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Sample records for integrating dna copy

  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. 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.

  5. 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.

  6. 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 ...

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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).

  12. 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.

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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.

  20. 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

  1. 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

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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.

  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. 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.

  10. 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

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. 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

  8. 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.

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

    DTIC Science & Technology

    2015-08-01

    and select 330 samples for CNV analysis. Months :1 - 2 • Subtask 2 Prepare sections (10 μm) for microdissection to ensure>80% tumor. Months 3 - 8...Subtask 3 DNA preparation from microdissected specimens. Months 3 - 8 Major Task 2: To determine the copy number gain and loss for early stage high...to prepare chip compatible samples. Months : 9 - 18 • Subtask 2 Genomic abnormality analysis by IlluminaHumanOmniExpress-FFPE BeadChip system. Months

  10. 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

  11. 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.

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. 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.

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

    PubMed Central

    Jorissen, Robert N.; Lipton, Lara; Gibbs, Peter; Chapman, Matthew; Desai, Jayesh; Jones, Ian T.; Yeatman, Timothy J.; East, Philip; Tomlinson, Ian P.M.; Verspaget, Hein W.; Aaltonen, Lauri A.; Kruhøffer, Mogens; Ørntoft, Torben F.; Andersen, Claus Lindbjerg; Sieber, Oliver M.

    2008-01-01

    Purpose About 15% of colorectal cancers (CRCs) harbor microsatellite instability (MSI). MSI-associated gene expression changes have been identified in CRCs, but little overlap exists between signatures hindering an assessment of overall consistency. Little is known about the causes and downstream effects of differential gene expression. Experimental Design DNA microarray data on 89 MSI and 140 MSS CRCs from this study, and 58 MSI and 77 MSS cases from three published reports were randomly divided into test and training sets. MSI-associated gene expression changes were assessed for cross-study consistency using training samples, and validated as MSI classifier using test samples. Differences in biological pathways were identified by functional category analysis. Causation of differential gene expression was investigated by comparison to DNA copy-number data. Results MSI-associated gene expression changes in CRCs were found to be highly consistent across multiple studies of primary tumors and cancer cell lines from patients of different ethnicities (P<0.001). Clustering based on consistent changes separated additional test cases by MSI status, and classification of individual samples predicted MSI status with a sensitivity of 96% and specificity of 85%. Genes associated with immune response were up-regulated in MSI cancers, whereas genes associated with cell-cell adhesion, ion-binding and regulation of metabolism were down-regulated. Differential gene expression was shown to reflect systematic differences in DNA copy-number aberrations between MSI and MSS tumors (P<0.001). Conclusions Our results demonstrate cross-study consistency of MSI-associated gene expression changes in CRCs. DNA copy-number alterations partly cause the differences in gene expression between MSI and MSS cancers. PMID:19088021

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. 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.

  6. 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

  7. 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

  8. Identification of single copy nuclear DNA markers for North American pit vipers.

    PubMed

    Gibbs, H Lisle; Diaz, Jose

    2010-01-01

    We describe 18 single copy nuclear DNA loci (10 loci cloned from a Sistrurus catenatus catenatus genomic library and eight intron-based loci amplified using conserved primers) that detect sequence variation in species from all genera (Sistrurus, Agkistrodon and Crotalus) of North American pit viper snakes. These loci (mean size in bp ± SE: 433 ± 51) show large numbers of phylogenetically informative sites across species (mean + SE: 10.2 ± 1.5), but limited variation within subspecies suggesting that they will be most useful for multilocus species-level phylogenetic analyses in these snakes.

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. 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

  14. 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.

  15. 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

  16. 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

  17. 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.

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. Integrated Sensing Using DNA Nanoarchitectures

    DTIC Science & Technology

    2014-05-20

    characterization of vibrational modes in artificially designed DNA monocrystal, Chemical Physics , (11 2013): 121. doi: 10.1016/j.chemphys.2013.08.015 Xiaoning...M. Rahman, M. L. Norton. Observation of terahertz absorption signatures in microliter DNA solutions, Applied Physics Letters, (01 2013): 23701...Modification of surface bound DNA nanoarchitectures with biomolecules and conjugates," ACS, Indianapolis, IN. (September 8, 2013). Rahman, M. (Author

  17. 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

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. Mitochondrial DNA copy number and future risk of B-cell lymphoma in a nested case-control study in the prospective EPIC cohort

    PubMed Central

    Hosnijeh, Fatemeh Saberi; Lan, Qing; Rothman, Nathaniel; San Liu, Chin; Cheng, Wen-Ling; Nieters, Alexandra; Guldberg, Per; Tjønneland, Anne; Campa, Daniele; Martino, Alessandro; Boeing, Heiner; Trichopoulou, Antonia; Lagiou, Pagona; Trichopoulos, Dimitrios; Krogh, Vittorio; Tumino, Rosario; Panico, Salvatore; Masala, Giovanna; Weiderpass, Elisabete; Huerta Castaño, José María; Ardanaz, Eva; Sala, Núria; Dorronsoro, Miren; Quirós, J. Ramón; Sánchez, María-José; Melin, Beatrice; Johansson, Ann Sofie; Malm, Johan; Borgquist, Signe; Peeters, Petra H.; Bueno-de-Mesquita, H. Bas; Wareham, Nick; Khaw, Kay-Tee; Travis, Ruth C.; Brennan, Paul; Siddiq, Afshan; Riboli, Elio; Vineis, Paolo

    2014-01-01

    It has been suggested that mitochondrial dysfunction and DNA damage are involved in lymphomagenesis. Increased copy number of mitochondrial DNA (mtDNA) as a compensatory mechanism of mitochondrial dysfunction previously has been associated with B-cell lymphomas, in particular chronic lymphocytic leukemia (CLL). However, current evidence is limited and based on a relatively small number of cases. Using a nested case-control study, we extended these findings with a focus on subtype-specific analyses. Relative mtDNA copy number was measured in the buffy coat of prospectively collected blood of 469 lymphoma cases and 469 matched controls. The association between mtDNA copy number and the risk of developing lymphoma and histologic subtypes was examined using logistic regression models. We found no overall association between mtDNA and risk of lymphoma. Subtype analyses revealed significant increased risks of CLL (n = 102) with increasing mtDNA copy number (odds ratio = 1.34, 1.44, and 1.80 for quartiles 2-4, respectively; P trend = .001). mtDNA copy number was not associated with follow-up time, suggesting that this observation is not strongly influenced by indolent disease status. This study substantially strengthens the evidence that mtDNA copy number is related to risk of CLL and supports the importance of mitochondrial dysfunction as a possible mechanistic pathway in CLL ontogenesis. PMID:24899624

  6. 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.

  7. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  9. 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.

  10. 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

  11. 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

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

    PubMed Central

    Hu, Liwen; Yao, Xinyue; Shen, Yi

    2016-01-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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.

  1. 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

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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.

  16. 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.

  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. 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

  2. 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

  3. [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.

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. [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.

  20. 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

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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)

  18. 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

  19. 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

  20. 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

  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. Integration of amplified differential gene expression (ADGE) and DNA microarray.

    PubMed

    Chen, Zhijian J; Gaté, Laurent; Davis, Warren; Ile, Kristina E; Tew, Kenneth D

    2002-12-01

    Amplified Differential Gene Expression (ADGE) provides a new concept that the ratios of differentially expressed genes are magnified before detection in order to improve both sensitivity and accuracy. This technology is now implemented with integration of DNA reassociation and PCR. The ADGE technique can be used either as a stand-alone method or in series with DNA microarray. ADGE is used in sample preprocessing and DNA microarray is used as a displaying system in the series combination. These two techniques are mutually synergistic: the quadratic magnification of ratios of differential gene expression achieved by ADGE improves the detection sensitivity and accuracy; the PCR amplification of templates enhances the signal intensity and reduces the requirement for large amounts of starting material; the high throughput for DNA microarray is maintained.

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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.

  14. 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.

  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. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. 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.

  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. 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.

  6. 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.

  7. 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

  8. 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

  9. DNA microarray technology. Introduction.

    PubMed

    Pollack, Jonathan R

    2009-01-01

    DNA microarray technology has revolutionized biological research by enabling genome-scale explorations. This chapter provides an overview of DNA microarray technology and its application to characterizing the physical genome, with a focus on cancer genomes. Specific areas discussed include investigations of DNA copy number alteration (and loss of heterozygosity), DNA methylation, DNA-protein (i.e., chromatin and transcription factor) interactions, DNA replication, and the integration of diverse genome-scale data types. Also provided is a perspective on recent advances and future directions in characterizing the physical genome.

  10. 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.

  11. 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

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. 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.

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

    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.

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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…

  20. 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

  1. 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.

  2. 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.

  3. 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

  4. 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

  5. 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.

  6. 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

  7. 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.

  8. Synthesis, integration, and restriction and modification of mycoplasma virus L2 DNA

    SciTech Connect

    Dybvig, K.

    1981-01-01

    Mycoplasma virus L2 is an enveloped, nonlytic virus containing double-stranded, superhelical DNA. The L2 virion contains about 7 to 8 major proteins identified by SDS-polyacrylamide gel electrophoresis, but the virion has no discernible capsid structure. It has been suggested that the L2 virion is a DNA-protein condensation surrounded by a lipid-protein membrane. The host for mycoplasma virus L2 is Acholeplasma laidlawii. A. laidlawii has no cell wall and contains a small genome, 1 x 10/sup 9/ daltons, which is two to three times smaller than that of most bacteria. Infection of A. laidlawii by L2 is nonlytic. The studies in this thesis show that L2 DNA synthesis begins at about 1 hour of infection and lasts throughout the infection. Viral DNA synthesis is inhibited by chloramphenicol, streptomycin, and novobiocin. Packaging of L2 DNA into progeny virus is also inhibited by chloramphenicol and novobiocin. It is concluded that protein synthesis and probably DNA gyrase activity are required for L2 DNA synthesis, and for packaging of L2 DNA into progeny virus. DNA-DNA hybridization studies demonstrate that L2 DNA integrates into the host cell during infection, and subsequent to infection the cells are mycoplasma virus L2 lysogens. The viral site of integration has been roughly mapped. L2 virus is restricted and modified by A. laidlawii strains JA1 and K2. The nature of the modification in strain K2 has been elucidated. Two L2 variants containing insertions in the viral DNA were identified in these studies. Restriction endonuclease cleavage maps of these variants have been determined. DNA from L2 and another isolate of L2, MV-Lg-L 172, are compared in these studies. 74 references, 33 figures, 6 tables. (ACR)

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

    SciTech Connect

    Brosey, Chris A; Yan, Chunli; Tsutakawa, Susan E; Heller, William T; Rambo, Robert P; Tainer, John A; Ivanov, Ivaylo; Chazin, Walter J

    2013-01-01

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

  10. Integration of rapid DNA hybridization and capillary zone electrophoresis using bidirectional isotachophoresis.

    PubMed

    Bahga, Supreet S; Han, Crystal M; Santiago, Juan G

    2013-01-07

    We present a method for rapid, sequence-specific detection of multiple DNA fragments by integrating isotachophoresis (ITP) based DNA hybridization and capillary zone electrophoresis (CZE) using bidirectional ITP. Our method leverages the high preconcentration ability of ITP to accelerate slow, second-order DNA hybridization kinetics, and the high resolving power of CZE to separate and identify reaction products. We demonstrate the speed and sensitivity of our assay by detecting 5 pM, 39 nt ssDNA target within 3 min, using a molecular beacon probe. We also demonstrate the feasibility of our assay for multiplexed detection of multiple-length ssDNA targets by simultaneously detecting 39 and 90 nt ssDNA targets.

  11. SMARCAL1 maintains telomere integrity during DNA replication.

    PubMed

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

    2015-12-01

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

  12. Association of mitochondrial antioxidant enzymes with mitochondrial DNA as integral nucleoid constituents

    PubMed Central

    Kienhöfer, Joachim; Häussler, Dagmar Johanna Franziska; Ruckelshausen, Florian; Muessig, Elisabeth; Weber, Klaus; Pimentel, David; Ullrich, Volker; Bürkle, Alexander; Bachschmid, Markus Michael

    2009-01-01

    Mitochondrial DNA (mtDNA) is organized in protein-DNA macrocomplexes called nucleoids. Average nucleoids contain 2–8 mtDNA molecules, which are organized by the histone-like mitochondrial transcription factor A. Besides well-characterized constituents, such as single-stranded binding protein or polymerase γ (Polγ), various other proteins with ill-defined functions have been identified. We report for the first time that mammalian nucleoids contain essential enzymes of an integral antioxidant system. Intact nucleoids were isolated with sucrose density gradients from rat and bovine heart as well as human Jurkat cells. Manganese superoxide dismutase (SOD2) was detected by Western blot in the nucleoid fractions. DNA, mitochondrial glutathione peroxidase (GPx1), and Polγ were coimmunoprecipitated with SOD2 from nucleoid fractions, which suggests that an antioxidant system composed of SOD2 and GPx1 are integral constituents of nucleoids. Interestingly, in cultured bovine endothelial cells the association of SOD2 with mtDNA was absent. Using a sandwich filter-binding assay, direct association of SOD2 by salt-sensitive ionic forces with a chemically synthesized mtDNA fragment was demonstrated. Increasing salt concentrations during nucleoid isolation on sucrose density gradients disrupted the association of SOD2 with mitochondrial nucleoids. Our biochemical data reveal that nucleoids contain an integral antioxidant system that may protect mtDNA from superoxide-induced oxidative damage.—Kienhöfer, J., Häussler, D. J. F., Ruckelshausen, F., Muessig, E., Weber, K., Pimentel, D., Ullrich, V., Bürkle, A., Bachschmid, M. M. Association of mitochondrial antioxidant enzymes with mitochondrial DNA as integral nucleoid constituents. PMID:19228881

  13. Pegylated Interferon Alfa-2a Monotherapy Results in Suppression of HIV Type 1 Replication and Decreased Cell-Associated HIV DNA Integration

    PubMed Central

    Azzoni, Livio; Foulkes, Andrea S.; Papasavvas, Emmanouil; Mexas, Angela M.; Lynn, Kenneth M.; Mounzer, Karam; Tebas, Pablo; Jacobson, Jeffrey M.; Frank, Ian; Busch, Michael P.; Deeks, Steven G.; Carrington, Mary; O'Doherty, Una; Kostman, Jay; Montaner, Luis J.

    2013-01-01

    Background. Antiretroviral therapy (ART)–mediated immune reconstitution fails to restore the capacity of the immune system to spontaneously control human immunodeficiency virus (HIV) replication. Methods. A total of 23 HIV type 1 (HIV-1)–infected, virologically suppressed subjects receiving ART (CD4+ T-cell count, >450 cells/μL) were randomly assigned to have 180 μg/week (for arm A) or 90 μg/week (for arm B) of pegylated (Peg) interferon alfa-2a added to their current ART regimen. After 5 weeks, ART was interrupted, and Peg–interferon alfa-2a was continued for up to 12 weeks (the primary end point), with an option to continue to 24 weeks. End points included virologic failure (viral load, ≥400 copies/mL) and adverse events. Residual viral load and HIV-1 DNA integration were also assessed. Results. At week 12 of Peg–interferon alfa-2a monotherapy, viral suppression was observed in 9 of 20 subjects (45%), a significantly greater proportion than expected (arm A, P = .0088; arm B, P = .0010; combined arms, P < .0001). Over 24 weeks, both arms had lower proportions of subjects who had viral load, compared with the proportion of subjects in a historical control group (arm A, P = .0046; arm B, P = .0011). Subjects who had a sustained viral load of <400 copies/mL had decreased levels of integrated HIV DNA (P = .0313) but increased residual viral loads (P = .0078), compared with subjects who experienced end-point failure. Conclusions. Peg–interferon alfa-2a immunotherapy resulted in control of HIV replication and decreased HIV-1 integration, supporting a role for immunomediated approaches in HIV suppression and/or eradication. Clinical Trials Registration. NCT00594880. PMID:23105144

  14. Detection of Prostate Cancer Progression by Serum DNA Integrity

    DTIC Science & Technology

    2009-04-01

    specimens from patients with PCa. A recent report demonstrated 0%– 18% rates of LOH for various chromosomal loci in plasma DNA; 44% of specimens tested...2004;351: 1802 –3. 4. Bock JL, Klee GG. How sensitive is a prostate- specific antigen measurement? How sensitive does it need to be? Arch Pathol Lab Med...in blood and bone marrow plasma from patients with prostate cancer. Int J Cancer 2007;120:1465–71. 28. Liu L, Yoon JH, Dammann R, Pfeifer GP. Frequent

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

    PubMed

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

    2017-01-25

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

  16. Copy-left and Copy-right

    NASA Astrophysics Data System (ADS)

    VanderPlas, Jacob

    2015-01-01

    Any discussion of open licensing almost invariably devolves into a debate between copy-left licenses and permissive licenses, both sides defending their views with a nearly religious fervor. Copy-left licenses, typified by the GPL family of licenses, require all derived products to maintain the open, GPL license. Permissive licenses, typified by the BSD family of licenses, do not impose such requirements. I'll briefly explore the common arguments put forth in favor of either approach, and discuss some concrete examples of where these approaches have helped or hindered the software packages that used them.

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

    PubMed

    De, Ananya; Campbell, Colin

    2007-02-15

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

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

    PubMed

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

    2015-05-05

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

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

    NASA Technical Reports Server (NTRS)

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

    2000-01-01

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

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

    PubMed

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

    2017-02-01

    Life dispersal between planets, planetary protection, and the search for biosignatures are main topics in astrobiology. Under the umbrella of the STARLIFE project, three Antarctic endolithic microorganisms, the melanized fungus Cryomyces antarcticus CCFEE 515, a hyaline strain of Umbilicaria sp. (CCFEE 6113, lichenized fungus), and a Stichococcus sp. strain (C45A, green alga), were exposed to high doses of space-relevant gamma radiation ((60)Co), up to 117.07 kGy. After irradiation survival, DNA integrity and ultrastructural damage were tested. The first was assessed by clonogenic test; viability and dose responses were reasonably described by the linear-quadratic formalism. DNA integrity was evaluated by PCR, and ultrastructural damage was observed by transmission electron microscopy. The most resistant among the tested organisms was C. antarcticus both in terms of colony formation and DNA preservation. Besides, results clearly demonstrate that DNA was well detectable in all the tested organisms even when microorganisms were dead. This high resistance provides support for the use of DNA as a possible biosignature during the next exploration campaigns. Implication in planetary protection and contamination during long-term space travel are put forward. Key Words: Biosignatures-Ionizing radiation-DNA integrity-Eukaryotic microorganisms-Fingerprinting-Mars exploration. Astrobiology 17, 126-135.

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

    PubMed

    Goldstein, Paul Z; DeSalle, Rob

    2011-02-01

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

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

    PubMed

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

    2015-12-01

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

  3. An Integrated Encyclopedia of DNA Elements in the Human Genome

    PubMed Central

    2012-01-01

    Summary The human genome encodes the blueprint of life, but the function of the vast majority of its nearly three billion bases is unknown. The Encyclopedia of DNA Elements (ENCODE) project has systematically mapped regions of transcription, transcription factor association, chromatin structure, and histone modification. These data enabled us to assign biochemical functions for 80% of the genome, in particular outside of the well-studied protein-coding regions. Many discovered candidate regulatory elements are physically associated with one another and with expressed genes, providing new insights into the mechanisms of gene regulation. The newly identified elements also show a statistical correspondence to sequence variants linked to human disease, and can thereby guide interpretation of this variation. Overall the project provides new insights into the organization and regulation of our genes and genome, and an expansive resource of functional annotations for biomedical research. PMID:22955616

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

    PubMed

    2012-09-06

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

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

    PubMed

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

    2017-01-01

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

  6. Hyperglycemia Differentially Affects Maternal and Fetal DNA Integrity and DNA Damage Response

    PubMed Central

    Moreli, Jusciele B.; Santos, Janine H.; Lorenzon-Ojea, Aline Rodrigues; Corrêa-Silva, Simone; Fortunato, Rodrigo S.; Rocha, Clarissa Ribeiro; Rudge, Marilza V.; Damasceno, Débora C.; Bevilacqua, Estela; Calderon, Iracema M.

    2016-01-01

    Objective: Investigate the DNA damage and its cellular response in blood samples from both mother and the umbilical cord of pregnancies complicated by hyperglycemia. Methods: A total of 144 subjects were divided into 4 groups: normoglycemia (ND; 46 cases), mild gestational hyperglycemia (MGH; 30 cases), gestational diabetes mellitus (GDM; 45 cases) and type-2 diabetes mellitus (DM2; 23 cases). Peripheral blood mononuclear cell (PBMC) isolation and/or leukocytes from whole maternal and umbilical cord blood were obtained from all groups at delivery. Nuclear and mitochondrial DNA damage were measured by gene-specific quantitative PCR, and the expression of mRNA and proteins involved in the base excision repair (BER) pathway were assessed by real-time qPCR and Western blot, respectively. Apoptosis was measured in vitro experiments by caspase 3/7 activity and ATP levels. Results: GDM and DM2 groups were characterized by an increase in oxidative stress biomarkers, an increase in nuclear and mitochondrial DNA damage, and decreased expression of mRNA (APE1, POLβ and FEN1) and proteins (hOGG1, APE1) involved in BER. The levels of hyperglycemia were associated with the in vitro apoptosis pathway. Blood levels of DNA damage in umbilical cord were similar among the groups. Newborns of diabetic mothers had increased expression of BER mRNA (APE1, POLβ and FEN1) and proteins (hOGG1, APE1, POLβ and FEN1). A diabetes-like environment was unable to induce apoptosis in the umbilical cord blood cells. Conclusions: Our data show relevant asymmetry between maternal and fetal blood cell susceptibility to DNA damage and apoptosis induction. Maternal cells seem to be more predisposed to changes in an adverse glucose environment. This may be due to differential ability in upregulating multiple genes involved in the activation of DNA repair response, especially the BER mechanism. However if this study shows a more effective adaptive response by the fetal organism, it also calls for

  7. Integrated Taxonomy and DNA Barcoding of Alpine Midges (Diptera: Chironomidae)

    PubMed Central

    Montagna, Matteo; Mereghetti, Valeria; Lencioni, Valeria; Rossaro, Bruno

    2016-01-01

    Rapid and efficient DNA-based tools are recommended for the evaluation of the insect biodiversity of high-altitude streams. In the present study, focused principally on larvae of the genus Diamesa Meigen 1835 (Diptera: Chironomidae), the congruence between morphological/molecular delimitation of species as well as performances in taxonomic assignments were evaluated. A fragment of the mitochondrial cox1 gene was obtained from 112 larvae, pupae and adults (Diamesinae, Orthocladiinae and Tanypodinae) that were collected in different mountain regions of the Alps and Apennines. On the basis of morphological characters 102 specimens were attributed to 16 species, and the remaining ten specimens were identified to the genus level. Molecular species delimitation was performed using: i) distance-based Automatic Barcode Gap Discovery (ABGD), with no a priori assumptions on species identification; and ii) coalescent tree-based approaches as the Generalized Mixed Yule Coalescent model, its Bayesian implementation and Bayesian Poisson Tree Processes. The ABGD analysis, estimating an optimal intra/interspecific nucleotide distance threshold of 0.7%-1.4%, identified 23 putative species; the tree-based approaches, identified between 25–26 entities, provided nearly identical results. All species belonging to zernyi, steinboecki, latitarsis, bertrami, dampfi and incallida groups, as well as outgroup species, are recovered as separate entities, perfectly matching the identified morphospecies. In contrast, within the cinerella group, cases of discrepancy arose: i) the two morphologically separate species D. cinerella and D. tonsa are neither monophyletic nor diagnosable exhibiting low values of between-taxa nucleotide mean divergence (0.94%); ii) few cases of larvae morphological misidentification were observed. Head capsule color is confirmed to be a valid character able to discriminate larvae of D. zernyi, D. tonsa and D. cinerella, but it is here better defined as a color

  8. The Agrobacterium tumefaciens virulence D2 protein is responsible for precise integration of T-DNA into the plant genome.

    PubMed Central

    Tinland, B; Schoumacher, F; Gloeckler, V; Bravo-Angel, A M; Hohn, B

    1995-01-01

    The VirD2 protein of Agrobacterium tumefaciens was shown to pilot T-DNA during its transfer to the plant cell nucleus. We analyze here its participation in the integration of T-DNA by using a virD2 mutant. This mutation reduces the efficiency of T-DNA transfer, but the efficiency of integration of T-DNA per se is unaffected. Southern and sequence analyses of integration events obtained with the mutated VirD2 protein revealed an aberrant pattern of integration. These results indicate that the wild-type VirD2 protein participates in ligation of the 5'-end of the T-strand to plant DNA and that this ligation step is not rate limiting for T-DNA integration. Images PMID:7628458

  9. Integrated digital error suppression for improved detection of circulating tumor DNA

    PubMed Central

    Kurtz, David M.; Chabon, Jacob J.; Scherer, Florian; Stehr, Henning; Liu, Chih Long; Bratman, Scott V.; Say, Carmen; Zhou, Li; Carter, Justin N.; West, Robert B.; Sledge, George W.; Shrager, Joseph B.; Loo, Billy W.; Neal, Joel W.; Wakelee, Heather A.; Diehn, Maximilian; Alizadeh, Ash A.

    2016-01-01

    High-throughput sequencing of circulating tumor DNA (ctDNA) promises to facilitate personalized cancer therapy. However, low quantities of cell-free DNA (cfDNA) in the blood and sequencing artifacts currently limit analytical sensitivity. To overcome these limitations, we introduce an approach for integrated digital error suppression (iDES). Our method combines in silico elimination of highly stereotypical background artifacts with a molecular barcoding strategy for the efficient recovery of cfDNA molecules. Individually, these two methods each improve the sensitivity of cancer personalized profiling by deep sequencing (CAPP-Seq) by ~3 fold, and synergize when combined to yield ~15-fold improvements. As a result, iDES-enhanced CAPP-Seq facilitates noninvasive variant detection across hundreds of kilobases. Applied to clinical non-small cell lung cancer (NSCLC) samples, our method enabled biopsy-free profiling of EGFR kinase domain mutations with 92% sensitivity and 96% specificity and detection of ctDNA down to 4 in 105 cfDNA molecules. We anticipate that iDES will aid the noninvasive genotyping and detection of ctDNA in research and clinical settings. PMID:27018799

  10. Differences in aggressive behavior and DNA copy number variants between BALB/cJ and BALB/cByJ substrains.

    PubMed

    Velez, Lady; Sokoloff, Greta; Miczek, Klaus A; Palmer, Abraham A; Dulawa, Stephanie C

    2010-03-01

    Some BALB/c substrains exhibit different levels of aggression. We compared aggression levels between male BALB/cJ and BALB/cByJ substrains using the resident intruder paradigm. These substrains were also assessed in other tests of emotionality and information processing including the open field, forced swim, fear conditioning, and prepulse inhibition tests. We also evaluated single nucleotide polymorphisms (SNPs) previously reported between these BALB/c substrains. Finally, we compared BALB/cJ and BALB/cByJ mice for genomic deletions or duplications, collectively termed copy number variants (CNVs), to identify candidate genes that might underlie the observed behavioral differences. BALB/cJ mice showed substantially higher aggression levels than BALB/cByJ mice; however, only minor differences in other behaviors were observed. None of the previously reported SNPs were verified. Eleven CNV regions were identified between the two BALB/c substrains. Our findings identify a robust difference in aggressive behavior between BALB/cJ and BALB/cByJ substrains, which could be the result of the identified CNVs.

  11. Kinetic versus Energetic Discrimination in Biological Copying

    NASA Astrophysics Data System (ADS)

    Sartori, Pablo; Pigolotti, Simone

    2013-05-01

    We study stochastic copying schemes in which discrimination between a right and a wrong match is achieved via different kinetic barriers or different binding energies of the two matches. We demonstrate that, in single-step reactions, the two discrimination mechanisms are strictly alternative and cannot be mixed to further reduce the error fraction. Close to the lowest error limit, kinetic discrimination results in a diverging copying velocity and dissipation per copied bit. On the other hand, energetic discrimination reaches its lowest error limit in an adiabatic regime where dissipation and velocity vanish. By analyzing experimentally measured kinetic rates of two DNA polymerases, T7 and Polγ, we argue that one of them operates in the kinetic and the other in the energetic regime. Finally, we show how the two mechanisms can be combined in copying schemes implementing error correction through a proofreading pathway.

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

    PubMed

    Wild, Jadwiga; Hradecna, Zdenka; Szybalski, Waclaw

    2002-09-01

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

  13. Comparison of DNA binding and integration half-site selection by avian myeloblastosis virus integrase.

    PubMed Central

    Grandgenett, D P; Inman, R B; Vora, A C; Fitzgerald, M L

    1993-01-01

    Insertion of the linear retrovirus DNA genome into the host DNA by the virus-encoded integrase (IN) is essential for efficient replication. We devised an efficient virus-like DNA plasmid integration assay which mimics the standard oligonucleotide assay for integration. It permitted us to study, by electron microscopy and sequence analysis, insertion of a single long terminal repeat terminus (LTR half-site) of one plasmid into another linearized plasmid. The reaction was catalyzed by purified avian myeloblastosis virus IN in the presence of Mg2+. The recombinant molecules were easily visualized and quantitated by agarose gel electrophoresis. Agarose gel-purified recombinants could be genetically selected by transformation of ligated recombinants into Escherichia coli HB101 cells. Electron microscopy also permitted the identification and localization of IN-DNA complexes on the virus-like substrate in the absence of the joining reaction. Intramolecular and intermolecular DNA looping by IN was visualized. Although IN preferentially bound to AT-rich regions in the absence of the joining reaction, there was a bias towards GC-rich regions for the joining reaction. Alignment of 70 target site sequences 5' of the LTR half-site insertions with 68 target sites previously identified for the concerted insertion of both LTR termini (LTR full-site reaction) indicated similar GC inflection patterns with both insertional events. Comparison of the data suggested that IN recognized only half of the target sequences necessary for integration with the LTR half-site reaction. Images PMID:8474165

  14. DNA damage responses by human ELG1 in S phase are important to maintain genomic integrity.

    PubMed

    Sikdar, Nilabja; Banerjee, Soma; Lee, Kyoo-young; Wincovitch, Stephen; Pak, Evgenia; Nakanishi, Koji; Jasin, Maria; Dutra, Amalia; Myung, Kyungjae

    2009-10-01

    Genomic integrity depends on DNA replication, recombination and repair, particularly in S phase. We demonstrate that a human homologue of yeast Elg1 plays an important role in S phase to preserve genomic stability. The level of ELG1 is induced during recovery from a variety of DNA damage. In response to DNA damage, ELG1 forms distinct foci at stalled DNA replication forks that are different from DNA double strand break foci. Targeted gene knockdown of ELG1 resulted in spontaneous foci formation of gamma-H2AX, 53BP1 and phosphorylated-ATM that mark chromosomal breaks. Abnormal chromosomes including fusions, inversions and hypersensitivity to DNA damaging agents were also observed in cells expressing low level of ELG1 by targeted gene knockdown. Knockdown of ELG1 by siRNA reduced homologous recombination frequency in the I-SceI induced double strand break-dependent assay. In contrast, spontaneous homologous recombination frequency and sister chromatin exchange rate were upregulated when ELG1 was silenced by shRNA. Taken together, we propose that ELG1 would be a new member of proteins involved in maintenance of genomic integrity.

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

    PubMed

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

    2010-03-01

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

  16. Integrating DNA strand displacement circuitry to the nonlinear hybridization chain reaction.

    PubMed

    Zhang, Zhuo; Fan, Tsz Wing; Hsing, I-Ming

    2017-02-23

    Programmable and modular attributes of DNA molecules allow one to develop versatile sensing platforms that can be operated isothermally and enzyme-free. In this work, we present an approach to integrate upstream DNA strand displacement circuits that can be turned on by a sequence-specific microRNA analyte with a downstream nonlinear hybridization chain reaction for a cascading hyperbranched nucleic acid assembly. This system provides a two-step amplification strategy for highly sensitive detection of the miRNA analyte, conducive for multiplexed detection. Multiple miRNA analytes were tested with our integrated circuitry using the same downstream signal amplification setting, showing the decoupling of nonlinear self-assembly with the analyte sequence. Compared with the reported methods, our signal amplification approach provides an additional control module for higher-order DNA self-assembly and could be developed into a promising platform for the detection of critical nucleic-acid based biomarkers.

  17. Mechanisms of change in gene copy number.

    PubMed

    Hastings, P J; Lupski, James R; Rosenberg, Susan M; Ira, Grzegorz

    2009-08-01

    Deletions and duplications of chromosomal segments (copy number variants, CNVs) are a major source of variation between individual humans and are an underlying factor in human evolution and in many diseases, including mental illness, developmental disorders and cancer. CNVs form at a faster rate than other types of mutation, and seem to do so by similar mechanisms in bacteria, yeast and humans. Here we review current models of the mechanisms that cause copy number variation. Non-homologous end-joining mechanisms are well known, but recent models focus on perturbation of DNA replication and replication of non-contiguous DNA segments. For example, cellular stress might induce repair of broken replication forks to switch from high-fidelity homologous recombination to non-homologous repair, thus promoting copy number change.

  18. Real-Time Tracking of Parental Histones Reveals Their Contribution to Chromatin Integrity Following DNA Damage.

    PubMed

    Adam, Salomé; Dabin, Juliette; Chevallier, Odile; Leroy, Olivier; Baldeyron, Céline; Corpet, Armelle; Lomonte, Patrick; Renaud, Olivier; Almouzni, Geneviève; Polo, Sophie E

    2016-10-06

    Chromatin integrity is critical for cell function and identity but is challenged by DNA damage. To understand how chromatin architecture and the information that it conveys are preserved or altered following genotoxic stress, we established a system for real-time tracking of parental histones, which characterize the pre-damage chromatin state. Focusing on histone H3 dynamics after local UVC irradiation in human cells, we demonstrate that parental histones rapidly redistribute around damaged regions by a dual mechanism combining chromatin opening and histone mobilization on chromatin. Importantly, parental histones almost entirely recover and mix with new histones in repairing chromatin. Our data further define a close coordination of parental histone dynamics with DNA repair progression through the damage sensor DDB2 (DNA damage-binding protein 2). We speculate that this mechanism may contribute to maintaining a memory of the original chromatin landscape and may help preserve epigenome stability in response to DNA damage.

  19. Supercoiled plasmid DNA purification by integrating membrane technology with a monolithic chromatography.

    PubMed

    Nunes, Catherine; Sousa, Angela; Nunes, José C; Morão, António M; Sousa, Fani; Queiroz, João A

    2014-06-01

    The present study describes the integration of membrane technology with monolithic chromatography to obtain plasmid DNA with high quality. Isolation and clarification of plasmid DNA lysate were first conducted by a microfiltration step, by using a hydrophilic nylon microfiltration membrane, avoiding the need of centrifugation. For the total elimination of the remaining impurities, a suitable purification step is required. Monolithic stationary phases have been successfully applied as an alternative to conventional supports. Thus, the sample recovered from the membrane process was applied into a nongrafted CarbonylDiImidazole disk. Throughout the global procedure, a reduced level of impurities such as proteins and RNA was obtained, and no genomic DNA was detectable in the plasmid DNA sample. The chromatographic process demonstrated an efficient performance on supercoiled plasmid DNA purity and recovery (100 and 84.44%, respectively). Thereby, combining the membrane technology to eliminate some impurities from lysate sample with an efficient chromatographic strategy to purify the supercoiled plasmid DNA arises as a powerful approach for industrial-scale systems aiming at plasmid DNA purification.

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

    PubMed

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

    2015-01-01

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

  1. Targeted DNA damage at individual telomeres disrupts their integrity and triggers cell death

    PubMed Central

    Sun, Luxi; Tan, Rong; Xu, Jianquan; LaFace, Justin; Gao, Ying; Xiao, Yanchun; Attar, Myriam; Neumann, Carola; Li, Guo-Min; Su, Bing; Liu, Yang; Nakajima, Satoshi; Levine, Arthur S.; Lan, Li

    2015-01-01

    Cellular DNA is organized into chromosomes and capped by a unique nucleoprotein structure, the telomere. Both oxidative stress and telomere shortening/dysfunction cause aging-related degenerative pathologies and increase cancer risk. However, a direct connection between oxidative damage to telomeric DNA, comprising <1% of the genome, and telomere dysfunction has not been established. By fusing the KillerRed chromophore with the telomere repeat binding factor 1, TRF1, we developed a novel approach to generate localized damage to telomere DNA and to monitor the real time damage response at the single telomere level. We found that DNA damage at long telomeres in U2OS cells is not repaired efficiently compared to DNA damage in non-telomeric regions of the same length in heterochromatin. Telomeric DNA damage shortens the average length of telomeres and leads to cell senescence in HeLa cells and cell death in HeLa, U2OS and IMR90 cells, when DNA damage at non-telomeric regions is undetectable. Telomere-specific damage induces chromosomal aberrations, including chromatid telomere loss and telomere associations, distinct from the damage induced by ionizing irradiation. Taken together, our results demonstrate that oxidative damage induces telomere dysfunction and underline the importance of maintaining telomere integrity upon oxidative damage. PMID:26082495

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

    PubMed

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

    2010-10-01

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

  3. A case of bilateral human herpes virus 6 panuveitis with genomic viral DNA integration

    PubMed Central

    2014-01-01

    Background We report a rare case of bilateral panuveitis from human herpes virus 6 (HHV-6) with genomic viral DNA integration in an immunocompromised man. Findings A 59-year-old man with history of multiple myeloma presented with altered mental status, bilateral eye redness, and blurry vision. Examination revealed bilateral diffuse keratic precipitates, 4+ anterior chamber cell, hypopyon, vitritis, and intraretinal hemorrhages. Intraocular fluid testing by polymerase chain reaction (PCR) was positive for HHV-6. The patient was successfully treated with intravitreal foscarnet and intravenous ganciclovir and foscarnet. Despite clinical improvement, his serum HHV-6 levels remained high, and it was concluded that he had HHV-6 chromosomal integration. Conclusions HHV-6 should be considered in the differential for infectious uveitis in immunocompromised hosts who may otherwise have a negative work-up. HHV-6 DNA integration may lead to difficulties in disease diagnosis and determining disease resolution. PMID:24995045

  4. Integration of human papillomavirus 18 DNA in esophageal carcinoma 109 cells

    PubMed Central

    Zhang, Ke; Li, Jin-Tao; Li, Shu-Ying; Zhu, Li-Hua; Zhou, Ling; Zeng, Yi

    2011-01-01

    AIM: To detect human papillomavirus (HPV) DNA in esophageal carcinoma (EC) 109 cells and investigate the relationship between HPV and EC. METHODS: Genomic DNA and total RNA from EC109 cells were isolated. HPV DNA was detected by polymerase chain reaction (PCR) with the general primer sets of My09/11 and GP5 +/6 + for the HPV L1 gene and type-specific primer sets for HPV18 E6 and HPV18 E6-E7. Reverse transcription (RT) of mRNA isolated from EC109 cells was performed to produce a cDNA. And then a PCR-based protocol for the amplification of papillomavirus oncogene transcripts was used to analyze HPV18 DNA and integrated transcripts of HPV18 in the chromosomes of EC109 cells. The final nested PCR products were cloned into a pMD-18T vector and sequenced to analyze the chromosomal location of HPV integration. RESULTS: HPV18 DNA was detected in EC109 cells by PCR using the general primer sets of My09/11 and GP5 +/6 + for HPV L1 and the type-specific primer sets for HPV18 E6 and E6-E7 to generate products of 450 bp, 150 bp, 335 bp and 944 bp, respectively. Approximately 600 bp of integrated HPV18-specific transcript was identified. The final nested PCR product of integrated HPV18 DNA was cloned into a pMD-18T vector and sequenced to analyze the chromosomal location of HPV integration. Sequence alignment showed that the HPV18 sequence from EC109 cells was identical to that of the encoded early protein E7-E1 of the standard HPV18 strain X05015, and another partial gene sequence was identical to a partial sequence of human chromosome 8. CONCLUSION: Integration of the HPV genome into the host cell chromosome suggests that persistent HPV infection is vital for malignant cell transformation and carcinogenesis. PMID:22072858

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

    PubMed

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

    2015-09-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2017-02-01

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

  7. Integrative analysis of DNA methylation and gene expression in butyrate-treated CHO cells.

    PubMed

    Wippermann, Anna; Rupp, Oliver; Brinkrolf, Karina; Hoffrogge, Raimund; Noll, Thomas

    2016-11-24

    The cellular mechanisms responsible for the versatile properties of CHO cells as the major production cell line for biopharmaceutical molecules are not entirely understood yet, although several 'omics' data facilitate the understanding of CHO cells and their reactions to environmental conditions. However, genome-wide studies of epigenetic processes such as DNA methylation are still limited. To prove the applicability and usefulness of integrating DNA methylation and gene expression data in a biotechnological context, we exemplarily analyzed the time course of cellular reactions upon butyrate addition in antibody-producing CHO cells by whole-genome bisulfite sequencing and CHO-specific cDNA microarrays. Gene expression and DNA methylation analyses showed that pathways known to be affected by butyrate, including cell cycle and apoptosis, as well as pathways potentially involved in butyrate-induced hyperproductivity such as central energy metabolism and protein biosynthesis were affected. Differentially methylated regions were furthermore found to contain binding-site motifs of specific transcription factors and were hypothesized to represent regulatory regions closely connected to the cellular response to butyrate. Generally, our experiment underlines the benefit of integrating DNA methylation and gene expression data, as it provided potential novel candidate genes for rational cell line development and allowed for new insights into the butyrate effect on CHO cells.

  8. Effects of Zidovudine Treatment on Heart mRNA Expression and Mitochondrial DNA Copy Number Associated with Alterations in Deoxynucleoside Triphosphate Composition in a Neonatal Rat Model.

    PubMed

    Snowdin, Jacob W; Hsiung, Chia-Heng; Kesterson, Daniel G; Kamath, Vasudeva G; McKee, Edward E

    2015-10-01

    The prevention of mother-to-child transmission (MTCT) of HIV is a crucial component in HIV therapy. Nucleoside reverse transcriptase inhibitors (NRTIs), primarily 3'-azido-3'-thymidine (AZT [zidovudine]), have been used to treat both mothers and neonates. While AZT is being replaced with less toxic drugs in treating mothers in MTCT prevention, it is still commonly used to treat neonates. Problems related to mitochondrial toxicity and potential mutagenesis associated with AZT treatment have been reported in treated cohorts. Yet little is known concerning the metabolism and potential toxicity of AZT on embryonic and neonatal tissues, especially considering that the enzymes of nucleoside metabolism change dramatically as many tissues convert from hyperplastic to hypertrophic growth during this period. AZT is known to inhibit thymidine phosphorylation and potentially alter deoxynucleoside triphosphate (dNTP) pools in adults. This study examines the effects of AZT on dNTP pools, mRNA expression of deoxynucleoside/deoxynucleotide metabolic enzymes, and mitochondrial DNA levels in a neonatal rat model. Results show that AZT treatment dramatically altered dNTP pools in the first 7 days of life after birth, which normalized to age-matched controls in the second and third weeks. Additionally, AZT treatment dramatically increased the mRNA levels of many enzymes involved in deoxynucleotide synthesis and mitochondrial biogenesis during the first week of life, which normalized to age-matched controls by the third week. These results were correlated with depletion of mitochondrial DNA noted in the second week. Taken together, results demonstrated that AZT treatment has a powerful effect on the deoxynucleotide synthesis pathways that may be associated with toxicity and mutagenesis.

  9. Sperm Chromatin Immaturity Observed in Short Abstinence Ejaculates Affects DNA Integrity and Longevity In Vitro

    PubMed Central

    Salian, Sujith Raj; Kumar, Dayanidhi; Singh, Vikram Jeet; D’Souza, Fiona; Kalthur, Guruprasad; Kamath, Asha; Adiga, Satish Kumar

    2016-01-01

    Background The influence of ejaculatory abstinence (EA) on semen parameters and subsequent reproductive outcome is still debatable; hence understanding the impact of EA on sperm structural and functional integrity may provide a valuable information on predicting successful clinical outcome. Objective To understand the influence of EA on sperm chromatin maturity, integrity, longevity and global methylation status. Methods This experimental prospective study included 76 ejaculates from 19 healthy volunteers who provided ejaculates after observing 1, 3, 5 and 7 days of abstinence. Sperm chromatin maturity, DNA integrity and global methylation status were assessed in the neat ejaculate. Sperm motility, DNA integrity and longevity were assessed in the processed fraction of the fresh and frozen-thawed ejaculates to determine their association with the length of EA. Results Spermatozoa from 1 day ejaculatory abstinence (EA-1) displayed significantly higher level of sperm chromatin immaturity in comparison to EA-3 (P < 0.05) and EA-5 (P < 0.01) whereas; the number of 5-methyl cytosine immunostained spermatozoa did not vary significantly across groups. On the other hand, in vitro incubation of processed ejaculate from EA-1 resulted in approximately 20 and 40 fold increase in the DNA fragmented spermatozoa at the end of 6 and 24h respectively (P < 0.01–0.001). Conclusion Use of short-term EA for therapeutic fertilization would be a clinically valuable strategy to improve the DNA quality. However, use of such spermatozoa after prolonged incubation in vitro should be avoided as it can carry a substantial risk of transmitting DNA fragmentation to the oocytes. PMID:27043437

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

    PubMed

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

    2015-01-01

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

  11. Integrated affinity capture, purification, and capillary electrophoresis microdevice for quantitative double-stranded DNA analysis.

    PubMed

    Toriello, Nicholas M; Liu, Chung N; Blazej, Robert G; Thaitrong, Numrin; Mathies, Richard A

    2007-11-15

    A novel injection method is developed that utilizes a thermally switchable oligonucleotide affinity capture gel to mediate the concentration, purification, and injection of dsDNA for quantitative microchip capillary electrophoresis analysis. The affinity capture matrix consists of a 20 base acrydite modified oligonucleotide copolymerized into a 6% linear polyacrylamide gel that captures ssDNA or dsDNA analyte including PCR amplicons and synthetic oligonucleotides. Double stranded PCR amplicons with complementarity to the capture probe up to 81 bases from their 5' terminus are reproducibly captured via helix invasion. By integrating the oligo capture matrix directly with the CE separation channel, the electrophoretically mobilized target fragments are quantitatively captured and injected after thermal release for unbiased, efficient, and quantitative analysis. The capture process exhibits optimal efficiency at 44 degrees C and 100 V/cm with a 20 microM affinity capture probe (TM = 57.7 degrees C). A dsDNA titration assay with 20 bp fragments validated that dsDNA is captured at the same efficiency as ssDNA. Dilution studies with a duplex 20mer show that targets can be successfully captured and analyzed with a limit of detection of 1 pM from 250 nL of solution (approximately 150,000 fluorescent molecules). Simultaneous capture and injection of amplicons from E. coli K12 and M13mp18 using a mixture of two different capture probes demonstrates the feasibility of multiplex target capture. Unlike the traditional cross-injector, this method enables efficient capture and injection of dsDNA amplicons which will facilitate the quantitative analysis of products from integrated nanoliter-scale PCR reactors.

  12. Staying in aerobic shape: how the structural integrity of mitochondria and mitochondrial DNA is maintained.

    PubMed

    Scott, Sidney V; Cassidy-Stone, Ann; Meeusen, Shelly L; Nunnari, Jodi

    2003-08-01

    The structure and integrity of the mitochondrial compartment are features essential for it to function efficiently. The maintenance of mitochondrial structure in cells ranging from yeast to humans has been shown to require both ongoing fission and fusion. Recent characterization of many of the molecular components that direct mitochondrial fission and fusion events have led to a more complete understanding of how these processes take place. Further, mitochondrial fragmentation observed when cells undergo apoptosis requires mitochondrial fission, underlying the importance of mitochondrial dynamics in cellular homeostasis. Mitochondrial structure also impacts mitochondrial DNA inheritance. Recent studies suggest that faithful transmission of mitochondrial DNA to daughter cells might require a mitochondrial membrane tethering apparatus.

  13. Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker

    PubMed Central

    Heap, John T.; Ehsaan, Muhammad; Cooksley, Clare M.; Ng, Yen-Kuan; Cartman, Stephen T.; Winzer, Klaus; Minton, Nigel P.

    2012-01-01

    Most bacteria can only be transformed with circular plasmids, so robust DNA integration methods for these rely upon selection of single-crossover clones followed by counter-selection of double-crossover clones. To overcome the limited availability of heterologous counter-selection markers, here we explore novel DNA integration strategies that do not employ them, and instead exploit (i) activation or inactivation of genes leading to a selectable phenotype, and (ii) asymmetrical regions of homology to control the order of recombination events. We focus here on the industrial biofuel-producing bacterium Clostridium acetobutylicum, which previously lacked robust integration tools, but the approach we have developed is broadly applicable. Large sequences can be delivered in a series of steps, as we demonstrate by inserting the chromosome of phage lambda (minus a region apparently unstable in Escherichia coli in our cloning context) into the chromosome of C. acetobutylicum in three steps. This work should open the way to reliable integration of DNA including large synthetic constructs in diverse microorganisms. PMID:22259038

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

    PubMed

    Fenech, Michael F

    2014-01-01

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

  15. Benefits of Selenium Supplementation on Leukocyte DNA Integrity Interact with Dietary Micronutrients: A Short Communication

    PubMed Central

    Karunasinghe, Nishi; Zhu, Shuotun; Ferguson, Lynnette R.

    2016-01-01

    A male cohort from New Zealand has previously shown variability in Selenium (Se) supplementation effects on measured biomarkers. The current analysis is to understand the reasons for variability of the H2O2-induced DNA damage recorded after Se supplementation. We have looked at the variation of demographic, lifestyle, medication, genetic and dietary factors and biomarkers measured at baseline and post-supplementation in these two extreme subgroups A and B. Group A showed increased H2O2-induced DNA damage and group B showed decreased damage after Se supplementation. We have also considered correlations of biomarkers and dietary factors in the complete dataset. The glutathione peroxidase (GPx) activity and DNA damage were significantly lower at post-supplementation in Group B compared to Group A. Post-supplementation, Group B showed a significant reduction in the GPx activity, while Group A showed a significant increase in DNA damage compared to baseline levels. Dietary methionine intake was significantly higher and folate intake was significantly lower in Group B compared to Group A. Se supplementation significantly increased the caspase-cleaved keratin 18 levels in both groups, indicating increased apoptotic potential of this supplement. Parameter correlation with the complete dataset showed dietary methionine to have a significant negative correlation with H2O2-induced DNA damage post-supplementation. The data suggest that Se supplementation is beneficial for the leukocyte DNA integrity only in interaction with the dietary methionine and folate intake. PMID:27128937

  16. Integrating DNA-strand-displacement circuitry with self-assembly of spherical nucleic acids.

    PubMed

    Yao, Dongbao; Song, Tingjie; Sun, Xianbao; Xiao, Shiyan; Huang, Fujian; Liang, Haojun

    2015-11-11

    Programmable and algorithmic behaviors of DNA molecules allow one to control the structures of DNA-assembled materials with nanometer precision and to construct complex networks with digital and analog behaviors. Here we developed a way of integrating a DNA-strand-displacement circuit with self-assembly of spherical nucleic acids, wherein a single DNA strand was used to initiate and catalyze the operation of upstream circuits to release a single strand that subsequently triggers self-assembly of spherical nucleic acids in downstream circuits, realizing a programmable kinetic control of self-assembly of spherical nucleic acids. Through utilizing this method, single-nucleotide polymorphisms or indels occurring at different positions of a sequence of oligonucleotide were unambiguously discriminated. We provide here a sophisticated way of combining the DNA-strand-displacement-based characteristic of DNA with the distinct assembly properties of inorganic nanoparticles, which may find broad potential applications in the fabrication of a wide range of complex multicomponent devices and architectures.

  17. The DNA Binding Domain of a Papillomavirus E2 Protein Programs a Chimeric Nuclease To Cleave Integrated Human Papillomavirus DNA in HeLa Cervical Carcinoma Cells▿

    PubMed Central

    Horner, Stacy M.; DiMaio, Daniel

    2007-01-01

    Viral DNA binding proteins that direct nucleases or other protein domains to viral DNA in lytically or latently infected cells may provide a novel approach to modulate viral gene expression or replication. Cervical carcinogenesis is initiated by high-risk human papillomavirus (HPV) infection, and viral DNA persists in the cancer cells. To test whether a DNA binding domain of a papillomavirus protein can direct a nuclease domain to cleave HPV DNA in cervical cancer cells, we fused the DNA binding domain of the bovine papillomavirus type 1 (BPV1) E2 protein to the catalytic domain of the FokI restriction endonuclease, generating a BPV1 E2-FokI chimeric nuclease (BEF). BEF introduced DNA double-strand breaks on both sides of an E2 binding site in vitro, whereas DNA binding or catalytic mutants of BEF did not. After expression of BEF in HeLa cervical carcinoma cells, we detected cleavage at E2 binding sites in the integrated HPV18 DNA in these cells and also at an E2 binding site in cellular DNA. BEF-expressing cells underwent senescence, which required the DNA binding activity of BEF, but not its nuclease activity. These results demonstrate that DNA binding domains of viral proteins can target effector molecules to cognate binding sites in virally infected cells. PMID:17392356

  18. DNA-PK-mediated phosphorylation of EZH2 regulates the DNA damage-induced apoptosis to maintain T-cell genomic integrity

    PubMed Central

    Wang, Y; Sun, H; Wang, J; Wang, H; Meng, L; Xu, C; Jin, M; Wang, B; Zhang, Y; Zhang, Y; Zhu, T

    2016-01-01

    EZH2 is a histone methyltransferase whose functions in stem cells and tumor cells are well established. Accumulating evidence shows that EZH2 has critical roles in T cells and could be a promising therapeutic target for several immune diseases. To further reveal the novel functions of EZH2 in human T cells, protein co-immunoprecipitation combined mass spectrometry was conducted and several previous unknown EZH2-interacting proteins were identified. Of them, we focused on a DNA damage responsive protein, Ku80, because of the limited knowledge regarding EZH2 in the DNA damage response. Then, we demonstrated that instead of being methylated by EZH2, Ku80 bridges the interaction between the DNA-dependent protein kinase (DNA-PK) complex and EZH2, thus facilitating EZH2 phosphorylation. Moreover, EZH2 histone methyltransferase activity was enhanced when Ku80 was knocked down or DNA-PK activity was inhibited, suggesting DNA-PK-mediated EZH2 phosphorylation impairs EZH2 histone methyltransferase activity. On the other hand, EZH2 inhibition increased the DNA damage level at the late phase of T-cell activation, suggesting EZH2 involved in genomic integrity maintenance. In conclusion, our study is the first to demonstrate that EZH2 is phosphorylated by the DNA damage responsive complex DNA-PK and regulates DNA damage-mediated T-cell apoptosis, which reveals a novel functional crosstalk between epigenetic regulation and genomic integrity. PMID:27468692

  19. Integration of woodchuck hepatitis virus (WHV) DNA at two chromosomal sites (Vk and gag-like) in a hepatocellular carcinoma.

    PubMed

    Yamazoe, M; Nakai, S; Ogasawara, N; Yoshikawa, H

    1991-04-01

    Integration of woodchuck hepatitis virus (WHV) DNA into the liver DNA of a woodchuck infected by the virus was investigated. Clonal viral integration was not detected three months before the appearance of four hepatocellular carcinomas (HCC). Integration of the viral DNA was detected in all four HCCs, of which one was chosen to determine the structure of the viral integration completely in a single tumor. The integration occurred in two sites. One part contains the viral DNA from the middle of the gene encoding surface antigen to two-thirds of the way through the gene encoding X protein (X) with no structural changes. The coding frame of the truncated X gene continues into the chromosomal sequence to make a possible fusion protein. The integration seems to have occurred by recombination within two direct repeats of the viral genome in one junction and by homologous recombination between viral DNA and chromosomal DNA in the other junction. The viral DNA is integrated into a spacer of the immunoglobulin L-chain Vk (IgVk) region without any chromosomal rearrangement accompanying the integration. The viral DNA at the second site has a complex structural rearrangement: part of the preS gene is duplicated and attached to the terminus of the gene encoding core antigen in a head-to-tail fashion, followed by three small fragments derived from other parts of the viral DNA. The integrated preS gene has its own 5' regulatory element and a coding frame consisting of the truncated preS gene, the other parts of viral DNA and the chromosomal sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

  20. A mitochondrial retroplasmid integrates into mitochondrial DNA by a novel mechanism involving the synthesis of a hybrid cDNA and homologous recombination.

    PubMed Central

    Chiang, C C; Kennell, J C; Wanner, L A; Lambowitz, A M

    1994-01-01

    The Mauriceville and Varkud mitochondrial plasmids of Neurospora spp. are closely related, small circular DNAs that propagate via an RNA intermediate and reverse transcription. Although the plasmids ordinarily replicate autonomously, they can also integrate into mitochondrial DNA (mtDNA), yielding defective mtDNAs that in some cases cause senescence. To investigate the integration mechanism, we analyzed four cases in which the Varkud plasmid integrated into the mitochondrial small rRNA gene, three in wild-type subcultures and one in a senescent mutant. Our analysis suggests that the integrations occurred by the plasmid reverse transcriptase template switching between the plasmid transcript and internal sequences in the mitochondrial small rRNA to yield hybrid cDNAs that circularized and recombined homologously with the mtDNA. The integrated plasmid sequences are transcribed, presumably from the mitochondrial small rRNA promoters, resulting in hybrid RNAs containing the 5' segment of the mitochondrial small rRNA linked head-to-tail to the full-length plasmid transcript. Analysis of additional senescent mutants revealed three cases in which the plasmid used the same mechanism to integrate at other locations in the mtDNA. In these cases, circular variant plasmids that had incorporated a mitochondrial tRNA or tRNA-like sequence by template switching integrated by homologous recombination at the site of the corresponding tRNA or tRNA-like sequence in mtDNA. This simple integration mechanism involving template switching to generate a hybrid cDNA that integrates homologously could have been used by primitive retroelements prior to the acquisition of a specialized integration machinery. Images PMID:7523850

  1. Integration of an insertion-type transferred DNA vector from Agrobacterium tumefaciens into the Saccharomyces cerevisiae genome by gap repair.

    PubMed Central

    Risseeuw, E; Franke-van Dijk, M E; Hooykaas, P J

    1996-01-01

    Recently, it was shown that Agrobacterium tumefaciens can transfer transferred DNA (T-DNA) to Saccharomyces cerevisiae and that this T-DNA, when used as a replacement vector, is integrated via homologous recombination into the yeast genome. To test whether T-DNA can be a suitable substrate for integration via the gap repair mechanism as well, a model system developed for detection of homologous recombination events in plants was transferred to S. cerevisiae. Analysis of the yeast transformants revealed that an insertion type T-DNA vector can indeed be integrated via gap repair. Interestingly, the transformation frequency and the type of recombination events turned out to depend strongly on the orientation of the insert between the borders in such an insertion type T-DNA vector. PMID:8816506

  2. Integration of hard copy and soft copy exploitation

    NASA Astrophysics Data System (ADS)

    Fultz, Roy C., Jr.

    1996-11-01

    Exploitation of remotely sensed and aerially derived imagery has, in the past, been primarily performed through the use of analog light tables, by displaying individual pieces or rolls of imagery over a brightly lit surface to allow light through the nonopaque surface of the film medium. The interpreter would then peer through optical viewing scopes allowing him (or her) to analyze the imagery. Over the course of the last two decades, digital data, or as it is better known, "softcopy imagery," has for many become the desired path which technology has dictated. Softcopy imagery offers many benefits, such as the ability to manipulate imagery in ways analog workstations cannot and were never designed to do. Functions which can be performed on softcopy imagery are endless and growing constantly: image spatial rectification, pixel manipulation, image contrast, and brightness enhancements. All are performed by the running of algorithmic equations to manipulate the digital data. It has become evident that in the future a large portion of imagery analysis will be performed by softcopy. However, studies indicate that aerial imagery will continue to be acquired via hardcopy means for many civil, educational, and commercial applications in the foreseeable future, making it clear that any large scale transformation from hardcopy to softcopy will not be feasible for a long time to come. A major issue dictating the slow-down in this transition is the over 35 years of hardcopy imagery archived and housed in facilities throughout the world, including the recently declassified "Corona" satellite imagery which will provide a wealth of hardcopy data for use by ecologists and conservationists. Yes, the technology to transfer hardcopy to softcopy exists, but the time and cost required to complete this task would be phenomenal and, in many cases, when digitization and storage become affordable, it still may prove beneficial to retain the imagery in a hardcopy form for retention of the highest quality resolution. An analogy which I feel best portrays this dilemma is the automobile-eventually all automobiles will be electric or hydrogen driven but the time and cost involved in the transformation predicts a slow progression. Since a predominate amount of imagery analysis, especially in the intelligence community, is the comparison ofnew imagery data to that of archived imagery in order to detect changes or to monitor progressions, it is conceivable that the majority of imagery analysts will be using a combination of hardcopy and softcopy workstations in order to facilitate analysis. The incorporation of hardcopy and softcopy functions into one workstation is the most cost effective and time essential means in which in-depth analysis can be performed.

  3. Borrowing Nuclear DNA Helicases to Protect Mitochondrial DNA

    PubMed Central

    Ding, Lin; Liu, Yilun

    2015-01-01

    In normal cells, mitochondria are the primary organelles that generate energy, which is critical for cellular metabolism. Mitochondrial dysfunction, caused by mitochondrial DNA (mtDNA) mutations or an abnormal mtDNA copy number, is linked to a range of human diseases, including Alzheimer’s disease, premature aging‎ and cancer. mtDNA resides in the mitochondrial lumen, and its duplication requires the mtDNA replicative helicase, Twinkle. In addition to Twinkle, many DNA helicases, which are encoded by the nuclear genome and are crucial for nuclear genome integrity, are transported into the mitochondrion to also function in mtDNA replication and repair. To date, these helicases include RecQ-like helicase 4 (RECQ4), petite integration frequency 1 (PIF1), DNA replication helicase/nuclease 2 (DNA2) and suppressor of var1 3-like protein 1 (SUV3). Although the nuclear functions of some of these DNA helicases have been extensively studied, the regulation of their mitochondrial transport and the mechanisms by which they contribute to mtDNA synthesis and maintenance remain largely unknown. In this review, we attempt to summarize recent research progress on the role of mammalian DNA helicases in mitochondrial genome maintenance and the effects on mitochondria-associated diseases. PMID:25984607

  4. Borrowing nuclear DNA helicases to protect mitochondrial DNA.

    PubMed

    Ding, Lin; Liu, Yilun

    2015-05-13

    In normal cells, mitochondria are the primary organelles that generate energy, which is critical for cellular metabolism. Mitochondrial dysfunction, caused by mitochondrial DNA (mtDNA) mutations or an abnormal mtDNA copy number, is linked to a range of human diseases, including Alzheimer's disease, premature aging‎ and cancer. mtDNA resides in the mitochondrial lumen, and its duplication requires the mtDNA replicative helicase, Twinkle. In addition to Twinkle, many DNA helicases, which are encoded by the nuclear genome and are crucial for nuclear genome integrity, are transported into the mitochondrion to also function in mtDNA replication and repair. To date, these helicases include RecQ-like helicase 4 (RECQ4), petite integration frequency 1 (PIF1), DNA replication helicase/nuclease 2 (DNA2) and suppressor of var1 3-like protein 1 (SUV3). Although the nuclear functions of some of these DNA helicases have been extensively studied, the regulation of their mitochondrial transport and the mechanisms by which they contribute to mtDNA synthesis and maintenance remain largely unknown. In this review, we attempt to summarize recent research progress on the role of mammalian DNA helicases in mitochondrial genome maintenance and the effects on mitochondria-associated diseases.

  5. Human immunodeficiency virus type 1 DNA integration: fine structure target analysis using synthetic oligonucleotides.

    PubMed Central

    Hong, T; Murphy, E; Groarke, J; Drlica, K

    1993-01-01

    The target specificity of DNA strand transfer mediated by human immunodeficiency virus type 1 integrase was examined in vitro with synthetic oligonucleotides. Although insertion occurred at most locations in the target, some sites were preferred over others by at least 15-fold. Changing the nucleotide sequence of the target changed the distribution of preferred sites in complex ways, some of which included changes in target preference distant from the sequence alteration. Alignment of target sequences revealed that adenosine is preferred adjacent to the insertion site. Strand transfer occurred to within 2 nucleotides of the 3' end and to within 3 nucleotides of the 5' end of the target. This suggests that only 2 or 3 nucleotides flanking the target site are required for integration; such restricted contact with target DNA would allow integrase to insert the two ends of viral DNA into two closely spaced sites in host DNA, consistent with the concerted in vivo integration reaction that generates a 5-bp target duplication. Images PMID:8419642

  6. Flow cytometry application in the assessment of sperm DNA integrity of men with asthenozoospermia.

    PubMed

    Piasecka, M; Gaczarzewicz, D; Laszczyńska, M; Starczewski, A; Brodowska, A

    2007-01-01

    Sperm genomic integrity and ultrastructural features of ejaculated spermatozoa contributing to the assessment of gamete fertility potential in patients with asthenozoospermia are discussed. The proportion of TUNEL-positive cells was significantly higher in the semen of patients with low sperm motility (n=40; p<0.01) as compared to men with normal sperm motility (n=54). Sperm DNA fragmentation negatively correlated (n=94) with sperm motility, sperm concentration, and integrity of the sperm cellular membrane (HOS-test). Two categories of patients were distinguished: (1) patients (23 out of 94 subjects) with < or = 4% of TUNEL-positive cells and (2) patients (71 subjects) with 4% of TUNEL-positive cells. A significant difference was noted in the sperm motility and HOS-test results between patients from both groups. Large numbers of immature spermatozoa with extensive cytoplasmic retention, ultrastructural chromatin and midpiece abnormalities, and conglomerates containing sperm fragments were present more frequently in the semen of asthenozoospermic subjects with >4% of TUNEL-positive sperm cells. Low sperm motility seems to be accompanied by serious defects of gamete chromatin expressed as diminished sperm genomic integrity and abnormal DNA condensation and by defects of sperm midpiece. These abnormalities may reflect developmental failure during the spermatogenic remodeling process. The DNA fragmentation test may be considered as an additional assay for the evaluation of spermatozoa beside standard analysis and taken together with electron microscopy may help to determine the actual number of "healthy" spermatozoa thereby playing an important role during diagnosis and treatment of male infertility.

  7. DyNAMiC Workbench: an integrated development environment for dynamic DNA nanotechnology.

    PubMed

    Grun, Casey; Werfel, Justin; Zhang, David Yu; Yin, Peng

    2015-10-06

    Dynamic DNA nanotechnology provides a promising avenue for implementing sophisticated assembly processes, mechanical behaviours, sensing and computation at the nanoscale. However, design of these systems is complex and error-prone, because the need to control the kinetic pathway of a system greatly increases the number of design constraints and possible failure modes for the system. Previous tools have automated some parts of the design workflow, but an integrated solution is lacking. Here, we present software implementing a three 'tier' design process: a high-level visual programming language is used to describe systems, a molecular compiler builds a DNA implementation and nucleotide sequences are generated and optimized. Additionally, our software includes tools for analysing and 'debugging' the designs in silico, and for importing/exporting designs to other commonly used software systems. The software we present is built on many existing pieces of software, but is integrated into a single package—accessible using a Web-based interface at http://molecular-systems.net/workbench. We hope that the deep integration between tools and the flexibility of this design process will lead to better experimental results, fewer experimental design iterations and the development of more complex DNA nanosystems.

  8. DyNAMiC Workbench: an integrated development environment for dynamic DNA nanotechnology

    PubMed Central

    Grun, Casey; Werfel, Justin; Zhang, David Yu; Yin, Peng

    2015-01-01

    Dynamic DNA nanotechnology provides a promising avenue for implementing sophisticated assembly processes, mechanical behaviours, sensing and computation at the nanoscale. However, design of these systems is complex and error-prone, because the need to control the kinetic pathway of a system greatly increases the number of design constraints and possible failure modes for the system. Previous tools have automated some parts of the design workflow, but an integrated solution is lacking. Here, we present software implementing a three ‘tier’ design process: a high-level visual programming language is used to describe systems, a molecular compiler builds a DNA implementation and nucleotide sequences are generated and optimized. Additionally, our software includes tools for analysing and ‘debugging’ the designs in silico, and for importing/exporting designs to other commonly used software systems. The software we present is built on many existing pieces of software, but is integrated into a single package—accessible using a Web-based interface at http://molecular-systems.net/workbench. We hope that the deep integration between tools and the flexibility of this design process will lead to better experimental results, fewer experimental design iterations and the development of more complex DNA nanosystems. PMID:26423437

  9. Microfluidic chip integrating high throughput continuous-flow PCR and DNA hybridization for bacteria analysis.

    PubMed

    Jiang, Xiran; Shao, Ning; Jing, Wenwen; Tao, Shengce; Liu, Sixiu; Sui, Guodong

    2014-05-01

    Rapid identification of clinical pathogens is the initial and essential step for antimicrobial therapy. Herein, we successfully developed a microfluidic device which combines high-throughput continuous-flow PCR and DNA hybridization for the detection of various bacterial pathogens. Universal primers were designed based on the conserved regions of bacterial 16S ribosomal DNA (16S rDNA), and specific probes were designed from a variable region of 16S rDNA within the amplicon sequences. In the chip operation, after the continuous flow PCR was achieved in the first microfluidic chip, the product was directly introduced into a hybridization chip integrated with microarray containing the immobilized DNA probes. The target-probe hybridization was completed within 1h at 55 °C, and fluorescence signals were obtained as the readout. The presented device is simple, versatile and with less sample consumption compared with traditional instruments. It can perform high-throughput bacteria detections continuously in a single assay, which makes it a promising platform for clinical bacteria identifications.

  10. Plasma cell-free DNA levels and integrity in patients with chest radiological findings: NSCLC versus benign lung nodules.

    PubMed

    Szpechcinski, Adam; Rudzinski, Piotr; Kupis, Wlodzimierz; Langfort, Renata; Orlowski, Tadeusz; Chorostowska-Wynimko, Joanna

    2016-05-01

    Effective discrimination between lung cancer and benign tumours is a common clinical problem in the differential diagnosis of solitary pulmonary nodules. The analysis of cell-free DNA (cfDNA) in blood may greatly aid the early detection of lung cancer by evaluating cancer-related alterations. The plasma cfDNA levels and integrity were analysed in 65 non-small cell lung cancer (NSCLC) patients, 28 subjects with benign lung tumours, and 16 healthy controls using real-time PCR. The NSCLC patients demonstrated significantly higher mean plasma cfDNA levels compared with those with benign tumours (P = 0.0009) and healthy controls (P < 0.0001). The plasma cfDNA integrity in healthy individuals was significantly different than that found in patients with NSCLC or benign lung tumours (P < 0.0003). In ROC curve analysis, plasma cfDNA levels >2.8 ng/ml provided 86.4% sensitivity and 61.4% specificity in discriminating NSCLC from benign lung pathologies and healthy controls. cfDNA integrity showed better discriminatory power (91% sensitivity, 68.2% specificity). These data demonstrate that plasma cfDNA concentration and integrity analyses can significantly differentiate between NSCLC and benign lung tumours. The diagnostic capacity of the quantitative cfDNA assay is comparable to the values presented by conventional imaging modalities used in clinical practice.

  11. Repair of oxidative DNA base damage in the host genome influences the HIV integration site sequence preference.

    PubMed

    Bennett, Geoffrey R; Peters, Ryan; Wang, Xiao-hong; Hanne, Jeungphill; Sobol, Robert W; Bundschuh, Ralf; Fishel, Richard; Yoder, Kristine E

    2014-01-01

    Host base excision repair (BER) proteins that repair oxidative damage enhance HIV infection. These proteins include the oxidative DNA damage glycosylases 8-oxo-guanine DNA glycosylase (OGG1) and mutY homolog (MYH) as well as DNA polymerase beta (Polβ). While deletion of oxidative BER genes leads to decreased HIV infection and integration efficiency, the mechanism remains unknown. One hypothesis is that BER proteins repair the DNA gapped integration intermediate. An alternative hypothesis considers that the most common oxidative DNA base damages occur on guanines. The subtle consensus sequence preference at HIV integration sites includes multiple G:C base pairs surrounding the points of joining. These observations suggest a role for oxidative BER during integration targeting at the nucleotide level. We examined the hypothesis that BER repairs a gapped integration intermediate by measuring HIV infection efficiency in Polβ null cell lines complemented with active site point mutants of Polβ. A DNA synthesis defective mutant, but not a 5'dRP lyase mutant, rescued HIV infection efficiency to wild type levels; this suggested Polβ DNA synthesis activity is not necessary while 5'dRP lyase activity is required for efficient HIV infection. An alternate hypothesis that BER events in the host genome influence HIV integration site selection was examined by sequencing integration sites in OGG1 and MYH null cells. In the absence of these 8-oxo-guanine specific glycosylases the chromatin elements of HIV integration site selection remain the same as in wild type cells. However, the HIV integration site sequence preference at G:C base pairs is altered at several positions in OGG1 and MYH null cells. Inefficient HIV infection in the absence of oxidative BER proteins does not appear related to repair of the gapped integration intermediate; instead oxidative damage repair may participate in HIV integration site preference at the sequence level.

  12. PennCNV: An integrated hidden Markov model designed for high-resolution copy number variation detection in whole-genome SNP genotyping data

    PubMed Central

    Wang, Kai; Li, Mingyao; Hadley, Dexter; Liu, Rui; Glessner, Joseph; Grant, Struan F.A.; Hakonarson, Hakon; Bucan, Maja

    2007-01-01

    Comprehensive identification and cataloging of copy number variations (CNVs) is required to provide a complete view of human genetic variation. The resolution of CNV detection in previous experimental designs has been limited to tens or hundreds of kilobases. Here we present PennCNV, a hidden Markov model (HMM) based approach, for kilobase-resolution detection of CNVs from Illumina high-density SNP genotyping data. This algorithm incorporates multiple sources of information, including total signal intensity and allelic intensity ratio at each SNP marker, the distance between neighboring SNPs, the allele frequency of SNPs, and the pedigree information where available. We applied PennCNV to genotyping data generated for 112 HapMap individuals; on average, we detected ∼27 CNVs for each individual with a median size of ∼12 kb. Excluding common rearrangements in lymphoblastoid cell lines, the fraction of CNVs in offspring not detected in parents (CNV-NDPs) was 3.3%. Our results demonstrate the feasibility of whole-genome fine-mapping of CNVs via high-density SNP genotyping. PMID:17921354

  13. An integrative approach to investigate the respective roles of single-nucleotide variants and copy-number variants in Attention-Deficit/Hyperactivity Disorder

    PubMed Central

    de Araújo Lima, Leandro; Feio-dos-Santos, Ana Cecília; Belangero, Sintia Iole; Gadelha, Ary; Bressan, Rodrigo Affonseca; Salum, Giovanni Abrahão; Pan, Pedro Mario; Moriyama, Tais Silveira; Graeff-Martins, Ana Soledade; Tamanaha, Ana Carina; Alvarenga, Pedro; Krieger, Fernanda Valle; Fleitlich-Bilyk, Bacy; Jackowski, Andrea Parolin; Brietzke, Elisa; Sato, João Ricardo; Polanczyk, Guilherme Vanoni; Mari, Jair de Jesus; Manfro, Gisele Gus; do Rosário, Maria Conceição; Miguel, Eurípedes Constantino; Puga, Renato David; Tahira, Ana Carolina; Souza, Viviane Neri; Chile, Thais; Gouveia, Gisele Rodrigues; Simões, Sérgio Nery; Chang, Xiao; Pellegrino, Renata; Tian, Lifeng; Glessner, Joseph T.; Hashimoto, Ronaldo Fumio; Rohde, Luis Augusto; Sleiman, Patrick M.A.; Hakonarson, Hakon; Brentani, Helena

    2016-01-01

    Many studies have attempted to investigate the genetic susceptibility of Attention-Deficit/Hyperactivity Disorder (ADHD), but without much success. The present study aimed to analyze both single-nucleotide and copy-number variants contributing to the genetic architecture of ADHD. We generated exome data from 30 Brazilian trios with sporadic ADHD. We also analyzed a Brazilian sample of 503 children/adolescent controls from a High Risk Cohort Study for the Development of Childhood Psychiatric Disorders, and also previously published results of five CNV studies and one GWAS meta-analysis of ADHD involving children/adolescents. The results from the Brazilian trios showed that cases with de novo SNVs tend not to have de novo CNVs and vice-versa. Although the sample size is small, we could also see that various comorbidities are more frequent in cases with only inherited variants. Moreover, using only genes expressed in brain, we constructed two “in silico” protein-protein interaction networks, one with genes from any analysis, and other with genes with hits in two analyses. Topological and functional analyses of genes in this network uncovered genes related to synapse, cell adhesion, glutamatergic and serotoninergic pathways, both confirming findings of previous studies and capturing new genes and genetic variants in these pathways. PMID:26947246

  14. An integrative approach to investigate the respective roles of single-nucleotide variants and copy-number variants in Attention-Deficit/Hyperactivity Disorder.

    PubMed

    Lima, Leandro de Araújo; Feio-dos-Santos, Ana Cecília; Belangero, Sintia Iole; Gadelha, Ary; Bressan, Rodrigo Affonseca; Salum, Giovanni Abrahão; Pan, Pedro Mario; Moriyama, Tais Silveira; Graeff-Martins, Ana Soledade; Tamanaha, Ana Carina; Alvarenga, Pedro; Krieger, Fernanda Valle; Fleitlich-Bilyk, Bacy; Jackowski, Andrea Parolin; Brietzke, Elisa; Sato, João Ricardo; Polanczyk, Guilherme Vanoni; Mari, Jair de Jesus; Manfro, Gisele Gus; do Rosário, Maria Conceição; Miguel, Eurípedes Constantino; Puga, Renato David; Tahira, Ana Carolina; Souza, Viviane Neri; Chile, Thais; Gouveia, Gisele Rodrigues; Simões, Sérgio Nery; Chang, Xiao; Pellegrino, Renata; Tian, Lifeng; Glessner, Joseph T; Hashimoto, Ronaldo Fumio; Rohde, Luis Augusto; Sleiman, Patrick M A; Hakonarson, Hakon; Brentani, Helena

    2016-03-07

    Many studies have attempted to investigate the genetic susceptibility of Attention-Deficit/Hyperactivity Disorder (ADHD), but without much success. The present study aimed to analyze both single-nucleotide and copy-number variants contributing to the genetic architecture of ADHD. We generated exome data from 30 Brazilian trios with sporadic ADHD. We also analyzed a Brazilian sample of 503 children/adolescent controls from a High Risk Cohort Study for the Development of Childhood Psychiatric Disorders, and also previously published results of five CNV studies and one GWAS meta-analysis of ADHD involving children/adolescents. The results from the Brazilian trios showed that cases with de novo SNVs tend not to have de novo CNVs and vice-versa. Although the sample size is small, we could also see that various comorbidities are more frequent in cases with only inherited variants. Moreover, using only genes expressed in brain, we constructed two "in silico" protein-protein interaction networks, one with genes from any analysis, and other with genes with hits in two analyses. Topological and functional analyses of genes in this network uncovered genes related to synapse, cell adhesion, glutamatergic and serotoninergic pathways, both confirming findings of previous studies and capturing new genes and genetic variants in these pathways.

  15. Copy Machine Art.

    ERIC Educational Resources Information Center

    Sommer, Jean

    1984-01-01

    Images created with copy machines make children feel successful, as their work acquires the authority of being printed. Students can learn advanced processes like electrostatic image-making and can get involved in projects like making collages. They acquire an appreciation of design and of two-dimensional composition. (CS)

  16. Modification of some biological properties of HeLa cells containing adeno-associated virus DNA integrated into chromosome 17.

    PubMed Central

    Walz, C; Schlehofer, J R

    1992-01-01

    Parvoviruses are known to interfere with cellular transformation and carcinogenesis. Since infecting adeno-associated virus (AAV) frequently integrates its DNA into the cellular genome, we analyzed whether this integration influences the transformed phenotype of the human tumor cell line HeLa. Analysis of three independent HeLa cell clones with integrated AAV DNA (HA-3x, HA-16, and HA-28) revealed the following phenotypic changes of these cells: (i) reduced growth rate, (ii) increased serum requirement, (iii) reduced capacity for colony formation in soft agar, (iv) reduced cloning efficiency on plastic, (v) elevated sensitivity to genotoxic agents (N-methyl-N'-nitro-N-nitrosoguanidine, 7,12-dimethylbenz[a]anthracene, human tumor necrosis factor alpha, UV irradiation [256 nm], and heat [42 degrees C]), and (vi) reduced sensitivity to the cytolytic effect of parvovirus H-1. Reduced growth rate and enhanced sensitivity to gamma irradiation were also observed in vivo when tumors from AAV DNA-containing HeLa cells were transplanted into nude mice. This alteration of the biological properties of HeLa cells was independent of the number of AAV genomes integrated, the physical structure of integrated AAV DNA, and the transcription of AAV genes. Integration of AAV DNA was found to occur preferentially on the long arm of chromosome 17 in the three HeLa cell clones analyzed. These findings demonstrate that genomic integration of AAV DNA can alter the biological properties of human tumor cells. Images PMID:1313913

  17. Charging YOYO-1 on capillary wall for online DNA intercalation and integrating this approach with multiplex PCR and bare narrow capillary-hydrodynamic chromatography for online DNA analysis.

    PubMed

    Chen, Huang; Zhu, Zaifang; Lu, Joann Juan; Liu, Shaorong

    2015-02-03

    Multiplex polymerase chain reaction (PCR) has been widely utilized for high-throughput pathogen identification. Often, a dye is used to intercalate the amplified DNA fragments, and identifications of the pathogens are carried out by DNA melting curve analysis or gel electrophoresis. Integrating DNA amplification and identification is a logic path toward maximizing the benefit of multiplex PCR. Although PCR and gel electrophoresis have been integrated, replenishing the gels after each run is tedious and time-consuming. In this technical note, we develop an approach to address this issue. We perform multiplex PCR inside a capillary, transfer the amplified fragments to a bare narrow capillary, and measure their lengths online using bare narrow capillary-hydrodynamic chromatography (BaNC-HDC), a new technique recently developed in our laboratory for free-solution DNA separation. To intercalate the DNA with YOYO-1 (a fluorescent dye) for BaNC-HDC, we flush the capillary column with a YOYO-1 solution; positively charged YOYO-1 is adsorbed (or charged) onto the negatively charged capillary wall. As DNA molecules are driven down the column for separation, they react with the YOYO-1 stored on the capillary wall and are online-intercalated with the dye. With a single YOYO-1 charging, the column can be used for more than 40 runs, although the fluorescence signal intensities of the DNA peaks decrease gradually. Although the dye-DNA intercalation occurs during the separation, it does not affect the retention times, separation efficiencies, or resolutions.

  18. Polycomb repressive complex 1 provides a molecular explanation for repeat copy number dependency in FSHD muscular dystrophy.

    PubMed

    Casa, Valentina; Runfola, Valeria; Micheloni, Stefano; Aziz, Arif; Dilworth, F Jeffrey; Gabellini, Davide

    2016-12-30

    Repression of repetitive elements is crucial to preserve genome integrity and has been traditionally ascribed to constitutive heterochromatin pathways. FacioScapuloHumeral Muscular Dystrophy (FSHD), one of the most common myopathies, is characterized by a complex interplay of genetic and epigenetic events. The main FSHD form is linked to a reduced copy number of the D4Z4 macrosatellite repeat on 4q35, causing loss of silencing and aberrant expression of the D4Z4-embedded DUX4 gene leading to disease. By an unknown mechanism, D4Z4 copy-number correlates with FSHD phenotype. Here we show that the DUX4 proximal promoter (DUX4p) is sufficient to nucleate the enrichment of both constitutive and facultative heterochromatin components and to mediate a copy-number dependent gene silencing. We found that both the CpG/GC dense DNA content and the repetitive nature of DUX4p arrays are important for their repressive ability. We showed that DUX4p mediates a copy number-dependent Polycomb Repressive Complex 1 (PRC1) recruitment, which is responsible for the copy-number dependent gene repression. Overall, we directly link genetic and epigenetic defects in FSHD by proposing a novel molecular explanation for the copy number-dependency in FSHD pathogenesis, and offer insight into the molecular functions of repeats in chromatin regulation.

  19. The Cellular DNA Helicase ChlR1 Regulates Chromatin and Nuclear Matrix Attachment of the Human Papillomavirus 16 E2 Protein and High-Copy-Number Viral Genome Establishment

    PubMed Central

    Harris, Leanne; McFarlane-Majeed, Laura; Campos-León, Karen; Roberts, Sally

    2016-01-01

    ABSTRACT In papillomavirus infections, the viral genome is established as a double-stranded DNA episome. To segregate the episomes into daughter cells during mitosis, they are tethered to cellular chromatin by the viral E2 protein. We previously demonstrated that the E2 proteins of diverse papillomavirus types, including bovine papillomavirus (BPV) and human papillomavirus 16 (HPV16), associate with the cellular DNA helicase ChlR1. This virus-host interaction is important for the tethering of BPV E2 to mitotic chromatin and the stable maintenance of BPV episomes. The role of the association between E2 and ChlR1 in the HPV16 life cycle is unresolved. Here we show that an HPV16 E2 Y131A mutant (E2Y131A) had significantly reduced binding to ChlR1 but retained transcriptional activation and viral origin-dependent replication functions. Subcellular fractionation of keratinocytes expressing E2Y131A showed a marked change in the localization of the protein. Compared to that of wild-type E2 (E2WT), the chromatin-bound pool of E2Y131A was decreased, concomitant with an increase in nuclear matrix-associated protein. Cell cycle synchronization indicated that the shift in subcellular localization of E2Y131A occurred in mid-S phase. A similar alteration between the subcellular pools of the E2WT protein occurred upon ChlR1 silencing. Notably, in an HPV16 life cycle model in primary human keratinocytes, mutant E2Y131A genomes were established as episomes, but at a markedly lower copy number than that of wild-type HPV16 genomes, and they were not maintained upon cell passage. Our studies indicate that ChlR1 is an important regulator of the chromatin association of E2 and of the establishment and maintenance of HPV16 episomes. IMPORTANCE Infections with high-risk human papillomaviruses (HPVs) are a major cause of anogenital and oropharyngeal cancers. During infection, the circular DNA genome of HPV persists within the nucleus, independently of the host cell chromatin. Persistence

  20. Integrated genomic DNA/RNA profiling of hematologic malignancies in the clinical setting

    PubMed Central

    He, Jie; Abdel-Wahab, Omar; Nahas, Michelle K.; Wang, Kai; Rampal, Raajit K.; Intlekofer, Andrew M.; Patel, Jay; Krivstov, Andrei; Frampton, Garrett M.; Young, Lauren E.; Zhong, Shan; Bailey, Mark; White, Jared R.; Roels, Steven; Deffenbaugh, Jason; Fichtenholtz, Alex; Brennan, Timothy; Rosenzweig, Mark; Pelak, Kimberly; Knapp, Kristina M.; Brennan, Kristina W.; Donahue, Amy L.; Young, Geneva; Garcia, Lazaro; Beckstrom, Selmira T.; Zhao, Mandy; White, Emily; Banning, Vera; Buell, Jamie; Iwanik, Kiel; Ross, Jeffrey S.; Morosini, Deborah; Younes, Anas; Hanash, Alan M.; Paietta, Elisabeth; Roberts, Kathryn; Mullighan, Charles; Dogan, Ahmet; Armstrong, Scott A.; Mughal, Tariq; Vergilio, Jo-Anne; Labrecque, Elaine; Erlich, Rachel; Vietz, Christine; Yelensky, Roman; Stephens, Philip J.; Miller, Vincent A.; van den Brink, Marcel R. M.; Otto, Geoff A.; Lipson, Doron

    2016-01-01

    The spectrum of somatic alterations in hematologic malignancies includes substitutions, insertions/deletions (indels), copy number alterations (CNAs), and a wide range of gene fusions; no current clinically available single assay captures the different types of alterations. We developed a novel next-generation sequencing-based assay to identify all classes of genomic alterations using archived formalin-fixed paraffin-embedded blood and bone marrow samples with high accuracy in a clinically relevant time frame, which is performed in our Clinical Laboratory Improvement Amendments–certified College of American Pathologists–accredited laboratory. Targeted capture of DNA/RNA and next-generation sequencing reliably identifies substitutions, indels, CNAs, and gene fusions, with similar accuracy to lower-throughput assays that focus on specific genes and types of genomic alterations. Profiling of 3696 samples identified recurrent somatic alterations that impact diagnosis, prognosis, and therapy selection. This comprehensive genomic profiling approach has proved effective in detecting all types of genomic alterations, including fusion transcripts, which increases the ability to identify clinically relevant genomic alterations with therapeutic relevance. PMID:26966091

  1. Integrating DNA-based data into bioassessments improves our understanding of species distributions and species habitat relationships

    EPA Science Inventory

    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 inv...

  2. Integrase residues that determine nucleotide preferences at sites of HIV-1 integration: implications for the mechanism of target DNA binding

    PubMed Central

    Serrao, Erik; Krishnan, Lavanya; Shun, Ming-Chieh; Li, Xiang; Cherepanov, Peter; Engelman, Alan; Maertens, Goedele N.

    2014-01-01

    Retroviruses favor target-DNA (tDNA) distortion and particular bases at sites of integration, but the mechanism underlying HIV-1 selectivity is unknown. Crystal structures revealed a network of prototype foamy virus (PFV) integrase residues that distort tDNA: Ala188 and Arg329 interact with tDNA bases, while Arg362 contacts the phosphodiester backbone. HIV-1 integrase residues Ser119, Arg231, and Lys258 were identified here as analogs of PFV integrase residues Ala188, Arg329 and Arg362, respectively. Thirteen integrase mutations were analyzed for effects on integrase activity in vitro and during virus infection, yielding a total of 1610 unique HIV-1 integration sites. Purine (R)/pyrimidine (Y) dinucleotide sequence analysis revealed HIV-1 prefers the tDNA signature (0)RYXRY(4), which accordingly favors overlapping flexible dinucleotides at the center of the integration site. Consistent with roles for Arg231 and Lys258 in sequence specific and non-specific binding, respectively, the R231E mutation altered integration site nucleotide preferences while K258E had no effect. S119A and S119T integrase mutations significantly altered base preferences at positions −3 and 7 from the site of viral DNA joining. The S119A preference moreover mimicked wild-type PFV selectivity at these positions. We conclude that HIV-1 IN residue Ser119 and PFV IN residue Ala188 contact analogous tDNA bases to effect virus integration. PMID:24520116

  3. Specific targeted integration of kanamycin resistance-associated nonselectable DNA in the genome of the yeast Saccharomyces cerevisiae.

    PubMed

    Waghmare, Sanjeev K; Caputo, Valentina; Radovic, Slobodanka; Bruschi, Carlo V

    2003-05-01

    Sophisticated genome manipulation requires the possibility to modify any intergenic or intragenic DNA sequence at will, without leaving large amounts of undesired vector DNA at the site of alteration. To this end, a series of vectors was developed from a previous gene knockout plasmid system to integrate nonselectable foreign DNA at any desired genomic location in yeast, with a minimum amount of residual plasmid DNA. These vectors have two mutated Flp recognition targets (FRT) sequences flanking the KanMX4 gene and multiple sites for subcloning the DNA fragment to be integrated. The selectable marker can be recycled by Flp site-specific excision between the identical FRTs, thereby allowing the integration of further DNA fragments. With this system, the NLS-tetR-GFP and DsRed genes were successfully integrated at the thr1 locus, and the RVB1 gene was tagged at the C-terminus with the V5-epitope-6-histidine tag. This plasmid system provides for a new molecular tool to integrate any DNA fragment at any genome location in [cir+] yeast strains. Moreover, the system can be extrapolated to other eukaryotic cells in which the FLP/FRT system functions efficiently.

  4. Spermatozoa bound to solid state hyaluronic acid show chromatin structure with high DNA chain integrity: an acridine orange fluorescence study.

    PubMed

    Yagci, Artay; Murk, William; Stronk, Jill; Huszar, Gabor

    2010-01-01

    During human spermiogenesis, the elongated spermatids undergo a plasma membrane remodeling step that facilitates formation of the zona pellucida and hyaluronic acid (HA) binding sites. Various biochemical sperm markers indicated that human sperm bound to HA exhibit attributes similar to that of zona pellucida-bound sperm, including minimal DNA fragmentation, normal shape, and low frequency of chromosomal aneuploidies. In this work, we tested the hypothesis that HA-bound sperm would be enhanced in sperm of high DNA chain integrity and green acridine orange fluorescence (AOF) compared with the original sperm in semen. Sperm DNA integrity in semen and in their respective HA-bound sperm fractions was studied in 50 men tested for fertility. In the semen samples, the proportions of sperm with green AOF (high DNA integrity) and red AOF (DNA breaks) were 54.9% ± 2.0% and 45.0% ± 1.9%, whereas in the HA-bound sperm fraction, the respective proportions were 99% and 1.0%, respectively. The data indeed demonstrated that HA shows a high degree of selectivity for sperm with high DNA integrity. These findings are important from the points of view of human sperm DNA integrity, sperm function, and the potential efficacy of HA-mediated sperm selection for intracytoplasmic sperm injection.

  5. Integration and scaling of UV-B radiation effects on plants: from DNA to leaf

    PubMed Central

    Suchar, Vasile Alexandru; Robberecht, Ronald

    2015-01-01

    A process-based model integrating the effects of UV-B radiation through epidermis, cellular DNA, and its consequences to the leaf expansion was developed from key parameters in the published literature. Enhanced UV-B radiation-induced DNA damage significantly delayed cell division, resulting in significant reductions in leaf growth and development. Ambient UV-B radiation-induced DNA damage significantly reduced the leaf growth of species with high relative epidermal absorbance at longer wavelengths and average/low pyrimidine cyclobutane dimers (CPD) photorepair rates. Leaf expansion was highly dependent on the number of CPD present in the DNA, as a result of UV-B radiation dose, quantitative and qualitative absorptive properties of epidermal pigments, and repair mechanisms. Formation of pyrimidine-pyrimidone (6-4) photoproducts (6-4PP) has no effect on the leaf expansion. Repair mechanisms could not solely prevent the UV-B radiation interference with the cell division. Avoidance or effective shielding by increased or modified qualitative epidermal absorptance was required. Sustained increased UV-B radiation levels are more detrimental than short, high doses of UV-B radiation. The combination of low temperature and increased UV-B radiation was more significant in the level of UV-B radiation-induced damage than UV-B radiation alone. Slow-growing leaves were more affected by increased UV-B radiation than fast-growing leaves. PMID:26257869

  6. Integrative taxonomy at work: DNA barcoding of taeniids harboured by wild and domestic cats.

    PubMed

    Galimberti, A; Romano, D F; Genchi, M; Paoloni, D; Vercillo, F; Bizzarri, L; Sassera, D; Bandi, C; Genchi, C; Ragni, B; Casiraghi, M

    2012-05-01

    In modern taxonomy, DNA barcoding is particularly useful where biometric parameters are difficult to determine or useless owing to the poor quality of samples. These situations are frequent in parasitology. Here, we present an integrated study, based on both DNA barcoding and morphological analysis, on cestodes belonging to the genus Taenia, for which biodiversity is still largely underestimated. In particular, we characterized cestodes from Italian wildcats (Felis silvestris silvestris), free-ranging domestic cats (Felis silvestris catus) and hybrids populations. Adult taeniids were collected by post-mortem examinations of the hosts and morphologically identified as Taenia taeniaeformis. We produced cox1 barcode sequences for all the analysed specimens, and we compared them with reference sequences of individuals belonging to the genus Taenia retrieved from GenBank. In order to evaluate the performance of a DNA barcoding approach to discriminate these parasites, the strength of correlation between species identification based on classical morphology and the molecular divergence of cox1 sequences was measured. Our study provides clear evidence that DNA barcoding is highly efficient to reveal the presence of cryptic lineages within already-described taeniid species. Indeed, we detected three well-defined molecular lineages within the whole panel of specimens morphologically identified as T. taeniaeformis. Two of these molecular groups were already identified by other authors and should be ranked at species level. The third molecular group encompasses only samples collected in Italy during this study, and it represents a third candidate species, still morphologically undescribed.

  7. Integration and scaling of UV-B radiation effects on plants: from DNA to leaf.

    PubMed

    Suchar, Vasile Alexandru; Robberecht, Ronald

    2015-07-01

    A process-based model integrating the effects of UV-B radiation through epidermis, cellular DNA, and its consequences to the leaf expansion was developed from key parameters in the published literature. Enhanced UV-B radiation-induced DNA damage significantly delayed cell division, resulting in significant reductions in leaf growth and development. Ambient UV-B radiation-induced DNA damage significantly reduced the leaf growth of species with high relative epidermal absorbance at longer wavelengths and average/low pyrimidine cyclobutane dimers (CPD) photorepair rates. Leaf expansion was highly dependent on the number of CPD present in the DNA, as a result of UV-B radiation dose, quantitative and qualitative absorptive properties of epidermal pigments, and repair mechanisms. Formation of pyrimidine-pyrimidone (6-4) photoproducts (6-4PP) has no effect on the leaf expansion. Repair mechanisms could not solely prevent the UV-B radiation interference with the cell division. Avoidance or effective shielding by increased or modified qualitative epidermal absorptance was required. Sustained increased UV-B radiation levels are more detrimental than short, high doses of UV-B radiation. The combination of low temperature and increased UV-B radiation was more significant in the level of UV-B radiation-induced damage than UV-B radiation alone. Slow-growing leaves were more affected by increased UV-B radiation than fast-growing leaves.

  8. Sufficient minimal model for DNA denaturation: Integration of harmonic scalar elasticity and bond energies.

    PubMed

    Singh, Amit Raj; Granek, Rony

    2016-10-14

    We study DNA denaturation by integrating elasticity - as described by the Gaussian network model - with bond binding energies, distinguishing between different base pairs and stacking energies. We use exact calculation, within the model, of the Helmholtz free-energy of any partial denaturation state, which implies that the entropy of all formed "bubbles" ("loops") is accounted for. Considering base pair bond removal single events, the bond designated for opening is chosen by minimizing the free-energy difference for the process, over all remaining base pair bonds. Despite of its great simplicity, for several known DNA sequences our results are in accord with available theoretical and experimental studies. Moreover, we report free-energy profiles along the denaturation pathway, which allow to detect stable or meta-stable partial denaturation states, composed of bubble, as local free-energy minima separated by barriers. Our approach allows to study very long DNA strands with commonly available computational power, as we demonstrate for a few random sequences in the range 200-800 base-pairs. For the latter, we also elucidate the self-averaging property of the system. Implications for the well known breathing dynamics of DNA are elucidated.

  9. Impact of radio frequency electromagnetic radiation on DNA integrity in the male germline.

    PubMed

    Aitken, R J; Bennetts, L E; Sawyer, D; Wiklendt, A M; King, B V

    2005-06-01

    Concern has arisen over human exposures to radio frequency electromagnetic radiation (RFEMR), including a recent report indicating that regular mobile phone use can negatively impact upon human semen quality. These effects would be particularly serious if the biological effects of RFEMR included the induction of DNA damage in male germ cells. In this study, mice were exposed to 900 MHz RFEMR at a specific absorption rate of approximately 90 mW/kg inside a waveguide for 7 days at 12 h per day. Following exposure, DNA damage to caudal epididymal spermatozoa was assessed by quantitative PCR (QPCR) as well as alkaline and pulsed-field gel electrophoresis. The treated mice were overtly normal and all assessment criteria, including sperm number, morphology and vitality were not significantly affected. Gel electrophoresis revealed no gross evidence of increased single- or double-DNA strand breakage in spermatozoa taken from treated animals. However, a detailed analysis of DNA integrity using QPCR revealed statistically significant damage to both the mitochondrial genome (p < 0.05) and the nuclear beta-globin locus (p < 0.01). This study suggests that while RFEMR does not have a dramatic impact on male germ cell development, a significant genotoxic effect on epididymal spermatozoa is evident and deserves further investigation.

  10. Sufficient minimal model for DNA denaturation: Integration of harmonic scalar elasticity and bond energies

    NASA Astrophysics Data System (ADS)

    Singh, Amit Raj; Granek, Rony

    2016-10-01

    We study DNA denaturation by integrating elasticity — as described by the Gaussian network model — with bond binding energies, distinguishing between different base pairs and stacking energies. We use exact calculation, within the model, of the Helmholtz free-energy of any partial denaturation state, which implies that the entropy of all formed "bubbles" ("loops") is accounted for. Considering base pair bond removal single events, the bond designated for opening is chosen by minimizing the free-energy difference for the process, over all remaining base pair bonds. Despite of its great simplicity, for several known DNA sequences our results are in accord with available theoretical and experimental studies. Moreover, we report free-energy profiles along the denaturation pathway, which allow to detect stable or meta-stable partial denaturation states, composed of bubble, as local free-energy minima separated by barriers. Our approach allows to study very long DNA strands with commonly available computational power, as we demonstrate for a few random sequences in the range 200-800 base-pairs. For the latter, we also elucidate the self-averaging property of the system. Implications for the well known breathing dynamics of DNA are elucidated.

  11. Selection of functional human sperm with higher DNA integrity and fewer reactive oxygen species

    PubMed Central

    Asghar, Waseem; Velasco, Vanessa; Kingsley, James L.; Shoukat, Muhammad S.; Shafiee, Hadi; Anchan, Raymond M.; Mutter, George L.; Tüzel, Erkan; Demirci, Utkan

    2014-01-01

    Fertilization and reproduction are central to the survival and propagation of a species. Couples who cannot reproduce naturally have to undergo in vitro clinical procedures. An integral part of these clinical procedures includes isolation of healthy sperm from raw semen. Existing sperm sorting methods are not efficient and isolate sperm having high DNA fragmentation and reactive oxygen species, and suffer from multiple manual steps and variations between embryologists. Inspired by in vivo natural sperm sorting mechanisms where vaginal mucus becomes less viscous to form microchannels to guide sperm towards egg, we present a chip that efficiently sorts healthy, motile and morphologically normal sperm without centrifugation. Higher percentage of sorted sperm show significantly lesser reactive oxygen species and DNA fragmentation than the conventional swim-up method. The presented chip is an easy-to-use high throughput sperm sorter that provides standardized sperm sorting assay with less reliance on embryologist’s skills, facilitating reliable operational steps. PMID:24753434

  12. Design of a Synthetic Integral Feedback Circuit: Dynamic Analysis and DNA Implementation.

    PubMed

    Briat, Corentin; Zechner, Christoph; Khammash, Mustafa

    2016-10-21

    The design and implementation of regulation motifs ensuring robust perfect adaptation are challenging problems in synthetic biology. Indeed, the design of high-yield robust metabolic pathways producing, for instance, drug precursors and biofuels, could be easily imagined to rely on such a control strategy in order to optimize production levels and reduce production costs, despite the presence of environmental disturbance and model uncertainty. We propose here a motif that ensures tracking and robust perfect adaptation for the controlled reaction network through integral feedback. Its metabolic load on the host is fully tunable and can be made arbitrarily close to the constitutive limit, the universal minimal metabolic load of all possible controllers. A DNA implementation of the controller network is finally provided. Computer simulations using realistic parameters demonstrate the good agreement between the DNA implementation and the ideal controller dynamics.

  13. Addressing RNA integrity to determine the impact of mitochondrial DNA mutations on brain mitochondrial function with age.

    PubMed

    Wang, Wei; Scheffler, Katja; Esbensen, Ying; Strand, Janne M; Stewart, James B; Bjørås, Magnar; Eide, Lars

    2014-01-01

    Mitochondrial DNA (mtDNA) mutations can result in mitochondrial dysfunction, but emerging experimental data question the fundamental role of mtDNA mutagenesis in age-associated mitochondrial impairment. The multicopy nature of mtDNA renders the impact of a given mtDNA mutation unpredictable. In this study, we compared mtDNA stability and mtRNA integrity during normal aging. Seven distinct sites in mouse brain mtDNA and corresponding mtRNA were analyzed. Accumulation of mtDNA mutations during aging was highly site-specific. The variation in mutation frequencies overrode the age-mediated increase by more than 100-fold and aging generally did not influence mtDNA mutagenesis. Errors introduced by mtRNA polymerase were also site-dependent and up to two hundred-fold more frequent than mtDNA mutations, and independent of mtDNA mutation frequency. We therefore conclude that mitochondrial transcription fidelity limits the impact of mtDNA mutations.

  14. Addressing RNA Integrity to Determine the Impact of Mitochondrial DNA Mutations on Brain Mitochondrial Function with Age

    PubMed Central

    Wang, Wei; Scheffler, Katja; Esbensen, Ying; Strand, Janne M.; Stewart, James B.; Bjørås, Magnar; Eide, Lars

    2014-01-01

    Mitochondrial DNA (mtDNA) mutations can result in mitochondrial dysfunction, but emerging experimental data question the fundamental role of mtDNA mutagenesis in age-associated mitochondrial impairment. The multicopy nature of mtDNA renders the impact of a given mtDNA mutation unpredictable. In this study, we compared mtDNA stability and mtRNA integrity during normal aging. Seven distinct sites in mouse brain mtDNA and corresponding mtRNA were analyzed. Accumulation of mtDNA mutations during aging was highly site-specific. The variation in mutation frequencies overrode the age-mediated increase by more than 100-fold and aging generally did not influence mtDNA mutagenesis. Errors introduced by mtRNA polymerase were also site-dependent and up to two hundred-fold more frequent than mtDNA mutations, and independent of mtDNA mutation frequency. We therefore conclude that mitochondrial transcription fidelity limits the impact of mtDNA mutations. PMID:24819950

  15. Prediction of the efficacy of immunotherapy by measuring the integrity of cell-free DNA in plasma in colorectal cancer.

    PubMed

    Kitahara, Masahiro; Hazama, Shoichi; Tsunedomi, Ryouichi; Takenouchi, Hiroko; Kanekiyo, Shinsuke; Inoue, Yuka; Nakajima, Masao; Tomochika, Shinobu; Tokuhisa, Yoshihiro; Iida, Michihisa; Sakamoto, Kazuhiko; Suzuki, Nobuaki; Takeda, Shigeru; Ueno, Tomio; Yamamoto, Shigeru; Yoshino, Shigefumi; Nagano, Hiroaki

    2016-12-01

    We previously reported a phase II study of a cancer vaccine using five novel peptides recognized by HLA-A*2402-restricted CTL in combination with oxaliplatin-containing chemotherapy (FXV study) as first-line therapy for patients with metastatic colorectal cancer and demonstrated the safety and promising potential of our five-peptide cocktail. The objective of this analysis was to identify predictive biomarkers for identifying patients who are likely to receive a clinical benefit from immunochemotherapy. Circulating cell-free DNA (cfDNA) in plasma has been reported to be a candidate molecular biomarker for the efficacy of anticancer therapy. Unlike uniformly truncated small-sized DNA released from apoptotic normal cells, DNA released from necrotic cancer cells varies in size. The integrity of plasma cfDNA (i.e. the ratio of longer fragments [400 bp] to shorter fragments [100 bp] of cfDNA), may be clinically useful for detecting colorectal cancer progression. We assessed plasma samples collected from 93 patients prior to receiving immunochemotherapy. The cfDNA levels and integrity were analyzed by semi-quantitative real-time PCR. Progression-free survival was significantly better in patients with a low plasma cfDNA integrity value than in those with a high value (P = 0.0027). Surprisingly, in the HLA-A*2402-matched group, patients with a low plasma cfDNA integrity value had significantly better progression-free survival than those with a high value (P = 0.0015). This difference was not observed in the HLA-A*2402-unmatched group. In conclusion, the integrity of plasma cfDNA may provide important clinical information and may be a useful predictive biomarker of the outcome of immunotherapy in metastatic colorectal cancer.

  16. Cell kinetics, DNA integrity, differentiation, and lipid fingerprinting analysis of rabbit adipose-derived stem cells.

    PubMed

    Barretto, Letícia Siqueira de Sá; Lessio, Camila; Sawaki e Nakamura, Ahy Natally; Lo Turco, Edson Guimarães; da Silva, Camila Gonzaga; Zambon, João Paulo; Gozzo, Fábio César; Pilau, Eduardo Jorge; de Almeida, Fernando Gonçalves

    2014-10-01

    Human adipose tissue has been described as a potential alternative reservoir for stem cells. Although studies have been performed in rabbits using autologous adipose-derived stem cells (ADSC), these cells have not been well characterized. The primary objectives of this study were to demonstrate the presence of adipose-derived stem cells isolated from rabbit inguinal fat pads and to characterize them through osteogenic and adipogenic in vitro differentiation and lipid fingerprinting analysis. The secondary objective was to evaluate cell behavior through growth kinetics, cell viability, and DNA integrity. Rabbit ADSCs were isolated to determine the in vitro growth kinetics and cell viability. DNA integrity was assessed by an alkaline Comet assay in passages 0 and 5. The osteogenic differentiation was evaluated by Von Kossa, and Alizarin Red S staining and adipogenic differentiation were assessed by Oil Red O staining. Lipid fingerprinting analyses of control, adipogenic, and osteogenic differentiated cells were performed by MALDI-TOF/MS. We demonstrate that rabbit ADSC have a constant growth rate at the early passages, with increased DNA fragmentation at or after passage 5. Rabbit ADSC viability was similar in passages 2 and 5 (90.7% and 86.6%, respectively), but there was a tendency to decreased cellular growth rate after passage 3. The ADSC were characterized by the expression of surface markers such as CD29 (67.4%) and CD44 (89.4%), using CD 45 (0.77%) as a negative control. ADSC from rabbits were successfully isolated form the inguinal region. These cells were capable to differentiate into osteogenic and adipogenic tissue when they were placed in inductive media. After each passage, there was a trend towards decreased cell growth. On the other hand, DNA fragmentation increased at each passage. ADSC had a different lipid profile when placed in control, adipogenic, or osteogenic media.

  17. Preparing Cellular DNA from Nuclei or Whole Cells.

    PubMed

    Nilsen, Timothy W

    2015-09-01

    It is often desirable to have cellular DNA on hand. DNA is stable and can be maintained intact for many years. This protocol describes the preparation of DNA from nuclei after the cytoplasmic extract has been removed. The resulting DNA is suitable for polymerase chain reactions and Southern blots to determine copy number and sites of integration of plasmids in stable cell lines. Quantitation of DNA may not be exact because RNA is not completely removed. The method can also be used on whole cells, but there will be more RNA contamination.

  18. Virus promoters determine interference by defective RNAs: selective amplification of mini-RNA vectors and rescue from cDNA by a 3' copy-back ambisense rabies virus.

    PubMed

    Finke, S; Conzelmann, K K

    1999-05-01

    Typical defective interfering (DI) RNAs are more successful in the competition for viral polymerase than the parental (helper) virus, which is mostly due to an altered DI promoter composition. Rabies virus (RV) internal deletion RNAs which possess the authentic RV terminal promoters, and which therefore are transcriptionally active and can be used as vectors for foreign gene expression, are poorly propagated in RV-infected cells and do not interfere with RV replication. To allow DI-like amplification and high-level gene expression from such mini-RNA vectors, we have used an engineered 3' copy-back (ambisense) helper RV in which the strong replication promoter of the antigenome was replaced with the 50-fold-weaker genome promoter. In cells coinfected with ambisense helper virus and mini-RNAs encoding chloramphenicol acetyltransferase (CAT) and luciferase, mini-RNAs were amplified to high levels. This was correlated with interference with helper virus replication, finally resulting in a clear predominance of mini-RNAs over helper virus. However, efficient successive passaging of mini-RNAs and high-level reporter gene activity could be achieved without adding exogenous helper virus, revealing a rather moderate degree of interference not precluding substantial HV propagation. Compared to infections with recombinant RV vectors expressing CAT, the availability of abundant mini-RNA templates led to increased levels of CAT mRNA such that CAT activities were augmented up to 250-fold, while virus gene transcription was kept to a minimum. We have also exploited the finding that internal deletion model RNAs behave like DI RNAs and are selectively amplified in the presence of ambisense helper virus to demonstrate for the first time RV-supported rescue of cDNA after transfection of mini-RNA cDNAs in ambisense RV-infected cells expressing T7 RNA polymerase.

  19. Microelectrophoresis devices with integrated fluorescence detectors and reactors for high-throughput DNA sequencing

    NASA Astrophysics Data System (ADS)

    Soper, Steven A.; Ford, Sean M.; Davies, Jack; Williams, Daryl C.; Cheng, Benxu; Klopf, J. Michael; Calderon, Gina M.; Saile, Volker

    1997-05-01

    This work describes the development of micro-devices for high-throughput DNA sequencing applications. Basically, two research efforts will be discussed; (1) fabrication and characterization of micro-reactors to prepare Sanger chain terminated DNA sequencing fragments on a nanoliter scale and; (2) x-ray photolithography of PMMA substrates for the high aspect ratio preparation of electrophoresis devices. The micro-reactor consisted of a 5'-biotinylated catfish olfactory gene, which was amplified by PCR, and attached to the interior wall of an aminoalkylisilane derivatized fused- silica capillary tube via a streptavidin/biotin linkage. Coverage of the interior capillary wall with biotinylated DNA averaged 77 percent. Stability of the anchored template under pressure and electroosmotic rinsing was favorable, requiring approximately 150 h of continuous rinsing to reduce the coverage by only 50 percent. The capillary micro- reactor was placed inside an air thermocycler to control temperature during Sanger ddNTP chain extension and directly coupled to a capillary separation column filled with a LPA solution via low dead volume capillary interlocks. The complimentary DNA fragments generated in the reactor were heat denatured from the immobilized template and directly injected onto a gel-filled capillary using electropumping for size fractionation and detection using NIR-LIF analysis. The total amount of termination fragments in the 31 nL reactor volume was estimated to be 5.2 X 1013 moles and sequencing was shown to produce read lengths on the order to 400 bases. Work will also be described concerning the development of micro-electrophoresis devices in x-ray sensitive photoresists using LIGA techniques. An electrophoresis device with an integrated fluorescence detector was constructed for the high resolution separation of DNA oligonucleotides. The choice of substrate for the electrophoresis was PMMA, due to its intrinsic low electroosmotic flow. Using x-ray lithography in

  20. Detecting DNA in Eukaryotic Cells Using an Integrated Microfluidics Electronic Sensor

    NASA Astrophysics Data System (ADS)

    Sohn, L. L.; Saleh, O. A.; Facer, G. R.; Carbeck, J. D.; Beavis, A.; Notterman, D. A.

    2000-03-01

    We have developed an integrated electronic sensor fabricated on a microfluidic chip which can measure the dielectric properties of material flowing through a micron-sized channel. We show that this sensor is advantageous over the usual optical detection techniques for microfluidics as it measures the inherent solid-state property of the analytes to be examined. In this talk, we show that we are able to not only identify small volumes of fluids but, more importantly, detect and diffentiate eukaryotic cells on the basis of DNA content.

  1. The epididymal sperm viability, motility and DNA integrity in dead mice maintained at 4-6oC

    PubMed Central

    Golshan Iranpour, Farhad; Rezazadeh Valojerdi, Mojtaba

    2013-01-01

    Background: When male animals die, spermatozoa within the body of animal will be degenerated. Because of unique chromatin structure of sperm, maybe this degeneration is different from other cells. However there is not any research which considered directly the integrity of sperm DNA by keeping the cadaver in refrigerator. Objective: The aim of this study was to assess viability, total motility and DNA integrity of sperm cells after death. Materials and Methods:In this experimental study, 24 male Swiss white mice were killed by cervical dislocation and then kept in refrigerator (4-6oC) for up to 12 days. On the 0 (immediately after death as control group), 1st, 2nd, 3rd, 5th, 7th, 10th and the 12th days after death cauda epididymides were removed and squeezed in Ham’s F10 medium. The proportion of viable, motile and double stranded DNA spermatozoa was examined. Viability and DNA integrity of sperm cells were examined consecutively by eosin nigrosin and acridine orange stainings. Results:The data obtained from this study showed that viability and total motility of sperm cells were significantly decreased during 12 days after death (p<0.001). In contrast with viability and motility, DNA integrity was without significant changes (even 12 days after death). Conclusion:This study suggests that integrity of sperm DNA would not change even after 12 days after death if the cadaver kept in refrigerator. PMID:24639746

  2. A Single-Array-Based Method for Detecting Copy Number Variants Using Affymetrix High Density SNP Arrays and its Application to Breast Cancer

    PubMed Central

    Li, Ming; Wen, Yalu; Fu, Wenjiang

    2014-01-01

    Cumulative evidence has shown that structural variations, due to insertions, deletions, and inversions of DNA, may contribute considerably to the development of complex human diseases, such as breast cancer. High-throughput genotyping technologies, such as Affymetrix high density single-nucleotide polymorphism (SNP) arrays, have produced large amounts of genetic data for genome-wide SNP genotype calling and copy number estimation. Meanwhile, there is a great need for accurate and efficient statistical methods to detect copy number variants. In this article, we introduce a hidden-Markov-model (HMM)-based method, referred to as the PICR-CNV, for copy number inference. The proposed method first estimates copy number abundance for each single SNP on a single array based on the raw fluorescence values, and then standardizes the estimated copy number abundance to achieve equal footing among multiple arrays. This method requires no between-array normalization, and thus, maintains data integrity and independence of samples among individual subjects. In addition to our efforts to apply new statistical technology to raw fluorescence values, the HMM has been applied to the standardized copy number abundance in order to reduce experimental noise. Through simulations, we show our refined method is able to infer copy number variants accurately. Application of the proposed method to a breast cancer dataset helps to identify genomic regions significantly associated with the disease. PMID:26279618

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

    PubMed

    Zheng, S J; Henken, B; Sofiari, E; Jacobsen, E; Krens, F A; Kik, C

    2001-06-01

    Genomic DNA blot hybridization is traditionally used to demonstrate that, via genetic transformation, foreign genes are integrated into host genomes. However, in large genome species, such as Allium cepa L., the use of genomic DNA blot hybridization is pushed towards its limits, because a considerable quantity of DNA is needed to obtain enough genome copies for a clear hybridization pattern. Furthermore, genomic DNA blot hybridization is a time-consuming method. Adaptor ligation PCR (AL-PCR) of genomic DNA flanking T-DNA borders does not have these drawbacks and seems to be an adequate alternative to genomic DNA blot hybridization. Using AL-PCR we proved that T-DNA was integrated into the A. cepa genome of three transgenic lines transformed with Agrobacterium tumefaciens EHA 105 (pCAMBIA 1301). The AL-PCR patterns obtained were specific and reproducible for a given transgenic line. The results showed that T-DNA integration took place and gave insight in the number of T-DNA copies present. Comparison of AL-PCR and previously obtained genomic DNA blot hybridization results pointed towards complex T-DNA integration patterns in some of the transgenic plants. After cloning and sequencing the AL-PCR products, the junctions between plant genomic DNA and the T-DNA insert could be analysed in great detail. For example it was shown that upon T-DNA integration a 66 bp genomic sequence was deleted, and no filler DNA was inserted. Primers located within the left and right flanking genomic DNA in transgenic shallot plants were used to recover the target site of T-DNA integration.

  4. Eclipse period of R1 plasmids during downshift from elevated copy number: Nonrandom selection of copies for replication.

    PubMed

    Olsson, Jan A; Berg, Otto; Nordström, Kurt; Dasgupta, Santanu

    2012-03-01

    The classical Meselson-Stahl density-shift method was used to study replication of pOU71, a runaway-replication derivative of plasmid R1 in Escherichia coli. The miniplasmid maintained the normal low copy number of R1 during steady growth at 30°C, but as growth temperatures were raised above 34°C, the copy number of the plasmid increased to higher levels, and at 42°C, it replicated without control in a runaway replication mode with lethal consequences for the host. The eclipse periods (minimum time between successive replication of the same DNA) of the plasmid shortened with rising copy numbers at increasing growth temperatures (Olsson et al., 2003). In this work, eclipse periods were measured during downshifts in copy number of pOU71 after it had replicated at 39 and 42°C, resulting in 7- and 50-fold higher than normal plasmid copy number per cell, respectively. Eclipse periods for plasmid replication, measured during copy number downshift, suggested that plasmid R1, normally selected randomly for replication, showed a bias such that a newly replicated DNA had a higher probability of replication compared to the bulk of the R1 population. However, even the unexpected nonrandom replication followed the copy number kinetics such that every generation, the plasmids underwent the normal inherited number of replication, n, independent of the actual number of plasmid copies in a newborn cell.

  5. Sperm DNA integrity testing: big halo is a good predictor of embryo quality and pregnancy after conventional IVF.

    PubMed

    Tandara, M; Bajić, A; Tandara, L; Bilić-Zulle, L; Šunj, M; Kozina, V; Goluža, T; Jukić, M

    2014-09-01

    Sperm DNA integrity is a sperm functional parameter of male fertility evaluation. Two parameters of sperm DNA integrity were observed: DNA damage expressed as DNA fragmentation index (DFI) and percentage of the DNA undamaged spermatozoa expressed as big halo. Halosperm test was used for sperm DNA integrity determination. The aim of this study was to evaluate which DNA integrity parameter is better as an embryo quality and pregnancy prognostic parameter after the conventional IVF. We evaluated two embryo groups (positive and negative group) according to the 3rd day cumulative embryo score. Big halo and DFI, as we expected, showed good correlation (r = -0.69; p < 0.001). Receiver operating characteristic (ROC) analyses show that DFI and big halo are significant (p < 0.001) as prognostic parameters of embryo quality. ROC curves comparison of DFI and big halo revealed the AUC value for big halo to be significantly higher (DFI AUC = 0.71 vs. big halo AUC = 0.83; p = 0.025) than for DFI. Big halo was found to be the only independent predictor of embryo quality. Sperm DNA integrity both parameters are good prognostic parameters of embryo quality after the conventional IVF where big halo seems to be better. ROC analyses show DFI and big halo as significant prognostic parameters for achieved pregnancy (AUC ± SE for DFI was 0.67 ± 0.06 and 0.75 ± 0.06 for big halo). To our knowledge, this is the first study demonstrating the correlation between sperm DNA undamaged rate expressed as big halo parameter and semen characteristics as well as the influence on fertilization rate, embryo quality and pregnancy in conventional IVF.

  6. Confirmed rare copy number variants implicate novel genes in schizophrenia.

    PubMed

    Tam, Gloria W C; van de Lagemaat, Louie N; Redon, Richard; Strathdee, Karen E; Croning, Mike D R; Malloy, Mary P; Muir, Walter J; Pickard, Ben S; Deary, Ian J; Blackwood, Douglas H R; Carter, Nigel P; Grant, Seth G N

    2010-04-01

    Understanding how cognitive processes including learning, memory, decision making and ideation are encoded by the genome is a key question in biology. Identification of sets of genes underlying human mental disorders is a path towards this objective. Schizophrenia is a common disease with cognitive symptoms, high heritability and complex genetics. We have identified genes involved with schizophrenia by measuring differences in DNA copy number across the entire genome in 91 schizophrenia cases and 92 controls in the Scottish population. Our data reproduce rare and common variants observed in public domain data from >3000 schizophrenia cases, confirming known disease loci as well as identifying novel loci. We found copy number variants in PDE10A (phosphodiesterase 10A), CYFIP1 [cytoplasmic FMR1 (Fragile X mental retardation 1)-interacting protein 1], K(+) channel genes KCNE1 and KCNE2, the Down's syndrome critical region 1 gene RCAN1 (regulator of calcineurin 1), cell-recognition protein CHL1 (cell adhesion molecule with homology with L1CAM), the transcription factor SP4 (specificity protein 4) and histone deacetylase HDAC9, among others (see http://www.genes2cognition.org/SCZ-CNV). Integrating the function of these many genes into a coherent model of schizophrenia and cognition is a major unanswered challenge.

  7. Lentivector integration sites in ependymal cells from a model of metachromatic leukodystrophy: non-B DNA as a new factor influencing integration.

    PubMed

    McAllister, Robert G; Liu, Jiahui; Woods, Matthew W; Tom, Sean K; Rupar, C Anthony; Barr, Stephen D

    2014-08-26

    The blood-brain barrier controls the passage of molecules from the blood into the central nervous system (CNS) and is a major challenge for treatment of neurological diseases. Metachromatic leukodystrophy is a neurodegenerative lysosomal storage disease caused by loss of arylsulfatase A (ARSA) activity. Gene therapy via intraventricular injection of a lentiviral vector is a potential approach to rapidly and permanently deliver therapeutic levels of ARSA to the CNS. We present the distribution of integration sites of a lentiviral vector encoding human ARSA (LV-ARSA) in murine brain choroid plexus and ependymal cells, administered via a single intracranial injection into the CNS. LV-ARSA did not exhibit a strong preference for integration in or near actively transcribed genes, but exhibited a strong preference for integration in or near satellite DNA. We identified several genomic hotspots for LV-ARSA integration and identified a consensus target site sequence characterized by two G-quadruplex-forming motifs flanking the integration site. In addition, our analysis identified several other non-B DNA motifs as new factors that potentially influence lentivirus integration, including human immunodeficiency virus type-1 in human cells. Together, our data demonstrate a clinically favorable integration site profile in the murine brain and identify non-B DNA as a potential new host factor that influences lentiviral integration in murine and human cells.

  8. The quality of sperm preparation medium affects the motility, viability, and DNA integrity of human spermatozoa

    PubMed Central

    Anbari, Fatemeh; Halvaei, Iman; Nabi, Ali; Ghazali, Shahin; Khalili, Mohammad Ali; Johansson, Lars

    2016-01-01

    AIM: The goal was to compare the effects of three different sperm preparation media on sperm motility, viability, and DNA integrity of semen samples from normozoospermic men. METHODS: A total of 15 normozoospermic males were included in the study. The semen analysis (SA) was performed in accordance with the WHO guidelines (2010). After SA, each sample was divided into three aliquots, and swim-up was performed with three different sperm preparation media (Sperm Preparation Media, Origio, Denmark; Ham's F10, Biochrome, Berlin, Germany; and VitaSperm™, Innovative Biotech, Iran). Sperm motility, viability, and DNA fragmentation were evaluated at 0, 1, 2, and 24 h after swim-up. RESULTS: There were no significant differences, at any time intervals, in the total sperm motility between the different sperm preparation media. However, the rate of progressive motility was significantly higher in spermatozoa prepared using the media from Origio in comparison with VitaSperm™ (P = 0.03), whereas no significant difference was found against Ham's F10 medium. No significant differences in sperm viability were seen between the media products. However, 1 h after swim-up, the extent of sperm DNA fragmentation was lower in the medium from Origio versus VitaSperm™ (P = 0.02). CONCLUSIONS: The data showed that the quality of medium for preparation of semen samples from normozoospermic men significantly affects the performance of spermatozoa in assisted conception programs. PMID:28216914

  9. Effects of mercuric chloride on antioxidant system and DNA integrity of the crab Charybdis japonica

    NASA Astrophysics Data System (ADS)

    Zhang, Hongxia; Pan, Luqing; Miao, Jingjing; Xu, Chaoqun

    2009-12-01

    Mercury (Hg) is one of the commonly encountered heavy metals, which is widespread in inshore sediments of China. In order to investigate the toxicity of Hg on marine invertebrates, we studied the effects of the divalent mercuricion (Hg2+) (at two final concentrations of 0.0025 and 0.0050 mg L-1, prepared with HgCl2) on metallothionein (MT) content, DNA integrity (DNA strand breaks) and catalase (CAT) in the gills and hepatopancreas, antioxidant enzyme activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), in the hemolymph, gills and hepatopancreas of the portunid crab Charybdis japonica for an experiment period up to 15 d. The results indicated that MT was significantly induced after 3 d, with a positive correlation with Hg2+ dose and time in the hepatopancreas and a negative correlation with Hg2+ dose and time in the gills. While CAT in the hemolymph was not detected, it increased in the hepatopancreas during the entire experiment; SOD and GPx in the three tissues were stimulated after 12 h, both attained peak value and then reduced during the experimental period. Meanwhile, DNA strand breaks were all induced significantly after 12 h. These results suggested the detoxification strategies against Hg2+ in three tissues of C. japonica.

  10. Integration of clinical point-of-care requirements in a DNA microarray genotyping test.

    PubMed

    Van Dorst, Bieke; Cremers, Amelieke; Jans, Karolien; Van Domburg, Trees; Steegen, Kim; Huang, Chengjun; Dorrer, Christian; Lagae, Liesbet; Ferwerda, Gerben; Stuyver, Lieven J

    2014-11-15

    Various proof-of-concept studies have shown the potential of biosensors with a high multiplex detection capability for the readout of DNA microarrays in a lab-on-a-chip. This is particularly interesting for the development of point-of-care genotyping tests, to screen for multiple pathogens and/or antibiotic resistance patterns. In this paper, an assay workflow is presented, suited for the development of novel lab-on-a-chips with an integrated DNA microarray. Besides the description of the different assay steps (DNA purification, amplification and detection), a control strategy is presented according to recommendations of the US Food and Drug Administration (FDA). To use a lab-on-a-chip for diagnostic applications, the optimization and evaluation of the assay performance with clinical samples is very important. Therefore, appropriate quantification methods are described, which allow optimization and evaluation of the separate assay steps, as well as total assay performance. In order to demonstrate and evaluate the total workflow, blood samples spiked with Streptococcus pneumoniae were tested. All blood samples with ≥ 10(3)CFU S. pneumoniae per ml of human blood were successfully detected by this genotyping assay.

  11. Integrative DNA, RNA, and protein evidence connects TREML4 to coronary artery calcification.

    PubMed

    Sen, Shurjo K; Boelte, Kimberly C; Barb, Jennifer J; Joehanes, Roby; Zhao, XiaoQing; Cheng, Qi; Adams, Lila; Teer, Jamie K; Accame, David S; Chowdhury, Soma; Singh, Larry N; Kavousi, Maryam; Peyser, Patricia A; Quigley, Laura; Priel, Debra Long; Lau, Karen; Kuhns, Douglas B; Yoshimura, Teizo; Johnson, Andrew D; Hwang, Shih-Jen; Chen, Marcus Y; Arai, Andrew E; Green, Eric D; Mullikin, James C; Kolodgie, Frank D; O'Donnell, Christopher J; Virmani, Renu; Munson, Peter J; McVicar, Daniel W; Biesecker, Leslie G

    2014-07-03

    Coronary artery calcification (CAC) is a heritable and definitive morphologic marker of atherosclerosis that strongly predicts risk for future cardiovascular events. To search for genes involved in CAC, we used an integrative transcriptomic, genomic, and protein expression strategy by using next-generation DNA sequencing in the discovery phase with follow-up studies using traditional molecular biology and histopathology techniques. RNA sequencing of peripheral blood from a discovery set of CAC cases and controls was used to identify dysregulated genes, which were validated by ClinSeq and Framingham Heart Study data. Only a single gene, TREML4, was upregulated in CAC cases in both studies. Further examination showed that rs2803496 was a TREML4 cis-eQTL and that the minor allele at this locus conferred up to a 6.5-fold increased relative risk of CAC. We characterized human TREML4 and demonstrated by immunohistochemical techniques that it is localized in macrophages surrounding the necrotic core of coronary plaques complicated by calcification (but not in arteries with less advanced disease). Finally, we determined by von Kossa staining that TREML4 colocalizes with areas of microcalcification within coronary plaques. Overall, we present integrative RNA, DNA, and protein evidence implicating TREML4 in coronary artery calcification. Our findings connect multimodal genomics data with a commonly used clinical marker of cardiovascular disease.

  12. An Integrated System for DNA Sequencing by Synthesis Using Novel Nucleotide Analogues

    PubMed Central

    Guo, Jia; Yu, Lin; Turro, Nicholas J.; Ju, Jingyue

    2010-01-01

    via click chemistry is unambiguously identified with this chip-SBS system. The second generation (G-2) SBS system was developed based on the concept that the closer the structures of the added nucleotide and the primer are to their natural counterparts, the more faithfully the polymerase would incorporate the nucleotide. In this approach, the polymerase reaction is performed with the combination of 3′-capped nucleotide reversible terminators (NRTs) and cleavable fluorescent dideoxynucleotides (ddNTPs). By sacrificing a small amount of the primers permanently terminated by ddNTPs, the majority of the primers extended by the reversible terminators are reverted to the natural ones after each sequencing cycle. We have also developed the 3′-capped nucleotide reversible terminators to solve the problem of deciphering the homopolymeric regions of the template in conventional pyrosequencing. The 3′-capping moiety on the DNA extension product temporarily terminates the polymerase reaction, which allows only one nucleotide to be incorporated during each sequencing cycle. Thus, the number of nucleotides in the homopolymeric regions are unambiguously determined using the 3′-capped NRTs. It has been established that millions of DNA templates can be immobilized on a chip surface through a variety of approaches. Therefore, the integration of these high-density DNA chips with the molecular-level SBS approaches described in this Account is expected to generate a high-throughput and accurate DNA sequencing system with wide applications in biological research and health care. PMID:20121268

  13. Comprehensive mapping of the human papillomavirus (HPV) DNA integration sites in cervical carcinomas by HPV capture technology

    PubMed Central

    Xu, Ruiping; Ke, Yang

    2016-01-01

    Integration of human papillomavirus (HPV) DNA into the host genome can be a driver mutation in cervical carcinoma. Identification of HPV integration at base resolution has been a longstanding technical challenge, largely due to sensitivity masking by HPV in episomes or concatenated forms. The aim was to enhance the understanding of the precise localization of HPV integration sites using an innovative strategy. Using HPV capture technology combined with next generation sequencing, HPV prevalence and the exact integration sites of the HPV DNA in 47 primary cervical cancer samples and 2 cell lines were investigated. A total of 117 unique HPV integration sites were identified, including HPV16 (n = 101), HPV18 (n = 7), and HPV58 (n = 9). We observed that the HPV16 integration sites were broadly located across the whole viral genome. In addition, either single or multiple integration events could occur frequently for HPV16, ranging from 1 to 19 per sample. The viral integration sites were distributed across almost all the chromosomes, except chromosome 22. All the cervical cancer cases harboring more than four HPV16 integration sites showed clinical diagnosis of stage III carcinoma. A significant enrichment of overlapping nucleotides shared between the human genome and HPV genome at integration breakpoints was observed, indicating that it may play an important role in the HPV integration process. The results expand on knowledge from previous findings on HPV16 and HPV18 integration sites and allow a better understanding of the molecular basis of the pathogenesis of cervical carcinoma. PMID:26735580

  14. Comprehensive mapping of the human papillomavirus (HPV) DNA integration sites in cervical carcinomas by HPV capture technology.

    PubMed

    Liu, Ying; Lu, Zheming; Xu, Ruiping; Ke, Yang

    2016-02-02

    Integration of human papillomavirus (HPV) DNA into the host genome can be a driver mutation in cervical carcinoma. Identification of HPV integration at base resolution has been a longstanding technical challenge, largely due to sensitivity masking by HPV in episomes or concatenated forms. The aim was to enhance the understanding of the precise localization of HPV integration sites using an innovative strategy. Using HPV capture technology combined with next generation sequencing, HPV prevalence and the exact integration sites of the HPV DNA in 47 primary cervical cancer samples and 2 cell lines were investigated. A total of 117 unique HPV integration sites were identified, including HPV16 (n = 101), HPV18 (n = 7), and HPV58 (n = 9). We observed that the HPV16 integration sites were broadly located across the whole viral genome. In addition, either single or multiple integration events could occur frequently for HPV16, ranging from 1 to 19 per sample. The viral integration sites were distributed across almost all the chromosomes, except chromosome 22. All the cervical cancer cases harboring more than four HPV16 integration sites showed clinical diagnosis of stage III carcinoma. A significant enrichment of overlapping nucleotides shared between the human genome and HPV genome at integration breakpoints was observed, indicating that it may play an important role in the HPV integration process. The results expand on knowledge from previous findings on HPV16 and HPV18 integration sites and allow a better understanding of the molecular basis of the pathogenesis of cervical carcinoma.

  15. Integrating multi-scale data on homologous recombination into a new recognition mechanism based on simulations of the RecA-ssDNA/dsDNA structure

    PubMed Central

    Yang, Darren; Boyer, Benjamin; Prévost, Chantal; Danilowicz, Claudia; Prentiss, Mara

    2015-01-01

    RecA protein is the prototypical recombinase. Members of the recombinase family can accurately repair double strand breaks in DNA. They also provide crucial links between pairs of sister chromatids in eukaryotic meiosis. A very broad outline of how these proteins align homologous sequences and promote DNA strand exchange has long been known, as are the crystal structures of the RecA-DNA pre- and postsynaptic complexes; however, little is known about the homology searching conformations and the details of how DNA in bacterial genomes is rapidly searched until homologous alignment is achieved. By integrating a physical model of recognition to new modeling work based on docking exploration and molecular dynamics simulation, we present a detailed structure/function model of homology recognition that reconciles extremely quick searching with the efficient and stringent formation of stable strand exchange products and which is consistent with a vast body of previously unexplained experimental results. PMID:26384422

  16. Circulating nucleic acids damage DNA of healthy cells by integrating into their genomes.

    PubMed

    Mittra, Indraneel; Khare, Naveen Kumar; Raghuram, Gorantla Venkata; Chaubal, Rohan; Khambatti, Fatema; Gupta, Deepika; Gaikwad, Ashwini; Prasannan, Preeti; Singh, Akshita; Iyer, Aishwarya; Singh, Ankita; Upadhyay, Pawan; Nair, Naveen Kumar; Mishra, Pradyumna Kumar; Dutt, Amit

    2015-03-01

    Whether nucleic acids that circulate in blood have any patho-physiological functions in the host have not been explored.We report here that far from being inert molecules, circulating nucleic acids have significant biological activities of their own that are deleterious to healthy cells of the body. Fragmented DNA and chromatin (DNAfs and Cfs) isolated from blood of cancer patients and healthy volunteers are readily taken up by a variety of cells in culture to be localized in their nuclei within a few minutes. The intra-nuclear DNAfs and Cfs associate themselves with host cell chromosomes to evoke a cellular DNA-damage-repair-response (DDR) followed by their incorporation into the host cell genomes. Whole genome sequencing detected the presence of tens of thousands of human sequence reads in the recipient mouse cells. Genomic incorporation of DNAfs and Cfs leads to dsDNA breaks and activation of apoptotic pathways in the treated cells. When injected intravenously into Balb/C mice, DNAfs and Cfs undergo genomic integration into cells of their vital organs resulting in activation of DDR and apoptotic proteins in the recipient cells. Cfs have significantly greater activity than DNAfs with respect to all parameters examined, while both DNAfs and Cfs isolated from cancer patients are more active than those from normal volunteers. All the above pathological actions of DNAfs and Cfs described above can be abrogated by concurrent treatment with DNase I and/or anti-histone antibody complexed nanoparticles both in vitro and in vivo. Taken together, our results suggest that circulating DNAfs and Cfs are physiological, continuously arising, endogenous DNA damaging agents with implications to ageing and a multitude of human pathologies including initiation of cancer.

  17. A multiplex real-time PCR-platform integrated into automated extraction method for the rapid detection and measurement of oncogenic HPV type-specific viral DNA load from cervical samples.

    PubMed

    Broccolo, Francesco

    2014-01-01

    The persistent infection with most frequent high-risk (HR)-HPV types (HPV-16, -18, -31, -33, -45, -52, and -58) is considered to be the true precursor of neoplastic progression. HR-HPV detection and genotyping is the most effective and accurate approach in screening of the early cervical lesions and cervical cancer, although also the HR-HPV DNA load is considered an ancillary marker for persistent HPV infection. Here, it is described an in-house multiplex quantitative real-time PCR (qPCR)-based typing system for the rapid detection and quantitation of the most common HR-HPV genotypes from cervical cytology screening tests. First, a separate qPCR assay to quantify a single-copy gene is recommended prior to screening (prescreening assay) to verify the adequate cellularity of the sample and the quality of DNA extracted and to normalize the HPV copy number per genomic DNA equivalent in the sample. Subsequently, to minimize the number of reactions, two multiplex qPCR assays (first line screening) are performed to detect and quantify HPV-16, -18, -31, -33, -45, -52, and -58 (HPV-18 and -45 are measured together by single-fluorophore). In addition, a multiplex qPCR assay specific for HPV-18 and HPV-45 is also available to type precisely the samples found to be positive for one of the two strains. Finally, two nucleic acid extraction methods are proposed by using a 96-well plate format: one manual method (supported by centrifuge or by vacuum) and one automated method integrated into a robotic liquid handler workstation to minimize material and hands-on time. In conclusion, this system provides a reliable high-throughput method for the rapid detection and quantitation of HR-HPV DNA load in cervical samples.

  18. Resistance against Friend leukemia virus-induced leukemogenesis in DNA-dependent protein kinase (DNA-PK)-deficient scid mice associated with defective viral integration at the Spi-1 and Fli-1 site.

    PubMed

    Hasegawa, Maki; Yamaguchi, Shuichi; Aizawa, Shiro; Ikeda, Hidetoshi; Tatsumi, Kouichi; Noda, Yuko; Hirokawa, Katsuiku; Kitagawa, Masanobu

    2005-08-01

    Retroviral DNA integration is mediated by the viral protein integrase. However, elements of the host DNA repair machinery such as the phosphatidylinositol 3-kinase (PI-3K)-related protein kinase family system would play a role in the integration of viral DNA into the host DNA. Here, we show that a host PI-3K-related protein kinase, DNA-dependent protein kinase (DNA-PK), plays a role in the specific integration of retroviral DNA and induction of retroviral diseases in vivo. DNA-PK-deficient scid mice inoculated with Friend leukemia virus (FLV) exhibited a random integration into their genomic DNA and expressed the viral envelope protein gp70. However, the specific integration of FLV at Spi-1 or Fli-1 sites did not occur in association with the significant resistance of scid mice to FLV-induced leukemogenesis. In contrast, the knockout of another member of the PI-3K-related protein kinase family, encoded by the ataxia telangiectasia mutated (ATM) gene, resulted in mice as sensitive to FLV-induced leukemogenesis as the wild type mice. FLV was specifically integrated into the DNA at Spi-1 and Fli-1 sites with significant expression of these transcription factors. These findings indicated that DNA-PK would be essential for controlling the in vivo integration of FLV at specific sites as well as the susceptibility to FLV-induced leukemogenesis.

  19. Stwl Modifies Chromatin Compaction and Is Required to Maintain DNA Integrity in the Presence of Perturbed DNA Replication

    PubMed Central

    Yi, Xia; de Vries, Hilda I.; Siudeja, Katarzyna; Rana, Anil; Lemstra, Willy; Brunsting, Jeanette F.; Kok, Rob M.; Smulders, Yvo M.; Schaefer, Matthias; Dijk, Freark; Shang, Yongfeng; Eggen, Bart J.L.; Kampinga, Harm H.

    2009-01-01

    Hydroxyurea, a well-known DNA replication inhibitor, induces cell cycle arrest and intact checkpoint functions are required to survive DNA replication stress induced by this genotoxic agent. Perturbed DNA synthesis also results in elevated levels of DNA damage. It is unclear how organisms prevent accumulation of this type of DNA damage that coincides with hampered DNA synthesis. Here, we report the identification of stonewall (stwl) as a novel hydroxyurea-hypersensitive mutant. We demonstrate that Stwl is required to prevent accumulation of DNA damage induced by hydroxyurea; yet, Stwl is not involved in S/M checkpoint regulation. We show that Stwl is a heterochromatin-associated protein with transcription-repressing capacities. In stwl mutants, levels of trimethylated H3K27 and H3K9 (two hallmarks of silent chromatin) are decreased. Our data provide evidence for a Stwl-dependent epigenetic mechanism that is involved in the maintenance of the normal balance between euchromatin and heterochromatin and that is required to prevent accumulation of DNA damage in the presence of DNA replication stress. PMID:19056684

  20. Rare Copy Number Variants

    PubMed Central

    Grozeva, Detelina; Kirov, George; Ivanov, Dobril; Jones, Ian R.; Jones, Lisa; Green, Elaine K.; St Clair, David M.; Young, Allan H.; Ferrier, Nicol; Farmer, Anne E.; McGuffin, Peter; Holmans, Peter A.; Owen, Michael J.; O’Donovan, Michael C.; Craddock, Nick

    2015-01-01

    Context Recent studies suggest that copy number variation in the human genome is extensive and may play an important role in susceptibility to disease, including neuropsychiatric disorders such as schizophrenia and autism. The possible involvement of copy number variants (CNVs) in bipolar disorder has received little attention to date. Objectives To determine whether large (>100 000 base pairs) and rare (found in <1% of the population) CNVs are associated with susceptibility to bipolar disorder and to compare with findings in schizophrenia. Design A genome-wide survey of large, rare CNVs in a case-control sample using a high-density microarray. Setting The Wellcome Trust Case Control Consortium. Participants There were 1697 cases of bipolar disorder and 2806 nonpsychiatric controls. All participants were white UK residents. Main Outcome Measures Overall load of CNVs and presence of rare CNVs. Results The burden of CNVs in bipolar disorder was not increased compared with controls and was significantly less than in schizophrenia cases. The CNVs previously implicated in the etiology of schizophrenia were not more common in cases with bipolar disorder. Conclusions Schizophrenia and bipolar disorder differ with respect to CNV burden in general and association with specific CNVs in particular. Our data are consistent with the possibility that possession of large, rare deletions may modify the phenotype in those at risk of psychosis: those possessing such events are more likely to be diagnosed as having schizophrenia, and those without them are more likely to be diagnosed as having bipolar disorder. PMID:20368508

  1. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

    Structural form, bonding scheme, and chromatin structure of and gene-modification experiments with deoxyribonucleic acid (DNA) are described. Indicates that DNA's double helix is variable and also flexible as it interacts with regulatory and other molecules to transfer hereditary messages. (DH)

  2. 11. Photographic copy of copy of Twin Lakes Outlet Works ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    11. Photographic copy of copy of Twin Lakes Outlet Works construction drawing dated January 15, 1951. Drawn by W.A. Doe for the Twin Lakes Reservoir and Canal Co. (copy in possession of Bureau of Reclamation, location of original unknown) 'AS CONSTRUCTED' PLANS OF 1949-1950, REHABILITATION OF TWIN LAKES RESERVOIR OUTLET WORKS, DETAILS OF UPSTREAM WING WALLS. - Twin Lakes Dam & Outlet Works, Beneath Twin Lakes Reservoir, T11S, R80W, S22, Twin Lakes, Lake County, CO

  3. 12. Photographic copy of copy of Twin Lakes Outlet Works ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    12. Photographic copy of copy of Twin Lakes Outlet Works construction drawing dated January 15, 1951. Drawn by W.A. Doe for the Twin Lakes Reservoir and Canal Co. (copy in possession of Bureau of Reclamation, location of original unknown) 'AS CONSTRUCTED' PLANS OF 1949-50, REHABILITATION OF TWIN LAKES RESERVOIR OUTLET WORKS, DETAILS OF DISCHARGE BASIN. - Twin Lakes Dam & Outlet Works, Beneath Twin Lakes Reservoir, T11S, R80W, S22, Twin Lakes, Lake County, CO

  4. Precise in-frame integration of exogenous DNA mediated by CRISPR/Cas9 system in zebrafish.

    PubMed

    Hisano, Yu; Sakuma, Tetsushi; Nakade, Shota; Ohga, Rie; Ota, Satoshi; Okamoto, Hitoshi; Yamamoto, Takashi; Kawahara, Atsuo

    2015-03-05

    The CRISPR/Cas9 system provides a powerful tool for genome editing in various model organisms, including zebrafish. The establishment of targeted gene-disrupted zebrafish (knockouts) is readily achieved by CRISPR/Cas9-mediated genome modification. Recently, exogenous DNA integration into the zebrafish genome via homology-independent DNA repair was reported, but this integration contained various mutations at the junctions of genomic and integrated DNA. Thus, precise genome modification into targeted genomic loci remains to be achieved. Here, we describe efficient, precise CRISPR/Cas9-mediated integration using a donor vector harbouring short homologous sequences (10-40 bp) flanking the genomic target locus. We succeeded in integrating with high efficiency an exogenous mCherry or eGFP gene into targeted genes (tyrosinase and krtt1c19e) in frame. We found the precise in-frame integration of exogenous DNA without backbone vector sequences when Cas9 cleavage sites were introduced at both sides of the left homology arm, the eGFP sequence and the right homology arm. Furthermore, we confirmed that this precise genome modification was heritable. This simple method enables precise targeted gene knock-in in zebrafish.

  5. Autoregulation of the dnaA-dnaN Operon and Effects of DnaA Protein Levels on Replication Initiation in Bacillus subtilis

    PubMed Central

    Ogura, Yoshitoshi; Imai, Yukiho; Ogasawara, Naotake; Moriya, Shigeki

    2001-01-01

    In Escherichia coli, the DnaA protein level appears to play a pivotal role in determining the timing of replication initiation. To examine the effects on replication initiation in B. subtilis, we constructed a strain in which a copy of the dnaA gene was integrated at the purA locus on the chromosome under the control of an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible promoter. However, increasing the DnaA level resulted in cell elongation and inhibition of cell growth by induction of the SOS response. Transcription of the native dnaA-dnaN operon was greatly reduced at high DnaA levels, but it was increased in a dnaA-null mutant, indicating autoregulation of the operon by DnaA. When a copy of the dnaN gene was added downstream of the additional dnaA gene at purA, the cells grew at high DnaA levels, suggesting that depletion of DnaN (β subunit of DNA polymerase III) within the cell by repression of the native dnaA-dnaN operon at high DnaA levels was the cause of the SOS induction. Flow cytometry of the cells revealed that the cell mass at initiation of replication increased at a lower DnaA level and decreased at DnaA levels higher than those of the wild type. Proper timing of replication initiation was observed at DnaA levels nearly comparable to the wild-type level. These results suggest that if the DnaA level increases with progression of the replication cycle, it could act as a rate-limiting factor of replication initiation in B. subtilis. PMID:11395445

  6. Autoregulation of the dnaA-dnaN operon and effects of DnaA protein levels on replication initiation in Bacillus subtilis.

    PubMed

    Ogura, Y; Imai, Y; Ogasawara, N; Moriya, S

    2001-07-01

    In Escherichia coli, the DnaA protein level appears to play a pivotal role in determining the timing of replication initiation. To examine the effects on replication initiation in B. subtilis, we constructed a strain in which a copy of the dnaA gene was integrated at the purA locus on the chromosome under the control of an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoter. However, increasing the DnaA level resulted in cell elongation and inhibition of cell growth by induction of the SOS response. Transcription of the native dnaA-dnaN operon was greatly reduced at high DnaA levels, but it was increased in a dnaA-null mutant, indicating autoregulation of the operon by DnaA. When a copy of the dnaN gene was added downstream of the additional dnaA gene at purA, the cells grew at high DnaA levels, suggesting that depletion of DnaN (beta subunit of DNA polymerase III) within the cell by repression of the native dnaA-dnaN operon at high DnaA levels was the cause of the SOS induction. Flow cytometry of the cells revealed that the cell mass at initiation of replication increased at a lower DnaA level and decreased at DnaA levels higher than those of the wild type. Proper timing of replication initiation was observed at DnaA levels nearly comparable to the wild-type level. These results suggest that if the DnaA level increases with progression of the replication cycle, it could act as a rate-limiting factor of replication initiation in B. subtilis.

  7. Arabidopsis DNA polymerase lambda mutant is mildly sensitive to DNA double strand breaks but defective in integration of a transgene.

    PubMed

    Furukawa, Tomoyuki; Angelis, Karel J; Britt, Anne B

    2015-01-01

    The DNA double-strand break (DSB) is a critical type of damage, and can be induced by both endogenous sources (e.g., errors of oxidative metabolism, transposable elements, programmed meiotic breaks, or perturbation of the DNA replication fork) and exogenous sources (e.g., ionizing radiation or radiomimetic chemicals). Although higher plants, like mammals, are thought to preferentially repair DSBs via nonhomologous end joining (NHEJ), much remains unclear about plant DSB repair pathways. Our reverse genetic approach suggests that DNA polymerase λ is involved in DSB repair in Arabidopsis. The Arabidopsis T-DNA insertion mutant (atpolλ-1) displayed sensitivity to both gamma-irradiation and treatment with radiomimetic reagents, but not to other DNA damaging treatments. The atpolλ-1 mutant showed a moderate sensitivity to DSBs, while Arabidopsis Ku70 and DNA ligase 4 mutants (atku70-3 and atlig4-2), both of which play critical roles in NHEJ, exhibited a hypersensitivity to these treatments. The atpolλ-1/atlig4-2 double mutant exhibited a higher sensitivity to DSBs than each single mutant, but the atku70/atpolλ-1 showed similar sensitivity to the atku70-3 mutant. We showed that transcription of the DNA ligase 1, DNA ligase 6, and Wee1 genes was quickly induced by BLM in several NHEJ deficient mutants in contrast to wild-type. Finally, the T-DNA transformation efficiency dropped in NHEJ deficient mutants and the lowest transformation efficiency was scored in the atpolλ-1/atlig4-2 double mutant. These results imply that AtPolλ is involved in both DSB repair and DNA damage response pathway.

  8. Arabidopsis DNA polymerase lambda mutant is mildly sensitive to DNA double strand breaks but defective in integration of a transgene

    PubMed Central

    Furukawa, Tomoyuki; Angelis, Karel J.; Britt, Anne B.

    2015-01-01

    The DNA double-strand break (DSB) is a critical type of damage, and can be induced by both endogenous sources (e.g., errors of oxidative metabolism, transposable elements, programmed meiotic breaks, or perturbation of the DNA replication fork) and exogenous sources (e.g., ionizing radiation or radiomimetic chemicals). Although higher plants, like mammals, are thought to preferentially repair DSBs via nonhomologous end joining (NHEJ), much remains unclear about plant DSB repair pathways. Our reverse genetic approach suggests that DNA polymerase λ is involved in DSB repair in Arabidopsis. The Arabidopsis T-DNA insertion mutant (atpolλ-1) displayed sensitivity to both gamma-irradiation and treatment with radiomimetic reagents, but not to other DNA damaging treatments. The atpolλ-1 mutant showed a moderate sensitivity to DSBs, while Arabidopsis Ku70 and DNA ligase 4 mutants (atku70-3 and atlig4-2), both of which play critical roles in NHEJ, exhibited a hypersensitivity to these treatments. The atpolλ-1/atlig4-2 double mutant exhibited a higher sensitivity to DSBs than each single mutant, but the atku70/atpolλ-1 showed similar sensitivity to the atku70-3 mutant. We showed that transcription of the DNA ligase 1, DNA ligase 6, and Wee1 genes was quickly induced by BLM in several NHEJ deficient mutants in contrast to wild-type. Finally, the T-DNA transformation efficiency dropped in NHEJ deficient mutants and the lowest transformation efficiency was scored in the atpolλ-1/atlig4-2 double mutant. These results imply that AtPolλ is involved in both DSB repair and DNA damage response pathway. PMID:26074930

  9. Comparison of Agrobacterium and particle bombardment using whole plasmid or minimal cassette for production of high-expressing, low-copy transgenic plants.

    PubMed

    Jackson, Mark A; Anderson, David J; Birch, Robert G

    2013-02-01

    Transgene integration complexity in the recipient genome can be an important determinant of transgene expression and field performance in transgenic crops. We provide the first direct comparison of Agrobacterium-mediated transformation (AMT) and particle bombardment using whole plasmid (WP) and excised minimal cassettes (MC), for transformation efficiency, transgene integration complexity and transgene expression in plants. To enable direct comparison, a selectable marker and a luciferase reporter gene were linked in identical configurations in plasmids suitable for AMT or direct gene transfer into sugarcane. Transformation efficiencies were similar between WP and MC when equal molar DNA quantities were delivered. When the MC concentration was reduced from 66 to 6.6 ng per shot, transformation efficiency dropped fourfold, to a level equivalent with AMT in amenable genotype Q117. The highest proportion of transformants combining low copy number (estimated below two integrated copies by qPCR) with expression of the non-selected reporter gene was obtained using AMT (55 %) or MC at low DNA concentration (30 %). In sugarcane, both of these methods yielded high-expressing, single-copy transgenic plant lines at a workable efficiency for practical plant improvement; but AMT is currently limited to a few amenable genotypes. These methods are best coupled with rapid early screens for desired molecular characteristics of transformants, e.g. PCR screens for low copy number and/or transcription of the gene of practical interest.

  10. An integrative model links multiple inputs and signaling pathways to the onset of DNA synthesis in hepatocytes

    PubMed Central

    Huard, Jérémy; Mueller, Stephanie; Gilles, Ernst D; Klingmüller, Ursula; Klamt, Steffen

    2012-01-01

    During liver regeneration, quiescent hepatocytes re-enter the cell cycle to proliferate and compensate for lost tissue. Multiple signals including hepatocyte growth factor, epidermal growth factor, tumor necrosis factor α, interleukin-6, insulin and transforming growth factor β orchestrate these responses and are integrated during the G1 phase of the cell cycle. To investigate how these inputs influence DNA synthesis as a measure for proliferation, we established a large-scale integrated logical model connecting multiple signaling pathways and the cell cycle. We constructed our model based upon established literature knowledge, and successively improved and validated its structure using hepatocyte-specific literature as well as experimental DNA synthesis data. Model analyses showed that activation of the mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways was sufficient and necessary for triggering DNA synthesis. In addition, we identified key species in these pathways that mediate DNA replication. Our model predicted oncogenic mutations that were compared with the COSMIC database, and proposed intervention targets to block hepatocyte growth factor-induced DNA synthesis, which we validated experimentally. Our integrative approach demonstrates that, despite the complexity and size of the underlying interlaced network, logical modeling enables an integrative understanding of signaling-controlled proliferation at the cellular level, and thus can provide intervention strategies for distinct perturbation scenarios at various regulatory levels. PMID:22443451

  11. An innovative and integrated approach based on DNA walking to identify unauthorised GMOs.

    PubMed

    Fraiture, Marie-Alice; Herman, Philippe; Taverniers, Isabel; De Loose, Marc; Deforce, Dieter; Roosens, Nancy H

    2014-03-15

    In the coming years, the frequency of unauthorised genetically modified organisms (GMOs) being present in the European food and feed chain will increase significantly. Therefore, we have developed a strategy to identify unauthorised GMOs containing a pCAMBIA family vector, frequently present in transgenic plants. This integrated approach is performed in two successive steps on Bt rice grains. First, the potential presence of unauthorised GMOs is assessed by the qPCR SYBR®Green technology targeting the terminator 35S pCAMBIA element. Second, its presence is confirmed via the characterisation of the junction between the transgenic cassette and the rice genome. To this end, a DNA walking strategy is applied using a first reverse primer followed by two semi-nested PCR rounds using primers that are each time nested to the previous reverse primer. This approach allows to rapidly identify the transgene flanking region and can easily be implemented by the enforcement laboratories.

  12. Integration and bioinformatics analysis of DNA-methylated genes associated with drug resistance in ovarian cancer

    PubMed Central

    YAN, BINGBING; YIN, FUQIANG; WANG, QI; ZHANG, WEI; LI, LI

    2016-01-01

    The main obstacle to the successful treatment of ovarian cancer is the development of drug resistance to combined chemotherapy. Among all the factors associated with drug resistance, DNA methylation apparently plays a critical role. In this study, we performed an integrative analysis of the 26 DNA-methylated genes associated with drug resistance in ovarian cancer, and the genes were further evaluated by comprehensive bioinformatics analysis including gene/protein interaction, biological process enrichment and annotation. The results from the protein interaction analyses revealed that at least 20 of these 26 methylated genes are present in the protein interaction network, indicating that they interact with each other, have a correlation in function, and may participate as a whole in the regulation of ovarian cancer drug resistance. There is a direct interaction between the phosphatase and tensin homolog (PTEN) gene and at least half of the other genes, indicating that PTEN may possess core regulatory functions among these genes. Biological process enrichment and annotation demonstrated that most of these methylated genes were significantly associated with apoptosis, which is possibly an essential way for these genes to be involved in the regulation of multidrug resistance in ovarian cancer. In addition, a comprehensive analysis of clinical factors revealed that the methylation level of genes that are associated with the regulation of drug resistance in ovarian cancer was significantly correlated with the prognosis of ovarian cancer. Overall, this study preliminarily explains the potential correlation between the genes with DNA methylation and drug resistance in ovarian cancer. This finding has significance for our understanding of the regulation of resistant ovarian cancer by methylated genes, the treatment of ovarian cancer, and improvement of the prognosis of ovarian cancer. PMID:27347118

  13. Integrated analysis of gene expression and DNA methylation changes induced by hepatocyte growth factor in human hepatocytes.

    PubMed

    Xie, Cheng-Rong; Sun, Hongguang; Wang, Fu-Qiang; Li, Zhao; Yin, Yi-Rui; Fang, Qin-Liang; Sun, Yu; Zhao, Wen-Xiu; Zhang, Sheng; Zhao, Wen-Xing; Wang, Xiao-Min; Yin, Zhen-Yu

    2015-09-01

    Hepatocellular carcinoma (HCC) is the one of most common malignant tumors. The tumor microenvironment has a role in not only supporting growth and survival of tumor cells, but also triggering tumor recurrence and metastasis. Hepatocyte growth factor (HGF), one of the important growth factors in the tumor microenvironment, has an important role in angiogenesis, tumorigenesis and regeneration. However, the exact mechanism by which HGF regulates HCC initiation and development via epigenetic reprogramming has remained elusive. The present study focused on the epigenetic modification and target tumor-suppressive genes of HGF treatment in HCC. Expression profiling and DNA methylation array were performed to investigate the function of HGF and examine global genomic DNA methylation changes, respectively. Integrated analysis of gene expression and DNA methylation revealed potential tumor suppressor genes (TSGs) in HCC. The present study showed the multiple functions of HGF in tumorous and non‑tumorous pathways and global genomic DNA methylation changes. HGF treatment upregulated the expression of DNA methyltransferase 1 (DNMT1). Overexpression of DNMT1 in HCC patients correlated with the malignant potential and poor prognosis of HCC. Furthermore, integration analysis of gene expression and DNA methylation changes revealed novel potential tumor suppressor genes TSGs including MYOCD, PANX2 and LHX9. The present study has provided mechanistic insight into epigenetic repression of TSGs through HGF‑induced DNA hypermethylation.

  14. Control of Genome Integrity by RFC Complexes; Conductors of PCNA Loading onto and Unloading from Chromatin during DNA Replication

    PubMed Central

    Shiomi, Yasushi; Nishitani, Hideo

    2017-01-01

    During cell division, genome integrity is maintained by faithful DNA replication during S phase, followed by accurate segregation in mitosis. Many DNA metabolic events linked with DNA replication are also regulated throughout the cell cycle. In eukaryotes, the DNA sliding clamp, proliferating cell nuclear antigen (PCNA), acts on chromatin as a processivity factor for DNA polymerases. Since its discovery, many other PCNA binding partners have been identified that function during DNA replication, repair, recombination, chromatin remodeling, cohesion, and proteolysis in cell-cycle progression. PCNA not only recruits the proteins involved in such events, but it also actively controls their function as chromatin assembles. Therefore, control of PCNA-loading onto chromatin is fundamental for various replication-coupled reactions. PCNA is loaded onto chromatin by PCNA-loading replication factor C (RFC) complexes. Both RFC1-RFC and Ctf18-RFC fundamentally function as PCNA loaders. On the other hand, after DNA synthesis, PCNA must be removed from chromatin by Elg1-RFC. Functional defects in RFC complexes lead to chromosomal abnormalities. In this review, we summarize the structural and functional relationships among RFC complexes, and describe how the regulation of PCNA loading/unloading by RFC complexes contributes to maintaining genome integrity. PMID:28134787

  15. Control of Genome Integrity by RFC Complexes; Conductors of PCNA Loading onto and Unloading from Chromatin during DNA Replication.

    PubMed

    Shiomi, Yasushi; Nishitani, Hideo

    2017-01-26

    During cell division, genome integrity is maintained by faithful DNA replication during S phase, followed by accurate segregation in mitosis. Many DNA metabolic events linked with DNA replication are also regulated throughout the cell cycle. In eukaryotes, the DNA sliding clamp, proliferating cell nuclear antigen (PCNA), acts on chromatin as a processivity factor for DNA polymerases. Since its discovery, many other PCNA binding partners have been identified that function during DNA replication, repair, recombination, chromatin remodeling, cohesion, and proteolysis in cell-cycle progression. PCNA not only recruits the proteins involved in such events, but it also actively controls their function as chromatin assembles. Therefore, control of PCNA-loading onto chromatin is fundamental for various replication-coupled reactions. PCNA is loaded onto chromatin by PCNA-loading replication factor C (RFC) complexes. Both RFC1-RFC and Ctf18-RFC fundamentally function as PCNA loaders. On the other hand, after DNA synthesis, PCNA must be removed from chromatin by Elg1-RFC. Functional defects in RFC complexes lead to chromosomal abnormalities. In this review, we summarize the structural and functional relationships among RFC complexes, and describe how the regulation of PCNA loading/unloading by RFC complexes contributes to maintaining genome integrity.

  16. An integrative approach to species discovery in odonates: from character-based DNA barcoding to ecology.

    PubMed

    Damm, Sandra; Schierwater, Bernd; Hadrys, Heike

    2010-09-01

    Modern taxonomy requires an analytical approach incorporating all lines of evidence into decision-making. Such an approach can enhance both species identification and species discovery. The character-based DNA barcode method provides a molecular data set that can be incorporated into classical taxonomic data such that the discovery of new species can be made in an analytical framework that includes multiple sources of data. We here illustrate such a corroborative framework in a dragonfly model system that permits the discovery of two new, but visually cryptic species. In the African dragonfly genus Trithemis three distinct genetic clusters can be detected which could not be identified by using classical taxonomic characters. In order to test the hypothesis of two new species, DNA-barcodes from different sequence markers (ND1 and COI) were combined with morphological, ecological and biogeographic data sets. Phylogenetic analyses and incorporation of all data sets into a scheme called taxonomic circle highly supports the hypothesis of two new species. Our case study suggests an analytical approach to modern taxonomy that integrates data sets from different disciplines, thereby increasing the ease and reliability of both species discovery and species assignment.

  17. Epididymis seleno-independent glutathione peroxidase 5 maintains sperm DNA integrity in mice

    PubMed Central

    Chabory, Eléonore; Damon, Christelle; Lenoir, Alain; Kauselmann, Gary; Kern, Hedrun; Zevnik, Branko; Garrel, Catherine; Saez, Fabrice; Cadet, Rémi; Henry-Berger, Joelle; Schoor, Michael; Gottwald, Ulrich; Habenicht, Ursula; Drevet, Joël R.; Vernet, Patrick

    2009-01-01

    The mammalian epididymis provides sperm with an environment that promotes their maturation and protects them from external stresses. For example, it harbors an array of antioxidants, including non-conventional glutathione peroxidase 5 (GPX5), to protect them from oxidative stress. To explore the role of GPX5 in the epididymis, we generated mice that lack epididymal expression of the enzyme. Histological analyses of Gpx5–/– epididymides and sperm cells revealed no obvious defects. Furthermore, there were no apparent differences in the fertilization rate of sexually mature Gpx5–/– male mice compared with WT male mice. However, a higher incidence of miscarriages and developmental defects were observed when WT female mice were mated with Gpx5-deficient males over 1 year old compared with WT males of the same age. Flow cytometric analysis of spermatozoa recovered from Gpx5-null and WT male mice revealed that sperm DNA compaction was substantially lower in the cauda epididymides of Gpx5-null animals and that they suffered from DNA oxidative attacks. Real-time PCR analysis of enzymatic scavengers expressed in the mouse epididymis indicated that the cauda epididymidis epithelium of Gpx5-null male mice mounted an antioxidant response to cope with an excess of ROS. These observations suggest that GPX5 is a potent antioxidant scavenger in the luminal compartment of the mouse cauda epididymidis that protects spermatozoa from oxidative injuries that could compromise their integrity and, consequently, embryo viability. PMID:19546506

  18. Automatic on-chip RNA-DNA hybridization assay with integrated phase change microvalves

    NASA Astrophysics Data System (ADS)

    Weng, Xuan; Jiang, Hai; Wang, Junsheng; Chen, Shu; Cao, Honghe; Li, Dongqing

    2012-07-01

    An RNA-DNA hybridization assay microfluidic chip integrated with electrothermally actuated phase change microvalves for detecting pathogenic bacteria is presented in this paper. In order to realize the sequential loading and washing processes required in such an assay, gravity-based pressure-driven flow and phase-change microvalves were used in the microfluidic chip. Paraffin wax was used as the phase change material in the valves and thin film heaters were used to electrothermally actuate microvalves. Light absorption measured by a photodetector to determine the concentrations of the samples. The automatic control of the complete assay was implemented by a self-coded LabVIEW program. To examine the performance of this chip, Salmonella was used as a sample pathogen. Significantly, reduction in reagent/sample consumption (up to 20 folds) was achieved by this on-chip assay, compared with using the commercial test kit following the same protocol in conventional labs. The experimental results show that the quantitative detection can be obtained in approximately 26 min, and the detection limit is as low as 103 CFU ml-1. This RNA-DNA hybridization assay microfluidic chip shows an excellent potential in the development of a portable device for point-of-testing applications.

  19. Outer membrane protein functions as integrator of protein import and DNA inheritance in mitochondria

    PubMed Central

    Käser, Sandro; Oeljeklaus, Silke; Týč, Jiří; Vaughan, Sue; Warscheid, Bettina; Schneider, André

    2016-01-01

    Trypanosomatids are one of the earliest diverging eukaryotes that have fully functional mitochondria. pATOM36 is a trypanosomatid-specific essential mitochondrial outer membrane protein that has been implicated in protein import. Changes in the mitochondrial proteome induced by ablation of pATOM36 and in vitro assays show that pATOM36 is required for the assembly of the archaic translocase of the outer membrane (ATOM), the functional analog of the TOM complex in other organisms. Reciprocal pull-down experiments and immunofluorescence analyses demonstrate that a fraction of pATOM36 interacts and colocalizes with TAC65, a previously uncharacterized essential component of the tripartite attachment complex (TAC). The TAC links the single-unit mitochondrial genome to the basal body of the flagellum and mediates the segregation of the replicated mitochondrial genomes. RNAi experiments show that pATOM36, in line with its dual localization, is not only essential for ATOM complex assembly but also for segregation of the replicated mitochondrial genomes. However, the two functions are distinct, as a truncated version of pATOM36 lacking the 75 C-terminal amino acids can rescue kinetoplast DNA missegregation but not the lack of ATOM complex assembly. Thus, pATOM36 has a dual function and integrates mitochondrial protein import with mitochondrial DNA inheritance. PMID:27436903

  20. Outer membrane protein functions as integrator of protein import and DNA inheritance in mitochondria.

    PubMed

    Käser, Sandro; Oeljeklaus, Silke; Týč, Jiří; Vaughan, Sue; Warscheid, Bettina; Schneider, André

    2016-08-02

    Trypanosomatids are one of the earliest diverging eukaryotes that have fully functional mitochondria. pATOM36 is a trypanosomatid-specific essential mitochondrial outer membrane protein that has been implicated in protein import. Changes in the mitochondrial proteome induced by ablation of pATOM36 and in vitro assays show that pATOM36 is required for the assembly of the archaic translocase of the outer membrane (ATOM), the functional analog of the TOM complex in other organisms. Reciprocal pull-down experiments and immunofluorescence analyses demonstrate that a fraction of pATOM36 interacts and colocalizes with TAC65, a previously uncharacterized essential component of the tripartite attachment complex (TAC). The TAC links the single-unit mitochondrial genome to the basal body of the flagellum and mediates the segregation of the replicated mitochondrial genomes. RNAi experiments show that pATOM36, in line with its dual localization, is not only essential for ATOM complex assembly but also for segregation of the replicated mitochondrial genomes. However, the two functions are distinct, as a truncated version of pATOM36 lacking the 75 C-terminal amino acids can rescue kinetoplast DNA missegregation but not the lack of ATOM complex assembly. Thus, pATOM36 has a dual function and integrates mitochondrial protein import with mitochondrial DNA inheritance.

  1. Trace levels of mitomycin C disrupt genomic integrity and lead to DNA damage response defect in long-term-cultured human embryonic stem cells.

    PubMed

    Zhou, Di; Lin, Ge; Zeng, Si-Cong; Xiong, Bo; Xie, Ping-Yuan; Cheng, De-Hua; Zheng, Qing; Ouyang, Qi; Zhou, Xiao-Ying; Tang, Wei-Ling; Sun, Yi; Lu, Guang-Ying; Lu, Guang-Xiu

    2015-01-01

    How to maintain the genetic integrity of cultured human embryonic stem (hES) cells is raising crucial concerns for future clinical use in regenerative medicine. Mitomycin C(MMC), a DNA damage agent, is widely used for preparation of feeder cells in many laboratories. However, to what extent MMC affects the karyotypic stability of hES cells is not clear. Here, we measured residual MMC using High Performance Liquid Chromatography-Mass Spectrometry/Mass Spectrometry following each step of feeder preparation and found that 2.26 ± 0.77 and 3.50 ± 0.92 ng/ml remained in mouse feeder cells and human feeder cells, respectively. In addition, different amounts of MMC caused different chromosomal aberrations in hES cells. In particular, one abnormality, dup(1)(p32p36), was the same identical to one we previously reported in another hES cell line. Using Affymetrix SNP 6.0 arrays, the copy number variation changes of the hES cells maintained on MMC-inactivated feeders (MMC-feeder) were significantly more than those cultured on γ-inactivated feeder (IR-feeder) cells. Furthermore, DNA damage response (DDR) genes were down-regulated during long-term culture in the MMC-containing system, leading to DDR defect and shortened telomeres of hES cells, a sign of genomic instability. Therefore, MMC-feeder and MMC-induced genomic variation present an important safety problem that would limit such hES from being applied for future clinic use and drug screening.

  2. Structural basis for HIV-1 DNA integration in the human genome, role of the LEDGF/P75 cofactor

    PubMed Central

    Michel, Fabrice; Crucifix, Corinne; Granger, Florence; Eiler, Sylvia; Mouscadet, Jean-François; Korolev, Sergei; Agapkina, Julia; Ziganshin, Rustam; Gottikh, Marina; Nazabal, Alexis; Emiliani, Stéphane; Benarous, Richard; Moras, Dino; Schultz, Patrick; Ruff, Marc

    2009-01-01

    Integration of the human immunodeficiency virus (HIV-1) cDNA into the human genome is catalysed by integrase. Several studies have shown the importance of the interaction of cellular cofactors with integrase for viral integration and infectivity. In this study, we produced a stable and functional complex between the wild-type full-length integrase (IN) and the cellular cofactor LEDGF/p75 that shows enhanced in vitro integration activity compared with the integrase alone. Mass spectrometry analysis and the fitting of known atomic structures in cryo negatively stain electron microscopy (EM) maps revealed that the functional unit comprises two asymmetric integrase dimers and two LEDGF/p75 molecules. In the presence of DNA, EM revealed the DNA-binding sites and indicated that, in each asymmetric dimer, one integrase molecule performs the catalytic reaction, whereas the other one positions the viral DNA in the active site of the opposite dimer. The positions of the target and viral DNAs for the 3′ processing and integration reaction shed light on the integration mechanism, a process with wide implications for the understanding of viral-induced pathologies. PMID:19229293

  3. Photocopy of copy of 1922 map, revised in 1936. Copy ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Photocopy of copy of 1922 map, revised in 1936. Copy in the Fitzsimons Army Medical Center Directorate of Public Works, building 118. - Fitzsimons General Hospital, Bounded by East Colfax to south, Peoria Street to west, Denver City/County & Adams County Line to north, & U.S. Route 255 to east, Aurora, Adams County, CO

  4. 10. Photographic copy of copy of original construction drawing, dated ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    10. Photographic copy of copy of original construction drawing, dated 1899?. Original in possession of Twin Lakes Reservoir and Canal Company, Ordway, Colorado. PLAN OF DAM AND HEAD GATES FOR THE TWIN LAKES RESERVOIR. - Twin Lakes Dam & Outlet Works, Beneath Twin Lakes Reservoir, T11S, R80W, S22, Twin Lakes, Lake County, CO

  5. Effects of freezing-thawing on DNA integrity of boar spermatozoa assessed by the neutral comet assay.

    PubMed

    Fraser, L; Strzezek, J

    2005-12-01

    A modified version of the neutral comet assay was employed to evaluate the effect of the freezing-thawing process on boar-sperm DNA integrity. The sperm-rich fractions were collected from four mature boars and frozen into aluminium tubes and straws after extension in lactose-hen egg yolk-glycerol extender (lactose-HEY-G) or an extender containing lactose, lyophilized lipoprotein fractions extracted from ostrich egg yolk and glycerol (lactose-LPFo-G). The semen samples were also frozen in a standard boar semen extender (Kortowo-3), without the addition of cryoprotective substances. Post-thaw sperm motility and plasma membrane integrity, assessed by SYBR-14/PI and Hoechst 33258 stains, declined (p < or = 0.05) with a corresponding increase (p < or = 0.05) in sperm DNA damage, regardless of the extender type and packaging material. Spermatozoa frozen in lactose-HEY-G or lactose-LPFo-G extender showed lower (p < or = 0.05) DNA damage than those frozen in the absence of cryoprotective substances. The addition of HEY or LPFo to the freezing extender helped reduce the rate of cryo-damage to sperm DNA, which varied among the boars. Inter-boar variations in post-thaw DNA damage were more pronounced in sperm samples frozen in lactose-HEY-G or lactose-LPFo-G extender. The results of this study show that the freezing-thawing process affects the DNA integrity of boar spermatozoa, irrespective of the extender type and packaging material. Furthermore, the use of whole hen egg yolk and ostrich lyophilized lipoprotein fractions in the freezing extender gave similar results regarding sperm DNA integrity. It can be concluded that the neutral comet assay can be used in conjunction with routine sperm parameters for assessment of post-thaw quality of boar semen.

  6. Post-translational modifications of proliferating cell nuclear antigen: A key signal integrator for DNA damage response (Review).

    PubMed

    Zhu, Qiong; Chang, Yuxiao; Yang, Jin; Wei, Quanfang

    2014-05-01

    Previous studies have shown that the post-translational modifications of proliferating cell nuclear antigen (PCNA) may be crucial in influencing the cellular choice between different pathways, such as the cell cycle checkpoint, DNA repair or apoptosis pathways, in order to maintain genomic stability. DNA damage leads to replication stress and the subsequent induction of PCNA modification by small ubiquitin (Ub)-related modifiers and Ub, which has been identified to affect multiple biological processes of genomic DNA. Thus far, much has been learned concerning the behavior of modified PCNA as a key signal integrator in response to DNA damage. In humans and yeast, modified PCNA activates DNA damage bypass via an error-prone or error-free pathway to prevent the breakage of DNA replication forks, which may potentially induce double-strand breaks and subsequent chromosomal rearrangements. However, the exact mechanisms by which these pathways work and by what means the modified PCNA is involved in these processes remain elusive. Thus, the improved understanding of PCNA modification and its implications for DNA damage response may provide us with more insight into the mechanisms by which human cells regulate aberrant recombination events, and cancer initiation and development. The present review focuses on the post-translational modifications of PCNA and its important functions in mediating mammalian cellular response to different types of DNA damage.

  7. Circulating Cell-Free DNA in Dogs with Mammary Tumors: Short and Long Fragments and Integrity Index

    PubMed Central

    Bedin, Chiara; Romualdi, Chiara; Mainenti, Marta; Mollo, Antonio; Cavicchioli, Laura; Ferro, Silvia; Trez, Davide; De Maria, Raffaella; Nitti, Donato; Saccani, Andrea; Campanella, Michelangelo; Agostini, Marco; Zappulli, Valentina

    2017-01-01

    Circulating cell-free DNA (cfDNA) has been considered an interesting diagnostic/prognostic plasma biomarker in tumor-bearing subjects. In cancer patients, cfDNA can hypothetically derive from tumor necrosis/apoptosis, lysed circulating cells, and some yet unrevealed mechanisms of active release. This study aimed to preliminarily analyze cfDNA in dogs with canine mammary tumors (CMTs). Forty-four neoplastic, 17 non-neoplastic disease-bearing, and 15 healthy dogs were recruited. Necrosis and apoptosis were also assessed as potential source of cfDNA on 78 CMTs diagnosed from the 44 dogs. The cfDNA fragments and integrity index significantly differentiated neoplastic versus non-neoplastic dogs (P<0.05), and allowed the distinction between benign and malignant lesions (P<0.05). Even if without statistical significance, the amount of cfDNA was also affected by tumor necrosis and correlated with tumor size and apoptotic markers expression. A significant (P<0.01) increase of Bcl-2 in malignant tumors was observed, and in metastatic CMTs the evasion of apoptosis was also suggested. This study, therefore, provides evidence that cfDNA could be a diagnostic marker in dogs carrying mammary nodules suggesting that its potential application in early diagnostic procedures should be further investigated. PMID:28081183

  8. Paternal transmission of mitochondrial DNA as an integral part of mitochondrial inheritance in metapopulations of Drosophila simulans.

    PubMed

    Wolff, J N; Nafisinia, M; Sutovsky, P; Ballard, J W O

    2013-01-01

    Maternal inheritance is one of the hallmarks of animal mitochondrial DNA (mtDNA) and central to its success as a molecular marker. This mode of inheritance and subsequent lack of heterologous recombination allows us to retrace evolutionary relationships unambiguously down the matriline and without the confounding effects of recombinant genetic information. Accumulating evidence of biparental inheritance of mtDNA (paternal leakage), however, challenges our current understanding of how this molecule is inherited. Here, using Drosophila simulans collected from an East African metapopulation exhibiting recurring mitochondrial heteroplasmy, we conducted single fly matings and screened F1 offspring for the presence of paternal mtDNA using allele-specific PCR assays (AS-PCR). In all, 27 out of 4092 offspring were identified as harboring paternal mtDNA, suggesting a frequency of 0.66% paternal leakage in this species. Our findings strongly suggest that recurring mtDNA heteroplasmy as observed in natural populations of Drosophila simulans is most likely caused by repeated paternal leakage. Our findings further suggest that this phenomenon to potentially be an integral part of mtDNA inheritance in these populations and consequently of significance for mtDNA as a molecular marker.

  9. Wafer-scale integration of sacrificial nanofluidic chips for detecting and manipulating single DNA molecules

    PubMed Central

    Wang, Chao; Nam, Sung-Wook; Cotte, John M.; Jahnes, Christopher V.; Colgan, Evan G.; Bruce, Robert L.; Brink, Markus; Lofaro, Michael F.; Patel, Jyotica V.; Gignac, Lynne M.; Joseph, Eric A.; Rao, Satyavolu Papa; Stolovitzky, Gustavo; Polonsky, Stanislav; Lin, Qinghuang

    2017-01-01

    Wafer-scale fabrication of complex nanofluidic systems with integrated electronics is essential to realizing ubiquitous, compact, reliable, high-sensitivity and low-cost biomolecular sensors. Here we report a scalable fabrication strategy capable of producing nanofluidic chips with complex designs and down to single-digit nanometre dimensions over 200 mm wafer scale. Compatible with semiconductor industry standard complementary metal-oxide semiconductor logic circuit fabrication processes, this strategy extracts a patterned sacrificial silicon layer through hundreds of millions of nanoscale vent holes on each chip by gas-phase Xenon difluoride etching. Using single-molecule fluorescence imaging, we demonstrate these sacrificial nanofluidic chips can function to controllably and completely stretch lambda DNA in a two-dimensional nanofluidic network comprising channels and pillars. The flexible nanofluidic structure design, wafer-scale fabrication, single-digit nanometre channels, reliable fluidic sealing and low thermal budget make our strategy a potentially universal approach to integrating functional planar nanofluidic systems with logic circuits for lab-on-a-chip applications. PMID:28112157

  10. Microfluidic device for integrated restriction digestion reaction and resulting DNA fragment analysis.

    PubMed

    Xie, Hua; Li, Bowei; Zhong, Runtao; Qin, Jianhua; Zhu, Yisheng; Lin, Bingcheng

    2008-12-01

    A microfluidic system combining temperature-controlled reactor, analyte delivery, chip electrophoresis (CE) separation, and fluorescence detection was developed, in which the heaters, resistance temperature detectors, enzymatic reactors, CE channels, and pneumatic valves/pumps were integrated onto a single glass-PDMS chip. The microdevice was used to perform the digestion reaction, followed by on-line electrophoresis separation and detection of the resulting fragments with endonuclease BamHI and FokI as models. Pneumatic valves/pumps served not only for isolating the reaction region from the separation medium to prevent contamination, but also for delivering and quantitatively diluting the fluid from the reaction chamber to the CE section. Thus enzymatic reaction and electrophoresis separation could be insulated and connected as needed. A dynamic coating procedure with the use of PVP and mannitol was firstly adopted for glass-PDMS hybrid chip-based DNA separations, leading to an improved separation efficiency with reproducible migration time and theoretical plates. The expected 263- and 287-bp digestion products of BamHI and FokI were definitely verified by the size-based electrophoretic separation and detection. The whole integrated reaction-CE system can be manipulated in a simple manner with good reproducibility, which is expected to be applied in other on-line analysis of various biochemical reactions.

  11. Wafer-scale integration of sacrificial nanofluidic chips for detecting and manipulating single DNA molecules

    NASA Astrophysics Data System (ADS)

    Wang, Chao; Nam, Sung-Wook; Cotte, John M.; Jahnes, Christopher V.; Colgan, Evan G.; Bruce, Robert L.; Brink, Markus; Lofaro, Michael F.; Patel, Jyotica V.; Gignac, Lynne M.; Joseph, Eric A.; Rao, Satyavolu Papa; Stolovitzky, Gustavo; Polonsky, Stanislav; Lin, Qinghuang

    2017-01-01

    Wafer-scale fabrication of complex nanofluidic systems with integrated electronics is essential to realizing ubiquitous, compact, reliable, high-sensitivity and low-cost biomolecular sensors. Here we report a scalable fabrication strategy capable of producing nanofluidic chips with complex designs and down to single-digit nanometre dimensions over 200 mm wafer scale. Compatible with semiconductor industry standard complementary metal-oxide semiconductor logic circuit fabrication processes, this strategy extracts a patterned sacrificial silicon layer through hundreds of millions of nanoscale vent holes on each chip by gas-phase Xenon difluoride etching. Using single-molecule fluorescence imaging, we demonstrate these sacrificial nanofluidic chips can function to controllably and completely stretch lambda DNA in a two-dimensional nanofluidic network comprising channels and pillars. The flexible nanofluidic structure design, wafer-scale fabrication, single-digit nanometre channels, reliable fluidic sealing and low thermal budget make our strategy a potentially universal approach to integrating functional planar nanofluidic systems with logic circuits for lab-on-a-chip applications.

  12. Wafer-scale integration of sacrificial nanofluidic chips for detecting and manipulating single DNA molecules.

    PubMed

    Wang, Chao; Nam, Sung-Wook; Cotte, John M; Jahnes, Christopher V; Colgan, Evan G; Bruce, Robert L; Brink, Markus; Lofaro, Michael F; Patel, Jyotica V; Gignac, Lynne M; Joseph, Eric A; Rao, Satyavolu Papa; Stolovitzky, Gustavo; Polonsky, Stanislav; Lin, Qinghuang

    2017-01-23

    Wafer-scale fabrication of complex nanofluidic systems with integrated electronics is essential to realizing ubiquitous, compact, reliable, high-sensitivity and low-cost biomolecular sensors. Here we report a scalable fabrication strategy capable of producing nanofluidic chips with complex designs and down to single-digit nanometre dimensions over 200 mm wafer scale. Compatible with semiconductor industry standard complementary metal-oxide semiconductor logic circuit fabrication processes, this strategy extracts a patterned sacrificial silicon layer through hundreds of millions of nanoscale vent holes on each chip by gas-phase Xenon difluoride etching. Using single-molecule fluorescence imaging, we demonstrate these sacrificial nanofluidic chips can function to controllably and completely stretch lambda DNA in a two-dimensional nanofluidic network comprising channels and pillars. The flexible nanofluidic structure design, wafer-scale fabrication, single-digit nanometre channels, reliable fluidic sealing and low thermal budget make our strategy a potentially universal approach to integrating functional planar nanofluidic systems with logic circuits for lab-on-a-chip applications.

  13. Integrated taxonomy: traditional approach and DNA barcoding for the identification of filarioid worms and related parasites (Nematoda)

    PubMed Central

    Ferri, Emanuele; Barbuto, Michela; Bain, Odile; Galimberti, Andrea; Uni, Shigehiko; Guerrero, Ricardo; Ferté, Hubert; Bandi, Claudio; Martin, Coralie; Casiraghi, Maurizio

    2009-01-01

    Background We compared here the suitability and efficacy of traditional morphological approach and DNA barcoding to distinguish filarioid nematodes species (Nematoda, Spirurida). A reliable and rapid taxonomic identification of these parasites is the basis for a correct diagnosis of important and widespread parasitic diseases. The performance of DNA barcoding with different parameters was compared measuring the strength of correlation between morphological and molecular identification approaches. Molecular distance estimation was performed with two different mitochondrial markers (coxI and 12S rDNA) and different combinations of data handling were compared in order to provide a stronger tool for easy identification of filarioid worms. Results DNA barcoding and morphology based identification of filarioid nematodes revealed high coherence. Despite both coxI and 12S rDNA allow to reach high-quality performances, only coxI revealed to be manageable. Both alignment algorithm, gaps treatment, and the criteria used to define the threshold value were found to affect the performance of DNA barcoding with 12S rDNA marker. Using coxI and a defined level of nucleotide divergence to delimit species boundaries, DNA barcoding can also be used to infer potential new species. Conclusion An integrated approach allows to reach a higher discrimination power. The results clearly show where DNA-based and morphological identifications are consistent, and where they are not. The coherence between DNA-based and morphological identification for almost all the species examined in our work is very strong. We propose DNA barcoding as a reliable, consistent, and democratic tool for species discrimination in routine identification of parasitic nematodes. PMID:19128479

  14. DNA integrity assessment in hemocytes of soft-shell clams (Mya arenaria) in the Saguenay Fjord (Québec, Canada).

    PubMed

    Debenest, T; Gagné, F; Burgeot, T; Blaise, C; Pellerin, J

    2013-02-01

    The purpose of this study was to examine the effects of pollution on DNA integrity in the feral soft-shell clam (Mya arenaria) in the Saguenay Fjord. Intertidal clams were collected downstream and upstream of the fjord at sites under anthropogenic pollution. DNA integrity was assessed by following changes in single- and double-stranded breaks, variation in DNA content and micro-nuclei (MN) incidence in hemocytes. The results revealed that clams collected at polluted sites had reduced DNA strand breaks (lower DNA repair activity), increased DNA content variation and MN frequency in hemocytes. The data revealed that DNA content variation was closely related to MN frequency and negatively with DNA strand breaks formation. Water conductivity was also related to reduced MN frequency and DNA content variation, indicating that, in addition to the effects of pollution, the gradual dilution of saltwater could compromise mussel health.

  15. When genome integrity and cell cycle decisions collide: roles of polo kinases in cellular adaptation to DNA damage.

    PubMed

    Serrano, Diego; D'Amours, Damien

    2014-09-01

    The drive to proliferate and the need to maintain genome integrity are two of the most powerful forces acting on biological systems. When these forces enter in conflict, such as in the case of cells experiencing DNA damage, feedback mechanisms are activated to ensure that cellular proliferation is stopped and no further damage is introduced while cells repair their chromosomal lesions. In this circumstance, the DNA damage response dominates over the biological drive to proliferate, and may even result in programmed cell death if the damage cannot be repaired efficiently. Interestingly, the drive to proliferate can under specific conditions overcome the DNA damage response and lead to a reactivation of the proliferative program in checkpoint-arrested cells. This phenomenon is known as adaptation to DNA damage and is observed in all eukaryotic species where the process has been studied, including normal and cancer cells in humans. Polo-like kinases (PLKs) are critical regulators of the adaptation response to DNA damage and they play key roles at the interface of cell cycle and checkpoint-related decisions in cells. Here, we review recent progress in defining the specific roles of PLKs in the adaptation process and how this conserved family of eukaryotic kinases can integrate the fundamental need to preserve genomic integrity with effective cellular proliferation.

  16. Mechanisms of COPI vesicle formation

    PubMed Central

    Hsu, Victor W.; Yang, Jia-Shu

    2009-01-01

    Coat Protein I (COPI) is one of the most intensely investigated coat complexes. Numerous studies have contributed to a general understanding of how coat proteins act to initiate intracellular vesicular transport. This review highlights key recent findings that have shaped our current understanding of how COPI vesicles are formed. PMID:19854177

  17. Counting copy number and calories.

    PubMed

    White, Stefan

    2015-08-01

    Copy number variation (CNV) at several genomic loci has been associated with different human traits and diseases, but in many cases the findings could not be replicated. A new study provides insights into the degree of variation present at the amylase locus and calls into question a previous association between amylase copy number and body mass index.

  18. Application of DNA fingerprinting in medicolegal practice.

    PubMed

    Raina, Anupuma; Dogra, T D

    2002-12-01

    Fingerprinting is thought to establish the identify of an individual in forensic cases. The technique is extensively used for forensic purposes. Deoxyribonucleic acid (DNA) is the vehicle of generational transference of heritable unit. While arching markers for genetic disease professor Alec Jeffreys discovered that certain regions of DNA showed variations in the number of tandem repeats known as variable number of tandem repeats (VNTRs). Thus DNA fingerprint was named by observing the number of repeated sequences which differ from individual to individual. The structure of DNA is quite flexible, within the nucleus of each cell resides an identical copy of the individual's genetic material, DNA. The coding regions of the genomic DNA are known as genes. The DNA fingerprinting in forensic science has generated considerable excitement in the criminal justice community. DNA fingerprinting can be applied to identify an individual in criminal and civil cases. Polymerase chain reaction has revolutionised molecular biology it has an ability to amplify (usually fewer than 3000 bp) a particular sequence of DNA into million of copies in a very short period. Consequently only a very tiny amount of an organism's DNA needs to be available originally. This property of polymerase chain reaction has enabled to analyse many forensic samples particularly which are degraded. Microsatellite DNA or commonly as short tandem repeats are scattered throughout the human genome and occur on an average of every 10,000 nucleotides. Microsatellite markers are considered to be the most powerful genetic markers. Collection, preservation and handling are the integral part of DNA fingerprinting analysis. There are various methods to isolate DNA from different biological materials but presently most of the laboratories prefer using FTA paper. The age of humans can be estimated by using DNA based on telomere shortening.

  19. EVALUATION OF DNA INTEGRITY USING TUNEL AND COMET ASSAY IN HUMAN SEMEN: IMMEDIATE- VERSUS DELAYED-FREEZING

    EPA Science Inventory

    EVALUATION OF DNA INTEGRITY USING TUNEL AND COMET ASSAY IN HUMAN SEMEN: IMMEDIATE- VERSUS DELAYED-FREEZING
    K. Young,* L. Xun,* S. Rothmann,? S. Perreault, ? W. Robbins*
    *University of California, Los Angeles, Los Angeles, California; ?Fertility Solutions Inc., Cleveland, ...

  20. DNA

    ERIC Educational Resources Information Center

    Stent, Gunther S.

    1970-01-01

    This history for molecular genetics and its explanation of DNA begins with an analysis of the Golden Jubilee essay papers, 1955. The paper ends stating that the higher nervous system is the one major frontier of biological inquiry which still offers some romance of research. (Author/VW)

  1. DNA methylome in spleen of avian pathogenic Escherichia coli-challenged broilers and integration with mRNA expression.

    PubMed

    Xu, Haiping; Zhu, Xuenong; Hu, Yongsheng; Li, Zhenhui; Zhang, Xiquan; Nie, Qinghua; Nolan, Lisa K; Lamont, Susan J

    2014-03-06

    Avian pathogenic Escherichia coli (APEC) are responsible for heavy economic losses in poultry industry. Here we investigate DNA methylome of spleen and identify functional DNA methylation changes related to host response to APEC among groups of non-challenged chickens (NC), challenged with mild (MD) and severe pathology (SV). DNA methylation was enriched in the gene bodies and repeats. Promoter and CGIs are hypomethylated. Integration analysis revealed 22, 87, and 9 genes exhibiting inversely changed DNA methylation and gene expression in NC vs. MD, NC vs. SV, and MD vs. SV, respectively. IL8, IL2RB, and IL1RAPL1 were included. Gene network analysis suggested that besides inflammatory response, other networks and pathways such as organismal injury and abnormalities, cell signaling and molecular transport, are probably related to host response to APEC infection. Moreover, methylation changes in cell cycle processes might contribute to the lesion phenotype differences between MD and SV.

  2. DNA methylome in spleen of avian pathogenic escherichia coli-challenged broilers and integration with mRNA expression

    PubMed Central

    Xu, Haiping; Zhu, Xuenong; Hu, Yongsheng; Li, Zhenhui; Zhang, Xiquan; Nie, Qinghua

    2014-01-01

    Avian pathogenic Escherichia coli (APEC) are responsible for heavy economic losses in poultry industry. Here we investigate DNA methylome of spleen and identify functional DNA methylation changes related to host response to APEC among groups of non-challenged chickens (NC), challenged with mild (MD) and severe pathology (SV). DNA methylation was enriched in the gene bodies and repeats. Promoter and CGIs are hypomethylated. Integration analysis revealed 22, 87, and 9 genes exhibiting inversely changed DNA methylation and gene expression in NC vs. MD, NC vs. SV, and MD vs. SV, respectively. IL8, IL2RB, and IL1RAPL1 were included. Gene network analysis suggested that besides inflammatory response, other networks and pathways such as organismal injury and abnormalities, cell signaling and molecular transport, are probably related to host response to APEC infection. Moreover, methylation changes in cell cycle processes might contribute to the lesion phenotype differences between MD and SV. PMID:24599154

  3. Integrating a DNA Strand Displacement Reaction with a Whispering Gallery Mode Sensor for Label-Free Mercury (II) Ion Detection

    PubMed Central

    Wu, Fengchi; Wu, Yuqiang; Niu, Zhongwei; Vollmer, Frank

    2016-01-01

    Mercury is an extremely toxic chemical pollutant of our environment. It has attracted the world’s attention due to its high mobility and the ease with which it accumulates in organisms. Sensitive devices and methods specific for detecting mercury ions are, hence, in great need. Here, we have integrated a DNA strand displacement reaction with a whispering gallery mode (WGM) sensor for demonstrating the detection of Hg2+ ions. Our approach relies on the displacement of a DNA hairpin structure, which forms after the binding of mercury ions to an aptamer DNA sequence. The strand displacement reaction of the DNA aptamer provides highly specific and quantitative means for determining the mercury ion concentration on a label-free WGM sensor platform. Our approach also shows the possibility for manipulating the kinetics of a strand displacement reaction with specific ionic species. PMID:27483277

  4. Molecular Dissection of Mycobacterium tuberculosis Integration Host Factor Reveals Novel Insights into the Mode of DNA Binding and Nucleoid Compaction*

    PubMed Central

    Sharadamma, Narayanaswamy; Harshavardhana, Yadumurthy; Ravishankar, Apoorva; Anand, Praveen; Chandra, Nagasuma; Muniyappa, K.

    2014-01-01

    The annotated whole-genome sequence of Mycobacterium tuberculosis revealed that Rv1388 (Mtihf) is likely to encode for a putative 20-kDa integration host factor (mIHF). However, very little is known about the functional properties of mIHF or the organization of the mycobacterial nucleoid. Molecular modeling of the mIHF three-dimensional structure, based on the cocrystal structure of Streptomyces coelicolor IHF duplex DNA, a bona fide relative of mIHF, revealed the presence of Arg-170, Arg-171, and Arg-173, which might be involved in DNA binding, and a conserved proline (Pro-150) in the tight turn. The phenotypic sensitivity of Escherichia coli ΔihfA and ΔihfB strains to UV and methyl methanesulfonate could be complemented with the wild-type Mtihf but not its alleles bearing mutations in the DNA-binding residues. Protein-DNA interaction assays revealed that wild-type mIHF, but not its DNA-binding variants, binds with high affinity to fragments containing attB and attP sites and curved DNA. Strikingly, the functionally important amino acid residues of mIHF and the mechanism(s) underlying its binding to DNA, DNA bending, and site-specific recombination are fundamentally different from that of E. coli IHFαβ. Furthermore, we reveal novel insights into IHF-mediated DNA compaction depending on the placement of its preferred binding sites; mIHF promotes DNA compaction into nucleoid-like or higher order filamentous structures. We therefore propose that mIHF is a distinct member of a subfamily of proteins that serve as essential cofactors in site-specific recombination and nucleoid organization and that these findings represent a significant advance in our understanding of the role(s) of nucleoid-associated proteins. PMID:25324543

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

    SciTech Connect

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

    2013-12-22

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

  6. Integrating stakeholder perspectives into the translation of cell-free fetal DNA testing for aneuploidy

    PubMed Central

    2012-01-01

    Background The translation of novel genomic technologies from bench to bedside enjoins the comprehensive consideration of the perspectives of all stakeholders who stand to influence, or be influenced by, the translational course. Non-invasive prenatal aneuploidy testing that utilizes cell-free fetal DNA (cffDNA) circulating in maternal blood is one example of an innovative technology that promises significant benefits for its intended end users; however, it is currently uncertain whether it will achieve widespread clinical implementation. We conducted qualitative interviews with 18 diverse stakeholders in this domain, including prospective users of the technology and healthcare personnel, researchers and developers, and experts in social, legal, and regulatory aspects of genetic technology, and a pilot survey of 62 obstetric healthcare providers. Analysis of interview and survey data was combined with a review of the proceedings of a full-day, multidisciplinary conference on the topic and published scientific and ethics literature surrounding this and other relevant technologies. Discussion We constructed potential pathways for technological implementation, identified broad stakeholder classes party to these translational processes, and performed a preliminary assessment of the viewpoints and interrelations among these diverse stakeholders. Some of the stakeholders whose priorities are critical to understand and integrate into translation include pregnant women and their families; healthcare providers; scientists, their institutions or companies, and the funding agencies that support them; regulatory and judicial bodies; third-party payers; professional societies; educational systems; disability rights communities; and other representatives from civil society. Stakeholder interviews, survey findings, and conference proceedings add complexity to these envisioned pathways and also demonstrate a paramount need to incorporate an iterative stakeholder analysis early and

  7. Accumulation of human immunodeficiency virus type 1 DNA in T cells: results of multiple infection events.

    PubMed Central

    Robinson, H L; Zinkus, D M

    1990-01-01

    Human immunodeficiency virus type 1 DNA synthesis was followed in a CD4+ line of T cells (C8166) grown in the presence or absence of a monoclonal antibody to CD4 that blocks infection By 48 h after infection, cultures grown in the presence of the antibody contained approximately 4 copies of human immunodeficiency virus type 1 DNA per cell, whereas those grown in the absence of the antibody contained approximately 80 copies of viral DNA per cell. Most of the viral DNA in cultures grown in the absence of the antibody was present in a broad smear of apparently incomplete viral sequences. In cultures grown in the presence or absence of the antibody, the 9.6-kilobase linear duplex of viral DNA appeared to undergo integration within 24 h of its appearance. These results demonstrate that T cells accumulate unintegrated human immunodeficiency virus type 1 DNA as a result of multiple virions entering cells. Images PMID:2398529

  8. Nonhomologous end joining and homologous recombination DNA repair pathways in integration mutagenesis in the xylose-fermenting yeast Pichia stipitis.

    PubMed

    Maassen, Nicole; Freese, Stefan; Schruff, Barbara; Passoth, Volkmar; Klinner, Ulrich

    2008-08-01

    Pichia stipitis integrates linear homologous DNA fragments mainly ectopically. High rates of randomly occurring integration allow tagging mutagenesis with high efficiency using simply PCR amplificates of suitable selection markers from the P. stipitis genome. Linearization of an autonomously replicating vector caused a distinct increase of the transformation efficiency compared with the circular molecule. Cotransformation of a restriction endonuclease further enhanced the transformation efficiency. This effect was also observed with integrative vector DNA. In most cases vector integration in chromosomal targets did not depend on microhomologies, indicating that restriction-enzyme-mediated integration (REMI) does not play an essential role in P. stipitis. Small deletions were observed at the ends of the integrated vectors and in the target sites. Disruption of the PsKU80 gene increased the frequency of homologous integration considerably but resulted in a remarkable decrease of the transformation efficiency. These results suggest that in P. stipitis the nonhomologous end joining (NHEJ) pathway obviously predominates the homologous recombination pathway of double-strand break repair.

  9. Extrachromosomal HPV-16 LCR transcriptional activation by HDACi opposed by cellular differentiation and DNA integration.

    PubMed

    Bojilova, Ekaterina Dimitrova; Weyn, Christine; Antoine, Marie-Hélène; Fontaine, Véronique

    2016-11-15

    Histone deacetylase inhibitors (HDACi) have been shown to render HPV-carrying cells susceptible to intrinsic and extrinsic apoptotic signals. As such, these epigenetic drugs have entered clinical trials in the effort to treat cervical cancer. Here, we studied the effect of common HDACi, with an emphasis on Trichostatin A (TSA), on the transcriptional activity of the HPV-16 Long Control Region (LCR) in order to better understand the impact of these agents in the context of the HPV life cycle and infection. HDACi strongly induced transcription of the firefly luciferase reporter gene under the control of the HPV-16 LCR in a variety of cell lines. In the HaCaT keratinocyte cell line undergoing differentiation induced by TSA, we observed a reduction in LCR-controlled transcription. Three major AP-1 binding sites in the HPV-16 LCR are involved in the regulation by TSA. However, whatever the status of differentiation of the HaCaT cells, TSA induced integration of extra-chromosomal transfected DNA into the cellular genome. Although these data suggest caution using HDACi in the treatment of HR HPV infection, further in vivo studies are necessary to better assess the risk.

  10. Extrachromosomal HPV-16 LCR transcriptional activation by HDACi opposed by cellular differentiation and DNA integration

    PubMed Central

    Bojilova, Ekaterina Dimitrova; Weyn, Christine; Antoine, Marie-Hélène; Fontaine, Véronique

    2016-01-01

    Histone deacetylase inhibitors (HDACi) have been shown to render HPV-carrying cells susceptible to intrinsic and extrinsic apoptotic signals. As such, these epigenetic drugs have entered clinical trials in the effort to treat cervical cancer. Here, we studied the effect of common HDACi, with an emphasis on Trichostatin A (TSA), on the transcriptional activity of the HPV-16 Long Control Region (LCR) in order to better understand the impact of these agents in the context of the HPV life cycle and infection. HDACi strongly induced transcription of the firefly luciferase reporter gene under the control of the HPV-16 LCR in a variety of cell lines. In the HaCaT keratinocyte cell line undergoing differentiation induced by TSA, we observed a reduction in LCR-controlled transcription. Three major AP-1 binding sites in the HPV-16 LCR are involved in the regulation by TSA. However, whatever the status of differentiation of the HaCaT cells, TSA induced integration of extra-chromosomal transfected DNA into the cellular genome. Although these data suggest caution using HDACi in the treatment of HR HPV infection, further in vivo studies are necessary to better assess the risk. PMID:27705914

  11. Miniaturized devices towards an integrated lab-on-a-chip platform for DNA diagnostics

    NASA Astrophysics Data System (ADS)

    Kaprou, G.; Papadakis, G.; Kokkoris, G.; Papadopoulos, V.; Kefala, I.; Papageorgiou, D.; Gizeli, E.; Tserepi, A.

    2015-06-01

    Microfluidics is an emerging technology enabling the development of Lab-on-a-chip (LOC) systems for clinical diagnostics, drug discovery and screening, food safety and environmental analysis. LOC systems integrate and scale down one or several laboratory functions on a single chip of a few mm2 to cm2 in size, and account for many advantages on biochemical analyses, such as low sample and reagent consumption, low cost, reduced analysis time, portability and point-of-need compatibility. Currently, available nucleic acid diagnostic tests take advantage of Polymerase Chain Reaction (PCR) that allows exponential amplification of portions of nucleic acid sequences that can be used as indicators for the identification of various diseases. Here, we present a comparison between static chamber and continuous flow miniaturized PCR devices, in terms of energy consumption for devices fabricated on the same material stack, with identical sample volume and channel dimensions. The comparison is implemented by a computational study coupling heat transfer in both solid and fluid, mass conservation of species, and joule heating. Based on the conclusions of this study, we develop low-cost and fast DNA amplification devices for both PCR and isothermal amplification, and we implement them in the detection of mutations related to breast cancer. The devices are fabricated by mass production amenable technologies on printed circuit board (PCB) substrates, where copper facilitates the incorporation of on-chip microheaters, defining the thermal zones necessary for PCR or isothermal amplification methods.

  12. Effect of trehalose on DNA integrity of freeze-dried boar sperm, fertilization, and embryo development after intracytoplasmic sperm injection.

    PubMed

    Men, Nguyen Thi; Kikuchi, Kazuhiro; Nakai, Michiko; Fukuda, Atsunori; Tanihara, Fuminori; Noguchi, Junko; Kaneko, Hiroyuki; Linh, Nguyen Viet; Nguyen, Bui Xuan; Nagai, Takashi; Tajima, Atsushi

    2013-12-01

    Freeze-drying (FD) medium containing ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) is reported to be beneficial for maintenance of sperm DNA integrity after FD. Recently, trehalose has also been reported to have notable ability to stabilize the protein structure and biomembranes of sperm in a dry state. In this study, we examined the effect of a combination of EGTA and different concentrations of trehalose in FD medium on sperm DNA integrity and the in vitro development of IVM porcine oocytes after intracytoplasmic sperm injection (ICSI) using freeze-dried boar sperm. Ejaculated sperm from a boar were suspended in basic FD medium supplemented with 0, 3.75, 7.5, 15, 30, 60, or 90 mM trehalose and freeze-dried. After rehydration, the sperm in all groups were subjected to DNA damage detection using a Halomax kit. It was found that the level of DNA damage in 15-mM group was significantly lower than that in 0-mM group, and no difference was observed between the 15-, 7.5-, and 3.75-mM groups. Moreover, there were no significant differences in the DNA damage level among 0, 3.75 mM, and other groups treated with trehalose. When freeze-dried sperm were used for ICSI, the fertilization rates and blastocyst formation rates (observed at 10 hours and 6 days of IVC after ICSI, respectively) in the 7.5- and 15-mM groups were not different from those in 0-mM group. These results suggest that FD medium supplemented with trehalose at appropriate concentrations improves sperm DNA integrity, but does not improve fertilization and preimplantation embryo development of IVM oocytes following ICSI.

  13. High Resolution Copy Number Variation Data in the NCI-60 Cancer Cell Lines from Whole Genome Microarrays Accessible through CellMiner

    PubMed Central

    Varma, Sudhir; Pommier, Yves; Sunshine, Margot; Weinstein, John N.; Reinhold, William C.

    2014-01-01

    Array-based comparative genomic hybridization (aCGH) is a powerful technique for detecting gene copy number variation. It is generally considered to be robust and convenient since it measures DNA rather than RNA. In the current study, we combine copy number estimates from four different platforms (Agilent 44 K, NimbleGen 385 K, Affymetrix 500 K and Illumina Human1Mv1_C) to compute a reliable, high-resolution, easy to understand output for the measure of copy number changes in the 60 cancer cells of the NCI-DTP (the NCI-60). We then relate the results to gene expression. We explain how to access that database using our CellMiner web-tool and provide an example of the ease of comparison with transcript expression, whole exome sequencing, microRNA expression and response to 20,000 drugs and other chemical compounds. We then demonstrate how the data can be analyzed integratively with transcript expression data for the whole genome (26,065 genes). Comparison of copy number and expression levels shows an overall medium high correlation (median r = 0.247), with significantly higher correlations (median r = 0.408) for the known tumor suppressor genes. That observation is consistent with the hypothesis that gene loss is an important mechanism for tumor suppressor inactivation. An integrated analysis of concurrent DNA copy number and gene expression change is presented. Limiting attention to focal DNA gains or losses, we identify and reveal novel candidate tumor suppressors with matching alterations in transcript level. PMID:24670534

  14. Identification of human papillomavirus (HPV) 16 DNA integration and the ensuing patterns of methylation in HPV-associated head and neck squamous cell carcinoma cell lines.

    PubMed

    Hatano, Takashi; Sano, Daisuke; Takahashi, Hideaki; Hyakusoku, Hiroshi; Isono, Yasuhiro; Shimada, Shoko; Sawakuma, Kae; Takada, Kentaro; Oikawa, Ritsuko; Watanabe, Yoshiyuki; Yamamoto, Hiroyuki; Itoh, Fumio; Myers, Jeffrey N; Oridate, Nobuhiko

    2017-04-01

    Recent studies showed that human papillomavirus (HPV) integration contributes to the genomic instability seen in HPV-associated head and neck squamous cell carcinoma (HPV-HNSCC). However, the epigenetic alterations induced after HPV integration remains unclear. To identify the molecular details of HPV16 DNA integration and the ensuing patterns of methylation in HNSCC, we performed next-generation sequencing using a target-enrichment method for the effective identification of HPV16 integration breakpoints as well as the characterization of genomic sequences adjacent to HPV16 integration breakpoints with three HPV16-related HNSCC cell lines. The DNA methylation levels of the integrated HPV16 genome and that of the adjacent human genome were also analyzed by bisulfite pyrosequencing. We found various integration loci, including novel integration sites. Integration loci were located predominantly in the intergenic region, with a significant enrichment of the microhomologous sequences between the human and HPV16 genomes at the integration breakpoints. Furthermore, various levels of methylation within both the human genome and the integrated HPV genome at the integration breakpoints in each integrant were observed. Allele-specific methylation analysis suggested that the HPV16 integrants remained hypomethylated when the flanking host genome was hypomethylated. After integration into highly methylated human genome regions, however, the HPV16 DNA became methylated. In conclusion, we found novel integration sites and methylation patterns in HPV-HNSCC using our unique method. These findings may provide insights into understanding of viral integration mechanism and virus-associated carcinogenesis of HPV-HNSCC.

  15. Location of the unique integration site on an Escherichia coli chromosome by bacteriophage lambda DNA in vivo

    PubMed Central

    Tal, Asaf; Arbel-Goren, Rinat; Costantino, Nina; Court, Donald L.; Stavans, Joel

    2014-01-01

    The search for specific sequences on long genomes is a key process in many biological contexts. How can specific target sequences be located with high efficiency, within physiologically relevant times? We addressed this question for viral integration, a fundamental mechanism of horizontal gene transfer driving prokaryotic evolution, using the infection of Escherichia coli bacteria with bacteriophage λ and following the establishment of a lysogenic state. Following the targeting process in individual live E. coli cells in real time revealed that λ DNA remains confined near the entry point of a cell following infection. The encounter between the 15-bp-long target sequence on the chromosome and the recombination site on the viral genome is facilitated by the directed motion of bacterial DNA generated during chromosome replication, in conjunction with constrained diffusion of phage DNA. Moving the native bacterial integration site to different locations on the genome and measuring the integration frequency in these strains reveals that the frequencies of the native site and a site symmetric to it relative to the origin are similar, whereas both are significantly higher than when the integration site is moved near the terminus, consistent with the replication-driven mechanism we propose. This novel search mechanism is yet another example of the exquisite coevolution of λ with its host. PMID:24799672

  16. Development of a real-world direct interface for integrated DNA extraction and amplification in a microfluidic device.

    PubMed

    Shaw, Kirsty J; Joyce, Domino A; Docker, Peter T; Dyer, Charlotte E; Greenway, Gillian M; Greenman, John; Haswell, Stephen J

    2011-02-07

    Integrated DNA extraction and amplification have been carried out in a microfluidic device using electro-osmotic pumping (EOP) for fluidic control. All the necessary reagents for performing both DNA extraction and polymerase chain reaction (PCR) amplification were pre-loaded into the microfluidic device following encapsulation in agarose gel. Buccal cells were collected using OmniSwabs [Whatman™, UK] and manually added to a chaotropic binding/lysis solution pre-loaded into the microfluidic device. The released DNA was then adsorbed onto a silica monolith contained within the DNA extraction chamber and the microfluidic device sealed using polymer electrodes. The washing and elution steps for DNA extraction were carried out using EOP, resulting in transfer of the eluted DNA into the PCR chamber. Thermal cycling, achieved using a Peltier element, resulted in amplification of the Amelogenin locus as confirmed using conventional capillary gel electrophoresis. It was demonstrated that the PCR reagents could be stored in the microfluidic device for at least 8 weeks at 4 °C with no significant loss of activity. Such methodology lends itself to the production of 'ready-to-use' microfluidic devices containing all the necessary reagents for sample processing, with many obvious applications in forensics and clinical medicine.

  17. Meta-Analysis of DNA Tumor-Viral Integration Site Selection Indicates a Role for Repeats, Gene Expression and Epigenetics.

    PubMed

    Doolittle-Hall, Janet M; Cunningham Glasspoole, Danielle L; Seaman, William T; Webster-Cyriaque, Jennifer

    2015-11-10

    Oncoviruses cause tremendous global cancer burden. For several DNA tumor viruses, human genome integration is consistently associated with cancer development. However, genomic features associated with tumor viral integration are poorly understood. We sought to define genomic determinants for 1897 loci prone to hosting human papillomavirus (HPV), hepatitis B virus (HBV) or Merkel cell polyomavirus (MCPyV). These were compared to HIV, whose enzyme-mediated integration is well understood. A comprehensive catalog of integration sites was constructed from the literature and experimentally-determined HPV integration sites. Features were scored in eight categories (genes, expression, open chromatin, histone modifications, methylation, protein binding, chromatin segmentation and repeats) and compared to random loci. Random forest models determined loci classification and feature selection. HPV and HBV integrants were not fragile site associated. MCPyV preferred integration near sensory perception genes. Unique signatures of integration-associated predictive genomic features were detected. Importantly, repeats, actively-transcribed regions and histone modifications were common tumor viral integration signatures.

  18. New insights into the transition pathway from nonspecific to specific complex of DNA with Escherichia coli integration host factor.

    PubMed

    Vivas, Paula; Kuznetsov, Serguei V; Ansari, Anjum

    2008-05-15

    To elucidate the nature of the transition-state ensemble along the reaction pathway from a nonspecific protein-DNA complex to the specific complex, we have carried out measurements of DNA bending/unbending dynamics on a cognate DNA substrate in complex with integration host factor (IHF), an architectural protein from E. coli that bends its cognate site by approximately 180 degrees . We use a laser temperature jump to perturb the IHF-DNA complex and monitor the relaxation kinetics with time-resolved FRET measurements on DNA substrates end-labeled with a FRET pair. Previously, we showed that spontaneous bending/kinking of DNA, from thermal disruption of base-pairing/-stacking interactions, may be the rate-limiting step in the formation of the specific complex (Kuznetsov, S. V.; Sugimura, S.; Vivas, P.; Crothers, D. M.; Ansari, A. Proc. Natl. Acad. Sci. USA 2006, 103, 18515). Here, we probe the effect of varying [KCl], which affects the stability of the complex, on this rate-limiting step. We find that below approximately 250 mM KCl, the observed relaxation kinetics are from the unimolecular bending/unbending of DNA, and the relaxation rate kr is independent of [KCl]. Above approximately 300 mM KCl, dissociation of the IHF-DNA complex becomes significant, and the observed relaxation process includes contributions from the association/dissociation step, with kr decreasing with increasing [KCl]. The DNA bending step occurs with a positive activation enthalpy, despite the large negative enthalpy change reported for the specific IHF-DNA complex (Holbrook, J. A.; Tsodikov, O. V.; Saecker, R. M.; Record, M. T., Jr. J. Mol. Biol. 2001, 310, 379). Our conclusion from these studies is that in the uphill climb to the transition state, the DNA is kinked, but with no release of ions, as indicated by the salt-independent behavior of k(r) at low [KCl]. Any release of ions in the unimolecular process, together with conformational changes in the protein-DNA complex that facilitate

  19. CONTRA: copy number analysis for targeted resequencing

    PubMed Central

    Li, Jason; Lupat, Richard; Amarasinghe, Kaushalya C.; Thompson, Ella R.; Doyle, Maria A.; Ryland, Georgina L.; Tothill, Richard W.; Halgamuge, Saman K.; Campbell, Ian G.; Gorringe, Kylie L.

    2012-01-01

    Motivation: In light of the increasing adoption of targeted resequencing (TR) as a cost-effective strategy to identify disease-causing variants, a robust method for copy number variation (CNV) analysis is needed to maximize the value of this promising technology. Results: We present a method for CNV detection for TR data, including whole-exome capture data. Our method calls copy number gains and losses for each target region based on normalized depth of coverage. Our key strategies include the use of base-level log-ratios to remove GC-content bias, correction for an imbalanced library size effect on log-ratios, and the estimation of log-ratio variations via binning and interpolation. Our methods are made available via CONTRA (COpy Number Targeted Resequencing Analysis), a software package that takes standard alignment formats (BAM/SAM) and outputs in variant call format (VCF4.0), for easy integration with other next-generation sequencing analysis packages. We assessed our methods using samples from seven different target enrichment assays, and evaluated our results using simulated data and real germline data with known CNV genotypes. Availability and implementation: Source code and sample data are freely available under GNU license (GPLv3) at http://contra-cnv.sourceforge.net/ Contact: Jason.Li@petermac.org Supplementary information: Supplementary data are available at Bioinformatics online. PMID:22474122

  20. From sample to PCR product in under 45 minutes: a polymeric integrated microdevice for clinical and forensic DNA analysis.

    PubMed

    Lounsbury, Jenny A; Karlsson, Anne; Miranian, Daniel C; Cronk, Stephen M; Nelson, Daniel A; Li, Jingyi; Haverstick, Doris M; Kinnon, Paul; Saul, David J; Landers, James P

    2013-04-07

    The extraction and amplification of DNA from biological samples is laborious and time-consuming, requiring numerous instruments and sample handling steps. An integrated, single-use, poly(methyl methacrylate) (PMMA) microdevice for DNA extraction and amplification would benefit clinical and forensic communities, providing a completely closed system with rapid sample-in-PCR-product-out capability. Here, we show the design and simple flow control required for enzyme-based DNA preparation and PCR from buccal swabs or liquid whole blood samples with an ~5-fold reduction in time. A swab containing cells or DNA could be loaded into a novel receptacle together with the DNA liberation reagents, heated using an infrared heating system, mixed with PCR reagents for one of three different target sets under syringe-driven flow, and thermally-cycled in less than 45 min, an ~6-fold reduction in analysis time as compared to conventional methods. The 4 : 1 PCR reagents : DNA ratio required to provide the correct final concentration of all PCR components for effective amplification was verified using image analysis of colored dyes in the PCR chamber. Novel single-actuation, 'normally-open' adhesive valves were shown to effectively seal the PCR chamber during thermal cycling, preventing air bubble expansion. The effectiveness of the device was demonstrated using three target sets: the sex-typing gene Amelogenin, co-amplification of the β-globin and gelsolin genes, and the amplification of 15 short tandem repeat (STR) loci plus Amelogenin. The use of the integrated microdevice was expanded to the analysis of liquid blood samples which, when incubated with the DNA liberation reagents, form a brown precipitate that inhibits PCR. A simple centrifugation of the integrated microchips (on a custom centrifuge), mobilized the precipitate away from the microchannel entrance, improving amplification of the β-globin and gelsolin gene fragments by ~6-fold. This plastic integrated microdevice

  1. Association between sperm DNA integrity and seminal plasma antioxidant levels in health workers occupationally exposed to ionizing radiation

    SciTech Connect

    Kumar, Dayanidhi; Salian, Sujith Raj; Kalthur, Guruprasad; Uppangala, Shubhashree; Kumari, Sandhya; Challapalli, Srinivas; Chandraguthi, Shrinidhi Gururajarao; Jain, Navya; Krishnamurthy, Hanumanthappa; Kumar, Pratap; Adiga, Satish Kumar

    2014-07-15

    There is a paucity of data regarding the association between occupational radiation exposure and risk to human fertility. Recently, we provided the first evidence on altered sperm functional characteristics, DNA damage and hypermethylation in radiation health workers. However, there is no report elucidating the association between seminal plasma antioxidants and sperm chromatin integrity in occupationally exposed subjects. Here, we assessed the seminal plasma antioxidants and lipid peroxidation level in 83 men who were occupationally exposed to ionizing radiation and then correlated with the sperm chromatin integrity. Flow cytometry based sperm chromatin integrity assay revealed a significant decline in αt value in the exposed group in comparison to the non-exposed group (P<0.0001). Similarly, both total and reduced glutathione levels and total antioxidant capacity in the seminal plasma were significantly higher in exposed group than the non-exposed group (P<0.01, 0.001 and 0.0001, respectively). However, superoxide dismutase level and malondialdehyde level, which is an indicator of lipid peroxidation in the seminal plasma, did not differ significantly between two groups. The total antioxidant capacity (TAC) and GSH level exhibited a positive correlation with sperm DNA integrity in exposed subjects. To conclude, this study distinctly shows that altered sperm chromatin integrity in radiation health workers is associated with increase in seminal plasma antioxidant level. Further, the increased seminal plasma GSH and TAC could be an adaptive measure to tackle the oxidative stress to protect genetic and functional sperm deformities in radiation health workers. - Highlights: • Seminal plasma antioxidants were measured in men occupationally exposed to radiation. • Sperm chromatin integrity was significantly affected in the exposed group. • Glutathione and total antioxidant capacity was significantly higher in exposed group. • Sperm DNA damage in exposed subjects

  2. Integration of the Reconfigurable Self-Healing eDNA Architecture in an Embedded System

    NASA Technical Reports Server (NTRS)

    Boesen, Michael Reibel; Keymeulen, Didier; Madsen, Jan; Lu, Thomas; Chao, Tien-Hsin

    2011-01-01

    In this work we describe the first real world case study for the self-healing eDNA (electronic DNA) architecture by implementing the control and data processing of a Fourier Transform Spectrometer (FTS) on an eDNA prototype. For this purpose the eDNA prototype has been ported from a Xilinx Virtex 5 FPGA to an embedded system consisting of a PowerPC and a Xilinx Virtex 5 FPGA. The FTS instrument features a novel liquid crystal waveguide, which consequently eliminates all moving parts from the instrument. The addition of the eDNA architecture to do the control and data processing has resulted in a highly fault-tolerant FTS instrument. The case study has shown that the early stage prototype of the autonomous self-healing eDNA architecture is expensive in terms of execution time.

  3. Influence of human papillomavirus type 16 (HPV-16) E2 polymorphism on quantification of HPV-16 episomal and integrated DNA in cervicovaginal lavages from women with cervical intraepithelial neoplasia.

    PubMed

    Azizi, Naoufel; Brazete, Jessica; Hankins, Catherine; Money, Deborah; Fontaine, Julie; Koushik, Anita; Rachlis, Anita; Pourreaux, Karina; Ferenczy, Alex; Franco, Eduardo; Coutlée, François

    2008-07-01

    Integrated human papillomavirus type 16 (HPV-16) viral loads are currently estimated by quantification with real-time PCR of HPV-16 E6 (RT-E6 and HPV-16 PG) and E2 (RT-E2-1) DNA. We assessed the influence of HPV-16 E2 polymorphism on quantification of integrated HPV-16 DNA in anogenital specimens. HPV-16 E2 was sequenced from 135 isolates (123 from European and 12 from non-European lineages). An assay targeting conserved HPV-16 E2 sequences (RT-E2-2) was optimized and applied with RT-E6 and RT-E2-1 on 139 HPV-16-positive cervicovaginal lavages collected from 74 women [58 human immunodeficiency virus (HIV)-seropositive and 16 HIV-seronegative]. Ratios of HPV-16 copies measured with RT-E2-2 and RT-E2-1 obtained with African 2 (median=3.23, range=1.92-3.49) or Asian-American (median=3.78, range=1.47-37) isolates were greater than those obtained with European isolates (median=1.02, range=0.64-1.80; P<0.02 for each comparison). The distribution of HPV-16 E2 copies measured in 139 samples with RT-E2-2 (median=6150) and RT-E2-1 (median=8960) were different (P<0.0001). The risk of high-grade cervical intraepithelial neoplasia (CIN-2,3) compared with women without CIN was increased with higher HPV-16 total [odds ratio (OR)=2.17, 95 % confidence interval (CI)=1.11-4.23], episomal (OR=2.14, 95 % CI=1.09-4.19), but not for HPV-16 integrated viral load (OR=1.71, 95 % CI=0.90-3.26), after controlling for age, race, CD4 count, HIV and HPV-16 polymorphism. The proportion of samples with an E6/E2 ratio >2 in women without squamous intraepithelial lesion (7 of 35) was similar to that of women with CIN-2,3 (5 of 11, P=0.24) or CIN-1 (5 of 14, P=0.50). HPV-16 E2 polymorphism was a significant factor that influenced measures of HPV-16 integrated viral load.

  4. Human copy number variation and complex genetic disease.

    PubMed

    Girirajan, Santhosh; Campbell, Catarina D; Eichler, Evan E

    2011-01-01

    Copy number variants (CNVs) play an important role in human disease and population diversity. Advancements in technology have allowed for the analysis of CNVs in thousands of individuals with disease in addition to thousands of controls. These studies have identified rare CNVs associated with neuropsychiatric diseases such as autism, schizophrenia, and intellectual disability. In addition, copy number polymorphisms (CNPs) are present at higher frequencies in the population, show high diversity in copy number, sequence, and structure, and have been associated with multiple phenotypes, primarily related to immune or environmental response. However, the landscape of copy number variation still remains largely unexplored, especially for smaller CNVs and those embedded within complex regions of the human genome. An integrated approach including characterization of single nucleotide variants and CNVs in a large number of individuals with disease and normal genomes holds the promise of thoroughly elucidating the genetic basis of human disease and diversity.

  5. Global analysis of ion dependence unveils hidden steps in DNA binding and bending by integration host factor

    NASA Astrophysics Data System (ADS)

    Vivas, Paula; Velmurugu, Yogambigai; Kuznetsov, Serguei V.; Rice, Phoebe A.; Ansari, Anjum

    2013-09-01

    Proteins that recognize and bind to specific sites on DNA often distort the DNA at these sites. The rates at which these DNA distortions occur are considered to be important in the ability of these proteins to discriminate between specific and nonspecific sites. These rates have proven difficult to measure for most protein-DNA complexes in part because of the difficulty in separating the kinetics of unimolecular conformational rearrangements (DNA bending and kinking) from the kinetics of bimolecular complex association and dissociation. A notable exception is the Integration Host Factor (IHF), a eubacterial architectural protein involved in chromosomal compaction and DNA recombination, which binds with subnanomolar affinity to specific DNA sites and bends them into sharp U-turns. The unimolecular DNA bending kinetics has been resolved using both stopped-flow and laser temperature-jump perturbation. Here we expand our investigation by presenting a global analysis of the ionic strength dependence of specific binding affinity and relaxation kinetics of an IHF-DNA complex. This analysis enables us to obtain each of the underlying elementary rates (DNA bending/unbending and protein-DNA association/dissociation), and their ionic strength dependence, even under conditions where the two processes are coupled. Our analysis indicates interesting differences in the ionic strength dependence of the bi- versus unimolecular steps. At moderate [KCl] (100-500 mM), nearly all the ionic strength dependence to the overall equilibrium binding affinity appears in the bimolecular association/dissociation of an initial, presumably weakly bent, encounter complex, with a slope SKbi ≈ 8 describing the loglog-dependence of the equilibrium constant to form this complex on [KCl]. In contrast, the unimolecular equilibrium constant to form the fully wrapped specific complex from the initial complex is nearly independent of [KCl], with SKuni < 0.5. This result is counterintuitive because there

  6. Principles and Concepts of DNA Replication in Bacteria, Archaea, and Eukarya

    PubMed Central

    O’Donnell, Michael; Langston, Lance; Stillman, Bruce

    2013-01-01

    The accurate copying of genetic information in the double helix of DNA is essential for inheritance of traits that define the phenotype of cells and the organism. The core machineries that copy DNA are conserved in all three domains of life: bacteria, archaea, and eukaryotes. This article outlines the general nature of the DNA replication machinery, but also points out important and key differences. The most complex organisms, eukaryotes, have to coordinate the initiation of DNA replication from many origins in each genome and impose regulation that maintains genomic integrity, not only for the sake of each cell, but for the organism as a whole. In addition, DNA replication in eukaryotes needs to be coordinated with inheritance of chromatin, developmental patterning of tissues, and cell division to ensure that the genome replicates once per cell division cycle. PMID:23818497

  7. Fabrication and characterization of high-K dielectric integrated silicon nanowire sensor for DNA sensing application (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Jayakumar, Ganesh; Legallais, Maxime; Hellström, Per-Erik; Mouis, Mireille; Stambouli, Valérie; Ternon, Céline; Östling, Mikael

    2016-09-01

    1D silicon nanowires (SiNW) are attractive for charge based DNA sensing applications due to their small size and large surface to volume ratio. An ideal portable biosensor is expected to have repeatable and reliable sensitivity, selectivity, low production cost and small feature size. Instead of using tools such as e-beam that are capital and time intensive, we propose a low cost CMOS self-aligned-double-patterning I-line lithography process to fabricate 60 nm wide SiNW. DNA probes are grafted on a thin dielectric layer that is deposited on top of the SiNW surface. Here we used HfO2 instead of the usual SiO2. Indeed, compared to SiO2, HfO2 has been reported to have higher amount of OH groups on its surface leading to enhanced signal quality. We also report preliminary biosensor characterizations. After HfO2 functionalization and single-stranded DNA probe grafting onto the SiNWs, the sensors were first put in contact with fluorophore labelled complementary DNA targets in order to test the efficiency of DNA hybridization optically. Then, a sequence of hybridization, de-hybridization and re-hybridization steps was followed by Id-Vg measurements in order to measure the electrical response of the sensors to target DNA as well as recycling capability. After each step, SiNW devices exhibited a threshold voltage shift larger than device-to-device dispersion, showing that both complementary DNA hybridization and de-hybridization can be electrically detected. These results are very encouraging as they open new frontiers for heterogeneous integration of liquid interacting array of nano sensors with CMOS circuits to fabricate a complete lab on chip.

  8. Impact of estrogenic compounds on DNA integrity in human spermatozoa: evidence for cross-linking and redox cycling activities.

    PubMed

    Bennetts, L E; De Iuliis, G N; Nixon, B; Kime, M; Zelski, K; McVicar, C M; Lewis, S E; Aitken, R J

    2008-05-10

    A great deal of circumstantial evidence has linked DNA damage in human spermatozoa with adverse reproductive outcomes including reduced fertility and high rates of miscarriage. Although oxidative stress is thought to make a significant contribution to DNA damage in the male germ line, the factors responsible for creating this stress have not been elucidated. One group of compounds that are thought to be active in this context are the estrogens, either generated as a result of the endogenous metabolism of androgens within the male reproductive tract or gaining access to the latter as a consequence of environmental exposure. In this study, a wide variety of estrogenic compounds were assessed for their direct effects on human spermatozoa in vitro. DNA integrity was assessed using the Comet and TUNEL assays, lesion frequencies were quantified by QPCR using targets within the mitochondrial and nuclear (beta-globin) genomes, DNA adducts were characterized by mass spectrometry and redox activity was monitored using dihydroethidium (DHE) as the probe. Of the estrogenic and estrogen analogue compounds evaluated, catechol estrogens, quercetin, diethylstilbestrol and pyrocatechol stimulated intense redox activity while genistein was only active at the highest doses tested. Other estrogens and estrogen analogues, such as 17beta-estradiol, nonylphenol, bisphenol A and 2,3-dihydroxynaphthalene were inactive. Estrogen-induced redox activity was associated with a dramatic loss of motility and, in the case of 2-hydroxyestradiol, the induction of significant DNA fragmentation. Mass spectrometry also indicated that catechol estrogens were capable of forming dimers that can cross-link the densely packed DNA strands in sperm chromatin, impairing nuclear decondensation. These results highlight the potential importance of estrogenic compounds in creating oxidative stress and DNA damage in the male germ line and suggest that further exploration of these compounds in the aetiology of male

  9. Chromosome Conformation Capture Carbon Copy (5C) in Budding Yeast.

    PubMed

    Belton, Jon-Matthew; Dekker, Job

    2015-06-01

    Chromosome conformation capture carbon copy (5C) is a high-throughput method for detecting ligation products of interest in a chromosome conformation capture (3C) library. 5C uses ligation-mediated amplification (LMA) to generate carbon copies of 3C ligation product junctions using single-stranded oligonucleotide probes. This procedure produces a 5C library of short DNA molecules which represent the interactions between the corresponding restriction fragments. The 5C library can be amplified using universal primers containing the Illumina paired-end adaptor sequences for subsequent high-throughput sequencing.

  10. Zero-Copy Objects System

    NASA Technical Reports Server (NTRS)

    Burleigh, Scott C.

    2011-01-01

    Zero-Copy Objects System software enables application data to be encapsulated in layers of communication protocol without being copied. Indirect referencing enables application source data, either in memory or in a file, to be encapsulated in place within an unlimited number of protocol headers and/or trailers. Zero-copy objects (ZCOs) are abstract data access representations designed to minimize I/O (input/output) in the encapsulation of application source data within one or more layers of communication protocol structure. They are constructed within the heap space of a Simple Data Recorder (SDR) data store to which all participating layers of the stack must have access. Each ZCO contains general information enabling access to the core source data object (an item of application data), together with (a) a linked list of zero or more specific extents that reference portions of this source data object, and (b) linked lists of protocol header and trailer capsules. The concatenation of the headers (in ascending stack sequence), the source data object extents, and the trailers (in descending stack sequence) constitute the transmitted data object constructed from the ZCO. This scheme enables a source data object to be encapsulated in a succession of protocol layers without ever having to be copied from a buffer at one layer of the protocol stack to an encapsulating buffer at a lower layer of the stack. For large source data objects, the savings in copy time and reduction in memory consumption may be considerable.

  11. Integration of DNA barcoding into an ongoing inventory of complex tropical biodiversity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The extensive use of DNA barcoding technology in a large inventory of Macrolepidoptera and their parasitoids is documented. The methodology used and its practical applications are summarized, and numerous examples of how DNA barcoding has untangled complexes of cryptic species of butterflies, moths...

  12. The pathological consequences of impaired genome integrity in humans; disorders of the DNA replication machinery.

    PubMed

    O'Driscoll, Mark

    2017-01-01

    Accurate and efficient replication of the human genome occurs in the context of an array of constitutional barriers, including regional topological constraints imposed by chromatin architecture and processes such as transcription, catenation of the helical polymer and spontaneously generated DNA lesions, including base modifications and strand breaks. DNA replication is fundamentally important for tissue development and homeostasis; differentiation programmes are intimately linked with stem cell division. Unsurprisingly, impairments of the DNA replication machinery can have catastrophic consequences for genome stability and cell division. Functional impacts on DNA replication and genome stability have long been known to play roles in malignant transformation through a variety of complex mechanisms, and significant further insights have been gained from studying model organisms in this context. Congenital hypomorphic defects in components of the DNA replication machinery have been and continue to be identified in humans. These disorders present with a wide range of clinical features. Indeed, in some instances, different mutations in the same gene underlie different clinical presentations. Understanding the origin and molecular basis of these features opens a window onto the range of developmental impacts of suboptimal DNA replication and genome instability in humans. Here, I will briefly overview the basic steps involved in DNA replication and the key concepts that have emerged from this area of research, before switching emphasis to the pathological consequences of defects within the DNA replication network; the human disorders. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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

    PubMed

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

    2003-08-15

    Genome-wide synthetic genetic interaction screens with mutants in the mus81 and mms4 replication fork-processing genes identified a novel replication factor C (RFC) homolog, Elg1, which forms an alternative RFC complex with Rfc2-5. This complex is distinct from the DNA replication RFC, the DNA damage checkpoint RFC and the sister chromatid cohesion RFC. As expected from its genetic interactions, elg1 mutants are sensitive to DNA damage. Elg1 is redundant with Rad24 in the DNA damage response and contributes to activation of the checkpoint kinase Rad53. We find that elg1 mutants display DNA replication defects and genome instability, including increased recombination and mutation frequencies, and minichromosome maintenance defects. Mutants in elg1 show genetic interactions with pathways required for processing of stalled replication forks, and are defective in recovery from DNA damage during S phase. We propose that Elg1-RFC functions both in normal DNA replication and in the DNA damage response.

  14. A model for the control of DNA integrity by the sperm nuclear matrix

    PubMed Central

    Gawecka, Joanna E; Ribas-Maynou, Jordi; Benet, Jordi; Ward, W Steven

    2015-01-01

    The highly condensed chromatin of mammalian spermatozoa is usually considered to be biologically inert before fertilization. However, we have demonstrated that even in this compacted state, sperm chromatin is subject to degradation at open configurations associated with the nuclear matrix through a process we have termed sperm chromatin fragmentation (SCF). This suggests that a mechanism exists to monitor the health of spermatozoa during transit through the male reproductive tract and to destroy the genome of defective sperm cells. The site of DNA damage in SCF, the matrix attachment sites, are the same that we hypothesize initiate DNA synthesis in the zygote. When sperm that have damaged DNA are injected into the oocyte, the newly created zygote responds by delaying DNA synthesis in the male pronucleus and, if the damage is severe enough, arresting the embryo's development. Here we present a model for paternal DNA regulation by the nuclear matrix that begins during sperm maturation and continues through early embryonic development. PMID:25926613

  15. Final Scientific/Technical Report, DE-FG02-06ER64171, Integrated Nucleic Acid System for In-Field Monitoring of Microbial Community Dynamics and Metabolic Activity – Subproject to Co-PI Eric E. Roden

    SciTech Connect

    Eric E. Roden

    2009-07-08

    This report summarizes research conducted in conjunction with a project entitled “Integrated Nucleic Acid System for In-Field Monitoring of Microbial Community Dynamics and Metabolic Activity”, which was funded throu