seeds were planted in the trial field after 4 days space flight on the shenzhou-6 Spaceship of China. Adult leaves of space-treated rice including 8 plants chosen randomly and 2 plants with phenotypic mutation were used for AFLP and MSAP analysis. Polymorphism of both DNA sequence and cytosine methylation were detected. For MSAP analysis, the average polymorphic frequency of the on-ground controls, space-treated plants and mutants are 1.3%, 3.1% and 11% respectively. For AFLP analysis, the average polymorphic frequencies are 1.4%, 2.9%and 8%respectively. Total 27 and 22 polymorphic fragments were cloned sequenced from MSAP and AFLP analysis respectively. Nine of the 27 fragments from MSAP analysis show homology to coding sequence. For the 22 polymorphic fragments from AFLP analysis, no one shows homology to mRNA sequence and eight fragments show homology to repeat region or retrotransposon sequence. These results suggest that although both genomic DNA sequence and cytosine methylation status can be effected by space flight, the genomic region homology to the fragments from genome DNA and cytosine methylation analysis were different.

  3. cDNA, genomic cloning and sequence analysis of ribosomal protein ...

    African Journals Online (AJOL)

    enoh

    2012-03-13

    Mar 13, 2012 ... Ribosomal protein S4X (RPS4X) is one of the 40S ribosomal proteins encoded by the RPS4X gene. The. cDNA and the genomic sequence of RPS4X were cloned successfully from giant panda (Ailuropoda melanoleuca) using reverse transcriptase-polymerase chain reaction (RT-PCR) and touchdown- ...

  4. Genome-wide analysis of DNA methylation in Arabidopsis using MeDIP-chip

    NARCIS (Netherlands)

    Cortijo, Sandra; Wardenaar, René; Colomé-Tatché, Maria; Johannes, Frank; Colot, Vincent

    2014-01-01

    DNA methylation is an epigenetic mark that is essential for preserving genome integrity and normal development in plants and mammals. Although this modification may serve a variety of purposes, it is best known for its role in stable transcriptional silencing of transposable elements and epigenetic

  5. cDNA, genomic cloning and sequence analysis of ribosomal protein ...

    African Journals Online (AJOL)

    Ribosomal protein S4X (RPS4X) is one of the 40S ribosomal proteins encoded by the RPS4X gene. The cDNA and the genomic sequence of RPS4X were cloned successfully from giant panda (Ailuropoda melanoleuca) using reverse transcriptase-polymerase chain reaction (RT-PCR) and touchdown-PCR technology ...

  6. Myeloperoxidase-produced Genomic DNA-centered Radicals and Protection by Resveratrol

    Science.gov (United States)

    Myeloperoxidase (MPO) released by activated neutrophils, production of hypochlorous acid (HOCI) and oxidation of the genomic DNA in epithelial cells is thought to initiate and promote carcinogenesis. In this study we applied the 5,5-dimethyl-l-pyrroline N-oxide (DMPO)-based i;nmu...

  7. Shotgun Bisulfite Sequencing of the Betula platyphylla Genome Reveals the Tree’s DNA Methylation Patterning

    Directory of Open Access Journals (Sweden)

    Chang Su

    2014-12-01

    Full Text Available DNA methylation plays a critical role in the regulation of gene expression. Most studies of DNA methylation have been performed in herbaceous plants, and little is known about the methylation patterns in tree genomes. In the present study, we generated a map of methylated cytosines at single base pair resolution for Betula platyphylla (white birch by bisulfite sequencing combined with transcriptomics to analyze DNA methylation and its effects on gene expression. We obtained a detailed view of the function of DNA methylation sequence composition and distribution in the genome of B. platyphylla. There are 34,460 genes in the whole genome of birch, and 31,297 genes are methylated. Conservatively, we estimated that 14.29% of genomic cytosines are methylcytosines in birch. Among the methylation sites, the CHH context accounts for 48.86%, and is the largest proportion. Combined transcriptome and methylation analysis showed that the genes with moderate methylation levels had higher expression levels than genes with high and low methylation. In addition, methylated genes are highly enriched for the GO subcategories of binding activities, catalytic activities, cellular processes, response to stimulus and cell death, suggesting that methylation mediates these pathways in birch trees.

  8. DNA copy number aberrations in breast cancer by array comparative genomic hybridization

    DEFF Research Database (Denmark)

    Li, J.; Wang, K.; Li, S.

    2009-01-01

    Array comparative genomic hybridization (CGH) has been popularly used for analyzing DNA copy number variations in diseases like cancer. In this study, we investigated 82 sporadic samples from 49 breast cancer patients using 1-Mb resolution bacterial artificial chromosome CGH arrays. A number...

  9. De novo reconstruction of plant RNA and DNA virus genomes from viral siRNAs

    Science.gov (United States)

    In antiviral defense, plants produce massive quantities of 21-24 nucleotide siRNAs. Here we demonstrate that the complete genomes of DNA and RNA viruses and viroids can be reconstructed by deep sequencing and de novo assembly of viral/viroid siRNAs from experimentally- and naturally-infected plants....

  10. The levels of yield and purity of genomic DNA from five tomato ...

    African Journals Online (AJOL)

    Isolation of good quality genomic DNA from different plant materials is an important prerequisite for many molecular techniques related to both basic and applied research in the areas of plant molecular biology, crop improvement, biodiversity studies and conservation of genetic materials. Therefore, the need to extract ...

  11. Archaeal virus with exceptional virion architecture and the largest single-stranded DNA genome

    Science.gov (United States)

    Mochizuki, Tomohiro; Krupovic, Mart; Pehau-Arnaudet, Gérard; Sako, Yoshihiko; Forterre, Patrick; Prangishvili, David

    2012-01-01

    Known viruses build their particles using a restricted number of redundant structural solutions. Here, we describe the Aeropyrum coil-shaped virus (ACV), of the hyperthermophilic archaeon Aeropyrum pernix, with a virion architecture not previously observed in the viral world. The nonenveloped, hollow, cylindrical virion is formed from a coiling fiber, which consists of two intertwining halves of a single circular nucleoprotein. The virus ACV is also exceptional for its genomic properties. It is the only virus with a single-stranded (ss) DNA genome among the known hyperthermophilic archaeal viruses. Moreover, the size of its circular genome, 24,893 nt, is double that of the largest known ssDNA genome, suggesting an efficient solution for keeping ssDNA intact at 90–95 °C, the optimal temperature range of A. pernix growth. The genome content of ACV is in line with its unique morphology and confirms that ACV is not closely related to any known virus. PMID:22826255

  12. Epigenetic Variation in Monozygotic Twins: A Genome-Wide Analysis of DNA Methylation in Buccal Cells

    Directory of Open Access Journals (Sweden)

    Jenny van Dongen

    2014-05-01

    Full Text Available DNA methylation is one of the most extensively studied epigenetic marks in humans. Yet, it is largely unknown what causes variation in DNA methylation between individuals. The comparison of DNA methylation profiles of monozygotic (MZ twins offers a unique experimental design to examine the extent to which such variation is related to individual-specific environmental influences and stochastic events or to familial factors (DNA sequence and shared environment. We measured genome-wide DNA methylation in buccal samples from ten MZ pairs (age 8–19 using the Illumina 450k array and examined twin correlations for methylation level at 420,921 CpGs after QC. After selecting CpGs showing the most variation in the methylation level between subjects, the mean genome-wide correlation (rho was 0.54. The correlation was higher, on average, for CpGs within CpG islands (CGIs, compared to CGI shores, shelves and non-CGI regions, particularly at hypomethylated CpGs. This finding suggests that individual-specific environmental and stochastic influences account for more variation in DNA methylation in CpG-poor regions. Our findings also indicate that it is worthwhile to examine heritable and shared environmental influences on buccal DNA methylation in larger studies that also include dizygotic twins.

  13. Horizontal transfer of DNA from the mitochondrial to the plastid genome and its subsequent evolution in milkweeds (Apocynaceae)

    Science.gov (United States)

    Shannon C.K. Straub; Richard C. Cronn; Christopher Edwards; Mark Fishbein; Aaron. Liston

    2013-01-01

    Horizontal gene transfer (HGT) of DNA from the plastid to the nuclear and mitochondrial genomes of higher plants is a common phenomenon; however, plastid genomes (plastomes) are highly conserved and have generally been regarded as impervious to HGT. We sequenced the 158 kb plastome and the 690 kb mitochondrial genome of common milkweed (Asclepias syriaca [Apocynaceae...

  14. The exceptional genomic word symmetry along DNA sequences

    OpenAIRE

    Afreixo, Vera; Rodrigues, Jo?o M. O. S.; Carlos A. C. Bastos; Silva, Raquel M.

    2016-01-01

    Background The second Chargaff?s parity rule and its extensions are recognized as universal phenomena in DNA sequences. However, parity of the frequencies of reverse complementary oligonucleotides could be a mere consequence of the single nucleotide parity rule, if nucleotide independence is assumed. Exceptional symmetry (symmetry beyond that expected under an independent nucleotide assumption) was proposed previously as a meaningful measure of the extension of the second parity rule to oligo...

  15. Extraction of PCR-amplifiable genomic DNA from Bacillus anthracisspores

    Energy Technology Data Exchange (ETDEWEB)

    Torok, Tamas

    2003-05-19

    Bacterial endospore disruption and nucleic acid extractionresulting in DNA of PCR-amplifiable quality and quantity are not trivial.Responding to the needs of the Hazardous Materials Response Unit (HMRU),Laboratory Division, Federal Bureau of Investigation, protocols weredeveloped to close these gaps. Effectiveness and reproducibility of thetechniques were validated with laboratory grown pure spores of Bacillusanthracis and its close phylogenetic neighbors, and with spiked soils anddamaged samples.

  16. 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.

  17. Site-selective scission of human genome using PNA-based artificial restriction DNA cutter.

    Science.gov (United States)

    Ito, Kenichiro; Komiyama, Makoto

    2014-01-01

    Site-selective scission of genomes is quite important for future biotechnology. However, naturally occurring restriction enzymes cut these huge DNAs at too many sites and cannot be used for this purpose. Recently, we have developed a completely chemistry-based artificial restriction DNA cutter (ARCUT) by combining a pair of pseudo-complementary PNA (pcPNA) strands (sequence recognition moiety) and Ce(IV)/EDTA complex (molecular scissors). The scission site of ARCUT and its scission specificity can be freely modulated in terms of the sequences and lengths of the pcPNA strands so that even huge genomes can be selectively cut at only one predetermined site. In this chapter, the method of site-selective scission of human genomic DNA using ARCUT is described in detail.

  18. P-Hint-Hunt: a deep parallelized whole genome DNA methylation detection tool.

    Science.gov (United States)

    Peng, Shaoliang; Yang, Shunyun; Gao, Ming; Liao, Xiangke; Liu, Jie; Yang, Canqun; Wu, Chengkun; Yu, Wenqiang

    2017-03-14

    The increasing studies have been conducted using whole genome DNA methylation detection as one of the most important part of epigenetics research to find the significant relationships among DNA methylation and several typical diseases, such as cancers and diabetes. In many of those studies, mapping the bisulfite treated sequence to the whole genome has been the main method to study DNA cytosine methylation. However, today's relative tools almost suffer from inaccuracies and time-consuming problems. In our study, we designed a new DNA methylation prediction tool ("Hint-Hunt") to solve the problem. By having an optimal complex alignment computation and Smith-Waterman matrix dynamic programming, Hint-Hunt could analyze and predict the DNA methylation status. But when Hint-Hunt tried to predict DNA methylation status with large-scale dataset, there are still slow speed and low temporal-spatial efficiency problems. In order to solve the problems of Smith-Waterman dynamic programming and low temporal-spatial efficiency, we further design a deep parallelized whole genome DNA methylation detection tool ("P-Hint-Hunt") on Tianhe-2 (TH-2) supercomputer. To the best of our knowledge, P-Hint-Hunt is the first parallel DNA methylation detection tool with a high speed-up to process large-scale dataset, and could run both on CPU and Intel Xeon Phi coprocessors. Moreover, we deploy and evaluate Hint-Hunt and P-Hint-Hunt on TH-2 supercomputer in different scales. The experimental results illuminate our tools eliminate the deviation caused by bisulfite treatment in mapping procedure and the multi-level parallel program yields a 48 times speed-up with 64 threads. P-Hint-Hunt gain a deep acceleration on CPU and Intel Xeon Phi heterogeneous platform, which gives full play of the advantages of multi-cores (CPU) and many-cores (Phi).

  19. Genetically contextual effects of smoking on genome wide DNA methylation.

    Science.gov (United States)

    Dogan, Meeshanthini V; Beach, Steven R H; Philibert, Robert A

    2017-09-01

    Smoking is the leading cause of death in the United States. It exerts its effects by increasing susceptibility to a variety of complex disorders among those who smoke, and if pregnant, to their unborn children. In prior efforts to understand the epigenetic mechanisms through which this increased vulnerability is conveyed, a number of investigators have conducted genome wide methylation analyses. Unfortunately, secondary to methodological limitations, these studies were unable to examine methylation in gene regions with significant amounts of genetic variation. Using genome wide genetic and epigenetic data from the Framingham Heart Study, we re-examined the relationship of smoking status to genome wide methylation status. When only methylation status is considered, smoking was significantly associated with differential methylation in 310 genes that map to a variety of biological process and cellular differentiation pathways. However, when SNP effects on the magnitude of smoking associated methylation changes are also considered, cis and trans-interaction effects were noted at a total of 266 and 4353 genes with no marked enrichment for any biological pathways. Furthermore, the SNP variation participating in the significant interaction effects is enriched for loci previously associated with complex medical illnesses. The enlarged scope of the methylome shown to be affected by smoking may better explicate the mediational pathways linking smoking with a myriad of smoking related complex syndromes. Additionally, these results strongly suggest that combined epigenetic and genetic data analyses may be critical for a more complete understanding of the relationship between environmental variables, such as smoking, and pathophysiological outcomes. © 2017 Wiley Periodicals, Inc.

  20. Genome Stability by DNA Polymerase β in Neural Progenitors Contributes to Neuronal Differentiation in Cortical Development.

    Science.gov (United States)

    Onishi, Kohei; Uyeda, Akiko; Shida, Mitsuhiro; Hirayama, Teruyoshi; Yagi, Takeshi; Yamamoto, Nobuhiko; Sugo, Noriyuki

    2017-08-30

    DNA repair is crucial for genome stability in the developing cortex, as somatic de novo mutations cause neurological disorders. However, how DNA repair contributes to neuronal development is largely unknown. To address this issue, we studied the spatiotemporal roles of DNA polymerase β (Polβ), a key enzyme in DNA base excision repair pathway, in the developing cortex using distinct forebrain-specific conditional knock-out mice, Emx1-Cre/Polβ (fl/fl) and Nex-Cre/Polβ (fl/fl) mice. Polβ expression was absent in both neural progenitors and postmitotic neurons in Emx1-Cre/Polβ (fl/fl) mice, whereas only postmitotic neurons lacked Polβ expression in Nex-Cre/Polβ (fl/fl) mice. We found that DNA double-strand breaks (DSBs) were frequently detected during replication in cortical progenitors of Emx1-Cre/Polβ (fl/fl) mice. Increased DSBs remained in postmitotic cells, which resulted in p53-mediated neuronal apoptosis. This neuronal apoptosis caused thinning of the cortical plate, although laminar structure was normal. In addition, accumulated DSBs also affected growth of corticofugal axons but not commissural axons. These phenotypes were not observed in Nex-Cre/Polβ (fl/fl) mice. Moreover, cultured Polβ-deficient neural progenitors exhibited higher sensitivity to the base-damaging agent methylmethanesulfonate, resulting in enhanced DSB formation. Similar damage was found by vitamin C treatment, which induces TET1-mediated DNA demethylation via 5-hydroxymethylcytosine. Together, genome stability mediated by Polβ-dependent base excision repair is crucial for the competence of neural progenitors, thereby contributing to neuronal differentiation in cortical development.SIGNIFICANCE STATEMENT DNA repair is crucial for development of the nervous system. However, how DNA polymerase β (Polβ)-dependent DNA base excision repair pathway contributes to the process is still unknown. We found that loss of Polβ in cortical progenitors rather than postmitotic neurons led to

  1. Coordination of DNA replication and recombination activities in the maintenance of genome stability.

    Science.gov (United States)

    Maher, Robyn L; Branagan, Amy M; Morrical, Scott W

    2011-10-01

    Across the evolutionary spectrum, living organisms depend on high-fidelity DNA replication and recombination mechanisms to maintain genome stability and thus to avoid mutation and disease. The repair of severe lesions in the DNA such as double-strand breaks or stalled replication forks requires the coordinated activities of both the homologous recombination (HR) and DNA replication machineries. Growing evidence indicates that so-called "accessory proteins" in both systems are essential for the effective coupling of recombination to replication which is necessary to restore genome integrity following severe DNA damage. In this article we review the major processes of homology-directed DNA repair (HDR), including the double Holliday Junction (dHJ), synthesis-dependent strand annealing (SDSA), break-induced replication (BIR), and error-free lesion bypass pathways. Each of these pathways involves the coupling of a HR event to DNA synthesis. We highlight two major classes of accessory proteins in recombination and replication that facilitate HDR: Recombination mediator proteins exemplified by T4 UvsY, Saccharomyces cerevisiae Rad52, and human BRCA2; and DNA helicases/translocases exemplified by T4 Gp41/Gp59, E. coli DnaB and PriA, and eukaryotic Mcm2-7, Rad54, and Mph1. We illustrate how these factors help to direct the flow of DNA and protein-DNA intermediates on the pathway from a double-strand break or stalled replication fork to a high-fidelity recombination-dependent replication apparatus that can accurately repair the damage. Copyright © 2011 Wiley-Liss, Inc.

  2. DHX9 helicase is involved in preventing genomic instability induced by alternatively structured DNA in human cells

    Science.gov (United States)

    Jain, Aklank; Bacolla, Albino; del Mundo, Imee M.; Zhao, Junhua; Wang, Guliang; Vasquez, Karen M.

    2013-01-01

    Sequences that have the capacity to adopt alternative (i.e. non-B) DNA structures in the human genome have been implicated in stimulating genomic instability. Previously, we found that a naturally occurring intra-molecular triplex (H-DNA) caused genetic instability in mammals largely in the form of DNA double-strand breaks. Thus, it is of interest to determine the mechanism(s) involved in processing H-DNA. Recently, we demonstrated that human DHX9 helicase preferentially unwinds inter-molecular triplex DNA in vitro. Herein, we used a mutation-reporter system containing H-DNA to examine the relevance of DHX9 activity on naturally occurring H-DNA structures in human cells. We found that H-DNA significantly increased mutagenesis in small-interfering siRNA-treated, DHX9-depleted cells, affecting mostly deletions. Moreover, DHX9 associated with H-DNA in the context of supercoiled plasmids. To further investigate the role of DHX9 in the recognition/processing of H-DNA, we performed binding assays in vitro and chromatin immunoprecipitation assays in U2OS cells. DHX9 recognized H-DNA, as evidenced by its binding to the H-DNA structure and enrichment at the H-DNA region compared with a control region in human cells. These composite data implicate DHX9 in processing H-DNA structures in vivo and support its role in the overall maintenance of genomic stability at sites of alternatively structured DNA. PMID:24049074

  3. Genomic DNA from animals shows contrasting strand bias in large and small subsequences

    Directory of Open Access Journals (Sweden)

    Evans Kenneth J

    2008-01-01

    Full Text Available Abstract Background For eukaryotes, there is almost no strand bias with regard to base composition, with exceptions for origins of replication and transcription start sites and transcribed regions. This paper revisits the question for subsequences of DNA taken at random from the genome. Results For a typical mammal, for example mouse or human, there is a small strand bias throughout the genomic DNA: there is a correlation between (G - C and (A - T on the same strand, (that is between the difference in the number of guanine and cytosine bases and the difference in the number of adenine and thymine bases. For small subsequences – up to 1 kb – this correlation is weak but positive; but for large windows – around 50 kb to 2 Mb – the correlation is strong and negative. This effect is largely independent of GC%. Transcribed and untranscribed regions give similar correlations both for small and large subsequences, but there is a difference in these regions for intermediate sized subsequences. An analysis of the human genome showed that position within the isochore structure did not affect these correlations. An analysis of available genomes of different species shows that this contrast between large and small windows is a general feature of mammals and birds. Further down the evolutionary tree, other organisms show a similar but smaller effect. Except for the nematode, all the animals analysed showed at least a small effect. Conclusion The correlations on the large scale may be explained by DNA replication. Transcription may be a modifier of these effects but is not the fundamental cause. These results cast light on how DNA mutations affect the genome over evolutionary time. At least for vertebrates, there is a broad relationship between body temperature and the size of the correlation. The genome of mammals and birds has a structure marked by strand bias segments.

  4. Mapping ribonucleotides in genomic DNA and exploring replication dynamics by polymerase usage sequencing (Pu-seq).

    Science.gov (United States)

    Keszthelyi, Andrea; Daigaku, Yasukazu; Ptasińska, Katie; Miyabe, Izumi; Carr, Antony M

    2015-11-01

    Ribonucleotides are frequently misincorporated into DNA during replication, and they are rapidly repaired by ribonucleotide excision repair (RER). Although ribonucleotides in template DNA perturb replicative polymerases and can be considered as DNA damage, they also serve positive biological functions, including directing the orientation of mismatch repair. Here we describe a method for ribonucleotide identification by high-throughput sequencing that allows mapping of the location of ribonucleotides across the genome. When combined with specific mutations in the replicative polymerases that incorporate ribonucleotides at elevated frequencies, our ribonucleotide identification method was adapted to map polymerase usage across the genome. Polymerase usage sequencing (Pu-seq) has been used to define, in unprecedented detail, replication dynamics in yeasts. Although other methods that examine replication dynamics provide direct measures of replication timing and indirect estimates of origin efficiency, Pu-seq directly ascertains origin efficiency. The Pu-seq protocol can be completed in 12-14 d.

  5. Ligation bias in illumina next-generation DNA libraries: implications for sequencing ancient genomes.

    Directory of Open Access Journals (Sweden)

    Andaine Seguin-Orlando

    Full Text Available Ancient DNA extracts consist of a mixture of endogenous molecules and contaminant DNA templates, often originating from environmental microbes. These two populations of templates exhibit different chemical characteristics, with the former showing depurination and cytosine deamination by-products, resulting from post-mortem DNA damage. Such chemical modifications can interfere with the molecular tools used for building second-generation DNA libraries, and limit our ability to fully characterize the true complexity of ancient DNA extracts. In this study, we first use fresh DNA extracts to demonstrate that library preparation based on adapter ligation at AT-overhangs are biased against DNA templates starting with thymine residues, contrarily to blunt-end adapter ligation. We observe the same bias on fresh DNA extracts sheared on Bioruptor, Covaris and nebulizers. This contradicts previous reports suggesting that this bias could originate from the methods used for shearing DNA. This also suggests that AT-overhang adapter ligation efficiency is affected in a sequence-dependent manner and results in an uneven representation of different genomic contexts. We then show how this bias could affect the base composition of ancient DNA libraries prepared following AT-overhang ligation, mainly by limiting the ability to ligate DNA templates starting with thymines and therefore deaminated cytosines. This results in particular nucleotide misincorporation damage patterns, deviating from the signature generally expected for authenticating ancient sequence data. Consequently, we show that models adequate for estimating post-mortem DNA damage levels must be robust to the molecular tools used for building ancient DNA libraries.

  6. Identification of Poxvirus Genome Uncoating and DNA Replication Factors with Mutually Redundant Roles.

    Science.gov (United States)

    Liu, Baoming; Panda, Debasis; Mendez-Rios, Jorge D; Ganesan, Sundar; Wyatt, Linda S; Moss, Bernard

    2018-01-17

    Genome uncoating is essential for replication of most viruses. For poxviruses, the process is divided into two stages: removal of the envelope allowing early gene expression, and breaching of the core wall allowing DNA release, replication and late gene expression. Subsequent studies showed that the host proteasome and the viral D5 protein, which has an essential role in DNA replication, are required for vaccinia virus (VACV) genome uncoating. In a search for additional VACV uncoating proteins, we noted a report that described a defect in DNA replication and late expression when the gene encoding a 68 kDa ankyrin-repeat/F box protein (68k-ank), associated with the cellular SCF ubiquitin ligase complex, was deleted from the attenuated modified vaccinia virus Ankara (MVA). Here we showed that the formation of DNA pre-replication sites and degradation of viral cores were severely diminished indicating that the 68k-ank deletion mutant had a defect in genome uncoating as well as an additional independent defect in DNA synthesis. Deletion of the 68k-ank homolog of VACV strain WR, however, was without effect suggesting the existence of compensating genes. By inserting VACV genes into a MVA 68k-ank deletion mutant, we discovered that M2, a member of the Poxvirus Immune Evasion (PIE) domain superfamily and a regulator of NF-κB, and that C5, a member of the BTB/kelch superfamily associated with cullin-3-based ligase complexes, independently rescued the 68k-ank deletion phenotype. Thus, poxvirus uncoating and DNA replication are intertwined processes involving at least three viral proteins with mutually redundant functions in addition to D5.IMPORTANCE Poxviruses comprise a family of large DNA viruses that infect vertebrates and invertebrates and cause diseases of medical and zoological importance. Poxviruses, unlike most other DNA viruses, replicate in the cytoplasm and their large genomes usually encode 200 or more proteins with diverse functions. About 90 genes may be

  7. The Conjugative Relaxase TrwC Promotes Integration of Foreign DNA in the Human Genome.

    Science.gov (United States)

    González-Prieto, Coral; Gabriel, Richard; Dehio, Christoph; Schmidt, Manfred; Llosa, Matxalen

    2017-06-15

    Bacterial conjugation is a mechanism of horizontal DNA transfer. The relaxase TrwC of the conjugative plasmid R388 cleaves one strand of the transferred DNA at the oriT gene, covalently attaches to it, and leads the single-stranded DNA (ssDNA) into the recipient cell. In addition, TrwC catalyzes site-specific integration of the transferred DNA into its target sequence present in the genome of the recipient bacterium. Here, we report the analysis of the efficiency and specificity of the integrase activity of TrwC in human cells, using the type IV secretion system of the human pathogen Bartonella henselae to introduce relaxase-DNA complexes. Compared to Mob relaxase from plasmid pBGR1, we found that TrwC mediated a 10-fold increase in the rate of plasmid DNA transfer to human cells and a 100-fold increase in the rate of chromosomal integration of the transferred DNA. We used linear amplification-mediated PCR and plasmid rescue to characterize the integration pattern in the human genome. DNA sequence analysis revealed mostly reconstituted oriT sequences, indicating that TrwC is active and recircularizes transferred DNA in human cells. One TrwC-mediated site-specific integration event was detected, proving that TrwC is capable of mediating site-specific integration in the human genome, albeit with very low efficiency compared to the rate of random integration. Our results suggest that TrwC may stabilize the plasmid DNA molecules in the nucleus of the human cell, probably by recircularization of the transferred DNA strand. This stabilization would increase the opportunities for integration of the DNA by the host machinery.IMPORTANCE Different biotechnological applications, including gene therapy strategies, require permanent modification of target cells. Long-term expression is achieved either by extrachromosomal persistence or by integration of the introduced DNA. Here, we studied the utility of conjugative relaxase TrwC, a bacterial protein with site

  8. A Multi-Breed Genome-Wide Association Analysis for Canine Hypothyroidism Identifies a Shared Major Risk Locus on CFA12.

    Directory of Open Access Journals (Sweden)

    Matteo Bianchi

    Full Text Available Hypothyroidism is a complex clinical condition found in both humans and dogs, thought to be caused by a combination of genetic and environmental factors. In this study we present a multi-breed analysis of predisposing genetic risk factors for hypothyroidism in dogs using three high-risk breeds--the Gordon Setter, Hovawart and the Rhodesian Ridgeback. Using a genome-wide association approach and meta-analysis, we identified a major hypothyroidism risk locus shared by these breeds on chromosome 12 (p = 2.1x10(-11. Further characterisation of the candidate region revealed a shared ~167 kb risk haplotype (4,915,018-5,081,823 bp, tagged by two SNPs in almost complete linkage disequilibrium. This breed-shared risk haplotype includes three genes (LHFPL5, SRPK1 and SLC26A8 and does not extend to the dog leukocyte antigen (DLA class II gene cluster located in the vicinity. These three genes have not been identified as candidate genes for hypothyroid disease previously, but have functions that could potentially contribute to the development of the disease. Our results implicate the potential involvement of novel genes and pathways for the development of canine hypothyroidism, raising new possibilities for screening, breeding programmes and treatments in dogs. This study may also contribute to our understanding of the genetic etiology of human hypothyroid disease, which is one of the most common endocrine disorders in humans.

  9. A Multi-Breed Genome-Wide Association Analysis for Canine Hypothyroidism Identifies a Shared Major Risk Locus on CFA12.

    Science.gov (United States)

    Bianchi, Matteo; Dahlgren, Stina; Massey, Jonathan; Dietschi, Elisabeth; Kierczak, Marcin; Lund-Ziener, Martine; Sundberg, Katarina; Thoresen, Stein Istre; Kämpe, Olle; Andersson, Göran; Ollier, William E R; Hedhammar, Åke; Leeb, Tosso; Lindblad-Toh, Kerstin; Kennedy, Lorna J; Lingaas, Frode; Rosengren Pielberg, Gerli

    2015-01-01

    Hypothyroidism is a complex clinical condition found in both humans and dogs, thought to be caused by a combination of genetic and environmental factors. In this study we present a multi-breed analysis of predisposing genetic risk factors for hypothyroidism in dogs using three high-risk breeds--the Gordon Setter, Hovawart and the Rhodesian Ridgeback. Using a genome-wide association approach and meta-analysis, we identified a major hypothyroidism risk locus shared by these breeds on chromosome 12 (p = 2.1x10(-11)). Further characterisation of the candidate region revealed a shared ~167 kb risk haplotype (4,915,018-5,081,823 bp), tagged by two SNPs in almost complete linkage disequilibrium. This breed-shared risk haplotype includes three genes (LHFPL5, SRPK1 and SLC26A8) and does not extend to the dog leukocyte antigen (DLA) class II gene cluster located in the vicinity. These three genes have not been identified as candidate genes for hypothyroid disease previously, but have functions that could potentially contribute to the development of the disease. Our results implicate the potential involvement of novel genes and pathways for the development of canine hypothyroidism, raising new possibilities for screening, breeding programmes and treatments in dogs. This study may also contribute to our understanding of the genetic etiology of human hypothyroid disease, which is one of the most common endocrine disorders in humans.

  10. Evidence of a genomic insertion in intron 2 of SOD1 causing allelic drop-out during routine diagnostic testing for canine degenerative myelopathy.

    Science.gov (United States)

    Turba, M E; Loechel, R; Rombolà, E; Gandini, G; Gentilini, F

    2017-06-01

    Degenerative myelopathy is a severe and progressive neurodegenerative disease and, in the majority of breeds, is associated with the c.118G>A substitution in exon 2 of the canine superoxide dismutase 1 (SOD1) gene. Our laboratories have been engaged in determining the cause of many discordant findings between the parental and the offspring genotypes found by different laboratories. In both cases, the discordant findings refer to actual heterozygous dogs that had been typed as homozygous for the variant allele. To that aim, the genomic context of the causative variant was investigated in two Hovawart dogs. An insertion of 54 nucleotides composed of a poly-T stretch and 15 nucleotides containing the duplication of the exon 2-intron 2 junction was found. The insertion was responsible for the partial mismatch of the reverse primer used for a direct sequencing assay. The mismatch hampered the amplification of the corresponding allele and caused an evident drop-out effect. The insertion is in complete linkage disequilibrium with the c.118G allele. The allele containing the insertion was highly prevalent in Hovawart dogs, accounting for the 26.6% of allele frequency. The insertion was also found in other unrelated breeds such as Rough Collies and Standard Poodles. In conclusion, the study illustrates the importance of correctly designing the primers to avoid inaccurate genotyping of the degenerative myelopathy causative variant in exon 2 of the SOD1 gene. © 2016 Stichting International Foundation for Animal Genetics.

  11. Realistic artificial DNA sequences as negative controls for computational genomics

    Science.gov (United States)

    Caballero, Juan; Smit, Arian F. A.; Hood, Leroy; Glusman, Gustavo

    2014-01-01

    A common practice in computational genomic analysis is to use a set of ‘background’ sequences as negative controls for evaluating the false-positive rates of prediction tools, such as gene identification programs and algorithms for detection of cis-regulatory elements. Such ‘background’ sequences are generally taken from regions of the genome presumed to be intergenic, or generated synthetically by ‘shuffling’ real sequences. This last method can lead to underestimation of false-positive rates. We developed a new method for generating artificial sequences that are modeled after real intergenic sequences in terms of composition, complexity and interspersed repeat content. These artificial sequences can serve as an inexhaustible source of high-quality negative controls. We used artificial sequences to evaluate the false-positive rates of a set of programs for detecting interspersed repeats, ab initio prediction of coding genes, transcribed regions and non-coding genes. We found that RepeatMasker is more accurate than PClouds, Augustus has the lowest false-positive rate of the coding gene prediction programs tested, and Infernal has a low false-positive rate for non-coding gene detection. A web service, source code and the models for human and many other species are freely available at http://repeatmasker.org/garlic/. PMID:24803667

  12. Realistic artificial DNA sequences as negative controls for computational genomics.

    Science.gov (United States)

    Caballero, Juan; Smit, Arian F A; Hood, Leroy; Glusman, Gustavo

    2014-07-01

    A common practice in computational genomic analysis is to use a set of 'background' sequences as negative controls for evaluating the false-positive rates of prediction tools, such as gene identification programs and algorithms for detection of cis-regulatory elements. Such 'background' sequences are generally taken from regions of the genome presumed to be intergenic, or generated synthetically by 'shuffling' real sequences. This last method can lead to underestimation of false-positive rates. We developed a new method for generating artificial sequences that are modeled after real intergenic sequences in terms of composition, complexity and interspersed repeat content. These artificial sequences can serve as an inexhaustible source of high-quality negative controls. We used artificial sequences to evaluate the false-positive rates of a set of programs for detecting interspersed repeats, ab initio prediction of coding genes, transcribed regions and non-coding genes. We found that RepeatMasker is more accurate than PClouds, Augustus has the lowest false-positive rate of the coding gene prediction programs tested, and Infernal has a low false-positive rate for non-coding gene detection. A web service, source code and the models for human and many other species are freely available at http://repeatmasker.org/garlic/. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Complete canine papillomavirus life cycle in pigmented lesions.

    Science.gov (United States)

    Lange, Christian E; Tobler, Kurt; Schraner, Elisabeth M; Vetsch, Elisabeth; Fischer, Nina M; Ackermann, Mathias; Favrot, Claude

    2013-03-23

    Canine papillomaviruses (CPVs) have been identified in various benign and malignant neoplastic skin disorders. The most frequent manifestations of CPV infections are classical warts and pigmented plaques. Although the etiology of canine oral papillomatosis is well established, knowledge about CPVs role in the development of pigmented plaques remains vague. Indeed, as CPV DNA may frequently be found on clinically healthy canine skin, its mere detection in lesions cannot be regarded as a sufficient indicator of causality. Whether CPVs are actually active in pigmented plaques, a requirement for any conceivable involvement, is consequently an open question. To inquire such viral activity, two distinct clinical cases of canine pigmented lesions were evaluated in greater detail. The histological findings in the two cases supported the clinical diagnosis of pigmented viral plaques. Sequencing of amplified DNA from these lesions revealed the genomes of two novel CPV types, i.e. CPV9 and CPV14, both putatively belonging to the genus Chi. Furthermore, transcription and splicing of corresponding CPV mRNA could be shown by RT-PCR in the respective lesions. Finally, viral particles were detected by electron microscopy in homogenates as well as in nuclei of keratinocytes in pigmented lesions. In conclusion, the results link clinical signs of pigmented plaques to histological changes, the presence of CPV specific DNA, viral gene transcription, and the presence of viral particles in and from the lesions. Thus, the findings outline the entire replicative cycle of CPVs in pigmented plaques, which might help understanding the relationship between these viruses and the associated disorders. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. High-throughput DNA sequencing: a genomic data manufacturing process.

    Science.gov (United States)

    Huang, G M

    1999-01-01

    The progress trends in automated DNA sequencing operation are reviewed. Technological development in sequencing instruments, enzymatic chemistry and robotic stations has resulted in ever-increasing capacity of sequence data production. This progress leads to a higher demand on laboratory information management and data quality assessment. High-throughput laboratories face the challenge of organizational management, as well as technology management. Engineering principles of process control should be adopted in this biological data manufacturing procedure. While various systems attempt to provide solutions to automate different parts of, or even the entire process, new technical advances will continue to change the paradigm and provide new challenges.

  15. Comparative genomics of DNA recombination and repair in cyanobacteria: biotechnological implications

    Directory of Open Access Journals (Sweden)

    Corinne Cassier-Chauvat

    2016-11-01

    Full Text Available Cyanobacteria are fascinating photosynthetic prokaryotes that are regarded as the ancestors of the plant chloroplast; the purveyors of oxygen and biomass for the food chain; and promising cell factories for an environmentally friendly production of chemicals. In colonizing most waters and soils of our planet, cyanobacteria are inevitably challenged by environmental stresses that generate DNA damages. Furthermore, many strains engineered for biotechnological purposes can use DNA recombination to stop synthesizing the biotechnological product. Hence, it is important to study DNA recombination and repair in cyanobacteria for both basic and applied research. This review reports what is known in a few widely studied model cyanobacteria and what can be inferred by mining the sequenced genomes of morphologically and physiologically diverse strains. We show that cyanobacteria possess many E. coli-like DNA recombination and repair genes, and possibly other genes not yet identified. E. coli-homolog genes are unevenly distributed in cyanobacteria, in agreement with their wide genome diversity. Many genes are extremely well conserved in cyanobacteria (mutMS, radA, recA, recFO, recG, recN, ruvABC, ssb and uvrABCD, even in small genomes, suggesting that they encode the core DNA repair process. In addition to these core genes, the marine Prochlorococcus and Synechococcus strains harbor recBCD (DNA recombination, umuCD (mutational DNA replication, as well as the key SOS genes lexA (regulation of the SOS system and sulA (postponing of cell division until completion of DNA reparation. Hence, these strains could possess an E. coli-type SOS system. In contrast, several cyanobacteria endowed with larger genomes lack typical SOS genes. For examples, the two studied Gloeobacter strains lack alkB, lexA and sulA; and Synechococcus PCC7942 has neither lexA nor recCD. Furthermore, the Synechocystis PCC6803 lexA product does not regulate DNA repair genes. Collectively

  16. Genome-wide DNA hypomethylation and RNA:DNA hybrid accumulation in Aicardi–Goutières syndrome

    Science.gov (United States)

    Lim, Yoong Wearn; Sanz, Lionel A; Xu, Xiaoqin; Hartono, Stella R; Chédin, Frédéric

    2015-01-01

    Aicardi–Goutières syndrome (AGS) is a severe childhood inflammatory disorder that shows clinical and genetic overlap with systemic lupus erythematosus (SLE). AGS is thought to arise from the accumulation of incompletely metabolized endogenous nucleic acid species owing to mutations in nucleic acid-degrading enzymes TREX1 (AGS1), RNase H2 (AGS2, 3 and 4), and SAMHD1 (AGS5). However, the identity and source of such immunogenic nucleic acid species remain undefined. Using genome-wide approaches, we show that fibroblasts from AGS patients with AGS1-5 mutations are burdened by excessive loads of RNA:DNA hybrids. Using MethylC-seq, we show that AGS fibroblasts display pronounced and global loss of DNA methylation and demonstrate that AGS-specific RNA:DNA hybrids often occur within DNA hypomethylated regions. Altogether, our data suggest that RNA:DNA hybrids may represent a common immunogenic form of nucleic acids in AGS and provide the first evidence of epigenetic perturbations in AGS, furthering the links between AGS and SLE. DOI: http://dx.doi.org/10.7554/eLife.08007.001 PMID:26182405

  17. Genome-wide DNA hypomethylation and RNA:DNA hybrid accumulation in Aicardi-Goutières syndrome.

    Science.gov (United States)

    Lim, Yoong Wearn; Sanz, Lionel A; Xu, Xiaoqin; Hartono, Stella R; Chédin, Frédéric

    2015-07-16

    Aicardi-Goutières syndrome (AGS) is a severe childhood inflammatory disorder that shows clinical and genetic overlap with systemic lupus erythematosus (SLE). AGS is thought to arise from the accumulation of incompletely metabolized endogenous nucleic acid species owing to mutations in nucleic acid-degrading enzymes TREX1 (AGS1), RNase H2 (AGS2, 3 and 4), and SAMHD1 (AGS5). However, the identity and source of such immunogenic nucleic acid species remain undefined. Using genome-wide approaches, we show that fibroblasts from AGS patients with AGS1-5 mutations are burdened by excessive loads of RNA:DNA hybrids. Using MethylC-seq, we show that AGS fibroblasts display pronounced and global loss of DNA methylation and demonstrate that AGS-specific RNA:DNA hybrids often occur within DNA hypomethylated regions. Altogether, our data suggest that RNA:DNA hybrids may represent a common immunogenic form of nucleic acids in AGS and provide the first evidence of epigenetic perturbations in AGS, furthering the links between AGS and SLE.

  18. RNA exosome regulates AID DNA mutator activity in the B cell genome

    Science.gov (United States)

    Pefanis, Evangelos; Basu, Uttiya

    2015-01-01

    The immunoglobulin diversification processes of somatic hypermutation and class switch recombination critically rely on transcription coupled targeting of AID to Ig loci in activated B lymphocytes. AID catalyzes deamination of cytidine deoxynucleotides on exposed single stranded DNA. In addition to driving immunoglobulin diversity, promiscuous targeting of AID mutagenic activity poses a deleterious threat to genomic stability. Recent genome-wide studies have uncovered pervasive AID activity throughout the B cell genome. It is increasingly apparent that AID activity is frequently targeted to genomic loci undergoing early transcription termination where RNA exosome promotes the resolution of stalled transcription complexes via co-transcriptional RNA degradation mechanisms. Here we review aspects and consequences of eukaryotic transcription that lead to early termination, RNA exosome recruitment, and ultimately targeting of AID mutagenic activity. PMID:26073986

  19. Segmentation of genomic DNA through entropic divergence: Power laws and scaling

    Science.gov (United States)

    Azad, Rajeev K.; Bernaola-Galván, Pedro; Ramaswamy, Ramakrishna; Rao, J. Subba

    2002-05-01

    Genomic DNA is fragmented into segments using the Jensen-Shannon divergence. Use of this criterion results in the fragments being entropically homogeneous to within a predefined level of statistical significance. Application of this procedure is made to complete genomes of organisms from archaebacteria, eubacteria, and eukaryotes. The distribution of fragment lengths in bacterial and primitive eukaryotic DNAs shows two distinct regimes of power-law scaling. The characteristic length separating these two regimes appears to be an intrinsic property of the sequence rather than a finite-size artifact, and is independent of the significance level used in segmenting a given genome. Fragment length distributions obtained in the segmentation of the genomes of more highly evolved eukaryotes do not have such distinct regimes of power-law behavior.

  20. Profiling the genome-wide DNA methylation pattern of porcine ovaries using reduced representation bisulfite sequencing.

    Science.gov (United States)

    Yuan, Xiao-Long; Gao, Ning; Xing, Yan; Zhang, Hai-Bin; Zhang, Ai-Ling; Liu, Jing; He, Jin-Long; Xu, Yuan; Lin, Wen-Mian; Chen, Zan-Mou; Zhang, Hao; Zhang, Zhe; Li, Jia-Qi

    2016-02-25

    Substantial evidence has shown that DNA methylation regulates the initiation of ovarian and sexual maturation. Here, we investigated the genome-wide profile of DNA methylation in porcine ovaries at single-base resolution using reduced representation bisulfite sequencing. The biological variation was minimal among the three ovarian replicates. We found hypermethylation frequently occurred in regions with low gene abundance, while hypomethylation in regions with high gene abundance. The DNA methylation around transcriptional start sites was negatively correlated with their own CpG content. Additionally, the methylation level in the bodies of genes was higher than that in their 5' and 3' flanking regions. The DNA methylation pattern of the low CpG content promoter genes differed obviously from that of the high CpG content promoter genes. The DNA methylation level of the porcine ovary was higher than that of the porcine intestine. Analyses of the genome-wide DNA methylation in porcine ovaries would advance the knowledge and understanding of the porcine ovarian methylome.

  1. Comparison of microbial DNA enrichment tools for metagenomic whole genome sequencing.

    Science.gov (United States)

    Thoendel, Matthew; Jeraldo, Patricio R; Greenwood-Quaintance, Kerryl E; Yao, Janet Z; Chia, Nicholas; Hanssen, Arlen D; Abdel, Matthew P; Patel, Robin

    2016-08-01

    Metagenomic whole genome sequencing for detection of pathogens in clinical samples is an exciting new area for discovery and clinical testing. A major barrier to this approach is the overwhelming ratio of human to pathogen DNA in samples with low pathogen abundance, which is typical of most clinical specimens. Microbial DNA enrichment methods offer the potential to relieve this limitation by improving this ratio. Two commercially available enrichment kits, the NEBNext Microbiome DNA Enrichment Kit and the Molzym MolYsis Basic kit, were tested for their ability to enrich for microbial DNA from resected arthroplasty component sonicate fluids from prosthetic joint infections or uninfected sonicate fluids spiked with Staphylococcus aureus. Using spiked uninfected sonicate fluid there was a 6-fold enrichment of bacterial DNA with the NEBNext kit and 76-fold enrichment with the MolYsis kit. Metagenomic whole genome sequencing of sonicate fluid revealed 13- to 85-fold enrichment of bacterial DNA using the NEBNext enrichment kit. The MolYsis approach achieved 481- to 9580-fold enrichment, resulting in 7 to 59% of sequencing reads being from the pathogens known to be present in the samples. These results demonstrate the usefulness of these tools when testing clinical samples with low microbial burden using next generation sequencing. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. DNA replication factor C1 mediates genomic stability and transcriptional gene silencing in Arabidopsis

    KAUST Repository

    Liu, Qian

    2010-07-01

    Genetic screening identified a suppressor of ros1-1, a mutant of REPRESSOR OF SILENCING1 (ROS1; encoding a DNA demethylation protein). The suppressor is a mutation in the gene encoding the largest subunit of replication factor C (RFC1). This mutation of RFC1 reactivates the unlinked 35S-NPTII transgene, which is silenced in ros1 and also increases expression of the pericentromeric Athila retrotransposons named transcriptional silent information in a DNA methylationindependent manner. rfc1 is more sensitive than the wild type to the DNA-damaging agent methylmethane sulphonate and to the DNA inter- and intra- cross-linking agent cisplatin. The rfc1 mutant constitutively expresses the G2/M-specific cyclin CycB1;1 and other DNA repair-related genes. Treatment with DNA-damaging agents mimics the rfc1 mutation in releasing the silenced 35S-NPTII, suggesting that spontaneously induced genomic instability caused by the rfc1 mutation might partially contribute to the released transcriptional gene silencing (TGS). The frequency of somatic homologous recombination is significantly increased in the rfc1 mutant. Interestingly, ros1 mutants show increased telomere length, but rfc1 mutants show decreased telomere length and reduced expression of telomerase. Our results suggest that RFC1 helps mediate genomic stability and TGS in Arabidopsis thaliana. © 2010 American Society of Plant Biologists.

  3. Novel extraction method of genomic DNA suitable for long-fragment amplification from small amounts of milk.

    Science.gov (United States)

    Liu, Y F; Gao, J L; Yang, Y F; Ku, T; Zan, L S

    2014-11-01

    Isolation of genomic DNA is a prerequisite for assessment of milk quality. As a source of genomic DNA, milk somatic cells from milking ruminants are practical, animal friendly, and cost-effective sources. Extracting DNA from milk can avoid the stress response caused by blood and tissue sampling of cows. In this study, we optimized a novel DNA extraction method for amplifying long (>1,000 bp) DNA fragments and used it to evaluate the isolation of DNA from small amounts of milk. The techniques used for the separation of milk somatic cell were explored and combined with a sodium dodecyl sulfate (SDS)-phenol method for optimizing DNA extraction from milk. Spectrophotometry was used to determine the concentration and purity of the extracted DNA. Gel electrophoresis and DNA amplification technologies were used for to determine DNA size and quality. The DNA of 112 cows was obtained from milk (samples of 13 ± 1 mL) and the corresponding optical density ratios at 260:280 nm were between 1.65 and 1.75. Concentrations were between 12 and 45 μg/μL and DNA size and quality were acceptable. The specific PCR amplification of 1,019- and 729-bp bovine DNA fragments was successfully carried out. This novel method can be used as a practical, fast, and economical mean for long genomic DNA extraction from a small amount of milk. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  4. Complete mitochondrial DNA genome of Pseudobagrus brevicaudatus (Siluriformes: Bagridae).

    Science.gov (United States)

    Liang, Hong-Wei; Meng, Yan; Li, Zhong; Zhang, Yan; Zou, Gui-wei

    2014-06-01

    The complete mitochondrial genome of Pseudobagrus brevicaudatus (Siluriformes: Bagridae) was sequenced in this study. The total length of the mitogenome is 16,533 bp, with the base composition of 31.6% A, 26.8% T, 15.0%G, 26.6% C. The gene order and genes were the same as that found in other previously reported catfishes, including 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and 1 non-coding control region. Except for ND6 gene and 8 tRNA genes, all other mitochondrial genes were encoded on the heavy strand. This complete mitogenome data provides the basis for taxonomic and conservation research of this and closely related species.

  5. Links between persistent DNA damage, genome instability, and aging

    Energy Technology Data Exchange (ETDEWEB)

    Dynan, William S. [Emory Univ., Atlanta, GA (United States). Dept. of Radiation Oncology

    2016-11-14

    The goal of this study was to examine long-term effects of low-dose radiation exposure. One of the hypotheses was that radiation exposure would accelerate the normal aging process. The study was jointly funded by NASA and examined both low-LET radiation (γ-rays) and high-LET radiation (1000 MeV/nucleon 56Fe ions) at doses of 0.1 Gy and up. The work used the Japanese medaka fish (Oryzias latipes), as a vertebrate model organism that can be maintained in large numbers at low cost for lifetime studies. Like other small laboratory fish, Japanese medaka share many anatomical and histological characteristics with other vertebrates, and a variety of genetic and genomic resources are available. Some work also used the zebrafish (Danio rerio), another widely used laboratory model organism.

  6. Vaccines for canine leishmaniasis.

    Science.gov (United States)

    Palatnik-de-Sousa, Clarisa B

    2012-01-01

    Leishmaniasis is the third most important vector-borne disease worldwide. Visceral leishmaniasis (VL) is a severe and frequently lethal protozoan disease of increasing incidence and severity due to infected human and dog migration, new geographical distribution of the insect due to global warming, coinfection with immunosuppressive diseases, and poverty. The disease is an anthroponosis in India and Central Africa and a canid zoonosis (ZVL) in the Americas, the Middle East, Central Asia, China, and the Mediterranean. The ZVL epidemic has been controlled by one or more measures including the culling of infected dogs, treatment of human cases, and insecticidal treatment of homes and dogs. However, the use of vaccines is considered the most cost-effective control tool for human and canine disease. Since the severity of the disease is related to the generation of T-cell immunosuppression, effective vaccines should be capable of sustaining or enhancing the T-cell immunity. In this review we summarize the clinical and parasitological characteristics of ZVL with special focus on the cellular and humoral canine immune response and review state-of-the-art vaccine development against human and canine VL. Experimental vaccination against leishmaniasis has evolved from the practice of leishmanization with living parasites to vaccination with crude lysates, native parasite extracts to recombinant and DNA vaccination. Although more than 30 defined vaccines have been studied in laboratory models no human formulation has been licensed so far; however three second-generation canine vaccines have already been registered. As expected for a zoonotic disease, the recent preventive vaccination of dogs in Brazil has led to a reduction in the incidence of canine and human disease. The recent identification of several Leishmania proteins with T-cell epitopes anticipates development of a multiprotein vaccine that will be capable of protecting both humans and dogs against VL.

  7. Vaccines for canine leishmaniasis

    Directory of Open Access Journals (Sweden)

    Clarisa B. Palatnik-De-Sousa

    2012-04-01

    Full Text Available Leishmaniasis is the third most important vector-borne disease worldwide. Visceral leishmaniasis (VL is a severe and frequently lethal protozoan disease of increasing incidence and severity due to infected human and dog migration, new geographical distribution of the insect due to global-warming, co-infection with immunosuppressive diseases and poverty. The disease is an anthroponosis in India and Central Africa and a canid zoonosis (ZVL in the Americas, the Middle East, Central Asia, China and the Mediterranean. The ZVL epidemic has been controlled by one or more measures including the culling of infected dogs, treatment of human cases and insecticidal treatment of homes and dogs. However, the use of vaccines is considered the most cost-effective control tool for human and canine disease. Since the severity of the disease is related to the generation of T-cell immunosuppression, effective vaccines should be capable of sustaining or enhancing the T-cell immunity. In this review we summarize the clinical and parasitological characteristics of ZVL with special focus on the cellular and humoral canine immune response and review state-of-the-art vaccine development against human and canine visceral leishmaniasis. Experimental vaccination against leishmaniasis has evolved from the practice of leishmanization with living parasites to vaccination with crude lysates, native parasite extracts to recombinant and DNA vaccination. Although more than 30 defined vaccines have been studied in laboratory models no human formulation has been licensed so far; however three second-generation canine vaccines have already been registered. As expected for a zoonotic disease, the recent preventive vaccination of dogs in Brazil has led to a reduction in the incidence of canine and human disease. The recent identification of several Leishmania proteins with T-cell epitopes anticipates development of a multiprotein vaccine that will be capable of protecting both humans

  8. Vaccines for Canine Leishmaniasis

    Science.gov (United States)

    Palatnik-de-Sousa, Clarisa B.

    2012-01-01

    Leishmaniasis is the third most important vector-borne disease worldwide. Visceral leishmaniasis (VL) is a severe and frequently lethal protozoan disease of increasing incidence and severity due to infected human and dog migration, new geographical distribution of the insect due to global warming, coinfection with immunosuppressive diseases, and poverty. The disease is an anthroponosis in India and Central Africa and a canid zoonosis (ZVL) in the Americas, the Middle East, Central Asia, China, and the Mediterranean. The ZVL epidemic has been controlled by one or more measures including the culling of infected dogs, treatment of human cases, and insecticidal treatment of homes and dogs. However, the use of vaccines is considered the most cost–effective control tool for human and canine disease. Since the severity of the disease is related to the generation of T-cell immunosuppression, effective vaccines should be capable of sustaining or enhancing the T-cell immunity. In this review we summarize the clinical and parasitological characteristics of ZVL with special focus on the cellular and humoral canine immune response and review state-of-the-art vaccine development against human and canine VL. Experimental vaccination against leishmaniasis has evolved from the practice of leishmanization with living parasites to vaccination with crude lysates, native parasite extracts to recombinant and DNA vaccination. Although more than 30 defined vaccines have been studied in laboratory models no human formulation has been licensed so far; however three second-generation canine vaccines have already been registered. As expected for a zoonotic disease, the recent preventive vaccination of dogs in Brazil has led to a reduction in the incidence of canine and human disease. The recent identification of several Leishmania proteins with T-cell epitopes anticipates development of a multiprotein vaccine that will be capable of protecting both humans and dogs against VL. PMID:22566950

  9. Mitochondrial genome of the moon jelly Aurelia aurita (Cnidaria, Scyphozoa): A linear DNA molecule encoding a putative DNA-dependent DNA polymerase.

    Science.gov (United States)

    Shao, Zhiyong; Graf, Shannon; Chaga, Oleg Y; Lavrov, Dennis V

    2006-10-15

    The 16,937-nuceotide sequence of the linear mitochondrial DNA (mt-DNA) molecule of the moon jelly Aurelia aurita (Cnidaria, Scyphozoa) - the first mtDNA sequence from the class Scypozoa and the first sequence of a linear mtDNA from Metazoa - has been determined. This sequence contains genes for 13 energy pathway proteins, small and large subunit rRNAs, and methionine and tryptophan tRNAs. In addition, two open reading frames of 324 and 969 base pairs in length have been found. The deduced amino-acid sequence of one of them, ORF969, displays extensive sequence similarity with the polymerase [but not the exonuclease] domain of family B DNA polymerases, and this ORF has been tentatively identified as dnab. This is the first report of dnab in animal mtDNA. The genes in A. aurita mtDNA are arranged in two clusters with opposite transcriptional polarities; transcription proceeding toward the ends of the molecule. The determined sequences at the ends of the molecule are nearly identical but inverted and lack any obvious potential secondary structures or telomere-like repeat elements. The acquisition of mitochondrial genomic data for the second class of Cnidaria allows us to reconstruct characteristic features of mitochondrial evolution in this animal phylum.

  10. Whole-genome resequencing of 100 healthy individuals using DNA pooling.

    Science.gov (United States)

    Wang, Xiaobin; Sui, Weiguo; Wu, Weiqing; Hou, Xianliang; Ou, Minglin; Xiang, Yueying; Dai, Yong

    2016-11-01

    With the advent of next-generation sequencing technology, the cost of sequencing has significantly decreased. However, sequencing costs remain high for large-scale studies. In the present study, DNA pooling was applied as a cost-effective strategy for sequencing. The sequencing results for 100 healthy individuals obtained via whole-genome resequencing and using DNA pooling are presented in the present study. In order to minimise the likelihood of systematic bias in sampling, paired-end libraries with an insert size of 500 bp were prepared for all samples and then subjected to whole-genome sequencing using four lanes for each library and resulting in at least a 30-fold haploid coverage for each sample. The NCBI human genome build37 (hg19) was used as a reference genome for the present study and the short reads were aligned to the reference genome achieving 99.84% coverage. In addition, the average sequencing depth was 32.76. In total, ~3 million single-nucleotide polymorphisms were identified, of which 99.88% were in the NCBI dbSNP database. Furthermore, ~600,000 small insertion/deletions, 500,000 structure variants, 5,000 copy number variations and 13,000 single nucleotide variants were identified. According to the present study, the whole genome has been sequenced for a small sample subjects from southern China for the first time. Furthermore, new variation sites were identified by comparing with the reference sequence, and new knowledge of the human genome variation was added to the human genomic databases. Furthermore, the particular distribution regions of variation were illustrated by analyzing various sites of variation, such as single-nucleotide polymorphisms.

  11. Genome-Wide Analyses Suggest Mechanisms Involving Early B-Cell Development in Canine IgA Deficiency.

    Directory of Open Access Journals (Sweden)

    Mia Olsson

    Full Text Available Immunoglobulin A deficiency (IgAD is the most common primary immune deficiency disorder in both humans and dogs, characterized by recurrent mucosal tract infections and a predisposition for allergic and other immune mediated diseases. In several dog breeds, low IgA levels have been observed at a high frequency and with a clinical resemblance to human IgAD. In this study, we used genome-wide association studies (GWAS to identify genomic regions associated with low IgA levels in dogs as a comparative model for human IgAD. We used a novel percentile groups-approach to establish breed-specific cut-offs and to perform analyses in a close to continuous manner. GWAS performed in four breeds prone to low IgA levels (German shepherd, Golden retriever, Labrador retriever and Shar-Pei identified 35 genomic loci suggestively associated (p <0.0005 to IgA levels. In German shepherd, three genomic regions (candidate genes include KIRREL3 and SERPINA9 were genome-wide significantly associated (p <0.0002 with IgA levels. A ~20kb long haplotype on CFA28, significantly associated (p = 0.0005 to IgA levels in Shar-Pei, was positioned within the first intron of the gene SLIT1. Both KIRREL3 and SLIT1 are highly expressed in the central nervous system and in bone marrow and are potentially important during B-cell development. SERPINA9 expression is restricted to B-cells and peaks at the time-point when B-cells proliferate into antibody-producing plasma cells. The suggestively associated regions were enriched for genes in Gene Ontology gene sets involving inflammation and early immune cell development.

  12. Phylogenetic and genome-wide deep-sequencing analyses of canine parvovirus reveal co-infection with field variants and emergence of a recent recombinant strain.

    Directory of Open Access Journals (Sweden)

    Ruben Pérez

    Full Text Available Canine parvovirus (CPV, a fast-evolving single-stranded DNA virus, comprises three antigenic variants (2a, 2b, and 2c with different frequencies and genetic variability among countries. The contribution of co-infection and recombination to the genetic variability of CPV is far from being fully elucidated. Here we took advantage of a natural CPV population, recently formed by the convergence of divergent CPV-2c and CPV-2a strains, to study co-infection and recombination. Complete sequences of the viral coding region of CPV-2a and CPV-2c strains from 40 samples were generated and analyzed using phylogenetic tools. Two samples showed co-infection and were further analyzed by deep sequencing. The sequence profile of one of the samples revealed the presence of CPV-2c and CPV-2a strains that differed at 29 nucleotides. The other sample included a minor CPV-2a strain (13.3% of the viral population and a major recombinant strain (86.7%. The recombinant strain arose from inter-genotypic recombination between CPV-2c and CPV-2a strains within the VP1/VP2 gene boundary. Our findings highlight the importance of deep-sequencing analysis to provide a better understanding of CPV molecular diversity.

  13. Phylogenetic and Genome-Wide Deep-Sequencing Analyses of Canine Parvovirus Reveal Co-Infection with Field Variants and Emergence of a Recent Recombinant Strain

    Science.gov (United States)

    Pérez, Ruben; Calleros, Lucía; Marandino, Ana; Sarute, Nicolás; Iraola, Gregorio; Grecco, Sofia; Blanc, Hervé; Vignuzzi, Marco; Isakov, Ofer; Shomron, Noam; Carrau, Lucía; Hernández, Martín; Francia, Lourdes; Sosa, Katia; Tomás, Gonzalo; Panzera, Yanina

    2014-01-01

    Canine parvovirus (CPV), a fast-evolving single-stranded DNA virus, comprises three antigenic variants (2a, 2b, and 2c) with different frequencies and genetic variability among countries. The contribution of co-infection and recombination to the genetic variability of CPV is far from being fully elucidated. Here we took advantage of a natural CPV population, recently formed by the convergence of divergent CPV-2c and CPV-2a strains, to study co-infection and recombination. Complete sequences of the viral coding region of CPV-2a and CPV-2c strains from 40 samples were generated and analyzed using phylogenetic tools. Two samples showed co-infection and were further analyzed by deep sequencing. The sequence profile of one of the samples revealed the presence of CPV-2c and CPV-2a strains that differed at 29 nucleotides. The other sample included a minor CPV-2a strain (13.3% of the viral population) and a major recombinant strain (86.7%). The recombinant strain arose from inter-genotypic recombination between CPV-2c and CPV-2a strains within the VP1/VP2 gene boundary. Our findings highlight the importance of deep-sequencing analysis to provide a better understanding of CPV molecular diversity. PMID:25365348

  14. Complexities due to single-stranded RNA during antibody detection of genomic rna:dna hybrids.

    Science.gov (United States)

    Zhang, Zheng Z; Pannunzio, Nicholas R; Hsieh, Chih-Lin; Yu, Kefei; Lieber, Michael R

    2015-04-08

    Long genomic R-loops in eukaryotes were first described at the immunoglobulin heavy chain locus switch regions using bisulfite sequencing and functional studies. A mouse monoclonal antibody called S9.6 has been used for immunoprecipitation (IP) to identify R-loops, based on the assumption that it is specific for RNA:DNA over other nucleic acid duplexes. However, recent work has demonstrated that a variable domain of S9.6 binds AU-rich RNA:RNA duplexes with a KD that is only 5.6-fold weaker than for RNA:DNA duplexes. Most IP protocols do not pre-clear the genomic nucleic acid with RNase A to remove free RNA. Fold back of ssRNA can readily generate RNA:RNA duplexes that may bind the S9.6 antibody, and adventitious binding of RNA may also create short RNA:DNA regions. Here we investigate whether RNase A is needed to obtain reliable IP with S9.6. As our test locus, we chose the most well-documented site for kilobase-long mammalian genomic R-loops, the immunoglobulin heavy chain locus (IgH) class switch regions. The R-loops at this locus can be induced by using cytokines to stimulate transcription from germline transcript promoters. We tested IP using S9.6 with and without various RNase treatments. The RNase treatments included RNase H to destroy the RNA in an RNA:DNA duplex and RNase A to destroy single-stranded (ss) RNA to prevent it from binding S9.6 directly (as duplex RNA) and to prevent the ssRNA from annealing to the genome, resulting in adventitious RNA:DNA hybrids. We find that optimal detection of RNA:DNA duplexes requires removal of ssRNA using RNase A. Without RNase A treatment, known regions of R-loop formation containing RNA:DNA duplexes can not be reliably detected. With RNase A treatment, a signal can be detected over background, but only within a limited 2 or 3-fold range, even with a stable kilobase-long genomic R-loop. Any use of the S9.6 antibody must be preceded by RNase A treatment to remove free ssRNA that may compete for the S9.6 binding by

  15. Buccal cells DNA extraction to obtain high quality human genomic DNA suitable for polymorphism genotyping by PCR-RFLP and Real-Time PCR

    National Research Council Canada - National Science Library

    Küchler, Erika Calvano; Tannure, Patricia Nivoloni; Falagan-Lotsch, Priscila; Lopes, Taliria Silva; Granjeiro, Jose Mauro; Amorim, Lidia Maria Fonte

    2012-01-01

    The aim of this study was to evaluate, by PCR-RFLP and real-time PCR, the yield and quality of genomic DNA collected from buccal cells by mouthwash after different storage times at room temperature...

  16. Whole genome sequence analysis of recently circulating Bluetongue virus serotype 11 strains from the United States including two domestic canine isolates

    Science.gov (United States)

    Bluetongue virus (BTV) is a vector-transmitted pathogen that that typically infects and causes disease in domestic and wild ruminants. BTV is also known to infect domestic canines as discovered when dogs were vaccinated with a BTV-contaminated vaccine. Canine BTV infections have been documented thro...

  17. Canine gastritis.

    Science.gov (United States)

    Webb, Craig; Twedt, David C

    2003-09-01

    Gastritis--inflammation of the stomach--is a frequently cited differential yet rarely characterized diagnosis in cases of canine anorexia and vomiting. Although the list of rule-outs for acute or chronic gastritis is extensive, a review of the veterinary literature reveals fewer than 15 articles that have focused on clinical cases of canine gastritis over the last 25 years. The dog frequently appears in the human literature as an experimentally manipulated model for the study of endoscopic techniques or the effect of medications on gastric mucosa. In the veterinary patient, cases of acute gastritis are rarely pursued with the complete diagnostic armamentarium, and cases of chronic gastritis are rarely found to occur as an entity isolated from the rest of the gastrointestinal tract. This article focuses on those findings most clinically relevant to cases of canine gastritis in veterinary medicine.

  18. Genome-Wide Discovery and Information Resource Development of DNA Polymorphisms in Cassava

    Science.gov (United States)

    Yoshida, Takuhiro; Akiyama, Kenji; Ishitani, Manabu; Seki, Motoaki; Shinozaki, Kazuo

    2013-01-01

    Cassava (Manihot esculenta Crantz) is an important crop that provides food security and income generation in many tropical countries, and is known for its adaptability to various environmental conditions. Its draft genome sequence and many expressed sequence tags are now publicly available, allowing the development of cassava polymorphism information. Here, we describe the genome-wide discovery of cassava DNA polymorphisms. Using the alignment of predicted transcribed sequences from the cassava draft genome sequence and ESTs from GenBank, we discovered 10,546 single-nucleotide polymorphisms and 647 insertions and deletions. To facilitate molecular marker development for cassava, we designed 9,316 PCR primer pairs to amplify the genomic region around each DNA polymorphism. Of the discovered SNPs, 62.7% occurred in protein-coding regions. Disease-resistance genes were found to have a significantly higher ratio of nonsynonymous-to-synonymous substitutions. We identified 24 read-through (changes of a stop codon to a coding codon) and 38 premature stop (changes of a coding codon to a stop codon) single-nucleotide polymorphisms, and found that the 5 gene ontology terms in biological process were significantly different in genes with read-through single-nucleotide polymorphisms compared with all cassava genes. All data on the discovered DNA polymorphisms were organized into the Cassava Online Archive database, which is available at http://cassava.psc.riken.jp/. PMID:24040164

  19. Predicting DNA Methylation State of CpG Dinucleotide Using Genome Topological Features and Deep Networks.

    Science.gov (United States)

    Wang, Yiheng; Liu, Tong; Xu, Dong; Shi, Huidong; Zhang, Chaoyang; Mo, Yin-Yuan; Wang, Zheng

    2016-01-22

    The hypo- or hyper-methylation of the human genome is one of the epigenetic features of leukemia. However, experimental approaches have only determined the methylation state of a small portion of the human genome. We developed deep learning based (stacked denoising autoencoders, or SdAs) software named "DeepMethyl" to predict the methylation state of DNA CpG dinucleotides using features inferred from three-dimensional genome topology (based on Hi-C) and DNA sequence patterns. We used the experimental data from immortalised myelogenous leukemia (K562) and healthy lymphoblastoid (GM12878) cell lines to train the learning models and assess prediction performance. We have tested various SdA architectures with different configurations of hidden layer(s) and amount of pre-training data and compared the performance of deep networks relative to support vector machines (SVMs). Using the methylation states of sequentially neighboring regions as one of the learning features, an SdA achieved a blind test accuracy of 89.7% for GM12878 and 88.6% for K562. When the methylation states of sequentially neighboring regions are unknown, the accuracies are 84.82% for GM12878 and 72.01% for K562. We also analyzed the contribution of genome topological features inferred from Hi-C. DeepMethyl can be accessed at http://dna.cs.usm.edu/deepmethyl/.

  20. Higher-Density Culture in Human Embryonic Stem Cells Results in DNA Damage and Genome Instability

    Science.gov (United States)

    Jacobs, Kurt; Zambelli, Filippo; Mertzanidou, Afroditi; Smolders, Ilse; Geens, Mieke; Nguyen, Ha Thi; Barbé, Lise; Sermon, Karen; Spits, Claudia

    2016-01-01

    Summary Human embryonic stem cells (hESC) show great promise for clinical and research applications, but their well-known proneness to genomic instability hampers the development to their full potential. Here, we demonstrate that medium acidification linked to culture density is the main cause of DNA damage and genomic alterations in hESC grown on feeder layers, and this even in the short time span of a single passage. In line with this, we show that increasing the frequency of the medium refreshments minimizes the levels of DNA damage and genetic instability. Also, we show that cells cultured on laminin-521 do not present this increase in DNA damage when grown at high density, although the (long-term) impact on their genomic stability remains to be elucidated. Our results explain the high levels of genome instability observed over the years by many laboratories worldwide, and show that the development of optimal culture conditions is key to solving this problem. PMID:26923824

  1. Impact of DNA damaging agents on genome-wide transcriptional profiles in two marine Synechococcus species

    Directory of Open Access Journals (Sweden)

    Sasha Gabrielle Tetu

    2013-08-01

    Full Text Available Marine microorganisms, particularly those residing in coastal areas, may come in contact with any number of chemicals of environmental or xenobiotic origin. The sensitivity and response of marine cyanobacteria to such chemicals is, at present, poorly understood. We have looked at the transcriptional response of well characterised Synechococcus open ocean (WH8102 and coastal (CC9311 isolates to two DNA damaging agents, mitomycin C and ethidium bromide, using whole-genome expression microarrays. The coastal strain showed differential regulation of a larger proportion of its genome following ‘shock’ treatment with each agent. Many of the orthologous genes in these strains, including those encoding sensor kinases, showed different transcriptional responses, with the CC9311 genes more likely to show significant changes in both treatments. While the overall response of each strain was considerably different, there were distinct transcriptional responses common to both strains observed for each DNA damaging agent, linked to the mode of action of each chemical. In both CC9311 and WH8102 there was evidence of SOS response induction under mitomycin C treatment, with genes recA, lexA and umuC significantly upregulated in this experiment but not under ethidium bromide treatment. Conversely, ethidium bromide treatment tended to result in upregulation of the DNA-directed RNA polymerase genes, not observed following mitomycin C treatment. Interestingly, a large number of genes residing on putative genomic island regions of each genome also showed significant upregulation under one or both chemical treatments.

  2. Predicting DNA Methylation State of CpG Dinucleotide Using Genome Topological Features and Deep Networks

    Science.gov (United States)

    Wang, Yiheng; Liu, Tong; Xu, Dong; Shi, Huidong; Zhang, Chaoyang; Mo, Yin-Yuan; Wang, Zheng

    2016-01-01

    The hypo- or hyper-methylation of the human genome is one of the epigenetic features of leukemia. However, experimental approaches have only determined the methylation state of a small portion of the human genome. We developed deep learning based (stacked denoising autoencoders, or SdAs) software named “DeepMethyl” to predict the methylation state of DNA CpG dinucleotides using features inferred from three-dimensional genome topology (based on Hi-C) and DNA sequence patterns. We used the experimental data from immortalised myelogenous leukemia (K562) and healthy lymphoblastoid (GM12878) cell lines to train the learning models and assess prediction performance. We have tested various SdA architectures with different configurations of hidden layer(s) and amount of pre-training data and compared the performance of deep networks relative to support vector machines (SVMs). Using the methylation states of sequentially neighboring regions as one of the learning features, an SdA achieved a blind test accuracy of 89.7% for GM12878 and 88.6% for K562. When the methylation states of sequentially neighboring regions are unknown, the accuracies are 84.82% for GM12878 and 72.01% for K562. We also analyzed the contribution of genome topological features inferred from Hi-C. DeepMethyl can be accessed at http://dna.cs.usm.edu/deepmethyl/.

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

    Directory of Open Access Journals (Sweden)

    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.

  4. Archaeal Genome Guardians Give Insights into Eukaryotic DNA Replication and Damage Response Proteins

    Directory of Open Access Journals (Sweden)

    David S. Shin

    2014-01-01

    Full Text Available As the third domain of life, archaea, like the eukarya and bacteria, must have robust DNA replication and repair complexes to ensure genome fidelity. Archaea moreover display a breadth of unique habitats and characteristics, and structural biologists increasingly appreciate these features. As archaea include extremophiles that can withstand diverse environmental stresses, they provide fundamental systems for understanding enzymes and pathways critical to genome integrity and stress responses. Such archaeal extremophiles provide critical data on the periodic table for life as well as on the biochemical, geochemical, and physical limitations to adaptive strategies allowing organisms to thrive under environmental stress relevant to determining the boundaries for life as we know it. Specifically, archaeal enzyme structures have informed the architecture and mechanisms of key DNA repair proteins and complexes. With added abilities to temperature-trap flexible complexes and reveal core domains of transient and dynamic complexes, these structures provide insights into mechanisms of maintaining genome integrity despite extreme environmental stress. The DNA damage response protein structures noted in this review therefore inform the basis for genome integrity in the face of environmental stress, with implications for all domains of life as well as for biomanufacturing, astrobiology, and medicine.

  5. Extreme heterogeneous composition of the Paramecium caudatum macronuclear genomic DNA between hemoglobin and nucleosome assembly protein-1 genes.

    Science.gov (United States)

    Nishiyama, Norihito; Mikami, Kazuyuki; Matsuoka, Ariki; Ochiai, Takehiko; Yamauchi, Kiyoshi

    2010-02-01

    The intergenic region between the hemoglobin (hb) and nucleosome assembly protein-1 (nap-1) genes in the Paramecium caudatum macronuclear genome was previously found to be heterogeneously composed. Cloning of this intergenic region from the macronuclear genomic DNA identified four unique DNA fragments of different sizes. Sequencing of the cloned fragments revealed extreme heterogeneity and characteristics of both internal eliminated sequence (IES) and imprecise internal deletion sequences (IIDSs) in the intergenic region. Missing sequences were an AT-rich and direct repeats existed in their boundaries. Southern blotting of the total genomic DNA and polymerase chain reaction (PCR) of the total genomic DNAs indicated that there exist a dozen DNA fragments of different sizes in this intergenic region. It is likely that the heterogeneity found in the P. caudatum macronuclear genome results from the variable removal of an intergenic region.

  6. Dysregulation of DNA methylation induced by past arsenic treatment causes persistent genomic instability in mammalian cells.

    Science.gov (United States)

    Mauro, Maurizio; Caradonna, Fabio; Klein, Catherine B

    2016-03-01

    The mechanisms by which arsenic-induced genomic instability is initiated and maintained are poorly understood. To investigate potential epigenetic mechanisms, in this study we evaluated global DNA methylation levels in V79 cells and human HaCaT keratinocytes at several time points during expanded growth of cell cultures following removal of arsenite exposures. We have found altered genomic methylation patterns that persisted up to 40 cell generations in HaCaT cells after the treatments were withdrawn. Moreover, mRNA expression levels were evaluated by RT-PCR for DNMT1, DNMT3A, DNMT3B, HMLH1, and HMSH2 genes, demonstrating that the down regulation of DNMT3A and DNMT3B genes, but not DNMT1, occurred in an arsenic dose-dependent manner, and persisted for many cell generations following removal of the arsenite, offering a plausible mechanism of persistently genotoxic arsenic action. Analyses of promoter methylation status of the DNA mismatch repair genes HMLH1 and HMSH2 show that HMSH2, but not HMLH1, was epigenetically regulated by promoter hypermethylation changes following arsenic treatment. The results reported here demonstrate that arsenic exposure promptly induces genome-wide global DNA hypomethylation, and some specific gene promoter methylation changes, that persist for many cell generations following withdrawal of arsenite, supporting the hypothesis that the cells undergo epigenetic reprogramming at both the gene and genome level that is durable over many cell generations in the absence of further arsenic treatment. These DNA methylation changes, in concert with other known epigenome alterations, are likely contributing to long-lasting arsenic-induced genomic instability that manifests in several ways, including aberrant chromosomal effects. © 2015 Wiley Periodicals, Inc.

  7. Characterisation of the changing genomic landscape of metastatic melanoma using cell free DNA.

    Science.gov (United States)

    Cutts, Anthony; Venn, Oliver; Dilthey, Alexander; Gupta, Avinash; Vavoulis, Dimitris; Dreau, Helene; Middleton, Mark; McVean, Gil; Taylor, Jenny C; Schuh, Anna

    2017-09-04

    Cancer is characterised by complex somatically acquired genetic aberrations that manifest as intra-tumour and inter-tumour genetic heterogeneity and can lead to treatment resistance. In this case study, we characterise the genome-wide somatic mutation dynamics in a metastatic melanoma patient during therapy using low-input (50 ng) PCR-free whole genome sequencing of cell-free DNA from pre-treatment and post-relapse blood samples. We identify de novo tumour-specific somatic mutations from cell-free DNA, while the sequence context of single nucleotide variants showed the characteristic UV-damage mutation signature of melanoma. To investigate the behaviour of individual somatic mutations during proto-oncogene B-Raf -targeted and immune checkpoint inhibition, amplicon-based deep sequencing was used to verify and track frequencies of 212 single nucleotide variants at 10 distinct time points over 13 months of treatment. Under checkpoint inhibition therapy, we observed an increase in mutant allele frequencies indicating progression on therapy 88 days before clinical determination of non-response positron emission tomogrophy-computed tomography. We also revealed mutations from whole genome sequencing of cell-free DNA that were not present in the tissue biopsy, but that later contributed to relapse. Our findings have potential clinical applications where high quality tumour-tissue derived DNA is not available.

  8. Genome rearrangements caused by depletion of essential DNA replication proteins in Saccharomyces cerevisiae.

    Science.gov (United States)

    Cheng, Edith; Vaisica, Jessica A; Ou, Jiongwen; Baryshnikova, Anastasia; Lu, Yong; Roth, Frederick P; Brown, Grant W

    2012-09-01

    Genetic screens of the collection of ~4500 deletion mutants in Saccharomyces cerevisiae have identified the cohort of nonessential genes that promote maintenance of genome integrity. Here we probe the role of essential genes needed for genome stability. To this end, we screened 217 tetracycline-regulated promoter alleles of essential genes and identified 47 genes whose depletion results in spontaneous DNA damage. We further showed that 92 of these 217 essential genes have a role in suppressing chromosome rearrangements. We identified a core set of 15 genes involved in DNA replication that are critical in preventing both spontaneous DNA damage and genome rearrangements. Mapping, classification, and analysis of rearrangement breakpoints indicated that yeast fragile sites, Ty retrotransposons, tRNA genes, early origins of replication, and replication termination sites are common features at breakpoints when essential replication genes that suppress chromosome rearrangements are downregulated. We propose mechanisms by which depletion of essential replication proteins can lead to double-stranded DNA breaks near these features, which are subsequently repaired by homologous recombination at repeated elements.

  9. Recent advances in the genome-wide study of DNA replication origins in yeast.

    Science.gov (United States)

    Peng, Chong; Luo, Hao; Zhang, Xi; Gao, Feng

    2015-01-01

    DNA replication, one of the central events in the cell cycle, is the basis of biological inheritance. In order to be duplicated, a DNA double helix must be opened at defined sites, which are called DNA replication origins (ORIs). Unlike in bacteria, where replication initiates from a single replication origin, multiple origins are utilized in the eukaryotic genomes. Among them, the ORIs in budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe have been best characterized. In recent years, advances in DNA microarray and next-generation sequencing technologies have increased the number of yeast species involved in ORIs research dramatically. The ORIs in some non-conventional yeast species such as Kluyveromyces lactis and Pichia pastoris have also been genome-widely identified. Relevant databases of replication origins in yeast were constructed, then the comparative genomic analysis can be carried out. Here, we review several experimental approaches that have been used to map replication origins in yeast and some of the available web resources related to yeast ORIs. We also discuss the sequence characteristics and chromosome structures of ORIs in the four yeast species, which can be utilized to improve yeast replication origins prediction.

  10. Recent advances in the genome-wide study of DNA replication origins in yeast

    Directory of Open Access Journals (Sweden)

    Chong ePeng

    2015-02-01

    Full Text Available DNA replication, one of the central events in the cell cycle, is the basis of biological inheritance. In order to be duplicated, a DNA double helix must be opened at defined sites, which are called DNA replication origins (ORIs. Unlike in bacteria, where replication initiates from a single replication origin, multiple origins are utilized in the eukaryotic genome. Among them, the ORIs in budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe have been best characterized. In recent years, advances in DNA microarray and next-generation sequencing technologies have increased the number of yeast species involved in ORIs research dramatically. The ORIs in some nonconventional yeast species such as Kluyveromyces lactis and Pichia pastoris have also been genome-widely identified. Relevant databases of replication origins in yeast were constructed, then the comparative genomic analysis can be carried out. Here, we review several experimental approaches that have been used to map replication origins in yeast and some of the available web resources related to yeast ORIs. We also discuss the sequence characteristics and chromosome structures of ORIs in the four yeast species, which can be utilized to improve the replication origins prediction.

  11. DNA Length Modulates the Affinity of Fragments of Genomic DNA for the Nuclear Matrix In Vitro.

    Science.gov (United States)

    García-Vilchis, David; Aranda-Anzaldo, Armando

    2017-12-01

    Classical observations have shown that during the interphase the chromosomal DNA of metazoans is organized in supercoiled loops attached to a compartment known as the nuclear matrix (NM). Fragments of chromosomal DNA able to bind the isolated NM in vitro are known as matrix associated/attachment/addressed regions or MARs. No specific consensus sequence or motif has been found that may constitute a universal, defining feature of MARs. On the other hand, high-salt resistant DNA-NM interactions in situ define true DNA loop anchorage regions or LARs, that might correspond to a subset of the potential MARs but are not necessarily identical to MARs characterized in vitro, since there are several examples of MARs able to bind the NM in vitro but which are not actually bound to the NM in situ. In the present work we assayed the capacity of two LARs, as well as of shorter fragments within such LARs, for binding to the NM in vitro. Paradoxically the isolated (≈2 kb) LARs cannot bind to the NM in vitro while their shorter (≈300 pb) sub-fragments and other non-related but equally short DNA fragments, bind to the NM in a high-salt resistant fashion. Our results suggest that the ability of a given DNA fragment for binding to the NM in vitro primarily depends on the length of the fragment, suggesting that binding to the NM is modulated by the local topology of the DNA fragment in suspension that it is known to depend on the DNA length. J. Cell. Biochem. 118: 4487-4497, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  12. Within-genome evolution of REPINs: a new family of miniature mobile DNA in bacteria.

    Directory of Open Access Journals (Sweden)

    Frederic Bertels

    2011-06-01

    Full Text Available Repetitive sequences are a conserved feature of many bacterial genomes. While first reported almost thirty years ago, and frequently exploited for genotyping purposes, little is known about their origin, maintenance, or processes affecting the dynamics of within-genome evolution. Here, beginning with analysis of the diversity and abundance of short oligonucleotide sequences in the genome of Pseudomonas fluorescens SBW25, we show that over-represented short sequences define three distinct groups (GI, GII, and GIII of repetitive extragenic palindromic (REP sequences. Patterns of REP distribution suggest that closely linked REP sequences form a functional replicative unit: REP doublets are over-represented, randomly distributed in extragenic space, and more highly conserved than singlets. In addition, doublets are organized as inverted repeats, which together with intervening spacer sequences are predicted to form hairpin structures in ssDNA or mRNA. We refer to these newly defined entities as REPINs (REP doublets forming hairpins and identify short reads from population sequencing that reveal putative transposition intermediates. The proximal relationship between GI, GII, and GIII REPINs and specific REP-associated tyrosine transposases (RAYTs, combined with features of the putative transposition intermediate, suggests a mechanism for within-genome dissemination. Analysis of the distribution of REPs in a range of RAYT-containing bacterial genomes, including Escherichia coli K-12 and Nostoc punctiforme, show that REPINs are a widely distributed, but hitherto unrecognized, family of miniature non-autonomous mobile DNA.

  13. Organization and evolution of primate centromeric DNA from whole-genome shotgun sequence data.

    Science.gov (United States)

    Alkan, Can; Ventura, Mario; Archidiacono, Nicoletta; Rocchi, Mariano; Sahinalp, S Cenk; Eichler, Evan E

    2007-09-01

    The major DNA constituent of primate centromeres is alpha satellite DNA. As much as 2%-5% of sequence generated as part of primate genome sequencing projects consists of this material, which is fragmented or not assembled as part of published genome sequences due to its highly repetitive nature. Here, we develop computational methods to rapidly recover and categorize alpha-satellite sequences from previously uncharacterized whole-genome shotgun sequence data. We present an algorithm to computationally predict potential higher-order array structure based on paired-end sequence data and then experimentally validate its organization and distribution by experimental analyses. Using whole-genome shotgun data from the human, chimpanzee, and macaque genomes, we examine the phylogenetic relationship of these sequences and provide further support for a model for their evolution and mutation over the last 25 million years. Our results confirm fundamental differences in the dispersal and evolution of centromeric satellites in the Old World monkey and ape lineages of evolution.

  14. G-quadruplex DNA sequences are evolutionarily conserved and associated with distinct genomic features in Saccharomyces cerevisiae.

    Science.gov (United States)

    Capra, John A; Paeschke, Katrin; Singh, Mona; Zakian, Virginia A

    2010-07-22

    G-quadruplex DNA is a four-stranded DNA structure formed by non-Watson-Crick base pairing between stacked sets of four guanines. Many possible functions have been proposed for this structure, but its in vivo role in the cell is still largely unresolved. We carried out a genome-wide survey of the evolutionary conservation of regions with the potential to form G-quadruplex DNA structures (G4 DNA motifs) across seven yeast species. We found that G4 DNA motifs were significantly more conserved than expected by chance, and the nucleotide-level conservation patterns suggested that the motif conservation was the result of the formation of G4 DNA structures. We characterized the association of conserved and non-conserved G4 DNA motifs in Saccharomyces cerevisiae with more than 40 known genome features and gene classes. Our comprehensive, integrated evolutionary and functional analysis confirmed the previously observed associations of G4 DNA motifs with promoter regions and the rDNA, and it identified several previously unrecognized associations of G4 DNA motifs with genomic features, such as mitotic and meiotic double-strand break sites (DSBs). Conserved G4 DNA motifs maintained strong associations with promoters and the rDNA, but not with DSBs. We also performed the first analysis of G4 DNA motifs in the mitochondria, and surprisingly found a tenfold higher concentration of the motifs in the AT-rich yeast mitochondrial DNA than in nuclear DNA. The evolutionary conservation of the G4 DNA motif and its association with specific genome features supports the hypothesis that G4 DNA has in vivo functions that are under evolutionary constraint.

  15. G-quadruplex DNA sequences are evolutionarily conserved and associated with distinct genomic features in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    John A Capra

    2010-07-01

    Full Text Available G-quadruplex DNA is a four-stranded DNA structure formed by non-Watson-Crick base pairing between stacked sets of four guanines. Many possible functions have been proposed for this structure, but its in vivo role in the cell is still largely unresolved. We carried out a genome-wide survey of the evolutionary conservation of regions with the potential to form G-quadruplex DNA structures (G4 DNA motifs across seven yeast species. We found that G4 DNA motifs were significantly more conserved than expected by chance, and the nucleotide-level conservation patterns suggested that the motif conservation was the result of the formation of G4 DNA structures. We characterized the association of conserved and non-conserved G4 DNA motifs in Saccharomyces cerevisiae with more than 40 known genome features and gene classes. Our comprehensive, integrated evolutionary and functional analysis confirmed the previously observed associations of G4 DNA motifs with promoter regions and the rDNA, and it identified several previously unrecognized associations of G4 DNA motifs with genomic features, such as mitotic and meiotic double-strand break sites (DSBs. Conserved G4 DNA motifs maintained strong associations with promoters and the rDNA, but not with DSBs. We also performed the first analysis of G4 DNA motifs in the mitochondria, and surprisingly found a tenfold higher concentration of the motifs in the AT-rich yeast mitochondrial DNA than in nuclear DNA. The evolutionary conservation of the G4 DNA motif and its association with specific genome features supports the hypothesis that G4 DNA has in vivo functions that are under evolutionary constraint.

  16. DNA end resection by CtIP and exonuclease 1 prevents genomic instability

    DEFF Research Database (Denmark)

    Eid, Wassim; Steger, Martin; El-Shemerly, Mahmoud

    2010-01-01

    ). In this study, we show that the localization of EXO1 to DSBs depends on both CtIP and MRN. We also establish that CtIP interacts with EXO1 and restrains its exonucleolytic activity in vitro. Finally, we show that on exposure to camptothecin, depletion of EXO1 in CtIP-deficient cells increases the frequency......End resection of DNA-which is essential for the repair of DNA double-strand breaks (DSBs) by homologous recombination-relies first on the partnership between MRE11-RAD50-NBS1 (MRN) and CtIP, followed by a processive step involving helicases and exonucleases such as exonuclease 1 (EXO1...... of DNA-PK-dependent radial chromosome formation. Thus, our study identifies new functions of CtIP and EXO1 in DNA end resection and provides new information on the regulation of DSB repair pathways, which is a key factor in the maintenance of genome integrity....

  17. Complete DNA sequence of the linear mitochondrial genome of the pathogenic yeast Candida parapsilosis

    DEFF Research Database (Denmark)

    Nosek, J.; Novotna, M.; Hlavatovicova, Z.

    2004-01-01

    spacers. In silico analysis predicted genes encoding fourteen protein subunits of complexes of the respiratory chain and ATP synthase, rRNAs of the large and small subunits of the mitochondrial ribosome, and twenty-four transfer RNAs. These genes are organized into two transcription units. In addition......, six intronic ORFs coding for homologues of RNA maturase, reverse transcriptase and DNA endonucleases were identified. In contrast to its overall molecular architecture, the coding sequences of the linear mitochondrial DNA of C. parapsilosis are highly similar to their counterparts in the circular......The complete sequence of the mitochondrial DNA of the opportunistic yeast pathogen Candida parapsilosis was determined. The mitochondrial genome is represented by linear DNA molecules terminating with tandem repeats of a 738-bp unit. The number of repeats varies, thus generating a population...

  18. Time- and Cost-Efficient Identification of T-DNA Insertion Sites through Targeted Genomic Sequencing

    Science.gov (United States)

    Lepage, Étienne; Zampini, Éric; Boyle, Brian; Brisson, Normand

    2013-01-01

    Forward genetic screens enable the unbiased identification of genes involved in biological processes. In Arabidopsis, several mutant collections are publicly available, which greatly facilitates such practice. Most of these collections were generated by agrotransformation of a T-DNA at random sites in the plant genome. However, precise mapping of T-DNA insertion sites in mutants isolated from such screens is a laborious and time-consuming task. Here we report a simple, low-cost and time efficient approach to precisely map T-DNA insertions simultaneously in many different mutants. By combining sequence capture, next-generation sequencing and 2D-PCR pooling, we developed a new method that allowed the rapid localization of T-DNA insertion sites in 55 out of 64 mutant plants isolated in a screen for gyrase inhibition hypersensitivity. PMID:23951038

  19. The transcription factor TFII-I promotes DNA translesion synthesis and genomic stability.

    Directory of Open Access Journals (Sweden)

    Farjana J Fattah

    2014-06-01

    Full Text Available Translesion synthesis (TLS enables DNA replication through damaged bases, increases cellular DNA damage tolerance, and maintains genomic stability. The sliding clamp PCNA and the adaptor polymerase Rev1 coordinate polymerase switching during TLS. The polymerases Pol η, ι, and κ insert nucleotides opposite damaged bases. Pol ζ, consisting of the catalytic subunit Rev3 and the regulatory subunit Rev7, then extends DNA synthesis past the lesion. Here, we show that Rev7 binds to the transcription factor TFII-I in human cells. TFII-I is required for TLS and DNA damage tolerance. The TLS function of TFII-I appears to be independent of its role in transcription, but requires homodimerization and binding to PCNA. We propose that TFII-I bridges PCNA and Pol ζ to promote TLS. Our findings extend the general principle of component sharing among divergent nuclear processes and implicate TLS deficiency as a possible contributing factor in Williams-Beuren syndrome.

  20. A Revised Genome Assembly of the Region 5' to Canine SOX9 Includes the RevSex Orthologous Region.

    Science.gov (United States)

    Rossi, Elena; Radi, Orietta; De Lorenzi, Lisa; Iannuzzi, Alessandra; Camerino, Giovanna; Zuffardi, Orsetta; Parma, Pietro

    2015-01-01

    The SOX gene family includes many genes that play a determinant role in several developmental pathways. The SOX9 gene has been identified as a major factor in testis development in mammals after it is activated by the SRY gene. However, duplication of the gene itself in some mammalian species, or of a well-delimited upstream 'RevSex' region in humans, has been shown to result in testis development in the absence of the SRY gene. In the current study, we present an accurate analysis of the genomic organization of the SOX9 locus in dogs by both in silico and FISH approaches. Contrary to what is observed in the current dog genome assembly, we found that the genomic organization is quite similar to that reported in humans and other mammalian species, including the position of the RevSex region in respect to SOX9. The analysis of the conserved sequences within this region in 7 mammalian species facilitated the highlighting of a consensus sequence for SRY binding. This new information could help in the identification of evolutionarily conserved elements relevant for SOX9 gene regulation, and could provide valid targets for mutation analysis in XY DSD patients. © 2015 S. Karger AG, Basel.

  1. Complete genome sequence of the mitochondrial DNA of the river lamprey, Lethenteron japonicum.

    Science.gov (United States)

    Kawai, Yuri L; Yura, Kei; Shindo, Miyuki; Kusakabe, Rie; Hayashi, Keiko; Hata, Kenichiro; Nakabayashi, Kazuhiko; Okamura, Kohji

    2015-01-01

    Lampreys are eel-like jawless fishes evolutionarily positioned between invertebrates and vertebrates, and have been used as model organisms to explore vertebrate evolution. In this study we determined the complete genome sequence of the mitochondrial DNA of the Japanese river lamprey, Lethenteron japonicum, using next-generation sequencers. The sequence was 16,272 bp in length. The gene content and order were identical to those of the sea lamprey, Petromyzon marinus, which has been the reference among lamprey species. However, the sequence similarity was less than 90%, suggesting the need for the whole-genome sequencing of L. japonicum.

  2. Plasmid DNA immunization with Trypanosoma cruzi genes induces cardiac and clinical protection against Chagas disease in the canine model

    Directory of Open Access Journals (Sweden)

    Rodríguez-Morales Olivia

    2012-11-01

    Full Text Available Abstract The only existing preventive measure against American trypanosomosis, or Chagas disease, is the control of the transmitting insect, which has only been effective in a few South American regions. Currently, there is no vaccine available to prevent this disease. Here, we present the clinical and cardiac levels of protection induced by expression to Trypanosoma cruzi genes encoding the TcSP and TcSSP4 proteins in the canine model. Physical examination, diagnostic chagasic serology, and serial electrocardiograms were performed before and after immunization, as well as after experimental infection. We found that immunization with recombinant plasmids prevented hyperthermia in the acute phase of experimental infection and produced lymphadenomegaly as an immunological response against the parasite and additionally prevented heart rate elevation (tachycardia in the acute and/or chronic stages of infection. Immunization with T. cruzi genes encoding the TcSP and TcSSP4 antigens diminished the quality and quantity of the electrocardiographic abnormalities, thereby avoiding progression to more severe developments such as right bundle branch block or ventricular premature complexes in a greater number of dogs.

  3. Seventeen new complete mtDNA sequences reveal extensive mitochondrial genome evolution within the Demospongiae.

    Directory of Open Access Journals (Sweden)

    Xiujuan Wang

    Full Text Available Two major transitions in animal evolution--the origins of multicellularity and bilaterality--correlate with major changes in mitochondrial DNA (mtDNA organization. Demosponges, the largest class in the phylum Porifera, underwent only the first of these transitions and their mitochondrial genomes display a peculiar combination of ancestral and animal-specific features. To get an insight into the evolution of mitochondrial genomes within the Demospongiae, we determined 17 new mtDNA sequences from this group and analyzing them with five previously published sequences. Our analysis revealed that all demosponge mtDNAs are 16- to 25-kbp circular molecules, containing 13-15 protein genes, 2 rRNA genes, and 2-27 tRNA genes. All but four pairs of sampled genomes had unique gene orders, with the number of shared gene boundaries ranging from 1 to 41. Although most demosponge species displayed low rates of mitochondrial sequence evolution, a significant acceleration in evolutionary rates occurred in the G1 group (orders Dendroceratida, Dictyoceratida, and Verticillitida. Large variation in mtDNA organization was also observed within the G0 group (order Homosclerophorida including gene rearrangements, loss of tRNA genes, and the presence of two introns in Plakortis angulospiculatus. While introns are rare in modern-day demosponge mtDNA, we inferred that at least one intron was present in cox1 of the common ancestor of all demosponges. Our study uncovered an extensive mitochondrial genomic diversity within the Demospongiae. Although all sampled mitochondrial genomes retained some ancestral features, including a minimally modified genetic code, conserved structures of tRNA genes, and presence of multiple non-coding regions, they vary considerably in their size, gene content, gene order, and the rates of sequence evolution. Some of the changes in demosponge mtDNA, such as the loss of tRNA genes and the appearance of hairpin-containing repetitive elements

  4. DNA damage response and spindle assembly checkpoint function throughout the cell cycle to ensure genomic integrity.

    Directory of Open Access Journals (Sweden)

    Katherine S Lawrence

    2015-04-01

    Full Text Available Errors in replication or segregation lead to DNA damage, mutations, and aneuploidies. Consequently, cells monitor these events and delay progression through the cell cycle so repair precedes division. The DNA damage response (DDR, which monitors DNA integrity, and the spindle assembly checkpoint (SAC, which responds to defects in spindle attachment/tension during metaphase of mitosis and meiosis, are critical for preventing genome instability. Here we show that the DDR and SAC function together throughout the cell cycle to ensure genome integrity in C. elegans germ cells. Metaphase defects result in enrichment of SAC and DDR components to chromatin, and both SAC and DDR are required for metaphase delays. During persistent metaphase arrest following establishment of bi-oriented chromosomes, stability of the metaphase plate is compromised in the absence of DDR kinases ATR or CHK1 or SAC components, MAD1/MAD2, suggesting SAC functions in metaphase beyond its interactions with APC activator CDC20. In response to DNA damage, MAD2 and the histone variant CENPA become enriched at the nuclear periphery in a DDR-dependent manner. Further, depletion of either MAD1 or CENPA results in loss of peripherally associated damaged DNA. In contrast to a SAC-insensitive CDC20 mutant, germ cells deficient for SAC or CENPA cannot efficiently repair DNA damage, suggesting that SAC mediates DNA repair through CENPA interactions with the nuclear periphery. We also show that replication perturbations result in relocalization of MAD1/MAD2 in human cells, suggesting that the role of SAC in DNA repair is conserved.

  5. A novel sandwich hybridization method for selecting cDNAs from large genomic regions: Identification of cDNAs from the cloned genomic DNA spanning the XLRP locus

    Energy Technology Data Exchange (ETDEWEB)

    Yan, D.; McHenry, C.; Fujita, R. [Univ. of Michigan, Ann Arbor, MI (United States)] [and others

    1994-09-01

    We have developed an efficient hybridization-based cDNA-selection method. A sandwich of three species - single-stranded cDNA, tagged RNA derived from genomic DNA, and biotinylated RNA complementary to the tag - allows specific retention of hybrids on an avidin-matrix. Previously, using model experiments, we demonstrated highly specific and efficient selection of a retinal gene, NRL, from complex mixtures of cDNA clones, using a sub-library from a 5 kb NRL genomic clone. We have now applied this selection strategy to isolate cDNAs from human adult retina and fetal eye libraries, with the {open_quotes}genomic RNA{close_quotes} derived from two YAC clones (OTC-C and 55B) spanning the region of X-linked retinitis pigmentosa (XLRP) locus RP3 at Xp21.1. Effectiveness of the selection-method was monitored by enrichment of TCTEX-1L gene that maps within the 55B YAC. Of the 15 selected cDNA clones that hybridized to the 55B YAC DNA, five appear to the map to specific cosmid clones derived from the 55B YAC. Inserts in these selected cDNA clones range from 0.5 to 2.3 kb in size. Additional clones are now being isolated and characterized. This procedure should be independent of the size or complexity of genomic DNA being used for selection, allow for the isolation of full-length cDNAs, and may have wider application.

  6. DPS – A rapid method for genome sequencing of DNA-containing bacteriophages directly from a single plaque

    DEFF Research Database (Denmark)

    Kot, Witold; Vogensen, Finn Kvist; Sørensen, Søren J.

    2014-01-01

    methods for characterization of phages is determination of the whole genome using high throughput sequencing approaches. Here a direct plaque sequencing (DPS) is described, which is a rapid method that allows easy full genome sequencing of DNA-containing phages using the Nextera XT™ kit. A combination...... of host-DNA removal followed by purification and concentration of the viral DNA, allowed the construction of Illumina-compatible sequencing libraries using the Nextera™ XT technology directly from single phage plaques without any whole genome amplification step. This method was tested on three...

  7. Dissecting the Satellite DNA Landscape in Three Cactophilic Drosophila Sequenced Genomes

    Directory of Open Access Journals (Sweden)

    Leonardo G. de Lima

    2017-08-01

    Full Text Available Eukaryote genomes are replete with repetitive DNAs. This class includes tandemly repeated satellite DNAs (satDNA which are among the most abundant, fast evolving (yet poorly studied genomic components. Here, we used high-throughput sequencing data from three cactophilic Drosophila species, D. buzzatii, D. seriema, and D. mojavensis, to access and study their whole satDNA landscape. In total, the RepeatExplorer software identified five satDNAs, three previously described (pBuM, DBC-150 and CDSTR198 and two novel ones (CDSTR138 and CDSTR130. Only pBuM is shared among all three species. The satDNA repeat length falls within only two classes, between 130 and 200 bp or between 340 and 390 bp. FISH on metaphase and polytene chromosomes revealed the presence of satDNA arrays in at least one of the following genomic compartments: centromeric, telomeric, subtelomeric, or dispersed along euchromatin. The chromosomal distribution ranges from a single chromosome to almost all chromosomes of the complement. Fiber-FISH and sequence analysis of contigs revealed interspersion between pBuM and CDSTR130 in the microchromosomes of D. mojavensis. Phylogenetic analyses showed that the pBuM satDNA underwent concerted evolution at both interspecific and intraspecific levels. Based on RNA-seq data, we found transcription activity for pBuM (in D. mojavensis and CDSTR198 (in D. buzzatii in all five analyzed developmental stages, most notably in pupae and adult males. Our data revealed that cactophilic Drosophila present the lowest amount of satDNAs (1.9–2.9% within the Drosophila genus reported so far. We discuss how our findings on the satDNA location, abundance, organization, and transcription activity may be related to functional aspects.

  8. DNA repair efficiency in germ cells and early mouse embryos and consequences for radiation-induced transgenerational genomic damage

    Energy Technology Data Exchange (ETDEWEB)

    Marchetti, Francesco; Wyrobek, Andrew J.

    2009-01-18

    Exposure to ionizing radiation and other environmental agents can affect the genomic integrity of germ cells and induce adverse health effects in the progeny. Efficient DNA repair during gametogenesis and the early embryonic cycles after fertilization is critical for preventing transmission of DNA damage to the progeny and relies on maternal factors stored in the egg before fertilization. The ability of the maternal repair machinery to repair DNA damage in both parental genomes in the fertilizing egg is especially crucial for the fertilizing male genome that has not experienced a DNA repair-competent cellular environment for several weeks prior to fertilization. During the DNA repair-deficient period of spermatogenesis, DNA lesions may accumulate in sperm and be carried into the egg where, if not properly repaired, could result in the formation of heritable chromosomal aberrations or mutations and associated birth defects. Studies with female mice deficient in specific DNA repair genes have shown that: (i) cell cycle checkpoints are activated in the fertilized egg by DNA damage carried by the sperm; and (ii) the maternal genotype plays a major role in determining the efficiency of repairing genomic lesions in the fertilizing sperm and directly affect the risk for abnormal reproductive outcomes. There is also growing evidence that implicates DNA damage carried by the fertilizing gamete as a mediator of postfertilization processes that contribute to genomic instability in subsequent generations. Transgenerational genomic instability most likely involves epigenetic mechanisms or error-prone DNA repair processes in the early embryo. Maternal and embryonic DNA repair processes during the early phases of mammalian embryonic development can have far reaching consequences for the genomic integrity and health of subsequent generations.

  9. History of plastid DNA insertions reveals weak deletion and at mutation biases in angiosperm mitochondrial genomes.

    Science.gov (United States)

    Sloan, Daniel B; Wu, Zhiqiang

    2014-11-21

    Angiosperm mitochondrial genomes exhibit many unusual properties, including heterogeneous nucleotide composition and exceptionally large and variable genome sizes. Determining the role of nonadaptive mechanisms such as mutation bias in shaping the molecular evolution of these unique genomes has proven challenging because their dynamic structures generally prevent identification of homologous intergenic sequences for comparative analyses. Here, we report an analysis of angiosperm mitochondrial DNA sequences that are derived from inserted plastid DNA (mtpts). The availability of numerous completely sequenced plastid genomes allows us to infer the evolutionary history of these insertions, including the specific nucleotide substitutions and indels that have occurred because their incorporation into the mitochondrial genome. Our analysis confirmed that many mtpts have a complex history, including frequent gene conversion and multiple examples of horizontal transfer between divergent angiosperm lineages. Nevertheless, it is clear that the majority of extant mtpt sequence in angiosperms is the product of recent transfer (or gene conversion) and is subject to rapid loss/deterioration, suggesting that most mtpts are evolving relatively free from functional constraint. The evolution of mtpt sequences reveals a pattern of biased mutational input in angiosperm mitochondrial genomes, including an excess of small deletions over insertions and a skew toward nucleotide substitutions that increase AT content. However, these mutation biases are far weaker than have been observed in many other cellular genomes, providing insight into some of the notable features of angiosperm mitochondrial architecture, including the retention of large intergenic regions and the relatively neutral GC content found in these regions. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  10. Systematic comparison of three genomic enrichment methods for massively parallel DNA sequencing.

    Science.gov (United States)

    Teer, Jamie K; Bonnycastle, Lori L; Chines, Peter S; Hansen, Nancy F; Aoyama, Natsuyo; Swift, Amy J; Abaan, Hatice Ozel; Albert, Thomas J; Margulies, Elliott H; Green, Eric D; Collins, Francis S; Mullikin, James C; Biesecker, Leslie G

    2010-10-01

    Massively parallel DNA sequencing technologies have greatly increased our ability to generate large amounts of sequencing data at a rapid pace. Several methods have been developed to enrich for genomic regions of interest for targeted sequencing. We have compared three of these methods: Molecular Inversion Probes (MIP), Solution Hybrid Selection (SHS), and Microarray-based Genomic Selection (MGS). Using HapMap DNA samples, we compared each of these methods with respect to their ability to capture an identical set of exons and evolutionarily conserved regions associated with 528 genes (2.61 Mb). For sequence analysis, we developed and used a novel Bayesian genotype-assigning algorithm, Most Probable Genotype (MPG). All three capture methods were effective, but sensitivities (percentage of targeted bases associated with high-quality genotypes) varied for an equivalent amount of pass-filtered sequence: for example, 70% (MIP), 84% (SHS), and 91% (MGS) for 400 Mb. In contrast, all methods yielded similar accuracies of >99.84% when compared to Infinium 1M SNP BeadChip-derived genotypes and >99.998% when compared to 30-fold coverage whole-genome shotgun sequencing data. We also observed a low false-positive rate with all three methods; of the heterozygous positions identified by each of the capture methods, >99.57% agreed with 1M SNP BeadChip, and >98.840% agreed with the whole-genome shotgun data. In addition, we successfully piloted the genomic enrichment of a set of 12 pooled samples via the MGS method using molecular bar codes. We find that these three genomic enrichment methods are highly accurate and practical, with sensitivities comparable to that of 30-fold coverage whole-genome shotgun data.

  11. Protocol: a simple method for extracting next-generation sequencing quality genomic DNA from recalcitrant plant species.

    Science.gov (United States)

    Healey, Adam; Furtado, Agnelo; Cooper, Tal; Henry, Robert J

    2014-01-01

    Next-generation sequencing technologies rely on high quality DNA that is suitable for library preparation followed by sequencing. Some plant species store large amounts of phenolics and polysaccharides within their leaf tissue making genomic DNA extraction difficult. While many DNA extraction methods exist that contend with the presence of phenolics and polysaccharides, these methods rely on long incubations, multiple precipitations or commercially available kits to produce high molecular weight and contaminant-free DNA. In this protocol, we describe simple modifications to the established CTAB- based extraction method that allows for reliable isolation of high molecular weight genomic DNA from difficult to isolate plant species Corymbia (a eucalypt) and Coffea (coffee). The simplified protocol does not require multiple clean up steps or commercial based kits, and the isolated DNA passed stringent quality control standards for whole genome sequencing on Illumina HiSeq and TruSeq sequencing platforms.

  12. GHK and DNA: Resetting the Human Genome to Health

    Directory of Open Access Journals (Sweden)

    Loren Pickart

    2014-01-01

    Full Text Available During human aging there is an increase in the activity of inflammatory, cancer promoting, and tissue destructive genes plus a decrease in the activity of regenerative and reparative genes. The human blood tripeptide GHK possesses many positive effects but declines with age. It improves wound healing and tissue regeneration (skin, hair follicles, stomach and intestinal linings, and boney tissue, increases collagen and glycosaminoglycans, stimulates synthesis of decorin, increases angiogenesis, and nerve outgrowth, possesses antioxidant and anti-inflammatory effects, and increases cellular stemness and the secretion of trophic factors by mesenchymal stem cells. Recently, GHK has been found to reset genes of diseased cells from patients with cancer or COPD to a more healthy state. Cancer cells reset their programmed cell death system while COPD patients’ cells shut down tissue destructive genes and stimulate repair and remodeling activities. In this paper, we discuss GHK’s effect on genes that suppress fibrinogen synthesis, the insulin/insulin-like system, and cancer growth plus activation of genes that increase the ubiquitin-proteasome system, DNA repair, antioxidant systems, and healing by the TGF beta superfamily. A variety of methods and dosages to effectively use GHK to reset genes to a healthier state are also discussed.

  13. PHOTOPROBER® Biotin: An Alternative Method for Labeling Archival DNA for Comparative Genomic Hybridization

    Directory of Open Access Journals (Sweden)

    Dirk Korinth

    2004-01-01

    Full Text Available Comparative genomic hybridization (CGH represents a powerful method for screening the entire genome of solid tumors for chromosomal imbalances. Particularly it enabled the molecular cytogenetic analysis of archival, formalin‐fixed, paraffin‐embedded (FFPE tissue. A well‐known dilemma, however, is the poor DNA quality of this material with fragment sizes below 1000 bp. Nick translation, the conventionally used enzymatic DNA labeling method in CGH, leads to even shorter fragments often below a critical limit for successful analysis. In this study we report the alternative application of non‐enzymatic, PHOTOPROBE® biotin labeling for conjugation of the hapten to the DNA prior to in situ hybridization and fluorescence detection. We analyzed 51 FFPE tumor samples mainly from the upper respiratory tract by both labeling methods. In 19 cases, both approaches were successful. The comparison of hybridized metaphases showed a distinct higher fluorescence signal of the PHOTOPROBE® samples sometimes with a discrete cytoplasm background which however did not interfere with specificity and sensitivity of the detected chromosomal imbalances. For further 32 cases characterized by an average DNA fragment size below 1000 bp, PHOTOPROBE® biotin was the only successful labeling technique thus offering a new option for CGH analysis of highly degraded DNA from archival material.

  14. Universal internucleotide statistics in full genomes: a footprint of the DNA structure and packaging?

    Science.gov (United States)

    Bogachev, Mikhail I; Kayumov, Airat R; Bunde, Armin

    2014-01-01

    Uncovering the fundamental laws that govern the complex DNA structural organization remains challenging and is largely based upon reconstructions from the primary nucleotide sequences. Here we investigate the distributions of the internucleotide intervals and their persistence properties in complete genomes of various organisms from Archaea and Bacteria to H. Sapiens aiming to reveal the manifestation of the universal DNA architecture. We find that in all considered organisms the internucleotide interval distributions exhibit the same [Formula: see text]-exponential form. While in prokaryotes a single [Formula: see text]-exponential function makes the best fit, in eukaryotes the PDF contains additionally a second [Formula: see text]-exponential, which in the human genome makes a perfect approximation over nearly 10 decades. We suggest that this functional form is a footprint of the heterogeneous DNA structure, where the first [Formula: see text]-exponential reflects the universal helical pitch that appears both in pro- and eukaryotic DNA, while the second [Formula: see text]-exponential is a specific marker of the large-scale eukaryotic DNA organization.

  15. Universal internucleotide statistics in full genomes: a footprint of the DNA structure and packaging?

    Directory of Open Access Journals (Sweden)

    Mikhail I Bogachev

    Full Text Available Uncovering the fundamental laws that govern the complex DNA structural organization remains challenging and is largely based upon reconstructions from the primary nucleotide sequences. Here we investigate the distributions of the internucleotide intervals and their persistence properties in complete genomes of various organisms from Archaea and Bacteria to H. Sapiens aiming to reveal the manifestation of the universal DNA architecture. We find that in all considered organisms the internucleotide interval distributions exhibit the same [Formula: see text]-exponential form. While in prokaryotes a single [Formula: see text]-exponential function makes the best fit, in eukaryotes the PDF contains additionally a second [Formula: see text]-exponential, which in the human genome makes a perfect approximation over nearly 10 decades. We suggest that this functional form is a footprint of the heterogeneous DNA structure, where the first [Formula: see text]-exponential reflects the universal helical pitch that appears both in pro- and eukaryotic DNA, while the second [Formula: see text]-exponential is a specific marker of the large-scale eukaryotic DNA organization.

  16. Capturing Genomic Evolution of Lung Cancers through Liquid Biopsy for Circulating Tumor DNA

    Directory of Open Access Journals (Sweden)

    Michael Offin

    2017-01-01

    Full Text Available Genetic sequencing of malignancies has become increasingly important to uncover therapeutic targets and capture the tumor’s dynamic changes to drug sensitivity and resistance through genomic evolution. In lung cancers, the current standard of tissue biopsy at the time of diagnosis and progression is not always feasible or practical and may underestimate intratumoral heterogeneity. Technological advances in genetic sequencing have enabled the use of circulating tumor DNA (ctDNA analysis to obtain information on both targetable mutations and capturing real-time Darwinian evolution of tumor clones and drug resistance mechanisms under selective therapeutic pressure. The ability to analyze ctDNA from plasma, CSF, or urine enables a comprehensive view of cancers as systemic diseases and captures intratumoral heterogeneity. Here, we describe these recent advances in the setting of lung cancers and advocate for further research and the incorporation of ctDNA analysis in clinical trials of targeted therapies. By capturing genomic evolution in a noninvasive manner, liquid biopsy for ctDNA analysis could accelerate therapeutic discovery and deliver the next leap forward in precision medicine for patients with lung cancers and other solid tumors.

  17. Isolation of genomic DNA using magnetic nanoparticles as a solid-phase support

    Energy Technology Data Exchange (ETDEWEB)

    Saiyed, Z M; Ramchand, C N [Kemin Nutritional Technologies (India) Pvt Ltd, Trapezium, Nelson Manickam Road, Chennai 600 029 (India); Telang, S D [Department of Biochemistry, Faculty of Science, M S University of Baroda, Sayajigunj, Vadodara 390 002 (India)], E-mail: cnramchand@yahoo.com, E-mail: ramchand.cn@kemin.com

    2008-05-21

    In recent years, techniques employing magnetizable solid-phase supports (MSPS) have found application in numerous biological fields. This magnetic separation procedure offers several advantages in terms of subjecting the analyte to very little mechanical stress compared to other methods. Secondly, these methods are non-laborious, cheap, and often highly scalable. The current paper details a genomic DNA isolation method optimized in our laboratory using magnetic nanoparticles as a solid-phase support. The quality and yields of the isolated DNA from all the samples using magnetic nanoparticles were higher or equivalent to the traditional DNA extraction procedures. Additionally, the magnetic method takes less than 15 min to extract polymerase chain reaction (PCR) ready genomic DNA as against several hours taken by traditional phenol-chloroform extraction protocols. Moreover, the isolated DNA was found to be compatible in PCR amplification and restriction endonuclease digestion. The developed procedure is quick, inexpensive, robust, and it does not require the use of organic solvents or sophisticated instruments, which makes it more amenable to automation and miniaturization.

  18. Isolation of genomic DNA using magnetic nanoparticles as a solid-phase support

    Science.gov (United States)

    Saiyed, Z. M.; Ramchand, C. N.; Telang, S. D.

    2008-05-01

    In recent years, techniques employing magnetizable solid-phase supports (MSPS) have found application in numerous biological fields. This magnetic separation procedure offers several advantages in terms of subjecting the analyte to very little mechanical stress compared to other methods. Secondly, these methods are non-laborious, cheap, and often highly scalable. The current paper details a genomic DNA isolation method optimized in our laboratory using magnetic nanoparticles as a solid-phase support. The quality and yields of the isolated DNA from all the samples using magnetic nanoparticles were higher or equivalent to the traditional DNA extraction procedures. Additionally, the magnetic method takes less than 15 min to extract polymerase chain reaction (PCR) ready genomic DNA as against several hours taken by traditional phenol-chloroform extraction protocols. Moreover, the isolated DNA was found to be compatible in PCR amplification and restriction endonuclease digestion. The developed procedure is quick, inexpensive, robust, and it does not require the use of organic solvents or sophisticated instruments, which makes it more amenable to automation and miniaturization.

  19. Origins of DNA Replication and Amplification in the Breast Cancer Genome

    Science.gov (United States)

    2013-09-01

    and Li J.J. (2005). Loss of rereplication control in Saccharomyces cerevisiae results in extensive DNA damage. Mol. Biol. Cell 16: 421-432...Green B.M., Morreale R.J., Ozaydin B., Derisi J.L. and Li J.J. (2006). Genome-wide mapping of DNA synthesis in Saccharomyces cerevisiae reveals that...Neu) gene, which encodes human epidermal growth factor 2, occurs in ~25% of invasive breast cancers and in 50-60% of ductal carcinoma in situ. HER2

  20. Effect of the Plasmid-DNA Vaccination on Macroscopic and Microscopic Damage Caused by the Experimental Chronic Trypanosoma cruzi Infection in the Canine Model

    Directory of Open Access Journals (Sweden)

    Olivia Rodríguez-Morales

    2013-01-01

    Full Text Available The dog is considered the main domestic reservoir for Trypanosoma cruzi infection and a suitable experimental animal model to study the pathological changes during the course of Chagas disease (CD. Vaccine development is one of CD prevention methods to protect people at risk. Two plasmids containing genes encoding a trans-sialidase protein (TcSP and an amastigote-specific glycoprotein (TcSSP4 were used as DNA vaccines in a canine model. Splenomegaly was not found in either of the recombinant plasmid-immunized groups; however, cardiomegaly was absent in animals immunized only with the plasmid containing the TcSSP4 gene. The inflammation of subendocardial and myocardial tissues was prevented only with the immunization with TcSSP4 gene. In conclusion, the vaccination with these genes has a partial protective effect on the enlargement of splenic and cardiac tissues during the chronic CD and on microscopic hearth damage, since both plasmids prevented splenomegaly but only one avoided cardiomegaly, and the lesions in heart tissue of dog immunized with plasmid containing the TcSSP4 gene covered only subepicardial tissue.

  1. Regulation of mitochondrial genome replication by hypoxia: The role of DNA oxidation in D-loop region.

    Science.gov (United States)

    Pastukh, Viktor M; Gorodnya, Olena M; Gillespie, Mark N; Ruchko, Mykhaylo V

    2016-07-01

    Mitochondria of mammalian cells contain multiple copies of mitochondrial (mt) DNA. Although mtDNA copy number can fluctuate dramatically depending on physiological and pathophysiologic conditions, the mechanisms regulating mitochondrial genome replication remain obscure. Hypoxia, like many other physiologic stimuli that promote growth, cell proliferation and mitochondrial biogenesis, uses reactive oxygen species as signaling molecules. Emerging evidence suggests that hypoxia-induced transcription of nuclear genes requires controlled DNA damage and repair in specific sequences in the promoter regions. Whether similar mechanisms are operative in mitochondria is unknown. Here we test the hypothesis that controlled oxidative DNA damage and repair in the D-loop region of the mitochondrial genome are required for mitochondrial DNA replication and transcription in hypoxia. We found that hypoxia had little impact on expression of mitochondrial proteins in pulmonary artery endothelial cells, but elevated mtDNA content. The increase in mtDNA copy number was accompanied by oxidative modifications in the D-loop region of the mitochondrial genome. To investigate the role of this sequence-specific oxidation of mitochondrial genome in mtDNA replication, we overexpressed mitochondria-targeted 8-oxoguanine glycosylase Ogg1 in rat pulmonary artery endothelial cells, enhancing the mtDNA repair capacity of transfected cells. Overexpression of Ogg1 resulted in suppression of hypoxia-induced mtDNA oxidation in the D-loop region and attenuation of hypoxia-induced mtDNA replication. Ogg1 overexpression also reduced binding of mitochondrial transcription factor A (TFAM) to both regulatory and coding regions of the mitochondrial genome without altering total abundance of TFAM in either control or hypoxic cells. These observations suggest that oxidative DNA modifications in the D-loop region during hypoxia are important for increased TFAM binding and ensuing replication of the mitochondrial

  2. Improved reproducibility in genome-wide DNA methylation analysis for PAXgene-fixed samples compared with restored formalin-fixed and paraffin-embedded DNA.

    Science.gov (United States)

    Andersen, Gitte Brinch; Hager, Henrik; Hansen, Lise Lotte; Tost, Jörg

    2015-01-01

    Formalin fixation has been the standard method for conservation of clinical specimens for decades. However, a major drawback is the high degradation of nucleic acids, which complicates its use in genome-wide analyses. Unbiased identification of biomarkers, however, requires genome-wide studies, precluding the use of the v