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  1. Integrated analysis of DNA copy number and gene expression microarray data using gene sets

    NARCIS (Netherlands)

    R.X. de Menezes (Renee); M. Boetzer (Marten); M. Sieswerda (Melle); G.J.B. van Ommen; J.M. Boer (Judith)

    2009-01-01

    textabstractBackground: Genes that play an important role in tumorigenesis are expected to show association between DNA copy number and RNA expression. Optimal power to find such associations can only be achieved if analysing copy number and gene expression jointly. Furthermore, some copy number

  2. TALE nickase mediates high efficient targeted transgene integration at the human multi-copy ribosomal DNA locus.

    Science.gov (United States)

    Wu, Yong; Gao, Tieli; Wang, Xiaolin; Hu, Youjin; Hu, Xuyun; Hu, Zhiqing; Pang, Jialun; Li, Zhuo; Xue, Jinfeng; Feng, Mai; Wu, Lingqian; Liang, Desheng

    2014-03-28

    Although targeted gene addition could be stimulated strikingly by a DNA double strand break (DSB) created by either zinc finger nucleases (ZFNs) or TALE nucleases (TALENs), the DSBs are really mutagenic and toxic to human cells. As a compromised solution, DNA single-strand break (SSB) or nick has been reported to mediate high efficient gene addition but with marked reduction of random mutagenesis. We previously demonstrated effective targeted gene addition at the human multicopy ribosomal DNA (rDNA) locus, a genomic safe harbor for the transgene with therapeutic potential. To improve the transgene integration efficiency by using TALENs while lowering the cytotoxicity of DSBs, we created both TALENs and TALE nickases (TALENickases) targeting this multicopy locus. A targeting vector which could integrate a GFP cassette at the rDNA locus was constructed and co-transfected with TALENs or TALENickases. Although the fraction of GFP positive cells using TALENs was greater than that using TALENickases during the first few days after transfection, it reduced to a level less than that using TALENickases after continuous culture. Our findings showed that the TALENickases were more effective than their TALEN counterparts at the multi-copy rDNA locus, though earlier studies using ZFNs and ZFNickases targeting the single-copy loci showed the reverse. Besides, TALENickases mediated the targeted integration of a 5.4 kb fragment at a frequency of up to 0.62% in HT1080 cells after drug selection, suggesting their potential application in targeted gene modification not being limited at the rDNA locus.

  3. Cancer gene prioritization by integrative analysis of mRNA expression and DNA copy number data: a comparative review

    CERN Document Server

    Lahti, Leo; Klein, Hans-Ulrich; Bicciato, Silvio; Dugas, Martin

    2011-01-01

    A variety of genome-wide profiling techniques are available to probe complementary aspects of genome structure and function. Integrative analysis of heterogeneous data sources can reveal higher-level interactions that cannot be detected based on individual observations. A standard integration task in cancer studies is to identify altered genomic regions that induce changes in the expression of the associated genes based on joint analysis of genome-wide gene expression and copy number profiling measurements. In this review, we provide a comparison among various modeling procedures for integrating genome-wide profiling data of gene copy number and transcriptional alterations and highlight common approaches to genomic data integration. A transparent benchmarking procedure is introduced to quantitatively compare the cancer gene prioritization performance of the alternative methods. The benchmarking algorithms and data sets are available at http://intcomp.r-forge.r-project.org

  4. Hacking DNA copy number for circuit engineering.

    Science.gov (United States)

    Wu, Feilun; You, Lingchong

    2017-07-27

    DNA copy number represents an essential parameter in the dynamics of synthetic gene circuits but typically is not explicitly considered. A new study demonstrates how dynamic control of DNA copy number can serve as an effective strategy to program robust oscillations in gene expression circuits.

  5. Discovery of common Asian copy number variants using integrated high-resolution array CGH and massively parallel DNA sequencing.

    Science.gov (United States)

    Park, Hansoo; Kim, Jong-Il; Ju, Young Seok; Gokcumen, Omer; Mills, Ryan E; Kim, Sheehyun; Lee, Seungbok; Suh, Dongwhan; Hong, Dongwan; Kang, Hyunseok Peter; Yoo, Yun Joo; Shin, Jong-Yeon; Kim, Hyun-Jin; Yavartanoo, Maryam; Chang, Young Wha; Ha, Jung-Sook; Chong, Wilson; Hwang, Ga-Ram; Darvishi, Katayoon; Kim, Hyeran; Yang, Song Ju; Yang, Kap-Seok; Kim, Hyungtae; Hurles, Matthew E; Scherer, Stephen W; Carter, Nigel P; Tyler-Smith, Chris; Lee, Charles; Seo, Jeong-Sun

    2010-05-01

    Copy number variants (CNVs) account for the majority of human genomic diversity in terms of base coverage. Here, we have developed and applied a new method to combine high-resolution array comparative genomic hybridization (CGH) data with whole-genome DNA sequencing data to obtain a comprehensive catalog of common CNVs in Asian individuals. The genomes of 30 individuals from three Asian populations (Korean, Chinese and Japanese) were interrogated with an ultra-high-resolution array CGH platform containing 24 million probes. Whole-genome sequencing data from a reference genome (NA10851, with 28.3x coverage) and two Asian genomes (AK1, with 27.8x coverage and AK2, with 32.0x coverage) were used to transform the relative copy number information obtained from array CGH experiments into absolute copy number values. We discovered 5,177 CNVs, of which 3,547 were putative Asian-specific CNVs. These common CNVs in Asian populations will be a useful resource for subsequent genetic studies in these populations, and the new method of calling absolute CNVs will be essential for applying CNV data to personalized medicine.

  6. Stable high-copy-number integration of Aspergillus oryzae alpha-AMYLASE cDNA in an industrial baker's yeast strain.

    Science.gov (United States)

    Nieto, A; Prieto, J A; Sanz, P

    1999-01-01

    The Aspergillus oryzae alpha-amylase cDNA was placed under the control of the Saccharomyces cerevisiae actin promoter (pACT1) and introduced into the ribosomal DNA locus of an industrial baker's yeast strain. To obtain a strain eligible for commercial use, we constructed an integrative cassette lacking bacterial DNA sequences but containing the alpha-amylase cDNA and ribosomal DNA sequences to target the integration to this locus. High-copy-number integrants were obtained including a defective TRP1d promoter in the integrative cassette. We selected one transformant, Rib-AMY (CECT10872), in which the multi-integrated sequences were stable even after 200 generations of growth in nonselective medium. This transformant also expressed and secreted high levels of alpha-amylase. Bread made with this strain had a higher volume, lower density, and softer crumbs than bread made with a control strain. The Rib-AMY transformant also was useful in retarding bread firming. This new strain fulfills all the requirements for commercial utilization and should reduce or eliminate the requirement for addition of exogenous alpha-amylase to the flour, reducing allergenic work-related symptoms due to this enzyme.

  7. DNA replication stress restricts ribosomal DNA copy number.

    Science.gov (United States)

    Salim, Devika; Bradford, William D; Freeland, Amy; Cady, Gillian; Wang, Jianmin; Pruitt, Steven C; Gerton, Jennifer L

    2017-09-15

    Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen the yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a "normal" rDNA copy number.

  8. Integrative analysis of DNA copy number and gene expression in metastatic oral squamous cell carcinoma identifies genes associated with poor survival.

    Science.gov (United States)

    Xu, Chang; Liu, Yan; Wang, Pei; Fan, Wenhong; Rue, Tessa C; Upton, Melissa P; Houck, John R; Lohavanichbutr, Pawadee; Doody, David R; Futran, Neal D; Zhao, Lue Ping; Schwartz, Stephen M; Chen, Chu; Méndez, Eduardo

    2010-06-11

    Lymphotropism in oral squamous cell carcinoma (OSCC) is one of the most important prognostic factors of 5-year survival. In an effort to identify genes that may be responsible for the initiation of OSCC lymphotropism, we examined DNA copy number gains and losses and corresponding gene expression changes from tumor cells in metastatic lymph nodes of patients with OSCC. We performed integrative analysis of DNA copy number alterations (CNA) and corresponding mRNA expression from OSCC cells isolated from metastatic lymph nodes of 20 patients using Affymetrix 250 K Nsp I SNP and U133 Plus 2.0 arrays, respectively. Overall, genome CNA accounted for expression changes in 31% of the transcripts studied. Genome region 11q13.2-11q13.3 shows the highest correlation between DNA CNA and expression. With a false discovery rate expression. Among these, we found two subsets that were significantly associated with OSCC (n = 122) when compared to controls, and with survival (n = 27), as tested using an independent dataset with genome-wide expression profiles for 148 primary OSCC and 45 normal oral mucosa. We fit Cox models to calculate a principal component analysis-derived risk-score for these two gene sets ('122-' or '27-transcript PC'). The models combining the 122- or 27-transcript PC with stage outperformed the model using stage alone in terms of the Area Under the Curve (AUC = 0.82 or 0.86 vs. 0.72, with p = 0.044 or 0.011, respectively). Genes exhibiting CNA-correlated expression may have biological impact on carcinogenesis and cancer progression in OSCC. Determination of copy number-associated transcripts associated with clinical outcomes in tumor cells with an aggressive phenotype (i.e., cells metastasized to the lymph nodes) can help prioritize candidate transcripts from high-throughput data for further studies.

  9. Interactive Data Integration through Smart Copy & Paste

    CERN Document Server

    Ives, Zachary; Minton, Steve; Jacob, Marie; Talukdar, Partha; Tuchinda, Rattapoom; Ambite, Jose Luis; Muslea, Maria; Gazen, Cenk

    2009-01-01

    In many scenarios, such as emergency response or ad hoc collaboration, it is critical to reduce the overhead in integrating data. Ideally, one could perform the entire process interactively under one unified interface: defining extractors and wrappers for sources, creating a mediated schema, and adding schema mappings ? while seeing how these impact the integrated view of the data, and refining the design accordingly. We propose a novel smart copy and paste (SCP) model and architecture for seamlessly combining the design-time and run-time aspects of data integration, and we describe an initial prototype, the CopyCat system. In CopyCat, the user does not need special tools for the different stages of integration: instead, the system watches as the user copies data from applications (including the Web browser) and pastes them into CopyCat?s spreadsheet-like workspace. CopyCat generalizes these actions and presents proposed auto-completions, each with an explanation in the form of provenance. The user provides f...

  10. Ribosomal DNA copy number loss and sequence variation in cancer.

    Science.gov (United States)

    Xu, Baoshan; Li, Hua; Perry, John M; Singh, Vijay Pratap; Unruh, Jay; Yu, Zulin; Zakari, Musinu; McDowell, William; Li, Linheng; Gerton, Jennifer L

    2017-06-01

    Ribosomal DNA is one of the most variable regions in the human genome with respect to copy number. Despite the importance of rDNA for cellular function, we know virtually nothing about what governs its copy number, stability, and sequence in the mammalian genome due to challenges associated with mapping and analysis. We applied computational and droplet digital PCR approaches to measure rDNA copy number in normal and cancer states in human and mouse genomes. We find that copy number and sequence can change in cancer genomes. Counterintuitively, human cancer genomes show a loss of copies, accompanied by global copy number co-variation. The sequence can also be more variable in the cancer genome. Cancer genomes with lower copies have mutational evidence of mTOR hyperactivity. The PTEN phosphatase is a tumor suppressor that is critical for genome stability and a negative regulator of the mTOR kinase pathway. Surprisingly, but consistent with the human cancer genomes, hematopoietic cancer stem cells from a Pten-/- mouse model for leukemia have lower rDNA copy number than normal tissue, despite increased proliferation, rRNA production, and protein synthesis. Loss of copies occurs early and is associated with hypersensitivity to DNA damage. Therefore, copy loss is a recurrent feature in cancers associated with mTOR activation. Ribosomal DNA copy number may be a simple and useful indicator of whether a cancer will be sensitive to DNA damaging treatments.

  11. Mitochondrial DNA copy number in peripheral blood and melanoma risk.

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    Jie Shen

    Full Text Available Mitochondrial DNA (mtDNA copy number in peripheral blood has been suggested as risk modifier in various types of cancer. However, its influence on melanoma risk is unclear. We evaluated the association between mtDNA copy number in peripheral blood and melanoma risk in 500 melanoma cases and 500 healthy controls from an ongoing melanoma study. The mtDNA copy number was measured using real-time polymerase chain reaction. Overall, mean mtDNA copy number was significantly higher in cases than in controls (1.15 vs 0.99, P<0.001. Increased mtDNA copy number was associated with a 1.45-fold increased risk of melanoma (95% confidence interval: 1.12-1.97. Significant joint effects between mtDNA copy number and variables related to pigmentation and history of sunlight exposure were observed. This study supports an association between increased mtDNA copy number and melanoma risk that is independent on the known melanoma risk factors (pigmentation and history of sunlight exposure.

  12. Mitochondrial DNA Copy Number in Sleep Duration Discordant Monozygotic Twins

    DEFF Research Database (Denmark)

    Wrede, Joanna E; Mengel-From, Jonas; Buchwald, Dedra

    2015-01-01

    STUDY OBJECTIVES: Mitochondrial DNA (mtDNA) copy number is an important component of mitochondrial function and varies with age, disease, and environmental factors. We aimed to determine whether mtDNA copy number varies with habitual differences in sleep duration within pairs of monozygotic twins....... SETTING: Academic clinical research center. PARTICIPANTS: 15 sleep duration discordant monozygotic twin pairs (30 twins, 80% female; mean age 42.1 years [SD 15.0]). DESIGN: Sleep duration was phenotyped with wrist actigraphy. Each twin pair included a "normal" (7-9 h/24) and "short" (sleeping...... twin. Fasting peripheral blood leukocyte DNA was assessed for mtDNA copy number via the n-fold difference between qPCR measured mtDNA and nuclear DNA creating an mtDNA measure without absolute units. We used generalized estimating equation linear regression models accounting for the correlated data...

  13. Number matters: control of mammalian mitochondrial DNA copy number.

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    Clay Montier, Laura L; Deng, Janice J; Bai, Yidong

    2009-03-01

    Regulation of mitochondrial biogenesis is essential for proper cellular functioning. Mitochondrial DNA (mtDNA) depletion and the resulting mitochondrial malfunction have been implicated in cancer, neurodegeneration, diabetes, aging, and many other human diseases. Although it is known that the dynamics of the mammalian mitochondrial genome are not linked with that of the nuclear genome, very little is known about the mechanism of mtDNA propagation. Nevertheless, our understanding of the mode of mtDNA replication has advanced in recent years, though not without some controversies. This review summarizes our current knowledge of mtDNA copy number control in mammalian cells, while focusing on both mtDNA replication and turnover. Although mtDNA copy number is seemingly in excess, we reason that mtDNA copy number control is an important aspect of mitochondrial genetics and biogenesis and is essential for normal cellular function.

  14. Getting DNA copy numbers without control samples

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    Ortiz-Estevez Maria

    2012-08-01

    Full Text Available Abstract Background The selection of the reference to scale the data in a copy number analysis has paramount importance to achieve accurate estimates. Usually this reference is generated using control samples included in the study. However, these control samples are not always available and in these cases, an artificial reference must be created. A proper generation of this signal is crucial in terms of both noise and bias. We propose NSA (Normality Search Algorithm, a scaling method that works with and without control samples. It is based on the assumption that genomic regions enriched in SNPs with identical copy numbers in both alleles are likely to be normal. These normal regions are predicted for each sample individually and used to calculate the final reference signal. NSA can be applied to any CN data regardless the microarray technology and preprocessing method. It also finds an optimal weighting of the samples minimizing possible batch effects. Results Five human datasets (a subset of HapMap samples, Glioblastoma Multiforme (GBM, Ovarian, Prostate and Lung Cancer experiments have been analyzed. It is shown that using only tumoral samples, NSA is able to remove the bias in the copy number estimation, to reduce the noise and therefore, to increase the ability to detect copy number aberrations (CNAs. These improvements allow NSA to also detect recurrent aberrations more accurately than other state of the art methods. Conclusions NSA provides a robust and accurate reference for scaling probe signals data to CN values without the need of control samples. It minimizes the problems of bias, noise and batch effects in the estimation of CNs. Therefore, NSA scaling approach helps to better detect recurrent CNAs than current methods. The automatic selection of references makes it useful to perform bulk analysis of many GEO or ArrayExpress experiments without the need of developing a parser to find the normal samples or possible batches within the

  15. An integrated Bayesian analysis of LOH and copy number data

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    Hutter Marcus

    2010-06-01

    Full Text Available Abstract Background Cancer and other disorders are due to genomic lesions. SNP-microarrays are able to measure simultaneously both genotype and copy number (CN at several Single Nucleotide Polymorphisms (SNPs along the genome. CN is defined as the number of DNA copies, and the normal is two, since we have two copies of each chromosome. The genotype of a SNP is the status given by the nucleotides (alleles which are present on the two copies of DNA. It is defined homozygous or heterozygous if the two alleles are the same or if they differ, respectively. Loss of heterozygosity (LOH is the loss of the heterozygous status due to genomic events. Combining CN and LOH data, it is possible to better identify different types of genomic aberrations. For example, a long sequence of homozygous SNPs might be caused by either the physical loss of one copy or a uniparental disomy event (UPD, i.e. each SNP has two identical nucleotides both derived from only one parent. In this situation, the knowledge of the CN can help in distinguishing between these two events. Results To better identify genomic aberrations, we propose a method (called gBPCR which infers the type of aberration occurred, taking into account all the possible influence in the microarray detection of the homozygosity status of the SNPs, resulting from an altered CN level. Namely, we model the distributions of the detected genotype, given a specific genomic alteration and we estimate the parameters involved on public reference datasets. The estimation is performed similarly to the modified Bayesian Piecewise Constant Regression, but with improved estimators for the detection of the breakpoints. Using artificial and real data, we evaluate the quality of the estimation of gBPCR and we also show that it outperforms other well-known methods for LOH estimation. Conclusions We propose a method (gBPCR for the estimation of both LOH and CN aberrations, improving their estimation by integrating both types

  16. DNA Copy Number Signature to Predict Recurrence in Early-Stage Ovarian Cancer

    Science.gov (United States)

    2015-08-01

    and clinical data inventory at MGH, 4) Sequenced RNA from these tumor samples. 2 Preliminary RNAseq analysis has indicated the need of analyzing...CNV on 300 samples, so that integration analysis with RNAseq can initiate. Plans for the next reporting period to accomplish the goals: Finish...analysis of DNA CNV on 300 samples and integrated analysis of the copy number variation result and the RNAseq results obtained from a paralleled DOD

  17. Genome-wide copy number analysis using copy number inferring tool (CNIT) and DNA pooling.

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    Lin, Chien-hsing; Huang, Mei-chu; Li, Ling-hui; Wu, Jer-yuarn; Chen, Yuan-tsong; Fann, Cathy S J

    2008-08-01

    Copy number variation (CNV) has become an important genomic structure element in the human population, and some CNVs are related to specific traits and diseases. Moreover, analysis of human genomes has been potentiated by the use of high-resolution microarrays that assess single nucleotide polymorphisms (SNPs). Although many programs have been designed to analyze data from Affymetrix SNP microarrays, they all have high false-positive rates (FPRs) in copy number (CN) analyses. Copy number analysis tool (CNAT) 4.0 is a recently developed program that offers improved CN estimation, but small amplifications and deletions are lost when using the smoothing procedure. Here, we propose a copy number inferring tool (CNIT) algorithm for the 100K SNP microarray to investigate CNVs at 29.6-kb resolution. CNIT estimated SNP allelic and total CN with reliable P values based on intensity data. In addition, the hidden Markov model (HMM) method was applied to predict regions having altered CN by considering contiguous SNPs. Based on a CN analysis of 23 unrelated Taiwanese and 30 HapMap Centre d'Etude du Polymorphisme Humain (CEPH) trios, CNIT showed higher accuracy and power than other programs. The FPRs and false-negative rates (FNRs) of CNIT were 0.1% and 0.16%, respectively. CNIT also showed better sensitivity for detecting small amplifications and deletions. Furthermore, DNA pooling of 10 and 30 normal unrelated individuals were applied to the 100K SNP microarray, respectively, and 12 common CN-variable regions were identified, suggesting that DNA pooling can be applied to discover common CNVs.

  18. Magellan: A Web Based System for the Integrated Analysis of Heterogeneous Biological Data and Annotations; Application to DNA Copy Number and Expression Data in Ovarian Cancer

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    Chris B. Kingsley

    2006-01-01

    Full Text Available Recent advances in high throughput biological methods allow researchers to generate enormous amounts of data from a single experiment. In order to extract meaningful conclusions from this tidal wave of data, it will be necessary to develop analytical methods of sufficient power and utility. It is particularly important that biologists themselves be able to perform many of these analyses, such that their background knowledge of the experimental system under study can be used to interpret results and direct further inquiries. We have developed a web-based system, Magellan, which allows the upload, storage, and analysis of multivariate data and textual or numerical annotations. Data and annotations are treated as abstract entities, to maximize the different types of information the system can store and analyze. Annotations can be used in analyses/visualizations, as a means of subsetting data to reduce dimensionality, or as a means of projecting variables from one data type or data set to another. Analytical methods are deployed within Magellan such that new functionalities can be added in a straightforward fashion. Using Magellan, we performed an integrated analysis of genome-wide comparative genomic hybridization (CGH, mRNA expression, and clinical data from ovarian tumors. Analyses included the use of permutation-based methods to identify genes whose mRNA expression levels correlated with patient survival, a nearest neighbor classifier to predict patient survival from CGH data, and curated annotations such as genomic position and derived annotations such as statistical computations to explore the quantitative relationship between CGH and mRNA expression data.

  19. Magellan: a web based system for the integrated analysis of heterogeneous biological data and annotations; application to DNA copy number and expression data in ovarian cancer.

    Science.gov (United States)

    Kingsley, Chris B; Kuo, Wen-Lin; Polikoff, Daniel; Berchuck, Andy; Gray, Joe W; Jain, Ajay N

    2007-02-05

    Recent advances in high throughput biological methods allow researchers to generate enormous amounts of data from a single experiment. In order to extract meaningful conclusions from this tidal wave of data, it will be necessary to develop analytical methods of sufficient power and utility. It is particularly important that biologists themselves be able to perform many of these analyses, such that their background knowledge of the experimental system under study can be used to interpret results and direct further inquiries. We have developed a web-based system, Magellan, which allows the upload, storage, and analysis of multivariate data and textual or numerical annotations. Data and annotations are treated as abstract entities, to maximize the different types of information the system can store and analyze. Annotations can be used in analyses/visualizations, as a means of subsetting data to reduce dimensionality, or as a means of projecting variables from one data type or data set to another. Analytical methods are deployed within Magellan such that new functionalities can be added in a straightforward fashion. Using Magellan, we performed an integrated analysis of genome-wide comparative genomic hybridization (CGH), mRNA expression, and clinical data from ovarian tumors. Analyses included the use of permutation-based methods to identify genes whose mRNA expression levels correlated with patient survival, a nearest neighbor classifier to predict patient survival from CGH data, and curated annotations such as genomic position and derived annotations such as statistical computations to explore the quantitative relationship between CGH and mRNA expression data.

  20. Reduction in mitochondrial DNA copy number in peripheral leukocytes after onset of Huntington's disease

    DEFF Research Database (Denmark)

    Petersen, Maria Hvidberg; Budtz-Jørgensen, Esben; Sørensen, Sven Asger;

    2014-01-01

    to the investigation of the mitochondrial DNA (mtDNA) copy number relative to nuclear DNA (nDNA) in leukocytes from carriers of the HD mutation compared to healthy individuals. We found significantly reduced mtDNA/nDNA in HD mutation carriers compared to controls. A longitudinal study of archive DNA sample pairs from...

  1. Analysis of T-DNA/Host-Plant DNA Junction Sequences in Single-Copy Transgenic Barley Lines

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    Joanne G. Bartlett

    2014-01-01

    Full Text Available Sequencing across the junction between an integrated transfer DNA (T-DNA and a host plant genome provides two important pieces of information. The junctions themselves provide information regarding the proportion of T-DNA which has integrated into the host plant genome, whilst the transgene flanking sequences can be used to study the local genetic environment of the integrated transgene. In addition, this information is important in the safety assessment of GM crops and essential for GM traceability. In this study, a detailed analysis was carried out on the right-border T-DNA junction sequences of single-copy independent transgenic barley lines. T-DNA truncations at the right-border were found to be relatively common and affected 33.3% of the lines. In addition, 14.3% of lines had rearranged construct sequence after the right border break-point. An in depth analysis of the host-plant flanking sequences revealed that a significant proportion of the T-DNAs integrated into or close to known repetitive elements. However, this integration into repetitive DNA did not have a negative effect on transgene expression.

  2. Simple binary segmentation frameworks for identifying variation in DNA copy number

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    Yang Tae Young

    2012-10-01

    Full Text Available Abstract Background Variation in DNA copy number, due to gains and losses of chromosome segments, is common. A first step for analyzing DNA copy number data is to identify amplified or deleted regions in individuals. To locate such regions, we propose a circular binary segmentation procedure, which is based on a sequence of nested hypothesis tests, each using the Bayesian information criterion. Results Our procedure is convenient for analyzing DNA copy number in two general situations: (1 when using data from multiple sources and (2 when using cohort analysis of multiple patients suffering from the same type of cancer. In the first case, data from multiple sources such as different platforms, labs, or preprocessing methods are used to study variation in copy number in the same individual. Combining these sources provides a higher resolution, which leads to a more detailed genome-wide survey of the individual. In this case, we provide a simple statistical framework to derive a consensus molecular signature. In the framework, the multiple sequences from various sources are integrated into a single sequence, and then the proposed segmentation procedure is applied to this sequence to detect aberrant regions. In the second case, cohort analysis of multiple patients is carried out to derive overall molecular signatures for the cohort. For this case, we provide another simple statistical framework in which data across multiple profiles is standardized before segmentation. The proposed segmentation procedure is then applied to the standardized profiles one at a time to detect aberrant regions. Any such regions that are common across two or more profiles are probably real and may play important roles in the cancer pathogenesis process. Conclusions The main advantages of the proposed procedure are flexibility and simplicity.

  3. Mitochondrial DNA copy number in whole blood and glioma risk: A case control study.

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    Shen, Jie; Song, Renduo; Lu, Zhimin; Zhao, Hua

    2016-12-01

    Alterations in mitochondrial DNA (mtDNA) copy number are observed in human gliomas. However, whether variations in mtDNA copy number in whole blood play any role in glioma carcinogenesis is still largely unknown. In current study with 395 glioma patients and 425 healthy controls, we intended to investigate the association between mtDNA copy number in whole blood and glioma risk. Overall, we found that levels of mtDNA copy number were significantly higher in glioma cases than healthy controls (mean: 1.48 vs. 1.32, P copy number were inversely correlated with age (P copy number than their counterparts (P = 0.02, P copy number levels were associated with a 1.63-fold increased risk of glioma (adjusted odds ratio (OR) = 1.63, 95% confidence interval (CI) = 1.23-2.14). In further quartile analysis, study subjects who had highest levels of mtNDA copy number had 1.75-fold increased risk of gliomas (adjOR = 1.75, 95%CI = 1.18-2.61). In brief, our findings support the role of mtDNA copy number in the glioma carcinogenesis. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  4. High-Resolution Analysis of Gene Copy Number Alterations in Human Prostate Cancer Using CGH on cDNA Microarrays: Impact of Copy Number on Gene Expression

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    Maija Wolf

    2004-05-01

    Full Text Available Identification of target genes for genetic rearrangements in prostate cancer and the impact of copy number changes on gene expression are currently not well understood. Here, we applied high-resolution comparative genomic hybridization (CGH on cDNA microarrays for analysis of prostate cancer cell lines. CGH microarrays identified most of the alterations detected by classical chromosomal CGH, as well as a number of previously unreported alterations. Specific recurrent regions of gain (28 and loss (18 were found, their boundaries defined with sub-megabasepair accuracy. The most common changes included copy number decreases at 13% and gains at iq and 5p. Refined mapping identified several sites, such as at 13q (33-44, 49-51, 74-76 Mbp from the p-telomere, which matched with minimal regions of loss seen in extensive loss of heterozygosity mapping studies of large numbers of tumors. Previously unreported recurrent changes were found at 2p, 2q, 3p, 17q (losses, at 3q, 5p, 6p (gains. Integration of genomic and transcriptomic data revealed the role of individual candidate target genes for genomic alterations as well as a highly significant (P < .0001 overall association between copy number levels and the percentage of differentially expressed genes. Across the genome, the overall impact of copy number on gene expression levels was, to a large extent, attributable to low-level gains and losses of copy number, corresponding to common deletions and gains of often large chromosomal regions.

  5. Peripheral blood mitochondrial DNA copy number is associated with prostate cancer risk and tumor burden.

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    Weimin Zhou

    Full Text Available Alterations of mitochondrial DNA (mtDNA have been associated with the risk of a number of human cancers; however, the relationship between mtDNA copy number in peripheral blood leukocytes (PBLs and the risk of prostate cancer (PCa has not been investigated. In a case-control study of 196 PCa patients and 196 age-paired healthy controls in a Chinese Han population, the association between mtDNA copy number in PBLs and PCa risk was evaluated. The relative mtDNA copy number was measured using quantitative real-time PCR; samples from three cases and two controls could not be assayed, leaving 193 cases and 194 controls for analysis. PCa patients had significantly higher mtDNA copy numbers than controls (medians 0.91 and 0.82, respectively; P<0.001. Dichotomized at the median value of mtDNA copy number in the controls, high mtDNA copy number was significantly associated with an increased risk of PCa (adjusted odds ratio= 1.85, 95% confidence interval: 1.21-2.83. A significant dose-response relationship was observed between mtDNA copy number and risk of PCa in quartile analysis (Ptrend = 0.011. Clinicopathological analysis showed that high mtDNA copy numbers in PCa patients were significantly associated with high Gleason score and advanced tumor stage, but not serum prostate-specific antigen level (P = 0.002, 0.012 and 0.544, respectively. These findings of the present study indicate that increased mtDNA copy number in PBLs is significantly associated with an increased risk of PCa and may be a reflection of tumor burden.

  6. Modulation of Mitochondrial DNA Copy Number to Induce Hepatocytic Differentiation of Human Amniotic Epithelial Cells.

    Science.gov (United States)

    Vaghjiani, Vijesh; Cain, Jason E; Lee, William; Vaithilingam, Vijayaganapathy; Tuch, Bernard E; St John, Justin C

    2017-09-05

    Mitochondrial deoxyribonucleic acid (mtDNA) copy number is tightly regulated during pluripotency and differentiation. There is increased demand of cellular adenosine triphosphate (ATP) during differentiation for energy-intensive cell types such as hepatocytes and neurons to meet the cell's functional requirements. During hepatocyte differentiation, mtDNA copy number should be synchronously increased to generate sufficient ATP through oxidative phosphorylation. Unlike bone marrow mesenchymal cells, mtDNA copy number failed to increase by 28 days of differentiation of human amniotic epithelial cells (hAEC) into hepatocyte-like cells (HLC) despite their expression of some end-stage hepatic markers. This was due to higher levels of DNA methylation at exon 2 of POLGA, the mtDNA-specific replication factor. Treatment with a DNA demethylation agent, 5-azacytidine, resulted in increased mtDNA copy number, reduced DNA methylation at exon 2 of POLGA, and reduced hepatic gene expression. Depletion of mtDNA followed by subsequent differentiation did not increase mtDNA copy number, but reduced DNA methylation at exon 2 of POLGA and increased expression of hepatic and pluripotency genes. We encapsulated hAEC in barium alginate microcapsules and subsequently differentiated them into HLC. Encapsulation resulted in no net increase of mtDNA copy number but a significant reduction in DNA methylation of POLGA. RNAseq analysis showed that differentiated HLC express hepatocyte-specific genes but also increased expression of inflammatory interferon genes. Differentiation in encapsulated cells showed suppression of inflammatory genes as well as increased expression of genes associated with hepatocyte function pathways and networks. This study demonstrates that an increase in classical hepatic gene expression can be achieved in HLC through encapsulation, although they fail to effectively regulate mtDNA copy number.

  7. Focal DNA copy number changes in neuroblastoma target MYCN regulated genes.

    Directory of Open Access Journals (Sweden)

    Candy Kumps

    Full Text Available Neuroblastoma is an embryonic tumor arising from immature sympathetic nervous system cells. Recurrent genomic alterations include MYCN and ALK amplification as well as recurrent patterns of gains and losses of whole or large partial chromosome segments. A recent whole genome sequencing effort yielded no frequently recurring mutations in genes other than those affecting ALK. However, the study further stresses the importance of DNA copy number alterations in this disease, in particular for genes implicated in neuritogenesis. Here we provide additional evidence for the importance of focal DNA copy number gains and losses, which are predominantly observed in MYCN amplified tumors. A focal 5 kb gain encompassing the MYCN regulated miR-17~92 cluster as sole gene was detected in a neuroblastoma cell line and further analyses of the array CGH data set demonstrated enrichment for other MYCN target genes in focal gains and amplifications. Next we applied an integrated genomics analysis to prioritize MYCN down regulated genes mediated by MYCN driven miRNAs within regions of focal heterozygous or homozygous deletion. We identified RGS5, a negative regulator of G-protein signaling implicated in vascular normalization, invasion and metastasis, targeted by a focal homozygous deletion, as a new MYCN target gene, down regulated through MYCN activated miRNAs. In addition, we expand the miR-17~92 regulatory network controlling TGFß signaling in neuroblastoma with the ring finger protein 11 encoding gene RNF11, which was previously shown to be targeted by the miR-17~92 member miR-19b. Taken together, our data indicate that focal DNA copy number imbalances in neuroblastoma (1 target genes that are implicated in MYCN signaling, possibly selected to reinforce MYCN oncogene addiction and (2 serve as a resource for identifying new molecular targets for treatment.

  8. Telomere length is correlated with mitochondrial DNA copy number in intestinal, but not diffuse, gastric cancer.

    Science.gov (United States)

    Jung, Soo-Jung; Cho, Ji-Hyoung; Park, Won-Jin; Heo, Yu-Ran; Lee, Jae-Ho

    2017-07-01

    A positive correlation between telomere length and mitochondrial DNA (mtDNA) copy number has previously been observed in healthy individuals, and in patients with psychiatric disorders. In the present study, telomere length and mtDNA copy number were evaluated in gastric cancer (GC) tissue samples. DNA was extracted from 109 GC samples (including 82 intestinal, and 27 diffuse cases), and the telomere length and mtDNA copy number were analyzed using a quantitative-polymerase chain reaction assay. The relative telomere length and mtDNA copy number in tumor tissue, as compared with in normal tissue, (mean ± standard deviation) in all GC samples were 11.48±1.14 and 14.86±1.35, respectively. Telomere length and mtDNA copy number were not identified as exhibiting clinical or prognostic value for GC. However, positive correlations between telomere length and mitochondrial DNA copy number were identified in GC (r=0.408, P<0.001) and in the adjacent normal mucosa (r=0.363; P<0.001). When stratified by Lauren classification, the correlation was identified in intestinal type GC samples (r=0.461; P<0.001), but not in diffuse type GC samples (r=0.225; P=0.260). This result indicated that loss of the correlation of telomeres and mitochondrial function may induce the initiation or progression of GC pathogenesis.

  9. Mitochondrial DNA copy number variation as a potential predictor of renal cell carcinoma.

    Science.gov (United States)

    Elsayed, Eman T; Hashad, Mohamed M; Elgohary, Iman E

    2017-07-24

    Peripheral blood mitochondrial DNA (mtDNA) copy number alteration has been suggested as a risk factor for several types of cancer. The aim of the present study was to assess the role of peripheral blood mtDNA copy number variation as a noninvasive biomarker in the prediction and early detection of renal cell carcinoma (RCC) in a cohort of Egyptian patients. Quantitative real-time polymerase chain reaction (qPCR) was used to measure peripheral blood mtDNA copy numbers in 57 patients with newly diagnosed, early-stage localized RCC and 60 age- and sex-matched healthy individuals as a control group. Median mtDNA copy number was significantly higher in RCC cases than in controls (166 vs. 91, pcopy number was associated with an 18-fold increased risk of RCC (95% confidence interval: 5.065-63.9). On receiver operating characteristic curve analysis, it was found that mtDNA could distinguish between RCC patients and healthy controls, with 86% sensitivity, 80% specificity, 80.3% positive predictive value and 85.7% negative predictive value at a cutoff value of 108.5. Our results showed that increased peripheral blood mtDNA copy number was associated with increased risk of RCC. Therefore, RCC might be considered as part of a range of potential tumors in cases with elevated blood mtDNA copy number.

  10. Reduced mtDNA copy number increases the sensitivity of tumor cells to chemotherapeutic drugs.

    Science.gov (United States)

    Mei, H; Sun, S; Bai, Y; Chen, Y; Chai, R; Li, H

    2015-04-02

    Many cancer drugs are toxic to cells by activating apoptotic pathways. Previous studies have shown that mitochondria have key roles in apoptosis in mammalian cells, but the role of mitochondrial DNA (mtDNA) copy number variation in the pathogenesis of tumor cell apoptosis remains largely unknown. We used the HEp-2, HNE2, and A549 tumor cell lines to explore the relationship between mtDNA copy number variation and cell apoptosis. We first induced apoptosis in three tumor cell lines and one normal adult human skin fibroblast cell line (HSF) with cisplatin (DDP) or doxorubicin (DOX) treatment and found that the mtDNA copy number significantly increased in apoptotic tumor cells, but not in HSF cells. We then downregulated the mtDNA copy number by transfection with shRNA-TFAM plasmids or treatment with ethidium bromide and found that the sensitivity of tumor cells to DDP or DOX was significantly increased. Furthermore, we observed that levels of reactive oxygen species (ROS) increased significantly in tumor cells with lower mtDNA copy numbers, and this might be related to a low level of antioxidant gene expression. Finally, we rescued the increase of ROS in tumor cells with lipoic acid or N-acetyl-L-cysteine and found that the apoptosis rate decreased. Our studies suggest that the increase of mtDNA copy number is a self-protective mechanism of tumor cells to prevent apoptosis and that reduced mtDNA copy number increases ROS levels in tumor cells, increases the tumor cells' sensitivity to chemotherapeutic drugs, and increases the rate of apoptosis. This research provides evidence that mtDNA copy number variation might be a promising new therapeutic target for the clinical treatment of tumors.

  11. Reduced mtDNA copy number increases the sensitivity of tumor cells to chemotherapeutic drugs

    Science.gov (United States)

    Mei, H; Sun, S; Bai, Y; Chen, Y; Chai, R; Li, H

    2015-01-01

    Many cancer drugs are toxic to cells by activating apoptotic pathways. Previous studies have shown that mitochondria have key roles in apoptosis in mammalian cells, but the role of mitochondrial DNA (mtDNA) copy number variation in the pathogenesis of tumor cell apoptosis remains largely unknown. We used the HEp-2, HNE2, and A549 tumor cell lines to explore the relationship between mtDNA copy number variation and cell apoptosis. We first induced apoptosis in three tumor cell lines and one normal adult human skin fibroblast cell line (HSF) with cisplatin (DDP) or doxorubicin (DOX) treatment and found that the mtDNA copy number significantly increased in apoptotic tumor cells, but not in HSF cells. We then downregulated the mtDNA copy number by transfection with shRNA-TFAM plasmids or treatment with ethidium bromide and found that the sensitivity of tumor cells to DDP or DOX was significantly increased. Furthermore, we observed that levels of reactive oxygen species (ROS) increased significantly in tumor cells with lower mtDNA copy numbers, and this might be related to a low level of antioxidant gene expression. Finally, we rescued the increase of ROS in tumor cells with lipoic acid or N-acetyl-L-cysteine and found that the apoptosis rate decreased. Our studies suggest that the increase of mtDNA copy number is a self-protective mechanism of tumor cells to prevent apoptosis and that reduced mtDNA copy number increases ROS levels in tumor cells, increases the tumor cells' sensitivity to chemotherapeutic drugs, and increases the rate of apoptosis. This research provides evidence that mtDNA copy number variation might be a promising new therapeutic target for the clinical treatment of tumors. PMID:25837486

  12. Construction of plasmids with tunable copy numbers in Saccharomyces cerevisiae and their applications in pathway optimization and multiplex genome integration.

    Science.gov (United States)

    Lian, Jiazhang; Jin, Run; Zhao, Huimin

    2016-11-01

    The CEN/ARS-based low-copy plasmids and 2 μ-based high-copy plasmids have been broadly used for both fundamental studies and practical applications in Saccharomyces cerevisiae. However, the relative low copy numbers and narrow dynamic range limit their applications in many cases. In this study, the expression level of the selection marker proteins was engineered to increase the plasmid copy numbers. A series of plasmids with step-wise increased copy numbers were constructed. The copy number of the plasmids with engineered dominant markers (5-100 copies per cell) showed a positive correlation with the concentration of antibiotics supplemented to the growth media. Based on this finding, we developed a simple yet highly efficient strategy, named Pathway Optimization by Tuning Antibiotic Concentrations (POTAC) to rapidly balance the flux of multi-gene pathways at the DNA level in S. cerevisiae. As proof of concept, POTAC was used to optimize the lycopene and n-butanol biosynthetic pathways, increasing the production of lycopene and n-butanol by 10- and 100-fold, respectively. Additionally, multiplex genome integration with controllable copy numbers was attempted by combining the engineered dominant markers with the CRISPR/Cas9 system. Biotechnol. Bioeng. 2016;113: 2462-2473. © 2016 Wiley Periodicals, Inc.

  13. Presequence-Independent Mitochondrial Import of DNA Ligase Facilitates Establishment of Cell Lines with Reduced mtDNA Copy Number.

    Directory of Open Access Journals (Sweden)

    Domenico Spadafora

    Full Text Available Due to the essential role played by mitochondrial DNA (mtDNA in cellular physiology and bioenergetics, methods for establishing cell lines with altered mtDNA content are of considerable interest. Here, we report evidence for the existence in mammalian cells of a novel, low- efficiency, presequence-independent pathway for mitochondrial protein import, which facilitates mitochondrial uptake of such proteins as Chlorella virus ligase (ChVlig and Escherichia coli LigA. Mouse cells engineered to depend on this pathway for mitochondrial import of the LigA protein for mtDNA maintenance had severely (up to >90% reduced mtDNA content. These observations were used to establish a method for the generation of mouse cell lines with reduced mtDNA copy number by, first, transducing them with a retrovirus encoding LigA, and then inactivating in these transductants endogenous Lig3 with CRISPR-Cas9. Interestingly, mtDNA depletion to an average level of one copy per cell proceeds faster in cells engineered to maintain mtDNA at low copy number. This makes a low-mtDNA copy number phenotype resulting from dependence on mitochondrial import of DNA ligase through presequence-independent pathway potentially useful for rapidly shifting mtDNA heteroplasmy through partial mtDNA depletion.

  14. Presequence-Independent Mitochondrial Import of DNA Ligase Facilitates Establishment of Cell Lines with Reduced mtDNA Copy Number.

    Science.gov (United States)

    Spadafora, Domenico; Kozhukhar, Natalia; Alexeyev, Mikhail F

    2016-01-01

    Due to the essential role played by mitochondrial DNA (mtDNA) in cellular physiology and bioenergetics, methods for establishing cell lines with altered mtDNA content are of considerable interest. Here, we report evidence for the existence in mammalian cells of a novel, low- efficiency, presequence-independent pathway for mitochondrial protein import, which facilitates mitochondrial uptake of such proteins as Chlorella virus ligase (ChVlig) and Escherichia coli LigA. Mouse cells engineered to depend on this pathway for mitochondrial import of the LigA protein for mtDNA maintenance had severely (up to >90%) reduced mtDNA content. These observations were used to establish a method for the generation of mouse cell lines with reduced mtDNA copy number by, first, transducing them with a retrovirus encoding LigA, and then inactivating in these transductants endogenous Lig3 with CRISPR-Cas9. Interestingly, mtDNA depletion to an average level of one copy per cell proceeds faster in cells engineered to maintain mtDNA at low copy number. This makes a low-mtDNA copy number phenotype resulting from dependence on mitochondrial import of DNA ligase through presequence-independent pathway potentially useful for rapidly shifting mtDNA heteroplasmy through partial mtDNA depletion.

  15. Characterization of an inducible promoter in different DNA copy number conditions.

    Science.gov (United States)

    Zucca, Susanna; Pasotti, Lorenzo; Mazzini, Giuliano; De Angelis, Maria Gabriella Cusella; Magni, Paolo

    2012-03-28

    The bottom-up programming of living organisms to implement novel user-defined biological capabilities is one of the main goals of synthetic biology. Currently, a predominant problem connected with the construction of even simple synthetic biological systems is the unpredictability of the genetic circuitry when assembled and incorporated in living cells. Copy number, transcriptional/translational demand and toxicity of the DNA-encoded functions are some of the major factors which may lead to cell overburdening and thus to nonlinear effects on system output. It is important to disclose the linearity working boundaries of engineered biological systems when dealing with such phenomena. The output of an N-3-oxohexanoyl-L-homoserine lactone (HSL)-inducible RFP-expressing device was studied in Escherichia coli in different copy number contexts, ranging from 1 copy per cell (integrated in the genome) to hundreds (via multicopy plasmids). The system is composed by a luxR constitutive expression cassette and a RFP gene regulated by the luxI promoter, which is activated by the HSL-LuxR complex. System output, in terms of promoter activity as a function of HSL concentration, was assessed relative to the one of a reference promoter in identical conditions by using the Relative Promoter Units (RPU) approach. Nonlinear effects were observed in the maximum activity, which is identical in single and low copy conditions, while it decreases for higher copy number conditions. In order to properly compare the luxI promoter strength among all the conditions, a mathematical modeling approach was used to relate the promoter activity to the estimated HSL-LuxR complex concentration, which is the actual activator of transcription. During model fitting, a correlation between the copy number and the dissociation constant of HSL-LuxR complex and luxI promoter was observed. Even in a simple inducible system, nonlinear effects are observed and non-trivial data processing is necessary to fully

  16. Change and Significance of Mitochondrial DNA Copy Number in Esophageal Squamous Cell Carcinoma

    Institute of Scientific and Technical Information of China (English)

    Zongwen Liu; Zhihua Zhao; Qiumin Zhao; Shenglei Li; Dongling Gao; Xia Pang; Kuisheng Chen; Yunhan Zhang

    2007-01-01

    OBJECTIVE To compare the differences of mitochondrial DNA (mtDNA)copies among the tissues of esophageal squamous cell carcinoma (ESCC),para-neoplastic tissue and normal mucous membrane of the esophagus,and to study the relationship between the mtDNA and the occurrence and development of esophageal squamous cell carcinoma.METHODS The mtDNA copies of 42 specimens with the ESCC,paraneoplastic mucous tissue and normal mucous membrane of the esophagus were determined using real-time fluorescence quantitative PCR.The mtDNA was analyzed using agarose gel electrophoresis.RESULTS The mtDNA from all of the tissues (42/42) from the ESCC,para-neoplastic tissue and normal esophageal mucous membranes was analyzed.showing thal there were an average mtDNA copy number of 27.1894x106 μg DNA.9.4102x106 μg DNA and 5.9347x106 μg DNA,from the respective tissues.There were significant differences (F=27.83,P<0.05) in mtDNA copy number among the three.A positive band was shown at 403 bp after qel electrophoresis of the PCR products.and the lane where the ESCC mtDNA located was rather bright.which was in accordance with the result of the real-time PCR determination.CONCLUSION An increase in the mtDNA copy number is related to the occurrence and development of ESCC.

  17. A prospective study of mitochondrial DNA copy number and the risk of prostate cancer.

    Science.gov (United States)

    Moore, Amy; Lan, Qing; Hofmann, Jonathan N; Liu, Chin-San; Cheng, Wen-Ling; Lin, Ta-Tsung; Berndt, Sonja I

    2017-06-01

    Evidence suggests that mitochondrial DNA (mtDNA) copy number increases in response to DNA damage. Increased mtDNA copy number has been observed in prostate cancer (PCa) cells, suggesting a role in PCa development, but this association has not yet been investigated prospectively. We conducted a nested case-control study (793 cases and 790 controls) of men randomized to the screening arm of the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO) to evaluate the association between pre-diagnosis mtDNA copy number, measured in peripheral blood leukocytes, and the risk of PCa. We used logistic regression to estimate odds ratios (OR) and 95% confidence intervals (CI) and polytomous logistic regression to analyze differences in associations by non-aggressive (Stage I/II AND Gleason grade copy number was not significantly associated with PCa risk overall (OR 1.23, 95% CI 0.97-1.55, p = 0.089), increasing mtDNA copy number was associated with an increased risk of non-aggressive PCa (OR 1.29, 95% CI 1.01-1.65, p = 0.044) compared to controls. No association was observed with aggressive PCa (OR 1.02, 95% CI 0.64-1.63, p = 0.933). Higher mtDNA copy number was also associated with increased PSA levels among controls (p = 0.014). These results suggest that alterations in mtDNA copy number may reflect disruption of the normal prostate glandular architecture seen in early-stage disease, as opposed to reflecting the large number of tumor cells seen with advanced PCa.

  18. The relationship between mitochondrial DNA copy number and stallion sperm function.

    Science.gov (United States)

    Darr, Christa R; Moraes, Luis E; Connon, Richard E; Love, Charles C; Teague, Sheila; Varner, Dickson D; Meyers, Stuart A

    2017-05-01

    Mitochondrial DNA (mtDNA) copy number has been utilized as a measure of sperm quality in several species including mice, dogs, and humans, and has been suggested as a potential biomarker of fertility in stallion sperm. The results of the present study extend this recent discovery using sperm samples from American Quarter Horse stallions of varying age. By determining copy number of three mitochondrial genes, cytochrome b (CYTB), NADH dehydrogenase 1 (ND1) and NADH dehydrogenase 4 (ND4), instead of a single gene, we demonstrate an improved understanding of mtDNA fate in stallion sperm mitochondria following spermatogenesis. Sperm samples from 37 stallions ranging from 3 to 24 years old were collected at four breeding ranches in north and central Texas during the 2015 breeding season. Samples were analyzed for sperm motion characteristics, nuclear DNA denaturability and mtDNA copy number. Mitochondrial DNA content in individual sperm was determined by real-time qPCR and normalized with a single copy nuclear gene, Beta actin. Exploratory correlation analysis revealed that total motility was negatively correlated with CYTB copy number and sperm chromatin structure. Stallion age did not have a significant effect on copy number for any of the genes. Copy number differences existed between the three genes with CYTB having the greatest number of copies (20.6 ± 1.2 copies, range: 6.0 to 41.1) followed by ND4 (15.5 ± 0.8 copies, range: 6.7 to 27.8) and finally ND1 (12.0 ± 1.0 copies, range: 0.4 to 26.6) (P copy number across mitochondrial genes is likely to be a result of mtDNA fragmentation and degradation since downregulation of sperm mtDNA occurs during spermatogenesis and may be important for normal sperm function. Beta regression analysis suggested that for every unit increase in mtDNA copy number of CYTB, there was a 4% decrease in the odds of sperm movement (P = 0.001). Influential analysis suggested that results are robust and not highly influenced by

  19. High copy number of mitochondrial DNA predicts poor prognosis in patients with advanced stage colon cancer.

    Science.gov (United States)

    Wang, Yun; He, Shuixiang; Zhu, Xingmei; Qiao, Wei; Zhang, Juan

    2016-12-23

    The aim of this investigation was to determine whether alterations in mitochondrial DNA (mtDNA) copy number in colon cancer were associated with clinicopathological parameters and postsurgical outcome. By quantitative real-time PCR assay, the mtDNA copy number was detected in a cohort of colon cancer and matched adjacent colon tissues (n = 162). The majority of patients had higher mtDNA content in colon cancer tissues than matched adjacent colon tissues. Moreover, high mtDNA content in tumor tissues was associated with larger tumor size, higher serum CEA level, advanced TNM stage, vascular emboli, and liver metastases. Further survival curve analysis showed that high mtDNA content was related to the worst survival in patients with colon cancer at advanced TNM stage. High mtDNA content is a potential effective factor of poor prognosis in patients with advanced stage colon cancer.

  20. Mitochondrial DNA Copy Number in Peripheral Blood Cells and Risk of Developing Breast Cancer.

    Science.gov (United States)

    Lemnrau, Alina; Brook, Mark N; Fletcher, Olivia; Coulson, Penny; Tomczyk, Katarzyna; Jones, Michael; Ashworth, Alan; Swerdlow, Anthony; Orr, Nick; Garcia-Closas, Montserrat

    2015-07-15

    Increased mitochondrial DNA (mtDNA) copy number in peripheral blood cells (PBC) has been associated with the risk of developing several tumor types. Here we evaluate sources of variation of this biomarker and its association with breast cancer risk in a prospective cohort study. mtDNA copy number was measured using quantitative real-time PCR on PBC DNA samples from participants in the UK-based Breakthrough Generations Study. Temporal and assay variation was evaluated in a serial study of 91 women, with two blood samples collected approximately 6-years apart. Then, associations with breast cancer risk factors and risk were evaluated in 1,108 cases and 1,099 controls using a nested case-control design. In the serial study, mtDNA copy number showed low assay variation but large temporal variation [assay intraclass correlation coefficient (ICC), 79.3%-87.9%; temporal ICC, 38.3%). Higher mtDNA copy number was significantly associated with younger age at blood collection, being premenopausal, having an older age at menopause, and never taking HRT, both in cases and controls. Based on measurements in a single blood sample taken on average 6 years before diagnosis, higher mtDNA copy number was associated with increased breast cancer risk [OR (95% CI) for highest versus lowest quartile, 1.37 (1.02-1.83); P trend = 0.007]. In conclusion, mtDNA copy number is associated with breast cancer risk and represents a promising biomarker for risk assessment. The relatively large temporal variation should be taken into account in future analyses.

  1. Loss of the Association between Telomere Length and Mitochondrial DNA Copy Number Contribute to Colorectal Carcinogenesis.

    Science.gov (United States)

    Lee, Hyunsu; Cho, Ji-Hyoung; Park, Won-Jin; Jung, Soo-Jung; Choi, In-Jang; Lee, Jae-Ho

    2017-05-09

    Positive association between telomere length and mitochondrial DNA (mtDNA) copy number were introduced in healthy and patients with psychiatric disorder. Based on frequent genetic changes of telomere and mitochondria in colorectal carcinomas (CRC), we studied their clinical characteristics and their association in colorectal carcinogenesis. DNA was extracted from 109 CRCs, 64 colorectal tubular adenomas (TAs), and 28 serrated polyps (SPs), and then, telomere length and mtDNA copy number were analyzed in these legions by using a real-time PCR assay. Telomere length and mtDNA copy number (mean ± S.D) in CRCs was 1.87 ± 1.52 and 1.61 ± 1.37, respectively. In TAs and SPs, relative mtDNA copy number was 0.92 ± 0.71 and 1.84 ± 1.06, respectively, shoing statistical difference (p = 0.017). However, telomere length was similar in these precancerous legions. Telomere length and mtDNA copy number did not show clinical and prognostic values in CRCs, however, positive correlation between telomere length and mitochondrial DNA copy number were found in CRC (r = 0.408, p < 0.001). However, this association was not shown in precancerous lesions (r = -0.031, p = 0.765). This result suggests that loss of co-regulation between telomeres and mitochondrial function may induce the initiation or play a role as trigger factor of colorectal carcinogenesis.

  2. Association between Leukocyte Mitochondrial DNA Copy Number and Regular Exercise in Postmenopausal Women.

    Science.gov (United States)

    Chang, Yu Kyung; Kim, Da Eun; Cho, Soo Hyun; Kim, Jung-Ha

    2016-11-01

    Previous studies suggest that habitual exercise can improve skeletal mitochondrial function; however, to date, the association between exercise and mitochondrial function in peripheral leukocytes has not been reported. The aim of this study was to evaluate the relationship between regular exercise and mitochondrial function by measuring leukocyte mitochondrial DNA (mtDNA) copy number in postmenopausal women. This cross-sectional study included 144 relatively healthy, non-diabetic, non-smoking, postmenopausal women. Clinical parameters, including anthropometric measurements and cardio-metabolic parameters, were assessed. Regular exercise was defined as at least 150 minutes per week of moderate-intensity activity, or an equivalent combination of moderate and vigorous-intensity activity, over a duration of at least 6 months. Leukocyte mtDNA copy numbers were measured using real-time polymerase chain reaction assays, and these were normalized to the β-globin copy number to give the relative mtDNA copy number. The mtDNA copy number of peripheral leukocytes was significantly greater in the exercise group (1.33±0.02) than in the no exercise group (1.05±0.02, Pcopy number (β=0.25, Pcopy number in postmenopausal women.

  3. Mitochondrial DNA copy number in peripheral blood cells declines with age and is associated with general health among elderly

    DEFF Research Database (Denmark)

    Mengel-From, Jonas; Thinggaard, Mikael; Dalgård, Christine

    2014-01-01

    compared to nuclear DNA, i.e. the mitochondrial DNA copy number, was measured by PCR technology and used as a proxy for the content of mitochondria copies. In 1,067 Danish twins and singletons (18-93 years of age), with the majority being elderly individuals, the estimated mean mitochondrial DNA copy...... an individual was more than twice as steep as observed in the cross-sectional analysis [decline of mtDNA content: -1.27; 95 % CI (-1.71; -0.82)]. Subjects with low mitochondrial DNA copy number had poorer outcomes in terms of cognitive performance, physical strength, self-rated health, and higher all......-cause mortality than subjects with high mitochondrial DNA copy number, also when age was controlled for. The copy number mortality association can contribute to the smaller decline in a cross-sectional sample of the population compared to the individual, longitudinal decline. This study suggests that high...

  4. Decreased mtDNA Copy Number of Gastric Cancer: a New Tumor Marker?

    Institute of Scientific and Technical Information of China (English)

    FanLi; XiaosongWang; ChengboHan; JieLin

    2004-01-01

    OBJECTIVE To explore the relationship between mtDNA (mitochondrial DNA) and gastric cancer by comparing the difference of mtDNA copy number in gastric cancers and paracancerous tissues.METHODS The HV1 (hypervariable region) and HV2 of the mitochondrial Dloop region from 20 cases of gastric cancer and 20 paracancerous tissues were amplified by PCR with 13-actin serving as a quantitative standard marker. The products were separated by polyacrylamide gel electrophoresis (PAGE) and silver stained in order to compare the difference in mtDNA copy number between gastric cancers and paracancerous tissues. The mtDNA copy number was determined for gastric cancer shaving various pathological characteristics and the results compared with previous immunohistochemical staininq of the tumors,RESULTS There was a significantly quantitative difference in HV1, HV2 (standardized with β-actin) between gastric cancers and paracancerous tissues (P0.05).CONCLUSION The occurrence of gastric cancer was closely associated with decreased mtDNA copy number, which may be a new tumor marker.

  5. Cardiometabolic phenotypes and mitochondrial DNA copy number in two cohorts of UK women.

    Science.gov (United States)

    Guyatt, Anna L; Burrows, Kimberley; Guthrie, Philip A I; Ring, Sue; McArdle, Wendy; Day, Ian N M; Ascione, Raimondo; Lawlor, Debbie A; Gaunt, Tom R; Rodriguez, Santiago

    2017-08-15

    The mitochondrial genome is present at variable copy number between individuals. Mitochondria are vulnerable to oxidative stress, and their dysfunction may be associated with cardiovascular disease. The association of mitochondrial DNA copy number with cardiometabolic risk factors (lipids, glycaemic traits, inflammatory markers, anthropometry and blood pressure) was assessed in two independent cohorts of European origin women, one in whom outcomes were measured at mean (SD) age 30 (4.3) years (N=2278) and the second at 69.4 (5.5) years (N=2872). Mitochondrial DNA copy number was assayed by quantitative polymerase chain reaction. Associations were adjusted for smoking, sociodemographic status, laboratory factors and white cell traits. Out of a total of 12 outcomes assessed in both cohorts, mitochondrial DNA copy number showed little or no association with the majority (point estimates were close to zero and nearly all p-values were >0.01). The strongest evidence was for an inverse association in the older cohort with insulin (standardised beta [95%CI]: -0.06, [-0.098, -0.022], p=0.002), but this association did not replicate in the younger cohort. Our findings do not provide support for variation in mitochondrial DNA copy number having an important impact on cardio-metabolic risk factors in European origin women. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  6. Mitochondrial DNA copy number is regulated by DNA methylation and demethylation of POLGA in stem and cancer cells and their differentiated progeny.

    Science.gov (United States)

    Lee, W; Johnson, J; Gough, D J; Donoghue, J; Cagnone, G L M; Vaghjiani, V; Brown, K A; Johns, T G; St John, J C

    2015-02-26

    Mitochondrial DNA (mtDNA) copy number is strictly regulated during differentiation so that cells with a high requirement for ATP generated through oxidative phosphorylation have high mtDNA copy number, whereas those with a low requirement have few copies. Using immunoprecipitation of DNA methylation on 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), which distinguish between de novo DNA methylation and demethylation, respectively, we set out to determine whether DNA methylation at exon 2 of the human mtDNA-specific polymerase (DNA polymerase gamma A (POLGA)) regulates cell-specific mtDNA copy number in highly proliferative and terminally differentiated cells. Highly proliferative cancer and pluripotent and multipotent cells possessed low mtDNA copy number and were highly methylated at exon 2 of POLGA in contrast to post-mitotic cells. Unlike neural stem cells, cancer cells were unable to differentiate and remained extensively DNA methylated at exon 2 of POLGA. However, mtDNA depletion of cancer cells reduced DNA methylation at exon 2 of POLGA as they replenished mtDNA to form tumours in mice. Glioblastoma cells treated with the DNA demethylation agent 5-azacytidine over 28 days of astrocyte-induced differentiation demethylated exon 2 of POLGA leading to increased mtDNA copy number and expression of the astrocyte endpoint marker glial fibrillary acidic protein (GFAP). However, the demethylation agent vitamin C (VitC) was unable to sustain increased mtDNA copy number and differentiation, as was the case when VitC was withdrawn after short-term treatment. These data demonstrate that DNA demethylation of POLGA is an essential regulator of mtDNA copy number and cellular fate and that cancer cells are only able to modulate DNA methylation of POLGA and mtDNA copy number in the presence of a DNA demethylation agent that inhibits de novo methyltransferase 1 activity.

  7. DNA copy number changes define spatial patterns of heterogeneity in colorectal cancer

    Science.gov (United States)

    Mamlouk, Soulafa; Childs, Liam Harold; Aust, Daniela; Heim, Daniel; Melching, Friederike; Oliveira, Cristiano; Wolf, Thomas; Durek, Pawel; Schumacher, Dirk; Bläker, Hendrik; von Winterfeld, Moritz; Gastl, Bastian; Möhr, Kerstin; Menne, Andrea; Zeugner, Silke; Redmer, Torben; Lenze, Dido; Tierling, Sascha; Möbs, Markus; Weichert, Wilko; Folprecht, Gunnar; Blanc, Eric; Beule, Dieter; Schäfer, Reinhold; Morkel, Markus; Klauschen, Frederick; Leser, Ulf; Sers, Christine

    2017-01-01

    Genetic heterogeneity between and within tumours is a major factor determining cancer progression and therapy response. Here we examined DNA sequence and DNA copy-number heterogeneity in colorectal cancer (CRC) by targeted high-depth sequencing of 100 most frequently altered genes. In 97 samples, with primary tumours and matched metastases from 27 patients, we observe inter-tumour concordance for coding mutations; in contrast, gene copy numbers are highly discordant between primary tumours and metastases as validated by fluorescent in situ hybridization. To further investigate intra-tumour heterogeneity, we dissected a single tumour into 68 spatially defined samples and sequenced them separately. We identify evenly distributed coding mutations in APC and TP53 in all tumour areas, yet highly variable gene copy numbers in numerous genes. 3D morpho-molecular reconstruction reveals two clusters with divergent copy number aberrations along the proximal–distal axis indicating that DNA copy number variations are a major source of tumour heterogeneity in CRC. PMID:28120820

  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 of h...

  9. A scale-space method for detecting recurrent DNA copy number changes with analytical false discovery rate control

    NARCIS (Netherlands)

    Van Dyk, E.; Reinders, M.J.T.; Wessels, L.F.A.

    2013-01-01

    Tumor formation is partially driven by DNA copy number changes, which are typically measured using array comparative genomic hybridization, SNP arrays and DNA sequencing platforms. Many techniques are available for detecting recurring aberrations across multiple tumor samples, including CMAR, STAC,

  10. Reduced rDNA Copy Number Does Not Affect “Competitive” Chromosome Pairing in XYY Males of Drosophila melanogaster

    OpenAIRE

    Keith A. Maggert

    2014-01-01

    The ribosomal DNA (rDNA) arrays are causal agents in X-Y chromosome pairing in meiosis I of Drosophila males. Despite broad variation in X-linked and Y-linked rDNA copy number, polymorphisms in regulatory/spacer sequences between rRNA genes, and variance in copy number of interrupting R1 and R2 retrotransposable elements, there is little evidence that different rDNA arrays affect pairing efficacy. I investigated whether induced rDNA copy number polymorphisms affect chromosome pairing in a “co...

  11. The potential role for use of mitochondrial DNA copy number as predictive biomarker in presbycusis

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    Falah M

    2016-10-01

    Full Text Available Masoumeh Falah,1,2 Massoud Houshmand,3 Mohammad Najafi,2 Maryam Balali,1 Saeid Mahmoudian,1 Alimohamad Asghari,4 Hessamaldin Emamdjomeh,1 Mohammad Farhadi1 1ENT and Head & Neck Research Center and Department, Iran University of Medical Sciences, Tehran, Iran; 2Cellular and Molecular Research Center, Biochemistry Department, Iran University of Medical Sciences, Tehran, Iran; 3Department of Medical Genetics, National Institute for Genetic Engineering and Biotechnology, Tehran, Iran; 4Skull base research center, Iran University of Medical Sciences, Tehran, Iran Objectives: Age-related hearing impairment, or presbycusis, is the most common communication disorder and neurodegenerative disease in the elderly. Its prevalence is expected to increase, due to the trend of growth of the elderly population. The current diagnostic test for detection of presbycusis is implemented after there has been a change in hearing sensitivity. Identification of a pre-diagnostic biomarker would raise the possibility of preserving hearing sensitivity before damage occurs. Mitochondrial dysfunction, including the production of reactive oxygen species and induction of expression of apoptotic genes, participates in the progression of presbycusis. Mitochondrial DNA sequence variation has a critical role in presbycusis. However, the nature of the relationship between mitochondrial DNA copy number, an important biomarker in many other diseases, and presbycusis is undetermined.Methods: Fifty-four subjects with presbycusis and 29 healthy controls were selected after ear, nose, throat examination and pure-tone audiometry. DNA was extracted from peripheral blood samples. The copy number of mitochondrial DNA relative to the nuclear genome was measured by quantitative real-time polymerase chain reaction.Results: Subjects with presbycusis had a lower median mitochondrial DNA copy number than healthy subjects and the difference was statistically significant (P=0.007. Mitochondrial DNA

  12. Integrated microfluidic systems for DNA analysis.

    Science.gov (United States)

    Njoroge, Samuel K; Chen, Hui-Wen; Witek, Małgorzata A; Soper, Steven A

    2011-01-01

    microfluidic systems that are composed of two or more microdevices directed toward DNA analyses. Our discussions will primarily be focused on the integration of various processing steps with microcapillary electrophoresis (μCE) or microarrays. The advantages afforded by fully integrated microfluidic systems to enable challenging applications, such as single-copy DNA sequencing, single-cell gene expression analysis, pathogen detection, and forensic DNA analysis in formats that provide high throughput and point-of-analysis capabilities will be discussed as well.

  13. Quantification of Fewer than Ten Copies of a DNA Biomarker without Amplification or Labeling.

    Science.gov (United States)

    Lee, Yoonhee; Kim, Youngkyu; Lee, Donggyu; Roy, Dhruvajyoti; Park, Joon Won

    2016-06-08

    Polymerase chain reaction (PCR) is a highly sensitive diagnosis technique for detection of nucleic acids and for monitoring residual disease; however, PCR can be unreliable for samples containing very few target molecules. Here, we describe a quantification method, using force-distance (FD) curve based atomic force microscopy (AFM) to detect a target DNA bound to small (1.4-1.9 μm diameter) probe DNA spots, allowing mapping of entire spots to nanometer resolution. Using a synthetic BCR-ABL fusion gene sequence target, we examined samples containing between one and 10 target copies. A high degree of correlation (r(2) = 0.994) between numbers of target copies and detected probe clusters was observed, and the approach could detect the BCR-ABL biomarker when only a single copy was present, although multiple screens were required. Our results clearly demonstrate that FD curve-based imaging is suitable for quantitative analysis of fewer than 10 copies of DNA biomarkers without amplification, modification, or labeling.

  14. A single copy integration vector that integrates at an engineered site on the Staphylococcus aureus chromosome

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    Lei Mei G

    2012-01-01

    Full Text Available Abstract Background Single-copy integration vectors based upon the site-specific recombination systems of bacteriophage are invaluable tools in the study of bacterial pathogenesis. The utility of such vectors is often limited, however, by the fact that integration often results in the inactivation of bacterial genes or has undesirable effects on gene transcription. The aim of this study is to develop an integration vector that does not have a detectable effect on gene transcription upon integration. Findings We have developed a single-copy integration system that enables the cloning vector to integrate at a specific engineered site, within an untranscribed intergenic region, in the chromosome of Staphylococcus aureus. This system is based on the lysogenic phage L54a site-specific recombination system in which the L54a phage (attP and chromosome (attB attachment sites, which share an 18-bp identical core sequence, were modified with identical mutations. The integration vector, pLL102, was constructed to contain the modified L54a attP site (attP2 that was altered at 5 nucleotide positions within the core sequence. In the recipient strain, the similarly modified attB site (attB2 was inserted in an intergenic region devoid of detectable transcription read-through. Integration of the vector, which is unable to replicate in S. aureus extrachromosomally, was achieved by providing the L54a integrase gene in a plasmid in the recipient. We showed that pLL102 integrated specifically at the engineered site rather than at the native L54a attB site and that integration did not have a significant effect on transcription of genes immediately upstream or downstream of the integration site. Conclusions In this work, we describe an E. coli-S. aureus shuttle vector that can be used to introduce any cloned gene into the S. aureus chromosome at a select site without affecting gene expression. The vector should be useful for genetic manipulation of S. aureus and for

  15. An integrated analysis of miRNA and gene copy numbers in xenografts of Ewing's sarcoma

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    Mosakhani Neda

    2012-03-01

    Full Text Available Abstract Background Xenografts have been shown to provide a suitable source of tumor tissue for molecular analysis in the absence of primary tumor material. We utilized ES xenograft series for integrated microarray analyses to identify novel biomarkers. Method Microarray technology (array comparative genomic hybridization (aCGH and micro RNA arrays was used to screen and identify copy number changes and differentially expressed miRNAs of 34 and 14 passages, respectively. Incubated cells used for xenografting (Passage 0 were considered to represent the primary tumor. Four important differentially expressed miRNAs (miR-31, miR-31*, miR-145, miR-106 were selected for further validation by real time polymerase chain reaction (RT-PCR. Integrated analysis of aCGH and miRNA data was performed on 14 xenograft passages by bioinformatic methods. Results The most frequent losses and gains of DNA copy number were detected at 9p21.3, 16q and at 8, 15, 17q21.32-qter, 1q21.1-qter, respectively. The presence of these alterations was consistent in all tumor passages. aCGH profiles of xenograft passages of each series resembled their corresponding primary tumors (passage 0. MiR-21, miR-31, miR-31*, miR-106b, miR-145, miR-150*, miR-371-5p, miR-557 and miR-598 showed recurrently altered expression. These miRNAS were predicted to regulate many ES-associated genes, such as genes of the IGF1 pathway, EWSR1, FLI1 and their fusion gene (EWS-FLI1. Twenty differentially expressed miRNAs were pinpointed in regions carrying altered copy numbers. Conclusion In the present study, ES xenografts were successfully applied for integrated microarray analyses. Our findings showed expression changes of miRNAs that were predicted to regulate many ES associated genes, such as IGF1 pathway genes, FLI1, EWSR1, and the EWS-FLI1 fusion genes.

  16. Assessing Mitochondrial DNA Variation and Copy Number in Lymphocytes of ~2,000 Sardinians Using Tailored Sequencing Analysis Tools.

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    Jun Ding

    2015-07-01

    Full Text Available DNA sequencing identifies common and rare genetic variants for association studies, but studies typically focus on variants in nuclear DNA and ignore the mitochondrial genome. In fact, analyzing variants in mitochondrial DNA (mtDNA sequences presents special problems, which we resolve here with a general solution for the analysis of mtDNA in next-generation sequencing studies. The new program package comprises 1 an algorithm designed to identify mtDNA variants (i.e., homoplasmies and heteroplasmies, incorporating sequencing error rates at each base in a likelihood calculation and allowing allele fractions at a variant site to differ across individuals; and 2 an estimation of mtDNA copy number in a cell directly from whole-genome sequencing data. We also apply the methods to DNA sequence from lymphocytes of ~2,000 SardiNIA Project participants. As expected, mothers and offspring share all homoplasmies but a lesser proportion of heteroplasmies. Both homoplasmies and heteroplasmies show 5-fold higher transition/transversion ratios than variants in nuclear DNA. Also, heteroplasmy increases with age, though on average only ~1 heteroplasmy reaches the 4% level between ages 20 and 90. In addition, we find that mtDNA copy number averages ~110 copies/lymphocyte and is ~54% heritable, implying substantial genetic regulation of the level of mtDNA. Copy numbers also decrease modestly but significantly with age, and females on average have significantly more copies than males. The mtDNA copy numbers are significantly associated with waist circumference (p-value = 0.0031 and waist-hip ratio (p-value = 2.4×10-5, but not with body mass index, indicating an association with central fat distribution. To our knowledge, this is the largest population analysis to date of mtDNA dynamics, revealing the age-imposed increase in heteroplasmy, the relatively high heritability of copy number, and the association of copy number with metabolic traits.

  17. Copy number of tandem direct repeats within the inverted repeats of Marek's disease virus DNA.

    Science.gov (United States)

    Kanamori, A; Nakajima, K; Ikuta, K; Ueda, S; Kato, S; Hirai, K

    1986-12-01

    We previously reported that DNA of the oncogenic strain BC-1 of Marek's disease virus serotype 1 (MDV1) contains three units of tandem direct repeats with 132 base pair (bp) repeats within the inverted repeats of the long regions of the MDV1 genome, whereas the attenuated, nononcogenic viral DNA contains multiple units of tandem direct repeats (Maotani et al., 1986). In the present study, the difference in the copy numbers of 132 bp repeats of oncogenic and nononcogenic MDV1 DNAs in other strains of MDV1 was investigated by Southern blot hybridization. The main copy numbers in different oncogenic MDV1 strains differed: those of BC-1, JM and highly oncogenic Md5 were 3, 5 to 12 and 2, respectively. The viral DNA population with two units of repeats was small, but detectable, in cells infected with either the oncogenic BC-1 or JM strain. The MDV1 DNA in various MD cell lines contained either two units or both two and three units of repeats. The significance of the copy number of repeats in oncogenicity of MDV1 is discussed.

  18. CNVkit: Genome-Wide Copy Number Detection and Visualization from Targeted DNA Sequencing.

    Directory of Open Access Journals (Sweden)

    Eric Talevich

    2016-04-01

    Full Text Available Germline copy number variants (CNVs and somatic copy number alterations (SCNAs are of significant importance in syndromic conditions and cancer. Massively parallel sequencing is increasingly used to infer copy number information from variations in the read depth in sequencing data. However, this approach has limitations in the case of targeted re-sequencing, which leaves gaps in coverage between the regions chosen for enrichment and introduces biases related to the efficiency of target capture and library preparation. We present a method for copy number detection, implemented in the software package CNVkit, that uses both the targeted reads and the nonspecifically captured off-target reads to infer copy number evenly across the genome. This combination achieves both exon-level resolution in targeted regions and sufficient resolution in the larger intronic and intergenic regions to identify copy number changes. In particular, we successfully inferred copy number at equivalent to 100-kilobase resolution genome-wide from a platform targeting as few as 293 genes. After normalizing read counts to a pooled reference, we evaluated and corrected for three sources of bias that explain most of the extraneous variability in the sequencing read depth: GC content, target footprint size and spacing, and repetitive sequences. We compared the performance of CNVkit to copy number changes identified by array comparative genomic hybridization. We packaged the components of CNVkit so that it is straightforward to use and provides visualizations, detailed reporting of significant features, and export options for integration into existing analysis pipelines. CNVkit is freely available from https://github.com/etal/cnvkit.

  19. Cell-free DNA copy number variations in plasma from colorectal cancer patients.

    Science.gov (United States)

    Li, Jian; Dittmar, Rachel L; Xia, Shu; Zhang, Huijuan; Du, Meijun; Huang, Chiang-Ching; Druliner, Brooke R; Boardman, Lisa; Wang, Liang

    2017-08-01

    To evaluate the clinical utility of cell-free DNA (cfDNA), we performed whole-genome sequencing to systematically examine plasma cfDNA copy number variations (CNVs) in a cohort of patients with colorectal cancer (CRC, n = 80), polyps (n = 20), and healthy controls (n = 35). We initially compared cfDNA yield in 20 paired serum-plasma samples and observed significantly higher cfDNA concentration in serum (median = 81.20 ng, range 7.18-500 ng·mL(-1) ) than in plasma (median = 5.09 ng, range 3.76-62.8 ng·mL(-1) ) (P copy number analysis showed common CNVs in multiple chromosomal regions, including amplifications on 1q, 8q, and 5q and deletions on 1p, 4q, 8p, 17p, 18q, and 22q. Copy number changes were also evident in genes critical to the cell cycle, DNA repair, and WNT signaling pathways. To evaluate whether cumulative copy number changes were associated with tumor stages, we calculated plasma genomic abnormality in colon cancer (PGA-C) score by summing the most significant CNVs. The PGA-C score showed predictive performance with an area under the curve from 0.54 to 0.84 for CRC stages I-IV. Locus-specific copy number analysis identified nine genomic regions where CNVs were significantly associated with survival in stage III-IV CRC patients. A multivariate model using six of nine genomic regions demonstrated a significant association of high-risk score with shorter survival (HR = 5.33, 95% CI = 6.76-94.44, P < 0.0001). Our study demonstrates the importance of using plasma (rather than serum) to test tumor-related genomic variations. Plasma cfDNA-based tests can capture tumor-specific genetic changes and may provide a measurable classifier for assessing clinical outcomes in advanced CRC patients. © 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

  20. Mitochondrial DNA copy numbers in pyramidal neurons are decreased and mitochondrial biogenesis transcriptome signaling is disrupted in Alzheimer's disease hippocampi.

    Science.gov (United States)

    Rice, Ann C; Keeney, Paula M; Algarzae, Norah K; Ladd, Amy C; Thomas, Ravindar R; Bennett, James P

    2014-01-01

    Alzheimer's disease (AD) is the major cause of adult-onset dementia and is characterized in its pre-diagnostic stage by reduced cerebral cortical glucose metabolism and in later stages by reduced cortical oxygen uptake, implying reduced mitochondrial respiration. Using quantitative PCR we determined the mitochondrial DNA (mtDNA) gene copy numbers from multiple groups of 15 or 20 pyramidal neurons, GFAP(+) astrocytes and dentate granule neurons isolated using laser capture microdissection, and the relative expression of mitochondrial biogenesis (mitobiogenesis) genes in hippocampi from 10 AD and 9 control (CTL) cases. AD pyramidal but not dentate granule neurons had significantly reduced mtDNA copy numbers compared to CTL neurons. Pyramidal neuron mtDNA copy numbers in CTL, but not AD, positively correlated with cDNA levels of multiple mitobiogenesis genes. In CTL, but not in AD, hippocampal cDNA levels of PGC1α were positively correlated with multiple downstream mitobiogenesis factors. Mitochondrial DNA copy numbers in pyramidal neurons did not correlate with hippocampal Aβ1-42 levels. After 48 h exposure of H9 human neural stem cells to the neurotoxic fragment Aβ25-35, mtDNA copy numbers were not significantly altered. In summary, AD postmortem hippocampal pyramidal neurons have reduced mtDNA copy numbers. Mitochondrial biogenesis pathway signaling relationships are disrupted in AD, but are mostly preserved in CTL. Our findings implicate complex alterations of mitochondria-host cell relationships in AD.

  1. Familial longevity study reveals a significant association of mitochondrial DNA copy number between centenarians and their offspring.

    Science.gov (United States)

    He, Yong-Han; Chen, Xiao-Qiong; Yan, Dong-Jing; Xiao, Fu-Hui; Lin, Rong; Liao, Xiao-Ping; Liu, Yao-Wen; Pu, Shao-Yan; Yu, Qin; Sun, Hong-Peng; Jiang, Jian-Jun; Cai, Wang-Wei; Kong, Qing-Peng

    2016-11-01

    Reduced mitochondrial function is an important cause of aging and age-related diseases. We previously revealed a relatively higher level of mitochondrial DNA (mtDNA) content in centenarians. However, it is still unknown whether such an mtDNA content pattern of centenarians could be passed on to their offspring and how it was regulated. To address these issues, we recruited 60 longevity families consisting of 206 family members (cohort 1) and explored their mtDNA copy number. The results showed that the first generation of the offspring (F1 offspring) had a higher level of mtDNA copy number than their spouses (p copy number in centenarians with that in F1 offspring (r = 0.54, p = 0.0008) but not with that in F1 spouses. These results were replicated in another independent cohort consisting of 153 subjects (cohort 2). RNA sequencing analysis suggests that the single-stranded DNA-binding protein 4 was significantly associated with mtDNA copy number and was highly expressed in centenarians as well as F1 offspring versus the F1 spouses, thus likely regulates the mtDNA copy number in the long-lived family members. In conclusion, our results suggest that the pattern of high mtDNA copy number is likely inheritable, which may act as a favorable factor to familial longevity through assuring adequate energy supply. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Copy number of the transposon, Pokey, in rDNA is positively correlated with rDNA copy number in Daphnia obtuse [corrected].

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    Kaitlynn LeRiche

    Full Text Available Pokey is a class II DNA transposon that inserts into 28S ribosomal RNA (rRNA genes and other genomic regions of species in the subgenus, Daphnia. Two divergent lineages, PokeyA and PokeyB have been identified. Recombination between misaligned rRNA genes changes their number and the number of Pokey elements. We used quantitative PCR (qPCR to estimate rRNA gene and Pokey number in isolates from natural populations of Daphnia obtusa, and in clonally-propagated mutation accumulation lines (MAL initiated from a single D. obtusa female. The change in direction and magnitude of Pokey and rRNA gene number did not show a consistent pattern across ∼ 87 generations in the MAL; however, Pokey and rRNA gene number changed in concert. PokeyA and 28S gene number were positively correlated in the isolates from both natural populations and the MAL. PokeyB number was much lower than PokeyA in both MAL and natural population isolates, and showed no correlation with 28S gene number. Preliminary analysis did not detect PokeyB outside rDNA in any isolates and detected only 0 to 4 copies of PokeyA outside rDNA indicating that Pokey may be primarily an rDNA element in D. obtusa. The recombination rate in this species is high and the average size of the rDNA locus is about twice as large as that in other Daphnia species such as D. pulicaria and D. pulex, which may have facilitated expansion of PokeyA to much higher numbers in D. obtusa rDNA than these other species.

  3. A robust penalized method for the analysis of noisy DNA copy number data

    Directory of Open Access Journals (Sweden)

    Huang Jian

    2010-09-01

    Full Text Available Abstract Background Deletions and amplifications of the human genomic DNA copy number are the causes of numerous diseases, such as, various forms of cancer. Therefore, the detection of DNA copy number variations (CNV is important in understanding the genetic basis of many diseases. Various techniques and platforms have been developed for genome-wide analysis of DNA copy number, such as, array-based comparative genomic hybridization (aCGH and high-resolution mapping with high-density tiling oligonucleotide arrays. Since complicated biological and experimental processes are often associated with these platforms, data can be potentially contaminated by outliers. Results We propose a penalized LAD regression model with the adaptive fused lasso penalty for detecting CNV. This method contains robust properties and incorporates both the spatial dependence and sparsity of CNV into the analysis. Our simulation studies and real data analysis indicate that the proposed method can correctly detect the numbers and locations of the true breakpoints while appropriately controlling the false positives. Conclusions The proposed method has three advantages for detecting CNV change points: it contains robustness properties; incorporates both spatial dependence and sparsity; and estimates the true values at each marker accurately.

  4. Laser Capture Microdissection of Cervical Human Papillomavirus Infections: Copy Number of the Virus in Cancerous and Normal Tissue and Heterogeneous DNA Methylation

    Science.gov (United States)

    Kalantari, Mina; Garcia-Carranca, Alejandro; Morales-Vazquez, Claudia Dalia; Zuna, Rosemary; Montiel, Delia Perez; Calleja-Macias, Itzel E.; Johansson, Bo; Andersson, Sonia; Bernard, Hans-Ulrich

    2009-01-01

    Research on the pathogenicity of human papillomaviruses (HPVs) during cervical carcinogenesis often relies on the study of homogenized tissue or cultured cells. This approach does not detect molecular heterogeneities within the infected tissue. It is desirable to understand molecular properties in specific histological contexts. We asked whether Laser Capture Microdissection (LCM) of archival cervical tumors in combination with real-time polymerase chain reaction and bisulfite sequencing permits (i) sensitive DNA diagnosis of small clusters of formalin fixed cells, (ii) quantification of HPV DNA in neoplastic and normal cells, and (iii) analysis of HPV DNA methylation, a marker of tumor progression. We analyzed 26 tumors containing HPV-16 or 18. We prepared DNA from LCM dissected thin sections of 100 to 2000 cells, and analyzed aliquots corresponding to between nine and 70 cells. We detected nine to 630 HPV-16 genome copies and one to 111 HPV-18 genome copies per tumor cell, respectively. In 17 of the 26 samples, HPV DNA existed in histologically normal cells distant from the margins of the tumors, but at much lower concentrations than in the tumor, suggesting that HPVs can infect at low levels without pathogenic changes. Methylation of HPV DNA, a biomarker of integration of the virus into cellular DNA, could be measured only in few samples due to limited sensitivity, and indicated heterogeneous methylation patterns in small clusters of cancerous and normal cells. LCM is powerful to study molecular parameters of cervical HPV infections like copy number, latency and epigenetics. PMID:19497607

  5. Mutation dependance of the mitochondrial DNA copy number in the first stages of human embryogenesis.

    Science.gov (United States)

    Monnot, Sophie; Samuels, David C; Hesters, Laetitia; Frydman, Nelly; Gigarel, Nadine; Burlet, Philippe; Kerbrat, Violaine; Lamazou, Frédéric; Frydman, René; Benachi, Alexandra; Feingold, Josué; Rotig, Agnes; Munnich, Arnold; Bonnefont, Jean-Paul; Steffann, Julie

    2013-05-01

    Mitochondrial DNA (mtDNA) content is thought to remain stable over the preimplantation period of human embryogenesis that is, therefore, suggested to be entirely dependent on ooplasm mtDNA capital. We have explored the impact of two disease-causing mutations [m.3243A>G myopathy, encephalopathy, lactic acidosis and stroke-like syndrome (MELAS) and m.8344A>G myoclonic epilepsy associated with ragged-red fibers (MERRF)] on mtDNA amounts in human oocytes and day 4-5 preimplantation embryos. The mtDNA amount was stable in MERRF and control materials, whereas gradually increasing from the germinal vesicle of oogenesis to the blastocyst stage of embryogenesis in MELAS cells, MELAS embryos carrying ∼3-fold higher mtDNA amount than control embryos (P = 0.0003). A correlation between mtDNA copy numbers and mutant loads was observed in MELAS embryos (R(2) = 0.42, P < 0.0013), suggestive of a compensation for the respiratory chain defect resulting from high mutation levels. These results suggest that mtDNA can replicate in early embryos and emphasize the need for sufficient amount of wild-type mtDNA to sustain embryonic development in humans.

  6. The association between DNA copy number aberrations at chromosome 5q22 and gastric cancer.

    Directory of Open Access Journals (Sweden)

    Pei-Chien Tsai

    Full Text Available BACKGROUND: Gastric cancer is common cancer. Discovering novel genetic biomarkers might help to identify high-risk individuals. Copy number variation (CNV has recently been shown to influence risk for several cancers. The aim of the present study was sought to test the association between copy number at a variant region and GC. METHODS: A total of 110 gastric cancer patients and 325 healthy volunteers were enrolled in this study. We searched for a CNV and found a CNV (Variation 7468 containing part of the APC gene, the SRP19 gene and the REEP5 gene. We chose four probes targeting at APC-intron8, APC-exon9, SRP19 and REEP5 to interrogate this CNV. Specific Taqman probes labeled by different reporter fluorophores were used in a real-time PCR platform to obtain copy number. Both the original non-integer data and transformed integer data on copy number were used for analyses. RESULTS: Gastric caner patients had a lower non-integer copy number than controls for the APC-exon9 probe (Adjusted p = 0.026 and SRP19 probe (Adjusted p = 0.002. The analysis of integer copy number yielded a similar pattern although less significant (Adjusted p = 0.07 for APC-exon9 probe and Adjusted p = 0.02 for SRP19 probe. CONCLUSIONS: Losses of a CNV at 5q22, especially in the DNA region surrounding APC-exon 9, may be associated with a higher risk of gastric cancer.

  7. Incorporating double copies of a chromatin insulator into lentiviral vectors results in less viral integrants

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    Rosenqvist Nina

    2009-02-01

    Full Text Available Abstract Background Lentiviral vectors hold great promise as gene transfer vectors in gene therapeutic settings. However, problems related to the risk of insertional mutagenesis, transgene silencing and positional effects have stalled the use of such vectors in the clinic. Chromatin insulators are boundary elements that can prevent enhancer-promoter interactions, if placed between these elements, and protect transgene cassettes from silencing and positional effects. It has been suggested that insulators can improve the safety and performance of lentiviral vectors. Therefore insulators have been incorporated into lentiviral vectors in order to enhance their safety profile and improve transgene expression. Commonly such insulator vectors are produced at lower titers than control vectors thus limiting their potential use. Results In this study we cloned in tandem copies of the chicken β-globin insulator (cHS4 on both sides of the transgene cassette in order to enhance the insulating effect. Our insulator vectors were produced at significantly lower titers compared to control vectors, and we show that this reduction in titer is due to a block during the transduction process that appears after reverse transcription but before integration of the viral DNA. This non-integrated viral DNA could be detected by PCR and, importantly, prevented efficient transduction of target cells. Conclusion These results have importance for the future use of insulator sequences in lentiviral vectors and might limit the use of insulators in vectors for in vivo use. Therefore, a careful analysis of the optimal design must be performed before insulators are included into clinical lentiviral vectors.

  8. Stability-based comparison of class discovery methods for DNA copy number profiles.

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    Isabel Brito

    Full Text Available MOTIVATION: Array-CGH can be used to determine DNA copy number, imbalances in which are a fundamental factor in the genesis and progression of tumors. The discovery of classes with similar patterns of array-CGH profiles therefore adds to our understanding of cancer and the treatment of patients. Various input data representations for array-CGH, dissimilarity measures between tumor samples and clustering algorithms may be used for this purpose. The choice between procedures is often difficult. An evaluation procedure is therefore required to select the best class discovery method (combination of one input data representation, one dissimilarity measure and one clustering algorithm for array-CGH. Robustness of the resulting classes is a common requirement, but no stability-based comparison of class discovery methods for array-CGH profiles has ever been reported. RESULTS: We applied several class discovery methods and evaluated the stability of their solutions, with a modified version of Bertoni's [Formula: see text]-based test [1]. Our version relaxes the assumption of independency required by original Bertoni's [Formula: see text]-based test. We conclude that Minimal Regions of alteration (a concept introduced by [2] for input data representation, sim [3] or agree [4] for dissimilarity measure and the use of average group distance in the clustering algorithm produce the most robust classes of array-CGH profiles. AVAILABILITY: The software is available from http://bioinfo.curie.fr/projects/cgh-clustering. It has also been partly integrated into "Visualization and analysis of array-CGH"(VAMP[5]. The data sets used are publicly available from ACTuDB [6].

  9. [Research on potential interaction between mitochondrial DNA copy number and related factors on risk of hypertension in coal miners].

    Science.gov (United States)

    Guo, J Y; Lei, L J; Qiao, N; Fan, G Q; Sun, C M; Huang, J J; Wang, T

    2017-01-10

    Objective: To investigate the effects of mitochondrial DNA (mtDNA) copy number in peripheral blood and related factors on the risk of hypertension in coal miners. Methods: A case-control study was conducted in 378 coal miners with hypertension and 325 healthy coal miners recruited from Datong Coal Mine Group. A standard questionnaire was used to collect their general information, such as demographic characteristics, habits and occupational history. Fluorescence quantitative PCR was performed to detect the copy number of mtDNA. Logistic regression model was applied for identifying the related risk factors of hypertension and analyzing the interaction between mtDNA copy number and risk factors. Results: The prevalence of hypertension of high mtDNA copy number was lower than mtDNA copy numberin 0-5.67 group, but the difference was not statistically significant (P=0.414). Alcohol drinking (OR=1.80, 95% CI: 1.26-2.56), family history of hypertension (OR=1.74, 95% CI: 1.20- 2.50), work shifts (OR=0.69, 95% CI: 0.48-0.99), education level (P=0.012) and family monthly income level (P=0.001) were related to the prevalence of hypertension. There were potential interactions between mtDNA copy number and alcohol drinking, family monthly income level, family history of hypertension, respectively. Alcohol drinking was a risk factor for hypertension [1.77 (1.25-2.50)]. Potential interactions between mtDNA copy number and alcohol drinking reduced the risk of hypertension (OR=1.20, 95% CI: 1.07-1.35). Family history of hypertension was a risk factor for hypertension [1.81(1.26-2.59)]. Potential interactions between mtDNA copy number and family history of hypertension reduced the risk of hypertension (OR=1.24, 95%CI: 1.09-1.41). Family monthly income level was a protect factor for hypertension [0.55(0.46-0.66)]. Potential interactions between mtDNA copy number and family monthly income level increased the protection role of hypertension (OR=0.90, 95% CI: 0.86-0.94). Conclusion: mtDNA

  10. Association of telomere length and mitochondrial DNA copy number in a community sample of healthy adults.

    Science.gov (United States)

    Tyrka, Audrey R; Carpenter, Linda L; Kao, Hung-Teh; Porton, Barbara; Philip, Noah S; Ridout, Samuel J; Ridout, Kathryn K; Price, Lawrence H

    2015-06-01

    Cellular aging plays a role in longevity and senescence, and has been implicated in medical and psychiatric conditions, including heart disease, cancer, major depression and posttraumatic stress disorder. Telomere shortening and mitochondrial dysfunction are thought to be central to the cellular aging process. The present study examined the association between mitochondrial DNA (mtDNA) copy number and telomere length in a sample of medically healthy adults. Participants (total n=392) were divided into 4 groups based on the presence or absence of early life adversity and lifetime psychopathology: No Adversity/No Disorder, n=136; Adversity/No Disorder, n=91; No Adversity/Disorder, n=46; Adversity/Disorder, n=119. Telomere length and mtDNA copy number were measured using quantitative polymerase chain reaction. There was a positive correlation between mtDNA and telomere length in the entire sample (r=0.120, ptelomere length in a large group of women and men both with and without early adversity and psychopathology, suggesting co-regulation of telomeres and mitochondrial function. The mechanisms underlying this association may be important in the pathophysiology of age-related medical conditions, such as heart disease and cancer, as well as for stress-associated psychiatric disorders.

  11. High Glucose-Induced Oxidative Stress Increases the Copy Number of Mitochondrial DNA in Human Mesangial Cells

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    Ghada Al-Kafaji

    2013-01-01

    Full Text Available Oxidative damage to mitochondrial DNA (mtDNA has been linked to the pathogenicity of diabetic nephropathy. We tested the hypothesis that mtDNA copy number may be increased in human mesangial cells in response to high glucose-induced reactive oxygen species (ROS to compensate for damaged mtDNA. The effect of manganese superoxide dismutase mimetic (MnTBAP on glucose-induced mtDNA copy number was also examined. The copy number of mtDNA was determined by real-time PCR in human mesangial cells cultured in 5 mM glucose, 25 mM glucose, and mannitol (osmotic control, as well as in cells cultured in 25 mM glucose in the presence and absence of 200 μM MnTBAP. Intracellular ROS was assessed by confocal microscopy and flow cytometry in human mesangial cells. The copy number of mtDNA was significantly increased when human mesangial cells were incubated with 25 mM glucose compared to 5 mM glucose and mannitol. In addition, 25 mM glucose rapidly generated ROS in the cells, which was not detected in 5 mM glucose. Furthermore, mtDNA copy number was significantly decreased and maintained to normal following treatment of cells with 25 mM glucose and MnTBAP compared to 25 mM glucose alone. Inclusion of MnTBAP during 25 mM glucose incubation inhibited mitochondrial superoxide in human mesangial cells. Increased mtDNA copy number in human mesangial cells by high glucose could contribute to increased mitochondrial superoxide, and prevention of mtDNA copy number could have potential in retarding the development of diabetic nephropathy.

  12. Mitochondrial DNA copy number in peripheral blood cells declines with age and is associated with general health among elderly.

    Science.gov (United States)

    Mengel-From, Jonas; Thinggaard, Mikael; Dalgård, Christine; Kyvik, Kirsten Ohm; Christensen, Kaare; Christiansen, Lene

    2014-09-01

    The role of the mitochondria in disease, general health and aging has drawn much attention over the years. Several attempts have been made to describe how the numbers of mitochondria correlate with age, although with inconclusive results. In this study, the relative quantity of mitochondrial DNA compared to nuclear DNA, i.e. the mitochondrial DNA copy number, was measured by PCR technology and used as a proxy for the content of mitochondria copies. In 1,067 Danish twins and singletons (18-93 years of age), with the majority being elderly individuals, the estimated mean mitochondrial DNA copy number in peripheral blood cells was similar for those 18-48 years of age [mean relative mtDNA content: 61.0; 95 % CI (52.1; 69.9)], but declined by -0.54 mtDNA 95 % CI (-0.63; -0.45) every year for those older than approximately 50 years of age. However, the longitudinal, yearly decline within an individual was more than twice as steep as observed in the cross-sectional analysis [decline of mtDNA content: -1.27; 95 % CI (-1.71; -0.82)]. Subjects with low mitochondrial DNA copy number had poorer outcomes in terms of cognitive performance, physical strength, self-rated health, and higher all-cause mortality than subjects with high mitochondrial DNA copy number, also when age was controlled for. The copy number mortality association can contribute to the smaller decline in a cross-sectional sample of the population compared to the individual, longitudinal decline. This study suggests that high mitochondrial DNA copy number in blood is associated with better health and survival among elderly.

  13. A case-control study of peripheral blood mitochondrial DNA copy number and risk of renal cell carcinoma.

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    Mark P Purdue

    Full Text Available BACKGROUND: Low mitochondrial DNA (mtDNA copy number is a common feature of renal cell carcinoma (RCC, and may influence tumor development. Results from a recent case-control study suggest that low mtDNA copy number in peripheral blood may be a marker for increased RCC risk. In an attempt to replicate that finding, we measured mtDNA copy number in peripheral blood DNA from a U.S. population-based case-control study of RCC. METHODOLOGY/PRINCIPAL FINDINGS: Relative mtDNA copy number was measured in triplicate by a quantitative real-time PCR assay using DNA extracted from peripheral whole blood. Cases (n = 603 had significantly lower mtDNA copy number than controls (n = 603; medians 0.85, 0.91 respectively; P = 0.0001. In multiple logistic regression analyses, the lowest quartile of mtDNA copy number was associated with a 60% increase in RCC risk relative to the highest quartile (OR = 1.6, 95% CI = 1.1-2.2; P(trend = 0.009. This association remained in analyses restricted to cases treated by surgery alone (OR (Q1 = 1.4, 95% CI = 1.0-2.1 and to localized tumors (2.0, 1.3-2.8. CONCLUSIONS/SIGNIFICANCE: Our findings from this investigation, to our knowledge the largest of its kind, offer important confirmatory evidence that low mtDNA copy number is associated with increased RCC risk. Additional research is needed to assess whether the association is replicable in prospective studies.

  14. Isolation, Mapping, DNA Sequence and RFLPs Studies of Random Single-Copy DNA Segments on Human X Chromosome

    Institute of Scientific and Technical Information of China (English)

    谭骏; 邱信芳; 薛京伦; 朱锡华; 纪贤文; 张冬梅; 秦世真

    1994-01-01

    Using the total human/mouse DNA as the probe, screening has been carried out three times with in situ plaque hybridization to obtain the single-copy DNA sequence from the human X chromosome genomic library. The effective rate of screening is 1. 45%. DNAs from clones containing single-copy inserts have been analyzed by a panel of hybrid cells with or without human X chromosome. Three segments, designated by DXFD52,73,75, are mapped to the X chromosome. DXFD52 has been precisely localized on Xq12-q13 with in situ chromosomal hybridization. DXFD52 has been partially sequenced. The results indicate that DXFD52 is a new isolated single-copy segment on the X chromosome. Great progress in the RFLPs study with DXFD52 has been achieved in the population of Chongqing, Sichuan Province. The results show that the DXFD52 can be used to detect the RFLP with Hind Ⅲ, Bgl Ⅱ, and Hinf Ⅰ. DXFD52 will be a potential "landmark" for the construction of the complete linkage map of human genome and the analysis of genomic s

  15. Identification of candidate growth promoting genes in ovarian cancer through integrated copy number and expression analysis.

    Science.gov (United States)

    Ramakrishna, Manasa; Williams, Louise H; Boyle, Samantha E; Bearfoot, Jennifer L; Sridhar, Anita; Speed, Terence P; Gorringe, Kylie L; Campbell, Ian G

    2010-04-08

    Ovarian cancer is a disease characterised by complex genomic rearrangements but the majority of the genes that are the target of these alterations remain unidentified. Cataloguing these target genes will provide useful insights into the disease etiology and may provide an opportunity to develop novel diagnostic and therapeutic interventions. High resolution genome wide copy number and matching expression data from 68 primary epithelial ovarian carcinomas of various histotypes was integrated to identify genes in regions of most frequent amplification with the strongest correlation with expression and copy number. Regions on chromosomes 3, 7, 8, and 20 were most frequently increased in copy number (> 40% of samples). Within these regions, 703/1370 (51%) unique gene expression probesets were differentially expressed when samples with gain were compared to samples without gain. 30% of these differentially expressed probesets also showed a strong positive correlation (r > or =0.6) between expression and copy number. We also identified 21 regions of high amplitude copy number gain, in which 32 known protein coding genes showed a strong positive correlation between expression and copy number. Overall, our data validates previously known ovarian cancer genes, such as ERBB2, and also identified novel potential drivers such as MYNN, PUF60 and TPX2.

  16. Identification of candidate growth promoting genes in ovarian cancer through integrated copy number and expression analysis.

    Directory of Open Access Journals (Sweden)

    Manasa Ramakrishna

    Full Text Available Ovarian cancer is a disease characterised by complex genomic rearrangements but the majority of the genes that are the target of these alterations remain unidentified. Cataloguing these target genes will provide useful insights into the disease etiology and may provide an opportunity to develop novel diagnostic and therapeutic interventions. High resolution genome wide copy number and matching expression data from 68 primary epithelial ovarian carcinomas of various histotypes was integrated to identify genes in regions of most frequent amplification with the strongest correlation with expression and copy number. Regions on chromosomes 3, 7, 8, and 20 were most frequently increased in copy number (> 40% of samples. Within these regions, 703/1370 (51% unique gene expression probesets were differentially expressed when samples with gain were compared to samples without gain. 30% of these differentially expressed probesets also showed a strong positive correlation (r > or =0.6 between expression and copy number. We also identified 21 regions of high amplitude copy number gain, in which 32 known protein coding genes showed a strong positive correlation between expression and copy number. Overall, our data validates previously known ovarian cancer genes, such as ERBB2, and also identified novel potential drivers such as MYNN, PUF60 and TPX2.

  17. DNA Copy Number Changes at 8q11–24 in Metastasized Colorectal Cancer

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    T. E. Buffart

    2005-01-01

    Full Text Available Background: C-Myc, a well-known oncogene located on 8q24.12–q24.23, is often amplified and over-expressed in both primary and metastasizing colorectal cancer. In addition, PRL-3 (also known as PTP4A3, a tyrosine phosphatase located on 8q24.3, is amplified in colorectal cancer metastasis. Beside PRL-3 and c-myc, other oncogenes located on the 8q23–24 region might be involved in this process. Therefore, the present study aims to correlate DNA copy number status of a series of genes at 8q23–24 in colorectal cancer at high resolution in correlation to metastatic disease. Materials and Methods: Thirty-two cases of colorectal cancer, 10 stage B1, 10 B2 and 12 D (Astler–Coller with their corresponding liver metastasis and one colorectal cell line (colo205, previously analyzed by array-CGH, were included in this study. A chromosome 8 specific MLPA probe mixture was used to analyze the presence of DNA copy number changes. The probe mixture contained 29 probes covering 25 genes on chromosome 8, as well as 6 control probes on other chromosomes. Results and Discussion: MLPA results obtained of the colo205 colorectal cell line were comparable with previous array-CGH results, thus validating the MLPA probe mixture. Astler–Coller B1 and B2 colorectal cancers differed significantly in DNA copy number of the genes, MOS (p = 0.04, MYC (p = 0.007, DDEF1 (p = 0.004, PTK2 (p = 0.02 and PTP4A3 (p = 0.04. When comparing these with Astler–Coller D primary tumors, significant differences were seen for several genes as well (MYC (p < 0.000, DDEF1 (p < 0.000, SLA (p < 0.000, PTK2 (p < 0.000, PTP4A3 (p = 0.002, and RECQL4 (p = 0.01. When comparing primary Astler–Coller D tumors and their corresponding liver metastases, a similar pattern of gains and losses was observed. Most of the liver metastases showed higher DNA copy number ratios than the corresponding primary tumors, but this difference was only significant for TPD52 (p = 0.02 and EIF3S6 (p = 0

  18. Construction of a food-grade multiple-copy integration system for Lactococcus lactis

    NARCIS (Netherlands)

    Leenhouts, K.; Bolhuis, A.; Venema, G.; Kok, J.

    A food-grade vector system was developed that allows stable integration of multiple plasmid copies in the chromosome of Lactococcus lactis. The vector consists of the plus origin of replication (Ori(+)) of the lactococcal plasmid pWV01, the sucrose genes of the lactic acid bacterium Pediococcus

  19. Polycyclic aromatic hydrocarbons exposure decreased sperm mitochondrial DNA copy number: A cross-sectional study (MARHCS) in Chongqing, China.

    Science.gov (United States)

    Ling, Xi; Zhang, Guowei; Sun, Lei; Wang, Zhi; Zou, Peng; Gao, Jianfang; Peng, Kaige; Chen, Qing; Yang, Huan; Zhou, Niya; Cui, Zhihong; Zhou, Ziyuan; Liu, Jinyi; Cao, Jia; Ao, Lin

    2017-01-01

    Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental pollutants that have adverse effects on the male reproductive function. Many studies have confirmed that PAHs preferentially accumulate in mitochondria DNA relative to nuclear DNA and disrupt mitochondrial functions. However, it is rare whether exposure to PAHs is associated with mitochondrial damage and dysfunction in sperm. To evaluate the effects of PAHs on sperm mitochondria, we measured mitochondrial membrane potential (MMP), mitochondrial DNA copy number (mtDNAcn) and mtDNA integrity in 666 individuals from the Male Reproductive Health in Chongqing College Students (MARHCS) study. PAHs exposure was estimated by measuring eight urinary PAH metabolites (1-OHNap, 2-OHNap, 1-OHPhe, 2-OHPhe, 3-OHPhe, 4-OHPhe, 2-OHFlu and 1-OHPyr). The subjects were divided into low, median and high exposure groups using the tertile levels of urinary PAH metabolites. In univariate analyses, the results showed that increased levels of 2-OHPhe, 3-OHPhe, ∑Phe metabolites and 2-OHFlu were found to be associated with decreased sperm mtDNAcn. After adjusting for potential confounders, significantly negative associations of these metabolites remained (p = 0.039, 0.012, 0.01, 0.035, respectively). Each 1 μg/g creatinine increase in 2-OHPhe, 3-OHPhe, ∑Phe metabolites and 2-OHFlu was associated with a decrease in sperm mtDNAcn of 9.427%, 11.488%, 9.635% and 11.692%, respectively. There were no significant associations between urinary PAH metabolites and sperm MMP or mtDNA integrity. The results indicated that the low exposure levels of PAHs can cause abnormities in sperm mitochondria.

  20. From DNA Copy Number to Gene Expression: Local aberrations, Trisomies and Monosomies

    Science.gov (United States)

    Shay, Tal

    The goal of my PhD research was to study the effect of DNA copy number changes on gene expression. DNA copy number aberrations may be local, encompassing several genes, or on the level of an entire chromosome, such as trisomy and monosomy. The main dataset I studied was of Glioblastoma, obtained in the framework of a collaboration, but I worked also with public datasets of cancer and Down's Syndrome. The molecular basis of expression changes in Glioblastoma. Glioblastoma is the most common and aggressive type of primary brain tumors in adults. In collaboration with Prof. Hegi (CHUV, Switzerland), we analyzed a rich Glioblastoma dataset including clinical information, DNA copy number (array CGH) and expression profiles. We explored the correlation between DNA copy number and gene expression at the level of chromosomal arms and local genomic aberrations. We detected known amplification and over expression of oncogenes, as well as deletion and down-regulation of tumor suppressor genes. We exploited that information to map alterations of pathways that are known to be disrupted in Glioblastoma, and tried to characterize samples that have no known alteration in any of the studied pathways. Identifying local DNA aberrations of biological significance. Many types of tumors exhibit chromosomal losses or gains and local amplifications and deletions. A region that is aberrant in many tumors, or whose copy number change is stronger, is more likely to be clinically relevant, and not just a by-product of genetic instability. We developed a novel method that defines and prioritizes aberrations by formalizing these intuitions. The method scores each aberration by the fraction of patients harboring it, its length and its amplitude, and assesses the significance of the score by comparing it to a null distribution obtained by permutations. This approach detects genetic locations that are significantly aberrant, generating a 'genomic aberration profile' for each sample. The 'genomic

  1. Microarray MAPH: accurate array-based detection of relative copy number in genomic DNA

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    Chan Alan

    2006-06-01

    Full Text Available Abstract Background Current methods for measurement of copy number do not combine all the desirable qualities of convenience, throughput, economy, accuracy and resolution. In this study, to improve the throughput associated with Multiplex Amplifiable Probe Hybridisation (MAPH we aimed to develop a modification based on the 3-Dimensional, Flow-Through Microarray Platform from PamGene International. In this new method, electrophoretic analysis of amplified products is replaced with photometric analysis of a probed oligonucleotide array. Copy number analysis of hybridised probes is based on a dual-label approach by comparing the intensity of Cy3-labelled MAPH probes amplified from test samples co-hybridised with similarly amplified Cy5-labelled reference MAPH probes. The key feature of using a hybridisation-based end point with MAPH is that discrimination of amplified probes is based on sequence and not fragment length. Results In this study we showed that microarray MAPH measurement of PMP22 gene dosage correlates well with PMP22 gene dosage determined by capillary MAPH and that copy number was accurately reported in analyses of DNA from 38 individuals, 12 of which were known to have Charcot-Marie-Tooth disease type 1A (CMT1A. Conclusion Measurement of microarray-based endpoints for MAPH appears to be of comparable accuracy to electrophoretic methods, and holds the prospect of fully exploiting the potential multiplicity of MAPH. The technology has the potential to simplify copy number assays for genes with a large number of exons, or of expanded sets of probes from dispersed genomic locations.

  2. Association between Salivary Mitochondrial DNA Copy Number and Chronic Fatigue according to Combined Symptoms in Korean Adults.

    Science.gov (United States)

    Shin, Jinyoung; Kim, Kyong Chol; Lee, Duk Chul; Lee, Hye Ree; Shim, Jae Yong

    2017-07-01

    We examined the association between salivary mitochondrial DNA (mtDNA) copy number and chronic fatigue combined with depression and insomnia. This cross-sectional study included 58 healthy adults with moderate to severe fatigue (Brief Fatigue Inventory [BFI] ≥4) for longer than 6 months. Subjects were classified as those without combined symptoms, with either depression (Beck Depression Inventory [BDI] ≥13) or insomnia (Pittsburgh Sleep Quality Index [PSQI] ≥5), or with both depression and insomnia. Salivary mtDNA copy number was measured by real-time quantitative polymerase chain reaction. The association was evaluated using a general linear model. About 76% of participants had either depression or insomnia as additional symptoms. These subjects were predominately female, drank more alcohol, and exercised less than those without combined symptoms (Pcopy number than those without combined symptoms (Pcopy number and usual fatigue were found in the group without combined symptoms, whereas the negative associations in the group with combined symptoms were attenuated. BDI and PSQI were not associated with mtDNA copy number. Chronic fatigue is negatively associated with salivary mtDNA copy number. Salivary mtDNA copy number may be a biological marker of fatigue with or without combined symptoms, indicating that a separate approach is necessary.

  3. DNA copy-number alterations underlie gene expression differences between microsatellite stable and unstable colorectal cancers

    DEFF Research Database (Denmark)

    Jorissen, Robert N; Lipton, Lara; Gibbs, Peter

    2008-01-01

    Purpose: About 15% of colorectal cancers harbor microsatellite instability (MSI). MSI-associated gene expression changes have been identified in colorectal cancers, but little overlap exists between signatures hindering an assessment of overall consistency. Little is known about the causes...... and downstream effects of differential gene expression. Experimental Design: DNA microarray data on 89 MSI and 140 microsatellite-stable (MSS) colorectal cancers from this study and 58 MSI and 77 MSS cases from three published reports were randomly divided into test and training sets. MSI-associated gene...... expression changes were assessed for cross-study consistency using training samples and validated as MSI classifier using test samples. Differences in biological pathways were identified by functional category analysis. Causation of differential gene expression was investigated by comparison to DNA copy...

  4. Transformation of Cowpea Vigna unguiculata with a Full-Length DNA Copy of Cowpea Mosaic Virus M-RNA

    NARCIS (Netherlands)

    Hille, Jacques; Goldbach, Rob

    1987-01-01

    A full-length DNA copy of the M-RNA of cowpea mosaic virus (CPMV), supplied with either the 35S promoter from cauliflower mosaic virus (CaMV) or the nopaline synthase promoter from Agrobacterium tumefaciens, was introduced into the T-DNA region of a Ti-plasmid-derived gene vector and transferred to

  5. Transformation of Cowpea Vigna unguiculata with a Full-Length DNA Copy of Cowpea Mosaic Virus M-RNA

    NARCIS (Netherlands)

    Hille, Jacques; Goldbach, Rob

    1987-01-01

    A full-length DNA copy of the M-RNA of cowpea mosaic virus (CPMV), supplied with either the 35S promoter from cauliflower mosaic virus (CaMV) or the nopaline synthase promoter from Agrobacterium tumefaciens, was introduced into the T-DNA region of a Ti-plasmid-derived gene vector and transferred to

  6. Mitochondrial DNA Copy Number in Peripheral Blood Is Independently Associated with Visceral Fat Accumulation in Healthy Young Adults

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    Jee-Yon Lee

    2014-01-01

    Full Text Available Aims. Visceral obesity is associated with an increased risk of cardiometabolic diseases and it is important to identify the underlying mechanisms. There is growing evidence that mitochondrial dysfunction is associated with metabolic disturbances related to visceral obesity. In addition, maintaining mitochondrial DNA (mtDNA copy number is important for preserving mitochondrial function. Therefore, we investigated the relationship between mtDNA copy number and visceral fat in healthy young adults. Methods. A total of 94 healthy young subjects were studied. Biomarkers of metabolic risk factors were assessed along with body composition by computed tomography. mtDNA copy number was measured in peripheral leukocytes using real-time polymerase chain reaction (PCR methods. Results. The mtDNA copy number correlated with BMI (r=-0.22, P=0.04, waist circumference (r=-0.23, P=0.03, visceral fat area (r=-0.28, P=-0.01, HDL-cholesterol levels (r=0.25, P=0.02, and hs-CRP (r=0.32, P=0.02 after adjusting for age and sex. Both stepwise and nonstepwise multiple regression analyses confirmed that visceral fat area was independently associated with mtDNA copy number (β=-0.33, P<0.01, β=0.32, and P=0.03, resp.. Conclusions. An independent association between mtDNA content and visceral adiposity was identified. These data suggest that mtDNA copy number is a potential predictive marker for metabolic disturbances. Further studies are required to understand the causality and clinical significance of our findings.

  7. Conserved Organisation of 45S rDNA Sites and rDNA Gene Copy Number among Major Clades of Early Land Plants.

    Science.gov (United States)

    Rosato, Marcela; Kovařík, Aleš; Garilleti, Ricardo; Rosselló, Josep A

    2016-01-01

    Genes encoding ribosomal RNA (rDNA) are universal key constituents of eukaryotic genomes, and the nuclear genome harbours hundreds to several thousand copies of each species. Knowledge about the number of rDNA loci and gene copy number provides information for comparative studies of organismal and molecular evolution at various phylogenetic levels. With the exception of seed plants, the range of 45S rDNA locus (encoding 18S, 5.8S and 26S rRNA) and gene copy number variation within key evolutionary plant groups is largely unknown. This is especially true for the three earliest land plant lineages Marchantiophyta (liverworts), Bryophyta (mosses), and Anthocerotophyta (hornworts). In this work, we report the extent of rDNA variation in early land plants, assessing the number of 45S rDNA loci and gene copy number in 106 species and 25 species, respectively, of mosses, liverworts and hornworts. Unexpectedly, the results show a narrow range of ribosomal locus variation (one or two 45S rDNA loci) and gene copies not present in vascular plant lineages, where a wide spectrum is recorded. Mutation analysis of whole genomic reads showed higher (3-fold) intragenomic heterogeneity of Marchantia polymorpha (Marchantiophyta) rDNA compared to Physcomitrella patens (Bryophyta) and two angiosperms (Arabidopsis thaliana and Nicotiana tomentosifomis) suggesting the presence of rDNA pseudogenes in its genome. No association between phylogenetic position, taxonomic adscription and the number of rDNA loci and gene copy number was found. Our results suggest a likely evolutionary rDNA stasis during land colonisation and diversification across 480 myr of bryophyte evolution. We hypothesise that strong selection forces may be acting against ribosomal gene locus amplification. Despite showing a predominant haploid phase and infrequent meiosis, overall rDNA homogeneity is not severely compromised in bryophytes.

  8. Conserved Organisation of 45S rDNA Sites and rDNA Gene Copy Number among Major Clades of Early Land Plants

    Science.gov (United States)

    Rosato, Marcela; Kovařík, Aleš; Garilleti, Ricardo; Rosselló, Josep A.

    2016-01-01

    Genes encoding ribosomal RNA (rDNA) are universal key constituents of eukaryotic genomes, and the nuclear genome harbours hundreds to several thousand copies of each species. Knowledge about the number of rDNA loci and gene copy number provides information for comparative studies of organismal and molecular evolution at various phylogenetic levels. With the exception of seed plants, the range of 45S rDNA locus (encoding 18S, 5.8S and 26S rRNA) and gene copy number variation within key evolutionary plant groups is largely unknown. This is especially true for the three earliest land plant lineages Marchantiophyta (liverworts), Bryophyta (mosses), and Anthocerotophyta (hornworts). In this work, we report the extent of rDNA variation in early land plants, assessing the number of 45S rDNA loci and gene copy number in 106 species and 25 species, respectively, of mosses, liverworts and hornworts. Unexpectedly, the results show a narrow range of ribosomal locus variation (one or two 45S rDNA loci) and gene copies not present in vascular plant lineages, where a wide spectrum is recorded. Mutation analysis of whole genomic reads showed higher (3-fold) intragenomic heterogeneity of Marchantia polymorpha (Marchantiophyta) rDNA compared to Physcomitrella patens (Bryophyta) and two angiosperms (Arabidopsis thaliana and Nicotiana tomentosifomis) suggesting the presence of rDNA pseudogenes in its genome. No association between phylogenetic position, taxonomic adscription and the number of rDNA loci and gene copy number was found. Our results suggest a likely evolutionary rDNA stasis during land colonisation and diversification across 480 myr of bryophyte evolution. We hypothesise that strong selection forces may be acting against ribosomal gene locus amplification. Despite showing a predominant haploid phase and infrequent meiosis, overall rDNA homogeneity is not severely compromised in bryophytes. PMID:27622766

  9. Integrated Analysis of Genome-Wide Copy Number Alterations and Gene Expression Profiling of Lung Cancer in Xuanwei, China

    Science.gov (United States)

    Zhang, Yanliang; Xue, Qiuyue; Pan, Guoqing; Meng, Qing H.; Tuo, Xiaoyu; Cai, Xuemei; Chen, Zhenghui; Li, Ya; Huang, Tao; Duan, Xincen; Duan, Yong

    2017-01-01

    Objectives Lung cancer in Xuanwei (LCXW), China, is known throughout the world for its distinctive characteristics, but little is known about its pathogenesis. The purpose of this study was to screen potential novel “driver genes” in LCXW. Methods Genome-wide DNA copy number alterations (CNAs) were detected by array-based comparative genomic hybridization and differentially expressed genes (DEGs) by gene expression microarrays in 8 paired LCXW and non-cancerous lung tissues. Candidate driver genes were screened by integrated analysis of CNAs and DEGs. The candidate genes were further validated by real-time quantitative polymerase chain reaction. Results Large numbers of CNAs and DEGs were detected, respectively. Some of the most frequently occurring CNAs included gains at 5p15.33-p15.32, 5p15.1-p14.3, and 5p14.3-p14.2 and losses at 11q24.3, 21q21.1, 21q22.12-q22.13, and 21q22.2. Integrated analysis of CNAs and DEGs identified 24 candidate genes with frequent copy number gains and concordant upregulation, which were considered potential oncogenes, including CREB3L4, TRIP13, and CCNE2. In addition, the analysis identified 19 candidate genes with a negative association between copy number change and expression change, considered potential tumor suppressor genes, including AHRR, NKD2, and KLF10. One of the most studied oncogenes, MYC, may not play a carcinogenic role in LCXW. Conclusions This integrated analysis of CNAs and DEGs identified several potential novel LCXW-related genes, laying an important foundation for further research on the pathogenesis of LCXW and identification of novel biomarkers or therapeutic targets. PMID:28056099

  10. mtDNA copy number in oocytes of different sizes from individual pre- and post-pubertal pigs

    DEFF Research Database (Denmark)

    Pedersen, Hanne Skovsgaard; Løvendahl, Peter; Larsen, Knud Erik

    2014-01-01

    Reproduction 131, 233–245). However, the correlation between size and mtDNA copy number in single oocytes has not been determined. This study describes the relation between oocytes of defined diameters from individual pre- and postpubertal pigs and mtDNA copy number. Cumulus-oocyte complexes were aspirated......Oocyte competence has been related to mtDNA copy number, but a large variation in mtDNA copy number between oocytes has been observed, caused by, e.g. oocyte donor and oocyte size (Sato et al. 2014 PLOS ONE 9, e94488; Cotterill et al. 2013 Mol. Hum. Reprod. 19, 444–450; El Shourbagy et al. 2006...... from ovaries of 10 pre- and 10 post-pubertal pigs. Cumulus cells were removed and the oocytes were measured (inside-ZP-diameter). Oocytes were transferred to DNAase-free tubes, snap-frozen, and stored at –80°C. The genes ND1 and COX1 were used to determine the mtDNA copy number. Plasmid preparations...

  11. Chloroplast DNA Copy Number May Link to Sex Determination in Leucadendron (Proteaceae

    Directory of Open Access Journals (Sweden)

    MADE PHARMAWATI

    2009-03-01

    Full Text Available Leucadendron (Proteaceae is a South African genus, the flowers of which have become a popular item in the Australian cut-flower industry. All species are dioecious. In general the female flowers are the more desirable as cut flowers. The availability of a molecular marker linked to sex determination is therefore needed both to maximize the efficiency of breeding programs and to supply markets with flowers from the preferred sex. The polymerase chain reaction-based method of suppression subtractive hybridization (SSH combined with mirror orientation selection (MOS were applied in an attempt to identify genome differences between male and female plants of Leucadendron discolor. Screening of 416 clones from a male-subtracted genomic DNA library and 282 clones from a female-subtracted library identified 13 candidates for male-specific genomic fragments. Sequence analyses of the 13 candidate DNA fragments showed that they were fragments of the chloroplast DNA, raising the possibility that chloroplast DNA copy number is linked to sex determination in Leucadendron.

  12. Germline DNA copy number aberrations identified as potential prognostic factors for breast cancer recurrence.

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    Yadav Sapkota

    Full Text Available Breast cancer recurrence (BCR is a common treatment outcome despite curative-intent primary treatment of non-metastatic breast cancer. Currently used prognostic and predictive factors utilize tumor-based markers, and are not optimal determinants of risk of BCR. Germline-based copy number aberrations (CNAs have not been evaluated as determinants of predisposition to experience BCR. In this study, we accessed germline DNA from 369 female breast cancer subjects who received curative-intent primary treatment following diagnosis. Of these, 155 experienced BCR and 214 did not, after a median duration of follow up after breast cancer diagnosis of 6.35 years (range = 0.60-21.78 and 8.60 years (range = 3.08-13.57, respectively. Whole genome CNA genotyping was performed on the Affymetrix SNP array 6.0 platform. CNAs were identified using the SNP-Fast Adaptive States Segmentation Technique 2 algorithm implemented in Nexus Copy Number 6.0. Six samples were removed due to poor quality scores, leaving 363 samples for further analysis. We identified 18,561 CNAs with ≥1 kb as a predefined cut-off for observed aberrations. Univariate survival analyses (log-rank tests identified seven CNAs (two copy number gains and five copy neutral-loss of heterozygosities, CN-LOHs showing significant differences (P<2.01×10(-5 in recurrence-free survival (RFS probabilities with and without CNAs.We also observed three additional but distinct CN-LOHs showing significant differences in RFS probabilities (P<2.86×10(-5 when analyses were restricted to stratified cases (luminal A, n = 208 only. After adjusting for tumor stage and grade in multivariate analyses (Cox proportional hazards models, all the CNAs remained strongly associated with the phenotype of BCR. Of these, we confirmed three CNAs at 17q11.2, 11q13.1 and 6q24.1 in representative samples using independent genotyping platforms. Our results suggest further investigations on the potential use of germline DNA

  13. Micro-Scale Genomic DNA Copy Number Aberrations as Another Means of Mutagenesis in Breast Cancer

    Science.gov (United States)

    Chao, Hann-Hsiang; He, Xiaping; Parker, Joel S.; Zhao, Wei; Perou, Charles M.

    2012-01-01

    Introduction In breast cancer, the basal-like subtype has high levels of genomic instability relative to other breast cancer subtypes with many basal-like-specific regions of aberration. There is evidence that this genomic instability extends to smaller scale genomic aberrations, as shown by a previously described micro-deletion event in the PTEN gene in the Basal-like SUM149 breast cancer cell line. Methods We sought to identify if small regions of genomic DNA copy number changes exist by using a high density, gene-centric Comparative Genomic Hybridizations (CGH) array on cell lines and primary tumors. A custom tiling array for CGH (244,000 probes, 200 bp tiling resolution) was created to identify small regions of genomic change, which was focused on previously identified basal-like-specific, and general cancer genes. Tumor genomic DNA from 94 patients and 2 breast cancer cell lines was labeled and hybridized to these arrays. Aberrations were called using SWITCHdna and the smallest 25% of SWITCHdna-defined genomic segments were called micro-aberrations (micro-aberrations, most of which are undetectable using typical-density genome-wide aCGH arrays. The basal-like subtype exhibited the highest incidence of these events. These micro-aberrations sometimes altered expression of the involved gene. We confirmed the presence of the PTEN micro-amplification in SUM149 and by mRNA-seq showed that this resulted in loss of expression of all exons downstream of this event. Micro-aberrations disproportionately affected the 5′ regions of the affected genes, including the promoter region, and high frequency of micro-aberrations was associated with poor survival. Conclusion Using a high-probe-density, gene-centric aCGH microarray, we present evidence of small-scale genomic aberrations that can contribute to gene inactivation. These events may contribute to tumor formation through mechanisms not detected using conventional DNA copy number analyses. PMID:23284754

  14. An evaluation of new and established methods to determine T-DNA copy number and homozygosity in transgenic plants.

    Science.gov (United States)

    Głowacka, Katarzyna; Kromdijk, Johannes; Leonelli, Lauriebeth; Niyogi, Krishna K; Clemente, Tom E; Long, Stephen P

    2016-04-01

    Stable transformation of plants is a powerful tool for hypothesis testing. A rapid and reliable evaluation method of the transgenic allele for copy number and homozygosity is vital in analysing these transformations. Here the suitability of Southern blot analysis, thermal asymmetric interlaced (TAIL-)PCR, quantitative (q)PCR and digital droplet (dd)PCR to estimate T-DNA copy number, locus complexity and homozygosity were compared in transgenic tobacco. Southern blot analysis and ddPCR on three generations of transgenic offspring with contrasting zygosity and copy number were entirely consistent, whereas TAIL-PCR often underestimated copy number. qPCR deviated considerably from the Southern blot results and had lower precision and higher variability than ddPCR. Comparison of segregation analyses and ddPCR of T1 progeny from 26 T0 plants showed that at least 19% of the lines carried multiple T-DNA insertions per locus, which can lead to unstable transgene expression. Segregation analyses failed to detect these multiple copies, presumably because of their close linkage. This shows the importance of routine T-DNA copy number estimation. Based on our results, ddPCR is the most suitable method, because it is as reliable as Southern blot analysis yet much faster. A protocol for this application of ddPCR to large plant genomes is provided.

  15. Variation in copy number of the 28S rDNA of Aspergillus fumigatus measured by droplet digital PCR and analog quantitative real-time PCR.

    Science.gov (United States)

    Alanio, Alexandre; Sturny-Leclère, Aude; Benabou, Marion; Guigue, Nicolas; Bretagne, Stéphane

    2016-08-01

    Droplet digital PCR (ddPCR) after DNA digestion yielded a 28S rDNA copy number of 61 to 86 copies/genome when testing 10 unrelated Aspergillus fumigatus isolates, higher than with quantitative PCR. Unfortunately, ddPCR after DNA digestion did not improve the sensitivity of our PCR assay when testing serum patients with invasive aspergillosis.

  16. Decreased peripheral mitochondrial DNA copy number is associated with the risk of heart failure and long-term outcome

    Institute of Scientific and Technical Information of China (English)

    HUANG Jin; TAN Lun; SHEN Ru-fei; ZHANG Lina; ZUO Hou-juan; WANG Dao-wen

    2016-01-01

    AIM:Mitochondrial DNA (mtDNA) copy number variation (CNV), which reflects the oxidant-induced cell damage, has been observed in a wide range of human diseases .However, whether it correlates with heart failure , which is closely related to oxidative stress, has never been elucidated before .We aimed to systematically investigate the association between leukocyte mtDNA CNV and heart failure risk and prognosis .METHODS: A total of 1 700 hospitalized patients with heart failure and 1 700 age-and gender-matched community population were consecutively enrolled in this observational study , as well as 1 638 ( 96.4%) patients were fol-lowed prospectively for a median of 17 months (12~24 months).The relative mtDNA copy number in leukocyte of peripheral blood or cardiac tissue was measured in triplicate by quantitative real-time PCR method .RESULTS:Patients with heart failure possessed much lower relative mtDNA copy number compared with control subjects (P mtDNA copy number depletion is an independent risk factor for heart failure and predicted higher risk of cardiovascular deaths in patients with heart failure .

  17. Jagged1 DNA Copy Number Variation Is Associated with Poor Outcome in Liver Cancer.

    Science.gov (United States)

    Kawaguchi, Kazunori; Honda, Masao; Yamashita, Taro; Okada, Hikari; Shirasaki, Takayoshi; Nishikawa, Masashi; Nio, Kouki; Arai, Kuniaki; Sakai, Yoshio; Yamashita, Tatsuya; Mizukoshi, Eishiro; Kaneko, Shuichi

    2016-08-01

    Notch signaling abnormalities are reported to be involved in the acceleration of malignancy in solid tumors and stem cell formation or regeneration in various organs. We analyzed specific genes for DNA copy number variations in liver cancer cells and investigated whether these factors relate to clinical outcome. Chromosome 20p, which includes the ligand for Notch pathways, Jagged1, was found to be amplified in several types of hepatoma cells, and its mRNA was up-regulated according to α-fetoprotein gene expression levels. Notch inhibition using Jagged1 shRNA and γ-secretase inhibitors produced significant suppression of cell growth in α-fetoprotein-producing cells with suppression of downstream genes. Using in vivo hepatoma models, the administration of γ-secretase inhibitors resulted in reduced tumor sizes and effective Notch inhibition with widespread apoptosis and necrosis of viable tumor cells. The γ-secretase inhibitors suppressed cell growth of the epithelial cell adhesion molecule-positive fraction in hepatoma cells, indicating that Notch inhibitors could suppress the stem cell features of liver cancer cells. Even in clinical liver cancer samples, the expression of α-fetoprotein and Jagged1 showed significant correlation, and amplification of the copy number of Jagged1 was associated with Jagged1 mRNA expression and poor survival after liver cancer surgical resection. In conclusion, amplification of Jagged1 contributed to mRNA expression that activates the Jagged1-Notch signaling pathway in liver cancer and led to poor outcome.

  18. Mitochondrial DNA copy number - but not a mitochondrial tandem CC to TT transition - is increased in sun-exposed skin.

    Science.gov (United States)

    Gebhard, Daniel; Mahler, Bettina; Matt, Katja; Burger, Katharina; Bergemann, Jörg

    2014-03-01

    Mitochondrial DNA (mtDNA) mutations are causatively associated with photo-ageing and are used as biomarkers of UV exposure. The most prominent mitochondrial mutation is the common deletion (CD), which is induced in many tissues by oxidative stress. More photo-specific mutations might be CC to TT tandem transitions which arise from UV-induced cyclobutane pyrimidine dimers. As nucleotide excision repair is absent in mitochondria, this DNA damage can presumably not be repaired resulting in high mitochondrial mutation levels. Here, we analysed levels of the CD, a mitochondrial and a chromosomal tandem transition in epidermis and dermis from exposed and less UV-exposed skin. We also analysed mtDNA copy number, for which changes as a result of oxidative stress have been described in different experimental settings. Whereas mitochondrial tandem transition levels were surprisingly low with no discernible correlation with UV exposure, mtDNA copy number and CD were significantly increased in UV-exposed samples.

  19. Low copy number DNA profiling from isolated sperm using the aureka®-micromanipulation system.

    Science.gov (United States)

    Schneider, C; Müller, U; Kilper, R; Siebertz, B

    2012-07-01

    A new cell isolation technique linked to the aureka® micromanipulation system (aureka®) was used to pick sperm from mixed samples containing sperm and epithelial cells. Both cell types were stained using the HY-LITER™ high-resolution, fluorescent staining kit. To isolate a single sperm of interest under a fluorescent microscope, a specific microsphere picking technique was used. This sensitive and reliable cell identification and isolation technique enables low-copy-number (LCN) DNA profiling, as few as 20 sperm are sufficient for obtaining a full short tandem repeat (STR) profile without any allelic drop out. The presented protocol covers the whole workflow, from sample staining and cell pick up to STR analysis.

  20. Integrated analyses of copy number variations and gene expression in lung adenocarcinoma.

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    Tzu-Pin Lu

    Full Text Available Numerous efforts have been made to elucidate the etiology and improve the treatment of lung cancer, but the overall five-year survival rate is still only 15%. Identification of prognostic biomarkers for lung cancer using gene expression microarrays poses a major challenge in that very few overlapping genes have been reported among different studies. To address this issue, we have performed concurrent genome-wide analyses of copy number variation and gene expression to identify genes reproducibly associated with tumorigenesis and survival in non-smoking female lung adenocarcinoma. The genomic landscape of frequent copy number variable regions (CNVRs in at least 30% of samples was revealed, and their aberration patterns were highly similar to several studies reported previously. Further statistical analysis for genes located in the CNVRs identified 475 genes differentially expressed between tumor and normal tissues (p<10(-5. We demonstrated the reproducibility of these genes in another lung cancer study (p = 0.0034, Fisher's exact test, and showed the concordance between copy number variations and gene expression changes by elevated Pearson correlation coefficients. Pathway analysis revealed two major dysregulated functions in lung tumorigenesis: survival regulation via AKT signaling and cytoskeleton reorganization. Further validation of these enriched pathways using three independent cohorts demonstrated effective prediction of survival. In conclusion, by integrating gene expression profiles and copy number variations, we identified genes/pathways that may serve as prognostic biomarkers for lung tumorigenesis.

  1. Copy number variation of ribosomal DNA and Pokey transposons in natural populations of Daphnia

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    Eagle Shannon HC

    2012-03-01

    possibility that many rDNA units do not contain a copy of both 18S and 28S genes suggests that rDNA is much more complicated than once thought, and warrants further study. In addition, the lack of correlation between rPokey, gPokey and rDNA unit numbers suggests that Pokey transposition rate is generally very low, and that recombination, in combination with natural selection, eliminates rPokey much faster than gPokey. Our results suggest that further research to determine the mechanisms by which Pokey has escaped complete inactivation by its host (the usual fate of DNA transposons, would provide important insights into transposon biology.

  2. DNA Copy Number Variants of Known Glaucoma Genes in Relation to Primary Open-Angle Glaucoma

    Science.gov (United States)

    Liu, Yutao; Garrett, Melanie E.; Yaspan, Brian L.; Bailey, Jessica Cooke; Loomis, Stephanie J.; Brilliant, Murray; Budenz, Donald L.; Christen, William G.; Fingert, John H.; Gaasterland, Douglas; Gaasterland, Terry; Kang, Jae H.; Lee, Richard K.; Lichter, Paul; Moroi, Sayoko E.; Realini, Anthony; Richards, Julia E.; Schuman, Joel S.; Scott, William K.; Singh, Kuldev; Sit, Arthur J.; Vollrath, Douglas; Weinreb, Robert; Wollstein, Gadi; Zack, Donald J.; Zhang, Kang; Pericak-Vance, Margaret A.; Haines, Jonathan L.; Pasquale, Louis R.; Wiggs, Janey L.; Allingham, R. Rand; Ashley-Koch, Allison E.; Hauser, Michael A.

    2014-01-01

    Purpose. We examined the role of DNA copy number variants (CNVs) of known glaucoma genes in relation to primary open angle glaucoma (POAG). Methods. Our study included DNA samples from two studies (NEIGHBOR and GLAUGEN). All the samples were genotyped with the Illumina Human660W_Quad_v1 BeadChip. After removing non–blood-derived and amplified DNA samples, we applied quality control steps based on the mean Log R Ratio and the mean B allele frequency. Subsequently, data from 3057 DNA samples (1599 cases and 1458 controls) were analyzed with PennCNV software. We defined CNVs as those ≥5 kilobases (kb) in size and interrogated by ≥5 consecutive probes. We further limited our investigation to CNVs in known POAG-related genes, including CDKN2B-AS1, TMCO1, SIX1/SIX6, CAV1/CAV2, the LRP12-ZFPM2 region, GAS7, ATOH7, FNDC3B, CYP1B1, MYOC, OPTN, WDR36, SRBD1, TBK1, and GALC. Results. Genomic duplications of CDKN2B-AS1 and TMCO1 were each found in a single case. Two cases carried duplications in the GAS7 region. Genomic deletions of SIX6 and ATOH7 were each identified in one case. One case carried a TBK1 deletion and another case carried a TBK1 duplication. No controls had duplications or deletions in these six genes. A single control had a duplication in the MYOC region. Deletions of GALC were observed in five cases and two controls. Conclusions. The CNV analysis of a large set of cases and controls revealed the presence of rare CNVs in known POAG susceptibility genes. Our data suggest that these rare CNVs may contribute to POAG pathogenesis and merit functional evaluation. PMID:25414181

  3. Prognostic impact of Epstein-Barr virus (EBV)-DNA copy number at diagnosis in chronic lymphocytic leukemia.

    Science.gov (United States)

    Liang, Jin-Hua; Gao, Rui; Xia, Yi; Gale, Robert Peter; Chen, Rui-Ze; Yang, Yu-Qiong; Wang, Li; Qu, Xiao-Yan; Qiu, Hai-Rong; Cao, Lei; Hong, Min; Wang, Rong; Wang, Yan; Fan, Lei; Chen, Yao-Yu; Hu, Zhi-Bin; Li, Jian-Yong; Xu, Wei

    2016-01-12

    Epstein-Barr virus (EBV)-DNA is detected in the blood of some persons with chronic lymphocytic leukemia (CLL) at diagnosis. Whether this is important in the development or progression of CLL is controversial. We interrogated associations between blood EBV-DNA copy number and biological and clinical variables in 243 new-diagnosed consecutive subjects with CLL. Quantification of EBV-DNA copies was done by real-time quantitative PCR (RQ-PCR). All subjects had serological evidence of prior EBV-infection. However, only 24 subjects (10%) had a EBV-DNA-positive test at diagnosis. EBV-DNA-positive subjects at diagnosis had lower hemoglobin concentrations and platelet levels, higher thymidine kinase-1 and serum ferritin levels, un-mutated IGHV genes and a greater risk of Richter transformation compared with EBV-DNA-negative subjects. Percent CD20-, CD148- and ZAP70-positive cells and mean fluorescence intensity (MFI) of each cluster designation were also increased in EBV-DNA-positive subjects at diagnosis. EBV-DNA test positivity was associated with a briefer time-to-treatment interval (HR 1.85; [95% confidence interval, 1.13, 3.03]; P=0.014) and worse survival (HR 2.77; [1.18, 6.49]; P=0.019). Reduction in EBV copies was significantly associated with therapy-response. A positive blood EBV-DNA test at diagnosis and sequential testing of EBV copies during therapy were significantly associated with biological and clinical variables, time-to-treatment, therapy-response and survival. If validated these data may be added to CLL prognostic scoring systems.

  4. Genetic variations related to maternal whole blood mitochondrial DNA copy number: a genome-wide and candidate gene study.

    Science.gov (United States)

    Workalemahu, Tsegaselassie; Enquobahrie, Daniel A; Tadesse, Mahlet G; Hevner, Karin; Gelaye, Bizu; Sanchez, Sixto E; Williams, Michelle A

    2017-10-01

    We conducted genome-wide (GWAS) and candidate gene association studies of maternal mitochondrial DNA copy number. Maternal peripheral blood was collected during labor and delivery admission from 471 participants of a placental abruption case-control study conducted in Lima, Peru. Single nucleotide polymorphism (SNP) genotyping was performed using the Illumina Cardio-Metabo Chip. Whole blood mitochondrial DNA (mtDNA) copy number was measured using qRT-PCR techniques. We evaluated 119,629 SNPs in the GWAS and 161 SNPs (in 29 mitochondrial biogenesis and oxidative phosphorylation genes) in the candidate association study. Top hits from GWAS and the candidate gene study were selected to compute weighted genetic risk scores (wGRS). Linear regression models were used to calculate effect size estimates and related nominal p values. The top hit in our GWAS was chr19:51063065 in FOXA3 (empirical p values = 2.20e - 6). A total of 134 SNPs had p values copy number (p values copy number was significantly associated with wGRS based on top GWAS hits (β = 0.49, 95% CI:0.38-0.60, p copy number.

  5. Integration of transcript expression, copy number and LOH analysis of infiltrating ductal carcinoma of the breast

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    Hawthorn Lesleyann

    2010-08-01

    Full Text Available Abstract Background A major challenge in the interpretation of genomic profiling data generated from breast cancer samples is the identification of driver genes as distinct from bystander genes which do not impact tumorigenesis. One way to assess the relative importance of alterations in the transcriptome profile is to combine parallel analyses that assess changes in the copy number alterations (CNAs. This integrated analysis permits the identification of genes with altered expression that map within specific chromosomal regions which demonstrate copy number alterations, providing a mechanistic approach to identify the 'driver genes'. Methods We have performed whole genome analysis of CNAs using the Affymetrix 250K Mapping array on 22 infiltrating ductal carcinoma samples (IDCs. Analysis of transcript expression alterations was performed using the Affymetrix U133 Plus2.0 array on 16 IDC samples. Fourteen IDC samples were analyzed using both platforms and the data integrated. We also incorporated data from loss of heterozygosity (LOH analysis to identify genes showing altered expression in LOH regions. Results Common chromosome gains and amplifications were identified at 1q21.3, 6p21.3, 7p11.2-p12.1, 8q21.11 and 8q24.3. A novel amplicon was identified at 5p15.33. Frequent losses were found at 1p36.22, 8q23.3, 11p13, 11q23, and 22q13. Over 130 genes were identified with concurrent increases or decreases in expression that mapped to these regions of copy number alterations. LOH analysis revealed three tumors with whole chromosome or p arm allelic loss of chromosome 17. Genes were identified that mapped to copy neutral LOH regions. LOH with accompanying copy loss was detected on Xp24 and Xp25 and genes mapping to these regions with decreased expression were identified. Gene expression data highlighted the PPARα/RXRα Activation Pathway as down-regulated in the tumor samples. Conclusion We have demonstrated the utility of the application of

  6. A Bayesian Analysis for Identifying DNA Copy Number Variations Using a Compound Poisson Process

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    Yiğiter Ayten

    2010-01-01

    Full Text Available To study chromosomal aberrations that may lead to cancer formation or genetic diseases, the array-based Comparative Genomic Hybridization (aCGH technique is often used for detecting DNA copy number variants (CNVs. Various methods have been developed for gaining CNVs information based on aCGH data. However, most of these methods make use of the log-intensity ratios in aCGH data without taking advantage of other information such as the DNA probe (e.g., biomarker positions/distances contained in the data. Motivated by the specific features of aCGH data, we developed a novel method that takes into account the estimation of a change point or locus of the CNV in aCGH data with its associated biomarker position on the chromosome using a compound Poisson process. We used a Bayesian approach to derive the posterior probability for the estimation of the CNV locus. To detect loci of multiple CNVs in the data, a sliding window process combined with our derived Bayesian posterior probability was proposed. To evaluate the performance of the method in the estimation of the CNV locus, we first performed simulation studies. Finally, we applied our approach to real data from aCGH experiments, demonstrating its applicability.

  7. Rapid detection of chromosome 18 copy number in buccal smears using DNA probes and FISH

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    Harris, C.; Nunez, M. [Univ. of Wisconsin, WI (United States); Giraldez, R. [ONCOR, Inc., Gaithersburg, MD (United States)

    1994-09-01

    Rapid diagnosis of trisomy 18 in newborns is often critical to clinical management decisions that must be made in a minimum of time. DNA probes combined with FISH can be used to accurately to determine the copy number of chromosome 18 in interphase cells. We have used the D18Z1 alpha satellite DNA probe to determine signal frequency in normal, previously karyotyped subjects, 12 females and 6 males. We also present one clinical case of trisomy 18, confirmed by karyotype, for comparison to the results obtained from normal subjects. Buccal smears, unlike cytogenetic preparations from peripheral blood, are quite resistant to penetration of probes and detection reagents resulting in higher levels of false monosomy. We have studied 19 individuals and have obtained consistent FISH results, ranging from 64 to 90% disomy. False monosomy rates ranged from 10 to 36%, while false trisomy or tetrasomy was less than 1% in all samples. High rates of false monosomy make this test questionable for detection of low order mosaicism for monosomy, but the extremely low false hyperploidy rate suggests that this is a dependable procedure for detection of trisomy 18, enabling the use of buccal epithelium which can be collected easily from even premature and tiny infants.

  8. DNA copy number changes in high-grade malignant peripheral nerve sheath tumors by array CGH

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    Bjerkehagen Bodil

    2008-06-01

    Full Text Available Abstract Background Malignant peripheral nerve sheath tumors (MPNSTs are rare and highly aggressive soft tissue tumors showing complex chromosomal aberrations. In order to identify recurrent chromosomal regions of gain and loss, and thereby novel gene targets of potential importance for MPNST development and/or progression, we have analyzed DNA copy number changes in seven high-grade MPNSTs using microarray-based comparative genomic hybridization (array CGH. Results Considerable more gains than losses were observed, and the most frequent minimal recurrent regions of gain included 1q24.1-q24.2, 1q24.3-q25.1, 8p23.1-p12, 9q34.11-q34.13 and 17q23.2-q25.3, all gained in five of seven samples. The 17q23.2-q25.3 region was gained in all five patients with poor outcome and not in the two patients with disease-free survival. cDNA microarray analysis and quantitative real-time reverse transcription PCR were used to investigate expression of genes located within these regions. The gene lysyl oxidase-like 2 (LOXL2 was identified as a candidate target for the 8p23.1-p12 gain. Within 17q, the genes topoisomerase II-α (TOP2A, ets variant gene 4 (E1A enhancer binding protein, E1AF (ETV4 and baculoviral IAP repeat-containing 5 (survivin (BIRC5 showed increased expression in all samples compared to two benign tumors. Increased expression of these genes has previously been associated with poor survival in other malignancies, and for TOP2A, in MPNSTs as well. In addition, we have analyzed the expression of five micro RNAs located within the 17q23.2-q25.3 region, but none of them showed high expression levels compared to the benign tumors. Conclusion Our study shows the potential of using DNA copy number changes obtained by array CGH to predict the prognosis of MPNST patients. Although no clear correlations between the expression level and patient outcome were observed, the genes TOP2A, ETV4 and BIRC5 are interesting candidate targets for the 17q gain associated

  9. Episomal and integrated maintenance of foreign DNA in Giardia lamblia.

    Science.gov (United States)

    Singer, S M; Yee, J; Nash, T E

    1998-04-01

    Giardia lamblia is an early diverging eukaryote which causes gastrointestinal disease throughout the world. Different subgroups of Giardia have been defined based on several biochemical and genetic criteria. We have developed a method for stably introducing DNA into the nuclei of the parasite using puromycin acetyltransferase (pac) as a dominant selectable marker. Transfected circular DNAs were maintained as episomes in the isolate WB, a representative of one Giardia subgroup. When input DNAs were linearized, integration was observed to occur by homologous recombination producing gene replacements in this isolate. In isolate GS, which represents a different subgroup, both linear and circular transfected DNAs were integrated into the genome by homologous recombination. In GS, linear DNA again produced gene replacements, while circular DNA produced duplicative integration events. The failure of GS to replicate episomes may reflect differences in the structure or recognition of DNA replication origins between these subgroups. A plasmid shuttle vector was also developed for expression of other genes in Giardia lamblia. Utilizing the green fluorescent protein as a reporter gene in the WB isolate, we show that gene expression from this vector correlated with plasmid copy number over a range of two orders of magnitude. Together these tools should greatly enhance our ability to study both the basic biology and the pathogenesis of this ubiquitous parasite.

  10. Mitochondrial DNA copy number in peripheral blood cell and hypertension risk among mining workers: a case-control study in Chinese coal miners.

    Science.gov (United States)

    Lei, L; Guo, J; Shi, X; Zhang, G; Kang, H; Sun, C; Huang, J; Wang, T

    2017-09-01

    Alteration of mitochondrial DNA (mtDNA) copy number, which reflects oxidant-induced cell damage, has been observed in a wide range of human diseases. However, whether it correlates with hypertension has not been elucidated. We aimed to explore the association between mtDNA copy number and the risk of hypertension in Chinese coal miners. A case-control study was performed with 378 hypertension patients and 325 healthy controls in a large coal mining group located in North China. Face-to-face interviews were conducted by trained staffs with necessary medical knowledge. The mtDNA copy number was measured by a quantitative real-time PCR assay using DNA extracted from peripheral blood. No significant differences in mtDNA copy number were observed between hypertension patients and healthy controls. However, in both case and control groups, the mtDNA copy number was statistically significantly lower in the elder population (≥45 years old) compared with the younger subjects (copy number could be found in hypertension patients consuming alcohol regularly compared with no alcohol consumption patients (7.09 vs 6.69); mtDNA copy number was also positively correlated with age and alcohol consumption. Hypertension was found significantly correlated with factors such as age, work duration, monthly family income and drinking status. Our results suggest that the mtDNA copy number is not associated with hypertension in coal miners.

  11. Construction and heterologous expression of a synthetic copy of the cutinase cDNA from Fusarium solani pisi

    NARCIS (Netherlands)

    Gemeren, I.A. van; Musters, W.; Hondel, C.A.M.J.J. van den; Verrips, C.T.

    1995-01-01

    A copy of the cutinase cDNA from Fusarium solani pisi was constructed starting from synthetic oligonucleotides. For this construction three separate cassettes were made, which were subsequently assembled to form the cutinase gene. Heterologous expression of the synthetic cutinase gene and the subseq

  12. Construction and heterologous expression of a synthetic copy of the cutinase cDNA from Fusarium solani pisi

    NARCIS (Netherlands)

    Gemeren, I.A. van; Musters, W.; Hondel, C.A.M.J.J. van den; Verrips, C.T.

    1995-01-01

    A copy of the cutinase cDNA from Fusarium solani pisi was constructed starting from synthetic oligonucleotides. For this construction three separate cassettes were made, which were subsequently assembled to form the cutinase gene. Heterologous expression of the synthetic cutinase gene and the

  13. Molecular cloning and expression of full-length DNA copies of the genomic RNAs of cowpea mosaic virus.

    NARCIS (Netherlands)

    Vos, P.A.J.

    1987-01-01

    The experiments described in this thesis were designed to unravel various aspects of the mechanism of gene expression of cowpea mosaic virus (CPMV). For this purpose full-length DNA copies of both genomic RNAs of CPMV were constructed. Using powerful invitro transcription systems RNA t

  14. Stability of single copy transgene expression in CHOK1 cells is affected by histone modifications but not by DNA methylation.

    Science.gov (United States)

    Spencer, Shawal; Gugliotta, Agustina; Koenitzer, Jennifer; Hauser, Hansjörg; Wirth, Dagmar

    2015-02-10

    Intraclonal heterogeneity of genetically modified mammalian cells has been observed as a phenomenon that has a strong impact on overall transgene expression levels and that limits the predictability of transgene expression in genetically modified cells, thereby hampering single cell based screening approaches. The underlying mechanism(s) leading to this variance are poorly understood. To study the dynamics and mechanisms of heterogeneity of early stage silencing we analyzed the expression in more than 100 independent clones of CHOK1 cells that harbour genetically stable integrates of single copy reporter cassettes driven by EF1α and CMV promoters. Single cell analysis showed intraclonal variability with heterogeneity in expression in genetically uniform populations. DNA methylation is a well known mechanism responsible for silencing of gene expression. Interestingly, loss of expression was not associated with DNA methylation of the CMV promoter. However, in most of the clonal populations expression could be increased by inhibitors of the histone deacetylases (HDACi) suggesting that heterogeneity of transgene expression is crucially governed by histone modifications. Further, to determine if the epigenetic status of transgene expression is governed by the chromosomal integration locus we targeted heterologous expression cassettes into two chromosomal sites using recombinase mediated cassette exchange (RMCE). The expression status of a particular clone was faithfully re-established when the same promoter used. In this way the problem of early stage cell clone instability can be bypassed. However, upon introduction of an unrelated promoter methylation-independent silencing was observed. Together, these results suggest that histone modifications are the relevant mechanisms by which epigenetic modulation of transgene expression cassettes is governed in the early phase of clone generation.

  15. Random DNA fragmentation allows detection of single-copy, single-exon alterations of copy number by oligonucleotide array CGH in clinical FFPE samples.

    Science.gov (United States)

    Hostetter, Galen; Kim, Su Young; Savage, Stephanie; Gooden, Gerald C; Barrett, Michael; Zhang, Jian; Alla, Lalitamba; Watanabe, April; Einspahr, Janine; Prasad, Anil; Nickoloff, Brian J; Carpten, John; Trent, Jeffrey; Alberts, David; Bittner, Michael

    2010-01-01

    Genomic technologies, such as array comparative genomic hybridization (aCGH), increasingly offer definitive gene dosage profiles in clinical samples. Historically, copy number profiling was limited to large fresh-frozen tumors where intact DNA could be readily extracted. Genomic analyses of pre-neoplastic tumors and diagnostic biopsies are often limited to DNA processed by formalin-fixation and paraffin-embedding (FFPE). We present specialized protocols for DNA extraction and processing from FFPE tissues utilizing DNase processing to generate randomly fragmented DNA. The protocols are applied to FFPE clinical samples of varied tumor types, from multiple institutions and of varied block age. Direct comparative analyses with regression coefficient were calculated on split-sample (portion fresh/portion FFPE) of colorectal tumor samples. We show equal detection of a homozygous loss of SMAD4 at the exon-level in the SW480 cell line and gene-specific alterations in the split tumor samples. aCGH application to a set of archival FFPE samples of skin squamous cell carcinomas detected a novel hemizygous deletion in INPP5A on 10q26.3. Finally we present data on derivative of log ratio, a particular sensitive detector of measurement variance, for 216 sequential hybridizations to assess protocol reliability over a wide range of FFPE samples.

  16. Intragenomic polymorphisms among high-copy loci: a genus-wide study of nuclear ribosomal DNA in Asclepias (Apocynaceae

    Directory of Open Access Journals (Sweden)

    Kevin Weitemier

    2015-01-01

    Full Text Available Despite knowledge that concerted evolution of high-copy loci is often imperfect, studies that investigate the extent of intragenomic polymorphisms and comparisons across a large number of species are rarely made. We present a bioinformatic pipeline for characterizing polymorphisms within an individual among copies of a high-copy locus. Results are presented for nuclear ribosomal DNA (nrDNA across the milkweed genus, Asclepias. The 18S-26S portion of the nrDNA cistron of Asclepias syriaca served as a reference for assembly of the region from 124 samples representing 90 species of Asclepias. Reads were mapped back to each individual’s consensus and at each position reads differing from the consensus were tallied using a custom perl script. Low frequency polymorphisms existed in all individuals (mean = 5.8%. Most nrDNA positions (91% were polymorphic in at least one individual, with polymorphic sites being less frequent in subunit regions and loops. Highly polymorphic sites existed in each individual, with highest abundance in the “noncoding” ITS regions. Phylogenetic signal was present in the distribution of intragenomic polymorphisms across the genus. Intragenomic polymorphisms in nrDNA are common in Asclepias, being found at higher frequency than any other study to date. The high and variable frequency of polymorphisms across species highlights concerns that phylogenetic applications of nrDNA may be error-prone. The new analytical approach provided here is applicable to other taxa and other high-copy regions characterized by low coverage genome sequencing (genome skimming.

  17. Intragenomic polymorphisms among high-copy loci: a genus-wide study of nuclear ribosomal DNA in Asclepias (Apocynaceae)

    Science.gov (United States)

    Straub, Shannon C.K.; Fishbein, Mark; Liston, Aaron

    2015-01-01

    Despite knowledge that concerted evolution of high-copy loci is often imperfect, studies that investigate the extent of intragenomic polymorphisms and comparisons across a large number of species are rarely made. We present a bioinformatic pipeline for characterizing polymorphisms within an individual among copies of a high-copy locus. Results are presented for nuclear ribosomal DNA (nrDNA) across the milkweed genus, Asclepias. The 18S-26S portion of the nrDNA cistron of Asclepias syriaca served as a reference for assembly of the region from 124 samples representing 90 species of Asclepias. Reads were mapped back to each individual’s consensus and at each position reads differing from the consensus were tallied using a custom perl script. Low frequency polymorphisms existed in all individuals (mean = 5.8%). Most nrDNA positions (91%) were polymorphic in at least one individual, with polymorphic sites being less frequent in subunit regions and loops. Highly polymorphic sites existed in each individual, with highest abundance in the “noncoding” ITS regions. Phylogenetic signal was present in the distribution of intragenomic polymorphisms across the genus. Intragenomic polymorphisms in nrDNA are common in Asclepias, being found at higher frequency than any other study to date. The high and variable frequency of polymorphisms across species highlights concerns that phylogenetic applications of nrDNA may be error-prone. The new analytical approach provided here is applicable to other taxa and other high-copy regions characterized by low coverage genome sequencing (genome skimming). PMID:25653903

  18. DNA COPY PROFILE IN NASOPHARYNGEAL CARCINOMA AND ITS CORRELATION WITH CLINICAL STAGING

    Institute of Scientific and Technical Information of China (English)

    鄢践; 方嬿; 梁启万; 曾益新

    2001-01-01

    To detect genetic alterations in nasopharyngeal carcinoma (NPC) in Cantonese, the population with the highest incidence of NPC, and to correlate the findings with clinical staging. Methods: Comparative genomic hybridization (CGH) was performed on 35 primary nasopharyngeal carcinomas and a nonparametric χ2 test was used to analyze relationship between chromosome changes and clinical staging. Results: The identified common chromosomal alterations in NPC included gain of chromosomes 12q (21 cases, 60%), 4q (19cases, 43%), 3q (18 cases, 51%), 1q (15 cases, 43%),8q (14 cases, 40%), and 2q (12 cases, 30%). The most frequently detected loss of chromosomal materials involved chromosome 1p (24 cases, 69%), chromosome 3p (21 cases, 60%), 11q (20 cases, 57%), 14q (18 cases, 51%), 16q (14 cases, 40%), 13(12 cases, 34%), and 9p(11 cases, 31%). The high frequency (>50%) 4q gain and 1p loss were novel findings. Compared by nonparametric χ2 test, gains on 12q and 8q were found mainly in stages Ⅲ/Ⅳ and there were significant differences between two clinical stage groups ( stagesⅠ/Ⅱvs stages Ⅲ/Ⅳ). Conclusions: Current analysis has revealed a comprehensive profile of the chromosomal regions showing DNA copy number changes, which may harbor oncogenes or tumor suppressor genes involved in the development of primary NPC.

  19. Effects of Integrating and Non-Integrating Reprogramming Methods on Copy Number Variation and Genomic Stability of Human Induced Pluripotent Stem Cells.

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    Xiangjin Kang

    Full Text Available Human-induced pluripotent stem cells (iPSCs are derived from differentiated somatic cells using defined factors and provide a renewable source of autologous cells for cell therapy. Many reprogramming methods have been employed to generate human iPSCs, including the use of integrating vectors and non-integrating vectors. Maintenance of the genomic integrity of iPSCs is highly desirable if the cells are to be used in clinical applications. Here, using the Affymetrix Cytoscan HD array, we investigated the genomic aberration profiles of 19 human cell lines: 5 embryonic stem cell (ESC lines, 6 iPSC lines derived using integrating vectors ("integrating iPSC lines", 6 iPSC lines derived using non-integrating vectors ("non-integrating iPSC lines", and the 2 parental cell lines from which the iPSCs were derived. The genome-wide copy number variation (CNV, loss of heterozygosity (LOH and mosaicism patterns of integrating and non-integrating iPSC lines were investigated. The maximum sizes of CNVs in the genomes of the integrating iPSC lines were 20 times higher than those of the non-integrating iPSC lines. Moreover, the total number of CNVs was much higher in integrating iPSC lines than in other cell lines. The average numbers of novel CNVs with a low degree of overlap with the DGV and of likely pathogenic CNVs with a high degree of overlap with the ISCA (International Symposium on Computer Architecture database were highest in integrating iPSC lines. Different single nucleotide polymorphisms (SNP calls revealed that, using the parental cell genotype as a reference, integrating iPSC lines displayed more single nucleotide variations and mosaicism than did non-integrating iPSC lines. This study describes the genome stability of human iPSCs generated using either a DNA-integrating or non-integrating reprogramming method, of the corresponding somatic cells, and of hESCs. Our results highlight the importance of using a high-resolution method to monitor genomic

  20. Effects of Integrating and Non-Integrating Reprogramming Methods on Copy Number Variation and Genomic Stability of Human Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Kang, Xiangjin; Yu, Qian; Huang, Yuling; Song, Bing; Chen, Yaoyong; Gao, Xingcheng; He, Wenyin; Sun, Xiaofang; Fan, Yong

    2015-01-01

    Human-induced pluripotent stem cells (iPSCs) are derived from differentiated somatic cells using defined factors and provide a renewable source of autologous cells for cell therapy. Many reprogramming methods have been employed to generate human iPSCs, including the use of integrating vectors and non-integrating vectors. Maintenance of the genomic integrity of iPSCs is highly desirable if the cells are to be used in clinical applications. Here, using the Affymetrix Cytoscan HD array, we investigated the genomic aberration profiles of 19 human cell lines: 5 embryonic stem cell (ESC) lines, 6 iPSC lines derived using integrating vectors ("integrating iPSC lines"), 6 iPSC lines derived using non-integrating vectors ("non-integrating iPSC lines"), and the 2 parental cell lines from which the iPSCs were derived. The genome-wide copy number variation (CNV), loss of heterozygosity (LOH) and mosaicism patterns of integrating and non-integrating iPSC lines were investigated. The maximum sizes of CNVs in the genomes of the integrating iPSC lines were 20 times higher than those of the non-integrating iPSC lines. Moreover, the total number of CNVs was much higher in integrating iPSC lines than in other cell lines. The average numbers of novel CNVs with a low degree of overlap with the DGV and of likely pathogenic CNVs with a high degree of overlap with the ISCA (International Symposium on Computer Architecture) database were highest in integrating iPSC lines. Different single nucleotide polymorphisms (SNP) calls revealed that, using the parental cell genotype as a reference, integrating iPSC lines displayed more single nucleotide variations and mosaicism than did non-integrating iPSC lines. This study describes the genome stability of human iPSCs generated using either a DNA-integrating or non-integrating reprogramming method, of the corresponding somatic cells, and of hESCs. Our results highlight the importance of using a high-resolution method to monitor genomic aberrations

  1. The FACT Complex Promotes Avian Leukosis Virus DNA Integration.

    Science.gov (United States)

    Winans, Shelby; Larue, Ross C; Abraham, Carly M; Shkriabai, Nikolozi; Skopp, Amelie; Winkler, Duane; Kvaratskhelia, Mamuka; Beemon, Karen L

    2017-04-01

    All retroviruses need to integrate a DNA copy of their genome into the host chromatin. Cellular proteins regulating and targeting lentiviral and gammaretroviral integration in infected cells have been discovered, but the factors that mediate alpharetroviral avian leukosis virus (ALV) integration are unknown. In this study, we have identified the FACT protein complex, which consists of SSRP1 and Spt16, as a principal cellular binding partner of ALV integrase (IN). Biochemical experiments with purified recombinant proteins show that SSRP1 and Spt16 are able to individually bind ALV IN, but only the FACT complex effectively stimulates ALV integration activity in vitro Likewise, in infected cells, the FACT complex promotes ALV integration activity, with proviral integration frequency varying directly with cellular expression levels of the FACT complex. An increase in 2-long-terminal-repeat (2-LTR) circles in the depleted FACT complex cell line indicates that this complex regulates the ALV life cycle at the level of integration. This regulation is shown to be specific to ALV, as disruption of the FACT complex did not inhibit either lentiviral or gammaretroviral integration in infected cells.IMPORTANCE The majority of human gene therapy approaches utilize HIV-1- or murine leukemia virus (MLV)-based vectors, which preferentially integrate near genes and regulatory regions; thus, insertional mutagenesis is a substantial risk. In contrast, ALV integrates more randomly throughout the genome, which decreases the risks of deleterious integration. Understanding how ALV integration is regulated could facilitate the development of ALV-based vectors for use in human gene therapy. Here we show that the FACT complex directly binds and regulates ALV integration efficiency in vitro and in infected cells. Copyright © 2017 American Society for Microbiology.

  2. The relationship between leukocyte mitochondrial DNA copy number and telomere length in community-dwelling elderly women.

    Directory of Open Access Journals (Sweden)

    Jung-Ha Kim

    Full Text Available PURPOSE: Both telomere length and mitochondrial function are accepted as reflective indices of aging. Recent studies have shown that telomere dysfunction may influence impaired mitochondrial biogenesis and function. However, there has been no study regarding the possible association between telomere and mitochondrial function in humans. Therefore, the purpose of the study was to identify any relationships between mitochondrial and telomere function. METHODS: The present study included 129 community-dwelling, elderly women. The leukocyte mitochondrial DNA copy number and telomere length were measured using a quantitative real-time polymerase chain reaction method. Anthropometric measurement, biochemical blood testing, a depression screening questionnaire using a 15-question geriatric depression scale (GDS-15, and a cognitive function test using the Korean version of the mini mental state examination (K-MMSE were performed. RESULTS: Leukocyte mtDNA copy number was positively associated with telomere length (r=0.39, p=<0.0001 and K-MMSE score (r=0.06, p=0.02. Additionally, leukocyte mtDNA copy number was negatively correlated with GDS-15 score (r=-0.17, p=0.04. Age (r=-0.15, p=0.09, waist circumference (r=-0.16, p=0.07, and serum ferritin level (r=-0.13, p=0.07 tended to be inversely correlated with leukocyte mtDNA copy number. With a stepwise multiple regression analysis, telomere length was found to be an independent factor associated with leukocyte mtDNA copy number after adjustment for confounding variables including age, body mass index, waist circumference, total cholesterol, HDL-cholesterol, LDL-cholesterol, triglycerides, hs-CRP, serum ferritin, HOMA-IR, K-MMSE, GDS-15, hypertension, diabetes, dyslipidemia, currently smoking, alcohol drinking, and regular exercise. CONCLUSIONS: This study showed that leukocyte mtDNA copy number was positively correlated with leukocyte telomere length in community-dwelling elderly women. Our findings suggest

  3. Increased mitochondrial DNA copy number in occupations associated with low-dose benzene exposure.

    Science.gov (United States)

    Carugno, Michele; Pesatori, Angela Cecilia; Dioni, Laura; Hoxha, Mirjam; Bollati, Valentina; Albetti, Benedetta; Byun, Hyang-Min; Bonzini, Matteo; Fustinoni, Silvia; Cocco, Pierluigi; Satta, Giannina; Zucca, Mariagrazia; Merlo, Domenico Franco; Cipolla, Massimo; Bertazzi, Pier Alberto; Baccarelli, Andrea

    2012-02-01

    Benzene is an established leukemogen at high exposure levels. Although low-level benzene exposure is widespread and may induce oxidative damage, no mechanistic biomarkers are available to detect biological dysfunction at low doses. Our goals were to determine in a large multicenter cross-sectional study whether low-level benzene is associated with increased blood mitochondrial DNA copy number (mtDNAcn, a biological oxidative response to mitochondrial DNA damage and dysfunction) and to explore potential links between mtDNAcn and leukemia-related epigenetic markers. We measured blood relative mtDNAcn by real-time polymerase chain reaction in 341 individuals selected from various occupational groups with low-level benzene exposures (> 100 times lower than the Occupational Safety and Health Administration/European Union standards) and 178 referents from three Italian cities (Genoa, Milan, Cagliari). In each city, benzene-exposed participants showed higher mtDNAcn than referents: mtDNAcn was 0.90 relative units in Genoa bus drivers and 0.75 in referents (p = 0.019); 0.90 in Milan gas station attendants, 1.10 in police officers, and 0.75 in referents (p-trend = 0.008); 1.63 in Cagliari petrochemical plant workers, 1.25 in referents close to the plant, and 0.90 in referents farther from the plant (p-trend = 0.046). Using covariate-adjusted regression models, we estimated that an interquartile range increase in personal airborne benzene was associated with percent increases in mtDNAcn equal to 10.5% in Genoa (p = 0.014), 8.2% (p = 0.008) in Milan, 7.5% in Cagliari (p = 0.22), and 10.3% in all cities combined (p < 0.001). Using methylation data available for the Milan participants, we found that mtDNAcn was associated with LINE-1 hypomethylation (-2.41%; p = 0.007) and p15 hypermethylation (+15.95%, p = 0.008). Blood MtDNAcn was increased in persons exposed to low benzene levels, potentially reflecting mitochondrial DNA damage and dysfunction.

  4. Genome-wide copy number profiling of single cells in S-phase reveals DNA-replication domains.

    Science.gov (United States)

    Van der Aa, Niels; Cheng, Jiqiu; Mateiu, Ligia; Zamani Esteki, Masoud; Kumar, Parveen; Dimitriadou, Eftychia; Vanneste, Evelyne; Moreau, Yves; Vermeesch, Joris Robert; Voet, Thierry

    2013-04-01

    Single-cell genomics is revolutionizing basic genome research and clinical genetic diagnosis. However, none of the current research or clinical methods for single-cell analysis distinguishes between the analysis of a cell in G1-, S- or G2/M-phase of the cell cycle. Here, we demonstrate by means of array comparative genomic hybridization that charting the DNA copy number landscape of a cell in S-phase requires conceptually different approaches to that of a cell in G1- or G2/M-phase. Remarkably, despite single-cell whole-genome amplification artifacts, the log2 intensity ratios of single S-phase cells oscillate according to early and late replication domains, which in turn leads to the detection of significantly more DNA imbalances when compared with a cell in G1- or G2/M-phase. Although these DNA imbalances may, on the one hand, be falsely interpreted as genuine structural aberrations in the S-phase cell's copy number profile and hence lead to misdiagnosis, on the other hand, the ability to detect replication domains genome wide in one cell has important applications in DNA-replication research. Genome-wide cell-type-specific early and late replicating domains have been identified by analyses of DNA from populations of cells, but cell-to-cell differences in DNA replication may be important in genome stability, disease aetiology and various other cellular processes.

  5. Wild-Type Mitochondrial DNA Copy Number in Urinary Cells as a Useful Marker for Diagnosing Severity of the Mitochondrial Diseases.

    Directory of Open Access Journals (Sweden)

    Hui Liu

    Full Text Available The genotype-phenotype relationship in diseases with mtDNA point mutations is still elusive. The maintenance of wild-type mtDNA copy number is essential to the normal mitochondrial oxidative function. This study examined the relationship between mtDNA copy number in blood and urine and disease severity of the patients harboring A3243G mutation. We recruited 115 A3243G patients, in which 28 were asymptomatic, 42 were oligo-symptomatic, and 45 were poly-symptomatic. Increase of total mtDNA copy number without correlation to the proportion of mutant mtDNA was found in the A3243G patients. Correlation analyses revealed that wild-type mtDNA copy number in urine was the most important factor correlated to disease severity, followed by proportion of mutant mtDNA in urine and proportion of mutant mtDNA in blood. Wild-type copy number in urine negatively correlated to the frequencies of several major symptoms including seizures, myopathy, learning disability, headache and stroke, but positively correlated to the frequencies of hearing loss and diabetes. Besides proportion of mutant mtDNA in urine, wild-type copy number in urine is also an important marker for disease severity of A3243G patients.

  6. Disentangling sources of variation in SSU rDNA sequences from single cell analyses of ciliates: impact of copy number variation and experimental error.

    Science.gov (United States)

    Wang, Chundi; Zhang, Tengteng; Wang, Yurui; Katz, Laura A; Gao, Feng; Song, Weibo

    2017-07-26

    Small subunit ribosomal DNA (SSU rDNA) is widely used for phylogenetic inference, barcoding and other taxonomy-based analyses. Recent studies indicate that SSU rDNA of ciliates may have a high level of sequence variation within a single cell, which impacts the interpretation of rDNA-based surveys. However, sequence variation can come from a variety of sources including experimental errors, especially the mutations generated by DNA polymerase in PCR. In the present study, we explore the impact of four DNA polymerases on sequence variation and find that low-fidelity polymerases exaggerate the estimates of single-cell sequence variation. Therefore, using a polymerase with high fidelity is essential for surveys of sequence variation. Another source of variation results from errors during amplification of SSU rDNA within the polyploidy somatic macronuclei of ciliates. To investigate further the impact of SSU rDNA copy number variation, we use a high-fidelity polymerase to examine the intra-individual SSU rDNA polymorphism in ciliates with varying levels of macronuclear amplification: Halteria grandinella, Blepharisma americanum and Strombidium stylifer We estimate the rDNA copy numbers of these three species by single-cell quantitative PCR. The results indicate that: (i) sequence variation of SSU rDNA within a single cell is authentic in ciliates, but the level of intra-individual SSU rDNA polymorphism varies greatly among species; (ii) rDNA copy numbers vary greatly among species, even those within the same class; (iii) the average rDNA copy number of Halteria grandinella is about 567 893 (s.d. = 165 481), which is the highest record of rDNA copy number in ciliates to date; and (iv) based on our data and the records from previous studies, it is not always true in ciliates that rDNA copy numbers are positively correlated with cell or genome size. © 2017 The Author(s).

  7. iGC-an integrated analysis package of gene expression and copy number alteration.

    Science.gov (United States)

    Lai, Yi-Pin; Wang, Liang-Bo; Wang, Wei-An; Lai, Liang-Chuan; Tsai, Mong-Hsun; Lu, Tzu-Pin; Chuang, Eric Y

    2017-01-14

    With the advancement in high-throughput technologies, researchers can simultaneously investigate gene expression and copy number alteration (CNA) data from individual patients at a lower cost. Traditional analysis methods analyze each type of data individually and integrate their results using Venn diagrams. Challenges arise, however, when the results are irreproducible and inconsistent across multiple platforms. To address these issues, one possible approach is to concurrently analyze both gene expression profiling and CNAs in the same individual. We have developed an open-source R/Bioconductor package (iGC). Multiple input formats are supported and users can define their own criteria for identifying differentially expressed genes driven by CNAs. The analysis of two real microarray datasets demonstrated that the CNA-driven genes identified by the iGC package showed significantly higher Pearson correlation coefficients with their gene expression levels and copy numbers than those genes located in a genomic region with CNA. Compared with the Venn diagram approach, the iGC package showed better performance. The iGC package is effective and useful for identifying CNA-driven genes. By simultaneously considering both comparative genomic and transcriptomic data, it can provide better understanding of biological and medical questions. The iGC package's source code and manual are freely available at https://www.bioconductor.org/packages/release/bioc/html/iGC.html .

  8. Multiplex Ligation-Dependent Probe Amplification Technique for Copy Number Analysis on Small Amounts of DNA Material

    DEFF Research Database (Denmark)

    Sørensen, Karina; Andersen, Paal; Larsen, Lars;

    2008-01-01

    The multiplex ligation-dependent probe amplification (MLPA) technique is a sensitive technique for relative quantification of up to 50 different nucleic acid sequences in a single reaction, and the technique is routinely used for copy number analysis in various syndromes and diseases. The aim...... of the study was to exploit the potential of MLPA when the DNA material is limited. The DNA concentration required in standard MLPA analysis is not attainable from dried blood spot samples (DBSS) often used in neonatal screening programs. A novel design of MLPA probes has been developed to permit for MLPA...... analysis on small amounts of DNA. Six patients with congenital adrenal hyperplasia (CAH) were used in this study. DNA was extracted from both whole blood and DBSS and subjected to MLPA analysis using normal and modified probes. Results were analyzed using GeneMarker and manual Excel analysis. A total...

  9. Advances in Research on Hepatitis B Virus DNA Integration

    Institute of Scientific and Technical Information of China (English)

    Ju-sheng LIN; Lin-lin GAO

    2008-01-01

    Since HBV DNA integration was discovered for the first time in 1980, various methods have been used to detect and study it, such as Southern Blot, in situ hybridization, polymerase chain reaction and so on. HBV DNA integration is thought to be random on the whole although some hot spots of integration were described by some researchers, one of which might be the repetitive sequences of the genomic DNA. Besides, DNA damage, especially double-strand breaks could promote HBV DNA integration into host genome. HBV DNA integration into cells may damage the stability of the genome, cause DNA rearrangement, promote DNA deletion and induce the formation of HCC.

  10. Ribosomal DNA copy number amplification and loss in human cancers is linked to tumor genetic context, nucleolus activity, and proliferation

    Science.gov (United States)

    2017-01-01

    Ribosomal RNAs (rRNAs) are transcribed from two multicopy DNA arrays: the 5S ribosomal DNA (rDNA) array residing in a single human autosome and the 45S rDNA array residing in five human autosomes. The arrays are among the most variable segments of the genome, exhibit concerted copy number variation (cCNV), encode essential components of the ribosome, and modulate global gene expression. Here we combined whole genome data from >700 tumors and paired normal tissues to provide a portrait of rDNA variation in human tissues and cancers of diverse mutational signatures, including stomach and lung adenocarcinomas, ovarian cancers, and others of the TCGA panel. We show that cancers undergo coupled 5S rDNA array expansion and 45S rDNA loss that is accompanied by increased estimates of proliferation rate and nucleolar activity. These somatic changes in rDNA CN occur in a background of over 10-fold naturally occurring rDNA CN variation across individuals and cCNV of 5S-45S arrays in some but not all tissues. Analysis of genetic context revealed associations between cancer rDNA CN amplification or loss and the presence of specific somatic alterations, including somatic SNPs and copy number gain/losses in protein coding genes across the cancer genome. For instance, somatic inactivation of the tumor suppressor gene TP53 emerged with a strong association with coupled 5S expansion / 45S loss in several cancers. Our results uncover frequent and contrasting changes in the 5S and 45S rDNA along rapidly proliferating cell lineages with high nucleolar activity. We suggest that 5S rDNA amplification facilitates increased proliferation, nucleolar activity, and ribosomal synthesis in cancer, whereas 45S rDNA loss emerges as a byproduct of transcription-replication conflict in rapidly replicating tumor cells. The observations raise the prospects of using the rDNA arrays as re-emerging targets for the design of novel strategies in cancer therapy. PMID:28880866

  11. Analysis of T-DNA integration and generative segregation in transgenic winter triticale (x Triticosecale Wittmack

    Directory of Open Access Journals (Sweden)

    Hensel Goetz

    2012-09-01

    Full Text Available Abstract Background While the genetic transformation of the major cereal crops has become relatively routine, to date only a few reports were published on transgenic triticale, and robust data on T-DNA integration and segregation have not been available in this species. Results Here, we present a comprehensive analysis of stable transgenic winter triticale cv. Bogo carrying the selectable marker gene HYGROMYCIN PHOSPHOTRANSFERASE (HPT and a synthetic green fluorescent protein gene (gfp. Progeny of four independent transgenic plants were comprehensively investigated with regard to the number of integrated T-DNA copies, the number of plant genomic integration loci, the integrity and functionality of individual T-DNA copies, as well as the segregation of transgenes in T1 and T2 generations, which also enabled us to identify homozygous transgenic lines. The truncation of some integrated T-DNAs at their left end along with the occurrence of independent segregation of multiple T-DNAs unintendedly resulted in a single-copy segregant that is selectable marker-free and homozygous for the gfp gene. The heritable expression of gfp driven by the maize UBI-1 promoter was demonstrated by confocal laser scanning microscopy. Conclusions The used transformation method is a valuable tool for the genetic engineering of triticale. Here we show that comprehensive molecular analyses are required for the correct interpretation of phenotypic data collected from the transgenic plants.

  12. Specific functions of the Rep and Rep' proteins of porcine circovirus during copy-release and rolling-circle DNA replication

    Science.gov (United States)

    The roles of two porcine circovirus replication initiator proteins, Rep and Rep', in generating copy-release and rolling-circle DNA replication intermediates were determined. Rep uses the supercoiled closed-circular genome (ccc) to initiate leading-strand synthesis (identical to copy-release replica...

  13. Inverse PCR and Quantitative PCR as Alternative Methods to Southern Blotting Analysis to Assess Transgene Copy Number and Characterize the Integration Site in Transgenic Woody Plants.

    Science.gov (United States)

    Stefano, Biricolti; Patrizia, Bogani; Matteo, Cerboneschi; Massimo, Gori

    2016-06-01

    One of the major unanswered questions with respect to the commercial use of genetic transformation in woody plants is the stability of the transgene expression over several decades within the same individual. Gene expression is strongly affected by the copy number which has been integrated into the plant genome and by the local DNA features close to the integration sites. Because woody plants cannot be subjected to selfing or backcrossing to modify the transgenic allelic structure without affecting the valuable traits of the cultivar, molecular characterization of the transformation event is therefore crucial. After assessing the transgene copy number of a set of apple transgenic clones with Southern blotting, we describe two alternative methods: the first is based on inverse PCR (i-PCR) and the second on the quantitative PCR (q-PCR). The methods produced comparable results with the exception of the data regarding a high copy number clone, but while the q-PCR-based system is rapid and easily adaptable to high throughput systems, the i-PCR-based method can provide information regarding the transformation event and the characteristics of the sequences flanking the transgenic construct.

  14. Relation of Mitochondrial DNA Copy Number in Peripheral Blood to Postoperative Atrial Fibrillation After Isolated Off-Pump Coronary Artery Bypass Grafting.

    Science.gov (United States)

    Zhang, Jian; Xu, Shu; Xu, Yinli; Liu, Yu; Li, Zhi; Zhang, Yuji; Jin, Yan; Xue, Xiaodong; Wang, Huishan

    2017-02-01

    Oxidative stress has been considered to be an important factor contributing to postoperative atrial fibrillation (PoAF). Mitochondrial DNA (mtDNA) copy number in peripheral blood has been found to be associated with a patient's oxidative stress. Therefore, we sought to determine whether there was association between mtDNA copy number and the onset of atrial fibrillation. mtDNA copy numbers were measured using the quantitative real-time polymerase chain reaction in peripheral blood from 485 consecutive patients with sinus rhythm undergoing coronary artery bypass grafting. The blood was collected before surgery. In the cohort, the incidence of PoAF was 20.8% (101/485). The mean mtDNA copy number was significantly higher in patents with PoAF than in those with sinus rhythm (36.43 vs 16.63, p copy number could predict PoAF with good sensitivity and specificity (area under the curve = 0.814, cutoff = 20.91, sensitivity = 70.3%, specificity = 80.2%, p copy number was shown to be a significant independent risk factor for PoAF (odds ratio = 10.01, p copy number and malondialdehyde in patients with PoAF (r = 0.449, p = 0.01). In conclusion, we showed that elevated mtDNA copy number in peripheral blood is associated with PoAF. Further investigation is needed to validate mtDNA copy number as a predictive biomarker for PoAF and to explore its potential role in arrhythmogenesis. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Removal of Contaminant DNA by Combined UV-EMA Treatment Allows Low Copy Number Detection of Clinically Relevant Bacteria Using Pan-Bacterial Real-Time PCR.

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    Bruce Humphrey

    Full Text Available More than two decades after its discovery, contaminant microbial DNA in PCR reagents continues to impact the sensitivity and integrity of broad-range PCR diagnostic techniques. This is particularly relevant to their use in the setting of human sepsis, where a successful diagnostic on blood samples needs to combine universal bacterial detection with sensitivity to 1-2 genome copies, because low levels of a broad range of bacteria are implicated.We investigated the efficacy of ethidium monoazide (EMA and propidium monoazide (PMA treatment as emerging methods for the decontamination of PCR reagents. Both treatments were able to inactivate contaminating microbial DNA but only at concentrations that considerably affected assay sensitivity. Increasing amplicon length improved EMA/PMA decontamination efficiency but at the cost of assay sensitivity. The same was true for UV exposure as an alternative decontamination strategy, likely due to damage sustained by oligonucleotide primers which were a significant source of contamination. However, a simple combination strategy with UV-treated PCR reagents paired with EMA-treated primers produced an assay capable of two genome copy detection and a <5% contamination rate. This decontamination strategy could have important utility in developing improved pan-bacterial assays for rapid diagnosis of low pathogen burden conditions such as in the blood of patients with suspected blood stream infection.

  16. Integrated analysis of gene expression, CpG island methylation, and gene copy number in breast cancer cells by deep sequencing.

    Directory of Open Access Journals (Sweden)

    Zhifu Sun

    Full Text Available We used deep sequencing technology to profile the transcriptome, gene copy number, and CpG island methylation status simultaneously in eight commonly used breast cell lines to develop a model for how these genomic features are integrated in estrogen receptor positive (ER+ and negative breast cancer. Total mRNA sequence, gene copy number, and genomic CpG island methylation were carried out using the Illumina Genome Analyzer. Sequences were mapped to the human genome to obtain digitized gene expression data, DNA copy number in reference to the non-tumor cell line (MCF10A, and methylation status of 21,570 CpG islands to identify differentially expressed genes that were correlated with methylation or copy number changes. These were evaluated in a dataset from 129 primary breast tumors. Gene expression in cell lines was dominated by ER-associated genes. ER+ and ER- cell lines formed two distinct, stable clusters, and 1,873 genes were differentially expressed in the two groups. Part of chromosome 8 was deleted in all ER- cells and part of chromosome 17 amplified in all ER+ cells. These loci encoded 30 genes that were overexpressed in ER+ cells; 9 of these genes were overexpressed in ER+ tumors. We identified 149 differentially expressed genes that exhibited differential methylation of one or more CpG islands within 5 kb of the 5' end of the gene and for which mRNA abundance was inversely correlated with CpG island methylation status. In primary tumors we identified 84 genes that appear to be robust components of the methylation signature that we identified in ER+ cell lines. Our analyses reveal a global pattern of differential CpG island methylation that contributes to the transcriptome landscape of ER+ and ER- breast cancer cells and tumors. The role of gene amplification/deletion appears to more modest, although several potentially significant genes appear to be regulated by copy number aberrations.

  17. Altered mitochondrial DNA copy number contributes to human cancer risk: evidence from an updated meta-analysis

    Science.gov (United States)

    Hu, Liwen; Yao, Xinyue; Shen, Yi

    2016-01-01

    Accumulating epidemiological evidence indicates that the quantitative changes in human mitochondrial DNA (mtDNA) copy number could affect the genetic susceptibility of malignancies in a tumor-specific manner, but the results are still elusive. To provide a more precise estimation on the association between mtDNA copy number and risk of diverse malignancies, a meta-analysis was conducted by calculating the pooled odds ratios (OR) and the 95% confidence intervals (95% CI). A total of 36 case-control studies involving 11,847 cases and 15,438 controls were finally included in the meta-analysis. Overall analysis of all studies suggested no significant association between mtDNA content and cancer risk (OR = 1.044, 95% CI = 0.866–1.260, P = 0.651). Subgroup analyses by cancer types showed an obvious positive association between mtDNA content and lymphoma and breast cancer (OR = 1.645, 95% CI = 1.117–2.421, P = 0.012; OR = 1.721, 95% CI = 1.130–2.622, P = 0.011, respectively), and a negative association for hepatic carcinoma. Stratified analyses by other confounding factors also found increased cancer risk in people with drinking addiction. Further analysis using studies of quartiles found that populations with the highest mtDNA content may be under more obvious risk of melanoma and that Western populations were more susceptible than Asians. PMID:27775013

  18. Integrated analysis of copy number variation and genome-wide expression profiling in colorectal cancer tissues.

    Science.gov (United States)

    Ali Hassan, Nur Zarina; Mokhtar, Norfilza Mohd; Kok Sin, Teow; Mohamed Rose, Isa; Sagap, Ismail; Harun, Roslan; Jamal, Rahman

    2014-01-01

    Integrative analyses of multiple genomic datasets for selected samples can provide better insight into the overall data and can enhance our knowledge of cancer. The objective of this study was to elucidate the association between copy number variation (CNV) and gene expression in colorectal cancer (CRC) samples and their corresponding non-cancerous tissues. Sixty-four paired CRC samples from the same patients were subjected to CNV profiling using the Illumina HumanOmni1-Quad assay, and validation was performed using multiplex ligation probe amplification method. Genome-wide expression profiling was performed on 15 paired samples from the same group of patients using the Affymetrix Human Gene 1.0 ST array. Significant genes obtained from both array results were then overlapped. To identify molecular pathways, the data were mapped to the KEGG database. Whole genome CNV analysis that compared primary tumor and non-cancerous epithelium revealed gains in 1638 genes and losses in 36 genes. Significant gains were mostly found in chromosome 20 at position 20q12 with a frequency of 45.31% in tumor samples. Examples of genes that were associated at this cytoband were PTPRT, EMILIN3 and CHD6. The highest number of losses was detected at chromosome 8, position 8p23.2 with 17.19% occurrence in all tumor samples. Among the genes found at this cytoband were CSMD1 and DLC1. Genome-wide expression profiling showed 709 genes to be up-regulated and 699 genes to be down-regulated in CRC compared to non-cancerous samples. Integration of these two datasets identified 56 overlapping genes, which were located in chromosomes 8, 20 and 22. MLPA confirmed that the CRC samples had the highest gains in chromosome 20 compared to the reference samples. Interpretation of the CNV data in the context of the transcriptome via integrative analyses may provide more in-depth knowledge of the genomic landscape of CRC.

  19. Integrated analysis of copy number variation and genome-wide expression profiling in colorectal cancer tissues.

    Directory of Open Access Journals (Sweden)

    Nur Zarina Ali Hassan

    Full Text Available Integrative analyses of multiple genomic datasets for selected samples can provide better insight into the overall data and can enhance our knowledge of cancer. The objective of this study was to elucidate the association between copy number variation (CNV and gene expression in colorectal cancer (CRC samples and their corresponding non-cancerous tissues. Sixty-four paired CRC samples from the same patients were subjected to CNV profiling using the Illumina HumanOmni1-Quad assay, and validation was performed using multiplex ligation probe amplification method. Genome-wide expression profiling was performed on 15 paired samples from the same group of patients using the Affymetrix Human Gene 1.0 ST array. Significant genes obtained from both array results were then overlapped. To identify molecular pathways, the data were mapped to the KEGG database. Whole genome CNV analysis that compared primary tumor and non-cancerous epithelium revealed gains in 1638 genes and losses in 36 genes. Significant gains were mostly found in chromosome 20 at position 20q12 with a frequency of 45.31% in tumor samples. Examples of genes that were associated at this cytoband were PTPRT, EMILIN3 and CHD6. The highest number of losses was detected at chromosome 8, position 8p23.2 with 17.19% occurrence in all tumor samples. Among the genes found at this cytoband were CSMD1 and DLC1. Genome-wide expression profiling showed 709 genes to be up-regulated and 699 genes to be down-regulated in CRC compared to non-cancerous samples. Integration of these two datasets identified 56 overlapping genes, which were located in chromosomes 8, 20 and 22. MLPA confirmed that the CRC samples had the highest gains in chromosome 20 compared to the reference samples. Interpretation of the CNV data in the context of the transcriptome via integrative analyses may provide more in-depth knowledge of the genomic landscape of CRC.

  20. Genomic DNA copy-number alterations of the let-7 family in human cancers.

    Directory of Open Access Journals (Sweden)

    Yanling Wang

    Full Text Available In human cancer, expression of the let-7 family is significantly reduced, and this is associated with shorter survival times in patients. However, the mechanisms leading to let-7 downregulation in cancer are still largely unclear. Since an alteration in copy-number is one of the causes of gene deregulation in cancer, we examined copy number alterations of the let-7 family in 2,969 cancer specimens from a high-resolution SNP array dataset. We found that there was a reduction in the copy number of let-7 genes in a cancer-type specific manner. Importantly, focal deletion of four let-7 family members was found in three cancer types: medulloblastoma (let-7a-2 and let-7e, breast cancer (let-7a-2, and ovarian cancer (let-7a-3/let-7b. For example, the genomic locus harboring let-7a-3/let-7b was deleted in 44% of the specimens from ovarian cancer patients. We also found a positive correlation between the copy number of let-7b and mature let-7b expression in ovarian cancer. Finally, we showed that restoration of let-7b expression dramatically reduced ovarian tumor growth in vitro and in vivo. Our results indicate that copy number deletion is an important mechanism leading to the downregulation of expression of specific let-7 family members in medulloblastoma, breast, and ovarian cancers. Restoration of let-7 expression in tumor cells could provide a novel therapeutic strategy for the treatment of cancer.

  1. To study the relationship between cadmium, zinc and mtDNA copy ...

    African Journals Online (AJOL)

    A. Abhishek

    2016-11-18

    Nov 18, 2016 ... Variation of metals and mtDNA in Prostate cancer. 127 distribution of ... hypothesize that Cd and Zn influence distribution pattern of mtDNA ..... of dioxins and heavy metals in cancer and neurological diseases using ROS-mediated ... biomolecules, modulation of DNA repair and genotoxic consequences.

  2. Cigarette toxicity triggers Leber's hereditary optic neuropathy by affecting mtDNA copy number, oxidative phosphorylation and ROS detoxification pathways

    Science.gov (United States)

    Giordano, L; Deceglie, S; d'Adamo, P; Valentino, M L; La Morgia, C; Fracasso, F; Roberti, M; Cappellari, M; Petrosillo, G; Ciaravolo, S; Parente, D; Giordano, C; Maresca, A; Iommarini, L; Del Dotto, V; Ghelli, A M; Salomao, S R; Berezovsky, A; Belfort, R; Sadun, A A; Carelli, V; Loguercio Polosa, P; Cantatore, P

    2015-01-01

    Leber's hereditary optic neuropathy (LHON), the most frequent mitochondrial disease, is associated with mitochondrial DNA (mtDNA) point mutations affecting Complex I subunits, usually homoplasmic. This blinding disorder is characterized by incomplete penetrance, possibly related to several genetic modifying factors. We recently reported that increased mitochondrial biogenesis in unaffected mutation carriers is a compensatory mechanism, which reduces penetrance. Also, environmental factors such as cigarette smoking have been implicated as disease triggers. To investigate this issue further, we first assessed the relationship between cigarette smoke and mtDNA copy number in blood cells from large cohorts of LHON families, finding that smoking was significantly associated with the lowest mtDNA content in affected individuals. To unwrap the mechanism of tobacco toxicity in LHON, we exposed fibroblasts from affected individuals, unaffected mutation carriers and controls to cigarette smoke condensate (CSC). CSC decreased mtDNA copy number in all cells; moreover, it caused significant reduction of ATP level only in mutated cells including carriers. This implies that the bioenergetic compensation in carriers is hampered by exposure to smoke derivatives. We also observed that in untreated cells the level of carbonylated proteins was highest in affected individuals, whereas the level of several detoxifying enzymes was highest in carriers. Thus, carriers are particularly successful in reactive oxygen species (ROS) scavenging capacity. After CSC exposure, the amount of detoxifying enzymes increased in all cells, but carbonylated proteins increased only in LHON mutant cells, mostly from affected individuals. All considered, it appears that exposure to smoke derivatives has a more deleterious effect in affected individuals, whereas carriers are the most efficient in mitigating ROS rather than recovering bioenergetics. Therefore, the identification of genetic modifiers that

  3. Cigarette toxicity triggers Leber's hereditary optic neuropathy by affecting mtDNA copy number, oxidative phosphorylation and ROS detoxification pathways.

    Science.gov (United States)

    Giordano, L; Deceglie, S; d'Adamo, P; Valentino, M L; La Morgia, C; Fracasso, F; Roberti, M; Cappellari, M; Petrosillo, G; Ciaravolo, S; Parente, D; Giordano, C; Maresca, A; Iommarini, L; Del Dotto, V; Ghelli, A M; Salomao, S R; Berezovsky, A; Belfort, R; Sadun, A A; Carelli, V; Loguercio Polosa, P; Cantatore, P

    2015-12-17

    Leber's hereditary optic neuropathy (LHON), the most frequent mitochondrial disease, is associated with mitochondrial DNA (mtDNA) point mutations affecting Complex I subunits, usually homoplasmic. This blinding disorder is characterized by incomplete penetrance, possibly related to several genetic modifying factors. We recently reported that increased mitochondrial biogenesis in unaffected mutation carriers is a compensatory mechanism, which reduces penetrance. Also, environmental factors such as cigarette smoking have been implicated as disease triggers. To investigate this issue further, we first assessed the relationship between cigarette smoke and mtDNA copy number in blood cells from large cohorts of LHON families, finding that smoking was significantly associated with the lowest mtDNA content in affected individuals. To unwrap the mechanism of tobacco toxicity in LHON, we exposed fibroblasts from affected individuals, unaffected mutation carriers and controls to cigarette smoke condensate (CSC). CSC decreased mtDNA copy number in all cells; moreover, it caused significant reduction of ATP level only in mutated cells including carriers. This implies that the bioenergetic compensation in carriers is hampered by exposure to smoke derivatives. We also observed that in untreated cells the level of carbonylated proteins was highest in affected individuals, whereas the level of several detoxifying enzymes was highest in carriers. Thus, carriers are particularly successful in reactive oxygen species (ROS) scavenging capacity. After CSC exposure, the amount of detoxifying enzymes increased in all cells, but carbonylated proteins increased only in LHON mutant cells, mostly from affected individuals. All considered, it appears that exposure to smoke derivatives has a more deleterious effect in affected individuals, whereas carriers are the most efficient in mitigating ROS rather than recovering bioenergetics. Therefore, the identification of genetic modifiers that

  4. Modeling DNA Dynamics by Path Integrals

    CERN Document Server

    Zoli, Marco

    2013-01-01

    Complementary strands in DNA double helix show temporary fluctuational openings which are essential to biological functions such as transcription and replication of the genetic information. Such large amplitude fluctuations, known as the breathing of DNA, are generally localized and, microscopically, are due to the breaking of the hydrogen bonds linking the base pairs (\\emph{bps}). I apply imaginary time path integral techniques to a mesoscopic Hamiltonian which accounts for the helicoidal geometry of a short circular DNA molecule. The \\emph{bps} displacements with respect to the ground state are interpreted as time dependent paths whose amplitudes are consistent with the model potential for the hydrogen bonds. The portion of the paths configuration space contributing to the partition function is determined by selecting the ensemble of paths which fulfill the second law of thermodynamics. Computations of the thermodynamics in the denaturation range show the energetic advantage for the equilibrium helicoidal g...

  5. SPERM DNA INTEGRITY IN BUFFALO, BULL AND STALLION

    OpenAIRE

    Serafini, Rosanna

    2015-01-01

    The interest in sperm DNA integrity evaluation and its relationship to subfertility and infertility loaded to development of several sperm DNA assays. The aim of this study was to compare several sperm DNA assays in buffaloes, bulls and stallions, and to identify the relationships between those DNA assays and traditional sperm features. In Italian Mediterranean Buffalo (IMB) bulls traditional sperm features (motility, viability, acrosome integrity and morphology), sperm DNA integrity (neutral...

  6. Systematic prioritization and integrative analysis of copy number variations in schizophrenia reveal key schizophrenia susceptibility genes.

    Science.gov (United States)

    Luo, Xiongjian; Huang, Liang; Han, Leng; Luo, Zhenwu; Hu, Fang; Tieu, Roger; Gan, Lin

    2014-11-01

    Schizophrenia is a common mental disorder with high heritability and strong genetic heterogeneity. Common disease-common variants hypothesis predicts that schizophrenia is attributable in part to common genetic variants. However, recent studies have clearly demonstrated that copy number variations (CNVs) also play pivotal roles in schizophrenia susceptibility and explain a proportion of missing heritability. Though numerous CNVs have been identified, many of the regions affected by CNVs show poor overlapping among different studies, and it is not known whether the genes disrupted by CNVs contribute to the risk of schizophrenia. By using cumulative scoring, we systematically prioritized the genes affected by CNVs in schizophrenia. We identified 8 top genes that are frequently disrupted by CNVs, including NRXN1, CHRNA7, BCL9, CYFIP1, GJA8, NDE1, SNAP29, and GJA5. Integration of genes affected by CNVs with known schizophrenia susceptibility genes (from previous genetic linkage and association studies) reveals that many genes disrupted by CNVs are also associated with schizophrenia. Further protein-protein interaction (PPI) analysis indicates that protein products of genes affected by CNVs frequently interact with known schizophrenia-associated proteins. Finally, systematic integration of CNVs prioritization data with genetic association and PPI data identifies key schizophrenia candidate genes. Our results provide a global overview of genes impacted by CNVs in schizophrenia and reveal a densely interconnected molecular network of de novo CNVs in schizophrenia. Though the prioritized top genes represent promising schizophrenia risk genes, further work with different prioritization methods and independent samples is needed to confirm these findings. Nevertheless, the identified key candidate genes may have important roles in the pathogenesis of schizophrenia, and further functional characterization of these genes may provide pivotal targets for future therapeutics and

  7. Restriction fragment length polymorphism and multiple copies of DNA sequences homologous with probes for P-fimbriae and hemolysin genes among uropathogenic Escherichia coli.

    Science.gov (United States)

    Hull, S I; Bieler, S; Hull, R A

    1988-03-01

    Hemolysin and P-fimbriae are two virulence traits frequently found together in uropathogenic Escherichia coli. Previous studies have discovered evidence both for linkage between the genes for these traits and for their duplication in the chromosomes of a limited number of strains. To test whether these observations are characteristic of uropathogenic Escherichia coli, the method of DNA hybridization to DNA restriction fragments separated by electrophoresis and transferred to nylon was used to determine copy number of genes for P-fimbriae (pap) among 51 E. coli strains isolated from symptomatic urinary tract infections. Twenty percent of the strains had more than one copy of pap homologous sequences. Fifteen strains, each representing a unique clone, were examined for the presence of sequences homologous with cloned hemolysin genes (hly). Samples of DNA from 14 of the 15 strains hybridized with hly probes. In eight strains the number of copies of pap equalled the number of copies of hly, including one strain with two apparent copies of each. Five strains appeared to have one more copy of pap than of hly, and one strain had an extra copy of hly.

  8. Helicase and Polymerase Move Together Close to the Fork Junction and Copy DNA in One-Nucleotide Steps

    Directory of Open Access Journals (Sweden)

    Manjula Pandey

    2014-03-01

    Full Text Available By simultaneously measuring DNA synthesis and dNTP hydrolysis, we show that T7 DNA polymerase and T7 gp4 helicase move in sync during leading-strand synthesis, taking one-nucleotide steps and hydrolyzing one dNTP per base-pair unwound/copied. The cooperative catalysis enables the helicase and polymerase to move at a uniformly fast rate without guanine:cytosine (GC dependency or idling with futile NTP hydrolysis. We show that the helicase and polymerase are located close to the replication fork junction. This architecture enables the polymerase to use its strand-displacement synthesis to increase the unwinding rate, whereas the helicase aids this process by translocating along single-stranded DNA and trapping the unwound bases. Thus, in contrast to the helicase-only unwinding model, our results suggest a model in which the helicase and polymerase are moving in one-nucleotide steps, DNA synthesis drives fork unwinding, and a role of the helicase is to trap the unwound bases and prevent DNA reannealing.

  9. A scale-space method for detecting recurrent DNA copy number changes with analytical false discovery rate control.

    Science.gov (United States)

    van Dyk, Ewald; Reinders, Marcel J T; Wessels, Lodewyk F A

    2013-05-01

    Tumor formation is partially driven by DNA copy number changes, which are typically measured using array comparative genomic hybridization, SNP arrays and DNA sequencing platforms. Many techniques are available for detecting recurring aberrations across multiple tumor samples, including CMAR, STAC, GISTIC and KC-SMART. GISTIC is widely used and detects both broad and focal (potentially overlapping) recurring events. However, GISTIC performs false discovery rate control on probes instead of events. Here we propose Analytical Multi-scale Identification of Recurrent Events, a multi-scale Gaussian smoothing approach, for the detection of both broad and focal (potentially overlapping) recurring copy number alterations. Importantly, false discovery rate control is performed analytically (no need for permutations) on events rather than probes. The method does not require segmentation or calling on the input dataset and therefore reduces the potential loss of information due to discretization. An important characteristic of the approach is that the error rate is controlled across all scales and that the algorithm outputs a single profile of significant events selected from the appropriate scales. We perform extensive simulations and showcase its utility on a glioblastoma SNP array dataset. Importantly, ADMIRE detects focal events that are missed by GISTIC, including two events involving known glioma tumor-suppressor genes: CDKN2C and NF1.

  10. GeneBreak: detection of recurrent DNA copy number aberration-associated chromosomal breakpoints within genes [version 2; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Evert van den Broek

    2017-07-01

    Full Text Available Development of cancer is driven by somatic alterations, including numerical and structural chromosomal aberrations. Currently, several computational methods are available and are widely applied to detect numerical copy number aberrations (CNAs of chromosomal segments in tumor genomes. However, there is lack of computational methods that systematically detect structural chromosomal aberrations by virtue of the genomic location of CNA-associated chromosomal breaks and identify genes that appear non-randomly affected by chromosomal breakpoints across (large series of tumor samples. ‘GeneBreak’ is developed to systematically identify genes recurrently affected by the genomic location of chromosomal CNA-associated breaks by a genome-wide approach, which can be applied to DNA copy number data obtained by array-Comparative Genomic Hybridization (CGH or by (low-pass whole genome sequencing (WGS. First, ‘GeneBreak’ collects the genomic locations of chromosomal CNA-associated breaks that were previously pinpointed by the segmentation algorithm that was applied to obtain CNA profiles. Next, a tailored annotation approach for breakpoint-to-gene mapping is implemented. Finally, dedicated cohort-based statistics is incorporated with correction for covariates that influence the probability to be a breakpoint gene. In addition, multiple testing correction is integrated to reveal recurrent breakpoint events. This easy-to-use algorithm, ‘GeneBreak’, is implemented in R (www.cran.r-project.org and is available from Bioconductor (www.bioconductor.org/packages/release/bioc/html/GeneBreak.html.

  11. Apparent Polyploidization after Gamma Irradiation: Pitfalls in the Use of Quantitative Polymerase Chain Reaction (qPCR) for the Estimation of Mitochondrial and Nuclear DNA Gene Copy Numbers

    Science.gov (United States)

    Kam, Winnie W. Y.; Lake, Vanessa; Banos, Connie; Davies, Justin; Banati, Richard

    2013-01-01

    Quantitative polymerase chain reaction (qPCR) has been widely used to quantify changes in gene copy numbers after radiation exposure. Here, we show that gamma irradiation ranging from 10 to 100 Gy of cells and cell-free DNA samples significantly affects the measured qPCR yield, due to radiation-induced fragmentation of the DNA template and, therefore, introduces errors into the estimation of gene copy numbers. The radiation-induced DNA fragmentation and, thus, measured qPCR yield varies with temperature not only in living cells, but also in isolated DNA irradiated under cell-free conditions. In summary, the variability in measured qPCR yield from irradiated samples introduces a significant error into the estimation of both mitochondrial and nuclear gene copy numbers and may give spurious evidence for polyploidization. PMID:23722662

  12. Copy number variation of ribosomal DNA and Pokey transposons in natural populations of Daphnia

    Science.gov (United States)

    2012-01-01

    Background Despite their ubiquity and high diversity in eukaryotic genomes, DNA transposons are rarely encountered in ribosomal DNA (rDNA). In contrast, R-elements, a diverse group of non-LTR retrotransposons, specifically target rDNA. Pokey is a DNA transposon that targets a specific rDNA site, but also occurs in many other genomic locations, unlike R-elements. However, unlike most DNA transposons, Pokey has been a stable component of Daphnia genomes for over 100 million years. Here we use qPCR to estimate the number of 18S and 28S ribosomal RNA genes and Pokey elements in rDNA (rPokey), as well as other genomic locations (gPokey) in two species of Daphnia. Our goals are to estimate the correlation between (1) the number of 18S and 28S rRNA genes, (2) the number of 28S genes and rPokey, and (3) the number of rPokey and gPokey. In addition, we ask whether Pokey number and distribution in both genomic compartments are affected by differences in life history between D. pulex and D. pulicaria. Results We found differences in 18S and 28S gene number within isolates that are too large to be explained by experimental variation. In general, Pokey number within isolates is modest (18S and 28S genes suggests that rDNA is much more complicated than once thought, and warrants further study. In addition, the lack of correlation between rPokey, gPokey and rDNA unit numbers suggests that Pokey transposition rate is generally very low, and that recombination, in combination with natural selection, eliminates rPokey much faster than gPokey. Our results suggest that further research to determine the mechanisms by which Pokey has escaped complete inactivation by its host (the usual fate of DNA transposons), would provide important insights into transposon biology. PMID:22390386

  13. DNA copy-number control through inhibition of replication fork progression

    NARCIS (Netherlands)

    J.T. Nordman (Jared T.); E. Kozhevnikova (Elena); C.P. Verrijzer (Peter); A.V. Pindyurin (Alexey); E.N. Andreyeva (Evgeniya); V.V. Shloma (Victor); I.F. Zhimulev (Igor); T. Orr-Weaver (T.)

    2014-01-01

    textabstractProper control of DNA replication is essential to ensure faithful transmission of genetic material and prevent chromosomal aberrations that can drive cancer progression and developmental disorders. DNA replication is regulated primarily at the level of initiation and is under strict

  14. DNA copy-number control through inhibition of replication fork progression

    NARCIS (Netherlands)

    J.T. Nordman (Jared T.); E. Kozhevnikova (Elena); C.P. Verrijzer (Peter); A.V. Pindyurin (Alexey); E.N. Andreyeva (Evgeniya); V.V. Shloma (Victor); I.F. Zhimulev (Igor); T. Orr-Weaver (T.)

    2014-01-01

    textabstractProper control of DNA replication is essential to ensure faithful transmission of genetic material and prevent chromosomal aberrations that can drive cancer progression and developmental disorders. DNA replication is regulated primarily at the level of initiation and is under strict cell

  15. DNA copy-number control through inhibition of replication fork progression

    NARCIS (Netherlands)

    J.T. Nordman (Jared T.); E. Kozhevnikova (Elena); C.P. Verrijzer (Peter); A.V. Pindyurin (Alexey); E.N. Andreyeva (Evgeniya); V.V. Shloma (Victor); I.F. Zhimulev (Igor); T. Orr-Weaver (T.)

    2014-01-01

    textabstractProper control of DNA replication is essential to ensure faithful transmission of genetic material and prevent chromosomal aberrations that can drive cancer progression and developmental disorders. DNA replication is regulated primarily at the level of initiation and is under strict cell

  16. Integrative analysis of copy number and gene expression in breast cancer using formalin-fixed paraffin-embedded core biopsy tissue: a feasibility study.

    Science.gov (United States)

    Iddawela, Mahesh; Rueda, Oscar; Eremin, Jenny; Eremin, Oleg; Cowley, Jed; Earl, Helena M; Caldas, Carlos

    2017-07-11

    An absence of reliable molecular markers has hampered individualised breast cancer treatments, and a major limitation for translational research is the lack of fresh tissue. There are, however, abundant banks of formalin-fixed paraffin-embedded (FFPE) tissue. This study evaluated two platforms available for the analysis of DNA copy number and gene expression using FFPE samples. The cDNA-mediated annealing, selection, extension, and ligation assay (DASL™) has been developed for gene expression analysis and the Molecular Inversion Probes assay (Oncoscan™), were used for copy number analysis using FFPE tissues. Gene expression and copy number were evaluated in core-biopsy samples from patients with breast cancer undergoing neoadjuvant chemotherapy (NAC). Forty-three core-biopsies were evaluated and characteristic copy number changes in breast cancers, gains in 1q, 8q, 11q, 17q and 20q and losses in 6q, 8p, 13q and 16q, were confirmed. Regions that frequently exhibited gains in tumours showing a pathological complete response (pCR) to NAC were 1q (55%), 8q (40%) and 17q (40%), whereas 11q11 (37%) gain was the most frequent change in non-pCR tumours. Gains associated with poor survival were 11q13 (62%), 8q24 (54%) and 20q (47%). Gene expression assessed by DASL correlated with immunohistochemistry (IHC) analysis for oestrogen receptor (ER) [area under the curve (AUC) = 0.95], progesterone receptor (PR)(AUC = 0.90) and human epidermal growth factor type-2 receptor (HER-2) (AUC = 0.96). Differential expression analysis between ER+ and ER- cancers identified over-expression of TTF1, LAF-4 and C-MYB (p ≤ 0.05), and between pCR vs non-pCRs, over-expression of CXCL9, AREG, B-MYB and under-expression of ABCG2. This study was an integrative analysis of copy number and gene expression using FFPE core biopsies and showed that molecular marker data from FFPE tissues were consistent with those in previous studies using fresh-frozen samples. FFPE tissue can provide

  17. Detection of the free living amoeba Naegleria fowleri by using conventional and real-time PCR based on a single copy DNA sequence.

    Science.gov (United States)

    Régoudis, Estelle; Pélandakis, Michel

    2016-02-01

    The amoeba-flagellate Naegleria fowleri is a causative agent of primary amoebic meningoencephalitis (PAM). This thermophilic species occurs worldwide and tends to proliferate in warm aquatic environment. The PAM cases remain rare but this infection is mostly fatal. Here, we describe a single copy region which has been cloned and sequenced, and was used for both conventional and real-time PCR. Targeting a single-copy DNA sequence allows to directly quantify the N. fowleri cells. The real-time PCR results give a detection limit of 1 copy per reaction with high reproducibility without the need of a Taqman probe. This procedure is of interest as compared to other procedures which are mostly based on the detection of multi-copy DNA associated with a Taqman probe.

  18. A novel technique for measuring variations in DNA copy-number: competitive genomic polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    Nakagawara Akira

    2007-07-01

    Full Text Available Background Changes in genomic copy number occur in many human diseases including cancer. Characterization of these changes is important for both basic understanding and diagnosis of these diseases. Microarrays have recently become the standard technique and are commercially available. However, it is useful to have an affordable technique to complement them. Results We describe a novel polymerase chain reaction (PCR-based technique, termed competitive genomic PCR (CGP. The main characteristic of CGP is that different adaptors are added to the sample and control genomic DNAs after appropriate restriction enzyme digestion. These adaptor-supplemented DNAs are subjected to competitive PCR using an adaptor-primer and a locus-specific primer. The amplified products are then separated according to size differences between the adaptors. CGP eliminates the tedious steps inherent in quantitative PCR and achieves moderate throughput. Assays with different X chromosome numbers showed that it can provide accurate quantification. High-resolution analysis of neuroblastoma cell lines around the MYCN locus revealed novel junctions for amplification, which were not detected by a commercial array. Conclusion CGP is a moderate throughput technique for analyzing changes in genomic copy numbers. Because CGP can measure any genomic locus using PCR primers, it is especially useful for detailed analysis of a genomic region of interest.

  19. Decreased mitochondrial DNA copy number in the hippocampus and peripheral blood during opiate addiction is mediated by autophagy and can be salvaged by melatonin.

    Science.gov (United States)

    Feng, Yue-Mei; Jia, Yun-Fang; Su, Ling-Yan; Wang, Dong; Lv, Li; Xu, Lin; Yao, Yong-Gang

    2013-09-01

    Drug addiction is a chronic brain disease that is a serious social problem and causes enormous financial burden. Because mitochondrial abnormalities have been associated with opiate addiction, we examined the effect of morphine on mtDNA levels in rat and mouse models of addiction and in cultured cells. We found that mtDNA copy number was significantly reduced in the hippocampus and peripheral blood of morphine-addicted rats and mice compared with control animals. Concordantly, decreased mtDNA copy number and elevated mtDNA damage were observed in the peripheral blood from opiate-addicted patients, indicating detrimental effects of drug abuse and stress. In cultured rat pheochromocytoma (PC12) cells and mouse neurons, morphine treatment caused many mitochondrial defects, including a reduction in mtDNA copy number that was mediated by autophagy. Knockdown of the Atg7 gene was able to counteract the loss of mtDNA copy number induced by morphine. The mitochondria-targeted antioxidant melatonin restored mtDNA content and neuronal outgrowth and prevented the increase in autophagy upon morphine treatment. In mice, coadministration of melatonin with morphine ameliorated morphine-induced behavioral sensitization, analgesic tolerance and mtDNA content reduction. During drug withdrawal in opiate-addicted patients and improvement of protracted abstinence syndrome, we observed an increase of serum melatonin level. Taken together, our study indicates that opioid addiction is associated with mtDNA copy number reduction and neurostructural remodeling. These effects appear to be mediated by autophagy and can be salvaged by melatonin.

  20. No association between body mass index and sperm DNA integrity

    DEFF Research Database (Denmark)

    Bandel, I; Bungum, M; Richtoff, J

    2015-01-01

    STUDY QUESTION: Is overweight associated with impaired sperm DNA integrity? SUMMARY ANSWER: High body mass index (BMI) is not associated with impaired sperm DNA integrity as assessed by the DNA Fragmentation Index (DFI). WHAT IS KNOWN ALREADY: Previous studies, based on fewer subjects and including...

  1. Noninvasive detection of cancer-associated genome-wide hypomethylation and copy number aberrations by plasma DNA bisulfite sequencing

    Science.gov (United States)

    Chan, K. C. Allen; Jiang, Peiyong; Chan, Carol W. M.; Sun, Kun; Wong, John; Hui, Edwin P.; Chan, Stephen L.; Chan, Wing Cheong; Hui, David S. C.; Ng, Simon S. M.; Chan, Henry L. Y.; Wong, Cesar S. C.; Ma, Brigette B. Y.; Chan, Anthony T. C.; Lai, Paul B. S.; Sun, Hao; Chiu, Rossa W. K.; Lo, Y. M. Dennis

    2013-01-01

    We explored the detection of genome-wide hypomethylation in plasma using shotgun massively parallel bisulfite sequencing as a marker for cancer. Tumor-associated copy number aberrations (CNAs) could also be observed from the bisulfite DNA sequencing data. Hypomethylation and CNAs were detected in the plasma DNA of patients with hepatocellular carcinoma, breast cancer, lung cancer, nasopharyngeal cancer, smooth muscle sarcoma, and neuroendocrine tumor. For the detection of nonmetastatic cancer cases, plasma hypomethylation gave a sensitivity and specificity of 74% and 94%, respectively, when a mean of 93 million reads per case were obtained. Reducing the sequencing depth to 10 million reads per case was found to have no adverse effect on the sensitivity and specificity for cancer detection, giving respective figures of 68% and 94%. This characteristic thus indicates that analysis of plasma hypomethylation by this sequencing-based method may be a relatively cost-effective approach for cancer detection. We also demonstrated that plasma hypomethylation had utility for monitoring hepatocellular carcinoma patients following tumor resection and for detecting residual disease. Plasma hypomethylation can be combined with plasma CNA analysis for further enhancement of the detection sensitivity or specificity using different diagnostic algorithms. Using the detection of at least one type of aberration to define an abnormality, a sensitivity of 87% could be achieved with a specificity of 88%. These developments have thus expanded the applications of plasma DNA analysis for cancer detection and monitoring. PMID:24191000

  2. Genome-Wide DNA Copy Number Analysis of Acute Lymphoblastic Leukemia Identifies New Genetic Markers Associated with Clinical Outcome.

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    Maribel Forero-Castro

    Full Text Available Identifying additional genetic alterations associated with poor prognosis in acute lymphoblastic leukemia (ALL is still a challenge.To characterize the presence of additional DNA copy number alterations (CNAs in children and adults with ALL by whole-genome oligonucleotide array (aCGH analysis, and to identify their associations with clinical features and outcome. Array-CGH was carried out in 265 newly diagnosed ALLs (142 children and 123 adults. The NimbleGen CGH 12x135K array (Roche was used to analyze genetic gains and losses. CNAs were analyzed with GISTIC and aCGHweb software. Clinical and biological variables were analyzed. Three of the patients showed chromothripsis (cth6, cth14q and cth15q. CNAs were associated with age, phenotype, genetic subtype and overall survival (OS. In the whole cohort of children, the losses on 14q32.33 (p = 0.019 and 15q13.2 (p = 0.04 were related to shorter OS. In the group of children without good- or poor-risk cytogenetics, the gain on 1p36.11 was a prognostic marker independently associated with shorter OS. In adults, the gains on 19q13.2 (p = 0.001 and Xp21.1 (p = 0.029, and the loss of 17p (p = 0.014 were independent markers of poor prognosis with respect to OS. In summary, CNAs are frequent in ALL and are associated with clinical parameters and survival. Genome-wide DNA copy number analysis allows the identification of genetic markers that predict clinical outcome, suggesting that detection of these genetic lesions will be useful in the management of patients newly diagnosed with ALL.

  3. T-DNA integration in plants results from polymerase-θ-mediated DNA repair.

    Science.gov (United States)

    van Kregten, Maartje; de Pater, Sylvia; Romeijn, Ron; van Schendel, Robin; Hooykaas, Paul J J; Tijsterman, Marcel

    2016-10-31

    Agrobacterium tumefaciens is a pathogenic bacterium, which transforms plants by transferring a discrete segment of its DNA, the T-DNA, to plant cells. The T-DNA then integrates into the plant genome. T-DNA biotechnology is widely exploited in the genetic engineering of model plants and crops. However, the molecular mechanism underlying T-DNA integration remains unknown(1). Here we demonstrate that in Arabidopsis thaliana T-DNA integration critically depends on polymerase theta (Pol θ). We find that TEBICHI/POLQ mutant plants (which have mutated Pol θ), although susceptible to Agrobacterium infection, are resistant to T-DNA integration. Characterization of >10,000 T-DNA-plant genome junctions reveals a distinct signature of Pol θ action and also indicates that 3' end capture at genomic breaks is the prevalent mechanism of T-DNA integration. The primer-template switching ability of Pol θ can explain the molecular patchwork known as filler DNA that is frequently observed at sites of integration. T-DNA integration signatures in other plant species closely resemble those of Arabidopsis, suggesting that Pol-θ-mediated integration is evolutionarily conserved. Thus, Pol θ provides the mechanism for T-DNA random integration into the plant genome, demonstrating a potential to disrupt random integration so as to improve the quality and biosafety of plant transgenesis.

  4. Phylogeny of the genus Chrysanthemum L.: evidence from single-copy nuclear gene and chloroplast DNA sequences.

    Science.gov (United States)

    Liu, Ping-Li; Wan, Qian; Guo, Yan-Ping; Yang, Ji; Rao, Guang-Yuan

    2012-01-01

    Chrysanthemum L. (Asteraceae-Anthemideae) is a genus with rapid speciation. It comprises about 40 species, most of which are distributed in East Asia. Many of these are narrowly distributed and habitat-specific. Considerable variations in morphology and ploidy are found in this genus. Some species have been the subjects of many studies, but the relationships between Chrysanthemum and its allies and the phylogeny of this genus remain poorly understood. In the present study, 32 species/varieties from Chrysanthemum and 11 from the allied genera were analyzed using DNA sequences of the single-copy nuclear CDS gene and seven cpDNA loci (psbA-trnH, trnC-ycf6, ycf6-psbM, trnY-rpoB, rpS4-trnT, trnL-F, and rpL16). The cpDNA and nuclear CDS gene trees both suggest that 1) Chrysanthemum is not a monophyletic taxon, and the affinity between Chrysanthemum and Ajania is so close that these two genera should be incorporated taxonomically; 2) Phaeostigma is more closely related to the Chrysanthemum+Ajania than other generic allies. According to pollen morphology and to the present cpDNA and CDS data, Ajania purpurea is a member of Phaeostigma. Species differentiation in Chrysanthemum appears to be correlated with geographic and environmental conditions. The Chinese Chrysanthemum species can be divided into two groups, the C. zawadskii group and the C. indicum group. The former is distributed in northern China and the latter in southern China. Many polyploid species, such as C. argyrophyllum, may have originated from allopolyploidization involving divergent progenitors. Considering all the evidence from present and previous studies, we conclude that geographic and ecological factors as well as hybridization and polyploidy play important roles in the divergence and speciation of the genus Chrysanthemum.

  5. Phylogeny of the genus Chrysanthemum L.: evidence from single-copy nuclear gene and chloroplast DNA sequences.

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    Ping-Li Liu

    Full Text Available Chrysanthemum L. (Asteraceae-Anthemideae is a genus with rapid speciation. It comprises about 40 species, most of which are distributed in East Asia. Many of these are narrowly distributed and habitat-specific. Considerable variations in morphology and ploidy are found in this genus. Some species have been the subjects of many studies, but the relationships between Chrysanthemum and its allies and the phylogeny of this genus remain poorly understood. In the present study, 32 species/varieties from Chrysanthemum and 11 from the allied genera were analyzed using DNA sequences of the single-copy nuclear CDS gene and seven cpDNA loci (psbA-trnH, trnC-ycf6, ycf6-psbM, trnY-rpoB, rpS4-trnT, trnL-F, and rpL16. The cpDNA and nuclear CDS gene trees both suggest that 1 Chrysanthemum is not a monophyletic taxon, and the affinity between Chrysanthemum and Ajania is so close that these two genera should be incorporated taxonomically; 2 Phaeostigma is more closely related to the Chrysanthemum+Ajania than other generic allies. According to pollen morphology and to the present cpDNA and CDS data, Ajania purpurea is a member of Phaeostigma. Species differentiation in Chrysanthemum appears to be correlated with geographic and environmental conditions. The Chinese Chrysanthemum species can be divided into two groups, the C. zawadskii group and the C. indicum group. The former is distributed in northern China and the latter in southern China. Many polyploid species, such as C. argyrophyllum, may have originated from allopolyploidization involving divergent progenitors. Considering all the evidence from present and previous studies, we conclude that geographic and ecological factors as well as hybridization and polyploidy play important roles in the divergence and speciation of the genus Chrysanthemum.

  6. Rare DNA copy number variants in cardiovascular malformations with extracardiac abnormalities.

    Science.gov (United States)

    Lalani, Seema R; Shaw, Chad; Wang, Xueqing; Patel, Ankita; Patterson, Lance W; Kolodziejska, Katarzyna; Szafranski, Przemyslaw; Ou, Zhishuo; Tian, Qi; Kang, Sung-Hae L; Jinnah, Amina; Ali, Sophia; Malik, Aamir; Hixson, Patricia; Potocki, Lorraine; Lupski, James R; Stankiewicz, Pawel; Bacino, Carlos A; Dawson, Brian; Beaudet, Arthur L; Boricha, Fatima M; Whittaker, Runako; Li, Chumei; Ware, Stephanie M; Cheung, Sau Wai; Penny, Daniel J; Jefferies, John Lynn; Belmont, John W

    2013-02-01

    Clinically significant cardiovascular malformations (CVMs) occur in 5-8 per 1000 live births. Recurrent copy number variations (CNVs) are among the known causes of syndromic CVMs, accounting for an important fraction of cases. We hypothesized that many additional rare CNVs also cause CVMs and can be detected in patients with CVMs plus extracardiac anomalies (ECAs). Through a genome-wide survey of 203 subjects with CVMs and ECAs, we identified 55 CNVs >50 kb in length that were not present in children without known cardiovascular defects (n=872). Sixteen unique CNVs overlapping these variants were found in an independent CVM plus ECA cohort (n=511), which were not observed in 2011 controls. The study identified 12/16 (75%) novel loci including non-recurrent de novo 16q24.3 loss (4/714) and de novo 2q31.3q32.1 loss encompassing PPP1R1C and PDE1A (2/714). The study also narrowed critical intervals in three well-recognized genomic disorders of CVM, such as the cat-eye syndrome region on 22q11.1, 8p23.1 loss encompassing GATA4 and SOX7 and 17p13.3-p13.2 loss. An analysis of protein-interaction databases shows that the rare inherited and de novo CNVs detected in the combined cohort are enriched for genes encoding proteins that are direct or indirect partners of proteins known to be required for normal cardiac development. Our findings implicate rare variants such as 16q24.3 loss and 2q31.3-q32.1 loss, and delineate regions within previously reported structural variants known to cause CVMs.

  7. Specific functions of the Rep and Rep׳ proteins of porcine circovirus during copy-release and rolling-circle DNA replication.

    Science.gov (United States)

    Cheung, Andrew K

    2015-07-01

    The roles of two porcine circovirus replication initiator proteins, Rep and Rep׳, in generating copy-release and rolling-circle DNA replication intermediates were determined. Rep uses the supercoiled closed-circular genome (ccc) to initiate leading-strand synthesis (identical to copy-release replication) and generates the single-stranded circular (ssc) genome from the displaced DNA strand. In the process, a minus-genome primer (MGP) necessary for complementary-strand synthesis, from ssc to ccc, is synthesized. Rep׳ cleaves the growing nascent-strand to regenerate the parent ccc molecule. In the process, a Rep׳-DNA hybrid containing the right palindromic sequence (at the origin of DNA replication) is generated. Analysis of the virus particle showed that it is composed of four components: ssc, MGP, capsid protein and a novel Rep-related protein (designated Protein-3).

  8. Mitochondrial DNA copy number is maintained during spermatogenesis and in the development of male larvae to sustain the doubly uniparental inheritance of mitochondrial DNA system in the blue mussel Mytilus galloprovincialis.

    Science.gov (United States)

    Sano, Natsumi; Obata, Mayu; Ooie, Yosiyasu; Komaru, Akira

    2011-08-01

    Doubly uniparental inheritance (DUI) of mitochondrial (mt) DNA has been reported in the blue mussel Mytilus galloprovincialis. In DUI, males inherit both paternal (M type) and maternal (F type) mtDNA. Here we investigated changes in M type mtDNA copy numbers and mitochondrial mass in testicular cells by real-time polymerase chain reaction and flow cytometry. The ratios of M type mtDNA copy numbers to nuclear DNA content were not different between haploid (1n), diploid (2n) and tetraploid (4n) spermatogenic cells. The mitochondrial mass decreased gradually during spermatogenesis. These results suggest that mtDNA and mitochondrial mass are maintained during spermatogenesis. We then traced M type mtDNA in larvae after fertilization. M type mtDNA was maintained up to 24 h after fertilization in the male-biased crosses, but decreased significantly in female-biased crosses (predicted by Mito Tracker staining pattern). These results are strikingly different from those reported for mammals and fish, where it is well known that the mitochondria and mtDNA are reduced during spermatogenesis and that sperm mitochondria and mtDNA are eliminated soon after fertilization. Thus, the M type mtDNA copy number is maintained during spermatogenesis and in the development of male larvae to sustain the DUI system in the blue mussel.

  9. T-DNA Integration Category and Mechanism in Rice Genome

    Institute of Scientific and Technical Information of China (English)

    Jiang WANG; Lin LI; Zhen-Ying SHI; Xin-Shan WAN; Lin-Sheng AN; Jing-Liu ZHANG

    2005-01-01

    T-DNA integration is a key step in the process of plant transformation, which is proven to be important for analyzing T-DNA integration mechanism. The structures of T-DNA right borders inserted into the rice (Oryza sativa L.) genome and their flanking sequences were analyzed. It was found that the integrated ends of the T-DNA right border occurred mainly on five nucleotides "TGACA" in inverse repeat (IR)sequence of 25 bp, especially on the third base "A". However, the integrated ends would sometimes lie inward of the IR sequence, which caused the IR sequence to be lost completely. Sometimes the right integrated ends appeared on the vector sequences rightward of the T-DNA right border, which made the TDNA, carrying vector sequences, integrated into the rice genome. These results seemingly suggest that the IR sequence of the right border plays an important role in the process of T-DNA integration into the rice genome, but is not an essential element. The appearance of vector sequences neighboring the T-DNA right border suggested that before being transferred into the plant cell from Agrobacterium, the entire T-DNA possibly began from the left border in synthesis and then read through at the right border. Several nucleotides in the T-DNA right border homologous with plant DNA and filler DNAs were frequently discovered in the integrated position ofT-DNA. Some small regions in the right border could match with the plant sequence, or form better matches, accompanied by the occurrence of filler DNA, through mutual twisting, and then the TDNA was integrated into plant chromosome through a partially homologous recombination mechanism. The appearance of filler DNA would facilitate T-DNA integration. The fragments flanking the T-DNA right border in transformed rice plants could derive from different parts of the inner T-DNA region; that is, disruption and recombination could occur at arbitrary positions in the entire T-DNA, in which the homologous area was comparatively

  10. Model-integrated estimation of normal tissue contamination for cancer SNP allelic copy number data.

    Science.gov (United States)

    Stjernqvist, Susann; Rydén, Tobias; Greenman, Chris D

    2011-01-01

    SNP allelic copy number data provides intensity measurements for the two different alleles separately. We present a method that estimates the number of copies of each allele at each SNP position, using a continuous-index hidden Markov model. The method is especially suited for cancer data, since it includes the fraction of normal tissue contamination, often present when studying data from cancer tumors, into the model. The continuous-index structure takes into account the distances between the SNPs, and is thereby appropriate also when SNPs are unequally spaced. In a simulation study we show that the method performs favorably compared to previous methods even with as much as 70% normal contamination. We also provide results from applications to clinical data produced using the Affymetrix genome-wide SNP 6.0 platform.

  11. DNA copy number analysis of fresh and formalin-fixed specimens by shallow whole-genome sequencing with identification and exclusion of problematic regions in the genome assembly

    NARCIS (Netherlands)

    Scheinin, I.; Sie, D.; Bengtsson, H.; Wiel, M.A. van de; Olshen, A.B.; Thuijl, H.F. van; Essen, H.F. van; Eijk, P.P.; Rustenburg, F.; Meijer, G.A.; Reijneveld, J.C.; Wesseling, P.; Pinkel, D.; Albertson, D.G.; Ylstra, B.

    2014-01-01

    Detection of DNA copy number aberrations by shallow whole-genome sequencing (WGS) faces many challenges, including lack of completion and errors in the human reference genome, repetitive sequences, polymorphisms, variable sample quality, and biases in the sequencing procedures. Formalin-fixed paraff

  12. Changes in mitochondrial DNA copy number and extracellular matrix (ECM proteins in the uterosacral ligaments of premenopausal women with pelvic organ prolapse

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    Mou-Jong Sun

    2016-02-01

    Conclusion: In the premenopausal state, higher BMI may be a stronger associate factor than vaginal birth for the development of POP. The higher mtDNA copy number, COL3α1, and MMP2 gene expressions are highly associated with POP in the UL of premenopausal women.

  13. Efficacy and limits of genotyping low copy number (LCN) DNA samples by multiplex PCR of STR loci.

    Science.gov (United States)

    Kloosterman, Ate D; Kersbergen, Paula

    2003-01-01

    In this study, we have evaluated the efficacy and the validity of the AmpFISTR SGM plus multiplex PCR typing system when Low Copy Number (LCN) amounts of DNA are processed. The characteristics of SGM plus profiles produced under LCN conditions were studied on the basis of heterozygote balance, between loci balance and stutter proportion based on profiles that were obtained from a variety of mock casework samples. These experiments clearly showed that LCN DNA profiles carry their own characteristic features, which must be taken into account during interpretation. Herewith, we confirmed the data of recent other studies that a comprehensive interpretation strategy is dependent upon multiple replication of the PCR using the same extract together with the proper use of extraction and amplification controls. The limitations of LCN DNA analysis were further studied in a series of single cell PCR experiments using an amplification regime of 34 PCR cycles. The allele dropout phenomenon was demonstrated to its full extent when single cells were analysed. However, the "consensus profile" which was obtained from separate single cell PCR experiments matched the actual profile of the cell donor. Single cell PCR experiments also showed that a further increase of the number of PCR cycles did not result in enhanced sensitivity and had a highly negative effect on the balance of this multiplex PCR system which hampered correct interpretation of the profile. Also, the potential of LCN typing in analysing mixtures of DNA was investigated. It was clearly shown that LCN typing had no advantages over 28 cycles amplification in the detection of the minor component of DNA-mixtures. In addition to the 34 cycles PCR amplification regime, the utility of a new approach that involved reamplification of the 28 cycle SGM plus PCR products with an extra 6 PCR cycles after the addition of fresh AmpliTaq Gold DNA Polymerase was investigated. This approach provides the scientist with an extra typing

  14. DNA polymerase ζ-dependent lesion bypass in Saccharomyces cerevisiae is accompanied by error-prone copying of long stretches of adjacent DNA.

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    Olga V Kochenova

    2015-03-01

    Full Text Available Translesion synthesis (TLS helps cells to accomplish chromosomal replication in the presence of unrepaired DNA lesions. In eukaryotes, the bypass of most lesions involves a nucleotide insertion opposite the lesion by either a replicative or a specialized DNA polymerase, followed by extension of the resulting distorted primer terminus by DNA polymerase ζ (Polζ. The subsequent events leading to disengagement of the error-prone Polζ from the primer terminus and its replacement with an accurate replicative DNA polymerase remain largely unknown. As a first step toward understanding these events, we aimed to determine the length of DNA stretches synthesized in an error-prone manner during the Polζ-dependent lesion bypass. We developed new in vivo assays to identify the products of mutagenic TLS through a plasmid-borne tetrahydrofuran lesion and a UV-induced chromosomal lesion. We then surveyed the region downstream of the lesion site (in respect to the direction of TLS for the presence of mutations indicative of an error-prone polymerase activity. The bypass of both lesions was associated with an approximately 300,000-fold increase in the mutation rate in the adjacent DNA segment, in comparison to the mutation rate during normal replication. The hypermutated tract extended 200 bp from the lesion in the plasmid-based assay and as far as 1 kb from the lesion in the chromosome-based assay. The mutation rate in this region was similar to the rate of errors produced by purified Polζ during copying of undamaged DNA in vitro. Further, no mutations downstream of the lesion were observed in rare TLS products recovered from Polζ-deficient cells. This led us to conclude that error-prone Polζ synthesis continues for several hundred nucleotides after the lesion bypass is completed. These results provide insight into the late steps of TLS and show that error-prone TLS tracts span a substantially larger region than previously appreciated.

  15. Determination of plasmid copy number reveals the total plasmid DNA amount is greater than the chromosomal DNA amount in Bacillus thuringiensis YBT-1520.

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    Chunying Zhong

    Full Text Available Bacillus thuringiensis is the most widely used bacterial bio-insecticide, and most insecticidal crystal protein-coding genes are located on plasmids. Most strains of B. thuringiensis harbor numerous diverse plasmids, although the plasmid copy numbers (PCNs of all native plasmids in this host and the corresponding total plasmid DNA amount remains unknown. In this study, we determined the PCNs of 11 plasmids (ranging from 2 kb to 416 kb in a sequenced B. thuringiensis subsp. kurstaki strain YBT-1520 using real-time qPCR. PCNs were found to range from 1.38 to 172, and were negatively correlated to plasmid size. The amount of total plasmid DNA (∼8.7 Mbp was 1.62-fold greater than the amount of chromosomal DNA (∼5.4 Mbp at the mid-exponential growth stage (OD(600 = 2.0 of the organism. Furthermore, we selected three plasmids with different sizes and replication mechanisms to determine the PCNs over the entire life cycle. We found that the PCNs dynamically shifted at different stages, reaching their maximum during the mid-exponential growth or stationary phases and remaining stable and close to their minimum after the prespore formation stage. The PCN of pBMB2062, which is the smallest plasmid (2062 bp and has the highest PCN of those tested, varied in strain YBT-1520, HD-1, and HD-136 (172, 115, and 94, respectively. These findings provide insight into both the total plasmid DNA amount of B. thuringiensis and the strong ability of the species to harbor plasmids.

  16. Physical state & copy number of high risk human papillomavirus type 16 DNA in progression of cervical cancer

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    Shirish Shukla

    2014-01-01

    Full Text Available Background & objectives: High-risk human papilloma virus (HR-HPV infection and its integration in host genome is a key event in malignant transformation of cervical cells. HPV16 being a dominant HR-HPV type, we undertook this study to analyze if viral load and physical state of the virus correlated with each other in the absence of other confounding variables and examined their potential as predictors of progressive cervical lesions. Methods: Both, viral load and integration status of HPV16 were determined by real time URR PCR and estimation of E2:E6 ratio in a total of 130 PGMY-RLB -confirmed, monotypic HPV16-infected cervical DNA samples from biopsies of cytology-confirmed low grade (LSIL, 30 and high grade (HSIL, 30, and invasive carcinoma, (squamous cell carcinoma SCC, 70 cases. Results: Investigation of DNA samples revealed a gradual increase in HPV16 viral load over several magnitudes and increased frequency of integration from LSIL to HSIL and HSIL to invasive cancer in relation to the severity of lesions in monotypic HPV16-infected cervical tissues. In a substantial number of precancer (11/60 and cancer cases (29/70, HPV16 was detected in concomitant mixed form. The concomitant form of HPV16 genome carried significantly higher viral load. Interpretation & conclusions: Overall, viral load and integration increased with disease severity and could be useful biomarkers in disease progression, at least, in HPV16-infected cervical pre-cancer and cancer lesions.

  17. Damaging the Integrated HIV Proviral DNA with TALENs.

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    Christy L Strong

    Full Text Available HIV-1 integrates its proviral DNA genome into the host genome, presenting barriers for virus eradication. Several new gene-editing technologies have emerged that could potentially be used to damage integrated proviral DNA. In this study, we use transcription activator-like effector nucleases (TALENs to target a highly conserved sequence in the transactivation response element (TAR of the HIV-1 proviral DNA. We demonstrated that TALENs cleave a DNA template with the HIV-1 proviral target site in vitro. A GFP reporter, under control of HIV-1 TAR, was efficiently inactivated by mutations introduced by transfection of TALEN plasmids. When infected cells containing the full-length integrated HIV-1 proviral DNA were transfected with TALENs, the TAR region accumulated indels. When one of these mutants was tested, the mutated HIV-1 proviral DNA was incapable of producing detectable Gag expression. TALEN variants engineered for degenerate recognition of select nucleotide positions also cleaved proviral DNA in vitro and the full-length integrated proviral DNA genome in living cells. These results suggest a possible design strategy for the therapeutic considerations of incomplete target sequence conservation and acquired resistance mutations. We have established a new strategy for damaging integrated HIV proviral DNA that may have future potential for HIV-1 proviral DNA eradication.

  18. Novel Candidate Key Drivers in the Integrative Network of Genes, MicroRNAs, Methylations, and Copy Number Variations in Squamous Cell Lung Carcinoma

    Directory of Open Access Journals (Sweden)

    Tao Huang

    2015-01-01

    Full Text Available The mechanisms of lung cancer are highly complex. Not only mRNA gene expression but also microRNAs, DNA methylation, and copy number variation (CNV play roles in tumorigenesis. It is difficult to incorporate so much information into a single model that can comprehensively reflect all these lung cancer mechanisms. In this study, we analyzed the 129 TCGA (The Cancer Genome Atlas squamous cell lung carcinoma samples with gene expression, microRNA expression, DNA methylation, and CNV data. First, we used variance inflation factor (VIF regression to build the whole genome integrative network. Then, we isolated the lung cancer subnetwork by identifying the known lung cancer genes and their direct regulators. This subnetwork was refined by the Bayesian method, and the directed regulations among mRNA genes, microRNAs, methylations, and CNVs were obtained. The novel candidate key drivers in this refined subnetwork, such as the methylation of ARHGDIB and HOXD3, microRNA let-7a and miR-31, and the CNV of AGAP2, were identified and analyzed. On three large public available lung cancer datasets, the key drivers ARHGDIB and HOXD3 demonstrated significant associations with the overall survival of lung cancer patients. Our results provide new insights into lung cancer mechanisms.

  19. A microfluidic chip integrating DNA extraction and real-time PCR for the detection of bacteria in saliva.

    Science.gov (United States)

    Oblath, Emily A; Henley, W Hampton; Alarie, Jean Pierre; Ramsey, J Michael

    2013-04-07

    A microfluidic chip integrating DNA extraction, amplification, and detection for the identification of bacteria in saliva is described. The chip design integrated a monolithic aluminum oxide membrane (AOM) for DNA extraction with seven parallel reaction wells for real-time polymerase chain reaction (rtPCR) amplification of the extracted DNA. Samples were first heated to lyse target organisms and then added to the chip and filtered through the nanoporous AOM to extract the DNA. PCR reagents were added to each of the wells and the chip was thermocycled. Identification of Streptococcus mutans in a saliva sample is demonstrated along with the detection of 300 fg (100-125 copies) of both methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) genomic DNA (gDNA) spiked into a saliva sample. Multiple target species and strains of bacteria can be simultaneously identified in the same sample by varying the primers and probes used in each of the seven reaction wells. In initial tests, as little as 30 fg (8-12 copies) of MSSA gDNA in buffer has been successfully amplified and detected with this device.

  20. Copy number variations of genes involved in stress responses reflect the redox state and DNA damage in brewing yeasts.

    Science.gov (United States)

    Adamczyk, Jagoda; Deregowska, Anna; Skoneczny, Marek; Skoneczna, Adrianna; Natkanska, Urszula; Kwiatkowska, Aleksandra; Rawska, Ewa; Potocki, Leszek; Kuna, Ewelina; Panek, Anita; Lewinska, Anna; Wnuk, Maciej

    2016-09-01

    The yeast strains of the Saccharomyces sensu stricto complex involved in beer production are a heterogeneous group whose genetic and genomic features are not adequately determined. Thus, the aim of the present study was to provide a genetic characterization of selected group of commercially available brewing yeasts both ale top-fermenting and lager bottom-fermenting strains. Molecular karyotyping revealed that the diversity of chromosome patterns and four strains with the most accented genetic variabilities were selected and subjected to genome-wide array-based comparative genomic hybridization (array-CGH) analysis. The differences in the gene copy number were found in five functional gene categories: (1) maltose metabolism and transport, (2) response to toxin, (3) siderophore transport, (4) cellular aldehyde metabolic process, and (5) L-iditol 2-dehydrogenase activity (p < 0.05). In the Saflager W-34/70 strain (Fermentis) with the most affected array-CGH profile, loss of aryl-alcohol dehydrogenase (AAD) gene dosage correlated with an imbalanced redox state, oxidative DNA damage and breaks, lower levels of nucleolar proteins Nop1 and Fob1, and diminished tolerance to fermentation-associated stress stimuli compared to other strains. We suggest that compromised stress response may not only promote oxidant-based changes in the nucleolus state that may affect fermentation performance but also provide novel directions for future strain improvement.

  1. Development and validation of InnoQuant(®) HY, a system for quantitation and quality assessment of total human and male DNA using high copy targets.

    Science.gov (United States)

    Loftus, Andrew; Murphy, Gina; Brown, Hiromi; Montgomery, Anne; Tabak, Jonathan; Baus, James; Carroll, Marion; Green, André; Sikka, Suresh; Sinha, Sudhir

    2017-07-01

    The development and validation of InnoQuant(®) HY, a real-time PCR system containing four DNA targets-two RE autosomal targets of different sizes, male specific targets, and an internal positive control target-are described herein. The ratio of the two autosomal targets provides a Degradation Index, or a quantitative value of a sample's degradation state. The male specific targets are multi-copy targets located on the Y chromosome, which provides information about a sample's male DNA composition. The experimental results demonstrate InnoQuant HY as a robust qPCR method producing accurate DNA quantitation results even at low dynamic ranges, with reproducibility among population groups. The system is human specific with low level higher primate cross reactivity and is able to consistently and reproducibly detect sub-picogram concentrations of human and human male DNA. The use of high copy number Alu and SVA (>1000 copies per genome) retrotransposable elements as the two autosomal targets significantly enhances both sensitivity and reproducibility of determination of DNA quantitation as well as DNA degradation in forensic samples. The inclusion of a sensitive multi-copy Y-chromosome specific target provides accurate quantitation of DNA from a male in challenging male-female mixtures (i.e. sexual assault samples). Even in the presence of a large excess of DNA from a female, accurate quantitation was achieved with a male to female ratio of 1:128,000. Population database studies reveal an average Short/Y target ratio of the quantification values across all four populations tested was 1.124±0.282, exhibiting the system's reproducibility across multiple populations. The results from InnoQuant HY provide a tool equipping a forensic analyst with crucial data about a sample's DNA quantitation, male:female ratio, degradation state, and the presence or absence of PCR inhibitors. With the information gained from the InnoQuant HY kit, a more streamlined and efficient workflow

  2. DNA minicircles clarify the specific role of DNA structure on retroviral integration.

    Science.gov (United States)

    Pasi, Marco; Mornico, Damien; Volant, Stevenn; Juchet, Anna; Batisse, Julien; Bouchier, Christiane; Parissi, Vincent; Ruff, Marc; Lavery, Richard; Lavigne, Marc

    2016-09-19

    Chromatin regulates the selectivity of retroviral integration into the genome of infected cells. At the nucleosome level, both histones and DNA structure are involved in this regulation. We propose a strategy that allows to specifically study a single factor: the DNA distortion induced by the nucleosome. This strategy relies on mimicking this distortion using DNA minicircles (MCs) having a fixed rotational orientation of DNA curvature, coupled with atomic-resolution modeling. Contrasting MCs with linear DNA fragments having identical sequences enabled us to analyze the impact of DNA distortion on the efficiency and selectivity of integration. We observed a global enhancement of HIV-1 integration in MCs and an enrichment of integration sites in the outward-facing DNA major grooves. Both of these changes are favored by LEDGF/p75, revealing a new, histone-independent role of this integration cofactor. PFV integration is also enhanced in MCs, but is not associated with a periodic redistribution of integration sites, thus highlighting its distinct catalytic properties. MCs help to separate the roles of target DNA structure, histone modifications and integrase (IN) cofactors during retroviral integration and to reveal IN-specific regulation mechanisms.

  3. ParseCNV integrative copy number variation association software with quality tracking.

    Science.gov (United States)

    Glessner, Joseph T; Li, Jin; Hakonarson, Hakon

    2013-03-01

    A number of copy number variation (CNV) calling algorithms exist; however, comprehensive software tools for CNV association studies are lacking. We describe ParseCNV, unique software that takes CNV calls and creates probe-based statistics for CNV occurrence in both case-control design and in family based studies addressing both de novo and inheritance events, which are then summarized based on CNV regions (CNVRs). CNVRs are defined in a dynamic manner to allow for a complex CNV overlap while maintaining precise association region. Using this approach, we avoid failure to converge and non-monotonic curve fitting weaknesses of programs, such as CNVtools and CNVassoc, and although Plink is easy to use, it only provides combined CNV state probe-based statistics, not state-specific CNVRs. Existing CNV association methods do not provide any quality tracking information to filter confident associations, a key issue which is fully addressed by ParseCNV. In addition, uncertainty in CNV calls underlying CNV associations is evaluated to verify significant results, including CNV overlap profiles, genomic context, number of probes supporting the CNV and single-probe intensities. When optimal quality control parameters are followed using ParseCNV, 90% of CNVs validate by polymerase chain reaction, an often problematic stage because of inadequate significant association review. ParseCNV is freely available at http://parsecnv.sourceforge.net.

  4. The DNA copy number of human endogenous retrovirus-W (MSRV-type is increased in multiple sclerosis patients and is influenced by gender and disease severity.

    Directory of Open Access Journals (Sweden)

    Marta Garcia-Montojo

    Full Text Available BACKGROUND: Multiple sclerosis is an autoimmune disease more prevalent in women than in men. Multiple Sclerosis Associated Retrovirus element (MSRV is a member of type-W endogenous retrovirus family (HERV-W, known to be associated to MS. Most HERVs are unable to replicate but MSRV expression associated with reverse-transcriptase activity in MS would explain reported DNA copy number increase in MS patients. A potential link between HERV-W copies on chromosome X and gender differential prevalence has been suggested. The present study addresses MSRV-type DNA load in relation with the gender differences and clinical status in MS and healthy controls. RESULTS: 178 MS patients (62.9% women and 124 controls (56.5% women were included. MSRV env load (copies/pg of DNA was analyzed by real time qPCR with specific primers and probe for its env gene, in DNA from peripheral blood mononuclear cells (PBMCs. MSRV load was more elevated in MS patients than in controls (p = 4.15e-7. MS women presented higher MSRV load than control women (p = 0.009 and MS men also had higher load than control men (p = 2.77e-6. Besides, women had higher levels than men, both among patients (p = 0.007 and controls (p = 1.24e-6. Concordantly, EDSS and MSSS scores were higher among female patients with an elevated MSRV load (p = 0.03 and p = 0.04, respectively. CONCLUSIONS: MSRV increases its copy number in PBMC of MS patients and particularly in women with high clinical scores. This may explain causes underlying the higher prevalence of MS in women. The association with the clinical severity calls for further investigations on MSRV load in PBMCs as a biomarker for MS.

  5. Detection of Clonal and Subclonal Copy-Number Variants in Cell-Free DNA from Patients with Breast Cancer Using a Massively Multiplexed PCR Methodology

    OpenAIRE

    Eser Kirkizlar; Bernhard Zimmermann; Tudor Constantin; Ryan Swenerton; Bin Hoang; Nicholas Wayham; Babiarz, Joshua E; Zachary Demko; Pelham, Robert J.; Stephanie Kareht; Simon, Alexander L.; Kristine N. Jinnett; Matthew Rabinowitz; Styrmir Sigurjonsson; Matthew Hill

    2015-01-01

    We demonstrate proof-of-concept for the use of massively multiplexed PCR and next-generation sequencing (mmPCR-NGS) to identify both clonal and subclonal copy-number variants (CNVs) in circulating tumor DNA. This is the first report of a targeted methodology for detection of CNVs in plasma. Using an in vitro model of cell-free DNA, we show that mmPCR-NGS can accurately detect CNVs with average allelic imbalances as low as 0.5%, an improvement over previously reported whole-genome sequencin...

  6. An Integrated Workflow for DNA Methylation Analysis

    Institute of Scientific and Technical Information of China (English)

    Pingchuan Li; Feray Demirci; Gayathri Mahalingam; Caghan Demirci; Mayumi Nakano; Blake C.Meyers

    2013-01-01

    The analysis of cytosine methylation provides a new way to assess and describe epigenetic regulation at a whole-genome level in many eukaryotes.DNA methylation has a demonstrated role in the genome stability and protection,regulation of gene expression and many other aspects of genome function and maintenance.BS-seq is a relatively unbiased method for profiling the DNA methylation,with a resolution capable of measuring methylation at individual cytosines.Here we describe,as an example,a workflow to handle DNA methylation analysis,from BS-seq library preparation to the data visualization.We describe some applications for the analysis and interpretation of these data.Our laboratory provides public access to plant DNA methylation data via visualization tools available at our "Next-Gen Sequence" websites (http://mpss.udel.edu),along with small RNA,RNA-seq and other data types.

  7. Integration of hepatitis B virus DNA into chromosomal DNA during acute hepatitis B

    Institute of Scientific and Technical Information of China (English)

    Gerald C Kimbi; Anna Kramvis; Michael C Kew

    2005-01-01

    AIM: To examine the serum from black African patients with acute hepatitis B to ascertain if integrants of viral DNA can be detected in fragments of cellular DNA leaking from damaged hepatocytes into the circulation.METHODS: DNA was extracted from the sera of five patients with uncomplicated acute hepatitis B and one with fulminant disease. Two subgenomic PCRs designed to amplify the complete genome of HBV were used and the resulting amplicons were cloned and sequenced.RESULTS: HBV and chromosomal DNA were amplified from the sera of all the patients. In one patient with uncomplicated disease, HBV DNA was integrated into host chromosome 7 q11.23 in the WBSCR1 gene. The viral DNA comprised 200 nucleotides covering the S and X genes in opposite orientation, with a 1 169 nudeotide deletion. The right virus/host junction was situated at nucleotide 1774 in the cohesive overlap region of the viral genome, at a preferred topoisomerase I cleavage motif. The chromosomal DNA was not rearranged.The patient made a full recovery and seroconverted to anti-HBs- and anti-HBe-positivity. Neither HBV nor chromosomal DNA could be amplified from his serum at that time.CONCLUSION: Integration of viral DNA into chromosomal DNA may occur rarely during acute hepatitis B and, with clonal propagation of the integrant, might play a role in hepatocarcinogenesis.

  8. Copy number analysis by low coverage whole genome sequencing using ultra low-input DNA from formalin-fixed paraffin embedded tumor tissue.

    Science.gov (United States)

    Kader, Tanjina; Goode, David L; Wong, Stephen Q; Connaughton, Jacquie; Rowley, Simone M; Devereux, Lisa; Byrne, David; Fox, Stephen B; Mir Arnau, Gisela; Tothill, Richard W; Campbell, Ian G; Gorringe, Kylie L

    2016-11-15

    Unlocking clinically translatable genomic information, including copy number alterations (CNA), from formalin-fixed paraffin-embedded (FFPE) tissue is challenging due to low yields and degraded DNA. We describe a robust, cost-effective low-coverage whole genome sequencing (LC WGS) method for CNA detection using 5 ng of FFPE-derived DNA. CN profiles using 100 ng or 5 ng input DNA were highly concordant and comparable with molecular inversion probe (MIP) array profiles. LC WGS improved CN profiles of samples that performed poorly using MIP arrays. Our technique enables identification of driver and prognostic CNAs in archival patient samples previously deemed unsuitable for genomic analysis due to DNA limitations.

  9. Regionalized pathology correlates with augmentation of mtDNA copy numbers in a patient with myoclonic epilepsy with ragged-red fibers (MERRF-syndrome.

    Directory of Open Access Journals (Sweden)

    Anja Brinckmann

    Full Text Available Human patients with myoclonic epilepsy with ragged-red fibers (MERRF suffer from regionalized pathology caused by a mutation in the mitochondrial DNA (m.8344A→G. In MERRF-syndrome brain and skeletal muscles are predominantly affected, despite mtDNA being present in any tissue. In the past such tissue-specificity could not be explained by varying mtDNA mutation loads. In search for a region-specific pathology in human individuals we determined the mtDNA/nDNA ratios along with the mutation loads in 43 different post mortem tissue samples of a 16-year-old female MERRF patient and in four previously healthy victims of motor vehicle accidents. In brain and muscle we further determined the quantity of mitochondrial proteins (COX subunits II and IV, transcription factors (NRF1 and TFAM, and VDAC1 (Porin as a marker for the mitochondrial mass. In the patient the mutation loads varied merely between 89-100%. However, mtDNA copy numbers were increased 3-7 fold in predominantly affected brain areas (e.g. hippocampus, cortex and putamen and in skeletal muscle. Similar increases were absent in unaffected tissues (e.g. heart, lung, kidney, liver, and gastrointestinal organs. Such mtDNA copy number increase was not paralleled by an augmentation of mitochondrial mass in some investigated tissues, predominantly in the most affected tissue regions of the brain. We thus conclude that "futile" stimulation of mtDNA replication per se or a secondary failure to increase the mitochondrial mass may contribute to the regionalized pathology seen in MERRF-syndrome.

  10. A new set of rDNA-NTS-based multiple integrative cassettes for the development of antibiotic-marker-free recombinant yeasts.

    Science.gov (United States)

    Moon, Hye Yun; Lee, Dong Wook; Sim, Gyu Hun; Kim, Hong-Jin; Hwang, Jee Youn; Kwon, Mun-Gyeong; Kang, Bo-Kyu; Kim, Jong Man; Kang, Hyun Ah

    2016-09-10

    The traditional yeast Saccharomyces cerevisiae has been widely used as a host system to produce recombinant proteins and metabolites of great commercial value. To engineer recombinant yeast that stably maintains expression cassettes without an antibiotic resistance gene, we developed new multiple integration cassettes by exploiting the non-transcribed spacer (NTS) of ribosomal DNA (rDNA) in combination with defective selection markers. The 5' and 3'-fragments of rDNA-NTS2 were used as flanking sequences for the expression cassettes carrying a set of URA3, LEU2, HIS3, and TRP1 selection markers with truncated promoters of different lengths. The integration numbers of NTS-based expression cassettes, ranging from one to ∼30 copies, showed a proportional increase with the extent of decreased expression of the auxotrophic markers. The NTS-based cassettes were used to construct yeast strains expressing the capsid protein of red-spotted grouper necrosis virus (RG-NNVCP) in a copy number-dependent manner. Oral administration of the recombinant yeast, harboring ∼30 copies of the integrated RG-NNVCP cassettes, provoked efficient immune responses in mice. In contrast, for the NTS cassettes expressing a truncated 3-hydroxyl-3-methylglutaryl-CoA reductase, the integrant carrying only 4 copies was screened as the highest producer of squalene, showing a 150-fold increase compared to that of the wild-type strain. The multiple integrated cassettes were stably retained under prolonged nonselective conditions. Altogether, our results strongly support that rDNA-NTS integrative cassettes are useful tools to construct recombinant yeasts carrying optimal copies of a desired expression cassette without an antibiotic marker gene, which are suitable as oral vaccines or feed additives for animal and human consumption.

  11. Exposure to Inorganic Arsenic Is Associated with Increased Mitochondrial DNA Copy Number and Longer Telomere Length in Peripheral Blood

    Science.gov (United States)

    Ameer, Syeda S.; Xu, YiYi; Engström, Karin; Li, Huiqi; Tallving, Pia; Nermell, Barbro; Boemo, Analia; Parada, Luis A.; Peñaloza, Lidia G.; Concha, Gabriela; Harari, Florencia; Vahter, Marie; Broberg, Karin

    2016-01-01

    Background: Exposure to inorganic arsenic (iAs) through drinking water causes cancer. Alterations in mitochondrial DNA copy number (mtDNAcn) and telomere length in blood have been associated with cancer risk. We elucidated if arsenic exposure alters mtDNAcn and telomere length in individuals with different arsenic metabolizing capacity. Methods: We studied two groups in the Salta province, Argentina, one in the Puna area of the Andes (N = 264, 89% females) and one in Chaco (N = 169, 75% females). We assessed arsenic exposure as the sum of arsenic metabolites [iAs, methylarsonic acid (MMA), dimethylarsinic acid (DMA)] in urine (U-As) using high-performance liquid chromatography coupled with hydride generation and inductively coupled plasma mass spectrometry. Efficiency of arsenic metabolism was expressed as percentage of urinary metabolites. MtDNAcn and telomere length were determined in blood by real-time PCR. Results: Median U-As was 196 (5–95 percentile: 21–537) μg/L in Andes and 80 (5–95 percentile: 15–1637) μg/L in Chaco. The latter study group had less-efficient metabolism, with higher %iAs and %MMA in urine compared with the Andean group. U-As was significantly associated with increased mtDNAcn (log2 transformed to improve linearity) in Chaco (β = 0.027 per 100 μg/L, p = 0.0085; adjusted for age and sex), but not in Andes (β = 0.025, p = 0.24). U-As was also associated with longer telomere length in Chaco (β = 0.016, p = 0.0066) and Andes (β = 0.0075, p = 0.029). In both populations, individuals with above median %iAs showed significantly higher mtDNAcn and telomere length compared with individuals with below median %iAs. Conclusions: Arsenic was associated with increased mtDNAcn and telomere length, particularly in individuals with less-efficient arsenic metabolism, a group who may have increased risk for arsenic-related cancer. PMID:27597942

  12. Exposure to inorganic arsenic is associated with increased mitochondrial DNA copy number and longer telomere length in peripheral blood.

    Directory of Open Access Journals (Sweden)

    Syeda Shegufta Ameer

    2016-08-01

    Full Text Available Background: Exposure to inorganic arsenic (iAs through drinking water causes cancer. Alterations in mitochondrial DNA copy number (mtDNAcn and telomere length in blood have been associated with cancer risk. We elucidated if arsenic exposure alters mtDNAcn and telomere length in individuals with different arsenic metabolizing capacity.Methods: We studied two groups in the Salta province, Argentina, one in the Puna area of the Andes (N=264, 89% females and one in Chaco (N=169, 75% females. We assessed arsenic exposure as the sum of arsenic metabolites [iAs, methylarsonic acid (MMA, dimethylarsinic acid (DMA] in urine (U-As using high-performance liquid chromatography coupled with hydride generation and inductively coupled plasma mass spectrometry. Efficiency of arsenic metabolism was expressed as percentage of urinary metabolites. MtDNAcn and telomere length were determined in blood by real-time PCR. Results: Median U-As was 196 (5 - 95 percentile: 21 - 537 µg/L in Andes and 80 (5 - 95 percentile: 15 - 1637 µg/L in Chaco. The latter study group had less-efficient metabolism, with higher %iAs and %MMA in urine compared with the Andean group. U-As was significantly associated with increased mtDNAcn (log2 transformed to improve linearity in Chaco (β=0.027 per 100 µg/L, p=0.0085; adjusted for age and sex, but not in Andes (β=0.025, p=0.24. U-As was also associated with longer telomere length in Chaco (β=0.016, p=0.0066 and Andes (β=0.0075, p=0.029. In both populations, individuals with above median %iAs showed significantly higher mtDNAcn and telomere length compared with individuals with below median %iAs. Conclusions: Arsenic was associated with increased mtDNAcn and telomere length, particularly in individuals with less-efficient arsenic metabolism, a group who may have increased risk for arsenic-related cancer.

  13. Prevalence and pathogen load estimates for the fungus Batrachochytrium dendrobatidis are impacted by ITS DNA copy number variation.

    Science.gov (United States)

    Rebollar, Eria A; Woodhams, Douglas C; LaBumbard, Brandon; Kielgast, Jos; Harris, Reid N

    2017-03-21

    The ribosomal gene complex is a multi-copy region that is widely used for phylogenetic analyses of organisms from all 3 domains of life. In fungi, the copy number of the internal transcribed spacer (ITS) is used to detect abundance of pathogens causing diseases such as chytridiomycosis in amphibians and white nose syndrome in bats. Chytridiomycosis is caused by the fungi Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), and is responsible for declines and extinctions of amphibians worldwide. Over a decade ago, a qPCR assay was developed to determine Bd prevalence and pathogen load. Here, we demonstrate the effect that ITS copy number variation in Bd strains can have on the estimation of prevalence and pathogen load. We used data sets from different amphibian species to simulate how ITS copy number affects prevalence and pathogen load. In addition, we tested 2 methods (gBlocks® synthetic standards and digital PCR) to determine ITS copy number in Bd strains. Our results show that assumptions about the ITS copy number can lead to under- or overestimation of Bd prevalence and pathogen load. The use of synthetic standards replicated previously published estimates of ITS copy number, whereas dPCR resulted in estimates that were consistently lower than previously published estimates. Standardizing methods will assist with comparison across studies and produce reliable estimates of prevalence and pathogen load in the wild, while using the same Bd strain for exposure experiments and zoospore standards in qPCR remains the best method for estimating parameters used in epidemiological studies.

  14. DNA integrity determination in marine invertebrates by Fast Micromethod

    Energy Technology Data Exchange (ETDEWEB)

    Jaksic, Zeljko; Batel, Renato

    2003-12-10

    This study was focused toward the adaptation of the previously developed Fast Micromethod for DNA damage determination to marine invertebrates for the establishment of biomonitoring assessment. The Fast Micromethod{sup TM} detects DNA damage (strand breaks, alkali-labile sites and incomplete excision repair) and determines DNA integrity in cell suspensions or tissue homogenates in single microplates. The procedure is based on the ability of the specific fluorochrome dye PicoGreen to preferentially interact with high integrity DNA molecules, dsDNA, in the presence of ssDNA and proteins in high alkaline medium, thereby allowing direct fluorometric measurements of dsDNA denaturation without sample handling and stepwise DNA separations. The results presented herein describe the influence of the DNA amount and the pH of the denaturation media on slopes of the kinetic denaturation curves and calculated strand scission factors (SSFs). The optimal amount of DNA in Mytilus galloprovincialis gills homogenate was found to be 100 ng ml{sup -1} and the greatest differences in DNA unwinding kinetics (slopes and SSF values) were reached at pH 11.5. The induction of DNA damage and loss of DNA integrity was measured in native DNA isolated from cotton-spinner Holothuria tubulosa, marine sponge Suberites domuncula cells and mussel M. galloprovincialis gills homogenate. DNA damage and loss of DNA integrity were detected after induction by different doses of ({gamma}-rays, generated by {sup 137}Cs 1800 Ci; 0-500 rad in marine sponge S. domuncula cells up to SSFx(-1) values 0.082{+-}0.012 for the highest radiation dose). Analysis by chemical xenobiotics based on the in vitro action of bleomycin (bleomycin-Fe(II) complex 0-50 or 0-83 {mu}g ml{sup -1} ({mu}M)) with native DNA from cotton-spinner H. tubulosa and mussel M. galloprovincialis gills homogenate yielded values of 0.537{+-}0.072 and 0.130{+-}0.018, respectively. In vivo experiments with mussel M. galloprovincialis gills

  15. Performance Evaluation of NIPT in Detection of Chromosomal Copy Number Variants Using Low-Coverage Whole-Genome Sequencing of Plasma DNA

    DEFF Research Database (Denmark)

    Liu, Hongtai; Gao, Ya; Hu, Zhiyang

    2016-01-01

    through Maternal Plasma Sequencing (FCAPS) to compare to the karyotyping/microarray results. Sensitivity, specificity and were evaluated. Results 33 samples with deletions/duplications ranging from 1 to 129 Mb were detected with the consistent CNV size and location to karyotyping/microarray results......Objectives The aim of this study was to assess the performance of noninvasively prenatal testing (NIPT) for fetal copy number variants (CNVs) in clinical samples, using a whole-genome sequencing method. Method A total of 919 archived maternal plasma samples with karyotyping/microarray results......, including 33 CNVs samples and 886 normal samples from September 1, 2011 to May 31, 2013, were enrolled in this study. The samples were randomly rearranged and blindly sequenced by low-coverage (about 7M reads) whole-genome sequencing of plasma DNA. Fetal CNVs were detected by Fetal Copy-number Analysis...

  16. Heterozygous deletion at the RLN1 locus in a family with testicular germ cell cancer identified by integrating copy number variation data with phenome and interactome information

    DEFF Research Database (Denmark)

    Edsgard, Stefan Daniel; Scheel, M.; Hansen, Niclas Tue

    2011-01-01

    To search for disease‐related copy number variations (CNVs) in families with a high frequency of germ cell tumours (GCT), we analysed 16 individuals from four families by array comparative genomic hybridization (aCGH) and applied an integrative systems biology algorithm that prioritizes risk...

  17. Viral Carcinogenesis: Factors Inducing DNA Damage and Virus Integration

    Directory of Open Access Journals (Sweden)

    Yan Chen

    2014-10-01

    Full Text Available Viruses are the causative agents of 10%–15% of human cancers worldwide. The most common outcome for virus-induced reprogramming is genomic instability, including accumulation of mutations, aberrations and DNA damage. Although each virus has its own specific mechanism for promoting carcinogenesis, the majority of DNA oncogenic viruses encode oncogenes that transform infected cells, frequently by targeting p53 and pRB. In addition, integration of viral DNA into the human genome can also play an important role in promoting tumor development for several viruses, including HBV and HPV. Because viral integration requires the breakage of both the viral and the host DNA, the integration rate is believed to be linked to the levels of DNA damage. DNA damage can be caused by both endogenous and exogenous factors, including inflammation induced by either the virus itself or by co-infections with other agents, environmental agents and other factors. Typically, cancer develops years to decades following the initial infection. A better understanding of virus-mediated carcinogenesis, the networking of pathways involved in transformation and the relevant risk factors, particularly in those cases where tumorigenesis proceeds by way of virus integration, will help to suggest prophylactic and therapeutic strategies to reduce the risk of virus-mediated cancer.

  18. DNA Ligase III is critical for mtDNA integrity but not Xrcc1-mediated nuclear DNA repair

    Science.gov (United States)

    Gao, Yankun; Katyal, Sachin; Lee, Youngsoo; Zhao, Jingfeng; Rehg, Jerold E.; Russell, Helen R.; McKinnon, Peter J.

    2011-01-01

    DNA replication and repair in mammalian cells involves three distinct DNA ligases; ligase I (Lig1), ligase III (Lig3) and ligase IV (Lig4)1. Lig3 is considered a key ligase during base excision repair because its stability depends upon its nuclear binding partner Xrcc1, a critical factor for this DNA repair pathway2,3. Lig3 is also present in the mitochondria where its role in mitochondrial DNA (mtDNA) maintenance is independent of Xrcc14. However, the biological role of Lig3 is unclear as inactivation of murine Lig3 results in early embryonic lethality5. Here we report that Lig3 is essential for mtDNA integrity but dispensable for nuclear DNA repair. Inactivation of Lig3 in the mouse nervous system resulted in mtDNA loss leading to profound mitochondrial dysfunction, disruption of cellular homeostasis and incapacitating ataxia. Similarly, inactivation of Lig3 in cardiac muscle resulted in mitochondrial dysfunction and defective heart pump function leading to heart failure. However, Lig3 inactivation did not result in nuclear DNA repair deficiency, indicating essential DNA repair functions of Xrcc1 can occur in the absence of Lig3. Instead, we found that Lig1 was critical for DNA repair, but in a cooperative manner with Lig3. Additionally, Lig3 deficiency did not recapitulate the hallmark features of neural Xrcc1 inactivation such as DNA damage-induced cerebellar interneuron loss6, further underscoring functional separation of these DNA repair factors. Therefore, our data reveal that the critical biological role of Lig3 is to maintain mtDNA integrity and not Xrcc1-dependent DNA repair. PMID:21390131

  19. Basal-like Breast cancer DNA copy number losses identify genes involved in genomic instability, response to therapy, and patient survival.

    Science.gov (United States)

    Weigman, Victor J; Chao, Hann-Hsiang; Shabalin, Andrey A; He, Xiaping; Parker, Joel S; Nordgard, Silje H; Grushko, Tatyana; Huo, Dezheng; Nwachukwu, Chika; Nobel, Andrew; Kristensen, Vessela N; Børresen-Dale, Anne-Lise; Olopade, Olufunmilayo I; Perou, Charles M

    2012-06-01

    Breast cancer is a heterogeneous disease with known expression-defined tumor subtypes. DNA copy number studies have suggested that tumors within gene expression subtypes share similar DNA Copy number aberrations (CNA) and that CNA can be used to further sub-divide expression classes. To gain further insights into the etiologies of the intrinsic subtypes, we classified tumors according to gene expression subtype and next identified subtype-associated CNA using a novel method called SWITCHdna, using a training set of 180 tumors and a validation set of 359 tumors. Fisher's exact tests, Chi-square approximations, and Wilcoxon rank-sum tests were performed to evaluate differences in CNA by subtype. To assess the functional significance of loss of a specific chromosomal region, individual genes were knocked down by shRNA and drug sensitivity, and DNA repair foci assays performed. Most tumor subtypes exhibited specific CNA. The Basal-like subtype was the most distinct with common losses of the regions containing RB1, BRCA1, INPP4B, and the greatest overall genomic instability. One Basal-like subtype-associated CNA was loss of 5q11-35, which contains at least three genes important for BRCA1-dependent DNA repair (RAD17, RAD50, and RAP80); these genes were predominantly lost as a pair, or all three simultaneously. Loss of two or three of these genes was associated with significantly increased genomic instability and poor patient survival. RNAi knockdown of RAD17, or RAD17/RAD50, in immortalized human mammary epithelial cell lines caused increased sensitivity to a PARP inhibitor and carboplatin, and inhibited BRCA1 foci formation in response to DNA damage. These data suggest a possible genetic cause for genomic instability in Basal-like breast cancers and a biological rationale for the use of DNA repair inhibitor related therapeutics in this breast cancer subtype.

  20. DNA copy number alterations, gene expression changes and disease-free survival in patients with colorectal cancer: a 10 year follow-up.

    Science.gov (United States)

    Bigagli, Elisabetta; De Filippo, Carlotta; Castagnini, Cinzia; Toti, Simona; Acquadro, Francesco; Giudici, Francesco; Fazi, Marilena; Dolara, Piero; Messerini, Luca; Tonelli, Francesco; Luceri, Cristina

    2016-12-01

    DNA copy number alterations (CNAs) and gene expression changes have amply been encountered in colorectal cancers (CRCs), but the extent at which CNAs affect gene expression, as well as their relevance for tumor development, are still poorly defined. Here we aimed at assessing the clinical relevance of these parameters in a 10 year follow-up study. Tumors and normal adjacent colon mucosa, obtained at primary surgery from 21 CRC patients, were subjected to (i) high-resolution array CGH (a-CGH) for the detection of CNAs and (ii) microarray-based transcriptome profiling for the detection of gene expression (GE) changes. Correlations between these genomic and transcriptomic changes and their associations with clinical and histopathological parameters were assessed with the aim to identify molecular signatures associated with disease-free survival of the CRC patients during a 10 year follow-up. DNA copy number gains were frequently detected in chromosomes 7, 8q, 13, 19, 20q and X, whereas DNA copy number losses were frequently detected in chromosomes 1p, 4, 8p, 15, 17p, 18, 19 and 22q. None of these alterations were observed in all samples. In addition, we found that 2,498 genes were up- and that 1,094 genes were down-regulated in the tumor samples compared to their corresponding normal mucosa (p copy number gains, whereas decreased expression levels of the MUC1, E2F2, HRAS and SIRT3 genes were associated with copy number losses. Pathways related to cell cycle progression, eicosanoid metabolism, and TGF-β and apoptosis signaling, were found to be most significantly affected. Our results suggest that CNAs in CRC tumor tissues are associated with concomitant changes in the expression of cancer-related genes. In other genes epigenetic mechanism may be at work. Up-regulation of the IL17RA, IGF2BP2 and ABCC2 genes, and of genes acting in the mTOR and cytokine receptor pathways, appear to be associated with a poor survival. These alterations may, in addition to Dukes

  1. Array-based comparative genomic hybridization for genome-wide screening of DNA copy number in bladder tumors.

    NARCIS (Netherlands)

    Veltman, J.A.; Fridlyand, J.; Pejavar, S.; Olshen, A.B.; Korkola, J.E.; Vries, S. de; Carroll, P.; Kuo, W.L.; Pinkel, D.; Albertson, D.; Cordon-Cardo, C.; Jain, A.N.; Waldman, F.M.

    2003-01-01

    Genome-wide copy number profiles were characterized in 41 primary bladder tumors using array-based comparative genomic hybridization (array CGH). In addition to previously identified alterations in large chromosomal regions, alterations were identified in many small genomic regions, some with high-l

  2. Identification of networks of co-occurring, tumor-related DNA copy number changes using a genome-wide scoring approach.

    Directory of Open Access Journals (Sweden)

    Christiaan Klijn

    2010-01-01

    Full Text Available Tumorigenesis is a multi-step process in which normal cells transform into malignant tumors following the accumulation of genetic mutations that enable them to evade the growth control checkpoints that would normally suppress their growth or result in apoptosis. It is therefore important to identify those combinations of mutations that collaborate in cancer development and progression. DNA copy number alterations (CNAs are one of the ways in which cancer genes are deregulated in tumor cells. We hypothesized that synergistic interactions between cancer genes might be identified by looking for regions of co-occurring gain and/or loss. To this end we developed a scoring framework to separate truly co-occurring aberrations from passenger mutations and dominant single signals present in the data. The resulting regions of high co-occurrence can be investigated for between-region functional interactions. Analysis of high-resolution DNA copy number data from a panel of 95 hematological tumor cell lines correctly identified co-occurring recombinations at the T-cell receptor and immunoglobulin loci in T- and B-cell malignancies, respectively, showing that we can recover truly co-occurring genomic alterations. In addition, our analysis revealed networks of co-occurring genomic losses and gains that are enriched for cancer genes. These networks are also highly enriched for functional relationships between genes. We further examine sub-networks of these networks, core networks, which contain many known cancer genes. The core network for co-occurring DNA losses we find seems to be independent of the canonical cancer genes within the network. Our findings suggest that large-scale, low-intensity copy number alterations may be an important feature of cancer development or maintenance by affecting gene dosage of a large interconnected network of functionally related genes.

  3. Original Copies

    DEFF Research Database (Denmark)

    Sørensen, Tim Flohr

    2013-01-01

    of similarity by looking at artefactual similarity as the results of prototyping and as a production of simulacra. In this light, the concept of copying turns out to be more than simply a matter of trying to imitate an exotic or prestigious original, and it fundamentally raises the question how different a copy...

  4. Presence and integration of HBV DNA in mouse oocytes

    Institute of Scientific and Technical Information of China (English)

    Tian-Hua Huang; Qing-Jian Zhang; Qing-Dong Xie; Li-Ping Zeng; Xi-Fan Zeng

    2005-01-01

    AIM: Hepatitis B is a worldwide public health problem. To explore the feasibility of hepatitis B virus (HBV) vertical transmission via oocytes, the presence and integration of HBV DNA in mouse oocytes were studied. METHODS: Genomic DNA was isolated and metaphases were prepared, respectively from mouse oocytes cocultured with pBR322-HBV DNA plasmids. PCR, Southern blot, dot hybridization and fluorescence in situ hybridization (FISH) were performed to explore the existence and integration of HBV DNA in oocytes.RESULTS: PCR detected positive bands in the tested samples, and then Southern blot revealed clear hybridization signals in PCR products. Final washing solutions were collected for dot hybridization and no signal for HBV DNA was observed, which excluded the possibility that contamination of washing solutions gave rise to positive results of PCR and Southern blot. FISH demonstrated that 36 of 1 000 metaphases presented positive signals. CONCLUSION: HBV DNA sequences are able to pass through the zona and oolemma to enter into oocytes and tointegrate into their chromosomes. HBV DNA sequences might be brought into embryo via oocytes as vectors when they are fertilized with normal spermatozoa.

  5. TP53 copy number expansion is associated with the evolution of increased body size and an enhanced DNA damage response in elephants.

    Science.gov (United States)

    Sulak, Michael; Fong, Lindsey; Mika, Katelyn; Chigurupati, Sravanthi; Yon, Lisa; Mongan, Nigel P; Emes, Richard D; Lynch, Vincent J

    2016-09-19

    A major constraint on the evolution of large body sizes in animals is an increased risk of developing cancer. There is no correlation, however, between body size and cancer risk. This lack of correlation is often referred to as 'Peto's Paradox'. Here, we show that the elephant genome encodes 20 copies of the tumor suppressor gene TP53 and that the increase in TP53 copy number occurred coincident with the evolution of large body sizes, the evolution of extreme sensitivity to genotoxic stress, and a hyperactive TP53 signaling pathway in the elephant (Proboscidean) lineage. Furthermore, we show that several of the TP53 retrogenes (TP53RTGs) are transcribed and likely translated. While TP53RTGs do not appear to directly function as transcription factors, they do contribute to the enhanced sensitivity of elephant cells to DNA damage and the induction of apoptosis by regulating activity of the TP53 signaling pathway. These results suggest that an increase in the copy number of TP53 may have played a direct role in the evolution of very large body sizes and the resolution of Peto's paradox in Proboscideans.

  6. Integrated and Total HIV-1 DNA Predict Ex Vivo Viral Outgrowth.

    Directory of Open Access Journals (Sweden)

    Maja Kiselinova

    2016-03-01

    Full Text Available The persistence of a reservoir of latently infected CD4 T cells remains one of the major obstacles to cure HIV. Numerous strategies are being explored to eliminate this reservoir. To translate these efforts into clinical trials, there is a strong need for validated biomarkers that can monitor the reservoir over time in vivo. A comprehensive study was designed to evaluate and compare potential HIV-1 reservoir biomarkers. A cohort of 25 patients, treated with suppressive antiretroviral therapy was sampled at three time points, with median of 2.5 years (IQR: 2.4-2.6 between time point 1 and 2; and median of 31 days (IQR: 28-36 between time point 2 and 3. Patients were median of 6 years (IQR: 3-12 on ART, and plasma viral load (<50 copies/ml was suppressed for median of 4 years (IQR: 2-8. Total HIV-1 DNA, unspliced (us and multiply spliced HIV-1 RNA, and 2LTR circles were quantified by digital PCR in peripheral blood, at 3 time points. At the second time point, a viral outgrowth assay (VOA was performed, and integrated HIV-1 DNA and relative mRNA expression levels of HIV-1 restriction factors were quantified. No significant change was found for long- and short-term dynamics of all HIV-1 markers tested in peripheral blood. Integrated HIV-1 DNA was associated with total HIV-1 DNA (p<0.001, R² = 0.85, us HIV-1 RNA (p = 0.029, R² = 0.40, and VOA (p = 0.041, R2 = 0.44. Replication-competent virus was detected in 80% of patients by the VOA and it correlated with total HIV-1 DNA (p = 0.039, R² = 0.54. The mean quantification difference between Alu-PCR and VOA was 2.88 log10, and 2.23 log10 between total HIV-1 DNA and VOA. The levels of usHIV-1 RNA were inversely correlated with mRNA levels of several HIV-1 restriction factors (TRIM5α, SAMHD1, MX2, SLFN11, pSIP1. Our study reveals important correlations between the viral outgrowth and total and integrated HIV-1 DNA measures, suggesting that the total pool of HIV-1 DNA may predict the size of the

  7. An integrated web medicinal materials DNA database: MMDBD (Medicinal Materials DNA Barcode Database

    Directory of Open Access Journals (Sweden)

    But Paul

    2010-06-01

    Full Text Available Abstract Background Thousands of plants and animals possess pharmacological properties and there is an increased interest in using these materials for therapy and health maintenance. Efficacies of the application is critically dependent on the use of genuine materials. For time to time, life-threatening poisoning is found because toxic adulterant or substitute is administered. DNA barcoding provides a definitive means of authentication and for conducting molecular systematics studies. Owing to the reduced cost in DNA authentication, the volume of the DNA barcodes produced for medicinal materials is on the rise and necessitates the development of an integrated DNA database. Description We have developed an integrated DNA barcode multimedia information platform- Medicinal Materials DNA Barcode Database (MMDBD for data retrieval and similarity search. MMDBD contains over 1000 species of medicinal materials listed in the Chinese Pharmacopoeia and American Herbal Pharmacopoeia. MMDBD also contains useful information of the medicinal material, including resources, adulterant information, medical parts, photographs, primers used for obtaining the barcodes and key references. MMDBD can be accessed at http://www.cuhk.edu.hk/icm/mmdbd.htm. Conclusions This work provides a centralized medicinal materials DNA barcode database and bioinformatics tools for data storage, analysis and exchange for promoting the identification of medicinal materials. MMDBD has the largest collection of DNA barcodes of medicinal materials and is a useful resource for researchers in conservation, systematic study, forensic and herbal industry.

  8. DNA integrity of human leukocytes after magnetic resonance imaging.

    Science.gov (United States)

    Szerencsi, Ágnes; Kubinyi, Györgyi; Váliczkó, Éva; Juhász, Péter; Rudas, Gábor; Mester, Ádám; Jánossy, Gábor; Bakos, József; Thuróczy, György

    2013-10-01

    This study focuses on the effects of high-field (3T) magnetic resonance imaging (MRI) scans on the DNA integrity of human leukocytes in vitro in order to validate the study where genotoxic effects were obtained and published by Lee et al. The scanning protocol and exposure situation were the same as those used under routine clinical brain MRI scan. Peripheral blood samples from healthy non-smoking male donors were exposed to electromagnetic fields (EMF) produced by 3T magnetic resonance imaging equipment for 0, 22, 45, 67, and 89 min during the scanning procedure. Samples of positive control were exposed to ionizing radiation (4 Gy of (60)Co-γ). Single breaks of DNA in leukocytes were detected by single-cell gel electrophoresis (Comet assay). Chromosome breakage, chromosome loss and micronuclei formations were detected by a micronucleus test (MN). Three independent experiments were performed. The data of comet tail DNA%, olive tail moment and micronucleus frequency showed no DNA damages due to MRI exposure. The results of the Comet assay and the micronucleus test indicate that the applied exposure of MRI does not appear to produce breaks in the DNA and has no significant effect on DNA integrity.

  9. Reticulate evolution in diploid and tetraploid species of Polystachya (Orchidaceae) as shown by plastid DNA sequences and low-copy nuclear genes

    Science.gov (United States)

    Russell, Anton; Samuel, Rosabelle; Klejna, Verena; Barfuss, Michael H. J.; Rupp, Barbara; Chase, Mark W.

    2010-01-01

    Background and Aims Here evidence for reticulation in the pantropical orchid genus Polystachya is presented, using gene trees from five nuclear and plastid DNA data sets, first among only diploid samples (homoploid hybridization) and then with the inclusion of cloned tetraploid sequences (allopolyploids). Two groups of tetraploids are compared with respect to their origins and phylogenetic relationships. Methods Sequences from plastid regions, three low-copy nuclear genes and ITS nuclear ribosomal DNA were analysed for 56 diploid and 17 tetraploid accessions using maximum parsimony and Bayesian inference. Reticulation was inferred from incongruence between gene trees using supernetwork and consensus network analyses and from cloning and sequencing duplicated loci in tetraploids. Key Results Diploid trees from individual loci showed considerable incongruity but little reticulation signal when support from more than one gene tree was required to infer reticulation. This was coupled with generally low support in the individual gene trees. Sequencing the duplicated gene copies in tetraploids showed clearer evidence of hybrid evolution, including multiple origins of one group of tetraploids included in the study. Conclusions A combination of cloning duplicate gene copies in allotetraploids and consensus network comparison of gene trees allowed a phylogenetic framework for reticulation in Polystachya to be built. There was little evidence for homoploid hybridization, but our knowledge of the origins and relationships of three groups of allotetraploids are greatly improved by this study. One group showed evidence of multiple long-distance dispersals to achieve a pantropical distribution; another showed no evidence of multiple origins or long-distance dispersal but had greater morphological variation, consistent with hybridization between more distantly related parents. PMID:20525745

  10. T-DNA transfer and integration as a tool for insertional mutagenesis in the taxol-producing fungus

    Institute of Scientific and Technical Information of China (English)

    Chi Yan; Ping Wenxiang; Li Shanshan; Zhu Jing; Ma Xi; Gao Fengshan; Zhou Dongpo

    2008-01-01

    Agrobacterium tumefaciens-mediated DNA transformation method was applied to transform Nodulisporium sylviforme fusant HDF-68, a taxol-producing fungus. We constructed a binary vector pBI121-43 carrying a hygromycin-resistant gene cassette between the right and left borders of T-DNA. Optimal co-cultivation of N.sylviforme with A.tumefaciens containing pBI121-43 led to 110~130 hygromycin-resistant transformants per million conidia. Putative transformants were found to be mitotically stable. The molecular analysis of transformants demonstrated the random integration of single copy of the T-DNA into the host genome. This transformation system serves as a basic tool for insertional mutagenesis in N.sylviforme fusant HDF-68, and the development of such system lays a solid foundation for constructing high-yied gene engineering strain and clarifying taxol biosynthesis pathway in this fungus.

  11. HIV Integration at Certain Sites in Host DNA Is Linked to the Expansion and Persistence of Infected Cells | Poster

    Science.gov (United States)

    Editor’s note: This article was originally published on the Center for Cancer Research website. When the Human Immunodeficiency Virus (HIV) infects a cell, the virus inserts a copy of its genetic material into the host cell’s DNA. The inserted genetic material, which is also called a provirus, is used to produce new viruses. Because the viral DNA can be inserted at many sites in the host cell DNA, the site of integration marks each infected cell. Patients infected with HIV are currently treated with combined antiretroviral therapy (cART), which prevents viral replication in the majority of treated patients. When cART is initiated, most HIV-infected cells die in one or two days, and more of the infected cells die over a period of weeks to months. However there are some long-lived infected cells that do not die, which prevents patients from being cured.

  12. Mitochondrial DNA copy number augments performance of A1C and oral glucose tolerance testing in the prediction of type 2 diabetes

    Science.gov (United States)

    Cho, Seong Beom; Koh, InSong; Nam, Hye-Young; Jeon, Jae-Pil; Lee, Hong Kyu; Han, Bok-Ghee

    2017-01-01

    Here, we tested the performance of the mitochondrial DNA copy number (mtDNA-CN) in predicting future type 2 diabetes (n = 1108). We used the baseline clinical data (age, sex, body mass index, waist-to-hip ratio, systolic and diastolic blood pressure) and the mtDNA-CN, hemoglobin A1c (A1C) levels and results of oral glucose tolerance test (OGTT) including fasting plasma glucose, 1-hour glucose, and 2-hour glucose levels, to predict future diabetes. We built a prediction model using the baseline data and the diabetes status at biannual follow-up of 8 years. The mean area under curve (AUC) for all follow-ups of the full model including all variables was 0.92 ± 0.04 (mean ± standard deviation), while that of the model excluding the mtDNA-CN was 0.90 ± 0.03. The sensitivity of the f4ull model was much greater than that of the model not including mtDNA-CN: the mean sensitivities of the model with and without mtDNA-CN were 0.60 ± 0.06 and 0.53 ± 0.04, respectively. We found that the mtDNA-CN of peripheral leukocytes is a biomarker that augments the predictive power for future diabetes of A1C and OGTT. We believe that these results could provide invaluable information for developing strategies for the management of diabetes. PMID:28251996

  13. A universal strategy to interpret DNA profiles that does not require a definition of low-copy-number.

    Science.gov (United States)

    Gill, Peter; Buckleton, John

    2010-07-01

    In this paper we critically examine the causes of the underlying confusion that relates to the issue of low-template (LT) DNA profile interpretation. Firstly, there is much difficulty in attempting to distinguish between LT-DNA vs. conventional DNA because there is no discrete 'cut-off' point that can be reasonably defined or evaluated. LT-DNA is loosely characterised by drop-out (where alleles may be missing) and drop-in (where additional alleles may be present). We have previously described probabilistic methods that can be used to incorporate these phenomena using likelihood ratio (LR) principles. This is preferred to the random man not excluded (RMNE) method, because we cannot identify a coherent way forward within the restrictions provided by this framework. Most LT-DNA profiles are interpreted using a 'consensus' profile method, we called this the 'biological model', where only those alleles that are duplicated in consecutive tests are reported. We recognise that there is an increased need for probabilistic models to take precedence over the biological model. These models are required for all kinds of DNA profiles, not just those that are believed to be low-template. We also recognise that there is a need for education and training if the methods we recommend are to be widely introduced.

  14. Hepatitis B virus DNA integration and transactivation of cellular genes

    Directory of Open Access Journals (Sweden)

    Vijay Kumar

    2007-02-01

    Full Text Available

    Chronic hepatitis B virus (HBV infection is etiologically related to human hepatocellular carcinoma (HCC. Most HCCs contain integrated HBV DNA in hepatocyte, suggesting that the integration may be involved in carcinogenesis. Available data on the integrants from human hepatocellular carcinomas seem to represent primary integrants as well as the products of secondary rearrangements. By means of structural analyses of the possible primary integrants, it has been observed that the replication intermediates of the viral genome are the preferred substrates for integration. The integrated HBV DNA and the target cellular DNA are invariably associated with deletions, possibly reflecting the substrate for, and the mechanism of, the integration reaction. The host DNA sequences as well as the target site of integration in chromosomes are selected randomly suggesting that HBV DNA integration should bring about random mutagenic effects. Analysis of the samples recovered from hepatocellular carcinomas show that the integrated HBV DNA can mediate secondary rearrangements of chromosomes, such as translocations, inversions, deletions and (possibly amplifications. The integration of HBV DNA into the host genome occurs at early steps of clonal tumor expansion. The integration has been shown in a number of cases to affect a variety of cancer-related genes and to exert insertional mutagenesis. However, in contrast to the woodchuck model, in which specific HBV-DNA integration is detectable in most cases, insertional activation or inactivation of cellular genes appears to be a rare event in man. The discovery of transactivating functions exerted by HBx and truncated HBs(urface proteins supports the notion that these could be relevant to hepatocarcinogenesis as these transactivator sequences have been found in a large number of HCC tumors or hepatoma-derived cell lines. The HBx

  15. META2: Intercellular DNA Methylation Pairwise Annotation and Integrative Analysis

    Directory of Open Access Journals (Sweden)

    Binhua Tang

    2016-01-01

    Full Text Available Genome-wide deciphering intercellular differential DNA methylation as well as its roles in transcriptional regulation remains elusive in cancer epigenetics. Here we developed a toolkit META2 for DNA methylation annotation and analysis, which aims to perform integrative analysis on differentially methylated loci and regions through deep mining and statistical comparison methods. META2 contains multiple versatile functions for investigating and annotating DNA methylation profiles. Benchmarked with T-47D cell, we interrogated the association within differentially methylated CpG (DMC and region (DMR candidate count and region length and identified major transition zones as clues for inferring statistically significant DMRs; together we validated those DMRs with the functional annotation. Thus META2 can provide a comprehensive analysis approach for epigenetic research and clinical study.

  16. Performance Evaluation of NIPT in Detection of Chromosomal Copy Number Variants Using Low-Coverage Whole-Genome Sequencing of Plasma DNA.

    Directory of Open Access Journals (Sweden)

    Hongtai Liu

    Full Text Available The aim of this study was to assess the performance of noninvasively prenatal testing (NIPT for fetal copy number variants (CNVs in clinical samples, using a whole-genome sequencing method.A total of 919 archived maternal plasma samples with karyotyping/microarray results, including 33 CNVs samples and 886 normal samples from September 1, 2011 to May 31, 2013, were enrolled in this study. The samples were randomly rearranged and blindly sequenced by low-coverage (about 7M reads whole-genome sequencing of plasma DNA. Fetal CNVs were detected by Fetal Copy-number Analysis through Maternal Plasma Sequencing (FCAPS to compare to the karyotyping/microarray results. Sensitivity, specificity and were evaluated.33 samples with deletions/duplications ranging from 1 to 129 Mb were detected with the consistent CNV size and location to karyotyping/microarray results in the study. Ten false positive results and two false negative results were obtained. The sensitivity and specificity of detection deletions/duplications were 84.21% and 98.42%, respectively.Whole-genome sequencing-based NIPT has high performance in detecting genome-wide CNVs, in particular >10Mb CNVs using the current FCAPS algorithm. It is possible to implement the current method in NIPT to prenatally screening for fetal CNVs.

  17. Collateral damage: Spread of repeat-induced point mutation from a duplicated DNA sequence into an adjoining single-copy gene in Neurospora crassa

    Indian Academy of Sciences (India)

    Meenal Vyas; Durgadas P Kasbekar

    2005-02-01

    Repeat-induced point mutation (RIP) is an unusual genome defense mechanism that was discovered in Neurospora crassa. RIP occurs during a sexual cross and induces numerous G : C to A : T mutations in duplicated DNA sequences and also methylates many of the remaining cytosine residues. We measured the susceptibility of the erg-3 gene, present in single copy, to the spread of RIP from duplications of adjoining sequences. Genomic segments of defined length (1, 1.5 or 2 kb) and located at defined distances (0, 0.5, 1 or 2 kb) upstream or downstream of the erg-3 open reading frame (ORF) were amplified by polymerase chain reaction (PCR), and the duplications were created by transformation of the amplified DNA. Crosses were made with the duplication strains and the frequency of erg-3 mutant progeny provided a measure of the spread of RIP from the duplicated segments into the erg-3 gene. Our results suggest that ordinarily RIP-spread does not occur. However, occasionally the mechanism that confines RIP to the duplicated segment seems to fail (frequency 0.1–0.8%) and then RIP can spread across as much as 1 kb of unduplicated DNA. Additionally, the bacterial hph gene appeared to be very susceptible to the spread of RIP-associated cytosine methylation.

  18. Potential use of low-copy nuclear genes in DNA barcoding: a comparison with plastid genes in two Hawaiian plant radiations.

    Science.gov (United States)

    Pillon, Yohan; Johansen, Jennifer; Sakishima, Tomoko; Chamala, Srikar; Barbazuk, W Brad; Roalson, Eric H; Price, Donald K; Stacy, Elizabeth A

    2013-02-09

    DNA barcoding of land plants has relied traditionally on a small number of markers from the plastid genome. In contrast, low-copy nuclear genes have received little attention as DNA barcodes because of the absence of universal primers for PCR amplification. From pooled-species 454 transcriptome data we identified two variable intron-less nuclear loci for each of two species-rich genera of the Hawaiian flora: Clermontia (Campanulaceae) and Cyrtandra (Gesneriaceae) and compared their utility as DNA barcodes with that of plastid genes. We found that nuclear genes showed an overall greater variability, but also displayed a high level of heterozygosity, intraspecific variation, and retention of ancient alleles. Thus, nuclear genes displayed fewer species-diagnostic haplotypes compared to plastid genes and no interspecies gaps. The apparently greater coalescence times of nuclear genes are likely to limit their utility as barcodes, as only a small proportion of their alleles were fixed and unique to individual species. In both groups, species-diagnostic markers from either genome were scarce on the youngest island; a minimum age of ca. two million years may be needed for a species flock to be barcoded. For young plant groups, nuclear genes may not be a superior alternative to slowly evolving plastid genes.

  19. Validation and development of interpretation guidelines for low copy number (LCN) DNA profiling in New Zealand using the AmpFlSTR SGM Plus multiplex.

    Science.gov (United States)

    Petricevic, Sue; Whitaker, Jonathan; Buckleton, John; Vintiner, Sue; Patel, Jayshree; Simon, Pauline; Ferraby, Helen; Hermiz, Waseem; Russell, Amanda

    2010-10-01

    The characteristics of STR profiles produced from approximately 1 ng starting template using the AMPFlSTR SGM Plus multiplex and 28 PCR cycles, are well documented. However, the analysis of samples perceived as low in starting template (less than 100 pg), and referred to as low template DNA (LTDNA), can require a test of higher sensitivity in order to achieve successful results. One way of increasing this sensitivity is to increase the number of PCR amplification cycles from 28 to 34. This type of analysis has become known as low copy number, or LCN, DNA profiling. Amplification of LTDNA under LCN conditions can result in increased incidents of profile characteristics such as allelic 'drop-in' and allelic 'drop-out'. Adopting a testing regime which includes duplicate analysis, and maintaining a laboratory environment of stringent and monitored cleanliness, enables the scientist to identify and control these phenomena for a reliable interpretation of the DNA profiling results. A recent court ruling has questioned the reliability of LCN analysis and commented on the paucity of publications surrounding the validation of the technique. We present data for the LCN validation undertaken in our laboratory, and describe the guidelines and working practices we have developed for the analysis and interpretation of profiles generated after LCN profiling. This study augments the published record relating to LCN validation and should act as a useful guide for other laboratories who are considering implementing LCN profiling.

  20. A multi-locus analysis of phylogenetic relationships within grass subfamily Pooideae (Poaceae) inferred from sequences of nuclear single copy gene regions compared with plastid DNA.

    Science.gov (United States)

    Hochbach, Anne; Schneider, Julia; Röser, Martin

    2015-06-01

    To investigate phylogenetic relationships within the grass subfamily Pooideae we studied about 50 taxa covering all recognized tribes, using one plastid DNA (cpDNA) marker (matK gene-3'trnK exon) and for the first time four nuclear single copy gene loci. DNA sequence information from two parts of the nuclear genes topoisomerase 6 (Topo6) spanning the exons 8-13 and 17-19, the exons 9-13 encoding plastid acetyl-CoA-carboxylase (Acc1) and the partial exon 1 of phytochrome B (PhyB) were generated. Individual and nuclear combined data were evaluated using maximum parsimony, maximum likelihood and Bayesian methods. All of the phylogenetic results show Brachyelytrum and the tribe Nardeae as earliest diverging lineages within the subfamily. The 'core' Pooideae (Hordeeae and the Aveneae/Poeae tribe complex) are also strongly supported, as well as the monophyly of the tribes Brachypodieae, Meliceae and Stipeae (except PhyB). The beak grass tribe Diarrheneae and the tribe Duthieeae are not monophyletic in some of the analyses. However, the combined nuclear DNA (nDNA) tree yields the highest resolution and the best delimitation of the tribes, and provides the following evolutionary hypothesis for the tribes: Brachyelytrum, Nardeae, Duthieeae, Meliceae, Stipeae, Diarrheneae, Brachypodieae and the 'core' Pooideae. Within the individual datasets, the phylogenetic trees obtained from Topo6 exon 8-13 shows the most interesting results. The divergent positions of some clone sequences of Ampelodesmos mauritanicus and Trikeraia pappiformis, for instance, may indicate a hybrid origin of these stipoid taxa. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Inactivation of Pol ? and C-NHEJ eliminates off-target integration of exogenous DNA

    OpenAIRE

    Zelensky, Alex N; Schimmel, Joost; Kool, Hanneke; Kanaar, Roland; Tijsterman, Marcel

    2017-01-01

    Off-target or random integration of exogenous DNA hampers precise genomic engineering and presents a safety risk in clinical gene therapy strategies. Genetic definition of random integration has been lacking for decades. Here, we show that the A-family DNA polymerase ? (Pol ?) promotes random integration, while canonical non-homologous DNA end joining plays a secondary role; cells double deficient for polymerase ? and canonical non-homologous DNA end joining are devoid of any integration even...

  2. Integrative bacterial artificial chromosomes for DNA integration into the Bacillus subtilis chromosome.

    Science.gov (United States)

    Juhas, Mario; Ajioka, James W

    2016-06-01

    Bacillus subtilis is a well-characterized model bacterium frequently used for a number of biotechnology and synthetic biology applications. Novel strategies combining the advantages of B. subtilis with the DNA assembly and editing tools of Escherichia coli are crucial for B. subtilis engineering efforts. We combined Gibson Assembly and λ red recombineering in E. coli with RecA-mediated homologous recombination in B. subtilis for bacterial artificial chromosome-mediated DNA integration into the well-characterized amyE target locus of the B. subtilis chromosome. The engineered integrative bacterial artificial chromosome iBAC(cav) can accept any DNA fragment for integration into B. subtilis chromosome and allows rapid selection of transformants by B. subtilis-specific antibiotic resistance and the yellow fluorescent protein (mVenus) expression. We used the developed iBAC(cav)-mediated system to integrate 10kb DNA fragment from E. coli K12 MG1655 into B. subtilis chromosome. iBAC(cav)-mediated chromosomal integration approach will facilitate rational design of synthetic biology applications in B. subtilis.

  3. Multiple Components of the VHL Tumor Suppressor Complex Are Frequently Affected by DNA Copy Number Loss in Pheochromocytoma

    Science.gov (United States)

    Rowbotham, David A.; Enfield, Katey S. S.; Martinez, Victor D.; Thu, Kelsie L.; Vucic, Emily A.; Stewart, Greg L.; Bennewith, Kevin L.; Lam, Wan L.

    2014-01-01

    Pheochromocytomas (PCC) are rare tumors that arise in chromaffin tissue of the adrenal gland. PCC are frequently inherited through predisposing mutations in genes such as the von Hippel-Lindau (VHL) tumor suppressor. VHL is part of the VHL elongin BC protein complex that also includes CUL2/5, TCEB1, TCEB2, and RBX1; in normoxic conditions this complex targets hypoxia-inducible factor 1 alpha (HIF1A) for degradation, thus preventing a hypoxic response. VHL inactivation by genetic mechanisms, such as mutation and loss of heterozygosity, inhibits HIF1A degradation, even in the presence of oxygen, and induces a pseudohypoxic response. However, the described <10% VHL mutation rate cannot account for the high frequency of hypoxic response observed. Indeed, little is known about genetic mechanisms disrupting other complex component genes. Here, we show that, in a panel of 171 PCC tumors, 59.6% harbored gene copy number loss (CNL) of at least one complex component. CNL significantly reduced gene expression and was associated with enrichment of gene targets controlled by HIF1. Interestingly, we show that VHL-related renal clear cell carcinoma harbored disruption of VHL alone. Our results indicate that VHL elongin BC protein complex components other than VHL could be important for PCC tumorigenesis and merit further investigation. PMID:25298778

  4. Multiple Components of the VHL Tumor Suppressor Complex Are Frequently Affected by DNA Copy Number Loss in Pheochromocytoma

    Directory of Open Access Journals (Sweden)

    David A. Rowbotham

    2014-01-01

    Full Text Available Pheochromocytomas (PCC are rare tumors that arise in chromaffin tissue of the adrenal gland. PCC are frequently inherited through predisposing mutations in genes such as the von Hippel-Lindau (VHL tumor suppressor. VHL is part of the VHL elongin BC protein complex that also includes CUL2/5, TCEB1, TCEB2, and RBX1; in normoxic conditions this complex targets hypoxia-inducible factor 1 alpha (HIF1A for degradation, thus preventing a hypoxic response. VHL inactivation by genetic mechanisms, such as mutation and loss of heterozygosity, inhibits HIF1A degradation, even in the presence of oxygen, and induces a pseudohypoxic response. However, the described <10% VHL mutation rate cannot account for the high frequency of hypoxic response observed. Indeed, little is known about genetic mechanisms disrupting other complex component genes. Here, we show that, in a panel of 171 PCC tumors, 59.6% harbored gene copy number loss (CNL of at least one complex component. CNL significantly reduced gene expression and was associated with enrichment of gene targets controlled by HIF1. Interestingly, we show that VHL-related renal clear cell carcinoma harbored disruption of VHL alone. Our results indicate that VHL elongin BC protein complex components other than VHL could be important for PCC tumorigenesis and merit further investigation.

  5. Multiple Components of the VHL Tumor Suppressor Complex Are Frequently Affected by DNA Copy Number Loss in Pheochromocytoma.

    Science.gov (United States)

    Rowbotham, David A; Enfield, Katey S S; Martinez, Victor D; Thu, Kelsie L; Vucic, Emily A; Stewart, Greg L; Bennewith, Kevin L; Lam, Wan L

    2014-01-01

    Pheochromocytomas (PCC) are rare tumors that arise in chromaffin tissue of the adrenal gland. PCC are frequently inherited through predisposing mutations in genes such as the von Hippel-Lindau (VHL) tumor suppressor. VHL is part of the VHL elongin BC protein complex that also includes CUL2/5, TCEB1, TCEB2, and RBX1; in normoxic conditions this complex targets hypoxia-inducible factor 1 alpha (HIF1A) for degradation, thus preventing a hypoxic response. VHL inactivation by genetic mechanisms, such as mutation and loss of heterozygosity, inhibits HIF1A degradation, even in the presence of oxygen, and induces a pseudohypoxic response. However, the described complex component genes. Here, we show that, in a panel of 171 PCC tumors, 59.6% harbored gene copy number loss (CNL) of at least one complex component. CNL significantly reduced gene expression and was associated with enrichment of gene targets controlled by HIF1. Interestingly, we show that VHL-related renal clear cell carcinoma harbored disruption of VHL alone. Our results indicate that VHL elongin BC protein complex components other than VHL could be important for PCC tumorigenesis and merit further investigation.

  6. Recurrent De Novo Dominant Mutations in SLC25A4 Cause Severe Early-Onset Mitochondrial Disease and Loss of Mitochondrial DNA Copy Number.

    Science.gov (United States)

    Thompson, Kyle; Majd, Homa; Dallabona, Christina; Reinson, Karit; King, Martin S; Alston, Charlotte L; He, Langping; Lodi, Tiziana; Jones, Simon A; Fattal-Valevski, Aviva; Fraenkel, Nitay D; Saada, Ann; Haham, Alon; Isohanni, Pirjo; Vara, Roshni; Barbosa, Inês A; Simpson, Michael A; Deshpande, Charu; Puusepp, Sanna; Bonnen, Penelope E; Rodenburg, Richard J; Suomalainen, Anu; Õunap, Katrin; Elpeleg, Orly; Ferrero, Ileana; McFarland, Robert; Kunji, Edmund R S; Taylor, Robert W

    2016-10-06

    Mutations in SLC25A4 encoding the mitochondrial ADP/ATP carrier AAC1 are well-recognized causes of mitochondrial disease. Several heterozygous SLC25A4 mutations cause adult-onset autosomal-dominant progressive external ophthalmoplegia associated with multiple mitochondrial DNA deletions, whereas recessive SLC25A4 mutations cause childhood-onset mitochondrial myopathy and cardiomyopathy. Here, we describe the identification by whole-exome sequencing of seven probands harboring dominant, de novo SLC25A4 mutations. All affected individuals presented at birth, were ventilator dependent and, where tested, revealed severe combined mitochondrial respiratory chain deficiencies associated with a marked loss of mitochondrial DNA copy number in skeletal muscle. Strikingly, an identical c.239G>A (p.Arg80His) mutation was present in four of the seven subjects, and the other three case subjects harbored the same c.703C>G (p.Arg235Gly) mutation. Analysis of skeletal muscle revealed a marked decrease of AAC1 protein levels and loss of respiratory chain complexes containing mitochondrial DNA-encoded subunits. We show that both recombinant AAC1 mutant proteins are severely impaired in ADP/ATP transport, affecting most likely the substrate binding and mechanics of the carrier, respectively. This highly reduced capacity for transport probably affects mitochondrial DNA maintenance and in turn respiration, causing a severe energy crisis. The confirmation of the pathogenicity of these de novo SLC25A4 mutations highlights a third distinct clinical phenotype associated with mutation of this gene and demonstrates that early-onset mitochondrial disease can be caused by recurrent de novo mutations, which has significant implications for the application and analysis of whole-exome sequencing data in mitochondrial disease. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  7. High-resolution copy number profiling by array CGH using DNA isolated from formalin-fixed, paraffin-embedded tissues.

    Science.gov (United States)

    van Essen, Hendrik F; Ylstra, Bauke

    2012-01-01

    We describe protocols to acquire high-quality DNA from formalin-fixed, paraffin-embedded (FFPE) tissues for the use in array comparative genome hybridization (CGH). Formalin fixation combined with paraffin embedding is routine procedure for solid malignancies in the diagnostic practice of the pathologist. As a consequence, large archives of FFPE tissues are available in pathology institutes across the globe. This archival material is for many research questions an invaluable resource, with long-term clinical follow-up and survival data available. FFPE is, thus, highly attractive for large genomics studies, including experiments requiring samples for test/learning and validation. Most larger array CGH studies have, therefore, made use of FFPE material and show that CNAs have tumor- and tissue-specific traits (Chin et al. Cancer Cell 10: 529-541, 2006; Fridlyand et al. BMC Cancer 6: 96, 2006; Weiss et al. Oncogene 22: 1872-1879, 2003; Jong et al. Oncogene 26: 1499-1506, 2007). The protocols described are tailored to array CGH of FFPE solid malignancies: from sectioning FFPE blocks to specific cynosures for pathological revisions of sections, DNA isolation, quality testing, and amplification. The protocols are technical in character and elaborate up to the labeling of isolated DNA while further processes and interpretation and data analysis are beyond the scope.

  8. Cascade of chromosomal rearrangements caused by a heterogeneous T-DNA integration supports the double-strand break repair model for T-DNA integration.

    Science.gov (United States)

    Hu, Yufei; Chen, Zhiyu; Zhuang, Chuxiong; Huang, Jilei

    2017-02-28

    Transferred DNA (T-DNA) from Agrobacterium tumefaciens can be integrated into the plant genome. The double-strand break repair (DSBR) pathway is a major model for T-DNA integration. From this model, we expect that two ends of a T-DNA molecule would invade into a single DNA double-strand break (DSB) or independent DSBs in the plant genome. We call the later phenomenon a heterogeneous T-DNA integration which has never been observed. In this work, we demonstrated it in an Arabidopsis T-DNA insertion mutant seb19. To resolve the chromosomal structural changes caused by T-DNA integration at both the nucleotide and chromosome levels, we performed inverse PCR, genome resequencing, fluorescence in situ hybridization and linkage analysis. We found, in seb19, a single T-DNA connected two different chromosomal loci and caused complex chromosomal rearrangements. The specific break-junction pattern in seb19 is consistent with the result of heterogeneous T-DNA integration but not of recombination between two T-DNA insertions. We demonstrated that, in seb19, heterogeneous T-DNA integration evoked a cascade of incorrect repair of seven DSBs on chromosome 4 and 5, and then produced translocation, inversion, duplication and deletion. Heterogeneous T-DNA integration supports the DSBR model and suggests that two ends of a T-DNA molecule could be integrated into the plant genome independently. Our results also show a new origin of chromosomal abnormalities. This article is protected by copyright. All rights reserved.

  9. Detection of Clonal and Subclonal Copy-Number Variants in Cell-Free DNA from Patients with Breast Cancer Using a Massively Multiplexed PCR Methodology

    Science.gov (United States)

    Kirkizlar, Eser; Zimmermann, Bernhard; Constantin, Tudor; Swenerton, Ryan; Hoang, Bin; Wayham, Nicholas; Babiarz, Joshua E.; Demko, Zachary; Pelham, Robert J.; Kareht, Stephanie; Simon, Alexander L.; Jinnett, Kristine N.; Rabinowitz, Matthew; Sigurjonsson, Styrmir; Hill, Matthew

    2015-01-01

    We demonstrate proof-of-concept for the use of massively multiplexed PCR and next-generation sequencing (mmPCR-NGS) to identify both clonal and subclonal copy-number variants (CNVs) in circulating tumor DNA. This is the first report of a targeted methodology for detection of CNVs in plasma. Using an in vitro model of cell-free DNA, we show that mmPCR-NGS can accurately detect CNVs with average allelic imbalances as low as 0.5%, an improvement over previously reported whole-genome sequencing approaches. Our method revealed differences in the spectrum of CNVs detected in tumor tissue subsections and matching plasma samples from 11 patients with stage II breast cancer. Moreover, we showed that liquid biopsies are able to detect subclonal mutations that may be missed in tumor tissue biopsies. We anticipate that this mmPCR-NGS methodology will have broad applicability for the characterization, diagnosis, and therapeutic monitoring of CNV-enriched cancers, such as breast, ovarian, and lung cancer. PMID:26500031

  10. Genome-wide analysis of DNA methylation, copy number variation, and gene expression in monozygotic twins discordant for primary biliary cirrhosis

    Directory of Open Access Journals (Sweden)

    Carlo eSelmi

    2014-03-01

    Full Text Available Primary biliary cirrhosis (PBC is an uncommon autoimmune disease with a homogeneous clinical phenotype that reflects incomplete disease concordance in monozygotic (MZ twins. We have taken advantage of a unique collection consisting of genomic DNA and mRNA from peripheral blood cells of female MZ twins (n=3 sets and sisters of similar age (n=8 pairs discordant for disease. We performed a genome-wide study to investigate differences in (i DNA methylation (using a custom tiled 4-plex array containing tiled 50-mers 19,084 randomly chosen methylation sites, (ii copy number variation (CNV (with a chip including markers derived from the 1000 Genomes Project, all three HapMap phases, and recently published studies, and/or (iii gene expression (by whole-genome expression arrays. Based on the results obtained from these three approaches we utilized quantitative PCR to compare the expression of candidate genes. Importantly, our data support consistent differences in discordant twins and siblings for the (i methylation profiles of 60 gene regions, (ii CNV of 10 genes, and (iii the expression of 2 interferon-dependent genes. Quantitative PCR analysis showed that 17 of these genes are differentially expressed in discordant sibling pairs. In conclusion, we report that MZ twins and sisters discordant for PBC manifest particular epigenetic differences and highlight the value of the epigenetic study of twins.

  11. Agrobacterium may delay plant nonhomologous end-joining DNA repair via XRCC4 to favor T-DNA integration.

    Science.gov (United States)

    Vaghchhipawala, Zarir E; Vasudevan, Balaji; Lee, Seonghee; Morsy, Mustafa R; Mysore, Kirankumar S

    2012-10-01

    Agrobacterium tumefaciens is a soilborne pathogen that causes crown gall disease in many dicotyledonous plants by transfer of a portion of its tumor-inducing plasmid (T-DNA) into the plant genome. Several plant factors that play a role in Agrobacterium attachment to plant cells and transport of T-DNA to the nucleus have been identified, but the T-DNA integration step during transformation is poorly understood and has been proposed to occur via nonhomologous end-joining (NHEJ)-mediated double-strand DNA break (DSB) repair. Here, we report a negative role of X-ray cross complementation group4 (XRCC4), one of the key proteins required for NHEJ, in Agrobacterium T-DNA integration. Downregulation of XRCC4 in Arabidopsis and Nicotiana benthamiana increased stable transformation due to increased T-DNA integration. Overexpression of XRCC4 in Arabidopsis decreased stable transformation due to decreased T-DNA integration. Interestingly, XRCC4 directly interacted with Agrobacterium protein VirE2 in a yeast two-hybrid system and in planta. VirE2-expressing Arabidopsis plants were more susceptible to the DNA damaging chemical bleomycin and showed increased stable transformation. We hypothesize that VirE2 titrates or excludes active XRCC4 protein available for DSB repair, thus delaying the closure of DSBs in the chromosome, providing greater opportunity for T-DNA to integrate.

  12. Plasma DNA integrity index as a potential molecular diagnostic marker for breast cancer.

    Science.gov (United States)

    Kamel, Azza M; Teama, Salwa; Fawzy, Amal; El Deftar, Mervat

    2016-06-01

    Plasma DNA integrity index is increased in various malignancies including breast cancer, the most common cancer in women worldwide; early detection is crucial for successful treatment. Current screening methods fail to detect many cases of breast cancer at an early stage. In this study, we evaluated the level of plasma DNA integrity index in 260 females (95 with breast cancer, 95 with benign breast lesions, and 70 healthy controls) to verify its potential value in discriminating malignant from benign breast lesions. The criteria of the American Joint Committee on Cancer were used for staging of breast cancer patients. DNA integrity index was measured by real-time PCR. DNA integrity index was significantly higher in breast cancer than in benign breast patients and healthy subjects (P = cancer group was 85.3 % at 0.55 DNA integrity index cutoff. In conclusion, the plasma DNA integrity index may be a promising molecular diagnostic marker of malignancy in breast lesions.

  13. A High Excision Potential of TALENs for Integrated DNA of HIV-Based Lentiviral Vector

    OpenAIRE

    Hirotaka Ebina; Yuka Kanemura; Naoko Misawa; Tetsushi Sakuma; Tomoko Kobayashi; Takashi Yamamoto; Yoshio Koyanagi

    2015-01-01

    DNA-editing technology has made it possible to rewrite genetic information in living cells. Human immunodeficiency virus (HIV) provirus, an integrated form of viral complementary DNA in host chromosomes, could be a potential target for this technology. We recently reported that HIV proviral DNA could be excised from the chromosomal DNA of HIV-based lentiviral DNA-transduced T cells after multiple introductions of a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 endonuc...

  14. Prevalence and pathogen load estimates for the fungus Batrachochytrium dendrobatidis are impacted by ITS DNA copy number variation

    DEFF Research Database (Denmark)

    Rebollar, Eria A.; Woodhams, Douglas C.; LaBumbard, Brandon

    2017-01-01

    The ribosomal gene complex is a multi-copy region that is widely used for phylogenetic analyses of organisms from all 3 domains of life. In fungi, the copy number of the internal transcribed spacer (ITS) is used to detect abundance of pathogens causing diseases such as chytridiomycosis in amphibi...

  15. Body fluid identification by integrated analysis of DNA methylation and body fluid-specific microbial DNA.

    Science.gov (United States)

    Choi, Ajin; Shin, Kyoung-Jin; Yang, Woo Ick; Lee, Hwan Young

    2014-01-01

    Identification of body fluids found at crime scenes provides important information that can support a link between sample donors and actual criminal acts. Previous studies have reported that DNA methylation analysis at several tissue-specific differentially methylated regions (tDMRs) enables successful identification of semen, and the detection of certain bacterial DNA can allow for identification of saliva and vaginal fluid. In the present study, a method for detecting bacterial DNA was integrated into a previously reported multiplex methylation-sensitive restriction enzyme-polymerase chain reaction. The developed multiplex PCR was modified by the addition of a new semen-specific marker and by including amplicons for the 16S ribosomal RNA gene of saliva- and vaginal fluid-specific bacteria to improve the efficacy to detect a specific type of body fluid. Using the developed multiplex system, semen was distinguishable by unmethylation at the USP49, DACT1, and PFN3 tDMRs and by hypermethylation at L81528, and saliva could be identified by detection of saliva-specific bacteria, Veillonella atypica and/or Streptococcus salivarius. Additionally, vaginal fluid and menstrual blood were differentiated from other body fluids by hypomethylation at the PFN3 tDMR and the presence of vaginal fluid-specific bacteria, Lactobacillus crispatus and/or Lactobacillus gasseri. Because the developed multiplex system uses the same biological source of DNA for individual identification profiling and simultaneously analyses various types of body fluid in one PCR reaction, this method will facilitate more efficient body fluid identification in forensic casework.

  16. Vertically integrated analysis of human DNA. Final technical report

    Energy Technology Data Exchange (ETDEWEB)

    Olson, M.

    1997-10-01

    This project has been oriented toward improving the vertical integration of the sequential steps associated with the large-scale analysis of human DNA. The central focus has been on an approach to the preparation of {open_quotes}sequence-ready{close_quotes} maps, which is referred to as multiple-complete-digest (MCD) mapping, primarily directed at cosmid clones. MCD mapping relies on simple experimental steps, supported by advanced image-analysis and map-assembly software, to produce extremely accurate restriction-site and clone-overlap maps. We believe that MCD mapping is one of the few high-resolution mapping systems that has the potential for high-level automation. Successful automation of this process would be a landmark event in genome analysis. Once other higher organisms, paving the way for cost-effective sequencing of these genomes. Critically, MCD mapping has the potential to provide built-in quality control for sequencing accuracy and to make possible a highly integrated end product even if there are large numbers of discontinuities in the actual sequence.

  17. DNA transformations of Candida tropicalis with replicating and integrative vectors.

    Science.gov (United States)

    Sanglard, D; Fiechter, A

    1992-12-01

    The alkane-assimilating yeast Candida tropicalis was used as a host for DNA transformations. A stable ade2 mutant (Ha900) obtained by UV-mutagenesis was used as a recipient for different vectors carrying selectable markers. A first vector, pMK16, that was developed for the transformation of C. albicans and carries an ADE2 gene marker and a Candida autonomously replicating sequence (CARS) element promoting autonomous replication, was compatible for transforming Ha900. Two transformant types were observed: (i) pink transformants which easily lose pMK16 under non-selective growth conditions; (ii) white transformants, in which the same plasmid exhibited a higher mitotic stability. In both cases pMK16 could be rescued from these cells in Escherichia coli. A second vector, pADE2, containing the isolated C. tropicalis ADE2, gene, was used to transform Ha900. This vector integrated in the yeast genome at homologous sites of the ade2 locus. Different integration types were observed at one or both ade2 alleles in single or in tandem repeats.

  18. Use of Quantitative Polymerase Chain Reaction for Determining Copy Numbers of Transgenes in Lesquerella fendleri

    Directory of Open Access Journals (Sweden)

    Grace Q. Chen

    2010-01-01

    Full Text Available Problem statement: In transgenic plants, the number of transgene copies could greatly influence the level of expression and genetic stability of the target gene, thus it is important to develop an efficient method for accurate estimation of transgene copies. The quantitative Polymerase Chain Reaction (qPCR technique is becoming more efficient nowadays to determine copy numbers of transgenes in transgenic plants, being used here, for the first time in quantifying copy numbers of transgenes in Lesquerella fendleri. Approach: The system utilized a known one copy gene, LfKCS4/5, from L. fendleri as an endogenous calibrator and the threshold Crossing point (Ct measured by Applied Biosystem 7500 system to calculate the copy numbers of transgenes in primary transgenic lines (T0 generation. Results: The qPCR condition was optimized and each primer set had a PCR efficiency of 0.99 or 1.01. Our data demonstrated unambiguous 2-fold discrimination of the copy number of β-glucuronidase gene (gusA and hygromycine phosphotransferase II (hptII genes in 12 T0 lines. Most of the lines contained one or two copies of each gene. Eight out of 12 samples (66.7% showed more copies of gusA gene than that of hptII gene, suggesting rearrangements of the Transferred (T-DNA. Possible modifications of the T-DNA cassette in L. fendleri are discussed based on main models of T-DNA integration in the plant genome. Conclusion: The qPCR described in this study is an efficient method and it is particularly useful in identification and selection of transgenic plants with desirable copy numbers at early stage.

  19. Characterization of copy numbers of 16S rDNA and 16S rRNA of Candidatus Liberibacter asiaticus and the implication in detection in planta using quantitative PCR

    Directory of Open Access Journals (Sweden)

    Wang Nian

    2009-03-01

    Full Text Available Abstract Background Citrus Huanglongbing (HLB is one of the most devastating diseases on citrus and is associated with Candidatus Liberibacter spp.. The pathogens are phloem limited and have not been cultured in vitro. The current management strategy of HLB is to remove infected citrus trees and reduce psyllid populations with insecticides to prevent the spreading. This strategy requires sensitive and reliable diagnostic methods for early detection. Results We investigated the copy numbers of the 16S rDNA and 16S rRNA of the HLB pathogen and the implication of improving the diagnosis of HLB for early detection using Quantitative PCR. We compared the detection of HLB with different Quantitative PCR based methods with primers/probe targeting either 16S rDNA, beta-operon DNA, 16S rRNA, or beta-operon RNA. The 16S rDNA copy number of Ca. Liberibacter asiaticus was estimated to be three times of that of the beta-operon region, thus allowing detection of lower titer of Ca. L. asiaticus. Quantitative reverse transcriptional PCR (QRT-PCR indicated that the 16S rRNA averaged 7.83 times more than that of 16S rDNA for the same samples. Dilution analysis also indicates that QRT-PCR targeting 16S rRNA is 10 time more sensitive than QPCR targeting 16S rDNA. Thus QRT-PCR was able to increase the sensitivity of detection by targeting 16S rRNA. Conclusion Our result indicates that Candidatus Liberibacter asiaticus contains three copies of 16S rDNA. The copy number of 16S rRNA of Ca. L. asiaticus in planta averaged about 7.8 times of 16S rDNA for the same set of samples tested in this study. Detection sensitivity of HLB could be improved through the following approaches: using 16S rDNA based primers/probe in the QPCR assays; and using QRT-PCR assays targeting 16S rRNA.

  20. Low-copy nuclear DNA, phylogeny and the evolution of dichogamy in the betel nut palms and their relatives (Arecinae; Arecaceae).

    Science.gov (United States)

    Loo, Adrian H B; Dransfield, John; Chase, Mark W; Baker, William J

    2006-06-01

    For the betel nut palm genus Areca and the other seven genera in subtribe Arecinae (Areceae; Arecoideae; Arecaceae) we collected DNA sequences from two low-copy nuclear genes, phosphoribulokinase (PRK) and the second largest subunit of RNA polymerase II (RPB2). The data were used to evaluate monophyly of the subtribe and its component genera, explore the radiation of the group across its range, and examine evolution of protandry and protogyny, which is particularly diverse in Arecinae. The subtribe and some genera are not monophyletic. Three lineages of Arecinae are recovered: one widespread, but centered on the Sunda Shelf, another endemic to the islands east of Wallace's line and a third, comprising the Sri Lanka endemic Loxococcus, that is most closely related to genera from outside subtribe Arecinae. Strong support is obtained for broadening the circumscription of the genus Hydriastele to include Gronophyllum, Gulubia and Siphokentia. In clarifying phylogenetic relationships, we have demonstrated that a perceived bimodal distribution of the subtribe across Wallace's line does not in fact exist. Character optimizations indicate that the evolution of protogyny, an unusual condition in palms, is potentially correlated with a large radiation in the genus Pinanga and possibly also to dramatic diversification in pollen morphology and genome size. The evolution of dichogamy in the clade endemic to the east of Wallace's line is complex and reveals a pattern of numerous transformations between protandry and protogyny that is in marked contrast with other Arecinae. We suggest that this contrast is most likely a reflection of differing geological histories and pollinator spectra in each region.

  1. Integration of copy number and transcriptomics provides risk stratification in prostate cancer: A discovery and validation cohort study

    Directory of Open Access Journals (Sweden)

    H. Ross-Adams

    2015-09-01

    Interpretation: For the first time in prostate cancer this study demonstrates the importance of integrated genomic analyses incorporating both benign and tumour tissue data in identifying molecular alterations leading to the generation of robust gene sets that are predictive of clinical outcome in independent patient cohorts.

  2. Highly informative single-copy nuclear microsatellite DNA markers developed using an AFLP-SSR approach in black spruce (Picea mariana and red spruce (P. rubens.

    Directory of Open Access Journals (Sweden)

    Yong-Zhong Shi

    Full Text Available Microsatellites or simple sequence repeats (SSRs are highly informative molecular markers for various biological studies in plants. In spruce (Picea and other conifers, the development of single-copy polymorphic genomic microsatellite markers is quite difficult, owing primarily to the large genome size and predominance of repetitive DNA sequences throughout the genome. We have developed highly informative single-locus genomic microsatellite markers in black spruce (Picea mariana and red spruce (Picea rubens using a simple but efficient method based on a combination of AFLP and microsatellite technologies.A microsatellite-enriched library was constructed from genomic AFLP DNA fragments of black spruce. Sequencing of the 108 putative SSR-containing clones provided 94 unique sequences with microsatellites. Twenty-two of the designed 34 primer pairs yielded scorable amplicons, with single-locus patterns. Fourteen of these microsatellite markers were characterized in 30 black spruce and 30 red spruce individuals drawn from many populations. The number of alleles at a polymorphic locus ranged from 2 to 18, with a mean of 9.3 in black spruce, and from 3 to 15, with a mean of 6.2 alleles in red spruce. The polymorphic information content or expected heterozygosity ranged from 0.340 to 0.909 (mean = 0.67 in black spruce and from 0.161 to 0.851 (mean = 0.62 in red spruce. Ten SSR markers showing inter-parental polymorphism inherited in a single-locus Mendelian mode, with two cases of distorted segregation. Primer pairs for almost all polymorphic SSR loci resolved microsatellites of comparable size in Picea glauca, P. engelmannii, P. sitchensis, and P. abies.The AFLP-based microsatellite-enriched library appears to be a rapid, cost-effective approach for isolating and developing single-locus informative genomic microsatellite markers in black spruce. The markers developed should be useful in black spruce, red spruce and other Picea species for

  3. Human telomeres that carry an integrated copy of human herpesvirus 6 are often short and unstable, facilitating release of the viral genome from the chromosome.

    Science.gov (United States)

    Huang, Yan; Hidalgo-Bravo, Alberto; Zhang, Enjie; Cotton, Victoria E; Mendez-Bermudez, Aaron; Wig, Gunjan; Medina-Calzada, Zahara; Neumann, Rita; Jeffreys, Alec J; Winney, Bruce; Wilson, James F; Clark, Duncan A; Dyer, Martin J; Royle, Nicola J

    2014-01-01

    Linear chromosomes are stabilized by telomeres, but the presence of short dysfunctional telomeres triggers cellular senescence in human somatic tissues, thus contributing to ageing. Approximately 1% of the population inherits a chromosomally integrated copy of human herpesvirus 6 (CI-HHV-6), but the consequences of integration for the virus and for the telomere with the insertion are unknown. Here we show that the telomere on the distal end of the integrated virus is frequently the shortest measured in somatic cells but not the germline. The telomere carrying the CI-HHV-6 is also prone to truncations that result in the formation of a short telomere at a novel location within the viral genome. We detected extra-chromosomal circular HHV-6 molecules, some surprisingly comprising the entire viral genome with a single fully reconstituted direct repeat region (DR) with both terminal cleavage and packaging elements (PAC1 and PAC2). Truncated CI-HHV-6 and extra-chromosomal circular molecules are likely reciprocal products that arise through excision of a telomere-loop (t-loop) formed within the CI-HHV-6 genome. In summary, we show that the CI-HHV-6 genome disrupts stability of the associated telomere and this facilitates the release of viral sequences as circular molecules, some of which have the potential to become fully functioning viruses.

  4. CRISPR Outsourcing: Commissioning IHF for Site-Specific Integration of Foreign DNA at the CRISPR Array.

    Science.gov (United States)

    Wei, Yunzhou; Terns, Michael P

    2016-06-16

    In this issue of Molecular Cell, Nuñez et al. (2016) report that site-specific integration of foreign DNA into CRISPR loci by the Cas1-Cas2 integrase complex is promoted by a host factor, IHF (integration host factor), that binds and bends CRISPR leader DNA.

  5. Modern human sperm freezing: Effect on DNA, chromatin and acrosome integrity

    Directory of Open Access Journals (Sweden)

    Tahereh Rahiminia

    2017-08-01

    Conclusion: Sperm in Vapour was healthier in terms of DNA, chromatin and acrosome integrity. In contrast of higher motility and normal morphology; DNA, chromatin and acrosome integrity were decreased in Vit. However, these findings were more acceptable in SSV or Vapour.

  6. Constructing Bayesian networks by integrating gene expression and copy number data identifies NLGN4Y as a novel regulator of prostate cancer progression.

    Science.gov (United States)

    Gong, Yixuan; Wang, Li; Chippada-Venkata, Uma; Dai, Xudong; Oh, William K; Zhu, Jun

    2016-10-18

    To understand the heterogeneity of prostate cancer (PCa) and identify novel underlying drivers, we constructed integrative molecular Bayesian networks (IMBNs) for PCa by integrating gene expression and copy number alteration data from published datasets. After demonstrating such IMBNs with superior network accuracy, we identified multiple sub-networks within IMBNs related to biochemical recurrence (BCR) of PCa and inferred the corresponding key drivers. The key drivers regulated a set of common effectors including genes preferentially expressed in neuronal cells. NLGN4Y-a protein involved in synaptic adhesion in neurons-was ranked as the top gene closely linked to key drivers of myogenesis subnetworks. Lower expression of NLGN4Y was associated with higher grade PCa and an increased risk of BCR. We show that restoration of the protein expression of NLGN4Y in PC-3 cells leads to decreased cell proliferation, migration and inflammatory cytokine expression. Our results suggest that NLGN4Y is an important negative regulator in prostate cancer progression. More importantly, it highlights the value of IMBNs in generating biologically and clinically relevant hypotheses about prostate cancer that can be validated by independent studies.

  7. Sperm quality and DNA integrity of coke oven workers exposed to polycyclic aromatic hydrocarbons.

    Science.gov (United States)

    Jeng, Hueiwang Anna; Pan, Chih-Hong; Chao, Mu-Rong; Chiu, Chien-Chih; Zhou, Guodong; Chou, Chon-Kit; Lin, Wen-Yi

    2016-11-18

    The objective of this study was to assess sperm quality and deoxyribonucleic acid (DNA) integrity of coke oven workers exposed to polycyclic aromatic hydrocarbons (PAHs) as compared to control subjects. The coke oven workers (N = 52) and administrative staff (N = 35) of a steel plant served as the exposed and control groups, respectively. Exposure to PAHs was assessed by measuring 1-hydroxypyren. Analysis of sperm quality (concentration, motility, vitality, and morphology) was performed simultaneously with sperm DNA integrity analysis, including DNA fragmentation, denaturation, bulky DNA adducts, and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo). A questionnaire was conducted to collect demographic and potential confounding data. The coke oven workers had lower percentages of sperm motility, vitality and normal morphology than the control group, but the difference was not significant. For DNA integrity, the coke oven workers had significantly higher concentrations of bulky DNA adducts and 8-oxo-dGuo than the control subjects (p = 0.009 and p = 0.048, respectively). However, DNA fragmentation percentages did not significantly increase as compared to those in the subjects from the control group (p = 0.232). There was no correlation between sperm quality parameters and DNA integrity indicators. Occupational exposure of the coke oven workers to PAHs was associated with decreased sperm DNA integrity. Int J Occup Med Environ Health 2016;29(6):915-926.

  8. Sperm quality and DNA integrity of coke oven workers exposed to polycyclic aromatic hydrocarbons

    Directory of Open Access Journals (Sweden)

    Hueiwang Anna Jeng

    2016-12-01

    Full Text Available Objectives: The objective of this study was to assess sperm quality and deoxyribonucleic acid (DNA integrity of coke oven workers exposed to polycyclic aromatic hydrocarbons (PAHs as compared to control subjects. Material and methods: The coke oven workers (N = 52 and administrative staff (N = 35 of a steel plant served as the exposed and control groups, respectively. Exposure to PAHs was assessed by measuring 1-hydroxypyren. Analysis of sperm quality (concentration, motility, vitality, and morphology was performed simultaneously with sperm DNA integrity analysis, including DNA fragmentation, denaturation, bulky DNA adducts, and 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxo-dGuo. A questionnaire was conducted to collect demographic and potential confounding data. Results: The coke oven workers had lower percentages of sperm motility, vitality and normal morphology than the control group, but the difference was not significant. For DNA integrity, the coke oven workers had significantly higher concentrations of bulky DNA adducts and 8-oxo-dGuo than the control subjects (p = 0.009 and p = 0.048, respectively. However, DNA fragmentation percentages did not significantly increase as compared to those in the subjects from the control group (p = 0.232. There was no correlation between sperm quality parameters and DNA integrity indicators. Conclusions: Occupational exposure of the coke oven workers to PAHs was associated with decreased sperm DNA integrity. Int J Occup Med Environ Health 2016;29(6:915–926

  9. A new structural framework for integrating replication protein A into DNA processing machinery

    Energy Technology Data Exchange (ETDEWEB)

    Brosey, Chris; Yan, Chunli; Tsutakawa, Susan; Heller, William; Rambo, Robert; Tainer, John; Ivanov, Ivaylo; Chazin, Walter

    2013-01-17

    By coupling the protection and organization of single-stranded DNA (ssDNA) with recruitment and alignment of DNA processing factors, replication protein A (RPA) lies at the heart of dynamic multi-protein DNA processing machinery. Nevertheless, how RPA coordinates biochemical functions of its eight domains remains unknown. We examined the structural biochemistry of RPA's DNA-binding activity, combining small-angle X-ray and neutron scattering with all-atom molecular dynamics simulations to investigate the architecture of RPA's DNA-binding core. The scattering data reveal compaction promoted by DNA binding; DNA-free RPA exists in an ensemble of states with inter-domain mobility and becomes progressively more condensed and less dynamic on binding ssDNA. Our results contrast with previous models proposing RPA initially binds ssDNA in a condensed state and becomes more extended as it fully engages the substrate. Moreover, the consensus view that RPA engages ssDNA in initial, intermediate and final stages conflicts with our data revealing that RPA undergoes two (not three) transitions as it binds ssDNA with no evidence for a discrete intermediate state. These results form a framework for understanding how RPA integrates the ssDNA substrate into DNA processing machinery, provides substrate access to its binding partners and promotes the progression and selection of DNA processing pathways.

  10. Reconstruction of putative DNA virus from endogenous rice tungro bacilliform virus-like sequences in the rice genome: implications for integration and evolution

    Directory of Open Access Journals (Sweden)

    Kishima Yuji

    2004-10-01

    Full Text Available Abstract Background Plant genomes contain various kinds of repetitive sequences such as transposable elements, microsatellites, tandem repeats and virus-like sequences. Most of them, with the exception of virus-like sequences, do not allow us to trace their origins nor to follow the process of their integration into the host genome. Recent discoveries of virus-like sequences in plant genomes led us to set the objective of elucidating the origin of the repetitive sequences. Endogenous rice tungro bacilliform virus (RTBV-like sequences (ERTBVs have been found throughout the rice genome. Here, we reconstructed putative virus structures from RTBV-like sequences in the rice genome and characterized to understand evolutionary implication, integration manner and involvements of endogenous virus segments in the corresponding disease response. Results We have collected ERTBVs from the rice genomes. They contain rearranged structures and no intact ORFs. The identified ERTBV segments were shown to be phylogenetically divided into three clusters. For each phylogenetic cluster, we were able to make a consensus alignment for a circular virus-like structure carrying two complete ORFs. Comparisons of DNA and amino acid sequences suggested the closely relationship between ERTBV and RTBV. The Oryza AA-genome species vary in the ERTBV copy number. The species carrying low-copy-number of ERTBV segments have been reported to be extremely susceptible to RTBV. The DNA methylation state of the ERTBV sequences was correlated with their copy number in the genome. Conclusions These ERTBV segments are unlikely to have functional potential as a virus. However, these sequences facilitate to establish putative virus that provided information underlying virus integration and evolutionary relationship with existing virus. Comparison of ERTBV among the Oryza AA-genome species allowed us to speculate a possible role of endogenous virus segments against its related disease.

  11. DNA Double-Strand Breaks,Potential Targets for HBV Integration

    Institute of Scientific and Technical Information of China (English)

    胡晓文; 林菊生; 谢琼慧; 任精华; 常莹; 吴文杰; 夏羽佳

    2010-01-01

    Hepatitis B virus(HBV)-induced hepatocellular carcinoma(HCC) is one of the most fre-quently occurring cancers.Hepadnaviral DNA integrations are considered to be essential agents which can promote the process of the hepatocarcinogenesis.More and more researches were designed to find the relationship of the two.In this study,we investigated whether HBV DNA integration occurred at sites of DNA double-strand breaks(DSBs),one of the most detrimental DNA damage.An 18-bp I-SceI homing endonuclease recognition site...

  12. Quantitative analysis of waterfowl parvoviruses in geese and Muscovy ducks by real-time polymerase chain reaction: correlation between age, clinical symptoms and DNA copy number of waterfowl parvoviruses

    Directory of Open Access Journals (Sweden)

    Woźniakowski Grzegorz

    2012-03-01

    Full Text Available Abstract Background Waterfowl parvoviruses cause serious loss in geese and ducks production. Goose parvovirus (GPV is infectious for geese and ducks while Muscovy duck parvovirus (MDPV infects Muscovy ducks only. So far, for these viruses' sensitive detection polymerase chain reaction (PCR and loop-mediated isothermal amplification (LAMP were applied. However, there was no molecular biology method for both waterfowl parvoviruses detection and quantification which could unify the laboratory procedures. The level of GPV and MDPV replication and distribution plays a significant role in the parvoviral infection progress and is strictly correlated to clinical symptoms. Meanwhile, experiments conducted previously on GPV distribution in geese, performed as animal trial, did not involve epidemiological data from the disease field cases. The study on the correlation between age, clinical symptoms and viral DNA copy number may be benefitable in understanding the GPV and MDPV infection. Such data may also aid in determination of the stage and severity of the infection with parvoviruses. Therefore the aim of this study was to develop quantitative real-time PCR for parallel detection of GPV and MDPV in geese and Muscovy ducks and to determine the correlation between the age of the infected birds, clinical symptoms and DNA copy number for the estimation of the disease stage or severity. Results In order to develop quantitative real-time PCR the viral material was collected from 13 farms of geese and 3 farms of Muscovy ducks. The designed primers and Taqman probe for real-time PCR were complementary to GPV and MDPV inverted terminal repeats region. The pITR plasmid was constructed, purified and used to prepare dilutions for standard curve preparation and DNA quantification. The applied method detected both GPV and MDPV in all the examined samples extracted from the heart and liver of the infected birds. The conducted correlation tests have shown relationship

  13. Quantitative analysis of waterfowl parvoviruses in geese and Muscovy ducks by real-time polymerase chain reaction: correlation between age, clinical symptoms and DNA copy number of waterfowl parvoviruses.

    Science.gov (United States)

    Woźniakowski, Grzegorz; Samorek-Salamonowicz, Elżbieta; Kozdruń, Wojciech

    2012-03-15

    Waterfowl parvoviruses cause serious loss in geese and ducks production. Goose parvovirus (GPV) is infectious for geese and ducks while Muscovy duck parvovirus (MDPV) infects Muscovy ducks only. So far, for these viruses' sensitive detection polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) were applied. However, there was no molecular biology method for both waterfowl parvoviruses detection and quantification which could unify the laboratory procedures. The level of GPV and MDPV replication and distribution plays a significant role in the parvoviral infection progress and is strictly correlated to clinical symptoms. Meanwhile, experiments conducted previously on GPV distribution in geese, performed as animal trial, did not involve epidemiological data from the disease field cases. The study on the correlation between age, clinical symptoms and viral DNA copy number may be benefitable in understanding the GPV and MDPV infection. Such data may also aid in determination of the stage and severity of the infection with parvoviruses. Therefore the aim of this study was to develop quantitative real-time PCR for parallel detection of GPV and MDPV in geese and Muscovy ducks and to determine the correlation between the age of the infected birds, clinical symptoms and DNA copy number for the estimation of the disease stage or severity. In order to develop quantitative real-time PCR the viral material was collected from 13 farms of geese and 3 farms of Muscovy ducks. The designed primers and Taqman probe for real-time PCR were complementary to GPV and MDPV inverted terminal repeats region. The pITR plasmid was constructed, purified and used to prepare dilutions for standard curve preparation and DNA quantification. The applied method detected both GPV and MDPV in all the examined samples extracted from the heart and liver of the infected birds. The conducted correlation tests have shown relationship between age, clinical symptoms during

  14. Scaling up Copy Detection

    OpenAIRE

    Li, Xian; Dong, Xin Luna; Lyons, Kenneth B.; Meng, Weiyi; Srivastava, Divesh

    2015-01-01

    Recent research shows that copying is prevalent for Deep-Web data and considering copying can significantly improve truth finding from conflicting values. However, existing copy detection techniques do not scale for large sizes and numbers of data sources, so truth finding can be slowed down by one to two orders of magnitude compared with the corresponding techniques that do not consider copying. In this paper, we study {\\em how to improve scalability of copy detection on structured data}. Ou...

  15. Exogenous DNA internalisation by sperm cells is improved by combining lipofection and restriction enzyme mediated integration.

    Science.gov (United States)

    Churchil, R R; Gupta, J; Singh, A; Sharma, D

    2011-06-01

    1. Three types of exogenous DNA inserts, i.e. complete linearised pVIVO2-GFP/LacZ vector (9620 bp), the LacZ gene (5317 bp) and the GFP gene (2152 bp) were used to transfect chicken spermatozoa through simple incubation of sperm cells with insert. 2. PCR assay, Dot Blot hybridisation and Southern hybridisation showed the successful internalisation of exogenous DNA by chicken sperm cells. 3. Lipofection and Restriction Enzyme Mediated Integration (REMI) were used to improve the rate of internalisation of exogenous DNA by sperm cells. 4. Results from dot blot as well as Southern hybridisation were semi-quantified and improved exogenous DNA uptake by sperm cells through lipofection and REMI. Stronger signals were observed from hybridisation of LacZ as well as GFP specific probe with the DNA from lipofected exogenous DNA transfected sperm DNA in comparison with those transfected with nude exogenous DNA.

  16. Inactivation of Pol θ and C-NHEJ eliminates off-target integration of exogenous DNA.

    Science.gov (United States)

    Zelensky, Alex N; Schimmel, Joost; Kool, Hanneke; Kanaar, Roland; Tijsterman, Marcel

    2017-07-07

    Off-target or random integration of exogenous DNA hampers precise genomic engineering and presents a safety risk in clinical gene therapy strategies. Genetic definition of random integration has been lacking for decades. Here, we show that the A-family DNA polymerase θ (Pol θ) promotes random integration, while canonical non-homologous DNA end joining plays a secondary role; cells double deficient for polymerase θ and canonical non-homologous DNA end joining are devoid of any integration events, demonstrating that these two mechanisms define random integration. In contrast, homologous recombination is not reduced in these cells and gene targeting is improved to 100% efficiency. Such complete reversal of integration outcome, from predominately random integration to exclusively gene targeting, provides a rational way forward to improve the efficacy and safety of DNA delivery and gene correction approaches.Random off-target integration events can impair precise gene targeting and poses a safety risk for gene therapy. Here the authors show that repression of polymerase θ and classical non-homologous recombination eliminates random integration.

  17. 猪卵母细胞线粒体分布及线粒体DNA拷贝数变化%Mitochondrial Distribution and Mitochondrial DNA Copy Number in Porcine Oocvte at Different Periods during in vitro Maturation

    Institute of Scientific and Technical Information of China (English)

    成文敏; 霍金龙; 信吉阁; 潘伟荣; 黄言; 魏红江; 曾养志

    2011-01-01

    To observe mitochondrial distribution and mitochondrial DNA copy number changes in porcine oocytes at different periods during in vitro maturation. Mito-Tracker Green and real-time Quantitative PCR were used to respectively detect the mitochondrial distribution and mitochondrial DNA copy number in porcine oocytes. The results showed that the mitochondrial distribution was gradually translocated from the cortex to the perinuclear area of oocytes, and then spread in the cytoplasm thoroughly, and mitochondrial clusters became larger, stain became deeper. Mitochondrial DNA copy number of 0, 11, 22 h were (2519. 52±940. 39), (3421. 47±345. 71) ,(9747. 58±1928. 24), respectively, and there was no significant difference (P>0. 05) among them. Mitochondrial DNA copy number of 33 h was (39913. 61±1180. 26), which was significantly higher than those of 0,11,22 h (P<0. 05). Mitochondrial DNA copy number of 44 h was (130074. 30±78119. 45), which was significantly higher than that of 33 h (P<0. 05). In conclusion, as the oocyte maturation process forward, mitochondrial activity enhanced and mitochondrial DNA copy number increased.%本研究旨在观察猪卵母细胞线粒体分布及线粒体DNA拷贝数变化,以期作为判定哺乳动物卵母细胞胞质成熟的指标,同时也为今后克隆技术的发展和相关基因表达调控的研究提供基础.运用线粒体分子探针标记技术检测体外成熟不同时期卵母细胞中线粒体的分布变化,运用实时荧光定量PCR技术检测其线粒体DNA拷贝数的变化趋势,揭示线粒体分布、线粒体DNA拷贝数变化与卵母细胞发育潜能的关系.结果表明,猪卵母细胞成熟前后,线粒体分布由未成熟的周边分布变为成熟后的均匀分布,并且线粒体簇变大,着色变深.卵母细胞成熟0、11、22 h的mtDNA拷贝数分别为(2 519.52士940.39)、(3 421.47士345.71)和(9 747.58士1 928.24),他们之间无显著性差异(P>0.05).卵母细胞成熟33 h

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

    Energy Technology Data Exchange (ETDEWEB)

    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

  19. Evaluation of the quality of the human spermatozoon: comparison between spermatic DNA integrity and semen variables

    National Research Council Canada - National Science Library

    Cruz, Ibis; Colmenares, Melisa; Berrueta-Carrillo, Leidith; Gomez-Perez, Roald; Montes, Henry; Berrueta, Lisbeth; Salmen, Siham; Osuna, Jesús Alfonso

    2010-01-01

    ... the sperm functional capability have been developed in the last decade, such as analysis of nuclear DNA integrity, which have improved considerably the clinical diagnosis of male infertility, and increased...

  20. Effect of repeated sequential ejaculation on sperm DNA integrity in subfertile males with asthenozoospermia.

    Science.gov (United States)

    Hussein, T M; Elariny, A F; Elabd, M M; Elgarem, Y F; Elsawy, M M

    2008-10-01

    The aim of this work was to study the possible beneficial effect of repeated sequential ejaculation on sperm DNA integrity in subfertile males and its possible implementation in assisted reproduction. The study included 20 infertile males with idiopathic asthenozoospermia or oligoasthenozoospermia. They underwent detailed history taking, complete clinical assessment and hormonal assessment. Patients were asked to bring two semen samples (taken within 1-3 h). Two consecutive samples were assessed with regard to semen volume, sperm count, motility grading, and morphology and sperm DNA integrity using the comet assay. There was a significant improvement in the sperm motility pattern and DNA integrity in the second sample in comparison with the first sample. Therefore, it is concluded that due to its positive impact on sperm motility and DNA integrity, repeated sequential ejaculation is recommended in subfertile males with idiopathic asthenozoospermia who pursue assisted reproduction.

  1. Reverse gyrase functions in genome integrity maintenance by protecting DNA breaks in vivo

    DEFF Research Database (Denmark)

    Han, Wenyuan; Feng, Xu; She, Qunxin

    2017-01-01

    Reverse gyrase introduces positive supercoils to circular DNA and is implicated in genome stability maintenance in thermophiles. The extremely thermophilic crenarchaeon Sulfolobus encodes two reverse gyrase proteins, TopR1 (topoisomerase reverse gyrase 1) and TopR2, whose functions in thermophili...... genomic DNA degradation during MMS treatment, accompanied by a higher rate of cell death. Taken together, these results indicate that TopR1 probably facilitates genome integrity maintenance by protecting DNA breaks from thermo-degradation in vivo....

  2. Blood DNA Yield but Not Integrity or Methylation Is Impacted After Long-Term Storage.

    Science.gov (United States)

    Bulla, Alexandre; De Witt, Brian; Ammerlaan, Wim; Betsou, Fay; Lescuyer, Pierre

    2016-02-01

    Collection of human whole blood for genomic DNA extraction is part of numerous clinical studies. Since DNA extraction cannot always be performed at the time of sample collection, whole blood samples may be stored for years before being processed. The use of appropriate storage conditions is then critical to obtain DNA in sufficient quantity and of adequate quality in order to obtain reliable results from the subsequent molecular biological analyses. In this study, EDTA whole blood samples were collected from 8 healthy volunteers, and different durations (up to 1 year) and temperatures (room temperature, 4°C, -20°C, and -80°C) of storage were compared. The effect of the addition of a DNA preservative agent was also assessed before and after storage. DNA concentrations measured by UV spectrophotometry and spectrofluorometry were used to calculate DNA extraction yields and double-strand DNA ratios. DNA integrity was controlled by agarose gel electrophoresis and long-range polymerase chain reaction. The impact of storage conditions on DNA methylation was also evaluated. Results showed that certain storage conditions have a significant impact on the DNA extraction yield but little or no effect on DNA integrity and methylation. Storage of EDTA blood at -80°C guarantees high-quality DNA with a good yield. Higher DNA extraction yields were obtained with the addition of a DNA preservative agent before thawing EDTA blood stored at -20°C or -80°C. Long-term storage at room temperature in the presence of a DNA preservative agent also appeared to be a reliable procedure.

  3. Multi-color fluorescent DNA analysis in an integrated optofluidic lab on a chip

    NARCIS (Netherlands)

    Dongre, C.

    2010-01-01

    Abstract: Sorting and sizing of DNA molecules within the human genome project has enabled the genetic mapping of various illnesses. Furthermore by employing tiny lab-on-a-chip device, integrated DNA sequencing and genetic diagnostics have become feasible. We present the combination of capillary

  4. Multi-color fluorescent DNA analysis in an integrated optofluidic lab on a chip

    NARCIS (Netherlands)

    Dongre, Chaitanya

    2010-01-01

    Sorting and sizing of DNA molecules within the human genome project has enabled the genetic mapping of various illnesses. Furthermore by employing tiny lab-on-a-chip device, integrated DNA sequencing and genetic diagnostics have become feasible. We present the combination of capillary electrophoresi

  5. DNA-enabled integrated molecular systems for computation and sensing.

    Science.gov (United States)

    LaBoda, Craig; Duschl, Heather; Dwyer, Chris L

    2014-06-17

    CONSPECTUS: Nucleic acids have become powerful building blocks for creating supramolecular nanostructures with a variety of new and interesting behaviors. The predictable and guided folding of DNA, inspired by nature, allows designs to manipulate molecular-scale processes unlike any other material system. Thus, DNA can be co-opted for engineered and purposeful ends. This Account details a small portion of what can be engineered using DNA within the context of computer architectures and systems. Over a decade of work at the intersection of DNA nanotechnology and computer system design has shown several key elements and properties of how to harness the massive parallelism created by DNA self-assembly. This work is presented, naturally, from the bottom-up beginning with early work on strand sequence design for deterministic, finite DNA nanostructure synthesis. The key features of DNA nanostructures are explored, including how the use of small DNA motifs assembled in a hierarchical manner enables full-addressability of the final nanostructure, an important property for building dense and complicated systems. A full computer system also requires devices that are compatible with DNA self-assembly and cooperate at a higher level as circuits patterned over many, many replicated units. Described here is some work in this area investigating nanowire and nanoparticle devices, as well as chromophore-based circuits called resonance energy transfer (RET) logic. The former is an example of a new way to bring traditional silicon transistor technology to the nanoscale, which is increasingly problematic with current fabrication methods. RET logic, on the other hand, introduces a framework for optical computing at the molecular level. This Account also highlights several architectural system studies that demonstrate that even with low-level devices that are inferior to their silicon counterparts and a substrate that harbors abundant defects, self-assembled systems can still

  6. Quantum copying: A review

    Directory of Open Access Journals (Sweden)

    Mark Hillery

    2000-07-01

    Full Text Available Quantum information is stored in two-level quantum systems known as qubits. The no-cloning theorem states that the state of an unknown qubit cannot be copied. This is in contrast to classical information which can be copied. If one drops the requirement that the copies be perfect it is possible to design quantum copiers. This paper presents a short review of the theory of quantum copying.

  7. An Integrated DNA Modified Dual-microelectrode Sensor Probe

    Institute of Scientific and Technical Information of China (English)

    Xiang Qin LIN; Li Ping LU; Xiao Hua JIANG

    2005-01-01

    A unique method for preparing a coaxial dual-microelectrode sensor by vaporizing the nano-thickness Au layer on the DNA modified carbon fiber micro-column electrode was illustrated.The dual-electrode showed particular merit for determination in biological systems.

  8. A new structural framework for integrating replication protein A into DNA processing machinery

    Energy Technology Data Exchange (ETDEWEB)

    Brosey, Chris A [Vanderbilt University; Yan, Chunli [Georgia State University, Atlanta; Tsutakawa, Susan E [Lawrence Berkeley National Laboratory (LBNL); Heller, William T [ORNL; Rambo, Robert P [Lawrence Berkeley National Laboratory (LBNL); Tainer, John A [Lawrence Berkeley National Laboratory, The Scripps Research Institite and The Skaggs Institute; Ivanov, Ivaylo [Georgia State University, Atlanta; Chazin, Walter J [Vanderbilt University

    2013-01-01

    By coupling the protection and organization of ssDNA with the recruitment and alignment of DNA processing factors, Replication Protein A (RPA) lies at the heart of dynamic multi-protein DNA processing machinery. Nevertheless, how RPA manages to coordinate the biochemical functions of its eight domains remains unknown. We examined the structural biochemistry of RPA s DNA binding activity, combining small-angle x-ray and neutron scattering with all-atom molecular dynamics simulations to investigate the architecture of RPA s DNA-binding core. It has been long held that RPA engages ssDNA in three stages, but our data reveal that RPA undergoes two rather than three transitions as it binds ssDNA. In contrast to previous models, RPA is more compact when fully engaged on 20-30 nucleotides of ssDNA than when DNA-free, and there is no evidence for significant population of a highly compacted structure in the initial 8-10 nucleotide binding mode. These results provide a new framework for understanding the integration of ssDNA into DNA processing machinery and how binding partners may manipulate RPA architecture to gain access to the substrate.

  9. Combinative exposure effect of radio frequency signals from CDMA mobile phones and aphidicolin on DNA integrity.

    Science.gov (United States)

    Tiwari, R; Lakshmi, N K; Surender, V; Rajesh, A D V; Bhargava, S C; Ahuja, Y R

    2008-01-01

    The aim of present study is to assess DNA integrity on the effect of exposure to a radio frequency (RF) signal from Code Division Multiple Access (CDMA) mobile phones. Whole blood samples from six healthy male individuals were exposed for RF signals from a CDMA mobile phone for 1 h. Alkaline comet assay was performed to assess the DNA damage. The combinative exposure effect of the RF signals and APC at two concentrations on DNA integrity was studied. DNA repair efficiency of the samples was also studied after 2 h of exposure. The RF signals and APC (0.2 microg/ml) alone or in synergism did not have any significant DNA damage as compared to sham exposed. However, univariate analysis showed that DNA damage was significantly different among combinative exposure of RF signals and APC at 0.2 microg/ml (p < 0.05) and at 2 microg/ml (p < 0.02). APC at 2 microg/ml concentration also showed significant damage levels (p < 0.05) when compared to sham exposed. DNA repair efficiency also varied in a significant way in combinative exposure sets (p < 0.05). From these results, it appears that the repair inhibitor APC enhances DNA breaks at 2 microg/ml concentration and that the damage is possibly repairable. Thus, it can be inferred that the in vitro exposure to RF signals induces reversible DNA damage in synergism with APC.

  10. Checkpoint for DNA integrity at the first mitosis after oocyte activation.

    Science.gov (United States)

    Liu, Lin; Trimarchi, James R; Smith, Peter J S; Keefe, David L

    2002-06-01

    Activation of oocytes, arrested at the meiosis II (MII) in mammals, initiates meiotic release, mitotic divisions, and development. Unlike most somatic cell types, MII arrested female germ cells lack an efficient DNA integrity checkpoint control. Here we present evidence showing a unique checkpoint for DNA integrity at first mitosis after oocyte activation. Mouse oocytes carrying intact DNA cleaved normally after meiotic release, whereas 50% of oocytes harboring damaged DNA manifested cytofragmentation, a morphological hallmark of apoptosis. If not activated, DNA-damaged MII oocytes did not show apoptotic fragmentation. Further, activated, enucleated oocytes or enucleated fertilized oocytes also underwent cytofragmentation, implicating cytoplasmic coordination of the fragmentation process, independent of the nucleus. Depolymerization of either actin filaments or microtubules induced no cytofragmentation, but inhibited fragmentation upon oocyte activation. During the process of fragmentation, microtubule networks formed, then microtubule asters congregated at discrete locations, around which fragmented cellular bodies formed. Mitotic spindles, however, were not formed inactivated oocytes with damaged or absent DNA; in contrast, normal mitotic spindles were formed in activated oocytes with intact DNA. These results demonstrate that damaged DNA or absence of DNA leads to cytofragmentation after oocyte activation. Further, we found a mechanism of cytoskeletal involvement in the process of cytofragmentation. In addition, possible implication of the present findings in somatic cell cloning and human clinical embryology is discussed.

  11. Specific Photocrosslinking of DNA-Protein Complexes: Identification of Contacts Between Integration Host Factor and Its Target DNA

    Science.gov (United States)

    Yang, Shu-Wei; Nash, Howard A.

    1994-12-01

    Azide moieties have been specifically placed in the backbone of DNA by chemical coupling between azidophenacyl bromide and uniquely positioned phosphorothioate residues. The derivatized DNA forms specific complexes with a DNA-binding protein and, following irradiation with 302-nm light, makes specific crosslinks to the protein. Isolation of this covalent complex, followed by tryptic digestion and Edman degradation of the resulting crosslinked peptide, identifies the portion of the protein that is near the derivatized segment of the target DNA. We use this method to probe the interaction between a specific DNA sequence and integration host factor (IHF) protein. A single IHF heterodimer is known to contact >25 bp of DNA and thereby introduce a sharp bend. Two segments of a typical IHF site were derivatized with aryl azide. Although the segments were separated by only 5 bp, they crosslinked to different subunits of IHF. The locations of the crosslinks support our current view for the way IHF protein binds to and bends its specific targets.

  12. Serum Xenohormone activity and DNA integrity of Europeans and Inuits

    DEFF Research Database (Denmark)

    Bonefeld-Jørgensen, Eva Cecilie

      Background. The toxicological assessment of the lipophilic persistent organic pollutants (POPs), including PCBs, pesticides and dioxins, is complicated since individuals are exposed to a complex mixture of contaminants. Therefore we developed ex vivo cell systems to determine the actual...... integrated level of  xenobiotic activity in the human serum fraction containing the POPs. Sperm DNA integrity is essential for the accurate transmission of genetic information and sperm DNA damage can cause decreased male fertility.    The AIM was to determine the integrated serum xenobiotic activity of POPs...

  13. Value of circulating DNA concentration and integrity as a screening ...

    African Journals Online (AJOL)

    Ebtsam R. Zaher

    2012-07-17

    Jul 17, 2012 ... a screening test for detection of cancer in an Egyptian cohort. Ebtsam R. ... CFDNA integrity was detected by conventional PCR for 100, 200, 400 and 800 bp. .... (excluding autoimmune diseases) of matched age and sex to.

  14. RECQL4 localizes to mitochondria and preserves mitochondrial DNA integrity

    DEFF Research Database (Denmark)

    Croteau, Deborah L; Rossi, Marie L; Canugovi, Chandrika

    2012-01-01

    RECQL4 is associated with Rothmund-Thomson Syndrome (RTS), a rare autosomal recessive disorder characterized by premature aging, genomic instability, and cancer predisposition. RECQL4 is a member of the RecQ helicase family, and has many similarities to WRN protein, which is also implicated...... in premature aging. There is no information about whether any of the RecQ helicases play roles in mitochondrial biogenesis, which is strongly implicated in the aging process. Here, we used microscopy to visualize RECQL4 in mitochondria. Fractionation of human and mouse cells also showed that RECQL4 was present...... in mitochondria. Q-PCR amplification of mitochondrial DNA demonstrated that mtDNA damage accumulated in RECQL4-deficient cells. Microarray analysis suggested that mitochondrial bioenergetic pathways might be affected in RTS. Measurements of mitochondrial bioenergetics showed a reduction in the mitochondrial...

  15. [Determination of the structural integrity of DNA from pathogenic enterobacteria].

    Science.gov (United States)

    Tets, V V; Ivanov, S D

    1985-06-01

    Among the enterobacterial strains under study, more organisms in the stationary phase of growth have been found to have nicks in their DNA than those in the exponential phase. Bacteria less sensitive to ultraviolet irradiation have the least number of nicks in each phase of growth. The number of nicks in different strains belonging to the serovar is sufficiently stable. Virulent and avirulent forms show no difference in this characteristic.

  16. SMARCAL1 maintains telomere integrity during DNA replication.

    Science.gov (United States)

    Poole, Lisa A; Zhao, Runxiang; Glick, Gloria G; Lovejoy, Courtney A; Eischen, Christine M; Cortez, David

    2015-12-01

    The SMARCAL1 (SWI/SNF related, matrix-associated, actin-dependent, regulator of chromatin, subfamily A-like 1) DNA translocase is one of several related enzymes, including ZRANB3 (zinc finger, RAN-binding domain containing 3) and HLTF (helicase-like transcription factor), that are recruited to stalled replication forks to promote repair and restart replication. These enzymes can perform similar biochemical reactions such as fork reversal; however, genetic studies indicate they must have unique cellular activities. Here, we present data showing that SMARCAL1 has an important function at telomeres, which present an endogenous source of replication stress. SMARCAL1-deficient cells accumulate telomere-associated DNA damage and have greatly elevated levels of extrachromosomal telomere DNA (C-circles). Although these telomere phenotypes are often found in tumor cells using the alternative lengthening of telomeres (ALT) pathway for telomere elongation, SMARCAL1 deficiency does not yield other ALT phenotypes such as elevated telomere recombination. The activity of SMARCAL1 at telomeres can be separated from its genome-maintenance activity in bulk chromosomal replication because it does not require interaction with replication protein A. Finally, this telomere-maintenance function is not shared by ZRANB3 or HLTF. Our results provide the first identification, to our knowledge, of an endogenous source of replication stress that requires SMARCAL1 for resolution and define differences between members of this class of replication fork-repair enzymes.

  17. Quality of human spermatozoa: relationship between high-magnification sperm morphology and DNA integrity.

    Science.gov (United States)

    Maettner, R; Sterzik, K; Isachenko, V; Strehler, E; Rahimi, G; Alabart, J L; Sánchez, R; Mallmann, P; Isachenko, E

    2014-06-01

    The aim of this work is to establish the relationship between the morphology of Intracytoplasmic Morphologically Selected Sperm Injection (IMSI)-selected spermatozoa and their DNA integrity. The 45 ejaculates were randomly distributed into three treatment groups: normozoospermic, oligoasthenozoospermic and oligoasthenotheratozoospermic samples. The evaluation of DNA integrity was performed using the sperm chromatin dispersion test. It was established that DNA integrity of spermatozoa is strongly dependent on ejaculate quality (P 0.1). With decreased ejaculate quality, the percentage of spermatozoa with nonfragmented DNA decreased significantly (P < 0.05) independent from morphological class. Nevertheless, the rate of IMSI-selected spermatozoa with fragmented DNA within of Class 1 in normozoospermic (Group 1), in oligoasthenozoospermic (Group 2) and in oligoasthenotheratozoospermic (Group 3) samples was 21.1%, 31.8% and 54.1%, respectively. In conclusion, there is a direct relationship between morphological parameters of spermatozoa and their DNA integrity. However, the IMSI technique alone is not enough for the selection of spermatozoa with intact nuclei.

  18. DNA Topoisomerase I Gene Copy Number and mRNA Expression Assessed as Predictive Biomarkers for Adjuvant Irinotecan in Stage II/III Colon Cancer

    DEFF Research Database (Denmark)

    Nygård, Sune Boris; Vainer, Ben; Nielsen, Signe L;

    2016-01-01

    (PETACC3) where patients were randomized to 5-fluorouracil/folinic acid with or without additional irinotecan. TOP1 copy number status was analyzed by fluorescence in situ hybridization (FISH) using a TOP1/CEN20 dual-probe combination. TOP1 mRNA data were available from previous analyses. RESULTS: TOP1......RNA data were available from 580 patients with stage III disease. Benefit of irinotecan was restricted to patients characterized by TOP1 mRNA expression ≥ 3rd quartile (RFS: HRadjusted, 0.59; P = .09; OS: HRadjusted, 0.44; P = 0.03). The treatment by TOP1 mRNA interaction was not statistically significant...

  19. 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.

  20. TRF2 binds branched DNA to safeguard telomere integrity.

    Science.gov (United States)

    Schmutz, Isabelle; Timashev, Leonid; Xie, Wei; Patel, Dinshaw J; de Lange, Titia

    2017-09-01

    Although t-loops protect telomeres, they are at risk of cleavage by Holliday junction (HJ) resolvases if branch migration converts the three-way t-loop junction into four-way HJs. T-loop cleavage is repressed by the TRF2 basic domain, which binds three- and four-way junctions and protects HJs in vitro. By replacing the basic domain with bacterial-protein domains binding three- and four-way junctions, we demonstrated the in vivo relevance of branched-DNA binding. Branched-DNA binding also repressed PARP1, presumably by masking the PARP1 site in the t-loop junction. Although PARP1 recruits HJ resolvases and promotes t-loop cleavage, PARP1 activation alone did not result in t-loop cleavage, thus suggesting that the basic domain also prevents formation of HJs. Concordantly, removal of HJs by BLM helicase mitigated t-loop cleavage in response to loss of the basic domain. We propose that TRF2 masks and stabilizes the t-loop three-way junction, thereby protecting telomeres from detrimental deletions and PARP1 activation.

  1. A novel non-integrative single-cycle chimeric HIV lentivector DNA vaccine.

    Science.gov (United States)

    Moussa, Maha; Arrode-Brusés, Géraldine; Manoylov, Iliyan; Malogolovkin, Alexander; Mompelat, Dimitri; Ishimwe, Honorine; Smaoune, Amel; Ouzrout, Bilel; Gagnon, Jean; Chebloune, Yahia

    2015-05-05

    Novel HIV vaccine vectors and strategies are needed to control HIV/AIDS epidemic in humans and eradicate the infection. DNA vaccines alone failed to induce immune responses robust enough to control HIV-1. Development of lentivirus-based DNA vaccines deficient for integration and with a limited replication capacity is an innovative and promising approach. This type of vaccine mimics the early stages of virus infection/replication like the live-attenuated viruses but lacks the inconvenient integration and persistence associated with disease. We developed a novel lentivector DNA vaccine "CAL-SHIV-IN(-)" that undergoes a single round of replication in the absence of integration resulting in augmented expression of vaccine antigens in vivo. Vaccine gene expression is under control of the LTRs of a naturally attenuated lentivirus, Caprine arthritis encephalitis virus (CAEV) the natural goat lentivirus. The safety of this vaccine prototype was increased by the removal of the integrase coding sequences from the pol gene. We examined the functional properties of this lentivector DNA in cell culture and the immunogenicity in mouse models. Viral proteins were expressed in transfected cells, assembled into viral particles that were able to transduce once target permissive cells. Unlike the parental replication-competent SHIV-KU2 that was detected in DNA samples from any of the serial passage infected cells, CAL-SHIV-IN(-) DNA was detected only in target cells of the first round of infection, hence demonstrating the single cycle replication of the vaccine. A single dose DNA immunization of humanized NOD/SCID/β2 mice showed a substantial increase of IFN-γ-ELISPOT in splenocytes compared to the former replication and integration defective Δ4SHIV-KU2 DNA vaccine. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. A novel interaction between DNA ligase III and DNA polymerase gamma plays an essential role in mitochondrial DNA stability.

    Science.gov (United States)

    De, Ananya; Campbell, Colin

    2007-02-15

    The data in the present study show that DNA polymerase gamma and DNA ligase III interact in mitochondrial protein extracts from cultured HT1080 cells. An interaction was also observed between the two recombinant proteins in vitro. Expression of catalytically inert versions of DNA ligase III that bind DNA polymerase gamma was associated with reduced mitochondrial DNA copy number and integrity. In contrast, overexpression of wild-type DNA ligase III had no effect on mitochondrial DNA copy number or integrity. Experiments revealed that wild-type DNA ligase III facilitates the interaction of DNA polymerase gamma with a nicked DNA substrate in vitro, and that the zinc finger domain of DNA ligase III is required for this activity. Mitochondrial protein extracts prepared from cells overexpressing a DNA ligase III protein that lacked the zinc finger domain had reduced base excision repair activity compared with extracts from cells overexpressing the wild-type protein. These data support the interpretation that the interaction of DNA ligase III and DNA polymerase gamma is required for proper maintenance of the mammalian mitochondrial genome.

  3. Integrating DNA barcode data and taxonomic practice: determination, discovery, and description.

    Science.gov (United States)

    Goldstein, Paul Z; DeSalle, Rob

    2011-02-01

    DNA barcodes, like traditional sources of taxonomic information, are potentially powerful heuristics in the identification of described species but require mindful analytical interpretation. The role of DNA barcoding in generating hypotheses of new taxa in need of formal taxonomic treatment is discussed, and it is emphasized that the recursive process of character evaluation is both necessary and best served by understanding the empirical mechanics of the discovery process. These undertakings carry enormous ramifications not only for the translation of DNA sequence data into taxonomic information but also for our comprehension of the magnitude of species diversity and its disappearance. This paper examines the potential strengths and pitfalls of integrating DNA sequence data, specifically in the form of DNA barcodes as they are currently generated and analyzed, with taxonomic practice.

  4. Expression of a chimeric human/salmon calcitonin gene integrated into the Saccharomyces cerevisiae genome using rDNA sequences as recombination sites.

    Science.gov (United States)

    Sun, Hengyi; Zang, Xiaonan; Liu, Yuantao; Cao, Xiaofei; Wu, Fei; Huang, Xiaoyun; Jiang, Minjie; Zhang, Xuecheng

    2015-12-01

    Calcitonin participates in controlling homeostasis of calcium and phosphorus and plays an important role in bone metabolism. The aim of this study was to endow an industrial strain of Saccharomyces cerevisiae with the ability to express chimeric human/salmon calcitonin (hsCT) without the use of antibiotics. To do so, a homologous recombination plasmid pUC18-rDNA2-ura3-P pgk -5hsCT-rDNA1 was constructed, which contains two segments of ribosomal DNA of 1.1 kb (rDNA1) and 1.4 kb (rDNA2), to integrate the heterologous gene into host rDNA. A DNA fragment containing five copies of a chimeric human/salmon calcitonin gene (5hsCT) under the control of the promoter for phosphoglycerate kinase (P pgk ) was constructed to express 5hsCT in S. cerevisiae using ura3 as a selectable auxotrophic marker gene. After digestion by restriction endonuclease HpaI, a linear fragment, rDNA2-ura3-P pgk -5hsCT-rDNA1, was obtained and transformed into the △ura3 mutant of S. cerevisiae by the lithium acetate method. The ura3-P pgk -5hsCT sequence was introduced into the genome at rDNA sites by homologous recombination, and the recombinant strain YS-5hsCT was obtained. Southern blot analysis revealed that the 5hsCT had been integrated successfully into the genome of S. cerevisiae. The results of Western blot and ELISA confirmed that the 5hsCT protein had been expressed in the recombinant strain YS-5hsCT. The expression level reached 2.04 % of total proteins. S. cerevisiae YS-5hsCT decreased serum calcium in mice by oral administration and even 0.01 g lyophilized S. cerevisiae YS-5hsCT/kg decreased serum calcium by 0.498 mM. This work has produced a commercial yeast strain potentially useful for the treatment of osteoporosis.

  5. An integrated encyclopedia of DNA elements in the human genome.

    Science.gov (United States)

    2012-09-01

    The human genome encodes the blueprint of life, but the function of the vast majority of its nearly three billion bases is unknown. The Encyclopedia of DNA Elements (ENCODE) project has systematically mapped regions of transcription, transcription factor association, chromatin structure and histone modification. These data enabled us to assign biochemical functions for 80% of the genome, in particular outside of the well-studied protein-coding regions. Many discovered candidate regulatory elements are physically associated with one another and with expressed genes, providing new insights into the mechanisms of gene regulation. The newly identified elements also show a statistical correspondence to sequence variants linked to human disease, and can thereby guide interpretation of this variation. Overall, the project provides new insights into the organization and regulation of our genes and genome, and is an expansive resource of functional annotations for biomedical research.

  6. Cheek swabs, SNP chips, and CNVs: Assessing the quality of copy number variant calls generated with subject-collected mail-in buccal brush DNA samples on a high-density genotyping microarray

    Directory of Open Access Journals (Sweden)

    Erickson Stephen W

    2012-06-01

    Full Text Available Abstract Background Multiple investigators have established the feasibility of using buccal brush samples to genotype single nucleotide polymorphisms (SNPs with high-density genome-wide microarrays, but there is currently no consensus on the accuracy of copy number variants (CNVs inferred from these data. Regardless of the source of DNA, it is more difficult to detect CNVs than to genotype SNPs using these microarrays, and it therefore remains an open question whether buccal brush samples provide enough high-quality DNA for this purpose. Methods To demonstrate the quality of CNV calls generated from DNA extracted from buccal samples, compared to calls generated from blood samples, we evaluated the concordance of calls from individuals who provided both sample types. The Illumina Human660W-Quad BeadChip was used to determine SNPs and CNVs of 39 Arkansas participants in the National Birth Defects Prevention Study (NBDPS, including 16 mother-infant dyads, who provided both whole blood and buccal brush DNA samples. Results We observed a 99.9% concordance rate of SNP calls in the 39 blood–buccal pairs. From the same dataset, we performed a similar analysis of CNVs. Each of the 78 samples was independently segmented into regions of like copy number using the Optimal Segmentation algorithm of Golden Helix SNP & Variation Suite 7. Across 640,663 loci on 22 autosomal chromosomes, segment-mean log R ratios had an average correlation of 0.899 between blood-buccal pairs of samples from the same individual, while the average correlation between all possible blood-buccal pairs of samples from unrelated individuals was 0.318. An independent analysis using the QuantiSNP algorithm produced average correlations of 0.943 between blood-buccal pairs from the same individual versus 0.332 between samples from unrelated individuals. Segment-mean log R ratios had an average correlation of 0.539 between mother-offspring dyads of buccal samples, which was not

  7. Capture of DNA in microfluidic channel using magnetic beads: increasing capture efficiency with integrated microfluidic mixer

    DEFF Research Database (Denmark)

    Lund-Olesen, Torsten; Dufva, Hans Martin; Hansen, Mikkel Fougt

    2007-01-01

    We have studied the hybridization of target DNA in solution with probe DNA on magnetic beads immobilized on the channel sidewalls in a magnetic bead separator. The hybridization is carried out under a liquid flow and is diffusion limited. Two systems are compared: one with a straight microfluidic...... channel and one with an integrated staggered herringbone mixer. Fluorescence microscopy studies show that the hybridization is much more efficient in the system with the integrated mixer. The results, which are discussed in terms of a simple model, are relevant for any diffusion-limited reaction taking...... place on the surface in a microfluidic system....

  8. Virtual Ribosome - a comprehensive DNA translation tool with support for integration of sequence feature annotation

    DEFF Research Database (Denmark)

    Wernersson, Rasmus

    2006-01-01

    Virtual Ribosome is a DNA translation tool with two areas of focus. ( i) Providing a strong translation tool in its own right, with an integrated ORF finder, full support for the IUPAC degenerate DNA alphabet and all translation tables defined by the NCBI taxonomy group, including the use...... of alternative start codons. ( ii) Integration of sequences feature annotation - in particular, native support for working with files containing intron/ exon structure annotation. The software is available for both download and online use at http://www.cbs.dtu.dk/services/VirtualRibosome/....

  9. Enhanced integration of large DNA into E. coli chromosome by CRISPR/Cas9.

    Science.gov (United States)

    Chung, Mu-En; Yeh, I-Hsin; Sung, Li-Yu; Wu, Meng-Ying; Chao, Yun-Peng; Ng, I-Son; Hu, Yu-Chen

    2017-01-01

    Metabolic engineering often necessitates chromosomal integration of multiple genes but integration of large genes into Escherichia coli remains difficult. CRISPR/Cas9 is an RNA-guided system which enables site-specific induction of double strand break (DSB) and programmable genome editing. Here, we hypothesized that CRISPR/Cas9-triggered DSB could enhance homologous recombination and augment integration of large DNA into E. coli chromosome. We demonstrated that CRISPR/Cas9 system was able to trigger DSB in >98% of cells, leading to subsequent cell death, and identified that mutagenic SOS response played roles in the cell survival. By optimizing experimental conditions and combining the λ-Red proteins and linear dsDNA, CRISPR/Cas9-induced DSB enabled homologous recombination of the donor DNA and replacement of lacZ gene in the MG1655 strain at efficiencies up to 99%, and allowed high fidelity, scarless integration of 2.4, 3.9, 5.4, and 7.0 kb DNA at efficiencies approaching 91%, 92%, 71%, and 61%, respectively. The CRISPR/Cas9-assisted gene integration also functioned in different E. coli strains including BL21 (DE3) and W albeit at different efficiencies. Taken together, our methodology facilitated precise integration of dsDNA as large as 7 kb into E. coli with efficiencies exceeding 60%, thus significantly ameliorating the editing efficiency and overcoming the size limit of integration using the commonly adopted recombineering approach. Biotechnol. Bioeng. 2017;114: 172-183. © 2016 Wiley Periodicals, Inc.

  10. The Art of Copying

    DEFF Research Database (Denmark)

    Christensen, Hans Dam

    2017-01-01

    This article discusses copies within the field of art museums by way of mapping strategies for copy practices. This mapping leans heavily towards parts of the writings of Jacques Derrida (1930–2004). Against the backdrop of this theoretical premise, the article distinguishes five main strategies....... An informational copy is just as unique as an original object of art, and at the same time, it defines the original and is itself defined by this opposition. Lastly, the strategy for the imagined relation between original and copy follows. This strategy is dependent upon several of the previous approaches, and...

  11. Premethylation of foreign DNA improves integrative transformation efficiency in Synechocystis sp. strain PCC 6803.

    Science.gov (United States)

    Wang, Bo; Yu, Jianping; Zhang, Weiwen; Meldrum, Deirdre R

    2015-12-01

    Restriction digestion of foreign DNA is one of the key biological barriers against genetic transformation in microorganisms. To establish a high-efficiency transformation protocol in the model cyanobacterium, Synechocystis sp. strain PCC 6803 (Synechocystis 6803), we investigated the effects of premethylation of foreign DNA on the integrative transformation of this strain. In this study, two type II methyltransferase-encoding genes, i.e., sll0729 (gene M) and slr0214 (gene C), were cloned from the chromosome of Synechocystis 6803 and expressed in Escherichia coli harboring an integration plasmid. After premethylation treatment in E. coli, the integration plasmid was extracted and used for transformation of Synechocystis 6803. The results showed that although expression of methyltransferase M had little impact on the transformation of Synechocystis 6803, expression of methyltransferase C resulted in 11- to 161-fold-higher efficiency in the subsequent integrative transformation of Synechocystis 6803. Effective expression of methyltransferase C, which could be achieved by optimizing the 5' untranslated region, was critical to efficient premethylation of the donor DNA and thus high transformation efficiency in Synechocystis 6803. Since premethylating foreign DNA prior to transforming Synechocystis avoids changing the host genetic background, the study thus provides an improved method for high-efficiency integrative transformation of Synechocystis 6803. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  12. Local repeat sequence organization of an intergenic spacer in the chloroplast genome of Chlamydomonas reinhardtii leads to DNA expansion and sequence scrambling: a complex mode of “copy-choice replication”?

    Indian Academy of Sciences (India)

    Mahendra D Wagle; Subhojit Sen; Basuthkar J Rao

    2001-12-01

    Parent-specific, randomly amplified polymorphic DNA (RAPD) markers were obtained from total genomic DNA of Chlamydomonas reinhardtii. Such parent-specific RAPD bands (genomic fingerprints) segregated uniparentally (through mt+) in a cross between a pair of polymorphic interfertile strains of Chlamydomonas (C. reinhardtii and C. minnesotti), suggesting that they originated from the chloroplast genome. Southern analysis mapped the RAPD-markers to the chloroplast genome. One of the RAPD-markers, ``P2” (1.6 kb) was cloned, sequenced and was fine mapped to the 3 kb region encompassing 3′ end of 23S, full 5S and intergenic region between 5S and psbA. This region seems divergent enough between the two parents, such that a specific PCR designed for a parental specific chloroplast sequence within this region, amplified a marker in that parent only and not in the other, indicating the utility of RAPD-scan for locating the genomic regions of sequence divergence. Remarkably, the RAPD-product, ``P2” seems to have originated from a PCR-amplification of a much smaller (about 600 bp), but highly repeat-rich (direct and inverted) domain of the 3 kb region in a manner that yielded no linear sequence alignment with its own template sequence. The amplification yielded the same uniquely ``sequence-scrambled” product, whether the template used for PCR was total cellular DNA, chloroplast DNA or a plasmid clone DNA corresponding to that region. The PCR product, a ``unique” new sequence, had lost the repetitive organization of the template genome where it had originated from and perhaps represented a ``complex path” of copy-choice replication.

  13. Comparison of repair of DNA double-strand breaks in identical sequences in primary human fibroblast and immortal hamster-human hybrid cells harboring a single copy of human chromosome 11

    Science.gov (United States)

    Fouladi, B.; Waldren, C. A.; Rydberg, B.; Cooper, P. K.; Chatterjee, A. (Principal Investigator)

    2000-01-01

    We have optimized a pulsed-field gel electrophoresis assay that measures induction and repair of double-strand breaks (DSBs) in specific regions of the genome (Lobrich et al., Proc. Natl. Acad. Sci. USA 92, 12050-12054, 1995). The increased sensitivity resulting from these improvements makes it possible to analyze the size distribution of broken DNA molecules immediately after the introduction of DSBs and after repair incubation. This analysis shows that the distribution of broken DNA pieces after exposure to sparsely ionizing radiation is consistent with the distribution expected from randomly induced DSBs. It is apparent from the distribution of rejoined DNA pieces after repair incubation that DNA ends continue to rejoin between 3 and 24 h postirradiation and that some of these rejoining events are in fact misrejoining events, since novel restriction fragments both larger and smaller than the original fragment are generated after repair. This improved assay was also used to study the kinetics of DSB rejoining and the extent of misrejoining in identical DNA sequences in human GM38 cells and human-hamster hybrid A(L) cells containing a single human chromosome 11. Despite the numerous differences between these cells, which include species and tissue of origin, levels of TP53, expression of telomerase, and the presence or absence of a homologous chromosome for the restriction fragments examined, the kinetics of rejoining of radiation-induced DSBs and the extent of misrejoining were similar in the two cell lines when studied in the G(1) phase of the cell cycle. Furthermore, DSBs were removed from the single-copy human chromosome in the hamster A(L) cells with similar kinetics and misrejoining frequency as at a locus on this hybrid's CHO chromosomes.

  14. Integration host factor of Mycobacterium tuberculosis, mIHF, compacts DNA by a bending mechanism.

    Directory of Open Access Journals (Sweden)

    Arpit Mishra

    Full Text Available The bacterial chromosomal DNA is folded into a compact structure called as 'nucleoid' so that the bacterial genome can be accommodated inside the cell. The shape and size of the nucleoid are determined by several factors including DNA supercoiling, macromolecular crowding and nucleoid associated proteins (NAPs. NAPs bind to different sites of the genome in sequence specific or non-sequence specific manner and play an important role in DNA compaction as well as regulation. Until recently, few NAPs have been discovered in mycobacteria owing to poor sequence similarities with other histone-like proteins of eubacteria. Several putative NAPs have now been identified in Mycobacteria on the basis of enriched basic residues or histone-like "PAKK" motifs. Here, we investigate mycobacterial Integration Host Factor (mIHF for its architectural roles as a NAP using atomic force microscopy and DNA compaction experiments. We demonstrate that mIHF binds DNA in a non-sequence specific manner and compacts it by a DNA bending mechanism. AFM experiments also indicate a dual architectural role for mIHF in DNA compaction as well as relaxation. These results suggest a convergent evolution in the mechanism of E. coli and mycobacterial IHF in DNA compaction.

  15. 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.

  16. Decreased Integrity, Content, and Increased Transcript Level of Mitochondrial DNA Are Associated with Keratoconus

    Science.gov (United States)

    Hao, Xiao-Dan; Chen, Zhao-Li; Qu, Ming-Li; Zhao, Xiao-Wen; Li, Su-Xia; Chen, Peng

    2016-01-01

    Oxidative stress may play an important role in the pathogenesis of keratoconus (KC). Mitochondrial DNA (mtDNA) is involved in mitochondrial function, and the mtDNA content, integrity, and transcript level may affect the generation of reactive oxygen species (ROS) and be involved in the pathogenesis of KC. We designed a case-control study to research the relationship between KC and mtDNA integrity, content and transcription. One-hundred ninety-eight KC corneas and 106 normal corneas from Chinese patients were studied. Quantitative real-time PCR was used to measure the relative mtDNA content, transcript levels of mtDNA and related genes. Long-extension PCR was used to detect mtDNA damage. ROS, mitochondrial membrane potential and ATP were measured by respective assay kit, and Mito-Tracker Green was used to label the mitochondria. The relative mtDNA content of KC corneas was significantly lower than that of normal corneas (P = 9.19×10−24), possibly due to decreased expression of the mitochondrial transcription factor A (TFAM) gene (P = 3.26×10−3). In contrast, the transcript levels of mtDNA genes were significantly increased in KC corneas compared with normal corneas (NADH dehydrogenase subunit 1 [ND1]: P = 1.79×10−3; cytochrome c oxidase subunit 1 [COX1]: P = 1.54×10−3; NADH dehydrogenase subunit 1, [ND6]: P = 4.62×10−3). The latter may be the result of increased expression levels of mtDNA transcription-related genes mitochondrial RNA polymerase (POLRMT) (P = 2.55×10−4) and transcription factor B2 mitochondrial (TFB2M) (P = 7.88×10−5). KC corneas also had increased mtDNA damage (P = 3.63×10−10), higher ROS levels, and lower mitochondrial membrane potential and ATP levels compared with normal corneas. Decreased integrity, content and increased transcript level of mtDNA are associated with KC. These changes may affect the generation of ROS and play a role in the pathogenesis of KC. PMID:27783701

  17. Scientific advice on the suitability of data for the assessment of DNA integration into the fish genome of a genetically modified DNA plasmid-based veterinary vaccine

    Directory of Open Access Journals (Sweden)

    European Food Safety Authority

    2013-05-01

    Full Text Available Pancreas disease caused by salmonid alphavirus in farmed Atlantic salmon (Salmo salar leads to high mortality rates post infection and histopathological lesions in several organs. As protection against pancreas disease, Novartis developed a prophylactic DNA plasmid-based vaccine to be administered to salmon as naked plasmid in a single intramuscular injection. In order to assess the legal status of the fish vaccinated with this new vaccine with regard to the legislation on genetically modified organisms, the European Commission suggested that the company carry out a scientific study on the integration/non-integration of the plasmid DNA into the fish genome. Subsequently, the European Commission requested EFSA to give scientific advice on the study design and the conclusions drawn by the company. PCR based analysis of genomic DNA from muscle samples, taken from at or around the injection site 436 days post vaccination, led the company to conclude that integration of plasmid DNA into the fish genome is extremely unlikely. After an assessment of the study, EFSA considers that the study presented by Novartis Animal Health on the integration/non-integration of DNA plasmid-based vaccine into the salmon genomic DNA provides insufficient information on the potential integration of plasmid DNA fragments into the fish genome due to a limited coverage of the plasmid DNA by the detection method provided, the limited number of samples analysed and an insufficient limit of detection and method validation. Therefore, EFSA is of the opinion that the results from the integration/non-integration study submitted by Novartis Animal Health are not sufficient to support the conclusion of non-integration of plasmid DNA into the fish genome drawn by the company.

  18. 水稻EPSP合酶cDNA克隆、序列分析及其拷贝数测定%Isolation of Rice EPSP Synthase cDNA and Its Sequence Analysis and Copy Number Determination

    Institute of Scientific and Technical Information of China (English)

    徐军望; 魏晓丽; 李旭刚; 陈蕾; 冯德江; 朱祯

    2002-01-01

    根据本室分离的水稻EPSP合酶基因的基因组序列设计一对引物,利用RT-PCR方法首次从水稻(Oryza sativa L. subsp. indica)叶片的RNA中扩增获得了水稻编码EPSP合酶的全长为1 585 bp的cDNA片段,它含有一个完整的开放读码框,编码511个氨基酸,包括444个氨基酸组成的成熟肽序列以及N端的67个氨基酸组成的叶绿体转运肽序列.成熟肽氨基酸序列对比表明,除真菌来源的EPSP合酶变异较大外,其他来源的EPSP合酶同源性较高,均在51%以上.而叶绿体转运肽氨基酸序列同源性较低.Southern杂交表明水稻EPSP合酶基因在水稻基因组中以单拷贝形式存在.RT-PCR分析表明,水稻EPSP合酶基因在根、未成熟种子和叶片中均有转录表达,在叶片中表达量最高.%In order to isolate the total cDNA of rice (Oryza sativa L.) epsps gene, RT-PCR was carried out with template of rice first-strand cDNA and primers designed according to rice EPSP synthase genomic sequence obtained in previous study. A 1 585-bp cDNA fragment was amplified and cloned. The 1 585-bp cDNA contains an open reading frame (ORF) comprising of 1 533 nucleotides (nt) which encodes a 511 residue polypepetides, including 67 amino acids chloroplast transit peptide and 444 amino acids EPSP synthase mature peptide. A comparison between the EPSP synthase of different sources indicates that the mature peptide shows more than 51% identity except for the fungi EPSP synthase and the transit peptide shows considerably less sequence conservation. The copy number of rice epsps gene is estimated to be one copy per haploid rice genome using southern blot. RT-PCR indicated that rice epsps gene is expressed in rice leaves, endosperms and roots and has the highest expression level in leaves.

  19. Frequency and character of alternative somatic recombination fates of paralogous genes during T-DNA integration.

    Science.gov (United States)

    Jelesko, John G; Carter, Kristy; Kinoshita, Yuki; Gruissem, Wilhelm

    2005-09-01

    A synthetic RBCSB gene cluster was transformed into Arabidopsis in order to simultaneously evaluate the frequency and character of somatic illegitimate recombination, homologous recombination, and targeted gene replacement events associated with T-DNA-mediated transformation. The most frequent type of recombination event observed was illegitimate integration of the T-DNA without activation of the silent DeltaRBCS1B: LUC transgene. Sixteen luc(+) (firefly luciferase positive) T1 plants were isolated. Six of these were due to illegitimate recombination events resulting in a gene trapping effect. Nine resulted from homologous recombination between paralogous RBCSB sequences associated with T-DNA integration. The frequency of somatic homologous recombination associated with T-DNA integration was almost 200 times higher than previously reported rates of meiotic homologous recombination with the same genes. The distribution of (somatic homologous) recombination resolution sites generally fits a fractional interval length model. However, a small region adjacent to an indel showed a significant over-representation of resolution sites, suggesting that DNA mismatch recognition may also play an important role in the positioning of somatic resolution sites. The frequency of somatic resolution within exon-2 was significantly different from that previously observed during meiotic recombination.

  20. [Evaluation of the quality of the human spermatozoon: comparison between spermatic DNA integrity and semen variables].

    Science.gov (United States)

    Cruz, Ibis; Colmenares, Melisa; Berrueta-Carrillo, Leidith; Gomez-Perez, Roald; Montes, Henry; Berrueta, Lisbeth; Salmen, Siham; Osuna, Jesús Alfonso

    2010-03-01

    Semen analysis does not have an absolute predictive value on fertility, however it is a reflection of male fertility potential, which is related to its spermatozoa quality and other semen variables. Great variability in human semen parameters has been demonstrated within a single individual, an observation that could explain why a male with low semen quality can successfully fertilize an egg. Although conventional semen analysis, such as sperm concentration, motility and morphology, provide important information about the clinical status of male fertility, new procedures to predict the sperm functional capability have been developed in the last decade, such as analysis of nuclear DNA integrity, which have improved considerably the clinical diagnosis of male infertility, and increased the knowledge about spermatozoa function. DNA fragmentation consist in interruptions, both in single and double DNA strains, that frequently occur in sperm samples from infertile patients. We have conducted a clinical study in semen samples from patients who have attended the Andrology laboratory of the University of Los Andes, between March 2007 and March 2009. The aim of this study was to compare sperm DNA integrity, analyzed by flow cytometry, with traditional semen parameters. Our results show remarkable correlations between conventional human semen variables and sperm chromatin integrity, contributing to asses an integral evaluation of sperm quality allowing the analysis of its fertilizing potential in clinical studies.

  1. On-Chip integration of sample pretreatment and Multiplex polymerase chain reaction (PCR) for DNA analysis

    DEFF Research Database (Denmark)

    Brivio, Monica; Snakenborg, Detlef; Søgaard, E.;

    2008-01-01

    In this paper we present a modular lab-on-a-chip system for integrated sample pre-treatment (PT) by magnetophoresis and DNA amplification by polymerase chain reaction (PCR). It consists of a polymer-based microfluidic chip mounted on a custom-made thermocycler (Figure 1) and includes a simple...

  2. Conditionally amplifiable BACs: switching from single-copy to high-copy vectors and genomic clones.

    Science.gov (United States)

    Wild, Jadwiga; Hradecna, Zdenka; Szybalski, Waclaw

    2002-09-01

    The widely used, very-low-copy BAC (bacterial artificial chromosome) vectors are the mainstay of present genomic research. The principal advantage of BACs is the high stability of inserted clones, but an important disadvantage is the low yield of DNA, both for vectors alone and when carrying genomic inserts. We describe here a novel class of single-copy/high-copy (SC/HC) pBAC/oriV vectors that retain all the advantages of low-copy BAC vectors, but are endowed with a conditional and tightly controlled oriV/TrfA amplification system that allows: (1) a yield of ~100 copies of the vector per host cell when conditionally induced with L-arabinose, and (2) analogous DNA amplification (only upon induction and with copy number depending on the insert size) of pBAC/oriV clones carrying >100-kb inserts. Amplifiable clones and libraries facilitate high-throughput DNA sequencing and other applications requiring HC plasmid DNA. To turn on DNA amplification, which is driven by the oriV origin of replication, we used copy-up mutations in the gene trfA whose expression was very tightly controlled by the araC-P(araBAD) promoter/regulator system. This system is inducible by L-arabinose, and could be further regulated by glucose and fucose. Amplification of DNA upon induction with L-arabinose and its modulation by glucose are robust and reliable. Furthermore, we discovered that addition of 0.2% D-glucose to the growth medium helped toward the objective of obtaining a real SC state for all BAC systems, thus enhancing the stability of their maintenance, which became equivalent to cloning into the host chromosome

  3. 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.

  4. A high excision potential of TALENs for integrated DNA of HIV-based lentiviral vector.

    Science.gov (United States)

    Ebina, Hirotaka; Kanemura, Yuka; Misawa, Naoko; Sakuma, Tetsushi; Kobayashi, Tomoko; Yamamoto, Takashi; Koyanagi, Yoshio

    2015-01-01

    DNA-editing technology has made it possible to rewrite genetic information in living cells. Human immunodeficiency virus (HIV) provirus, an integrated form of viral complementary DNA in host chromosomes, could be a potential target for this technology. We recently reported that HIV proviral DNA could be excised from the chromosomal DNA of HIV-based lentiviral DNA-transduced T cells after multiple introductions of a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 endonuclease system targeting HIV long terminal repeats (LTR). Here, we generated a more efficient strategy that enables the excision of HIV proviral DNA using customized transcription activator-like effector nucleases (TALENs) targeting the same HIV LTR site. A single transfection of TALEN-encoding mRNA, prepared from in vitro transcription, resulted in more than 80% of lentiviral vector DNA being successfully removed from the T cell lines. Furthermore, we developed a lentiviral vector system that takes advantage of the efficient proviral excision with TALENs and permits the simple selection of gene-transduced and excised cells in T cell lines.

  5. A high excision potential of TALENs for integrated DNA of HIV-based lentiviral vector.

    Directory of Open Access Journals (Sweden)

    Hirotaka Ebina

    Full Text Available DNA-editing technology has made it possible to rewrite genetic information in living cells. Human immunodeficiency virus (HIV provirus, an integrated form of viral complementary DNA in host chromosomes, could be a potential target for this technology. We recently reported that HIV proviral DNA could be excised from the chromosomal DNA of HIV-based lentiviral DNA-transduced T cells after multiple introductions of a clustered regularly interspaced short palindromic repeat (CRISPR/Cas9 endonuclease system targeting HIV long terminal repeats (LTR. Here, we generated a more efficient strategy that enables the excision of HIV proviral DNA using customized transcription activator-like effector nucleases (TALENs targeting the same HIV LTR site. A single transfection of TALEN-encoding mRNA, prepared from in vitro transcription, resulted in more than 80% of lentiviral vector DNA being successfully removed from the T cell lines. Furthermore, we developed a lentiviral vector system that takes advantage of the efficient proviral excision with TALENs and permits the simple selection of gene-transduced and excised cells in T cell lines.

  6. Seasonal Liza aurata tissue-specific DNA integrity in a multi-contaminated coastal lagoon (Ria de Aveiro, Portugal).

    Science.gov (United States)

    Oliveira, M; Maria, V L; Ahmad, I; Pacheco, M; Santos, M A

    2010-10-01

    In this study, the DNA integrity of golden grey mullet (Liza aurata) collected in differently contaminated sites of a coastal lagoon, Ria de Aveiro (Portugal), was assessed, over the period of 1 year, using the DNA alkaline unwinding assay, in four different tissues (gill, kidney, liver and blood) and compared to a reference site. The four tissues displayed different DNA integrity basal levels, clearly affected by seasonal factors. Gill and kidney were, respectively, the most and least sensitive tissues. All sites demonstrated the capacity to interfere with DNA integrity. The sites displaying the highest and lowest DNA damage capability were, respectively, Barra (subject to naval traffic) and Vagos (contaminated with polycyclic aromatic hydrocarbons). In terms of seasonal variability, autumn seems to be the more critical season (more DNA damage) unlike summer when no DNA damage was found in any tissue. Data recommend the continued monitoring of this aquatic system.

  7. Long-term administration of valacyclovir reduces the number of Epstein-Barr virus (EBV)-infected B cells but not the number of EBV DNA copies per B cell in healthy volunteers.

    Science.gov (United States)

    Hoshino, Yo; Katano, Harutaka; Zou, Ping; Hohman, Patricia; Marques, Adriana; Tyring, Stephen K; Follmann, Dean; Cohen, Jeffrey I

    2009-11-01

    Epstein-Barr virus (EBV) establishes a latent infection in B cells in the blood, and the latent EBV load in healthy individuals is generally stable over time, maintaining a "set point." It is unknown if the EBV load changes after long-term antiviral therapy in healthy individuals. We treated volunteers with either valacyclovir (valaciclovir) or no antiviral therapy for 1 year and measured the amount of EBV DNA in B cells every 3 months with a novel, highly sensitive assay. The number of EBV-infected B cells decreased in subjects receiving valacyclovir (half-life of 11 months; P = 0.02) but not in controls (half-life of 31 years; P = 0.86). The difference in the slopes of the lines for the number of EBV-infected B cells over time for the valacyclovir group versus the control group approached significance (P = 0.054). In contrast, the number of EBV DNA copies per B cell remained unchanged in both groups (P = 0.62 and P = 0.92 for the control and valacyclovir groups, respectively). Valacyclovir reduces the frequency of EBV-infected B cells when administered over a long period and, in theory, might allow eradication of EBV from the body if reinfection does not occur.

  8. Tensor GSVD of Patient- and Platform-Matched Tumor and Normal DNA Copy-Number Profiles Uncovers Chromosome Arm-Wide Patterns of Tumor-Exclusive Platform-Consistent Alterations Encoding for Cell Transformation and Predicting Ovarian Cancer Survival

    Science.gov (United States)

    Sankaranarayanan, Preethi; Schomay, Theodore E.; Aiello, Katherine A.; Alter, Orly

    2015-01-01

    The number of large-scale high-dimensional datasets recording different aspects of a single disease is growing, accompanied by a need for frameworks that can create one coherent model from multiple tensors of matched columns, e.g., patients and platforms, but independent rows, e.g., probes. We define and prove the mathematical properties of a novel tensor generalized singular value decomposition (GSVD), which can simultaneously find the similarities and dissimilarities, i.e., patterns of varying relative significance, between any two such tensors. We demonstrate the tensor GSVD in comparative modeling of patient- and platform-matched but probe-independent ovarian serous cystadenocarcinoma (OV) tumor, mostly high-grade, and normal DNA copy-number profiles, across each chromosome arm, and combination of two arms, separately. The modeling uncovers previously unrecognized patterns of tumor-exclusive platform-consistent co-occurring copy-number alterations (CNAs). We find, first, and validate that each of the patterns across only 7p and Xq, and the combination of 6p+12p, is correlated with a patient’s prognosis, is independent of the tumor’s stage, the best predictor of OV survival to date, and together with stage makes a better predictor than stage alone. Second, these patterns include most known OV-associated CNAs that map to these chromosome arms, as well as several previously unreported, yet frequent focal CNAs. Third, differential mRNA, microRNA, and protein expression consistently map to the DNA CNAs. A coherent picture emerges for each pattern, suggesting roles for the CNAs in OV pathogenesis and personalized therapy. In 6p+12p, deletion of the p21-encoding CDKN1A and p38-encoding MAPK14 and amplification of RAD51AP1 and KRAS encode for human cell transformation, and are correlated with a cell’s immortality, and a patient’s shorter survival time. In 7p, RPA3 deletion and POLD2 amplification are correlated with DNA stability, and a longer survival. In Xq

  9. Tensor GSVD of patient- and platform-matched tumor and normal DNA copy-number profiles uncovers chromosome arm-wide patterns of tumor-exclusive platform-consistent alterations encoding for cell transformation and predicting ovarian cancer survival.

    Directory of Open Access Journals (Sweden)

    Preethi Sankaranarayanan

    Full Text Available The number of large-scale high-dimensional datasets recording different aspects of a single disease is growing, accompanied by a need for frameworks that can create one coherent model from multiple tensors of matched columns, e.g., patients and platforms, but independent rows, e.g., probes. We define and prove the mathematical properties of a novel tensor generalized singular value decomposition (GSVD, which can simultaneously find the similarities and dissimilarities, i.e., patterns of varying relative significance, between any two such tensors. We demonstrate the tensor GSVD in comparative modeling of patient- and platform-matched but probe-independent ovarian serous cystadenocarcinoma (OV tumor, mostly high-grade, and normal DNA copy-number profiles, across each chromosome arm, and combination of two arms, separately. The modeling uncovers previously unrecognized patterns of tumor-exclusive platform-consistent co-occurring copy-number alterations (CNAs. We find, first, and validate that each of the patterns across only 7p and Xq, and the combination of 6p+12p, is correlated with a patient's prognosis, is independent of the tumor's stage, the best predictor of OV survival to date, and together with stage makes a better predictor than stage alone. Second, these patterns include most known OV-associated CNAs that map to these chromosome arms, as well as several previously unreported, yet frequent focal CNAs. Third, differential mRNA, microRNA, and protein expression consistently map to the DNA CNAs. A coherent picture emerges for each pattern, suggesting roles for the CNAs in OV pathogenesis and personalized therapy. In 6p+12p, deletion of the p21-encoding CDKN1A and p38-encoding MAPK14 and amplification of RAD51AP1 and KRAS encode for human cell transformation, and are correlated with a cell's immortality, and a patient's shorter survival time. In 7p, RPA3 deletion and POLD2 amplification are correlated with DNA stability, and a longer survival

  10. Early hCG addition to rFSH for ovarian stimulation in IVF provides better results and the cDNA copies of the hCG receptor may be an indicator of successful stimulation

    Directory of Open Access Journals (Sweden)

    Paraskevis Dimitris

    2009-10-01

    Full Text Available Abstract A simple, safe and cost-effective treatment protocol in ovarian stimulation is of great importance in IVF practice, especially in the case of previous unsuccessful attempts. hCG has been used as a substitute of LH because of the degree of homology between the two hormones. The main aim of this prospective randomized study was to determine, for the first time, whether low dose hCG added to rFSH for ovarian stimulation could produce better results compared to the addition of rLH in women entering IVF-ET, especially in those women that had previous IVF failures. An additional aim was to find an indicator that would allow us to follow-up ovarian stimulation and, possibly, modify it in order to achieve a better IVF outcome; and that indicator may be the cDNA copies of the LH/hCG receptor. Group A patients (n = 58 were administered hCG and Group B rLH (n = 56 in addition to rFSH in the first days of ovarian stimulation. The number of follicles and oocytes and, most importantly, implantation and pregnancy rates were shown to be statistically significantly higher in the hCG group. This study has also determined, for the first time to our best knowledge, m-RNA for LH/hCG receptors in the lymphocytes of peripheral blood 40 h before ovum pick-up. cDNA levels of the hCG receptor after ovarian stimulation were significantly higher among women receiving hCG compared to those receiving LH. In addition, higher levels were encountered among women with pregnancy compared to those without, although this was not statistically significant due to the small number of pregnancies. It seems that hCG permits a highly effective and more stable occupancy of rLH/hCG receptors and gives more follicles and more oocytes. The determination of cDNA copies could be, in the future, a marker during ovulation induction protocols and of course a predictor for the outcome of ART in the special subgroup of patients with previous failures.

  11. Herpesvirus telomeric repeats facilitate genomic integration into host telomeres and mobilization of viral DNA during reactivation.

    Science.gov (United States)

    Kaufer, Benedikt B; Jarosinski, Keith W; Osterrieder, Nikolaus

    2011-03-14

    Some herpesviruses, particularly lymphotropic viruses such as Marek's disease virus (MDV) and human herpesvirus 6 (HHV-6), integrate their DNA into host chromosomes. MDV and HHV-6, among other herpesviruses, harbor telomeric repeats (TMRs) identical to host telomeres at either end of their linear genomes. Using MDV as a natural virus-host model, we show that herpesvirus TMRs facilitate viral genome integration into host telomeres and that integration is important for establishment of latency and lymphoma formation. Integration into host telomeres also aids in reactivation from the quiescent state of infection. Our results and the presence of TMRs in many herpesviruses suggest that integration mediated by viral TMRs is a conserved mechanism, which ensures faithful virus genome maintenance in host cells during cell division and allows efficient mobilization of dormant viral genomes. This finding is of particular importance as reactivation is critical for virus spread between susceptible individuals and is necessary for continued herpesvirus evolution and survival.

  12. The Art of Copying

    DEFF Research Database (Denmark)

    Christensen, Hans Dam

    2017-01-01

    This article discusses copies within the field of art museums by way of mapping strategies for copy practices. This mapping leans heavily towards parts of the writings of Jacques Derrida (1930–2004). Against the backdrop of this theoretical premise, the article distinguishes five main strategies...... for the informational originalcopy will be discussed as it has a vital function in terms of talking about museum originals and copies. This is the strategy which grants the original artifacts their status as museum objects. An informational originalcopy is just as unique as an original object of art, and at the same...

  13. Oral antioxidant treatment partly improves integrity of human sperm DNA in infertile grade I varicocele patients.

    Science.gov (United States)

    Gual-Frau, Josep; Abad, Carlos; Amengual, María J; Hannaoui, Naim; Checa, Miguel A; Ribas-Maynou, Jordi; Lozano, Iris; Nikolaou, Alexandros; Benet, Jordi; García-Peiró, Agustín; Prats, Juan

    2015-09-01

    Infertile males with varicocele have the highest percentage of sperm cells with damaged DNA, compared to other infertile groups. Antioxidant treatment is known to enhance the integrity of sperm DNA; however, there are no data on the effects in varicocele patients. We thus investigated the potential benefits of antioxidant treatment specifically in grade I varicocele males. Twenty infertile patients with grade I varicocele were given multivitamins (1500 mg L-Carnitine, 60 mg vitamin C, 20 mg coenzyme Q10, 10 mg vitamin E, 200 μg vitamin B9, 1 μg vitamin B12, 10 mg zinc, 50 μg selenium) daily for three months. Semen parameters including total sperm count, concentration, progressive motility, vitality, and morphology were determined before and after treatment. In addition, sperm DNA fragmentation and the amount of highly degraded sperm cells were analyzed by Sperm Chromatin Dispersion. After treatment, patients showed an average relative reduction of 22.1% in sperm DNA fragmentation (p = 0.02) and had 31.3% fewer highly degraded sperm cells (p = 0.07). Total numbers of sperm cells were increased (p = 0.04), but other semen parameters were unaffected. These data suggest that sperm DNA integrity in grade I varicocele patients may be improved by oral antioxidant treatment.

  14. Survival, DNA Integrity, and Ultrastructural Damage in Antarctic Cryptoendolithic Eukaryotic Microorganisms Exposed to Ionizing Radiation

    Science.gov (United States)

    Pacelli, Claudia; Selbmann, Laura; Zucconi, Laura; Raguse, Marina; Moeller, Ralf; Shuryak, Igor; Onofri, Silvano

    2017-02-01

    Life dispersal between planets, planetary protection, and the search for biosignatures are main topics in astrobiology. Under the umbrella of the STARLIFE project, three Antarctic endolithic microorganisms, the melanized fungus Cryomyces antarcticus CCFEE 515, a hyaline strain of Umbilicaria sp. (CCFEE 6113, lichenized fungus), and a Stichococcus sp. strain (C45A, green alga), were exposed to high doses of space-relevant gamma radiation (60Co), up to 117.07 kGy. After irradiation survival, DNA integrity and ultrastructural damage were tested. The first was assessed by clonogenic test; viability and dose responses were reasonably described by the linear-quadratic formalism. DNA integrity was evaluated by PCR, and ultrastructural damage was observed by transmission electron microscopy. The most resistant among the tested organisms was C. antarcticus both in terms of colony formation and DNA preservation. Besides, results clearly demonstrate that DNA was well detectable in all the tested organisms even when microorganisms were dead. This high resistance provides support for the use of DNA as a possible biosignature during the next exploration campaigns. Implication in planetary protection and contamination during long-term space travel are put forward.

  15. Nucleolar organization, ribosomal DNA array stability, and acrocentric chromosome integrity are linked to telomere function.

    Directory of Open Access Journals (Sweden)

    Kaitlin M Stimpson

    Full Text Available The short arms of the ten acrocentric human chromosomes share several repetitive DNAs, including ribosomal RNA genes (rDNA. The rDNA arrays correspond to nucleolar organizing regions that coalesce each cell cycle to form the nucleolus. Telomere disruption by expressing a mutant version of telomere binding protein TRF2 (dnTRF2 causes non-random acrocentric fusions, as well as large-scale nucleolar defects. The mechanisms responsible for acrocentric chromosome sensitivity to dysfunctional telomeres are unclear. In this study, we show that TRF2 normally associates with the nucleolus and rDNA. However, when telomeres are crippled by dnTRF2 or RNAi knockdown of TRF2, gross nucleolar and chromosomal changes occur. We used the controllable dnTRF2 system to precisely dissect the timing and progression of nucleolar and chromosomal instability induced by telomere dysfunction, demonstrating that nucleolar changes precede the DNA damage and morphological changes that occur at acrocentric short arms. The rDNA repeat arrays on the short arms decondense, and are coated by RNA polymerase I transcription binding factor UBF, physically linking acrocentrics to one another as they become fusogenic. These results highlight the importance of telomere function in nucleolar stability and structural integrity of acrocentric chromosomes, particularly the rDNA arrays. Telomeric stress is widely accepted to cause DNA damage at chromosome ends, but our findings suggest that it also disrupts chromosome structure beyond the telomere region, specifically within the rDNA arrays located on acrocentric chromosomes. These results have relevance for Robertsonian translocation formation in humans and mechanisms by which acrocentric-acrocentric fusions are promoted by DNA damage and repair.

  16. Super DNAging-New insights into DNA integrity, genome stability and telomeres in the oldest old.

    Science.gov (United States)

    Franzke, Bernhard; Neubauer, Oliver; Wagner, Karl-Heinz

    2015-01-01

    Reductions in DNA integrity, genome stability, and telomere length are strongly associated with the aging process, age-related diseases as well as the age-related loss of muscle mass. However, in people reaching an age far beyond their statistical life expectancy the prevalence of diseases, such as cancer, cardiovascular disease, diabetes or dementia, is much lower compared to "averagely" aged humans. These inverse observations in nonagenarians (90-99 years), centenarians (100-109 years) and super-centenarians (110 years and older) require a closer look into dynamics underlying DNA damage within the oldest old of our society. Available data indicate improved DNA repair and antioxidant defense mechanisms in "super old" humans, which are comparable with much younger cohorts. Partly as a result of these enhanced endogenous repair and protective mechanisms, the oldest old humans appear to cope better with risk factors for DNA damage over their lifetime compared to subjects whose lifespan coincides with the statistical life expectancy. This model is supported by study results demonstrating superior chromosomal stability, telomere dynamics and DNA integrity in "successful agers". There is also compelling evidence suggesting that life-style related factors including regular physical activity, a well-balanced diet and minimized psycho-social stress can reduce DNA damage and improve chromosomal stability. The most conclusive picture that emerges from reviewing the literature is that reaching "super old" age appears to be primarily determined by hereditary/genetic factors, while a healthy lifestyle additionally contributes to achieving the individual maximum lifespan in humans. More research is required in this rapidly growing population of super old people. In particular, there is need for more comprehensive investigations including short- and long-term lifestyle interventions as well as investigations focusing on the mechanisms causing DNA damage, mutations, and telomere

  17. Integrated data analysis reveals potential drivers and pathways disrupted by DNA methylation in papillary thyroid carcinomas

    DEFF Research Database (Denmark)

    Beltrami, Caroline Moraes; Dos Reis, Mariana Bisarro; Barros-Filho, Mateus Camargo

    2017-01-01

    BACKGROUND: Papillary thyroid carcinoma (PTC) is a common endocrine neoplasm with a recent increase in incidence in many countries. Although PTC has been explored by gene expression and DNA methylation studies, the regulatory mechanisms of the methylation on the gene expression was poorly clarified......-validated by the The Cancer Genome Atlas data. The majority of these probes was found in non-promoters regions, distant from CGI and enriched by enhancers. The integrative analysis between gene expression and DNA methylation revealed 185 and 38 genes (mainly in the promoter and body regions, respectively) with negative...

  18. On-Chip integration of sample pretreatment and Multiplex polymerase chain reaction (PCR) for DNA analysis

    DEFF Research Database (Denmark)

    Brivio, Monica; Snakenborg, Detlef; Søgaard, E.

    2008-01-01

    In this paper we present a modular lab-on-a-chip system for integrated sample pre-treatment (PT) by magnetophoresis and DNA amplification by polymerase chain reaction (PCR). It consists of a polymer-based microfluidic chip mounted on a custom-made thermocycler (Figure 1) and includes a simple...... and efficient method for switching the liquid flow between the PT and PCR chamber. Purification of human genomic DNA from EDTA-treated blood and multiplex PCR were successfully carried out on-chip using the developed lab-on-a-chip system....

  19. Molecular verification of the integration of Tripsacum dactyloides DNA into wheat genome through wide hybridization

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    RAPD and RFLP analyses of double haploid lines which derived from hybridization between hexaploid wheat (Triticum aestivum L.2n=42) and eastern gamagrass (Tripsacum dactyloides L.2n=4x=72) are reported.Two of the 340 Operon primers have been screened,which stably amplified Tripsacum dactyloides (male parent) specific bands in the double haploid lines.These results confirm the fact that Tripsacum dactyloides DNA has been integrated into wheat genome by sexual hybridization at molecular level.This idea has been further testified by RFLP analysis.Application and potentials of transferring Tripsacum dactyloides DNA into wheat genome by sexual hybridization in wheat breeding are discussed.

  20. Pentoxifylline increase sperm motility in devitrified spermatozoa from asthenozoospermic patient without damage chromatin and DNA integrity.

    Science.gov (United States)

    Nabi, Ali; Khalili, Mohammad Ali; Fesahat, Farzaneh; Talebi, Alireza; Ghasemi-Esmailabad, Saeed

    2017-06-01

    The freeze-thaw process results in reduced motility, viability and fertilization potential of human spermatozoa. So, a variety of substances were evaluated in order to enhance human sperm resistance to the stress of cryopreservation, such as Pentoxifylline (PTX) for improving the Intracytoplasmic sperm injection (ICSI) outcomes. The aim was to investigate the effect of PTX on sperm parameters and chromatin/DNA integrity of asthenozoospermic semen post vitrification. A total of 30 semen specimens were obtained from infertile men with asthenozoospermia. The cryoprotectant-free vitrification was performed for the samples after assessment of sperm parameters. After warming, each sample was exposed for 30 min to 3.6 mmol/l PTX in experimental group and the control group without any treatment apposing at 37 °C for 30 min in regard, to repeat all in vitro analysis (sperm parameters and DNA integrity assay). Regardless of the vitrification devastating impacts on sperm parameters, incubation of post vitrified samples with PTX increased the rate of progressive motility (P integrity of asthenozoospermic sperm samples. The data showed that PTX was able to improve sperm movement without any adverse effects on sperm chromatin/DNA integrity in vitrification program. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Retroviral DNA integration: viral and cellular determinants of target-site selection.

    Directory of Open Access Journals (Sweden)

    Mary K Lewinski

    2006-06-01

    Full Text Available Retroviruses differ in their preferences for sites for viral DNA integration in the chromosomes of infected cells. Human immunodeficiency virus (HIV integrates preferentially within active transcription units, whereas murine leukemia virus (MLV integrates preferentially near transcription start sites and CpG islands. We investigated the viral determinants of integration-site selection using HIV chimeras with MLV genes substituted for their HIV counterparts. We found that transferring the MLV integrase (IN coding region into HIV (to make HIVmIN caused the hybrid to integrate with a specificity close to that of MLV. Addition of MLV gag (to make HIVmGagmIN further increased the similarity of target-site selection to that of MLV. A chimeric virus with MLV Gag only (HIVmGag displayed targeting preferences different from that of both HIV and MLV, further implicating Gag proteins in targeting as well as IN. We also report a genome-wide analysis indicating that MLV, but not HIV, favors integration near DNase I-hypersensitive sites (i.e., +/- 1 kb, and that HIVmIN and HIVmGagmIN also favored integration near these features. These findings reveal that IN is the principal viral determinant of integration specificity; they also reveal a new role for Gag-derived proteins, and strengthen models for integration targeting based on tethering of viral IN proteins to host proteins.

  2. Retroviral DNA integration: ASLV, HIV, and MLV show distinct target site preferences.

    Directory of Open Access Journals (Sweden)

    Rick S Mitchell

    2004-08-01

    Full Text Available The completion of the human genome sequence has made possible genome-wide studies of retroviral DNA integration. Here we report an analysis of 3,127 integration site sequences from human cells. We compared retroviral vectors derived from human immunodeficiency virus (HIV, avian sarcoma-leukosis virus (ASLV, and murine leukemia virus (MLV. Effects of gene activity on integration targeting were assessed by transcriptional profiling of infected cells. Integration by HIV vectors, analyzed in two primary cell types and several cell lines, strongly favored active genes. An analysis of the effects of tissue-specific transcription showed that it resulted in tissue-specific integration targeting by HIV, though the effect was quantitatively modest. Chromosomal regions rich in expressed genes were favored for HIV integration, but these regions were found to be interleaved with unfavorable regions at CpG islands. MLV vectors showed a strong bias in favor of integration near transcription start sites, as reported previously. ASLV vectors showed only a weak preference for active genes and no preference for transcription start regions. Thus, each of the three retroviruses studied showed unique integration site preferences, suggesting that virus-specific binding of integration complexes to chromatin features likely guides site selection.

  3. Nutriomes and personalised nutrition for DNA damage prevention, telomere integrity maintenance and cancer growth control.

    Science.gov (United States)

    Fenech, Michael F

    2014-01-01

    DNA damage at the base sequence and chromosome level is a fundamental cause of developmental and degenerative diseases. Multiple micronutrients and their interactions with the inherited and/or acquired genome determine DNA damage and genomic instability rates. The challenge is to identify for each individual the combination of micronutrients and their doses (i.e. the nutriome) that optimises genome stability, including telomere integrity and functionality and DNA repair. Using nutrient array systems with high-content analysis diagnostics of DNA damage, cell death and cell growth, it is possible to define, on an individual basis, the optimal nutriome for DNA damage prevention and cancer growth control. This knowledge can also be used to improve culture systems for cells used in therapeutics such as stem cells to ensure that they are not genetically aberrant when returned to the body. Furthermore, this information could be used to design dietary patterns that deliver the micronutrient combinations and concentrations required for preventing DNA damage by micronutrient deficiency or excess. Using this approach, new knowledge could be obtained to identify the dietary restrictions and/or supplementations required to control specific cancers, which is particularly important given that reliable validated advice is not yet available for those diagnosed with cancer.

  4. Detection of Clonal and Subclonal Copy-Number Variants in Cell-Free DNA from Patients with Breast Cancer Using a Massively Multiplexed PCR Methodology

    Directory of Open Access Journals (Sweden)

    Eser Kirkizlar

    2015-10-01

    Using an in vitro model of cell-free DNA, we show that mmPCR-NGS can accurately detect CNVs with average allelic imbalances as low as 0.5%, an improvement over previously reported whole-genome sequencing approaches. Our method revealed differences in the spectrum of CNVs detected in tumor tissue subsections and matching plasma samples from 11 patients with stage II breast cancer. Moreover, we showed that liquid biopsies are able to detect subclonal mutations that may be missed in tumor tissue biopsies. We anticipate that this mmPCR-NGS methodology will have broad applicability for the characterization, diagnosis, and therapeutic monitoring of CNV-enriched cancers, such as breast, ovarian, and lung cancer.

  5. Analysis on HBV DNA copies in the serum and breast milk of parturients with HBsAg positive%HBsAg阳性产妇血清和乳汁中HBV DNA载量分析

    Institute of Scientific and Technical Information of China (English)

    邹军; 汪永强

    2015-01-01

    Objective To study the relationship and correlation between serum HBV DNA and breast milk HBV DNA of HBsAg(+) parturients, and to provide evidence of guiding breast feeding for women infected by chronic hepatitis B virus (HBV). Methods A total of 148 parturients with HBsAg(+) of HBV were divided into the four groups according to the copies of HBV DNA in serum:<420 IU/ml group, 420~104 IU/ml group, 105~106 IU/ml group, 107~108 IU/ml group. The HBV DNA copies in the serum and breast milk from the four groups of women were detect-ed by fluorescence quantitative PCR (FQ-PCR) technique respectively. And the relationship and correlation of the load of HBV DNA were performed between in serum and breast milk from the four groups. Results The HBV positive rate of women with HBsAg (+) was 31.8%(47/148) in serum and 16.2%(24/148) in breast milk (χ2=7.355, P<0.05). In the parturients with serum HBV DNA<420 IU/ml, the corresponding breast milk was negative for the expression of HBV DNA. In the women with serum HBV DNA of 420~104 IU/ml, 105~106 IU/ml, 107~108 IU/ml, the corresponding HBV DNA positive rate in breast milk was 14.3%(3/18), 71.4%(5/7) and 84.2%(16/19), respectively (χ2=20.88, P<0.05). When serum HBV DNA load increased, the content of breast milk increased accordingly, which showed a mod-erate correlation (r=0.628, P<0.05). Conclusion HBV DNA is detected in breast milk of HBsAg(+) parturients. Its content is lower than that in the corresponding serum, and it increases when the load of HBV DNA in serum increases, which indicates that baby may be get infected through breast feeding.%目的:探讨HBsAg(+)产妇血清和乳汁中HBV DNA载量关系及相关性,为慢性乙型肝炎产妇母乳喂养提供实验依据。方法对148例HBsAg(+)产妇血清和乳汁中乙肝病毒采用实时荧光定量PCR (FQ-PCR)方法检测HBV DNA,按产妇血清HBV载量分为HBV DNA载量<420 IU/ml组、420~104 IU/ml组、105~106 IU/ml组、107~108 IU/ml组,并同

  6. Single-copy insertion of transgenes in Caenorhabditis elegans

    DEFF Research Database (Denmark)

    Frøkjaer-Jensen, Christian; Davis, M Wayne; Hopkins, Christopher E;

    2008-01-01

    At present, transgenes in Caenorhabditis elegans are generated by injecting DNA into the germline. The DNA assembles into a semistable extrachromosomal array composed of many copies of injected DNA. These transgenes are typically overexpressed in somatic cells and silenced in the germline. We have...

  7. Integrated DNA walking system to characterize a broad spectrum of GMOs in food/feed matrices.

    Science.gov (United States)

    Fraiture, Marie-Alice; Herman, Philippe; Lefèvre, Loic; Taverniers, Isabel; De Loose, Marc; Deforce, Dieter; Roosens, Nancy H

    2015-08-14

    In order to provide a system fully integrated with qPCR screening, usually used in GMO routine analysis, as well as being able to detect, characterize and identify a broad spectrum of GMOs in food/feed matrices, two bidirectional DNA walking methods targeting p35S or tNOS, the most common transgenic elements found in GM crops, were developed. These newly developed DNA walking methods are completing the previously implemented DNA walking method targeting the t35S pCAMBIA element. Food/feed matrices containing transgenic crops (Bt rice or MON863 maize) were analysed using the integrated DNA walking system. First, the newly developed DNA walking methods, anchored on the sequences used for the p35S or tNOS qPCR screening, were tested on Bt rice that contains these two transgenic elements. Second, the methods were assessed on a maize sample containing a low amount of the GM MON863 event, representing a more complex matrix in terms of genome size and sensitivity. Finally, to illustrate its applicability in GMO routine analysis by enforcement laboratories, the entire workflow of the integrated strategy, including qPCR screening to detect the potential presence of GMOs and the subsequent DNA walking methods to characterize and identify the detected GMOs, was applied on a GeMMA Scheme Proficiency Test matrix. Via the characterization of the transgene flanking region between the transgenic cassette and the plant genome as well as of a part of the transgenic cassette, the presence of GMOs was properly confirmed or infirmed in all tested samples. Due to their simple procedure and their short time-frame to get results, the developed DNA walking methods proposed here can be easily implemented in GMO routine analysis by the enforcement laboratories. In providing crucial information about the transgene flanking regions and/or the transgenic cassettes, this DNA walking strategy is a key molecular tool to prove the presence of GMOs in any given food/feed matrix.

  8. Flow cytometry application in the assessment of sperm DNA integrity of men with asthenozoospermia.

    Science.gov (United States)

    Piasecka, M; Gaczarzewicz, D; Laszczyńska, M; Starczewski, A; Brodowska, A

    2007-01-01

    Sperm genomic integrity and ultrastructural features of ejaculated spermatozoa contributing to the assessment of gamete fertility potential in patients with asthenozoospermia are discussed. The proportion of TUNEL-positive cells was significantly higher in the semen of patients with low sperm motility (n=40; p4% of TUNEL-positive sperm cells. Low sperm motility seems to be accompanied by serious defects of gamete chromatin expressed as diminished sperm genomic integrity and abnormal DNA condensation and by defects of sperm midpiece. These abnormalities may reflect developmental failure during the spermatogenic remodeling process. The DNA fragmentation test may be considered as an additional assay for the evaluation of spermatozoa beside standard analysis and taken together with electron microscopy may help to determine the actual number of "healthy" spermatozoa thereby playing an important role during diagnosis and treatment of male infertility.

  9. Spontaneous HBsAg loss in Korean patients: relevance of viral genotypes, S gene mutations, and covalently closed circular DNA copy numbers

    Science.gov (United States)

    Chang, Hye-Young; Park, Jun Yong; Park, Eun-Sook; Park, Yong Kwang; Han, Kwang-Hyub

    2014-01-01

    Background/Aims Occult HBV infection can persist following HBsAg loss and be transmitted, but the virological features are not well defined. Methods Here we investigated 25 Korean patients who lost HBsAg during follow up, either spontaneously or subsequent to therapy. Results Whereas subtype adr (genotype C) was found in 96% of HBsAg positive patients, 75 % of patients who lost HBsAg spontaneously were seemed to be infected with the ayw subtype with sequence similar to genotype D. Mutations in the major hydrophilic region (MHR) of HBsAg were found in 7 patients who lost HBsAg spontaneously. The mutations include T123S, M125I/N, C139R, D144E, V177A, L192F, and W196L, some of which have not been reported before. Functional analysis via transfection experiments indicate that the C139R and D144E mutations drastically reduced HBsAg antigenicity, while the Y225del mutation found in one interferon-treated patient impaired HBsAg secretion. Conclusions Lack of detectable HBsAg in patient serum could be explained by low level of ccc DNA in liver tissue, low antigenicity of the surface protein, or its secretion defect. PMID:25320728

  10. Integrated biochip for PCR-based DNA amplification and detection on capacitive biosensors

    Science.gov (United States)

    Moschou, D.; Vourdas, N.; Filippidou, M. K.; Tsouti, V.; Kokkoris, G.; Tsekenis, G.; Zergioti, I.; Chatzandroulis, S.; Tserepi, A.

    2013-05-01

    Responding to an increasing demand for LoC devices to perform bioanalytical protocols for disease diagnostics, the development of an integrated LoC device consisting of a μPCR module integrated with resistive microheaters and a biosensor array for disease diagnostics is presented. The LoC is built on a Printed Circuit Board (PCB) platform, implementing both the amplification of DNA samples and DNA detection/identification on-chip. The resistive microheaters for PCR and the wirings for the sensor read-out are fabricated by means of standard PCB technology. The microfluidic network is continuous-flow, designed to perform 30 PCR cycles with heated zones at constant temperatures, and is built onto the PCB utilizing commercial photopatternable polyimide layers. Following DNA amplification, the product is driven in a chamber where a Si-based biosensor array is placed for DNA detection through hybridization. The sensor array is tested for the detection of mutations of the KRAS gene, responsible for colon cancer.

  11. Impact of foreign DNA integration on tumor biology and on evolution via epigenetic alterations.

    Science.gov (United States)

    Doerfler, Walter

    2012-02-01

    The insertion of foreign DNA into mammalian genomes can alter their methylation and transcription patterns at remote sites from the locus of foreign DNA integration. The mechanisms leading to these fundamental changes and their frequencies are unknown. Sites and extent of changes in the recipient cells might depend on the location of foreign DNA integration. In the second part of this review, it will be hypothesized that the insertion event itself, for example, of tumor viral DNA via its epigenetic genome-wide consequences, plays an important role in oncogenesis. During evolution, the impact of ancient retrotransposon or retroviral genomes and the ensuing epigenetic alterations in the recipient genomes might have generated cells with completely different transcriptional profiles. Due to the continued presence of the transgenomes these alterations were genetically stable and were selected for or against by the environmental conditions prevalent at the time. These evolutionary effects are very different from those postulated for insertional mutagenesis, added genetic information or regulatory elements placed into the vicinity of cellular functions.

  12. Integrated Analysis for Identifying Radix Astragali and Its Adulterants Based on DNA Barcoding

    OpenAIRE

    Sihao Zheng; Dewang Liu; Weiguang Ren; Juan Fu; Linfang Huang; Shilin Chen

    2014-01-01

    Radix Astragali is a popular herb used in traditional Chinese medicine for its proimmune and antidiabetic properties. However, methods are needed to help distinguish Radix Astragali from its varied adulterants. DNA barcoding is a widely applicable molecular method used to identify medicinal plants. Yet, its use has been hampered by genetic distance, base variation, and limitations of the bio-NJ tree. Herein, we report the validation of an integrated analysis method for plant species identific...

  13. DyNAMiC Workbench: an integrated development environment for dynamic DNA nanotechnology

    OpenAIRE

    Grun, Casey; Werfel, Justin; Zhang, David Yu; Yin, Peng

    2015-01-01

    Dynamic DNA nanotechnology provides a promising avenue for implementing sophisticated assembly processes, mechanical behaviours, sensing and computation at the nanoscale. However, design of these systems is complex and error-prone, because the need to control the kinetic pathway of a system greatly increases the number of design constraints and possible failure modes for the system. Previous tools have automated some parts of the design workflow, but an integrated solution is lacking. Here, w...

  14. DyNAMiC Workbench: an integrated development environment for dynamic DNA nanotechnology.

    Science.gov (United States)

    Grun, Casey; Werfel, Justin; Zhang, David Yu; Yin, Peng

    2015-10-06

    Dynamic DNA nanotechnology provides a promising avenue for implementing sophisticated assembly processes, mechanical behaviours, sensing and computation at the nanoscale. However, design of these systems is complex and error-prone, because the need to control the kinetic pathway of a system greatly increases the number of design constraints and possible failure modes for the system. Previous tools have automated some parts of the design workflow, but an integrated solution is lacking. Here, we present software implementing a three 'tier' design process: a high-level visual programming language is used to describe systems, a molecular compiler builds a DNA implementation and nucleotide sequences are generated and optimized. Additionally, our software includes tools for analysing and 'debugging' the designs in silico, and for importing/exporting designs to other commonly used software systems. The software we present is built on many existing pieces of software, but is integrated into a single package—accessible using a Web-based interface at http://molecular-systems.net/workbench. We hope that the deep integration between tools and the flexibility of this design process will lead to better experimental results, fewer experimental design iterations and the development of more complex DNA nanosystems.

  15. As a Nucleus Enters a Small Pore, Chromatin Stretches and Maintains Integrity, Even with DNA Breaks.

    Science.gov (United States)

    Irianto, Jerome; Xia, Yuntao; Pfeifer, Charlotte R; Greenberg, Roger A; Discher, Dennis E

    2017-02-07

    As a cell pushes or pulls its nucleus through a small constriction, the chromatin must distort and somehow maintain genomic stability despite ever-present double-strand breaks in the DNA. Here we visualize within a living cell the pore-size dependent deformation of a specific locus engineered into chromosome-1 and cleaved. An mCherry-tagged nuclease targets the submicron locus, causing DNA cleavage and recruiting repair factors such as GFP-53BP1 to a large region around the locus. Aspiration of a cell and its nucleus into a micropipette shows that chromatin aligns and stretches parallel to the pore. Extension is largest in small pores, increasing >10-fold but remaining 30-fold shorter than the DNA contour length in the locus. Brochard and de Gennes' blob model for tube geometry fits the data, with a simple modification for chromatin crowding. Continuity of the highly extended, cleaved chromatin is also maintained, consistent with folding and cross bridging of the DNA. Surprisingly, extensional integrity is unaffected by an inhibitor of the DNA repair scaffold. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  16. Modern human sperm freezing: Effect on DNA, chromatin and acrosome integrity.

    Science.gov (United States)

    Rahiminia, Tahereh; Hosseini, Akram; Anvari, Morteza; Ghasemi-Esmailabad, Saeed; Talebi, Ali Reza

    2017-08-01

    Presence of vitrification method in sperm freezing and the introduction of solid surface vitrification beside rapid freezing in vapour, opens an easy and safe way to help infertility centres. While the effects of cryopreservation on motility, morphology and viability of sperm are documented, the question of the probable alteration of sperm DNA, chromatin and acrosome integrity after freezing and thawing procedures in different methods is still controversial. Normal sample were collected according to WHO strict criteria. Sperm suspensions were mixed 1:1 with 0.5 M sucrose and divided into four equal aliquots for freezing: fresh, nitrogen direct immersion vitrification (Vit), solid surface vitrification (SSV) and in vapour (Vapour). Sperm suspensions were transferred into a 0.25 ml sterile plastic. Then straw was inserted inside the 0.5 ml straw. For thawing, the straws were immersed in a 42 °C water bath. Beside the sperm parameters, we assessed the acrosome reaction by double staining, chromatin integrity by toluidine blue (Tb) and chromomycin A3 (CMA3) and DNA integrity by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) respectively. In progressive motility, the highest rate occurred in Vit (39.9 ± 13.3). Moreover, the lowest rate of immotile sperm was in Vit (32.7 ± 16.3). In normal morphology, the group Vit was similar to the fresh, while SSV and Vapour were significantly different from the fresh. The percentage of acrosome-reacted sperms was more in Vit (81.3 ± 10.2) than the fresh group. TUNEL+ results showed that DNA fragmentation was significantly increased in Vit (p-value = 0.025). While in SSV and Vapour results were comparable to fresh. There was a significant correlation between TUNEL+ and normal morphology, TB, CMA3 and presence of intact acrosome. Sperm in Vapour was healthier in terms of DNA, chromatin and acrosome integrity. In contrast of higher motility and normal morphology; DNA, chromatin and acrosome

  17. Elite suppressors harbor low levels of integrated HIV DNA and high levels of 2-LTR circular HIV DNA compared to HIV+ patients on and off HAART.

    Directory of Open Access Journals (Sweden)

    Erin H Graf

    2011-02-01

    Full Text Available Elite suppressors (ES are a rare population of HIV-infected individuals that are capable of naturally controlling the infection without the use of highly active anti-retroviral therapy (HAART. Patients on HAART often achieve viral control to similar (undetectable levels. Accurate and sensitive methods to measure viral burden are needed to elucidate important differences between these two patient populations in order to better understand their mechanisms of control. Viral burden quantification in ES patients has been limited to measurements of total DNA in PBMC, and estimates of Infectious Units per Million cells (IUPM. There appears to be no significant difference in the level of total HIV DNA between cells from ES patients and patients on HAART. However, recovering infectious virus from ES patient samples is much more difficult, suggesting their reservoir size should be much smaller than that in patients on HAART. Here we find that there is a significant difference in the level of integrated HIV DNA in ES patients compared to patients on HAART, providing an explanation for the previous results. When comparing the level of total to integrated HIV DNA in these samples we find ES patients have large excesses of unintegrated HIV DNA. To determine the composition of unintegrated HIV DNA in these samples, we measured circular 2-LTR HIV DNA forms and found ES patients frequently have high levels of 2-LTR circles in PBMC. We further show that these high levels of 2-LTR circles are not the result of inefficient integration in ES cells, since HIV integrates with similar efficiency in ES and normal donor cells. Our findings suggest that measuring integration provides a better surrogate of viral burden than total HIV DNA in ES patients. Moreover, they add significantly to our understanding of the mechanisms that allow viral control and reservoir maintenance in this unique patient population.

  18. Elite suppressors harbor low levels of integrated HIV DNA and high levels of 2-LTR circular HIV DNA compared to HIV+ patients on and off HAART.

    Science.gov (United States)

    Graf, Erin H; Mexas, Angela M; Yu, Jianqing J; Shaheen, Farida; Liszewski, Megan K; Di Mascio, Michele; Migueles, Stephen A; Connors, Mark; O'Doherty, Una

    2011-02-01

    Elite suppressors (ES) are a rare population of HIV-infected individuals that are capable of naturally controlling the infection without the use of highly active anti-retroviral therapy (HAART). Patients on HAART often achieve viral control to similar (undetectable) levels. Accurate and sensitive methods to measure viral burden are needed to elucidate important differences between these two patient populations in order to better understand their mechanisms of control. Viral burden quantification in ES patients has been limited to measurements of total DNA in PBMC, and estimates of Infectious Units per Million cells (IUPM). There appears to be no significant difference in the level of total HIV DNA between cells from ES patients and patients on HAART. However, recovering infectious virus from ES patient samples is much more difficult, suggesting their reservoir size should be much smaller than that in patients on HAART. Here we find that there is a significant difference in the level of integrated HIV DNA in ES patients compared to patients on HAART, providing an explanation for the previous results. When comparing the level of total to integrated HIV DNA in these samples we find ES patients have large excesses of unintegrated HIV DNA. To determine the composition of unintegrated HIV DNA in these samples, we measured circular 2-LTR HIV DNA forms and found ES patients frequently have high levels of 2-LTR circles in PBMC. We further show that these high levels of 2-LTR circles are not the result of inefficient integration in ES cells, since HIV integrates with similar efficiency in ES and normal donor cells. Our findings suggest that measuring integration provides a better surrogate of viral burden than total HIV DNA in ES patients. Moreover, they add significantly to our understanding of the mechanisms that allow viral control and reservoir maintenance in this unique patient population.

  19. Stwl modifies chromatin compaction and is required to maintain DNA integrity in the presence of perturbed DNA replication

    NARCIS (Netherlands)

    Yi, X.; Vries, de H.I.; Siudeja, K.; Rana, A.; Lemstra, W.; Brunsting, J.F.; Kok, R.J.M.; Smulders, Y.M.; Schaefer, M.; Dijk, F.; Shang, Y.F.; Eggen, B.J.L.; Kampinga, H.H.; Sibon, O.C.M.

    2009-01-01

    Hydroxyurea, a well-known DNA replication inhibitor, induces cell cycle arrest and intact checkpoint functions are required to survive DNA replication stress induced by this genotoxic agent. Perturbed DNA synthesis also results in elevated levels of DNA damage. It is unclear how organisms prevent ac

  20. Stwl Modifies Chromatin Compaction and Is Required to Maintain DNA Integrity in the Presence of Perturbed DNA Replication

    NARCIS (Netherlands)

    Yi, Xia; Vries, Hilda I. de; Siudeja, Katarzyna; Rana, Anil; Lemstra, Willy; Brunsting, Jeanette F.; Kok, Rob M.; Smulders, Yvo M.; Schaefer, Matthias; Dijk, Freark; Shang, Yongfeng; Eggen, Bart J.L.; Kampinga, Harm H.; Sibon, Ody C.M.

    2009-01-01

    Hydroxyurea, a well-known DNA replication inhibitor, induces cell cycle arrest and intact checkpoint functions are required to survive DNA replication stress induced by this genotoxic agent. Perturbed DNA synthesis also results in elevated levels of DNA damage. It is unclear how organisms prevent ac

  1. An integrative approach to investigate the respective roles of single-nucleotide variants and copy-number variants in Attention-Deficit/Hyperactivity Disorder.

    Science.gov (United States)

    Lima, Leandro de Araújo; Feio-dos-Santos, Ana Cecília; Belangero, Sintia Iole; Gadelha, Ary; Bressan, Rodrigo Affonseca; Salum, Giovanni Abrahão; Pan, Pedro Mario; Moriyama, Tais Silveira; Graeff-Martins, Ana Soledade; Tamanaha, Ana Carina; Alvarenga, Pedro; Krieger, Fernanda Valle; Fleitlich-Bilyk, Bacy; Jackowski, Andrea Parolin; Brietzke, Elisa; Sato, João Ricardo; Polanczyk, Guilherme Vanoni; Mari, Jair de Jesus; Manfro, Gisele Gus; do Rosário, Maria Conceição; Miguel, Eurípedes Constantino; Puga, Renato David; Tahira, Ana Carolina; Souza, Viviane Neri; Chile, Thais; Gouveia, Gisele Rodrigues; Simões, Sérgio Nery; Chang, Xiao; Pellegrino, Renata; Tian, Lifeng; Glessner, Joseph T; Hashimoto, Ronaldo Fumio; Rohde, Luis Augusto; Sleiman, Patrick M A; Hakonarson, Hakon; Brentani, Helena

    2016-03-07

    Many studies have attempted to investigate the genetic susceptibility of Attention-Deficit/Hyperactivity Disorder (ADHD), but without much success. The present study aimed to analyze both single-nucleotide and copy-number variants contributing to the genetic architecture of ADHD. We generated exome data from 30 Brazilian trios with sporadic ADHD. We also analyzed a Brazilian sample of 503 children/adolescent controls from a High Risk Cohort Study for the Development of Childhood Psychiatric Disorders, and also previously published results of five CNV studies and one GWAS meta-analysis of ADHD involving children/adolescents. The results from the Brazilian trios showed that cases with de novo SNVs tend not to have de novo CNVs and vice-versa. Although the sample size is small, we could also see that various comorbidities are more frequent in cases with only inherited variants. Moreover, using only genes expressed in brain, we constructed two "in silico" protein-protein interaction networks, one with genes from any analysis, and other with genes with hits in two analyses. Topological and functional analyses of genes in this network uncovered genes related to synapse, cell adhesion, glutamatergic and serotoninergic pathways, both confirming findings of previous studies and capturing new genes and genetic variants in these pathways.

  2. Copy number and loss of heterozygosity detected by SNP array of formalin-fixed tissues using whole-genome amplification.

    Science.gov (United States)

    Stokes, Angela; Drozdov, Ignat; Guerra, Eliete; Ouzounis, Christos A; Warnakulasuriya, Saman; Gleeson, Michael J; McGurk, Mark; Tavassoli, Mahvash; Odell, Edward W

    2011-01-01

    The requirement for large amounts of good quality DNA for whole-genome applications prohibits their use for small, laser capture micro-dissected (LCM), and/or rare clinical samples, which are also often formalin-fixed and paraffin-embedded (FFPE). Whole-genome amplification of DNA from these samples could, potentially, overcome these limitations. However, little is known about the artefacts introduced by amplification of FFPE-derived DNA with regard to genotyping, and subsequent copy number and loss of heterozygosity (LOH) analyses. Using a ligation adaptor amplification method, we present data from a total of 22 Affymetrix SNP 6.0 experiments, using matched paired amplified and non-amplified DNA from 10 LCM FFPE normal and dysplastic oral epithelial tissues, and an internal method control. An average of 76.5% of SNPs were called in both matched amplified and non-amplified DNA samples, and concordance was a promising 82.4%. Paired analysis for copy number, LOH, and both combined, showed that copy number changes were reduced in amplified DNA, but were 99.5% concordant when detected, amplifications were the changes most likely to be 'missed', only 30% of non-amplified LOH changes were identified in amplified pairs, and when copy number and LOH are combined ∼50% of gene changes detected in the unamplified DNA were also detected in the amplified DNA and within these changes, 86.5% were concordant for both copy number and LOH status. However, there are also changes introduced as ∼20% of changes in the amplified DNA are not detected in the non-amplified DNA. An integrative network biology approach revealed that changes in amplified DNA of dysplastic oral epithelium localize to topologically critical regions of the human protein-protein interaction network, suggesting their functional implication in the pathobiology of this disease. Taken together, our results support the use of amplification of FFPE-derived DNA, provided sufficient samples are used to increase power

  3. A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome.

    Science.gov (United States)

    Gleave, A P

    1992-12-01

    A versatile gene expression cartridge and binary vector system was constructed for use in Agrobacterium-mediated plant transformation. The expression cartridge of the primary cloning vector, pART7, comprises of cauliflower mosaic virus Cabb B-JI isolate 35S promoter, a multiple cloning site and the transcriptional termination region of the octopine synthase gene. The entire cartridge can be removed from pART7 as a Not I fragment and introduced directly into the binary vector, pART27, recombinants being selected by blue/white screening for beta-galactosidase. pART27 carries the RK2 minimal replicon for maintenance in Agrobacterium, the ColE1 origin of replication for high-copy maintenance in Escherichia coli and the Tn7 spectinomycin/streptomycin resistance gene as a bacterial selectable marker. The organisational structure of the T-DNA of pART27 has been constructed taking into account the right to left border, 5' to 3' model of T-DNA transfer. The T-DNA carries the chimaeric kanamycin resistance gene (nopaline synthase promoter-neomycin phosphotransferase-nopaline synthase terminator) distal to the right border relative to the lacZ' region. Utilisation of these vectors in Agrobacterium-mediated transformation of tobacco demonstrated efficient T-DNA transfer to the plant genome.

  4. Integrity and Quantity of Total Cell-Free DNA in the Diagnosis of Thyroid Cancer: Correlation with Cytological Classification.

    Science.gov (United States)

    Salvianti, Francesca; Giuliani, Corinna; Petrone, Luisa; Mancini, Irene; Vezzosi, Vania; Pupilli, Cinzia; Pinzani, Pamela

    2017-06-24

    Cell-free DNA (cfDNA) quantity and quality in plasma has been investigated as a non-invasive biomarker in cancer. Previous studies have demonstrated increased cfDNA amount and length in different types of cancer with respect to healthy controls. The present study aims to test the hypothesis that the presence of longer DNA strands circulating in plasma can be considered a biomarker for tumor presence in thyroid cancer. We adopted a quantitative real-time PCR (qPCR) approach based on the quantification of two amplicons of different length (67 and 180 bp respectively) to evaluate the integrity index 180/67. Cell-free DNA quantity and integrity were higher in patients affected by nodular thyroid diseases than in healthy controls. Importantly, cfDNA integrity index was higher in patients with cytological diagnosis of thyroid carcinoma (Thy4/Thy5) than in subjects with benign nodules (Thy2). Therefore, cfDNA integrity index 180/67 is a suitable parameter for monitoring cfDNA fragmentation in thyroid cancer patients and a promising circulating biomarker in the diagnosis of thyroid nodules.

  5. High-resolution electrophoretic separation and integrated-waveguide excitation of fluorescent DNA molecules in a lab on a chip

    NARCIS (Netherlands)

    Dongre, Chaitanya; Weerd, van Jasper; Besselink, Geert A.J.; Weeghel, van Rob; Martinez-Vazquez, Rebecca; Osellame, Roberto; Cerullo, Giulio; Cretich, Marina; Chiari, Marcella; Hoekstra, Hugo J.W.M.; Pollnau, Markus

    2010-01-01

    By applying integrated-waveguide laser excitation to an optofluidic chip, fluorescently labeled DNA molecules of 12 or 17 different sizes are separated by CE with high operating speed and low sample consumption of ~600 pL. When detecting the fluorescence signals of migrating DNA molecules with a PMT

  6. Polycomb repressive complex 1 provides a molecular explanation for repeat copy number dependency in FSHD muscular dystrophy.

    Science.gov (United States)

    Casa, Valentina; Runfola, Valeria; Micheloni, Stefano; Aziz, Arif; Dilworth, F Jeffrey; Gabellini, Davide

    2017-02-15

    Repression of repetitive elements is crucial to preserve genome integrity and has been traditionally ascribed to constitutive heterochromatin pathways. FacioScapuloHumeral Muscular Dystrophy (FSHD), one of the most common myopathies, is characterized by a complex interplay of genetic and epigenetic events. The main FSHD form is linked to a reduced copy number of the D4Z4 macrosatellite repeat on 4q35, causing loss of silencing and aberrant expression of the D4Z4-embedded DUX4 gene leading to disease. By an unknown mechanism, D4Z4 copy-number correlates with FSHD phenotype. Here we show that the DUX4 proximal promoter (DUX4p) is sufficient to nucleate the enrichment of both constitutive and facultative heterochromatin components and to mediate a copy-number dependent gene silencing. We found that both the CpG/GC dense DNA content and the repetitive nature of DUX4p arrays are important for their repressive ability. We showed that DUX4p mediates a copy number-dependent Polycomb Repressive Complex 1 (PRC1) recruitment, which is responsible for the copy-number dependent gene repression. Overall, we directly link genetic and epigenetic defects in FSHD by proposing a novel molecular explanation for the copy number-dependency in FSHD pathogenesis, and offer insight into the molecular functions of repeats in chromatin regulation. © The Author 2016. Published by Oxford University Press.

  7. Evaluation of Sperm Quality, Maturation and DNA Integrity in Adult Mice Treated with Sulpiride

    Directory of Open Access Journals (Sweden)

    S Salami

    2012-07-01

    Full Text Available Background: Use of certain antipsychotic drugs has severe effects on fertility in males. Hypothalamus and hypophysial impressions and changes in plasma hormones concentration like prolactin, LH and FSH can affect sperm production. In this study, we investigated the effects of sulpiride on sperm quality, maturation and DNA damage. Methods: Twenty for adult male mice (age: 6-8 weeks were divided into three groups. The treatment group received 40 mg/kg sulpiride solution and the control sham group was given carrier of the drug intraperitoneally (IP daily for 45 days but the control group received nothing. Finally, all the mice were sacrificed by cervical dislocation and their cauda epididymis were removed surgically. The excised specimens were placed in 1 ml HTF medium and incubated for 30 min in CO2 incubator to allow the spermatozoa to swim out. Later, sperm count, motility and viability were analyzed. Additionally, sperm chromatin quality and DNA integrity were assessed by aniline blue and acridine orange staining. Results: Significant decrease in sperm motility and count were observed in the treatment group while the number of abnormal sperm increased as compared with the other two groups. Sperm viability and DNA maturation showed significant reduction and the rate of DNA damage increased in comparison with the control sham and the control groups (P<0.05. Conclusion: The study showed that sulpiride has negative effects on sperm parameters in treated animals and in some cases it could cause secondary infertility.

  8. Integrated Analysis for Identifying Radix Astragali and Its Adulterants Based on DNA Barcoding

    Directory of Open Access Journals (Sweden)

    Sihao Zheng

    2014-01-01

    Full Text Available Radix Astragali is a popular herb used in traditional Chinese medicine for its proimmune and antidiabetic properties. However, methods are needed to help distinguish Radix Astragali from its varied adulterants. DNA barcoding is a widely applicable molecular method used to identify medicinal plants. Yet, its use has been hampered by genetic distance, base variation, and limitations of the bio-NJ tree. Herein, we report the validation of an integrated analysis method for plant species identification using DNA barcoding that focuses on genetic distance, identification efficiency, inter- and intraspecific variation, and barcoding gap. We collected 478 sequences from six candidate DNA barcodes (ITS2, ITS, psbA-trnH, rbcL, matK, and COI from 29 species of Radix Astragali and adulterants. The internal transcribed spacer (ITS sequence was demonstrated as the optimal barcode for identifying Radix Astragali and its adulterants. This new analysis method is helpful in identifying Radix Astragali and expedites the utilization and data mining of DNA barcoding.

  9. Sufficient minimal model for DNA denaturation: Integration of harmonic scalar elasticity and bond energies.

    Science.gov (United States)

    Singh, Amit Raj; Granek, Rony

    2016-10-14

    We study DNA denaturation by integrating elasticity - as described by the Gaussian network model - with bond binding energies, distinguishing between different base pairs and stacking energies. We use exact calculation, within the model, of the Helmholtz free-energy of any partial denaturation state, which implies that the entropy of all formed "bubbles" ("loops") is accounted for. Considering base pair bond removal single events, the bond designated for opening is chosen by minimizing the free-energy difference for the process, over all remaining base pair bonds. Despite of its great simplicity, for several known DNA sequences our results are in accord with available theoretical and experimental studies. Moreover, we report free-energy profiles along the denaturation pathway, which allow to detect stable or meta-stable partial denaturation states, composed of bubble, as local free-energy minima separated by barriers. Our approach allows to study very long DNA strands with commonly available computational power, as we demonstrate for a few random sequences in the range 200-800 base-pairs. For the latter, we also elucidate the self-averaging property of the system. Implications for the well known breathing dynamics of DNA are elucidated.

  10. Sufficient minimal model for DNA denaturation: Integration of harmonic scalar elasticity and bond energies

    Science.gov (United States)

    Singh, Amit Raj; Granek, Rony

    2016-10-01

    We study DNA denaturation by integrating elasticity — as described by the Gaussian network model — with bond binding energies, distinguishing between different base pairs and stacking energies. We use exact calculation, within the model, of the Helmholtz free-energy of any partial denaturation state, which implies that the entropy of all formed "bubbles" ("loops") is accounted for. Considering base pair bond removal single events, the bond designated for opening is chosen by minimizing the free-energy difference for the process, over all remaining base pair bonds. Despite of its great simplicity, for several known DNA sequences our results are in accord with available theoretical and experimental studies. Moreover, we report free-energy profiles along the denaturation pathway, which allow to detect stable or meta-stable partial denaturation states, composed of bubble, as local free-energy minima separated by barriers. Our approach allows to study very long DNA strands with commonly available computational power, as we demonstrate for a few random sequences in the range 200-800 base-pairs. For the latter, we also elucidate the self-averaging property of the system. Implications for the well known breathing dynamics of DNA are elucidated.

  11. Integrative taxonomy at work: DNA barcoding of taeniids harboured by wild and domestic cats.

    Science.gov (United States)

    Galimberti, A; Romano, D F; Genchi, M; Paoloni, D; Vercillo, F; Bizzarri, L; Sassera, D; Bandi, C; Genchi, C; Ragni, B; Casiraghi, M

    2012-05-01

    In modern taxonomy, DNA barcoding is particularly useful where biometric parameters are difficult to determine or useless owing to the poor quality of samples. These situations are frequent in parasitology. Here, we present an integrated study, based on both DNA barcoding and morphological analysis, on cestodes belonging to the genus Taenia, for which biodiversity is still largely underestimated. In particular, we characterized cestodes from Italian wildcats (Felis silvestris silvestris), free-ranging domestic cats (Felis silvestris catus) and hybrids populations. Adult taeniids were collected by post-mortem examinations of the hosts and morphologically identified as Taenia taeniaeformis. We produced cox1 barcode sequences for all the analysed specimens, and we compared them with reference sequences of individuals belonging to the genus Taenia retrieved from GenBank. In order to evaluate the performance of a DNA barcoding approach to discriminate these parasites, the strength of correlation between species identification based on classical morphology and the molecular divergence of cox1 sequences was measured. Our study provides clear evidence that DNA barcoding is highly efficient to reveal the presence of cryptic lineages within already-described taeniid species. Indeed, we detected three well-defined molecular lineages within the whole panel of specimens morphologically identified as T. taeniaeformis. Two of these molecular groups were already identified by other authors and should be ranked at species level. The third molecular group encompasses only samples collected in Italy during this study, and it represents a third candidate species, still morphologically undescribed.

  12. Analysis of DNA sequences by an optical time-integrating correlator.

    Science.gov (United States)

    Brousseau, N; Brousseau, R; Salt, J W; Gutz, L; Tucker, M D

    1992-08-10

    The analysis of the molecular structure called DNA is of particular interest for the understanding of the basic processes governing life. Correlation techniques implemented on digital computers are currently used to do this analysis, but the process is so slow that the mapping and sequencing of the entire human genome requires a computational breakthrough. This paper presents a new method of performing the analysis of DNA sequences with an optical time-integrating correlator. The method is characterized by short processing times that make the analysis of the entire human genome a tractable enterprise. A processing strategy and the resultant processing times are presented. Experimental proofs of concept for the two types of analysis specified by the strategy are also included.

  13. Sensitivity and fidelity of DNA microarray improved with integration of Amplified Differential Gene Expression (ADGE

    Directory of Open Access Journals (Sweden)

    Ile Kristina E

    2003-07-01

    Full Text Available Abstract Background The ADGE technique is a method designed to magnify the ratios of gene expression before detection. It improves the detection sensitivity to small change of gene expression and requires small amount of starting material. However, the throughput of ADGE is low. We integrated ADGE with DNA microarray (ADGE microarray and compared it with regular microarray. Results When ADGE was integrated with DNA microarray, a quantitative relationship of a power function between detected and input ratios was found. Because of ratio magnification, ADGE microarray was better able to detect small changes in gene expression in a drug resistant model cell line system. The PCR amplification of templates and efficient labeling reduced the requirement of starting material to as little as 125 ng of total RNA for one slide hybridization and enhanced the signal intensity. Integration of ratio magnification, template amplification and efficient labeling in ADGE microarray reduced artifacts in microarray data and improved detection fidelity. The results of ADGE microarray were less variable and more reproducible than those of regular microarray. A gene expression profile generated with ADGE microarray characterized the drug resistant phenotype, particularly with reference to glutathione, proliferation and kinase pathways. Conclusion ADGE microarray magnified the ratios of differential gene expression in a power function, improved the detection sensitivity and fidelity and reduced the requirement for starting material while maintaining high throughput. ADGE microarray generated a more informative expression pattern than regular microarray.

  14. Embryonic stem cells or induced pluripotent stem cells? A DNA integrity perspective.

    Science.gov (United States)

    Bai, Qiang; Desprat, Romain; Klein, Bernard; Lemaître, Jean-Marc; De Vos, John

    2013-04-01

    Induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) are two types of pluripotent stem cells that hold great promise for biomedical research and medical applications. iPSCs were initially favorably compared to ESCs. This view was first based on ethical arguments (the generation of iPSCs does not require the destruction of an embryo) and on immunological reasons (it is easier to derive patient HLA-matched iPSCs than ESCs). However, several reports suggest that iPSCs might be characterized by higher occurrence of epigenetic and genetic aberrations than ESCs as a consequence of the reprogramming process. We focus here on the DNA integrity of pluripotent stem cells and examine the three main sources of genomic abnormalities in iPSCs: (1) genomic variety of the parental cells, (2) cell reprogramming, and (3) in vitro cell culture. Recent reports claim that it is possible to generate mouse or human iPSC lines with a mutation level similar to that of the parental cells, suggesting that "genome-friendly" reprogramming techniques can be developed. The issue of iPSC DNA integrity clearly highlights the crucial need of guidelines to define the acceptable level of genomic integrity of pluripotent stem cells for biomedical applications. We discuss here the main issues that such guidelines should address.

  15. Addressing RNA Integrity to Determine the Impact of Mitochondrial DNA Mutations on Brain Mitochondrial Function with Age

    Science.gov (United States)

    Wang, Wei; Scheffler, Katja; Esbensen, Ying; Strand, Janne M.; Stewart, James B.; Bjørås, Magnar; Eide, Lars

    2014-01-01

    Mitochondrial DNA (mtDNA) mutations can result in mitochondrial dysfunction, but emerging experimental data question the fundamental role of mtDNA mutagenesis in age-associated mitochondrial impairment. The multicopy nature of mtDNA renders the impact of a given mtDNA mutation unpredictable. In this study, we compared mtDNA stability and mtRNA integrity during normal aging. Seven distinct sites in mouse brain mtDNA and corresponding mtRNA were analyzed. Accumulation of mtDNA mutations during aging was highly site-specific. The variation in mutation frequencies overrode the age-mediated increase by more than 100-fold and aging generally did not influence mtDNA mutagenesis. Errors introduced by mtRNA polymerase were also site-dependent and up to two hundred-fold more frequent than mtDNA mutations, and independent of mtDNA mutation frequency. We therefore conclude that mitochondrial transcription fidelity limits the impact of mtDNA mutations. PMID:24819950

  16. Prediction of the efficacy of immunotherapy by measuring the integrity of cell-free DNA in plasma in colorectal cancer.

    Science.gov (United States)

    Kitahara, Masahiro; Hazama, Shoichi; Tsunedomi, Ryouichi; Takenouchi, Hiroko; Kanekiyo, Shinsuke; Inoue, Yuka; Nakajima, Masao; Tomochika, Shinobu; Tokuhisa, Yoshihiro; Iida, Michihisa; Sakamoto, Kazuhiko; Suzuki, Nobuaki; Takeda, Shigeru; Ueno, Tomio; Yamamoto, Shigeru; Yoshino, Shigefumi; Nagano, Hiroaki

    2016-12-01

    We previously reported a phase II study of a cancer vaccine using five novel peptides recognized by HLA-A*2402-restricted CTL in combination with oxaliplatin-containing chemotherapy (FXV study) as first-line therapy for patients with metastatic colorectal cancer and demonstrated the safety and promising potential of our five-peptide cocktail. The objective of this analysis was to identify predictive biomarkers for identifying patients who are likely to receive a clinical benefit from immunochemotherapy. Circulating cell-free DNA (cfDNA) in plasma has been reported to be a candidate molecular biomarker for the efficacy of anticancer therapy. Unlike uniformly truncated small-sized DNA released from apoptotic normal cells, DNA released from necrotic cancer cells varies in size. The integrity of plasma cfDNA (i.e. the ratio of longer fragments [400 bp] to shorter fragments [100 bp] of cfDNA), may be clinically useful for detecting colorectal cancer progression. We assessed plasma samples collected from 93 patients prior to receiving immunochemotherapy. The cfDNA levels and integrity were analyzed by semi-quantitative real-time PCR. Progression-free survival was significantly better in patients with a low plasma cfDNA integrity value than in those with a high value (P = 0.0027). Surprisingly, in the HLA-A*2402-matched group, patients with a low plasma cfDNA integrity value had significantly better progression-free survival than those with a high value (P = 0.0015). This difference was not observed in the HLA-A*2402-unmatched group. In conclusion, the integrity of plasma cfDNA may provide important clinical information and may be a useful predictive biomarker of the outcome of immunotherapy in metastatic colorectal cancer.

  17. Flow cytometry application in the assessment of sperm DNA integrity of men with asthenozoospermia.

    Directory of Open Access Journals (Sweden)

    A Brodowska

    2008-04-01

    Full Text Available Sperm genomic integrity and ultrastructural features of ejaculated spermatozoa contributing to the assessment of gamete fertility potential in patients with asthenozoospermia are discussed. The proportion of TUNEL-positive cells was significantly higher in the semen of patients with low sperm motility (n=40; p<0.01 as compared to men with normal sperm motility (n=54. Sperm DNA fragmentation negatively correlated (n=94 with sperm motility, sperm concentration, and integrity of the sperm cellular membrane (HOS-test. Two categories of patients were distinguished: (1 patients (23 out of 94 subjects with < or = 4% of TUNEL-positive cells and (2 patients (71 subjects with 4% of TUNEL-positive cells. A significant difference was noted in the sperm motility and HOS-test results between patients from both groups. Large numbers of immature spermatozoa with extensive cytoplasmic retention, ultrastructural chromatin and midpiece abnormalities, and conglomerates containing sperm fragments were present more frequently in the semen of asthenozoospermic subjects with >4% of TUNEL-positive sperm cells. Low sperm motility seems to be accompanied by serious defects of gamete chromatin expressed as diminished sperm genomic integrity and abnormal DNA condensation and by defects of sperm midpiece. These abnormalities may reflect developmental failure during the spermatogenic remodeling process. The DNA fragmentation test may be considered as an additional assay for the evaluation of spermatozoa beside standard analysis and taken together with electron microscopy may help to determine the actual number of "healthy" spermatozoa thereby playing an important role during diagnosis and treatment of male infertility.

  18. Effects of indium chloride exposure on sperm morphology and DNA integrity in rats

    Directory of Open Access Journals (Sweden)

    Kuo-Hsin Lee

    2015-03-01

    Full Text Available Indium, a Group IIIA element of the periodic chart and a rare earth metal characterized by high plasticity, corrosion resistance, and a low melting point, is widely used in the electronics industry where released streams can contaminate the environment. Consequently, indium can reach humans mainly by natural ways, which could result in a health hazard. Although reproductive toxicities have been surveyed in some studies in animal models, the infertility effects of sperm function induced by indium compounds have not been greatly investigated. We designed a study to investigate the toxicities of subacute exposure to indium compounds on male sperm function and the process of spermatogenesis in a rodent model. Fourteen Sprague-Dawley rats on postnatal Day (PND 84 were randomly divided into exposure and control groups, and weekly received intraperitoneal injections of indium chloride (1.5 mg/kg body weight and normal saline, respectively, for 8 weeks. Cauda epididymal sperm count, motility, morphology, chromatin DNA integrity, mitochondrial membrane potential (MMP, reactive oxygen species (ROS generation, and testis DNA content were investigated. The indium chloride exposed group showed significant toxicity to sperm function, as well as an increased percentage of sperm morphological abnormality and chromatin DNA damage. Furthermore, positive correlations between abnormal sperm morphology, chromatin DNA damage, and superoxide anion generation were also noted. The results of this study demonstrated the toxic effect of subacute low dose indium exposure during sexual maturation on sperm function, resulting in sperm chromatin DNA damage through an increase in sperm ROS generation in a rodent model.

  19. Learning protein-DNA interaction landscapes by integrating experimental data through computational models.

    Science.gov (United States)

    Zhong, Jianling; Wasson, Todd; Hartemink, Alexander J

    2014-10-15

    Transcriptional regulation is directly enacted by the interactions between DNA and many proteins, including transcription factors (TFs), nucleosomes and polymerases. A critical step in deciphering transcriptional regulation is to infer, and eventually predict, the precise locations of these interactions, along with their strength and frequency. While recent datasets yield great insight into these interactions, individual data sources often provide only partial information regarding one aspect of the complete interaction landscape. For example, chromatin immunoprecipitation (ChIP) reveals the binding positions of a protein, but only for one protein at a time. In contrast, nucleases like MNase and DNase can be used to reveal binding positions for many different proteins at once, but cannot easily determine the identities of those proteins. Currently, few statistical frameworks jointly model these different data sources to reveal an accurate, holistic view of the in vivo protein-DNA interaction landscape. Here, we develop a novel statistical framework that integrates different sources of experimental information within a thermodynamic model of competitive binding to jointly learn a holistic view of the in vivo protein-DNA interaction landscape. We show that our framework learns an interaction landscape with increased accuracy, explaining multiple sets of data in accordance with thermodynamic principles of competitive DNA binding. The resulting model of genomic occupancy provides a precise mechanistic vantage point from which to explore the role of protein-DNA interactions in transcriptional regulation. The C source code for compete and Python source code for MCMC-based inference are available at http://www.cs.duke.edu/∼amink. amink@cs.duke.edu Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  20. Measurement of DNA integrity in marine gastropods as biomarker of genotoxicity

    Digital Repository Service at National Institute of Oceanography (India)

    Sarkar, A.; Vashistha, D.; Gupta, N.; Malik, K.; Gaitonde, D.C.S.

    stream_size 15539 stream_content_type text/plain stream_name Env_Pollut_Ecol_impacts_Health_Issu_Mgmt_108a.pdf.txt stream_source_info Env_Pollut_Ecol_impacts_Health_Issu_Mgmt_108a.pdf.txt Content-Encoding UTF-8 Content-Type text.../plain; charset=UTF-8 Author version: Environmental pollution: Ecological impacts, health issues and management. Eds. by: Bhattacharya, B.; Ghosh, A.; Majumdar, S.K.Mudrakar; Kolkata; India; 2011; 108-112 Measurement of DNA Integrity in Marine...

  1. Pleural fluid cell-free DNA integrity index to identify cytologically negative malignant pleural effusions including mesotheliomas

    Directory of Open Access Journals (Sweden)

    Sriram Krishna B

    2012-09-01

    Full Text Available Abstract Background The diagnosis of malignant pleural effusions (MPE is often clinically challenging, especially if the cytology is negative for malignancy. DNA integrity index has been reported to be a marker of malignancy. The aim of this study was to evaluate the utility of pleural fluid DNA integrity index in the diagnosis of MPE. Methods We studied 75 pleural fluid and matched serum samples from consecutive subjects. Pleural fluid and serum ALU DNA repeats [115bp, 247bp and 247bp/115bp ratio (DNA integrity index] were assessed by real-time quantitative PCR. Pleural fluid and serum mesothelin levels were quantified using ELISA. Results Based on clinico-pathological evaluation, 52 subjects had MPE (including 16 mesotheliomas and 23 had benign effusions. Pleural fluid DNA integrity index was higher in MPE compared with benign effusions (1.2 vs. 0.8; p Conclusion Pleural fluid DNA integrity index is a promising diagnostic biomarker for identification of MPEs, including mesothelioma. This biomarker may be particularly useful in cases of MPE where pleural aspirate cytology is negative, and could guide the decision to undertake more invasive definitive testing. A prospective validation study is being undertaken to validate our findings and test the clinical utility of this biomarker for altering clinical practice.

  2. Watermark copy attack

    OpenAIRE

    Kutter, Martin; Voloshynovskyy, Svyatoslav; Herrigel, Alexander

    2000-01-01

    Research in digital watermarking has progressed along two paths. While new watermarking technologies are being developed, some researchers are also investigating di erent ways of attacking digital watermarks. Common attacks to watermarks usually aim to destroy theembedded watermark or to impair its detection. In this paper we propose a conceptually new attack for digitally watermarked images. The proposed attack doesnot destroy anembedded watermark, but copies it from one image to a di erent ...

  3. A Single-Array-Based Method for Detecting Copy Number Variants Using Affymetrix High Density SNP Arrays and its Application to Breast Cancer.

    Science.gov (United States)

    Li, Ming; Wen, Yalu; Fu, Wenjiang

    2014-01-01

    Cumulative evidence has shown that structural variations, due to insertions, deletions, and inversions of DNA, may contribute considerably to the development of complex human diseases, such as breast cancer. High-throughput genotyping technologies, such as Affymetrix high density single-nucleotide polymorphism (SNP) arrays, have produced large amounts of genetic data for genome-wide SNP genotype calling and copy number estimation. Meanwhile, there is a great need for accurate and efficient statistical methods to detect copy number variants. In this article, we introduce a hidden-Markov-model (HMM)-based method, referred to as the PICR-CNV, for copy number inference. The proposed method first estimates copy number abundance for each single SNP on a single array based on the raw fluorescence values, and then standardizes the estimated copy number abundance to achieve equal footing among multiple arrays. This method requires no between-array normalization, and thus, maintains data integrity and independence of samples among individual subjects. In addition to our efforts to apply new statistical technology to raw fluorescence values, the HMM has been applied to the standardized copy number abundance in order to reduce experimental noise. Through simulations, we show our refined method is able to infer copy number variants accurately. Application of the proposed method to a breast cancer dataset helps to identify genomic regions significantly associated with the disease.

  4. DNA replication and spindle checkpoints cooperate during S phase to delay mitosis and preserve genome integrity.

    Science.gov (United States)

    Magiera, Maria M; Gueydon, Elisabeth; Schwob, Etienne

    2014-01-20

    Deoxyribonucleic acid (DNA) replication and chromosome segregation must occur in ordered sequence to maintain genome integrity during cell proliferation. Checkpoint mechanisms delay mitosis when DNA is damaged or upon replication stress, but little is known on the coupling of S and M phases in unperturbed conditions. To address this issue, we postponed replication onset in budding yeast so that DNA synthesis is still underway when cells should enter mitosis. This delayed mitotic entry and progression by transient activation of the S phase, G2/M, and spindle assembly checkpoints. Disabling both Mec1/ATR- and Mad2-dependent controls caused lethality in cells with deferred S phase, accompanied by Rad52 foci and chromosome missegregation. Thus, in contrast to acute replication stress that triggers a sustained Mec1/ATR response, multiple pathways cooperate to restrain mitosis transiently when replication forks progress unhindered. We suggest that these surveillance mechanisms arose when both S and M phases were coincidently set into motion by a unique ancestral cyclin-Cdk1 complex.

  5. The quality of sperm preparation medium affects the motility, viability, and DNA integrity of human spermatozoa

    Science.gov (United States)

    Anbari, Fatemeh; Halvaei, Iman; Nabi, Ali; Ghazali, Shahin; Khalili, Mohammad Ali; Johansson, Lars

    2016-01-01

    AIM: The goal was to compare the effects of three different sperm preparation media on sperm motility, viability, and DNA integrity of semen samples from normozoospermic men. METHODS: A total of 15 normozoospermic males were included in the study. The semen analysis (SA) was performed in accordance with the WHO guidelines (2010). After SA, each sample was divided into three aliquots, and swim-up was performed with three different sperm preparation media (Sperm Preparation Media, Origio, Denmark; Ham's F10, Biochrome, Berlin, Germany; and VitaSperm™, Innovative Biotech, Iran). Sperm motility, viability, and DNA fragmentation were evaluated at 0, 1, 2, and 24 h after swim-up. RESULTS: There were no significant differences, at any time intervals, in the total sperm motility between the different sperm preparation media. However, the rate of progressive motility was significantly higher in spermatozoa prepared using the media from Origio in comparison with VitaSperm™ (P = 0.03), whereas no significant difference was found against Ham's F10 medium. No significant differences in sperm viability were seen between the media products. However, 1 h after swim-up, the extent of sperm DNA fragmentation was lower in the medium from Origio versus VitaSperm™ (P = 0.02). CONCLUSIONS: The data showed that the quality of medium for preparation of semen samples from normozoospermic men significantly affects the performance of spermatozoa in assisted conception programs. PMID:28216914

  6. The quality of sperm preparation medium affects the motility, viability, and DNA integrity of human spermatozoa

    Directory of Open Access Journals (Sweden)

    Fatemeh Anbari

    2016-01-01

    Full Text Available Aim: The goal was to compare the effects of three different sperm preparation media on sperm motility, viability, and DNA integrity of semen samples from normozoospermic men. Methods: A total of 15 normozoospermic males were included in the study. The semen analysis (SA was performed in accordance with the WHO guidelines (2010. After SA, each sample was divided into three aliquots, and swim-up was performed with three different sperm preparation media (Sperm Preparation Media, Origio, Denmark; Ham′s F10, Biochrome, Berlin, Germany; and VitaSperm TM , Innovative Biotech, Iran. Sperm motility, viability, and DNA fragmentation were evaluated at 0, 1, 2, and 24 h after swim-up. Results: There were no significant differences, at any time intervals, in the total sperm motility between the different sperm preparation media. However, the rate of progressive motility was significantly higher in spermatozoa prepared using the media from Origio in comparison with VitaSperm TM (P = 0.03, whereas no significant difference was found against Ham′s F10 medium. No significant differences in sperm viability were seen between the media products. However, 1 h after swim-up, the extent of sperm DNA fragmentation was lower in the medium from Origio versus VitaSperm TM (P = 0.02. Conclusions: The data showed that the quality of medium for preparation of semen samples from normozoospermic men significantly affects the performance of spermatozoa in assisted conception programs.

  7. The quality of sperm preparation medium affects the motility, viability, and DNA integrity of human spermatozoa.

    Science.gov (United States)

    Anbari, Fatemeh; Halvaei, Iman; Nabi, Ali; Ghazali, Shahin; Khalili, Mohammad Ali; Johansson, Lars

    2016-01-01

    The goal was to compare the effects of three different sperm preparation media on sperm motility, viability, and DNA integrity of semen samples from normozoospermic men. A total of 15 normozoospermic males were included in the study. The semen analysis (SA) was performed in accordance with the WHO guidelines (2010). After SA, each sample was divided into three aliquots, and swim-up was performed with three different sperm preparation media (Sperm Preparation Media, Origio, Denmark; Ham's F10, Biochrome, Berlin, Germany; and VitaSperm™, Innovative Biotech, Iran). Sperm motility, viability, and DNA fragmentation were evaluated at 0, 1, 2, and 24 h after swim-up. There were no significant differences, at any time intervals, in the total sperm motility between the different sperm preparation media. However, the rate of progressive motility was significantly higher in spermatozoa prepared using the media from Origio in comparison with VitaSperm™ (P = 0.03), whereas no significant difference was found against Ham's F10 medium. No significant differences in sperm viability were seen between the media products. However, 1 h after swim-up, the extent of sperm DNA fragmentation was lower in the medium from Origio versus VitaSperm™ (P = 0.02). The data showed that the quality of medium for preparation of semen samples from normozoospermic men significantly affects the performance of spermatozoa in assisted conception programs.

  8. Integrated solid-state nanopore platform for nanopore fabrication via dielectric breakdown, DNA-speed deceleration and noise reduction

    Science.gov (United States)

    Goto, Yusuke; Yanagi, Itaru; Matsui, Kazuma; Yokoi, Takahide; Takeda, Ken-Ichi

    2016-08-01

    The practical use of solid-state nanopores for DNA sequencing requires easy fabrication of the nanopores, reduction of the DNA movement speed and reduction of the ionic current noise. Here, we report an integrated nanopore platform with a nanobead structure that decelerates DNA movement and an insulating polyimide layer that reduces noise. To enable rapid nanopore fabrication, we introduced a controlled dielectric breakdown (CDB) process into our system. DNA translocation experiments revealed that single nanopores were created by the CDB process without sacrificing performance in reducing DNA movement speed by up to 10 μs/base or reducing noise up to 600 pArms at 1 MHz. Our platform provides the essential components for proceeding to the next step in the process of DNA sequencing.

  9. Sperm DNA Integrity Assessment: A New Tool in Diagnosis and Treatment of Fertility

    Directory of Open Access Journals (Sweden)

    Mona Bungum

    2012-01-01

    Full Text Available Infertility affects 15% of all couples. Although male infertility factors with reduced semen quality are contributing to about half of all involuntary childlessness, the value of standard semen parameters in prediction of fertility in vivo and choice of proper method for assisted reproduction is limited. In the search for better markers of male fertility, during the last 10 years, assessment of sperm DNA integrity has emerged as a strong new biomarker of semen quality that may have the potential to discriminate between infertile and fertile men. Sperm DNA Fragmentation Index (DFI as assessed by the flow cytometric Sperm Chromatin Structure Assay (SCSA can be used for evaluation of sperm chromatin integrity. The biological background for abnormal DFI is not completely known, but clinical data show that DFI above 30% is associated with very low chance for achieving pregnancy in natural way or by insemination, but not in vitro. Already when the DFI is above 20%, the chance of natural pregnancy may be reduced, despite other sperm parameters being normal. Thus this method may explain a significant proportion of cases of unexplained infertility and can be beneficial in counselling involuntary childless couples need of in vitro fertilisation.

  10. Eclipse period of R1 plasmids during downshift from elevated copy number: Nonrandom selection of copies for replication.

    Science.gov (United States)

    Olsson, Jan A; Berg, Otto; Nordström, Kurt; Dasgupta, Santanu

    2012-03-01

    The classical Meselson-Stahl density-shift method was used to study replication of pOU71, a runaway-replication derivative of plasmid R1 in Escherichia coli. The miniplasmid maintained the normal low copy number of R1 during steady growth at 30°C, but as growth temperatures were raised above 34°C, the copy number of the plasmid increased to higher levels, and at 42°C, it replicated without control in a runaway replication mode with lethal consequences for the host. The eclipse periods (minimum time between successive replication of the same DNA) of the plasmid shortened with rising copy numbers at increasing growth temperatures (Olsson et al., 2003). In this work, eclipse periods were measured during downshifts in copy number of pOU71 after it had replicated at 39 and 42°C, resulting in 7- and 50-fold higher than normal plasmid copy number per cell, respectively. Eclipse periods for plasmid replication, measured during copy number downshift, suggested that plasmid R1, normally selected randomly for replication, showed a bias such that a newly replicated DNA had a higher probability of replication compared to the bulk of the R1 population. However, even the unexpected nonrandom replication followed the copy number kinetics such that every generation, the plasmids underwent the normal inherited number of replication, n, independent of the actual number of plasmid copies in a newborn cell.

  11. Reliable transgene-independent method for determining Sleeping Beauty transposon copy numbers

    Directory of Open Access Journals (Sweden)

    Kolacsek Orsolya

    2011-03-01

    Full Text Available Abstract Background The transposon-based gene delivery technique is emerging as a method of choice for gene therapy. The Sleeping Beauty (SB system has become one of the most favored methods, because of its efficiency and its random integration profile. Copy-number determination of the delivered transgene is a crucial task, but a universal method for measuring this is lacking. In this paper, we show that a real-time quantitative PCR-based, transgene-independent (qPCR-TI method is able to determine SB transposon copy numbers regardless of the genetic cargo. Results We designed a specific PCR assay to amplify the left inverted repeat-direct repeat region of SB, and used it together with the single-copy control gene RPPH1 and a reference genomic DNA of known copy number. The qPCR-TI method allowed rapid and accurate determination of SB transposon copy numbers in various cell types, including human embryonic stem cells. We also found that this sensitive, rapid, highly reproducible and non-radioactive method is just as accurate and reliable as the widely used blotting techniques or the transposon display method. Because the assay is specific for the inverted repeat region of the transposon, it could be used in any system where the SB transposon is the genetic vehicle. Conclusions We have developed a transgene-independent method to determine copy numbers of transgenes delivered by the SB transposon system. The technique is based on a quantitative real-time PCR detection method, offering a sensitive, non-radioactive, rapid and accurate approach, which has a potential to be used for gene therapy.

  12. Confirmed rare copy number variants implicate novel genes in schizophrenia.

    Science.gov (United States)

    Tam, Gloria W C; van de Lagemaat, Louie N; Redon, Richard; Strathdee, Karen E; Croning, Mike D R; Malloy, Mary P; Muir, Walter J; Pickard, Ben S; Deary, Ian J; Blackwood, Douglas H R; Carter, Nigel P; Grant, Seth G N

    2010-04-01

    Understanding how cognitive processes including learning, memory, decision making and ideation are encoded by the genome is a key question in biology. Identification of sets of genes underlying human mental disorders is a path towards this objective. Schizophrenia is a common disease with cognitive symptoms, high heritability and complex genetics. We have identified genes involved with schizophrenia by measuring differences in DNA copy number across the entire genome in 91 schizophrenia cases and 92 controls in the Scottish population. Our data reproduce rare and common variants observed in public domain data from >3000 schizophrenia cases, confirming known disease loci as well as identifying novel loci. We found copy number variants in PDE10A (phosphodiesterase 10A), CYFIP1 [cytoplasmic FMR1 (Fragile X mental retardation 1)-interacting protein 1], K(+) channel genes KCNE1 and KCNE2, the Down's syndrome critical region 1 gene RCAN1 (regulator of calcineurin 1), cell-recognition protein CHL1 (cell adhesion molecule with homology with L1CAM), the transcription factor SP4 (specificity protein 4) and histone deacetylase HDAC9, among others (see http://www.genes2cognition.org/SCZ-CNV). Integrating the function of these many genes into a coherent model of schizophrenia and cognition is a major unanswered challenge.

  13. Circulating nucleic acids damage DNA of healthy cells by integrating into their genomes

    Indian Academy of Sciences (India)

    Indraneel Mittra; Naveen Kumar Khare; Gorantla Venkata Raghuram; Rohan Chaubal; Fatema Khambatti; Deepika Gupta; Ashwini Gaikwad; Preeti Prasannan; Akshita Singh; Aishwarya Iyer; Ankita Singh; Pawan Upadhyay; Naveen Kumar Nair; Pradyumna Kumar Mishra; Amit Dutt

    2015-03-01

    Whether nucleic acids that circulate in blood have any patho-physiological functions in the host have not been explored. We report here that far from being inert molecules, circulating nucleic acids have significant biological activities of their own that are deleterious to healthy cells of the body. Fragmented DNA and chromatin (DNAfs and Cfs) isolated from blood of cancer patients and healthy volunteers are readily taken up by a variety of cells in culture to be localized in their nuclei within a few minutes. The intra-nuclear DNAfs and Cfs associate themselves with host cell chromosomes to evoke a cellular DNA-damage-repair-response (DDR) followed by their incorporation into the host cell genomes. Whole genome sequencing detected the presence of tens of thousands of human sequence reads in the recipient mouse cells. Genomic incorporation of DNAfs and Cfs leads to dsDNA breaks and activation of apoptotic pathways in the treated cells. When injected intravenously into Balb/C mice, DNAfs and Cfs undergo genomic integration into cells of their vital organs resulting in activation of DDR and apoptotic proteins in the recipient cells. Cfs have significantly greater activity than DNAfs with respect to all parameters examined, while both DNAfs and Cfs isolated from cancer patients are more active than those from normal volunteers. All the above pathological actions of DNAfs and Cfs described above can be abrogated by concurrent treatment with DNase I and/or anti-histone antibody complexed nanoparticles both in vitro and in vivo. Taken together, our results that circulating DNAfs and Cfs are physiological, continuously arising, endogenous DNA damaging agents with implications to ageing and a multitude of human pathologies including initiation of cancer.

  14. In vitro analysis of integrated global high-resolution DNA methylation profiling with genomic imbalance and gene expression in osteosarcoma.

    Directory of Open Access Journals (Sweden)

    Bekim Sadikovic

    Full Text Available Genetic and epigenetic changes contribute to deregulation of gene expression and development of human cancer. Changes in DNA methylation are key epigenetic factors regulating gene expression and genomic stability. Recent progress in microarray technologies resulted in developments of high resolution platforms for profiling of genetic, epigenetic and gene expression changes. OS is a pediatric bone tumor with characteristically high level of numerical and structural chromosomal changes. Furthermore, little is known about DNA methylation changes in OS. Our objective was to develop an integrative approach for analysis of high-resolution epigenomic, genomic, and gene expression profiles in order to identify functional epi/genomic differences between OS cell lines and normal human osteoblasts. A combination of Affymetrix Promoter Tilling Arrays for DNA methylation, Agilent array-CGH platform for genomic imbalance and Affymetrix Gene 1.0 platform for gene expression analysis was used. As a result, an integrative high-resolution approach for interrogation of genome-wide tumour-specific changes in DNA methylation was developed. This approach was used to provide the first genomic DNA methylation maps, and to identify and validate genes with aberrant DNA methylation in OS cell lines. This first integrative analysis of global cancer-related changes in DNA methylation, genomic imbalance, and gene expression has provided comprehensive evidence of the cumulative roles of epigenetic and genetic mechanisms in deregulation of gene expression networks.

  15. Robust Adaptable Video Copy Detection

    DEFF Research Database (Denmark)

    Assent, Ira; Kremer, Hardy

    2009-01-01

    Video copy detection should be capable of identifying video copies subject to alterations e.g. in video contrast or frame rates. We propose a video copy detection scheme that allows for adaptable detection of videos that are altered temporally (e.g. frame rate change) and/or visually (e.g. change...... in contrast). Our query processing combines filtering and indexing structures for efficient multistep computation of video copies under this model. We show that our model successfully identifies altered video copies and does so more reliably than existing models....

  16. Robust Adaptable Video Copy Detection

    DEFF Research Database (Denmark)

    Assent, Ira; Kremer, Hardy

    2009-01-01

    Video copy detection should be capable of identifying video copies subject to alterations e.g. in video contrast or frame rates. We propose a video copy detection scheme that allows for adaptable detection of videos that are altered temporally (e.g. frame rate change) and/or visually (e.g. change...... in contrast). Our query processing combines filtering and indexing structures for efficient multistep computation of video copies under this model. We show that our model successfully identifies altered video copies and does so more reliably than existing models....

  17. Critical evaluation of HPV16 gene copy number quantification by SYBR green PCR.

    Science.gov (United States)

    Roberts, Ian; Ng, Grace; Foster, Nicola; Stanley, Margaret; Herdman, Michael T; Pett, Mark R; Teschendorff, Andrew; Coleman, Nicholas

    2008-07-24

    Human papilloma virus (HPV) load and physical status are considered useful parameters for clinical evaluation of cervical squamous cell neoplasia. However, the errors implicit in HPV gene quantification by PCR are not well documented. We have undertaken the first rigorous evaluation of the errors that can be expected when using SYBR green qPCR for quantification of HPV type 16 gene copy numbers. We assessed a modified method, in which external calibration curves were generated from a single construct containing HPV16 E2, HPV16 E6 and the host gene hydroxymethylbilane synthase in a 1:1:1 ratio. When testing dilutions of mixed HPV/host DNA in replicate runs, we observed errors in quantifying E2 and E6 amplicons of 5-40%, with greatest error at the lowest DNA template concentration (3 ng/microl). Errors in determining viral copy numbers per diploid genome were 13-53%. Nevertheless, in cervical keratinocyte cell lines we observed reasonable agreement between viral loads determined by qPCR and Southern blotting. The mean E2/E6 ratio in episome-only cells was 1.04, but with a range of 0.76-1.32. In three integrant-only lines the mean E2/E6 ratios were 0.20, 0.72 and 2.61 (values confirmed by gene-specific Southern blotting). When E2/E6 ratios in fourteen HPV16-positive cervical carcinomas were analysed, conclusions regarding viral physical state could only be made in three cases, where the E2/E6 ratio was unavoidable inaccuracies that should be allowed for when quantifying HPV gene copy number. While E6 copy numbers can be considered to provide a useable indication of viral loads, the E2/E6 ratio is of limited value. Previous studies may have overestimated the frequency of mixed episomal/integrant HPV infections.

  18. CasEMBLR: Cas9-Facilitated Multiloci Genomic Integration of in Vivo Assembled DNA Parts in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Jakociunas, Tadas; Rajkumar, Arun Stephen; Zhang, Jie

    2015-01-01

    , we present a method for marker-free multiloci integration of in vivo assembled DNA parts. By the use of CRISPR/Cas9-mediated one-step double-strand breaks at single, double and triple integration sites we report the successful in vivo assembly and chromosomal integration of DNA parts. We call our...

  19. Insertion mutagenesis of the yeast Candida famata (Debaryomyces hansenii) by random integration of linear DNA fragments.

    Science.gov (United States)

    Dmytruk, Kostyantyn V; Voronovsky, Andriy Y; Sibirny, Andriy A

    2006-09-01

    The feasibility of using random insertional mutagenesis to isolate mutants of the flavinogenic yeast Candida famata was explored. Mutagenesis was performed by transformation of the yeast with an integrative plasmid containing the Saccharomyces cerevisiae LEU2 gene as a selective marker. The addition of restriction enzyme together with the plasmid (restriction enzyme-mediated integration, REMI) increased the transformation frequency only slightly. Integration of the linearized plasmid occurred randomly in the C. famata genome. To investigate the potential of insertional mutagenesis, it was used for tagging genes involved in positive regulation of riboflavin synthesis in C. famata. Partial DNA sequencing of tagged genes showed that they were homologous to the S. cerevisiae genes RIB1, MET2, and SEF1. Intact orthologs of these genes isolated from Debaryomyces hansenii restored the wild phenotype of the corresponding mutants, i.e., the ability to overproduce riboflavin under iron limitation. The Staphylococcus aureus ble gene conferring resistance to phleomycin was used successfully in the study as a dominant selection marker for C. famata. The results obtained indicate that insertional mutagenesis is a powerful tool for tagging genes in C. famata.

  20. CNARA: reliability assessment for genomic copy number profiles.

    Science.gov (United States)

    Ai, Ni; Cai, Haoyang; Solovan, Caius; Baudis, Michael

    2016-10-12

    DNA copy number profiles from microarray and sequencing experiments sometimes contain wave artefacts which may be introduced during sample preparation and cannot be removed completely by existing preprocessing methods. Besides, large derivative log ratio spread (DLRS) of the probes correlating with poor DNA quality is sometimes observed in genome screening experiments and may lead to unreliable copy number profiles. Depending on the extent of these artefacts and the resulting misidentification of copy number alterations/variations (CNA/CNV), it may be desirable to exclude such samples from analyses or to adapt the downstream data analysis strategy accordingly. Here, we propose a method to distinguish reliable genomic copy number profiles from those containing heavy wave artefacts and/or large DLRS. We define four features that adequately summarize the copy number profiles for reliability assessment, and train a classifier on a dataset of 1522 copy number profiles from various microarray platforms. The method can be applied to predict the reliability of copy number profiles irrespective of the underlying microarray platform and may be adapted for those sequencing platforms from which copy number estimates could be computed as a piecewise constant signal. Further details can be found at https://github.com/baudisgroup/CNARA . We have developed a method for the assessment of genomic copy number profiling data, and suggest to apply the method in addition to and after other state-of-the-art noise correction and quality control procedures. CNARA could be instrumental in improving the assessment of data used for genomic data mining experiments and support the reliable functional attribution of copy number aberrations especially in cancer research.

  1. Arabidopsis DNA polymerase lambda mutant is mildly sensitive to DNA double strand breaks but defective in integration ofa transgene.

    Directory of Open Access Journals (Sweden)

    Tomoyuki eFurukawa

    2015-05-01

    Full Text Available The DNA double-strand break (DSB is a critical type of damage, and can be induced by both endogenous sources (e.g. errors of oxidative metabolism, transposable elements, programmed meiotic breaks, or perturbation of the DNA replication fork and exogenous sources (e.g. ionizing radiation or radiomimetic chemicals. Although higher plants, like mammals, are thought to preferentially repair DSBs via nonhomologous end joining (NHEJ, much remains unclear about plant DSB repair pathways. Our reverse genetic approach suggests that DNA polymerase λ is involved in DSB repair in Arabidopsis. The Arabidopsis T-DNA insertion mutant (atpolλ-1 displayed sensitivity to both gamma-irradiation and treatment with radiomimetic reagents, but not to other DNA damaging treatments. The atpolλ-1 mutant showed a moderate sensitivity to DSBs, while Arabidopsis Ku70 and DNA ligase 4 mutants (atku70-3 and atlig4-2, both of which play critical roles in NHEJ, exhibited a hypersensitivity to these treatments. The atpolλ-1/atlig4-2 double mutant exhibited a higher sensitivity to DSBs than each single mutant, but the atku70/atpolλ-1 showed similar sensitivity to the atku70-3 mutant. We showed that transcription of the DNA ligase 1, DNA ligase 6, and Wee1 genes was quickly induced by BLM in several NHEJ deficient mutants in contrast to wild-type. Finally, the T-DNA transformation efficiency dropped in NHEJ deficient mutants and the lowest transformation efficiency was scored in the atpolλ-1/atlig4-2 double mutant. These results imply that AtPolλ is involved in both DSB repair and DNA damage response pathway.

  2. Autoregulation of the dnaA-dnaN operon and effects of DnaA protein levels on replication initiation in Bacillus subtilis.

    Science.gov (United States)

    Ogura, Y; Imai, Y; Ogasawara, N; Moriya, S

    2001-07-01

    In Escherichia coli, the DnaA protein level appears to play a pivotal role in determining the timing of replication initiation. To examine the effects on replication initiation in B. subtilis, we constructed a strain in which a copy of the dnaA gene was integrated at the purA locus on the chromosome under the control of an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoter. However, increasing the DnaA level resulted in cell elongation and inhibition of cell growth by induction of the SOS response. Transcription of the native dnaA-dnaN operon was greatly reduced at high DnaA levels, but it was increased in a dnaA-null mutant, indicating autoregulation of the operon by DnaA. When a copy of the dnaN gene was added downstream of the additional dnaA gene at purA, the cells grew at high DnaA levels, suggesting that depletion of DnaN (beta subunit of DNA polymerase III) within the cell by repression of the native dnaA-dnaN operon at high DnaA levels was the cause of the SOS induction. Flow cytometry of the cells revealed that the cell mass at initiation of replication increased at a lower DnaA level and decreased at DnaA levels higher than those of the wild type. Proper timing of replication initiation was observed at DnaA levels nearly comparable to the wild-type level. These results suggest that if the DnaA level increases with progression of the replication cycle, it could act as a rate-limiting factor of replication initiation in B. subtilis.

  3. Integral parametrization of the Kinetics of Crosslink production in plasmid DNA as a function of 8-methoxypsoralen concentration

    Energy Technology Data Exchange (ETDEWEB)

    Vidania, R. de; Paramio, J. M.; Bauluz, C.

    1986-07-01

    In this paper we present results of crosslink production in pBR322 DNA along a wide range of 8-methoxypsoralen (8-MOP) concentration. Experimental data were obtained as DNA renaturation percentages, from the shift in hyperchromicity after a temperature-dependent denaturation-renaturation process. the experimental results showed a three-stage profile when represented as a function of the natural logarithms of 8-MOP concentration. an integral parametrization which allows a simultaneous fit of the three observed stages is presented here. the theoretical values of crosslink production determined from the fit are useful to asses the genotoxicity of psoralen-induced crosslinks in plasmid DNA. (Author) 24 refs.

  4. An integrated disposable device for DNA extraction and helicase dependent amplification.

    Science.gov (United States)

    Mahalanabis, Madhumita; Do, Jaephil; ALMuayad, Hussam; Zhang, Jane Y; Klapperich, Catherine M

    2010-04-01

    Here we report the demonstration of an integrated microfluidic chip that performs helicase dependent amplification (HDA) on samples containing live bacteria. Combined chip-based sample preparation and isothermal amplification are attractive for world health applications, since the need for instrumentation to control flow rate and temperature changes are reduced or eliminated. Bacteria lysis, nucleic acid extraction, and DNA amplification with a fluorescent reporter are incorporated into a disposable polymer cartridge format. Smart passive fluidic control using a flap valve and a hydrophobic vent (with a nanoporous PTFE membrane) with a simple on-chip mixer eliminates multiple user operations. The device is able to detect as few as ten colony forming units (CFU) of E. coli in growth medium.

  5. A Micro Polymerase Chain Reaction Module for Integrated and Portable DNA Analysis Systems

    Directory of Open Access Journals (Sweden)

    Elisa Morganti

    2011-01-01

    Full Text Available This work deals with the design, fabrication, and thermal characterization of a disposable miniaturized Polymerase Chain Reaction (PCR module that will be integrated in a portable and fast DNA analysis system. It is composed of two independent parts: a silicon substrate with embedded heater and thermometers and a PDMS (PolyDiMethylSiloxane chamber reactor as disposable element; the contact between the two parts is assured by a mechanical clamping obtained using a Plastic Leaded Chip Carrier (PLCC. This PLCC is also useful, avoid the PCR mix evaporation during the thermal cycles. Finite Element Analysis was used to evaluate the thermal requirements of the device. The thermal behaviour of the device was characterized revealing that the temperature can be controlled with a precision of ±0.5°C. Different concentrations of carbon nanopowder were mixed to the PDMS curing agent in order to increase the PDMS thermal conductivity and so the temperature control accuracy.

  6. Control of Genome Integrity by RFC Complexes; Conductors of PCNA Loading onto and Unloading from Chromatin during DNA Replication.

    Science.gov (United States)

    Shiomi, Yasushi; Nishitani, Hideo

    2017-01-26

    During cell division, genome integrity is maintained by faithful DNA replication during S phase, followed by accurate segregation in mitosis. Many DNA metabolic events linked with DNA replication are also regulated throughout the cell cycle. In eukaryotes, the DNA sliding clamp, proliferating cell nuclear antigen (PCNA), acts on chromatin as a processivity factor for DNA polymerases. Since its discovery, many other PCNA binding partners have been identified that function during DNA replication, repair, recombination, chromatin remodeling, cohesion, and proteolysis in cell-cycle progression. PCNA not only recruits the proteins involved in such events, but it also actively controls their function as chromatin assembles. Therefore, control of PCNA-loading onto chromatin is fundamental for various replication-coupled reactions. PCNA is loaded onto chromatin by PCNA-loading replication factor C (RFC) complexes. Both RFC1-RFC and Ctf18-RFC fundamentally function as PCNA loaders. On the other hand, after DNA synthesis, PCNA must be removed from chromatin by Elg1-RFC. Functional defects in RFC complexes lead to chromosomal abnormalities. In this review, we summarize the structural and functional relationships among RFC complexes, and describe how the regulation of PCNA loading/unloading by RFC complexes contributes to maintaining genome integrity.

  7. Control of Genome Integrity by RFC Complexes; Conductors of PCNA Loading onto and Unloading from Chromatin during DNA Replication

    Directory of Open Access Journals (Sweden)

    Yasushi Shiomi

    2017-01-01

    Full Text Available During cell division, genome integrity is maintained by faithful DNA replication during S phase, followed by accurate segregation in mitosis. Many DNA metabolic events linked with DNA replication are also regulated throughout the cell cycle. In eukaryotes, the DNA sliding clamp, proliferating cell nuclear antigen (PCNA, acts on chromatin as a processivity factor for DNA polymerases. Since its discovery, many other PCNA binding partners have been identified that function during DNA replication, repair, recombination, chromatin remodeling, cohesion, and proteolysis in cell-cycle progression. PCNA not only recruits the proteins involved in such events, but it also actively controls their function as chromatin assembles. Therefore, control of PCNA-loading onto chromatin is fundamental for various replication-coupled reactions. PCNA is loaded onto chromatin by PCNA-loading replication factor C (RFC complexes. Both RFC1-RFC and Ctf18-RFC fundamentally function as PCNA loaders. On the other hand, after DNA synthesis, PCNA must be removed from chromatin by Elg1-RFC. Functional defects in RFC complexes lead to chromosomal abnormalities. In this review, we summarize the structural and functional relationships among RFC complexes, and describe how the regulation of PCNA loading/unloading by RFC complexes contributes to maintaining genome integrity.

  8. An integrative approach to species discovery in odonates: from character-based DNA barcoding to ecology.

    Science.gov (United States)

    Damm, Sandra; Schierwater, Bernd; Hadrys, Heike

    2010-09-01

    Modern taxonomy requires an analytical approach incorporating all lines of evidence into decision-making. Such an approach can enhance both species identification and species discovery. The character-based DNA barcode method provides a molecular data set that can be incorporated into classical taxonomic data such that the discovery of new species can be made in an analytical framework that includes multiple sources of data. We here illustrate such a corroborative framework in a dragonfly model system that permits the discovery of two new, but visually cryptic species. In the African dragonfly genus Trithemis three distinct genetic clusters can be detected which could not be identified by using classical taxonomic characters. In order to test the hypothesis of two new species, DNA-barcodes from different sequence markers (ND1 and COI) were combined with morphological, ecological and biogeographic data sets. Phylogenetic analyses and incorporation of all data sets into a scheme called taxonomic circle highly supports the hypothesis of two new species. Our case study suggests an analytical approach to modern taxonomy that integrates data sets from different disciplines, thereby increasing the ease and reliability of both species discovery and species assignment.

  9. An integrative multi-scale analysis of the dynamic DNA methylation landscape in aging.

    Directory of Open Access Journals (Sweden)

    Tian Yuan

    2015-02-01

    Full Text Available Recent studies have demonstrated that the DNA methylome changes with age. This epigenetic drift may have deep implications for cellular differentiation and disease development. However, it remains unclear how much of this drift is functional or caused by underlying changes in cell subtype composition. Moreover, no study has yet comprehensively explored epigenetic drift at different genomic length scales and in relation to regulatory elements. Here we conduct an in-depth analysis of epigenetic drift in blood tissue. We demonstrate that most of the age-associated drift is independent of the increase in the granulocyte to lymphocyte ratio that accompanies aging and that enrichment of age-hypermethylated CpG islands increases upon adjustment for cellular composition. We further find that drift has only a minimal impact on in-cis gene expression, acting primarily to stabilize pre-existing baseline expression levels. By studying epigenetic drift at different genomic length scales, we demonstrate the existence of mega-base scale age-associated hypomethylated blocks, covering approximately 14% of the human genome, and which exhibit preferential hypomethylation in age-matched cancer tissue. Importantly, we demonstrate the feasibility of integrating Illumina 450k DNA methylation with ENCODE data to identify transcription factors with key roles in cellular development and aging. Specifically, we identify REST and regulatory factors of the histone methyltransferase MLL complex, whose function may be disrupted in aging. In summary, most of the epigenetic drift seen in blood is independent of changes in blood cell type composition, and exhibits patterns at different genomic length scales reminiscent of those seen in cancer. Integration of Illumina 450k with appropriate ENCODE data may represent a fruitful approach to identify transcription factors with key roles in aging and disease.

  10. Paternal transmission of mitochondrial DNA as an integral part of mitochondrial inheritance in metapopulations of Drosophila simulans.

    Science.gov (United States)

    Wolff, J N; Nafisinia, M; Sutovsky, P; Ballard, J W O

    2013-01-01

    Maternal inheritance is one of the hallmarks of animal mitochondrial DNA (mtDNA) and central to its success as a molecular marker. This mode of inheritance and subsequent lack of heterologous recombination allows us to retrace evolutionary relationships unambiguously down the matriline and without the confounding effects of recombinant genetic information. Accumulating evidence of biparental inheritance of mtDNA (paternal leakage), however, challenges our current understanding of how this molecule is inherited. Here, using Drosophila simulans collected from an East African metapopulation exhibiting recurring mitochondrial heteroplasmy, we conducted single fly matings and screened F1 offspring for the presence of paternal mtDNA using allele-specific PCR assays (AS-PCR). In all, 27 out of 4092 offspring were identified as harboring paternal mtDNA, suggesting a frequency of 0.66% paternal leakage in this species. Our findings strongly suggest that recurring mtDNA heteroplasmy as observed in natural populations of Drosophila simulans is most likely caused by repeated paternal leakage. Our findings further suggest that this phenomenon to potentially be an integral part of mtDNA inheritance in these populations and consequently of significance for mtDNA as a molecular marker.

  11. Wafer-scale integration of sacrificial nanofluidic chips for detecting and manipulating single DNA molecules

    Science.gov (United States)

    Wang, Chao; Nam, Sung-Wook; Cotte, John M.; Jahnes, Christopher V.; Colgan, Evan G.; Bruce, Robert L.; Brink, Markus; Lofaro, Michael F.; Patel, Jyotica V.; Gignac, Lynne M.; Joseph, Eric A.; Rao, Satyavolu Papa; Stolovitzky, Gustavo; Polonsky, Stanislav; Lin, Qinghuang

    2017-01-01

    Wafer-scale fabrication of complex nanofluidic systems with integrated electronics is essential to realizing ubiquitous, compact, reliable, high-sensitivity and low-cost biomolecular sensors. Here we report a scalable fabrication strategy capable of producing nanofluidic chips with complex designs and down to single-digit nanometre dimensions over 200 mm wafer scale. Compatible with semiconductor industry standard complementary metal-oxide semiconductor logic circuit fabrication processes, this strategy extracts a patterned sacrificial silicon layer through hundreds of millions of nanoscale vent holes on each chip by gas-phase Xenon difluoride etching. Using single-molecule fluorescence imaging, we demonstrate these sacrificial nanofluidic chips can function to controllably and completely stretch lambda DNA in a two-dimensional nanofluidic network comprising channels and pillars. The flexible nanofluidic structure design, wafer-scale fabrication, single-digit nanometre channels, reliable fluidic sealing and low thermal budget make our strategy a potentially universal approach to integrating functional planar nanofluidic systems with logic circuits for lab-on-a-chip applications.

  12. Integrated taxonomy: traditional approach and DNA barcoding for the identification of filarioid worms and related parasites (Nematoda

    Directory of Open Access Journals (Sweden)

    Bandi Claudio

    2009-01-01

    Full Text Available Abstract Background We compared here the suitability and efficacy of traditional morphological approach and DNA barcoding to distinguish filarioid nematodes species (Nematoda, Spirurida. A reliable and rapid taxonomic identification of these parasites is the basis for a correct diagnosis of important and widespread parasitic diseases. The performance of DNA barcoding with different parameters was compared measuring the strength of correlation between morphological and molecular identification approaches. Molecular distance estimation was performed with two different mitochondrial markers (coxI and 12S rDNA and different combinations of data handling were compared in order to provide a stronger tool for easy identification of filarioid worms. Results DNA barcoding and morphology based identification of filarioid nematodes revealed high coherence. Despite both coxI and 12S rDNA allow to reach high-quality performances, only coxI revealed to be manageable. Both alignment algorithm, gaps treatment, and the criteria used to define the threshold value were found to affect the performance of DNA barcoding with 12S rDNA marker. Using coxI and a defined level of nucleotide divergence to delimit species boundaries, DNA barcoding can also be used to infer potential new species. Conclusion An integrated approach allows to reach a higher discrimination power. The results clearly show where DNA-based and morphological identifications are consistent, and where they are not. The coherence between DNA-based and morphological identification for almost all the species examined in our work is very strong. We propose DNA barcoding as a reliable, consistent, and democratic tool for species discrimination in routine identification of parasitic nematodes.

  13. Integration of the Reconfigurable Self-Healing eDNA Architecture in an Embedded System

    DEFF Research Database (Denmark)

    Boesen, Michael Reibel; Keymeulen, Didier; Madsen, Jan

    2011-01-01

    In this work we describe the first real world case study for the self-healing eDNA (electronic DNA) architecture by implementing the control and data processing of a Fourier Transform Spectrometer (FTS) on an eDNA prototype. For this purpose the eDNA prototype has been ported from a Xilinx Virtex...

  14. An integrative analysis of DNA methylation and RNA-Seq data for human heart, kidney and liver

    Directory of Open Access Journals (Sweden)

    Xie Linglin

    2011-12-01

    Full Text Available Abstract Background Many groups, including our own, have proposed the use of DNA methylation profiles as biomarkers for various disease states. While much research has been done identifying DNA methylation signatures in cancer vs. normal etc., we still lack sufficient knowledge of the role that differential methylation plays during normal cellular differentiation and tissue specification. We also need thorough, genome level studies to determine the meaning of methylation of individual CpG dinucleotides in terms of gene expression. Results In this study, we have used (insert statistical method here to compile unique DNA methylation signatures from normal human heart, lung, and kidney using the Illumina Infinium 27 K methylation arraysand compared those to gene expression by RNA sequencing. We have identified unique signatures of global DNA methylation for human heart, kidney and liver, and showed that DNA methylation data can be used to correctly classify various tissues. It indicates that DNA methylation reflects tissue specificity and may play an important role in tissue differentiation. The integrative analysis of methylation and RNA-Seq data showed that gene methylation and its transcriptional levels were comprehensively correlated. The location of methylation markers in terms of distance to transcription start site and CpG island showed no effects on the regulation of gene expression by DNA methylation in normal tissues. Conclusions This study showed that an integrative analysis of methylation array and RNA-Seq data can be utilized to discover the global regulation of gene expression by DNA methylation and suggests that DNA methylation plays an important role in normal tissue differentiation via modulation of gene expression.

  15. When genome integrity and cell cycle decisions collide: roles of polo kinases in cellular adaptation to DNA damage.

    Science.gov (United States)

    Serrano, Diego; D'Amours, Damien

    2014-09-01

    The drive to proliferate and the need to maintain genome integrity are two of the most powerful forces acting on biological systems. When these forces enter in conflict, such as in the case of cells experiencing DNA damage, feedback mechanisms are activated to ensure that cellular proliferation is stopped and no further damage is introduced while cells repair their chromosomal lesions. In this circumstance, the DNA damage response dominates over the biological drive to proliferate, and may even result in programmed cell death if the damage cannot be repaired efficiently. Interestingly, the drive to proliferate can under specific conditions overcome the DNA damage response and lead to a reactivation of the proliferative program in checkpoint-arrested cells. This phenomenon is known as adaptation to DNA damage and is observed in all eukaryotic species where the process has been studied, including normal and cancer cells in humans. Polo-like kinases (PLKs) are critical regulators of the adaptation response to DNA damage and they play key roles at the interface of cell cycle and checkpoint-related decisions in cells. Here, we review recent progress in defining the specific roles of PLKs in the adaptation process and how this conserved family of eukaryotic kinases can integrate the fundamental need to preserve genomic integrity with effective cellular proliferation.

  16. 儿童传染性单核细胞增多症的热程与EB病毒DNA阳性率和拷贝数相关性及临床意义的探讨%Correlations of fever' s periods and positive percents of EBV infection detected by DNA copies, and the value for diagnosis of children Infectious Mononucleosis

    Institute of Scientific and Technical Information of China (English)

    伏洁; 周士新; 崔红; 刘晓红; 张英超; 丁瑛雪; 姜丽娜; 王新宝

    2012-01-01

    Objective To explore correlations of fever' s periods and positive percents of Epstein-Barr virus ( EBV ) infection detected by DNA copies, and the value for diagnosis of children Infectious Mononucleosis (IM). Methods We analyzed retrospectively 32 children IM cases of suffering high fever in our department from May 2011 to May 2012. The days of fever starting were analyzed to observe whether existed a relationship with percents and copies of positive EBV-DNA infections. Results The positive percent of EBV-DNA was 68. 75% (20/32). The number of DNA copies was 1. 39×107/ml ( mean) or 8. 36x 10 /ml ( median) . The time of detection were 6. 6 days ( mean) or 8.0 days (median). The positive percents of EBV-DNA before treatment were 91. 67% for fever day 1 to 4, 61. 54% for fever day 5 to 8 , 33. 3% for fever day 9 to 12, 0 for fever day 13 to 16, respectively. The copies and positive percents of EBV-DNA for fever day 1 to 4 were significantly higher than other groups of fever days ( P< 0. 05). The positive percents of EBV-DNA decreased with a linear pattern when fever' s period prolonged. Correlation coefficient R was 0.99 (P<0.01). Conclusion Detection of EBV-DNA on early fever stage of children (from 1 to 4 days) had a high value for diagnosis of IM. However, this detection for EBV-DNA was less diagnosis value when children fever time was over 9 days.%目的 探讨儿童传染性单核细胞增多症(IM)的热程与EB病毒DNA(EBV-DNA)检测阳性率的相关性及临床诊断意义.方法 回顾性分析2011年5月至2012年5月住院的IM患儿32例,分析其热程与EBV-DNA阳性率和拷贝数的关系.结果 儿童IM治疗前EBV-DNA的阳性率为68.75% (20/32),拷贝数平均为1.39×107/ml(中位数8.36×104/ml),检测时发病时间平均6.6天(中位数8.0天).患儿治疗前EBV-DNA的阳性率分别为1~4天91.67%、5~8天61.54%、9~12天33.33%、13~16天0.热程1~4天EBV-DNA的检出阳性率和拷贝数均高于其

  17. Integrating a DNA Strand Displacement Reaction with a Whispering Gallery Mode Sensor for Label-Free Mercury (II) Ion Detection.

    Science.gov (United States)

    Wu, Fengchi; Wu, Yuqiang; Niu, Zhongwei; Vollmer, Frank

    2016-07-29

    Mercury is an extremely toxic chemical pollutant of our environment. It has attracted the world's attention due to its high mobility and the ease with which it accumulates in organisms. Sensitive devices and methods specific for detecting mercury ions are, hence, in great need. Here, we have integrated a DNA strand displacement reaction with a whispering gallery mode (WGM) sensor for demonstrating the detection of Hg(2+) ions. Our approach relies on the displacement of a DNA hairpin structure, which forms after the binding of mercury ions to an aptamer DNA sequence. The strand displacement reaction of the DNA aptamer provides highly specific and quantitative means for determining the mercury ion concentration on a label-free WGM sensor platform. Our approach also shows the possibility for manipulating the kinetics of a strand displacement reaction with specific ionic species.

  18. 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.

  19. DNA

    Science.gov (United States)

    Stent, Gunther S.

    1970-01-01

    This history for molecular genetics and its explanation of DNA begins with an analysis of the Golden Jubilee essay papers, 1955. The paper ends stating that the higher nervous system is the one major frontier of biological inquiry which still offers some romance of research. (Author/VW)

  20. DNA Delivery and Genomic Integration into Mammalian Target Cells through Type IV A and B Secretion Systems of Human Pathogens

    Directory of Open Access Journals (Sweden)

    Dolores L. Guzmán-Herrador

    2017-08-01

    Full Text Available We explore the potential of bacterial secretion systems as tools for genomic modification of human cells. We previously showed that foreign DNA can be introduced into human cells through the Type IV A secretion system of the human pathogen Bartonella henselae. Moreover, the DNA is delivered covalently attached to the conjugative relaxase TrwC, which promotes its integration into the recipient genome. In this work, we report that this tool can be adapted to other target cells by using different relaxases and secretion systems. The promiscuous relaxase MobA from plasmid RSF1010 can be used to deliver DNA into human cells with higher efficiency than TrwC. MobA also promotes DNA integration, albeit at lower rates than TrwC. Notably, we report that DNA transfer to human cells can also take place through the Type IV secretion system of two intracellular human pathogens, Legionella pneumophila and Coxiella burnetii, which code for a distantly related Dot/Icm Type IV B secretion system. This suggests that DNA transfer could be an intrinsic ability of this family of secretion systems, expanding the range of target human cells. Further analysis of the DNA transfer process showed that recruitment of MobA by Dot/Icm was dependent on the IcmSW chaperone, which may explain the higher DNA transfer rates obtained. Finally, we observed that the presence of MobA negatively affected the intracellular replication of C. burnetii, suggesting an interference with Dot/Icm translocation of virulence factors.

  1. Nonhomologous end joining and homologous recombination DNA repair pathways in integration mutagenesis in the xylose-fermenting yeast Pichia stipitis.

    Science.gov (United States)

    Maassen, Nicole; Freese, Stefan; Schruff, Barbara; Passoth, Volkmar; Klinner, Ulrich

    2008-08-01

    Pichia stipitis integrates linear homologous DNA fragments mainly ectopically. High rates of randomly occurring integration allow tagging mutagenesis with high efficiency using simply PCR amplificates of suitable selection markers from the P. stipitis genome. Linearization of an autonomously replicating vector caused a distinct increase of the transformation efficiency compared with the circular molecule. Cotransformation of a restriction endonuclease further enhanced the transformation efficiency. This effect was also observed with integrative vector DNA. In most cases vector integration in chromosomal targets did not depend on microhomologies, indicating that restriction-enzyme-mediated integration (REMI) does not play an essential role in P. stipitis. Small deletions were observed at the ends of the integrated vectors and in the target sites. Disruption of the PsKU80 gene increased the frequency of homologous integration considerably but resulted in a remarkable decrease of the transformation efficiency. These results suggest that in P. stipitis the nonhomologous end joining (NHEJ) pathway obviously predominates the homologous recombination pathway of double-strand break repair.

  2. The chromatin response to DNA breaks: leaving a mark on genome integrity.

    Science.gov (United States)

    Smeenk, Godelieve; van Attikum, Haico

    2013-01-01

    Genetic, biochemical, and cellular studies have uncovered many of the molecular mechanisms underlying the signaling and repair of chromosomal DNA breaks. However, efficient repair of DNA damage is complicated in that genomic DNA is packaged, through histone and nonhistone proteins, into chromatin. The DNA repair machinery has to overcome this physical barrier to gain access to damaged DNA and repair DNA lesions. Posttranslational modifications of chromatin as well as ATP-dependent chromatin remodeling factors help to overcome this barrier and facilitate access to damaged DNA by altering chromatin structure at sites of DNA damage. Here we review and discuss our current knowledge of and recent advances in chromatin changes induced by chromosome breakage in mammalian cells and their implications for genome stability and human disease.

  3. Integrated Analysis of DNA Methylation and mRNA Expression Profiles to Identify Key Genes in Severe Oligozoospermia

    Directory of Open Access Journals (Sweden)

    Zhiming Li

    2017-05-01

    Full Text Available Severe oligozoospermia (SO is a complex disorder, whose etiology is the combined effect of genetic factors and epigenetic conditions. In this study, we examined DNA methylation and mRNA expression status in a set of testicular tissues of SO patients (n = 3, and compared methylated data with those derived from obstructive azoospermia (OA patients (n = 3 with normal spermatogenesis phenotype. We identified 1,960 differentially methylated CpG sites showing significant alterations in SO vs. OA using the Illumina Infinium HumanMethylation450 bead array. By integrating above DNA methylation data and mRNA expression results, we totally identified 72 methylated CpG sites located in 65 genes with anti-correlation between DNA methylation and mRNA expression. Integrated pathways analysis indicates that these genes are involved in response to hormone stimulus, activation of protein kinase activity, and apoptotic process, among others. We also observed some genes with inversely correlated difference is novel in male infertility field, including PTPRN2, EPHX1, SERPINB9, SLIT3, etc. Our results lay a groundwork for further biological study of SO. Moreover, we generated a workflow for integrated analysis of DNA methylation and mRNA expression, which is expandable to other study types.

  4. Trypanosoma brucei RAP1 maintains telomere and subtelomere integrity by suppressing TERRA and telomeric RNA:DNA hybrids

    Science.gov (United States)

    Nanavaty, Vishal; Sandhu, Ranjodh; Jehi, Sanaa E.; Pandya, Unnati M.

    2017-01-01

    Abstract Trypanosoma brucei causes human African trypanosomiasis and regularly switches its major surface antigen, VSG, thereby evading the host's immune response. VSGs are monoallelically expressed from subtelomeric expression sites (ESs), and VSG switching exploits subtelomere plasticity. However, subtelomere integrity is essential for T. brucei viability. The telomeric transcript, TERRA, was detected in T. brucei previously. We now show that the active ES-adjacent telomere is transcribed. We find that TbRAP1, a telomere protein essential for VSG silencing, suppresses VSG gene conversion-mediated switching. Importantly, TbRAP1 depletion increases the TERRA level, which appears to result from longer read-through into the telomere downstream of the active ES. Depletion of TbRAP1 also results in more telomeric RNA:DNA hybrids and more double strand breaks (DSBs) at telomeres and subtelomeres. In TbRAP1-depleted cells, expression of excessive TbRNaseH1, which cleaves the RNA strand of the RNA:DNA hybrid, brought telomeric RNA:DNA hybrids, telomeric/subtelomeric DSBs and VSG switching frequency back to WT levels. Therefore, TbRAP1-regulated appropriate levels of TERRA and telomeric RNA:DNA hybrid are fundamental to subtelomere/telomere integrity. Our study revealed for the first time an important role of a long, non-coding RNA in antigenic variation and demonstrated a link between telomeric silencing and subtelomere/telomere integrity through TbRAP1-regulated telomere transcription. PMID:28334836

  5. Miniaturized devices towards an integrated lab-on-a-chip platform for DNA diagnostics

    Science.gov (United States)

    Kaprou, G.; Papadakis, G.; Kokkoris, G.; Papadopoulos, V.; Kefala, I.; Papageorgiou, D.; Gizeli, E.; Tserepi, A.

    2015-06-01

    Microfluidics is an emerging technology enabling the development of Lab-on-a-chip (LOC) systems for clinical diagnostics, drug discovery and screening, food safety and environmental analysis. LOC systems integrate and scale down one or several laboratory functions on a single chip of a few mm2 to cm2 in size, and account for many advantages on biochemical analyses, such as low sample and reagent consumption, low cost, reduced analysis time, portability and point-of-need compatibility. Currently, available nucleic acid diagnostic tests take advantage of Polymerase Chain Reaction (PCR) that allows exponential amplification of portions of nucleic acid sequences that can be used as indicators for the identification of various diseases. Here, we present a comparison between static chamber and continuous flow miniaturized PCR devices, in terms of energy consumption for devices fabricated on the same material stack, with identical sample volume and channel dimensions. The comparison is implemented by a computational study coupling heat transfer in both solid and fluid, mass conservation of species, and joule heating. Based on the conclusions of this study, we develop low-cost and fast DNA amplification devices for both PCR and isothermal amplification, and we implement them in the detection of mutations related to breast cancer. The devices are fabricated by mass production amenable technologies on printed circuit board (PCB) substrates, where copper facilitates the incorporation of on-chip microheaters, defining the thermal zones necessary for PCR or isothermal amplification methods.

  6. Human Sperm Cryopreservation: Update on Techniques, Effect on DNA Integrity, and Implications for ART

    Directory of Open Access Journals (Sweden)

    Marlea Di Santo

    2012-01-01

    Full Text Available Cryopreservation of human spermatozoa—introduced in the 1960's—has been recognized as an efficient procedure for management of male fertility before therapy for malignant diseases, vasectomy or surgical infertility treatments, to store donor and partner spermatozoa before assisted reproduction treatments and to ensure the recovery of a small number of spermatozoa in severe male factor infertility. Despite the usefulness of it, cryopreservation may lead to deleterious changes of sperm structure and function: while the effects of cryopreservation on cells are well documented, to date there is no agreement in the literature on whether or not cryopreservation affects sperm chromatin integrity or on the use of a unique and functional protocol for the freezing-thawing procedure. Therefore, sperm cryopreservation is an important component of fertility management and much of its successful application seems to affect the reproductive outcome of assisted reproduction technologies (ART: appropriate use of cryoprotectants before and sperm selection technologies after cryopreservation seem to have the greatest impact on preventing DNA fragmentation, thus improving sperm cryosurvival rates.

  7. An innovative and integrated approach based on DNA walking to identify unauthorised GMOs.

    Science.gov (United States)

    Fraiture, Marie-Alice; Herman, Philippe; Taverniers, Isabel; De Loose, Marc; Deforce, Dieter; Roosens, Nancy H

    2014-03-15

    In the coming years, the frequency of unauthorised genetically modified organisms (GMOs) being present in the European food and feed chain will increase significantly. Therefore, we have developed a strategy to identify unauthorised GMOs containing a pCAMBIA family vector, frequently present in transgenic plants. This integrated approach is performed in two successive steps on Bt rice grains. First, the potential presence of unauthorised GMOs is assessed by the qPCR SYBR®Green technology targeting the terminator 35S pCAMBIA element. Second, its presence is confirmed via the characterisation of the junction between the transgenic cassette and the rice genome. To this end, a DNA walking strategy is applied using a first reverse primer followed by two semi-nested PCR rounds using primers that are each time nested to the previous reverse primer. This approach allows to rapidly identify the transgene flanking region and can easily be implemented by the enforcement laboratories. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

  8. Global analysis of ion dependence unveils hidden steps in DNA binding and bending by integration host factor

    Science.gov (United States)

    Vivas, Paula; Velmurugu, Yogambigai; Kuznetsov, Serguei V.; Rice, Phoebe A.; Ansari, Anjum

    2013-09-01

    Proteins that recognize and bind to specific sites on DNA often distort the DNA at these sites. The rates at which these DNA distortions occur are considered to be important in the ability of these proteins to discriminate between specific and nonspecific sites. These rates have proven difficult to measure for most protein-DNA complexes in part because of the difficulty in separating the kinetics of unimolecular conformational rearrangements (DNA bending and kinking) from the kinetics of bimolecular complex association and dissociation. A notable exception is the Integration Host Factor (IHF), a eubacterial architectural protein involved in chromosomal compaction and DNA recombination, which binds with subnanomolar affinity to specific DNA sites and bends them into sharp U-turns. The unimolecular DNA bending kinetics has been resolved using both stopped-flow and laser temperature-jump perturbation. Here we expand our investigation by presenting a global analysis of the ionic strength dependence of specific binding affinity and relaxation kinetics of an IHF-DNA complex. This analysis enables us to obtain each of the underlying elementary rates (DNA bending/unbending and protein-DNA association/dissociation), and their ionic strength dependence, even under conditions where the two processes are coupled. Our analysis indicates interesting differences in the ionic strength dependence of the bi- versus unimolecular steps. At moderate [KCl] (100-500 mM), nearly all the ionic strength dependence to the overall equilibrium binding affinity appears in the bimolecular association/dissociation of an initial, presumably weakly bent, encounter complex, with a slope SKbi ≈ 8 describing the loglog-dependence of the equilibrium constant to form this complex on [KCl]. In contrast, the unimolecular equilibrium constant to form the fully wrapped specific complex from the initial complex is nearly independent of [KCl], with SKuni water molecules, which is osmotically favored by

  9. Early and late effects of Ibuprofen on mouse sperm parameters, chromatin condensation, and DNA integrity in mice

    Directory of Open Access Journals (Sweden)

    Fatemeh Roodbari

    2015-11-01

    Full Text Available Background: There are few studies indicating the detrimental effects of ibuprofen on sperm fertility potential and DNA integrity. Objective: To determine the effects of Ibuprofen on sperm parameters, chromatin condensation and DNA integrity of mice. Materials and Methods: In this experimental study, 36 adult male mice with average weight 37 gr were divided into three groups, including control (group I, n=12, normal dosage of ibuprofen (group II, n=12 and high dosage (group III, n=12. Ibuprofen with different doses was dissolved in daily water of animals. After 35, 70 and 105 days, the cauda epididymis of mice were cut and incubated in Ham’s F10 media. Sperm samples were analyzed for parameters (motility, morphology and count, DNA integrity (SCD test and chromatin condensation (chromomycin A3 and Aniline blue staining. Results: After 35 days, in addition to above mentioned sperm parameters, all of the treated mice showed statistically significant increase in spermatozoa with immature chromatin (P<0.05. However, after 70 days, the rate of sperm DNA fragmentation assessed by SCD was increased in group II (66.5±0.7 and the percentage of immature spermatozoa (AB+ and CMA3+ was higher in group III (77.5±0.7 and 49.5±6.3 respectively than other groups. After 105 days, the AB+ spermatozoa were increased in both normal dose and high dose groups. Conclusion: Ibuprofen may cause a significant reduction in sperm parameters and sperm chromatin/DNA integrity in mice. It should be noted that these deleterious effects are dose-dependent and can be seen in early and late stage of drug treatments.

  10. Identification of human papillomavirus (HPV) 16 DNA integration and the ensuing patterns of methylation in HPV-associated head and neck squamous cell carcinoma cell lines.

    Science.gov (United States)

    Hatano, Takashi; Sano, Daisuke; Takahashi, Hideaki; Hyakusoku, Hiroshi; Isono, Yasuhiro; Shimada, Shoko; Sawakuma, Kae; Takada, Kentaro; Oikawa, Ritsuko; Watanabe, Yoshiyuki; Yamamoto, Hiroyuki; Itoh, Fumio; Myers, Jeffrey N; Oridate, Nobuhiko

    2017-04-01

    Recent studies showed that human papillomavirus (HPV) integration contributes to the genomic instability seen in HPV-associated head and neck squamous cell carcinoma (HPV-HNSCC). However, the epigenetic alterations induced after HPV integration remains unclear. To identify the molecular details of HPV16 DNA integration and the ensuing patterns of methylation in HNSCC, we performed next-generation sequencing using a target-enrichment method for the effective identification of HPV16 integration breakpoints as well as the characterization of genomic sequences adjacent to HPV16 integration breakpoints with three HPV16-related HNSCC cell lines. The DNA methylation levels of the integrated HPV16 genome and that of the adjacent human genome were also analyzed by bisulfite pyrosequencing. We found various integration loci, including novel integration sites. Integration loci were located predominantly in the intergenic region, with a significant enrichment of the microhomologous sequences between the human and HPV16 genomes at the integration breakpoints. Furthermore, various levels of methylation within both the human genome and the integrated HPV genome at the integration breakpoints in each integrant were observed. Allele-specific methylation analysis suggested that the HPV16 integrants remained hypomethylated when the flanking host genome was hypomethylated. After integration into highly methylated human genome regions, however, the HPV16 DNA became methylated. In conclusion, we found novel integration sites and methylation patterns in HPV-HNSCC using our unique method. These findings may provide insights into understanding of viral integration mechanism and virus-associated carcinogenesis of HPV-HNSCC.

  11. Genome wide copy number analysis of single cells

    Science.gov (United States)

    Baslan, Timour; Kendall, Jude; Rodgers, Linda; Cox, Hilary; Riggs, Mike; Stepansky, Asya; Troge, Jennifer; Ravi, Kandasamy; Esposito, Diane; Lakshmi, B.; Wigler, Michael; Navin, Nicholas; Hicks, James

    2016-01-01

    Summary Copy number variation (CNV) is increasingly recognized as an important contributor to phenotypic variation in health and disease. Most methods for determining CNV rely on admixtures of cells, where information regarding genetic heterogeneity is lost. Here, we present a protocol that allows for the genome wide copy number analysis of single nuclei isolated from mixed populations of cells. Single nucleus sequencing (SNS), combines flow sorting of single nuclei based on DNA content, whole genome amplification (WGA), followed by next generation sequencing to quantize genomic intervals in a genome wide manner. Multiplexing of single cells is discussed. Additionally, we outline informatic approaches that correct for biases inherent in the WGA procedure and allow for accurate determination of copy number profiles. All together, the protocol takes ~3 days from flow cytometry to sequence-ready DNA libraries. PMID:22555242

  12. Mate Choice Copying in Humans.

    Science.gov (United States)

    Waynforth, D

    2007-09-01

    There is substantial evidence that in human mate choice, females directly select males based on male display of both physical and behavioral traits. In non-humans, there is additionally a growing literature on indirect mate choice, such as choice through observing and subsequently copying the mating preferences of conspecifics (mate choice copying). Given that humans are a social species with a high degree of sharing information, long-term pair bonds, and high parental care, it is likely that human females could avoid substantial costs associated with directly searching for information about potential males by mate choice copying. The present study was a test of whether women perceived men to be more attractive when men were presented with a female date or consort than when they were presented alone, and whether the physical attractiveness of the female consort affected women's copying decisions. The results suggested that women's mate choice decision rule is to copy only if a man's female consort is physically attractive. Further analyses implied that copying may be a conditional female mating tactic aimed at solving the problem of informational constraints on assessing male suitability for long-term sexual relationships, and that lack of mate choice experience, measured as reported lifetime number of sex partners, is also an important determinant of copying.

  13. Meta-Analysis of DNA Tumor-Viral Integration Site Selection Indicates a Role for Repeats, Gene Expression and Epigenetics

    Directory of Open Access Journals (Sweden)

    Janet M. Doolittle-Hall

    2015-11-01

    Full Text Available Oncoviruses cause tremendous global cancer burden. For several DNA tumor viruses, human genome integration is consistently associated with cancer development. However, genomic features associated with tumor viral integration are poorly understood. We sought to define genomic determinants for 1897 loci prone to hosting human papillomavirus (HPV, hepatitis B virus (HBV or Merkel cell polyomavirus (MCPyV. These were compared to HIV, whose enzyme-mediated integration is well understood. A comprehensive catalog of integration sites was constructed from the literature and experimentally-determined HPV integration sites. Features were scored in eight categories (genes, expression, open chromatin, histone modifications, methylation, protein binding, chromatin segmentation and repeats and compared to random loci. Random forest models determined loci classification and feature selection. HPV and HBV integrants were not fragile site associated. MCPyV preferred integration near sensory perception genes. Unique signatures of integration-associated predictive genomic features were detected. Importantly, repeats, actively-transcribed regions and histone modifications were common tumor viral integration signatures.

  14. Development of a real-world direct interface for integrated DNA extraction and amplification in a microfluidic device.

    Science.gov (United States)

    Shaw, Kirsty J; Joyce, Domino A; Docker, Peter T; Dyer, Charlotte E; Greenway, Gillian M; Greenman, John; Haswell, Stephen J

    2011-02-07

    Integrated DNA extraction and amplification have been carried out in a microfluidic device using electro-osmotic pumping (EOP) for fluidic control. All the necessary reagents for performing both DNA extraction and polymerase chain reaction (PCR) amplification were pre-loaded into the microfluidic device following encapsulation in agarose gel. Buccal cells were collected using OmniSwabs [Whatman™, UK] and manually added to a chaotropic binding/lysis solution pre-loaded into the microfluidic device. The released DNA was then adsorbed onto a silica monolith contained within the DNA extraction chamber and the microfluidic device sealed using polymer electrodes. The washing and elution steps for DNA extraction were carried out using EOP, resulting in transfer of the eluted DNA into the PCR chamber. Thermal cycling, achieved using a Peltier element, resulted in amplification of the Amelogenin locus as confirmed using conventional capillary gel electrophoresis. It was demonstrated that the PCR reagents could be stored in the microfluidic device for at least 8 weeks at 4 °C with no significant loss of activity. Such methodology lends itself to the production of 'ready-to-use' microfluidic devices containing all the necessary reagents for sample processing, with many obvious applications in forensics and clinical medicine.

  15. Repetitive, Marker-Free, Site-Specific Integration as a Novel Tool for Multiple Chromosomal Integration of DNA

    DEFF Research Database (Denmark)

    Petersen, Kia Vest; Martinussen, Jan; Jensen, Peter Ruhdal

    2013-01-01

    We present a tool for repetitive, marker-free, site-specific integration in Lactococcus lactis, in which a nonreplicating plasmid vector (pKV6) carrying a phage attachment site (attP) can be integrated into a bacterial attachment site (attB). The novelty of the tool described here is the inclusio...

  16. Association between sperm DNA integrity and seminal plasma antioxidant levels in health workers occupationally exposed to ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Dayanidhi; Salian, Sujith Raj; Kalthur, Guruprasad; Uppangala, Shubhashree; Kumari, Sandhya [Division of Clinical Embryology, Department of Obstetrics and Gynecology, Kasturba Medical College, Manipal University, Manipal 576104 (India); Challapalli, Srinivas [Department of Radiotherapy, Kasturba Medical College, Mangalore (India); Chandraguthi, Shrinidhi Gururajarao [Department of Radiotherapy and Oncology, Kasturba Medical College, Manipal (India); Jain, Navya; Krishnamurthy, Hanumanthappa [National Centre for Biological Sciences, Bangalore (India); Kumar, Pratap [Department of Obstetrics and Gynecology, Kasturba Medical College, Manipal University, Manipal (India); Adiga, Satish Kumar, E-mail: satish.adiga@manipal.edu [Division of Clinical Embryology, Department of Obstetrics and Gynecology, Kasturba Medical College, Manipal University, Manipal 576104 (India)

    2014-07-15

    There is a paucity of data regarding the association between occupational radiation exposure and risk to human fertility. Recently, we provided the first evidence on altered sperm functional characteristics, DNA damage and hypermethylation in radiation health workers. However, there is no report elucidating the association between seminal plasma antioxidants and sperm chromatin integrity in occupationally exposed subjects. Here, we assessed the seminal plasma antioxidants and lipid peroxidation level in 83 men who were occupationally exposed to ionizing radiation and then correlated with the sperm chromatin integrity. Flow cytometry based sperm chromatin integrity assay revealed a significant decline in αt value in the exposed group in comparison to the non-exposed group (P<0.0001). Similarly, both total and reduced glutathione levels and total antioxidant capacity in the seminal plasma were significantly higher in exposed group than the non-exposed group (P<0.01, 0.001 and 0.0001, respectively). However, superoxide dismutase level and malondialdehyde level, which is an indicator of lipid peroxidation in the seminal plasma, did not differ significantly between two groups. The total antioxidant capacity (TAC) and GSH level exhibited a positive correlation with sperm DNA integrity in exposed subjects. To conclude, this study distinctly shows that altered sperm chromatin integrity in radiation health workers is associated with increase in seminal plasma antioxidant level. Further, the increased seminal plasma GSH and TAC could be an adaptive measure to tackle the oxidative stress to protect genetic and functional sperm deformities in radiation health workers. - Highlights: • Seminal plasma antioxidants were measured in men occupationally exposed to radiation. • Sperm chromatin integrity was significantly affected in the exposed group. • Glutathione and total antioxidant capacity was significantly higher in exposed group. • Sperm DNA damage in exposed subjects

  17. Photon-induced cell migration and integrin expression promoted by DNA integration of HPV16 genome

    Energy Technology Data Exchange (ETDEWEB)

    Rieken, Stefan; Simon, Florian; Habermehl, Daniel; Dittmar, Jan Oliver; Combs, Stephanie E.; Weber, Klaus; Debus, Juergen; Lindel, Katja [University Hospital of Heidelberg, Department of Radiation Therapy and Radiation Oncology, Heidelberg (Germany)

    2014-10-15

    Persistent human papilloma virus 16 (HPV16) infections are a major cause of cervical cancer. The integration of the viral DNA into the host genome causes E2 gene disruption which prevents apoptosis and increases host cell motility. In cervical cancer patients, survival is limited by local infiltration and systemic dissemination. Surgical control rates are poor in cases of parametrial infiltration. In these patients, radiotherapy (RT) is administered to enhance local control. However, photon irradiation itself has been reported to increase cell motility. In cases of E2-disrupted cervical cancers, this phenomenon would impose an additional risk of enhanced tumor cell motility. Here, we analyze mechanisms underlying photon-increased migration in keratinocytes with differential E2 gene status. Isogenic W12 (intact E2 gene status) and S12 (disrupted E2 gene status) keratinocytes were analyzed in fibronectin-based and serum-stimulated migration experiments following single photon doses of 0, 2, and 10 Gy. Quantitative FACS analyses of integrin expression were performed. Migration and adhesion are increased in E2 gene-disrupted keratinocytes. E2 gene disruption promotes attractability by serum components, therefore, effectuating the risk of local infiltration and systemic dissemination. In S12 cells, migration is further increased by photon RT which leads to enhanced expression of fibronectin receptor integrins. HPV16-associated E2 gene disruption is a main predictor of treatment-refractory cancer virulence. E2 gene disruption promotes cell motility. Following photon RT, E2-disrupted tumors bear the risk of integrin-related infiltration and dissemination. (orig.) [German] Persistierende Infektionen mit humanen Papillomaviren 16 (HPV16) sind ein Hauptausloeser des Zervixkarzinoms. Die Integration der viralen DNS in das Wirtszellgenom fuehrt zum Integritaetsverlust des E2-Gens, wodurch in der Wirtszelle Apoptose verhindert und Motilitaet gesteigert werden. In

  18. Integration of the Reconfigurable Self-Healing eDNA Architecture in an Embedded System

    Science.gov (United States)

    Boesen, Michael Reibel; Keymeulen, Didier; Madsen, Jan; Lu, Thomas; Chao, Tien-Hsin

    2011-01-01

    In this work we describe the first real world case study for the self-healing eDNA (electronic DNA) architecture by implementing the control and data processing of a Fourier Transform Spectrometer (FTS) on an eDNA prototype. For this purpose the eDNA prototype has been ported from a Xilinx Virtex 5 FPGA to an embedded system consisting of a PowerPC and a Xilinx Virtex 5 FPGA. The FTS instrument features a novel liquid crystal waveguide, which consequently eliminates all moving parts from the instrument. The addition of the eDNA architecture to do the control and data processing has resulted in a highly fault-tolerant FTS instrument. The case study has shown that the early stage prototype of the autonomous self-healing eDNA architecture is expensive in terms of execution time.

  19. The effects of pyridaben pesticide on the DNA integrity of sperms and early in vitro embryonic development in mice

    Directory of Open Access Journals (Sweden)

    Ghodrat Ebadi Manas

    2013-01-01

    Full Text Available Background: Pyridaben, a pyridazinone derivative, is a new acaricide and insecticide for control of mites and some insects such as white flies, aphids and thrips. Objective: This study was designed to elucidate how pyridaben can affect the sperms' morphological parameters, its DNA integrity, and to estimate the effect of various quantities of pyridaben on in vitro fertilization rate. Materials and Methods: In this study, 80 adult male Balb/C strain mice were used. Animals were divided into control and two test groups. Control group received distilled water. The test group was divided into two subgroups, viz, high dose (212 mg/kg/day and low dose (53 mg/kg/day and they received the pyridaben, orally for duration of 45 days. The spermatozoa were obtained from caudae epididymides on day 45 in all groups. Sperm viability, protamin compression (nuclear maturity, DNA double-strand breaks, and in vitro fertilizing (IVF ability were examined. Results: The pyridaben treatment provoked a significant decrease in sperm population and viability in epididymides. The data obtained from this experiment revealed that, the pyridaben brings about negative impact on the sperm maturation and DNA integrity in a time-dependent manner, which consequently caused a significant (p<0.05 reduction in IVF capability. Embryo developing arrest was significantly (p<0.05 higher in treated than the control group. Conclusion: Theses results confirmed that, the pyridaben is able to induce DNA damage and chromatin abnormalities in spermatozoa which were evident by low IVF rate.

  20. Analysis on HBV DNA copies in the serum, colostrum and mature milk of women with chronic infection of hepatitis B virus%慢性HBV感染产妇血清、初乳和成熟乳中HBV DNA含量分析及临床意义

    Institute of Scientific and Technical Information of China (English)

    汪雪玲; 易为; 李春梅; 王士俊; 刘雪净; 刘建云; 刘亚楠; 万钢

    2014-01-01

    Objective To investigate the evidence of guiding the breast feeding for women infected by chronic hepatitis B virus (HBV) by detection the load of HBV DNA in the serum, colostrum, and mature milk.Methods Total of 211 women with infection of HBV were classified into the three groups: the negative group (< 5 × 102copies/ml), the minor group (500-9.99 × 105 copies/ml) and the major group (≥1.0 × 106 copies/ml) according to the copies of HBV DNA in serum. The HBV DNA copies in the serum, colostrum and mature milk from the three groups of women were detected by lfuorescence quantitative PCR technique, respectively. And the statistical analysis of the load of HBV DNA were performed between in serum, colostrum and mature milk from the three groups.Results The negative group was 71 cases, the corresponding colostrum and mature milk was negative expression of HBV DNA. The minor group was 75 cases, the positive rates in the corresponding colostrum and mature milk was separately 4% and 0%, respectively. There was statistically significant differences between serum and colostrum (χ2 = 112.55, P< 0.001). However, the positive rate in colostrum was compared with the mature milk without statistical signiifcance (P = 0.24). There were 65 cases in the major group, and the positive rate in the corresponding colostrum and mature milk was separately 75.4% and 15.4%, respectively. The differences between in the serum and the colostrum or between in the colostrum and the mature milk were both with statitstical signiifcance (χ2 = 16.99,P < 0.001;χ2 = 47.2,P < 0.001). When the load of HBV DNA of serum were more than 1.0 × 107 copies/ml, the positive rate of HBV DNA in the mature milk was 24.3% and the load of HBV DNA was low (< 104 copies/ml). The colostrum and the serum or the mature milk and the colostrum were both the positive correlation (r = 0.70,P < 0.001;r = 0.29, P < 0.001).Conclusions HBV DNA copies in the corresponding colostrum and mature milk were both increased when

  1. Fabrication and characterization of high-K dielectric integrated silicon nanowire sensor for DNA sensing application (Conference Presentation)

    Science.gov (United States)

    Jayakumar, Ganesh; Legallais, Maxime; Hellström, Per-Erik; Mouis, Mireille; Stambouli, Valérie; Ternon, Céline; Östling, Mikael

    2016-09-01

    1D silicon nanowires (SiNW) are attractive for charge based DNA sensing applications due to their small size and large surface to volume ratio. An ideal portable biosensor is expected to have repeatable and reliable sensitivity, selectivity, low production cost and small feature size. Instead of using tools such as e-beam that are capital and time intensive, we propose a low cost CMOS self-aligned-double-patterning I-line lithography process to fabricate 60 nm wide SiNW. DNA probes are grafted on a thin dielectric layer that is deposited on top of the SiNW surface. Here we used HfO2 instead of the usual SiO2. Indeed, compared to SiO2, HfO2 has been reported to have higher amount of OH groups on its surface leading to enhanced signal quality. We also report preliminary biosensor characterizations. After HfO2 functionalization and single-stranded DNA probe grafting onto the SiNWs, the sensors were first put in contact with fluorophore labelled complementary DNA targets in order to test the efficiency of DNA hybridization optically. Then, a sequence of hybridization, de-hybridization and re-hybridization steps was followed by Id-Vg measurements in order to measure the electrical response of the sensors to target DNA as well as recycling capability. After each step, SiNW devices exhibited a threshold voltage shift larger than device-to-device dispersion, showing that both complementary DNA hybridization and de-hybridization can be electrically detected. These results are very encouraging as they open new frontiers for heterogeneous integration of liquid interacting array of nano sensors with CMOS circuits to fabricate a complete lab on chip.

  2. Impact of estrogenic compounds on DNA integrity in human spermatozoa: Evidence for cross-linking and redox cycling activities

    Energy Technology Data Exchange (ETDEWEB)

    Bennetts, L.E.; De Iuliis, G.N.; Nixon, B.; Kime, M.; Zelski, K. [ARC Centre of Excellence in Biotechnology and Development and Discipline of Biological Sciences, University of Newcastle, NSW (Australia); McVicar, C.M.; Lewis, S.E. [Obstetrics and Gynaecology, Queen' s University, Belfast (United Kingdom); Aitken, R.J. [ARC Centre of Excellence in Biotechnology and Development and Discipline of Biological Sciences, University of Newcastle, NSW (Australia)], E-mail: jaitken@mail.newcastle.edu.au

    2008-05-10

    A great deal of circumstantial evidence has linked DNA damage in human spermatozoa with adverse reproductive outcomes including reduced fertility and high rates of miscarriage. Although oxidative stress is thought to make a significant contribution to DNA damage in the male germ line, the factors responsible for creating this stress have not been elucidated. One group of compounds that are thought to be active in this context are the estrogens, either generated as a result of the endogenous metabolism of androgens within the male reproductive tract or gaining access to the latter as a consequence of environmental exposure. In this study, a wide variety of estrogenic compounds were assessed for their direct effects on human spermatozoa in vitro. DNA integrity was assessed using the Comet and TUNEL assays, lesion frequencies were quantified by QPCR using targets within the mitochondrial and nuclear ({beta}-globin) genomes, DNA adducts were characterized by mass spectrometry and redox activity was monitored using dihydroethidium (DHE) as the probe. Of the estrogenic and estrogen analogue compounds evaluated, catechol estrogens, quercetin, diethylstilbestrol and pyrocatechol stimulated intense redox activity while genistein was only active at the highest doses tested. Other estrogens and estrogen analogues, such as 17{beta}-estradiol, nonylphenol, bisphenol A and 2,3-dihydroxynaphthalene were inactive. Estrogen-induced redox activity was associated with a dramatic loss of motility and, in the case of 2-hydroxyestradiol, the induction of significant DNA fragmentation. Mass spectrometry also indicated that catechol estrogens were capable of forming dimers that can cross-link the densely packed DNA strands in sperm chromatin, impairing nuclear decondensation. These results highlight the potential importance of estrogenic compounds in creating oxidative stress and DNA damage in the male germ line and suggest that further exploration of these compounds in the aetiology of

  3. Human copy number variation and complex genetic disease.

    Science.gov (United States)

    Girirajan, Santhosh; Campbell, Catarina D; Eichler, Evan E

    2011-01-01

    Copy number variants (CNVs) play an important role in human disease and population diversity. Advancements in technology have allowed for the analysis of CNVs in thousands of individuals with disease in addition to thousands of controls. These studies have identified rare CNVs associated with neuropsychiatric diseases such as autism, schizophrenia, and intellectual disability. In addition, copy number polymorphisms (CNPs) are present at higher frequencies in the population, show high diversity in copy number, sequence, and structure, and have been associated with multiple phenotypes, primarily related to immune or environmental response. However, the landscape of copy number variation still remains largely unexplored, especially for smaller CNVs and those embedded within complex regions of the human genome. An integrated approach including characterization of single nucleotide variants and CNVs in a large number of individuals with disease and normal genomes holds the promise of thoroughly elucidating the genetic basis of human disease and diversity.

  4. 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

  5. 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.

  6. Centrosomes split in the presence of impaired DNA integrity during mitosis

    NARCIS (Netherlands)

    Hut, HMJ; Lemstra, W; Blaauw, EH; van Cappellen, GWA; Kampinga, HH; Sibon, OCM

    A well-established function of centrosomes is their role in accomplishing a successful mitosis that gives rise to a pair of identical daughter cells. We recently showed that DNA replication defects and DNA damage in Drosophila embryos trigger centrosomal changes, but it remained unclear whether

  7. Centrosomes split in the presence of impaired DNA integrity during mitosis

    NARCIS (Netherlands)

    Hut, HMJ; Lemstra, W; Blaauw, EH; van Cappellen, GWA; Kampinga, HH; Sibon, OCM

    2003-01-01

    A well-established function of centrosomes is their role in accomplishing a successful mitosis that gives rise to a pair of identical daughter cells. We recently showed that DNA replication defects and DNA damage in Drosophila embryos trigger centrosomal changes, but it remained unclear whether comp

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

    Science.gov (United States)

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

    2003-08-15

    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 damage checkpoint RFC and the sister chromatid cohesion RFC. As expected from its genetic interactions, elg1 mutants are sensitive to DNA damage. Elg1 is redundant with Rad24 in the DNA damage response and contributes to activation of the checkpoint kinase Rad53. We find that elg1 mutants display DNA replication defects and genome instability, including increased recombination and mutation frequencies, and minichromosome maintenance defects. Mutants in elg1 show genetic interactions with pathways required for processing of stalled replication forks, and are defective in recovery from DNA damage during S phase. We propose that Elg1-RFC functions both in normal DNA replication and in the DNA damage response.

  9. 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 damag

  10. Mycobacterium avium Possesses Extracellular DNA that Contributes to Biofilm Formation, Structural Integrity, and Tolerance to Antibiotics.

    Science.gov (United States)

    Rose, Sasha J; Babrak, Lmar M; Bermudez, Luiz E

    2015-01-01

    Mycobacterium avium subsp. hominissuis is an opportunistic pathogen that is associated with biofilm-related infections of the respiratory tract and is difficult to treat. In recent years, extracellular DNA (eDNA) has been found to be a major component of bacterial biofilms, including many pathogens involved in biofilm-associated infections. To date, eDNA has not been described as a component of mycobacterial biofilms. In this study, we identified and characterized eDNA in a high biofilm-producing strain of Mycobacterium avium subsp. hominissuis (MAH). In addition, we surveyed for presence of eDNA in various MAH strains and other nontuberculous mycobacteria. Biofilms of MAH A5 (high biofilm-producing strain) and MAH 104 (reference strain) were established at 22°C and 37°C on abiotic surfaces. Acellular biofilm matrix and supernatant from MAH A5 7 day-old biofilms both possess abundant eDNA, however very little eDNA was found in MAH 104 biofilms. A survey of MAH clinical isolates and other clinically relevant nontuberculous mycobacterial species revealed many species and strains that also produce eDNA. RAPD analysis demonstrated that eDNA resembles genomic DNA. Treatment with DNase I reduced the biomass of MAH A5 biofilms when added upon biofilm formation or to an already established biofilm both on abiotic surfaces and on top of human pharyngeal epithelial cells. Furthermore, co-treatment of an established biofilm with DNase 1 and either moxifloxacin or clarithromycin significantly increased the susceptibility of the bacteria within the biofilm to these clinically used antimicrobials. Collectively, our results describe an additional matrix component of mycobacterial biofilms and a potential new target to help treat biofilm-associated nontuberculous mycobacterial infections.

  11. Cytonuclear conflict in interpopulation hybrids: the role of RNA polymerase in mtDNA transcription and replication.

    Science.gov (United States)

    Ellison, C K; Burton, R S

    2010-03-01

    Organismal fitness requires functional integration of nuclear and mitochondrial genomes. Structural and regulatory elements coevolve within lineages and several studies have found that interpopulation hybridization disrupts mitonuclear interactions. Because mitochondrial RNA polymerase (mtRPOL) plays key roles in both mitochondrial DNA (mtDNA) replication and transcription, the interaction between mtRPOL and coevolved regulatory sites in the mtDNA may be central to mitonuclear integration. Here, we generate interpopulation hybrids between divergent populations of the copepod Tigriopus californicus to obtain lines having different combinations of mtRPOL and mtDNA. Lines were scored for mtDNA copy number and ATP6 (mtDNA) gene expression. We find that there is a genotype-dependent negative association between mitochondrial transcriptional response and mtDNA copy number. We argue that an observed increase in mtDNA copy number and reduced mtDNA transcription in hybrids reflects the regulatory role of mtRPOL; depending on the mitonuclear genotype, hybridization may disrupt the normal balance between transcription and replication of the mitochondrial genome.

  12. Integrity of the human centromere DNA repeats is protected by CENP-A, CENP-C, and CENP-T.

    Science.gov (United States)

    Giunta, Simona; Funabiki, Hironori

    2017-02-21

    Centromeres are highly specialized chromatin domains that enable chromosome segregation and orchestrate faithful cell division. Human centromeres are composed of tandem arrays of α-satellite DNA, which spans up to several megabases. Little is known about the mechanisms that maintain integrity of the long arrays of α-satellite DNA repeats. Here, we monitored centromeric repeat stability in human cells using chromosome-orientation fluorescent in situ hybridization (CO-FISH). This assay detected aberrant centromeric CO-FISH patterns consistent with sister chromatid exchange at the frequency of 5% in primary tissue culture cells, whereas higher levels were seen in several cancer cell lines and during replicative senescence. To understand the mechanism(s) that maintains centromere integrity, we examined the contribution of the centromere-specific histone variant CENP-A and members of the constitutive centromere-associated network (CCAN), CENP-C, CENP-T, and CENP-W. Depletion of CENP-A and CCAN proteins led to an increase in centromere aberrations, whereas enhancing chromosome missegregation by alternative methods did not, suggesting that CENP-A and CCAN proteins help maintain centromere integrity independently of their role in chromosome segregation. Furthermore, superresolution imaging of centromeric CO-FISH using structured illumination microscopy implied that CENP-A protects α-satellite repeats from extensive rearrangements. Our study points toward the presence of a centromere-specific mechanism that actively maintains α-satellite repeat integrity during human cell proliferation.

  13. Sperm DNA integrity in frozen-thawed semen from Italian Mediterranean Buffalo bulls and its relationship to in vivo fertility.

    Science.gov (United States)

    Serafini, Rosanna; Love, Charles C; Coletta, Angelo; Mari, Gaetano; Mislei, Beatrice; Caso, Chiara; Di Palo, Rossella

    2016-09-01

    The relationship among sperm attributes of DNA integrity, sperm motility, morphology, viability, acrosome integrity and in vivo fertility of frozen-thawed Italian Mediterranean Buffalo (IMB) sperm has not been reported. Straws of frozen-thawed semen samples from three bulls were examined. Sperm DNA assays (i.e., neutral Comet assay, Sperm Bos Halomax-SBH and Sperm Chromatin Structure Assay-SCSA) were not correlated to each other (P>0.05). Many neutral Comet assay measures were correlated to total sperm motility-TMOT (% head-H-DNA, r=0.74; Olive moment, r=-0.76; P<0.05) and coiled tails (r-values ranged from% H-DNA, r=-0.80 to tail length, r=-0.71; P<0.05). The COMP-αt was negatively correlated to viable acrosome intact (VAI) sperm, and distal droplets (r=-0.60 and -0.61; P<0.05), whereas Mean-αt and Mode-αt were positively correlated to bent midpieces (r=0.63 and 0.61; P<0.05). The SBH assay was positively correlated to non-viable acrosome damaged (NVAD) sperm (r=0.60; P<0.05) and negatively correlated to viable acrosome damaged (VAD) sperm (r=-0.63; P<0.05). The overall pregnancy rate (PR-at 30 and 45d post artificial insemination-AI) and the calving rate were 57%, 55% and 45%, respectively. Among sperm features analyzed the area under the Receiver Operating Characteristic (ROC) Curve was significant (P<0.05) for TMOT, NVAD, Standard Deviation-αt (SD-αt) and neutral comet measures (Olive tail moment and tail moment, % H- DNA and tail area) in estimating pregnancy.

  14. Generation of Glyphosate-resistant Transgenic Rice Harboring Single Copy of 2mG2-epsps Gene by Clean DNA Transformation%洁净DNA转化获得2mG2-epsps 基因单拷贝整合的抗草甘膦水稻

    Institute of Scientific and Technical Information of China (English)

    赵艳; 邓春泉; 邓丽蝶

    2014-01-01

    selection with transformation frequency at 17.20%.Southern blot analysis revealed that the 2mG2-epsps gene cassette was all integrated into rice genome in single copy and 52.17%(12/23)transgenic lines can render 12-50 mmol/L glyphosate.The present research provided the foundation for breeding appliance of the glyphosate resistant transgenic rices.

  15. Chromosome Conformation Capture Carbon Copy (5C) in Budding Yeast.

    Science.gov (United States)

    Belton, Jon-Matthew; Dekker, Job

    2015-06-01

    Chromosome conformation capture carbon copy (5C) is a high-throughput method for detecting ligation products of interest in a chromosome conformation capture (3C) library. 5C uses ligation-mediated amplification (LMA) to generate carbon copies of 3C ligation product junctions using single-stranded oligonucleotide probes. This procedure produces a 5C library of short DNA molecules which represent the interactions between the corresponding restriction fragments. The 5C library can be amplified using universal primers containing the Illumina paired-end adaptor sequences for subsequent high-throughput sequencing.

  16. Final Scientific/Technical Report, DE-FG02-06ER64171, Integrated Nucleic Acid System for In-Field Monitoring of Microbial Community Dynamics and Metabolic Activity – Subproject to Co-PI Eric E. Roden

    Energy Technology Data Exchange (ETDEWEB)

    Eric E. Roden

    2009-07-08

    This report summarizes research conducted in conjunction with a project entitled “Integrated Nucleic Acid System for In-Field Monitoring of Microbial Community Dynamics and Metabolic Activity”, which was funded through the Integrative Studies Element of the former NABIR Program (now the Environmental Remediation Sciences Program) within the Office of Biological and Environmental Research. Dr. Darrell Chandler (originally at Argonne National Laboratory, now with Akonni Biosystems) was the overall PI/PD for the project. The overall project goals were to (1) apply a model iron-reducer and sulfate-reducer microarray and instrumentation systems to sediment and groundwater samples from the Scheibe et al. FRC Area 2 field site, UMTRA sediments, and other DOE contaminated sites; (2) continue development and expansion of a 16S rRNA/rDNA¬-targeted probe suite for microbial community dynamics as new sequences are obtained from DOE-relevant sites; and (3) address the fundamental molecular biology and analytical chemistry associated with the extraction, purification and analysis of functional genes and mRNA in environmental samples. Work on the UW subproject focused on conducting detailed batch and semicontinuous culture reactor experiments with uranium-contaminated FRC Area 2 sediment. The reactor experiments were designed to provide coherent geochemical and microbiological data in support of microarray analyses of microbial communities in Area 2 sediments undergoing biostimulation with ethanol. A total of four major experiments were conducted (one batch and three semicontinuous culture), three of which (the batch and two semicontinuous culture) provided samples for DNA microarray analysis. A variety of other molecular analyses (clone libraries, 16S PhyloChip, RT-PCR, and T-RFLP) were conducted on parallel samples from the various experiments in order to provide independent information on microbial community response to biostimulation.

  17. HPV integration detection in CaSki and SiHa using detection of integrated papillomavirus sequences and restriction-site PCR.

    Science.gov (United States)

    Raybould, Rachel; Fiander, Alison; Wilkinson, Gavin W G; Hibbitts, Sam

    2014-09-01

    Human Papillomavirus (HPV) infection is the primary cause of cervical neoplasia. HPV DNA is integrated into the human genome in the majority of cervical cancers. The nature of integration may differ with integration incorporating a single copy of HPV or occurring in concatenated form. Our understanding of HPV tumorigenesis is largely based on studies using characterised cell lines with defined integration sites; these cell lines provide an invaluable standard for validation of diagnostic assays. Cell lines also further understanding of integration mechanisms in clinical samples. The objective of this study was to explore integration assays and to investigate integration events in cell lines where HPV is integrated in concatenated form. Restriction site PCR and detection of integrated papillomavirus sequences were performed on DNA from SiHa and CaSki. A novel integration site on Xq27.3 and HPV genome rearrangements were detected in CaSki DNA. However, where integration was previously detected by FISH in CaSki, and reported to be integrated in concatenated form, integration was not detected by DIPS or RS-PCR. The data presented illustrate that HPV copy number can hinder integration detection; this needs consideration when interpreting results from tests applied to clinical samples.

  18. Zero-Copy Objects System

    Science.gov (United States)

    Burleigh, Scott C.

    2011-01-01

    Zero-Copy Objects System software enables application data to be encapsulated in layers of communication protocol without being copied. Indirect referencing enables application source data, either in memory or in a file, to be encapsulated in place within an unlimited number of protocol headers and/or trailers. Zero-copy objects (ZCOs) are abstract data access representations designed to minimize I/O (input/output) in the encapsulation of application source data within one or more layers of communication protocol structure. They are constructed within the heap space of a Simple Data Recorder (SDR) data store to which all participating layers of the stack must have access. Each ZCO contains general information enabling access to the core source data object (an item of application data), together with (a) a linked list of zero or more specific extents that reference portions of this source data object, and (b) linked lists of protocol header and trailer capsules. The concatenation of the headers (in ascending stack sequence), the source data object extents, and the trailers (in descending stack sequence) constitute the transmitted data object constructed from the ZCO. This scheme enables a source data object to be encapsulated in a succession of protocol layers without ever having to be copied from a buffer at one layer of the protocol stack to an encapsulating buffer at a lower layer of the stack. For large source data objects, the savings in copy time and reduction in memory consumption may be considerable.

  19. Budding Yeast Rif1 Controls Genome Integrity by Inhibiting rDNA Replication.

    Science.gov (United States)

    Shyian, Maksym; Mattarocci, Stefano; Albert, Benjamin; Hafner, Lukas; Lezaja, Aleksandra; Costanzo, Michael; Boone, Charlie; Shore, David

    2016-11-01

    The Rif1 protein is a negative regulator of DNA replication initiation in eukaryotes. Here we show that budding yeast Rif1 inhibits DNA replication initiation at the rDNA locus. Absence of Rif1, or disruption of its interaction with PP1/Glc7 phosphatase, leads to more intensive rDNA replication. The effect of Rif1-Glc7 on rDNA replication is similar to that of the Sir2 deacetylase, and the two would appear to act in the same pathway, since the rif1Δ sir2Δ double mutant shows no further increase in rDNA replication. Loss of Rif1-Glc7 activity is also accompanied by an increase in rDNA repeat instability that again is not additive with the effect of sir2Δ. We find, in addition, that the viability of rif1Δ cells is severely compromised in combination with disruption of the MRX or Ctf4-Mms22 complexes, both of which are implicated in stabilization of stalled replication forks. Significantly, we show that removal of the rDNA replication fork barrier (RFB) protein Fob1, alleviation of replisome pausing by deletion of the Tof1/Csm3 complex, or a large deletion of the rDNA repeat array all rescue this synthetic growth defect of rif1Δ cells lacking in addition either MRX or Ctf4-Mms22 activity. These data suggest that the repression of origin activation by Rif1-Glc7 is important to avoid the deleterious accumulation of stalled replication forks at the rDNA RFB, which become lethal when fork stability is compromised. Finally, we show that Rif1-Glc7, unlike Sir2, has an important effect on origin firing outside of the rDNA locus that serves to prevent activation of the DNA replication checkpoint. Our results thus provide insights into a mechanism of replication control within a large repetitive chromosomal domain and its importance for the maintenance of genome stability. These findings may have important implications for metazoans, where large blocks of repetitive sequences are much more common.

  20. Budding Yeast Rif1 Controls Genome Integrity by Inhibiting rDNA Replication.

    Directory of Open Access Journals (Sweden)

    Maksym Shyian

    2016-11-01

    Full Text Available The Rif1 protein is a negative regulator of DNA replication initiation in eukaryotes. Here we show that budding yeast Rif1 inhibits DNA replication initiation at the rDNA locus. Absence of Rif1, or disruption of its interaction with PP1/Glc7 phosphatase, leads to more intensive rDNA replication. The effect of Rif1-Glc7 on rDNA replication is similar to that of the Sir2 deacetylase, and the two would appear to act in the same pathway, since the rif1Δ sir2Δ double mutant shows no further increase in rDNA replication. Loss of Rif1-Glc7 activity is also accompanied by an increase in rDNA repeat instability that again is not additive with the effect of sir2Δ. We find, in addition, that the viability of rif1Δ cells is severely compromised in combination with disruption of the MRX or Ctf4-Mms22 complexes, both of which are implicated in stabilization of stalled replication forks. Significantly, we show that removal of the rDNA replication fork barrier (RFB protein Fob1, alleviation of replisome pausing by deletion of the Tof1/Csm3 complex, or a large deletion of the rDNA repeat array all rescue this synthetic growth defect of rif1Δ cells lacking in addition either MRX or Ctf4-Mms22 activity. These data suggest that the repression of origin activation by Rif1-Glc7 is important to avoid the deleterious accumulation of stalled replication forks at the rDNA RFB, which become lethal when fork stability is compromised. Finally, we show that Rif1-Glc7, unlike Sir2, has an important effect on origin firing outside of the rDNA locus that serves to prevent activation of the DNA replication checkpoint. Our results thus provide insights into a mechanism of replication control within a large repetitive chromosomal domain and its importance for the maintenance of genome stability. These findings may have important implications for metazoans, where large blocks of repetitive sequences are much more common.

  1. Performance of Molecular Inversion Probes (MIP) in Allele CopyNumber Determination

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yuker; Moorhead, Martin; Karlin-Neumann, George; Wang,Nicolas J.; Ireland, James; Lin, Steven; Chen, Chunnuan; Heiser, LauraM.; Chin, Koei; Esserman, Laura; Gray, Joe W.; Spellman, Paul T.; Faham,Malek

    2007-05-14

    We have developed a new protocol for using MolecularInversion Probes (MIP) to accurately and specifically measure allele copynumber (ACN). The new protocol provides for significant improvementsincluding the reduction of input DNA (from 2?g) by more than 25 fold (to75ng total genomic DNA), higher overall precision resulting in one orderof magnitude lower false positive rate, and greater dynamic range withaccurate absolute copy number up to 60 copies.

  2. Human Papilloma Viral DNA Replicates as a Stable Episome in Cultured Epidermal Keratinocytes

    Science.gov (United States)

    Laporta, Robert F.; Taichman, Lorne B.

    1982-06-01

    Human papilloma virus (HPV) is poorly understood because systems for its growth in tissue culture have not been developed. We report here that cultured human epidermal keratinocytes could be infected with HPV from plantar warts and that the viral DNA persisted and replicated as a stable episome. There were 50-200 copies of viral DNA per cell and there was no evidence to indicate integration of viral DNA into the cellular genome. There was also no evidence to suggest that viral DNA underwent productive replication. We conclude that cultured human epidermal keratinocytes may be a model for the study of certain aspects of HPV biology.

  3. Predicting functionality of protein-DNA interactions by integrating diverse evidence

    DEFF Research Database (Denmark)

    Ucar, Duygu; Beyer, A.; Parthasarathy, S.

    2009-01-01

    Chromatin immunoprecipitation (ChIP-chip) experiments enable capturing physical interactions between regulatory proteins and DNA in vivo. However, measurement of chromatin binding alone is not sufficient to detect regulatory interactions. A detected binding event may not be biologically relevant...

  4. Thermostable and site-specific DNA binding of the gene product ORF56 from the Sulfolobus islandicus plasmid pRN1, a putative archael plasmid copy control protein

    Science.gov (United States)

    Lipps, Georg; Stegert, Mario; Krauss, Gerhard

    2001-01-01

    There is still a lack of information on the specific characteristics of DNA-binding proteins from hyperthermophiles. Here we report on the product of the gene orf56 from plasmid pRN1 of the acidophilic and thermophilic archaeon Sulfolobus islandicus. orf56 has not been characterised yet but low sequence similarily to several eubacterial plasmid-encoded genes suggests that this 6.5 kDa protein is a sequence-specific DNA-binding protein. The DNA-binding properties of ORF56, expressed in Escherichia coli, have been investigated by EMSA experiments and by fluorescence anisotropy measurements. Recombinant ORF56 binds to double-stranded DNA, specifically to an inverted repeat located within the promoter of orf56. Binding to this site could down-regulate transcription of the orf56 gene and also of the overlapping orf904 gene, encoding the putative initiator protein of plasmid replication. By gel filtration and chemical crosslinking we have shown that ORF56 is a dimeric protein. Stoichiometric fluorescence anisotropy titrations further indicate that ORF56 binds as a tetramer to the inverted repeat of its target binding site. CD spectroscopy points to a significant increase in ordered secondary structure of ORF56 upon binding DNA. ORF56 binds without apparent cooperativity to its target DNA with a dissociation constant in the nanomolar range. Quantitative analysis of binding isotherms performed at various salt concentrations and at different temperatures indicates that approximately seven ions are released upon complex formation and that complex formation is accompanied by a change in heat capacity of –6.2 kJ/mol. Furthermore, recombinant ORF56 proved to be highly thermostable and is able to bind DNA up to 85°C. PMID:11160922

  5. Paired quantitative and qualitative assessment of the replication-competent HIV-1 reservoir and comparison with integrated proviral DNA.

    Science.gov (United States)

    Lorenzi, Julio C C; Cohen, Yehuda Z; Cohn, Lillian B; Kreider, Edward F; Barton, John P; Learn, Gerald H; Oliveira, Thiago; Lavine, Christy L; Horwitz, Joshua A; Settler, Allison; Jankovic, Mila; Seaman, Michael S; Chakraborty, Arup K; Hahn, Beatrice H; Caskey, Marina; Nussenzweig, Michel C

    2016-12-06

    HIV-1-infected individuals harbor a latent reservoir of infected CD4(+) T cells that is not eradicated by antiretroviral therapy (ART). This reservoir presents the greatest barrier to an HIV-1 cure and has remained difficult to characterize, in part, because the vast majority of integrated sequences are defective and incapable of reactivation. To characterize the replication-competent reservoir, we have combined two techniques, quantitative viral outgrowth and qualitative sequence analysis of clonal outgrowth viruses. Leukapheresis samples from four fully ART-suppressed, chronically infected individuals were assayed at two time points separated by a 4- to 6-mo interval. Overall, 54% of the viruses emerging from the latent reservoir showed gp160 env sequences that were identical to at least one other virus. Moreover, 43% of the env sequences from viruses emerging from the reservoir were part of identical groups at the two time points. Groups of identical expanded sequences made up 54% of proviral DNA, and, as might be expected, the sequences of replication-competent viruses in the active reservoir showed limited overlap with integrated proviral DNA, most of which is known to represent defective viruses. Finally, there was an inverse correlation between proviral DNA clone size and the probability of reactivation, suggesting that replication-competent viruses are less likely to be found among highly expanded provirus-containing cell clones.

  6. Agrobacterium tumefaciens T-DNA Integration and Gene Targeting in Arabidopsis thaliana Non-Homologous End-Joining Mutants

    Directory of Open Access Journals (Sweden)

    Qi Jia

    2012-01-01

    Full Text Available In order to study the role of AtKu70 and AtKu80 in Agrobacterium-mediated transformation and gene targeting, plant lines with a T-DNA insertion in AtKu80 or AtKu70 genes were functionally characterized. Such plant lines lacked both subunits, indicating that heterodimer formation between AtKu70 and AtKu80 is needed for the stability of the proteins. Homozygous mutants were phenotypically indistinguishable from wild-type plants and were fertile. However, they were hypersensitive to the genotoxic agent bleomycin, resulting in more DSBs as quantified in comet assays. They had lower end-joining efficiency, suggesting that NHEJ is a critical pathway for DSB repair in plants. Both Atku mutants and a previously isolated Atmre11 mutant were impaired in Agrobacterium T-DNA integration via floral dip transformation, indicating that AtKu70, AtKu80, and AtMre11 play an important role in T-DNA integration in Arabidopsis. The frequency of gene targeting was not significantly increased in the Atku80 and Atku70 mutants, but it was increased at least 10-fold in the Atmre11 mutant compared with the wild type.

  7. Evaluation of impairment of DNA integrity in marine gastropods (Cronia contracta) as a biomarker of genotoxic contaminants in coastal water around Goa, west coast of India

    Digital Repository Service at National Institute of Oceanography (India)

    Sarkar, A.; Gaitonde, D.C.S.; Sarkar, Amit; Vashistha, D.; DeSilva, C.; Dalal, S.G.

    was evaluated in terms of the loss of DNA integrity (expressed as the value of 'I') in marine snails with respect to those from the reference site (Palolem) over a period from April 2004 to May 2005 using the technique of alkaline unwinding assay. The DNA...

  8. Direct immobilization of DNA probes on non-modified plastics by UV irradiation and integration in microfluidic devices for rapid bioassay

    DEFF Research Database (Denmark)

    Yi, Sun; Perch-Nielsen, Ivan R.; Dufva, Martin

    2012-01-01

    and parallel identification of Avian Influenza Virus by DNA hybridization. The one-step, cost-effective DNA-linking method on non-modified polymers significantly simplifies microarray fabrication procedures and permits great flexibility to plastic material selection, thus making it convenient to integrate...

  9. Integrating Optical Tweezers, DNA Tightropes, and Single-Molecule Fluorescence Imaging: Pitfalls and Traps.

    Science.gov (United States)

    Wang, J; Barnett, J T; Pollard, M R; Kad, N M

    2017-01-01

    Fluorescence imaging is one of the c