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  1. Integration of DNA Copy Number Alterations and Transcriptional Expression Analysis in Human Gastric Cancer

    PubMed Central

    Coral, Ho; Yuen, Siu Tsan; Chu, Kent Man; Law, Simon; Zhang, Lianhai; Ji, Jiafu; Leung, Suet Yi; Chen, Xin

    2012-01-01

    Background Genomic instability with frequent DNA copy number alterations is one of the key hallmarks of carcinogenesis. The chromosomal regions with frequent DNA copy number gain and loss in human gastric cancer are still poorly defined. It remains unknown how the DNA copy number variations contributes to the changes of gene expression profiles, especially on the global level. Principal Findings We analyzed DNA copy number alterations in 64 human gastric cancer samples and 8 gastric cancer cell lines using bacterial artificial chromosome (BAC) arrays based comparative genomic hybridization (aCGH). Statistical analysis was applied to correlate previously published gene expression data obtained from cDNA microarrays with corresponding DNA copy number variation data to identify candidate oncogenes and tumor suppressor genes. We found that gastric cancer samples showed recurrent DNA copy number variations, including gains at 5p, 8q, 20p, 20q, and losses at 4q, 9p, 18q, 21q. The most frequent regions of amplification were 20q12 (7/72), 20q12–20q13.1 (12/72), 20q13.1–20q13.2 (11/72) and 20q13.2–20q13.3 (6/72). The most frequent deleted region was 9p21 (8/72). Correlating gene expression array data with aCGH identified 321 candidate oncogenes, which were overexpressed and showed frequent DNA copy number gains; and 12 candidate tumor suppressor genes which were down-regulated and showed frequent DNA copy number losses in human gastric cancers. Three networks of significantly expressed genes in gastric cancer samples were identified by ingenuity pathway analysis. Conclusions This study provides insight into DNA copy number variations and their contribution to altered gene expression profiles during human gastric cancer development. It provides novel candidate driver oncogenes or tumor suppressor genes for human gastric cancer, useful pathway maps for the future understanding of the molecular pathogenesis of this malignancy, and the construction of new therapeutic

  2. Integrative analysis of DNA copy number, DNA methylation and gene expression in multiple myeloma reveals alterations related to relapse

    PubMed Central

    Krzeminski, Patryk; Corchete, Luis A.; García, Juan L.; López-Corral, Lucía; Fermiñán, Encarna; García, Eva M.; Martín, Ana A.; Hernández-Rivas, Jesús M.; García-Sanz, Ramón; Miguel, Jesús F. San; Gutiérrez, Norma C.

    2016-01-01

    Multiple myeloma (MM) remains incurable despite the introduction of novel agents, and a relapsing course is observed in most patients. Although the development of genomic technologies has greatly improved our understanding of MM pathogenesis, the mechanisms underlying relapse have been less thoroughly investigated. In this study, an integrative analysis of DNA copy number, DNA methylation and gene expression was conducted in matched diagnosis and relapse samples from MM patients. Overall, the acquisition of abnormalities at relapse was much more frequent than the loss of lesions present at diagnosis, and DNA losses were significantly more frequent in relapse than in diagnosis samples. Interestingly, copy number abnormalities involving more than 100 Mb of DNA at relapse significantly affect the gene expression of these samples, provoking a particular deregulation of the IL-8 pathway. On the other hand, no significant modifications of gene expression were observed in those samples with less than 100 Mb affected by chromosomal changes. Although several statistical approaches were used to identify genes whose abnormal expression at relapse was regulated by methylation, only two genes that were significantly deregulated in relapse samples (SORL1 and GLT1D1) showed a negative correlation between methylation and expression. Further analysis revealed that DNA methylation was involved in regulating SORL1 expression in MM. Finally, relevant changes in gene expression observed in relapse samples, such us downregulation of CD27 and P2RY8, were most likely not preceded by alterations in the corresponding DNA. Taken together, these results suggest that the genomic heterogeneity described at diagnosis remains at relapse. PMID:27811368

  3. Integrative analysis of DNA copy number, DNA methylation and gene expression in multiple myeloma reveals alterations related to relapse.

    PubMed

    Krzeminski, Patryk; Corchete, Luis A; García, Juan L; López-Corral, Lucía; Fermiñán, Encarna; García, Eva M; Martín, Ana A; Hernández-Rivas, Jesús M; García-Sanz, Ramón; San Miguel, Jesús F; Gutiérrez, Norma C

    2016-12-06

    Multiple myeloma (MM) remains incurable despite the introduction of novel agents, and a relapsing course is observed in most patients. Although the development of genomic technologies has greatly improved our understanding of MM pathogenesis, the mechanisms underlying relapse have been less thoroughly investigated. In this study, an integrative analysis of DNA copy number, DNA methylation and gene expression was conducted in matched diagnosis and relapse samples from MM patients. Overall, the acquisition of abnormalities at relapse was much more frequent than the loss of lesions present at diagnosis, and DNA losses were significantly more frequent in relapse than in diagnosis samples. Interestingly, copy number abnormalities involving more than 100 Mb of DNA at relapse significantly affect the gene expression of these samples, provoking a particular deregulation of the IL-8 pathway. On the other hand, no significant modifications of gene expression were observed in those samples with less than 100 Mb affected by chromosomal changes. Although several statistical approaches were used to identify genes whose abnormal expression at relapse was regulated by methylation, only two genes that were significantly deregulated in relapse samples (SORL1 and GLT1D1) showed a negative correlation between methylation and expression. Further analysis revealed that DNA methylation was involved in regulating SORL1 expression in MM. Finally, relevant changes in gene expression observed in relapse samples, such us downregulation of CD27 and P2RY8, were most likely not preceded by alterations in the corresponding DNA. Taken together, these results suggest that the genomic heterogeneity described at diagnosis remains at relapse.

  4. High copy and stable expression of the xylanase XynHB in Saccharomyces cerevisiae by rDNA-mediated integration.

    PubMed

    Fang, Cheng; Wang, Qinhong; Selvaraj, Jonathan Nimal; Zhou, Yuling; Ma, Lixin; Zhang, Guimin; Ma, Yanhe

    2017-08-18

    Xylanase is a widely-used additive in baking industry for enhancing dough and bread quality. Several xylanases used in baking industry were expressed in different systems, but their expression in antibiotic free vector system is highly essential and safe. In the present study, an alternative rDNA-mediated technology was developed to increase the copy number of target gene by integrating it into Saccharomyces cerevisiae genome. A xylanase-encoding gene xynHB from Bacillus sp. was cloned into pHBM367H and integrated into S. cerevisiae genome through rDNA-mediated recombination. Exogenous XynHB expressed by recombinant S. cerevisiae strain A13 exhibited higher degradation activity towards xylan than other transformants. The real-time PCR analysis on A13 genome revealed the presence of 13.64 copies of xynHB gene. Though no antibiotics have been used, the genetic stability and the xylanase activity of xynHB remained stable up to 1,011 generations of cultivation. S. cerevisiae strain A13 expressing xylanase reduced the required kneading time and increased the height and diameter of the dough size, which would be safe and effective in baking industry as no antibiotics-resistance risk. The new effective rDNA-mediated technology without using antibiotics here provides a way to clone other food related industrial enzymes for applications.

  5. Hacking DNA copy number for circuit engineering.

    PubMed

    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.

  6. Integrated DNA methylation and copy-number profiling identify three clinically and biologically relevant groups of anaplastic glioma.

    PubMed

    Wiestler, Benedikt; Capper, David; Sill, Martin; Jones, David T W; Hovestadt, Volker; Sturm, Dominik; Koelsche, Christian; Bertoni, Anna; Schweizer, Leonille; Korshunov, Andrey; Weiß, Elisa K; Schliesser, Maximilian G; Radbruch, Alexander; Herold-Mende, Christel; Roth, Patrick; Unterberg, Andreas; Hartmann, Christian; Pietsch, Torsten; Reifenberger, Guido; Lichter, Peter; Radlwimmer, Bernhard; Platten, Michael; Pfister, Stefan M; von Deimling, Andreas; Weller, Michael; Wick, Wolfgang

    2014-10-01

    The outcome of patients with anaplastic gliomas varies considerably. Whether a molecular classification of anaplastic gliomas based on large-scale genomic or epigenomic analyses is superior to histopathology for reflecting distinct biological groups, predicting outcomes and guiding therapy decisions has yet to be determined. Epigenome-wide DNA methylation analysis, using a platform which also allows the detection of copy-number aberrations, was performed in a cohort of 228 patients with anaplastic gliomas (astrocytomas, oligoastrocytomas, and oligodendrogliomas), including 115 patients of the NOA-04 trial. We further compared these tumors with a group of 55 glioblastomas. Unsupervised clustering of DNA methylation patterns revealed two main groups correlated with IDH status: CpG island methylator phenotype (CIMP) positive (77.5 %) or negative (22.5 %). CIMP(pos) (IDH mutant) tumors showed a further separation based on copy-number status of chromosome arms 1p and 19q. CIMP(neg) (IDH wild type) tumors showed hallmark copy-number alterations of glioblastomas, and clustered together with CIMP(neg) glioblastomas without forming separate groups based on WHO grade. Notably, there was no molecular evidence for a distinct biological entity representing anaplastic oligoastrocytoma. Tumor classification based on CIMP and 1p/19q status was significantly associated with survival, allowing a better prediction of outcome than the current histopathological classification: patients with CIMP(pos) tumors with 1p/19q codeletion (CIMP-codel) had the best prognosis, followed by patients with CIMP(pos) tumors but intact 1p/19q status (CIMP-non-codel). Patients with CIMP(neg) anaplastic gliomas (GBM-like) had the worst prognosis. Collectively, our data suggest that anaplastic gliomas can be grouped by IDH and 1p/19q status into three molecular groups that show clear links to underlying biology and a significant association with clinical outcome in a prospective trial cohort.

  7. Single copy DNA homology in sea stars.

    PubMed

    Smith, M J; Nicholson, R; Stuerzl, M; Lui, A

    1982-01-01

    The sequence homology in the single copy DNA of sea stars has been measured. Labeled single copy DNA from Pisaster ochraceus was reannealed with excess genomic DNA from P. brevispinus, Evasterias troschelii, Pycnopodia helianthoides, Solaster stimpsoni, and Dermasterias imbricata. Reassociation reactions were performed under two criteria of salt and temperature. The extent of reassociation and thermal denaturation characteristics of hybrid single copy DNA molecules follow classical taxonomic lines. P. brevispinus DNA contains essentially all of the sequences present in P. ochraceus single copy tracer while Evasterias and Pycnopodia DNAs contain 52% and 46% of such sequences respectively. Reciprocal reassociation reactions with labeled Evasterias single copy DNA confirm the amount and fidelity of the sequence homology. There is a small definite reaction of uncertain homology between P. ochraceus single copy DNA and Solaster or Dermasterias DNA. Similarly Solaster DNA contains sequences homologous to approximately 18% of Dermasterias unique DNA. The thermal denaturation temperatures of heteroduplexes indicate that the genera Pisaster and Evasterias diverged shortly after the divergence of the subfamilies Pycnopodiinae and Asteriinae. The two Pisaster species diverged more recently, probably in the most recent quarter of the interval since the separation of the genera Pisaster and Evasterias.

  8. DNA replication stress restricts ribosomal DNA copy number

    PubMed Central

    Salim, Devika; Bradford, William D.; Freeland, Amy; Cady, Gillian; Wang, Jianmin

    2017-01-01

    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 a 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. PMID:28915237

  9. DNA replication stress restricts ribosomal DNA copy number.

    PubMed

    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.

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

    PubMed Central

    2010-01-01

    Background 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. Results 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 < 1%, 530 transcripts (461 genes) demonstrated a correlation between CNA and 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). Conclusions 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. PMID:20537188

  11. Integrated immunohistochemical and DNA copy number profiling analysis provides insight into the molecular pathogenesis of canine follicular lymphoma.

    PubMed

    Thomas, R; Demeter, Z; Kennedy, K A; Borst, L; Singh, K; Valli, V E; Le Boedec, K; Breen, M

    2017-09-01

    Follicular lymphomas (FLs) typically exhibit a chromosome translocation that induces constitutive expression of the anti-apoptotic bcl2 protein and accumulation of additional molecular defects. This rearrangement offers a promising therapeutic target, but its nature as a fundamental driver of FL pathogenesis remains unclear as 15% of cases lack the translocation. We performed an integrated immunohistochemical and genomic investigation of 10 naturally occurring FL cases from domestic dogs, showing that, as with human tumours, they exhibit marked heterogeneity in the frequency and intensity of bcl2 protein expression. Genomic copy number aberrations were infrequent and broadly consistent with those of other canine B-cell lymphoma subtypes. None of the canine FL specimens exhibited a rearrangement consistent with the hallmark translocation of human FL, despite their remarkable histomorphologic similarity. Parallel exploration of canine and human cases may reveal alternative tumour-initiating mechanisms other than BCL2 disruption, yielding a more complete definition of the molecular pathogenesis of FL. © 2016 John Wiley & Sons Ltd.

  12. Identification of Novel Breast Cancer Subtype-Specific Biomarkers by Integrating Genomics Analysis of DNA Copy Number Aberrations and miRNA-mRNA Dual Expression Profiling.

    PubMed

    Li, Dongguo; Xia, Hong; Li, Zhen-ya; Hua, Lin; Li, Lin

    2015-01-01

    Breast cancer is a heterogeneous disease with well-defined molecular subtypes. Currently, comparative genomic hybridization arrays (aCGH) techniques have been developed rapidly, and recent evidences in studies of breast cancer suggest that tumors within gene expression subtypes share similar DNA copy number aberrations (CNA) which can be used to further subdivide subtypes. Moreover, subtype-specific miRNA expression profiles are also proposed as novel signatures for breast cancer classification. The identification of mRNA or miRNA expression-based breast cancer subtypes is considered an instructive means of prognosis. Here, we conducted an integrated analysis based on copy number aberrations data and miRNA-mRNA dual expression profiling data to identify breast cancer subtype-specific biomarkers. Interestingly, we found a group of genes residing in subtype-specific CNA regions that also display the corresponding changes in mRNAs levels and their target miRNAs' expression. Among them, the predicted direct correlation of BRCA1-miR-143-miR-145 pairs was selected for experimental validation. The study results indicated that BRCA1 positively regulates miR-143-miR-145 expression and miR-143-miR-145 can serve as promising novel biomarkers for breast cancer subtyping. In our integrated genomics analysis and experimental validation, a new frame to predict candidate biomarkers of breast cancer subtype is provided and offers assistance in order to understand the potential disease etiology of the breast cancer subtypes.

  13. Most Cancers Caused by Random DNA Copying Errors

    MedlinePlus

    ... fullstory_164252.html Most Cancers Caused by Random DNA Copying Errors While habits, environment can be key ... factors, genes inherited from parents, or simply random DNA copying errors. From their calculations, the researchers now ...

  14. Retroviral DNA Integration

    PubMed Central

    2016-01-01

    The integration of a DNA copy of the viral RNA genome into host chromatin is the defining step of retroviral replication. This enzymatic process is catalyzed by the virus-encoded integrase protein, which is conserved among retroviruses and LTR-retrotransposons. Retroviral integration proceeds via two integrase activities: 3′-processing of the viral DNA ends, followed by the strand transfer of the processed ends into host cell chromosomal DNA. Herein we review the molecular mechanism of retroviral DNA integration, with an emphasis on reaction chemistries and architectures of the nucleoprotein complexes involved. We additionally discuss the latest advances on anti-integrase drug development for the treatment of AIDS and the utility of integrating retroviral vectors in gene therapy applications. PMID:27198982

  15. Chloroplast DNA Copy Number Changes during Plant Development in Organelle DNA Polymerase Mutants

    PubMed Central

    Morley, Stewart A.; Nielsen, Brent L.

    2016-01-01

    Chloroplast genome copy number is very high in leaf tissue, with upwards of 10,000 or more copies of the chloroplast DNA (ctDNA) per leaf cell. This is often promoted as a major advantage for engineering the plastid genome, as it provides high gene copy number and thus is expected to result in high expression of foreign proteins from integrated genes. However, it is also known that ctDNA copy number and ctDNA integrity decrease as cells age. Quantitative PCR (qPCR) allows measurement of organelle DNA levels relative to a nuclear gene target. We have used this approach to determine changes in copy number of ctDNA relative to the nuclear genome at different ages of Arabidopsis plant growth and in organellar DNA polymerase mutants. The mutant plant lines have T-DNA insertions in genes encoding the two organelle localized DNA polymerases (PolIA and PolIB). Each of these mutant lines exhibits some delay in plant growth and development as compared to wild-type plants, with the PolIB plants having a more pronounced delay. Both mutant lines develop to maturity and produce viable seeds. Mutants for both proteins were observed to have a reduction in ctDNA and mtDNA copy number relative to wild type plants at all time points as measured by qPCR. Both DNA polymerase mutants had a fairly similar decrease in ctDNA copy number, while the PolIB mutant had a greater effect of reduction in mtDNA levels. However, despite similar decreases in genome copy number, RT-PCR analysis of PolIA mutants show that PolIB expression remains unchanged, suggesting that PolIA may not be essential to plant survival. Furthermore, genotypic analysis of plants from heterozygous parents display a strong pressure to maintain two functioning copies of PolIB. These results indicate that the two DNA polymerases are both important in ctDNA replication, and they are not fully redundant to each other, suggesting each has a specific function in plant organelles. PMID:26870072

  16. Chloroplast DNA Copy Number Changes during Plant Development in Organelle DNA Polymerase Mutants.

    PubMed

    Morley, Stewart A; Nielsen, Brent L

    2016-01-01

    Chloroplast genome copy number is very high in leaf tissue, with upwards of 10,000 or more copies of the chloroplast DNA (ctDNA) per leaf cell. This is often promoted as a major advantage for engineering the plastid genome, as it provides high gene copy number and thus is expected to result in high expression of foreign proteins from integrated genes. However, it is also known that ctDNA copy number and ctDNA integrity decrease as cells age. Quantitative PCR (qPCR) allows measurement of organelle DNA levels relative to a nuclear gene target. We have used this approach to determine changes in copy number of ctDNA relative to the nuclear genome at different ages of Arabidopsis plant growth and in organellar DNA polymerase mutants. The mutant plant lines have T-DNA insertions in genes encoding the two organelle localized DNA polymerases (PolIA and PolIB). Each of these mutant lines exhibits some delay in plant growth and development as compared to wild-type plants, with the PolIB plants having a more pronounced delay. Both mutant lines develop to maturity and produce viable seeds. Mutants for both proteins were observed to have a reduction in ctDNA and mtDNA copy number relative to wild type plants at all time points as measured by qPCR. Both DNA polymerase mutants had a fairly similar decrease in ctDNA copy number, while the PolIB mutant had a greater effect of reduction in mtDNA levels. However, despite similar decreases in genome copy number, RT-PCR analysis of PolIA mutants show that PolIB expression remains unchanged, suggesting that PolIA may not be essential to plant survival. Furthermore, genotypic analysis of plants from heterozygous parents display a strong pressure to maintain two functioning copies of PolIB. These results indicate that the two DNA polymerases are both important in ctDNA replication, and they are not fully redundant to each other, suggesting each has a specific function in plant organelles.

  17. PCR-Based Detection of DNA Copy Number Variation.

    PubMed

    Mehrotra, Meenakshi

    2016-01-01

    Copy number variations are important polymorphisms that can influence gene expression within and close to the rearranged region, and results in phenotypic variation. Techniques that detect abnormalities in DNA copy number are therefore useful for studying the associations between DNA aberrations and disease phenotype and for locating critical genes. PCR-based detection of copy number of target gene using TaqMan copy number assay offers a reliable method to measure copy number variation in human genome.

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

    PubMed Central

    Xu, Baoshan; Li, Hua; Perry, John M.; Singh, Vijay Pratap; Yu, Zulin; Zakari, Musinu; Li, Linheng

    2017-01-01

    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. PMID:28640831

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

    PubMed

    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.

  20. Integrated Genome-wide DNA Copy Number and Expression Analysis Identifies Distinct Mechanisms of Primary Chemo-resistance in Ovarian Carcinomas

    PubMed Central

    Etemadmoghadam, Dariush; deFazio, Anna; Beroukhim, Rameen; Mermel, Craig; George, Joshy; Getz, Gaddy; Tothill, Richard; Okamoto, Aikou; Raeder, Maria B; Harnett, Paul; Lade, Stephen; Akslen, Lars A; Tinker, Anna; Locandro, Bianca; Alsop, Kathryn; Chiew, Yoke-Eng; Traficante, Nadia; Fereday, Sian; Johnson, Daryl; Fox, Stephen; Sellers, William; Urashima, Mitsuyoshi; Salvesen, Helga B; Meyerson, Matthew; Bowtell, David

    2009-01-01

    Purpose A significant number of women with serous ovarian cancer are intrinsically refractory to platinum-taxol based treatment. We analyzed somatic DNA copy number variation (CNV) and gene expression data to identify key mechanisms associated with primary resistance in advanced-stage serous cancers. Experimental Design Genome-wide CNV was measured in 118 ovarian tumors using high-resolution oligonucleotide microarrays. A well-defined subset of 85 advanced-stage serous tumors was then used to relate CNV to primary resistance to treatment. The discovery-based approach was complemented by quantitative-PCR copy number analysis of twelve candidate genes as independent validation of previously reported associations with clinical outcome. Likely CNV targets and tumor molecular subtypes were further characterized by gene expression profiling. Results Amplification of 19q12, containing Cyclin E (CCNE1) and 20q11.22-q13.12, mapping immediately adjacent to the steroid receptor co-activator NCOA3, were significantly associated with poor response to primary treatment. Other genes previously associated with CNV and clinical outcome in ovarian cancer were not associated with primary treatment resistance. Chemoresistant tumors with high CCNE1 copy number and protein expression were associated with increased cellular proliferation but so too were a subset of treatment responsive patients, suggesting a cell-cycle independent role for CCNE1 in modulating chemoresponse. Patients with a poor clinical outcome without CCNE1 amplification over expressed genes involved in extracellular matrix deposition. Conclusions We have identified two distinct mechanisms of primary treatment failure in serous ovarian cancer, involving CCNE1 amplification and enhanced extracellular matrix deposition. CCNE1 copy number is validated as a dominant marker of patient outcome in ovarian cancer. PMID:19193619

  1. Mitochondrial DNA Copy Number Is Associated with Breast Cancer Risk

    PubMed Central

    Thyagarajan, Bharat; Wang, Renwei; Nelson, Heather; Barcelo, Helene; Koh, Woon-Puay; Yuan, Jian-Min

    2013-01-01

    Mitochondrial DNA (mtDNA) copy number in peripheral blood is associated with increased risk of several cancers. However, data from prospective studies on mtDNA copy number and breast cancer risk are lacking. We evaluated the association between mtDNA copy number in peripheral blood and breast cancer risk in a nested case-control study of 183 breast cancer cases with pre-diagnostic blood samples and 529 individually matched controls among participants of the Singapore Chinese Health Study. The mtDNA copy number was measured using real time PCR. Conditional logistic regression analyses showed that there was an overall positive association between mtDNA copy number and breast cancer risk (Ptrend = 0.01). The elevated risk for higher mtDNA copy numbers was primarily seen for women with <3 years between blood draw and cancer diagnosis; ORs (95% CIs) for 2nd, 3rd, 4th, and 5th quintile of mtDNA copy number were 1.52 (0.61, 3.82), 2.52 (1.03, 6.12), 3.12 (1.31, 7.43), and 3.06 (1.25, 7.47), respectively, compared with the 1st quintile (Ptrend = 0.004). There was no association between mtDNA copy number and breast cancer risk among women who donated a blood sample ≥3 years before breast cancer diagnosis (Ptrend = 0.41). This study supports a prospective association between increased mtDNA copy number and breast cancer risk that is dependent on the time interval between blood collection and breast cancer diagnosis. Future studies are warranted to confirm these findings and to elucidate the biological role of mtDNA copy number in breast cancer risk. PMID:23776581

  2. No association between mitochondrial DNA copy number and colorectal adenomas.

    PubMed

    Thyagarajan, Bharat; Guan, Weihua; Fedirko, Veronika; Barcelo, Helene; Tu, Huakang; Gross, Myron; Goodman, Michael; Bostick, Roberd M

    2016-08-01

    Despite previously reported associations between peripheral blood mtDNA copy number and colorectal cancer, it remains unclear whether altered mtDNA copy number in peripheral blood is a risk factor for colorectal cancer or a biomarker for undiagnosed colorectal cancer. Though colorectal adenomas are well-recognized precursor lesions to colorectal cancer, no study has evaluated an association between mtDNA copy number and colorectal adenoma risk. Hence, we investigated an association between peripheral blood mtDNA copy number and incident, sporadic colorectal adenoma in 412 colorectal adenoma cases and 526 cancer-free controls pooled from three colonoscopy-based case-control studies that used identical methods for case ascertainment, risk factor determination, and biospecimen collection. We also evaluated associations between relative mtDNA copy number and markers of oxidative stress, including circulating F2 -isoprostanes, carotenoids, and fluorescent oxidation products. We measured mtDNA copy number using a quantitative real time polymerase chain reaction (PCR). We used unconditional logistic regression to analyze the association between mtDNA copy number and colorectal adenoma risk after multivariable adjustment. We found no association between logarithmically transformed relative mtDNA copy number, analyzed as a continuous variable, and colorectal adenoma risk (odds ratio = 1.02, 95%CI: 0.82-1.27; P = 0.86). There were no statistically significant associations between relative mtDNA copy number and other markers of oxidative stress. Our findings, taken together with those from previous studies, suggest that relative mtDNA copy number in peripheral blood may more likely be a marker of early colorectal cancer than of risk for the disease or of in vivo oxidative stress. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

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

    PubMed

    Wrede, Joanna E; Mengel-From, Jonas; Buchwald, Dedra; Vitiello, Michael V; Bamshad, Michael; Noonan, Carolyn; Christiansen, Lene; Christensen, Kaare; Watson, Nathaniel F

    2015-10-01

    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. Academic clinical research center. 15 sleep duration discordant monozygotic twin pairs (30 twins, 80% female; mean age 42.1 years [SD 15.0]). Sleep duration was phenotyped with wrist actigraphy. Each twin pair included a "normal" (7-9 h/24) and "short" (< 7 h/24) 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 structure to assess within-pair effects of sleep duration on mtDNA copy number. Mean within-pair sleep duration difference per 24 hours was 94.3 minutes (SD 62.6 min). We found reduced sleep duration (β = 0.06; 95% CI 0.004, 0.12; P < 0.05) and sleep efficiency (β = 0.51; 95% CI 0.06, 0.95; P < 0.05) were significantly associated with reduced mtDNA copy number within twin pairs. Thus every 1-minute decrease in actigraphy-defined sleep duration was associated with a decrease in mtDNA copy number of 0.06. Likewise, a 1% decrease in actigraphy-defined sleep efficiency was associated with a decrease in mtDNA copy number of 0.51. Reduced sleep duration and sleep efficiency were associated with reduced mitochondrial DNA copy number in sleep duration discordant monozygotic twins offering a potential mechanism whereby short sleep impairs health and longevity through mitochondrial stress. © 2015 Associated Professional Sleep Societies, LLC.

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

    PubMed

    Shen, Jie; Gopalakrishnan, Vancheswaran; Lee, Jeffrey E; Fang, Shenying; Zhao, Hua

    2015-01-01

    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).

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

    PubMed

    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.

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

    PubMed Central

    Wrede, Joanna E.; Mengel-From, Jonas; Buchwald, Dedra; Vitiello, Michael V.; Bamshad, Michael; Noonan, Carolyn; Christiansen, Lene; Christensen, Kaare; Watson, Nathaniel F.

    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” (< 7 h/24) 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 structure to assess within-pair effects of sleep duration on mtDNA copy number. Measurements and Results: Mean within-pair sleep duration difference per 24 hours was 94.3 minutes (SD 62.6 min). We found reduced sleep duration (β = 0.06; 95% CI 0.004, 0.12; P < 0.05) and sleep efficiency (β = 0.51; 95% CI 0.06, 0.95; P < 0.05) were significantly associated with reduced mtDNA copy number within twin pairs. Thus every 1-minute decrease in actigraphy-defined sleep duration was associated with a decrease in mtDNA copy number of 0.06. Likewise, a 1% decrease in actigraphy-defined sleep efficiency was associated with a decrease in mtDNA copy number of 0.51. Conclusions: Reduced sleep duration and sleep efficiency were associated with reduced mitochondrial DNA copy number in sleep duration discordant monozygotic twins offering a potential mechanism whereby short sleep impairs health and longevity through mitochondrial stress. Citation: Wrede JE, Mengel-From J, Buchwald D, Vitiello MV, Bamshad M, Noonan C, Christiansen L, Christensen K, Watson NF. Mitochondrial DNA copy number

  7. Systematic biases in DNA copy number originate from isolation procedures

    PubMed Central

    2013-01-01

    Background The ability to accurately detect DNA copy number variation in both a sensitive and quantitative manner is important in many research areas. However, genome-wide DNA copy number analyses are complicated by variations in detection signal. Results While GC content has been used to correct for this, here we show that coverage biases are tissue-specific and independent of the detection method as demonstrated by next-generation sequencing and array CGH. Moreover, we show that DNA isolation stringency affects the degree of equimolar coverage and that the observed biases coincide with chromatin characteristics like gene expression, genomic isochores, and replication timing. Conclusion These results indicate that chromatin organization is a main determinant for differential DNA retrieval. These findings are highly relevant for germline and somatic DNA copy number variation analyses. PMID:23618369

  8. Getting DNA copy numbers without control samples

    PubMed Central

    2012-01-01

    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 data. The method is

  9. Getting DNA copy numbers without control samples.

    PubMed

    Ortiz-Estevez, Maria; Aramburu, Ander; Rubio, Angel

    2012-08-16

    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. 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. 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 data. The method is available in the open-source R package

  10. Mitochondrial DNA copy number variation across human cancers

    PubMed Central

    Reznik, Ed; Miller, Martin L; Şenbabaoğlu, Yasin; Riaz, Nadeem; Sarungbam, Judy; Tickoo, Satish K; Al-Ahmadie, Hikmat A; Lee, William; Seshan, Venkatraman E; Hakimi, A Ari; Sander, Chris

    2016-01-01

    Mutations, deletions, and changes in copy number of mitochondrial DNA (mtDNA), are observed throughout cancers. Here, we survey mtDNA copy number variation across 22 tumor types profiled by The Cancer Genome Atlas project. We observe a tendency for some cancers, especially of the bladder, breast, and kidney, to be depleted of mtDNA, relative to matched normal tissue. Analysis of genetic context reveals an association between incidence of several somatic alterations, including IDH1 mutations in gliomas, and mtDNA content. In some but not all cancer types, mtDNA content is correlated with the expression of respiratory genes, and anti-correlated to the expression of immune response and cell-cycle genes. In tandem with immunohistochemical evidence, we find that some tumors may compensate for mtDNA depletion to sustain levels of respiratory proteins. Our results highlight the extent of mtDNA copy number variation in tumors and point to related therapeutic opportunities. DOI: http://dx.doi.org/10.7554/eLife.10769.001 PMID:26901439

  11. Reconstructing DNA copy number by joint segmentation of multiple sequences

    PubMed Central

    2012-01-01

    Background Variations in DNA copy number carry information on the modalities of genome evolution and mis-regulation of DNA replication in cancer cells. Their study can help localize tumor suppressor genes, distinguish different populations of cancerous cells, and identify genomic variations responsible for disease phenotypes. A number of different high throughput technologies can be used to identify copy number variable sites, and the literature documents multiple effective algorithms. We focus here on the specific problem of detecting regions where variation in copy number is relatively common in the sample at hand. This problem encompasses the cases of copy number polymorphisms, related samples, technical replicates, and cancerous sub-populations from the same individual. Results We present a segmentation method named generalized fused lasso (GFL) to reconstruct copy number variant regions. GFL is based on penalized estimation and is capable of processing multiple signals jointly. Our approach is computationally very attractive and leads to sensitivity and specificity levels comparable to those of state-of-the-art specialized methodologies. We illustrate its applicability with simulated and real data sets. Conclusions The flexibility of our framework makes it applicable to data obtained with a wide range of technology. Its versatility and speed make GFL particularly useful in the initial screening stages of large data sets. PMID:22897923

  12. DNA Copy Number Control Through Inhibition of Replication Fork Progression

    PubMed Central

    Nordman, Jared T.; Kozhevnikova, Elena N.; Verrijzer, C. Peter; Pindyurin, Alexey V.; Andreyeva, Evgeniya N.; Shloma, Victor V.; Zhimulev, Igor F.; Orr-Weaver, Terry L.

    2014-01-01

    Summary Proper control of DNA replication is essential to ensure faithful transmission of genetic material and to 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 cycle regulation. Importantly, DNA replication is highly influenced by developmental cues. In Drosophila, specific regions of the genome are repressed for DNA replication during differentiation by the SNF2 domain-containing protein SUUR through an unknown mechanism. We demonstrate that SUUR is recruited to active replication forks and mediates repression of DNA replication by directly inhibiting replication fork progression instead of functioning as a replication fork barrier. Mass-spec identification of SUUR associated proteins identified the replicative helicase member CDC45 as a SUUR-associated protein, supporting a role for SUUR directly at replication forks. Our results reveal that control of eukaryotic DNA copy number can occur through inhibition of replication fork progression. PMID:25437540

  13. Rates of single-copy DNA evolution in herons.

    PubMed

    Sheldon, F H

    1987-01-01

    DNA-DNA hybridization was used to discover the extent of single-copy DNA similarity among 13 species of herons and one ibis. Genetic distances among taxa were summarized as Tm values in a folded matrix. From this matrix, trees with the same branching pattern were constructed by least squares under one of two assumptions: (1) that sister branches are equal in length and (2) that sister branches are not necessarily equal in length. The residual sums of squares of these trees were compared by F-test to see whether the branches of the tree built under assumption (2) fit the matrix data significantly better than those of the tree built under assumption (1). By this method the existence of different rates of DNA evolution in different heron lineages was established. Bittern single-copy DNA has evolved at a rate approximately 25% faster, and boat-billed heron (Cochearius) and rufescent tiger heron (Tigrisoma lineatum) DNA has evolved approximately 19% slower, than that of day and night herons. It appears that the differences in rates of DNA evolution may increase proportionally with genealogical distance.

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

    PubMed

    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.

  15. Integrative genomics identifies distinct molecular classes of neuroblastoma and shows that multiple genes are targeted by regional alterations in DNA copy number.

    PubMed

    Wang, Qun; Diskin, Sharon; Rappaport, Eric; Attiyeh, Edward; Mosse, Yael; Shue, Daniel; Seiser, Eric; Jagannathan, Jayanti; Shusterman, Suzanne; Bansal, Manisha; Khazi, Deepa; Winter, Cynthia; Okawa, Erin; Grant, Gregory; Cnaan, Avital; Zhao, Huaqing; Cheung, Nai-Kong; Gerald, William; London, Wendy; Matthay, Katherine K; Brodeur, Garrett M; Maris, John M

    2006-06-15

    Neuroblastoma is remarkable for its clinical heterogeneity and is characterized by genomic alterations that are strongly correlated with tumor behavior. The specific genes that influence neuroblastoma biology and are targeted by genomic alterations remain largely unknown. We quantified mRNA expression in a highly annotated series of 101 prospectively collected diagnostic neuroblastoma primary tumors using an oligonucleotide-based microarray. Genomic copy number status at the prognostically relevant loci 1p36, 2p24 (MYCN), 11q23, and 17q23 was determined by PCR and was aberrant in 26, 20, 40, and 38 cases, respectively. In addition, 72 diagnostic neuroblastoma primary tumors assayed in a different laboratory were used as an independent validation set. Unsupervised hierarchical clustering showed that gene expression was highly correlated with genomic alterations and clinical markers of tumor behavior. The vast majority of samples with MYCN amplification and 1p36 loss of heterozygosity (LOH) clustered together on a terminal node of the sample dendrogram, whereas the majority of samples with 11q deletion clustered separately and both of these were largely distinct from the copy number neutral group of tumors. Genes involved in neurodevelopment were broadly overrepresented in the more benign tumors, whereas genes involved in RNA processing and cellular proliferation were highly represented in the most malignant cases. By combining transcriptomic and genomic data, we showed that LOH at 1p and 11q was associated with significantly decreased expression of 122 (61%) and 88 (27%) of the genes mapping to 1p35-36 and all of 11q, respectively, suggesting that multiple genes may be targeted by LOH events. A total of 71 of the 1p35-36 genes were also differentially expressed in the independent validation data set, providing a prioritized list of candidate neuroblastoma suppressor genes. Taken together, these data are consistent with the hypotheses that the neuroblastoma

  16. Molecular Poltergeists: Mitochondrial DNA Copies (numts) in Sequenced Nuclear Genomes

    PubMed Central

    Hazkani-Covo, Einat; Zeller, Raymond M.; Martin, William

    2010-01-01

    The natural transfer of DNA from mitochondria to the nucleus generates nuclear copies of mitochondrial DNA (numts) and is an ongoing evolutionary process, as genome sequences attest. In humans, five different numts cause genetic disease and a dozen human loci are polymorphic for the presence of numts, underscoring the rapid rate at which mitochondrial sequences reach the nucleus over evolutionary time. In the laboratory and in nature, numts enter the nuclear DNA via non-homolgous end joining (NHEJ) at double-strand breaks (DSBs). The frequency of numt insertions among 85 sequenced eukaryotic genomes reveal that numt content is strongly correlated with genome size, suggesting that the numt insertion rate might be limited by DSB frequency. Polymorphic numts in humans link maternally inherited mitochondrial genotypes to nuclear DNA haplotypes during the past, offering new opportunities to associate nuclear markers with mitochondrial markers back in time. PMID:20168995

  17. PCR-based analysis of mitochondrial DNA copy number, mitochondrial DNA damage, and nuclear DNA damage

    PubMed Central

    Gonzalez-Hunt, Claudia P.; Rooney, John P.; Ryde, Ian T.; Anbalagan, Charumathi; Joglekar, Rashmi

    2016-01-01

    Because of the role DNA damage and depletion play in human disease, it is important to develop and improve tools to assess these endpoints. This unit describes PCR-based methods to measure nuclear and mitochondrial DNA damage and copy number. Long amplicon quantitative polymerase chain reaction (LA-QPCR) is used to detect DNA damage by measuring the number of polymerase-inhibiting lesions present based on the amount of PCR amplification; real-time PCR (RT-PCR) is used to calculate genome content. In this unit we provide step-by-step instructions to perform these assays in Homo sapiens, Mus musculus, Rattus norvegicus, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Oryzias latipes, Fundulus grandis, and Fundulus heteroclitus, and discuss the advantages and disadvantages of these assays. PMID:26828332

  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.

    PubMed

    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.

  19. Mitochondrial DNA Copy Number and Exposure to Polycyclic Aromatic Hydrocarbons

    PubMed Central

    Pavanello, Sofia; Dioni, Laura; Hoxha, Mirjam; Fedeli, Ugo; Mielzynska-Švach, Danuta; Baccarelli, Andrea A.

    2013-01-01

    Background Increased mitochondrial DNA copy number (mtDNAcn) is a biological response to mtDNA damage and dysfunction predictive of lung cancer risk. Polycyclic aromatic hydrocarbons (PAHs) are established lung carcinogens and may cause mitochondrial toxicity. Whether PAH exposure and PAH-related nuclear DNA (nDNA) genotoxic effects are linked with increased mtDNAcn has never been evaluated. Methods We investigated the effect of chronic exposure to PAHs on mtDNAcn in peripheral blood lymphocytes (PBLs) of 46 Polish male non-current smoking cokeoven workers and 44 matched controls, who were part of a group of 94 study individuals examined in our previous work. Subjects PAH exposure and genetic alterations were characterized through measures of internal dose (urinary 1-pyrenol), target dose [anti-benzo[a]pyrene diolepoxide (anti-BPDE)-DNA adduct], genetic instability (micronuclei, MN and telomere length [TL]) and DNA methylation [p53 promoter] in PBLs. mtDNAcn (MT/S) was measured using a validated real-time PCR method. Results Workers with PAH exposure above the median value (>3 µmol 1-pyrenol/mol creatinine) showed higher mtDNAcn [geometric means (GM) of 1.06 (unadjusted) and 1.07 (age-adjusted)] compared to controls [GM 0.89 (unadjusted); 0.89 (age-adjusted)] (p=0.029 and 0.016), as well as higher levels of genetic and chromosomal [i.e. anti-BPDE-DNA adducts (p<0.001), MN (p<0.001) and TL (p=0.053)] and epigenetic [i.e., p53 gene-specific promoter methylation (p<0.001)] alterations in the nDNA. In the whole study population, unadjusted and age-adjusted mtDNAcn was positively correlated with 1-pyrenol (p=0.043 and 0.032) and anti-BPDE-DNA adducts (p=0.046 and 0.049). Conclusions PAH exposure and PAH-related nDNA genotoxicity are associated with increased mtDNAcn. Impact The present study is suggestive of potential roles of mtDNAcn in PAH-induced carcinogenesis. PMID:23885040

  20. RECONSTRUCTING DNA COPY NUMBER BY PENALIZED ESTIMATION AND IMPUTATION.

    PubMed

    Zhang, Zhongyang; Lange, Kenneth; Ophoff, Roel; Sabatti, Chiara

    2010-12-01

    Recent advances in genomics have underscored the surprising ubiquity of DNA copy number variation (CNV). Fortunately, modern genotyping platforms also detect CNVs with fairly high reliability. Hidden Markov models and algorithms have played a dominant role in the interpretation of CNV data. Here we explore CNV reconstruction via estimation with a fused-lasso penalty as suggested by Tibshirani and Wang [Biostatistics 9 (2008) 18-29]. We mount a fresh attack on this difficult optimization problem by the following: (a) changing the penalty terms slightly by substituting a smooth approximation to the absolute value function, (b) designing and implementing a new MM (majorization-minimization) algorithm, and (c) applying a fast version of Newton's method to jointly update all model parameters. Together these changes enable us to minimize the fused-lasso criterion in a highly effective way.We also reframe the reconstruction problem in terms of imputation via discrete optimization. This approach is easier and more accurate than parameter estimation because it relies on the fact that only a handful of possible copy number states exist at each SNP. The dynamic programming framework has the added bonus of exploiting information that the current fused-lasso approach ignores. The accuracy of our imputations is comparable to that of hidden Markov models at a substantially lower computational cost.

  1. Reconstructing labroid evolution with single-copy nuclear DNA.

    PubMed Central

    Streelman, J T; Karl, S A

    1997-01-01

    Fifteen per cent of all living fishes are united in a single suborder (Labroidei) and display a dazzling array of behavioural and ecological traits. The labroids are considered monophyletic and members share a pharyngeal jaw apparatus (PJA) modified for crushing and processing prey. Outside of the explicitly functional PJA, there is no corroborative evidence for a monophyletic Labroidei. Here, we report the first molecular phylogenetic analysis of the suborder. Contrary to morphology-based phylogenies, our single-copy nuclear DNA data do not support labroid families as a natural group. Our data indicate that pharyngognathy has evolved independently among labroid families and that characters of the PJA are not reliable markers of perciform evolution. This work 'crushes' conventional views of fish phylogeny and should engender novel concepts of piscine life history evolution. PMID:9263469

  2. Loss of the antioxidant enzyme CuZnSOD (Sod1) mimics an age-related increase in absolute mitochondrial DNA copy number in the skeletal muscle.

    PubMed

    Masser, Dustin R; Clark, Nicholas W; Van Remmen, Holly; Freeman, Willard M

    2016-08-01

    Mitochondria contain multiple copies of the circular mitochondrial genome (mtDNA) that encodes ribosomal RNAs and proteins locally translated for oxidative phosphorylation. Loss of mtDNA integrity, both altered copy number and increased mutations, is implicated in cellular dysfunction with aging. Published data on mtDNA copy number and aging is discordant which may be due to methodological limitations for quantifying mtDNA copy number. Existing quantitative PCR (qPCR) mtDNA copy number quantification methods provide only relative abundances and are problematic to normalize to different template input amounts and across tissues/sample types. As well, existing methods cannot quantify mtDNA copy number in subcellular isolates, such as isolated mitochondria and neuronal synaptic terminals, which lack nuclear genomic DNA for normalization. We have developed and validated a novel absolute mtDNA copy number quantitation method that uses chip-based digital polymerase chain reaction (dPCR) to count the number of copies of mtDNA and used this novel method to assess the literature discrepancy in which there is no clear consensus whether mtDNA numbers change with aging in skeletal muscle. Skeletal muscle in old mice was found to have increased absolute mtDNA numbers compared to young controls. Furthermore, young Sod1 (-/-) mice were assessed and show an age-mimicking increase in skeletal muscle mtDNA. These findings reproduce a number of previous studies that demonstrate age-related increases in mtDNA. This simple and cost effective dPCR approach should enable precise and accurate mtDNA copy number quantitation in mitochondrial studies, eliminating contradictory studies of mitochondrial DNA content with aging.

  3. Retroviral DNA Integration Directed by HIV Integration Protein in Vitro

    NASA Astrophysics Data System (ADS)

    Bushman, Frederic D.; Fujiwara, Tamio; Craigie, Robert

    1990-09-01

    Efficient retroviral growth requires integration of a DNA copy of the viral RNA genome into a chromosome of the host. As a first step in analyzing the mechanism of integration of human immunodeficiency virus (HIV) DNA, a cell-free system was established that models the integration reaction. The in vitro system depends on the HIV integration (IN) protein, which was partially purified from insect cells engineered to express IN protein in large quantities. Integration was detected in a biological assay that scores the insertion of a linear DNA containing HIV terminal sequences into a λ DNA target. Some integration products generated in this assay contained five-base pair duplications of the target DNA at the recombination junctions, a characteristic of HIV integration in vivo; the remaining products contained aberrant junctional sequences that may have been produced in a variation of the normal reaction. These results indicate that HIV IN protein is the only viral protein required to insert model HIV DNA sequences into a target DNA in vitro.

  4. Lower mitochondrial DNA copy number in peripheral blood leukocytes increases the risk of endometrial cancer.

    PubMed

    Sun, Yuhui; Zhang, Liren; Ho, Simon S; Wu, Xifeng; Gu, Jian

    2016-06-01

    Mitochondria are the primary source of energy generation in human cells. Low mitochondrial DNA (mtDNA) copy number in peripheral blood leukocytes (PBLs) has been associated with obesity and increased risks of several cancers. Since obesity is a significant risk factor for endometrial cancer, we hypothesize that low mtDNA copy number in PBLs is associated with an increased susceptibility to endometrial cancer. Using a Caucasian case-control study, we measured mtDNA copy number in PBLs from 139 endometrial cancer patients and 139 age-matched controls and determined the association of mtDNA copy number with the risk of endometrial cancer using multivariate logistic regression analysis. The normalized mtDNA copy number was significantly lower in endometrial cancer cases (median, 0.84; range, 0.24-2.00) than in controls (median, 1.06; range, 0.64-1.96) (P < 0.001). Dichotomized into high and low groups based on the median mtDNA copy number value in the controls, individuals with low mtDNA copy number had a significantly increased risk of endometrial cancer (adjusted OR, 5.59; 95%CI, 3.05-10.25; P < 0.001) compared to those with high mtDNA copy number. There was a significant dose-response association in tertile analysis. In addition, there was a significant joint effect between lower mtDNA copy number and never smoking, hypertension, diabetes, and obesity in elevating the risk of endometrial cancer. Low mtDNA copy number in PBLs is significantly associated with an increased risk of endometrial cancer in Caucasians. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  5. Increased leukocyte mitochondrial DNA copy number is associated with oral premalignant lesions: an epidemiology study.

    PubMed

    He, Yonggang; Gong, Yilei; Gu, Jian; Lee, J Jack; Lippman, Scott M; Wu, Xifeng

    2014-08-01

    Although changes in the mitochondrial DNA (mtDNA) copy number in peripheral blood leukocytes (PBLs) have been linked to increased susceptibility to several cancers, the relationship between the mtDNA copy number in PBLs and the risk of cancer precursors has not been investigated. In this study, we measured the relative mtDNA copy number in PBLs of 143 patients with histologically confirmed oral premalignant lesions (OPLs) and of 357 healthy controls that were frequency-matched to patients according to age, sex and race. OPL patients had a significantly higher mtDNA copy number than the controls (1.36 ± 0.74 versus 1.11 ± 0.32; P < 0.001). In analyses stratified by sex, race, alcohol consumption and smoking status, the mtDNA copy number was higher in the OPL patients than in the controls in all the strata. Using the median mtDNA copy number in the control group as a cutoff, we found that individuals with a high mtDNA copy number had significantly higher risk of having OPLs than individuals with a low mtDNA copy number (adjusted odds ratio, 1.93; 95% confidence interval, 1.23-3.05, P = 0.004). Analysis of the joint effect of alcohol consumption and smoking revealed even greater risk for OPLs. Our results suggest that high mtDNA copy number in PBLs is significantly associated with having OPLs. To our knowledge, this is the first epidemiologic study to show that the mtDNA copy number may indicate the risk of cancer precursors.

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

    PubMed

    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, p<0.001). Increased mtDNA copy 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.

  7. DNA Copy Number Variations Characterize Benign and Malignant Thyroid Tumors

    PubMed Central

    Liu, Yan; Cope, Leslie; Sun, Wenyue; Wang, Yongchun; Prasad, Nijaguna; Sangenario, Lauren; Talbot, Kristen; Somervell, Helina; Westra, William; Bishop, Justin; Califano, Joseph; Zeiger, Martha

    2013-01-01

    Context: Fine-needle aspiration (FNA) is the best diagnostic tool for preoperative evaluation of thyroid nodules but is often inconclusive as a guide for surgical management. Objective: Our hypothesis was that thyroid tumor subtypes may show characteristic DNA copy number variation (CNV) patterns, which may further improve the preoperative classification. Design: Our study cohorts included benign follicular adenomas (FAs), classic papillary thyroid carcinomas (PTCs), and follicular variant PTCs (FVPTCs), the three subtypes most commonly associated with inconclusive preoperative cytopathology. Setting: Tissue and FNA samples were obtained at an academic tertiary referral center. Patients: Cases were identified that underwent partial or complete thyroidectomy for malignant or indeterminate thyroid lesions between 2000 and 2008 and had adequate snap-frozen tissue. Interventions: Pairs of tumor tissue and matching normal thyroid tissue-derived DNA were compared using 550K single-nucleotide polymorphism arrays. Main Outcome Measure: Statistically significant differences in CNV patterns between tumor subtypes were identified. Results: Segmental amplifications in chromosomes (Ch) 7 and 12 were more common in FAs than in PTCs or FVPTCs. Additionally, a subset of FAs and FVPTCs showed deletions in Ch22. We identified the 5 CNV-associated genes best at discriminating between FAs and PTCs/FVPTCs, which correctly classified 90% of cases. These 5 Ch12 genes were validated by quantitative genomic PCR and gene expression array analyses on the same patient cohort. The 5-gene signature was then successfully validated against an independent test cohort of benign and malignant tumor samples. Finally, we performed a feasibility study on matched FA-derived intraoperative FNA samples and were able to correctly identify FAs harboring the Ch12 amplification signature, whereas FAs without amplification showed a normal Ch12 signature. Conclusions: Thyroid tumor subtypes possess

  8. Global Genetic Determinants of Mitochondrial DNA Copy Number

    PubMed Central

    Zhang, Hengshan; Singh, Keshav K.

    2014-01-01

    Many human diseases including development of cancer is associated with depletion of mitochondrial DNA (mtDNA) content. These diseases are collectively described as mitochondrial DNA depletion syndrome (MDS). High similarity between yeast and human mitochondria allows genomic study of the budding yeast to be used to identify human disease genes. In this study, we systematically screened the pre-existing respiratory-deficient Saccharomyces cerevisiae yeast strains using fluorescent microscopy and identified 102 nuclear genes whose deletions result in a complete mtDNA loss, of which 52 are not reported previously. Strikingly, these genes mainly encode protein products involved in mitochondrial protein biosynthesis process (54.9%). The rest of these genes either encode protein products associated with nucleic acid metabolism (14.7%), oxidative phosphorylation (3.9%), or other protein products (13.7%) responsible for bud-site selection, mitochondrial intermembrane space protein import, assembly of cytochrome-c oxidase, vacuolar protein sorting, protein-nucleus import, calcium-mediated signaling, heme biosynthesis and iron homeostasis. Thirteen (12.7%) of the genes encode proteins of unknown function. We identified human orthologs of these genes, conducted the interaction between the gene products and linked them to human mitochondrial disorders and other pathologies. In addition, we screened for genes whose defects affect the nuclear genome integrity. Our data provide a systematic view of the nuclear genes involved in maintenance of mitochondrial DNA. Together, our studies i) provide a global view of the genes regulating mtDNA content; ii) provide compelling new evidence toward understanding novel mechanism involved in mitochondrial genome maintenance and iii) provide useful clues in understanding human diseases in which mitochondrial defect and in particular depletion of mitochondrial genome plays a critical role. PMID:25170845

  9. Integrated Reproduction of Human Motion Components by Motion Copying System

    NASA Astrophysics Data System (ADS)

    Tsunashima, Noboru; Katsura, Seiichiro

    Currently, the development of leading-edge technology for recording and loading human motion on the basis of haptic information is required in the field of manufacturing and human support. Human movement is an assembly of motion components. Since human movements should be supported by a robot in real time, it is necessary to integrate the morion components, which were saved earlier. Once such motion integration is realized, future technology for use in daily human life is developed. This paper proposes the integrated reproduction of the decomposed components of human motion by using a motion copying system. This system is the key technology for the realization of the acquisition, saving and reproduction of the real-world haptic information. By the proposed method, it is possible not only to achieve expert skill acquisition, skill transfer to robots, and power assist for each motion component but also to open up new areas of applications.

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

    DTIC Science & Technology

    2015-08-01

    AWARD NUMBER: W81XWH-14-1-0194 TITLE: DNA Copy Number Signature to Predict Recurrence in Early-Stage Ovarian Cancer PRINCIPAL INVESTIGATOR...SUBTITLE 5a. CONTRACT NUMBER DNA Copy Number Signature to Predict Recurrence in Early Stage Ovarian Cancer 5b. GRANT NUMBER W81XWH-14-1-0194 5c...tasks Major Task 1: Obtain DNA samples from consortium specimens • Subtask 1 Pathological review of 592 early-stage high-grade ovarian cancer specimens

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

    PubMed

    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.

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

    PubMed

    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 < 0.01). In both cases and controls, levels of mtDNA copy number were inversely correlated with age (P < 0.01, respectively). And in cases, newly diagnosed, glioblastoma (GBM), and high grade glioma patients had significantly lower mtDNA copy number than their counterparts (P = 0.02, P < 0.01, and P = 0.04, respectively). In the multivariate analysis, elevated mtDNA 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.

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

    PubMed

    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.

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

    PubMed Central

    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

  15. Mitochondrial DNA copy number, but not haplogroup is associated with keratoconus in Han Chinese population.

    PubMed

    Hao, Xiao-Dan; Chen, Peng; Wang, Ye; Li, Su-Xia; Xie, Li-Xin

    2015-03-01

    Oxidative stress may play a role in the pathogenesis of keratoconus (KC). Mitochondrial DNA (mtDNA) is closely related to mitochondrion function, and variations may affect the generation of reactive oxygen species (ROS) and be involved in the pathogenesis of KC. To test whether mtDNA background and copy number confer genetic susceptibility to KC in the Han Chinese population, we performed this association study. We analyzed mtDNA sequence variations in 210 KC patients and 309 matched individuals from China, and classified each subject by haplogroup. Mitochondrial DNA copy number was measured in a subset of these subjects (193 patients and 103 controls). Comparison of matrilineal components of the cases and control populations revealed no significant difference. However, measurement of mtDNA copy number showed that KC patients had significantly lower mtDNA copy numbers than controls (P = 0.0002), even when age, gender, and mtDNA background were considered. Our results suggest that mtDNA copy number, but not haplogroup, is associated with keratoconus, and may contribute to its pathogenesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

    PubMed

    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. © 2016 Wiley Periodicals, Inc.

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

    PubMed Central

    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. PMID:27031233

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

    PubMed

    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.

  19. Porcine oocyte mtDNA copy number is high or low depending on the donor.

    PubMed

    Pedersen, Hanne Skovsgaard; Løvendahl, Peter; Larsen, Knud; Madsen, Lone Bruhn; Callesen, Henrik

    2016-08-01

    Oocyte capacity is relevant in understanding decreasing female fertility and in the use of assisted reproductive technologies in human and farm animals. Mitochondria are important to the development of a functionally good oocyte and the oocyte mtDNA copy number has been introduced as a useful parameter for prediction of oocyte competence. The aim of this study was to investigate: (i) if the oocyte donor has an influence on its oocyte's mtDNA copy number; and (ii) the relation between oocyte size and mtDNA copy number using pre- and postpubertal pig oocytes. Cumulus-oocyte complexes were collected from individual donor pigs. The oocytes were allocated into different size-groups, snap-frozen and single-oocyte mtDNA copy number was estimated by quantitative real-time PCR using the genes ND1 and COX1. Results showed that mean mtDNA copy number in oocytes from any individual donor could be categorized as either 'high' (≥100,000) or 'low' (<100,000) with no difference in threshold between pre- and postpubertal oocytes. No linear correlation was detected between oocyte size and mtDNA copy number within pre- and postpubertal oocytes. This study demonstrates the importance of the oocyte donor in relation to oocyte mtDNA copy number, irrespectively of the donor's puberty status and the oocyte's growth stage. Observations from this study facilitate both further investigations of the importance of mtDNA copy number and the unravelling of relations between different mitochondrial parameters and oocyte competence.

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

    PubMed Central

    2012-01-01

    Background 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. Results 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. Conclusions Even in a simple inducible system, nonlinear effects are observed and non-trivial data

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

    PubMed

    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

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

    PubMed

    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 < 8) or aggressive (Stage III/IV OR Gleason grade ≥ 8) PCa. Although mtDNA 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.

  3. Organellar genome copy number variation and integrity during moderate maturation of roots and leaves of maize seedlings.

    PubMed

    Ma, Jin; Li, Xiu-Qing

    2015-11-01

    Little information is available about organellar genome copy numbers and integrity in plant roots, although it was reported recently that the plastid and mitochondrial genomes were damaged under light, resulting in non-functional fragments in green seedling leaves in a maize line. In the present study, we investigated organellar genome copy numbers and integrity, after assessing the cellular ploidy, in seedling leaves and roots of two elite maize (Zea mays) cultivars using both long-fragment polymerase chain reaction (long-PCR) and real-time quantitative polymerase chain reaction (qPCR, a type of short-PCR). Since maize leaf and root cells are mainly diploid according to chromosome number counting and the literature, the DNA amount ratio between the organellar genomes and the nuclear genome could be used to estimate average organellar genome copy numbers per cell. In the present study, both long-PCR and qPCR analyses found that green leaves had dramatically more plastid DNA and less mitochondrial DNA than roots had in both cultivars. The similarity in results from long-PCR and qPCR suggests that green leaves and roots during moderate maturation have largely intact plastid and mitochondrial genomes. The high resolution of qPCR led to the detection of an increase in copies in the plastid genome and a decrease in copies in the analyzed mitochondrial sub-genomes during the moderate maturation of seedling leaves and roots. These results suggest that green seedling leaves and roots of these two maize cultivars during moderate maturation had essentially intact organellar genomes, an increased copy number of the plastid genome, and decreased copy numbers of certain mitochondrial sub-genomes.

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

    PubMed

    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 < 0.05). Varying 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

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

    PubMed

    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.

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

    PubMed

    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, P<0.01). Stepwise multiple regression analysis showed that regular exercise was independently associated with mtDNA copy number (β=0.25, P<0.01) after adjusting for the variables age, body mass index, waist-to-hip ratio, systolic and diastolic blood pressure, homeostasis model assessment of insulin resistance value, and levels of high-density lipoprotein cholesterol, triglycerides, and homocysteine. Regular exercise is associated with greater leukocyte mtDNA copy number in postmenopausal women.

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

    PubMed Central

    Chang, Yu Kyung; Kim, Da Eun; Cho, Soo Hyun

    2016-01-01

    Background 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. Methods 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. Results 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, P<0.01). Stepwise multiple regression analysis showed that regular exercise was independently associated with mtDNA copy number (β=0.25, P<0.01) after adjusting for the variables age, body mass index, waist-to-hip ratio, systolic and diastolic blood pressure, homeostasis model assessment of insulin resistance value, and levels of high-density lipoprotein cholesterol, triglycerides, and homocysteine. Conclusion Regular exercise is associated with greater leukocyte mtDNA copy number in postmenopausal women. PMID:27900071

  8. Ribosomal DNA and Stellate gene copy number variation on the Y chromosome of Drosophila melanogaster.

    PubMed

    Lyckegaard, E M; Clark, A G

    1989-03-01

    Multigene families on the Y chromosome face an unusual array of evolutionary forces. Both ribosomal DNA and Stellate, the two families examined here, have multiple copies of similar sequences on the X and Y chromosomes. Although the rate of sequence divergence on the Y chromosome depends on rates of mutation, gene conversion and exchange with the X chromosome, as well as purifying selection, the regulation of gene copy number may also depend on other pleiotropic functions, such as maintenance of chromosome pairing. Gene copy numbers were estimated for a series of 34 Y chromosome replacement lines using densitometric measurements of slot blots of genomic DNA from adult Drosophila melanogaster. Scans of autoradiographs of the same blots probed with the cloned alcohol dehydrogenase gene, a single copy gene, served as internal standards. Copy numbers span a 6-fold range for ribosomal DNA and a 3-fold range for Stellate DNA. Despite this magnitude of variation, there was no association between copy number and segregation variation of the sex chromosomes.

  9. GeneBreak: detection of recurrent DNA copy number aberration-associated chromosomal breakpoints within genes.

    PubMed

    van den Broek, Evert; van Lieshout, Stef; Rausch, Christian; Ylstra, Bauke; van de Wiel, Mark A; Meijer, Gerrit A; Fijneman, Remond J A; Abeln, Sanne

    2016-01-01

    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).

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

    PubMed

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

    2016-09-08

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

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

    PubMed Central

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

    2016-01-01

    The Saccharomyces cerevisiae ribosomal DNA (rDNA) locus is known to exhibit greater instability relative to the rest of the genome. However, wild-type cells preferentially maintain a stable number of rDNA copies, suggesting underlying genetic control of the size of this locus. We performed a screen of a subset of the Yeast Knock-Out (YKO) single gene deletion collection to identify genetic regulators of this locus and to determine if rDNA copy number correlates with yeast replicative lifespan. While we found no correlation between replicative lifespan and rDNA size, we identified 64 candidate strains with significant rDNA copy number differences. However, in the process of validating candidate rDNA variants, we observed that independent isolates of our de novo gene deletion strains had unsolicited but significant changes in rDNA copy number. Moreover, we were not able to recapitulate rDNA phenotypes from the YKO yeast deletion collection. Instead, we found that the standard lithium acetate transformation protocol is a significant source of rDNA copy number variation, with lithium acetate exposure being the treatment causing variable rDNA copy number events after transformation. As the effects of variable rDNA copy number are being increasingly reported, our finding that rDNA is affected by lithium acetate exposure suggested that rDNA copy number variants may be influential passenger mutations in standard strain construction in S. cerevisiae. PMID:27449518

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

    PubMed Central

    Falah, Masoumeh; Houshmand, Massoud; Najafi, Mohammad; Balali, Maryam; Mahmoudian, Saeid; Asghari, Alimohamad; Emamdjomeh, Hessamaldin; Farhadi, Mohammad

    2016-01-01

    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 copy number was also significantly associated with degree of hearing impairment (P=0.025) and audiogram configuration (P=0.022). Conclusion The findings of this study suggest that lower mitochondrial DNA copy number is responsible for presbycusis through alteration of mitochondrial function. Moreover, the significant association of mitochondrial DNA copy number in peripheral blood samples with the degree of hearing impairment and audiogram configuration has potential for use as a standard test for presbycusis, providing the possibility of the development of an easy

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

    PubMed

    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.

  14. Integrative genome-wide analysis reveals a robust genomic glioblastoma signature associated with copy number driving changes in gene expression.

    PubMed

    de Tayrac, Marie; Etcheverry, Amandine; Aubry, Marc; Saïkali, Stephan; Hamlat, Abderrahmane; Quillien, Veronique; Le Treut, André; Galibert, Marie-Dominique; Mosser, Jean

    2009-01-01

    Glioblastoma multiforme shows multiple chromosomal aberrations, the impact of which on gene expression remains unclear. To investigate this relationship and to identify putative initiating genomic events, we integrated a paired copy number and gene expression survey in glioblastoma using whole human genome arrays. Loci of recurrent copy number alterations were combined with gene expression profiles obtained on the same tumor samples. We identified a set of 406 "cis-acting DNA targeted genes" corresponding to genomic aberrations with direct copy-number-driving changes in gene expression, defined as genes with either significantly concordant or correlated changes in DNA copy number and expression. Functional annotation revealed that these genes participate in key processes of cancer cell biology, providing insights into the genetic mechanisms driving glioblastoma. The robustness of the gene selection was validated on an external microarray data set including 81 glioblastomas and 23 non-neoplastic brain samples. The integration of array CGH and gene expression data highlights a robust cis-acting DNA targeted genes signature that may be critical for glioblastoma progression, with two tumor suppressor genes PCDH9 and STARD13 that could be involved in tumor invasiveness and resistance to etoposide.

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

    PubMed

    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.

  16. Quantitation of DNA copy number in individual mitochondrial particles by capillary electrophoresis.

    PubMed

    Navratil, Marian; Poe, Bobby G; Arriaga, Edgar A

    2007-10-15

    Here, we present a direct method for determining mitochondrial DNA (mtDNA) copy numbers in individual mitochondrial particles, isolated from cultured cells, by means of capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection. We demonstrate that this method can detect a single molecule of PicoGreen-stained mtDNA in intact DsRed2-labeled mitochondrial particles isolated from human osteosarcoma 143B cells. This ultimate limit of mtDNA detection made it possible to confirm that an individual mitochondrial nucleoid, the genetic unit of mitochondrial inheritance, can contain a single copy of mtDNA. The validation of this approach was achieved via monitoring chemically induced mtDNA depletion and comparing the CE-LIF results to those obtained by quantitative microscopy imaging and multiplex real-time PCR analysis. Owing to its sensitivity, the CE-LIF method may become a powerful tool for investigating the copy number and organization of mtDNA in mitochondrial disease and aging, and in molecular biology techniques requiring manipulation and quantitation of DNA molecules such as plasmids.

  17. Determination of the molecular mass of bacterial genomic DNA and plasmid copy number by high-pressure liquid chromatography.

    PubMed Central

    Genthner, F J; Hook, L A; Strohl, W R

    1985-01-01

    Relatively rapid methods for the determination of relative genome molecular mass (Mr) and the estimation of plasmid copy number have been developed. These methods are based on the ability of the Bio-Rad high-pressure liquid chromatography hydroxylapatite column to separate and quantify single-stranded DNA, double-stranded DNA, and plasmid DNA. Genome Mr values were calculated from reassociation kinetics of single-stranded DNA as measured with the hydroxylapatite column. Bacteriophage T4 DNA was used to establish a C0t (moles of nucleotides times seconds per liter), or standard reassociation value. From this C0t value, C0t values for Escherichia coli B, Beggiatoa alba B18LD, and Streptomyces coelicolor were determined by comparative calculations. From those calculated C0t values, the Mr values of 1.96 X 10(9) for E. coli, 2.02 X 10(9) for B. alba, and 3.28 X 10(9) for S. coelicolor were estimated. Plasmid concentration was determined from cleared lysates by comparing the integrated area under the phosphate buffer-eluted plasmid peak to values obtained with known amounts of plasmid. The plasmid copy number was estimated by multiplying the ratio between the amounts of plasmid and chromosomal DNA by the ratio between the Mr values of the chromosome and the plasmid. A copy number of 29 was obtained from a culture of E. coli HB101 harboring pBR322 grown to a culture density of 1.6 X 10(9) CFU . ml-1. PMID:4083871

  18. Human TOP3: a single-copy gene encoding DNA topoisomerase III.

    PubMed Central

    Hanai, R; Caron, P R; Wang, J C

    1996-01-01

    A human cDNA encoding a protein homologous to the Escherichia coli DNA topoisomerase I subfamily of enzymes has been identified through cloning and sequencing. Expressing the cloned human cDNA in yeast (delta)top1 cells lacking endogenous DNA topoisomerase I yielded an activity in cell extracts that specifically reduces the number of supercoils in a highly negatively supercoiled DNA. On the basis of these results, the human gene containing the cDNA sequence has been denoted TOP3, and the protein it encodes has been denoted DNA topoisomerase III. Screening of a panel of human-rodent somatic hybrids and fluorescence in situ hybridization of cloned TOP3 genomic DNA to metaphase chromosomes indicate that human TOP3 is a single-copy gene located at chromosome 17p11.2-12. Images Fig. 2 PMID:8622991

  19. Allele-specific copy number profiling by next-generation DNA sequencing.

    PubMed

    Chen, Hao; Bell, John M; Zavala, Nicolas A; Ji, Hanlee P; Zhang, Nancy R

    2015-02-27

    The progression and clonal development of tumors often involve amplifications and deletions of genomic DNA. Estimation of allele-specific copy number, which quantifies the number of copies of each allele at each variant loci rather than the total number of chromosome copies, is an important step in the characterization of tumor genomes and the inference of their clonal history. We describe a new method, falcon, for finding somatic allele-specific copy number changes by next generation sequencing of tumors with matched normals. falcon is based on a change-point model on a bivariate mixed Binomial process, which explicitly models the copy numbers of the two chromosome haplotypes and corrects for local allele-specific coverage biases. By using the Binomial distribution rather than a normal approximation, falcon more effectively pools evidence from sites with low coverage. A modified Bayesian information criterion is used to guide model selection for determining the number of copy number events. Falcon is evaluated on in silico spike-in data and applied to the analysis of a pre-malignant colon tumor sample and late-stage colorectal adenocarcinoma from the same individual. The allele-specific copy number estimates obtained by falcon allows us to draw detailed conclusions regarding the clonal history of the individual's colon cancer.

  20. Copy number variations in urine cell free DNA as biomarkers in advanced prostate cancer.

    PubMed

    Xia, Yun; Huang, Chiang-Ching; Dittmar, Rachel; Du, Meijun; Wang, Yuan; Liu, Hongyan; Shenoy, Niraj; Wang, Liang; Kohli, Manish

    2016-06-14

    Genetic profiling of urine cell free DNA (cfDNA) has not been evaluated in advanced prostate cancer. We performed whole genome sequencing of urine cfDNAs to identify tumor-associated copy number variations in urine before and after initiating androgen deprivation therapy in HSPC stage and docetaxel chemotherapy in CRPC stage. A log2 ratio-based copy number analysis detected common genomic abnormalities in prostate cancer including AR amplification in 5/10 CRPC patients. Other abnormalities identified included TMPRSS2-ERG fusion, PTEN gene deletion, NOTCH1 locus amplification along with genomic amplifications at 8q24.3, 9q34.3, 11p15.5 and 14q11.2, and deletions at 4q35.2, 5q31.3, 7q36.3, 12q24.33, and 16p11.2. By comparing copy number between pre- and post-treatment, we found significant copy number changes in 34 genomic loci. To estimate the somatic tumor DNA fraction in urine cfDNAs, we developed a Urine Genomic Abnormality (UGA) score algorithm that summed the top ten most significant segments with copy number changes. The UGA scores correlated with tumor burden and the change in UGA score after stage-specific therapies reflected disease progression status and overall survival. The study demonstrates the potential clinical utility of urine cfDNAs in predicting treatment response and monitoring disease progression.

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

    PubMed Central

    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

  2. Comparison of dna-copying fidelity during repair and semiconservative synthesis by radioactive precursor distribution analysis

    SciTech Connect

    Nemirovskii, L.E.; Vasil'ev, V.K.

    1986-04-01

    The authors compare the fidelity of DNA copying during semiconservative and reparative synthesis under normal conditions and during cortisol-induced activation of free-radical processes, by examining the distribution of radioactivity among DNA pyrimidine isopliths. Radioactivity of nucleotide material in the isopliths was measured by counting in appropriate zones of the chromatograms in toluene scintillator. The investigation shows that injury to DNA of different organs, both directly and as a result of faulty repair, leads to shortening of the pyrimidine isopliths, i.e., to changes in the primary structure of DNA. These data help to explain the simultaneously cytostatic, carcinostatic, and mutagenic action of irradiation, cortisol and hydroxyurea.

  3. Detection of DNA copy number alterations in cancer by array comparative genomic hybridization.

    PubMed

    Michels, Evi; De Preter, Katleen; Van Roy, Nadine; Speleman, Frank

    2007-09-01

    Over the past few years, various reliable platforms for high-resolution detection of DNA copy number changes have become widely available. Together with optimized protocols for labeling and hybridization and algorithms for data analysis and representation, this has lead to a rapid increase in the application of this technology in the study of copy number variation in the human genome in normal cells and copy number imbalances in genetic diseases, including cancer. In this review, we briefly discuss specific technical issues relevant for array comparative genomic hybridization analysis in cancer tissues. We specifically focus on recent successes of array comparative genomic hybridization technology in the progress of our understanding of oncogenesis in a variety of cancer types. A third section highlights the potential of sensitive genome-wide detection of patterns of DNA imbalances or molecular portraits for class discovery and therapeutic stratification.

  4. Sequential Model Selection based Segmentation to Detect DNA Copy Number Variation

    PubMed Central

    Hu, Jianhua; Zhang, Liwen; Wang, Huixia Judy

    2016-01-01

    Summary Array-based CGH experiments are designed to detect genomic aberrations or regions of DNA copy-number variation that are associated with an outcome, typically a state of disease. Most of the existing statistical methods target on detecting DNA copy number variations in a single sample or array. We focus on the detection of group effect variation, through simultaneous study of multiple samples from multiple groups. Rather than using direct segmentation or smoothing techniques, as commonly seen in existing detection methods, we develop a sequential model selection procedure that is guided by a modified Bayesian information criterion. This approach improves detection accuracy by accumulatively utilizing information across contiguous clones, and has computational advantage over the existing popular detection methods. Our empirical investigation suggests that the performance of the proposed method is superior to that of the existing detection methods, in particular, in detecting small segments or separating neighboring segments with differential degrees of copy-number variation. PMID:26954760

  5. Genome-wide DNA copy number analysis in pancreatic cancer using high-density single nucleotide polymorphism arrays

    PubMed Central

    Harada, T; Chelala, C; Bhakta, V; Chaplin, T; Caulee, K; Baril, P; Young, BD; Lemoine, NR

    2008-01-01

    To identify genomic abnormalities characteristic of pancreatic ductal adenocarcinoma (PDAC) in vivo, a panel of 27 microdissected PDAC specimens were analysed using high-density microarrays representing ∼116 000 single nucleotide polymorphism (SNP) loci. We detected frequent gains of 1q, 2, 3, 5, 7p, 8q, 11, 14q and 17q (≥78% of cases), and losses of 1p, 3p, 6, 9p, 13q, 14q, 17p and 18q (≥44%). Although the results were comparable with those from array CGH, regions of those genetic changes were defined more accurately by SNP arrays. Integrating the Ensembl public data, we have generated ‘gene’ copy number indices that facilitate the search for novel candidates involved in pancreatic carcinogenesis. Copy numbers in a subset of the genes were validated using quantitative real-time PCR. The SKAP2/SCAP2 gene (7p15.2), which belongs to the src family kinases, was most frequently (63%) amplified in our sample set and its recurrent overexpression (67%) was confirmed by reverse transcription–PCR. Furthermore, fluorescence in situ hybridization and in situ RNA hybridization analyses for this gene have demonstrated a significant correlation between DNA copy number and mRNA expression level in an independent sample set (P<0.001). These findings indicate that the dysregulation of SKAP2/SCAP2, which is mostly caused by its increased gene copy number, is likely to be associated with the development of PDAC. PMID:17952125

  6. Comparison of four digital PCR platforms for accurate quantification of DNA copy number of a certified plasmid DNA reference material

    PubMed Central

    Dong, Lianhua; Meng, Ying; Sui, Zhiwei; Wang, Jing; Wu, Liqing; Fu, Boqiang

    2015-01-01

    Digital polymerase chain reaction (dPCR) is a unique approach to measurement of the absolute copy number of target DNA without using external standards. However, the comparability of different dPCR platforms with respect to measurement of DNA copy number must be addressed before dPCR can be classified fundamentally as an absolute quantification technique. The comparability of four dPCR platforms with respect to accuracy and measurement uncertainty was investigated by using a certified plasmid reference material. Plasmid conformation was found to have a significant effect on droplet-based dPCR (QX100 and RainDrop) not shared with chip-based QuantStudio 12k or BioMark. The relative uncertainty of partition volume was determined to be 0.7%, 0.8%, 2.3% and 2.9% for BioMark, QX100, QuantStudio 12k and RainDrop, respectively. The measurements of the certified pNIM-001 plasmid made using the four dPCR platforms were corrected for partition volume and closely consistent with the certified value within the expended uncertainty. This demonstrated that the four dPCR platforms are of comparable effectiveness in quantifying DNA copy number. These findings provide an independent assessment of this method of determining DNA copy number when using different dPCR platforms and underline important factors that should be taken into consideration in the design of dPCR experiments. PMID:26302947

  7. Integrated microfluidic systems for DNA analysis.

    PubMed

    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.

  8. DiNAMIC: a method to identify recurrent DNA copy number aberrations in tumors

    PubMed Central

    Walter, Vonn; Nobel, Andrew B.; Wright, Fred A.

    2011-01-01

    Motivation: DNA copy number gains and losses are commonly found in tumor tissue, and some of these aberrations play a role in tumor genesis and development. Although high resolution DNA copy number data can be obtained using array-based techniques, no single method is widely used to distinguish between recurrent and sporadic copy number aberrations. Results: Here we introduce Discovering Copy Number Aberrations Manifested In Cancer (DiNAMIC), a novel method for assessing the statistical significance of recurrent copy number aberrations. In contrast to competing procedures, the testing procedure underlying DiNAMIC is carefully motivated, and employs a novel cyclic permutation scheme. Extensive simulation studies show that DiNAMIC controls false positive discoveries in a variety of realistic scenarios. We use DiNAMIC to analyze two publicly available tumor datasets, and our results show that DiNAMIC detects multiple loci that have biological relevance. Availability: Source code implemented in R, as well as text files containing examples and sample datasets are available at http://www.bios.unc.edu/research/genomic_software/DiNAMIC. Contact: vwalter@email.unc.edu; fwright@bios.unc.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:21183584

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

    PubMed

    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.

  10. Mitochondrial DNA detection and copy number determination in the spermatozoa of the sea urchin Arbacia lixula.

    PubMed

    De Giorgi, C; D'Alessandro, A; Saccone, C

    1992-02-14

    The Polymerase Chain reaction technique has been used in order to detect and amplify a specific region of mtDNA, in a total DNA preparation extracted from the sperm of the sea urchin Arbacia lixula. The amplified fragment is the D-loop region which hybridizes with the homologous region extracted from the egg mtDNA. The results demonstrate that mtDNA is present in sperm cell, and, since the replication origin is present it is potentially able to replicate in the zygote. Furthermore, the technique used allowed us to estimate mtDNA copy number in sea urchin sperm, which has never been done before. Our results are that sea urchin sperm cell contains between 4 and 28 mtDNA molecules.

  11. High interindividual restriction fragment length and copy number of polymorphism of a TVRI family in moderate human DNA repeats

    SciTech Connect

    Rogaev, E.I.; Shapiro, Yu.A.

    1987-06-01

    The authors describe the selection of cloned human DNA sequences, with a copy number not exceeding 1000 copies per diploid genome, and their testing for interindividual restriction fragment lengths and copy number of polymorphism (RFLCP). As a result of the investigation a DNA clone was found (TVRI-6), about 2.8 kilobase-pairs in size, for which an unusually high level of interindividual RFLCP was discovered. The TVRI-6 sequence was obtained from a bank of Pst I restriction fragments of human placental nuclear DNA cloned in pBR 322. The bank was analyzed by hybridization of colonies with phosphorus 32-labelled human nuclear DNA.

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

    PubMed Central

    Ding, Jun; Sidore, Carlo; Butler, Thomas J.; Wing, Mary Kate; Qian, Yong; Meirelles, Osorio; Busonero, Fabio; Tsoi, Lam C.; Maschio, Andrea; Angius, Andrea; Kang, Hyun Min; Nagaraja, Ramaiah; Cucca, Francesco; Abecasis, Gonçalo R.; Schlessinger, David

    2015-01-01

    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. PMID:26172475

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

    PubMed

    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 < 0.0001). However, tumor-derived cfDNA content was significantly lower in serum than in matched plasma samples tested. With ~10 million reads per sample, the sequencing-based 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.

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

    PubMed

    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 (P<0.05). The group with both depression and insomnia exhibited significantly higher BFI and lower mtDNA copy number than those without combined symptoms (P<0.05). After adjusting for confounding factors, significant negative associations between mtDNA copy 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.

  15. Alterations of Mitochondrial DNA Copy Number and Telomere Length with Early Adversity and Psychopathology

    PubMed Central

    Tyrka, Audrey R.; Parade, Stephanie H.; Price, Lawrence H.; Kao, Hung-Teh; Porton, Barbara; Philip, Noah S.; Welch, Emma S.; Carpenter, Linda L.

    2015-01-01

    Background Telomere shortening and alterations of mitochondrial biogenesis are involved in cellular aging. Childhood adversity is associated with telomere shortening, and several investigations have shown short telomeres in psychiatric disorders. Recent studies have examined whether mitochondria might be involved in neuropsychiatric conditions; findings are limited and no prior work has examined this in relation to stress exposure. Methods Two-hundred and ninety healthy adults provided information on childhood parental loss and maltreatment and completed diagnostic interviews. Participants were categorized into four groups based upon the presence or absence of childhood adversity and the presence or absence of lifetime psychopathology (depressive, anxiety, and substance use disorders). Telomere length and mtDNA copy number were measured from leukocyte DNA by qPCR. Results Childhood adversity and lifetime psychopathology were each associated with shorter telomeres (p < .01) and higher mtDNA copy numbers (p < .001). Significantly higher mtDNA copy numbers and shorter telomeres were seen in individuals with major depression, depressive disorders, and anxiety disorders, as well as those with parental loss and childhood maltreatment. A history of substance disorders was also associated with significantly higher mtDNA copy numbers. Conclusion This study provides the first evidence of an alteration of mitochondrial biogenesis with early life stress and with anxiety and substance use disorders. We replicate prior work on telomere length and psychopathology, and show that this effect is not secondary to medication use or comorbid medical illness. Finally, we show that early life stress and psychopathology are each associated with these markers of cellular aging. PMID:25749099

  16. Mutation of the little finger domain in human DNA polymerase η alters fidelity when copying undamaged DNA.

    PubMed

    Beardslee, Renee A; Suarez, Samuel C; Toffton, Shannon M; McCulloch, Scott D

    2013-10-01

    DNA polymerase η (pol η) synthesizes past cyclobutane pyrimidine dimer and possibly 7,8-dihydro-8-oxoguanine (8-oxoG) lesions during DNA replication. Loss of pol η is associated with an increase in mutation rate, demonstrating its indispensable role in mutation suppression. It has been recently reported that β-strand 12 (amino acids 316-324) of the little finger region correctly positions the template strand with the catalytic core of the enzyme. The authors hypothesized that modification of β-strand 12 residues would disrupt correct enzyme-DNA alignment and alter pol η's activity and fidelity. To investigate this, the authors purified proteins containing the catalytic core of the polymerase, incorporated single amino acid changes to select β-strand 12 residues, and evaluated DNA synthesis activity for each pol η. Lesion bypass efficiencies and replication fidelities when copying DNA-containing cis-syn cyclobutane thymine-thymine dimer and 8-oxoG lesions were determined and compared with the corresponding values for the wild-type polymerase. The results confirm the importance of the β-strand in polymerase function and show that fidelity is most often altered when undamaged DNA is copied. Additionally, it is shown that DNA-protein contacts distal to the active site can significantly affect the fidelity of synthesis.

  17. Individual and combined effects of DNA methylation and copy number alterations on miRNA expression in breast tumors

    PubMed Central

    2013-01-01

    Background The global effect of copy number and epigenetic alterations on miRNA expression in cancer is poorly understood. In the present study, we integrate genome-wide DNA methylation, copy number and miRNA expression and identify genetic mechanisms underlying miRNA dysregulation in breast cancer. Results We identify 70 miRNAs whose expression was associated with alterations in copy number or methylation, or both. Among these, five miRNA families are represented. Interestingly, the members of these families are encoded on different chromosomes and are complementarily altered by gain or hypomethylation across the patients. In an independent breast cancer cohort of 123 patients, 41 of the 70 miRNAs were confirmed with respect to aberration pattern and association to expression. In vitro functional experiments were performed in breast cancer cell lines with miRNA mimics to evaluate the phenotype of the replicated miRNAs. let-7e-3p, which in tumors is found associated with hypermethylation, is shown to induce apoptosis and reduce cell viability, and low let-7e-3p expression is associated with poorer prognosis. The overexpression of three other miRNAs associated with copy number gain, miR-21-3p, miR-148b-3p and miR-151a-5p, increases proliferation of breast cancer cell lines. In addition, miR-151a-5p enhances the levels of phosphorylated AKT protein. Conclusions Our data provide novel evidence of the mechanisms behind miRNA dysregulation in breast cancer. The study contributes to the understanding of how methylation and copy number alterations influence miRNA expression, emphasizing miRNA functionality through redundant encoding, and suggests novel miRNAs important in breast cancer. PMID:24257477

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

    PubMed Central

    Shain, A. Hunter; Botton, Thomas; Bastian, Boris C.

    2016-01-01

    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. PMID:27100738

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

    PubMed

    Talevich, Eric; Shain, A Hunter; Botton, Thomas; Bastian, Boris C

    2016-04-01

    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.

  20. Association between Mitochondrial DNA Copy Number in Peripheral Blood and Incident CKD in the Atherosclerosis Risk in Communities Study.

    PubMed

    Tin, Adrienne; Grams, Morgan E; Ashar, Foram N; Lane, John A; Rosenberg, Avi Z; Grove, Megan L; Boerwinkle, Eric; Selvin, Elizabeth; Coresh, Josef; Pankratz, Nathan; Arking, Dan E

    2016-08-01

    Mitochondrial dysfunction in kidney cells has been implicated in the pathogenesis of CKD. Mitochondrial DNA (mtDNA) copy number is a surrogate measure of mitochondrial function, and higher mtDNA copy number in peripheral blood has been associated with lower risk of two important risk factors for CKD progression, diabetes and microalbuminuria. We evaluated whether mtDNA copy number in peripheral blood associates with incident CKD in a population-based cohort of middle-aged adults. We estimated mtDNA copy number using 25 high-quality mitochondrial single nucleotide polymorphisms from the Affymetrix 6.0 array. Among 9058 participants, those with higher mtDNA copy number had a lower rate of prevalent diabetes and lower C-reactive protein levels and white blood cell counts. Baseline eGFR did not differ significantly by mtDNA copy number. Over a median follow-up of 19.6 years, 1490 participants developed CKD. Higher mtDNA copy number associated with lower risk of incident CKD (highest versus lowest quartile: hazard ratio 0.65; 95% confidence interval, 0.56 to 0.75; P<0.001) after adjusting for age, sex, and race. After adjusting for additional risk factors of CKD, including prevalent diabetes, hypertension, C-reactive protein level, and white blood cell count, this association remained significant (highest versus lowest quartile: hazard ratio 0.75; 95% confidence interval, 0.64 to 0.87; P<0.001). In conclusion, higher mtDNA copy number associated with lower incidence of CKD independent of traditional risk factors and inflammation biomarker levels in this cohort. Further research on modifiable factors influencing mtDNA copy number may lead to improvement in the prevention and treatment of CKD. Copyright © 2016 by the American Society of Nephrology.

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

    PubMed

    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.

  2. Copy Number of the Transposon, Pokey, in rDNA Is Positively Correlated with rDNA Copy Number in Daphnia obtusa

    PubMed Central

    LeRiche, Kaitlynn; Eagle, Shannon H. C.; Crease, Teresa J.

    2014-01-01

    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. PMID:25490398

  3. Mutation of the Little Finger Domain in Human DNA Polymerase η Alters Fidelity When Copying Undamaged DNA

    PubMed Central

    Beardslee, Renee A.; Suarez, Samuel C.; Toffton, Shannon M.; McCulloch, Scott D.

    2014-01-01

    DNA polymerase η (pol η) synthesizes past cyclobutane pyrimidine dimer and possibly 7,8-dihydro-8-oxoguanine (8-oxoG) lesions during DNA replication. Loss of pol η is associated with an increase in mutation rate, demonstrating its indispensable role in mutation suppression. It has been recently reported that β-strand 12 (amino acids 316–324) of the little finger region correctly positions the template strand with the catalytic core of the enzyme. The authors hypothesized that modification of β-strand 12 residues would disrupt correct enzyme–DNA alignment and alter pol η’s activity and fidelity. To investigate this, the authors purified proteins containing the catalytic core of the polymerase, incorporated single amino acid changes to select β-strand 12 residues, and evaluated DNA synthesis activity for each pol η. Lesion bypass efficiencies and replication fidelities when copying DNA-containing cis-syn cyclobutane thymine-thymine dimer and 8-oxoG lesions were determined and compared with the corresponding values for the wild-type polymerase. The results confirm the importance of the β-strand in polymerase function and show that fidelity is most often altered when undamaged DNA is copied. Additionally, it is shown that DNA–protein contacts distal to the active site can significantly affect the fidelity of synthesis. PMID:23913529

  4. Integrated small copy number variations and epigenome maps of disorders of sex development

    PubMed Central

    Amarillo, Ina E; Nievera, Isabelle; Hagan, Andrew; Huchthagowder, Vishwa; Heeley, Jennifer; Hollander, Abby; Koenig, Joel; Austin, Paul; Wang, Ting

    2016-01-01

    Small copy number variations (CNVs) have typically not been analyzed or reported in clinical settings and hence have remained underrepresented in databases and the literature. Here, we focused our investigations on these small CNVs using chromosome microarray analysis (CMA) data previously obtained from patients with atypical characteristics or disorders of sex development (DSD). Using our customized CMA track targeting 334 genes involved in the development of urogenital and reproductive structures and a less stringent analysis filter, we uncovered small genes with recurrent and overlapping CNVs as small as 1 kb, and small regions of homozygosity (ROHs), imprinting and position effects. Detailed analysis of these high-resolution data revealed CNVs and ROHs involving structural and functional domains, repeat elements, active transcription sites and regulatory regions. Integration of these genomic data with DNA methylation, histone modification and predicted RNA expression profiles in normal testes and ovaries suggested spatiotemporal and tissue-specific gene regulation. This study emphasized a DSD-specific and gene-targeted CMA approach that uncovered previously unanalyzed or unreported small genes and CNVs, contributing to the growing resources on small CNVs and facilitating the narrowing of the genomic gap for identifying candidate genes or regions. This high-resolution analysis tool could improve the diagnostic utility of CMA, not only in patients with DSD but also in other clinical populations. These integrated data provided a better genomic-epigenomic landscape of DSD and greater opportunities for downstream research. PMID:27340555

  5. A novel satellite DNA sequence in the Peromyscus genome (PMSat): Evolution via copy number fluctuation.

    PubMed

    Louzada, Sandra; Vieira-da-Silva, Ana; Mendes-da-Silva, Ana; Kubickova, Svatava; Rubes, Jiri; Adega, Filomena; Chaves, Raquel

    2015-11-01

    Satellite DNAs (satDNA) are tandemly arrayed repeated sequences largely present in eukaryotic genomes, which play important roles in genome evolution and function, and therefore, their analysis is vital. Here, we describe the isolation of a novel satellite DNA family (PMSat) from the rodent Peromyscus eremicus (Cricetidae, Rodentia), which is located in pericentromeric regions and exhibits a typical satellite DNA genome organization. Orthologous PMSat sequences were isolated and characterized from three species belonging to Cricetidae: Cricetus cricetus, Phodopus sungorus and Microtus arvalis. In these species, PMSat is highly conserved, with the absence of fixed species-specific mutations. Strikingly, different numbers of copies of this sequence were found among the species, suggesting evolution by copy number fluctuation. Repeat units of PMSat were also found in the Peromyscus maniculatus bairdii BioProject, but our results suggest that these repeat units are from genome regions outside the pericentromere. The remarkably high evolutionary sequence conservation along with the preservation of a few numbers of copies of this sequence in the analyzed genomes may suggest functional significance but a different sequence nature/organization. Our data highlight that repeats are difficult to analyze due to the limited tools available to dissect genomes and the fact that assemblies do not cover regions of constitutive heterochromatin.

  6. Mitochondrial DNA Copy Number and Risk of Oral Cancer: A Report from Northeast India

    PubMed Central

    Mondal, Rosy; Ghosh, Sankar Kumar; Choudhury, Javed Hussain; Seram, Anil; Sinha, Kavita; Hussain, Marine; Laskar, Ruhina Shirin; Rabha, Bijuli; Dey, Pradip; Ganguli, Sabitri; NathChoudhury, Monisha; Talukdar, Fazlur Rahman; Chaudhuri, Biswadeep; Dhar, Bishal

    2013-01-01

    Background Oral squamous cell carcinoma (OSCC) is the sixth most common cancer globally. Tobacco consumption and HPV infection, both are the major risk factor for the development of oral cancer and causes mitochondrial dysfunction. Genetic polymorphisms in xenobiotic-metabolizing enzymes modify the effect of environmental exposures, thereby playing a significant role in gene–environment interactions and hence contributing to the individual susceptibility to cancer. Here, we have investigated the association of tobacco - betel quid chewing, HPV infection, GSTM1-GSTT1 null genotypes, and tumour stages with mitochondrial DNA (mtDNA) content variation in oral cancer patients. Methodology/Principal Findings The study comprised of 124 cases of OSCC and 140 control subjects to PCR based detection was done for high-risk HPV using a consensus primer and multiplex PCR was done for detection of GSTM1-GSTT1 polymorphism. A comparative ΔCt method was used for determination of mtDNA content. The risk of OSCC increased with the ceased mtDNA copy number (Ptrend = 0.003). The association between mtDNA copy number and OSCC risk was evident among tobacco – betel quid chewers rather than tobacco – betel quid non chewers; the interaction between mtDNA copy number and tobacco – betel quid was significant (P = 0.0005). Significant difference was observed between GSTM1 - GSTT1 null genotypes (P = 0.04, P = 0.001 respectively) and HPV infection (P<0.001) with mtDNA content variation in cases and controls. Positive correlation was found with decrease in mtDNA content with the increase in tumour stages (P<0.001). We are reporting for the first time the association of HPV infection and GSTM1-GSTT1 null genotypes with mtDNA content in OSCC. Conclusion Our results indicate that the mtDNA content in tumour tissues changes with tumour stage and tobacco-betel quid chewing habits while low levels of mtDNA content suggests invasive thereby serving as a biomarker in detection

  7. Laser capture microdissection of cervical human papillomavirus infections: copy number of the virus in cancerous and normal tissue and heterogeneous DNA methylation.

    PubMed

    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

    2009-08-01

    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.

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

    PubMed

    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.

  9. Concerted copy number variation balances ribosomal DNA dosage in human and mouse genomes

    PubMed Central

    Gibbons, John G.; Branco, Alan T.; Godinho, Susana A.; Yu, Shoukai; Lemos, Bernardo

    2015-01-01

    Tandemly repeated ribosomal DNA (rDNA) arrays are among the most evolutionary dynamic loci of eukaryotic genomes. The loci code for essential cellular components, yet exhibit extensive copy number (CN) variation within and between species. CN might be partly determined by the requirement of dosage balance between the 5S and 45S rDNA arrays. The arrays are nonhomologous, physically unlinked in mammals, and encode functionally interdependent RNA components of the ribosome. Here we show that the 5S and 45S rDNA arrays exhibit concerted CN variation (cCNV). Despite 5S and 45S rDNA elements residing on different chromosomes and lacking sequence similarity, cCNV between these loci is strong, evolutionarily conserved in humans and mice, and manifested across individual genotypes in natural populations and pedigrees. Finally, we observe that bisphenol A induces rapid and parallel modulation of 5S and 45S rDNA CN. Our observations reveal a novel mode of genome variation, indicate that natural selection contributed to the evolution and conservation of cCNV, and support the hypothesis that 5S CN is partly determined by the requirement of dosage balance with the 45S rDNA array. We suggest that human disease variation might be traced to disrupted rDNA dosage balance in the genome. PMID:25583482

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

    PubMed

    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

  11. A High-Resolution Map of Segmental DNA Copy Number Variation in the Mouse Genome

    PubMed Central

    Graubert, Timothy A; Selzer, Rebecca R; Richmond, Todd A; Eis, Peggy S; Shannon, William D; Li, Xia; McLeod, Howard L; Cheverud, James M; Ley, Timothy J

    2007-01-01

    Submicroscopic (less than 2 Mb) segmental DNA copy number changes are a recently recognized source of genetic variability between individuals. The biological consequences of copy number variants (CNVs) are largely undefined. In some cases, CNVs that cause gene dosage effects have been implicated in phenotypic variation. CNVs have been detected in diverse species, including mice and humans. Published studies in mice have been limited by resolution and strain selection. We chose to study 21 well-characterized inbred mouse strains that are the focus of an international effort to measure, catalog, and disseminate phenotype data. We performed comparative genomic hybridization using long oligomer arrays to characterize CNVs in these strains. This technique increased the resolution of CNV detection by more than an order of magnitude over previous methodologies. The CNVs range in size from 21 to 2,002 kb. Clustering strains by CNV profile recapitulates aspects of the known ancestry of these strains. Most of the CNVs (77.5%) contain annotated genes, and many (47.5%) colocalize with previously mapped segmental duplications in the mouse genome. We demonstrate that this technique can identify copy number differences associated with known polymorphic traits. The phenotype of previously uncharacterized strains can be predicted based on their copy number at these loci. Annotation of CNVs in the mouse genome combined with sequence-based analysis provides an important resource that will help define the genetic basis of complex traits. PMID:17206864

  12. Microfluidic-integrated DNA nanobiosensors.

    PubMed

    Ansari, M I Haque; Hassan, Shabir; Qurashi, Ahsanulhaq; Khanday, Firdous Ahmad

    2016-11-15

    Over the last few decades, an increased demand has emerged for integrating biosensors with microfluidic- and nanofluidic-based lab-on-chip (LOC) devices for point-of-care (POC) diagnostics, in the medical industry and environmental monitoring of pathogenic threat agents. Such a merger of microfluidics with biosensing technologies allows for the precise control of volumes, as low as one nanolitre and the integration of various types of bioassays on a single miniaturized platform. This integration offers several favorable advantages, such as low reagent consumption, automation of sample preparation, reduction in processing time, low cost analysis, minimal handling of hazardous materials, high detection accuracy, portability and disposability. This review provides a synopsis of the most recent developments in the microfluidic-integrated biosensing field by delineating the fundamental theory of microfluidics, fabrication techniques and a detailed account of the various transduction methods that are employed. Lastly, the review discusses state-of-the-art DNA biosensors with a focus on optical DNA biosensors.

  13. DNA copy number aberrations associated with lymphovascular invasion in upper urinary tract urothelial carcinoma.

    PubMed

    Misumi, Taku; Yamamoto, Yoshiaki; Miyachika, Yoshihiro; Eguchi, Satoshi; Chochi, Yasuyo; Nakao, Motonao; Nagao, Kazuhiro; Hara, Takahiko; Sakano, Shigeru; Furuya, Tomoko; Oga, Atsunori; Kawauchi, Shigeto; Sasaki, Kohsuke; Matsuyama, Hideyasu

    2012-06-01

    Recent studies have reported that lymphovascular invasion (LVI) is a predictor of patient prognosis in upper urinary tract urothelial carcinoma (UUTUC). DNA copy number aberrations (DCNAs) identified by array-based comparative genomic hybridization (aCGH) had not previously been examined in UUTUC. We therefore examined DCNAs in UUTUC and compared them with DCNAs in LVI. We applied aCGH technology using DNA chips spotted with 4,030 BAC clones to 32 UUTUC patients. Frequent copy number gains were detected on chromosomal regions 8p23.1 and 20q13.12, whereas frequent copy number losses were detected on chromosomal regions 13q21.1, 17p13.1, 6q16.3, and 17p11.2. DCNAs occurred more frequently in tumors with LVI than in those without it (P = 0.0002), and this parameter was more closely associated with LVI than with the tumor grade or pT stage. Disease-specific survival rate was higher in tumors without LVI than in those with it (P = 0.0120); however, tumor grade and stage were not significant prognostic factors of patient outcome. These data support our hypothesis that tumors with LVI have more genetic alterations in terms of total numbers of DCNAs than those without, and provide proof that aggressive adjuvant therapy should be considered for UUTUC patients with LVI.

  14. An initiator protein for plasmid R6K DNA replication. Mutations affecting the copy-number control.

    PubMed

    Inuzuka, M; Wada, Y

    1988-02-08

    Two kinds of mutations affecting the copy-number control of plasmid R6K were isolated and identified in an initiator pi protein by DNA sequencing. Firstly, a temperature-sensitive replication mutation, ts22, with decreased copy number results in a substitution of threonine to isoleucine at position 138 of the 305-amino-acid pi protein. Secondly, a high-copy-number (cop21) mutant was isolated from this ts mutant and was identified by an alteration of alanine to serine at position 162. This cop21 mutation suppressed the Ts character and was recessive to the wild-type allele in the copy control.

  15. DNA is hypomethylated at repetitive and single copy loci in patients with ICF syndrome

    SciTech Connect

    Schuffenhauer, S.; Buchholz, B.; Neitinger, T.

    1994-09-01

    ICF syndrome (immunodeficiency, centromeric heterochromatin instability, facial anomaly) is a very rare genetic disorder, reported in only 12 cases. Chromosomal rearrangements occur predominantly in the heterochromatic regions of HC 1 and 16 and include stretching, whole arm deletions and multibranched configurations. The molecular defect of these abnormalities is not known. Similar abnormalities have been found in cell cultures treated with viruses or 5-acacytidine, agents which cause DNA hypomethylation. Because heterochromatic DNA is known to be highly methylated, we hypothesise that DNA hypomethylation and subsequent disturbance of heterochromatin structure may play a role in the chromosomal rearrangements of ICF syndrome. Methylation studies in DNA from peripheral lymphocytes of two non-related ICF patients revealed hypomethylation of satellite-2 DNA localized in the heterochromatic regions of HC 1 and 16. DNA hypomethylation was also found at single copy loci, e.g. D15S63, D15S9, H19 and DXS255. Differences between the two patients suggest a random distribution of DNA hypomethylation. While a causal relationship between the molecular and cytogenetic abnormalities is likely, the postulated relationship between hypomethylation and the clinical symptoms in ICF syndrome remains to be elucidated.

  16. Role of Mitochondrial DNA Copy Number Alteration in Human Renal Cell Carcinoma †

    PubMed Central

    Lin, Chen-Sung; Lee, Hui-Ting; Lee, Ming-Huei; Pan, Siao-Cian; Ke, Chen-Yeh; Chiu, Allen Wen-Hsiang; Wei, Yau-Huei

    2016-01-01

    We investigated the role of mitochondrial DNA (mtDNA) copy number alteration in human renal cell carcinoma (RCC). The mtDNA copy numbers of paired cancer and non-cancer parts from five resected RCC kidneys after radical nephrectomy were determined by quantitative polymerase chain reaction (Q-PCR). An RCC cell line, 786-O, was infected by lentiviral particles to knock down mitochondrial transcriptional factor A (TFAM). Null target (NT) and TFAM-knockdown (TFAM-KD) represented the control and knockdown 786-O clones, respectively. Protein or mRNA expression levels of TFAM; mtDNA-encoded NADH dehydrogenase subunit 1 (ND1), ND6 and cytochrome c oxidase subunit 2 (COX-2); nuclear DNA (nDNA)-encoded succinate dehydrogenase subunit A (SDHA); v-akt murine thymoma viral oncogene homolog 1 gene (AKT)-encoded AKT and v-myc myelocytomatosis viral oncogene homolog gene (c-MYC)-encoded MYC; glycolytic enzymes including hexokinase II (HK-II), glucose 6-phosphate isomerase (GPI), phosphofructokinase (PFK), and lactate dehydrogenase subunit A (LDHA); and hypoxia-inducible factors the HIF-1α and HIF-2α, pyruvate dehydrogenase kinase 1 (PDK1), and pyruvate dehydrogenase E1 component α subunit (PDHA1) were analyzed by Western blot or Q-PCR. Bioenergetic parameters of cellular metabolism, basal mitochondrial oxygen consumption rate (mOCRB) and basal extracellular acidification rate (ECARB), were measured by a Seahorse XFe-24 analyzer. Cell invasiveness was evaluated by a trans-well migration assay and vimentin expression. Doxorubicin was used as a chemotherapeutic agent. The results showed a decrease of mtDNA copy numbers in resected RCC tissues (p = 0.043). The TFAM-KD clone expressed lower mtDNA copy number (p = 0.034), lower mRNA levels of TFAM (p = 0.008), ND1 (p = 0.007), and ND6 (p = 0.017), and lower protein levels of TFAM and COX-2 than did the NT clone. By contrast, the protein levels of HIF-2α, HK-II, PFK, LDHA, AKT, MYC and vimentin; trans-well migration activity (p = 0

  17. Role of Mitochondrial DNA Copy Number Alteration in Human Renal Cell Carcinoma.

    PubMed

    Lin, Chen-Sung; Lee, Hui-Ting; Lee, Ming-Huei; Pan, Siao-Cian; Ke, Chen-Yeh; Chiu, Allen Wen-Hsiang; Wei, Yau-Huei

    2016-05-25

    We investigated the role of mitochondrial DNA (mtDNA) copy number alteration in human renal cell carcinoma (RCC). The mtDNA copy numbers of paired cancer and non-cancer parts from five resected RCC kidneys after radical nephrectomy were determined by quantitative polymerase chain reaction (Q-PCR). An RCC cell line, 786-O, was infected by lentiviral particles to knock down mitochondrial transcriptional factor A (TFAM). Null target (NT) and TFAM-knockdown (TFAM-KD) represented the control and knockdown 786-O clones, respectively. Protein or mRNA expression levels of TFAM; mtDNA-encoded NADH dehydrogenase subunit 1 (ND1), ND6 and cytochrome c oxidase subunit 2 (COX-2); nuclear DNA (nDNA)-encoded succinate dehydrogenase subunit A (SDHA); v-akt murine thymoma viral oncogene homolog 1 gene (AKT)-encoded AKT and v-myc myelocytomatosis viral oncogene homolog gene (c-MYC)-encoded MYC; glycolytic enzymes including hexokinase II (HK-II), glucose 6-phosphate isomerase (GPI), phosphofructokinase (PFK), and lactate dehydrogenase subunit A (LDHA); and hypoxia-inducible factors the HIF-1α and HIF-2α, pyruvate dehydrogenase kinase 1 (PDK1), and pyruvate dehydrogenase E1 component α subunit (PDHA1) were analyzed by Western blot or Q-PCR. Bioenergetic parameters of cellular metabolism, basal mitochondrial oxygen consumption rate (mOCRB) and basal extracellular acidification rate (ECARB), were measured by a Seahorse XF(e)-24 analyzer. Cell invasiveness was evaluated by a trans-well migration assay and vimentin expression. Doxorubicin was used as a chemotherapeutic agent. The results showed a decrease of mtDNA copy numbers in resected RCC tissues (p = 0.043). The TFAM-KD clone expressed lower mtDNA copy number (p = 0.034), lower mRNA levels of TFAM (p = 0.008), ND1 (p = 0.007), and ND6 (p = 0.017), and lower protein levels of TFAM and COX-2 than did the NT clone. By contrast, the protein levels of HIF-2α, HK-II, PFK, LDHA, AKT, MYC and vimentin; trans-well migration activity (p = 0

  18. Two Drosophila retrotransposon gypsy subfamilies differ in ability to produce new DNA copies via reverse transcription in Drosophila cultured cells.

    PubMed Central

    Lyubomirskaya, N V; Avedisov, S N; Surkov, S A; Ilyin, Y V

    1993-01-01

    Plasmid DNA constructs containing 5' end truncated retrotransposon gypsy were introduced into Drosophila cultured cells. Appearance of new complete DNA copies with reconstructed via reverse transcription 5'LTR were detected by PCR after transient expression and by Southern blot analysis of genome DNA of stably transformed cells. Two gypsy subfamilies supposed to be different in transpositional activity were analyzed in terms of their ability to produce new DNA copies via reverse transcription in D. hydei cultured cells. It was demonstrated that both gypsy variants undergo retrotransposition but with different efficiency. Images PMID:7688116

  19. The effects of mitochondrial DNA deletion and copy number variations on different exercise intensities in highly trained swimmers.

    PubMed

    Baykara, O; Sahin, S K; Akbas, F; Guven, M; Onaran, I

    2016-10-31

    It has been suggested that heavy exercise might increase oxidative stress, causing mitochondrial DNA (mtDNA) mutations as well as DNA mutations and changes in the mtDNA copy number in cells. mtDNA4977 deletion is one of the most common deletions seen on mitochondria. We hypothesize association between exercise induced oxidative stress and mtDNA damage in peripheral blood lymphocytes (PBLs) of highly trained swimmers. Therefore we studied the mtDNA4977 deletion level, mtDNA copy number and their relationship with cellular ATP and oxidative stress status in PBLs of swimmers. 8 highly trained and 8 normal trained swimmers and 8 non-athlete subjects were included in the study. The mtDNA4977 deletion and amount of mtDNA were measured using RT-PCR method whereas dichlorohydrofluoroscein (DCF) assay method was used to assess cellular oxidative stress and ATP levels were measured using bioluminescence method. Even though an increase in mtDNA4977 deletion was found in all study groups, the difference was not statistically significant (p=0.98). The mtDNA copy numbers were found to be surprisingly high in highly trained swimmers compared to normal trained swimmers and non-athlete subjects by 4.03 fold (p= 0.0002) and 5.58 fold (p=0.0003), respectively. No significant differences were found between groups by means of intracellular ATP levels (p=0.406) and oxidative stress (p=0.430).  No correlation was found between mtDNA copy number and intracellular ATP content of the PBLs (p=0.703). Our results suggest that heavy training does not have a specific effect on mtDNA4977 deletion but it may be affecting mitochondrial copy numbers which may act as a compensatory mechanism related to ATP levels in blood.

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

    PubMed

    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.

  1. Identification of Candidate Growth Promoting Genes in Ovarian Cancer through Integrated Copy Number and Expression Analysis

    PubMed Central

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

    2010-01-01

    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≥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. PMID:20386695

  2. QUANTITATIVE SCREENING OF SINGLE COPIES OF HUMAN PAPILLOMA VIRAL DNA WITHOUT AMPLIFICATION

    PubMed Central

    Li, Jiangwei; Lee, Ji-Young; Yeung, Edward S.

    2008-01-01

    We describe a novel quantitative viral screening method based on single-molecule detection that does not require amplification. DNA of human papilloma virus (HPV), the major etiological agent of cervical cancer, served as the screening target in this study. Eight 100-nucleotide (nt) single-stranded (ss)-DNA probes were designed complementary to the E6-E7 gene of HPV-16 DNA. The probes were covalently stained with Alexa Fluor 532 and hybridized to the target in solution. The individual hybridized molecules were imaged with an intensified charge-coupled device (ICCD) in two ways. In the single-color mode, target molecules were detected via fluorescence from hybridized probes only. This system could detect HPV-16 DNA in the presence of human genomic DNA down to 0.7 copy/cell, and had a linear dynamic range of over six orders of magnitude. In the dual-color mode, we employed fluorescence resonance energy transfer (FRET) and added YOYO-3 dye as the acceptor. The two colors from Alexa Fluor 532 and YOYO-3 were dispersed by a transmission grating located in front of the ICCD. With this reinforced criteria for identifying the hybridized molecules, zero false-positive count was achieved. We also showed that DNA extracts from Pap test specimens did not interfere with the measurements. PMID:16970325

  3. Classification of human cancers based on DNA copy number amplification modeling

    PubMed Central

    Myllykangas, Samuel; Tikka, Jarkko; Böhling, Tom; Knuutila, Sakari; Hollmén, Jaakko

    2008-01-01

    Background DNA amplifications alter gene dosage in cancer genomes by multiplying the gene copy number. Amplifications are quintessential in a considerable number of advanced cancers of various anatomical locations. The aims of this study were to classify human cancers based on their amplification patterns, explore the biological and clinical fundamentals behind their amplification-pattern based classification, and understand the characteristics in human genomic architecture that associate with amplification mechanisms. Methods We applied a machine learning approach to model DNA copy number amplifications using a data set of binary amplification records at chromosome sub-band resolution from 4400 cases that represent 82 cancer types. Amplification data was fused with background data: clinical, histological and biological classifications, and cytogenetic annotations. Statistical hypothesis testing was used to mine associations between the data sets. Results Probabilistic clustering of each chromosome identified 111 amplification models and divided the cancer cases into clusters. The distribution of classification terms in the amplification-model based clustering of cancer cases revealed cancer classes that were associated with specific DNA copy number amplification models. Amplification patterns – finite or bounded descriptions of the ranges of the amplifications in the chromosome – were extracted from the clustered data and expressed according to the original cytogenetic nomenclature. This was achieved by maximal frequent itemset mining using the cluster-specific data sets. The boundaries of amplification patterns were shown to be enriched with fragile sites, telomeres, centromeres, and light chromosome bands. Conclusions Our results demonstrate that amplifications are non-random chromosomal changes and specifically selected in tumor tissue microenvironment. Furthermore, statistical evidence showed that specific chromosomal features co-localize with

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

    PubMed

    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.

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

    PubMed

    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, p<0.001) and in each of the four groups of participants (No Adversity/No Disorder, r=0.291, p=0.001; Adversity/No Disorder r=0.279, p=0.007; No Adversity/Disorder r=0.449, p=0.002; Adversity/Disorder, r=0.558, p<0.001). These correlations remained significant when controlling for age, smoking, and body mass index and establish an association between mtDNA and telomere 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.

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

    PubMed

    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 < 0.05) independent of a gender effect. In addition, we found a positive association of mtDNA 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.

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

    NASA Astrophysics Data System (ADS)

    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

  8. A highly efficient single-step, markerless strategy for multi-copy chromosomal integration of large biochemical pathways in Saccharomyces cerevisiae.

    PubMed

    Shi, Shuobo; Liang, Youyun; Zhang, Mingzi M; Ang, Ee Lui; Zhao, Huimin

    2016-01-01

    Despite recent advances in genome editing capabilities for the model organism Saccharomyces cerevisiae, the chromosomal integration of large biochemical pathways for stable industrial production remains challenging. In this work, we developed a simple platform for high-efficiency, single-step, markerless, multi-copy chromosomal integration of full biochemical pathways in Saccharomyces cerevisiae. In this Di-CRISPR (delta integration CRISPR-Cas) platform based on the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated systems (Cas), we specifically designed guide RNA sequences to target multiple delta sites in the yeast genome. The generation of double stranded breaks at the delta sites allowed simultaneous integration of multiple copies of linearized donor DNA containing large biochemical pathways. With our newly developed Di-CRISPR platform, we were able to attain highly efficient and markerless integration of large biochemical pathways and achieve an unprecedented 18-copy genomic integration of a 24 kb combined xylose utilization and (R,R)-2,3-butanediol (BDO) production pathway in a single step, thus generating a strain that was able to produce BDO directly from xylose. The simplicity and high efficiency of the Di-CRISPR platform could provide a superior alternative to high copy plasmids and would render this platform an invaluable tool for genome editing and metabolic engineering in S. cerevisiae. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  9. Copy Number Variation of Mitochondrial DNA Genes in Pneumocystis jirovecii According to the Fungal Load in BAL Specimens

    PubMed Central

    Valero, Clara; Buitrago, María José; Gits-Muselli, Maud; Benazra, Marion; Sturny-Leclère, Aude; Hamane, Samia; Guigue, Nicolas; Bretagne, Stéphane; Alanio, Alexandre

    2016-01-01

    Pneumocystis jirovecii is an unculturable fungus and the causative agent of Pneumocystis pneumonia, a life-threatening opportunistic infection. Although molecular diagnosis is often based on the detection of mtLSU rRNA mitochondrial gene, the number of copies of mitochondrial genes had not been investigated. We developed and optimized six real-time PCR assays in order to determine the copy number of four mitochondrial genes (mtSSU rRNA, mtLSU rRNA, NAD1, and CYTB) in comparison to nuclear genome (DHPS and HSP70) and tested 84 bronchoalveolar fluids of patients at different stages of the infection. Unexpectedly, we found that copy number of mitochondrial genes varied from gene to gene with mtSSU rRNA gene being more represented (37 copies) than NAD1 (23 copies), mtLSU rRNA (15 copies) and CYTB (6 copies) genes compared to nuclear genome. Hierarchical clustering analysis (HCA) allowed us to define five major clusters, significantly associated with fungal load (p = 0.029), in which copy number of mitochondrial genes was significantly different among them. More importantly, copy number of mtLSU rRNA, NAD1, and CYTB but not mtSSU rRNA differed according to P. jirovecii physiological state with a decreased number of copies when the fungal load is low. This suggests the existence of a mixture of various subspecies of mtDNA that can harbor different amplification rates. Overall, we revealed here an unexpected variability of P. jirovecii mtDNA copy number that fluctuates according to P. jirovecii’s physiological state, except for mtSSU that is the most stable and the most present mitochondrial gene. PMID:27672381

  10. DNA copy number amplifications in human neoplasms: review of comparative genomic hybridization studies.

    PubMed Central

    Knuutila, S.; Björkqvist, A. M.; Autio, K.; Tarkkanen, M.; Wolf, M.; Monni, O.; Szymanska, J.; Larramendy, M. L.; Tapper, J.; Pere, H.; El-Rifai, W.; Hemmer, S.; Wasenius, V. M.; Vidgren, V.; Zhu, Y.

    1998-01-01

    This review summarizes reports of recurrent DNA sequence copy number amplifications in human neoplasms detected by comparative genomic hybridization. Some of the chromosomal areas with recurrent DNA copy number amplifications (amplicons) of 1p22-p31, 1p32-p36, 1q, 2p13-p16, 2p23-p25, 2q31-q33, 3q, 5p, 6p12-pter, 7p12-p13, 7q11.2, 7q21-q22, 8p11-p12, 8q, 11q13-q14, 12p, 12q13-q21, 13q14, 13q22-qter, 14q13-q21, 15q24-qter, 17p11.2-p12, 17q12-q21, 17q22-qter, 18q, 19p13.2-pter, 19cen-q13.3, 20p11.2-p12, 20q, Xp11.2-p21, and Xp11-q13 and genes therein are presented in more detail. The paper with more than 150 references and two tables can be accessed from our web site http://www.helsinki.fi/lglvwww/CMG.html. The data will be updated biannually until the year 2001. PMID:9588877

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

    PubMed Central

    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

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

    PubMed

    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.

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

    PubMed Central

    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

  14. Mathematical Analysis of Copy Number Variation in a DNA Sample Using Digital PCR on a Nanofluidic Device

    PubMed Central

    Dube, Simant; Qin, Jian; Ramakrishnan, Ramesh

    2008-01-01

    Copy Number Variations (CNVs) of regions of the human genome have been associated with multiple diseases. We present an algorithm which is mathematically sound and computationally efficient to accurately analyze CNV in a DNA sample utilizing a nanofluidic device, known as the digital array. This numerical algorithm is utilized to compute copy number variation and the associated statistical confidence interval and is based on results from probability theory and statistics. We also provide formulas which can be used as close approximations. PMID:18682853

  15. Elevated Mitochondrial DNA Copy Number in Peripheral Blood and Tissue Predict the Opposite Outcome of Cancer: A Meta-Analysis

    PubMed Central

    Chen, Nan; Wen, Shu; Sun, Xiaoru; Fang, Qian; Huang, Lin; Liu, Shuai; Li, Wanling; Qiu, Meng

    2016-01-01

    Previous studies have suggested that mitochondrial DNA (mtDNA) copy number was associated with cancer risk. However, no solid conclusion revealed the potential predictive value of mtDNA copy number for cancer prognosis. The present meta-analysis was performed to clarify the problem. Hence, we performed a systematic search in PubMed, EmBase, Web of Science databases independently and a total of eighteen studies comprising 3961 cases satisfied the criteria and finally enrolled. Our results didn’t show the association between them but significant heterogeneity in overall analysis (OS: HR = 0.923, 95% CI: 0.653–1.306, p = 0.652; DFS: HR = 0.997, 95% CI: 0.599–1.659, p = 0.99). However, subgroup analysis stratified by sample came to the opposite conclusion. High level mitochondrial DNA copy number in peripheral blood predicted a poor cancer prognosis (OS: HR = 1.624, 95% CI: 1.211–2.177, p = 0.001; DFS: HR = 1.582, 95% CI: 1.026–2.439, p = 0.038) while patients with high level mitochondrial DNA copy number in tumor tissue exhibited better outcomes (OS: HR = 0.604 95% CI: 0.406–0.899, p = 0.013; DFS: HR = 0.593, 95% CI: 0.411–0.857, p = 0.005). These findings were further proved in detailed analyses in blood or tissue subgroup. In conclusion, our study suggested the elevated mtDNA copy number in peripheral blood predicted a poor cancer prognosis while the better outcome was presented among patients with elevated mtDNA copy number in tumor tissue. PMID:27857175

  16. Mitochondrial DNA copy number and biogenesis in different tissues of early- and late-lactating dairy cows.

    PubMed

    Laubenthal, L; Hoelker, M; Frahm, J; Dänicke, S; Gerlach, K; Südekum, K-H; Sauerwein, H; Häussler, S

    2016-02-01

    Energy balance in dairy cows changes during the course of lactation due to alterations in voluntary feed intake and energy required for milk synthesis. To adapt to the demands of lactation, energy metabolism needs to be regulated and coordinated in key organs such as adipose tissue (AT), liver, and mammary gland. Mitochondria are the main sites of energy production in mammalian cells and their number varies depending on age, organ, and physiological condition. The copy number of the mitochondrial genome, the mitochondrial DNA (mtDNA), reflects the abundance of mitochondria within a cell and is regulated by transcriptional and translational factors. Environmental, physiological, and energetic conditions change during lactation and we thus hypothesized that these changes may influence the mtDNA copy number and the abundance of genes regulating mitochondrial biogenesis. Therefore, we aimed to provide an overview of mitochondrial biogenesis in liver, subcutaneous (sc)AT, mammary gland, and peripheral blood cells during early and late lactation in dairy cows. German Holstein cows (n=21) were fed according to their requirements, and biopsies from scAT, liver, mammary gland, and blood were collected in early and late lactation and assayed for relative mtDNA copy numbers and the mRNA abundance of genes regulating mitochondrial biogenesis, such as nuclear-respiratory factor 1 and 2 (NRF-1, NRF-2), mitochondrial transcription factor A (TFAM), and peroxisome proliferator-activated receptor-gamma coactivator 1-α (PGC-1α). The number of mtDNA copies increased from early to late lactation in all tissues, whereas that in peripheral blood cells was greater in early compared with late lactation. Moreover, mitochondrial activity enzymes (i.e., citrate synthase and cytochrome c oxidase) increased from early to late lactation in scAT. Comparing the number of mtDNA copies between tissues and blood in dairy cows, the highest mtDNA content was observed in liver. The mRNA abundance of

  17. A rapid and reliable strategy for chromosomal integration of gene(s) with multiple copies

    PubMed Central

    Gu, Pengfei; Yang, Fan; Su, Tianyuan; Wang, Qian; Liang, Quanfeng; Qi, Qingsheng

    2015-01-01

    Direct optimization of the metabolic pathways on the chromosome requires tools that can fine tune the overexpression of a desired gene or optimize the combination of multiple genes. Although plasmid-dependent overexpression has been used for this task, fundamental issues concerning its genetic stability and operational repeatability have not been addressed. Here, we describe a rapid and reliable strategy for chromosomal integration of gene(s) with multiple copies (CIGMC), which uses the flippase from the yeast 2-μm plasmid. Using green fluorescence protein as a model, we verified that the fluorescent intensity was in accordance with the integration copy number of the target gene. When a narrow-host-range replicon, R6K, was used in the integrative plasmid, the maximum integrated copy number of Escherichia coli reached 15. Applying the CIGMC method to optimize the overexpression of single or multiple genes in amino acid biosynthesis, we successfully improved the product yield and stability of the production. As a flexible strategy, CIGMC can be used in various microorganisms other than E. coli. PMID:25851494

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

    PubMed Central

    Głowacka, Katarzyna; Kromdijk, Johannes; Leonelli, Lauriebeth; Niyogi, Krishna K.; Clemente, Tom E.

    2016-01-01

    Abstract 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. PMID:26670088

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed Central

    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 (<64 contiguous probes, ∼ 15 kb). Results Our data showed that primary tumor breast cancer genomes frequently contained many small-scale copy number gains and losses, termed 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

  2. EGFR-activating mutations, DNA copy number abundance of ErbB family, and prognosis in lung adenocarcinoma.

    PubMed

    Chen, Hsuan-Yu; Liu, Chia-Hsin; Chang, Ya-Hsuan; Yu, Sung-Liang; Ho, Bing-Ching; Hsu, Chung-Ping; Yang, Tsung-Ying; Chen, Kun-Chieh; Hsu, Kuo-Hsuan; Tseng, Jeng-Sen; Hsia, Jiun-Yi; Chuang, Cheng-Yen; Chang, Chi-Sheng; Li, Yu-Cheng; Li, Ker-Chau; Chang, Gee-Chen; Yang, Pan-Chyr

    2016-02-23

    In this study, EGFR-activating mutation status and DNA copy number abundances of members of ErbB family were measured in 261 lung adenocarcinomas. The associations between DNA copy number abundances of ErbB family, EGFR-activating mutation status, and prognosis were explored. Results showed that DNA copy number abundances of EGFR, ERBB2, ERBB3, and ERBB4 had associations with overall survival in lung adenocarcinoma with EGFR-activating mutations. In the stratification analysis, only ERBB2 showed significant discrepancy in patients carrying wild type EGFR and other members of ErbB family in patients carrying EGFR-activating mutation. This indicated that CNAs of ErbB family had effect modifications of EGFR-activating mutation status. Findings of this study demonstrate potential molecular guidance of patient management of lung adenocarcinoma with or without EGFR-activating mutations.

  3. Integrity Verification for Multiple Data Copies in Cloud Storage Based on Spatiotemporal Chaos

    NASA Astrophysics Data System (ADS)

    Long, Min; Li, You; Peng, Fei

    Aiming to strike for a balance between the security, efficiency and availability of the data verification in cloud storage, a novel integrity verification scheme based on spatiotemporal chaos is proposed for multiple data copies. Spatiotemporal chaos is implemented for node calculation of the binary tree, and the location of the data in the cloud is verified. Meanwhile, dynamic operation can be made to the data. Furthermore, blind information is used to prevent a third-party auditor (TPA) leakage of the users’ data privacy in a public auditing process. Performance analysis and discussion indicate that it is secure and efficient, and it supports dynamic operation and the integrity verification of multiple copies of data. It has a great potential to be implemented in cloud storage services.

  4. Mitochondrial DNA Copy Number and Pancreatic Cancer in the Alpha-Tocopherol Beta-Carotene Cancer Prevention (ATBC) Study

    PubMed Central

    Lynch, Shannon M.; Weinstein, Stephanie J.; Virtamo, Jarmo; Lan, Qing; Lui, Chin-San; Cheng, Wen-Ling; Rothman, Nathaniel; Albanes, Demetrius; Stolzenberg-Solomon, Rachael Z.

    2011-01-01

    Background Diabetes, obesity, and cigarette smoke, consistent risk factors for pancreatic cancer, are sources of oxidative stress in humans that could cause mitochondrial DNA (mtDNA) damage and increase mtDNA copy number. Methods To test whether higher mtDNA copy number is associated with increased incident pancreatic cancer, we conducted a nested case-control study in the Alpha-Tocopherol Beta Carotene Cancer Prevention (ATBC) Study cohort of male smokers, aged 50-69 years at baseline. Between 1992 and 2004, 203 incident cases of pancreatic adenocarcinoma occurred (follow-up: 12 years) among participants with whole blood samples used for mtDNA extraction. For these cases and 656 controls, we calculated odds ratios (OR) and 95% confidence intervals using unconditional logistic regression, adjusting for age, smoking, and diabetes history. All statistical tests were two-sided. Results Higher mtDNA copy number was significantly associated with increased pancreatic cancer risk (highest vs. lowest mtDNA copy number quintile, OR=1.64, 95%CI=1.01-2.67, continuous OR=1.14, 95% CI 1.06-1.23), particularly for cases diagnosed during the first 7 years of follow-up (OR=2.14,95% CI=1.16-3.96, p-trend=0.01, continuous OR=1.21, 95% CI 1.10-1.33), but not for cases occurring during follow-up of 7 years or greater (OR= 1.14, 95% CI=0.53-2.45, continuous OR=1.05, 95% CI 0.93-1.18). Conclusion Our results support the hypothesis that mtDNA copy number is associated with pancreatic cancer and could possibly serve as a biomarker for pancreatic cancer development. PMID:21859925

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

    PubMed

    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.

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

    PubMed Central

    2012-01-01

    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. PMID:22390386

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

    PubMed

    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 < 0.001 including rs17111633 in CNNM1 (p values = 6.32e - 6) and chr19:51083059 in MYPOP (p values = 3.23e - 5). In the candidate association study, several SNPs in PPARG, PRKCA, SP1 and THRB were associated with mtDNA copy number (p values < 0.05). mtDNA copy number was significantly associated with wGRS based on top GWAS hits (β = 0.49, 95% CI:0.38-0.60, p < 0.001). Variations in nuclear DNA are potentially associated with maternal mtDNA copy number.

  8. Decreased Peripheral Mitochondrial DNA Copy Number is Associated with the Risk of Heart Failure and Long-term Outcomes.

    PubMed

    Huang, Jin; Tan, Lun; Shen, Rufei; Zhang, Lina; Zuo, Houjuan; Wang, Dao W

    2016-04-01

    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 associations between leukocyte mtDNA CNV and heart failure risk and prognosis. A total of 1700 hospitalized patients with heart failure and 1700 age- and sex-matched community population were consecutively enrolled in this observational study, as well as 1638 (96.4%) patients were followed prospectively for a median of 17 months (12-24 months). The relative mtDNA copy number of leukocyte of peripheral blood or cardiac tissue was measured in triplicate by quantitative real-time PCR method. Patients with heart failure possessed much lower relative mtDNA copy number compared with control subjects (median 0.83, interquartile range [IQR] 0.60-1.16 vs median 1.00, IQR 0.47-2.20; P < 0.001), especially for the patients with ischemic etiology (median, 0.77 for ischemic and 0.91 for non-ischemic, P < 0.001). Patients with lower mtDNA copy number exhibited 1.7 times higher risk of heart failure (odds ratio 1.71, 95% confidence interval [CI] 1.48-1.97, P < 0.001). Long-term follow-up (median of 17 months) showed that decreased mtDNA copy number was significant associated with both increased cardiovascular deaths (hazard ratio [HR] 1.58, 95% CI 1.16-2.16, P = 0.004) and cardiovascular rehospitalization (HR 1.48, 95% CI 1.21-1.82, P < 0.001). After adjusting for the conventional risk factors and medications, lower mtDNA copy numbers were still significantly associated with 50% higher cardiovascular mortality (P = 0.035). In conclusion, mtDNA copy number depletion is an independent risk factor for heart failure and predicts higher cardiovascular mortality in patients with heart failure.

  9. DNA copy number variants of known glaucoma genes in relation to primary open-angle glaucoma.

    PubMed

    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

    2014-11-20

    We examined the role of DNA copy number variants (CNVs) of known glaucoma genes in relation to primary open angle glaucoma (POAG). 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. 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. 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. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

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

    PubMed Central

    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

  11. Elevation of Pollen Mitochondrial DNA Copy Number by WHIRLY2: Altered Respiration and Pollen Tube Growth in Arabidopsis.

    PubMed

    Cai, Qiang; Guo, Liang; Shen, Zhao-Rui; Wang, Dan-Yang; Zhang, Quan; Sodmergen

    2015-09-01

    In plants, the copy number of the mitochondrial DNA (mtDNA) can be much lower than the number of mitochondria. The biological significance and regulatory mechanisms of this phenomenon remain poorly understood. Here, using the pollen vegetative cell, we examined the role of the Arabidopsis (Arabidopsis thaliana) mtDNA-binding protein WHIRLY2 (AtWHY2). AtWHY2 decreases during pollen development, in parallel with the rapid degradation of mtDNA; to examine the importance of this decrease, we used the pollen vegetative cell-specific promoter Lat52 to express AtWHY2. The transgenic plants (LWHY2) had very high mtDNA levels in pollen, more than 10 times more than in the wild type (ecotype Columbia-0). LWHY2 plants were fertile, morphologically normal, and set seeds; however, reciprocal crosses with heterozygous plants showed reduced transmission of LWHY2-1 through the male and slower growth of LWHY2-1 pollen tubes. We found that LWHY2-1 pollen had significantly more reactive oxygen species and less ATP compared with the wild type, indicating an effect on mitochondrial respiration. These findings reveal that AtWHY2 affects mtDNA copy number in pollen and suggest that low mtDNA copy numbers might be the normal means by which plant cells maintain mitochondrial genetic information. © 2015 American Society of Plant Biologists. All Rights Reserved.

  12. Elevation of Pollen Mitochondrial DNA Copy Number by WHIRLY2: Altered Respiration and Pollen Tube Growth in Arabidopsis1

    PubMed Central

    Cai, Qiang; Guo, Liang; Shen, Zhao-Rui; Wang, Dan-Yang; Zhang, Quan; Sodmergen

    2015-01-01

    In plants, the copy number of the mitochondrial DNA (mtDNA) can be much lower than the number of mitochondria. The biological significance and regulatory mechanisms of this phenomenon remain poorly understood. Here, using the pollen vegetative cell, we examined the role of the Arabidopsis (Arabidopsis thaliana) mtDNA-binding protein WHIRLY2 (AtWHY2). AtWHY2 decreases during pollen development, in parallel with the rapid degradation of mtDNA; to examine the importance of this decrease, we used the pollen vegetative cell-specific promoter Lat52 to express AtWHY2. The transgenic plants (LWHY2) had very high mtDNA levels in pollen, more than 10 times more than in the wild type (ecotype Columbia-0). LWHY2 plants were fertile, morphologically normal, and set seeds; however, reciprocal crosses with heterozygous plants showed reduced transmission of LWHY2-1 through the male and slower growth of LWHY2-1 pollen tubes. We found that LWHY2-1 pollen had significantly more reactive oxygen species and less ATP compared with the wild type, indicating an effect on mitochondrial respiration. These findings reveal that AtWHY2 affects mtDNA copy number in pollen and suggest that low mtDNA copy numbers might be the normal means by which plant cells maintain mitochondrial genetic information. PMID:26195569

  13. DNA biochip using a phototransistor integrated circuit.

    PubMed

    Vo-Dinh, T; Alarie, J P; Isola, N; Landis, D; Wintenberg, A L; Ericson, M N

    1999-01-15

    This work describes the development of an integrated biosensor based on phototransistor integrated circuits (IC) for use in medical detection, DNA diagnostics, and gene mapping. The evaluation of various system components developed for an integrated biosensor microchip is discussed. Methods to develop a microarray of DNA probes on nitrocellulose substrate are discussed. The biochip device has sensors, amplifiers, discriminators, and logic circuitry on board. Integration of light-emitting diodes into the device is also possible. To achieve improved sensitivity, we have designed an IC system having each phototransistor sensing element composed of 220 phototransistor cells connected in parallel. Measurements of fluorescent-labeled DNA probe microarrays and hybridization experiments with a sequence-specific DNA probe for the human immunodeficiency virus 1 system on nitrocellulose substrates illustrate the usefulness and potential of the DNA biochip.

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

    SciTech Connect

    Harris, C.; Nunez, M.; Giraldez, R.

    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.

  15. Excessive genomic DNA copy number variation in the Li–Fraumeni cancer predisposition syndrome

    PubMed Central

    Shlien, Adam; Tabori, Uri; Marshall, Christian R.; Pienkowska, Malgorzata; Feuk, Lars; Novokmet, Ana; Nanda, Sonia; Druker, Harriet; Scherer, Stephen W.; Malkin, David

    2008-01-01

    DNA copy number variations (CNVs) are a significant and ubiquitous source of inherited human genetic variation. However, the importance of CNVs to cancer susceptibility and tumor progression has not yet been explored. Li–Fraumeni syndrome (LFS) is an autosomal dominantly inherited disorder characterized by a strikingly increased risk of early-onset breast cancer, sarcomas, brain tumors and other neoplasms in individuals harboring germline TP53 mutations. Known genetic determinants of LFS do not fully explain the variable clinical phenotype in affected family members. As part of a wider study of CNVs and cancer, we conducted a genome-wide profile of germline CNVs in LFS families. Here, by examining DNA from a large healthy population and an LFS cohort using high-density oligonucleotide arrays, we show that the number of CNVs per genome is well conserved in the healthy population, but strikingly enriched in these cancer-prone individuals. We found a highly significant increase in CNVs among carriers of germline TP53 mutations with a familial cancer history. Furthermore, we identified a remarkable number of genomic regions in which known cancer-related genes coincide with CNVs, in both LFS families and healthy individuals. Germline CNVs may provide a foundation that enables the more dramatic chromosomal changes characteristic of TP53-related tumors to be established. Our results suggest that screening families predisposed to cancer for CNVs may identify individuals with an abnormally high number of these events. PMID:18685109

  16. Transfer of Large Contiguous DNA Fragments onto a Low Copy Plasmid or into the Bacterial Chromosome

    PubMed Central

    Reeves, Analise Z; Lesser, Cammie F

    2017-01-01

    Bacterial pathogenicity islands and other contiguous operons can be difficult to clone using conventional methods due to their large size. Here we describe a robust 3-step method to transfer large defined fragments of DNA from virulence plasmids or cosmids onto smaller autonomously replicating plasmids or directly into defined sites in the bacterial chromosome that incorporates endogenous yeast and λ Red homologous recombination systems. This methodology has been successfully used to isolate and integrate at least 31 kb of contiguous DNA and can be readily adapted for the recombineering of E. coli and its close relatives. PMID:28203614

  17. High-resolution genomic copy number profiling of glioblastoma multiforme by single nucleotide polymorphism DNA microarray.

    PubMed

    Yin, Dong; Ogawa, Seishi; Kawamata, Norihiko; Tunici, Patrizia; Finocchiaro, Gaetano; Eoli, Marica; Ruckert, Christian; Huynh, Thien; Liu, Gentao; Kato, Motohiro; Sanada, Masashi; Jauch, Anna; Dugas, Martin; Black, Keith L; Koeffler, H Phillip

    2009-05-01

    Glioblastoma multiforme (GBM) is an extremely malignant brain tumor. To identify new genomic alterations in GBM, genomic DNA of tumor tissue/explants from 55 individuals and 6 GBM cell lines were examined using single nucleotide polymorphism DNA microarray (SNP-Chip). Further gene expression analysis relied on an additional 56 GBM samples. SNP-Chip results were validated using several techniques, including quantitative PCR (Q-PCR), nucleotide sequencing, and a combination of Q-PCR and detection of microsatellite markers for loss of heterozygosity with normal copy number [acquired uniparental disomy (AUPD)]. Whole genomic DNA copy number in each GBM sample was profiled by SNP-Chip. Several signaling pathways were frequently abnormal. Either the p16(INK4A)/p15(INK4B)-CDK4/6-pRb or p14(ARF)-MDM2/4-p53 pathways were abnormal in 89% (49 of 55) of cases. Simultaneous abnormalities of both pathways occurred in 84% (46 of 55) samples. The phosphoinositide 3-kinase pathway was altered in 71% (39 of 55) GBMs either by deletion of PTEN or amplification of epidermal growth factor receptor and/or vascular endothelial growth factor receptor/platelet-derived growth factor receptor alpha. Deletion of chromosome 6q26-27 often occurred (16 of 55 samples). The minimum common deleted region included PARK2, PACRG, QKI, and PDE10A genes. Further reverse transcription Q-PCR studies showed that PARK2 expression was decreased in another collection of GBMs at a frequency of 61% (34 of 56) of samples. The 1p36.23 region was deleted in 35% (19 of 55) of samples. Notably, three samples had homozygous deletion encompassing this site. Also, a novel internal deletion of a putative tumor suppressor gene, LRP1B, was discovered causing an aberrant protein. AUPDs occurred in 58% (32 of 55) of the GBM samples and five of six GBM cell lines. A common AUPD was found at chromosome 17p13.3-12 (included p53 gene) in 13 of 61 samples and cell lines. Single-strand conformational polymorphism and nucleotide

  18. Effect of location, organization, and repeat-copy number in satellite-DNA evolution.

    PubMed

    Navajas-Pérez, R; Quesada del Bosque, M E; Garrido-Ramos, M A

    2009-10-01

    Here, we analyze the evolutionary dynamics of a satellite-DNA family in an attempt to understand the effect of factors such as location, organization, and repeat-copy number in the molecular drive process leading to the concerted-evolution pattern found in this type of repetitive sequences. The presence of RAE180 satellite-DNA in the dioecious species of the plant genus Rumex is a noteworthy feature at this respect, as RAE180 satellite repeats have accumulated differentially, showing a distinct distribution pattern in different species. The evolution of dioecious Rumex gave rise to two phylogenetic clades: one clade composed of species with an ancestral XX/XY sex chromosome system and a second, derived clade of species with a multiple sex-chromosome system XX/XY(1)Y(2). While in the XX/XY dioecious species, the RAE180 satellite-DNA is located only in a small autosomal locus, the RAE180 repeats are present also in a small autosomal locus and additionally have been massively amplified in the Y chromosomes of XX/XY(1)Y(2) species. Here, we have found that the RAE180 repeats of the autosomal locus of XX/XY species are characterized by intra-specific sequence homogeneity and inter-specific divergence and that the comparison of individual nucleotide positions between related species shows a general pattern of concerted evolution. On the contrary, both in the autosomal and the Y-linked loci of XX/XY(1)Y(2) species, ancestral variability has remained with reduced rates of sequence homogenization and of evolution. Thus, this study demonstrates that molecular mechanisms of non-reciprocal exchange are key factors in the molecular drive process; the satellite DNAs in the non-recombining Y chromosomes show low rates of concerted evolution and intra-specific variability increase with no inter-specific divergence. By contrast, freely recombining loci undergo concerted evolution with genetic differentiation between species as occurred in the autosomal locus of XX/XY species

  19. Integrated Sensing Using DNA Nanoarchitectures

    DTIC Science & Technology

    2014-05-20

    Norton. Thiolated Dendrimers as Multi-Point Binding Headgroups for DNA Immobilization on Gold, Langmuir, (10 2011): 0. doi: 10.1021/la202444s...Figure 6, uses dendrimers to provide multipoint adhesion of a single stranded DNA component on a surface. Figure 6 Process for immobilizing... dendrimer (shown as a round species). These dendrimer species are Generation 3 PAMAM dendrimers with ~ 30 thiol groups to bind the dendrimer /DNA construct

  20. Integrative Analysis of Transcriptional Regulatory Network and Copy Number Variation in Intrahepatic Cholangiocarcinoma

    PubMed Central

    Li, Ling; Lian, Baofeng; Li, Chao; Li, Wei; Li, Jing; Zhang, Yuannv; He, Xianghuo; Li, Yixue; Xie, Lu

    2014-01-01

    Background Transcriptional regulatory network (TRN) is used to study conditional regulatory relationships between transcriptional factors and genes. However few studies have tried to integrate genomic variation information such as copy number variation (CNV) with TRN to find causal disturbances in a network. Intrahepatic cholangiocarcinoma (ICC) is the second most common hepatic carcinoma with high malignancy and poor prognosis. Research about ICC is relatively limited comparing to hepatocellular carcinoma, and there are no approved gene therapeutic targets yet. Method We first constructed TRN of ICC (ICC-TRN) using forward-and-reverse combined engineering method, and then integrated copy number variation information with ICC-TRN to select CNV-related modules and constructed CNV-ICC-TRN. We also integrated CNV-ICC-TRN with KEGG signaling pathways to investigate how CNV genes disturb signaling pathways. At last, unsupervised clustering method was applied to classify samples into distinct classes. Result We obtained CNV-ICC-TRN containing 33 modules which were enriched in ICC-related signaling pathways. Integrated analysis of the regulatory network and signaling pathways illustrated that CNV might interrupt signaling through locating on either genomic sites of nodes or regulators of nodes in a signaling pathway. In the end, expression profiles of nodes in CNV-ICC-TRN were used to cluster the ICC patients into two robust groups with distinct biological function features. Conclusion Our work represents a primary effort to construct TRN in ICC, also a primary effort to try to identify key transcriptional modules based on their involvement of genetic variations shown by gene copy number variations (CNV). This kind of approach may bring the traditional studies of TRN based only on expression data one step further to genetic disturbance. Such kind of approach can easily be extended to other disease samples with appropriate data. PMID:24897108

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

    PubMed

    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.

  2. Reduced rDNA copy number does not affect "competitive" chromosome pairing in XYY males of Drosophila melanogaster.

    PubMed

    Maggert, Keith A

    2014-03-20

    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 "competitive" situation in which complex pairing configurations were possible using males with XYY constitution. Using a common normal X chromosome, one of two different full-length Y chromosomes, and a third chromosome from a series of otherwise-isogenic rDNA deletions, I detected no differences in X-Y or Y-Y pairing or chromosome segregation frequencies that could not be attributed to random variation alone. This work was performed in the context of an undergraduate teaching program at Texas A&M University, and I discuss the pedagogical utility of this and other such experiments.

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

    PubMed Central

    Maggert, Keith A.

    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 “competitive” situation in which complex pairing configurations were possible using males with XYY constitution. Using a common normal X chromosome, one of two different full-length Y chromosomes, and a third chromosome from a series of otherwise-isogenic rDNA deletions, I detected no differences in X-Y or Y-Y pairing or chromosome segregation frequencies that could not be attributed to random variation alone. This work was performed in the context of an undergraduate teaching program at Texas A&M University, and I discuss the pedagogical utility of this and other such experiments. PMID:24449686

  4. The effects of a wheat germ rich diet on oxidative mtDNA damage, mtDNA copy number and antioxidant enzyme activities in aging Drosophila.

    PubMed

    Mutlu, Ayse Gul

    2013-03-01

    The free radical theory of aging posits that the accumulation of macromolecular damage induced by toxic reactive oxygen species plays a central role in the aging process. Therefore consumption of dietary antioxidants appears to be of great importance. Wheat germ have strong antioxidant properties. Aim of this study is investigate the effects of a wheat germ rich diet on oxidative mtDNA damage, mtDNA copy number and antioxidant enzyme activities in Drosophila. Current results suggested that dietary wheat germ enhances the activities of antioxidant enzymes in Drosophila. There was no statistically difference in mtDNA damage and mtDNA copy number results of "Wheat Germ" and "Refined White Flour" feed groups. mtDNA damage slightly increased with aging in both groups but these changes were no statistically different.

  5. FACETS: allele-specific copy number and clonal heterogeneity analysis tool for high-throughput DNA sequencing

    PubMed Central

    Shen, Ronglai; Seshan, Venkatraman E.

    2016-01-01

    Allele-specific copy number analysis (ASCN) from next generation sequencing (NGS) data can greatly extend the utility of NGS beyond the identification of mutations to precisely annotate the genome for the detection of homozygous/heterozygous deletions, copy-neutral loss-of-heterozygosity (LOH), allele-specific gains/amplifications. In addition, as targeted gene panels are increasingly used in clinical sequencing studies for the detection of ‘actionable’ mutations and copy number alterations to guide treatment decisions, accurate, tumor purity-, ploidy- and clonal heterogeneity-adjusted integer copy number calls are greatly needed to more reliably interpret NGS-based cancer gene copy number data in the context of clinical sequencing. We developed FACETS, an ASCN tool and open-source software with a broad application to whole genome, whole-exome, as well as targeted panel sequencing platforms. It is a fully integrated stand-alone pipeline that includes sequencing BAM file post-processing, joint segmentation of total- and allele-specific read counts, and integer copy number calls corrected for tumor purity, ploidy and clonal heterogeneity, with comprehensive output and integrated visualization. We demonstrate the application of FACETS using The Cancer Genome Atlas (TCGA) whole-exome sequencing of lung adenocarcinoma samples. We also demonstrate its application to a clinical sequencing platform based on a targeted gene panel. PMID:27270079

  6. Specific patterns of DNA copy number gains and losses in eight new glioblastoma multiforme cell lines.

    PubMed

    Ramirez, Tzutzuy; Thoma, Karen; Taja-Chayeb, Lucia; Efferth, Thomas; Herrera, Luis A; Halatsch, Marc-Eric; Gebhart, Erich

    2003-08-01

    Eight cell lines newly established from glioblastoma multiforme were examined by comparative genomic hybridization for their patterns of genomic imbalance. The total number of DNA copy number alterations (CNAs) found in the eight cell lines varied between 15 and 24. This characterized the examined cell lines (or the tumors they were derived from) as distinctly progressed in karyotypic evolution. The most frequent CNAs were gains of the entire chromosome 6 or, at least, parts of it, and of 7p22, which were found in all eight cell lines. Other changes present in seven of the eight cell lines were gains of 3q26qter and the entire chromosome 7 and losses of segments on chromosome 4q (e.g., 4q34q35) and of the short arm of chromosome 10. Enh(3q21q25), dim(4q22q33) and dim(4qter), dim(13q22), enh(15q14), and enh(18q22q23) were found in six of the eight cell lines. Several other CNAs [e.g., dim(9p21)] were found in common in five or less of the eight lines. Using a hierarchical cluster analysis, the specific patterns of genomic imbalance allowed various groupings of the examined cell lines. Although a close relation could be confirmed among all examined lines on the basis of shared CNAs, two main groups could be roughly differentiated. Among those there were also more or less closely related subgroups. However, also alterations which were restricted to one single cell line each were found, e.g., dim(1q41qter), dim(2q22qter), enh(4p), dim(5p), dim(4p13pter), dim(8q21qter), enh(9p), dim(9q), dim(11p14pter), enh(12q15q23), enh(13q21), dim(14q21qter), dim(15q21qter), dim(19q), and enh(22q). The comparison of the obtained data on gains and losses of DNA copy numbers in specific chromosomal segments with the data on localization of genes possibly associated with the biology of glioblastoma multiforme additionally shows high conformity but also disparity of the examined cell lines among each other, as well as compared to primary glioblastoma multiforme. Eventually, each of the

  7. Transcription reporters that shuttle cloned DNA between high-copy Escherichia coli plasmids and low-copy broad-host-range plasmids.

    PubMed

    Ouimet, M C; Marczynski, G T

    2000-09-01

    We describe and apply lacZ transcription reporter plasmids designed for both biochemical analyses requiring high DNA yield and physiological studies requiring low gene dosage. Standard DNA ligations are performed at seven unique restriction sites 5' to the lacZ gene on high-copy ColE1 plasmids suitable for double- or single-strand DNA sequencing. A divergent gusA transcription reporter is included and serves as an internal control. Rec(+) Escherichia coli cells readily shuttle DNA placed between gusA and lacZ by allelic exchange with pRK290-based plasmids that subsequently conjugate and replicate in most gram-negative bacteria. We applied this system to study Caulobacter crescentus cell cycle promoters directed by the CtrA response-regulator protein. Synthetic oligonucleotides were ligated to create altered CtrA binding sites and corresponding promoters with varied transcription strength. We also document the phenomenon of long-range promoter interference. A strong promoter can repress up to twofold the transcription from a divergent promoter located 100 bp away. However, the cell cycle timing of both promoters is not changed. Additional applications of our system and theoretical aspects of promoter organization are discussed. Copyright 2000 Academic Press.

  8. Fluorescence imaging of single-copy DNA sequences within the human genome using PNA-directed padlock probe assembly

    PubMed Central

    Yaroslavsky, Anastasia I.; Smolina, Irina V.

    2013-01-01

    SUMMARY We present a novel approach for fluorescent in situ detection of short, single-copy sequences within genomic DNA in human cells. The single copy sensitivity and single base specificity of our method is achieved due to the combination of three components. First, a peptide nucleic acid (PNA) probe locally opens a chosen target site, which allows a padlock DNA probe to access the site and become ligated. Second, rolling circle amplification (RCA) generates thousands of single-stranded copies of the target sequence. Finally, fluorescent in situ hybridization (FISH) is used to visualize the amplified DNA. We validate this new technique by successfully detecting six unique target sites on human mitochondrial and autosomal DNA. We also demonstrate the high specificity of this method by detecting X- and Y- specific sequences on human sex chromosomes and by simultaneously detecting three unique target sites. Finally, we discriminate two target sites that differ by two nucleotides. The PNA-RCA-FISH approach is a unique in situ hybridization method capable of multi-target visualization within human chromosomes and nuclei that does not require DNA denaturation and is extremely sequence specific. PMID:23521801

  9. The FACT complex promotes avian leukosis virus DNA integration.

    PubMed

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

    2017-01-25

    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. Here, 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-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.

  10. Simultaneous quantification of mitochondrial DNA copy number and deletion ratio: A multiplex real-time PCR assay

    PubMed Central

    Phillips, Nicole R.; Sprouse, Marc L.; Roby, Rhonda K.

    2014-01-01

    Mitochondrial dysfunction is implicated in a vast array of diseases and conditions, such as Alzheimer's disease, cancer, and aging. Alterations in mitochondrial DNA (mtDNA) may provide insight into the processes that either initiate or propagate this dysfunction. Here, we describe a unique multiplex assay which simultaneously provides assessments of mtDNA copy number and the proportion of genomes with common large deletions by targeting two mitochondrial sites and one nuclear locus. This probe-based, single-tube multiplex provides high specificity while eliminating well-to-well variability that results from assaying nuclear and mitochondrial targets individually. PMID:24463429

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

    PubMed Central

    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

  12. Mitochondrial DNA integrity changes with age but does not correlate with learning performance in honey bees.

    PubMed

    Hystad, E M; Amdam, G V; Eide, L

    2014-01-01

    The honey bee is a well-established model organism to study aging, learning and memory. Here, we used young and old forager honey bees to investigate whether age-related learning capacity correlates with mitochondrial function. The bees were selected for age and learning performance and mitochondrial function was evaluated by measuring mtDNA integrity, mtDNA copy number and mitochondrial gene expression. Quite unexpectedly, mtDNA from young bees showed more damage than mtDNA from older bees, but neither mtDNA integrity, nor mtDNA copy number nor mitochondrial gene expression correlated with learning performance. Although not statistically significant (p=0.07) the level of L-rRNA increased with age in good learners whereas it decreased in poor learners. Our results show that learning performance in honey bee does not correlate with absolute mitochondrial parameters like mtDNA damage, copy number or expression of mitochondrial genes, but may be associated with the ability to regulate mitochondrial activity.

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

    PubMed

    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.

  14. Increased Mitochondrial DNA Copy Number in Occupations Associated with Low-Dose Benzene Exposure

    PubMed Central

    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

    2011-01-01

    Background: 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. Objectives: 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. Methods: 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). Results: 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). Conclusions: Blood MtDNAcn was increased in persons exposed to low benzene levels, potentially reflecting mitochondrial

  15. DNA copy number aberrations in small-cell lung cancer reveal activation of the focal adhesion pathway

    PubMed Central

    Ocak, S; Yamashita, H; Udyavar, AR; Miller, AN; Gonzalez, AL; Zou, Y; Jiang, A; Yi, Y; Shyr, Y; Estrada, L; Quaranta, V; Massion, PP

    2015-01-01

    Small-cell lung cancer (SCLC) is the most aggressive subtype of lung cancer in its clinical behavior, with a 5-year overall survival as low as 5%. Despite years of research in the field, molecular determinants of SCLC behavior are still poorly understood, and this deficiency has translated into an absence of specific diagnostics and targeted therapeutics. We hypothesized that tumor DNA copy number alterations would allow the identification of molecular pathways involved in SCLC progression. Array comparative genomic hybridization was performed on DNA extracted from 46 formalin-fixed paraffin-embedded SCLC tissue specimens. Genomic profiling of tumor and sex-matched control DNA allowed the identification of 70 regions of copy number gain and 55 regions of copy number loss. Using molecular pathway analysis, we found a strong enrichment in these regions of copy number alterations for 11 genes associated with the focal adhesion pathway. We verified these findings at the genomic, gene expression and protein level. Focal Adhesion Kinase (FAK), one of the central genes represented in this pathway, was commonly expressed in SCLC tumors and constitutively phosphorylated in SCLC cell lines. Those were poorly adherent to most substrates but not to laminin-322. Inhibition of FAK phosphorylation at Tyr397 by a small-molecule inhibitor, PF-573,228, induced a dose-dependent decrease of adhesion and an increase of spreading in SCLC cell lines on laminin-322. Cells that tended to spread also showed a decrease in focal adhesions, as demonstrated by a decreased vinculin expression. These results support the concept that pathway analysis of genes in regions of copy number alterations may uncover molecular mechanisms of disease progression and demonstrate a new role of FAK and associated adhesion pathways in SCLC. Further investigations of FAK at the functional level may lead to a better understanding of SCLC progression and may have therapeutic implications. PMID:20802517

  16. Localization of single-copy T-DNA insertion in transgenic shallots (Allium cepa) by using ultra-sensitive FISH with tyramide signal amplification.

    PubMed

    Khrustaleva, L I; Kik, C

    2001-03-01

    The sensitivity of fluorescence in situ hybridization (FISH) for mapping plant chromosomes of single-copy DNA sequences is limited. We have adapted for plant cytogenetics a new signal-amplification method termed tyramide-FISH (Tyr-FISH). Until present this technique has only been applied to human chromosomes. The method is based on enzymatic deposition of fluorochrome-conjugated tyramide. With Tyr-FISH it was possible to detect target T-DNA sequences on plant metaphase chromosomes as small as 710 bp without using a cooled CCD camera. Short detection time and high sensitivity, in combination with a low background, make the Tyr-FISH method very suitable for routine application in plant cytogenetic research. With Tyr-FISH we analysed the position of T-DNA inserts in transgenic shallots. We found that the inserts were preferentially located in the distal region of metaphase chromosomes. Sequential fluorescence in situ hybridization with a 375 bp satellite sequence suggested that a specific T-DNA insert was located within the satellite sequence hybridization region on a metaphase chromosome. Analysis of less-condensed prophase and interphase chromosomes revealed that the T-DNA was integrated outside the satellite DNA-hybridization region in a more proximal euchromatin region.

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

    PubMed Central

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

    2013-01-01

    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. PMID:23295674

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

    PubMed

    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.

  19. Integrated genomic, transcriptomic, and RNA-interference analysis of genes in somatic copy number gains in pancreatic ductal adenocarcinoma.

    PubMed

    Samuel, Nardin; Sayad, Azin; Wilson, Gavin; Lemire, Mathieu; Brown, Kevin R; Muthuswamy, Lakshmi; Hudson, Thomas J; Moffat, Jason

    2013-08-01

    This study used an integrated analysis of copy number, gene expression, and RNA interference screens for identification of putative driver genes harbored in somatic copy number gains in pancreatic ductal adenocarcinoma (PDAC). Somatic copy number gain data on 60 PDAC genomes were extracted from public data sets to identify genomic loci that are recurrently gained. Array-based data from a panel of 29 human PDAC cell lines were used to quantify associations between copy number and gene expression for the set of genes found in somatic copy number gains. The most highly correlated genes were assessed in a compendium of pooled short hairpin RNA screens on 27 of the same human PDAC cell lines. A catalog of 710 protein-coding and 46 RNA genes mapping to 20 recurrently gained genomic loci were identified. The gene set was further refined through stringent integration of copy number, gene expression, and RNA interference screening data to uncover 34 candidate driver genes. Among the candidate genes from the integrative analysis, ECT2 was found to have significantly higher essentiality in specific PDAC cell lines with genomic gains at the 3q26.3 locus, which harbors this gene, suggesting that ECT2 may play an oncogenic role in the PDAC neoplastic process.

  20. Relationship between mitochondrial DNA Copy Number and SIRT1 Expression in Porcine Oocytes

    PubMed Central

    Sato, Daichi; Itami, Nobuhiko; Tasaki, Hidetaka; Takeo, Shun; Kuwayama, Takehito; Iwata, Hisataka

    2014-01-01

    The present study assessed the effect of resveratrol on the expression of SIRT1 and mitochondrial quality and quantity in porcine oocytes. Supplementing the maturation medium with 20 µM resveratrol increased the expression of SIRT1, and enhanced mitochondrial functions, as observed from the increased ATP content and mitochondrial membrane potential. Addition of resveratrol also improved the ability of oocytes to develop into the blastocyst stage following activation. The effects of resveratrol on mitochondrial number were examined by comparing the mitochondrial DNA copy number (Mt number) between group of oocytes collected from the same donor gilt ovaries. Supplementing the maturation medium with only resveratrol did not affect the Mt number in the oocytes. However, supplementing the maturation medium with 10 µM MG132, a proteasome inhibitor, significantly increased the amount of ubiquitinated proteins and Mt number by 12 and 14%, respectively. In addition, when resveratrol was added to the medium containing MG132, the Mt number increased significantly by 39%, this effect was diminished by the addition of the SIRT1 inhibitor EX527. Furthermore, supplementing the medium with MG132 and EX527 did not affect Mt number. The mean SIRT1 expression in 20 oocytes was significantly and positively correlated with the Mt number in oocytes collected from the same donor. This study suggests that the expression of SIRT1 is associated with the Mt number in oocytes. In addition, activation of SIRT1 by resveratrol enhances the biosynthesis and degradation of mitochondria in oocytes, thereby replenishing and improving mitochondrial function and the developmental ability of oocytes. PMID:24747689

  1. Discovering subgroups of patients from DNA copy number data using NMF on compacted matrices.

    PubMed

    de Campos, Cassio P; Rancoita, Paola M V; Kwee, Ivo; Zucca, Emanuele; Zaffalon, Marco; Bertoni, Francesco

    2013-01-01

    In the study of complex genetic diseases, the identification of subgroups of patients sharing similar genetic characteristics represents a challenging task, for example, to improve treatment decision. One type of genetic lesion, frequently investigated in such disorders, is the change of the DNA copy number (CN) at specific genomic traits. Non-negative Matrix Factorization (NMF) is a standard technique to reduce the dimensionality of a data set and to cluster data samples, while keeping its most relevant information in meaningful components. Thus, it can be used to discover subgroups of patients from CN profiles. It is however computationally impractical for very high dimensional data, such as CN microarray data. Deciding the most suitable number of subgroups is also a challenging problem. The aim of this work is to derive a procedure to compact high dimensional data, in order to improve NMF applicability without compromising the quality of the clustering. This is particularly important for analyzing high-resolution microarray data. Many commonly used quality measures, as well as our own measures, are employed to decide the number of subgroups and to assess the quality of the results. Our measures are based on the idea of identifying robust subgroups, inspired by biologically/clinically relevance instead of simply aiming at well-separated clusters. We evaluate our procedure using four real independent data sets. In these data sets, our method was able to find accurate subgroups with individual molecular and clinical features and outperformed the standard NMF in terms of accuracy in the factorization fitness function. Hence, it can be useful for the discovery of subgroups of patients with similar CN profiles in the study of heterogeneous diseases.

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

    PubMed

    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).

  3. Activities of Human Immunodeficiency Virus (HIV) Integration Protein In vitro: Specific Cleavage and Integration of HIV DNA

    NASA Astrophysics Data System (ADS)

    Bushman, Frederic D.; Craigie, Robert

    1991-02-01

    Growth of human immunodeficiency virus (HIV) after infection requires the integration of a DNA copy of the viral RNA genome into a chromosome of the host. Here we present a simple in vitro system that carries out the integration reaction and the use of this system to probe the mechanism of integration. The only HIV protein necessary is the integration (IN) protein, which has been overexpressed in insect cells and then partially purified. DNA substrates are supplied as oligonucleotides that match the termini of the linear DNA product of reverse transcription. In the presence of HIV IN protein, oligonucleotide substrates are cleaved to generate the recessed 3' ends that are the precursor for integration, and the cleaved molecules are efficiently inserted into a DNA target. Analysis of reaction products reveals that HIV IN protein joins 3' ends of the viral DNA to 5' ends of cuts made by IN protein in the DNA target. We have also used this assay to characterize the sequences at the ends of the viral DNA involved in integration. The assay provides a simple screen for testing candidate inhibitors of HIV IN protein; some such inhibitors might have useful antiviral activity.

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

    PubMed

    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 .

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

    PubMed

    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 (<45 years old; 7.17 vs 6.64, P=0.005 and 7.21 vs 6.84, P=0.036). A significantly higher mtDNA 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.

  6. High Mitochondrial DNA Copy Number Is a Protective Factor From Vision Loss in Heteroplasmic Leber's Hereditary Optic Neuropathy (LHON).

    PubMed

    Bianco, Angelica; Bisceglia, Luigi; Russo, Luciana; Palese, Luigi L; D'Agruma, Leonardo; Emperador, Sonia; Montoya, Julio; Guerriero, Silvana; Petruzzella, Vittoria

    2017-04-01

    Leber's hereditary optic neuropathy (LHON) is a mitochondrial disease that typically causes bilateral blindness in young men. It is characterized by as yet undisclosed genetic and environmental factors affecting the incomplete penetrance. We identified 27 LHON subjects who possess heteroplasmic primary LHON mutations. Mitochondrial DNA (mtDNA) copy number was evaluated. The presence of centrocecal scotoma, an edematous, hyperemic optic nerve head, and vascular tortuosity, as well as telangiectasia was recognized in affected subjects. We found higher cellular mtDNA content in peripheral blood cells of unaffected heteroplasmic mutation carriers with respect to the affected. The increase of cellular mtDNA content prevents complete loss of vision despite the presence of a heteroplasmic state of LHON primary mutation, suggesting that it is a key factor responsible for penetrance of LHON.

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

    PubMed Central

    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

  8. RUBIC identifies driver genes by detecting recurrent DNA copy number breaks

    PubMed Central

    van Dyk, Ewald; Hoogstraat, Marlous; ten Hoeve, Jelle; Reinders, Marcel J. T.; Wessels, Lodewyk F. A.

    2016-01-01

    The frequent recurrence of copy number aberrations across tumour samples is a reliable hallmark of certain cancer driver genes. However, state-of-the-art algorithms for detecting recurrent aberrations fail to detect several known drivers. In this study, we propose RUBIC, an approach that detects recurrent copy number breaks, rather than recurrently amplified or deleted regions. This change of perspective allows for a simplified approach as recursive peak splitting procedures and repeated re-estimation of the background model are avoided. Furthermore, we control the false discovery rate on the level of called regions, rather than at the probe level, as in competing algorithms. We benchmark RUBIC against GISTIC2 (a state-of-the-art approach) and RAIG (a recently proposed approach) on simulated copy number data and on three SNP6 and NGS copy number data sets from TCGA. We show that RUBIC calls more focal recurrent regions and identifies a much larger fraction of known cancer genes. PMID:27396759

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

    USDA-ARS?s Scientific Manuscript database

    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...

  10. 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.

    PubMed

    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.

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

    PubMed Central

    Humphrey, Bruce; McLeod, Neil; Turner, Carrie; Sutton, J. Mark; Dark, Paul M.; Warhurst, Geoffrey

    2015-01-01

    Background 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. Results 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. PMID:26172943

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

    PubMed

    Humphrey, Bruce; McLeod, Neil; Turner, Carrie; Sutton, J Mark; Dark, Paul M; Warhurst, Geoffrey

    2015-01-01

    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.

  13. Leukocyte mitochondrial DNA copy number, anthropometric indices, and weight change in US women

    PubMed Central

    Meng, Shasha; Wu, Shaowei; Liang, Liming; Liang, Geyu; Giovannucci, Edward; Vivo, Immaculata De; Nan, Hongmei

    2016-01-01

    Objectives To examine the association between leukocyte mitochondrial DNA copy number (mtCN) and different anthropometric indices as well as weight changes; and to compare mtCN and telomere length with respect to their associations with BMI and age. Design Population based cohort study. Setting Nurses' Health Study, an ongoing prospective cohort study of 121,700 nurses enrolled in 1976; in 1989-1990 a subset of 32,826 women provided blood samples. Participants 1,700 disease-free US women from case-control studies nested within the Nurses' Health Study with mtCN and telomere length measured who also have anthropometric measurements. Main outcome measure Relative mtCN and telomere lengths in peripheral blood leukocytes measured by quantitative real time polymerase chain reaction and various anthropometric measurements data from initial questionnaire. Results Leukocyte mtCN was inversely associated with current weight (LS means Q1-Q4: 0.07, 0.04, 0.03, −0.17; P trend =0.002), waist size (LS means Q1-Q4: 0.06, 0.05, −0.04, −0.06; P trend = 0.04), BMI (LS means normal light, normal heavy, overweight, pre-obese, obese: 0.11, −0.01, −0.04, 0.04, −0.25; P trend<0.0001), and waist-hip ratio (WHR) (LS means Q1-Q4: 0.06, 0.08, −0.04, −0.06; P trend = 0.03). A one-unit decrease in mtCN z score was equivalent to approximately 3.5 pounds of weight gain for an adult of 5′10″. In addition, weight gain was bi-directionally and inversely associated with mtCN. Moreover, mtCN was strongly positively correlated with telomere length (LS means Q1-Q4: −0.02, 0.09, 0.11, 0.33; P trend <0.0001). MtCN was inversely associated with BMI even after adjusting for telomere length (P trend =0.003), while telomere length was not associated with BMI. On the other hand, telomere length was inversely associated with age after adjusting for mtCN (P trend =0.04), while mtCN was not associated with age. Conclusions Our results provide compelling evidence for a potential bi

  14. Coordinating DNA replication to produce one copy of the genome requires genes that act in ubiquitin metabolism.

    PubMed Central

    Singer, J D; Manning, B M; Formosa, T

    1996-01-01

    We have developed a genetic screen of the yeast Saccharomyces cerevisiae to identify genes that act to coordinate DNA replication so that each part of the genome is copied exactly once per cell cycle. A mutant was recovered in this screen that accumulates aberrantly high DNA contents but does not complete a second round of synthesis. The mutation principally responsible for this phenotype is in the DOA4 gene, which encodes a ubiquitin hydrolase, one of several yeast genes that encode enzymes that can remove the signalling polypeptide ubiquitin hydrolase, one of several yeast genes that encode enzymes that can remove the signaling polypeptide ubiquitin from its covalently linked conjugated forms. DOA4 is nonessential, and deleting this gene causes uncoordinated replication. Overreplication does not occur in cells with limiting amounts of Cdc7 protein kinase, suggesting that entry into S phase is required for this phenotype. The DNA formed in doa4 mutants is not highly unusual in the sense that mitotic recombination rates are normal, implying that a high level of repair is not induced. The temperature sensitivity of doa4 mutations is partially suppressed by extra copies of the polyubiquitin gene UB14, but overreplication still occurs in the presence of this suppressor. Mutations in DOA4 cause loss of the free ubiquitin pool in cells under heat stress conditions, and extra copies of UB14 restore this pool without restoring coordination of replication. We conclude that a ubiquitin-mediated signaling event directly involving the ubiquitin hydrolase encoded by DOA4 is needed in S. cerevisiae to prevent uncoordinated DNA replication. PMID:8657109

  15. Optimal design of oligonucleotide microarrays for measurement of DNA copy-number.

    PubMed

    Sharp, Andrew J; Itsara, Andy; Cheng, Ze; Alkan, Can; Schwartz, Stuart; Eichler, Evan E

    2007-11-15

    Copy-number variants (CNVs) occur frequently within the human genome, and may be associated with many human phenotypes. If disease association studies of CNVs are to be performed routinely, it is essential that the copy-number status be accurately genotyped. We systematically assessed the dynamic range response of an oligonucleotide microarray platform to accurately predict copy-number in a set of seven patients who had previously been shown to carry between 1 and 6 copies of an approximately 4 Mb region of 15q12.2-q13.1. We identify probe uniqueness, probe length, uniformity of probe melting temperature, overlap with SNPs and common repeats (particularly Alu elements) and guanine homopolymer content as parameters that significantly affect probe performance. Further, we prove the influence of these criteria on array performance by using these parameters to prospectively filter data from a second array design covering an independent genomic region and observing significant improvements in data quality. The informed selection of probes which have superior performance characteristics allows the prospective design of oligonucleotide arrays which show increased sensitivity and specificity compared with current designs. Although based on the analysis of data from comparative genomic hybridization experiments, we anticipate that our results are relevant to the design of improved oligonucleotide arrays for high-throughput copy-number genotyping of complex regions of the human genome.

  16. High-resolution analysis of DNA copy number alterations in patients with isolated sporadic keratoconus

    PubMed Central

    Hellani, Ali M.; Al Mansouri, Sameer M.; Kalantan, Hatem; Al-Muammar, Abdulrahman M.

    2011-01-01

    Purpose To determine whether patients with sporadic, non-familial keratoconus and no pathogenic mutations in the visual system homeobox 1 (VSX1) gene have evidence of chromosomal copy number alterations. Methods Twenty Saudi Arabian patients with isolated keratoconus, no family history of the disease and no mutations in VSX1 were recruited. Additionally, 10 ethnically-matched healthy controls were also recruited for this study. We screened patients for chromosomal copy number aberrations using the Agilent Human Genome CGH 244A Oligo Microarray Chip. Results None of the keratoconus patients screened had evidence of chromosomal copy number alterations when compared to normal ethnically matched controls. Conclusions Chromosomal deletions and/or duplications were not detected in any of the patients tested here. Other chromosomal imbalances such as translocations, inversions, and some ploidies cannot be detected by current array CGH technology and other nuclear genetic or epigenetic factors cannot be excluded as a possible contributing factor to keratoconus pathogenesis. PMID:21528002

  17. High-resolution analysis of DNA copy number alterations in patients with isolated sporadic keratoconus.

    PubMed

    Abu-Amero, Khaled K; Hellani, Ali M; Al Mansouri, Sameer M; Kalantan, Hatem; Al-Muammar, Abdulrahman M

    2011-03-30

    To determine whether patients with sporadic, non-familial keratoconus and no pathogenic mutations in the visual system homeobox 1 (VSX1) gene have evidence of chromosomal copy number alterations. Twenty Saudi Arabian patients with isolated keratoconus, no family history of the disease and no mutations in VSX1 were recruited. Additionally, 10 ethnically-matched healthy controls were also recruited for this study. We screened patients for chromosomal copy number aberrations using the Agilent Human Genome CGH 244A Oligo Microarray Chip. None of the keratoconus patients screened had evidence of chromosomal copy number alterations when compared to normal ethnically matched controls. Chromosomal deletions and/or duplications were not detected in any of the patients tested here. Other chromosomal imbalances such as translocations, inversions, and some ploidies cannot be detected by current array CGH technology and other nuclear genetic or epigenetic factors cannot be excluded as a possible contributing factor to keratoconus pathogenesis.

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

    PubMed

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

    USDA-ARS?s Scientific Manuscript database

    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...

  20. Induction of direct repeat recombination by psoralen-DNA adducts in Saccharomyces cerevisiae: defects in DNA repair increase gene copy number variation.

    PubMed

    Saffran, Wilma A; Ahmed, Anam; Binyaminov, Olga; Gonzalez, Cynthia; Gupta, Amita; Fajardo, Manuel A; Kishun, Devindra; Nandram, Ashana; Reyes, Kenneth; Scalercio, Karina; Senior, Charles W

    2014-09-01

    Psoralen photoreaction produces covalent monoadducts and interstrand crosslinks in DNA. The interstrand DNA crosslinks are complex double strand lesions that require the involvement of multiple pathways for repair. Homologous recombination, which can carry out error-free repair, is a major pathway for crosslink repair; however, some recombination pathways can also produce DNA rearrangements. Psoralen photoreaction-induced recombination in yeast was measured using direct repeat substrates that can detect gene conversions, a form of conservative recombination, as well as deletions and triplications, which generate gene copy number changes. In repair-proficient cells the major products of recombination were gene conversions, along with substantial fractions of deletions. Deficiencies in DNA repair pathways increased non-conservative recombination products. Homologous recombination-deficient rad51, rad54, and rad57 strains had low levels of crosslink-induced recombination, and most products were deletions produced by single strand annealing. Nucleotide excision repair-deficient rad1 and rad2 yeast had increased levels of triplications, and rad1 cells had lower crosslink-induced recombination. Deficiencies in post-replication repair increased crosslink-induced recombination and gene copy number changes. Loss of REV3 function, in the error-prone branch, and of RAD5 and UBC13, in the error-free branch, produced moderate increases in deletions and triplications; rad18 cells, deficient in both post-replication repair sub-pathways, exhibited hyperrecombination, with primarily non-conservative products. Proper functioning of all the DNA repair pathways tested was required to maintain genomic stability and avoid gene copy number variation in response to interstrand crosslinks. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Human Herpesvirus 6 DNA Levels in Cerebrospinal Fluid Due to Primary Infection Differ from Those Due to Chromosomal Viral Integration and Have Implications for Diagnosis of Encephalitis▿

    PubMed Central

    Ward, Katherine N.; Leong, Hoe Nam; Thiruchelvam, Anton D.; Atkinson, Claire E.; Clark, Duncan A.

    2007-01-01

    The prevalence and concentration of human herpesvirus 6 (HHV-6) DNA in the cerebrospinal fluid (CSF) of the immunocompetent in primary infection was compared with that in viral chromosomal integration. Samples from 510 individuals with suspected encephalitis, 200 young children and 310 older children and/or adults, and 12 other patients were tested. HHV-6 DNA concentration (log10 copies/ml) was measured in CSF, serum, and whole blood using PCR. Serum HHV-6 immunoglobulin G antibody was measured by indirect immunofluorescence. Primary infection was defined by antibody seroconversion and/or a low concentration of HHV-6 DNA (<3.0 log10 copies/ml) in a seronegative serum. Chromosomal integration was defined by a high concentration of viral DNA in serum (≥3.5 log10 copies/ml) or whole blood (≥6.0 log10 copies/ml). The prevalences of CSF HHV-6 DNA in primary infection and chromosomal integration were 2.5% and 2.0%, respectively, in the young children (<2 years) and 0% and 1.3%, respectively, in the older children and/or adults. The mean concentration of CSF HHV-6 DNA in 9 children with primary infection (2.4 log10 copies/ml) was significantly lower than that of 21 patients with viral chromosomal integration (4.0 log10 copies/ml). Only HHV-6B DNA was found in primary infection, whereas in viral integration, 4 patients had HHV-6A and 17 patients HHV-6B. Apart from primary infection, chromosomal integration is the most likely cause of HHV-6 DNA in the CSF of the immunocompetent. Our results show that any diagnosis of HHV-6 encephalitis or other type of active central nervous system infection should not be made without first excluding chromosomal HHV-6 integration by measuring DNA load in CSF, serum, and/or whole blood. PMID:17229866

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

    PubMed Central

    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. Effective use of the TSPY gene-specific copy number in determining fetal DNA in the maternal blood of cynomolgus monkeys.

    PubMed

    Yasmin, Lubna; Takano, Jun-Ichiro; Sankai, Tadashi

    2016-08-01

    Since the available concentration of single-copy fetal genes in maternal blood DNA is sometimes lower than detection limits by PCR methods, the development of specific and quantitative PCR detection methods for fetal DNA in maternal blood is anticipated, which may broaden the methods that can be used to monitor pregnancy. We used the TaqMan qPCR amplification for DYS14 multi-copy sequence and the SRY gene in maternal blood plasma (cell-free DNA) and fractional precipitated blood cells (cellular DNA) from individual cynomolgus monkeys at 22 weeks of pregnancy. The availability of cell-free fetal DNA was higher in maternal blood plasma than that of cellular DNA from fractional precipitated blood cells. There was a significantly higher (P < 0.001) mean copy number of fetal male DYS14 from maternal plasma (4.4 × 10(4) copies/mL) than that of detected fetal cellular DNA from fractional blood cell pellets. The sensitivity of the DYS14 PCR assay was found to be higher than that of the SRY assay for the detection of fetal DNA when its presence was at a minimum. The DYS14 assay is an improved method for quantifying male fetal DNA in circulating maternal blood in the primate model.

  4. Deconvolving tumor purity and ploidy by integrating copy number alterations and loss of heterozygosity.

    PubMed

    Li, Yi; Xie, Xiaohui

    2014-08-01

    Next-generation sequencing (NGS) has revolutionized the study of cancer genomes. However, the reads obtained from NGS of tumor samples often consist of a mixture of normal and tumor cells, which themselves can be of multiple clonal types. A prominent problem in the analysis of cancer genome sequencing data is deconvolving the mixture to identify the reads associated with tumor cells or a particular subclone of tumor cells. Solving the problem is, however, challenging because of the so-called 'identifiability problem', where different combinations of tumor purity and ploidy often explain the sequencing data equally well. We propose a new model to resolve the identifiability problem by integrating two types of sequencing information-somatic copy number alterations and loss of heterozygosity-within a unified probabilistic framework. We derive algorithms to solve our model, and implement them in a software package called PyLOH. We benchmark the performance of PyLOH using both simulated data and 12 breast cancer sequencing datasets and show that PyLOH outperforms existing methods in disambiguating the identifiability problem and estimating tumor purity. The PyLOH package is written in Python and is publicly available at https://github.com/uci-cbcl/PyLOH. xhx@ics.uci.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.

  5. Microfabricated structures for integrated DNA analysis.

    PubMed Central

    Burns, M A; Mastrangelo, C H; Sammarco, T S; Man, F P; Webster, J R; Johnsons, B N; Foerster, B; Jones, D; Fields, Y; Kaiser, A R; Burke, D T

    1996-01-01

    Photolithographic micromachining of silicon is a candidate technology for the construction of high-throughput DNA analysis devices. However, the development of complex silicon microfabricated systems has been hindered in part by the lack of a simple, versatile pumping method for integrating individual components. Here we describe a surface-tension-based pump able to move discrete nanoliter drops through enclosed channels using only local heating. This thermocapillary pump can accurately mix, measure, and divide drops by simple electronic control. In addition, we have constructed thermal-cycling chambers, gel electrophoresis channels, and radiolabeled DNA detectors that are compatible with the fabrication of thermocapillary pump channels. Since all of the components are made by conventional photolithographic techniques, they can be assembled into more complex integrated systems. The combination of pump and components into self-contained miniaturized devices may provide significant improvements in DNA analysis speed, portability, and cost. The potential of microfabricated systems lies in the low unit cost of silicon-based construction and in the efficient sample handling afforded by component integration. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8643614

  6. Systematic Prioritization and Integrative Analysis of Copy Number Variations in Schizophrenia Reveal Key Schizophrenia Susceptibility Genes

    PubMed Central

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

    2014-01-01

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

    PubMed

    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

  8. Integrative analysis of gene expression and copy number alterations using canonical correlation analysis

    PubMed Central

    2010-01-01

    Background With the rapid development of new genetic measurement methods, several types of genetic alterations can be quantified in a high-throughput manner. While the initial focus has been on investigating each data set separately, there is an increasing interest in studying the correlation structure between two or more data sets. Multivariate methods based on Canonical Correlation Analysis (CCA) have been proposed for integrating paired genetic data sets. The high dimensionality of microarray data imposes computational difficulties, which have been addressed for instance by studying the covariance structure of the data, or by reducing the number of variables prior to applying the CCA. In this work, we propose a new method for analyzing high-dimensional paired genetic data sets, which mainly emphasizes the correlation structure and still permits efficient application to very large data sets. The method is implemented by translating a regularized CCA to its dual form, where the computational complexity depends mainly on the number of samples instead of the number of variables. The optimal regularization parameters are chosen by cross-validation. We apply the regularized dual CCA, as well as a classical CCA preceded by a dimension-reducing Principal Components Analysis (PCA), to a paired data set of gene expression changes and copy number alterations in leukemia. Results Using the correlation-maximizing methods, regularized dual CCA and PCA+CCA, we show that without pre-selection of known disease-relevant genes, and without using information about clinical class membership, an exploratory analysis singles out two patient groups, corresponding to well-known leukemia subtypes. Furthermore, the variables showing the highest relevance to the extracted features agree with previous biological knowledge concerning copy number alterations and gene expression changes in these subtypes. Finally, the correlation-maximizing methods are shown to yield results which are more

  9. Integrative analysis of copy number and transcriptional expression profiles in esophageal cancer to identify a novel driver gene for therapy

    PubMed Central

    Dong, Gaochao; Mao, Qixing; Yu, Decai; Zhang, Yi; Qiu, Mantang; Dong, Gaoyue; Chen, Qiang; Xia, Wenjie; Wang, Jie; Xu, Lin; Jiang, Feng

    2017-01-01

    An increasing amount of evidence has highlighted the critical roles that copy number variants play in cancer progression. Here, we systematically analyzed the copy number alterations and differentially transcribed genes. Integrative analysis of the association between copy number variants and differential gene expression suggested that copy number variants will lead to aberrant expression of the corresponding genes. We performed a KEGG pathway and GO analysis, which revealed that cell cycle may have an effective role in the progression of esophageal cancer. FAM60A was then screened out as a potential prognostic factor through survival analysis and correlation analysis with clinical-pathological parameters. We subsequently showed that silencing of FAM60A could inhibit esophageal carcinoma tumor cell growth, migration and invasion in vitro. Through the bioinformatic analysis, we predict that FAM60A may act as a transcriptional factor to regulate genes that are correlated with each cell cycle. In summary, we comprehensively analyzed copy number segments and transcriptional expression profiles, which provided a novel approach to identify clinical biomarkers and therapeutic targets of esophageal carcinoma. PMID:28169357

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

    PubMed

    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 <0.001). The receiver operating characteristic analysis proved that the mtDNA 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 <0.001). On multivariate logistic and Cox regression analysis, mtDNA copy number was shown to be a significant independent risk factor for PoAF (odds ratio = 10.01, p <0.001 and hazard ratio = 7.011, p = 0.004). There was a strong positive correlation between mtDNA 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.

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

    PubMed Central

    van Dyk, Ewald; Reinders, Marcel J.T.; Wessels, Lodewyk 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, 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. PMID:23476020

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

    PubMed

    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.

  13. 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

    PubMed Central

    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

  14. Sites of retroviral DNA integration: From basic research to clinical applications.

    PubMed

    Serrao, Erik; Engelman, Alan N

    2016-01-01

    One of the most crucial steps in the life cycle of a retrovirus is the integration of the viral DNA (vDNA) copy of the RNA genome into the genome of an infected host cell. Integration provides for efficient viral gene expression as well as for the segregation of viral genomes to daughter cells upon cell division. Some integrated viruses are not well expressed, and cells latently infected with human immunodeficiency virus type 1 (HIV-1) can resist the action of potent antiretroviral drugs and remain dormant for decades. Intensive research has been dedicated to understanding the catalytic mechanism of integration, as well as the viral and cellular determinants that influence integration site distribution throughout the host genome. In this review, we summarize the evolution of techniques that have been used to recover and map retroviral integration sites, from the early days that first indicated that integration could occur in multiple cellular DNA locations, to current technologies that map upwards of millions of unique integration sites from single in vitro integration reactions or cell culture infections. We further review important insights gained from the use of such mapping techniques, including the monitoring of cell clonal expansion in patients treated with retrovirus-based gene therapy vectors, or patients with acquired immune deficiency syndrome (AIDS) on suppressive antiretroviral therapy (ART). These insights span from integrase (IN) enzyme sequence preferences within target DNA (tDNA) at the sites of integration, to the roles of host cellular proteins in mediating global integration distribution, to the potential relationship between genomic location of vDNA integration site and retroviral latency.

  15. Sites of Retroviral DNA Integration: From Basic Research to Clinical Applications

    PubMed Central

    Serrao, Erik; Engelman, Alan N.

    2016-01-01

    One of the most crucial steps in the life cycle of a retrovirus is the integration of the viral DNA (vDNA) copy of the RNA genome into the genome of an infected host cell. Integration provides for efficient viral gene expression as well as for the segregation of the viral genomes to daughter cells upon cell division. Some integrated viruses are not well expressed, and cells latently infected with HIV-1 can resist the action of potent antiretroviral drugs and remain dormant for decades. Intensive research has been dedicated to understanding the catalytic mechanism of integration, as well as the viral and cellular determinants that influence integration site distribution throughout the host genome. In this review we summarize the evolution of techniques that have been used to recover and map retroviral integration sites, from the early days that first indicated that integration could occur in multiple cellular DNA locations, to current technologies that map upwards of millions of unique integration sites from single in vitro integration reactions or cell culture infections. We further review important insights gained from the use of such mapping techniques, including the monitoring of cell clonal expansion in patients treated with retrovirus-based gene therapy vectors, or AIDS patients on suppressive antiretroviral therapy (ART). These insights span from integrase (IN) enzyme sequence preferences within target DNA (tDNA) at the sites of integration, to the roles of host cellular proteins in mediating global integration distribution, to the potential relationship between genomic location of vDNA integration site and retroviral latency. PMID:26508664

  16. Genome-wide DNA Copy-number Analysis in ACTS-CC Trial of Adjuvant Chemotherapy for Stage III Colonic Cancer.

    PubMed

    Ishikawa, Toshiaki; Uetake, Hiroyuki; Murotani, Kenta; Kobunai, Takashi; Ishiguro, Megumi; Matsui, Shigeyuki; Sugihara, Kenichi

    2016-03-01

    The adjuvant chemotherapy trial of TS-1 for colon cancer phase III trial was designed to validate the non-inferiority of the oral fluoropyrimidine S-1 to uracil and tegafur/leucovorin as adjuvant chemotherapy for stage III colonic cancer. As a prospective biomarker study of this trial, DNA copy number was studied using formalin-fixed, paraffin-embedded specimens. FFPE blocks were obtained from 795 patients of the 1,535 patients enrolled in the study. The quality of extracted DNA was assessed using arbitrarily primed polymerase chain reaction and microfluidic analysis. Genomic copy-number alterations in cancer were analyzed by high-density single-nucleotide polymorphism arrays. Copy-number changes in Japanese patients with colonic cancer were compared with those in Western countries using data from a previously reported meta-analysis. We then compared genome-wide segment copy number and clinicopathological features of colorectal cancer. Genome-wide copy number was analyzed in 161 samples and DNA copy-number alteration profiles showed frequent DNA copy-number gains at chromosome 7, 8q and 13, and losses at 4, 5q, 8p, 17p and 18q. The weighted kappa statistic from comparing copy-number alteration status with data from Western countries was 0.828 (95% confidence interval=0.786 -0.871). DNA copy-number alterations of 8,684 segments were compared with clinicopathological features in 161 patients. Location of the tumor correlated with genomic segments of chromosome 4, 5, 7, 8, 13, 14, 18 and 20. Differentiation of the tumor correlated with segments in chromosome 4, 6, 8, 11, 13, 14,15, 16, 17 and 20. Somatic copy-number alteration profiles of stage III colonic cancer in the Japanese ACTS-CC trial closely agreed with the results of previous Western studies. Location and differentiation of the tumor correlated with DNA copy-number alterations. Our findings will facilitate understanding the characteristics of colonic cancer. Further investigation may contribute to the

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

    PubMed

    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

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

    PubMed

    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.

  19. Optical Imaging of Paramagnetic Bead-DNA Aggregation Inhibition Allows for Low Copy Number Detection of Infectious Pathogens

    PubMed Central

    DuVall, Jacquelyn A.; Borba, Juliane C.; Shafagati, Nazly; Luzader, Deborah; Shukla, Nishant; Li, Jingyi; Kehn-Hall, Kylene; Kendall, Melissa M.; Feldman, Sanford H.; Landers, James P.

    2015-01-01

    DNA-paramagnetic silica bead aggregation in a rotating magnetic field facilitates the quantification of DNA with femtogram sensitivity, but yields no sequence-specific information. Here we provide an original description of aggregation inhibition for the detection of DNA and RNA in a sequence-specific manner following loop-mediated isothermal amplification (LAMP). The fragments generated via LAMP fail to induce chaotrope-mediated bead aggregation; however, due to their ability to passivate the bead surface, they effectively inhibit bead aggregation by longer ‘trigger’ DNA. We demonstrate the utility of aggregation inhibition as a method for the detection of bacterial and viral pathogens with sensitivity that approaches single copies of the target. We successfully use this methodology for the detection of notable food-borne pathogens Escherichia coli O157:H7 and Salmonella enterica, as well as Rift Valley fever virus, a weaponizable virus of national security concern. We also show the concentration dependence of aggregation inhibition, suggesting the potential for quantification of target nucleic acid in clinical and environmental samples. Lastly, we demonstrate the ability to rapidly detect infectious pathogens by utilizing a cell phone and custom-written application (App), making this novel detection modality fully portable for point-of-care use. PMID:26068926

  20. Optical Imaging of Paramagnetic Bead-DNA Aggregation Inhibition Allows for Low Copy Number Detection of Infectious Pathogens.

    PubMed

    DuVall, Jacquelyn A; Borba, Juliane C; Shafagati, Nazly; Luzader, Deborah; Shukla, Nishant; Li, Jingyi; Kehn-Hall, Kylene; Kendall, Melissa M; Feldman, Sanford H; Landers, James P

    2015-01-01

    DNA-paramagnetic silica bead aggregation in a rotating magnetic field facilitates the quantification of DNA with femtogram sensitivity, but yields no sequence-specific information. Here we provide an original description of aggregation inhibition for the detection of DNA and RNA in a sequence-specific manner following loop-mediated isothermal amplification (LAMP). The fragments generated via LAMP fail to induce chaotrope-mediated bead aggregation; however, due to their ability to passivate the bead surface, they effectively inhibit bead aggregation by longer 'trigger' DNA. We demonstrate the utility of aggregation inhibition as a method for the detection of bacterial and viral pathogens with sensitivity that approaches single copies of the target. We successfully use this methodology for the detection of notable food-borne pathogens Escherichia coli O157:H7 and Salmonella enterica, as well as Rift Valley fever virus, a weaponizable virus of national security concern. We also show the concentration dependence of aggregation inhibition, suggesting the potential for quantification of target nucleic acid in clinical and environmental samples. Lastly, we demonstrate the ability to rapidly detect infectious pathogens by utilizing a cell phone and custom-written application (App), making this novel detection modality fully portable for point-of-care use.

  1. Proliferation of mitochondria in chronically stimulated rabbit skeletal muscle--transcription of mitochondrial genes and copy number of mitochondrial DNA.

    PubMed

    Schultz, J; Wiesner, R J

    2000-12-01

    Mitochondrial proliferation was studied in chronically stimulated rabbit skeletal muscle over a period of 50 days. After this time, subunits of COX had increased about fourfold. Corresponding mRNAs, encoded on mitochondrial DNA as well as on nuclear genes, were unchanged when related to total tissue RNA, however, they were elevated two- to fivefold when the massive increase of ribosomes per unit mass of muscle was taken into account. The same was true for the mRNA encoding mitochondrial transcription factor A. Surprisingly, tissue levels of mtTFA protein were reduced about twofold, together with mitochondrial DNA. In conclusion, mitochondria are able to maintain high rates of mitochondrial transcription even in the presence of reduced mtTFA protein and mtDNA levels. Therefore, stimulated mtTFA gene expression accompanies stimulated mitochondrial transcription, as in other models, but it is not sufficient for an increase of mtDNA copy number and other, yet unknown, factors have to be postulated.

  2. The human specialized DNA polymerases and non-B DNA: vital relationships to preserve genome integrity.

    PubMed

    Boyer, Anne-Sophie; Grgurevic, Srdana; Cazaux, Christophe; Hoffmann, Jean-Sébastien

    2013-11-29

    In addition to the canonical right-handed double helix, DNA molecule can adopt several other non-B DNA structures. Readily formed in the genome at specific DNA repetitive sequences, these secondary conformations present a distinctive challenge for progression of DNA replication forks. Impeding normal DNA synthesis, cruciforms, hairpins, H DNA, Z DNA and G4 DNA considerably impact the genome stability and in some instances play a causal role in disease development. Along with previously discovered dedicated DNA helicases, the specialized DNA polymerases emerge as major actors performing DNA synthesis through these distorted impediments. In their new role, they are facilitating DNA synthesis on replication stalling sites formed by non-B DNA structures and thereby helping the completion of DNA replication, a process otherwise crucial for preserving genome integrity and concluding normal cell division. This review summarizes the evidence gathered describing the function of specialized DNA polymerases in replicating DNA through non-B DNA structures.

  3. Measurement and relevance of neuroblastoma DNA copy number changes in the post-genome era.

    PubMed

    Mosse, Yael P; Greshock, Joel; Weber, Barbara L; Maris, John M

    2005-10-18

    The completion of the human genome sequence and the development of high throughput technology present exciting opportunities for the study of cancer cells. High-resolution analysis of chromosomal aberrations provides a global framework for understanding complex patterns in cancer cells, allowing us to ask hypothesis-driven questions. Genome-wide analysis of amplification and deletion of genomic regions is a critical step to resolving the mechanisms of neuroblastoma tumorigenesis. We used a high-resolution aCGH system that has over 4000 human BAC clones, resulting in an average coverage of 1Mb across the genome, to define whole genome copy number aberrations (CNAs) in a panel of human neuroblastoma-derived cell lines. By combining the aCGH data with meticulous regional validation studies, we showed that array CGH could reliably detect known aberrations including single copy gain or loss, that data correlate well with standard techniques used for the detection of these genetic changes, and that this technique can be used to identify novel regions of genomic imbalance.

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

    PubMed

    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.

  5. Tetramethylpyrazine blocks TFAM degradation and up-regulates mitochondrial DNA copy number by interacting with TFAM

    PubMed Central

    Lan, Linhua; Guo, Miaomiao; Ai, Yong; Chen, Fuhong; Zhang, Ya; Xia, Lei; Huang, Dawei; Niu, Lili; Zheng, Ying; Suzuki, Carolyn K.

    2017-01-01

    The natural small molecule compound: 2,3,5,6-tetramethylpyrazine (TMP), is a major component of the Chinese medicine Chuanxiong, which has wide clinical applications in dilating blood vessels, inhibiting platelet aggregation and treating thrombosis. Recent work suggests that TMP is also an antitumour agent. Despite its chemotherapeutic potential, the mechanism(s) underlying TMP action are unknown. Herein, we demonstrate that TMP binds to mitochondrial transcription factor A (TFAM) and blocks its degradation by the mitochondrial Lon protease. TFAM is a key regulator of mtDNA replication, transcription and transmission. Our previous work showed that when TFAM is not bound to DNA, it is rapidly degraded by the ATP-dependent Lon protease, which is essential for mitochondrial proteostasis. In cultured cells, TMP specifically blocks Lon-mediated degradation of TFAM, leading to TFAM accumulation and subsequent up-regulation of mtDNA content in cells with substantially low levels of mtDNA. In vitro protease assays show that TMP does not directly inhibit mitochondrial Lon, rather interacts with TFAM and blocks degradation. Pull-down assays show that biotinylated TMP interacts with TFAM. These findings suggest a novel mechanism whereby TMP stabilizes TFAM and confers resistance to Lon-mediated degradation, thereby promoting mtDNA up-regulation in cells with low mtDNA content. PMID:28465355

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

    PubMed

    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 < 0.01). The expression of 65 genes was found to be significantly associated with prognosis (p < 0.01). Specifically, we found that up-regulation of the IL17RA, IGF2BP2 and ABCC2 genes, and of genes acting in the mTOR and cytokine receptor pathways, were strongly associated with a poor survival. Subsequent integrated analyses revealed that increased expression levels of the MMP9, BMP7, UBE2C, I-CAM, NOTCH3, NOTCH1, PTGES2, HMGB1 and ERBB3 genes were associated with 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

  7. The effect of leaving groups on binding and reactivity in enzyme-free copying of DNA and RNA

    PubMed Central

    Kervio, Eric; Sosson, Marilyne; Richert, Clemens

    2016-01-01

    The template-directed incorporation of nucleotides at the terminus of a growing primer is the basis of the transmission of genetic information. Nature uses polymerases-catalyzed reactions, but enzyme-free versions exist that employ nucleotides with organic leaving groups. The leaving group affects yields, but it was not clear whether inefficient extensions are due to poor binding, low reactivity toward the primer, or rapid hydrolysis. We have measured the binding of a total of 15 different activated nucleotides to DNA or RNA sequences. Further, we determined rate constants for the chemical step of primer extension involving methylimidazolides or oxyazabenzotriazolides of deoxynucleotides or ribonucleotides. Binding constants range from 10 to >500 mM and rate constants from 0.1 to 370 M−1 h−1. For aminoterminal primers, a fast covalent step and slow hydrolysis are the main factors leading to high yields. For monomers with weakly pairing bases, the leaving group can improve binding significantly. A detailed mechanistic picture emerges that explains why some enzyme-free primer extensions occur in high yield, while others remain recalcitrant to copying without enzymatic catalysis. A combination of tight binding and rapid extension, coupled with slow hydrolysis induces efficient enzyme-free copying. PMID:27235418

  8. DNA copy number profiling reveals extensive genomic loss in hereditary BRCA1 and BRCA2 ovarian carcinomas

    PubMed Central

    Kamieniak, M M; Muñoz-Repeto, I; Rico, D; Osorio, A; Urioste, M; García-Donas, J; Hernando, S; Robles-Díaz, L; Ramón y Cajal, T; Cazorla, A; Sáez, R; García-Bueno, J M; Domingo, S; Borrego, S; Palacios, J; van de Wiel, M A; Ylstra, B; Benítez, J; García, M J

    2013-01-01

    Background: Few studies have attempted to characterise genomic changes occurring in hereditary epithelial ovarian carcinomas (EOCs) and inconsistent results have been obtained. Given the relevance of DNA copy number alterations in ovarian oncogenesis and growing clinical implications of the BRCA-gene status, we aimed to characterise the genomic profiles of hereditary and sporadic ovarian tumours. Methods: High-resolution array Comparative Genomic Hybridisation profiling of 53 familial (21 BRCA1, 6 BRCA2 and 26 non-BRCA1/2) and 15 sporadic tumours in combination with supervised and unsupervised analysis was used to define common and/or specific copy number features. Results: Unsupervised hierarchical clustering did not stratify tumours according to their familial or sporadic condition or to their BRCA1/2 mutation status. Common recurrent changes, spanning genes potentially fundamental for ovarian carcinogenesis, regardless of BRCA mutations, and several candidate subtype-specific events were defined. Despite similarities, greater contribution of losses was revealed to be a hallmark of BRCA1 and BRCA2 tumours. Conclusion: Somatic alterations occurring in the development of familial EOCs do not differ substantially from the ones occurring in sporadic carcinomas. However, some specific features like extensive genomic loss observed in BRCA1/2 tumours may be of clinical relevance helping to identify BRCA-related patients likely to respond to PARP inhibitors. PMID:23558894

  9. The application of ultraviolet irradiation to exogenous sources of DNA in plasticware and water for the amplification of low copy number DNA.

    PubMed

    Tamariz, Jeannie; Voynarovska, Kristina; Prinz, Mechthild; Caragine, Theresa

    2006-07-01

    Using high sensitivity forensic STR polymerase chain reaction (PCR) typing procedures, we have found low concentrations of DNA contamination in plasticware and water assumed to be sterile, which is not detected by standard DNA procedures. One technique commonly used to eliminate the presence of DNA is ultraviolet (UV) irradiation; we optimized such a protocol used in the treatment of water, tubes, plates, and tips for low copy number DNA (LCN) amplification. UV light from a Stratalinker((R)) 2400 was administered to 0.2, 1.5 mL tubes, and PCR plates contaminated with up to 500 pg of DNA. They were subsequently quantified with an ALU-based real-time PCR method using the Rotorgene 3000. Overall, there was a decrease in concentration of DNA recovered as the duration of treatment increased. Nonetheless, following 45 min of irradiating a PCR plate with 500 pg of DNA, nearly 6 pg were still detected. However, when the plate was raised within an inch of the UV source, less than 0.2 pg of DNA was detected. Additionally, lining the area around the samples with aluminum foil further reduced the amount of time necessary for irradiation, as only 30 min eliminated the presence DNA in the raised PCR plate. Similar experiments were conducted using tubes filled with a solution of DNA and water in equivalent concentrations for 50, 15, and 1.5 mL tubes with comparative results. It is plausible that the aluminum foil increased the amount of reflection in the area thereby enhancing penetration of UV rays through the walls of the plasticware. This protocol was tested for the possibility of inhibitors produced from irradiation of plastic tubes. As our protocols require less irradiation time than previous studies, PCR sensitivity was not affected. Moreover, the lifespan of the UV lamps was extended. Our findings demonstrate that this method is useful as an additional precautionary measure to prevent amplification of extraneous DNA from plasticware and water without compromising the

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

    PubMed Central

    Forero-Castro, Maribel; Robledo, Cristina; Benito, Rocío; Abáigar, María; África Martín, Ana; Arefi, Maryam; Fuster, José Luis; de las Heras, Natalia; Rodríguez, Juan N.; Quintero, Jonathan; Riesco, Susana; Hermosín, Lourdes; de la Fuente, Ignacio; Recio, Isabel; Ribera, Jordi; Labrador, Jorge; Alonso, José M.; Olivier, Carmen; Sierra, Magdalena; Megido, Marta; Corchete-Sánchez, Luis A.; Ciudad Pizarro, Juana; García, Juan Luis; Ribera, José M.; Hernández-Rivas, Jesús M.

    2016-01-01

    Identifying additional genetic alterations associated with poor prognosis in acute lymphoblastic leukemia (ALL) is still a challenge. Aims: 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. PMID:26872047

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

    PubMed

    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.

  12. Recurrent DNA copy number alterations in intestinal-type sinonasal adenocarcinoma.

    PubMed

    Perez-Escuredo, J; Lopez-Hernandez, A; Costales, M; Lopez, F; Ares, S P; Vivanco, B; Llorente, J L; Hermsen, M A

    2016-09-01

    Intestinal-type sinonasal adenocarcinoma (ITAC) is a rare tumour related to occupational wood dust exposure. Few studies have described recurrent genetic changes on a genome-wide scale. The aim of this study was to obtain a high resolution map of recurrent genetic alterations in ITAC. Copy number alterations were evaluated by microarray CGH and MLPA in 37 primary tumours. The results were correlated with pathological characteristics and clinical outcome. Microarray CGH identified the following recurrent aberrations, in descending order: gains at 5p15 (22 cases, 60%), 8q24 (21 cases, 57%), 20q13 (20 cases, 54%), 20q11, and 8q21 (19 cases, 51%), 20p13, and 7p11 (16 cases, 43%), and losses at 5q11-qter, 8p12-pter, and 18q12-23 (15 cases, 40%), and 17p13, and 19p13 (13 cases, 35%). MLPA analysis confirmed this global pattern of gains and losses. Chromosomal loss at 4q32-ter and gains at 1q22, 6p22 and 3q29, as well as deletion of TIMP2 and CRK correlated with unfavourable clinical outcome. ITACs have a unique pattern of chromosomal abnormalities. The four different histological subtypes of ITAC appeared genetically similar. Four chromosomal gains and losses and two specific genes showed prognostic value and may be involved in tumour progression.

  13. Integrated microfluidic biochips for DNA microarray analysis.

    PubMed

    Liu, Robin Hui; Dill, Kilian; Fuji, H Sho; McShea, Andy

    2006-03-01

    A fully integrated and self-contained microfluidic biochip device has been developed to automate the fluidic handling steps required to perform a gene expression study of the human leukemia cell line (K-562). The device consists of a DNA microarray semiconductor chip with 12,000 features and a microfluidic cartridge that consists of microfluidic pumps, mixers, valves, fluid channels and reagent storage chambers. Microarray hybridization and subsequent fluidic handling and reactions (including a number of washing and labeling steps) were performed in this fully automated and miniature device before fluorescent image scanning of the microarray chip. Electrochemical micropumps were integrated in the cartridge to provide pumping of liquid solutions. A micromixing technique based on gas bubbling generated by electrochemical micropumps was developed. Low-cost check valves were implemented in the cartridge to prevent cross-talk of the stored reagents. A single-color transcriptional analysis of K-562 cells with a series of calibration controls (spiked-in controls) was performed to characterize this new platform with regard to sensitivity, specificity and dynamic range. The device detected sample RNAs with a concentration as low as 0.375 pM. Detection was quantitative over more than 3 orders of magnitude. Experiments also demonstrated that chip-to-chip variability was low, indicating that the integrated microfluidic devices eliminate manual fluidic handling steps that can be a significant source of variability in genomic analysis.

  14. Deficiency of the multi-copy mouse Y gene Sly causes sperm DNA damage and abnormal chromatin packaging.

    PubMed

    Riel, Jonathan M; Yamauchi, Yasuhiro; Sugawara, Atsushi; Li, Ho Yan J; Ruthig, Victor; Stoytcheva, Zoia; Ellis, Peter J I; Cocquet, Julie; Ward, Monika A

    2013-02-01

    In mouse and man Y chromosome deletions are frequently associated with spermatogenic defects. Mice with extensive deletions of non-pairing Y chromosome long arm (NPYq) are infertile and produce sperm with grossly misshapen heads, abnormal chromatin packaging and DNA damage. The NPYq-encoded multi-copy gene Sly controls the expression of sex chromosome genes after meiosis and Sly deficiency results in a remarkable upregulation of sex chromosome genes. Sly deficiency has been shown to be the underlying cause of the sperm head anomalies and infertility associated with NPYq gene loss, but it was not known whether it recapitulates sperm DNA damage phenotype. We produced and examined mice with transgenically (RNAi) silenced Sly and demonstrated that these mice have increased incidence of sperm with DNA damage and poorly condensed and insufficiently protaminated chromatin. We also investigated the contribution of each of the two Sly-encoded transcript variants and noted that the phenotype was only observed when both variants were knocked down, and that the phenotype was intermediate in severity compared with mice with severe NPYq deficiency. Our data demonstrate that Sly deficiency is responsible for the sperm DNA damage/chromatin packaging defects observed in mice with NPYq deletions and point to SLY proteins involvement in chromatin reprogramming during spermiogenesis, probably through their effect on the post-meiotic expression of spermiogenic genes. Considering the importance of the sperm epigenome for embryonic and fetal development and the possibility of its inter-generational transmission, our results are important for future investigations of the molecular mechanisms of this biologically and clinically important process.

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

    PubMed

    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.

  16. Gene copy silencing and DNA methylation in natural and artificially produced allopolyploid fish.

    PubMed

    Matos, Isa M N; Coelho, Maria M; Schartl, Manfred

    2016-10-01

    Allelic silencing is an important mechanism for coping with gene dosage changes in polyploid organisms that is well known in allopolyploid plants. Only recently, it was shown in the allotriploid fish Squalius alburnoides that this process also occurs in vertebrates. However, it is still unknown whether this silencing mechanism is common to other allopolyploid fish, and which mechanisms might be responsible for allelic silencing. We addressed these questions in a comparative study between Squalius alburnoides and another allopolyploid complex, the Amazon molly (Poecilia formosa). We examined the allelic expression patterns for three target genes in four somatic tissues of natural allo-anorthoploids and laboratory-produced tri-genomic hybrids of S. alburnoides and P. formosa. Also, for both complexes, we evaluated the correlation between total DNA methylation level and the ploidy status and genomic composition of the individuals. We found that allelic silencing also occurs in other allopolyploid organisms besides the single one that was previously known. We found and discuss disparities within and between the two considered complexes concerning the pattern of allele-specific expression and DNA methylation levels. Disparities might be due to intrinsic characteristics of each genome involved in the hybridization process. Our findings also support the idea that long-term evolutionary processes have an effect on the allele expression patterns and possibly also on DNA methylation levels. © 2016. Published by The Company of Biologists Ltd.

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

    PubMed Central

    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

    2013-01-01

    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. PMID:22929023

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

    PubMed

    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).

  19. Methods for Applying Accurate Digital PCR Analysis on Low Copy DNA Samples

    PubMed Central

    Whale, Alexandra S.; Cowen, Simon; Foy, Carole A.; Huggett, Jim F.

    2013-01-01

    Digital PCR (dPCR) is a highly accurate molecular approach, capable of precise measurements, offering a number of unique opportunities. However, in its current format dPCR can be limited by the amount of sample that can be analysed and consequently additional considerations such as performing multiplex reactions or pre-amplification can be considered. This study investigated the impact of duplexing and pre-amplification on dPCR analysis by using three different assays targeting a model template (a portion of the Arabidopsis thaliana alcohol dehydrogenase gene). We also investigated the impact of different template types (linearised plasmid clone and more complex genomic DNA) on measurement precision using dPCR. We were able to demonstrate that duplex dPCR can provide a more precise measurement than uniplex dPCR, while applying pre-amplification or varying template type can significantly decrease the precision of dPCR. Furthermore, we also demonstrate that the pre-amplification step can introduce measurement bias that is not consistent between experiments for a sample or assay and so could not be compensated for during the analysis of this data set. We also describe a model for estimating the prevalence of molecular dropout and identify this as a source of dPCR imprecision. Our data have demonstrated that the precision afforded by dPCR at low sample concentration can exceed that of the same template post pre-amplification thereby negating the need for this additional step. Our findings also highlight the technical differences between different templates types containing the same sequence that must be considered if plasmid DNA is to be used to assess or control for more complex templates like genomic DNA. PMID:23472156

  20. 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.

    PubMed

    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.

  1. Assessment of DNA Integrity, Applications for Cancer Research.

    PubMed

    Zonta, Eleonora; Nizard, Philippe; Taly, Valérie

    2015-01-01

    Many methods have been developed for DNA integrity assessment including electrophoresis-based procedures, quantitative PCR, and, more recently, microfluidics-based procedures. DNA integrity evaluation can be employed for characterizing biological samples quality before extensive genomic analysis and also finds applications in reproductive medicine, prenatal diagnostics, or cancer research. In this chapter, we will focus on the assessment of DNA integrity in cancer research. In particular, we will present the application of the determination of DNA integrity for tracking of circulating tumor DNA. Finally, we will conclude by illustrating the potential innovative application of DNA integrity as a biomarker in clinical research, especially for prognostic purposes, patient follow-up, or early diagnosis. © 2015 Elsevier Inc. All rights reserved.

  2. DNA Copy Number Aberrations, and Human Papillomavirus Status in Penile Carcinoma. Clinico-Pathological Correlations and Potential Driver Genes

    PubMed Central

    Lambros, Maryou; Stankiewicz, Elzbieta; Ng, Charlotte K. Y.; Weigelt, Britta; Rajab, Ramzi; Tinwell, Brendan; Corbishley, Cathy; Watkin, Nick; Berney, Dan; Reis-Filho, Jorge S.

    2016-01-01

    Penile squamous cell carcinoma is a rare disease, in which somatic genetic aberrations have yet to be characterized. We hypothesized that gene copy aberrations might correlate with human papillomavirus status and clinico-pathological features. We sought to determine the spectrum of gene copy number aberrations in a large series of PSCCs and to define their correlations with human papillomavirus, histopathological subtype, and tumor grade, stage and lymph node status. Seventy formalin-fixed, paraffin embedded penile squamous cell carcinomas were centrally reviewed by expert uropathologists. DNA was extracted from micro-dissected samples, subjected to PCR-based human papillomavirus assessment and genotyping (INNO-LiPA human papillomavirus Genotyping Extra Assay) and microarray-based comparative genomic hybridization using a 32K Bacterial Artificial Chromosome array platform. Sixty-four samples yielded interpretable results. Recurrent gains were observed in chromosomes 1p13.3-q44 (88%), 3p12.3-q29 (86%), 5p15.33-p11 (67%) and 8p12-q24.3 (84%). Amplifications of 5p15.33-p11 and 11p14.1-p12 were found in seven (11%) and four (6%) cases, respectively. Losses were observed in chromosomes 2q33-q37.3 (86%), 3p26.3-q11.1 (83%) and 11q12.2-q25 (81%). Although many losses and gains were similar throughout the cohort, there were small significant differences observed at specific loci, between human papillomavirus positive and negative tumors, between tumor types, and tumor grade and nodal status. These results demonstrate that despite the diversity of genetic aberrations in penile squamous cell carcinomas, there are significant correlations between the clinico-pathological data and the genetic changes that may play a role in disease natural history and progression and highlight potential driver genes, which may feature in molecular pathways for existing therapeutic agents. PMID:26901676

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

    PubMed

    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

  4. [R1 and R2 retrotransposons of German cockroach Blattella germanica: comparative analysis of 5' truncated copies integrated into genome].

    PubMed

    Kagramanova, A S; Kapelinskaia, T V; Korolev, A L; Mukha, D V

    2007-01-01

    This is the first report providing results on identification, cloning, and sequencing of extended fragments (5'-truncated copies) of R1 and R2 retrotransposons integrated into Blattella germanica genome. Comparative structural analysis of the received clones revealed two distinct subfamilies of R1 elements. However, all B. germanica R1 clones have two common features: poly(T) tails and similar target site duplications. Nucleotide structure and organization of five sequenced R2 fragments was similar. Analysis of R2 nucleotide sequences revealed typical deletions at the 3'end of target sites and lack of homopolynucleotides tails.

  5. Co-transcribed 3' host sequences augment expression of integrated hepatitis B virus DNA.

    PubMed Central

    von Loringhoven, A F; Koch, S; Hofschneider, P H; Koshy, R

    1985-01-01

    We have previously reported the cloning and structural analysis of integrated hepatitis B virus DNA copies from the human hepatoma cell line PLC/PRF/5. Here we show that the cloned DNA fragments of 10.7 kb and 10.5 kb contain intact coding sequences for HBsAg since Ltk- cells transfected with these DNAs secrete considerable amounts of HBsAg. We show for the 10.7-kb fragment that multiple readthrough messages composed of viral as well as cellular sequences are transcribed. These RNAs differ only in their 3' sequences. Furthermore, the 10.7-kb insert leads to a substantial increase in HBsAg produced compared with HBV DNA and with the 10.5-kb insert. We provide evidence that the different 3' sequences on the HBsAg transcripts account for the augmentation of expression. Images Fig. 2. Fig. 3. Fig. 5. PMID:2990895

  6. Effects of acetyl-L-carnitine on lamb oocyte blastocyst rate, ultrastructure, and mitochondrial DNA copy number.

    PubMed

    Reader, Karen L; Cox, Neil R; Stanton, Jo-Ann L; Juengel, Jennifer L

    2015-06-01

    Viable lambs can be produced after transfer of in vitro-derived embryos from oocytes harvested from prepubertal lambs. However, this occurs at a much lower efficiency than from adult ewe oocyte donors. The reduced competence of prepubertal oocytes is believed to be due, at least in part, to deficiencies in cytoplasmic maturation. Differences in the cytoplasmic ultrastructure between prepubertal and adult oocytes have been described in the sheep, pig, and cow. Prepubertal lamb oocytes have been shown to have a different distribution of mitochondria and lipid droplets, and less mitochondria and storage vesicles than their adult counterparts. L-carnitine plays a role in supplying energy to the cell by transporting long-chain fatty acids into mitochondria for β-oxidation to produce ATP. Both L-carnitine and its derivative acetyl-L-carnitine have been reported to increase the blastocyst rate of oocytes from mice, cows, and pigs, treated during IVM. L-carnitine has also been shown to increase mitochondrial biogenesis in adipose cells. Therefore, the aims of this study were to determine if treatment of oocytes from prepubertal lambs with acetyl-L-carnitine during IVM could increase the blastocyst rate and alter mitochondria, vesicle, or lipid droplet number, volume, or distribution. The blastocyst rate was doubled in prepubertal lamb oocytes treated with acetyl-L-carnitine when compared to untreated oocytes (10.0% and 4.6%, respectively; P = 0.028). Light microscopy, scanning electron microscopy, and stereology techniques were used to quantify organelles in untreated and acetyl-L-carnitine-treated lamb oocytes, and quantitative polymerase chain reaction methods were used to measure the mitochondrial DNA copy number. There were no differences in mitochondrial volume, number, or mitochondrial DNA copy number. Acetyl-L-carnitine treatment increased the cytoplasmic volume (P = 0.015) of the oocytes, and there were trends toward an increase in the vesicle volume (P = 0

  7. Quantification of plasmid DNA copies in the nucleus after lipoplex and polyplex transfection.

    PubMed

    Cohen, Richard N; van der Aa, Marieke A E M; Macaraeg, Nichole; Lee, Ai Ping; Szoka, Francis C

    2009-04-17

    Nuclear uptake of plasmid DNA is one of the many cellular barriers that limit the efficiency of non-viral gene delivery systems. We have determined the number of plasmids that reach the nucleus of a transfected cell using an internally standardized quantitative PCR (qPCR) assay. We isolated nuclei using two different protocols: a density gradient technique and a detergent-based method. The density gradient procedure yielded nuclei with substantially less adhering plasmids on the outside of the nuclei. Using the density gradient protocol we determined that cells transfected with Lipofectamine lipoplexes or polyethylenimine polyplexes contained between 75 and 50,000 plasmids/nucleus, depending on the applied plasmid dose. Any increase above 3000 plasmids/nucleus resulted in only marginal increases in transgene expression. Furthermore, lipoplex-delivered plasmids were more efficiently expressed, on the basis of protein expression per plasmid number in the nucleus, than polyplex-delivered plasmids. This indicates that polymer may remain bound to some plasmids in the nucleus. Lastly, by sorting transfected cells into high- and low-expressing sub-populations, we observe that a sub-population of cells contain 3x greater plasmids/nucleus but express nearly 100x more transgene than other cells within a single transfection reaction. Taken together these results suggest the importance of considering the processes downstream from nuclear entry for strategies to improve the efficiency of gene transfer reagents.

  8. A whole-genome mouse BAC microarray with 1-Mb resolution for analysis of DNA copy number changes by array comparative genomic hybridization.

    PubMed

    Chung, Yeun-Jun; Jonkers, Jos; Kitson, Hannah; Fiegler, Heike; Humphray, Sean; Scott, Carol; Hunt, Sarah; Yu, Yuejin; Nishijima, Ichiko; Velds, Arno; Holstege, Henne; Carter, Nigel; Bradley, Allan

    2004-01-01

    Microarray-based comparative genomic hybridization (CGH) has become a powerful method for the genome-wide detection of chromosomal imbalances. Although BAC microarrays have been used for mouse CGH studies, the resolving power of these analyses was limited because high-density whole-genome mouse BAC microarrays were not available. We therefore developed a mouse BAC microarray containing 2803 unique BAC clones from mouse genomic libraries at 1-Mb intervals. For the general amplification of BAC clone DNA prior to spotting, we designed a set of three novel degenerate oligonucleotide-primed (DOP) PCR primers that preferentially amplify mouse genomic sequences while minimizing unwanted amplification of contaminating Escherichia coli DNA. The resulting 3K mouse BAC microarrays reproducibly identified DNA copy number alterations in cell lines and primary tumors, such as single-copy deletions, regional amplifications, and aneuploidy.

  9. Damaging the Integrated HIV Proviral DNA with TALENs

    PubMed Central

    Strong, Christy L.; Guerra, Horacio P.; Mathew, Kiran R.; Roy, Nervik; Simpson, Lacy R.; Schiller, Martin R.

    2015-01-01

    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. PMID:25946221

  10. A DNA target-enrichment approach to detect mutations, copy number changes and immunoglobulin translocations in multiple myeloma

    PubMed Central

    Bolli, N; Li, Y; Sathiaseelan, V; Raine, K; Jones, D; Ganly, P; Cocito, F; Bignell, G; Chapman, M A; Sperling, A S; Anderson, K C; Avet-Loiseau, H; Minvielle, S; Campbell, P J; Munshi, N C

    2016-01-01

    Genomic lesions are not investigated during routine diagnostic workup for multiple myeloma (MM). Cytogenetic studies are performed to assess prognosis but with limited impact on therapeutic decisions. Recently, several recurrently mutated genes have been described, but their clinical value remains to be defined. Therefore, clinical-grade strategies to investigate the genomic landscape of myeloma samples are needed to integrate new and old prognostic markers. We developed a target-enrichment strategy followed by next-generation sequencing (NGS) to streamline simultaneous analysis of gene mutations, copy number changes and immunoglobulin heavy chain (IGH) translocations in MM in a high-throughput manner, and validated it in a panel of cell lines. We identified 548 likely oncogenic mutations in 182 genes. By integrating published data sets of NGS in MM, we retrieved a list of genes with significant relevance to myeloma and found that the mutational spectrum of primary samples and MM cell lines is partially overlapping. Gains and losses of chromosomes, chromosomal segments and gene loci were identified with accuracy comparable to conventional arrays, allowing identification of lesions with known prognostic significance. Furthermore, we identified IGH translocations with high positive and negative predictive value. Our approach could allow the identification of novel biomarkers with clinical relevance in myeloma. PMID:27588520

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

    PubMed

    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.

  12. FISH Oracle: a web server for flexible visualization of DNA copy number data in a genomic context.

    PubMed

    Mader, Malte; Simon, Ronald; Steinbiss, Sascha; Kurtz, Stefan

    2011-07-28

    The rapidly growing amount of array CGH data requires improved visualization software supporting the process of identifying candidate cancer genes. Optimally, such software should work across multiple microarray platforms, should be able to cope with data from different sources and should be easy to operate. We have developed a web-based software FISH Oracle to visualize data from multiple array CGH experiments in a genomic context. Its fast visualization engine and advanced web and database technology supports highly interactive use. FISH Oracle comes with a convenient data import mechanism, powerful search options for genomic elements (e.g. gene names or karyobands), quick navigation and zooming into interesting regions, and mechanisms to export the visualization into different high quality formats. These features make the software especially suitable for the needs of life scientists. FISH Oracle offers a fast and easy to use visualization tool for array CGH and SNP array data. It allows for the identification of genomic regions representing minimal common changes based on data from one or more experiments. FISH Oracle will be instrumental to identify candidate onco and tumor suppressor genes based on the frequency and genomic position of DNA copy number changes. The FISH Oracle application and an installed demo web server are available at http://www.zbh.uni-hamburg.de/fishoracle.

  13. FISH Oracle: a web server for flexible visualization of DNA copy number data in a genomic context

    PubMed Central

    2011-01-01

    Background The rapidly growing amount of array CGH data requires improved visualization software supporting the process of identifying candidate cancer genes. Optimally, such software should work across multiple microarray platforms, should be able to cope with data from different sources and should be easy to operate. Results We have developed a web-based software FISH Oracle to visualize data from multiple array CGH experiments in a genomic context. Its fast visualization engine and advanced web and database technology supports highly interactive use. FISH Oracle comes with a convenient data import mechanism, powerful search options for genomic elements (e.g. gene names or karyobands), quick navigation and zooming into interesting regions, and mechanisms to export the visualization into different high quality formats. These features make the software especially suitable for the needs of life scientists. Conclusions FISH Oracle offers a fast and easy to use visualization tool for array CGH and SNP array data. It allows for the identification of genomic regions representing minimal common changes based on data from one or more experiments. FISH Oracle will be instrumental to identify candidate onco and tumor suppressor genes based on the frequency and genomic position of DNA copy number changes. The FISH Oracle application and an installed demo web server are available at http://www.zbh.uni-hamburg.de/fishoracle. PMID:21884636

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

    PubMed

    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.

  15. Identification of DNA copy-number aberrations by array-comparative genomic hybridization in patients with schizophrenia.

    PubMed

    Moon, Ho Jin; Yim, Sung-Vin; Lee, Woon Kyu; Jeon, Yang-Whan; Kim, Young Hoon; Ko, Young Jin; Lee, Kwang-Soo; Lee, Kweon-Haeng; Han, Sang-Ick; Rha, Hyoung Kyun

    2006-06-02

    Chromosomal abnormalities are implicated as important markers for the pathogenesis in patients with schizophrenia. In this study, with using bacterial artificial chromosome (BAC) array-based comparative genomic hybridization (CGH), we analyzed DNA copy-number changes among 30 patients with schizophrenia. The most frequent changes were partial gain of Xq23 (52%) and loss of 3q13.12 (32%). Other frequent gains were found in: 1p, 6q, 10p, 11p, 11q, 14p, and 15q regions, and frequent losses were found in: 2p, 9q, 10q, 14q, 20q, and 22q regions. The set of abnormal regions was confirmed by real-time PCR (9q12, 9q34.2, 11p15.4, 14q32.33, 15q15.1, 22q11.21, and Xq23). All real-time PCR results were consistent with the array-CGH results. Therefore, it is suggested that array-CGH and real-time PCR analysis could be used as powerful tools in screening for schizophrenia-related genes. Our results might be useful for further exploration of candidate genomic regions in the pathogenesis of schizophrenia.

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

    PubMed Central

    Ross-Adams, H.; Lamb, A.D.; Dunning, M.J.; Halim, S.; Lindberg, J.; Massie, C.M.; Egevad, L.A.; Russell, R.; Ramos-Montoya, A.; Vowler, S.L.; Sharma, N.L.; Kay, J.; Whitaker, H.; Clark, J.; Hurst, R.; Gnanapragasam, V.J.; Shah, N.C.; Warren, A.Y.; Cooper, C.S.; Lynch, A.G.; Stark, R.; Mills, I.G.; Grönberg, H.; Neal, D.E.

    2015-01-01

    Background Understanding the heterogeneous genotypes and phenotypes of prostate cancer is fundamental to improving the way we treat this disease. As yet, there are no validated descriptions of prostate cancer subgroups derived from integrated genomics linked with clinical outcome. Methods In a study of 482 tumour, benign and germline samples from 259 men with primary prostate cancer, we used integrative analysis of copy number alterations (CNA) and array transcriptomics to identify genomic loci that affect expression levels of mRNA in an expression quantitative trait loci (eQTL) approach, to stratify patients into subgroups that we then associated with future clinical behaviour, and compared with either CNA or transcriptomics alone. Findings We identified five separate patient subgroups with distinct genomic alterations and expression profiles based on 100 discriminating genes in our separate discovery and validation sets of 125 and 103 men. These subgroups were able to consistently predict biochemical relapse (p = 0.0017 and p = 0.016 respectively) and were further validated in a third cohort with long-term follow-up (p = 0.027). We show the relative contributions of gene expression and copy number data on phenotype, and demonstrate the improved power gained from integrative analyses. We confirm alterations in six genes previously associated with prostate cancer (MAP3K7, MELK, RCBTB2, ELAC2, TPD52, ZBTB4), and also identify 94 genes not previously linked to prostate cancer progression that would not have been detected using either transcript or copy number data alone. We confirm a number of previously published molecular changes associated with high risk disease, including MYC amplification, and NKX3-1, RB1 and PTEN deletions, as well as over-expression of PCA3 and AMACR, and loss of MSMB in tumour tissue. A subset of the 100 genes outperforms established clinical predictors of poor prognosis (PSA, Gleason score), as well as previously published gene

  17. The activity and copy number of mitochondrial DNA in ovine oocytes throughout oogenesis in vivo and during oocyte maturation in vitro.

    PubMed

    Cotterill, Matthew; Harris, Sarah E; Collado Fernandez, Esther; Lu, Jianping; Huntriss, John D; Campbell, Bruce K; Picton, Helen M

    2013-07-01

    Mitochondria are responsible for the production of ATP, which drives cellular metabolic and biosynthetic processes. This is the first study to quantify the mtDNA copy number across all stages of oogenesis in a large monovulatory species, it includes assessment of the activity of mitochondria in germinal vesicle (GV) and metaphase II (MII) oocytes through JC1 staining. Primordial to early antral follicles (n = 249) were isolated from the sheep ovarian cortex following digestion at 37°C for 1 h and all oocytes were disaggregated from their somatic cells. Germinal vesicle oocytes (n = 133) were aspirated from 3- to 5-mm diameter antral follicles, and mature MII oocytes (n = 71) were generated following in vitro maturation (IVM). The mtDNA copy number in each oocyte was quantified using real-time PCR and showed a progressive, but variable increase in the amount of mtDNA in oocytes from primordial follicles (605 ± 205, n = 8) to mature MII oocytes (744 633 ± 115 799, n = 13; P < 0.05). Mitochondrial activity (P > 0.05) was not altered during meiotic progression from GV to MII during IVM. The observed increase in the mtDNA copy number across oogenesis reflects the changing ATP demands needed to orchestrate cytoskeletal and cytoplasmic reorganization during oocyte growth and maturation and the need to fuel the resumption of meiosis in mature oocytes following the pre-ovulatory gonadotrophin surge.

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

    PubMed

    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

  19. Human Immunodeficiency Virus Integration Protein Expressed in Escherichia Coli Possesses Selective DNA Cleaving Activity

    NASA Astrophysics Data System (ADS)

    Sherman, Paula A.; Fyfe, James A.

    1990-07-01

    The human immunodeficiency virus (HIV) integration protein, a potential target for selective antiviral therapy, was expressed in Escherichia coli. The purified protein, free of detectable contaminating endonucleases, selectively cleaved double-stranded DNA oligonucleotides that mimic the U3 and the U5 termini of linear HIV DNA. Two nucleotides were removed from the 3' ends of both the U5 plus strand and the U3 minus strand; in both cases, cleavage was adjacent to a conserved CA dinucleotide. The reaction was metal-ion dependent, with a preference for Mn2+ over Mg2+. Reaction selectivity was further demonstrated by the lack of cleavage of an HIV U5 substrate on the complementary (minus) strand, an analogous substrate that mimics the U3 terminus of an avian retrovirus, and an HIV U5 substrate in which the conserved CA dinucleotide was replaced with a TA dinucleotide. Such an integration protein-mediated cleavage reaction is expected to occur as part of the integration event in the retroviral life cycle, in which a double-stranded DNA copy of the viral RNA genome is inserted into the host cell DNA.

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

    PubMed

    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.

  1. HBV DNA Integration: Molecular Mechanisms and Clinical Implications

    PubMed Central

    Tu, Thomas; Budzinska, Magdalena A.; Shackel, Nicholas A.; Urban, Stephan

    2017-01-01

    Chronic infection with the Hepatitis B Virus (HBV) is a major cause of liver-related morbidity and mortality. One peculiar observation in cells infected with HBV (or with closely‑related animal hepadnaviruses) is the presence of viral DNA integration in the host cell genome, despite this form being a replicative dead-end for the virus. The frequent finding of somatic integration of viral DNA suggests an evolutionary benefit for the virus; however, the mechanism of integration, its functions, and the clinical implications remain unknown. Here we review the current body of knowledge of HBV DNA integration, with particular focus on the molecular mechanisms and its clinical implications (including the possible consequences of replication-independent antigen expression and its possible role in hepatocellular carcinoma). HBV DNA integration is likely to influence HBV replication, persistence, and pathogenesis, and so deserves greater attention in future studies. PMID:28394272

  2. Site-Specific Integration of Foreign DNA into Minimal Bacterial and Human Target Sequences Mediated by a Conjugative Relaxase

    PubMed Central

    Agúndez, Leticia; González-Prieto, Coral; Machón, Cristina; Llosa, Matxalen

    2012-01-01

    Background Bacterial conjugation is a mechanism for horizontal DNA transfer between bacteria which requires cell to cell contact, usually mediated by self-transmissible plasmids. A protein known as relaxase is responsible for the processing of DNA during bacterial conjugation. TrwC, the relaxase of conjugative plasmid R388, is also able to catalyze site-specific integration of the transferred DNA into a copy of its target, the origin of transfer (oriT), present in a recipient plasmid. This reaction confers TrwC a high biotechnological potential as a tool for genomic engineering. Methodology/Principal Findings We have characterized this reaction by conjugal mobilization of a suicide plasmid to a recipient cell with an oriT-containing plasmid, selecting for the cointegrates. Proteins TrwA and IHF enhanced integration frequency. TrwC could also catalyze integration when it is expressed from the recipient cell. Both Y18 and Y26 catalytic tyrosil residues were essential to perform the reaction, while TrwC DNA helicase activity was dispensable. The target DNA could be reduced to 17 bp encompassing TrwC nicking and binding sites. Two human genomic sequences resembling the 17 bp segment were accepted as targets for TrwC-mediated site-specific integration. TrwC could also integrate the incoming DNA molecule into an oriT copy present in the recipient chromosome. Conclusions/Significance The results support a model for TrwC-mediated site-specific integration. This reaction may allow R388 to integrate into the genome of non-permissive hosts upon conjugative transfer. Also, the ability to act on target sequences present in the human genome underscores the biotechnological potential of conjugative relaxase TrwC as a site-specific integrase for genomic modification of human cells. PMID:22292089

  3. Analysis of chromatin integrity and DNA damage of buffalo spermatozoa

    PubMed Central

    Mahmoud, K. Gh. M.; El-Sokary, A. A. E.; Abdel-Ghaffar, A. E.; Abou El-Roos, M. E. A.; Ahmed, Y. F.

    2015-01-01

    This study was conducted to determine chromatin integrity and DNA damage by DNA electrophoresis and comet assays of buffalo fresh and frozen semen. Semen samples were collected from four buffalo bulls and evaluated after freezing for semen motility, viability, sperm abnormalities, chromatin integrity and DNA damage. A significant variation was found in semen parameters after thawing. Highly significant differences (P<0.001) in chromatin integrity were observed between fresh and frozen semen. For the fresh semen, there was no significant difference between the bulls for chromatin integrity; however, a significant variation (P<0.05) was detected in their frozen semen. No DNA fragmentation was observed by agarose gel electrophoresis. The percentage of sperm with damaged DNA detected by comet assay differed significantly between fresh and frozen semen. A significant negative correlation was recorded between motility and DNA damage (r=-0.68, P<0.05). Sperm abnormalities and DNA fragmentation were significantly positively correlated (r=0.59, P<0.05). In conclusion, DNA damage evaluation can provide reassurance about genomic normalcy and guide the development of improved methods of selecting spermatozoa with intact DNA to be used in artificial insemination. PMID:27175169

  4. Impact of copy number variations burden on coding genome in humans using integrated high resolution arrays.

    PubMed

    Veerappa, Avinash M; Lingaiah, Kusuma; Vishweswaraiah, Sangeetha; Murthy, Megha N; Suresh, Raviraj V; Manjegowda, Dinesh S; Ramachandra, Nallur B

    2014-12-16

    Copy number variations (CNVs) alter the transcriptional and translational levels of genes by disrupting the coding structure and this burden of CNVs seems to be a significant contributor to phenotypic variations. Therefore it was necessary to assess the complexities of CNV burden on the coding genome. A total of 1715 individuals from 12 populations were used for CNV analysis in the present investigation. Analysis was performed using Affymetrix Genome-Wide Human SNP Array 6·0 chip and CytoScan High-Density arrays. CNVs were more frequently observed in the coding region than in the non-coding region. CNVs were observed vastly more frequently in the coding region than the non-coding region. CNVs were found to be enriched in the regions containing functional genes (83-96%) compared with the regions containing pseudogenes (4-17%). CNVs across the genome of an individual showed multiple hits across many genes, whose proteins interact physically and function under the same pathway. We identified varying numbers of proteins and degrees of interactions within protein complexes of single individual genomes. This study represents the first draft of a population-specific CNV genes map as well as a cross-populational map. The complex relationship of CNVs on genes and their physically interacting partners unravels many complexities involved in phenotype expression. This study identifies four mechanisms contributing to the complexities caused by the presence of multiple CNVs across many genes in the coding part of the genome.

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

    PubMed

    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.

  6. Mechanism of spacer integration links the CRISPR/Cas system to transposition as a form of mobile DNA.

    PubMed

    Dyda, Fred; Hickman, Alison B

    2015-01-01

    It has recently become clear that many bacterial and archaeal species possess adaptive immune systems. These are typified by multiple copies of DNA sequences known as clustered regularly interspaced short palindromic repeats (CRISPRs). These CRISPR repeats are the sites at which short spacers containing sequences of previously encountered foreign DNA are integrated, and the spacers serve as the molecular memory of previous invaders. In vivo work has demonstrated that two CRISPR-associated proteins - Cas1 and Cas2 - are required for spacer integration, but the mechanism by which this is accomplished remained unclear. Here we review a recent paper describing the in vitro reconstitution of CRISPR spacer integration using purified Cas1 and Cas2 and place the results in context of similar DNA transposition reactions and the crystal structure of the Cas1/Cas2 complex.

  7. Retroviral Integrase Proteins and HIV-1 DNA Integration*

    PubMed Central

    Krishnan, Lavanya; Engelman, Alan

    2012-01-01

    Retroviral integrases catalyze two reactions, 3′-processing of viral DNA ends, followed by integration of the processed ends into chromosomal DNA. X-ray crystal structures of integrase-DNA complexes from prototype foamy virus, a member of the Spumavirus genus of Retroviridae, have revealed the structural basis of integration and how clinically relevant integrase strand transfer inhibitors work. Underscoring the translational potential of targeting virus-host interactions, small molecules that bind at the host factor lens epithelium-derived growth factor/p75-binding site on HIV-1 integrase promote dimerization and inhibit integrase-viral DNA assembly and catalysis. Here, we review recent advances in our knowledge of HIV-1 DNA integration, as well as future research directions. PMID:23043109

  8. A Double-Layered Mixture Model for the Joint Analysis of DNA Copy Number and Gene Expression Data

    PubMed Central

    Choi, Hyungwon; Qin, Zhaohui S.

    2010-01-01

    Abstract Copy number aberration is a common form of genomic instability in cancer. Gene expression is closely tied to cytogenetic events by the central dogma of molecular biology, and serves as a mediator of copy number changes in disease phenotypes. Accordingly, it is of interest to develop proper statistical methods for jointly analyzing copy number and gene expression data. This work describes a novel Bayesian inferential approach for a double-layered mixture model (DLMM) which directly models the stochastic nature of copy number data and identifies abnormally expressed genes due to aberrant copy number. Simulation studies were conducted to illustrate the robustness of DLMM under various settings of copy number aberration frequency, confounding effects, and signal-to-noise ratio in gene expression data. Analysis of a real breast cancer data shows that DLMM is able to identify expression changes specifically attributable to copy number aberration in tumors and that a sample-specific index built based on the selected genes is correlated with relevant clinical information. PMID:20170400

  9. Preservation of DNA integrity and neuronal degeneration.

    PubMed

    Francisconi, Simona; Codenotti, Mara; Ferrari-Toninelli, Giulia; Uberti, Daniela; Memo, Maurizio

    2005-04-01

    The mismatch repair system (MMR) is an important member of the DNA checkpoint, that includes a number of protein deputed to control genomic stability through cell cycle arrest, DNA repair, and apoptosis. Here we summarize some recent data from our and other groups underlining the contribution to neurodegeneration of MSH2, perhaps the most relevant component of the MMR system. These data suggest that this protein participates not only in the cancer prevention machinery for the body but also in neurodegenerative processes.

  10. [Length polymorphism of integrated copies of R1 and R2 retrotransposons in the German cockroach (Blattella germanica) as a potential marker for population and phylogenetic studies].

    PubMed

    Kagramanova, A S; Korolev, A L; Schal, C; Mukha, D V

    2006-04-01

    Using polymerase chain reaction technique with primers flanking target sites of retrotransposons R1 and R2, integrated copies of these transposable elements were amplified in various cockroach species (Blattodea). It was shown that each species has a unique pattern of "5'-undertranscripts" with the definite set of amplified fragments of different lengths. Intraspecies polymorphism was revealed in analysis of German cockroach specimens obtained upon individual mating. This is the first report providing results of identifying, cloning, and sequencing extended fragments (5'-truncated copies) of Blatella germanica R1 and R2 retrotransposons. It may be assumed that patterns of 5'-truncated copies of R1 and R2 elements can be used as markers in population and phylogenetic studies. Moreover, cloned and sequenced fragments will be employed in our further studies for screening of the German cockroach genomic library in order to detect full-length copies in this class transposable elements.

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

    PubMed

    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.

  12. Development and validation of InnoQuant™, a sensitive human DNA quantitation and degradation assessment method for forensic samples using high copy number mobile elements Alu and SVA.

    PubMed

    Pineda, Gina M; Montgomery, Anne H; Thompson, Robyn; Indest, Brooke; Carroll, Marion; Sinha, Sudhir K

    2014-11-01

    There is a constant need in forensic casework laboratories for an improved way to increase the first-pass success rate of forensic samples. The recent advances in mini STR analysis, SNP, and Alu marker systems have now made it possible to analyze highly compromised samples, yet few tools are available that can simultaneously provide an assessment of quantity, inhibition, and degradation in a sample prior to genotyping. Currently there are several different approaches used for fluorescence-based quantification assays which provide a measure of quantity and inhibition. However, a system which can also assess the extent of degradation in a forensic sample will be a useful tool for DNA analysts. Possessing this information prior to genotyping will allow an analyst to more informatively make downstream decisions for the successful typing of a forensic sample without unnecessarily consuming DNA extract. Real-time PCR provides a reliable method for determining the amount and quality of amplifiable DNA in a biological sample. Alu are Short Interspersed Elements (SINE), approximately 300bp insertions which are distributed throughout the human genome in large copy number. The use of an internal primer to amplify a segment of an Alu element allows for human specificity as well as high sensitivity when compared to a single copy target. The advantage of an Alu system is the presence of a large number (>1000) of fixed insertions in every human genome, which minimizes the individual specific variation possible when using a multi-copy target quantification system. This study utilizes two independent retrotransposon genomic targets to obtain quantification of an 80bp "short" DNA fragment and a 207bp "long" DNA fragment in a degraded DNA sample in the multiplex system InnoQuant™. The ratio of the two quantitation values provides a "Degradation Index", or a qualitative measure of a sample's extent of degradation. The Degradation Index was found to be predictive of the observed loss

  13. Alterations in mitochondrial DNA copy number and the activities of electron transport chain complexes and pyruvate dehydrogenase in the frontal cortex from subjects with autism.

    PubMed

    Gu, F; Chauhan, V; Kaur, K; Brown, W T; LaFauci, G; Wegiel, J; Chauhan, A

    2013-09-03

    Autism is a neurodevelopmental disorder associated with social deficits and behavioral abnormalities. Recent evidence suggests that mitochondrial dysfunction and oxidative stress may contribute to the etiology of autism. This is the first study to compare the activities of mitochondrial electron transport chain (ETC) complexes (I-V) and pyruvate dehydrogenase (PDH), as well as mitochondrial DNA (mtDNA) copy number in the frontal cortex tissues from autistic and age-matched control subjects. The activities of complexes I, V and PDH were most affected in autism (n=14) being significantly reduced by 31%, 36% and 35%, respectively. When 99% confidence interval (CI) of control group was taken as a reference range, impaired activities of complexes I, III and V were observed in 43%, 29% and 43% of autistic subjects, respectively. Reduced activities of all five ETC complexes were observed in 14% of autistic cases, and the activities of multiple complexes were decreased in 29% of autistic subjects. These results suggest that defects in complexes I and III (sites of mitochondrial free radical generation) and complex V (adenosine triphosphate synthase) are more prevalent in autism. PDH activity was also reduced in 57% of autistic subjects. The ratios of mtDNA of three mitochondrial genes ND1, ND4 and Cyt B (that encode for subunits of complexes I and III) to nuclear DNA were significantly increased in autism, suggesting a higher mtDNA copy number in autism. Compared with the 95% CI of the control group, 44% of autistic children showed higher copy numbers of all three mitochondrial genes examined. Furthermore, ND4 and Cyt B deletions were observed in 44% and 33% of autistic children, respectively. This study indicates that autism is associated with mitochondrial dysfunction in the brain.

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

    PubMed

    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.

  15. The adenovirus type 12 - mouse cell system: permissivity and analysis of integration patterns of viral DNA in tumor cells.

    PubMed Central

    Starzinski-Powitz, A; Schulz, M; Esche, H; Mukai, N; Doerfler, W

    1982-01-01

    The integration patterns of persisting adenovirus type 12 (Ad12) DNA were analyzed in two Ad12-induced tumors of Balb/c and CBA/J mice and in one tumor cell line derived from an Ad12-induced retinoblastoma of C3H origin. In all three tumors the Ad12 genome was integrated colinearly and various copy numbers of viral DNA were found. Analysis of the Ad12 integration patterns revealed relatively simple offsize band patterns regardless of Ad12 copy numbers. The degree of methylation at the 5'-CCGG-3' sites in the inserted Ad12 genome was determined using the isoschizomeric restriction endonuclease pair HpaII and MspI. Methylation was rather incomplete in the primary tumor tissues but almost complete in the retinoblastoma line carried in culture for many passages. The levels of expression of the viral genome in the Balb/c tumor and in the retinoblastoma line were determined by in vitro translation of RNA isolated from these cells and selected with appropriate restriction endonuclease fragments of Ad12 DNA. In both instances the 59 K, 19 K, and 17 K proteins of the E1b region were expressed. Proteins of the E1a region appeared very faint in the size class between 22 K and 42 K. The permissivity of Ad12 and the replication of Ad12 DNA in mouse cells were investigated by blotting restricted DNA from cells soon after, and a long time after, infection and by hybridization with 32P-labeled Ad12 DNA. Neither primary mouse kidney cells nor the established L929 mouse cell line supported viral DNA replication. These results raise the question to what extent host cell factors determine Ad12 DNA replication in mammalian cells. Images Fig. 2. Fig. 3. Fig. 4. PMID:7188349

  16. Super-resolution microscopy reveals that mammalian mitochondrial nucleoids have a uniform size and frequently contain a single copy of mtDNA.

    PubMed

    Kukat, Christian; Wurm, Christian A; Spåhr, Henrik; Falkenberg, Maria; Larsson, Nils-Göran; Jakobs, Stefan

    2011-08-16

    Mammalian mtDNA is packaged in DNA-protein complexes denoted mitochondrial nucleoids. The organization of the nucleoid is a very fundamental question in mitochondrial biology and will determine tissue segregation and transmission of mtDNA. We have used a combination of stimulated emission depletion microscopy, enabling a resolution well below the diffraction barrier, and molecular biology to study nucleoids in a panel of mammalian tissue culture cells. We report that the nucleoids labeled with antibodies against DNA, mitochondrial transcription factor A (TFAM), or incorporated BrdU, have a defined, uniform mean size of ∼100 nm in mammals. Interestingly, the nucleoid frequently contains only a single copy of mtDNA (average ∼1.4 mtDNA molecules per nucleoid). Furthermore, we show by molecular modeling and volume calculations that TFAM is a main constituent of the nucleoid, besides mtDNA. These fundamental insights into the organization of mtDNA have broad implications for understanding mitochondrial dysfunction in disease and aging.

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

    PubMed Central

    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

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

    PubMed

    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.

  19. Polydnavirus DNA is integrated in the DNA of its parasitoid wasp host.

    PubMed Central

    Fleming, J G; Summers, M D

    1991-01-01

    The polydnavirus Campoletis sonorensis virus (CsV) is present in the oviducts of all adult C. sonorensis female wasps and appears to be required for these wasps to parasitize hosts successfully. Physical mapping, Southern blot analysis, and nucleotide sequence analysis demonstrate that the viral DNA B-specific sequences in cloned wasp DNA are colinear with viral genomic segment DNA B from nucleocapsids and are covalently linked to nonviral wasp sequences. Integrated DNA B terminates in 59-nucleotide imperfect direct repeats, but a single repeat exists in the extrachromosomal superhelical viral DNA B. Sequences near each junction form imperfect inverted repeats with sequences near the ends of an internal viral 540-base-pair repeat element gene. CsV appears to be the first documented integrated, nonretroviral DNA virus of insects and probably is vertically transmitted as a provirus. Images PMID:1946402

  20. Integrating DNA strand-displacement circuitry with DNA tile self-assembly

    PubMed Central

    Zhang, David Yu; Hariadi, Rizal F.; Choi, Harry M.T.; Winfree, Erik

    2013-01-01

    DNA nanotechnology has emerged as a reliable and programmable way of controlling matter at the nanoscale through the specificity of Watson–Crick base pairing, allowing both complex self-assembled structures with nanometer precision and complex reaction networks implementing digital and analog behaviors. Here we show how two well-developed frameworks, DNA tile self-assembly and DNA strand-displacement circuits, can be systematically integrated to provide programmable kinetic control of self-assembly. We demonstrate the triggered and catalytic isothermal self-assembly of DNA nanotubes over 10 μm long from precursor DNA double-crossover tiles activated by an upstream DNA catalyst network. Integrating more sophisticated control circuits and tile systems could enable precise spatial and temporal organization of dynamic molecular structures. PMID:23756381

  1. DNase I sensitivity of integrated simian virus 40 DNA.

    PubMed Central

    Blanck, G; Chen, S; Pollack, R

    1984-01-01

    We undertook an analysis of integrated simian virus 40 (SV40) DNA to learn whether the DNase I-sensitive region is retained in the integrated array of mouse transformants. Our results indicate that full-length integrated SV40 chromatin retains a DNase I-hypersensitive region at the same point as in nonintegrated SV40 chromatin. Thus, the lack of a DNase I-hypersensitive region is not likely to be the reason for nonpermissivity of SV40 in mouse cells. In addition, results reported here indicate that a deletion of about 200 base pairs of DNA in the region of the DNase I-hypersensitive site severely reduces the sensitivity of integrated SV40 chromatin. This result is similar to a previously reported result obtained with deletion mutants of SV40 analyzed in the lytic cycle. It is the first report of a DNA lesion affecting DNase I hypersensitivity of a mammalian chromosome. Images PMID:6325884

  2. DNA integrity determination in marine invertebrates by Fast Micromethod.

    PubMed

    Jaksić, 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 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(-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 137Cs 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 microg ml(-1) (microM)) 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 homogenate by 4

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

    PubMed Central

    Chen, Yan; Williams, Vonetta; Filippova, Maria; Filippov, Valery; Duerksen-Hughes, Penelope

    2014-01-01

    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. PMID:25340830

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

    PubMed Central

    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

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

    PubMed

    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.

  6. Binding of nuclear factors to a satellite DNA of retroviral origin with marked differences in copy number among species of the rodent Ctenomys.

    PubMed Central

    Pesce, C G; Rossi, M S; Muro, A F; Reig, O A; Zorzópulos, J; Kornblihtt, A R

    1994-01-01

    The major satellite DNA of the subterranean rodent Ctenomys, named RPCS, contains several consensus sequences characteristic of the U3 region of retroviral long terminal repeats (LTRs), such as a polypurine tract, CCAAT boxes, binding sites for the CCAAT/enhancer-binding protein (C/EBP), a TATA box and putative polyadenylation signals. RPCS presents an enormous variation in abundance between species of the same genus: while C. australis or C. talarum have approximately 3 x 10(6) copies per genome, C. opimus has none. A sequence (RPCS-I) with identity to the SV40-enhancer core element, present in all the repeating units of the satellite is specifically protected in DNase I footprintings. Competitions of band-shift assays with different transcription factor binding sites indicate that binding to RPCS-I is specific and involves CCAAT proteins related to NF-1, but not to C/EBP. By the use of quantitative protein/DNA binding assays we determined that, despite of their conspicuous difference in RPCS copy number, C. talarum and C. opimus have equivalent amounts and identical quality of RPCS-binding proteins. These results are consistent with the observation, by in situ hybridization, that RPCS is clustered in heterochromatic regions, where it might have restricted accessibility to transcription factors in vivo. This is the first report of the binding of transcription factors to a satellite DNA of retroviral origin. Images PMID:8127714

  7. On the question of the integration of exogenous bacterial DNA into plant DNA.

    PubMed Central

    Kleinhofs, A; Eden, F C; Chilton, M D; Bendich, A J

    1975-01-01

    Extensive studies with pea, tomato, and barley failed to confirm the evidence presented by previous investigators for integration or replication of exogenously applied bacterial DNA in these plants. Labeled DNA of buoyant density in CsCl intermediate between that of high density donor bacterial DNA and of plant DNA was never observed with axenic plants. Intermediate peaks, similar to those used as evidence for recombination by earlier investigators, were observed only when the plants were contaminated with bacteria. Plant DNA prepared by a published procedure [Ledoux, L. & Huart, R. (1969) J. Mol. Biol. 43, 243-262] was found to be contaminated with unidentified impurities. Such DNA was partially protected from the action of DNase and produced aberrant banding patterns in CsCl after shearing. Much of the published evidence for integration of foreign DNA in plants is based upon experiments with plant DNA prepared by this procedure. We conclude that contamination is the likely explanation for what has been interpreted as evidence for integration. PMID:809769

  8. Cross-species DNA copy number analyses identifies multiple 1q21-q23 subtype-specific driver genes for breast cancer.

    PubMed

    Silva, Grace O; He, Xiaping; Parker, Joel S; Gatza, Michael L; Carey, Lisa A; Hou, Jack P; Moulder, Stacy L; Marcom, Paul K; Ma, Jian; Rosen, Jeffrey M; Perou, Charles M

    2015-07-01

    A large number of DNA copy number alterations (CNAs) exist in human breast cancers, and thus characterizing the most frequent CNAs is key to advancing therapeutics because it is likely that these regions contain breast tumor 'drivers' (i.e., cancer causal genes). This study aims to characterize the genomic landscape of breast cancer CNAs and identify potential subtype-specific drivers using a large set of human breast tumors and genetically engineered mouse (GEM) mammary tumors. Using a novel method called SWITCHplus, we identified subtype-specific DNA CNAs occurring at a 15% or greater frequency, which excluded many well-known breast cancer-related drivers such as amplification of ERBB2, and deletions of TP53 and RB1. A comparison of CNAs between mouse and human breast tumors identified regions with shared subtype-specific CNAs. Additional criteria that included gene expression-to-copy number correlation, a DawnRank network analysis, and RNA interference functional studies highlighted candidate driver genes that fulfilled these multiple criteria. Numerous regions of shared CNAs were observed between human breast tumors and GEM mammary tumor models that shared similar gene expression features. Specifically, we identified chromosome 1q21-23 as a Basal-like subtype-enriched region with multiple potential driver genes including PI4KB, SHC1, and NCSTN. This step-wise computational approach based on a cross-species comparison is applicable to any tumor type for which sufficient human and model system DNA copy number data exist, and in this instance, highlights that a single region of amplification may in fact harbor multiple driver genes.

  9. The COG and COPI complexes interact to control the abundance of GEARs, a subset of Golgi integral membrane proteins.

    PubMed

    Oka, Toshihiko; Ungar, Daniel; Hughson, Frederick M; Krieger, Monty

    2004-05-01

    The conserved oligomeric Golgi (COG) complex is a soluble hetero-octamer associated with the cytoplasmic surface of the Golgi. Mammalian somatic cell mutants lacking the Cog1 (ldlB) or Cog2 (ldlC) subunits exhibit pleiotropic defects in Golgi-associated glycoprotein and glycolipid processing that suggest COG is involved in the localization, transport, and/or function of multiple Golgi processing proteins. We have identified a set of COG-sensitive, integral membrane Golgi proteins called GEARs (mannosidase II, GOS-28, GS15, GPP130, CASP, giantin, and golgin-84) whose abundances were reduced in the mutant cells and, in some cases, increased in COG-overexpressing cells. In the mutants, some GEARs were abnormally localized in the endoplasmic reticulum and were degraded by proteasomes. The distributions of the GEARs were altered by small interfering RNA depletion of epsilon-COP in wild-type cells under conditions in which COG-insensitive proteins were unaffected. Furthermore, synthetic phenotypes arose in mutants deficient in both epsilon-COP and either Cog1 or Cog2. COG and COPI may work in concert to ensure the proper retention or retrieval of a subset of proteins in the Golgi, and COG helps prevent the endoplasmic reticulum accumulation and degradation of some GEARs.

  10. Quadruplex Integrated DNA (QuID) Nanosensors for Monitoring Dopamine

    PubMed Central

    Morales, Jennifer M.; Skipwith, Christopher G.; Clark, Heather A.

    2015-01-01

    Dopamine is widely innervated throughout the brain and critical for many cognitive and motor functions. Imbalances or loss in dopamine transmission underlie various psychiatric disorders and degenerative diseases. Research involving cellular studies and disease states would benefit from a tool for measuring dopamine transmission. Here we show a Quadruplex Integrated DNA (QuID) nanosensor platform for selective and dynamic detection of dopamine. This nanosensor exploits DNA technology and enzyme recognition systems to optically image dopamine levels. The DNA quadruplex architecture is designed to be compatible in physically constrained environments (110 nm) with high flexibility, homogeneity, and a lower detection limit of 110 µM. PMID:26287196

  11. A supramolecular assembly mediates lentiviral DNA integration

    PubMed Central

    Serrao, Erik; Locke, Julia; Swuec, Paolo; Jónsson, Stefán R.; Kotecha, Abhay; Cook, Nicola J.; Pye, Valerie E.; Taylor, Ian A.; Andrésdóttir, Valgerdur; Engelman, Alan N.; Costa, Alessandro; Cherepanov, Peter

    2017-01-01

    Retroviral integrase (IN) functions within the intasome nucleoprotein complex to catalyze insertion of viral DNA into cellular chromatin. Using cryo-electron microscopy, we now visualize the functional maedi-visna lentivirus intasome at 4.9 Å resolution. The intasome comprises a homo-hexadecamer of IN with a tetramer-of-tetramers architecture featuring eight structurally distinct types of IN protomers supporting two catalytically competent subunits. The conserved intasomal core, previously observed in simpler retroviral systems, is formed between two IN tetramers, with a pair of C-terminal domains from flanking tetramers completing the synaptic interface. Our results explain how HIV-1 IN, which self-associates into higher order multimers, can form a functional intasome, reconcile the bulk of early HIV-1 IN biochemical and structural data, and provide a lentiviral platform for design of HIV-1 IN inhibitors. PMID:28059770

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

    PubMed Central

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

    2016-01-01

    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. DOI: http://dx.doi.org/10.7554/eLife.11994.001 PMID:27642012

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

    PubMed

    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.

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

    PubMed Central

    Kiselinova, Maja; De Spiegelaere, Ward; Buzon, Maria Jose; Malatinkova, Eva; Lichterfeld, Mathias; Vandekerckhove, Linos

    2016-01-01

    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

  15. Integrating DNA-based data into bioassessments improves ...

    EPA Pesticide Factsheets

    The integration of DNA-based identification methods into bioassessments could result in more accurate representations of species distributions and species-habitat relationships. DNA-based approaches may be particularly informative for tracking the distributions of rare and/or invasive species that can comprise a small proportion of samples or are difficult to identify morphologically. In 2012 and 2013, we used a combination of morphological and DNA-based methods (meta-barcoding) to identify fish eggs and larvae collected in the St. Louis River estuary area, Minnesota. We found a large proportion of cases where a lack of agreement occurred between species identified at a site using morphological versus DNA identification, prompting a discussion of how to best reconcile these differences. Choices made during sampling collection, DNA amplification/extraction, and bioinformatics processing influence the DNA-morphology match. The distribution of some species (including several invasives) and their relationships to habitat changed after DNA-data was incorporated. Results highlight how incorporating of DNA-data may get us closer to the “truth”, which has large ramifications in the search for rare species and when understanding the environmental drivers of species distributions is important for management. not applicable

  16. Integrating prokaryotes and eukaryotes: DNA transposases in light of structure

    PubMed Central

    Hickman, Alison Burgess; Chandler, Michael; Dyda, Fred

    2011-01-01

    DNA rearrangements are important in genome function and evolution. Genetic material can be rearranged inadvertently during processes such as DNA repair, or can be moved in a controlled manner by enzymes specifically dedicated to the task. DNA transposases comprise one class of such enzymes. These move DNA segments known as transposons to new locations, without the need for sequence homology between transposon and target site. Several biochemically distinct pathways have evolved for DNA transposition, and genetic and biochemical studies have provided valuable insights into many of these. However, structural information on transposases – particularly with DNA substrates – has proven elusive in most cases. On the other hand, large-scale genome sequencing projects have led to an explosion in the number of annotated prokaryotic and eukaryotic mobile elements. Here, we briefly review biochemical and mechanistic aspects of DNA transposition, and propose that integrating sequence information with structural information using bioinformatics tools such as secondary structure prediction and protein threading can lead not only to an additional level of understanding but possibly also to testable hypotheses regarding transposition mechanisms. Detailed understanding of transposition pathways is a prerequisite for the long-term goal of exploiting DNA transposons as genetic tools and as a basis for genetic medical applications. PMID:20067338

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

    PubMed Central

    2010-01-01

    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. PMID:20576098

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

    PubMed

    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.

  19. International Comparison of Enumeration-Based Quantification of DNA Copy-Concentration Using Flow Cytometric Counting and Digital Polymerase Chain Reaction.

    PubMed

    Yoo, Hee-Bong; Park, Sang-Ryoul; Dong, Lianhua; Wang, Jing; Sui, Zhiwei; Pavšič, Jernej; Milavec, Mojca; Akgoz, Muslum; Mozioğlu, Erkan; Corbisier, Philippe; Janka, Mátrai; Cosme, Bruno; de V Cavalcante, Janaina J; Flatshart, Roberto Becht; Burke, Daniel; Forbes-Smith, Michael; McLaughlin, Jacob; Emslie, Kerry; Whale, Alexandra S; Huggett, Jim F; Parkes, Helen; Kline, Margaret C; Harenza, Jo Lynne; Vallone, Peter M

    2016-12-20

    Enumeration-based determination of DNA copy-concentration was assessed through an international comparison among national metrology institutes (NMIs) and designated institutes (DIs). Enumeration-based quantification does not require a calibration standard thereby providing a route to "absolute quantification", which offers the potential for reliable value assignments of DNA reference materials, and International System of Units (SI) traceability to copy number 1 through accurate counting. In this study, 2 enumeration-based methods, flow cytometric (FCM) counting and the digital polymerase chain reaction (dPCR), were compared to quantify a solution of the pBR322 plasmid at a concentration of several thousand copies per microliter. In addition, 2 orthogonal chemical-analysis methods based on nucleotide quantification, isotope-dilution mass spectrometry (IDMS) and capillary electrophoresis (CE) were applied to quantify a more concentrated solution of the plasmid. Although 9 dPCR results from 8 laboratories showed some dispersion (relative standard deviation [RSD] = 11.8%), their means were closely aligned with those of the FCM-based counting method and the orthogonal chemical-analysis methods, corrected for gravimetric dilution factors. Using the means of dPCR results, the RSD of all 4 methods was 1.8%, which strongly supported the validity of the recent enumeration approaches. Despite a good overall agreement, the individual dPCR results were not sufficiently covered by the reported measurement uncertainties. These findings suggest that some laboratories may not have considered all factors contributing to the measurement uncertainty of dPCR, and further investigation of this possibility is warranted.

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

    PubMed Central

    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

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

    Cancer.gov

    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.

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

    Cancer.gov

    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.

  3. Chopping Copy.

    ERIC Educational Resources Information Center

    Bush, Don

    1994-01-01

    Discusses ways an editor can cut out words to help the reader understand quickly. Discusses dead wood, redundancy, redundancy in thought, smothered verbs, false precision, editing and academia, and making copy smoother. (SR)

  4. Applications of DNA integrating elements: Facing the bias bully

    PubMed Central

    de Jong, Johann; Wessels, Lodewyk F A; van Lohuizen, Maarten; de Ridder, Jeroen; Akhtar, Waseem

    2014-01-01

    Retroviruses and DNA transposons are an important part of molecular biologists' toolbox. The applications of these elements range from functional genomics to oncogene discovery and gene therapy. However, these elements do not integrate uniformly across the genome, which is an important limitation to their use. A number of genetic and epigenetic factors have been shown to shape the integration preference of these elements. Insight into integration bias can significantly enhance the analysis and interpretation of results obtained using these elements. For three different applications, we outline how bias can affect results, and can potentially be addressed. PMID:26442173

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

    PubMed Central

    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

  6. Maternal age and ovarian stimulation independently affect oocyte mtDNA copy number and cumulus cell gene expression in bovine clones.

    PubMed

    Cree, Lynsey M; Hammond, Elizabeth R; Shelling, Andrew N; Berg, Martin C; Peek, John C; Green, Mark P

    2015-06-01

    Does maternal ageing and ovarian stimulation alter mitochondrial DNA (mtDNA) copy number and gene expression of oocytes and cumulus cells from a novel bovine model for human IVF? Oocytes collected from females with identical nuclear genetics show decreased mtDNA copy number and increased expression of an endoplasmic reticulum (ER) stress gene with repect to ovarian stimulation, whilst differences in the expression of genes involved in mitochondrial function, antioxidant protection and apoptosis were evident in relation to maternal ageing and the degree of ovarian stimulation in cumulus cells. Oocyte quality declines with advancing maternal age; however, the underlying mechanism, as well as the effects of ovarian stimulation are poorly understood. Human studies investigating these effects are often limited by differences in age and ovarian stimulation regimens within a patient cohort, as well as genetic and environmental variability. A novel bovine cross-sectional maternal age model for human IVF was undertaken. Follicles were aspirated from young (3 years of age; n = 7 females) and old (10 years of age; n = 5 females) Holstein Freisian clones following multiple unstimulated, mild and standard ovarian stimulation cycles. These bovine cloned females were generated by the process of somatic cell nuclear transfer (SCNT) from the same founder and represent a homogeneous population with reduced genetic and environmental variability. Maternal age and ovarian stimulation effects were investigated in relation to mtDNA copy number, and the expression of 19 genes involved in mitochondrial function, antioxidant protection, oocyte-cumulus cell signalling and follicle development in both oocytes and cumulus cells. Young (3 years of age; n = 7 females) and old (10 years of age; n = 5 females) Holstein Freisian bovine clones were maintained as one herd. Stimulation cycles were based on the long GnRH agonist down-regulation regimen used in human fertility clinics. Follicle growth

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

    PubMed

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

    2014-12-01

    The aims of this study were to evaluate: (1) the effect of cryopreservation on DNA fragmentation of canine epididymal spermatozoa, and (2) the potential protective effect of melatonin on post-thaw sperm quality (motility, morphology, acrosomal and DNA integrity). Epididymal spermatozoa were collected after orchiectomy of ten dogs. Sperm samples were frozen in the presence or absence of melatonin (1mM). DNA fragmentation index (percentage of spermatozoa with fragmented DNA) was similar in fresh samples (3.3±3.6) and samples frozen with (4.2±3.8) or without (3.6±3.7) melatonin. Sperm motility was significantly (p<0.0001) higher in fresh compared to frozen samples. The presence of melatonin in the freezing extender did not affect the sperm motility. Proportions of spermatozoa with normal morphology were similar in fresh and frozen samples, irrespective of the presence of melatonin in the extender. Acrosome integrity was significantly decreased (p<0.01) by cryopreservation, and melatonin did not exert any beneficial effects. In conclusion, DNA fragmentation of canine epididymal spermatozoa was not affected by the freezing procedure, and the presence of melatonin did not preserve motility and acrosome integrity which were adversely affected by cryopreservation. The evaluation of DNA status of thawed gametes is particularly relevant for epididymal spermatozoa since these spermatozoa are usually stored and used in assisted reproductive techniques. Copyright © 2014 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  8. Additional Copies of the Proteolipid Protein Gene Causing Pelizaeus-Merzbacher Disease Arise by Separate Integration into the X Chromosome

    PubMed Central

    Hodes, M. E.; Woodward, Karen; Spinner, Nancy B.; Emanuel, Beverly S.; Enrico-Simon, Agnes; Kamholz, John; Stambolian, Dwight; Zackai, Elaine H.; Pratt, Victoria M.; Thomas, I. T.; Crandall, Kerry; Dlouhy, Stephen R.; Malcolm, Sue

    2000-01-01

    The proteolipid protein gene (PLP) is normally present at chromosome Xq22. Mutations and duplications of this gene are associated with Pelizaeus-Merzbacher disease (PMD). Here we describe two new families in which males affected with PMD were found to have a copy of PLP on the short arm of the X chromosome, in addition to a normal copy on Xq22. In the first family, the extra copy was first detected by the presence of heterozygosity of the AhaII dimorphism within the PLP gene. The results of FISH analysis showed an additional copy of PLP in Xp22.1, although no chromosomal rearrangements could be detected by standard karyotype analysis. Another three affected males from the family had similar findings. In a second unrelated family with signs of PMD, cytogenetic analysis showed a pericentric inversion of the X chromosome. In the inv(X) carried by several affected family members, FISH showed PLP signals at Xp11.4 and Xq22. A third family has previously been reported, in which affected members had an extra copy of the PLP gene detected at Xq26 in a chromosome with an otherwise normal banding pattern. The identification of three separate families in which PLP is duplicated at a noncontiguous site suggests that such duplications could be a relatively common but previously undetected cause of genetic disorders. PMID:10827108

  9. Additional copies of the proteolipid protein gene causing Pelizaeus-Merzbacher disease arise by separate integration into the X chromosome.

    PubMed

    Hodes, M E; Woodward, K; Spinner, N B; Emanuel, B S; Enrico-Simon, A; Kamholz, J; Stambolian, D; Zackai, E H; Pratt, V M; Thomas, I T; Crandall, K; Dlouhy, S R; Malcolm, S

    2000-07-01

    The proteolipid protein gene (PLP) is normally present at chromosome Xq22. Mutations and duplications of this gene are associated with Pelizaeus-Merzbacher disease (PMD). Here we describe two new families in which males affected with PMD were found to have a copy of PLP on the short arm of the X chromosome, in addition to a normal copy on Xq22. In the first family, the extra copy was first detected by the presence of heterozygosity of the AhaII dimorphism within the PLP gene. The results of FISH analysis showed an additional copy of PLP in Xp22.1, although no chromosomal rearrangements could be detected by standard karyotype analysis. Another three affected males from the family had similar findings. In a second unrelated family with signs of PMD, cytogenetic analysis showed a pericentric inversion of the X chromosome. In the inv(X) carried by several affected family members, FISH showed PLP signals at Xp11.4 and Xq22. A third family has previously been reported, in which affected members had an extra copy of the PLP gene detected at Xq26 in a chromosome with an otherwise normal banding pattern. The identification of three separate families in which PLP is duplicated at a noncontiguous site suggests that such duplications could be a relatively common but previously undetected cause of genetic disorders.

  10. On-chip pressure injection for integration of infrared-mediated DNA amplification with electrophoretic separation.

    PubMed

    Easley, Christopher J; Karlinsey, James M; Landers, James P

    2006-05-01

    Poly(dimethylsiloxane) (PDMS) membrane valves were utilized for diaphragm pumping on a PDMS-glass hybrid microdevice in order to couple infrared-mediated DNA amplification with electrophoretic separation of the products in a single device. Specific amplification products created during non-contact, infrared (IR) mediated polymerase chain reaction (PCR) were injected via chip-based diaphragm pumping into an electrophoretic separation channel. Channel dimensions were designed for injection plug shaping via preferential flow paths, which aided in minimizing the plug widths. Unbiased injection of sample could be achieved in as little as 190 ms, decreasing the time required with electrokinetic injection by two orders of magnitude. Additionally, sample stacking was promoted using laminar or biased-laminar loading to co-inject either water or low ionic strength DNA marker solution along with the PCR-amplified sample. Complete baseline resolution (Res = 2.11) of the 80- and 102-bp fragments of pUC-18 DNA marker solution was achieved, with partially resolved 257- and 267-bp fragments (Res = 0.56), in a separation channel having an effective length of only 3.0 cm. This resolution was deemed adequate for many PCR amplicon separations, with the added advantage of short separation time-typically complete in <120 s. Decreasing the amount of glass surrounding the PCR chamber reduced the DNA amplification time, yielding a further enhancement in analysis speed, with heating and cooling rates as high as 13.4 and -6.4 degrees C s(-1), respectively. With the time requirements greatly reduced for each step, it was possible to seamlessly couple IR-mediated amplification, sample injection, and separation/detection of a 278-bp fragment from the invA gene of <1000 starting copies of Salmonella typhimurium DNA in approximately 12 min on a single device, representing the fastest PCR-ME integration achieved to date.

  11. Mitochondrial DNA integrity is not dependent on DNA polymerase-beta activity.

    PubMed

    Hansen, Alexis B; Griner, Nicholas B; Anderson, Jon P; Kujoth, Greg C; Prolla, Tomas A; Loeb, Lawrence A; Glick, Eitan

    2006-01-05

    Mutations in mitochondrial DNA (mtDNA) are involved in a variety of pathologies, including cancer and neurodegenerative diseases, as well as in aging. mtDNA mutations result predominantly from damage by reactive oxygen species (ROS) that is not repaired prior to replication. Repair of ROS-damaged bases occurs mainly via base excision repair (BER) in mitochondria and nuclei. In nuclear BER, the two penultimate steps are carried out by DNA polymerase-beta (Polbeta), which exhibits both 5'-deoxyribose-5-phosphate (5'-dRP) lyase and DNA polymerase activities. In mitochondria, DNA polymerase-gamma (Polgamma) is believed to be the sole polymerase and is therefore assumed to function in mitochondrial BER. However, a recent report suggested the presence of Polbeta or a "Polbeta-like" enzyme in bovine mitochondria. Consequently, in the present work, we tested the hypothesis that Polbeta is present and functions in mammalian mitochondria. Initially we identified two DNA polymerase activities, one corresponding to Polgamma and the other to Polbeta, in mitochondrial preparations obtained by differential centrifugation and discontinuous sucrose density gradient centrifugation. However, upon further fractionation in linear Percoll gradients, we were able to separate Polbeta from mitochondria and to show that intact mitochondria, identified by electron microscopy, lacked Polbeta activity. In a functional test for the presence of Polbeta function in mitochondria, we used a new assay for detection of random (i.e., non-clonal) mutations in single mtDNA molecules. We did not detect enhanced mutation frequency in mtDNA from Polbeta null cells. In contrast, mtDNA from cells harboring mutations in the Polgamma exonuclease domain that abolish proofreading displayed a >or=17-fold increase in mutation frequency. We conclude that Polbeta is not an essential component of the machinery that maintains mtDNA integrity.

  12. Therapeutic Targeting of the Mitochondria Initiates Excessive Superoxide Production and Mitochondrial Depolarization Causing Decreased mtDNA Integrity

    PubMed Central

    Pokrzywinski, Kaytee L.; Biel, Thomas G.; Kryndushkin, Dmitry; Rao, V. Ashutosh

    2016-01-01

    Mitochondrial dysregulation is closely associated with excessive reactive oxygen species (ROS) production. Altered redox homeostasis has been implicated in the onset of several diseases including cancer. Mitochondrial DNA (mtDNA) and proteins are particularly sensitive to ROS as they are in close proximity to the respiratory chain (RC). Mitoquinone (MitoQ), a mitochondria-targeted redox agent, selectively damages breast cancer cells possibly through damage induced via enhanced ROS production. However, the effects of MitoQ and other triphenylphosphonium (TPP+) conjugated agents on cancer mitochondrial homeostasis remain unknown. The primary objective of this study was to determine the impact of mitochondria-targeted agent [(MTAs) conjugated to TPP+: mitoTEMPOL, mitoquinone and mitochromanol-acetate] on mitochondrial physiology and mtDNA integrity in breast (MDA-MB-231) and lung (H23) cancer cells. The integrity of the mtDNA was assessed by quantifying the degree of mtDNA fragmentation and copy number, as well as by measuring mitochondrial proteins essential to mtDNA stability and maintenance (TFAM, SSBP1, TWINKLE, POLG and POLRMT). Mitochondrial status was evaluated by measuring superoxide production, mitochondrial membrane depolarization, oxygen consumption, extracellular acidification and mRNA or protein levels of the RC complexes along with TCA cycle activity. In this study, we demonstrated that all investigated MTAs impair mitochondrial health and decrease mtDNA integrity in MDA-MB-231 and H23 cells. However, differences in the degree of mitochondrial damage and mtDNA degradation suggest unique properties among each MTA that may be cell line, dose and time dependent. Collectively, our study indicates the potential for TPP+ conjugated molecules to impair breast and lung cancer cells by targeting mitochondrial homeostasis. PMID:28030582

  13. Validation of a digital PCR method for quantification of DNA copy number concentrations by using a certified reference material.

    PubMed

    Deprez, Liesbet; Corbisier, Philippe; Kortekaas, Anne-Marie; Mazoua, Stéphane; Beaz Hidalgo, Roxana; Trapmann, Stefanie; Emons, Hendrik

    2016-09-01

    Digital PCR has become the emerging technique for the sequence-specific detection and quantification of nucleic acids for various applications. During the past years, numerous reports on the development of new digital PCR methods have been published. Maturation of these developments into reliable analytical methods suitable for diagnostic or other routine testing purposes requires their validation for the intended use. Here, the results of an in-house validation of a droplet digital PCR method are presented. This method is intended for the quantification of the absolute copy number concentration of a purified linearized plasmid in solution with a nucleic acid background. It has been investigated which factors within the measurement process have a significant effect on the measurement results, and the contribution to the overall measurement uncertainty has been estimated. A comprehensive overview is provided on all the aspects that should be investigated when performing an in-house method validation of a digital PCR method.

  14. T-DNA integration into the Arabidopsis genome depends on sequences of pre-insertion sites

    PubMed Central

    Brunaud, Véronique; Balzergue, Sandrine; Dubreucq, Bertrand; Aubourg, Sébastien; Samson, Franck; Chauvin, Stéphanie; Bechtold, Nicole; Cruaud, Corinne; DeRose, Richard; Pelletier, Georges; Lepiniec, Loïc; Caboche, Michel; Lecharny, Alain

    2002-01-01

    A statistical analysis of 9000 flanking sequence tags characterizing transferred DNA (T-DNA) transformants in Arabidopsis sheds new light on T-DNA insertion by illegitimate recombination. T-DNA integration is favoured in plant DNA regions with an A-T-rich content. The formation of a short DNA duplex between the host DNA and the left end of the T-DNA sets the frame for the recombination. The sequence immediately downstream of the plant A-T-rich region is the master element for setting up the DNA duplex, and deletions into the left end of the integrated T-DNA depend on the location of a complementary sequence on the T-DNA. Recombination at the right end of the T-DNA with the host DNA involves another DNA duplex, 2–3 base pairs long, that preferentially includes a G close to the right end of the T-DNA. PMID:12446565

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

    PubMed

    Tang, Binhua

    2016-01-01

    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. META2: Intercellular DNA Methylation Pairwise Annotation and Integrative Analysis

    PubMed Central

    2016-01-01

    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. PMID:28116291

  17. Cloning of monomeric human papillomavirus type 16 DNA integrated within cell DNA from a cervical carcinoma

    SciTech Connect

    Matsukura, T.; Kanda, T.; Furuno, A.; Yoshikawa, H.; Kawana, T.; Yoshiike, K.

    1986-06-01

    The authors have molecularly cloned and characterized monomeric human papillomavirus type 16 DNA with flanking cell DNA sequences from a cervical carcinoma. Determination of nucleotide sequence around the junctions of human papillomavirus and cell DNAs revealed that at the site of integration within cell DNA the cloned viral DNA had a deletion between nucleotides 1284 and 4471 (numbering system from K. Seedorf, G. Kraemmer, M. Duerst, S. Suhai, and W.G. Roewkamp), which includes the greater part of E1 gene and the entire E2 gene. In the remaining part of the E1 gene, three guanines were found at the location where two guanines at nucleotides 1137 and 1138 have been recorded. This additional guanine shifted the reading frame and erased an interruption in the E1 gene. The data strongly suggest that, like other papillomaviruses, human papillomavirus type 16 has an uninterrupted E1 gene.

  18. An impedance-based integrated biosensor for suspended DNA characterization

    PubMed Central

    Ma, Hanbin; Wallbank, Richard W. R.; Chaji, Reza; Li, Jiahao; Suzuki, Yuji; Jiggins, Chris; Nathan, Arokia

    2013-01-01

    Herein, we describe a novel integrated biosensor for performing dielectric spectroscopy to analyze biological samples. We analyzed biomolecule samples with different concentrations and demonstrated that the solution's impedance is highly correlated with the concentration, indicating that it may be possible to use this sensor as a concentration sensor. In contrast with standard spectrophotometers, this sensor offers a low-cost and purely electrical solution for the quantitative analysis of biomolecule solutions. In addition to determining concentrations, we found that the sample solution impedance is highly correlated with the length of the DNA fragments, indicating that the sizes of PCR products could be validated with an integrated chip-based, sample-friendly system within a few minutes. The system could be the basis of a rapid, low-cost platform for DNA characterization with broad applications in cancer and genetic disease research. PMID:24060937

  19. Microfabricated bioprocessor for integrated nanoliter-scale Sanger DNA sequencing.

    PubMed

    Blazej, Robert G; Kumaresan, Palani; Mathies, Richard A

    2006-05-09

    An efficient, nanoliter-scale microfabricated bioprocessor integrating all three Sanger sequencing steps, thermal cycling, sample purification, and capillary electrophoresis, has been developed and evaluated. Hybrid glass-polydimethylsiloxane (PDMS) wafer-scale construction is used to combine 250-nl reactors, affinity-capture purification chambers, high-performance capillary electrophoresis channels, and pneumatic valves and pumps onto a single microfabricated device. Lab-on-a-chip-level integration enables complete Sanger sequencing from only 1 fmol of DNA template. Up to 556 continuous bases were sequenced with 99% accuracy, demonstrating read lengths required for de novo sequencing of human and other complex genomes. The performance of this miniaturized DNA sequencer provides a benchmark for predicting the ultimate cost and efficiency limits of Sanger sequencing.

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

    PubMed Central

    Lin, Linhua; Yin, Xuyang; Wang, Jun; Chen, Dayang; Chen, Fang; Jiang, Hui; Ren, Jinghui; Wang, Wei

    2016-01-01

    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 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 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. Conclusion 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. PMID:27415003

  1. A single amino acid alteration in the initiation protein is responsible for the DNA overproduction phenotype of copy number mutants of plasmid R6K.

    PubMed Central

    Inuzuka, M; Wada, Y

    1985-01-01

    A novel type of high copy-number (cop) mutants of a mini-R6K plasmid were isolated. The mutations were mapped in the pir gene which encodes the pi initiation protein for plasmid R6K DNA replication. They resulted in an alteration by substitution of a single amino acid: threonine to isoleucine at the 108th position for the cop41, and proline to serine at the 113th position for the cop50, of the 305 amino acid pi protein. The cop41 mutation in the pi protein was found to be trans-dominant over the wild-type allele in the copy control of plasmid R6K. Moreover, it was shown that the altered pi protein was not overproduced in maxicells carrying this mutant plasmid and had a higher affinity to the repeated sequence which is present in the pir promoter region. Most likely the mutated pi protein also interacts more efficiently with the same repeated sequences, a target of pi, in the replication origin region and increases the frequency of the initiation event per cell division. Images Fig. 2. Fig. 5. PMID:3000771

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

    PubMed Central

    2013-01-01

    Background 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. Results 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. Conclusions 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. PMID:23394592

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

    PubMed

    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.

  4. B chromosomes in the grasshopper Eyprepocnemis plorans are present in all body parts analyzed and show extensive variation for rDNA copy number.

    PubMed

    Ruiz-Estévez, Mercedes; Cabrero, Josefa; Camacho, Juan Pedro M; López-León, María Dolores

    2014-01-01

    B chromosomes in the grasshopper Eyprepocnemis plorans are considered to be mitotically stable, because all meiotic (primary spermatocytes and oocytes) or mitotic (embryos, ovarioles, and gastric caecum) cells analyzed within the same individual show the same B chromosome number. Nothing is known, however, about body parts with somatic tissues with no mitotic activity in adult individuals, constituting the immense majority of their body. Therefore, we investigated whether B chromosomes are present in 8 non-mitotically active somatic body parts from both sexes in addition to ovarioles and testes by PCR analysis of 2 B-specific molecular markers. We also elucidated the number of B chromosomes that an individual carried through quantifying the B-located rDNA copy number by qPCR. Our results indicated the amplification of both B-specific markers in all analyzed body parts. However, we found high variation between males for the estimated number of rDNA units in the B chromosomes. These results demonstrate the presence of B chromosomes in all body parts from the same individual and suggest a high variation in the rDNA content of the B chromosomes carried by different individuals from the same population, presumably due to unequal crossovers during meiosis. © 2014 S. Karger AG, Basel.

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

    PubMed

    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.

  6. 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.

    PubMed

    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.

  7. Analysis of DNA Copy Number Alterations in Ovarian Serous Tumors Identifies New Molecular Genetic Changes in Low-grade and High-grade Carcinomas

    PubMed Central

    Kuo, Kuan-Ting; Guan, Bin; Feng, Yuanjian; Mao, Tsui-Lien; Chen, Xu; Jinawath, Natini; Wang, Yue; Kurman, Robert J.; Shih, Ie-Ming; Wang, Tian-Li

    2009-01-01

    Ovarian serous carcinoma, the most common and lethal type of ovarian cancer, was thought to develop from two distinct molecular pathways. High-grade (HG) serous carcinomas contain frequent TP53 mutations while low-grade (LG) carcinomas arise from serous borderline tumors (SBT) and harbor mutations in KRAS/BRAF/ERBB2 pathway. However, the molecular alterations involved in the progression from SBT to LG carcinoma remain largely unknown. As well, the extent of deletion of tumor suppressors in ovarian serous carcinomas has not been well-studied. To further address these two issues, we assessed DNA copy number changes among affinity-purified tumor cells from 37 ovarian serous neoplasms including SBT, LG and HG tumors using high density 250K SNP arrays. Chromosomal instability index as measured by changes in DNA copy number was significantly higher in HG than in LG serous carcinomas. Hemizygous ch1p36 deletion was common in LG serous carcinomas but was rarely seen in SBT. This region contains several candidate tumor suppressors including miR-34a. In contrast, in HG serous carcinomas, significant numbers of amplifications and deletions including homozygous deletions were identified. Among homozygous deletions, loci containing Rb1, CDKN2A/B, CSMD1, and DOCK4 were most common, being present in 10.6%, 6.4%, 6.4% and 4.3%, respectively, in independent 47 affinity-purified HG serous carcinomas. Except the CDKN2A/B region, these homozygous deletions were not present in either SBT or LG tumors. Our study provides a genome-wide homozygous deletion profiles in HG serous carcinomas, serving as a molecular foundation to study tumor suppressors in ovarian cancer. PMID:19383911

  8. Effects of ginseng on peripheral blood mitochondrial DNA copy number and hormones in men with metabolic syndrome: A randomized clinical and pilot study.

    PubMed

    Jung, Dong-Hyuk; Lee, Yong-Jae; Kim, Chun-Bae; Kim, Jang-Young; Shin, Seung-Hun; Park, Jong-Ku

    2016-02-01

    It has been observed that mitochondrial dysfunction is associated with an increased risk of metabolic syndrome. There is growing evidence that hyperactivity of the hypothalamus-pituitary-adrenal (HPA) axis and hormone (testosterone and growth hormone) deficiency may lead to metabolic syndrome. Recent studies have reported that ginseng treatment improves mitochondrial and HPA-axis function and increases anabolic hormone secretion. The objective of this study was to investigate the effect of red ginseng (RG) on metabolic syndrome, hormones, and mitochondrial function using leukocyte mitochondrial DNA copy number in men with metabolic syndrome. We performed a randomized, double-blind, placebo-controlled study in 62 subjects who were not taking drugs that could affect their metabolic function. A total of 62 men with metabolic syndrome were randomly assigned to either an RG group (3.0g/day) or a placebo group for 4 weeks. We analyzed changes in metabolic syndrome components, leukocyte mitochondrial DNA copy number, hormones (total testosterone, IGF-1, cortisol, and DHEAS) and inflammatory markers (C-reactive protein, ferritin) from baseline to 4 weeks. Significant improvement in mitochondrial function (95% CI -44.9 to -1.3) and an increase in total testosterone (95% CI -70.1 to -1.0) and IGF-1(P=0.01) levels were observed in the RG group when compared with the placebo group. Diastolic blood pressure (95% CI 2.0-9.4) and serum cortisol (95% CI 1.1-5.5) significantly decreased in the RG group. We found evidence that RG had a favorable effect on mitochondrial function and hormones in men with metabolic syndrome. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

    PubMed

    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. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  10. Intracellular Forms of Adenovirus DNA III. Integration of the DNA of Adenovirus Type 2 into Host DNA in Productively Infected Cells

    PubMed Central

    Burger, Harold; Doerfler, Walter

    1974-01-01

    KB cells productively infected with human adenovirus type 2 contain an alkalistable class of viral DNA sedimenting in a broad zone between 50 and 90S as compared to 34S for virion DNA. This type of DNA is characterized as viral by DNA-DNA hybridization. It is extremely sensitive to shear fragmentation. Extensive control experiments demonstrate that the fast-sedimenting viral DNA is not due to artifactual drag of viral DNA mechanically trapped in cellular DNA or to association of viral DNA with protein or RNA. Furthermore, the fast-sedimenting DNA is found after infection with multiplicities between 1 and 1,000 PFU/cell and from 6 to 8 h postinfection until very late in infection (24 h). Analysis in dye-buoyant density gradients eliminates the possibility that the fast-sedimenting viral DNA represents supercoiled circular molecules. Upon equilibrium centrifugation in alkaline CsCl density gradients, the fast-sedimenting viral DNA bands in a density stratum intermediate between that of cellular and viral DNA. In contrast, the 34S virion DNA isolated and treated in the same manner as the fast-sedimenting DNA cobands with viral marker DNA. After ultrasonic treatment of the fast-sedimenting viral DNA, it shifts to the density positions of viral DNA and to a lesser extent to that of cellular DNA. The evidence presented here demonstrates that the 50 to 90S viral DNA represents adenovirus DNA covalently integrated into cell DNA. PMID:4824714

  11. Personal exposure to fine particulate matter and benzo[a]pyrene from indoor air pollution and leukocyte mitochondrial DNA copy number in rural China.

    PubMed

    Wong, Jason Y Y; Hu, Wei; Downward, George S; Seow, Wei Jie; Bassig, Bryan A; Ji, Bu-Tian; Wei, Fusheng; Wu, Guoping; Li, Jihua; He, Jun; Liu, Chin-San; Cheng, Wen-Ling; Huang, Yunchao; Yang, Kaiyun; Chen, Ying; Rothman, Nathaniel; Vermeulen, Roel C; Lan, Qing

    2017-09-01

    Households in Xuanwei and Fuyuan, China, possess hazardous levels of fine particulate matter with an aerodynamic diameter <2.5 microns (PM2.5) and polycyclic aromatic hydrocarbons (PAHs) from coal combustion. Previous studies found that increased exposure to PM2.5 and benzo[a]pyrene (BaP; a PAH) were associated with decreased mitochondrial DNA copy number (mtDNAcn), a marker of oxidative stress. We further evaluated these associations in a cross-sectional study of 148 healthy non-smoking women from Xuanwei and Fuyuan. Personal exposure to PM2.5 and BaP was measured using portable devices. MtDNAcn was measured using qPCR amplification of leukocyte DNA that was collected after air measurements. Linear regression models were used to estimate the associations between personal exposure to PM2.5 and BaP, and mtDNAcn adjusted for age, body mass index (BMI) and fuel type. We found inverse associations between exposure to PM2.5 and BaP, and mtDNAcn. Each incremental log-μg/m3 increase in PM2.5 was associated with a significant decrease in mtDNAcn of -10.3 copies per cell [95% confidence interval (95% CI): -18.6, -2.0; P = 0.02]. Additionally, each log-ng/m3 increase in BaP was associated with a significant decrease in mtDNAcn of -5.4 copies per cell (95% CI: -9.9, -0.8, P = 0.02). Age, BMI, fuel type and coal mine type were not significantly associated with mtDNAcn. Exposure to PM2.5 and BaP may alter mitochondrial dynamics in non-smoking Chinese women. MtDNAcn may be a potential mediator of indoor air pollution on chronic disease development. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  12. Retroviruses integrate into a shared, non-palindromic DNA motif.

    PubMed

    Kirk, Paul D W; Huvet, Maxime; Melamed, Anat; Maertens, Goedele N; Bangham, Charles R M

    2016-11-14

    Many DNA-binding factors, such as transcription factors, form oligomeric complexes with structural symmetry that bind to palindromic DNA sequences(1). Palindromic consensus nucleotide sequences are also found at the genomic integration sites of retroviruses(2-6) and other transposable elements(7-9), and it has been suggested that this palindromic consensus arises as a consequence of the structural symmetry in the integrase complex(2,3). However, we show here that the palindromic consensus sequence is not present in individual integration sites of human T-cell lymphotropic virus type 1 (HTLV-1) and human immunodeficiency virus type 1 (HIV-1), but arises in the population average as a consequence of the existence of a non-palindromic nucleotide motif that occurs in approximately equal proportions on the plus strand and the minus strand of the host genome. We develop a generally applicable algorithm to sort the individual integration site sequences into plus-strand and minus-strand subpopulations, and use this to identify the integration site nucleotide motifs of five retroviruses of different genera: HTLV-1, HIV-1, murine leukaemia virus (MLV), avian sarcoma leucosis virus (ASLV) and prototype foamy virus (PFV). The results reveal a non-palindromic motif that is shared between these retroviruses.

  13. Drosophila bloom helicase maintains genome integrity by inhibiting recombination between divergent DNA sequences.

    PubMed

    Kappeler, Michael; Kranz, Elisabeth; Woolcock, Katrina; Georgiev, Oleg; Schaffner, Walter

    2008-12-01

    DNA double strand breaks (DSB) can be repaired either via a sequence independent joining of DNA ends or via homologous recombination. We established a detection system in Drosophila melanogaster to investigate the impact of sequence constraints on the usage of the homology based DSB repair via single strand annealing (SSA), which leads to recombination between direct repeats with concomitant loss of one repeat copy. First of all, we find the SSA frequency to be inversely proportional to the spacer length between the repeats, for spacers up to 2.4 kb in length. We further show that SSA between divergent repeats (homeologous SSA) is suppressed in cell cultures and in vivo in a sensitive manner, recognizing sequence divergences smaller than 0.5%. Finally, we demonstrate that the suppression of homeologous SSA depends on the Bloom helicase (Blm), encoded by the Drosophila gene mus309. Suppression of homeologous recombination is a novel function of Blm in ensuring genomic integrity, not described to date in mammalian systems. Unexpectedly, distinct from its function in Saccharomyces cerevisiae, the mismatch repair factor Msh2 encoded by spel1 does not suppress homeologous SSA in Drosophila.

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

    PubMed

    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.

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

    PubMed Central

    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

  16. Types, levels and patterns of low-copy DNA sequence divergence, and phylogenetic implications, for Gossypium genome types

    PubMed Central

    Rong, J; Wang, X; Schulze, S R; Compton, R O; Williams-Coplin, T D; Goff, V; Chee, P W; Paterson, A H

    2012-01-01

    To explore types, levels and patterns of genetic divergence among diploid Gossypium (cotton) genomes, 780 cDNA, genomic DNA and simple sequence repeat (SSR) loci were re-sequenced in Gossypium herbaceum (A1 genome), G. arboreum (A2), G. raimondii (D5), G. trilobum (D8), G. sturtianum (C1) and an outgroup, Gossypioides kirkii. Divergence among these genomes ranged from 7.32 polymorphic base pairs per 100 between G. kirkii and G. herbaceum (A1) to only 1.44 between G. herbaceum (A1) and G. arboreum (A2). SSR loci are least conserved with 12.71 polymorphic base pairs and 3.77 polymorphic sites per 100 base pairs, whereas expressed sequence tags are most conserved with 3.96 polymorphic base pairs and 2.06 sites. SSR loci also exhibit the highest percentage of ‘extended polymorphisms' (spanning multiple consecutive nucleotides). The A genome lineage was particularly rapidly evolving, with the D genome also showing accelerated evolution relative to the C genome. Unexpected asymmetry in mutation rates was found, with much more transition than transversion mutation in the D genome after its divergence from a common ancestor shared with the A genome. This large quantity of orthologous DNA sequence strongly supports a phylogeny in which A–C divergence is more recent than A–D divergence, a subject that is of much importance in view of A–D polyploid formation being key to the evolution of the most productive and finest-quality cottons. Loci that are monomorphic within A or D genome types, but polymorphic between genome types, may be of practical importance for identifying locus-specific DNA markers in tetraploid cottons including leading cultivars. PMID:22511177

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

    PubMed

    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.

  18. Episomal High Copy Number Maintenance of Hairpin-capped DNA Bearing a Replication Initiation Region in Human Cells*

    PubMed Central

    Harada, Seiyu; Uchida, Masafumi; Shimizu, Noriaki

    2009-01-01

    We previously found that a plasmid bearing a replication initiation region efficiently initiates gene amplification in mammalian cells and that it generates extrachromosomal double minutes and/or chromosomal homogeneously staining regions. During analysis of the underlying mechanism, we serendipitously found that hairpin-capped linear DNA was stably maintained as numerous extrachromosomal tiny episomes for more than a few months in a human cancer cell line. Generation of such episomes depended on the presence of the replication initiation region in the original plasmid. Despite extrachromosomal maintenance, episomal gene expression was epigenetically suppressed. The Southern blot analysis of the DNA of cloned cells revealed that the region around the hairpin end was diversified between the clones. Furthermore, the bisulfite-modified PCR and the sequencing analyses revealed that the palindrome sequence that derived from the original hairpin end or its end-resected structure were well preserved during clonal long term growth. From these data, we propose a model that explains the formation and maintenance of these episomes, in which replication of the hairpin-capped DNA and cruciform formation and its resolution play central roles. Our findings may be relevant for the dissection of mammalian replicator sequences. PMID:19617622

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

    PubMed

    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.

  20. Screening somatic cell nuclear transfer parameters for generation of transgenic cloned cattle with intragenomic integration of additional gene copies that encode bovine adipocyte-type fatty acid-binding protein (A-FABP).

    PubMed

    Guo, Yong; Li, Hejuan; Wang, Ying; Yan, Xingrong; Sheng, Xihui; Chang, Di; Qi, Xiaolong; Wang, Xiangguo; Liu, Yunhai; Li, Junya; Ni, Hemin

    2017-02-01

    Somatic cell nuclear transfer (SCNT) is frequently used to produce transgenic cloned livestock, but it is still associated with low success rates. To our knowledge, we are the first to report successful production of transgenic cattle that overexpress bovine adipocyte-type fatty acid binding proteins (A-FABPs) with the aid of SCNT. Intragenomic integration of additional A-FABP gene copies has been found to be positively correlated with the intramuscular fat content in different farm livestock species. First, we optimized the cloning parameters to produce bovine embryos integrated with A-FABP by SCNT, such as applied voltage field strength and pulse duration for electrofusion, morphology and size of donor cells, and number of donor cells passages. Then, bovine fibroblast cells from Qinchuan cattle were transfected with A-FABP and used as donor cells for SCNT. Hybrids of Simmental and Luxi local cattle were selected as the recipient females for A-FABP transgenic SCNT-derived embryos. The results showed that a field strength of 2.5 kV/cm with two 10-μs duration electrical pulses was ideal for electrofusion, and 4-6th generation circular smooth type donor cells with diameters of 15-25 μm were optimal for producing transgenic bovine embryos by SCNT, and resulted in higher fusion (80%), cleavage (73%), and blastocyst (27%) rates. In addition, we obtained two transgenic cloned calves that expressed additional bovine A-FABP gene copies, as detected by PCR-amplified cDNA sequencing. We proposed a set of optimal protocols to produce transgenic SCNT-derived cattle with intragenomic integration of ectopic A-FABP-inherited exon sequences.

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

    PubMed

    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.

  2. Stably integrated mouse mammary tumor virus long terminal repeat DNA requires the octamer motifs for basal promoter activity.

    PubMed Central

    Buetti, E

    1994-01-01

    In the mouse mammary tumor virus promoter, a tandem of octamer motifs, recognized by ubiquitous and tissue-restricted Oct transcription factors, is located upstream of the TATA box and next to a binding site for the transcription factor nuclear factor I (NF-I). Their function was investigated with mutant long terminal repeats under different transfection conditions in mouse Ltk- cells and quantitative S1 nuclease mapping of the transcripts. In stable transfectants, which are most representative of the state of proviral DNA with respect to both number of integrated DNA templates and chromatin organization, a long terminal repeat mutant of both octamer sites showed an average 50-fold reduction of the basal transcription level, while the dexamethasone-stimulated level was unaffected. DNase I in vitro footprinting assays with L-cell nuclear protein extracts showed that the mutant DNA was unable to bind octamer factors but had a normal footprint in the NF-I site. I conclude that mouse mammary tumor virus employs the tandem octamer motifs of the viral promoter, recognized by the ubiquitous transcription factor Oct-1, for its basal transcriptional activity and the NF-I binding site, as previously shown, for glucocorticoid-stimulated transcription. A deletion mutant with only one octamer site showed a marked base-level reduction at high copy number but little reduction at low copies of integrated plasmids. The observed transcription levels may depend both on the relative ratio of transcription factors to DNA templates and on the relative affinity of binding sites, as determined by oligonucleotide competition footprinting. Images PMID:8289800

  3. Alterations of telomere length and mtDNA copy number are associated with overall survival in hepatocellular carcinoma patients treated with transarterial chemoembolization.

    PubMed

    Bao, Dengke; Ba, Yanna; Zhou, Feng; Zhao, Jing; Yang, Qi; Ge, Naijian; Guo, Xu; Wu, Zhenbiao; Zhang, Hongxin; Yang, Hushan; Wan, Shaogui; Xing, Jinliang

    2016-10-01

    Increasing evidence suggests that alterations in mitochondrial DNA (mtDNA) copy number (mtDNAcn) and relative telomere length (RTL) may be implicated in the tumorigenesis of several malignancies. Alterations of both RTL and mtDNAcn are generally accepted as independent biomarkers for predicting risk and prognosis in various cancers. The aim of this study was to evaluate the prognostic value of combining leukocyte RTL with mtDNAcn (RTL-mtDNAcn) in hepatocellular carcinoma (HCC). RTL and mtDNAcn in peripheral blood leukocytes (PBLs) were measured using a real-time PCR-based method in a total of 250 HCC patients treated with transcatheter arterial chemoembolization (TACE). We evaluated the associations between RTL and/or mtDNAcn and HCC overall survival using Kaplan-Meier curve analysis and Cox proportional hazards regression model. We found that patients with longer leukocyte RTL or lower mtDNAcn had shorter overall survival time. The univariate analysis (HR 1.63, 95 % CI 1.23-2.17, P = 7.7 × 10(-4)) and multivariate analysis (HR 1.78, 95 % CI 1.31-2.42, P = 2.4 × 10(-4)) indicated that longer leukocyte RTL was significantly associated with poorer OS in HCC patients. Kaplan-Meier curve analysis showed that patients with longer RTL had shorter overall survival time than those with shorter RTL (log-rank P = 0.001). Patients with lower mtDNA copy number was significantly associated with poorer OS by Cox proportional hazards model using both univariate (HR 1.60, 95 % CI 1.21-2.13, P = 0.001) and multivariate analyses (HR 1.77, 95 % CI 1.30-2.41, P = 2.8 × 10(-4)). Kaplan-Meier curve analysis showed that patients with lower mtDNA content had significantly shorter overall survival time than those with higher mtDNA content (log-rank P = 0.001). Furthermore, combination of leukocyte RTL and mtDNAcn significantly improved the efficacy of predicting HCC prognosis. Patients with longer RTL and lower mtDNAcn exhibited a significantly poorer overall

  4. Genome-Wide Loss of Heterozygosity and DNA Copy Number Aberration in HPV-Negative Oral Squamous Cell Carcinoma and Their Associations with Disease-Specific Survival.

    PubMed

    Chen, Chu; Zhang, Yuzheng; Loomis, Melissa M; Upton, Melissa P; Lohavanichbutr, Pawadee; Houck, John R; Doody, David R; Mendez, Eduardo; Futran, Neal; Schwartz, Stephen M; Wang, Pei

    2015-01-01

    Oral squamous cell cancer of the oral cavity and oropharynx (OSCC) is associated with high case-fatality. For reasons that are largely unknown, patients with the same clinical and pathologic staging have heterogeneous response to treatment and different probability of recurrence and survival, with patients with Human Papillomavirus (HPV)-positive oropharyngeal tumors having the most favorable survival. To gain insight into the complexity of OSCC and to identify potential chromosomal changes that may be associated with OSCC mortality, we used Affymtrix 6.0 SNP arrays to examine paired DNA from peripheral blood and tumor cell populations isolated by laser capture microdissection to assess genome-wide loss of heterozygosity (LOH) and DNA copy number aberration (CNA) and their associations with risk factors, tumor characteristics, and oral cancer-specific mortality among 75 patients with HPV-negative OSCC. We found a highly heterogeneous and complex genomic landscape of HPV-negative tumors, and identified regions in 4q, 8p, 9p and 11q that seem to play an important role in oral cancer biology and survival from this disease. If confirmed, these findings could assist in designing personalized treatment or in the creation of models to predict survival in patients with HPV-negative OSCC.

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

    PubMed

    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

    2015-10-01

    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. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Loss of 6q or 8p23 is associated with the total number of DNA copy number aberrations in adenoid cystic carcinoma.

    PubMed

    Oga, Atsunori; Uchida, Kenichiro; Nakao, Motonao; Kawauchi, Shigeto; Furuya, Tomoko; Chochi, Yasuyo; Ikemoto, Kenzo; Okada, Takae; Ueyama, Yoshiya; Sasaki, Kohsuke; Yousefpour, Fatemeh

    2011-12-01

    We analyzed 10 adenoid cystic carcinomas (ACCs) of the salivary glands by array-based comparative genomic hybridization (a-CGH) using DNA chips spotted with 4,030 bacterial artificial chromosome clones. After the data smoothing procedure was applied, a total of 88 DNA copy number aberrations (DCNAs) were detected. The frequent (≥30%) DCNAs were loss of 6q23-27 and 8p23, and gains of 6p, 6q23, 8p23 and 22q13. High-level gains were detected on 12q15, including MDM2 in two cases. These two cases showed an immunohistochemically high-level (>50%) expression of MDM2 and a low-level expression of p53 (<20%). Furthermore, the total number of DCNAs was significantly greater in ACCs with loss of 6q compared to other ACCs, and in ACCs without the loss of 8p23 compared to other ACCs, respectively. Although limitations exist, a-CGH detected several candidate chromosomal imbalances associated with accumulation of DCNAs in ACCs.

  7. Distinct patterns of DNA copy number alteration are associated with different clinicopathological features and gene-expression subtypes of breast cancer.

    PubMed

    Bergamaschi, Anna; Kim, Young H; Wang, Pei; Sørlie, Therese; Hernandez-Boussard, Tina; Lonning, Per E; Tibshirani, Robert; Børresen-Dale, Anne-Lise; Pollack, Jonathan R

    2006-11-01

    Breast cancer is a leading cause of cancer-death among women, where the clinicopathological features of tumors are used to prognosticate and guide therapy. DNA copy number alterations (CNAs), which occur frequently in breast cancer and define key pathogenetic events, are also potentially useful prognostic or predictive factors. Here, we report a genome-wide array-based comparative genomic hybridization (array CGH) survey of CNAs in 89 breast tumors from a patient cohort with locally advanced disease. Statistical analysis links distinct cytoband loci harboring CNAs to specific clinicopathological parameters, including tumor grade, estrogen receptor status, presence of TP53 mutation, and overall survival. Notably, distinct spectra of CNAs also underlie the different subtypes of breast cancer recently defined by expression-profiling, implying these subtypes develop along distinct genetic pathways. In addition, higher numbers of gains/losses are associated with the "basal-like" tumor subtype, while high-level DNA amplification is more frequent in "luminal-B" subtype tumors, suggesting also that distinct mechanisms of genomic instability might underlie their pathogenesis. The identified CNAs may provide a basis for improved patient prognostication, as well as a starting point to define important genes to further our understanding of the pathobiology of breast cancer. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat

  8. Agrobacterium tumefaciens-mediated creeping bentgrass (Agrostis stolonifera L.) transformation using phosphinothricin selection results in a high frequency of single-copy transgene integration.

    PubMed

    Luo, H; Hu, Q; Nelson, K; Longo, C; Kausch, A P; Chandlee, J M; Wipff, J K; Fricker, C R

    2004-04-01

    Genetic transformation of creeping bentgrass mediated by Agrobacterium tumefaciens has been achieved. Embryogenic callus initiated from seeds (cv. Penn-A-4) was infected with an A. tumefaciens strain (LBA4404) harboring a super-binary vector that contained an herbicide-resistant bar gene driven either by the CaMV 35S promoter or a rice ubiquitin promoter. Plants were regenerated from 219 independent transformation events. The overall stable transformation efficiency ranged from 18% to 45%. Southern blot and genetic analysis confirmed transgene integration in the creeping bentgrass genome and normal transmission and stable expression of the transgene in the T1 generation. All independent transformation events carried one to three copies of the transgene, and a majority (60-65%) contained only a single copy of the foreign gene with no apparent rearrangements. We report here the successful use of Agrobacterium for the large-scale production of transgenic creeping bentgrass plants with a high frequency of a single-copy transgene insertion that exhibit stable inheritance patterns.

  9. Mobile elements and viral integrations prompt considerations for bacterial DNA integration as a novel carcinogen

    PubMed Central

    Robinson, Kelly M.; Hotopp, Julie C. Dunning

    2014-01-01

    Insertional mutagenesis has been repeatedly demonstrated in cancer genomes and has a role in oncogenesis. Mobile genetic elements can induce cancer development by random insertion into cancer related genes or by inducing translocations. L1s are typically implicated in cancers of an epithelial cell origin, while Alu elements have been implicated in leukemia as well as epithelial cell cancers. Likewise, viral infections have a significant role in cancer development predominantly through integration into the human genome and mutating or deregulating cancer related genes. Human papilloma virus is the best-known example of viral integrations contributing to carcinogenesis. However, hepatitis B virus, Epstein-Barr virus, and Merkel cell polyomavirus also integrate into the human genome and disrupt cancer related genes. Thus far, the role of microbes in cancer has primarily been attributed to mutations induced through chronic inflammation or toxins, as is the case with Helicobacter pylori and enterotoxigenic Bacteroides fragilis. We hypothesize that like mobile elements and viral DNA, bacterial and parasitic DNA may also integrate into the human somatic genome and be oncogenic. Until recently it was believed that bacterial DNA could not integrate into the human genome, but new evidence demonstrates that bacterial insertional mutagenesis may occur in cancer cells. Although this work does not show causation between bacterial insertions and cancer, it prompts more research in this area. Promising new sequencing technologies may reduce the risk of artifactual chimeric sequences, thus diminishing some of the challenges of identifying novel insertions in the somatic human genome. PMID:24956175

  10. Genome-wide analysis of abdominal and pleural malignant mesothelioma with DNA arrays reveals both common and distinct regions of copy number alteration

    PubMed Central

    Borczuk, Alain C.; Pei, Jianming; Taub, Robert N.; Levy, Brynn; Nahum, Odelia; Chen, Jinli; Chen, Katherine; Testa, Joseph R.

    2016-01-01

    ABSTRACT Malignant mesothelioma (MM) is an aggressive tumor arising from mesothelial linings of the serosal cavities. Pleural space is the most common site, accounting for about 80% of cases, while peritoneum makes up the majority of the remaining 20%. While histologically similar, tumors from these sites are epidemiologically and clinically distinct and their attribution to asbestos exposure differs. We compared DNA array-based findings from 48 epithelioid peritoneal MMs and 41 epithelioid pleural MMs to identify similarities and differences in copy number alterations (CNAs). Losses in 3p (BAP1 gene), 9p (CDKN2A) and 22q (NF2) were seen in tumors from both tumor sites, although CDKN2A and NF2 losses were seen at a higher rate in pleural disease (p<0.01). Overall, regions of copy number gain were more common in peritoneal MM, whereas losses were more common in pleural MM, with regions of loss containing known tumor suppressor genes and regions of gain encompassing genes encoding receptor tyrosine kinase pathway members. Cases with known asbestos causation (n = 32 ) were compared with those linked to radiation exposure (n = 9 ). Deletions in 6q, 14q, 17p and 22q, and gain of 17q were seen in asbestos-associated but not radiation-related cases. As reported in post-radiation sarcoma, gains outnumbered losses in radiation-associated MM. The patterns of genomic imbalances suggest overlapping and distinct molecular pathways in MM of the pleura and peritoneum, and that differences in causation (i.e., asbestos vs. radiation) may account for some of these site-dependent differences PMID:26853494

  11. Transgene constructs in coho salmon (Oncorhynchus kisutch) are repeated in a head-to-tail fashion and can be integrated adjacent to horizontally-transmitted parasite DNA.

    PubMed

    Uh, Mitchell; Khattra, Jaswinder; Devlin, Robert H

    2006-12-01

    Currently, little information is available regarding the molecular organization of integrated transgenes in genetically-engineered fish. We performed a detailed structural analysis of an inserted transgene in one strain (M77) of transgenic coho salmon (Oncorhynchus kisutch) containing a salmon growth hormone gene construct (OnMTGH1). Microinjected DNA was found to have inserted into a single site in the coho salmon genome, and was organized with four complete internal copies and two partial terminal copies of the OnMTGH1 construct. All construct copies were organized in a direct-tandem (head-to-tail) repeat fashion in strain M77 and five additional strains (one also possessed a second recombinant junction fragment). For strain M77, the junctions between the transgene insert and the insertion point within the wild-type genome were cloned from strain-specific cosmid libraries and sequenced, revealing that the transgene insertion was accompanied by a deletion of 587 bp of wild-type DNA as well as a small insertion (19 bp) of unknown DNA upstream and a 14 bp direct- tandem duplication of sequence downstream. Upstream and downstream wild-type DNA sequence contained several repetitive sequence elements based on Southern blot analysis and homology to repetitive sequences in GenBank. In the downstream flank, a pseudogene sequence was also identified which has high homology to the CA membrane protein gene from Schistosoma japonicum, a parasite closely related to Sanguinicola sp. parasites which infect salmonids. Whether the presence of an inserted transgene and the presence of potentially horizontally-transmitted DNA are indicative of a genomic region with a predisposition for insertion of foreign DNA requires further study. The information derived from this transgene structure provides information useful for comparison to other transgenic organisms and for determination of the mechanism of transgene integration in lower vertebrates.

  12. RECQL4 LOCALIZES TO MITOCHONDRIA AND PRESERVES MITOCHONDRIAL DNA INTEGRITY

    PubMed Central

    Croteau, Deborah L.; Rossi, Marie L.; Canugovi, Chandrika; Tian, Jane; Sykora, Peter; Ramamoorthy, Mahesh; Wang, ZhengMing; Singh, Dharmendra Kumar; Akbari, Mansour; Kasiviswanathan, Rajesh; Copeland, William C.; Bohr, Vilhelm A.

    2012-01-01

    SUMMARY 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 reserve capacity after lentiviral knockdown of RECQL4 in two different primary cell lines. Additionally, biochemical assays with RECQL4, mitochondrial transcription factor A and mitochondrial DNA polymerase γ showed that the polymerase inhibited RECQL4’s helicase activity. RECQL4 is the first 3′ to 5′ RecQ helicase to be found in both human and mouse mitochondria and the loss of RECQL4 alters mitochondrial integrity. PMID:22296597

  13. Accelerated DNA Methylation Age: Associations with PTSD and Neural Integrity

    PubMed Central

    Wolf, Erika J.; Logue, Mark W.; Hayes, Jasmeet P.; Sadeh, Naomi; Schichman, Steven A.; Stone, Annjanette; Salat, David H.; Milberg, William; McGlinchey, Regina; Miller, Mark W.

    2015-01-01

    Background Accumulating evidence suggests that post traumatic stress disorder (PTSD) may accelerate cellular aging and lead to premature morbidity and neurocognitive decline. Methods This study evaluated associations between PTSD and DNA methylation (DNAm) age using recently developed algorithms of cellular age by Horvath (2013) and Hannum et al. (2013). These estimates reflect accelerated aging when they exceed chronological age. We also examined if accelerated cellular age manifested in degraded neural integrity, indexed via diffusion tensor imaging. Results Among 281 male and female veterans of the conflicts in Iraq and Afghanistan, DNAm age was strongly related to chronological age (rs ~.88). Lifetime PTSD severity was associated with Hannum DNAm age estimates residualized for chronological age (β = .13, p= .032). Advanced DNAm age was associated with reduced integrity in the genu of the corpus callosum (β = −.17, p= .009) and indirectly linked to poorer working memory performance via this region (indirect β = − .05, p= .029). Horvath DNAm age estimates were not associated with PTSD or neural integrity. Conclusions Results provide novel support for PTSD-related accelerated aging in DNAm and extend the evidence base of known DNAm age correlates to the domains of neural integrity and cognition. PMID:26447678

  14. Accelerated DNA methylation age: Associations with PTSD and neural integrity.

    PubMed

    Wolf, Erika J; Logue, Mark W; Hayes, Jasmeet P; Sadeh, Naomi; Schichman, Steven A; Stone, Annjanette; Salat, David H; Milberg, William; McGlinchey, Regina; Miller, Mark W

    2016-01-01

    Accumulating evidence suggests that posttraumatic stress disorder (PTSD) may accelerate cellular aging and lead to premature morbidity and neurocognitive decline. This study evaluated associations between PTSD and DNA methylation (DNAm) age using recently developed algorithms of cellular age by Horvath (2013) and Hannum et al. (2013). These estimates reflect accelerated aging when they exceed chronological age. We also examined if accelerated cellular age manifested in degraded neural integrity, indexed via diffusion tensor imaging. Among 281 male and female veterans of the conflicts in Iraq and Afghanistan, DNAm age was strongly related to chronological age (rs ∼.88). Lifetime PTSD severity was associated with Hannum DNAm age estimates residualized for chronological age (β=.13, p=.032). Advanced DNAm age was associated with reduced integrity in the genu of the corpus callosum (β=-.17, p=.009) and indirectly linked to poorer working memory performance via this region (indirect β=-.05, p=.029). Horvath DNAm age estimates were not associated with PTSD or neural integrity. Results provide novel support for PTSD-related accelerated aging in DNAm and extend the evidence base of known DNAm age correlates to the domains of neural integrity and cognition. Published by Elsevier Ltd.

  15. Correlations of scores on the developmental test of visual-motor integration and copying test in a South African multi-ethnic preschool sample.

    PubMed

    Dunn, Munita; Loxton, Helene; Naidoo, Anthony

    2006-12-01

    This study assessed the intercorrelations of scores on the Developmental Test of Visual-Motor Integration, the locally standardized Copying Test, and teachers' ratings of scholastic skills in a South African multi-ethnic preschool sample. The study also investigated whether cultural and socioeconomic factors might influence test data. Participants were 71 Black, 101 Coloured, and 66 White children attending preschools in a semirural district. Participants' ages ranged from 4 yr., 9 mo. to 7yr., 0 mo. (M=5.8 yr., SD= 0.3 yr.). Analysis yielded a correlation of .75 between the test scores and supports the suitability of the widely used Developmental Test of Visual-Motor Integration in a multi-ethnic sample. Scores on the Copying Test correlated higher with teachers' ratings. However, significant differences in test performance among groups by race and socioeconomic status suggest the rate of perceptual-motor development may be related to cultural factors. Normative data are reported for groups by race and socioeconomic status.

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

    PubMed

    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.

  17. Haplotype Detection from Next-Generation Sequencing in High-Ploidy-Level Species: 45S rDNA Gene Copies in the Hexaploid Spartina maritima

    PubMed Central

    Boutte, Julien; Aliaga, Benoît; Lima, Oscar; Ferreira de Carvalho, Julie; Ainouche, Abdelkader; Macas, Jiri; Rousseau-Gueutin, Mathieu; Coriton, Olivier; Ainouche, Malika; Salmon, Armel

    2015-01-01

    Gene and whole-genome duplications are widespread in plant nuclear genomes, resulting in sequence heterogeneity. Identification of duplicated genes may be particularly challenging in highly redundant genomes, especially when there are no diploid parents as a reference. Here, we developed a pipeline to detect the different copies in the ribosomal RNA gene family in the hexaploid grass Spartina maritima from next-generation sequencing (Roche-454) reads. The heterogeneity of the different domains of the highly repeated 45S unit was explored by identifying single nucleotide polymorphisms (SNPs) and assembling reads based on shared polymorphisms. SNPs were validated using comparisons with Illumina sequence data sets and by cloning and Sanger (re)sequencing. Using this approach, 29 validated polymorphisms and 11 validated haplotypes were reported (out of 34 and 20, respectively, that were initially predicted by our program). The rDNA domains of S. maritima have similar lengths as those found in other Poaceae, apart from the 5′-ETS, which is approximately two-times longer in S. maritima. Sequence homogeneity was encountered in coding regions and both internal transcribed spacers (ITS), whereas high intragenomic variability was detected in the intergenic spacer (IGS) and the external transcribed spacer (ETS). Molecular cytogenetic analysis by fluorescent in situ hybridization (FISH) revealed the presence of one pair of 45S rDNA signals on the chromosomes of S. maritima instead of three expected pairs for a hexaploid genome, indicating loss of duplicated homeologous loci through the diploidization process. The procedure developed here may be used at any ploidy level and using different sequencing technologies. PMID:26530424

  18. Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases

    PubMed Central

    Pinto, Cosimo; Kasaciunaite, Kristina; Seidel, Ralf; Cejka, Petr

    2016-01-01

    Human DNA2 (hDNA2) contains both a helicase and a nuclease domain within the same polypeptide. The nuclease of hDNA2 is involved in a variety of DNA metabolic processes. Little is known about the role of the hDNA2 helicase. Using bulk and single-molecule approaches, we show that hDNA2 is a processive helicase capable of unwinding kilobases of dsDNA in length. The nuclease activity prevents the engagement of the helicase by competing for the same substrate, hence prominent DNA unwinding by hDNA2 alone can only be observed using the nuclease-deficient variant. We show that the helicase of hDNA2 functionally integrates with BLM or WRN helicases to promote dsDNA degradation by forming a heterodimeric molecular machine. This collectively suggests that the hDNA2 motor promotes the enzyme's capacity to degrade dsDNA in conjunction with BLM or WRN and thus promote the repair of broken DNA. DOI: http://dx.doi.org/10.7554/eLife.18574.001 PMID:27612385

  19. Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases.

    PubMed

    Pinto, Cosimo; Kasaciunaite, Kristina; Seidel, Ralf; Cejka, Petr

    2016-09-09

    Human DNA2 (hDNA2) contains both a helicase and a nuclease domain within the same polypeptide. The nuclease of hDNA2 is involved in a variety of DNA metabolic processes. Little is known about the role of the hDNA2 helicase. Using bulk and single-molecule approaches, we show that hDNA2 is a processive helicase capable of unwinding kilobases of dsDNA in length. The nuclease activity prevents the engagement of the helicase by competing for the same substrate, hence prominent DNA unwinding by hDNA2 alone can only be observed using the nuclease-deficient variant. We show that the helicase of hDNA2 functionally integrates with BLM or WRN helicases to promote dsDNA degradation by forming a heterodimeric molecular machine. This collectively suggests that the hDNA2 motor promotes the enzyme's capacity to degrade dsDNA in conjunction with BLM or WRN and thus promote the repair of broken DNA.

  20. Agrobacterium T-DNA integration in Arabidopsis is correlated with DNA sequence compositions that occur frequently in gene promoter regions.

    PubMed

    Schneeberger, Richard G; Zhang, Ke; Tatarinova, Tatiana; Troukhan, Max; Kwok, Shing F; Drais, Josh; Klinger, Kevin; Orejudos, Francis; Macy, Kimberly; Bhakta, Amit; Burns, James; Subramanian, Gopal; Donson, Jonathan; Flavell, Richard; Feldmann, Kenneth A

    2005-10-01

    Mobile insertion elements such as transposons and T-DNA generate useful genetic variation and are important tools for functional genomics studies in plants and animals. The spectrum of mutations obtained in different systems can be highly influenced by target site preferences inherent in the mechanism of DNA integration. We investigated the target site preferences of Agrobacterium T-DNA insertions in the chromosomes of the model plant Arabidopsis thaliana. The relative frequencies of insertions in genic and intergenic regions of the genome were calculated and DNA composition features associated with the insertion site flanking sequences were identified. Insertion frequencies across the genome indicate that T-strand integration is suppressed near centromeres and rDNA loci, progressively increases towards telomeres, and is highly correlated with gene density. At the gene level, T-DNA integration events show a statistically significant preference for insertion in the 5' and 3' flanking regions of protein coding sequences as well as the promoter region of RNA polymerase I transcribed rRNA gene repeats. The increased insertion frequencies in 5' upstream regions compared to coding sequences are positively correlated with gene expression activity and DNA sequence composition. Analysis of the relationship between DNA sequence composition and gene activity further demonstrates that DNA sequences with high CG-skew ratios are consistently correlated with T-DNA insertion site preference and high gene expression. The results demonstrate genomic and gene-specific preferences for T-strand integration and suggest that DNA sequences with a pronounced transition in CG- and AT-skew ratios are preferred targets for T-DNA integration.

  1. During Stably Suppressive Antiretroviral Therapy Integrated HIV-1 DNA Load in Peripheral Blood is Associated with the Frequency of CD8 Cells Expressing HLA-DR/DP/DQ

    PubMed Central

    Ruggiero, Alessandra; De Spiegelaere, Ward; Cozzi-Lepri, Alessandro; Kiselinova, Maja; Pollakis, Georgios; Beloukas, Apostolos; Vandekerckhove, Linos; Strain, Matthew; Richman, Douglas; Phillips, Andrew; Geretti, Anna Maria; Vitiello, Paola; Mackie, Nicola; Ainsworth, Jonathan; Waters, Anele; Post, Frank; Edwards, Simon; Fox, Julie

    2015-01-01

    Background Characterising the correlates of HIV persistence improves understanding of disease pathogenesis and guides the design of curative strategies. This study investigated factors associated with integrated HIV-1 DNA load during consistently suppressive first-line antiretroviral therapy (ART). Method Total, integrated, and 2-long terminal repeats (LTR) circular HIV-1 DNA, residual plasma HIV-1 RNA, T-cell activation markers, and soluble CD14 (sCD14) were measured in peripheral blood of 50 patients that had received 1–14 years of efavirenz-based or nevirapine-based therapy. Results Integrated HIV-1 DNA load (per 106 peripheral blood mononuclear cells) was median 1.9 log10 copies (interquartile range 1.7–2.2) and showed a mean difference of 0.2 log10 copies per 10 years of suppressive ART (95% confidence interval − 0.2, 0.6; p = 0.28). It was positively correlated with total HIV-1 DNA load and frequency of CD8+HLA-DR/DP/DQ+ cells, and was also higher in subjects with higher sCD14 levels, but showed no correlation with levels of 2-LTR circular HIV-1 DNA and residual plasma HIV-1 RNA, or the frequency of CD4+CD38+ and CD8+CD38+ cells. Adjusting for pre-ART viral load, duration of suppressive ART, CD4 cell counts, residual plasma HIV-1 RNA levels, and sCD14 levels, integrated HIV-1 DNA load was mean 0.5 log10 copies higher for each 50% higher frequency of CD8+HLA-DR/DP/DQ+ cells (95% confidence interval 0.2, 0.9; p = 0.01). Conclusions The observed positive association between integrated HIV-1 DNA load and frequency of CD8+DR/DP/DQ+ cells indicates that a close correlation between HIV persistence and immune activation continues during consistently suppressive therapy. The inducers of the distinct activation profile warrant further investigation. PMID:26498496

  2. Integration of mRNA expression profile, copy number alterations, and microRNA expression levels in breast cancer to improve grade definition.

    PubMed

    Cava, Claudia; Bertoli, Gloria; Ripamonti, Marilena; Mauri, Giancarlo; Zoppis, Italo; Della Rosa, Pasquale Anthony; Gilardi, Maria Carla; Castiglioni, Isabella

    2014-01-01

    Defining the aggressiveness and growth rate of a malignant cell population is a key step in the clinical approach to treating tumor disease. The correct grading of breast cancer (BC) is a fundamental part in determining the appropriate treatment. Biological variables can make it difficult to elucidate the mechanisms underlying BC development. To identify potential markers that can be used for BC classification, we analyzed mRNAs expression profiles, gene copy numbers, microRNAs expression and their association with tumor grade in BC microarray-derived datasets. From mRNA expression results, we found that grade 2 BC is most likely a mixture of grade 1 and grade 3 that have been misclassified, being described by the gene signature of either grade 1 or grade 3. We assessed the potential of the new approach of integrating mRNA expression profile, copy number alterations, and microRNA expression levels to select a limited number of genomic BC biomarkers. The combination of mRNA profile analysis and copy number data with microRNA expression levels led to the identification of two gene signatures of 42 and 4 altered genes (FOXM1, KPNA4, H2AFV and DDX19A) respectively, the latter obtained through a meta-analytical procedure. The 42-based gene signature identifies 4 classes of up- or down-regulated microRNAs (17 microRNAs) and of their 17 target mRNA, and the 4-based genes signature identified 4 microRNAs (Hsa-miR-320d, Hsa-miR-139-5p, Hsa-miR-567 and Hsa-let-7c). These results are discussed from a biological point of view with respect to pathological features of BC. Our identified mRNAs and microRNAs were validated as prognostic factors of BC disease progression, and could potentially facilitate the implementation of assays for laboratory validation, due to their reduced number.

  3. ADP-ribosylation is involved in the integration of foreign DNA into the mammalian cell genome.

    PubMed Central

    Farzaneh, F; Panayotou, G N; Bowler, L D; Hardas, B D; Broom, T; Walther, C; Shall, S

    1988-01-01

    The most commonly used DNA transfection method, which employs the calcium phosphate co-precipitation of the donor DNA, involves several discrete steps (1,2). These include the uptake of the donor DNA by the recipient cells, the transport of the DNA to the nucleus, transient expression prior to integration into the host cell genome, concatenation and integration of the transfected DNA into the host cell genome and finally the stable expression of the integrated genes (2,3). Both the concatenation and the integration of the donor DNA into the host genome involve the formation and ligation of DNA strand-breaks. In the present study we demonstrate that the nuclear enzyme, adenosine diphosphoribosyl transferase (ADPRT, E.C. 2.4.2.30), which is dependent on the presence of DNA strand breaks for its activity (4,5) and necessary for the efficient ligation of DNA strand-breaks in eukaryotic cells (4,6), is required for the integration of donor DNA into the host genome. However, ADPRT activity does not influence the uptake of DNA into the cell, its episomal maintenance or replication, nor its expression either before or after integration into the host genome. These observations strongly suggest the involvement of ADPRT activity in eukaryotic DNA recombination events. Images PMID:3144706

  4. Retrospective analysis of varicella zoster virus (VZV) copy DNA numbers in plasma of immunocompetent patients with herpes zoster, of immunocompromised patients with disseminated VZV disease, and of asymptomatic solid organ transplant recipients.

    PubMed

    Kronenberg, A; Bossart, W; Wuthrich, R P; Cao, C; Lautenschlager, S; Wiegand, N D; Mullhaupt, B; Noll, G; Mueller, N J; Speck, R F

    2005-01-01

    Varicella zoster virus (VZV) causes significant morbidity and mortality in immunocompromised patients. Subclinical reactivation has been described in solid organ recipients and has been associated with graft versus host disease in bone marrow transplantation. Newer studies assessing the prevalence and impact of subclinical VZV reactivation in solid organ transplant (SOT) recipients are lacking. In a first step we developed a highly sensitive quantitative polymerase chain reaction (qPCR) assay for VZV DNA with a detection limit of < or = 20 copies/mL. Using this assay, we retrospectively analyzed plasma samples of different patient groups for VZV DNA. VZV DNA was found in 10/10 plasma samples of immunocompetent patients with herpes zoster (VZV copy numbers/mL: mean+/-SEM 1710+/-1018), in 1/1 sample of a human immunodeficiency virus-infected patient with primary VZV disease (15,192 copies/mL) and in 4/4 plasma samples of immunocompromised patients with visceral VZV disease (mean of first value 214,214+/-178,572). All 108 plasma samples of asymptomatic SOT recipients off any antiviral therapy, randomly sampled over 1 year, were negative for VZV DNA. Our qPCR assay proved to be highly sensitive (100%) in symptomatic VZV disease. We did not detect subclinical reactivation in asymptomatic SOT recipients during the first post-transplant year. Thus, subclinical VZV reactivation is either a rare event or does not exist. These data need to be confirmed in larger prospective trials.

  5. Integrative CAGE and DNA Methylation Profiling Identify Epigenetically Regulated Genes in NSCLC.

    PubMed

    Horie, Masafumi; Kaczkowski, Bogumil; Ohshima, Mitsuhiro; Matsuzaki, Hirotaka; Noguchi, Satoshi; Mikami, Yu; Lizio, Marina; Itoh, Masayoshi; Kawaji, Hideya; Lassmann, Timo; Carninci, Piero; Hayashizaki, Yoshihide; Forrest, Alistair R R; Takai, Daiya; Yamaguchi, Yoko; Micke, Patrick; Saito, Akira; Nagase, Takahide

    2017-10-01

    Lung cancer is the leading cause of cancer-related deaths worldwide. The majority of cancer driver mutations have been identified; however, relevant epigenetic regulation involved in tumorigenesis has only been fragmentarily analyzed. Epigenetically regulated genes have a great theranostic potential, especially in tumors with no apparent driver mutations. Here, epigenetically regulated genes were identified in lung cancer by an integrative analysis of promoter-level expression profiles from Cap Analysis of Gene Expression (CAGE) of 16 non-small cell lung cancer (NSCLC) cell lines and 16 normal lung primary cell specimens with DNA methylation data of 69 NSCLC cell lines and 6 normal lung epithelial cells. A core set of 49 coding genes and 10 long noncoding RNAs (lncRNA), which are upregulated in NSCLC cell lines due to promoter hypomethylation, was uncovered. Twenty-two epigenetically regulated genes were validated (upregulated genes with hypomethylated promoters) in the adenocarcinoma and squamous cell cancer subtypes of lung cancer using The Cancer Genome Atlas data. Furthermore, it was demonstrated that multiple copies of the REP522 DNA repeat family are prominently upregulated due to hypomethylation in NSCLC cell lines, which leads to cancer-specific expression of lncRNAs, such as RP1-90G24.10, AL022344.4, and PCAT7. Finally, Myeloma Overexpressed (MYEOV) was identified as the most promising candidate. Functional studies demonstrated that MYEOV promotes cell proliferation, survival, and invasion. Moreover, high MYEOV expression levels were associated with poor prognosis.Implications: This report identifies a robust list of 22 candidate driver genes that are epigenetically regulated in lung cancer; such genes may complement the known mutational drivers.Visual Overview: http://mcr.aacrjournals.org/content/early/2017/10/01/1354-1365.MCR-17-0191-ET/F1.large.jpg Mol Cancer Res; 15(10); 1354-65. ©2017 AACR. ©2017 American Association for Cancer Research.

  6. Evaluation of the persistence, integration, histopathology and environmental release of DNA vaccine encoding Eimeria tenella TA4 and chicken IL-2.

    PubMed

    Song, Xiaokai; Zhang, Zeyang; Liu, Chang; Xu, Lixin; Yan, Ruofeng; Li, Xiangrui

    2016-10-15

    In a previous study, the construction of the Eimeria tenella DNA vaccine pVAX1.0-TA4-IL-2 which provides effective protection against coccidiosis was described and the immunization procedure was optimized. However, the persistence, integration, histopathology and environmental release of the DNA vaccine remain unknown. In this study, the persistence, integration and histopathology of the DNA vaccine pVAX1.0-TA4-IL-2 was evaluated in chickens in the following immunization studies: (1) single-dose immunization in one-day-old chickens; (2) repeat-dose immunization in chickens; and (3) single-high-dose immunization of three batches of plasmid in chickens. The persistence, integration, histopathology of the DNA vaccine was also evaluated in mice. At 1, 1.5, 2-4 months post immunization, blood, duodenum, heart, liver, spleen, kidneys and the immunized muscle tissue were collected from ten animals of each group. Persistence and integration were evaluated using PCR with a confirmed sensitivity of 30 plasmid copies. Hematoxylin and eosin stained sections were examined for the presence of inflammation or abnormalities that may result from vaccination. Water and fecal samples were also collected from the chicken enclosures to evaluate the potential for environmental release of the DNA vaccine. Testing various tissues by PCR confirmed that plasmid DNA persisted 1.5 months in blood, heart, liver and spleen, 2 months in kidneys and muscle of injected site. Furthermore, the vaccine did not integrate with the host genome. The histopathological examinations did not show obvious inflammation or pathological damage in any tissue of the immunized chickens. Similar results were observed in mice. Moreover, the DNA vaccine was not released into the surrounding environment. These results indicate that the DNA vaccine pVAX1.0-TA4-IL-2 has potential as safe vaccine against coccidiosis. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Integrated analysis of somatic mutations and focal copy-number changes identifies key genes and pathways in hepatocellular carcinoma

    PubMed Central

    Guichard, Cécile; Amaddeo, Giuliana; Imbeaud, Sandrine; Ladeiro, Yannick; Pelletier, Laura; Maad, Ichrafe Ben; Calderaro, Julien; Bioulac-Sage, Paulette; Letexier, Mélanie; Degos, Françoise; Clément, Bruno; Balabaud, Charles; Chevet, Eric; Laurent, Alexis; Couchy, Gabrielle; Letouzé, Eric; Calvo, Fabien; Zucman-Rossi, Jessica

    2012-01-01

    Hepatocellular carcinoma (HCC) is the most common primary liver malignancy. High-resolution copy number analysis of 125 tumors of which 24 were subjected to whole-exome sequencing identified 135 homozygous deletions and 994 somatic gene mutations with predicted functional consequences. We identified new recurrent alterations in 6 genes (ARID1A, RPS6KA3, NFE2L2, IRF2, CDH8 and PROKR2) not previously described in HCC. Functional analyses demonstrated tumor suppressor properties for IRF2 whose inactivation, exclusively found in hepatitis B virus related tumors, leads to impaired TP53 function. Alternatively, inactivation of proteins involved in chromatin remodeling was frequent and predominant in alcohol related tumors. Moreover, activation of the oxidative stress metabolism and inactivation of RPS6KA3 were new pathways associated with WNT/β-catenin activation, thereby suggesting a cooperative effect in tumorigenesis. This study shows the dramatic somatic genetic diversity in HCC, it reveals interactions between oncogene and tumor suppressor gene mutations markedly related to specific risk factors. PMID:22561517

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

    PubMed

    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.

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

    PubMed

    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.

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

    SciTech Connect

    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.

  11. Seminal parameters of chronic male genital inflammation are associated with disturbed sperm DNA integrity.

    PubMed

    Haidl, F; Haidl, G; Oltermann, I; Allam, J P

    2015-05-01

    Definition of chronic male genital tract inflammation and its impact on male infertility is still a matter of debate. In particular, DNA integrity has been reported to be disturbed in subfertile men. Thus, the aim of this study was to investigate an association of DNA integrity to altered standard semen parameters as well as inflammatory parameters such as peroxidase-positive cells, macrophages and seminal interleukin-6 concentration. Macrophages were detected by CD18/HLA-Dr staining, and DNA integrity was analysed by acridine orange staining using flow cytometry. Interleukin-6 was detected by ELISA. Normal DNA integrity showed a significant correlation to sperm number and progressive motility. Moreover, a significant inverse correlation of DNA integrity to Interleukin-6 and macrophages could be demonstrated. Further on, seminal interleukin-6 also significantly correlated to macrophages. No association has been observed between the number of peroxidase-positive cells and normal DNA integrity. As disturbed DNA integrity has been reported to negatively influence spermatozoon-egg interaction and even fertilisation rates following ICSI, and as early miscarriages have been associated with sperm DNA damage, it should be screened very carefully for male genital tract inflammations in couples undergoing infertility treatment. Measuring Interleukin-6 seems superior to assessment of the number of leucocytes alone and additional assessment of DNA integrity into the diagnostic work-up should be considered. © 2015 Blackwell Verlag GmbH.

  12. Developmental exposure to second-hand smoke increases adult atherogenesis and alters mitochondrial DNA copy number and deletions in apoE(-/-) mice.

    PubMed

    Fetterman, Jessica L; Pompilius, Melissa; Westbrook, David G; Uyeminami, Dale; Brown, Jamelle; Pinkerton, Kent E; Ballinger, Scott W

    2013-01-01

    Cardiovascular disease is a major cause of morbidity and mortality in the United States. While many studies have focused upon the effects of adult second-hand smoke exposure on cardiovascular disease development, disease development occurs over decades and is likely influenced by childhood exposure. The impacts of in utero versus neonatal second-hand smoke exposure on adult atherosclerotic disease development are not known. The objective of the current study was to determine the effects of in utero versus neonatal exposure to a low dose (1 mg/m(3) total suspended particulate) of second-hand smoke on adult atherosclerotic lesion development using the apolipoprotein E null mouse model. Consequently, apolipoprotein E null mice were exposed to either filtered air or second-hand smoke: (i) in utero from gestation days 1-19, or (ii) from birth until 3 weeks of age (neonatal). Subsequently, all animals were exposed to filtered air and sacrificed at 12-14 weeks of age. Oil red-O staining of whole aortas, measures of mitochondrial damage, and oxidative stress were performed. Results show that both in utero and neonatal second-hand smoke exposure significantly increased adult atherogenesis in mice compared to filtered air controls. These changes were associated with changes in aconitase and mitochondrial superoxide dismutase activities consistent with increased oxidative stress in the aorta, changes in mitochondrial DNA copy number and deletion levels. These studies show that in utero or neonatal exposure to second-hand smoke significantly influences adult atherosclerotic lesion development and results in significant alterations to the mitochondrion and its genome that may contribute to atherogenesis.

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

    PubMed

    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.

  14. Integrating Multi-omics Data to Dissect Mechanisms of DNA repair Dysregulation in Breast Cancer.

    PubMed

    Liu, Chao; Rohart, Florian; Simpson, Peter T; Khanna, Kum Kum; Ragan, Mark A; Lê Cao, Kim-Anh

    2016-09-26

    DNA repair genes and pathways that are transcriptionally dysregulated in cancer provide the first line of evidence for the altered DNA repair status in tumours, and hence have been explored intensively as a source for biomarker discovery. The molecular mechanisms underlying DNA repair dysregulation, however, have not been systematically investigated in any cancer type. In this study, we performed a statistical analysis to dissect the roles of DNA copy number alteration (CNA), DNA methylation (DM) at gene promoter regions and the expression changes of transcription factors (TFs) in the differential expression of individual DNA repair genes in normal versus tumour breast samples. These gene-level results were summarised at pathway level to assess whether different DNA repair pathways are affected in distinct manners. Our results suggest that CNA and expression changes of TFs are major causes of DNA repair dysregulation in breast cancer, and that a subset of the identified TFs may exert global impacts on the dysregulation of multiple repair pathways. Our work hence provides novel insights into DNA repair dysregulation in breast cancer. These insights improve our understanding of the molecular basis of the DNA repair biomarkers identified thus far, and have potential to inform future biomarker discovery.

  15. Integrating Multi-omics Data to Dissect Mechanisms of DNA repair Dysregulation in Breast Cancer

    PubMed Central

    Liu, Chao; Rohart, Florian; Simpson, Peter T.; Khanna, Kum Kum; Ragan, Mark A.; Lê Cao, Kim-Anh

    2016-01-01

    DNA repair genes and pathways that are transcriptionally dysregulated in cancer provide the first line of evidence for the altered DNA repair status in tumours, and hence have been explored intensively as a source for biomarker discovery. The molecular mechanisms underlying DNA repair dysregulation, however, have not been systematically investigated in any cancer type. In this study, we performed a statistical analysis to dissect the roles of DNA copy number alteration (CNA), DNA methylation (DM) at gene promoter regions and the expression changes of transcription factors (TFs) in the differential expression of individual DNA repair genes in normal versus tumour breast samples. These gene-level results were summarised at pathway level to assess whether different DNA repair pathways are affected in distinct manners. Our results suggest that CNA and expression changes of TFs are major causes of DNA repair dysregulation in breast cancer, and that a subset of the identified TFs may exert global impacts on the dysregulation of multiple repair pathways. Our work hence provides novel insights into DNA repair dysregulation in breast cancer. These insights improve our understanding of the molecular basis of the DNA repair biomarkers identified thus far, and have potential to inform future biomarker discovery. PMID:27666291

  16. Highly Informative Single-Copy Nuclear Microsatellite DNA Markers Developed Using an AFLP-SSR Approach in Black Spruce (Picea mariana) and Red Spruce (P. rubens)

    PubMed Central

    Shi, Yong-Zhong; Forneris, Natascha; Rajora, Om P.

    2014-01-01

    Background 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. Principal Findings 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. Significance 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

  17. 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.

    PubMed

    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.

  18. Expression of a chromosomally integrated, single-copy GFP gene in Candida albicans, and its use as a reporter of gene regulation.

    PubMed

    Morschhäuser, J; Michel, S; Hacker, J

    1998-02-01

    Genetically engineered versions of the GFP gene, which encodes the green fluorescent protein of Aequorea victoria, were placed under the control of the constitutively active Candida albicans ACT1 promoter and integrated in single copy into the genome of this pathogenic yeast. Integrative transformants in which one of the two ACT1 alleles had been replaced by a GFP gene exhibited a homogeneous, constitutive fluorescent phenotype. Cells expressing GFP with the wild-type chromophore exhibited very weak fluorescence compared to those GFP proteins with the S65T or S65A, V68L, S72A (GFPmut2) chromophore mutations. Substitution of the CTG codon, which specifies serine instead of leucine in C. albicans, by TTG was absolutely necessary for GFP expression. Although GFP mRNA levels in cells containing a GFP gene with the CTG codon were comparable to those of transformants containing GFP with the TTG substitution, only the latter produced GFP protein, as detected by Western blotting, suggesting that the frequent failure to express heterologous genes in C. albicans is principally due to the noncanonical codon usage. Transformants expressing the modified GFP gene from the promoter of the SAP2 gene, which encodes one of the secreted acid proteinases of C. albicans, showed fluorescence only under conditions which promote proteinase expression, thereby demonstrating the utility of stable, chromosomally integrated GFP reporter genes for the study of gene activation in C. albicans.

  19. HIV Integration at Certain Sites in Host DNA is Linked to the Expansion and Persistence of Infected Cells | Center for Cancer Research

    Cancer.gov

    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.

  20. DNA Sequences from Formalin-Fixed Nematodes: Integrating Molecular and Morphological Approaches to Taxonomy

    PubMed Central

    Thomas, W. Kelley; Vida, J. T.; Frisse, Linda M.; Mundo, Manuel; Baldwin, James G.

    1997-01-01

    To effectively integrate DNA sequence analysis and classical nematode taxonomy, we must be able to obtain DNA sequences from formalin-fixed specimens. Microdissected sections of nematodes were removed from specimens fixed in formalin, using standard protocols and without destroying morphological features. The fixed sections provided sufficient template for multiple polymerase chain reaction-based DNA sequence analyses. PMID:19274156

  1. Detection and chromosomal assignment of SV40-DNA integration in Chinese hamster cell lines by chromosome sorting and dot blot hybridization.

    PubMed

    Hutter, K J; Klefenz, H; Goerttler, K

    1990-01-01

    A combination of cytometric (chromosome sorting), molecular (dot blot hybridization using radio-active and/or biotinylated DNA probes) and cytogenetic (G-banding) evaluation is described which allows the rapid identification of single copy and repetitive viral integrates and their assignment to chromosome groups or even individual chromosomes. In the case of Chinese hamster cell line CO 631 it could be demonstrated that SV40 DNA was solely integrated into a submetacentric marker chromosome. Such a cytometric/molecular/cytogenetic "identogram" may prove to be a useful tool in many areas of cell and tumor biology. Furthermore, amounts of chromosomes sufficient for analysis as well as subsequent cloning experiments can be accumulated.

  2. Integrity and Biological Activity of DNA after UV Exposure

    NASA Astrophysics Data System (ADS)

    Lyon, Delina Y.; Monier, Jean-Michel; Dupraz, Sébastien; Freissinet, Caroline; Simonet, Pascal; Vogel, Timothy M.

    2010-04-01

    The field of astrobiology lacks a universal marker with which to indicate the presence of life. This study supports the proposal to use nucleic acids, specifically DNA, as a signature of life (biosignature). In addition to its specificity to living organisms, DNA is a functional molecule that can confer new activities and characteristics to other organisms, following the molecular biology dogma, that is, DNA is transcribed to RNA, which is translated into proteins. Previous criticisms of the use of DNA as a biosignature have asserted that DNA molecules would be destroyed by UV radiation in space. To address this concern, DNA in plasmid form was deposited onto different surfaces and exposed to UVC radiation. The surviving DNA was quantified via the quantitative polymerase chain reaction (qPCR). Results demonstrate increased survivability of DNA attached to surfaces versus non-adsorbed DNA. The DNA was also tested for biological activity via transformation into the bacterium Acinetobacter sp. and assaying for antibiotic resistance conferred by genes encoded by the plasmid. The success of these methods to detect DNA and its gene products after UV exposure (254 nm, 3.5 J/m2s) not only supports the use of the DNA molecule as a biosignature on mineral surfaces but also demonstrates that the DNA retained biological activity.

  3. Lymphoma-type adult T-cell leukaemia-lymphoma with a bulky cutaneous tumour showing multiple human T-lymphotropic virus-1 DNA integration.

    PubMed

    Kato, N; Sugawara, H; Aoyagi, S; Mayuzumi, M

    2001-06-01

    Human T-lymphotropic virus-1 (HTLV-1) is considered to be the cause of adult T-cell leukaemia-lymphoma (ATL). Monoclonal integration of HTLV-1 proviral DNA, as is analysed by Southern blotting, has been demonstrated in ATL patients. Unusual integration patterns of HTLV-1 proviral DNA have occasionally been described, and it is suggested that the patterns have clinical implications for ATL pathophysiology. Multiple, complete and defective types of integration patterns, in that order, are apparently associated with prognoses from good to poor. We report a 73-year-old Japanese woman with lymphoma-type ATL and a bulky cutaneous tumour on the left thigh. Four bands of slightly differing intensity were seen after EcoRI digestion of skin and lymph node samples on Southern blot analysis of HTLV-1 proviral DNA. Analysis for T-cell receptor-beta gene revealed five novel bands after restriction enzyme digestion with HindIII, indicating that the patient has four separate tumour cell clones, each of which carries one copy of the provirus. She was treated with chemotherapy and radiation and remains under reasonable control despite some relapsing cutaneous nodules. The indolent course in this present case could be related to the multiple integration pattern of HTLV-1 proviral DNA detected.

  4. Urine Cell-Free DNA Integrity Analysis for Early Detection of Prostate Cancer Patients.

    PubMed

    Salvi, Samanta; Gurioli, Giorgia; Martignano, Filippo; Foca, Flavia; Gunelli, Roberta; Cicchetti, Giacomo; De Giorgi, Ugo; Zoli, Wainer; Calistri, Daniele; Casadio, Valentina

    2015-01-01

    The detection of tumor-specific markers in urine has paved the way for new early noninvasive diagnostic approaches for prostate cancer. We evaluated the DNA integrity in urine supernatant to verify its capacity to discriminate between prostate cancer and benign diseases of the urogenital tract. A total of 131 individuals were enrolled: 67 prostate cancer patients and 64 patients with benign diseases of the urogenital tract (control group). Prostate-specific antigen (PSA) levels were determined. Urine cell-free (UCF) DNA was isolated and sequences longer than 250 bp corresponding to 3 genes (c-MYC, HER2, and AR) were quantified by Real-Time PCR to assess UCF-DNA integrity. UCF-DNA was quantifiable in all samples, while UCF-DNA integrity was evaluable in all but 16 samples. Receiver operating characteristic analysis showed an area under the curve of 0.5048 for UCF-DNA integrity and 0.8423 for PSA. Sensitivity was 0.58 and 0.95 for UCF-DNA integrity and PSA, respectively. Specificity was 0.44 and 0.69, respectively. UCF-DNA integrity showed lower accuracy than PSA and would not seem to be a reliable marker for early prostate cancer diagnosis. Despite this, we believe that UCF-DNA could represent a source of other biomarkers and could detect gene alterations.

  5. Removal of Integrated Hepatitis B Virus DNA Using CRISPR-Cas9

    PubMed Central

    Li, Hao; Sheng, Chunyu; Wang, Shan; Yang, Lang; Liang, Yuan; Huang, Yong; Liu, Hongbo; Li, Peng; Yang, Chaojie; Yang, Xiaoxia; Jia, Leili; Xie, Jing; Wang, Ligui; Hao, Rongzhang; Du, Xinying; Xu, Dongping; Zhou, Jianjun; Li, Mingzhen; Sun, Yansong; Tong, Yigang; Li, Qiao; Qiu, Shaofu; Song, Hongbin

    2017-01-01

    The presence of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) and the permanent integration of HBV DNA into the host genome confers the risk of viral reactivation and hepatocellular carcinoma. Nucleoside/nucleotide analogs alone have little or no capacity to eliminate replicative HBV templates consisting of cccDNA or integrated HBV DNA. Recently, CRISPR/Cas9 technology has been widely applied as a promising genome-editing tool, and HBV-specific CRISPR-Cas9 systems were shown to effectively mediate HBV cccDNA disruption. However, the integrated HBV DNA fragments are considered as important pro-oncogenic properties and it serves as an important template for viral replication and expression in stable HBV cell line. In this study, we completely excised a full-length 3,175-bp integrated HBV DNA fragment and disrupted HBV cccDNA in a stable HBV cell line. In HBV-excised cell line, the HBV cccDNA inside cells, supernatant HBV DNA, HBsAg, and HBeAg remained below the negative critical values for more than 10 months. Besides, by whole genome sequencing, we analyzed off-target effects and excluded cell contamination. It is the first time that the HBV infection has been fully eradicated in a stable HBV cell line. These findings demonstrate that the CRISPR-Cas9 system is a potentially powerful tool capable of promoting a radical or “sterile” HBV cure. PMID:28382278

  6. Removal of Integrated Hepatitis B Virus DNA Using CRISPR-Cas9.

    PubMed

    Li, Hao; Sheng, Chunyu; Wang, Shan; Yang, Lang; Liang, Yuan; Huang, Yong; Liu, Hongbo; Li, Peng; Yang, Chaojie; Yang, Xiaoxia; Jia, Leili; Xie, Jing; Wang, Ligui; Hao, Rongzhang; Du, Xinying; Xu, Dongping; Zhou, Jianjun; Li, Mingzhen; Sun, Yansong; Tong, Yigang; Li, Qiao; Qiu, Shaofu; Song, Hongbin

    2017-01-01

    The presence of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) and the permanent integration of HBV DNA into the host genome confers the risk of viral reactivation and hepatocellular carcinoma. Nucleoside/nucleotide analogs alone have little or no capacity to eliminate replicative HBV templates consisting of cccDNA or integrated HBV DNA. Recently, CRISPR/Cas9 technology has been widely applied as a promising genome-editing tool, and HBV-specific CRISPR-Cas9 systems were shown to effectively mediate HBV cccDNA disruption. However, the integrated HBV DNA fragments are considered as important pro-oncogenic properties and it serves as an important template for viral replication and expression in stable HBV cell line. In this study, we completely excised a full-length 3,175-bp integrated HBV DNA fragment and disrupted HBV cccDNA in a stable HBV cell line. In HBV-excised cell line, the HBV cccDNA inside cells, supernatant HBV DNA, HBsAg, and HBeAg remained below the negative critical values for more than 10 months. Besides, by whole genome sequencing, we analyzed off-target effects and excluded cell contamination. It is the first time that the HBV infection has been fully eradicated in a stable HBV cell line. These findings demonstrate that the CRISPR-Cas9 system is a potentially powerful tool capable of promoting a radical or "sterile" HBV cure.

  7. Homologous recombination maintenance of genome integrity during DNA damage tolerance

    PubMed Central

    Prado, Félix

    2014-01-01

    The DNA strand exchange protein Rad51 provides a safe mechanism for the repair of DNA breaks using the information of a homologous DNA template. Homologous recombination (HR) also plays a key role in the response to DNA damage that impairs the advance of the replication forks by providing mechanisms to circumvent the lesion and fill in the tracks of single-stranded DNA that are generated during the process of lesion bypass. These activities postpone repair of the blocking lesion to ensure that DNA replication is completed in a timely manner. Experimental evidence generated over the last few years indicates that HR participates in this DNA damage tolerance response together with additional error-free (template switch) and error-prone (translesion synthesis) mechanisms through intricate connections, which are presented here. The choice between repair and tolerance, and the mechanism of tolerance, is critical to avoid increased mutagenesis and/or genome rearrangements, which are both hallmarks of cancer. PMID:27308329

  8. Adeno-associated virus (AAV) Rep proteins mediate complex formation between AAV DNA and its integration site in human DNA.

    PubMed Central

    Weitzman, M D; Kyöstiö, S R; Kotin, R M; Owens, R A

    1994-01-01

    AAV is unique among eukaryotic viruses in the ability of its DNA to integrate preferentially into a specific region of the human genome. Understanding AAV integration may aid in developing gene therapy systems with predictable integration sites. Using a gel mobility-shift assay, we have identified a DNA sequence within the AAV integration locus on human chromosome 19 which is specifically bound by the AAV Rep78 and Rep68 proteins. This Rep recognition sequence is a GCTC repeating motif very similar to sequences within the inverted terminal repeats of the AAV genome which are also bound by Rep78 and Rep68. Cloned oligonucleotides containing the recognition sequence can direct specific binding by Rep proteins. Binding assays with mutant Rep proteins show that the amino-terminal portion of Rep78 and Rep68 can direct binding to either the AAV terminal repeat hairpin DNA or chromosome 19. This human genomic DNA can be complexed with AAV DNA by Rep proteins as demonstrated by a dual-label (32P/biotin) assay. These results suggest a role for Rep in targeting viral integration. Images PMID:8016070

  9. Characterization of three active transposable elements recently inserted in three independent DFR-A alleles and one high-copy DNA transposon isolated from the Pink allele of the ANS gene in onion (Allium cepa L.).

    PubMed

    Kim, Sunggil; Park, Jee Young; Yang, Tae-Jin

    2015-06-01

    Intact retrotransposon and DNA transposons inserted in a single gene were characterized in onions (Allium cepa) and their transcription and copy numbers were estimated in this study. While analyzing diverse onion germplasm, large insertions in the DFR-A gene encoding dihydroflavonol 4-reductase (DFR) involved in the anthocyanin biosynthesis pathway were found in two accessions. A 5,070-bp long terminal repeat (LTR) retrotransposon inserted in the active DFR-A (R4) allele was identified from one of the large insertions and designated AcCOPIA1. An intact ORF encoded typical domains of copia-like LTR retrotransposons. However, AcCOPIA1 contained atypical 'TG' and 'TA' dinucleotides at the ends of the LTRs. A 4,615-bp DNA transposon was identified in the other large insertion. This DNA transposon, designated AcCACTA1, contained an ORF coding for a transposase showing homology with the CACTA superfamily transposable elements (TEs). Another 5,073-bp DNA transposon was identified from the DFR-A (TRN) allele. This DNA transposon, designated AchAT1, belonged to the hAT superfamily with short 4-bp terminal inverted repeats (TIRs). Finally, a 6,258-bp non-autonomous DNA transposon, designated AcPINK, was identified in the ANS-p allele encoding anthocyanidin synthase, the next downstream enzyme to DFR in the anthocyanin biosynthesis pathway. AcPINK also possessed very short 3-bp TIRs. Active transcription of AcCOPIA1, AcCACTA1, and AchAT1 was observed through RNA-Seq analysis and RT-PCR. The copy numbers of AcPINK estimated by mapping the genomic DNA reads produced by NextSeq 500 were predominantly high compared with the other TEs. A series of evidence indicated that these TEs might have transposed in these onion genes very recently, providing a stepping stone for elucidation of enormously large-sized onion genome structure.

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

    PubMed

    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.

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

    SciTech Connect

    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.

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

    PubMed

    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 = <0.001). DNA integrity index is correlated with TNM stage. Given 100 % specificity, the highest sensitivity achieved in detecting 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. 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.

    PubMed

    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. DNA integrity of onion root cells under catechol influence.

    PubMed

    Petriccione, Milena; Forte, Valentina; Valente, Diego; Ciniglia, Claudia

    2013-07-01

    Catechol is a highly toxic organic pollutant, usually abundant in the waste effluents of industrial processes and agricultural activities. The environmental sources of catechol include pesticides, wood preservatives, tanning lotion, cosmetic creams, dyes, and synthetic intermediates. Genotoxicity of catechol at a concentration range 5 × 10(-1)-5 mM was evaluated by applying random amplified polymorphic DNA (RAPD) and time-lapse DNA laddering tests using onion (Allium cepa) root cells as the assay system. RAPD analysis revealed polymorphisms in the nucleotidic sequence of DNA that reflected the genotoxic potential of catechol to provoke point mutations, or deletions, or chromosomal rearrangements. Time-lapse DNA laddering test provided evidence that catechol provoked DNA necrosis and apoptosis. Acridine orange/ethidium bromide staining could distinguish apoptotic from necrotic cells in root cells of A. cepa.

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

    PubMed

    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. GPHMM: an integrated hidden Markov model for identification of copy number alteration and loss of heterozygosity in complex tumor samples using whole genome SNP arrays

    PubMed Central

    Li, Ao; Liu, Zongzhi; Lezon-Geyda, Kimberly; Sarkar, Sudipa; Lannin, Donald; Schulz, Vincent; Krop, Ian; Winer, Eric; Harris, Lyndsay; Tuck, David

    2011-01-01

    There is an increasing interest in using single nucleotide polymorphism (SNP) genotyping arrays for profiling chromosomal rearrangements in tumors, as they allow simultaneous detection of copy number and loss of heterozygosity with high resolution. Critical issues such as signal baseline shift due to aneuploidy, normal cell contamination, and the presence of GC content bias have been reported to dramatically alter SNP array signals and complicate accurate identification of aberrations in cancer genomes. To address these issues, we propose a novel Global Parameter Hidden Markov Model (GPHMM) to unravel tangled genotyping data generated from tumor samples. In contrast to other HMM methods, a distinct feature of GPHMM is that the issues mentioned above are quantitatively modeled by global parameters and integrated within the statistical framework. We developed an efficient EM algorithm for parameter estimation. We evaluated performance on three data sets and show that GPHMM can correctly identify chromosomal aberrations in tumor samples containing as few as 10% cancer cells. Furthermore, we demonstrated that the estimation of global parameters in GPHMM provides information about the biological characteristics of tumor samples and the quality of genotyping signal from SNP array experiments, which is helpful for data quality control and outlier detection in cohort studies. PMID:21398628

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

    PubMed

    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.

  18. Integrated Copy Number and Expression Analysis Identifies Profiles of Whole-Arm Chromosomal Alterations and Subgroups with Favorable Outcome in Ovarian Clear Cell Carcinomas

    PubMed Central

    Uehara, Yuriko; Oda, Katsutoshi; Ikeda, Yuji; Koso, Takahiro; Tsuji, Shingo; Yamamoto, Shogo; Asada, Kayo; Sone, Kenbun; Kurikawa, Reiko; Makii, Chinami; Hagiwara, Otoe; Tanikawa, Michihiro; Maeda, Daichi; Hasegawa, Kosei; Nakagawa, Shunsuke; Wada-Hiraike, Osamu; Kawana, Kei; Fukayama, Masashi; Fujiwara, Keiichi; Yano, Tetsu; Osuga, Yutaka; Fujii, Tomoyuki; Aburatani, Hiroyuki

    2015-01-01

    Ovarian clear cell carcinoma (CCC) is generally associated with chemoresistance and poor clinical outcome, even with early diagnosis; whereas high-grade serous carcinomas (SCs) and endometrioid carcinomas (ECs) are commonly chemosensitive at advanced stages. Although an integrated genomic analysis of SC has been performed, conclusive views on copy number and expression profiles for CCC are still limited. In this study, we performed single nucleotide polymorphism analysis with 57 epithelial ovarian cancers (31 CCCs, 14 SCs, and 12 ECs) and microarray expression analysis with 55 cancers (25 CCCs, 16 SCs, and 14 ECs). We then evaluated PIK3CA mutations and ARID1A expression in CCCs. SNP array analysis classified 13% of CCCs into a cluster with high frequency and focal range of copy number alterations (CNAs), significantly lower than for SCs (93%, P < 0.01) and ECs (50%, P = 0.017). The ratio of whole-arm to all CNAs was higher in CCCs (46.9%) than SCs (21.7%; P < 0.0001). SCs with loss of heterozygosity (LOH) of BRCA1 (85%) also had LOH of NF1 and TP53, and LOH of BRCA2 (62%) coexisted with LOH of RB1 and TP53. Microarray analysis classified CCCs into three clusters. One cluster (CCC-2, n = 10) showed more favorable prognosis than the CCC-1 and CCC-3 clusters (P = 0.041). Coexistent alterations of PIK3CA and ARID1A were more common in CCC-1 and CCC-3 (7/11, 64%) than in CCC-2 (0/10, 0%; P < 0.01). Being in cluster CCC-2 was an independent favorable prognostic factor in CCC. In conclusion, CCC was characterized by a high ratio of whole-arm CNAs; whereas CNAs in SC were mainly focal, but preferentially caused LOH of well-known tumor suppressor genes. As such, expression profiles might be useful for sub-classification of CCC, and might provide useful information on prognosis. PMID:26043110

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

    SciTech Connect

    Peng, Jamy C.

    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

  20. An electronic DNA sensor chip using integrated capacitive read-out circuit.

    PubMed

    Lee, Byunghun; Lee, Kang-Ho; Lee, Jeong-Oen; Sohn, Mi-Jin; Choi, Suk-Hwan; Wang, Se-Won; Yoon, Jun-Bo; Cho, Gyu-Hyeong

    2010-01-01

    This paper presents fully integrated label-free DNA recognition circuit based on capacitance measurement. A CMOS-based DNA sensor is implemented for the electrical detection of DNA hybridization. The proposed architecture detects the difference of capacitance through the integration of current mismatch of capacitance between reference electrodes functionalized with only single-stranded DNA and sensing electrodes bound with complementary DNA strands specifically. In addition, to minimize the effects of parallel resistance between electrodes and DNA layers, the compensation technique of leakage current through the use of constant current charging and discharging is implemented in the proposed detection circuit. The chip was fabricated in 0.35um 4-metal 2-poly CMOS process, and 16 × 8 sensing electrode arrays were fabricated by post-processing steps.

  1. Evaluation of gene expression and DNA copy number profiles of adipose tissue-derived stromal cells and consecutive neurosphere-like cells generated from dogs with naturally occurring spinal cord injury.

    PubMed

    Lim, Ji-Hey; Koh, Sehwon; Thomas, Rachael; Breen, Matthew; Olby, Natasha J

    2017-03-01

    OBJECTIVE To evaluate gene expression and DNA copy number in adipose tissue-derived stromal cells (ADSCs) and in ADSC-derived neurosphere-like cell clusters (ADSC-NSCs) generated from tissues of chronically paraplegic dogs. ANIMALS 14 client-owned paraplegic dogs. PROCEDURES Dorsal subcutaneous adipose tissue (< 1 cm(3)) was collected under general anesthesia; ADSCs were isolated and cultured. Third-passage ADSCs were cultured in neural cell induction medium to generate ADSC-NSCs. Relative gene expression of mesenchymal cell surface marker CD90 and neural progenitor marker nestin was assessed in ADSCs and ADSC-NSCs from 3 dogs by quantitative real-time PCR assay; expression of these and various neural lineage genes was evaluated for the same dogs by reverse transcription PCR assay. Percentages of cells expressing CD90, nestin, glial fibrillary acidic protein (GFAP), and tubulin β 3 class III (TUJ1) proteins were determined by flow cytometry for all dogs. The DNA copy number stability (in samples from 6 dogs) and neural cell differentiation (14 dogs) were assessed with array-comparative genomic hybridization analysis and immunocytochemical evaluation, respectively. RESULTS ADSCs and ADSC-NSCs expressed neural cell progenitor and differentiation markers; GFAP and microtubule-associated protein 2 were expressed by ADSC-NSCs but not ADSCs. Relative gene expression of CD90 and nestin was subjectively higher in ADSC-NSCs than in ADSCs. Percentages of ADSC-NSCs expressing nestin, GFAP, and TUJ1 proteins were substantially higher than those of ADSCs. Cells expressing neuronal and glial markers were generated from ADSC-NSCs and had no DNA copy number instability detectable by the methods used. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested ADSCs can potentially be a safe and clinically relevant autologous source for canine neural progenitor cells. Further research is needed to verify these findings.

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

    PubMed

    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.

  3. Genomic landscape of human, bat, and ex vivo DNA transposon integrations.

    PubMed

    Campos-Sánchez, Rebeca; Kapusta, Aurélie; Feschotte, Cédric; Chiaromonte, Francesca; Makova, Kateryna D

    2014-07-01

    The integration and fixation preferences of DNA transposons, one of the major classes of eukaryotic transposable elements, have never been evaluated comprehensively on a genome-wide scale. Here, we present a detailed study of the distribution of DNA transposons in the human and bat genomes. We studied three groups of DNA transposons that integrated at different evolutionary times: 1) ancient (>40 My) and currently inactive human elements, 2) younger (<40 My) bat elements, and 3) ex vivo integrations of piggyBat and Sleeping Beauty elements in HeLa cells. Although the distribution of ex vivo elements reflected integration preferences, the distribution of human and (to a lesser extent) bat elements was also affected by selection. We used regression techniques (linear, negative binomial, and logistic regression models with multiple predictors) applied to 20-kb and 1-Mb windows to investigate how the genomic landscape in the vicinity of DNA transposons contributes to their integration and fixation. Our models indicate that genomic landscape explains 16-79% of variability in DNA transposon genome-wide distribution. Importantly, we not only confirmed previously identified predictors (e.g., DNA conformation and recombination hotspots) but also identified several novel predictors (e.g., signatures of double-strand breaks and telomere hexamer). Ex vivo integrations showed a bias toward actively transcribed regions. Older DNA transposons were located in genomic regions scarce in most conserved elements-likely reflecting purifying selection. Our study highlights how DNA transposons are integral to the evolution of bat and human genomes, and has implications for the development of DNA transposon assays for gene therapy and mutagenesis applications.

  4. Developing an Integrated DNA Sequencing System for Research and Education at Virginia State University

    DTIC Science & Technology

    2016-01-31

    SECURITY CLASSIFICATION OF: The funded project to establish an integrated DNA sequencing system for education and research at Virginia State...University has been successfully completed. A facility consisting of two next-generation DNA sequencers, which work in complement to each other, as well as a...series of instruments essential for DNA sample preparation and quality control, has been established in a dedicated laboratory at Virginia State

  5. The Conjugative Relaxase TrwC Promotes Integration of Foreign DNA in the Human Genome.

    PubMed

    González-Prieto, Coral; Gabriel, Richard; Dehio, Christoph; Schmidt, Manfred; Llosa, Matxalen

    2017-06-15

    Bacterial conjugation is a mechanism of horizontal DNA transfer. The relaxase TrwC of the conjugative plasmid R388 cleaves one strand of the transferred DNA at the oriT gene, covalently attaches to it, and leads the single-stranded DNA (ssDNA) into the recipient cell. In addition, TrwC catalyzes site-specific integration of the transferred DNA into its target sequence present in the genome of the recipient bacterium. Here, we report the analysis of the efficiency and specificity of the integrase activity of TrwC in human cells, using the type IV secretion system of the human pathogen Bartonella henselae to introduce relaxase-DNA complexes. Compared to Mob relaxase from plasmid pBGR1, we found that TrwC mediated a 10-fold increase in the rate of plasmid DNA transfer to human cells and a 100-fold increase in the rate of chromosomal integration of the transferred DNA. We used linear amplification-mediated PCR and plasmid rescue to characterize the integration pattern in the human genome. DNA sequence analysis revealed mostly reconstituted oriT sequences, indicating that TrwC is active and recircularizes transferred DNA in human cells. One TrwC-mediated site-specific integration event was detected, proving that TrwC is capable of mediating site-specific integration in the human genome, albeit with very low efficiency compared to the rate of random integration. Our results suggest that TrwC may stabilize the plasmid DNA molecules in the nucleus of the human cell, probably by recircularization of the transferred DNA strand. This stabilization would increase the opportunities for integration of the DNA by the host machinery.IMPORTANCE Different biotechnological applications, including gene therapy strategies, require permanent modification of target cells. Long-term expression is achieved either by extrachromosomal persistence or by integration of the introduced DNA. Here, we studied the utility of conjugative relaxase TrwC, a bacterial protein with site

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

    PubMed

    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.

  7. Urine cell-free DNA integrity as a marker for early bladder cancer diagnosis: preliminary data.

    PubMed

    Casadio, Valentina; Calistri, Daniele; Tebaldi, Michela; Bravaccini, Sara; Gunelli, Roberta; Martorana, Giuseppe; Bertaccini, Alessandro; Serra, Luigi; Scarpi, Emanuela; Amadori, Dino; Silvestrini, Rosella; Zoli, Wainer

    2013-11-01

    Urine cell-free (UCF) DNA has recently been proposed as a potential marker for early bladder cancer diagnosis. It is known that normal apoptotic cells produce highly fragmented DNA while cancer cells release longer DNA. Therefore, we verified the potential role of UCF DNA integrity in early bladder cancer diagnosis. UCF DNA was isolated from 51 bladder cancer patients, 46 symptomatic patients, and 32 healthy volunteers. To verify UCF DNA integrity, sequences longer than 250 bp, c-Myc, BCAS1, and HER2, were quantified by real time PCR. At the best cutoff value of 0.1 ng/μl, UCF DNA integrity analysis showed a sensitivity of 0.73 (95% CI 0.61-0.85), and a specificity of 0.84 (95% CI 0.71-0.97) in healthy individuals and 0.83 (95% CI 0.72-0.94) in symptomatic patients. Receiver operating characteristic (ROC) curve analysis revealed an area under the curve of 0.834 (95% CI 0.739-0.930) for healthy individuals and 0.796 (95% CI 0.707-0.885) for symptomatic patients. These preliminary data suggest that UCF DNA integrity is a potentially good marker for early noninvasive diagnosis of bladder cancer. Its diagnostic performance does not seem to vary significantly, even in an "at risk" population of symptomatic individuals. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. Novel use of polymerase chain reaction to amplify cellular DNA adjacent to an integrated provirus.

    PubMed Central

    Silver, J; Keerikatte, V

    1989-01-01

    We describe a modification of the polymerase chain reaction technique which allows amplification of cellular DNA adjacent to an integrated provirus given sequence information for the provirus only. The modified technique should be generally useful for studies of insertional mutagenesis and other situations in which one wishes to isolate DNA adjacent to a region of known sequence. Images PMID:2704070

  9. Non-homologous end-joining proteins are required for Agrobacterium T-DNA integration

    PubMed Central

    van Attikum, Haico; Bundock, Paul; Hooykaas, Paul J.J.

    2001-01-01

    Agrobacterium tumefaciens causes crown gall disease in dicotyledonous plants by introducing a segment of DNA (T-DNA), derived from its tumour-inducing (Ti) plasmid, into plant cells at infection sites. Besides these natural hosts, Agrobacterium can deliver the T-DNA also to monocotyledonous plants, yeasts and fungi. The T-DNA integrates randomly into one of the chromosomes of the eukaryotic host by an unknown process. Here, we have used the yeast Saccharomyces cerevisiae as a T-DNA recipient to demonstrate that the non-homologous end-joining (NHEJ) proteins Yku70, Rad50, Mre11, Xrs2, Lig4 and Sir4 are required for the integration of T-DNA into the host genome. We discovered a minor pathway for T-DNA integration at the telomeric regions, which is still operational in the absence of Rad50, Mre11 or Xrs2, but not in the absence of Yku70. T-DNA integration at the telomeric regions in the rad50, mre11 and xrs2 mutants was accompanied by gross chromosomal rearrangements. PMID:11707425

  10. Replication of a low-copy-number plasmid by a plasmid DNA-membrane complex extracted from minicells of Escherichia coli.

    PubMed Central

    Firshein, W; Strumph, P; Benjamin, P; Burnstein, K; Kornacki, J

    1982-01-01

    A DNA-membrane complex was extracted from minicells of an Escherichia coli mutant harboring a "miniplasmid" derivative (11.2 kilobases) of the low-copynumber plasmid RK2 (56 kilobases). The complex contained various species of supercoiled and intermediate forms of plasmid DNA, of which approximately 20% was bound firmly to the membrane after centrifugation in a CsCl density gradient. The plasmid DNA-membrane complex synthesized new plasmid DNA without the addition of exogenous template, enzymes, or other proteins. DNA synthesis appeared to proceed semi-conservatively, was dependent on the four deoxynucleoside triphosphates, partially dependent on ribonucleoside triphosphates, and was sensitive to rifampin, an antibiotic known to inhibit initiation of replication. Novobiocin and nalidixic acid also inhibited synthesis, as did the omission of ATP, N-Ethylmaleimide, an inhibitor of DNA polymerase II and III activity, but not DNA polymerase I activity, also partially inhibited the synthetic reaction, as did chloramphenicol. The plasmid DNA synthetic product was analyzed by alkaline sucrose and dye-CsCl gradient centrifugation, as well as by agarose gel electrophoresis. In each case, the product consisted of parental and intermediate forms of plasmid DNA. Some chromosomal DNA was also synthesized by a contaminating bacterial DNA-membrane complex, but this synthesis was rifampin insensitive and could be separated from plasmid DNA synthesis. PMID:7042688

  11. Breaking and Entering: Copying and Copy Protection.

    ERIC Educational Resources Information Center

    Westlake, Wayne; And Others

    1985-01-01

    Describes several commercially-available computer programs which allow users to make copies of "protected" software. Current costs, program features, and ordering information are provided for these "encryption" programs. Also describes a monthly journal (The HARDCORE Computist) which focuses on unlocking copy-protected…

  12. Breaking and Entering: Copying and Copy Protection.

    ERIC Educational Resources Information Center

    Westlake, Wayne; And Others

    1985-01-01

    Describes several commercially-available computer programs which allow users to make copies of "protected" software. Current costs, program features, and ordering information are provided for these "encryption" programs. Also describes a monthly journal (The HARDCORE Computist) which focuses on unlocking copy-protected…

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

    PubMed

    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

  14. Integrative modelling of tumour DNA methylation quantifies the contribution of metabolism

    PubMed Central

    Mehrmohamadi, Mahya; Mentch, Lucas K.; Clark, Andrew G.; Locasale, Jason W.

    2016-01-01

    Altered DNA methylation is common in cancer and often considered an early event in tumorigenesis. However, the sources of heterogeneity of DNA methylation among tumours remain poorly defined. Here we capitalize on the availability of multi-platform data on thousands of human tumours to build integrative models of DNA methylation. We quantify the contribution of clinical and molecular factors in explaining intertumoral variability in DNA methylation. We show that the levels of a set of metabolic genes involved in the methionine cycle is predictive of several features of DNA methylation in tumours, including the methylation of cancer genes. Finally, we demonstrate that patients whose DNA methylation can be predicted from the methionine cycle exhibited improved survival over cases where this regulation is disrupted. This study represents a comprehensive analysis of the determinants of methylation and demonstrates the surprisingly large interaction between metabolism and DNA methylation variation. Together, our results quantify links between tumour metabolism and epigenetics and outline clinical implications. PMID:27966532

  15. Increased frequency of de novo copy number variants in congenital heart disease by integrative analysis of single nucleotide polymorphism array and exome sequence data.

    PubMed

    Glessner, Joseph T; Bick, Alexander G; Ito, Kaoru; Homsy, Jason; Rodriguez-Murillo, Laura; Fromer, Menachem; Mazaika, Erica; Vardarajan, Badri; Italia, Michael; Leipzig, Jeremy; DePalma, Steven R; Golhar, Ryan; Sanders, Stephan J; Yamrom, Boris; Ronemus, Michael; Iossifov, Ivan; Willsey, A Jeremy; State, Matthew W; Kaltman, Jonathan R; White, Peter S; Shen, Yufeng; Warburton, Dorothy; Brueckner, Martina; Seidman, Christine; Goldmuntz, Elizabeth; Gelb, Bruce D; Lifton, Richard; Seidman, Jonathan; Hakonarson, Hakon; Chung, Wendy K

    2014-10-24

    Congenital heart disease (CHD) is among the most common birth defects. Most cases are of unknown pathogenesis. To determine the contribution of de novo copy number variants (CNVs) in the pathogenesis of sporadic CHD. We studied 538 CHD trios using genome-wide dense single nucleotide polymorphism arrays and whole exome sequencing. Results were experimentally validated using digital droplet polymerase chain reaction. We compared validated CNVs in CHD cases with CNVs in 1301 healthy control trios. The 2 complementary high-resolution technologies identified 63 validated de novo CNVs in 51 CHD cases. A significant increase in CNV burden was observed when comparing CHD trios with healthy trios, using either single nucleotide polymorphism array (P=7×10(-5); odds ratio, 4.6) or whole exome sequencing data (P=6×10(-4); odds ratio, 3.5) and remained after removing 16% of de novo CNV loci previously reported as pathogenic (P=0.02; odds ratio, 2.7). We observed recurrent de novo CNVs on 15q11.2 encompassing CYFIP1, NIPA1, and NIPA2 and single de novo CNVs encompassing DUSP1, JUN, JUP, MED15, MED9, PTPRE SREBF1, TOP2A, and ZEB2, genes that interact with established CHD proteins NKX2-5 and GATA4. Integrating de novo variants in whole exome sequencing and CNV data suggests that ETS1 is the pathogenic gene altered by 11q24.2-q25 deletions in Jacobsen syndrome and that CTBP2 is the pathogenic gene in 10q subtelomeric deletions. We demonstrate a significantly increased frequency of rare de novo CNVs in CHD patients compared with healthy controls and suggest several novel genetic loci for CHD. © 2014 American Heart Association, Inc.

  16. Increased Frequency of De Novo Copy Number Variations in Congenital Heart Disease by Integrative Analysis of SNP Array and Exome Sequence Data

    PubMed Central

    Rodriguez-Murillo, Laura; Fromer, Menachem; Mazaika, Erica; Vardarajan, Badri; Italia, Michael; Leipzig, Jeremy; DePalma, Steven R.; Golhar, Ryan; Sanders, Stephan J.; Yamrom, Boris; Ronemus, Michael; Iossifov, Ivan; Willsey, A. Jeremy; State, Matthew W.; Kaltman, Jonathan R.; White, Peter S.; Shen, Yufeng; Warburton, Dorothy; Brueckner, Martina; Seidman, Christine; Goldmuntz, Elizabeth; Gelb, Bruce D.; Lifton, Richard; Seidman, Jonathan; Hakonarson, Hakon; Chung, Wendy K.

    2014-01-01

    Rationale Congenital heart disease (CHD) is among the most common birth defects. Most cases are of unknown etiology. Objective To determine the contribution of de novo copy number variants (CNVs) in the etiology of sporadic CHD. Methods and Results We studied 538 CHD trios using genome-wide dense single nucleotide polymorphism (SNP) arrays and/or whole exome sequencing (WES). Results were experimentally validated using digital droplet PCR. We compared validated CNVs in CHD cases to CNVs in 1,301 healthy control trios. The two complementary high-resolution technologies identified 63 validated de novo CNVs in 51 CHD cases. A significant increase in CNV burden was observed when comparing CHD trios with healthy trios, using either SNP array (p=7x10−5, Odds Ratio (OR)=4.6) or WES data (p=6x10−4, OR=3.5) and remained after removing 16% of de novo CNV loci previously reported as pathogenic (p=0.02, OR=2.7). We observed recurrent de novo CNVs on 15q11.2 encompassing CYFIP1, NIPA1, and NIPA2 and single de novo CNVs encompassing DUSP1, JUN, JUP, MED15, MED9, PTPRE SREBF1, TOP2A, and ZEB2, genes that interact with established CHD proteins NKX2-5 and GATA4. Integrating de novo variants in WES and CNV data suggests that ETS1 is the pathogenic gene altered by 11q24.2-q25 deletions in Jacobsen syndrome and that CTBP2 is the pathogenic gene in 10q sub-telomeric deletions. Conclusions We demonstrate a significantly increased frequency of rare de novo CNVs in CHD patients compared with healthy controls and suggest several novel genetic loci for CHD. PMID:25205790

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

    PubMed

    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 < 0.05). The count of spermatozoa with nonfragmented DNA in normozoospermic samples was high and independent from IMSI-morphological classes (Class 1 versus Class 3, respectively) (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. Retroviral intasomes search for a target DNA by 1D diffusion which rarely results in integration

    PubMed Central

    Jones, Nathan D.; Lopez Jr, Miguel A.; Hanne, Jeungphill; Peake, Mitchell B.; Lee, Jong-Bong; Fishel, Richard; Yoder, Kristine E.

    2016-01-01

    Retroviruses must integrate their linear viral cDNA into the host genome for a productive infection. Integration is catalysed by the retrovirus-encoded integrase (IN), which forms a tetramer or octamer complex with the viral cDNA long terminal repeat (LTR) ends termed an intasome. IN removes two 3′-nucleotides from both LTR ends and catalyses strand transfer of the recessed 3′-hydroxyls into the target DNA separated by 4–6 bp. Host DNA repair restores the resulting 5′-Flap and single-stranded DNA (ssDNA) gap. Here we have used multiple single molecule imaging tools to determine that the prototype foamy virus (PFV) retroviral intasome searches for an integration site by one-dimensional (1D) rotation-coupled diffusion along DNA. Once a target site is identified, the time between PFV strand transfer events is 470 ms. The majority of PFV intasome search events were non-productive. These observations identify new dynamic IN functions and suggest that target site-selection limits retroviral integration. PMID:27108531

  19. Paternal influence of sperm DNA integrity on early embryonic development.

    PubMed

    Simon, L; Murphy, K; Shamsi, M B; Liu, L; Emery, B; Aston, K I; Hotaling, J; Carrell, D T

    2014-11-01

    Does sperm DNA damage affect early embryonic development? Increased sperm DNA damage adversely affects embryo quality starting at Day 2 of early embryonic development and continuing after embryo transfer, resulting in reduced implantation rates and pregnancy outcomes. Abnormalities in the sperm DNA in the form of single and double strand breaks can be assessed by an alkaline Comet assay. Some prior studies have shown a strong paternal effect of sperm DNA damage on IVF outcome, including reduced fertilization, reduced embryo quality and cleavage rates, reduced numbers of embryos developing into blastocysts, increased percentage of embryos undergoing developmental arrest, and reduced implantation and pregnancy rates. A cross-sectional study of 215 men from infertile couples undergoing assisted reproduction techniques at the University of Utah Center for Reproductive Medicine. Sperm from men undergoing ART were analyzed for DNA damage using an alkaline Comet assay and classified into three groups: 'low damage' (0-30%), 'intermediate damage' (31-70%) and 'high damage' (71-100%). The cause of couples' infertility was categorized into one of the three types (male, female or unexplained). Each embryo was categorized as 'good', 'fair' or 'poor' quality, based on the number and grade of blastomeres. The influence of sperm DNA damage on early embryonic development was observed and classified into four stages: peri-fertilization effect (fertilization rate), early paternal effect (embryonic days 1-2), late paternal effect (embryonic days 3-5) and implantation stage effect. The paternal effect of sperm DNA damage was observed at each stage of early embryonic development. The peri-fertilization effect was higher in oocytes from patients with female infertility (20.85%) compared with male (8.22%; P < 0.001) and unexplained (7.30%; P < 0.001) infertility factors. In both the early and late paternal effect stages, the low DNA damage group had a higher percentage of good quality

  20. Assembly of prototype foamy virus strand transfer complexes on product DNA bypassing catalysis of integration.

    PubMed

    Yin, Zhiqi; Lapkouski, Mikalai; Yang, Wei; Craigie, Robert

    2012-12-01

    Integrase is the key enzyme that mediates integration of retroviral DNA into cellular DNA which is essential for viral replication. Inhibitors of HIV-1 that target integrase recognize the nucleoprotein complexes formed by integrase and viral DNA substrate (intasomes) rather than the free enzyme. Atomic resolution structures of HIV-1 intasomes are therefore required to understand the mechanisms of inhibition and drug resistance. To date, prototype foamy virus (PFV) is the only retrovirus for which such structures have been determined. We show that PFV strand transfer complexes (STC) can be assembled on product DNA without going through the normal forward reaction pathway. The finding that a retroviral STC can be assembled in this way may provide a powerful tool to alleviate the obstacles that impede structural studies of nucleoprotein intermediates in HIV-1 DNA integration.