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Sample records for integrating dna copy

  1. Enhanced single copy integration events in corn via particle bombardment using low quantities of DNA.

    PubMed

    Lowe, Brenda A; Shiva Prakash, N; Way, Melissa; Mann, Michael T; Spencer, T Michael; Boddupalli, Raghava S

    2009-12-01

    Transgene copy number is an important criterion for determining the utility of transgenic events. Single copy integration events are highly desirable when the objective is to produce marker free plants through segregation or when it is necessary to introgress different transgenes into commercial cultivars from different transgenic events. In contrast multi-copy events are advocated by several authors for higher expression of the transgene. Till recently, it was thought that employment of the particle gun for transformation results in the production of a high proportion of multi-copy events often with complex integration pattern when compared to Agrobacterium-mediated transformation. However, it has been demonstrated that usage of cassette DNA for bombardment in place of whole plasmids would result in simple insertion pattern of the transgenes. While investigating the effect of varying the cassette DNA amount on stable transformation, the frequency of occurrence of low copy events was observed to increase when lower doses of cassette DNA was employed for bombardment. Large scale experimentation with rigorous statistical analysis performed to verify the above observations employing Helium gun and the Electric discharge gun for gene delivery confirmed the above observations. Helium gun experiments involving production of more than 1,600 corn events consistently yielded single copy events at higher frequencies at lower cassette DNA load (46% at 2.5 ng/shot) as compared to higher cassette DNA load (29% at 25 ng/shot) across 18 independent experiments. Results were nearly identical with the Electric discharge particle gun device where single copy events were recovered at frequencies of 54% at 2.5 ng cassettes DNA per shot as compared to 18% at 25 ng cassette DNA per shot. The transformation frequency declined from 41 to 34% (Helium gun) and from 48 to 31% (Electric discharge gun) with reduction in cassette DNA quantity from 25 to 2.5 ng per shot. This reduction in the

  2. Changes in DNA damage, molecular integrity, and copy number for plastid DNA and mitochondrial DNA during maize development.

    PubMed

    Kumar, Rachana A; Oldenburg, Delene J; Bendich, Arnold J

    2014-12-01

    The amount and structural integrity of organellar DNAs change during plant development, although the mechanisms of change are poorly understood. Using PCR-based methods, we quantified DNA damage, molecular integrity, and genome copy number for plastid and mitochondrial DNAs of maize seedlings. A DNA repair assay was also used to assess DNA impediments. During development, DNA damage increased and molecules with impediments that prevented amplification by Taq DNA polymerase increased, with light causing the greatest change. DNA copy number values depended on the assay method, with standard real-time quantitative PCR (qPCR) values exceeding those determined by long-PCR by 100- to 1000-fold. As the organelles develop, their DNAs may be damaged in oxidative environments created by photo-oxidative reactions and photosynthetic/respiratory electron transfer. Some molecules may be repaired, while molecules with unrepaired damage may be degraded to non-functional fragments measured by standard qPCR but not by long-PCR. PMID:25261192

  3. Changes in DNA damage, molecular integrity, and copy number for plastid DNA and mitochondrial DNA during maize development

    PubMed Central

    Kumar, Rachana A.; Oldenburg, Delene J.; Bendich, Arnold J.

    2014-01-01

    The amount and structural integrity of organellar DNAs change during plant development, although the mechanisms of change are poorly understood. Using PCR-based methods, we quantified DNA damage, molecular integrity, and genome copy number for plastid and mitochondrial DNAs of maize seedlings. A DNA repair assay was also used to assess DNA impediments. During development, DNA damage increased and molecules with impediments that prevented amplification by Taq DNA polymerase increased, with light causing the greatest change. DNA copy number values depended on the assay method, with standard real-time quantitative PCR (qPCR) values exceeding those determined by long-PCR by 100- to 1000-fold. As the organelles develop, their DNAs may be damaged in oxidative environments created by photo-oxidative reactions and photosynthetic/respiratory electron transfer. Some molecules may be repaired, while molecules with unrepaired damage may be degraded to non-functional fragments measured by standard qPCR but not by long-PCR. PMID:25261192

  4. Directed gene copy number amplification in Pichia pastoris by vector integration into the ribosomal DNA locus.

    PubMed

    Marx, Hans; Mecklenbräuker, Astrid; Gasser, Brigitte; Sauer, Michael; Mattanovich, Diethard

    2009-12-01

    The yeast Pichia pastoris is a widely used host organism for heterologous protein production. One of the basic steps for strain improvement is to ensure a sufficient level of transcription of the heterologous gene, based on promoter strength and gene copy number. To date, high-copy-number integrants of P. pastoris are achievable only by screening of random events or by cloning of gene concatemers. Methods for rapid and reliable multicopy integration of the expression cassette are therefore desirable. Here we present such a method based on vector integration into the rDNA locus and post-transformational vector amplification by repeated selection on increased antibiotic concentrations. Data are presented for two exemplary products: human serum albumin, which is secreted into the supernatant, and human superoxide dismutase, which is accumulated in the cytoplasm of the cells. The striking picture evolving is that intracellular protein production is tightly correlated with gene copy number, while use of the secretory pathway introduces a high clonal variability and the correlation with gene copy number is valid only for low gene copy numbers. PMID:19799640

  5. Gene copy number variations in the leukocyte genome of hepatocellular carcinoma patients with integrated hepatitis B virus DNA

    PubMed Central

    Xu, Guixia; Cheng, Kai; Cao, Guangwen; Wu, Mengchao; Cheng, Shuqun; Liu, Shanrong

    2016-01-01

    Integration of hepatitis B virus (HBV) DNA into the human liver cell genome is believed to promote HBV-related carcinogenesis. This study aimed to quantify the integration of HBV DNA into the leukocyte genome in hepatocellular carcinoma (HCC) patients in order to identify potential biomarkers for HBV-related diseases. Whole-genome comparative genomic hybridization (CGH) chip array analyses were performed to screen gene copy number variations (CNV) in the leukocyte genome, and the results were confirmed by quantitative polymerase chain reaction (qPCR). The commonly detected regions included chromosome arms 19p, 5q, 1q and 15p, where 200 copy number gain events and 270 copy number loss events were noted. In particular, gains were observed in 5q35.3 (OR4F3) and 19p13.3 (OR4F17) in 90% of the samples. Successful homologous recombination of OR4F3 and the HBV P gene was demonstrated, and the amplification at 5q35.3 is potentially associated with the integration of HBV P gene into natural killer cells isolated from peripheral blood mononuclear cells (PBMCs). Receiver operating characteristic (ROC) curve analysis indicated that the combination of OR4F3 and OR4F17 a novel potential biomarker of HBV-related diseases. PMID:26769853

  6. Variable copy number DNA sequences in rice.

    PubMed

    Kikuchi, S; Takaiwa, F; Oono, K

    1987-12-01

    We have cloned two types of variable copy number DNA sequences from the rice embryo genome. One of these sequences, which was cloned in pRB301, was amplified about 50-fold during callus formation and diminished in copy number to the embryonic level during regeneration. The other clone, named pRB401, showed the reciprocal pattern. The copy numbers of both sequences were changed even in the early developmental stage and eliminated from nuclear DNA along with growth of the plant. Sequencing analysis of the pRB301 insert revealed some open reading frames and direct repeat structures, but corresponding sequences were not identified in the EMBL and LASL DNA databases. Sequencing of the nuclear genomic fragment cloned in pRB401 revealed the presence of the 3'rps12-rps7 region of rice chloroplast DNA. Our observations suggest that during callus formation (dedifferentiation), regeneration and the growth process the copy numbers of some DNA sequences are variable and that nuclear integrated chloroplast DNA acts as a variable copy number sequence in the rice genome. Based on data showing a common sequence in mitochondria and chloroplast DNA of maize (Stern and Lonsdale 1982) and that the rps12 gene of tobacco chloroplast DNA is a divided gene (Torazawa et al. 1986), it is suggested that the sequence on the inverted repeat structure of chloroplast DNA may have the character of a movable genetic element. PMID:3481021

  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. DNA copy number losses in human neoplasms.

    PubMed

    Knuutila, S; Aalto, Y; Autio, K; Björkqvist, A M; El-Rifai, W; Hemmer, S; Huhta, T; Kettunen, E; Kiuru-Kuhlefelt, S; Larramendy, M L; Lushnikova, T; Monni, O; Pere, H; Tapper, J; Tarkkanen, M; Varis, A; Wasenius, V M; Wolf, M; Zhu, Y

    1999-09-01

    This review summarizes reports of recurrent DNA sequence copy number losses in human neoplasms detected by comparative genomic hybridization. Recurrent losses that affect each of the chromosome arms in 73 tumor types are tabulated from 169 reports. The tables are available online at http://www.amjpathol.org and http://www. helsinki.fi/ approximately lglvwww/CMG.html. The genes relevant to the lost regions are discussed for each of the chromosomes. The review is supplemented also by a list of known and putative tumor suppressor genes and DNA repair genes (see Table 1, online). Losses are found in all chromosome arms, but they seem to be relatively rare at 1q, 2p, 3q, 5p, 6p, 7p, 7q, 8q, 12p, and 20q. Losses and their minimal common overlapping areas that were present in a great proportion of the 73 tumor entities reported in Table 2 (see online) are (in descending order of frequency): 9p23-p24 (48%), 13q21 (47%), 6q16 (44%), 6q26-q27 (44%), 8p23 (37%), 18q22-q23 (37%), 17p12-p13 (34%), 1p36.1 (34%), 11q23 (33%), 1p22 (32%), 4q32-qter (31%), 14q22-q23 (25%), 10q23 (25%), 10q25-qter (25%),15q21 (23%), 16q22 (23%), 5q21 (23%), 3p12-p14 (22%), 22q12 (22%), Xp21 (21%), Xq21 (21%), and 10p12 (20%). The frequency of losses at chromosomes 7 and 20 was less than 10% in all tumors. The chromosomal regions in which the most frequent losses are found implicate locations of essential tumor suppressor genes and DNA repair genes that may be involved in the pathogenesis of several tumor types. PMID:10487825

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

    PubMed

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

    2016-08-01

    Despite previously reported associations between peripheral blood mtDNA copy number and colorectal cancer, it remains unclear whether altered mtDNA copy number in peripheral blood is a risk factor for colorectal cancer or a biomarker for undiagnosed colorectal cancer. Though colorectal adenomas are well-recognized precursor lesions to colorectal cancer, no study has evaluated an association between mtDNA copy number and colorectal adenoma risk. Hence, we investigated an association between peripheral blood mtDNA copy number and incident, sporadic colorectal adenoma in 412 colorectal adenoma cases and 526 cancer-free controls pooled from three colonoscopy-based case-control studies that used identical methods for case ascertainment, risk factor determination, and biospecimen collection. We also evaluated associations between relative mtDNA copy number and markers of oxidative stress, including circulating F2 -isoprostanes, carotenoids, and fluorescent oxidation products. We measured mtDNA copy number using a quantitative real time polymerase chain reaction (PCR). We used unconditional logistic regression to analyze the association between mtDNA copy number and colorectal adenoma risk after multivariable adjustment. We found no association between logarithmically transformed relative mtDNA copy number, analyzed as a continuous variable, and colorectal adenoma risk (odds ratio = 1.02, 95%CI: 0.82-1.27; P = 0.86). There were no statistically significant associations between relative mtDNA copy number and other markers of oxidative stress. Our findings, taken together with those from previous studies, suggest that relative mtDNA copy number in peripheral blood may more likely be a marker of early colorectal cancer than of risk for the disease or of in vivo oxidative stress. © 2015 Wiley Periodicals, Inc. PMID:26258394

  10. Mitochondrial DNA Copy Number in Peripheral Blood and Melanoma Risk

    PubMed Central

    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). PMID:26110424

  11. DNA sequence copy number analysis by Comparative Genomic Hybridization (CGH)

    SciTech Connect

    Pinkel, D.; Kallioniemi, A.; Kallioniemi, O.; Waldman, F.; Sudar, D.; Gray, I. ); Rutovitz, D.; Piper, I. )

    1993-01-01

    Comparative Genomic Hybridization (CGH) uses the kinetics of in situ hybridization to compare the copy numbers of different DNA sequences within the same genome and the copy numbers of the same sequences among different genomes. In a typical application genomic DNA from a tumor and from normal cells are differentially labeled and simultaneously hybridized to normal metaphase chromosomes, and detected with different fluorochromes. Properly registered images of each fluorochrome are obtained using a microscope equipped with multi-band filters and a CCD camera. Digital image analysis permits measurement of intensity ratio profiles along each of the target chromosomes. Studies of cells with known aberrations indicate that the intensity ratio at each position is proportional to the ratio of the copy numbers of the sequences that bind there in the tumor and normal genomes. Analytical challenges posed by the need to efficiently obtain copy number karyotypes are discussed.

  12. 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

  13. 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

  14. Mitochondrial DNA Copy Number in Spermatozoa of Fertile Stallions.

    PubMed

    Orsztynowicz, M; Pawlak, P; Podstawski, Z; Nizanski, W; Partyka, A; Gotowiecka, M; Kosiniak-Kamysz, K; Lechniak, D

    2016-06-01

    Predicting male fertility on non-invasive sperm traits is of big importance to human and animal reproduction strategies. Combining the wide range of parameters monitored by computer-assisted sperm analysis (CASA) with some molecular traits (e.g. mtDNA content) may help to identify markers of the male fertility. The aim of this study was to characterize variation in the mtDNA copy number in equine sperm and to investigate whether mtDNA content is correlated with quality traits of stallion spermatozoa and the age of the male. Ejaculates collected from 53 fertile stallions were divided into four age groups (3-5, 6-10, 11-14 and >15 years) and were subjected to a complex investigation including conventional analysis, CASA, flow cytometry and mtDNA content (real-time PCR). The mean (±SD) number of mtDNA copies equalled 14 ± 9 and varied from 3 to 64. Considering the great number of sperm parameters monitored in this study, only few of them were correlated with the mtDNA content: ejaculate volume (a positive correlation), the amplitude of lateral head displacement (ALH; a negative correlation) and the high mitochondrial activity index (a negative correlation). The stallion age was not correlated with the mtDNA copy number. This study provides the first set of data on mtDNA content in equine sperm and confirms phenomena previously described for humans and dog on associations between sperm mtDNA content and selected motility parameters monitored by the CASA. Basing our study on spermatozoa from fertile stallions could however limit the extent of detected associations. PMID:27037507

  15. RESOLUTION OF COMPLEX INTEGRATION PATTERNS TO OBTAIN SINGLE COPY TRANSGENES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The present invention provides methods for producing a transgenic cell having a stably integrated, single copy of an exogenous polynucleotide sequence. The method, which resolves repeated insertions of the introduced polynucleotide sequence into a single copy, involves introducing into a genetic loc...

  16. Mitochondrial DNA copy number and replication in reprogramming and differentiation.

    PubMed

    St John, Justin C

    2016-04-01

    Until recently, it was thought that the role of the mitochondrial genome was confined to encoding key proteins that generate ATP through the process of oxidative phosphorylation in the electron transfer chain. However, with increasing new evidence, it is apparent that the mitochondrial genome has a major role to play in a number of diseases and phenotypes. For example, mitochondrial variants and copy number have been implicated in the processes of fertilisation outcome and development and the onset of tumorigenesis. On the other hand, mitochondrial DNA (mtDNA) haplotypes have been implicated in a variety of diseases and most likely account for the adaptation that our ancestors achieved in order that they were fit for their environments. The mechanisms, which enable the mitochondrial genome to either protect or promote the disease phenotype, require further elucidation. However, there appears to be significant 'crosstalk' between the chromosomal and mitochondrial genomes that enable this to take place. One such mechanism is the regulation of DNA methylation by mitochondrial DNA, which is often perturbed in reprogrammed cells that have undergone dedifferentiation and affects mitochondrial DNA copy number. Furthermore, it appears that the mitochondrial genome interacts with the chromosomal genome to regulate the transcription of key genes at certain stages during development. Additionally, the mitochondrial genome can accumulate a series of mtDNA variants, which can lead to diseases such as cancer. It is likely that a combination of certain mitochondrial variants and aberrant patterns of mtDNA copy number could indeed account for many diseases that have previously been unaccounted for. This review focuses on the role that the mitochondrial genome plays especially during early stages of development and in cancer. PMID:26827792

  17. PCR-Based Analysis of Mitochondrial DNA Copy Number, Mitochondrial DNA Damage, and Nuclear DNA Damage.

    PubMed

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

    2016-01-01

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

  18. 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

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

    PubMed Central

    Bartlett, Joanne G.; Smedley, Mark A.; Harwood, Wendy A.

    2014-01-01

    Sequencing across the junction between an integrated transfer DNA (T-DNA) and a host plant genome provides two important pieces of information. The junctions themselves provide information regarding the proportion of T-DNA which has integrated into the host plant genome, whilst the transgene flanking sequences can be used to study the local genetic environment of the integrated transgene. In addition, this information is important in the safety assessment of GM crops and essential for GM traceability. In this study, a detailed analysis was carried out on the right-border T-DNA junction sequences of single-copy independent transgenic barley lines. T-DNA truncations at the right-border were found to be relatively common and affected 33.3% of the lines. In addition, 14.3% of lines had rearranged construct sequence after the right border break-point. An in depth analysis of the host-plant flanking sequences revealed that a significant proportion of the T-DNAs integrated into or close to known repetitive elements. However, this integration into repetitive DNA did not have a negative effect on transgene expression. PMID:24833334

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

    PubMed Central

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

    2013-01-01

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

  1. PCR Based Determination of Mitochondrial DNA Copy Number in Multiple Species

    PubMed Central

    Rooney, JP; Ryde, IT; Sanders, LH; Howlett, EH; Colton, MD; Germ, KE; Mayer, GD; Greenamyre, JT; Meyer, JN

    2015-01-01

    Summary Mitochondrial DNA (mtDNA) copy number is a critical component of overall mitochondrial health. In this chapter we describe methods for isolation of both mtDNA and nuclear DNA (nucDNA), and measurement of their respective copy numbers using quantitative PCR. Methods differ depending on the species and cell type of the starting material, and availability of specific PCR reagents. PMID:25308485

  2. RECONSTRUCTING DNA COPY NUMBER BY PENALIZED ESTIMATION AND IMPUTATION

    PubMed Central

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

    2011-01-01

    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. PMID:21572975

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

    NASA Astrophysics Data System (ADS)

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

    1990-09-01

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

  4. Creation of low-copy integrated transgenic lines in Caenorhabditis elegans.

    PubMed Central

    Praitis, V; Casey, E; Collar, D; Austin, J

    2001-01-01

    In Caenorhabditis elegans, transgenic lines are typically created by injecting DNA into the hermaphrodite germline to form multicopy extrachromosomal DNA arrays. This technique is a reliable means of expressing transgenes in C. elegans, but its use has limitations. Because extrachromosomal arrays are semistable, only a fraction of the animals in a transgenic extrachromosomal array line are transformed. In addition, because extrachromosomal arrays can contain hundreds of copies of the transforming DNA, transgenes may be overexpressed, misexpressed, or silenced. We have developed an alternative method for C. elegans transformation, using microparticle bombardment, that produces single- and low-copy chromosomal insertions. Using this method, we find that it is possible to create integrated transgenic lines that reproducibly express GFP reporter constructs without the variations in expression level and pattern frequently exhibited by extrachromosomal array lines. In addition, we find that low-copy integrated lines can also be used to express transgenes in the C. elegans germline, where conventional extrachromosomal arrays typically fail to express due to germline silencing. PMID:11238406

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

    NASA Astrophysics Data System (ADS)

    Tsunashima, Noboru; Katsura, Seiichiro

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

  6. Focal DNA Copy Number Changes in Neuroblastoma Target MYCN Regulated Genes

    PubMed Central

    Mestdagh, Pieter; Menten, Björn; Lefever, Steve; Pattyn, Filip; De Brouwer, Sara; Sante, Tom; Schulte, Johannes Hubertus; Schramm, Alexander; Van Roy, Nadine; Van Maerken, Tom; Noguera, Rosa; Combaret, Valérie; Devalck, Christine; Westermann, Frank; Laureys, Geneviève; Eggert, Angelika; Vandesompele, Jo; De Preter, Katleen; Speleman, Frank

    2013-01-01

    Neuroblastoma is an embryonic tumor arising from immature sympathetic nervous system cells. Recurrent genomic alterations include MYCN and ALK amplification as well as recurrent patterns of gains and losses of whole or large partial chromosome segments. A recent whole genome sequencing effort yielded no frequently recurring mutations in genes other than those affecting ALK. However, the study further stresses the importance of DNA copy number alterations in this disease, in particular for genes implicated in neuritogenesis. Here we provide additional evidence for the importance of focal DNA copy number gains and losses, which are predominantly observed in MYCN amplified tumors. A focal 5 kb gain encompassing the MYCN regulated miR-17∼92 cluster as sole gene was detected in a neuroblastoma cell line and further analyses of the array CGH data set demonstrated enrichment for other MYCN target genes in focal gains and amplifications. Next we applied an integrated genomics analysis to prioritize MYCN down regulated genes mediated by MYCN driven miRNAs within regions of focal heterozygous or homozygous deletion. We identified RGS5, a negative regulator of G-protein signaling implicated in vascular normalization, invasion and metastasis, targeted by a focal homozygous deletion, as a new MYCN target gene, down regulated through MYCN activated miRNAs. In addition, we expand the miR-17∼92 regulatory network controlling TGFß signaling in neuroblastoma with the ring finger protein 11 encoding gene RNF11, which was previously shown to be targeted by the miR-17∼92 member miR-19b. Taken together, our data indicate that focal DNA copy number imbalances in neuroblastoma (1) target genes that are implicated in MYCN signaling, possibly selected to reinforce MYCN oncogene addiction and (2) serve as a resource for identifying new molecular targets for treatment. PMID:23308108

  7. Improved Statistical Analysis for Array CGH-Based DNA Copy Number Aberrations

    PubMed Central

    Jiang, Hongmei; Zhu, Zhong-Zheng; Yu, Yue; Lin, Simon; Hou, Lifang

    2011-01-01

    Array-based comparative genomic hybridization (aCGH) allows measuring DNA copy number at the whole genome scale. In cancer studies, one may be interested in identifying DNA copy number aberrations (CNAs) associated with certain clinicopathological characteristics such as cancer metastasis. We proposed to define test regions based on copy number pattern profiles across multiple samples, using either smoothed log2-ratio or discrete data of copy number gain/loss calls. Association test performed on the refined test regions instead of the probes has improved power due to reduced number of tests. We also compared three types of measurement of copy number levels, normalized log2-ratio, smoothed log2-ratio, and copy number gain or loss calls in statistical hypothesis testing. The relative strengths and weaknesses of the proposed method were demonstrated using both simulation studies and real data analysis of a liver cancer study. PMID:22084565

  8. Reduced mitochondrial DNA copy number is a biomarker of Parkinson's disease

    PubMed Central

    Pyle, Angela; Anugrha, Haidyan; Kurzawa-Akanbi, Marzena; Yarnall, Alison; Burn, David; Hudson, Gavin

    2016-01-01

    Like any organ, the brain is susceptible to the march of time and a reduction in mitochondrial biogenesis is a hallmark of the aging process. In the largest investigation of mitochondrial copy number in Parkinson's disease (PD) to date and by using multiple tissues, we demonstrate that reduced Parkinson DNA (mitochondrial DNA mtDNA) copy number is a biomarker for the etiology of PD. We used established methods of mtDNA quantification to assess the copy number of mtDNA in n = 363 peripheral blood samples, n = 151 substantia nigra pars compacta tissue samples and n = 120 frontal cortex tissue samples from community-based PD cases fulfilling UK-PD Society brain bank criteria for the diagnosis of PD. Accepting technical limitations, our data show that PD patients suffer a significant reduction in mtDNA copy number in both peripheral blood and the vulnerable substantia nigra pars compacta when compared to matched controls. Our study indicates that reduced mtDNA copy number is restricted to the affected brain tissue, but is also reflected in the peripheral blood, suggesting that mtDNA copy number may be a viable diagnostic predictor of PD. PMID:26639155

  9. 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

  10. Leukocyte Mitochondrial DNA Copy Number Is Associated with Chronic Obstructive Pulmonary Disease

    PubMed Central

    Liu, Shih-Feng; Kuo, Ho-Chang; Tseng, Ching-Wan; Huang, Hung-Tu; Chen, Yung-Che; Tseng, Chia-Cheng; Lin, Meng-Chih

    2015-01-01

    Background Oxidative stress is known to be involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). Evidence suggests that leukocytes mitochondria DNA (mtDNA) is susceptible to undergo mutations, insertions, or depletion in response to reactive oxidative stress (ROS). We hypothesize that mtDNA copy number is associated with the development of COPD. Methodology/Principal Findings Relative mtDNA copy number was measured by a quantitative real-time PCR assay using DNA extracted from peripheral leukocytes. MtDNA copy number of peripheral leukocytes in the COPD group (n = 86) is significantly decreased compared with non-smoker group (n = 77) (250.3± 21.5 VS. 464.2± 49.9, P<0.001). MtDNA copy number in the COPD group was less than that in the healthy smoking group, but P value nearly achieved significance (250.3± 21.5 VS. 404.0± 76.7, P = 0.08) MtDNA copy number has no significance with age, gender, body mass index, current smoking, and pack-years in COPD group, healthy smoker group and no smoker group, respectively. Serum glutathione level in the COPD group is significantly decreased compared with healthy smoker and non-smoker groups (4.5± 1.3 VS. 6.2± 1.9 and 4.5± 1.3 VS. 7.1±1.1 mU/mL; P<0.001 respectively). Pearson correlation test shows a significant liner correlation between mtDNA copy number and serum glutathione level (R = 0.2, P = 0.009). Conclusions/Significance COPD is associated with decreased leukocyte mtDNA copy number and serum glutathione. COPD is a regulatory disorder of leukocytes mitochondria. However, further studies are needed to determine the real mechanisms about the gene and the function of mitochondria. PMID:26394041

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

    PubMed

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

    2016-08-01

    Oocyte capacity is relevant in understanding decreasing female fertility and in the use of assisted reproductive technologies in human and farm animals. Mitochondria are important to the development of a functionally good oocyte and the oocyte mtDNA copy number has been introduced as a useful parameter for prediction of oocyte competence. The aim of this study was to investigate: (i) if the oocyte donor has an influence on its oocyte's mtDNA copy number; and (ii) the relation between oocyte size and mtDNA copy number using pre- and postpubertal pig oocytes. Cumulus-oocyte complexes were collected from individual donor pigs. The oocytes were allocated into different size-groups, snap-frozen and single-oocyte mtDNA copy number was estimated by quantitative real-time PCR using the genes ND1 and COX1. Results showed that mean mtDNA copy number in oocytes from any individual donor could be categorized as either 'high' (≥100,000) or 'low' (<100,000) with no difference in threshold between pre- and postpubertal oocytes. No linear correlation was detected between oocyte size and mtDNA copy number within pre- and postpubertal oocytes. This study demonstrates the importance of the oocyte donor in relation to oocyte mtDNA copy number, irrespectively of the donor's puberty status and the oocyte's growth stage. Observations from this study facilitate both further investigations of the importance of mtDNA copy number and the unravelling of relations between different mitochondrial parameters and oocyte competence. PMID:26679989

  12. Low Mitochondrial DNA Copy Number is Associated With Adverse Clinical Outcomes in Peritoneal Dialysis Patients.

    PubMed

    Yoon, Chang-Yun; Park, Jung Tak; Kee, Youn Kyung; Han, Seung Gyu; Han, In Mee; Kwon, Young Eun; Park, Kyoung Sook; Lee, Mi Jung; Han, Seung Hyeok; Kang, Shin-Wook; Yoo, Tae-Hyun

    2016-02-01

    Mitochondrial dysfunction may play an important role in abnormal glucose metabolism and systemic inflammation. We aimed to investigate the relationship between mitochondrial DNA (mtDNA) copy number and clinical outcomes in peritoneal dialysis (PD) patients. We recruited 120 prevalent PD patients and determined mtDNA copy number by PCR. Primary outcome was all-cause mortality, whereas secondary outcomes included cardiovascular events, technical PD failure, and incident malignancy. Cox proportional hazards analysis determined the independent association of mtDNA copy number with outcomes. The mean patient age was 52.3 years; 42.5% were men. The mean log mtDNA copy number was 3.30 ± 0.50. During a follow-up period of 35.4 ± 19.3 months, all-cause mortality and secondary outcomes were observed in 20.0% and 59.2% of patients, respectively. Secondary outcomes were significantly lower in the highest mtDNA copy number group than in the lower groups. In multiple Cox analysis, the mtDNA copy number was not associated with all-cause mortality (lower two vs highest tertile: hazard ratio [HR] = 1.208, 95% confidence interval [CI] = 0.477-3.061). However, the highest tertile group was significantly associated with lower incidences of secondary outcomes (lower two vs highest tertile: HR [95% CI] = 0.494 [0.277-0.882]) after adjusting for confounding factors. The decreased mtDNA copy number was significantly associated with adverse clinical outcomes in PD patients. PMID:26886611

  13. Low Mitochondrial DNA Copy Number is Associated With Adverse Clinical Outcomes in Peritoneal Dialysis Patients

    PubMed Central

    Yoon, Chang-Yun; Park, Jung Tak; Kee, Youn Kyung; Han, Seung Gyu; Han, In Mee; Kwon, Young Eun; Park, Kyoung Sook; Lee, Mi Jung; Han, Seung Hyeok; Kang, Shin-Wook; Yoo, Tae-Hyun

    2016-01-01

    Abstract Mitochondrial dysfunction may play an important role in abnormal glucose metabolism and systemic inflammation. We aimed to investigate the relationship between mitochondrial DNA (mtDNA) copy number and clinical outcomes in peritoneal dialysis (PD) patients. We recruited 120 prevalent PD patients and determined mtDNA copy number by PCR. Primary outcome was all-cause mortality, whereas secondary outcomes included cardiovascular events, technical PD failure, and incident malignancy. Cox proportional hazards analysis determined the independent association of mtDNA copy number with outcomes. The mean patient age was 52.3 years; 42.5% were men. The mean log mtDNA copy number was 3.30 ± 0.50. During a follow-up period of 35.4 ± 19.3 months, all-cause mortality and secondary outcomes were observed in 20.0% and 59.2% of patients, respectively. Secondary outcomes were significantly lower in the highest mtDNA copy number group than in the lower groups. In multiple Cox analysis, the mtDNA copy number was not associated with all-cause mortality (lower two vs highest tertile: hazard ratio [HR] = 1.208, 95% confidence interval [CI] = 0.477–3.061). However, the highest tertile group was significantly associated with lower incidences of secondary outcomes (lower two vs highest tertile: HR [95% CI] = 0.494 [0.277–0.882]) after adjusting for confounding factors. The decreased mtDNA copy number was significantly associated with adverse clinical outcomes in PD patients. PMID:26886611

  14. Retroelements contribute to the excess low-copy-number DNA in pine.

    PubMed

    Elsik, C G; Williams, C G

    2000-09-01

    Excess DNA in the single-copy component is rarely recognized as a contributor to the C-value paradox yet the single-copy component of the pine genome is reported to comprise over 3000 Mb of DNA, in large excess over the estimated 100 Mb required for gene expression. Two hypotheses regarding the factors that might contribute to the excess low-copy-number DNA were tested. The first hypothesis proposes that the excess low-copy kinetic component is actually overestimated by reassociation data analysis. To test this, a previously published C0t curve for Pinus strobus was reanalyzed using a new estimate of genome size based on laser flow cytometry. Part of the excess low-copy-number DNA in the pine genome could be attributed to the choice of parameters used in the analysis of the reassociation data. The second hypothesis holds that diverged retrotransposons contribute to the excess low-copy DNA. Sequences randomly sampled from single-copy and low-repetitive kinetic components of the P. taeda genome were characterized. Twelve of 46 fragments cloned from these fractions were found to show sequence similarity to retroelements: hence diverged retroelements contribute to the excess low-repetitive kinetic component in the pine genome. Similarity search was shown to be a conservative method for identifying retroelements, and thus the number of retroelements in the low-copy component was actually underestimated. Most of the retroelements in this fraction were nonfunctional. divergent from known retroelement families and previously reported only for flowering plants. Divergent retrotransposons are thus a major factor contributing to the expansion of the low-repetitive DNA component in higher plants. PMID:11016832

  15. Mitochondrial DNA copy number and lung cancer risk in a prospective cohort study

    PubMed Central

    Hosgood, H.Dean; Liu, Chin-San; Rothman, Nathaniel; Weinstein, Stephanie J.; Bonner, Matthew R.; Shen, Min; Lim, Unhee; Virtamo, Jarmo; Cheng, Wen-ling; Albanes, Demetrius; Lan, Qing

    2010-01-01

    Mitochondria are eukaryotic organelles responsible for energy production. Mitochondrial DNA (mtDNA) lack introns and protective histones, have limited DNA repair capacity and compensate for damage by increasing the number of mtDNA copies. As a consequence, mitochondria are more susceptible to reactive oxygen species, an important determinant of cancer risk, and it is hypothesized that increased mtDNA copy number may be associated with carcinogenesis. We assessed the association of mtDNA copy number and lung cancer risk in 227 prospectively collected cases and 227 matched controls from the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study. Conditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs), adjusting for age at randomization, smoking years and number of cigarettes smoked per day. There was suggestion of a dose-dependent relationship between mtDNA copy number and subsequent risk of lung cancer, with a prominent effect observed in the highest mtDNA copy number quartile [ORs (95% CI) by quartile: 1.0 (reference), 1.3 (0.7–2.5), 1.1 (0.6–2.2) and 2.4 (1.1–5.1); Ptrend = 0.008]. This is the first report, to the best of our knowledge, to suggest that mtDNA copy number may be positively associated with subsequent risk of lung cancer in a prospective cohort study; however, replication is needed in other studies and populations. PMID:20176654

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

    Lyckegaard, E M; Clark, A G

    1989-01-01

    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. Images PMID:2494656

  18. Urinary Mitochondrial DNA Copy Number Identifies Chronic Renal Injury in Hypertensive Patients.

    PubMed

    Eirin, Alfonso; Saad, Ahmed; Tang, Hui; Herrmann, Sandra M; Woollard, John R; Lerman, Amir; Textor, Stephen C; Lerman, Lilach O

    2016-08-01

    Mitochondrial injury contributes to renal dysfunction in several models of renal disease, but its involvement in human hypertension remains unknown. Fragments of the mitochondrial genome released from dying cells are considered surrogate markers of mitochondrial injury. We hypothesized that hypertension would be associated with increased urine mitochondrial DNA (mtDNA) copy numbers. We prospectively measured systemic and urinary copy number of the mtDNA genes cytochrome-c oxidase-3 and NADH dehydrogenase subunit-1 by quantitative polymerase chain reaction in essential (n=25) and renovascular (RVH, n=34) hypertensive patients and compared them with healthy volunteers (n=22). Urinary kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin served as indices of renal injury. Renal blood flow and oxygenation were assessed by multidetector computed tomography and blood oxygen level-dependent magnetic resonance imaging. Blood pressure, urinary neutrophil gelatinase-associated lipocalin, and kidney injury molecule-1 were similarly elevated in essential hypertension and RVH, and estimated glomerular filtration rate was lower in RVH versus healthy volunteers and essential hypertension. Renal blood flow was lower in RVH compared with essential hypertension. Urinary mtDNA copy number was higher in hypertension compared with healthy volunteers, directly correlated with urinary neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 and inversely with estimated glomerular filtration rate. In RVH, urinary mtDNA copy number correlated directly with intrarenal hypoxia. Furthermore, in an additional validation cohort, urinary mtDNA copy number was higher in RVH compared with healthy volunteers (n=10 each). The change in serum creatinine levels and estimated glomerular filtration rate 3 months after medical therapy without or with revascularization correlated with the change in urinary mtDNA. Therefore, elevated urinary mtDNA copy numbers in

  19. Real-time PCR designs to estimate nuclear and mitochondrial DNA copy number in forensic and ancient DNA studies.

    PubMed

    Alonso, Antonio; Martín, Pablo; Albarrán, Cristina; García, Pilar; García, Oscar; de Simón, Lourdes Fernández; García-Hirschfeld, Julia; Sancho, Manuel; de La Rúa, Concepción; Fernández-Piqueras, Jose

    2004-01-28

    We explore different designs to estimate both nuclear and mitochondrial human DNA (mtDNA) content based on the detection of the 5' nuclease activity of the Taq DNA polymerase using fluorogenic probes and a real-time quantitative PCR detection system. Human mtDNA quantification was accomplished by monitoring the real-time progress of the PCR-amplification of two different fragment sizes (113 and 287 bp) within the hypervariable region I (HV1) of the mtDNA control region, using two fluorogenic probes to specifically determine the mtDNA copy of each fragment size category. This mtDNA real-time PCR design has been used to assess the mtDNA preservation (copy number and degradation state) of DNA samples retrieved from 500 to 1500 years old human remains that showed low copy number and highly degraded mtDNA. The quantification of nuclear DNA was achieved by real-time PCR of a segment of the X-Y homologous amelogenin (AMG) gene that allowed the simultaneous estimation of a Y-specific fragment (AMGY: 112 bp) and a X-specific fragment (AMGX: 106 bp) making possible not only haploid or diploid DNA quantitation but also sex determination. The AMG real-time PCR design has been used to quantify a set of 57 DNA samples from 4-5 years old forensic bone remains with improved sensitivity compared with the slot-blot hybridization method. The potential utility of this technology to improve the quality of some PCR-based forensic and ancient DNA studies (microsatellite typing and mtDNA sequencing) is discussed. PMID:15040907

  20. Peripheral blood mitochondrial DNA copy number, length heteroplasmy and breast cancer risk: a replication study.

    PubMed

    Shen, Jie; Wan, Jie; Song, Renduo; Zhao, Hua

    2015-11-01

    Oxidative stress has consistently been linked to breast carcinogenesis, and mitochondria play a significant role in regulating reactive oxygen species generation. In our previous study, we found that increased levels of mitochondrial DNA (mtDNA) copy number and the presence of mitochondrial length heteroplasmies in the hypervariable (HV) regions 1 and 2 (HV1 and HV2) in peripheral blood are associated with increased risk of breast cancer. In current study with 1000 breast cancer cases and 1000 healthy controls, we intended to replicate our previous findings. Overall, levels of mtDNA copy number were significantly higher in breast cancer cases than healthy controls (mean: 1.17 versus 0.94, P < 0.001). In the multivariate linear regression analysis, increased mtDNA copy number levels were associated with a 1.32-fold increased risk of breast cancer [adjusted odds ratio (OR) = 1.32, 95% confidence interval (CI) = 1.15-1.67]. Breast cancer cases were more likely to have HV1 and HV2 region length heteroplasmies than healthy controls (P < 0.001, respectively). The existence of HV1 and HV2 length heteroplasmies was associated with 2.01- and 1.63-folds increased risk of breast cancer (for HV1: OR = 2.01, 95% CI = 1.66-2.42; for HV2: OR = 1.63, 95% CI = 1.34-1.92). Additionally, joint effects among mtDNA copy number, HV1 and HV2 length heteroplasmies were observed. Our results are consistent with our previous findings and further support the roles of mtDNA copy number and mtDNA length heteroplasmies that may play in the development of breast cancer. PMID:26363030

  1. 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

  2. The relationship between altered mitochondrial DNA copy number and cancer risk: a meta-analysis.

    PubMed

    Mi, Jia; Tian, Geng; Liu, Shuang; Li, Xianglin; Ni, Tianhui; Zhang, Liwei; Wang, Bin

    2015-01-01

    Currently, a comprehensive assessment between mitochondrial DNA (mtDNA) content and cancer risk is lacking. We designed this meta-analysis to test the hypothesis that altered mtDNA copy number might influence genetic susceptibility to some specific types of cancer. The processes of literature search, eligibility appraisal and data retrieval were independently completed in duplicate. The mtDNA copy number which was dichotomized or classified into tertiles was compared between cancer cases and controls. Twenty-six articles with 38 study groups were analyzed among 6682 cases and 9923 controls. When dichotomizing mtDNA copy number at the median value, there was an 11% increased cancer risk for carriers of high mtDNA content (P = 0.320). By cancer type, high mtDNA content was associated with an increased risk for lymphoma (OR = 1.76; P = 0.023) but a reduced risk for skeleton cancer (OR = 0.39; P = 0.001). Carriers of the 2(nd) and 3(rd) tertiles of mtDNA copy number had an 1.74-fold (P = 0.010) and 2.07-fold (P = 0.021) increased risk of lymphoma, respectively. By contrast, there was correspondingly a 56% (P < 0.001) and 80% (P < 0.001) reduced risk of skeleton cancer. Our findings suggested that elevated mtDNA content was associated with a higher risk for lymphoma, but a lower risk for skeleton cancer. PMID:25952580

  3. CONSERTING: integrating copy number analysis with structural variation detection

    PubMed Central

    Chen, Xiang; Gupta, Pankaj; Wang, Jianmin; Nakitandwe, Joy; Roberts, Kathryn; Dalton, James D.; Parker, Matthew; Patel, Samir; Holmfeldt, Linda; Payne, Debbie; Easton, John; Ma, Jing; Rusch, Michael; Wu, Gang; Patel, Aman; J. Baker, Suzanne; Dyer, Michael A.; Shurtleff, Sheila; Espy, Stephen; Pounds, Stanley; Downing, James R.; Ellison, David W.; Mullighan, Charles G.; Zhang, Jinghui

    2015-01-01

    We developed Copy Number Segmentation by Regression Tree in Next Generation Sequencing (CONSERTING), a novel algorithm for detecting somatic copy number alteration (CNA) using whole-genome sequencing (WGS) data. CONSERTING performs iterative analysis of segmentation by read depth change and localized structural variation detection, achieving high accuracy and sensitivity. Analysis of 43 pediatric and adult cancer genomes revealed novel oncogenic CNAs, complex re-arrangements and subclonal CNAs missed by alternative approaches. PMID:25938371

  4. 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. PMID:25477383

  5. 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

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

    PubMed Central

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

    2015-01-01

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

  7. DNA Copy Number Profiles in Affinity-Purified Ovarian Clear Cell Carcinoma

    PubMed Central

    Kuo, Kuan-Ting; Mao, Tsui-Lien; Chen, Xu; Feng, Yuanjian; Nakayama, Kentaro; Wang, Yue; Glas, Ruth; Ma, M. Joe; Kurman, Robert J.; Shih, Ie-Ming; Wang, Tian-Li

    2010-01-01

    Purpose Advanced ovarian clear cell carcinoma (CCC) is one of the most malignant ovarian malignancies, in part because it tends to be resistant to platinum-based chemotherapy. At present, little is known about the molecular genetic alterations in CCCs except that there are frequent activating mutations in PIK3CA. The purpose of this study is to comprehensively define the genomic changes in CCC based on DNA copy number alterations. Experimental Design We performed 250K high-density SNP array analysis in 12 affinity-purified CCCs and 10 CCC cell lines. Discrete regions of amplification and deletion were also analyzed in additional 21 affinity-purified CCCs using quantitative real-time PCR. Results The level of chromosomal instability in CCC as defined by the extent of DNA copy number changes is similar to those previously reported in low-grade ovarian serous carcinoma but much less than those in high-grade serous carcinoma. The most remarkable region with DNA copy number gain is at chr20 which harbors a potential oncogene, ZNF217. This is observed in 36% of CCCs but rarely detected in serous carcinomas regardless of grade. In addition, homozygous deletions are detected at CDKN2A/2B and LZTS1 loci. Interestingly, the DNA copy number changes observed in fresh CCC tissues are rarely detected in the established CCC cell lines. Conclusions This study provides the first high resolution, genome-wide view of DNA copy number alterations in ovarian CCC. The findings provide a genomic landscape for future studies aimed at elucidating the pathogenesis and developing new target-based therapies for CCCs. PMID:20233889

  8. 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. PMID:21607848

  9. Screen for mitochondrial DNA copy number maintenance genes reveals essential role for ATP synthase

    PubMed Central

    Fukuoh, Atsushi; Cannino, Giuseppe; Gerards, Mike; Buckley, Suzanne; Kazancioglu, Selena; Scialo, Filippo; Lihavainen, Eero; Ribeiro, Andre; Dufour, Eric; Jacobs, Howard T

    2014-01-01

    The machinery of mitochondrial DNA (mtDNA) maintenance is only partially characterized and is of wide interest due to its involvement in disease. To identify novel components of this machinery, plus other cellular pathways required for mtDNA viability, we implemented a genome-wide RNAi screen in Drosophila S2 cells, assaying for loss of fluorescence of mtDNA nucleoids stained with the DNA-intercalating agent PicoGreen. In addition to previously characterized components of the mtDNA replication and transcription machineries, positives included many proteins of the cytosolic proteasome and ribosome (but not the mitoribosome), three proteins involved in vesicle transport, some other factors involved in mitochondrial biogenesis or nuclear gene expression, > 30 mainly uncharacterized proteins and most subunits of ATP synthase (but no other OXPHOS complex). ATP synthase knockdown precipitated a burst of mitochondrial ROS production, followed by copy number depletion involving increased mitochondrial turnover, not dependent on the canonical autophagy machinery. Our findings will inform future studies of the apparatus and regulation of mtDNA maintenance, and the role of mitochondrial bioenergetics and signaling in modulating mtDNA copy number. PMID:24952591

  10. Extraction of single-copy nuclear DNA from forensic specimens with a variety of postmortem histories.

    PubMed

    Evison, M P; Smillie, D M; Chamberlain, A T

    1997-11-01

    Specimens of human bone, teeth and dried blood spots from 3 months to 91 years old, with a variety of postmortem histories, were used in a comparative study of recovery of single-copy nuclear DNA sequences from forensic material. Sequences of the amelogenin and HLA-DPB1 genes were chosen for their value in sexing and identification. Sequences of the mitochondrial non-coding region V were also amplified to compare the recovery of mitochondrial and single-copy nuclear DNA. A variation of the silica method for DNA extraction was refined for application to the forensic specimens in this sample. Single-copy nuclear DNA was amplified from 100% of recent postoperative bone specimens (n = 6), 80% of forensic teeth and bone specimens (n = 10), 78% of recently extracted teeth (n = 18), 78% of exhumed bone up to 91 years old (n = 37) and 69% of 15 year old bone specimens fixed in 10% formalin (n = 20). Amelogenin sexing was correct in 85% of cases (n = 74) in which the sex of the donor had been recorded. There was no correlation between the age of the specimen and the extent of DNA preservation. PMID:9397544

  11. 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

    2016-06-01

    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. PMID:27175474

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

    PubMed

    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

    2015-07-01

    DNA sequencing identifies common and rare genetic variants for association studies, but studies typically focus on variants in nuclear DNA and ignore the mitochondrial genome. In fact, analyzing variants in mitochondrial DNA (mtDNA) sequences presents special problems, which we resolve here with a general solution for the analysis of mtDNA in next-generation sequencing studies. The new program package comprises 1) an algorithm designed to identify mtDNA variants (i.e., homoplasmies and heteroplasmies), incorporating sequencing error rates at each base in a likelihood calculation and allowing allele fractions at a variant site to differ across individuals; and 2) an estimation of mtDNA copy number in a cell directly from whole-genome sequencing data. We also apply the methods to DNA sequence from lymphocytes of ~2,000 SardiNIA Project participants. As expected, mothers and offspring share all homoplasmies but a lesser proportion of heteroplasmies. Both homoplasmies and heteroplasmies show 5-fold higher transition/transversion ratios than variants in nuclear DNA. Also, heteroplasmy increases with age, though on average only ~1 heteroplasmy reaches the 4% level between ages 20 and 90. In addition, we find that mtDNA copy number averages ~110 copies/lymphocyte and is ~54% heritable, implying substantial genetic regulation of the level of mtDNA. Copy numbers also decrease modestly but significantly with age, and females on average have significantly more copies than males. The mtDNA copy numbers are significantly associated with waist circumference (p-value = 0.0031) and waist-hip ratio (p-value = 2.4×10-5), but not with body mass index, indicating an association with central fat distribution. To our knowledge, this is the largest population analysis to date of mtDNA dynamics, revealing the age-imposed increase in heteroplasmy, the relatively high heritability of copy number, and the association of copy number with metabolic traits. PMID:26172475

  13. The copy number of rice CACTA DNA transposons carrying MIR820 does not correlate with MIR820 expression

    PubMed Central

    Nosaka, Misuzu; Ishiwata, Aiko; Shimizu-Sato, Sae; Ono, Akemi; Ishimoto, Kiyoe; Noda, Yusaku; Sato, Yutaka

    2013-01-01

    miR820 is a small RNA species (22 and 24 nucleotides), produced from transcripts originated from a region inside CACTA DNA transposons in rice. Because MIR820 is a transposon gene, its expression may depend on the transposon copy number. Here, we investigated the copy number of MIR820 and its expression levels in various cultivars and wild species of rice. We found no correlation between copy number and expression level, suggesting that MIR820 transcription is regulated not by the copy dosage but by the epigenetic state of each copy. PMID:23733074

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

    PubMed

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

    2016-08-01

    Mitochondrial dysfunction in kidney cells has been implicated in the pathogenesis of CKD. Mitochondrial DNA (mtDNA) copy number is a surrogate measure of mitochondrial function, and higher mtDNA copy number in peripheral blood has been associated with lower risk of two important risk factors for CKD progression, diabetes and microalbuminuria. We evaluated whether mtDNA copy number in peripheral blood associates with incident CKD in a population-based cohort of middle-aged adults. We estimated mtDNA copy number using 25 high-quality mitochondrial single nucleotide polymorphisms from the Affymetrix 6.0 array. Among 9058 participants, those with higher mtDNA copy number had a lower rate of prevalent diabetes and lower C-reactive protein levels and white blood cell counts. Baseline eGFR did not differ significantly by mtDNA copy number. Over a median follow-up of 19.6 years, 1490 participants developed CKD. Higher mtDNA copy number associated with lower risk of incident CKD (highest versus lowest quartile: hazard ratio 0.65; 95% confidence interval, 0.56 to 0.75; P<0.001) after adjusting for age, sex, and race. After adjusting for additional risk factors of CKD, including prevalent diabetes, hypertension, C-reactive protein level, and white blood cell count, this association remained significant (highest versus lowest quartile: hazard ratio 0.75; 95% confidence interval, 0.64 to 0.87; P<0.001). In conclusion, higher mtDNA copy number associated with lower incidence of CKD independent of traditional risk factors and inflammation biomarker levels in this cohort. Further research on modifiable factors influencing mtDNA copy number may lead to improvement in the prevention and treatment of CKD. PMID:26794963

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

    PubMed Central

    2013-01-01

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

  16. 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

  17. Generation of Backbone-Free, Low Transgene Copy Plants by Launching T-DNA from the Agrobacterium Chromosome1[W][OA

    PubMed Central

    Oltmanns, Heiko; Frame, Bronwyn; Lee, Lan-Ying; Johnson, Susan; Li, Bo; Wang, Kan; Gelvin, Stanton B.

    2010-01-01

    In both applied and basic research, Agrobacterium-mediated transformation is commonly used to introduce genes into plants. We investigated the effect of three Agrobacterium tumefaciens strains and five transferred (T)-DNA origins of replication on transformation frequency, transgene copy number, and the frequency of integration of non-T-DNA portions of the T-DNA-containing vector (backbone) into the genome of Arabidopsis (Arabidopsis thaliana) and maize (Zea mays). Launching T-DNA from the picA locus of the Agrobacterium chromosome increases the frequency of single transgene integration events and almost eliminates the presence of vector backbone sequences in transgenic plants. Along with novel Agrobacterium strains we have developed, our findings are useful for improving the quality of T-DNA integration events. PMID:20023148

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

    PubMed Central

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

    2014-01-01

    Pokey is a class II DNA transposon that inserts into 28S ribosomal RNA (rRNA) genes and other genomic regions of species in the subgenus, Daphnia. Two divergent lineages, PokeyA and PokeyB have been identified. Recombination between misaligned rRNA genes changes their number and the number of Pokey elements. We used quantitative PCR (qPCR) to estimate rRNA gene and Pokey number in isolates from natural populations of Daphnia obtusa, and in clonally-propagated mutation accumulation lines (MAL) initiated from a single D. obtusa female. The change in direction and magnitude of Pokey and rRNA gene number did not show a consistent pattern across ∼87 generations in the MAL; however, Pokey and rRNA gene number changed in concert. PokeyA and 28S gene number were positively correlated in the isolates from both natural populations and the MAL. PokeyB number was much lower than PokeyA in both MAL and natural population isolates, and showed no correlation with 28S gene number. Preliminary analysis did not detect PokeyB outside rDNA in any isolates and detected only 0 to 4 copies of PokeyA outside rDNA indicating that Pokey may be primarily an rDNA element in D. obtusa. The recombination rate in this species is high and the average size of the rDNA locus is about twice as large as that in other Daphnia species such as D. pulicaria and D. pulex, which may have facilitated expansion of PokeyA to much higher numbers in D. obtusa rDNA than these other species. PMID:25490398

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

    PubMed

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

    2014-01-01

    Pokey is a class II DNA transposon that inserts into 28S ribosomal RNA (rRNA) genes and other genomic regions of species in the subgenus, Daphnia. Two divergent lineages, PokeyA and PokeyB have been identified. Recombination between misaligned rRNA genes changes their number and the number of Pokey elements. We used quantitative PCR (qPCR) to estimate rRNA gene and Pokey number in isolates from natural populations of Daphnia obtusa, and in clonally-propagated mutation accumulation lines (MAL) initiated from a single D. obtusa female. The change in direction and magnitude of Pokey and rRNA gene number did not show a consistent pattern across ∼ 87 generations in the MAL; however, Pokey and rRNA gene number changed in concert. PokeyA and 28S gene number were positively correlated in the isolates from both natural populations and the MAL. PokeyB number was much lower than PokeyA in both MAL and natural population isolates, and showed no correlation with 28S gene number. Preliminary analysis did not detect PokeyB outside rDNA in any isolates and detected only 0 to 4 copies of PokeyA outside rDNA indicating that Pokey may be primarily an rDNA element in D. obtusa. The recombination rate in this species is high and the average size of the rDNA locus is about twice as large as that in other Daphnia species such as D. pulicaria and D. pulex, which may have facilitated expansion of PokeyA to much higher numbers in D. obtusa rDNA than these other species. PMID:25490398

  20. 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. PMID:26103000

  1. 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

  2. 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. PMID:23390135

  3. DNA copy number concentration measured by digital and droplet digital quantitative PCR using certified reference materials.

    PubMed

    Corbisier, Philippe; Pinheiro, Leonardo; Mazoua, Stéphane; Kortekaas, Anne-Marie; Chung, Pui Yan Jenny; Gerganova, Tsvetelina; Roebben, Gert; Emons, Hendrik; Emslie, Kerry

    2015-03-01

    The value assignment for properties of six certified reference materials (ERM-AD623a-f), each containing a plasmid DNA solution ranging from 1 million to 10 copies per μL, by using digital PCR (dPCR) with the BioMark™ HD System (Fluidigm) has been verified by applying droplet digital PCR (ddPCR) using the QX100 system (Bio-Rad). One of the critical factors in the measurement of copy number concentrations by digital PCR is the partition volume. Therefore, we determined the average droplet volume by optical microscopy, revealing an average droplet volume that is 8 % smaller than the droplet volume used as the defined parameter in the QuantaSoft software version 1.3.2.0 (Bio-Rad) to calculate the copy number concentration. This observation explains why copy number concentrations estimated with ddPCR and using an average droplet volume predefined in the QuantaSoft software were systematically lower than those measured by dPCR, creating a significant bias between the values obtained by these two techniques. The difference was not significant anymore when the measured droplet volume of 0.834 nL was used to estimate copy number concentrations. A new version of QuantaSoft software (version 1.6.6.0320), which has since been released with Bio-Rad's new QX200 systems and QX100 upgrades, uses a droplet volume of 0.85 nL as a defined parameter to calculate copy number concentration. PMID:25600685

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

    PubMed

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

    2015-02-24

    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

  5. 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

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

    PubMed Central

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

    2015-01-01

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

  7. 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. PMID:27179566

  8. 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

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

    PubMed

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

    2016-01-01

    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 XF(e)-24 analyzer. Cell invasiveness was evaluated by a trans-well migration assay and vimentin expression. Doxorubicin was used as a chemotherapeutic agent. The results showed a decrease of mtDNA copy numbers in resected RCC tissues (p = 0.043). The TFAM-KD clone expressed lower mtDNA copy number (p = 0.034), lower mRNA levels of TFAM (p = 0.008), ND1 (p = 0.007), and ND6 (p = 0.017), and lower protein levels of TFAM and COX-2 than did the NT clone. By contrast, the protein levels of HIF-2α, HK-II, PFK, LDHA, AKT, MYC and vimentin; trans-well migration activity (p = 0

  10. Increased mitochondrial DNA deletions and copy number in transfusion-dependent thalassemia

    PubMed Central

    Lal, Ashutosh; Gomez, Esteban; Calloway, Cassandra

    2016-01-01

    BACKGROUND Iron overload is the primary cause of morbidity in transfusion-dependent thalassemia. Increase in iron causes mitochondrial dysfunction under experimental conditions, but the occurrence and significance of mitochondrial damage is not understood in patients with thalassemia. METHODS Mitochondrial DNA (mtDNA) to nuclear DNA copy number (Mt/N) and frequency of the common 4977-bp mitochondrial deletion (ΔmtDNA4977) were quantified using a quantitative PCR assay on whole blood samples from 38 subjects with thalassemia who were receiving regular transfusions. RESULTS Compared with healthy controls, Mt/N and ΔmtDNA4977 frequency were elevated in thalassemia (P = 0.038 and P < 0.001, respectively). ΔmtDNA4977 was increased in the presence of either liver iron concentration > 15 mg/g dry-weight or splenectomy, with the highest levels observed in subjects who had both risk factors (P = 0.003). Myocardial iron (MRI T2* < 20 ms) was present in 0%, 22%, and 46% of subjects with ΔmtDNA4977 frequency < 20, 20–40, and > 40/1 × 107 mtDNA, respectively (P = 0.025). Subjects with Mt/N values below the group median had significantly lower Matsuda insulin sensitivity index (5.76 ± 0.53) compared with the high Mt/N group (9.11 ± 0.95, P = 0.008). CONCLUSION Individuals with transfusion-dependent thalassemia demonstrate age-related increase in mtDNA damage in leukocytes. These changes are markedly amplified by splenectomy and are associated with extrahepatic iron deposition. Elevated mtDNA damage in blood cells may predict the risk of iron-associated organ damage in thalassemia. PMID:27583305

  11. 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. PMID:25845980

  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. Hybridization modeling of oligonucleotide SNP arrays for accurate DNA copy number estimation

    PubMed Central

    Wan, Lin; Sun, Kelian; Ding, Qi; Cui, Yuehua; Li, Ming; Wen, Yalu; Elston, Robert C.; Qian, Minping; Fu, Wenjiang J

    2009-01-01

    Affymetrix SNP arrays have been widely used for single-nucleotide polymorphism (SNP) genotype calling and DNA copy number variation inference. Although numerous methods have achieved high accuracy in these fields, most studies have paid little attention to the modeling of hybridization of probes to off-target allele sequences, which can affect the accuracy greatly. In this study, we address this issue and demonstrate that hybridization with mismatch nucleotides (HWMMN) occurs in all SNP probe-sets and has a critical effect on the estimation of allelic concentrations (ACs). We study sequence binding through binding free energy and then binding affinity, and develop a probe intensity composite representation (PICR) model. The PICR model allows the estimation of ACs at a given SNP through statistical regression. Furthermore, we demonstrate with cell-line data of known true copy numbers that the PICR model can achieve reasonable accuracy in copy number estimation at a single SNP locus, by using the ratio of the estimated AC of each sample to that of the reference sample, and can reveal subtle genotype structure of SNPs at abnormal loci. We also demonstrate with HapMap data that the PICR model yields accurate SNP genotype calls consistently across samples, laboratories and even across array platforms. PMID:19586935

  14. Array-based comparative genomic hybridization for the detection of DNA sequence copy number changes in Barrett's adenocarcinoma.

    PubMed

    Albrecht, Bettina; Hausmann, Michael; Zitzelsberger, Horst; Stein, Hubert; Siewert, Jörg Rüdiger; Hopt, Ulrich; Langer, Rupert; Höfler, Heinz; Werner, Martin; Walch, Axel

    2004-07-01

    Array-based comparative genomic hybridization (aCGH) allows the identification of DNA sequence copy number changes at high resolution by co-hybridizing differentially labelled test and control DNAs to a micro-array of genomic clones. The present study has analysed a series of 23 formalin-fixed, paraffin wax-embedded tissue samples of Barrett's adenocarcinoma (BCA, n = 18) and non-neoplastic squamous oesophageal (n = 2) and gastric cardia mucosa (n = 3) by aCGH. The micro-arrays used contained 287 genomic targets covering oncogenes, tumour suppressor genes, and DNA sequences localized within chromosomal regions previously reported to be altered in BCA. DNA sequence copy number changes for a panel of approximately 50 genes were identified, most of which have not been previously described in BCA. DNA sequence copy number gains (mean 41 +/- 25/BCA) were more frequent than DNA sequence copy number losses (mean 20 +/- 15/BCA). The highest frequencies for DNA sequence copy number gains were detected for SNRPN (61%); GNLY (44%); NME1 (44%); DDX15, ABCB1 (MDR), ATM, LAMA3, MYBL2, ZNF217, and TNFRSF6B (39% each); and MSH2, TERC, SERPINE1, AFM137XA11, IGF1R, and PTPN1 (33% each). DNA sequence copy number losses were identified for PDGFB (44%); D17S125 (39%); AKT3 (28%); and RASSFI, FHIT, CDKN2A (p16), and SAS (CDK4) (28% each). In all non-neoplastic tissue samples of squamous oesophageal and gastric cardia mucosa, the measured mean ratios were 1.00 (squamous oesophageal mucosa) or 1.01 (gastric mucosa), indicating that no DNA sequence copy number chances were present. For validation, the DNA sequence copy number changes of selected clones (SNRPN, CMYC, HER2, ZNF217) detected by aCGH were confirmed by fluorescence in situ hybridization (FISH). These data show the sensitivity of aCGH for the identification of DNA sequence copy number changes at high resolution in BCA. The newly identified genes may include so far unknown biomarkers in BCA and are therefore a starting point for

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

    PubMed

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

    2014-09-01

    Canine hemangiosarcoma is a highly aggressive vascular neoplasm associated with extensive clinical and anatomical heterogeneity and a grave prognosis. Comprehensive molecular characterization of hemangiosarcoma may identify novel therapeutic targets and advanced clinical management strategies, but there are no published reports of tumor-associated genome instability and disrupted gene dosage in this cancer. We performed genome-wide microarray-based somatic DNA copy number profiling of 75 primary intra-abdominal hemangiosarcomas from five popular dog breeds that are highly predisposed to this disease. The cohort exhibited limited global genomic instability, compared to other canine sarcomas studied to date, and DNA copy number aberrations (CNAs) were predominantly of low amplitude. Recurrent imbalances of several key cancer-associated genes were evident; however, the global penetrance of any single CNA was low and no distinct hallmark aberrations were evident. Copy number gains of dog chromosomes 13, 24, and 31, and loss of chromosome 16, were the most recurrent CNAs involving large chromosome regions, but their relative distribution within and between cases suggests they most likely represent passenger aberrations. CNAs involving CDKN2A, VEGFA, and the SKI oncogene were identified as potential driver aberrations of hemangiosarcoma development, highlighting potential targets for therapeutic modulation. CNA profiles were broadly conserved between the five breeds, although subregional variation was evident, including a near twofold lower incidence of VEGFA gain in Golden Retrievers versus other breeds (22 versus 40 %). These observations support prior transcriptional studies suggesting that the clinical heterogeneity of this cancer may reflect the existence of multiple, molecularly distinct subtypes of canine hemangiosarcoma. PMID:24599718

  16. 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. PMID:27622766

  17. Towards a DNA Nanoprocessor: Reusable Tile-Integrated DNA Circuits.

    PubMed

    Gerasimova, Yulia V; Kolpashchikov, Dmitry M

    2016-08-22

    Modern electronic microprocessors use semiconductor logic gates organized on a silicon chip to enable efficient inter-gate communication. Here, arrays of communicating DNA logic gates integrated on a single DNA tile were designed and used to process nucleic acid inputs in a reusable format. Our results lay the foundation for the development of a DNA nanoprocessor, a small and biocompatible device capable of performing complex analyses of DNA and RNA inputs. PMID:27430161

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

    PubMed

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

    2016-02-01

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

  19. Age-Related and Heteroplasmy-Related Variation in Human mtDNA Copy Number

    PubMed Central

    Li, Mingkun; Madea, Burkhard; Stoneking, Mark

    2016-01-01

    The mitochondrial (mt) genome is present in many copies in human cells, and intra-individual variation in mtDNA sequences is known as heteroplasmy. Recent studies found that heteroplasmies are highly tissue-specific, site-specific, and allele-specific, however the functional implications have not been explored. This study investigates variation in mtDNA copy numbers (mtCN) in 12 different tissues obtained at autopsy from 152 individuals (ranging in age from 3 days to 96 years). Three different methods to estimate mtCN were compared: shotgun sequencing (in 4 tissues), capture-enriched sequencing (in 12 tissues) and droplet digital PCR (ddPCR, in 2 tissues). The highest precision in mtCN estimation was achieved using shotgun sequencing data. However, capture-enrichment data provide reliable estimates of relative (albeit not absolute) mtCNs. Comparisons of mtCN from different tissues of the same individual revealed that mtCNs in different tissues are, with few exceptions, uncorrelated. Hence, each tissue of an individual seems to regulate mtCN in a tissue-related rather than an individual-dependent manner. Skeletal muscle (SM) samples showed an age-related decrease in mtCN that was especially pronounced in males, while there was an age-related increase in mtCN for liver (LIV) samples. MtCN in SM samples was significantly negatively correlated with both the total number of heteroplasmic sites and with minor allele frequency (MAF) at two heteroplasmic sites, 408 and 16327. Heteroplasmies at both sites are highly specific for SM, accumulate with aging and are part of functional elements that regulate mtDNA replication. These data support the hypothesis that selection acting on these heteroplasmic sites is reducing mtCN in SM of older individuals. PMID:26978189

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

    PubMed

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

    2016-08-01

    Droplet digital PCR (ddPCR) after DNA digestion yielded a 28S rDNA copy number of 61 to 86 copies/genome when testing 10 unrelated Aspergillus fumigatus isolates, higher than with quantitative PCR. Unfortunately, ddPCR after DNA digestion did not improve the sensitivity of our PCR assay when testing serum patients with invasive aspergillosis. PMID:27316653

  1. 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. PMID:26670088

  2. Patterns of integration of DNA microinjected into cultured mammalian cells: evidence for homologous recombination between injected plasmid DNA molecules.

    PubMed Central

    Folger, K R; Wong, E A; Wahl, G; Capecchi, M R

    1982-01-01

    We examined the fate of DNA microinjected into nuclei of cultured mammalian cells. The sequence composition and the physical form of the vector carrying the selectable gene affected the efficiency of DNA-mediated transformation. Introduction of sequences near the simian virus 40 origin of DNA replication or in the long terminal repeat of avian sarcoma provirus into a recombinant plasmid containing the herpes simplex virus thymidine kinase gene. (pBR322/HSV-tk) enhanced the frequency of transformation of LMtk- and RAT-2tk- cells to the TK+ phenotype 20- to 40-fold. In cells receiving injections of only a few plasmid DNA molecules, the transformation frequency was 40-fold higher after injection of linear molecules than after injection of supercoiled molecules. By controlling the number of gene copies injected into a recipient cell, we could obtain transformants containing a single copy or as many as 50 to 100 copies of the selectable gene. Multiple copies of the transforming gene were not scattered throughout the host genome but were integrated as a concatemer at one or a very few sites in the host chromosome. Independent transformants contained the donated genes in different chromosomes. The orientation of the gene copies within the concatemer was not random; rather, the copies were organized as tandem head-to-tail arrays. By analyzing transformants obtained by coinjecting two vectors which were identical except that in one a portion of the vector was inverted, we were able to conclude that the head-to-tail concatemers were generated predominantly by homologous recombination. Surprisingly, these head-to-tail concatemers were found in transformants obtained by injecting either supercoiled or linear plasmid DNA. Even though we demonstrated that cultured mammalian cells contain the enzymes for ligating two DNA molecules very efficiently irrespective of the sequences or topology at their ends, we found that even linear plasmid DNA was recruited into the concatemer by

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

    PubMed Central

    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

    2016-01-01

    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. PMID:26824984

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

    PubMed

    Weitemier, Kevin; 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

  5. Invited review DNA copy number changes as diagnostic tools for lung cancer.

    PubMed

    Bowcock, Anne M

    2014-05-01

    Lung cancer usually presents as advanced stage disease and there is a need for early diagnosis so that appropriate treatments can be provided prior to tumour progression. Copy number variation is frequently detected in tumours and can contribute to tumour progression. This is because regions harbouring DNA imbalance can contain genes encoding critical proteins whose altered dosage contributes to the neoplastic process. Three copy number variations (CNVs) from chromosomes 3p26-p11.1 (loss), 3q26.2-29 (gain) and 6q25.3-24.3 (loss) have previously been described in individuals presenting with endobronchial squamous metaplasia. These CNVs were predictors of cancer diagnosed within 44 months with 97% accuracy. An evaluation of this CNV-based classifier with an independent set of 12 samples (10 men and 2 women), each with a carcinoma in situ or invasive carcinoma at the same site at follow-up demonstrated 92% prediction accuracy. The negative predictive value of this classifier was 89%. The gain at 3q26.2-q29 contributed the most to the classification, being present in virtually all lesions. This region harbours the PIK3CA gene and evaluation of the number of copies of this gene gave very similar results to those from array comparative genomic hybridisation. This type of test can be performed on sputum or bronchial brushings. Larger cohorts now need to be examined to confirm this finding and to possibly refine the regions of CNV. This type of approach paves the way for future molecular analyses to assist in selecting subjects with endobronchial squamous metaplastic or dysplastic lesions who might benefit from more aggressive therapeutic intervention or surveillance. PMID:24188925

  6. Is mitochondrial DNA copy number associated with clinical characteristics and prognosis in gastric cancer?

    PubMed

    Lee, Hyunsu; Lee, Jae-Ho; Kim, Dong-Choon; Hwang, IlSeon; Kang, Yu-Na; Gwon, Gi-Jeong; Choi, In-Jang; Kim, Shin

    2015-01-01

    Alterations in mitochondrial DNA (mtDNA) have been studied in various cancers. However, the clinical value of mtDNA copy number (mtCN) alterations in gastric cancer (GC) is poorly understood. In the present study, we investigated whether alterations in mtCNs might be associated with clinicopathological parameters in GC cases. mtCN was measured in 109 patients with GC by quantitative real-time PCR. Then, correlations with clinicopathological characteristics were analyzed. mtCN was elevated in 64.2% of GC tissues compared with paired, adjacent, non-cancerous tissue. However, the observed alterations in mtCN were not associated with any clinicopathological characteristics, including age, gender, TN stage, Lauren classification, lymph node metastasis, and depth of invasion. Moreover, Kaplan-Meier survival curves revealed that mtCN was not significantly associated with the survival of GC patients. In this study, we demonstrated that mtCN was not a significant marker for predicting clinical characteristics or prognosis in GC. PMID:25640396

  7. Estimation of correlations between copy-number variants in non-coding DNA.

    PubMed

    Stamoulis, Catherine

    2011-01-01

    Allelic DNA aberrations across our genome have been associated with normal human genetic heterogeneity as well as with a number of diseases and disorders. When copy-number variations (CNVs) occur in gene-coding regions, known relationships between genes may help us understand correlations between CNVs. However, a large number of these aberrations occur in non-coding, extragenic regions and their correlations may be characterized only quantitatively, e.g., probabilistically, but not functionally. Using a signal processing approach to CNV detection, we identified distributed CNVs in short, non-coding regions across chromosomes and investigated their potential correlations. We estimated predominantly local correlations between CNVs within the same chromosome, and a small number of apparently random long-distance correlations. PMID:22255599

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

    PubMed Central

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

    2016-01-01

    Abstract Mitochondrial DNA (mtDNA) copy number variation (CNV), which reflects the oxidant-induced cell damage, has been observed in a wide range of human diseases. However, whether it correlates with heart failure, which is closely related to oxidative stress, has never been elucidated before. We aimed to systematically investigate the associations between leukocyte mtDNA CNV and heart failure risk and prognosis. A total of 1700 hospitalized patients with heart failure and 1700 age- and sex-matched community population were consecutively enrolled in this observational study, as well as 1638 (96.4%) patients were followed prospectively for a median of 17 months (12–24 months). The relative mtDNA copy number of leukocyte of peripheral blood or cardiac tissue was measured in triplicate by quantitative real-time PCR method. Patients with heart failure possessed much lower relative mtDNA copy number compared with control subjects (median 0.83, interquartile range [IQR] 0.60–1.16 vs median 1.00, IQR 0.47–2.20; P < 0.001), especially for the patients with ischemic etiology (median, 0.77 for ischemic and 0.91 for non-ischemic, P < 0.001). Patients with lower mtDNA copy number exhibited 1.7 times higher risk of heart failure (odds ratio 1.71, 95% confidence interval [CI] 1.48–1.97, P < 0.001). Long-term follow-up (median of 17 months) showed that decreased mtDNA copy number was significant associated with both increased cardiovascular deaths (hazard ratio [HR] 1.58, 95% CI 1.16–2.16, P = 0.004) and cardiovascular rehospitalization (HR 1.48, 95% CI 1.21–1.82, P < 0.001). After adjusting for the conventional risk factors and medications, lower mtDNA copy numbers were still significantly associated with 50% higher cardiovascular mortality (P = 0.035). In conclusion, mtDNA copy number depletion is an independent risk factor for heart failure and predicts higher cardiovascular mortality in patients with heart failure. PMID:27082579

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

    PubMed

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

    2016-01-12

    Epstein-Barr virus (EBV)-DNA is detected in the blood of some persons with chronic lymphocytic leukemia (CLL) at diagnosis. Whether this is important in the development or progression of CLL is controversial. We interrogated associations between blood EBV-DNA copy number and biological and clinical variables in 243 new-diagnosed consecutive subjects with CLL. Quantification of EBV-DNA copies was done by real-time quantitative PCR (RQ-PCR). All subjects had serological evidence of prior EBV-infection. However, only 24 subjects (10%) had a EBV-DNA-positive test at diagnosis. EBV-DNA-positive subjects at diagnosis had lower hemoglobin concentrations and platelet levels, higher thymidine kinase-1 and serum ferritin levels, un-mutated IGHV genes and a greater risk of Richter transformation compared with EBV-DNA-negative subjects. Percent CD20-, CD148- and ZAP70-positive cells and mean fluorescence intensity (MFI) of each cluster designation were also increased in EBV-DNA-positive subjects at diagnosis. EBV-DNA test positivity was associated with a briefer time-to-treatment interval (HR 1.85; [95% confidence interval, 1.13, 3.03]; P=0.014) and worse survival (HR 2.77; [1.18, 6.49]; P=0.019). Reduction in EBV copies was significantly associated with therapy-response. A positive blood EBV-DNA test at diagnosis and sequential testing of EBV copies during therapy were significantly associated with biological and clinical variables, time-to-treatment, therapy-response and survival. If validated these data may be added to CLL prognostic scoring systems. PMID:26539641

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

    PubMed Central

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

    2016-01-01

    Epstein-Barr virus (EBV)-DNA is detected in the blood of some persons with chronic lymphocytic leukemia (CLL) at diagnosis. Whether this is important in the development or progression of CLL is controversial. We interrogated associations between blood EBV-DNA copy number and biological and clinical variables in 243 new-diagnosed consecutive subjects with CLL. Quantification of EBV-DNA copies was done by real-time quantitative PCR (RQ-PCR). All subjects had serological evidence of prior EBV-infection. However, only 24 subjects (10%) had a EBV-DNA-positive test at diagnosis. EBV-DNA-positive subjects at diagnosis had lower hemoglobin concentrations and platelet levels, higher thymidine kinase-1 and serum ferritin levels, un-mutated IGHV genes and a greater risk of Richter transformation compared with EBV-DNA-negative subjects. Percent CD20-, CD148- and ZAP70-positive cells and mean fluorescence intensity (MFI) of each cluster designation were also increased in EBV-DNA-positive subjects at diagnosis. EBV-DNA test positivity was associated with a briefer time-to-treatment interval (HR 1.85; [95% confidence interval, 1.13, 3.03]; P=0.014) and worse survival (HR 2.77; [1.18, 6.49]; P=0.019). Reduction in EBV copies was significantly associated with therapy-response. A positive blood EBV-DNA test at diagnosis and sequential testing of EBV copies during therapy were significantly associated with biological and clinical variables, time-to-treatment, therapy-response and survival. If validated these data may be added to CLL prognostic scoring systems. PMID:26539641

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

    PubMed

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

    2015-09-01

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

  12. LoComatioN: a software tool for the analysis of low copy number DNA profiles.

    PubMed

    Gill, Peter; Kirkham, Amanda; Curran, James

    2007-03-01

    Previously, the interpretation of low copy number (LCN) STR profiles has been carried out using the biological or 'consensus' method-essentially, alleles are not reported, unless duplicated in separate PCR analyses [P. Gill, J. Whitaker, C. Flaxman, N. Brown, J. Buckleton, An investigation of the rigor of interpretation rules for STRs derived from less than 100 pg of DNA, Forens. Sci. Int. 112 (2000) 17-40]. The method is now widely used throughout Europe. Although a probabilistic theory was simultaneously introduced, its time-consuming complexity meant that it could not be easily applied in practice. The 'consensus' method is not as efficient as the probabilistic approach, as the former wastes information in DNA profiles. However, the theory was subsequently extended to allow for DNA mixtures and population substructure in a programmed solution by Curran et al. [J.M. Curran, P. Gill, M.R. Bill, Interpretation of repeat measurement DNA evidence allowing for multiple contributors and population substructure, Forens. Sci. Int. 148 (2005) 47-53]. In this paper, we describe an expert interpretation system (LoComatioN) which removes this computational burden, and enables application of the full probabilistic method. This is the first expert system that can be used to rapidly evaluate numerous alternative explanations in a likelihood ratio approach, greatly facilitating court evaluation of the evidence. This would not be possible with manual calculation. Finally, the Gill et al. and Curran et al. papers both rely on the ability of the user to specify two quantities: the probability of allelic drop-out, and the probability of allelic contamination ("drop-in"). In this paper, we offer some guidelines on how these quantities may be specified. PMID:16759831

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

    PubMed Central

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

    2014-01-01

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

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

    SciTech Connect

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

    1994-09-01

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

  15. Soluble normal and mutated DNA sequences from single-copy genes in human blood.

    PubMed

    Sorenson, G D; Pribish, D M; Valone, F H; Memoli, V A; Bzik, D J; Yao, S L

    1994-01-01

    Healthy individuals have soluble (extracellular) DNA in their blood, and increased amounts are present in cancer patients. Here we report the detection of specific sequences of the cystic fibrosis and K-ras genes in plasma DNA from normal donors by amplification with the polymerase chain reaction. In addition, mutated K-ras sequences are identified by polymerase chain reaction utilizing allele-specific primers in the plasma or serum from three patients with pancreatic carcinoma that contain mutated K-ras genes. The mutations are confirmed by direct sequencing. These results indicate that sequences of single-copy genes can be identified in normal plasma and that the sequences of mutated oncogenes can be detected and identified with allele-specific amplification by polymerase chain reaction in plasma or serum from patients with malignant tumors containing identical mutated genes. Mutated oncogenes in plasma and serum may represent tumor markers that could be useful for diagnosis, determining response to treatment, and predicting prognosis. PMID:8118388

  16. High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number

    PubMed Central

    2011-01-01

    Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of ∼2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow. Three applications demonstrate that the massive partitioning afforded by our ddPCR system provides orders of magnitude more precision and sensitivity than real-time PCR. First, we show the accurate measurement of germline copy number variation. Second, for rare alleles, we show sensitive detection of mutant DNA in a 100 000-fold excess of wildtype background. Third, we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR system will allow researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new era of molecular diagnostics. PMID:22035192

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

    PubMed

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

    2016-08-01

    Notch signaling abnormalities are reported to be involved in the acceleration of malignancy in solid tumors and stem cell formation or regeneration in various organs. We analyzed specific genes for DNA copy number variations in liver cancer cells and investigated whether these factors relate to clinical outcome. Chromosome 20p, which includes the ligand for Notch pathways, Jagged1, was found to be amplified in several types of hepatoma cells, and its mRNA was up-regulated according to α-fetoprotein gene expression levels. Notch inhibition using Jagged1 shRNA and γ-secretase inhibitors produced significant suppression of cell growth in α-fetoprotein-producing cells with suppression of downstream genes. Using in vivo hepatoma models, the administration of γ-secretase inhibitors resulted in reduced tumor sizes and effective Notch inhibition with widespread apoptosis and necrosis of viable tumor cells. The γ-secretase inhibitors suppressed cell growth of the epithelial cell adhesion molecule-positive fraction in hepatoma cells, indicating that Notch inhibitors could suppress the stem cell features of liver cancer cells. Even in clinical liver cancer samples, the expression of α-fetoprotein and Jagged1 showed significant correlation, and amplification of the copy number of Jagged1 was associated with Jagged1 mRNA expression and poor survival after liver cancer surgical resection. In conclusion, amplification of Jagged1 contributed to mRNA expression that activates the Jagged1-Notch signaling pathway in liver cancer and led to poor outcome. PMID:27315779

  18. 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

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

    PubMed Central

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

    2014-01-01

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

  20. 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

  1. 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

  2. Analysis of changes in DNA sequence copy number by comparative genomic hybridization in archival paraffin-embedded tumor samples.

    PubMed Central

    Isola, J.; DeVries, S.; Chu, L.; Ghazvini, S.; Waldman, F.

    1994-01-01

    Analysis of previously unknown genetic aberrations in solid tumors has become possible through the use of comparative genomic hybridization (CGH), which is based on competitive binding of tumor and control DNA to normal metaphase chromosomes. CGH allows detection of DNA sequence copy number changes (deletions, gains, and amplifications) on a genome-wide scale in a single hybridization. We describe here an improved CGH technique, which enables reliable detection of copy number changes in archival formalin-fixed paraffin-embedded tumor samples. The technique includes a modified DNA extraction protocol, which produces high molecular weight DNA which is necessary for high quality CGH. The DNA extraction includes a 3-day digestion with proteinase K, which remarkably improves the yield of high molecular weight DNA. Labeling of the test DNA with a directly fluorescein-conjugated nucleotide (instead of biotin labeling) improved significantly the quality of hybridization. Using the paraffin-block technique, we could analyze 70 to 90% of paraffin blocks, including very old samples as well as samples taken at autopsy. CGH from paraffin blocks was highly concordant (95%) with analyses done from matched freshly frozen tumor samples (n = 5 sample pairs; kappa coefficient = 0.83). The method described here has wide applicability in tumor pathology, allowing large retrospective prognostic studies of genetic aberrations as well as studies on genetic pathogenesis of solid tumors, inasmuch as premalignant lesions and primary and metastatic tumors can be analyzed by using archival paraffin-embedded samples. Images Figure 1 Figure 3 PMID:7992835

  3. High sensitivity detection and quantitation of DNA copy number and single nucleotide variants with single color droplet digital PCR.

    PubMed

    Miotke, Laura; Lau, Billy T; Rumma, Rowza T; Ji, Hanlee P

    2014-03-01

    In this study, we present a highly customizable method for quantifying copy number and point mutations utilizing a single-color, droplet digital PCR platform. Droplet digital polymerase chain reaction (ddPCR) is rapidly replacing real-time quantitative PCR (qRT-PCR) as an efficient method of independent DNA quantification. Compared to quantative PCR, ddPCR eliminates the needs for traditional standards; instead, it measures target and reference DNA within the same well. The applications for ddPCR are widespread including targeted quantitation of genetic aberrations, which is commonly achieved with a two-color fluorescent oligonucleotide probe (TaqMan) design. However, the overall cost and need for optimization can be greatly reduced with an alternative method of distinguishing between target and reference products using the nonspecific DNA binding properties of EvaGreen (EG) dye. By manipulating the length of the target and reference amplicons, we can distinguish between their fluorescent signals and quantify each independently. We demonstrate the effectiveness of this method by examining copy number in the proto-oncogene FLT3 and the common V600E point mutation in BRAF. Using a series of well-characterized control samples and cancer cell lines, we confirmed the accuracy of our method in quantifying mutation percentage and integer value copy number changes. As another novel feature, our assay was able to detect a mutation comprising less than 1% of an otherwise wild-type sample, as well as copy number changes from cancers even in the context of significant dilution with normal DNA. This flexible and cost-effective method of independent DNA quantification proves to be a robust alternative to the commercialized TaqMan assay. PMID:24483992

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

    PubMed Central

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

    2014-01-01

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

  5. 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.

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

    2012-01-01

    Background While the genetic transformation of the major cereal crops has become relatively routine, to date only a few reports were published on transgenic triticale, and robust data on T-DNA integration and segregation have not been available in this species. Results Here, we present a comprehensive analysis of stable transgenic winter triticale cv. Bogo carrying the selectable marker gene HYGROMYCIN PHOSPHOTRANSFERASE (HPT) and a synthetic green fluorescent protein gene (gfp). Progeny of four independent transgenic plants were comprehensively investigated with regard to the number of integrated T-DNA copies, the number of plant genomic integration loci, the integrity and functionality of individual T-DNA copies, as well as the segregation of transgenes in T1 and T2 generations, which also enabled us to identify homozygous transgenic lines. The truncation of some integrated T-DNAs at their left end along with the occurrence of independent segregation of multiple T-DNAs unintendedly resulted in a single-copy segregant that is selectable marker-free and homozygous for the gfp gene. The heritable expression of gfp driven by the maize UBI-1 promoter was demonstrated by confocal laser scanning microscopy. Conclusions The used transformation method is a valuable tool for the genetic engineering of triticale. Here we show that comprehensive molecular analyses are required for the correct interpretation of phenotypic data collected from the transgenic plants. PMID:23006412

  8. Integrated analysis of copy number and loss of heterozygosity in primary breast carcinomas using high-density SNP array.

    PubMed

    Ching, Ho Ching; Naidu, Rakesh; Seong, Mun Kein; Har, Yip Cheng; Taib, Nur Aishah Mohd

    2011-09-01

    Breast cancer is a heterogeneous disease, marked by extensive chromosomal aberrations. In this study, we aimed to explicate the underlying chromosomal copy number (CN) alterations and loss of heterozygosity (LOH) implicated in a cohort of Malaysian hospital-based primary breast carcinoma samples using a single nucleotide polymorphism (SNP) array platform. The analysis was conducted by hybridizing the extracted DNA of 70 primary breast carcinomas and 37 normal peripheral blood samples to the Affymetrix 250K Sty SNP arrays. Locus-specific CN aberrations and LOH were statistically summarized using the binary segmentation algorithm and hidden Markov model. Selected genes from the SNP array analysis were also validated using quantitative real-time PCR. The merging of CN and LOH data fabricated distinctive integrated alteration profiles, which were comprised of finely demarcated minimal sites of aberrations. The most prevalent gains (≥ 30%) were detected at the 8q arm: 8q23.1, 8q23.3, 8q24.11, 8q24.13, 8q24.21, 8q24.22, 8q24.23 and 8q24.3, whilst the most ubiquitous losses (≥ 20%) were noted at the 8p12, 8p21.1, 8p21.2, 8p21.1-p21.2, 8p21.3, 8p22, 8p23.1, 8p23.1‑p23.2, 8p23.3, 17p11.2, 17p12, 17p11.2-p12, 17p13.1 and 17p13.2 regions. Copy-neutral LOH was characterized as the most prevailing LOH event, in which the most frequent distributions (≥ 30%) were revealed at 3p21.31, 5q33.2, 12q24.12, 12q24.12‑q24.13 and 14q23.1. These findings offer compre-hensive genome-wide views on breast cancer genomic changes, where the most recurrent gain, loss and copy-neutral LOH events were harboured within the 8q24.21, 8p21.1 and 14q23.1 loci, respectively. This will facilitate the uncovering of true driver genes pertinent to breast cancer biology and the develop-ment of prospective therapeutics. PMID:21687935

  9. Multiple copies of Shope virus DNA are present in cells of benign and malignant non-virus-producing neoplasms.

    PubMed Central

    Stevens, J G; Wettstein, F O

    1979-01-01

    In an initial efforts to characterize the virological basis of neoplasia in the Shope papilloma-carcinoma system, the extent to which the viral genome is present in non-virus-producing benign and malignant tumors in domestic rabbits was established. Employing nick-translated radioactive viral DNA purified from productively infected papillomas on cotton tail rabbits as a probe, it was found that (i) papillomas, primary carcinomas, and metastatic carcinomas contain 10 to about 100 copies of the viral genome per diploid cell equivalent of DNA and (ii) viral DNA is present in detectable amounts in essentially all neoplastic cells. These results are consistent with the suggestion that continued presence of the viral genome is necessary for induction and maintenance of malignant as well as benign neoplasms. Images PMID:225548

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

    PubMed

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

    2016-06-01

    One of the major unanswered questions with respect to the commercial use of genetic transformation in woody plants is the stability of the transgene expression over several decades within the same individual. Gene expression is strongly affected by the copy number which has been integrated into the plant genome and by the local DNA features close to the integration sites. Because woody plants cannot be subjected to selfing or backcrossing to modify the transgenic allelic structure without affecting the valuable traits of the cultivar, molecular characterization of the transformation event is therefore crucial. After assessing the transgene copy number of a set of apple transgenic clones with Southern blotting, we describe two alternative methods: the first is based on inverse PCR (i-PCR) and the second on the quantitative PCR (q-PCR). The methods produced comparable results with the exception of the data regarding a high copy number clone, but while the q-PCR-based system is rapid and easily adaptable to high throughput systems, the i-PCR-based method can provide information regarding the transformation event and the characteristics of the sequences flanking the transgenic construct. PMID:26895172

  11. Effect of Maternal Age on the Ratio of Cleavage and Mitochondrial DNA Copy Number in Early Developmental Stage Bovine Embryos

    PubMed Central

    TAKEO, Shun; GOTO, Hiroya; KUWAYAMA, Takehito; MONJI, Yasunori; IWATA, Hisataka

    2012-01-01

    Abstract Age-associated deterioration in both the quality and quantity of mitochondria occurs in older women. The main aim of this study was to examine the effect of age on mitochondrial DNA copy number (mtDNA number) in early developmental stage bovine embryos as well as the dynamics of mtDNA number during early embryo development. Real-time PCR was used to determine mtDNA number. In vitro-produced embryos 48 h after insemination derived from Japanese black cows, ranging in age from 25 to 209 months were categorized based on their cleavage status. There was an overall negative relationship between the age of the cow and cleavage status, to the extent that the ratio of embryos cleaved over the 4-cell stage was greater in younger cows. The mtDNA number did not differ among the cleaved status of embryos. In the next experiment, oocytes collected from each donor cow were divided into 2 groups containing 10 oocytes each, in order to compare the mtDNA number of mature oocytes and early developmental stage embryos within individuals. Upon comparing the mtDNA number between oocytes at the M2 stage and early developmental stage 48 h post insemination, mtDNA number was found to decrease in most cows, but was found to increase in some cows. In conclusion, age affects the cleaving ability of oocytes, and very old cows (> 180 months) tend to have lower mtDNA numbers in their oocytes. The change in mtDNA number during early development varied among individual cows, although overall, it showed a tendency to decrease. PMID:23269452

  12. A nested case-control study of leukocyte mitochondrial DNA copy number and renal cell carcinoma in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial.

    PubMed

    Hofmann, Jonathan N; Hosgood, H Dean; Liu, Chin-San; Chow, Wong-Ho; Shuch, Brian; Cheng, Wen-Ling; Lin, Ta-Tsung; Moore, Lee E; Lan, Qing; Rothman, Nathaniel; Purdue, Mark P

    2014-05-01

    Mitochondrial DNA (mtDNA) is vulnerable to mutations, and the number of copies of mtDNA per cell may increase to compensate for DNA damage. Case-control studies have reported associations between altered mtDNA copy number and risk of renal cell carcinoma (RCC); however, this association has not been investigated prospectively. We conducted a nested case-control study (252 cases and 504 controls) of RCC risk in relation to pre-diagnostic leukocyte mtDNA copy number in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial. mtDNA copy number was measured in triplicate using a fluorescence-based quantitative PCR assay; samples from 22 cases and 36 controls could not be assayed, leaving 230 cases and 468 controls for analysis. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using unconditional logistic regression. High mtDNA copy number was associated with an increased risk of RCC, both overall (highest quartile versus lowest: OR = 2.0, 95% CI = 1.2-3.2; P trend = 0.002) and among cases diagnosed ≥6 years after blood collection (OR = 2.6, 95% CI = 1.4-5.0; P trend = 0.003). These findings did not differ significantly by sex, body mass index, history of hypertension or smoking status (P interaction ≥ 0.3). Results of this study suggest that high pre-diagnostic leukocyte mtDNA copy number, a suspected marker of oxidative DNA damage and mitochondrial dysfunction, is associated with increased future RCC risk. PMID:24398668

  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. 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

  15. Integrating data from heterogeneous DNA microarray platforms.

    PubMed

    Valente, Eduardo; Rocha, Miguel

    2015-01-01

    DNA microarrays are one of the most used technologies for gene expression measurement. However, there are several distinct microarray platforms, from different manufacturers, each with its own measurement protocol, resulting in data that can hardly be compared or directly integrated. Data integration from multiple sources aims to improve the assertiveness of statistical tests, reducing the data dimensionality problem. The integration of heterogeneous DNA microarray platforms comprehends a set of tasks that range from the re-annotation of the features used on gene expression, to data normalization and batch effect elimination. In this work, a complete methodology for gene expression data integration and application is proposed, which comprehends a transcript-based re-annotation process and several methods for batch effect attenuation. The integrated data will be used to select the best feature set and learning algorithm for a brain tumor classification case study. The integration will consider data from heterogeneous Agilent and Affymetrix platforms, collected from public gene expression databases, such as The Cancer Genome Atlas and Gene Expression Omnibus. PMID:26673932

  16. An integrative segmentation method for detecting germline copy number variations in SNP arrays.

    PubMed

    Shi, Jianxin; Li, Peng

    2012-05-01

    Germline copy number variations (CNVs) are a major source of genetic variation in humans. In large-scale studies of complex diseases, CNVs are usually detected from data generated by single nucleotide polymorphism (SNP) genotyping arrays. In this paper, we develop an integrative segmentation method, SegCNV, for detecting CNVs integrating both log R ratio (LRR) and B allele frequency (BAF). Based on simulation studies, SegCNV had modestly better power to detect deletions and substantially better power to detect duplications compared with circular binary segmentation (CBS) that relies purely on LRRs; and it had better power to detect deletions and a comparable performance to detect duplications compared with PennCNV and QuantiSNP. In two Hapmap subjects with deep sequence data available as a gold standard, SegCNV detected more true short deletions than PennCNV and QuantiSNP. For 21 short duplications validated experimentally in the AGRE dataset, SegCNV, QuantiSNP, and PennCNV detected all of them while CBS detected only three. SegCNV is much faster than the HMM-based (where HMM is hidden Markov model) methods, taking only several seconds to analyze genome-wide data for one subject. PMID:22539397

  17. 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

  18. 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

  19. De-methylation of displacement loop of mitochondrial DNA is associated with increased mitochondrial copy number and nicotinamide adenine dinucleotide subunit 2 expression in colorectal cancer.

    PubMed

    Gao, Jinhang; Wen, Shilei; Zhou, Hongying; Feng, Shi

    2015-11-01

    DNA methylation occurs in the displacement loop (D-loop) region of mammals; however, D-loop regions of certain tumor tissue types were found to be de‑methylated. Whether hypomethylation of the D‑loop region is involved in the regulation of the mitochondrial DNA (mtDNA) copy number and nicotinamide adenine dinucleotide subunit 2 (ND‑2) expressions in colorectal cancer has remained elusive. In the present study, the association between methylation status of the D‑loop region, mtDNA copy number and ND‑2 expression was investigated in 65 colorectal cancer specimens and their corresponding non‑cancerous tissues. In addition, a de‑methylation experiment was performed on the Caco‑2 colorectal cancer cell line by using 5‑aza-2'-deoxycytidine (5‑Aza). The methylation rate of the D‑loop region in all 65 colorectal cancer tissues was markedly reduced when compared with that of their corresponding non‑cancerous tissues (13.8 vs. 81.5%; P<0.05). Furthermore, the methylation rate of the D‑loop region in colorectal cancer tissues was markedly decreased in clinicopathological stages III and IV compared with that in clinicopathological stages I and II (7.1 and 0% vs. 25 and 16%; P<0.05). In addition, the mean relative mtDNA copy number and ND‑2 expression in colorectal cancer tissues were increased compared with those in the corresponding non‑cancerous tissues. De‑methylation of the D‑loop region was associated with an elevated mtDNA copy number and an increased ND‑2 expression. Furthermore, the mtDNA copy number and ND‑2 expression in Caco‑2 cells were significantly increased after 5‑Aza treatment. In conclusion, de‑methylation of the D‑loop region is likely to be involved in the regulation of the mtDNA copy number and ND-2 expression. PMID:26323487

  20. 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. PMID:26688582

  1. 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

  2. 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. PMID:26068926

  3. Impacts of low temperature preservation on mitochondrial DNA copy number in oocytes of the hard coral Echinopora sp.

    PubMed

    Tsai, Sujune; Thongpooe, Preeyanuch; Kuo, Fu-Wen; Lin, Chiahsin

    2016-07-01

    Given the current threats to coral reefs worldwide, there is an urgent need to develop protocols for the cryopreservation of reef-building corals. However, chilling may alter coral mitochondrial distribution and membrane potential, resulting in reduced ATP production. The aim of this study was to investigate the impacts of chilling on mitochondrial DNA copy number (CN) in oocytes of the hard coral Echinopora sp. Oocytes were exposed to 0.5 M, 1 M or 2 M methanol at 5, 0 or -5 °C for 2, 4, 8 and 16 h. When oocytes were chilled with no cryoprotectant (CPT) or 1 M methanol at 5 or 0 °C, the mtDNA CNs initially increased at hour 2 of incubation, although it decreased significantly over the 16 h of incubation in chilled oocytes at -5 °C. The mtDNA CN increased and picked in 0.5 M methanol at 5 °C and 0 °C at hour 8 of incubation in chilled oocytes indicating that the high mtDNA CN of these oocytes is probably responsible for withstanding high chilling sensitivity. We currently propose that 0.5 M methanol is the optimal CPT for oocytes of Echinopora sp., and potentially other reef corals. PMID:25901989

  4. 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

  5. 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. PMID:26508664

  6. The role of mitochondrial DNA copy number, variants, and haplotypes in farm animal developmental outcome.

    PubMed

    Tsai, Tesha; St John, Justin C

    2016-07-01

    The vast majority of cellular energy is generated through the process of oxidative phosphorylation, which takes place in the electron transport chain in the mitochondria. The electron transport chain is encoded by 2 genomes, the chromosomal and the mitochondrial genomes. Mitochondrial DNA is associated with a number of traits, which include tolerance to heat, growth and physical performance, meat and milk quality, and fertility. Mitochondrial genomes can be clustered into groups known as mtDNA haplotypes. Mitochondrial DNA haplotypes are a potential genetic source for manipulating phenotypes in farm animals. The use of assisted reproductive technologies, such as nuclear transfer, allows favorable chromosomal genetic traits to be mixed and matched with sought after mtDNA haplotype traits. As a result super breeds can be generated. PMID:27345311

  7. A novel T-DNA integration in rice involving two interchromosomal translocations.

    PubMed

    Majhi, Bharat Bhusan; Shah, Jasmine M; Veluthambi, Karuppannan

    2014-06-01

    A male sterile transgenic rice plant TC-19 harboured a novel T-DNA integration in chromosome 8 with two interchromosomal translocations of 6.55 kb chromosome 3 and 29.8 kb chromosome 9 segments. We report a complex Agrobacterium T-DNA integration in rice (Oryza sativa) associated with two interchromosomal translocations. The T-DNA-tagged rice mutant TC-19, which harboured a single copy of the T-DNA, displayed male sterile phenotype in the homozygous condition. Analysis of the junctions between the T-DNA ends and the rice genome by genome walking showed that the right border is flanked by a chromosome 3 sequence and the left border is flanked by a chromosome 9 sequence. Upon further walking on chromosome 3, a chromosome 3/chromosome 8 fusion was detected. Genome walking from the opposite end of the chromosome 8 break point revealed a chromosome 8/chromosome 9 fusion. Our findings revealed that the T-DNA, together with a 6.55-kb region of chromosome 3 and a 29.8-kb region of chromosome 9, was translocated to chromosome 8. Southern blot analysis of the homozygous TC-19 mutant revealed that the native sequences of chromosome 3 and 9 were restored but the disruption of chromosome 8 in the first intron of the gene Os08g0152500 was not restored. The integration of the complex T-DNA in chromosome 8 caused male sterility. PMID:24487649

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

    PubMed

    Kervio, Eric; Sosson, Marilyne; Richert, Clemens

    2016-07-01

    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

  9. 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

  10. One-step cloning and chromosomal integration of DNA.

    PubMed

    St-Pierre, François; Cui, Lun; Priest, David G; Endy, Drew; Dodd, Ian B; Shearwin, Keith E

    2013-09-20

    We describe "clonetegration", a method for integrating DNA into prokaryotic chromosomes that approaches the simplicity of cloning DNA within extrachromosomal vectors. Compared to existing techniques, clonetegration drastically decreases the time and effort needed for integration of single or multiple DNA fragments. Additionally, clonetegration facilitates cloning and expression of genetic elements that are impossible to propagate within typical multicopy plasmids. PMID:24050148

  11. 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

  12. Integrated adenovirus type 12 DNA in the transformed hamster cell line T637: sequence arrangements at the termini of viral DNA and mode of amplification.

    PubMed Central

    Eick, D; Doerfler, W

    1982-01-01

    Approximately 20 to 22 copies of adenovirus type 12 (Ad12) DNA per cell were integrated into the genome of the cell line T637. Only a few of these copies seemed to remain intact and colinear with virion DNA. All other persisting viral genomes exhibited deletions or inversions or both in the right-hand part of Ad12 DNA. Spontaneously arising morphological revertants of T637 cells has lost viral DNA. In most of the revertant cell lines only the intact or a part of the intact viral genome was preserved; other revertant cell lines has lost all viral DNA. In three other Ad12-transformed hamster cell lines, HA12/7, A2497-3, and CLAC3 (Stabel et al., J. Virol. 36:22-40, 1980), major rearrangements at the right end of the integrated Ad12 DNA were not found. These studies were performed to investigate the phenomena of amplification, rearrangements, and deletions of Ad12 DNA in hamster cells. Images PMID:6283150

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

    PubMed

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

    2012-01-01

    Chrysanthemum L. (Asteraceae-Anthemideae) is a genus with rapid speciation. It comprises about 40 species, most of which are distributed in East Asia. Many of these are narrowly distributed and habitat-specific. Considerable variations in morphology and ploidy are found in this genus. Some species have been the subjects of many studies, but the relationships between Chrysanthemum and its allies and the phylogeny of this genus remain poorly understood. In the present study, 32 species/varieties from Chrysanthemum and 11 from the allied genera were analyzed using DNA sequences of the single-copy nuclear CDS gene and seven cpDNA loci (psbA-trnH, trnC-ycf6, ycf6-psbM, trnY-rpoB, rpS4-trnT, trnL-F, and rpL16). The cpDNA and nuclear CDS gene trees both suggest that 1) Chrysanthemum is not a monophyletic taxon, and the affinity between Chrysanthemum and Ajania is so close that these two genera should be incorporated taxonomically; 2) Phaeostigma is more closely related to the Chrysanthemum+Ajania than other generic allies. According to pollen morphology and to the present cpDNA and CDS data, Ajania purpurea is a member of Phaeostigma. Species differentiation in Chrysanthemum appears to be correlated with geographic and environmental conditions. The Chinese Chrysanthemum species can be divided into two groups, the C. zawadskii group and the C. indicum group. The former is distributed in northern China and the latter in southern China. Many polyploid species, such as C. argyrophyllum, may have originated from allopolyploidization involving divergent progenitors. Considering all the evidence from present and previous studies, we conclude that geographic and ecological factors as well as hybridization and polyploidy play important roles in the divergence and speciation of the genus Chrysanthemum. PMID:23133665

  14. Phylogeny of the Genus Chrysanthemum L.: Evidence from Single-Copy Nuclear Gene and Chloroplast DNA Sequences

    PubMed Central

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

    2012-01-01

    Chrysanthemum L. (Asteraceae-Anthemideae) is a genus with rapid speciation. It comprises about 40 species, most of which are distributed in East Asia. Many of these are narrowly distributed and habitat-specific. Considerable variations in morphology and ploidy are found in this genus. Some species have been the subjects of many studies, but the relationships between Chrysanthemum and its allies and the phylogeny of this genus remain poorly understood. In the present study, 32 species/varieties from Chrysanthemum and 11 from the allied genera were analyzed using DNA sequences of the single-copy nuclear CDS gene and seven cpDNA loci (psbA-trnH, trnC-ycf6, ycf6-psbM, trnY-rpoB, rpS4-trnT, trnL-F, and rpL16). The cpDNA and nuclear CDS gene trees both suggest that 1) Chrysanthemum is not a monophyletic taxon, and the affinity between Chrysanthemum and Ajania is so close that these two genera should be incorporated taxonomically; 2) Phaeostigma is more closely related to the Chrysanthemum+Ajania than other generic allies. According to pollen morphology and to the present cpDNA and CDS data, Ajania purpurea is a member of Phaeostigma. Species differentiation in Chrysanthemum appears to be correlated with geographic and environmental conditions. The Chinese Chrysanthemum species can be divided into two groups, the C. zawadskii group and the C. indicum group. The former is distributed in northern China and the latter in southern China. Many polyploid species, such as C. argyrophyllum, may have originated from allopolyploidization involving divergent progenitors. Considering all the evidence from present and previous studies, we conclude that geographic and ecological factors as well as hybridization and polyploidy play important roles in the divergence and speciation of the genus Chrysanthemum. PMID:23133665

  15. 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). PMID:25768890

  16. Human subtelomeric copy number gains suggest a DNA replication mechanism for formation: beyond breakage – fusion - bridge for telomere stabilization

    PubMed Central

    Yatsenko, Svetlana A.; Hixson, Patricia; Roney, Erin K.; Scott, Daryl A.; Schaaf, Christian P.; Ng, Yu-tze; Palmer, Robbin; Fisher, Richard B.; Patel, Ankita; Cheung, Sau Wai; Lupski, James R.

    2012-01-01

    Constitutional deletions of distal 9q34 encompassing the EHMT1 (euchromatic histone methyltransferase 1) gene, or loss-of-function point mutations in EHMT1, are associated with the 9q34.3 microdeletion, also known as Kleefstra syndrome [MIM#610253]. We now report further evidence for genomic instability of the subtelomeric 9q34.3 region as evidenced by copy number gains of this genomic interval that include duplications, triplications, derivative chromosomes and complex rearrangements. Comparisons between the observed shared clinical features and molecular analyses in 20 subjects suggest that increased dosage of EHMT1 may be responsible for the neurodevelopmental impairment, speech delay, and autism spectrum disorders revealing the dosage sensitivity of yet another chromatin remodeling protein in human disease. Five patients had 9q34 genomic abnormalities resulting in complex deletion-duplication or duplication-triplication rearrangements; such complex triplications were also observed in six other subtelomeric intervals. Based on the specific structure of these complex genomic rearrangements (CGR) a DNA replication mechanism is proposed confirming recent findings in C elegans telomere healing. The end-replication challenges of subtelomeric genomic intervals may make them particularly prone to rearrangements generated by errors in DNA replication. PMID:22890305

  17. Functions of the high mobility group protein, Abf2p, in mitochondrial DNA segregation, recombination and copy number in Saccharomyces cerevisiae.

    PubMed Central

    Zelenaya-Troitskaya, O; Newman, S M; Okamoto, K; Perlman, P S; Butow, R A

    1998-01-01

    Previous studies have established that the mitochondrial high mobility group (HMG) protein, Abf2p, of Saccharomyces cerevisiae influences the stability of wild-type (rho+) mitochondrial DNA (mtDNA) and plays an important role in mtDNA organization. Here we report new functions for Abf2p in mtDNA transactions. We find that in homozygous deltaabf2 crosses, the pattern of sorting of mtDNA and mitochondrial matrix protein is altered, and mtDNA recombination is suppressed relative to homozygous ABF2 crosses. Although Abf2p is known to be required for the maintenance of mtDNA in rho+ cells growing on rich dextrose medium, we find that it is not required for the maintenance of mtDNA in p cells grown on the same medium. The content of both rho+ and rho- mtDNAs is increased in cells by 50-150% by moderate (two- to threefold) increases in the ABF2 copy number, suggesting that Abf2p plays a role in mtDNA copy control. Overproduction of Abf2p by > or = 10-fold from an ABF2 gene placed under control of the GAL1 promoter, however, leads to a rapid loss of rho+ mtDNA and a quantitative conversion of rho+ cells to petites within two to four generations after a shift of the culture from glucose to galactose medium. Overexpression of Abf2p in rho- cells also leads to a loss of mtDNA, but at a slower rate than was observed for rho+ cells. The mtDNA instability phenotype is related to the DNA-binding properties of Abf2p because a mutant Abf2p that contains mutations in residues of both HMG box domains known to affect DNA binding in vitro, and that binds poorly to mtDNA in vivo, complements deltaabf2 cells only weakly and greatly lessens the effect of overproduction on mtDNA instability. In vivo binding was assessed by colocalization to mtDNA of fusions between mutant or wild-type Abf2p and green fluorescent protein.These findings are discussed in the context of a model relating mtDNA copy number control and stability to mtDNA recombination. PMID:9581629

  18. 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

  19. Path integral method for DNA denaturation

    NASA Astrophysics Data System (ADS)

    Zoli, Marco

    2009-04-01

    The statistical physics of homogeneous DNA is investigated by the imaginary time path integral formalism. The base pair stretchings are described by an ensemble of paths selected through a macroscopic constraint, the fulfillment of the second law of thermodynamics. The number of paths contributing to the partition function strongly increases around and above a specific temperature Tc∗ , whereas the fraction of unbound base pairs grows continuously around and above Tc∗ . The latter is identified with the denaturation temperature. Thus, the separation of the two complementary strands appears as a highly cooperative phenomenon displaying a smooth crossover versus T . The thermodynamical properties have been computed in a large temperature range by varying the size of the path ensemble at the lower bound of the range. No significant physical dependence on the system size has been envisaged. The entropy grows continuously versus T while the specific heat displays a remarkable peak at Tc∗ . The location of the peak versus T varies with the stiffness of the anharmonic stacking interaction along the strand. The presented results suggest that denaturation in homogeneous DNA has the features of a second-order phase transition. The method accounts for the cooperative behavior of a very large number of degrees of freedom while the computation time is kept within a reasonable limit.

  20. 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

  1. 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

  2. Functional integration of PCR amplification and capillary electrophoresis in a microfabricated DNA analysis device.

    PubMed

    Woolley, A T; Hadley, D; Landre, P; deMello, A J; Mathies, R A; Northrup, M A

    1996-12-01

    Microfabricated silicon PCR reactors and glass capillary electrophoresis (CE) chips have been successfully coupled to form an integrated DNA analysis system. This construct combines the rapid thermal cycling capabilities of microfabricated PCR devices (10 degrees C/s heating, 2.5 degrees C/s cooling) with the high-speed (< 120 s) DNA separations provided by microfabricated CE chips. The PCR chamber and the CE chip were directly linked through a photolithographically fabricated channel filled with hydroxyethylcellulose sieving matrix. Electrophoretic injection directly from the PCR chamber through the cross injection channel was used as an "electrophoretic valve" to couple the PCR and CE devices on-chip. To demonstrate the functionality of this system, a 15 min PCR amplification of a beta-globin target cloned in M13 was immediately followed by high-speed CE chip separation in under 120 s, providing a rapid PCR-CE analysis in under 20 min. A rapid assay for genomic Salmonella DNA was performed in under 45 min, demonstrating that challenging amplifications of diagnostically interesting targets can also be performed. Real-time monitoring of PCR target amplification in these integrated PCR-CE devices is also feasible. Amplification of the beta-globin target as a function of cycle number was directly monitored for two different reactions starting with 4 x 10(7) and 4 x 10(5) copies of DNA template. This work establishes the feasibility of performing high-speed DNA analyses in microfabricated integrated fluidic systems. PMID:8946790

  3. Postoperative hepatitis B virus reactivation in hepatitis B virus-related hepatocellular carcinoma patients with hepatitis B virus DNA levels <500 copies/mL

    PubMed Central

    Xie, Zhi-Bo; Wang, Xiao-Bo; Fu, De-Liang; Zhong, Jian-Hong; Yang, Xia-Wei; Li, Le-Qun

    2016-01-01

    Background Patients with hepatocellular carcinoma have the risk of postoperative hepatitis B virus (HBV) reactivation (PHR). Antiviral therapy was given to patients with detectable HBV DNA levels but not to patients with undetectable HBV DNA levels. Methods In this retrospective study, 258 patients were enrolled (HBV DNA levels <500 copies/mL group, n=159, and HBV DNA levels >500 copies/mL group, n=99). Results A total of 50 patients (19.4%) had PHR. The following significant factors related to PHR were found: without antiviral therapy (hazard ratio [HR] =0.17, 95% confidence interval [CI] 0.031–0.911), hepatitis B e antigen positivity (HR =5.20, 95% CI 1.931–14.007), hepatitis B core antigen S1 positivity (HR =2.54, 95% CI 1.116–5.762), preoperative HBV DNA levels ≥500 copies/mL (HR =1.28, 95% CI 1.085–2.884), hepatic inflow occlusion (HR =3.60, 95% CI 1.402–9.277), moderate liver cirrhosis or more (HR =2.26, 95% CI 1.001–5.121), and blood transfusion (HR =2.89, 95% CI 0.836–10.041). Recurrence-free survival time was significantly shorter in patients with PHR (23.06±2.46 months) than in patients without PHR (29.30±1.27 months). Conclusion Antiviral therapy could efficiently decrease the incidence of PHR. Patients with HBV DNA levels <500 copies/mL still have the risk of PHR. PHR remained as a prognostic risk factor for hepatocellular carcinoma recurrence and recurrence-free survival. PMID:27524913

  4. 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. PMID:27299603

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

    PubMed

    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

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

    PubMed Central

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

    2013-01-01

    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. PMID:23468533

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

    PubMed Central

    Cai, Yu-dong

    2015-01-01

    The mechanisms of lung cancer are highly complex. Not only mRNA gene expression but also microRNAs, DNA methylation, and copy number variation (CNV) play roles in tumorigenesis. It is difficult to incorporate so much information into a single model that can comprehensively reflect all these lung cancer mechanisms. In this study, we analyzed the 129 TCGA (The Cancer Genome Atlas) squamous cell lung carcinoma samples with gene expression, microRNA expression, DNA methylation, and CNV data. First, we used variance inflation factor (VIF) regression to build the whole genome integrative network. Then, we isolated the lung cancer subnetwork by identifying the known lung cancer genes and their direct regulators. This subnetwork was refined by the Bayesian method, and the directed regulations among mRNA genes, microRNAs, methylations, and CNVs were obtained. The novel candidate key drivers in this refined subnetwork, such as the methylation of ARHGDIB and HOXD3, microRNA let-7a and miR-31, and the CNV of AGAP2, were identified and analyzed. On three large public available lung cancer datasets, the key drivers ARHGDIB and HOXD3 demonstrated significant associations with the overall survival of lung cancer patients. Our results provide new insights into lung cancer mechanisms. PMID:25802847

  8. 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.

  9. Early integration of high copy HPV16 detectable in women with normal and low grade cervical cytology and histology

    PubMed Central

    Kulmala, S‐M A; Syrjänen, S M; Gyllensten, U B; Shabalova, I P; Petrovichev, N; Tosi, P; Syrjänen, K J; Johansson, B C

    2006-01-01

    Background Integration of human papillomavirus (HPV) DNA has been considered a late event in cervical carcinogenesis. However, integrated forms of HPV were recently detected in cancer precursor lesions using a new real time polymerase chain reaction (PCR) to detect the deletions at the 3362–3443 region of HPV16 E2 Objective To study the frequency of HPV16 DNA integration in cervical lesions and compare the sensitivity of an additional upstream region of the E2 ORF (2962–3138) in detecting HPV integration. Methods Using the TaqMan based PCR, HPV16 positive DNA samples were analysed in 164 cervical scrapings from women participating in a multicentre screening trial. Biopsy confirmation was available in 62 cases. Results Primers targeting the 3362–3443 region detected the majority of E2 deletions. In only 23% of the samples was the E2 upstream region equal or better target than the 3362–3443 region. Mixed (episomal/integrated) pattern was the most prevalent physical state of HPV16, also present in PAP smears with normal morphology. Pure integrated form was most prevalent in HSIL and cancer lesions, but also detectable in low grade abnormalities (NSIL, ASC‐US, LSIL). Women with only integrated HPV16 were almost 10 years older than those with episomal HPV16. Viral load of integrated HPV16 was related to cytological abnormality (p = 0.003) but not to histology. Conclusions Integrated HPV16 is present in low grade cervical lesions, mostly mixed with the episomal form. Women with the pure integrated form of HPV16 are older than those with the other forms. PMID:16484445

  10. Mitotic stability and nuclear inheritance of integrated viral cDNA in engineered hypovirulent strains of the chestnut blight fungus.

    PubMed Central

    Chen, B; Choi, G H; Nuss, D L

    1993-01-01

    Transmissible hypovirulence is a novel form of biological control in which virulence of a fungal pathogen is attenuated by an endogenous RNA virus. The feasibility of engineering hypovirulence was recently demonstrated by transformation of the chestnut blight fungus, Cryphonectria parasitica, with a full-length cDNA copy of a hypovirulence-associated viral RNA. Engineered hypovirulent transformants were found to contain both a chromsomally integrated cDNA copy of the viral genome and a resurrected cytoplasmically replicating double-stranded RNA form. We now report stable maintenance of integrated viral cDNA through repeated rounds of asexual sporulation and passages on host plant tissue. We also demonstrate stable nuclear inheritance of the integrated viral cDNA and resurrection of the cytoplasmic viral double-stranded RNA form in progeny resulting from the mating of an engineered hypovirulent C. parasitica strain and a vegetatively incompatible virulent strain. Mitotic stability of the viral cDNA ensures highly efficient transmission of the hypovirulence phenotype through conidia. Meiotic transmission, a mode not observed for natural hypovirulent strains, introduces virus into ascospore progeny representing a spectrum of vegetative compatibility groups, thereby circumventing barriers to anastomosis-mediated transmission imposed by the fungal vegetative incompatibility system. These transmission properties significantly enhance the potential of engineered hypovirulent C. parasitica strains as effective biocontrol agents. Images PMID:8344241

  11. Increase in the frequency of hepadnavirus DNA integrations by oxidative DNA damage and inhibition of DNA repair.

    PubMed Central

    Petersen, J; Dandri, M; Bürkle, A; Zhang, L; Rogler, C E

    1997-01-01

    Persistent hepadnavirus infection leads to oxidative stress and DNA damage through increased production of toxic oxygen radicals. In addition, hepadnaviral DNA integrations into chromosomal DNA can promote the process of hepatocarcinogenesis (M. Feitelson, Clin. Microbiol. Rev. 5:275-301, 1992). While previous studies have identified preferred integration sites in hepadnaviral genomes and suggested integration mechanisms (M. A. Buendia, Adv. Cancer Res. 59:167-226, 1992; C. E. Rogler, Curr. Top. Microbiol. Immunol. 168:103-141, 1991; C. Shih et al., J. Virol. 61:3491-3498, 1987), very little is known about the effects of agents which damage chromosomal DNA on the frequency of hepadnaviral DNA integrations. Using a recently developed subcloning approach to detect stable new integrations of duck hepatitis B virus (DHBV) (S. S. Gong, A. D. Jensen, and C. E. Rogler, J. Virol. 70:2000-2007, 1996), we tested the effects of increased chromosomal DNA damage induced by H2O2, or of the disturbance in DNA repair due to the inhibition of poly(ADP-ribose) polymerase (PARP), on the frequency of DHBV DNA integrations. Subclones of LMH-D21-6 cells, which replicate DHBV, were grown in the presence of various H2O2 concentrations and exhibited up to a threefold increase in viral DNA integration frequency in a dose-dependent manner. Moreover, inhibition of PARP, which plays a role in cellular responses to DNA breakage, by 3-aminobenzamide (3-AB) resulted in a sevenfold increase in the total number of new DHBV DNA integrations into host chromosomal DNA. Removal of either H2O2 or 3-AB from the culture medium in a subsequent cycle of subcloning was accompanied by a reversion back towards the original lower frequency of stable DHBV DNA integrations for LMH-D21-6 cells. These data support the hypothesis that DNA damage sites can serve as sites for hepadnaviral DNA integration, and that increasing the number of DNA damage sites dramatically increases viral integration frequency. PMID

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  14. 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

  15. An integrated workflow for DNA methylation analysis.

    PubMed

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

    2013-05-20

    The analysis of cytosine methylation provides a new way to assess and describe epigenetic regulation at a whole-genome level in many eukaryotes. DNA methylation has a demonstrated role in the genome stability and protection, regulation of gene expression and many other aspects of genome function and maintenance. BS-seq is a relatively unbiased method for profiling the DNA methylation, with a resolution capable of measuring methylation at individual cytosines. Here we describe, as an example, a workflow to handle DNA methylation analysis, from BS-seq library preparation to the data visualization. We describe some applications for the analysis and interpretation of these data. Our laboratory provides public access to plant DNA methylation data via visualization tools available at our "Next-Gen Sequence" websites (http://mpss.udel.edu), along with small RNA, RNA-seq and other data types. PMID:23706300

  16. An integrated approach to reveal miRNAs' impacts on the functional consequence of copy number alterations in cancer.

    PubMed

    Li, Kening; Liu, Yongjing; Zhou, Yuanshuai; Zhang, Rui; Zhao, Ning; Yan, Zichuang; Zhang, Qiang; Zhang, Shujuan; Qiu, Fujun; Xu, Yan

    2015-01-01

    Copy number alteration (CNA) is known to induce gene expression changes mainly through dosage effect, and therefore affect the initiation and progression of tumor. However, tumor samples exhibit heterogeneity in gene dosage sensitivity due to the complicated mechanisms of transcriptional regulation. Currently, no high-throughput method has been available for identifying the regulatory factors affecting the functional consequences of CNA, and determining their effects on cancer. In view of the important regulatory role of miRNA, we investigated the influence of miRNAs on the dosage sensitivities of genes within the CNA regions. By integrating copy number, mRNA expression, miRNA expression profiles of three kinds of cancer, we observed a tendency for high dosage-sensitivity genes to be more targeted by miRNAs in cancer, and identified the miRNAs regulating the dosage sensitivity of amplified/deleted target genes. The results show that miRNAs can modulate oncogenic biological functions by regulating the genes within the CNA regions, and thus play a role as a trigger or balancer in cancer, affecting cancer processes, even survival. This work provided a framework for analyzing the regulation of dosage effect, which will shed a light on understanding the oncogenic and tumor suppressive mechanisms of CNA. Besides, new cancer-related miRNAs were identified. PMID:26099552

  17. 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

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

    PubMed

    Brinckmann, Anja; Weiss, Claudia; Wilbert, Friederike; von Moers, Arpad; Zwirner, Angelika; Stoltenburg-Didinger, Gisela; Wilichowski, Ekkehard; Schuelke, Markus

    2010-01-01

    Human patients with myoclonic epilepsy with ragged-red fibers (MERRF) suffer from regionalized pathology caused by a mutation in the mitochondrial DNA (m.8344A→G). In MERRF-syndrome brain and skeletal muscles are predominantly affected, despite mtDNA being present in any tissue. In the past such tissue-specificity could not be explained by varying mtDNA mutation loads. In search for a region-specific pathology in human individuals we determined the mtDNA/nDNA ratios along with the mutation loads in 43 different post mortem tissue samples of a 16-year-old female MERRF patient and in four previously healthy victims of motor vehicle accidents. In brain and muscle we further determined the quantity of mitochondrial proteins (COX subunits II and IV), transcription factors (NRF1 and TFAM), and VDAC1 (Porin) as a marker for the mitochondrial mass. In the patient the mutation loads varied merely between 89-100%. However, mtDNA copy numbers were increased 3-7 fold in predominantly affected brain areas (e.g. hippocampus, cortex and putamen) and in skeletal muscle. Similar increases were absent in unaffected tissues (e.g. heart, lung, kidney, liver, and gastrointestinal organs). Such mtDNA copy number increase was not paralleled by an augmentation of mitochondrial mass in some investigated tissues, predominantly in the most affected tissue regions of the brain. We thus conclude that "futile" stimulation of mtDNA replication per se or a secondary failure to increase the mitochondrial mass may contribute to the regionalized pathology seen in MERRF-syndrome. PMID:20976001

  19. Array CGH identifies distinct DNA copy number profiles of oncogenes and tumor suppressor genes in chromosomal- and microsatellite-unstable sporadic colorectal carcinomas.

    PubMed

    Lassmann, Silke; Weis, Roland; Makowiec, Frank; Roth, Jasmine; Danciu, Mihai; Hopt, Ulrich; Werner, Martin

    2007-03-01

    DNA copy number changes represent molecular fingerprints of solid tumors and are as such relevant for better understanding of tumor development and progression. In this study, we applied genome-wide array comparative genomic hybridization (aCGH) to identify gene-specific DNA copy number changes in chromosomal (CIN)- and microsatellite (MIN)-unstable sporadic colorectal cancers (sCRC). Genomic DNA was extracted from microdissected, matching normal colorectal epithelium and invasive tumor cells of formalin-fixed and paraffin-embedded tissues of 22 cases with colorectal cancer (CIN = 11, MIN = 11). DNA copy number changes were determined by aCGH for 287 target sequences in tumor cell DNAs, using pooled normal DNAs as reference. aCGH data of tumor cell DNAs was confirmed by fluorescence in situ hybridization (FISH) for three genes on serial tissues as those used for aCGH. aCGH revealed DNA copy number changes previously described by metaphase CGH (gains 7, 8q, 13q, and 20q; losses 8p, 15q, 18q, and 17p). However, chromosomal regions 20q, 13q, 7, and 17p were preferentially altered in CIN-type tumors and included DNA amplifications of eight genes on chromosome 20q (TOP1, AIB1, MYBL2, CAS, PTPN1, STK15, ZNF217, and CYP24), two genes on chromosome 13q (BRCA2 and D13S25), and three genes on chromosome 7 (IL6, CYLN2, and MET) as well as DNA deletions of two genes on chromosome 17p (HIC1 and LLGL1). Finally, additional CIN-tumor-associated DNA amplifications were identified for EXT1 (8q24.11) and MYC (8q24.12) as well as DNA deletions for MAP2K5 (15q23) and LAMA3 (18q11.2). In contrast, distinct MIN-tumor-associated DNA amplifications were detected for E2F5 (8p22-q21.3), GARP (11q13.5-q14), ATM (11q22.3), KAL (Xp22.3), and XIST (Xq13.2) as well as DNA deletions for RAF1 (3p25), DCC (18q21.3), and KEN (21q tel). aCGH revealed distinct DNA copy number changes of oncogenes and tumor suppressor genes in CIN- and MIN-type sporadic colorectal carcinomas. The identified candidate

  20. Repair of gaps in retroviral DNA integration intermediates.

    PubMed

    Yoder, K E; Bushman, F D

    2000-12-01

    Diverse mobile DNA elements are believed to pirate host cell enzymes to complete DNA transfer. Prominent examples are provided by retroviral cDNA integration and transposon insertion. These reactions initially involve the attachment of each element 3' DNA end to staggered sites in the host DNA by element-encoded integrase or transposase enzymes. Unfolding of such intermediates yields DNA gaps at each junction. It has been widely assumed that host DNA repair enzymes complete attachment of the remaining DNA ends, but the enzymes involved have not been identified for any system. We have synthesized DNA substrates containing the expected gap and 5' two-base flap structure present in retroviral integration intermediates and tested candidate enzymes for the ability to support repair in vitro. We find three required activities, two of which can be satisfied by multiple enzymes. These are a polymerase (polymerase beta, polymerase delta and its cofactor PCNA, or reverse transcriptase), a nuclease (flap endonuclease), and a ligase (ligase I, III, or IV and its cofactor XRCC4). A proposed pathway involving retroviral integrase and reverse transcriptase did not carry out repair under the conditions tested. In addition, prebinding of integrase protein to gapped DNA inhibited repair reactions, indicating that gap repair in vivo may require active disassembly of the integrase complex. PMID:11070016

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

    PubMed Central

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

    2011-01-01

    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. PMID:21808029

  2. 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. PMID:27411901

  3. High-level expression and characterization of a novel serine protease in Pichia pastoris by multi-copy integration.

    PubMed

    Shu, Min; Shen, Wei; Yang, Shihui; Wang, Xiaojuan; Wang, Fei; Wang, Yaping; Ma, Lixin

    2016-10-01

    A novel serine protease from Trichoderma koningii (SPTK) was synthesized and expressed in Pichia pastoris. The recombinant SPTK was completely inhibited by phenyl methyl sulfonyl fluoride (PMSF), suggesting that SPTK belonged to the subgroup of serine proteases. The optimum pH and temperature for the recombinant SPTK reaction were 6.0 and 55°C, respectively. SPTK performed a tolerance to most organic solvents and metal ions, and the addition of Triton X-100 exhibited an activation of SPTK up to 243% of its initial activity but SDS strongly inhibited. Moreover, our study showed that a portion of SPTK was N-glycosylated during fermentation. The activity and thermal stability of the recombinant SPTK were improved after the removal of glycosylation, and the N-glycosylation of SPTK could be efficiently removed through co-culture with P. pastoris strains expressing Endo-β-N-acetylglucosaminidase H. We constructed expression vectors harboring from one to four repeats of Sptk-expressing cassettes via an in vitro BioBrick assembly approach. And the result of quantitative polymerase chain reaction (qPCR) indicated that the tandem expression cassettes were integrated into the genome of P. pastoris through a single recombination event. These strains were used to study the correlation between the gene copy number and the expression level of SPTK. The results of qPCR and enzyme activity assays indicated that the copy number variation of Sptk gene generally had a positive effect on the expression level of SPTK, while an increase in integration of target gene did not guarantee its high expression. The maximum yield and specific activity of SPTK in P. pastoris were obtained from the recombinant yeast strain harboring two-copy tandem Sptk-expressing cassettes, the yield reached 0.48g/l after a 6-d induction using menthol in shake flasks and 3.2g/l in high-density fermentation with specific activity of 5200U/mg. In addition, the recombinant SPTK could efficiently degrade chicken

  4. Roles of DNA helicases in the maintenance of genome integrity

    PubMed Central

    Bochman, Matthew L

    2014-01-01

    Genome integrity is achieved and maintained by the sum of all of the processes in the cell that ensure the faithful duplication and repair of DNA, as well as its genetic transmission from one cell division to the next. As central players in virtually all of the DNA transactions that occur in vivo, DNA helicases (molecular motors that unwind double-stranded DNA to produce single-stranded substrates) represent a crucial enzyme family that is necessary for genomic stability. Indeed, mutations in many human helicase genes are linked to a variety of diseases with symptoms that can be generally described as genomic instability, such as predispositions to cancers. This review focuses on the roles of both DNA replication helicases and recombination/repair helicases in maintaining genome integrity and provides a brief overview of the diseases related to defects in these enzymes. PMID:27308340

  5. Inhalable particulate matter and mitochondrial DNA copy number in highly exposed individuals in Beijing, China: a repeated-measure study

    PubMed Central

    2013-01-01

    Background Mitochondria are both a sensitive target and a primary source of oxidative stress, a key pathway of air particulate matter (PM)-associated diseases. Mitochondrial DNA copy number (MtDNAcn) is a marker of mitochondrial damage and malfunctioning. We evaluated whether ambient PM exposure affects MtDNAcn in a highly-exposed population in Beijing, China. Methods The Beijing Truck Driver Air Pollution Study was conducted shortly before the 2008 Beijing Olympic Games (June 15-July 27, 2008) and included 60 truck drivers and 60 office workers. Personal PM2.5 and elemental carbon (EC, a tracer of traffic particles) were measured during work hours using portable monitors. Post-work blood samples were obtained on two different days. Ambient PM10 was averaged from 27 monitoring stations in Beijing. Blood MtDNAcn was determined by real-time PCR and examined in association with particle levels using mixed-effect models. Results In all participants combined, MtDNAcn was negatively associated with personal EC level measured during work hours (β=−0.059, 95% CI: -0.011; -0.0006, p=0.03); and 5-day (β=−0.017, 95% CI: -0.029;-0.005, p=0.01) and 8-day average ambient PM10 (β=−0.008, 95% CI: -0.043; -0.008, p=0.004) after adjusting for possible confounding factors, including study groups. MtDNAcn was also negatively associated among office workers with EC (β=−0.012, 95% CI: -0.022;-0.002, p=0.02) and 8-day average ambient PM10 (β=−0.030, 95% CI: -0.051;-0.008, p=0.007). Conclusions We observed decreased blood MtDNAcn in association with increased exposure to EC during work hours and recent ambient PM10 exposure. Our results suggest that MtDNAcn may be influenced by particle exposures. Further studies are required to determine the roles of MtDNAcn in the etiology of particle-related diseases. PMID:23628000

  6. 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

  7. Mitochondrial DNA copy number in peripheral blood leukocytes and the risk of clear cell renal cell carcinoma.

    PubMed

    Melkonian, Stephanie C; Wang, Xin; Gu, Jian; Matin, Surena F; Tannir, Nizar M; Wood, Christopher G; Wu, Xifeng

    2015-02-01

    Variation of mitochondrial DNA copy number (mtDNAcn) in peripheral blood leukocytes has been associated with the risk of various cancers, including renal cell carcinoma (RCC). We assessed the association between mtDNAcn and clear cell RCC (ccRCC) risk in 608 cases and 629 controls frequency-matched on age and gender. Unconditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) adjusting for age, gender, body mass index, smoking status, history of hypertension, total energy intake and physical activity. Our results suggest an association between low mtDNAcn and ccRCC risk (OR = 1.28, 95% CI: 0.97-1.68, P = 0.09). Lower mtDNAcn was associated with increased ccRCC risk in younger individuals (age <60, OR = 1.68, 95% CI: 1.13-2.49, P = 0.01), women (OR = 1.66, 95% CI: 1.03-2.73, P = 0.04), individuals without history of hypertension (OR = 1.62, 95% CI: 1.09-2.41, P = 0.02) and individuals with low physical activity levels (OR = 1.55, 95% CI: 1.02-2.37, P = 0.05). We observed significant and marginally significant interactions between both age and history of hypertension and mtDNAcn in elevating ccRCC risk (P for interaction = 0.04 and 0.07, respectively). Additionally, low mtDNAcn was associated with ccRCC risk in younger individuals with low levels of physical activity [ORs and 95% CI for medium and low physical activity levels, respectively, 2.31 (1.18-4.52) and 2.09 (1.17-3.75), P interaction = 0.04]. To our knowledge, this is the first report to investigate the role of mtDNAcn in the ccRCC subtype and the first to suggest that this association may be modified by risk factors including age, gender, history of hypertension and physical activity. PMID:25524925

  8. Mitochondrial DNA copy number in peripheral blood leukocytes and the risk of clear cell renal cell carcinoma

    PubMed Central

    Melkonian, Stephanie C.; Wang, Xin; Gu, Jian; Matin, Surena F.; Tannir, Nizar M.; Wood, Christopher G.; Wu, Xifeng

    2015-01-01

    Variation of mitochondrial DNA copy number (mtDNAcn) in peripheral blood leukocytes has been associated with the risk of various cancers, including renal cell carcinoma (RCC). We assessed the association between mtDNAcn and clear cell RCC (ccRCC) risk in 608 cases and 629 controls frequency-matched on age and gender. Unconditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) adjusting for age, gender, body mass index, smoking status, history of hypertension, total energy intake and physical activity. Our results suggest an association between low mtDNAcn and ccRCC risk (OR = 1.28, 95% CI: 0.97–1.68, P = 0.09). Lower mtDNAcn was associated with increased ccRCC risk in younger individuals (age <60, OR = 1.68, 95% CI: 1.13–2.49, P = 0.01), women (OR = 1.66, 95% CI: 1.03–2.73, P = 0.04), individuals without history of hypertension (OR = 1.62, 95% CI: 1.09–2.41, P = 0.02) and individuals with low physical activity levels (OR = 1.55, 95% CI: 1.02–2.37, P = 0.05). We observed significant and marginally significant interactions between both age and history of hypertension and mtDNAcn in elevating ccRCC risk (P for interaction = 0.04 and 0.07, respectively). Additionally, low mtDNAcn was associated with ccRCC risk in younger individuals with low levels of physical activity [ORs and 95% CI for medium and low physical activity levels, respectively, 2.31 (1.18–4.52) and 2.09 (1.17–3.75), P interaction = 0.04]. To our knowledge, this is the first report to investigate the role of mtDNAcn in the ccRCC subtype and the first to suggest that this association may be modified by risk factors including age, gender, history of hypertension and physical activity. PMID:25524925

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

    PubMed Central

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

    2013-01-01

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

  10. 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

  11. Estimation of copy number using SYBR Green: confounding by AT-rich DNA and by variation in amplicon length.

    PubMed

    Colborn, James M; Byrd, Brian D; Koita, Ousmane A; Krogstad, Donald J

    2008-12-01

    Although SYBR Green is used to estimate copy number, its fluorescence varies with amplicon length and adenine/thymine (AT) content. As a result, threshold cycle (Ct) values obtained using real-time polymerase chain reaction (PCR) are lower for longer amplicons (P<0.001) and amplicons with greater AT content (P<0.001). In contrast, neither amplicon length nor AT content affects the Ct with TaqMan probes or LUX-labeled primers. Because SYBR Green yields lower Cts with AT-rich templates and longer templates, it overestimates copy number for those templates. Therefore, sequence-specific methods such as TaqMan probes or LUX-labeled primers should be considered when using real-time PCR to estimate copy number if the amplicons generated are AT-rich or vary in length. PMID:19052298

  12. 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

  13. 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

  14. RADIA: RNA and DNA Integrated Analysis for Somatic Mutation Detection

    PubMed Central

    Radenbaugh, Amie J.; Ma, Singer; Ewing, Adam; Stuart, Joshua M.; Collisson, Eric A.; Zhu, Jingchun; Haussler, David

    2014-01-01

    The detection of somatic single nucleotide variants is a crucial component to the characterization of the cancer genome. Mutation calling algorithms thus far have focused on comparing the normal and tumor genomes from the same individual. In recent years, it has become routine for projects like The Cancer Genome Atlas (TCGA) to also sequence the tumor RNA. Here we present RADIA (RNA and DNA Integrated Analysis), a novel computational method combining the patient-matched normal and tumor DNA with the tumor RNA to detect somatic mutations. The inclusion of the RNA increases the power to detect somatic mutations, especially at low DNA allelic frequencies. By integrating an individual’s DNA and RNA, we are able to detect mutations that would otherwise be missed by traditional algorithms that examine only the DNA. We demonstrate high sensitivity (84%) and very high precision (98% and 99%) for RADIA in patient data from endometrial carcinoma and lung adenocarcinoma from TCGA. Mutations with both high DNA and RNA read support have the highest validation rate of over 99%. We also introduce a simulation package that spikes in artificial mutations to patient data, rather than simulating sequencing data from a reference genome. We evaluate sensitivity on the simulation data and demonstrate our ability to rescue back mutations at low DNA allelic frequencies by including the RNA. Finally, we highlight mutations in important cancer genes that were rescued due to the incorporation of the RNA. PMID:25405470

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

    PubMed

    Radenbaugh, Amie J; Ma, Singer; Ewing, Adam; Stuart, Joshua M; Collisson, Eric A; Zhu, Jingchun; Haussler, David

    2014-01-01

    The detection of somatic single nucleotide variants is a crucial component to the characterization of the cancer genome. Mutation calling algorithms thus far have focused on comparing the normal and tumor genomes from the same individual. In recent years, it has become routine for projects like The Cancer Genome Atlas (TCGA) to also sequence the tumor RNA. Here we present RADIA (RNA and DNA Integrated Analysis), a novel computational method combining the patient-matched normal and tumor DNA with the tumor RNA to detect somatic mutations. The inclusion of the RNA increases the power to detect somatic mutations, especially at low DNA allelic frequencies. By integrating an individual's DNA and RNA, we are able to detect mutations that would otherwise be missed by traditional algorithms that examine only the DNA. We demonstrate high sensitivity (84%) and very high precision (98% and 99%) for RADIA in patient data from endometrial carcinoma and lung adenocarcinoma from TCGA. Mutations with both high DNA and RNA read support have the highest validation rate of over 99%. We also introduce a simulation package that spikes in artificial mutations to patient data, rather than simulating sequencing data from a reference genome. We evaluate sensitivity on the simulation data and demonstrate our ability to rescue back mutations at low DNA allelic frequencies by including the RNA. Finally, we highlight mutations in important cancer genes that were rescued due to the incorporation of the RNA. PMID:25405470

  16. Site-specific T-DNA integration in Arabidopsis thaliana mediated by the combined action of CRE recombinase and ϕC31 integrase.

    PubMed

    De Paepe, Annelies; De Buck, Sylvie; Nolf, Jonah; Van Lerberge, Els; Depicker, Ann

    2013-07-01

    Random T-DNA integration into the plant host genome can be problematic for a variety of reasons, including potentially variable transgene expression as a result of different integration positions and multiple T-DNA copies, the risk of mutating the host genome and the difficulty of stacking well-defined traits. Therefore, recombination systems have been proposed to integrate the T-DNA at a pre-selected site in the host genome. Here, we demonstrate the capacity of the ϕC31 integrase (INT) for efficient targeted T-DNA integration. Moreover, we show that the iterative site-specific integration system (ISSI), which combines the activities of the CRE recombinase and INT, enables the targeting of genes to a pre-selected site with the concomitant removal of the resident selectable marker. To begin, plants expressing both the CRE and INT recombinase and containing the target attP site were constructed. These plants were supertransformed with a T-DNA vector harboring the loxP site, the attB sites, a selectable marker and an expression cassette encoding a reporter protein. Three out of the 35 transformants obtained (9%) showed transgenerational site-specific integration (SSI) of this T-DNA and removal of the resident selectable marker, as demonstrated by PCR, Southern blot and segregation analysis. In conclusion, our results show the applicability of the ISSI system for precise and targeted Agrobacterium-mediated integration, allowing the serial integration of transgenic DNA sequences in plants. PMID:23574114

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

    PubMed

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

    2016-03-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2010-02-01

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

  20. Single mammalian cells compensate for differences in cellular volume and DNA copy number through independent global transcriptional mechanisms

    PubMed Central

    Padovan-Merhar, Olivia; Nair, Gautham P.; Biaesch, Andrew; Mayer, Andreas; Scarfone, Steven; Foley, Shawn W.; Wu, Angela R.; Churchman, L. Stirling; Singh, Abhyudai; Raj, Arjun

    2015-01-01

    Summary Individual mammalian cells exhibit large variability in cellular volume even with the same absolute DNA content and so must compensate for differences in DNA concentration in order to maintain constant concentration of gene expression products. Using single molecule counting and computational image analysis, we show that transcript abundance correlates with cellular volume at the single cell level due to increased global transcription in larger cells. Cell fusion experiments establish that increased cellular content itself can directly increase transcription. Quantitative analysis shows that this mechanism measures the ratio of cellular volume to DNA content, mostly likely through sequestration of a transcriptional factor to DNA. Analysis of transcriptional bursts reveals a separate mechanism for gene dosage compensation after DNA replication that enables proper transcriptional output during early and late S-phase. Our results provide a framework for quantitatively understanding the relationships between DNA content, cell size and gene expression variability in single cells. PMID:25866248

  1. An integrative genomic and transcriptomic analysis reveals molecular pathways and networks regulated by copy number aberrations in basal-like, HER2 and luminal cancers.

    PubMed

    Natrajan, Rachael; Weigelt, Britta; Mackay, Alan; Geyer, Felipe C; Grigoriadis, Anita; Tan, David S P; Jones, Chris; Lord, Christopher J; Vatcheva, Radost; Rodriguez-Pinilla, Socorro M; Palacios, Jose; Ashworth, Alan; Reis-Filho, Jorge S

    2010-06-01

    Breast cancer is a heterogeneous disease caused by the accumulation of genetic changes in neoplastic cells. We hypothesised that molecular subtypes of breast cancer may be driven by specific constellations of genes whose expression is regulated by gene copy number aberrations. To address this question, we analysed a series of 48 microdissected grade III ductal carcinomas using high resolution microarray comparative genomic hybridisation and mRNA expression arrays. There were 5,931 genes whose expression significantly correlates with copy number identified; out of these, 1,897 genes were significantly differentially expressed between basal-like, HER2 and luminal tumours. Ingenuity Pathway Analysis (IPA) revealed that 'G1/S cell cycle regulation' and 'BRCA1 in DNA damage control' pathways were significantly enriched for genes whose expression correlates with copy number and are differentially expressed between the molecular subtypes of breast cancer. IPA of genes whose expression significantly correlates with copy number in each molecular subtype individually revealed that canonical pathways involved in oestrogen receptor (ER) signalling and DNA repair are enriched for these genes. We also identified 32, 157 and 265 genes significantly overexpressed when amplified in basal-like, HER2 and luminal cancers, respectively. These lists include known and novel potential therapeutic targets (e.g. HER2 and PPM1D in HER2 cancers). Our results provide strong circumstantial evidence that different patterns of genetic aberrations in distinct molecular subtypes of breast cancer contribute to their specific transcriptomic profiles and that biological phenomena characteristic of each subtype (e.g. proliferation, HER2 and ER signalling) may be driven by specific patterns of copy number aberrations. PMID:19688261

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

    Cancer.gov

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

  3. 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.

  4. Single-step co-integration of multiple expressible heterologous genes into the ribosomal DNA of the methylotrophic yeast Hansenula polymorpha.

    PubMed

    Klabunde, J; Diesel, A; Waschk, D; Gellissen, G; Hollenberg, C P; Suckow, M

    2002-05-01

    We have investigated the methylotrophic yeast Hansenula polymorpha as a host for the co-integration and expression of multiple heterologous genes using an rDNA integration approach. The ribosomal DNA (rDNA) of H. polymorpha was found to consist of a single rDNA cluster of about 50-60 repeats of an 8-kb unit located on chromosome II. A 2.4-kb segment of H. polymorpha rDNA encompassing parts of the 25S, the complete 5S and the non-transcribed spacer region between 25S and 18S rDNA was isolated and inserted into conventional integrative H. polymorpha plasmids harboring the Saccharomyces- cerevisiae-derived URA3 gene for selection. These rDNA plasmids integrated homologously into the rDNA repeats of a H. polymorpha (odc1) host as several independent clusters. Anticipating that this mode of multiple-cluster integration could be used for the simultaneous integration of several distinct rDNA plasmids, the host strain was co-transformed with a mixture of up to three different plasmids, all bearing the same URA3 selection marker. Transformations indeed resulted in mitotically stable strains harboring one, two, or all three plasmids integrated into the rDNA. The overall copy number of the plasmids integrated did not exceed the number of rDNA repeats present in the untransformed host strain, irrespective of the number of different plasmids involved. Strains harboring different plasmids co-expressed the introduced genes, resulting in functional proteins. Thus, this approach provides a new and attractive tool for the rapid generation of recombinant strains that simultaneously co-produce several proteins in desired stoichiometric ratios. PMID:12021801

  5. 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)

  6. An integrated lateral flow assay for effective DNA amplification and detection at the point of care.

    PubMed

    Choi, Jane Ru; Hu, Jie; Gong, Yan; Feng, Shangsheng; Wan Abas, Wan Abu Bakar; Pingguan-Murphy, Belinda; Xu, Feng

    2016-05-10

    Lateral flow assays (LFAs) have been extensively explored in nucleic acid testing (NAT) for medical diagnostics, food safety analysis and environmental monitoring. However, the amount of target nucleic acid in a raw sample is usually too low to be directly detected by LFAs, necessitating the process of amplification. Even though cost-effective paper-based amplification techniques have been introduced, they have always been separately performed from LFAs, hence increasing the risk of reagent loss and cross-contaminations. To date, integrating paper-based nucleic acid amplification into colorimetric LFA in a simple, portable and cost-effective manner has not been introduced. Herein, we developed an integrated LFA with the aid of a specially designed handheld battery-powered system for effective amplification and detection of targets in resource-poor settings. Interestingly, using the integrated paper-based loop-mediated isothermal amplification (LAMP)-LFA, we successfully performed highly sensitive and specific target detection, achieving a detection limit of as low as 3 × 10(3) copies of target DNA, which is comparable to the conventional tube-based LAMP-LFA in an unintegrated format. The device may serve in conjunction with a simple paper-based sample preparation to create a fully integrated paper-based sample-to-answer diagnostic device for point-of-care testing (POCT) in the near future. PMID:27010033

  7. Patterns of integration of DNA microinjected into cultured mammalian cells: Evidence for homologous recombination between injected plasmid DNA molecules

    SciTech Connect

    Folger, K.R.; Wong, E.A.; Wahl, G.; Capecchi, M.R.

    1982-11-01

    The authors examined the fate of DNA microinjected into nuclei of cultured mammalian cells. The sequence composition and the physical form of the vector carrying the selectable gene affected the efficiency of DNA-mediated transformation. Introduction of sequences near the simian virus 40 origin of DNA replication or in the long terminal repeat of avian sarcoma provirus into a recombinant plasmid containing the herpes simplex virus thymidine kinase gene (pBR322/HSV-tk) enhanced the frequency of transformation of LMtk/sup -/ and RAT-2tk/sup -/ cells to the TK/sup +/ phenotype 20- to 40-fold. In cells receiving injections of only a few plasmid DNA molecules, the transformation frequency was 40-fold higher after injection of linear molecules than after injection of supercoiled molecules. By controlling the number of gene copies injected into a recipient cell, we could obtain transformants containing a single copy or as many as 50 to 100 copies of the selectable gene. By analyzing transformants obtained by coinjecting two vectors which were identical except that in one a portion of the vector was inverted, the authors were able to conclude that the head-to-tail concatemers were generated predominantly by homologous recombination. Surprisingly, these head-to-tail concatemers were found in transformants obtained by injecting either supercoiled or linear plasmid DNA.

  8. Subspecies of DNA polymerase alpha from calf thymus with different fidelity in copying synthetic template-primers.

    PubMed Central

    Brosius, S; Grosse, F; Krauss, G

    1983-01-01

    Three different subspecies of DNA polymerase alpha from calf thymus sedimenting at 9 S, 7 S and 5.7 S have been investigated with respect to their accuracy of in vitro DNA synthesis on poly (dA) (dT)16 and poly d(AT) as template-primers. Our results indicate that the structure of DNA polymerase alpha has a strong influence on the accuracy of DNA synthesis. The 9 S enzyme shows a misincorporation frequency of about 1:100 000. An error rate of 1:15 000 is measured for the 7 S species. The 5.7 S enzyme for which an error rate of 1:3 000 is determined, has to be considered as error prone. Lowering the rate of DNA synthesis leads to a decrease in fidelity. The single stranded DNA binding protein from E.coli increases the accuracy of the 5.7 S and the 7 S enzyme by a factor of two. Mn2+ decreases the fidelity of all three subspecies in a concentration dependent manner. PMID:6866763

  9. 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.

  10. Detection of low copy human papilloma virus DNA and mRNA in routine paraffin sections of cervix by non-isotopic in situ hybridisation.

    PubMed Central

    Burns, J; Graham, A K; Frank, C; Fleming, K A; Evans, M F; McGee, J O

    1987-01-01

    In analysing human papilloma virus (HPV) infection of the cervix in formalin fixed paraffin sections by non-isotopic in situ hybridisation two main problems were found: detachment of sections from the glass during hybridisation and probe detection; inadequate sensitivity and inability to assess sensitivity of the in situ procedure. The first problem was investigated by assessing the efficiency of various tissue adhesives individually and in combination. The second problem was addressed by optimising conditions for DNA unmasking, hybridisation, and biotinylated probe detection. Sensitivity of the final in situ procedure developed was assessed by using the detection of pHY2.1 repeats as a built-in control. Extrapolation of data showed that less than 10 copies of HPV DNA can be visualised by these procedures. HPV nucleic acid, mainly in the form of DNA, was detected not only in koilocytic nuclei but also in suprabasal cells in condylomas and CIN lesions. HPV mRNA was also visualised in the cytoplasm (and probably also nuclei) of the same cell types. These non-isotopic in situ procedures give results comparable to those obtained with radiolabelled probes, but they are less time consuming and provide better morphological resolution. Images Fig 1 Fig 2 Fig 3 Fig 4 Fig 5 Fig 6 Fig 7 Fig 8 PMID:2821078

  11. 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. PMID:27617230

  12. DNA copy number aberrations and disruption of the p16Ink4a/Rb pathway in radiation-induced and spontaneous rat mammary carcinomas.

    PubMed

    Iizuka, Daisuke; Imaoka, Tatsuhiko; Takabatake, Takashi; Nishimura, Mayumi; Kakinuma, Shizuko; Nishimura, Yukiko; Shimada, Yoshiya

    2010-08-01

    Chromosomal amplifications and deletions are thought to be important events in spontaneous and radiation-induced carcinogenesis. To clarify how ionizing radiation induces mammary carcinogenesis, we characterized genomic copy number aberrations for gamma-ray-induced rat mammary carcinomas using microarray-based comparative genomic hybridization. We examined 14 carcinomas induced by gamma radiation (2 Gy) and found 26 aberrations, including trisomies of chromosomes 4 and 10 for three and one carcinomas, respectively, an amplification of the chromosomal region 1q12 in two carcinomas, and deletions of the chromosomal regions 3q35q36, 5q32 and 7q11 in two, two and four carcinomas, respectively. These aberrations were not observed in seven spontaneous mammary carcinomas. The expression of p16Ink4a and p19Arf, which are located in the chromosomal region 5q32, was always up-regulated except for a carcinoma with a homozygous deletion of region 5q32. The up-regulation was not accounted for by gene mutations or promoter hypomethylation. However, the amounts of Rb and its mRNA were down-regulated in these carcinomas, indicating a disruption of the p16Ink4a/Rb pathway. This is the first report of array CGH analysis for radiation-induced mammary tumors, which reveals that they show distinct DNA copy number aberration patterns that are different from those of spontaneous tumors and those reported previously for chemically induced tumors. PMID:20681787

  13. 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

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

    PubMed Central

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

    2013-01-01

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

  15. Mechanism of integrating foreign DNA during transformation of Bacillus subtilis.

    PubMed Central

    Duncan, C H; Wilson, G A; Young, F E

    1978-01-01

    Genes encoding thymidylate synthetase from Bacillus subtilis bacteriophages were cloned in Escherichia coli. Chimeric plasmids pCD1 and pCD3 were constructed from site-specific endonuclease digests of bacteriophage phi3T DNA cloned in pMB9 in E. coli. Similar cloning techniques with bacteriophage beta22 DNA yielded chimeric plasmids pCD4, pCD5, and pCD6. Endonuclease digests of DNA from pCD1 and pCD3 propagated in E. coli or from DNA isolated from bacteriophage phi3T propagated in B. subtilis transformed B. subtilis from Thy- to Thy+. Intact DNA from bacteriophage beta22, endonuclease digests of beta22 DNA, and a chimeric plasmid (pCD5) composed only of the thybeta22 gene and pMB9 did not transform B. subtilis from Thy- to Thy+ even though pCD5 could transform Thy- E. coli to Thy+. However, if the thybeta22 fragment from pCD5 was introduced into another chimeric plasmid, pCD2, that contains a region of homology to the chromosome of B. subtilis in addition to pMB9, transformation of Thy- clones of B. subtilis was possible. Furthermore, Southern hybridization analyses of the digests of chromosomal DNA from the Thy+ transformants established that the entire chimeric plasmid was incorporated into the chromosome of B. subtilis. Treatment of these plasmids with site-specific endonucleases abolished transformation. These results indicated that the entire chimeric plasmid can be incorporated into the chromosome of B. subtilis by a Campbell-like model. Therefore, an additional mechanism for transformation exists whereby plasmids can be integrated if sufficient chromosomal homology is maintained. Images PMID:99740

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

    PubMed

    Juhas, Mario; Ajioka, James W

    2016-06-01

    Bacillus subtilis is a well-characterized model bacterium frequently used for a number of biotechnology and synthetic biology applications. Novel strategies combining the advantages of B. subtilis with the DNA assembly and editing tools of Escherichia coli are crucial for B. subtilis engineering efforts. We combined Gibson Assembly and λ red recombineering in E. coli with RecA-mediated homologous recombination in B. subtilis for bacterial artificial chromosome-mediated DNA integration into the well-characterized amyE target locus of the B. subtilis chromosome. The engineered integrative bacterial artificial chromosome iBAC(cav) can accept any DNA fragment for integration into B. subtilis chromosome and allows rapid selection of transformants by B. subtilis-specific antibiotic resistance and the yellow fluorescent protein (mVenus) expression. We used the developed iBAC(cav)-mediated system to integrate 10kb DNA fragment from E. coli K12 MG1655 into B. subtilis chromosome. iBAC(cav)-mediated chromosomal integration approach will facilitate rational design of synthetic biology applications in B. subtilis. PMID:27033694

  17. Identification of Multiple DNA Copy Number Alterations Including Frequent 8p11.22 Amplification in Conjunctival Squamous Cell Carcinoma

    PubMed Central

    Asnaghi, Laura; Alkatan, Hind; Mahale, Alka; Othman, Maha; Alwadani, Saeed; Al-Hussain, Hailah; Jastaneiah, Sabah; Yu, Wayne; Maktabi, Azza; Edward, Deepak P.; Eberhart, Charles G.

    2014-01-01

    Purpose. Little is known about the molecular alterations that drive formation and growth of conjunctival squamous cell carcinoma (cSCC). We therefore sought to identify genetic changes that could be used as diagnostic markers or therapeutic targets. Methods. The DNA extracted from 10 snap-frozen cSCC tumor specimens and 2 in situ carcinomas was analyzed using array-based comparative genomic hybridization (aCGH), and further examined with NanoString and quantitative PCR. Results. The number of regions of DNA loss ranged from 1 to 23 per tumor, whereas gains and amplifications ranged from 1 to 15 per tumor. Most large regions of chromosomal gain and loss were confirmed by NanoString karyotype analysis. The commonest alteration was amplification of 8p11.22 in 9 tumors (75%), and quantitative PCR analysis revealed 100-fold or greater overexpression of ADAM3A mRNA from 8p11.22 locus. In addition, recurring losses were observed at 14q13.2 and 22q11.23, both lost in 5 (42%) of the 12 tumors, and at 12p13.31, lost in 4 (33%) of the 12 samples. Of the eight loci associated with the DNA damage repair syndrome xeroderma pigmentosum, three showed loss of at least one allele in our aCGH analysis, including XPA (9q22.33, one tumor), XPE/DDB2 (11p11.2, one tumor) and XPG/ERCC5 (13q33.1, three tumors). Conclusions. Conjunctival SCC contains a range of chromosomal alterations potentially important in tumor formation and growth. Amplification of 8p11.22 and overexpression of ADAM3A suggests a potential role for this protease. Our findings also suggest that defects in DNA repair loci are important in sporadic cSCC. PMID:25491297

  18. Anthocyanidins modulate the activity of human DNA topoisomerases I and II and affect cellular DNA integrity.

    PubMed

    Habermeyer, Michael; Fritz, Jessica; Barthelmes, Hans U; Christensen, Morten O; Larsen, Morten K; Boege, Fritz; Marko, Doris

    2005-09-01

    In the present study, we investigated the effect of anthocyanidins on human topoisomerases I and II and its relevance for DNA integrity within human cells. Anthocyanidins bearing vicinal hydroxy groups at the B-ring (delphinidin, DEL; cyanidin, CY) were found to potently inhibit the catalytic activity of human topoisomerases I and II, without discriminating between the IIalpha and the IIbeta isoforms. However, in contrast to topoisomerase poisons, DEL and CY did not stabilize the covalent DNA-topoisomerase intermediates (cleavable complex) of topoisomerase I or II. Using recombinant topoisomerase I, the presence of CY or DEL (> or = 1 microM) effectively prohibited the stabilization of the cleavable complex by the topoisomerase I poison camptothecin. We furthermore investigated whether the potential protective effect vs topoisomerase I poisons is reflected also on the cellular level, affecting the DNA damaging properties of camptothecin. Indeed, in HT29 cells, low micromolar concentrations of DEL (1-10 microM) significantly diminished the DNA strand breaking effect of camptothecin (100 microM). However, at concentrations > or = 50 microM, all anthocyanidins tested (delphinidin, cyanidin, malvidin, pelargonidin, and paeonidin), including those not interfering with topoisomerases, were found to induce DNA strand breaks in the comet assay. All of these analogues were able to compete with ethidium bromide for the intercalation into calf thymus DNA and to replace the minor groove binder Hoechst 33258. These data indicate substantial affinity to double-stranded DNA, which might contribute at least to the DNA strand breaking effect of anthocyanidins at higher concentrations (> or = 50 microM). PMID:16167831

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

    PubMed Central

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

    2015-01-01

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

  20. 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 Central

    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. PMID:26247464

  1. Microarray analysis of copy number variation in single cells.

    PubMed

    Konings, Peter; Vanneste, Evelyne; Jackmaert, Sigrun; Ampe, Michèle; Verbeke, Geert; Moreau, Yves; Vermeesch, Joris Robert; Voet, Thierry

    2012-02-01

    We present a protocol for reliably detecting DNA copy number aberrations in a single human cell. Multiple displacement-amplified DNAs of a cell are hybridized to a 3,000-bacterial artificial chromosome (BAC) array and to an Affymetrix 250,000 (250K)-SNP array. Subsequent copy number calling is based on the integration of BAC probe-specific copy number probabilities that are estimated by comparing probe intensities with a single-cell whole-genome amplification (WGA) reference model for diploid chromosomes, as well as SNP copy number and loss-of-heterozygosity states estimated by hidden Markov models (HMM). All methods for detecting DNA copy number aberrations in single human cells have difficulty in confidently discriminating WGA artifacts from true genetic variants. Furthermore, some methods lack thorough validation for segmental DNA imbalance detection. Our protocol minimizes false-positive variant calling and enables uniparental isodisomy detection in single cells. Additionally, it provides quality assessment, allowing the exclusion of uninterpretable single-cell WGA samples. The protocol takes 5-7 d. PMID:22262009

  2. Brown Planthopper Nudivirus DNA Integrated in Its Host Genome

    PubMed Central

    Cheng, Ruo-Lin; Xi, Yu; Lou, Yi-Han; Wang, Zhuo; Xu, Ji-Yu; Xu, Hai-Jun

    2014-01-01

    ABSTRACT The brown planthopper (BPH), Nilaparvata lugens (Hemiptera:Delphacidae), is one of the most destructive insect pests of rice crops in Asia. Nudivirus-like sequences were identified during the whole-genome sequencing of BPH. PCR examination showed that the virus sequences were present in all of the 22 BPH populations collected from East, Southeast, and South Asia. Thirty-two of the 33 nudivirus core genes were identified, including 20 homologues of baculovirus core genes. In addition, several gene clusters that were arranged collinearly with those of other nudiviruses were found in the partial virus genome. In a phylogenetic tree constructed using the supermatrix method, the original virus was grouped with other nudiviruses and was closely related to polydnavirus. Taken together, these data indicated that the virus sequences belong to a new member of the family Nudiviridae. More specifically, the virus sequences were integrated into the chromosome of its insect host during coevolution. This study is the first report of a large double-stranded circular DNA virus genome in a sap-sucking hemipteran insect. IMPORTANCE This is the first report of a large double-stranded DNA virus integrated genome in the planthopper, a plant sap-sucking hemipteran insect. It is an exciting addition to the evolutionary story of bracoviruses (polydnaviruses), nudiviruses, and baculoviruses. The results on the virus sequences integrated in the chromosomes of its insect host also represent a story of successful coevolution of an invertebrate virus and a plant sap-sucking insect. PMID:24574410

  3. T-DNA transfer and T-DNA integration efficiencies upon Arabidopsis thaliana root explant cocultivation and floral dip transformation.

    PubMed

    Ghedira, Rim; De Buck, Sylvie; Van Ex, Frédéric; Angenon, Geert; Depicker, Ann

    2013-12-01

    T-DNA transfer and integration frequencies during Agrobacterium-mediated root explant cocultivation and floral dip transformations of Arabidopsis thaliana were analyzed with and without selection for transformation-competent cells. Based on the presence or absence of CRE recombinase activity without or with the CRE T-DNA being integrated, transient expression versus stable transformation was differentiated. During root explant cocultivation, continuous light enhanced the number of plant cells competent for interaction with Agrobacterium and thus the number of transient gene expression events. However, in transformation competent plant cells, continuous light did not further enhance cotransfer or cointegration frequencies. Upon selection for root transformants expressing a first T-DNA, 43-69 % of these transformants showed cotransfer of another non-selected T-DNA in two different light regimes. However, integration of the non-selected cotransferred T-DNA occurred only in 19-46 % of these transformants, indicating that T-DNA integration in regenerating root cells limits the transformation frequencies. After floral dip transformation, transient T-DNA expression without integration could not be detected, while stable T-DNA transformation occurred in 0.5-1.3 % of the T1 seedlings. Upon selection for floral dip transformants with a first T-DNA, 8-34 % of the transformants showed cotransfer of the other non-selected T-DNA and in 93-100 % of them, the T-DNA was also integrated. Therefore, a productive interaction between the agrobacteria and the female gametophyte, rather than the T-DNA integration process, restricts the floral dip transformation frequencies. PMID:23975012

  4. Ploidy status and copy number aberrations in primary glioblastomas defined by integrated analysis of allelic ratios, signal ratios and loss of heterozygosity using 500K SNP Mapping Arrays

    PubMed Central

    Gardina, Paul J; Lo, Ken C; Lee, Walter; Cowell, John K; Turpaz, Yaron

    2008-01-01

    Background Genomic hybridization platforms, including BAC-CGH and genotyping arrays, have been used to estimate chromosome copy number (CN) in tumor samples by detecting the relative strength of genomic signal. The methods rely on the assumption that the predominant chromosomal background of the samples is diploid, an assumption that is frequently incorrect for tumor samples. In addition to generally greater resolution, an advantage of genotyping arrays over CGH arrays is the ability to detect signals from individual alleles, allowing estimation of loss-of-heterozygosity (LOH) and allelic ratios to enhance the interpretation of copy number alterations. Copy number events associated with LOH potentially have the same genetic consequences as deletions. Results We have utilized allelic ratios to detect patterns that are indicative of higher ploidy levels. An integrated analysis using allelic ratios, total signal and LOH indicates that many or most of the chromosomes from 24 glioblastoma tumors are in fact aneuploid. Some putative whole-chromosome losses actually represent trisomy, and many apparent sub-chromosomal losses are in fact relative losses against a triploid or tetraploid background. Conclusion These results suggest a re-interpretation of previous findings based only on total signal ratios. One interesting observation is that many single or multiple-copy deletions occur at common putative tumor suppressor sites subsequent to chromosomal duplication; these losses do not necessarily result in LOH, but nonetheless occur in conspicuous patterns. The 500 K Mapping array was also capable of detecting many sub-mega base losses and gains that were overlooked by CGH-BAC arrays, and was superior to CGH-BAC arrays in resolving regions of complex CN variation. PMID:18928532

  5. Replicational release of geminivirus genomes from tandemly repeated copies: evidence for rolling-circle replication of a plant viral DNA.

    PubMed

    Stenger, D C; Revington, G N; Stevenson, M C; Bisaro, D M

    1991-09-15

    Agrobacterium-mediated inoculation of Nicotiana benthamiana plants with Ti plasmids containing tandem genome repeats derived from different strains of the gemini-virus beet curly top virus (BCTV) resulted in the production of unit-length recombinant progeny genomes in systemically infected plants. When two putative plus-strand origins of replication were present in constructs used as inocula, a replicational escape mechanism was favored that resulted in progeny genomes of a single predominant genotype. The genotype was dependent upon the arrangement of repeated parental genomes in the inocula. Sequencing across the junction between parental BCTV strains in the recombinant progeny allowed mapping of the plus-strand origin of replication to a 20-base-pair sequence within the conserved hairpin found in all geminivirus genomes. In contrast, when inocula contained tandemly repeated BCTV genome sequences but only a single conserved hairpin, a number of different progeny genotypes were simultaneously replicated in infected plants, a result expected if unit-length viral genomes were generated by random intramolecular recombination events. These results and other considerations indicate that geminivirus DNA replication occurs by a rolling-circle mechanism. PMID:1896448

  6. 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

  7. Spectrum of T-DNA integrations for insertional mutagenesis of Histoplasma capsulatum

    PubMed Central

    Kemski, Megan M.; Stevens, Bryan; Rappleye, Chad A.

    2012-01-01

    Agrobacterium-mediated transformation is being increasingly used for insertional mutagenesis of fungi. To better evaluate its effectiveness as a mutagen for the fungal pathogen Histoplasma capsulatum, we analyzed a collection of randomly selected T-DNA insertion mutants. Testing of different T-DNA element vectors engineered for transformation of fungi showed that pBHt2 provides the highest transformation efficiency and the lowest rate of vector backbone carryover. Sixty-eight individual T-DNA integrations were characterized by recovery of T-DNA ends and flanking genomic sequences. The right border end of the T-DNA is largely preserved whereas the left border end is frequently truncated. Analysis of T-DNA insertion sites confirms the lack of any integration hotspots in the Histoplasma genome. Relative to genes, T-DNA integrations show significant bias towards promoter regions at the expense of coding sequences. With consideration for potential promoter interruption and the demonstrated efficacy of intronic insertions, 61% of mapped T-DNA insertions should impair gene expression or function. Mapping of T-DNA flanking sequences demonstrates 67% of T-DNA integrations are integrations at a single chromosomal site and 31% of T-DNA integrations are associated with large-scale chromosomal rearrangements. This characterization of T-DNA insertions in mutants selected without regard to phenotype supports application of Agrobacterium-mediated transformation as an insertional mutagen for genome-based screens and functional discovery of genes in Histoplasma. PMID:23332832

  8. Turning the corner in fertility: high DNA integrity of boundary-following sperm.

    PubMed

    Eamer, Lise; Vollmer, Marion; Nosrati, Reza; San Gabriel, Maria C; Zeidan, Krista; Zini, Armand; Sinton, David

    2016-07-01

    We present a passive microfluidic sperm selection strategy that collects motile sperm based on their preference to follow boundaries and turn corners. Clinical assessment of selected human sperm from the device revealed a strong correlation between high DNA integrity and the tendency for sperm to follow boundaries. Human sperm with preference to follow boundaries on the left- or right-hand sides have higher (>51%) DNA integrity than straight swimmers and significantly higher (>67%) DNA integrity than sperm in raw semen. Boundary following behaviour offers a strategy to selecting sperm with the highest DNA integrity to improve the success rate of assisted reproduction. PMID:27241827

  9. 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. PMID:24052059

  10. 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. PMID:24866763

  11. Analysis of the distribution and structure of integrated Banana streak virus DNA in a range of Musa cultivars.

    PubMed

    Geering, A D; Olszewski, N E; Dahal, G; Thomas, J E; Lockhart, B E

    2001-07-01

    Summary Banana streak virus strain OL (BSV-OL) commonly infects new Musa hybrids, and this infection is thought to arise de novo from integrated virus sequences present in the nuclear genome of the plant. Integrated DNA (Musa6+8 sequence) containing the whole genome of the virus has previously been cloned from cv. Obino l'Ewai (Musa AAB group), a parent of many of the hybrids. Using a Southern blot hybridization assay, we have examined the distribution and structure of integrated BSV-OL sequences in a range of Musa cultivars. For cv. Obino l'Ewai, almost every restriction fragment hybridizing to BSV-OL was predicted from the Musa6+8 sequence, suggesting that this is the predominant type of BSV-OL integrant in the genome. Furthermore, since only two junction fragments of Musa/BSV sequence were detected, and the Musa6+8 sequence is believed to be integrated as multiple copies in a tandem array, then the internal Musa spacer sequences must be highly conserved. Similarly sized restriction fragments were detected in four BB group cultivars, but not in six AA or AAA group cultivars, suggesting that the BSV-OL sequences are linked to the B-genome of Musa. We also provide evidence that cv. Williams (Musa AAA group) contains a distinct badnavirus integrant that is closely related to the 'dead' virus integrant previously characterized from Calcutta 4 (Musa acuminata ssp. burmannicoides). Our results suggest that the virus integrant from cv. Williams is linked to the A-genome, and the complexity of the hybridization patterns suggest multiple sites of integration and/or variation in sequence and structure of the integrants. PMID:20573008

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

    PubMed

    Sanglard, D; Fiechter, A

    1992-12-01

    The alkane-assimilating yeast Candida tropicalis was used as a host for DNA transformations. A stable ade2 mutant (Ha900) obtained by UV-mutagenesis was used as a recipient for different vectors carrying selectable markers. A first vector, pMK16, that was developed for the transformation of C. albicans and carries an ADE2 gene marker and a Candida autonomously replicating sequence (CARS) element promoting autonomous replication, was compatible for transforming Ha900. Two transformant types were observed: (i) pink transformants which easily lose pMK16 under non-selective growth conditions; (ii) white transformants, in which the same plasmid exhibited a higher mitotic stability. In both cases pMK16 could be rescued from these cells in Escherichia coli. A second vector, pADE2, containing the isolated C. tropicalis ADE2, gene, was used to transform Ha900. This vector integrated in the yeast genome at homologous sites of the ade2 locus. Different integration types were observed at one or both ade2 alleles in single or in tandem repeats. PMID:1293885

  13. 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.

  14. A high-resolution integrated map of copy number polymorphisms within and between breeds of the modern domesticated dog

    PubMed Central

    2011-01-01

    Background Structural variation contributes to the rich genetic and phenotypic diversity of the modern domestic dog, Canis lupus familiaris, although compared to other organisms, catalogs of canine copy number variants (CNVs) are poorly defined. To this end, we developed a customized high-density tiling array across the canine genome and used it to discover CNVs in nine genetically diverse dogs and a gray wolf. Results In total, we identified 403 CNVs that overlap 401 genes, which are enriched for defense/immunity, oxidoreductase, protease, receptor, signaling molecule and transporter genes. Furthermore, we performed detailed comparisons between CNVs located within versus outside of segmental duplications (SDs) and find that CNVs in SDs are enriched for gene content and complexity. Finally, we compiled all known dog CNV regions and genotyped them with a custom aCGH chip in 61 dogs from 12 diverse breeds. These data allowed us to perform the first population genetics analysis of canine structural variation and identify CNVs that potentially contribute to breed specific traits. Conclusions Our comprehensive analysis of canine CNVs will be an important resource in genetically dissecting canine phenotypic and behavioral variation. PMID:21846351

  15. Retroposons do jump: a B2 element recently integrated in an 18S rDNA gene.

    PubMed Central

    Oberbäumer, I

    1992-01-01

    Several cDNA clones were isolated from cDNA libraries constructed with mRNA longer than 28S RNA from the murine cell line PYS-2/12. The plasmids have inserts containing 1-1.2 kb of the ribosomal 5' external transcribed spacer followed by nearly 700 nt of sequence for 18S rRNA and ending with a B2 element (retroposon). The cloned sequence differed in a few positions from published ribosomal sequences. The 3' adjacent genomic sequence was obtained by polymerase chain reaction (PCR) and showed that the B2 element has a poly(A) tail of about 50 nt and is surrounded by perfect direct repeats of 15 nt. Analysis of genomic DNA from several murine cell lines revealed that PYS cells contain at least one copy of 18S RNA with the B2 element which is not present in the genome of other murine cell lines derived from the same teratocarcinoma. Similarly, rRNA transcripts containing the B2 element were only detected in PYS cells. According to the publication dates of the different cell lines, the B2 element must have been integrated into an rRNA transcription unit during the years 1970 through 1974 thus proving that retroposons (SINEs) can still be inserted into the genome in our times. Images PMID:1311830

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

    PubMed

    Wei, Yunzhou; Terns, Michael P

    2016-06-16

    In this issue of Molecular Cell, Nuñez et al. (2016) report that site-specific integration of foreign DNA into CRISPR loci by the Cas1-Cas2 integrase complex is promoted by a host factor, IHF (integration host factor), that binds and bends CRISPR leader DNA. PMID:27315553

  17. 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.

  18. Integrity and biological activity of DNA after UV exposure.

    PubMed

    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/m(2)s) not only supports the use of the DNA molecule as a biosignature on mineral surfaces but also demonstrates that the DNA retained biological activity. PMID:20446869

  19. Single copy insertion of transgenes in C. elegans

    PubMed Central

    Frøkjær-Jensen, Christian; Davis, M. Wayne; Hopkins, Christopher E.; Newman, Blake; Thummel, Jason M.; Olesen, Søren-Peter; Grunnet, Morten; Jorgensen, Erik M.

    2009-01-01

    Currently transgenes in C. elegans are generated by injecting DNA into the germline. The DNA assembles into a semi-stable extrachromosomal array composed of many copies of injected DNA. These transgenes are typically overexpressed in somatic cells and silenced in the germline. We have developed a method called MosSCI (Mos1-mediated Single Copy Insertion) that inserts a single copy of a transgene into a defined site. Mobilization of a Mos1 transposon generates a double strand break in non-coding DNA. The break is repaired by copying DNA from an extrachromosomal template into the chromosomal site. Homozygous single copy insertions can be obtained in less than two weeks by injecting approximately twenty animals. We have successfully inserted transgenes as long as 9 kb and verified that single copies are inserted at the targeted site. Single copy transgenes are expressed at endogenous levels and can be expressed in the female and male germlines. PMID:18953339

  20. Urine Cell-Free DNA Integrity Analysis for Early Detection of Prostate Cancer Patients

    PubMed Central

    Salvi, Samanta; Gurioli, Giorgia; Martignano, Filippo; Foca, Flavia; Gunelli, Roberta; Cicchetti, Giacomo; De Giorgi, Ugo; Zoli, Wainer; Calistri, Daniele; Casadio, Valentina

    2015-01-01

    Introduction. The detection of tumor-specific markers in urine has paved the way for new early noninvasive diagnostic approaches for prostate cancer. We evaluated the DNA integrity in urine supernatant to verify its capacity to discriminate between prostate cancer and benign diseases of the urogenital tract. Patients and Methods. A total of 131 individuals were enrolled: 67 prostate cancer patients and 64 patients with benign diseases of the urogenital tract (control group). Prostate-specific antigen (PSA) levels were determined. Urine cell-free (UCF) DNA was isolated and sequences longer than 250 bp corresponding to 3 genes (c-MYC, HER2, and AR) were quantified by Real-Time PCR to assess UCF-DNA integrity. Results. UCF-DNA was quantifiable in all samples, while UCF-DNA integrity was evaluable in all but 16 samples. Receiver operating characteristic analysis showed an area under the curve of 0.5048 for UCF-DNA integrity and 0.8423 for PSA. Sensitivity was 0.58 and 0.95 for UCF-DNA integrity and PSA, respectively. Specificity was 0.44 and 0.69, respectively. Conclusions. UCF-DNA integrity showed lower accuracy than PSA and would not seem to be a reliable marker for early prostate cancer diagnosis. Despite this, we believe that UCF-DNA could represent a source of other biomarkers and could detect gene alterations. PMID:26412928

  1. 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

  2. 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

  3. Integrating S-phase Checkpoint Signaling with Trans-Lesion Synthesis of Bulky DNA Adducts

    PubMed Central

    Barkley, Laura R.; Ohmori, Haruo; Vaziri, Cyrus

    2011-01-01

    Bulky adducts are DNA lesions generated in response to environmental agents including benzo[a]pyrene (a combustion product) and solar ultraviolet radiation. Error-prone replication of adducted DNA can cause mutations, which may result in cancer. To minimize the detrimental effects of bulky adducts and other DNA lesions, S-phase checkpoint mechanisms sense DNA damage and integrate DNA repair with ongoing DNA replication. The essential protein kinase Chk1 mediates the S-phase checkpoint, inhibiting initiation of new DNA synthesis and promoting stabilization and recovery of stalled replication forks. Here we review the mechanisms by which Chk1 is activated in response to bulky adducts and potential mechanisms by which Chk1 signaling inhibits the initiation stage of DNA synthesis. Additionally, we discuss mechanisms by which Chk1 signaling facilitates bypass of bulky lesions by specialized Y-family DNA polymerases, thereby attenuating checkpoint signaling and allowing resumption of normal cell cycle progression. PMID:17652783

  4. 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. PMID:25515665

  5. 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.

  6. Copy number variation and mutation

    NASA Astrophysics Data System (ADS)

    Clark, Brian; Weidner, Jacob; Wabick, Kevin

    2009-11-01

    Until very recently, the standard model of DNA included two genes for each trait. This dated model has given way to a model that includes copies of some genes well in excess of the canonical two. Copy number variations in the human genome play critical roles in causing or aggravating a number of syndromes and diseases while providing increased resistance to others. We explore the role of mutation, crossover, inversion, and reproduction in determining copy number variations in a numerical simulation of a population. The numerical model consists of a population of individuals, where each individual is represented by a single strand of DNA with the same number of genes. Each gene is initially assigned to one of two traits. Fitness of the individual is determined by the two most fit genes for trait one, and trait two genetic material is treated as a reservoir of junk DNA. After a sufficient number of generations, during which the genetic distribution is allowed to reach a steady-state, the mean numberof genes per trait and the copy number variation are recorded. Here, we focus on the role of mutation and compare simulation results to theory.

  7. Integrated copy number and gene expression profiling analysis of Epstein-Barr virus-positive diffuse large B-cell lymphoma.

    PubMed

    Yoon, Heejei; Park, Sanghui; Ju, Hyunjeong; Ha, Sang Yun; Sohn, InSuk; Jo, Jisuk; Do, In-Gu; Min, Sookee; Kim, Seok Jin; Kim, Won Seog; Yoo, Hae Yong; Ko, Young Hyeh

    2015-06-01

    Viral oncogenes and host immunosenescence have been suggested as causes of Epstein-Barr virus-positive diffuse large B-cell lymphoma (EBV + DLBCL) of the elderly. To investigate the molecular genetic basis of immune evasion and tumor outgrowth, we analyzed copy number alterations (CNAs) and gene expression profiles in EBV + DLBCL samples compared with EBV - DLBCL. There were relatively few genomic alterations in EBV + DLBCL compared with those detected in EBV-negative DLBCL. The most frequent CNAs (>30%) in EBV + DLBCLs were gains at 1q23.2-23.3, 1q23.3, 1q32.1, 5p15.3, 8q22.3, 8q24.1-24.2, and 9p24.1; losses at 6q27, 7q11.2, and 7q36.2-36.3 were also recurrent. A gene expression profile analysis identified the host immune response as a key molecular signature in EBV + DLBCL. Antiviral response genes, proinflammatory cytokines, and chemokines associated with the innate immune response were overexpressed, indicating the presence of a virusinduced inflammatory microenvironment. Genes associated with the B-cell receptor signaling pathway were downregulated. An integrated analysis indicated that SLAMF1 and PDL2 were key targets of the gains detected at 1q23.2-23.3 and 9p24.1. The chromosomal gain at 9p24.1 was associated with poor overall survival. Taken together, our results led to the identification of recurrent copy number alterations and distinct gene expression associated with the host immune response in EBV + DLBCL. We suggest that the upregulation of PDL2 on 9p24.1 promotes immune evasion and is associated with poor prognosis in EBV + DLBCL. PMID:25832818

  8. 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. PMID:26684805

  9. 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. PMID:23307075

  10. Local chromatin structure of heterochromatin regulates repeatedDNA stability, nucleolus structure, and genome integrity

    SciTech Connect

    Peng, Jamy C.

    2007-05-05

    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

  11. Sperm global DNA methylation level: association with semen parameters and genome integrity.

    PubMed

    Montjean, D; Zini, A; Ravel, C; Belloc, S; Dalleac, A; Copin, H; Boyer, P; McElreavey, K; Benkhalifa, M

    2015-03-01

    Sperm DNA methylation abnormalities have been detected in oligozoospermic men. However, the association between sperm DNA methylation defects, sperm parameters and sperm DNA, and chromatin integrity remains poorly understood. This study was designed to clarify this issue. We recruited a cohort of 92 men (62 normozoospermic and 30 oligoasthenozoospermic) presenting for infertility evaluation during a 1-year period. Sperm global DNA methylation was evaluated by an ELISA-like method, DNA fragmentation was evaluated by flow cytometry-based terminal transferase dUTP nick end-labeling (TUNEL) assay (reported as DNA fragmentation index or DFI), and sperm denaturation was evaluated by aniline blue staining (reported as sperm denaturation index or SDI, a marker of chromatin compaction). We found a significant positive association between sperm global DNA methylation level and conventional sperm parameters (sperm concentration and motility), supported by the results of methylation analysis on H19-DMR. We also identified significant inverse relationships between sperm global DNA methylation, and, both DFI and SDI. However, sperm global DNA methylation level was not related to sperm vitality or morphology. Our findings suggest that global sperm DNA methylation levels are related to conventional sperm parameters, as well as, sperm chromatin and DNA integrity. PMID:25755112

  12. Computational method for estimating DNA copy numbers in normal samples, cancer cell lines, and solid tumors using array comparative genomic hybridization.

    PubMed

    Abkevich, Victor; Iliev, Diana; Timms, Kirsten M; Tran, Thanh; Skolnick, Mark; Lanchbury, Jerry S; Gutin, Alexander

    2010-01-01

    Genomic copy number variations are a typical feature of cancer. These variations may influence cancer outcomes as well as effectiveness of treatment. There are many computational methods developed to detect regions with deletions and amplifications without estimating actual copy numbers (CN) in these regions. We have developed a computational method capable of detecting regions with deletions and amplifications as well as estimating actual copy numbers in these regions. The method is based on determining how signal intensity from different probes is related to CN, taking into account changes in the total genome size, and incorporating into analysis contamination of the solid tumors with benign tissue. Hidden Markov Model is used to obtain the most likely CN solution. The method has been implemented for Affymetrix 500K GeneChip arrays and Agilent 244K oligonucleotide arrays. The results of CN analysis for normal cell lines, cancer cell lines, and tumor samples are presented. The method is capable of detecting copy number alterations in tumor samples with up to 80% contamination with benign tissue. Analysis of 178 cancer cell lines reveals multiple regions of common homozygous deletions and strong amplifications encompassing known tumor suppressor genes and oncogenes as well as novel cancer related genes. PMID:20706610

  13. Functional profiling and gene expression analysis of chromosomal copy number alterations

    PubMed Central

    Conde, Lucía; Montaner, David; Burguet-Castell, Jordi; Tárraga, Joaquín; Al-Shahrour, Fátima; Dopazo, Joaquín

    2007-01-01

    Contrarily to the traditional view in which only one or a few key genes were supposed to be the causative factors of diseases, we discuss the importance of considering groups of functionally related genes in the study of pathologies characterised by chromosomal copy number alterations. Recent observations have reported the existence of regions in higher eukaryotic chromosomes (including humans) containing genes of related function that show a high degree of coregulation. Copy number alterations will consequently affect to clusters of functionally related genes, which will be the final causative agents of the diseased phenotype, in many cases. Therefore, we propose that the functional profiling of the regions affected by copy number alterations must be an important aspect to take into account in the understanding of this type of pathologies. To illustrate this, we present an integrated study of DNA copy number variations, gene expression along with the functional profiling of chromosomal regions in a case of multiple myeloma. PMID:17597935

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

    PubMed

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

    2016-02-01

    Collection of human whole blood for genomic DNA extraction is part of numerous clinical studies. Since DNA extraction cannot always be performed at the time of sample collection, whole blood samples may be stored for years before being processed. The use of appropriate storage conditions is then critical to obtain DNA in sufficient quantity and of adequate quality in order to obtain reliable results from the subsequent molecular biological analyses. In this study, EDTA whole blood samples were collected from 8 healthy volunteers, and different durations (up to 1 year) and temperatures (room temperature, 4°C, -20°C, and -80°C) of storage were compared. The effect of the addition of a DNA preservative agent was also assessed before and after storage. DNA concentrations measured by UV spectrophotometry and spectrofluorometry were used to calculate DNA extraction yields and double-strand DNA ratios. DNA integrity was controlled by agarose gel electrophoresis and long-range polymerase chain reaction. The impact of storage conditions on DNA methylation was also evaluated. Results showed that certain storage conditions have a significant impact on the DNA extraction yield but little or no effect on DNA integrity and methylation. Storage of EDTA blood at -80°C guarantees high-quality DNA with a good yield. Higher DNA extraction yields were obtained with the addition of a DNA preservative agent before thawing EDTA blood stored at -20°C or -80°C. Long-term storage at room temperature in the presence of a DNA preservative agent also appeared to be a reliable procedure. PMID:26812548

  15. Opto-electronic DNA chip-based integrated card for clinical diagnostics.

    PubMed

    Marchand, Gilles; Broyer, Patrick; Lanet, Véronique; Delattre, Cyril; Foucault, Frédéric; Menou, Lionel; Calvas, Bernard; Roller, Denis; Ginot, Frédéric; Campagnolo, Raymond; Mallard, Frédéric

    2008-02-01

    Clinical diagnostics is one of the most promising applications for microfluidic lab-on-a-chip or lab-on-card systems. DNA chips, which provide multiparametric data, are privileged tools for genomic analysis. However, automation of molecular biology protocol and use of these DNA chips in fully integrated systems remains a great challenge. Simplicity of chip and/or card/instrument interfaces is amongst the most critical issues to be addressed. Indeed, current detection systems for DNA chip reading are often complex, expensive, bulky and even limited in terms of sensitivity or accuracy. Furthermore, for liquid handling in the lab-on-cards, many devices use complex and bulky systems, either to directly manipulate fluids, or to ensure pneumatic or mechanical control of integrated valves. All these drawbacks prevent or limit the use of DNA-chip-based integrated systems, for point-of-care testing or as a routine diagnostics tool. We present here a DNA-chip-based protocol integration on a plastic card for clinical diagnostics applications including: (1) an opto-electronic DNA-chip, (2) fluid handling using electrically activated embedded pyrotechnic microvalves with closing/opening functions. We demonstrate both fluidic and electric packaging of the optoelectronic DNA chip without major alteration of its electronical and biological functionalities, and fluid control using novel electrically activable pyrotechnic microvalves. Finally, we suggest a complete design of a card dedicated to automation of a complex biological protocol with a fully electrical fluid handling and DNA chip reading. PMID:17636395

  16. Aptamer-integrated DNA nanostructures for biosensing, bioimaging and cancer therapy.

    PubMed

    Meng, Hong-Min; Liu, Hui; Kuai, Hailan; Peng, Ruizi; Mo, Liuting; Zhang, Xiao-Bing

    2016-05-01

    The combination of nanostructures with biomolecules leading to the generation of functional nanosystems holds great promise for biotechnological and biomedical applications. As a naturally occurring biomacromolecule, DNA exhibits excellent biocompatibility and programmability. Also, scalable synthesis can be readily realized through automated instruments. Such unique properties, together with Watson-Crick base-pairing interactions, make DNA a particularly promising candidate to be used as a building block material for a wide variety of nanostructures. In the past few decades, various DNA nanostructures have been developed, including one-, two- and three-dimensional nanomaterials. Aptamers are single-stranded DNA or RNA molecules selected by Systematic Evolution of Ligands by Exponential Enrichment (SELEX), with specific recognition abilities to their targets. Therefore, integrating aptamers into DNA nanostructures results in powerful tools for biosensing and bioimaging applications. Furthermore, owing to their high loading capability, aptamer-modified DNA nanostructures have also been altered to play the role of drug nanocarriers for in vivo applications and targeted cancer therapy. In this review, we summarize recent progress in the design of aptamers and related DNA molecule-integrated DNA nanostructures as well as their applications in biosensing, bioimaging and cancer therapy. To begin with, we first introduce the SELEX technology. Subsequently, the methodologies for the preparation of aptamer-integrated DNA nanostructures are presented. Then, we highlight their applications in biosensing and bioimaging for various targets, as well as targeted cancer therapy applications. Finally, we discuss several challenges and further opportunities in this emerging field. PMID:26954935

  17. The COG and COPI Complexes Interact to Control the Abundance of GEARs, a Subset of Golgi Integral Membrane ProteinsD⃞

    PubMed Central

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

    2004-01-01

    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 ε-COP in wild-type cells under conditions in which COG-insensitive proteins were unaffected. Furthermore, synthetic phenotypes arose in mutants deficient in both ε-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. PMID:15004235

  18. A spreadable, non-integrative and high copy number shuttle vector for Sulfolobus solfataricus based on the genetic element pSSVx from Sulfolobus islandicus.

    PubMed

    Aucelli, Tiziana; Contursi, Patrizia; Girfoglio, Michele; Rossi, Mosè; Cannio, Raffaele

    2006-01-01

    The pSSVx genetic element from Sulfolobus islandicus REY15/4 is a hybrid between a plasmid and a fusellovirus, able to be maintained in non-integrative form and to spread when the helper SSV2 virus is present in the cells. In this work, the satellite virus was engineered to obtain an Escherichia coli-Sulfolobus solfataricus shuttle vector for gene transfer and expression in S.solfataricus by fusing site-specifically the pSSVx chromosome with an E.coli plasmid replicon and the ampicillin resistance gene. The pSSVx-based vector was proven functional like the parental virus, namely it was able to spread efficiently through infected S.solfataricus cells. Moreover, the hybrid plasmid stably transformed S.solfataricus and propagated with no rearrangement, recombination or integration into the host chromosome. The high copy number of the artificial genetic element was found comparable with that calculated for the wild-type pSSVx in the new host cells, with no need of genetic markers for vector maintenance in the cells and for transfomant enrichment. The newly constructed vector was also shown to be an efficient cloning vehicle for the expression of passenger genes in S.solfataricus. In fact, a derivative plasmid carrying an expression cassette of the lacS gene encoding the beta-glycosidase from S.solfataricus under the control of the Sulfolobus chaperonine (thermosome tf55) heat shock promoter was also able to drive the expression of a functional enzyme. Complementation of the beta-galactosidase deficiency in a deletion mutant strain of S.solfataricus demonstrated that lacS gene was an efficient marker for selection of single transformants on solid minimal lactose medium. PMID:16971457

  19. Multiplex Identification of Human Papillomavirus 16 DNA Integration Sites in Cervical Carcinomas

    PubMed Central

    Xu, Bo; Chotewutmontri, Sasithorn; Wolf, Stephan; Klos, Ursula; Schmitz, Martina; Dürst, Matthias; Schwarz, Elisabeth

    2013-01-01

    Cervical cancer is caused by high-risk human papillomaviruses (HPV), in more than half of the worldwide cases by HPV16. Viral DNA integration into the host genome is a frequent mutation in cervical carcinogenesis. Because integration occurs into different genomic locations, it creates unique viral-cellular DNA junctions in every single case. This singularity complicates the precise identification of HPV integration sites enormously. We report here the development of a novel multiplex strategy for sequence determination of HPV16 DNA integration sites. It includes DNA fragmentation and adapter tagging, PCR enrichment of the HPV16 early region, Illumina next-generation sequencing, data processing, and validation of candidate integration sites by junction-PCR. This strategy was performed with 51 cervical cancer samples (47 primary tumors and 4 cell lines). Altogether 75 HPV16 integration sites (3′-junctions) were identified and assigned to the individual samples. By comparing the DNA junctions with the presence of viral oncogene fusion transcripts, 44 tumors could be classified into four groups: Tumors with one transcriptionally active HPV16 integrate (n = 12), tumors with transcribed and silent DNA junctions (n = 8), tumors carrying episomal HPV16 DNA (n = 10), and tumors with one to six DNA junctions, but without fusion transcripts (n = 14). The 3′-breakpoints of integrated HPV16 DNA show a statistically significant (p<0.05) preferential distribution within the early region segment upstream of the major splice acceptor underscoring the importance of deregulated viral oncogene expression for carcinogenesis. Half of the mapped HPV16 integration sites target cellular genes pointing to a direct influence of HPV integration on host genes (insertional mutagenesis). In summary, the multiplex strategy for HPV16 integration site determination worked very efficiently. It will open new avenues for comprehensive mapping of HPV integration sites and for the

  20. Integrated Copy Number and Expression Analysis Identifies Profiles of Whole-Arm Chromosomal Alterations and Subgroups with Favorable Outcome in Ovarian Clear Cell Carcinomas

    PubMed Central

    Uehara, Yuriko; Oda, Katsutoshi; Ikeda, Yuji; Koso, Takahiro; Tsuji, Shingo; Yamamoto, Shogo; Asada, Kayo; Sone, Kenbun; Kurikawa, Reiko; Makii, Chinami; Hagiwara, Otoe; Tanikawa, Michihiro; Maeda, Daichi; Hasegawa, Kosei; Nakagawa, Shunsuke; Wada-Hiraike, Osamu; Kawana, Kei; Fukayama, Masashi; Fujiwara, Keiichi; Yano, Tetsu; Osuga, Yutaka; Fujii, Tomoyuki; Aburatani, Hiroyuki

    2015-01-01

    Ovarian clear cell carcinoma (CCC) is generally associated with chemoresistance and poor clinical outcome, even with early diagnosis; whereas high-grade serous carcinomas (SCs) and endometrioid carcinomas (ECs) are commonly chemosensitive at advanced stages. Although an integrated genomic analysis of SC has been performed, conclusive views on copy number and expression profiles for CCC are still limited. In this study, we performed single nucleotide polymorphism analysis with 57 epithelial ovarian cancers (31 CCCs, 14 SCs, and 12 ECs) and microarray expression analysis with 55 cancers (25 CCCs, 16 SCs, and 14 ECs). We then evaluated PIK3CA mutations and ARID1A expression in CCCs. SNP array analysis classified 13% of CCCs into a cluster with high frequency and focal range of copy number alterations (CNAs), significantly lower than for SCs (93%, P < 0.01) and ECs (50%, P = 0.017). The ratio of whole-arm to all CNAs was higher in CCCs (46.9%) than SCs (21.7%; P < 0.0001). SCs with loss of heterozygosity (LOH) of BRCA1 (85%) also had LOH of NF1 and TP53, and LOH of BRCA2 (62%) coexisted with LOH of RB1 and TP53. Microarray analysis classified CCCs into three clusters. One cluster (CCC-2, n = 10) showed more favorable prognosis than the CCC-1 and CCC-3 clusters (P = 0.041). Coexistent alterations of PIK3CA and ARID1A were more common in CCC-1 and CCC-3 (7/11, 64%) than in CCC-2 (0/10, 0%; P < 0.01). Being in cluster CCC-2 was an independent favorable prognostic factor in CCC. In conclusion, CCC was characterized by a high ratio of whole-arm CNAs; whereas CNAs in SC were mainly focal, but preferentially caused LOH of well-known tumor suppressor genes. As such, expression profiles might be useful for sub-classification of CCC, and might provide useful information on prognosis. PMID:26043110

  1. 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 software. (JN)

  2. Isolation and characterization of a set of disease resistance-gene analogs (RGAs) from wild rice, Zizania latifolia Griseb. I. Introgression, copy number lability, sequence change, and DNA methylation alteration in several rice-Zizania introgression lines.

    PubMed

    Chen, Yu; Long, Likun; Lin, Xiuyun; Guo, Wanli; Liu, Bao

    2006-02-01

    Eight resistance-gene analogs (RGAs) were isolated from wild rice, Zizania latifolia Griseb., by degenerate primers designed according to conserved motifs at or around the nucleotide-binding site (NBS) of known NBS-containing plant resistance genes. The 8 RGAs were classified into 6 distinct groups based on their deduced amino acid sequence similarity of 60% or greater. Gel-blot hybridization of each of the RGAs to 4 rice - Z. latifolia intro gression lines indicated an array of changes at either introgressed Zizania RGAs or, more likely, their rice homologs. The changes included dramatic increase in copy number, modification at the primary DNA sequence, and alteration in DNA methylation patterns. PMID:16498465

  3. 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

  4. Retroviral intasomes search for a target DNA by 1D diffusion which rarely results in integration.

    PubMed

    Jones, Nathan D; Lopez, 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

  5. Array comparative genomic hybridization identifies a distinct DNA copy number profile in renal cell cancer associated with hereditary leiomyomatosis and renal cell cancer.

    PubMed

    Koski, Taru A; Lehtonen, Heli J; Jee, Kowan J; Ninomiya, Shinsuke; Joosse, Simon A; Vahteristo, Pia; Kiuru, Maija; Karhu, Auli; Sammalkorpi, Heli; Vanharanta, Sakari; Lehtonen, Rainer; Edgren, Henrik; Nederlof, Petra M; Hietala, Marja; Aittomäki, Kristiina; Herva, Riitta; Knuutila, Sakari; Aaltonen, Lauri A; Launonen, Virpi

    2009-07-01

    Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a tumor predisposition syndrome with cutaneous and uterine leiomyomatosis as well as renal cell cancer (RCC) as its clinical manifestations. HLRCC is caused by heterozygous germline mutations in the fumarate hydratase (fumarase) gene. In this study, we used array comparative genomic hybridization to identify the specific copy number changes characterizing the HLRCC-associated RCCs. The study material comprised formalin-fixed paraffin-embedded renal tumors obtained from Finnish patients with HLRCC. All 11 investigated tumors displayed the papillary type 2 histopathology typical for HLRCC renal tumors. The most frequent copy number changes detected in at least 3/11 (27%) of the tumors were gains in chromosomes 2, 7, and 17, and losses in 13q12.3-q21.1, 14, 18, and X. These findings provide genetic evidence for a distinct copy number profile in HLRCC renal tumors compared with sporadic RCC tumors of the same histopathological subtype, and delineate chromosomal regions that associate with this very aggressive form of RCC. PMID:19373782

  6. Uracil DNA glycosylase initiates degradation of HIV-1 cDNA containing misincorporated dUTP and prevents viral integration

    PubMed Central

    Weil, Amy F.; Ghosh, Devlina; Zhou, Yan; Seiple, Lauren; McMahon, Moira A.; Spivak, Adam M.; Siliciano, Robert F.; Stivers, James T.

    2013-01-01

    HIV-1 reverse transcriptase discriminates poorly between dUTP and dTTP, and accordingly, viral DNA products become heavily uracilated when viruses infect host cells that contain high ratios of dUTP:dTTP. Uracilation of invading retroviral DNA is thought to be an innate immunity barrier to retroviral infection, but the mechanistic features of this immune pathway and the cellular fate of uracilated retroviral DNA products is not known. Here we developed a model system in which the cellular dUTP:dTTP ratio can be pharmacologically increased to favor dUTP incorporation, allowing dissection of this innate immunity pathway. When the virus-infected cells contained elevated dUTP levels, reverse transcription was found to proceed unperturbed, but integration and viral protein expression were largely blocked. Furthermore, successfully integrated proviruses lacked detectable uracil, suggesting that only nonuracilated viral DNA products were integration competent. Integration of the uracilated proviruses was restored using an isogenic cell line that had no detectable human uracil DNA glycosylase (hUNG2) activity, establishing that hUNG2 is a host restriction factor in cells that contain high dUTP. Biochemical studies in primary cells established that this immune pathway is not operative in CD4+ T cells, because these cells have high dUTPase activity (low dUTP), and only modest levels of hUNG activity. Although monocyte-derived macrophages have high dUTP levels, these cells have low hUNG activity, which may diminish the effectiveness of this restriction pathway. These findings establish the essential elements of this pathway and reconcile diverse observations in the literature. PMID:23341616

  7. 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)

  8. 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.

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

    PubMed

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

    2008-01-01

    The aim of present study is to assess DNA integrity on the effect of exposure to a radio frequency (RF) signal from Code Division Multiple Access (CDMA) mobile phones. Whole blood samples from six healthy male individuals were exposed for RF signals from a CDMA mobile phone for 1 h. Alkaline comet assay was performed to assess the DNA damage. The combinative exposure effect of the RF signals and APC at two concentrations on DNA integrity was studied. DNA repair efficiency of the samples was also studied after 2 h of exposure. The RF signals and APC (0.2 microg/ml) alone or in synergism did not have any significant DNA damage as compared to sham exposed. However, univariate analysis showed that DNA damage was significantly different among combinative exposure of RF signals and APC at 0.2 microg/ml (p < 0.05) and at 2 microg/ml (p < 0.02). APC at 2 microg/ml concentration also showed significant damage levels (p < 0.05) when compared to sham exposed. DNA repair efficiency also varied in a significant way in combinative exposure sets (p < 0.05). From these results, it appears that the repair inhibitor APC enhances DNA breaks at 2 microg/ml concentration and that the damage is possibly repairable. Thus, it can be inferred that the in vitro exposure to RF signals induces reversible DNA damage in synergism with APC. PMID:19037791

  10. Integration of DNA marker information into breeding value predictions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Calves from seven breeds including 20 herds were genotyped with a reduced DNA marker panel for weaning weight. The marker panel used was derived using USMARC Cycle VII animals. The results from the current study suggest marker effects are not robust across breeds and that methodology exists to integ...

  11. Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites.

    PubMed

    Serrao, Erik; Cherepanov, Peter; Engelman, Alan N

    2016-01-01

    Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients. PMID:27023428

  12. Interactions between the Werner syndrome helicase and DNA polymerase delta specifically facilitate copying of tetraplex and hairpin structures of the d(CGG)n trinucleotide repeat sequence.

    PubMed

    Kamath-Loeb, A S; Loeb, L A; Johansson, E; Burgers, P M; Fry, M

    2001-05-11

    Werner syndrome (WS) is an inherited disorder characterized by premature aging and genomic instability. The protein encoded by the WS gene, WRN, possesses intrinsic 3' --> 5' DNA helicase and 3' --> 5' DNA exonuclease activities. WRN helicase resolves alternate DNA structures including tetraplex and triplex DNA, and Holliday junctions. Thus, one function of WRN may be to unwind secondary structures that impede cellular DNA transactions. We report here that hairpin and G'2 bimolecular tetraplex structures of the fragile X expanded sequence, d(CGG)(n), effectively impede synthesis by three eukaryotic replicative DNA polymerases (pol): pol alpha, pol delta, and pol epsilon. The constraints imposed on pol delta-catalyzed synthesis are relieved, however, by WRN; WRN facilitates pol delta to traverse these template secondary structures to synthesize full-length DNA products. The alleviatory effect of WRN is limited to pol delta; neither pol alpha nor pol epsilon can traverse template d(CGG)(n) hairpin and tetraplex structures in the presence of WRN. Alleviation of pausing by pol delta is observed with Escherichia coli RecQ but not with UvrD helicase, suggesting a concerted action of RecQ helicases and pol delta. Our findings suggest a possible role of WRN in rescuing pol delta-mediated replication at forks stalled by unusual DNA secondary structures. PMID:11279038

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

    PubMed Central

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

    2014-01-01

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

  14. Increased Frequency of De Novo Copy Number Variations in Congenital Heart Disease by Integrative Analysis of SNP Array and Exome Sequence Data

    PubMed Central

    Rodriguez-Murillo, Laura; Fromer, Menachem; Mazaika, Erica; Vardarajan, Badri; Italia, Michael; Leipzig, Jeremy; DePalma, Steven R.; Golhar, Ryan; Sanders, Stephan J.; Yamrom, Boris; Ronemus, Michael; Iossifov, Ivan; Willsey, A. Jeremy; State, Matthew W.; Kaltman, Jonathan R.; White, Peter S.; Shen, Yufeng; Warburton, Dorothy; Brueckner, Martina; Seidman, Christine; Goldmuntz, Elizabeth; Gelb, Bruce D.; Lifton, Richard; Seidman, Jonathan; Hakonarson, Hakon; Chung, Wendy K.

    2014-01-01

    Rationale Congenital heart disease (CHD) is among the most common birth defects. Most cases are of unknown etiology. Objective To determine the contribution of de novo copy number variants (CNVs) in the etiology of sporadic CHD. Methods and Results We studied 538 CHD trios using genome-wide dense single nucleotide polymorphism (SNP) arrays and/or whole exome sequencing (WES). Results were experimentally validated using digital droplet PCR. We compared validated CNVs in CHD cases to CNVs in 1,301 healthy control trios. The two complementary high-resolution technologies identified 63 validated de novo CNVs in 51 CHD cases. A significant increase in CNV burden was observed when comparing CHD trios with healthy trios, using either SNP array (p=7x10−5, Odds Ratio (OR)=4.6) or WES data (p=6x10−4, OR=3.5) and remained after removing 16% of de novo CNV loci previously reported as pathogenic (p=0.02, OR=2.7). We observed recurrent de novo CNVs on 15q11.2 encompassing CYFIP1, NIPA1, and NIPA2 and single de novo CNVs encompassing DUSP1, JUN, JUP, MED15, MED9, PTPRE SREBF1, TOP2A, and ZEB2, genes that interact with established CHD proteins NKX2-5 and GATA4. Integrating de novo variants in WES and CNV data suggests that ETS1 is the pathogenic gene altered by 11q24.2-q25 deletions in Jacobsen syndrome and that CTBP2 is the pathogenic gene in 10q sub-telomeric deletions. Conclusions We demonstrate a significantly increased frequency of rare de novo CNVs in CHD patients compared with healthy controls and suggest several novel genetic loci for CHD. PMID:25205790

  15. The right hand copy of the nopaline Ti-plasmid 25 bp repeat is required for tumour formation.

    PubMed

    Shaw, C H; Watson, M D; Carter, G H; Shaw, C H

    1984-08-10

    At either end of the nopaline Ti-plasmid T-region resides a copy of a 25 bp repeated element. The normal T-DNA endpoint is 1 bp internal of the right copy, with the transcription initiation site of the nopaline synthase (nos) gene being approximately 300 bp away in the same direction. Here we describe results which demonstrate that deletion of any combination of sequences between the nos initiation site and the right copy of the 25 bp repeat does not affect oncogenicity. Thus a mutant retaining the right copy and only 3 bp internal of it is indistinguishable from the wild type parent in its oncogenic properties. However deletion of a further 39 bp, including complete removal of the right copy abolishes crown gall tumour formation on Kalanchöe and tobacco. From these results we infer that unlike the left border, the right copy of the 25 bp repeat is required for T-DNA transfer and/or integration. This is the first conclusive demonstration of the involvement of a copy of the repeats in this process. PMID:6473100

  16. 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. PMID:26578802

  17. 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. PMID:26254786

  18. 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.

  19. Specific in situ hepatitis B viral double mutation (HBVDM) detection in urine with 60 copies ml(-1) analytical sensitivity in a background of 250-fold wild type without DNA isolation and amplification.

    PubMed

    Kirimli, Ceyhun E; Shih, Wei-Heng; Shih, Wan Y

    2015-03-01

    We have examined in situ detection of hepatitis B virus 1762T/1764A double mutation (HBVDM) in urine using a (Pb(Mg(1/3)Nb(2/3))O3)(0.65)(PbTiO3)(0.35) (PMN-PT) piezoelectric plate sensor (PEPS) coated with a 16-nucleotide (nt) probe DNA (pDNA) complementary to the HBVDM. The in situ mutation (MT) detection was carried out in a flow with the PEPS vertically situated at the center of the flow in a background of wild type (WT). For validation, this detection was followed by detection in the mixture of MT fluorescent reporter microspheres (FRMs) (MT FRMs) and WT FRMs that emitted different fluorescence colours and were designed to specifically bind to MT and WT, respectively. At 30 °C and 4 ml min(-1), a PEPS was shown to specifically detect HBVDM in situ with 60 copies ml(-1) analytical sensitivity in a background of clinically-relevant 250-fold more WT in 30 min without DNA isolation, amplification, or labelling as validated by the visualization of the captured MT FRMs and WT FRMs following FRM detection where the captured MT FRMs outnumbered the WT FRMs by a factor of 5 to 1. PMID:25599103

  20. A continental-wide perspective: the genepool of nuclear encoded ribosomal DNA and single-copy gene sequences in North American Boechera (Brassicaceae).

    PubMed

    Kiefer, Christiane; Koch, Marcus A

    2012-01-01

    74 of the currently accepted 111 taxa of the North American genus Boechera (Brassicaceae) were subject to pyhlogenetic reconstruction and network analysis. The dataset comprised 911 accessions for which ITS sequences were analyzed. Phylogenetic analyses yielded largely unresolved trees. Together with the network analysis confirming this result this can be interpreted as an indication for multiple, independent, and rapid diversification events. Network analyses were superimposed with datasets describing i) geographical distribution, ii) taxonomy, iii) reproductive mode, and iv) distribution history based on phylogeographic evidence. Our results provide first direct evidence for enormous reticulate evolution in the entire genus and give further insights into the evolutionary history of this complex genus on a continental scale. In addition two novel single-copy gene markers, orthologues of the Arabidopsis thaliana genes At2g25920 and At3g18900, were analyzed for subsets of taxa and confirmed the findings obtained through the ITS data. PMID:22606266

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

    PubMed

    2012-09-01

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

  2. 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

  3. 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

  4. Homology of pCS1 Plasmid Sequences with Chromosomal DNA in Clavibacter michiganense subsp. sepedonicum: Evidence for the Presence of a Repeated Sequence and Plasmid Integration

    PubMed Central

    Mogen, Bradley D.; Oleson, Arland E.

    1987-01-01

    Restriction fragments of pCS1, a 50.6-kilobase (kb) plasmid present in many strains of Clavibacter michiganense subsp. sepedonicum (“Corynebacterium sepedonicum”), have been cloned in an M13mp11 phage vector. Radiolabeled forms of these cloned fragments have been used as Southern hybridization probes for the presence of plasmid sequences in chromosomal DNA of this organism. These studies have shown that all tested strains lacking the covalently closed circular form of pCS1 contain the plasmid in integrated form. In each case the site of integration exists on a single plasmid restriction fragment with a size of 5.1 kb. Southern hybridizations with these probes have also revealed the existence of a major repeated sequence in C. michiganense subsp. sepedonicum. Hybridizations of chromosomal DNA with deletion subclones of a 2.9-kb plasmid fragment containing the repeated sequence indicate that the size of the repeated sequence is approximately 1.3 kb. One of the copies of the repeated sequence is on the plasmid fragment containing the site of integration. Images PMID:16347464

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

    PubMed

    Newman, Aaron M; Lovejoy, Alexander F; Klass, Daniel M; 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

    2016-05-01

    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 about threefold, 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 non-small cell lung cancer (NSCLC) patients, our method enabled biopsy-free profiling of EGFR kinase domain mutations with 92% sensitivity and >99.99% specificity at the variant level, and with 90% sensitivity and 96% specificity at the patient level. In addition, our approach allowed monitoring of NSCLC ctDNA down to 4 in 10(5) cfDNA molecules. We anticipate that iDES will aid the noninvasive genotyping and detection of ctDNA in research and clinical settings. PMID:27018799

  6. Genomic DNA extraction from cells by electroporation on an integrated microfluidic platform

    PubMed Central

    Geng, Tao; Bao, Ning; Sriranganathanw, Nammalwar; Li, Liwu; Lu, Chang

    2012-01-01

    The vast majority of genetic analysis of cells involves chemical lysis for release of DNA molecules. However, chemical reagents required in the lysis interfere with downstream molecular biology and often require removal after the step. Electrical lysis based on irreversible electroporation is a promising technique to prepare samples for genetic analysis due to its purely physical nature, fast speed, and simple operation. However, there has been no experimental confirmation on whether electrical lysis extracts genomic DNA from cells in a reproducible and efficient fashion in comparison to chemical lysis, especially for eukaryotic cells that have most of DNA enclosed in the nucleus. In this work, we construct an integrated microfluidic chip that physically traps a low number of cells, lyses the cells using electrical pulses rapidly, then purifies and concentrates genomic DNA. We demonstrate that electrical lysis offers high efficiency for DNA extraction from both eukaryotic cells (up to ~36% for Chinese hamster ovary cells) and bacterial cells (up to ~45% for Salmonella typhimurium) that is comparable to the widely-used chemical lysis. The DNA extraction efficiency has dependence on both electric parameters and relative amount of beads used for DNA adsorption. We envision that electroporation-based DNA extraction will find use in ultrasensitive assays that benefit from minimal dilution and simple procedure. PMID:23061629

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

    PubMed

    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 gradient

  9. 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.

  10. Effect of cooled storage on quality and DNA integrity of Asian elephant (Elephas maximus) spermatozoa.

    PubMed

    Imrat, P; Mahasawangkul, S; Gosálvez, J; Suthanmapinanth, P; Sombutputorn, P; Jansittiwate, S; Thongtip, N; Pinyopummin, A; Colenbrander, B; Holt, W V; Stout, T A E

    2012-01-01

    Artificial insemination (AI) is a potentially useful tool for breeding captive elephants because it facilitates efforts to minimise inbreeding. However, cooled storage of elephant semen markedly reduces fertility. This study compared the effects on semen-quality parameters, including sperm DNA fragmentation, of storing elephant semen at 4°C or 15°C in a commonly-used diluent (TEST) or a diluent developed to protect against sperm DNA damage (BullMax). Storing elephant semen for >24 h in either extender at either temperature resulted in decreases in sperm motility, viability, acrosome integrity and DNA integrity (P < 0.05); the decrease in motility was especially rapid. A subjective impression of circular sperm movement in TEST was confirmed by a higher curvilinear velocity and amplitude of lateral head displacement, but lower straight-line velocity and linearity than in BullMax. Initial percentages of spermatozoa with fragmented DNA (%SDF) did not differ between extenders or temperatures, but the rate of increase in %SDF during a 48-h incubation at 37°C was higher in TEST than in BullMax (P < 0.05). In conclusion, BullMax allows more linear movement and better preserves DNA stability of stored elephant spermatozoa than TEST. Sperm DNA stability during incubation at 37°C is a promising, discriminative parameter for selecting semen storage conditions of bulls for elephant AI. PMID:22951013

  11. The structure of adenovirus type 12 DNA integration sites in the hamster cell genome.

    PubMed Central

    Knoblauch, M; Schröer, J; Schmitz, B; Doerfler, W

    1996-01-01

    Foreign DNA can integrate into the genomes of mammalian cells, and this process plays major roles in viral oncogenesis and in the generation of transgenic organisms and will be important in evolving regimens for human somatic gene therapy. In the present study, the insertion sites of adenovirus type 12 (Ad12) DNA genomes have been analyzed in detail in the Ad12-transformed hamster cell line T637, its revertants, which have lost most of the >20 Ad12 genome equivalents integrated chromosomally in cell line T637, and in the Ad12-induced tumor T191. Some of these junction sites have been molecularly cloned, and the nucleotide sequences at the sites of transition between viral and cellular DNAs have been determined. The sites of linkage between the hamster cellular and the foreign (viral) DNA are characterized by the frequent occurrence of patch homologies between the recombination partners. The cellular junction sites investigated here are not transcriptionally active. One of the cellular DNA sequences abutting the right Ad12 DNA terminus in cell line T637 (os2) is represented only once in the hamster genome and has a strikingly low abundance of 5'-CG-3' dinucleotide sequences. One 5'-GCGC-3' sequence close to the Ad12 DNA integration site is heavily methylated in normal cells, Ad12-transformed cells, and Ad12-induced tumor cells. The second such sequence is more remote from the junction site, is partly methylated in BHK21 hamster cells, and shows differences in methylation in different Ad12-transformed cell lines. This site is unmethylated in liver DNA. The cellular DNA sequence at the site of Ad12 linkage in the tumor T191 exhibits homologies to highly repetitive sequences of the Alu family and to an origin of hamster DNA replication containing an Alu element. A number of junction sites between Ad12 DNA and hamster or mouse DNA in Ad12-transformed cell lines or Ad12-induced tumor cell lines, investigated here and previously, are characterized by stem-loop structures

  12. An integrated microfluidic system for bovine DNA purification and digital PCR detection.

    PubMed

    Tian, Qingchang; Mu, Ying; Xu, Yanan; Song, Qi; Yu, Bingwen; Ma, Congcong; Jin, Wei; Jin, Qinhan

    2015-12-15

    In this paper, we described an integrated modularized microfluidic system that contained two distinct functional modules, one for nucleic acids (NA) extraction and the other for digital PCR (dPCR), allowing for detecting the bovine DNA in ovine tissue. PMID:26364950

  13. 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

  14. An integrated system for enzymatic cleavage and electrostretching of freely-suspended single DNA molecules.

    PubMed

    Lam, Liza; Sakakihara, Shouichi; Ishizuka, Koji; Takeuchi, Shoji; Noji, Hiroyuki

    2007-12-01

    A novel polyacrylamide gel-based femtolitre microchamber system for performing single-molecule restriction enzyme assay on freely-suspended DNA molecules and subsequent DNA electrostretching by applying an alternating electric field has been developed. We attempted the integration by firstly initiating restriction enzyme reaction on a fluorescent-stained lambdaDNA molecule, encapsulated in a microchamber, using magnesium as an external trigger. Upon complete digestion, the cleaved DNA fragments were electrostretched to analyze the DNA lengths optically. The critical parameters for electrostretching of encapsulated DNA were investigated and optimum stretching was achieved by using 1.5 kHz pulses with electric field strength in the order of 10(3) V cm(-1) in 7% linear polyacrylamide (LPA) solution. LPA was adopted to minimize the adverse effects of ionic thermal agitation on molecular dielectrophoretic elongation in the microchamber. In our experiments, as the fragments were not immobilized throughout the entire protocol, it was found from repeated tests that digestion always occurred, producing the expected number of cleaved fragments. This versatile microchamber approach realized direct observation of these biological reactions on real-time basis at a single-molecule level. Furthermore, with the employment of porous polyacrylamide gel, the effective manipulation of DNA assays and the ability to combine conventionally independent bioanalytical processes have been demonstrated. PMID:18030395

  15. DNA Damage Response and Spindle Assembly Checkpoint Function throughout the Cell Cycle to Ensure Genomic Integrity

    PubMed Central

    Lawrence, Katherine S.; Chau, Thinh; Engebrecht, JoAnne

    2015-01-01

    Errors in replication or segregation lead to DNA damage, mutations, and aneuploidies. Consequently, cells monitor these events and delay progression through the cell cycle so repair precedes division. The DNA damage response (DDR), which monitors DNA integrity, and the spindle assembly checkpoint (SAC), which responds to defects in spindle attachment/tension during metaphase of mitosis and meiosis, are critical for preventing genome instability. Here we show that the DDR and SAC function together throughout the cell cycle to ensure genome integrity in C. elegans germ cells. Metaphase defects result in enrichment of SAC and DDR components to chromatin, and both SAC and DDR are required for metaphase delays. During persistent metaphase arrest following establishment of bi-oriented chromosomes, stability of the metaphase plate is compromised in the absence of DDR kinases ATR or CHK1 or SAC components, MAD1/MAD2, suggesting SAC functions in metaphase beyond its interactions with APC activator CDC20. In response to DNA damage, MAD2 and the histone variant CENPA become enriched at the nuclear periphery in a DDR-dependent manner. Further, depletion of either MAD1 or CENPA results in loss of peripherally associated damaged DNA. In contrast to a SAC-insensitive CDC20 mutant, germ cells deficient for SAC or CENPA cannot efficiently repair DNA damage, suggesting that SAC mediates DNA repair through CENPA interactions with the nuclear periphery. We also show that replication perturbations result in relocalization of MAD1/MAD2 in human cells, suggesting that the role of SAC in DNA repair is conserved. PMID:25898113

  16. Integration of the simian virus 40 genome into cellular DNA in temperature-sensitive (N) and temperature-insensitive (A) transformants of 3T3 rat and Chinese hamster lung cells.

    PubMed Central

    Chepelinsky, A B; Seif, R; Martin, R G

    1980-01-01

    We studied the pattern of integration of the simian virus 40 (SV40) genome into the cellular DNA of N-transformants (temperature sensitive) and A-transformants (temperature insensitive) derived from 3T3-Fisher rat and Chinese hamster lung cells. The SV40 DNA was covalently linked to the cellular DNA in both types of transformants. In the rat cells, most N-transformants contained SV40 sequences integrated at a single site; most A-transformants contained SV40 sequences integrated at two to five sites. In the Chinese hamster cells, no significant correlation between the number of integration sites and the phenotype of the transformant was found; one of three integration sites were observed for both the N- and A-transformants. Single copies and tandem repeats of SV40 sequences were observed in A- and N-transformants derived from rat cells. A-transformants arise neither by amplification of the SV40 genome nor by integration at a unique site. Images PMID:6251267

  17. 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.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

    The number of large-scale high-dimensional datasets recording different aspects of a single disease is growing, accompanied by a need for frameworks that can create one coherent model from multiple tensors of matched columns, e.g., patients and platforms, but independent rows, e.g., probes. We define and prove the mathematical properties of a novel tensor generalized singular value decomposition (GSVD), which can simultaneously find the similarities and dissimilarities, i.e., patterns of varying relative significance, between any two such tensors. We demonstrate the tensor GSVD in comparative modeling of patient- and platform-matched but probe-independent ovarian serous cystadenocarcinoma (OV) tumor, mostly high-grade, and normal DNA copy-number profiles, across each chromosome arm, and combination of two arms, separately. The modeling uncovers previously unrecognized patterns of tumor-exclusive platform-consistent co-occurring copy-number alterations (CNAs). We find, first, and validate that each of the patterns across only 7p and Xq, and the combination of 6p+12p, is correlated with a patient's prognosis, is independent of the tumor's stage, the best predictor of OV survival to date, and together with stage makes a better predictor than stage alone. Second, these patterns include most known OV-associated CNAs that map to these chromosome arms, as well as several previously unreported, yet frequent focal CNAs. Third, differential mRNA, microRNA, and protein expression consistently map to the DNA CNAs. A coherent picture emerges for each pattern, suggesting roles for the CNAs in OV pathogenesis and personalized therapy. In 6p+12p, deletion of the p21-encoding CDKN1A and p38-encoding MAPK14 and amplification of RAD51AP1 and KRAS encode for human cell transformation, and are correlated with a cell's immortality, and a patient's shorter survival time. In 7p, RPA3 deletion and POLD2 amplification are correlated with DNA stability, and a longer survival. In Xq, PABPC5

  20. Actinophage R4 integrase-based site-specific chromosomal integration of non-replicative closed circular DNA.

    PubMed

    Miura, Takamasa; Nishizawa, Akito; Nishizawa, Tomoyasu; Asayama, Munehiko; Shirai, Makoto

    2016-06-01

    The actinophage R4 integrase (Sre)-based molecular genetic engineering system was developed for the chromosomal integration of multiple genes in Escherichia coli. A cloned DNA fragment containing two attP sites, green fluorescent protein (gfp) as a first transgene, and an antibiotic resistance gene as a selection marker was self-ligated to generate non-replicative closed circular DNA (nrccDNA) for integration. nrccDNA was introduced into attB-inserted E. coli cells harboring the plasmid expressing Sre by electroporation. The expressed Sre catalyzed site-specific integration between one of the two attP sites on nrccDNA and the attB site on the E. coli chromosome. The integration frequency was affected by the chromosomal location of the target site. A second nrccDNA containing two attB sites, lacZα encoding the alpha fragment of β-galactosidase as a transgene, and another antibiotic resistance gene was integrated into the residual attP site on the gfp-integrated E. coli chromosome via one of the two attB sites according to reiterating site-specific recombination. The integrants clearly exhibited β-galactosidase activity and green fluorescence, suggesting the simultaneous expression of multiple recombinant proteins in E. coli. The results of the present study showed that a step-by-step integration procedure using nrccDNA achieved the chromosomal integration of multiple genes. PMID:26870903

  1. 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. PMID:26090928

  2. MitBASE : a comprehensive and integrated mitochondrial DNA database. The present status

    PubMed Central

    Attimonelli, M.; Altamura, N.; Benne, R.; Brennicke, A.; Cooper, J. M.; D’Elia, D.; Montalvo, A. de; Pinto, B. de; De Robertis, M.; Golik, P.; Knoop, V.; Lanave, C.; Lazowska, J.; Licciulli, F.; Malladi, B. S.; Memeo, F.; Monnerot, M.; Pasimeni, R.; Pilbout, S.; Schapira, A. H. V.; Sloof, P.; Saccone, C.

    2000-01-01

    MitBASE is an integrated and comprehensive database of mitochondrial DNA data which collects, under a single interface, databases for Plant, Vertebrate, Invertebrate, Human, Protist and Fungal mtDNA and a Pilot database on nuclear genes involved in mitochondrial biogenesis in Saccharomyces cerevisiae. MitBASE reports all available information from different organisms and from intraspecies variants and mutants. Data have been drawn from the primary databases and from the literature; value adding information has been structured, e.g., editing information on protist mtDNA genomes, pathological information for human mtDNA variants, etc. The different databases, some of which are structured using commercial packages (Microsoft Access, File Maker Pro) while others use a flat-file format, have been integrated under ORACLE. Ad hoc retrieval systems have been devised for some of the above listed databases keeping into account their peculiarities. The database is resident at the EBI and is available at the following site: http://www3.ebi.ac.uk/Research/Mitbase/mitbase.pl . The impact of this project is intended for both basic and applied research. The study of mitochondrial genetic diseases and mitochondrial DNA intraspecies diversity are key topics in several biotechnological fields. The database has been funded within the EU Biotechnology programme. PMID:10592207

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

    PubMed

    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

  4. 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

  5. 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

  6. Integrative DNA methylation and gene expression analysis in high-grade soft tissue sarcomas

    PubMed Central

    2013-01-01

    Background High-grade soft tissue sarcomas are a heterogeneous, complex group of aggressive malignant tumors showing mesenchymal differentiation. Recently, soft tissue sarcomas have increasingly been classified on the basis of underlying genetic alterations; however, the role of aberrant DNA methylation in these tumors is not well understood and, consequently, the usefulness of methylation-based classification is unclear. Results We used the Infinium HumanMethylation27 platform to profile DNA methylation in 80 primary, untreated high-grade soft tissue sarcomas, representing eight relevant subtypes, two non-neoplastic fat samples and 14 representative sarcoma cell lines. The primary samples were partitioned into seven stable clusters. A classification algorithm identified 216 CpG sites, mapping to 246 genes, showing different degrees of DNA methylation between these seven groups. The differences between the clusters were best represented by a set of eight CpG sites located in the genes SPEG, NNAT, FBLN2, PYROXD2, ZNF217, COL14A1, DMRT2 and CDKN2A. By integrating DNA methylation and mRNA expression data, we identified 27 genes showing negative and three genes showing positive correlation. Compared with non-neoplastic fat, NNAT showed DNA hypomethylation and inverse gene expression in myxoid liposarcomas, and DNA hypermethylation and inverse gene expression in dedifferentiated and pleomorphic liposarcomas. Recovery of NNAT in a hypermethylated myxoid liposarcoma cell line decreased cell migration and viability. Conclusions Our analysis represents the first comprehensive integration of DNA methylation and transcriptional data in primary high-grade soft tissue sarcomas. We propose novel biomarkers and genes relevant for pathogenesis, including NNAT as a potential tumor suppressor in myxoid liposarcomas. PMID:24345474

  7. 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

  8. 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

  9. 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. PMID:24114494

  10. Cellular integrity is required for inhibition of initiation of cellular DNA synthesis by reovirus type 3.

    PubMed Central

    Roner, M R; Cox, D C

    1985-01-01

    Synchronized HeLa cells, primed for entry into the synthesis phase by amethopterin, were prevented from initiating DNA synthesis 9 h after infection with reovirus type 3. However, nuclei isolated from synchronized cells infected with reovirus for 9 or 16 h demonstrated a restored ability to synthesize DNA. The addition of enucleated cytoplasmic extracts from infected or uninfected cells did not affect this restored capacity for synthesis. The addition of ribonucleotide triphosphates to nuclei isolated from infected cells stimulated additional DNA synthesis, suggesting that these nuclei were competent to initiate new rounds of DNA replication. Permeabilization of infected cells did not restore the ability of these cells to synthesize DNA. Nucleoids isolated from intact or permeabilized cells, infected for 9 or 16 h displayed an increased rate of sedimentation when compared with nucleoids isolated from uninfected cells. Nucleoids isolated from the nuclei of infected cells demonstrated a rate of sedimentation similar to that of nucleoids isolated from the nuclei of uninfected cells. The inhibition of initiation of cellular DNA synthesis by reovirus type 3 appears not to have been due to a permanent alteration of the replication complex, but this inhibition could be reversed by the removal of that complex from factors unique to the structural or metabolic integrity of the infected cell. Images PMID:3968718

  11. 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. PMID:26485090

  12. Borrowing nuclear DNA helicases to protect mitochondrial DNA.

    PubMed

    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

  13. 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 Central

    Snowdin, Jacob W.; Hsiung, Chia-Heng; Kesterson, Daniel G.; Kamath, Vasudeva G.

    2015-01-01

    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. PMID:26248377

  14. 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

  15. Urine Cell-Free DNA Integrity as a Marker for Early Prostate Cancer Diagnosis: A Pilot Study

    PubMed Central

    Casadio, Valentina; Calistri, Daniele; Salvi, Samanta; Gunelli, Roberta; Carretta, Elisa; Amadori, Dino; Silvestrini, Rosella; Zoli, Wainer

    2013-01-01

    Circulating cell-free DNA has been recognized as an accurate marker for the diagnosis of prostate cancer, whereas the role of urine cell-free DNA (UCF DNA) has never been evaluated in this setting. It is known that normal apoptotic cells produce highly fragmented DNA while cancer cells release longer DNA. We thus verified the potential role of UCF DNA integrity for early prostate cancer diagnosis. UCF DNA was isolated from 29 prostate cancer patients and 25 healthy volunteers. Sequences longer than 250 bp (c-Myc, BCAS1, and HER2) were quantified by real-time PCR to verify UCF DNA integrity. Receiver operating characteristic (ROC) curve analysis revealed an area under the curve of 0.7959 (95% CI 0.6729–0.9188). At the best cut-off value of 0.04 ng/μL, UCF DNA integrity analysis showed a sensitivity of 0.79 (95% CI 0.62–0.90) and a specificity of 0.84 (95% CI 0.65–0.94). These preliminary findings indicate that UCF DNA integrity could be a promising noninvasive marker for the early diagnosis of prostate cancer and pave the way for further research into this area. PMID:23509700

  16. Integration of hepatitis B virus DNA in chromosome-specific satellite sequences

    SciTech Connect

    Shaul, Y.; Garcia, P.D.; Schonberg, S.; Rutter, W.J.

    1986-09-01

    The authors previously reported the cloning and detailed analysis of the integrated hepatitis B virus sequences in a human hepatoma cell line. They report here the integration of at least one of hepatitis B virus at human satellite DNA sequences. The majority of the cellular sequences identified by this satellite were organized as a multimeric composition of a 0.6-kilobase EcoRI fragment. This clone hybridized in situ almost exclusively to the centromeric heterochromatin of chromosomes 1 and 16 and to a lower extent to chromosome 2 and to the heterochromatic region of the Y chromosome. The immediate flanking host sequence appeared as a hierarchy of repeating units which were almost identical to a previously reported human satellite III DNA sequence.

  17. Integrated biochip for PCR-based DNA amplification and detection on capacitive biosensors

    NASA Astrophysics Data System (ADS)

    Moschou, D.; Vourdas, N.; Filippidou, M. K.; Tsouti, V.; Kokkoris, G.; Tsekenis, G.; Zergioti, I.; Chatzandroulis, S.; Tserepi, A.

    2013-05-01

    Responding to an increasing demand for LoC devices to perform bioanalytical protocols for disease diagnostics, the development of an integrated LoC device consisting of a μPCR module integrated with resistive microheaters and a biosensor array for disease diagnostics is presented. The LoC is built on a Printed Circuit Board (PCB) platform, implementing both the amplification of DNA samples and DNA detection/identification on-chip. The resistive microheaters for PCR and the wirings for the sensor read-out are fabricated by means of standard PCB technology. The microfluidic network is continuous-flow, designed to perform 30 PCR cycles with heated zones at constant temperatures, and is built onto the PCB utilizing commercial photopatternable polyimide layers. Following DNA amplification, the product is driven in a chamber where a Si-based biosensor array is placed for DNA detection through hybridization. The sensor array is tested for the detection of mutations of the KRAS gene, responsible for colon cancer.

  18. Gene copy number and cell cycle arrest

    NASA Astrophysics Data System (ADS)

    Ghosh, Bhaswar; Bose, Indrani

    2006-03-01

    The cell cycle is an orderly sequence of events which ultimately lead to the division of a single cell into two daughter cells. In the case of DNA damage by radiation or chemicals, the damage checkpoints in the G1 and G2 phases of the cell cycle are activated. This results in an arrest of the cell cycle so that the DNA damage can be repaired. Once this is done, the cell continues with its usual cycle of activity. We study a mathematical model of the DNA damage checkpoint in the G2 phase which arrests the transition from the G2 to the M (mitotic) phase of the cell cycle. The tumor suppressor protein p53 plays a key role in activating the pathways leading to cell cycle arrest in mammalian systems. If the DNA damage is severe, the p53 proteins activate other pathways which bring about apoptosis, i.e., programmed cell death. Loss of the p53 gene results in the proliferation of cells containing damaged DNA, i.e., in the growth of tumors which may ultimately become cancerous. There is some recent experimental evidence which suggests that the mutation of a single copy of the p53 gene (in the normal cell each gene has two identical copies) is sufficient to trigger the formation of tumors. We study the effect of reducing the gene copy number of the p53 and two other genes on cell cycle arrest and obtain results consistent with experimental observations.

  19. Integrated on-line system for DNA sequencing by capillary electrophoresis: From template to called bases

    SciTech Connect

    Ton, H.; Yeung, E.S.

    1997-02-15

    An integrated on-line prototype for coupling a microreactor to capillary electrophoresis for DNA sequencing has been demonstrated. A dye-labeled terminator cycle-sequencing reaction is performed in a fused-silica capillary. Subsequently, the sequencing ladder is directly injected into a size-exclusion chromatographic column operated at nearly 95{degree}C for purification. On-line injection to a capillary for electrophoresis is accomplished at a junction set at nearly 70{degree}C. High temperature at the purification column and injection junction prevents the renaturation of DNA fragments during on-line transfer without affecting the separation. The high solubility of DNA in and the relatively low ionic strength of 1 x TE buffer permit both effective purification and electrokinetic injection of the DNA sample. The system is compatible with highly efficient separations by a replaceable poly(ethylene oxide) polymer solution in uncoated capillary tubes. Future automation and adaptation to a multiple-capillary array system should allow high-speed, high-throughput DNA sequencing from templates to called bases in one step. 32 refs., 5 figs.

  20. 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

  1. Study on optics integrated manufacture technology based on Windows DNA-OM

    NASA Astrophysics Data System (ADS)

    Yu, Min; Yang, Li; Wang, Yong-jan

    2006-02-01

    As the shortcoming and insufficiency of CNC systems applied in optics manufacturing, a design and application based on Windows DNA-OM is presented. After introducing the prime principles and logic structures of Windows DNA-OM, a specific solution of optics integrated manufacture based on this framework is discussed in detail. In this solution, the OPC(OLE for Process Control) technology based on COM(Component Object Model) of Microsoft company is used for CNC equipment in the workshop, and this could provide a general interface for communication of hetero-structure CNC equipment of workshop and so all hetero-structure CNC can be accessed in a simple way. The experience and knowledge of optic manufacturing is stored and integrated in Windows DNA-OM Data part, this data part can guide optics manufacture. By completing this data part, an optics manufacture expert system can be realized. Then the prime characteristics of the integrated manufacture system are given. Finally this system is proved to has a good flexibility and opening, ease realizing, low cost and high feasibility.

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

    PubMed

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

    2015-10-01

    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

  3. 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. PMID:26854716

  4. DNA Storage under High Temperature Conditions Does Not Affect Performance in Human Leukocyte Antigen Genotyping via Next-Generation Sequencing (DNA Integrity Maintained in Extreme Conditions)

    PubMed Central

    McDevitt, Shana L; Hogan, Michael E; Pappas, Derek J; Wong, Lily Y

    2014-01-01

    Background: Stable dry-state storage of DNA is desirable to minimize required storage space and to reduce electrical and shipping costs. DNA purified from various commercially available dry-state stabilization matrices has been used successfully in downstream molecular applications (e.g., quantitative polymerase chain reaction [qPCR], microarray, and sequence-based genotyping). However, standard DNA storage conditions still include freezing of DNA eluted in aqueous buffers or nuclease-free water. Broad implementation of dry-state, long-term DNA storage requires enhancement of such dry-state DNA stabilization products to control for temperature fluctuations at specimen collection, transit, and storage. This study tested the integrity of genomic DNA subjected to long-term storage on GenTegra™ DNA stabilization matrices (GenTegra LLC, Pleasanton, CA) at extreme conditions, as defined by a 4-year storage period at ambient temperature with an initial incubation for 7 months at 37°C, 56°C, or ambient temperature. Subsequently, purified DNA performance and integrity were measured by qPCR and next-generation sequencing (NGS)-based human leokocyte antigen (HLA) genotyping. Results: High molecular weight genomic DNA samples were recovered from the GenTegra product matrix and exhibited integrity comparable to a highly characterized commercial standard under assessment by qPCR. Samples were genotyped for classical HLA loci using next generation sequencing-based methodolgy on the Roche 454 GS Junior instrument. Amplification efficiency, sequence coverage, and sequence quality were all comparable with those produced from a cell line DNA sequenced as a control. No significant differences were observed in the mean, median, or mode quality scores between samples and controls (p≥0.4). Conclusions: Next generation HLA genotyping was chosen to test the integrity of GenTegra-treated genomic DNA due to the requirment for long sequence reads to genotype the highly polymorphic

  5. Impact of a novel homozygous mutation in nicotinamide nucleotide transhydrogenase on mitochondrial DNA integrity in a case of familial glucocorticoid deficiency

    PubMed Central

    Fujisawa, Yasuko; Napoli, Eleonora; Wong, Sarah; Song, Gyu; Yamaguchi, Rie; Matsui, Toshiharu; Nagasaki, Keisuke; Ogata, Tsutomu; Giulivi, Cecilia

    2014-01-01

    Background Familial glucocorticoid deficiency (FGD) is a rare autosomal recessive disorder that is characterized by isolated glucocorticoid deficiency. Recently, mutations in the gene encoding for the mitochondrial nicotinamide nucleotide transhydrogenase (NNT) have been identified as a causative gene for FGD; however, no NNT activities have been reported in FGD patients carrying NNT mutations. Methods Clinical, biochemical and molecular analyses of lymphocytes from FDG homozygous and heterozygous carriers for the F215S NNT mutation were performed. Results In this study, we described an FGD-affected Japanese patient carrying a novel NNT homozygous mutation (c.644T>C; F215S) with a significant loss-of-function (NNT activity = 31% of healthy controls) in peripheral blood cells' mitochondria. The NNT activities of the parents, heterozygous for the mutation, were 61% of the controls. Conclusions Our results indicated that (i) mitochondrial biogenesis (citrate synthase activity) and/or mtDNA replication (mtDNA copy number) were affected at ≤ 60% NNT activity because these parameters were affected in individuals carrying either one or both mutated alleles; and (ii) other outcomes (mtDNA deletions, protein tyrosine nitration, OXPHOS capacity) were affected at ≤ 30% NNT activity as also observed in murine cerebellar mitochondria from C57BL/6J (NNT−/−) vs. C57BL/6JN (NNT+/+) substrains. General significance By studying a family affected with a novel point mutation in the NNT gene, a gene–dose response was found for various mitochondrial outcomes providing for novel insights into the role of NNT in the maintenance of mtDNA integrity beyond that described for preventing oxidative stress. PMID:26309815

  6. Resistance of Spiroplasma citri Lines to the Virus SVTS2 Is Associated with Integration of Viral DNA Sequences into Host Chromosomal and Extrachromosomal DNA

    PubMed Central

    Sha, Y.; Melcher, U.; Davis, R. E.; Fletcher, J.

    1995-01-01

    Spiroplasmavirus SVTS2, isolated from Spiroplasma melliferum TS2, produces plaques when inoculated onto lawns of Spiroplasma citri M200H, a derivative of the type strain Maroc R8A2. S. citri strains MR2 and MR3, originally selected as colonies growing within plaques on a lawn of M200H inoculated with SVTS2, were resistant to SVTS2. Genomic DNA fingerprints and electrophoretic protein profiles of M200H, MR2, and MR3 were similar, but three proteins present in M200H were missing or significantly reduced in both resistant lines. None of these three polypeptides reacted with antiserum against S. citri membrane proteins, indicating that they probably are not surface-located virus receptors. Electroporation with SVTS2 DNA produced 1.5 x 10(sup5) transfectants per (mu)g of DNA in M200H but none in MR2 or MR3, suggesting that resistance may result from inhibition of viral replication. The digestion patterns of the extrachromosomal double-stranded (ds) DNA of these lines were similar. Three TaqI fragments of MR2 extrachromosomal DNA that were not present in M200H extrachromosomal DNA hybridized strongly to an SVTS2 probe, and two of these fragments plus an additional one hybridized with the MR3 extrachromosomal DNA, indicating that a fragment of SVTS2 DNA was present in the extrachromosomal ds DNA of MR2 and MR3 but not of M200H. When the restricted genomes of all three lines were probed with SVTS2 DNA, strong hybridization to two EcoRI fragments of chromosomal MR2 and MR3 DNA but not M200H DNA indicated that SVTS2 DNA had integrated into the genomes of MR2 and MR3 but not of M200H. When MR3 extrachromosomal ds DNA containing a 2.1-kb SVTS2 DNA fragment was transfected into M200H, the transformed spiroplasmas were resistant to SVTS2. These results suggest that SVTS2 DNA fragments, possibly integrated into the chromosomal or extrachromosomal DNA of a previously susceptible spiroplasma, may function as viral incompatibility elements, providing resistance to superinfection by

  7. 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

  8. Mitochondrial copy number and risk of breast cancer: a pilot study.

    PubMed

    Shen, Jie; Platek, Mary; Mahasneh, Amjad; Ambrosone, Christine B; Zhao, Hua

    2010-01-01

    It has been proposed that the copy number of mitochondria DNA (mtDNA) per cell reflects gene-environment interactions between unknown hereditary factors and exposures affecting levels of oxidative stress. However, whether copy number of mtDNA could be a risk predictor of oxidative stress-related human cancers, such as breast cancer, remains to be determined. To explore the role of mtDNA copy number in breast cancer etiology, we analyzed mtDNA copy number in whole blood from 103 patients with breast cancer and 103 matched control subjects and examined in relation to endogenous antioxidants. Case patients with breast cancer had a statistically significantly higher mtDNA copy number than control subjects (median: 1.29 vs. 0.80, P<0.01). High mtDNA copy number (above the median in controls) was associated with a statistically significantly increased risk of breast cancer, compared with low copy number (Odds ratio (OR)=4.67, 95% CI: 2.45-8.92), with a statistically significant dose-response relationship in trend analysis (P<0.01). Moreover, mtDNA copy number was significantly inversely associated with several important endogenous oxidants and antioxidants in blood in either the cases (total glutathione, CuZn-SOD activity and myeloperoxidase (MPO)) or the controls (catalase (CAT) activity). These results suggest the mtDNA copy number could be associated with risk of breast cancer, perhaps through an oxidative stress mechanism. PMID:19788937

  9. Mutational analysis of the transcription factor IIIB-DNA target of Ty3 retroelement integration.

    PubMed

    Yieh, Lynn; Hatzis, Heather; Kassavetis, George; Sandmeyer, Suzanne B

    2002-07-19

    The Ty3 retrovirus-like element inserts preferentially at the transcription initiation sites of genes transcribed by RNA polymerase III. The requirements for transcription factor (TF) IIIC and TFIIIB in Ty3 integration into the two initiation sites of the U6 gene carried on pU6LboxB were previously examined. Ty3 integrates at low but detectable frequencies in the presence of TFIIIB subunits Brf1 and TATA-binding protein. Integration increases in the presence of the third subunit, Bdp1. TFIIIC is not essential, but the presence of TFIIIC specifies an orientation of TFIIIB for transcriptional initiation and directs integration to the U6 gene-proximal initiation site. In the current study, recombinant wild type TATA-binding protein, wild type and mutant Brf1, and Bdp1 proteins and highly purified TFIIIC were used to investigate the roles of specific protein domains in Ty3 integration. The amino-terminal half of Brf1, which contains a TFIIB-like repeat, contributed more strongly than the carboxyl-terminal half of Brf1 to Ty3 targeting. Each half of Bdp1 split at amino acid 352 enhanced integration. In the presence of TFIIIB and TFIIIC, the pattern of integration extended downstream by several base pairs compared with the pattern observed in vitro in the absence of TFIIIC and in vivo, suggesting that TFIIIC may not be present on genes targeted by Ty3 in vivo. Mutations in Bdp1 that affect its interaction with TFIIIC resulted in TFIIIC-independent patterns of Ty3 integration. Brf1 zinc ribbon and Bdp1 internal deletion mutants that are competent for polymerase III recruitment but defective in promoter opening were competent for Ty3 integration irrespective of the state of DNA supercoiling. These results extend the similarities between the TFIIIB domains required for transcription and Ty3 integration and also reveal requirements that are specific to transcription. PMID:11994300

  10. 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

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

    PubMed

    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; Levine, Ross L

    2016-06-16

    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

  12. 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. PMID:20133967

  13. 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. PMID:26257869

  14. 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

  15. Micromachined pipettes integrated in a flow channel for single DNA molecule study by optical trapping

    NASA Astrophysics Data System (ADS)

    Rusu, Cristina R.; van't Oever, Ronny; de Boer, Meint J.; Jansen, Henri V.; Berenschot, Erwin; Elwenspoek, Miko C.; Bennink, Martin L.; Kanger, Johannes S.; de Grooth, Bart G.; Greve, Jan; Brugger, Juergen P.; van den Berg, Albert

    2000-03-01

    We have developed a micromachined flow cell consisting of a flow channel integrated with micropipettes. The flow cell is used in combination with an optical trap set-up (optical tweezers) to study mechanical and structural properties of (lambda) -DNA molecules. The flow cell was realized using silicon micromachining including the so-called buried channel technology to fabricate the micropipettes, the wet etching of glass to create the flow channel, and the powder blasting of glass to create the fluid connections. The volume of the flow cell is 2 (mu) l. The pipettes have a length of 130 micrometer, a width of 5 - 10 micrometer, a round opening of 1 micron and can be processed with different shapes. Using this flow cell we stretched single molecules ((lambda) -DNA) showing typical force-extension curves also found with conventional techniques.

  16. DNAzymes in DNA Nanomachines and DNA Analysis

    NASA Astrophysics Data System (ADS)

    He, Yu; Tian, Ye; Chen, Yi; Mao, Chengde

    This chapter discusses our efforts in using DNAzymes in DNA nano-machines and DNA analysis systems. 10-23 DNAzymes can cleave specific phos-phodiester bonds in RNA. We use them to construct an autonomous DNA-RNA chimera nanomotor, which constantly extracts chemical energy from RNA substrates and transduces the energy into a mechanical motion: cycles of contraction and extension. The motor's motion can be reversibly turned on and off by a DNA analogue (brake) of the RNA substrate. Addition and removal of the brake stops and restarts, respectively, the motor's motion. Furthermore, when the RNA substrates are preorganized into a one-dimensional track, a DNAzyme can continuously move along the track so long as there are substrates available ahead. Based on a similar mechanism, a novel DNA detection system has been developed. A target DNA activates a DNAzyme to cleave RNA-containing molecular beacons (MB), which generates an enhanced fluorescence signal. A following work integrates two steps of signal amplifications: a rolling-circle amplification (RCA) to synthesize multiple copies of DNAzymes, and the DNAzymes catalyze a chemical reaction to generate a colorimetric signal. This method allows detection of DNA analytes whose concentration is as low as 1 pM.

  17. Embryonic stem cells or induced pluripotent stem cells? A DNA integrity perspective

    PubMed Central

    Bai, Qiang; Desprat, Romain; Klein, Bernard; Lemaitre, Jean-Marc; De Vos, John

    2013-01-01

    Induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) are two types of pluripotent stem cells that hold great promise for biomedical research and medical applications. iPSCs were initially favorably compared to ESCs. This view was first based on ethical arguments (the generation of iPSCs does not require the destruction of an embryo) and on immunological reasons (it is easier to derive patient HLA-matched iPSCs than ESCs). However, several reports suggest that iPSCs might be characterized by higher occurrence of epigenetic and genetic aberrations than ESCs as a consequence of the reprogramming process. We focus here on the DNA integrity of pluripotent stem cells and examine the three main sources of genomic abnormalities in iPSCs: (1) genomic variety of the parental cells, (2) cell reprogramming, and (3) in vitro cell culture. Recent reports claim that it is possible to generate mouse or human iPSC lines with a mutation level similar to that of the parental cells, suggesting that “genome-friendly” reprogramming techniques can be developed. The issue of iPSC DNA integrity clearly highlights the crucial need of guidelines to define the acceptable level of genomic integrity of pluripotent stem cells for biomedical applications. We discuss here the main issues that such guidelines should address. PMID:23317057

  18. 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

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

    PubMed

    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

  20. Copying could be costly.

    PubMed

    Allen, A

    1992-02-01

    Thanks to modern technology, photocopying is quick, easy, and inexpensive. Or is it? One of the largest awards ever made for copyright infringement was levied in a case involving the use of material copied from publications without permission. This article presents a short discussion about US and international copyright protection, fair use, and work for hire. Suggestions for additional information are offered. PMID:1735873

  1. Creating Single-Copy Genetic Circuits.

    PubMed

    Lee, Jeong Wook; Gyorgy, Andras; Cameron, D Ewen; Pyenson, Nora; Choi, Kyeong Rok; Way, Jeffrey C; Silver, Pamela A; Del Vecchio, Domitilla; Collins, James J

    2016-07-21

    Synthetic biology is increasingly used to develop sophisticated living devices for basic and applied research. Many of these genetic devices are engineered using multi-copy plasmids, but as the field progresses from proof-of-principle demonstrations to practical applications, it is important to develop single-copy synthetic modules that minimize consumption of cellular resources and can be stably maintained as genomic integrants. Here we use empirical design, mathematical modeling, and iterative construction and testing to build single-copy, bistable toggle switches with improved performance and reduced metabolic load that can be stably integrated into the host genome. Deterministic and stochastic models led us to focus on basal transcription to optimize circuit performance and helped to explain the resulting circuit robustness across a large range of component expression levels. The design parameters developed here provide important guidance for future efforts to convert functional multi-copy gene circuits into optimized single-copy circuits for practical, real-world use. PMID:27425413

  2. 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

  3. 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

  4. 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)

  5. Integrated solid-state nanopore platform for nanopore fabrication via dielectric breakdown, DNA-speed deceleration and noise reduction.

    PubMed

    Goto, Yusuke; Yanagi, Itaru; Matsui, Kazuma; Yokoi, Takahide; Takeda, Ken-Ichi

    2016-01-01

    The practical use of solid-state nanopores for DNA sequencing requires easy fabrication of the nanopores, reduction of the DNA movement speed and reduction of the ionic current noise. Here, we report an integrated nanopore platform with a nanobead structure that decelerates DNA movement and an insulating polyimide layer that reduces noise. To enable rapid nanopore fabrication, we introduced a controlled dielectric breakdown (CDB) process into our system. DNA translocation experiments revealed that single nanopores were created by the CDB process without sacrificing performance in reducing DNA movement speed by up to 10 μs/base or reducing noise up to 600 pArms at 1 MHz. Our platform provides the essential components for proceeding to the next step in the process of DNA sequencing. PMID:27499264

  6. Identification of regulators of the innate immune response to cytosolic DNA and retroviral infection by an integrative approach

    PubMed Central

    Lee, Mark N; Roy, Matthew; Ong, Shao-En; Mertins, Philipp; Villani, Alexandra-Chloé; Li, Weibo; Dotiwala, Farokh; Sen, Jayita; Doench, John G; Orzalli, Megan H; Kramnik, Igor; Knipe, David M; Lieberman, Judy; Carr, Steven A; Hacohen, Nir

    2013-01-01

    The innate immune system senses viral DNA that enters mammalian cells, or in aberrant situations self-DNA, and triggers type I interferon production. Here we present an integrative approach that combines quantitative proteomics, genomics and small molecule perturbations to identify genes involved in this pathway. We silenced 809 candidate genes, measured the response to dsDNA and connected resulting hits with the known signaling network. We identified ABCF1 as a critical protein that associates with dsDNA and the DNA-sensing components HMGB2 and IFI204. We also found that CDC37 regulates the stability of the signaling molecule TBK1 and that chemical inhibition of the CDC37-HSP90 interaction and several other pathway regulators potently modulates the innate immune response to DNA and retroviral infection. PMID:23263557

  7. Integrated solid-state nanopore platform for nanopore fabrication via dielectric breakdown, DNA-speed deceleration and noise reduction

    PubMed Central

    Goto, Yusuke; Yanagi, Itaru; Matsui, Kazuma; Yokoi, Takahide; Takeda, Ken-ichi

    2016-01-01

    The practical use of solid-state nanopores for DNA sequencing requires easy fabrication of the nanopores, reduction of the DNA movement speed and reduction of the ionic current noise. Here, we report an integrated nanopore platform with a nanobead structure that decelerates DNA movement and an insulating polyimide layer that reduces noise. To enable rapid nanopore fabrication, we introduced a controlled dielectric breakdown (CDB) process into our system. DNA translocation experiments revealed that single nanopores were created by the CDB process without sacrificing performance in reducing DNA movement speed by up to 10 μs/base or reducing noise up to 600 pArms at 1 MHz. Our platform provides the essential components for proceeding to the next step in the process of DNA sequencing. PMID:27499264

  8. Integrated solid-state nanopore platform for nanopore fabrication via dielectric breakdown, DNA-speed deceleration and noise reduction

    NASA Astrophysics Data System (ADS)

    Goto, Yusuke; Yanagi, Itaru; Matsui, Kazuma; Yokoi, Takahide; Takeda, Ken-Ichi

    2016-08-01

    The practical use of solid-state nanopores for DNA sequencing requires easy fabrication of the nanopores, reduction of the DNA movement speed and reduction of the ionic current noise. Here, we report an integrated nanopore platform with a nanobead structure that decelerates DNA movement and an insulating polyimide layer that reduces noise. To enable rapid nanopore fabrication, we introduced a controlled dielectric breakdown (CDB) process into our system. DNA translocation experiments revealed that single nanopores were created by the CDB process without sacrificing performance in reducing DNA movement speed by up to 10 μs/base or reducing noise up to 600 pArms at 1 MHz. Our platform provides the essential components for proceeding to the next step in the process of DNA sequencing.

  9. 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. 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

  11. 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

  12. 69. Photographic copy of blueprint copy of original elevations drawing ...

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

    69. Photographic copy of blueprint copy of original elevations drawing (C. Howard Crane, Kenneth Franzheim, architects, Chicago: sheet $6, 1922). Blueprint copy located at William J. Bachman & Partners (Architects)), 5930 Hohman Avenue, Hammond, Indiana 46230, (219) 932-6006. - Indiana Hotel, 5116 Hohman Avenue, Hammond, Lake County, IN

  13. 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

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

    PubMed

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

    2015-12-01

    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

  15. Sperm chromatin condensation, DNA integrity, and apoptosis in men with spinal cord injury

    PubMed Central

    Talebi, Ali Reza; Khalili, Mohammad Ali; Vahidi, Serajodin; Ghasemzadeh, Jalal; Tabibnejad, Nasim

    2013-01-01

    Objectives To evaluate the effect of cord injury on (1) sperm parameters and (2) DNA chromatin status. Design Case–control study. Setting Data were collected from men referred to Research and Clinical Center for Infertility, Yazd, Iran. Participants Thirty infertile men with the presence of any level of spinal cord injury (SCI) were compared with 30 healthy donors with definite fertility and normal sperm parameters. Interventions Not applicable. Outcome measures Sperm chromatin integrity was assessed using aniline blue (AB), chromomycin A3 (CMA3), toluidine blue (TB), and acridine orange (AO) assays. The rate of apoptotic spermatozoa was evaluated with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) staining. Results Sperm concentration, motility, and morphology in men with SCI were significantly decreased compared with control group (P < 0.05). In addition, with regard to cytochemical staining and TUNEL test, the rate of reacted spermatozoa was increased significantly in SCI group when compared with the controls (P < 0.05). The majority of AB, TB, AO, and CMA3-reacted spermatozoa were higher than the “cut-off” value in men with SCI, as were the number of apoptotic spermatozoa stained with TUNEL. Conclusion Results showed that SCI disturbs sperm parameters, nuclear maturity, and DNA integrity of spermatozoa. Therefore, the production of spermatozoa with less condensed chromatin and more apoptotic rate increases after cord injury and this may be one possible cause of infertility following SCI. PMID:23809529

  16. Outer domains of integrase within retroviral intasomes are dispensible for catalysis of DNA integration.

    PubMed

    Li, Min; Lin, Shiqiang; Craigie, Robert

    2016-02-01

    Retroviral DNA integration is mediated by nucleoprotein complexes (intasomes) comprising a pair of viral DNA ends synapsed by a tetramer of integrase. Current integrase inhibitors act on intasomes rather than free integrase protein. Structural and functional studies of intasomes are essential to understand their mechanism of action and how the virus can escape by mutation. To date, prototype foamy virus (PFV) is the only retrovirus for which high-resolution structures of intasomes have been determined. In the PFV intasome structure, only the core domains of the outer subunits are ordered; the N-terminal domain, C-terminal domain, and N-terminal extension domain are disordered. Are these "missing domains" required for function or are they dispensable? We have devised a strategy to assemble "hetero-intasomes" in which the outer domains are not present as a tool to assess the functional role of the missing domains for catalysis of integration. We find that the disordered domains of outer subunits are not required for intasome assembly or catalytic activity as catalytic core domains can substitute for the outer subunits in the case of both PFV and HIV-1 intasomes. PMID:26537415

  17. Integrative DNA, RNA, and Protein Evidence Connects TREML4 to Coronary Artery Calcification

    PubMed Central

    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.

    2014-01-01

    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. PMID:24975946

  18. HIV-1 Integrase-DNA Recognition Mechanisms

    PubMed Central

    Kessl, Jacques J.; McKee, Christopher J.; Eidahl, Jocelyn O.; Shkriabai, Nikolozi; Katz, Ari; Kvaratskhelia, Mamuka

    2009-01-01

    Integration of a reverse transcribed DNA copy of the HIV viral genome into the host chromosome is essential for virus replication. This process is catalyzed by the virally encoded protein integrase. The catalytic activities, which involve DNA cutting and joining steps, have been recapitulated in vitro using recombinant integrase and synthetic DNA substrates. Biochemical and biophysical studies of these model reactions have been pivotal in advancing our understanding of mechanistic details for how IN interacts with viral and target DNAs, and are the focus of the present review. PMID:21994566

  19. Copying Machine Improvement

    NASA Technical Reports Server (NTRS)

    1981-01-01

    Manufacturer of the Model 2210 copying machine was looking for a plastic valve bushing material that could be produced by a low-cost injection molding process to replace the unsuitable valve bushing they were using. NERAC conducted a computer search of the NASA database and was able to supply Nashua Corporation with several technical reports in their area of interest. Information aided the company's development of a urethane valve bushing which solved the problem and created a dramatic reduction in unit cost.

  20. A Simple Sperm DNA Toroid Integrity Test and Risk of Miscarriage

    PubMed Central

    Chan, Philip J.; Orzylowska, Eliza M.; Corselli, Johannah U.; Jacobson, John D.; Wei, Albert K.

    2015-01-01

    Current methods of analyzing sperm chromatin competency overlook the inner sperm compartment which is inaccessible to probes and reagents. By breaking the molecular protamine disulfide bridges, the DNA toroids are exposed to integrity analysis. The aim was to develop a simple nuclear toroid test and determine its association with fertilization, pregnancy, and miscarriage. The approach involved treating washed sperm remaining after ICSI procedures (N = 35 cases) with acidified Triton X-100 and dithiothreitol (DTT) before Diff-Quik staining. Percentages of sperm with normal chromatin indicated by light-colored nuclei were assessed. The toroid integrity test showed more sperm with normal chromatin in the pregnant group (73.6 ± 1.7%, mean ± SEM) when compared with the miscarriage (51.2 ± 6.6%) or nonpregnant groups (60.9 ± 4.8%). Furthermore, the toroid results were correlated with ICSI fertilization (R = 0.32, P = 0.04) and pregnancy outcome (pregnant cases 73.6 ± 1.7% versus nonpregnant 58.0 ± 3.9%, P = 0.001). ROC calculated cut-off was >70.0% for normal toroid integrity (sensitivity 0.98, specificity 0.33, and diagnostic accuracy 78.3%). An association between normal sperm toroid integrity and miscarriage was evident when the staining procedure included acidified detergent DTT pretreatment. PMID:25649376

  1. Chronic oxidative stress increases the integration frequency of foreign DNA and human papillomavirus 16 in human keratinocytes

    PubMed Central

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

    2016-01-01

    Cervical cancer is the second most common cancer, and the fourth most common cause of cancer death in women worldwide. Nearly all of these cases are caused by high-risk HPVs (HR HPVs), of which HPV16 is the most prevalent type. In most cervical cancer specimens, HR HPVs are found integrated into the human genome, indicating that integration is a key event in cervical tumor development. An understanding of the mechanisms that promote integration may therefore represent a unique opportunity to intercept carcinogenesis. To begin identifying these mechanisms, we tested the hypothesis that chronic oxidative stress (OS) induced by virus- and environmentallymediated factors can induce DNA damage, and thereby increase the frequency with which HPV integrates into the host genome. We found that virus-mediated factors are likely involved, as expression of E6*, a splice isoform of HPV16 E6, increased the levels of reactive oxygen species (ROS), caused oxidative DNA damage, and increased the frequency of plasmid DNA integration as assessed by colony formation assays. To assess the influence of environmentally induced chronic OS, we used L-Buthionine-sulfoximine (BSO) to lower the level of the intracellular antioxidant glutathione. Similar to our observations with E6*, glutathione depletion by BSO also increased ROS levels, caused oxidative DNA damage and increased the integration frequency of plasmid DNA. Finally, under conditions of chronic OS, we were able to induce and characterize a few independent events in which episomal HPV16 integrated into the host genome of cervical keratinocytes. Our results support a chain of events leading from induction of oxidative stress, to DNA damage, to viral integration, and ultimately to carcinogenesis. PMID:27186429

  2. Chronic oxidative stress increases the integration frequency of foreign DNA and human papillomavirus 16 in human keratinocytes.

    PubMed

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

    2016-01-01

    Cervical cancer is the second most common cancer, and the fourth most common cause of cancer death in women worldwide. Nearly all of these cases are caused by high-risk HPVs (HR HPVs), of which HPV16 is the most prevalent type. In most cervical cancer specimens, HR HPVs are found integrated into the human genome, indicating that integration is a key event in cervical tumor development. An understanding of the mechanisms that promote integration may therefore represent a unique opportunity to intercept carcinogenesis. To begin identifying these mechanisms, we tested the hypothesis that chronic oxidative stress (OS) induced by virus- and environmentallymediated factors can induce DNA damage, and thereby increase the frequency with which HPV integrates into the host genome. We found that virus-mediated factors are likely involved, as expression of E6*, a splice isoform of HPV16 E6, increased the levels of reactive oxygen species (ROS), caused oxidative DNA damage, and increased the frequency of plasmid DNA integration as assessed by colony formation assays. To assess the influence of environmentally induced chronic OS, we used L-Buthionine-sulfoximine (BSO) to lower the level of the intracellular antioxidant glutathione. Similar to our observations with E6*, glutathione depletion by BSO also increased ROS levels, caused oxidative DNA damage and increased the integration frequency of plasmid DNA. Finally, under conditions of chronic OS, we were able to induce and characterize a few independent events in which episomal HPV16 integrated into the host genome of cervical keratinocytes. Our results support a chain of events leading from induction of oxidative stress, to DNA damage, to viral integration, and ultimately to carcinogenesis. PMID:27186429

  3. Reliable transgene-independent method for determining Sleeping Beauty transposon copy numbers

    PubMed Central

    2011-01-01

    Background The transposon-based gene delivery technique is emerging as a method of choice for gene therapy. The Sleeping Beauty (SB) system has become one of the most favored methods, because of its efficiency and its random integration profile. Copy-number determination of the delivered transgene is a crucial task, but a universal method for measuring this is lacking. In this paper, we show that a real-time quantitative PCR-based, transgene-independent (qPCR-TI) method is able to determine SB transposon copy numbers regardless of the genetic cargo. Results We designed a specific PCR assay to amplify the left inverted repeat-direct repeat region of SB, and used it together with the single-copy control gene RPPH1 and a reference genomic DNA of known copy number. The qPCR-TI method allowed rapid and accurate determination of SB transposon copy numbers in various cell types, including human embryonic stem cells. We also found that this sensitive, rapid, highly reproducible and non-radioactive method is just as accurate and reliable as the widely used blotting techniques or the transposon display method. Because the assay is specific for the inverted repeat region of the transposon, it could be used in any system where the SB transposon is the genetic vehicle. Conclusions We have developed a transgene-independent method to determine copy numbers of transgenes delivered by the SB transposon system. The technique is based on a quantitative real-time PCR detection method, offering a sensitive, non-radioactive, rapid and accurate approach, which has a potential to be used for gene therapy. PMID:21371313

  4. The genetic organization of integrated hepatitis B virus DNA in the human hepatoma cell line PLC/PRF/5.

    PubMed Central

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

    1984-01-01

    Hepatitis B virus (HBV) DNA is often found integrated in the genome of infected human liver cells and is supposed to be related to the development of primary liver carcinoma (PLC). Four clones of HBV DNA-containing sequences derived from DNA of the human PLC-derived cell line PLC/PRF/5 are discussed. The viral sequences show no intricate rearrangements excepting for a duplication and an inversion in one case, and a deletion in another. In all cases integration of the viral DNA was seen to be in a region which is single-stranded in the unintegrated HBV DNA. Sequence homologies between human and viral DNA flanking the integration sites have been detected. That may have a functional role in integration. Nucleotide sequence analyses of regions encompassing the viral-human junctions reveal open reading frames which consist of viral and/or human information. The possible expression of chimeric or cellular proteins may play a role in tumour development, and offers directions for further investigations. Images PMID:6091042

  5. Genome-Wide Analysis of Transposon and Retroviral Insertions Reveals Preferential Integrations in Regions of DNA Flexibility

    PubMed Central

    Vrljicak, Pavle; Tao, Shijie; Varshney, Gaurav K.; Quach, Helen Ngoc Bao; Joshi, Adita; LaFave, Matthew C.; Burgess, Shawn M.; Sampath, Karuna

    2016-01-01

    DNA transposons and retroviruses are important transgenic tools for genome engineering. An important consideration affecting the choice of transgenic vector is their insertion site preferences. Previous large-scale analyses of Ds transposon integration sites in plants were done on the basis of reporter gene expression or germ-line transmission, making it difficult to discern vertebrate integration preferences. Here, we compare over 1300 Ds transposon integration sites in zebrafish with Tol2 transposon and retroviral integration sites. Genome-wide analysis shows that Ds integration sites in the presence or absence of marker selection are remarkably similar and distributed throughout the genome. No strict motif was found, but a preference for structural features in the target DNA associated with DNA flexibility (Twist, Tilt, Rise, Roll, Shift, and Slide) was observed. Remarkably, this feature is also found in transposon and retroviral integrations in maize and mouse cells. Our findings show that structural features influence the integration of heterologous DNA in genomes, and have implications for targeted genome engineering. PMID:26818075

  6. Genome-Wide Analysis of Transposon and Retroviral Insertions Reveals Preferential Integrations in Regions of DNA Flexibility.

    PubMed

    Vrljicak, Pavle; Tao, Shijie; Varshney, Gaurav K; Quach, Helen Ngoc Bao; Joshi, Adita; LaFave, Matthew C; Burgess, Shawn M; Sampath, Karuna

    2016-01-01

    DNA transposons and retroviruses are important transgenic tools for genome engineering. An important consideration affecting the choice of transgenic vector is their insertion site preferences. Previous large-scale analyses of Ds transposon integration sites in plants were done on the basis of reporter gene expression or germ-line transmission, making it difficult to discern vertebrate integration preferences. Here, we compare over 1300 Ds transposon integration sites in zebrafish with Tol2 transposon and retroviral integration sites. Genome-wide analysis shows that Ds integration sites in the presence or absence of marker selection are remarkably similar and distributed throughout the genome. No strict motif was found, but a preference for structural features in the target DNA associated with DNA flexibility (Twist, Tilt, Rise, Roll, Shift, and Slide) was observed. Remarkably, this feature is also found in transposon and retroviral integrations in maize and mouse cells. Our findings show that structural features influence the integration of heterologous DNA in genomes, and have implications for targeted genome engineering. PMID:26818075

  7. 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

    2016-02-01

    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

  8. The Sperm Chromatin Structure Assay (SCSA(®)) and other sperm DNA fragmentation tests for evaluation of sperm nuclear DNA integrity as related to fertility.

    PubMed

    Evenson, Donald P

    2016-06-01

    Thirty-five years ago the pioneering paper in Science (240:1131) on the relationship between sperm DNA integrity and pregnancy outcome was featured as the cover issue showing a fluorescence photomicrograph of red and green stained sperm. The flow cytometry data showed a very significant difference in sperm DNA integrity between fertile and subfertile bulls and men. This study utilized heat (100°C, 5min) to denature DNA at sites of DNA strand breaks followed by staining with acridine orange (AO) and measurements of 5000 individual sperm of green double strand (ds) DNA and red single strand (ss) DNA fluorescence. Later, the heat protocol was changed to a low pH protocol to denature the DNA at sites of strand breaks; the heat and acid procedures produced the same results. SCSA data are very advantageously dual parameter with 1024 channels (degrees) of both red and green fluorescence. Hundreds of publications on the use of the SCSA test in animals and humans have validated the SCSA as a highly useful test for determining male breeding soundness. The SCSA test is a rapid, non-biased flow cytometer machine measurement providing robust statistical data with exceptional precision and repeatability. Many genotoxic experiments showed excellent dose response data with very low coefficient of variation that further validated the SCSA as being a highly powerful assay for sperm DNA integrity. Twelve years following the introduction of the SCSA test, the terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labelling (TUNEL) test (1993) for sperm was introduced as the only other flow cytometric assay for sperm DNA fragmentation. However, the TUNEL test can also be done by light microscopy with much less statistical robustness. The COMET (1998) and Sperm Chromatin Dispersion (SCD; HALO) (2003) tests were introduced as light microscope tests that don't require a flow cytometer. Since these tests measure only 50-200 sperm per sample, they suffer from the lack of

  9. An Integrated Approach for RNA-seq Data Normalization

    PubMed Central

    Yang, Shengping; Mercante, Donald E.; Zhang, Kun; Fang, Zhide

    2016-01-01

    BACKGROUND DNA copy number alteration is common in many cancers. Studies have shown that insertion or deletion of DNA sequences can directly alter gene expression, and significant correlation exists between DNA copy number and gene expression. Data normalization is a critical step in the analysis of gene expression generated by RNA-seq technology. Successful normalization reduces/removes unwanted nonbiological variations in the data, while keeping meaningful information intact. However, as far as we know, no attempt has been made to adjust for the variation due to DNA copy number changes in RNA-seq data normalization. RESULTS In this article, we propose an integrated approach for RNA-seq data normalization. Comparisons show that the proposed normalization can improve power for downstream differentially expressed gene detection and generate more biologically meaningful results in gene profiling. In addition, our findings show that due to the effects of copy number changes, some housekeeping genes are not always suitable internal controls for studying gene expression. CONCLUSIONS Using information from DNA copy number, integrated approach is successful in reducing noises due to both biological and nonbiological causes in RNA-seq data, thus increasing the accuracy of gene profiling. PMID:27385909

  10. 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

  11. Differences in susceptibility of Arabidopsis ecotypes to crown gall disease may result from a deficiency in T-DNA integration.

    PubMed Central

    Nam, J; Matthysse, A G; Gelvin, S B

    1997-01-01

    We show that among ecotypes of Arabidopsis, there is considerable variation in their susceptibility to crown gall disease. Differences in susceptibility are heritable and, in one ecotype, segregate as a single major contributing locus. In several ecotypes, recalcitrance to tumorigenesis results from decreased binding of Agrobacterium to inoculated root explants. The recalcitrance of another ecotype occurs at a late step in T-DNA transfer. Transient expression of a T-DNA-encoded beta-glucuronidase gusA gene is efficient, but the ecotype is deficient in crown gall tumorigenesis, transformation to kanamycin resistance, and stable GUS expression. This ecotype is also more sensitive to gamma radiation than is a susceptible ecotype. DNA gel blot analysis showed that after infection by Agrobacterium, less T-DNA was integrated into the genome of the recalcitrant ecotype than was integrated into the genome of a highly susceptible ecotype. PMID:9090878

  12. Acridine Orange and Flow Cytometry: Which Is Better to Measure the Effect of Varicocele on Sperm DNA Integrity?

    PubMed Central

    Mohammed, Essam-Elden M.; Mosad, Eman; Zahran, Asmaa M.; Hameed, Diaa A.; Taha, Emad A.; Mohamed, Mohamed A.

    2015-01-01

    We evaluated the effect of varicocelectomy on semen parameters and levels of sperm DNA damage in infertile men. A total of 75 infertile men with varicocele and 40 fertile men (controls) were included in this study. Semen analysis and sperm DNA damage expressed as the DNA fragmentation index using acridine orange staining and chromatin condensation test by flow cytometry were assessed before and 6 months after varicocelectomy. The patients were also followed up for 1 year for pregnancy outcome. Semen parameters were significantly lower in varicocele patients compared to controls (P < 0.05). Mean percentages of sperm DNA fragmentation and sperm DNA chromatin condensation in patients were significantly higher than those in controls (P < 0.05). After varicocelectomy, sperm DNA fragmentation improved significantly, whereas sperm chromatin condensation was not significantly changed. In 15 out of 75 varicocele patients, clinical pregnancy was diagnosed; those with positive pregnancy outcome had significant improvement in sperm count, progressive sperm motility, and sperm DNA fragmentation, but there was no significant difference in sperm DNA condensation compared to negative pregnancy outcome patients. We concluded from this study that acridine orange stain is more reliable method than flow cytometry in the evaluation of sperm DNA integrity after varicocelectomy. PMID:26681938

  13. 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

    2015-01-01

    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. PMID:25740433

  14. Automated and integrated system for high-throughput DNA genotyping directly from blood.

    PubMed

    Zhang, N; Tan, H; Yeung, E S

    1999-03-15

    An automated and integrated system for DNA typing directly from blood samples has been developed. The multiplexed eight-array system is based on capillary microfluidics and capillary array electrophoresis. Three short-tandem-repeat loci, vWA, THO1, and TPOX, are coamplified simultaneously in a fused-silica capillary by a hot-air thermocycler. Blood is directly used as the sample for polymerase chain reaction (PCR) without any pretreatment. Modifications of standard protocols are necessary for direct PCR from blood. A programmable syringe pump plus a set of multiplexed liquid nitrogen freeze/thaw switching valves are employed for liquid handling in the fluid distribution network. The system fully integrates sample loading, PCR, addition of an absolute standard, on-line injection of sample and standards, separation and detection. The genotypes from blood samples can be clearly identified in eight parallel channels when the electropherograms are compared with that of the standard allelic ladder by itself. Regeneration and cleaning of the entire system prior to subsequent runs are also integrated into the instrument. The instrumentation is compatible with future expansion to hundreds of capillaries to achieve even higher throughput. PMID:10093495

  15. Insertion mutagenesis of the yeast Candida famata (Debaryomyces hansenii) by random integration of linear DNA fragments.

    PubMed

    Dmytruk, Kostyantyn V; Voronovsky, Andriy Y; Sibirny, Andriy A

    2006-09-01

    The feasibility of using random insertional mutagenesis to isolate mutants of the flavinogenic yeast Candida famata was explored. Mutagenesis was performed by transformation of the yeast with an integrative plasmid containing the Saccharomyces cerevisiae LEU2 gene as a selective marker. The addition of restriction enzyme together with the plasmid (restriction enzyme-mediated integration, REMI) increased the transformation frequency only slightly. Integration of the linearized plasmid occurred randomly in the C. famata genome. To investigate the potential of insertional mutagenesis, it was used for tagging genes involved in positive regulation of riboflavin synthesis in C. famata. Partial DNA sequencing of tagged genes showed that they were homologous to the S. cerevisiae genes RIB1, MET2, and SEF1. Intact orthologs of these genes isolated from Debaryomyces hansenii restored the wild phenotype of the corresponding mutants, i.e., the ability to overproduce riboflavin under iron limitation. The Staphylococcus aureus ble gene conferring resistance to phleomycin was used successfully in the study as a dominant selection marker for C. famata. The results obtained indicate that insertional mutagenesis is a powerful tool for tagging genes in C. famata. PMID:16770625

  16. Elevated metals compromise repair of oxidative DNA damage via the base excision repair pathway: implications of pathologic iron overload in the brain on integrity of neuronal DNA.

    PubMed

    Li, Hui; Swiercz, Rafal; Englander, Ella W

    2009-09-01

    Tissue-specific iron content is tightly regulated to simultaneously satisfy specialized metabolic needs and avoid cytotoxicity. In the brain, disruption of iron homeostasis may occur in acute as well as progressive injuries associated with neuronal dysfunction and death. We hypothesized that adverse effects of disrupted metal homeostasis on brain function may involve impairment of DNA repair processes. Because in the brain, the base excision repair (BER) pathway is central for handling oxidatively damaged DNA, we investigated effects of elevated iron and zinc on key BER enzymes. In vitro DNA repair assays revealed inhibitory effects of metals on BER activities, including the incision of abasic sites, 5'-flap cleavage, gap filling DNA synthesis and ligation. Using the comet assay, we showed that while metals at concentrations which inhibit BER activities in in vitro assays, did not induce direct genomic damage in cultured primary neurons, they significantly delayed repair of genomic DNA damage induced by sublethal exposure to H(2)O(2). Thus, in the brain even a mild transient metal overload, may adversely affect the DNA repair capacity and thereby compromise genomic integrity and initiate long-term deleterious sequelae including neuronal dysfunction and death. PMID:19619136

  17. Pressure dissociation of integration host factor–DNA complexes reveals flexibility-dependent structural variation at the protein–DNA interface

    PubMed Central

    Senear, Donald F.; Tretyachenko-Ladokhina, Vira; Opel, Michael L.; Aeling, Kimberly A.; Wesley Hatfield, G.; Franklin, Laurie M.; Darlington, Reuben C.

    2007-01-01

    E. coli Integration host factor (IHF) condenses the bacterial nucleoid by wrapping DNA. Previously, we showed that DNA flexibility compensates for structural characteristics of the four consensus recognition elements associated with specific binding (Aeling et al., J. Biol. Chem. 281, 39236–39248, 2006). If elements are missing, high-affinity binding occurs only if DNA deformation energy is low. In contrast, if all elements are present, net binding energy is unaffected by deformation energy. We tested two hypotheses for this observation: in complexes containing all elements, (1) stiff DNA sequences are less bent upon binding IHF than flexible ones; or (2) DNA sequences with differing flexibility have interactions with IHF that compensate for unfavorable deformation energy. Time-resolved Förster resonance energy transfer (FRET) shows that global topologies are indistinguishable for three complexes with oligonucleotides of different flexibility. However, pressure perturbation shows that the volume change upon binding is smaller with increasing flexibility. We interpret these results in the context of Record and coworker's model for IHF binding (J. Mol. Biol. 310, 379–401, 2001). We propose that the volume changes reflect differences in hydration that arise from structural variation at IHF–DNA interfaces while the resulting energetic compensation maintains the same net binding energy. PMID:17324943

  18. 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

  19. 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. PMID:24206686

  20. Modes of integration of heterologous plasmid DNA into the chromosome of Streptococcus pneumoniae

    SciTech Connect

    Pozzi, G.; Guild, W.R.

    1985-03-01

    The authors compared the efficiencies of two different processes that can direct integration of plasmids into chromosomes of recipient cells during transformation. A donor-recipient system was constructed to allow a single donor plasmid to use either flanking homology, involving an apparent double crossover, or the insertion duplication process that has been described as due to a Campbell-like single crossover between the chromosome and a circular duplex. The latter process gave 600-fold fewer insertions that did the former, confirming expectations from prior work showing that insertion of heterologous DNA by use of flanking homology is highly efficient. It has some advantages for cloning and mapping purposes and can be exploited once it is recognized.

  1. Additional deleterious effects of alcohol consumption on sperm parameters and DNA integrity in diabetic mice.

    PubMed

    Pourentezari, M; Talebi, A R; Mangoli, E; Anvari, M; Rahimipour, M

    2016-06-01

    The aim of this study was to survey the impact of alcohol consumption on sperm parameters and DNA integrity in experimentally induced diabetic mice. A total of 32 adult male mice were divided into four groups: mice of group 1 served as control fed on basal diet, group 2 received streptozotocin (STZ) (200 mg kg(-1) , single dose, intraperitoneal) and basal diet, group 3 received alcohol (10 mg kg(-1) , water soluble) and basal diet, and group 4 received STZ and alcohol for 35 days. The cauda epididymidis of each mouse was dissected and placed in 1 ml of pre-warm Ham's F10 culture medium for 30 min. The swim-out spermatozoa were analysed for count, motility, morphology and viability. Sperm chromatin quality was evaluated with aniline blue, toluidine blue, acridine orange and chromomycin A3 staining. The results showed that all sperm parameters had significant differences (P < 0.05), also when sperm chromatin was assessed with cytochemical tests. There were significant differences (P < 0.001) between the groups. According to our results, alcohol and diabetes can cause abnormalities in sperm parameters and chromatin quality. In addition, alcohol consumption in diabetic mice can intensify sperm chromatin/DNA damage. PMID:26358836

  2. Increasing storage time of extended boar semen reduces sperm DNA integrity.

    PubMed

    Boe-Hansen, Gry B; Ersbøll, Annette K; Greve, Torben; Christensen, Preben

    2005-04-15

    There is an extensive use of artificial insemination (AI) in the pig industry. Extended liquid boar semen may be used for insemination for up to 5 days after collection. The objective of this study was to determine the changes in sperm quality, when boar semen was extended and stored at 18 degrees C for up to 72 h post-collection. The study included three ejaculates from five boars, for each of the four breeds: Duroc, Hampshire, Landrace and Danish Large White (n=60 ejaculates). The sperm chromatin structure assay (SCSA) showed an increase in DNA fragmentation index (DFI) after 72 h of incubation (P<0.001), with no differences between breeds (P=0.07). For two Hampshire boars, all ejaculates had a large increase in DFI after 24 h of incubation. The standard deviation of DFI (SD-DFI) differed between breeds, with the SD-DFI for Hampshire being significantly greater than for the other breeds. The SD-DFI did not change during the 72 h of storage. Sperm viability was determined using SYBR-14 and propidium iodide in combination with flow cytometry. The sperm viability did not differ between breeds (P=0.21), but a difference in viability during storage (P<0.001) was detected. In conclusion, the SCSA cytogram patterns were consistent for different ejaculates within boars and storage of extended boar semen at 18 degrees C for 72 h significantly decreased the integrity of sperm DNA. PMID:15823356

  3. 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.

  4. 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

    2016-08-01

    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

  5. Pluchea lanceolata protects against Benzo(a) pyrene induced renal toxicity and loss of DNA integrity.

    PubMed

    Jahangir, Tamanna; Safhi, Mohammed M; Sultana, Sarwat; Ahmad, Sayeed

    2013-03-01

    Evidence from epidemiological, experimental and clinical trial data indicates that a plant based diet can reduce the risk of chronic diseases and reduces toxic effects. In the present study, we report the antioxidant and anticlastogenic activity of Pluchea lanceolata (PL), an important medicinal plant, in both in vitro and in vivo model. Benzo(a)pyrene (B(a)P) administration leads to depletion of renal glutathione and its metabolizing enzymes. Pretreatment with PL (100 and 200 mg /kg b.wt) restored renal glutathione content and its dependent enzymes significantly (p<0.001) with simultaneous increase in catalase(CAT), quinone reductase(QR) in mouse kidney. Prophylactic administration of PL prior to B (a) P administration significantly decreased the malondialdehyde(MDA), H2O2 and xanthineoxidase (XO) levels at a significance of p<0.001, at both the doses. PL extract pretreated groups showed marked inhibition in B(a)P induced micronuclei formation in mouse bone marrow cells with simultaneous restoration of DNA integrity, viz. alkaline unwinding assay and DNA damage shown by gel-electrophoresis. HPTLC confirms the presence of quercetin in plant extract which could be responsible for PL protecting efficacy. In conclusion, the present findings strongly support the antioxidant efficacy of PL, possibly by modulation of antioxidant armory. PMID:24170979

  6. An Integrative Multi-scale Analysis of the Dynamic DNA Methylation Landscape in Aging

    PubMed Central

    Yuan, Tian; Jiao, Yinming; de Jong, Simone; Ophoff, Roel A.; Beck, Stephan; Teschendorff, Andrew E.

    2015-01-01

    Recent studies have demonstrated that the DNA methylome changes with age. This epigenetic drift may have deep implications for cellular differentiation and disease development. However, it remains unclear how much of this drift is functional or caused by underlying changes in cell subtype composition. Moreover, no study has yet comprehensively explored epigenetic drift at different genomic length scales and in relation to regulatory elements. Here we conduct an in-depth analysis of epigenetic drift in blood tissue. We demonstrate that most of the age-associated drift is independent of the increase in the granulocyte to lymphocyte ratio that accompanies aging and that enrichment of age-hypermethylated CpG islands increases upon adjustment for cellular composition. We further find that drift has only a minimal impact on in-cis gene expression, acting primarily to stabilize pre-existing baseline expression levels. By studying epigenetic drift at different genomic length scales, we demonstrate the existence of mega-base scale age-associated hypomethylated blocks, covering approximately 14% of the human genome, and which exhibit preferential hypomethylation in age-matched cancer tissue. Importantly, we demonstrate the feasibility of integrating Illumina 450k DNA methylation with ENCODE data to identify transcription factors with key roles in cellular development and aging. Specifically, we identify REST and regulatory factors of the histone methyltransferase MLL complex, whose function may be disrupted in aging. In summary, most of the epigenetic drift seen in blood is independent of changes in blood cell type composition, and exhibits patterns at different genomic length scales reminiscent of those seen in cancer. Integration of Illumina 450k with appropriate ENCODE data may represent a fruitful approach to identify transcription factors with key roles in aging and disease. PMID:25692570

  7. 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

  8. Multiple integration site of hepatitis B virus DNA in hepatocellular carcinoma and chronic active hepatitis tissues from children.

    PubMed Central

    Yaginuma, K; Kobayashi, H; Kobayashi, M; Morishima, T; Matsuyama, K; Koike, K

    1987-01-01

    Attention was directed to hepatitis B virus (HBV) integration in tissues obtained from an hepatocellular carcinoma (HCC) of an 11-year-old boy and from the liver of his 6-year-old brother, who had chronic active hepatitis. Multiple HBV DNA integration sites were demonstrated in both tissues. Cell population(s) in the HCC and liver from the patient with chronic active hepatitis were assumed to be heterogeneous with regard to HBV integration. The integrated forms in the two tissues showed similar genetic organization without gross rearrangement. The location of one of the virus-chromosomal junctions was restricted to the 5'-end region of the minus-strand DNA of HBV. The experimental results support our previous model for the mechanism of HBV integration, in which minus-strand replicative intermediates integrate into chromosomal DNA. The integrated HBV DNAs were conserved in the same region of the viral genome, spanning from the C gene through the S gene to the X gene, which contains intrinsic promoter-enhancer sequences. Images PMID:3033312

  9. 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)

  10. 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

  11. 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

  12. 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

  13. 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. PMID:22744162

  14. Paper electrode integrated lateral flow immunosensor for quantitative analysis of oxidative stress induced DNA damage

    PubMed Central

    Zhu, Xuena; Shah, Pratikkumar; Stoff, Susan; Liu, Hongyun; Li, Chen-zhong

    2014-01-01

    A novel device combining electrochemical and colorimetric detection is developed for the rapid measurement of 8-hydroxy-2′-deoxyguanosine (8-OHdG), a DNA oxidative damage biomarker. The device takes advantage of the speed and low cost of the conventional strip test as well as the high reliability and accuracy of electrochemical assay. Competitive immunoreactions were performed on the lateral flow strip, and the captured 8-OHdG on the control line was determined by chronoamperometric measurement with carbon nanotubes paper as the working electrode. At the same time, the color intensity of the test line was measured by a scanner and analyzed by the ImageJ software. The device was able to detect 8-OHdG concentrations in PBS as low as 2.07 ng mL−1 by the colorimetric method and 3.11 ng mL−1 by the electrochemical method. Furthermore, the device was successfully utilized to detect 8-OHdG in urine with a detection limit of 5.76 ng mL−1 (colorimetric method) and 8.85 ng mL−1 (electrochemical method), respectively. In conclusion, the integrated device with dual detections can provide a rapid, visual, quantitative and feasible detection method for 8-OHdG. The integration of these two methods holds two major advantages over tests based on single method. Firstly, it can provide double confidence on the same assay. Secondly, by involving two methods that differ in principle, the integration could potentially avoid false results coming from one method. In addition, these methods do not require expensive equipment or trained personnel, deeming it suitable for use as a simple, economical, portable field kit for on-site monitoring of 8-OHdG in a variety of clinical settings. PMID:24733353

  15. 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, ...

  16. 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

  17. Transgene integration and organization in cotton (Gossypium hirsutum L.) genome.

    PubMed

    Zhang, Jun; Cai, Lin; Cheng, Jiaqin; Mao, Huizhu; Fan, Xiaoping; Meng, Zhaohong; Chan, Ka Man; Zhang, Huijun; Qi, Jianfei; Ji, Lianghui; Hong, Yan

    2008-04-01

    While genetically modified upland cotton (Gossypium hirsutum L.) varieties are ranked among the most successful genetically modified organisms (GMO), there is little knowledge on transgene integration in the cotton genome, partly because of the difficulty in obtaining large numbers of transgenic plants. In this study, we analyzed 139 independently derived T0 transgenic cotton plants transformed by Agrobacterium tumefaciens strain AGL1 carrying a binary plasmid pPZP-GFP. It was found by PCR that as many as 31% of the plants had integration of vector backbone sequences. Of the 110 plants with good genomic Southern blot results, 37% had integration of a single T-DNA, 24% had two T-DNA copies and 39% had three or more copies. Multiple copies of the T-DNA existed either as repeats in complex loci or unlinked loci. Our further analysis of two T1 populations showed that segregants with a single T-DNA and no vector sequence could be obtained from T0 plants having multiple T-DNA copies and vector sequence. Out of the 57 T-DNA/T-DNA junctions cloned from complex loci, 27 had canonical T-DNA tandem repeats, the rest (30) had deletions to T-DNAs or had inclusion of vector sequences. Overlapping micro-homology was present for most of the T-DNA/T-DNA junctions (38/57). Right border (RB) ends of the T-DNA were precise while most left border (LB) ends (64%) had truncations to internal border sequences. Sequencing of collinear vector integration outside LB in 33 plants gave evidence that collinear vector sequence was determined in agrobacterium culture. Among the 130 plants with characterized flanking sequences, 12% had the transgene integrated into coding sequences, 12% into repetitive sequences, 7% into rDNAs. Interestingly, 7% had the transgene integrated into chloroplast derived sequences. Nucleotide sequence comparison of target sites in cotton genome before and after T-DNA integration revealed overlapping microhomology between target sites and the T-DNA (8/8), deletions to

  18. 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

  19. 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

    2014-01-01

    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

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

    PubMed

    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 Hg(2+) ions. Our approach relies on the displacement of a DNA hairpin structure, which forms after the binding of mercury ions to an aptamer DNA sequence. The strand displacement reaction of the DNA aptamer provides highly specific and quantitative means for determining the mercury ion concentration on a label-free WGM sensor platform. Our approach also shows the possibility for manipulating the kinetics of a strand displacement reaction with specific ionic species. PMID:27483277

  1. 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.

  2. Comparison of sperm quality and DNA integrity in mouse sperm exposed to various cooling velocities and osmotic stress.

    PubMed

    Yildiz, Cengiz; Law, Napoleon; Ottaviani, Palma; Jarvi, Keith; McKerlie, Colin

    2010-11-01

    The first objective was to compare sperm quality following conventional manual sperm freezing (cryovials held 1, 2, 3, and 4 cm, respectively, above liquid nitrogen (LN(2)) for 10 min, resulting in cooling velocities of approximately -14.9, -10.1, -6.6, and -5.1 °C/min, respectively), and cooling velocities of -5, -20, -40, and -100 °C/min in a programmed automated freezer, for sperm recovered from CD-1, B6129SF1, and C57BL/6NCrlBR mice. Furthermore, using these strains, as well as 129S/SvPaslco, and DBA/2NCrlBR mice, the second objective was to determine the effects on DNA integrity of sperm exposed to hyposmotic (1 mOsm/L) and hyperosmotic (2400 mOsm/L) solutions, compared to an isosmotic control (300 mOsm/L). For freezing above LN(2) or in an automated freezer, 2 cm above LN(2) and -100 °C/min, respectively, were optimal (P < 0.05-0.01), with no significant differences between these two approaches for post-thaw progressive motility, DNA integrity, and in vitro rates of fertilization and blastocyst formation. Both manual and automated freezing techniques increased post-thaw sperm DNA fragmentation (P < 0.01); the DNA integrity of post-thaw sperm was significantly affected by cooling velocity and strain background. Relative to isosmotic controls, a hyposmotic solution was more deleterious (P < 0.05-0.01) to sperm DNA integrity than a hyperosmotic solution for CD-1, B6129SF1, C57BL/6, and DBA mice (there were strain-dependent differences). In conclusion, optimization of freezing distance and cooling velocity (manual and automated freezing, respectively) were significant factors for efficient cryopreservation and re-derivation of mice from frozen-thawed sperm. Additionally, osmotically-driven volume changes in mouse sperm increased DNA fragmentation, with susceptibility affected by background strain. PMID:20728931

  3. 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

  4. 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

  5. ZINBA integrates local covariates with DNA-seq data to identify broad and narrow regions of enrichment, even within amplified genomic regions

    PubMed Central

    2011-01-01

    ZINBA (Zero-Inflated Negative Binomial Algorithm) identifies genomic regions enriched in a variety of ChIP-seq and related next-generation sequencing experiments (DNA-seq), calling both broad and narrow modes of enrichment across a range of signal-to-noise ratios. ZINBA models and accounts for factors that co-vary with background or experimental signal, such as G/C content, and identifies enrichment in genomes with complex local copy number variations. ZINBA provides a single unified framework for analyzing DNA-seq experiments in challenging genomic contexts. Software website: http://code.google.com/p/zinba/ PMID:21787385

  6. 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. PMID:26220133

  7. MethBank: a database integrating next-generation sequencing single-base-resolution DNA methylation programming data.

    PubMed

    Zou, Dong; Sun, Shixiang; Li, Rujiao; Liu, Jiang; Zhang, Jing; Zhang, Zhang

    2015-01-01

    DNA methylation plays crucial roles during embryonic development. Here we present MethBank (http://dnamethylome.org), a DNA methylome programming database that integrates the genome-wide single-base nucleotide methylomes of gametes and early embryos in different model organisms. Unlike extant relevant databases, MethBank incorporates the whole-genome single-base-resolution methylomes of gametes and early embryos at multiple different developmental stages in zebrafish and mouse. MethBank allows users to retrieve methylation levels, differentially methylated regions, CpG islands, gene expression profiles and genetic polymorphisms for a specific gene or genomic region. Moreover, it offers a methylome browser that is capable of visualizing high-resolution DNA methylation profiles as well as other related data in an interactive manner and thus is of great helpfulness for users to investigate methylation patterns and changes of gametes and early embryos at different developmental stages. Ongoing efforts are focused on incorporation of methylomes and related data from other organisms. Together, MethBank features integration and visualization of high-resolution DNA methylation data as well as other related data, enabling identification of potential DNA methylation signatures in different developmental stages and accordingly providing an important resource for the epigenetic and developmental studies. PMID:25294826

  8. An Overview of the Molecular Mechanisms of Recombinational DNA Repair.

    PubMed

    Kowalczykowski, Stephen C

    2015-11-01

    Recombinational DNA repair is a universal aspect of DNA metabolism and is essential for genomic integrity. It is a template-directed process that uses a second chromosomal copy (sister, daughter, or homolog) to ensure proper repair of broken chromosomes. The key steps of recombination are conserved from phage through human, and an overview of those steps is provided in this review. The first step is resection by helicases and nucleases to produce single-stranded DNA (ssDNA) that defines the homologous locus. The ssDNA is a scaffold for assembly of the RecA/RAD51 filament, which promotes the homology search. On finding homology, the nucleoprotein filament catalyzes exchange of DNA strands to form a joint molecule. Recombination is controlled by regulating the fate of both RecA/RAD51 filaments and DNA pairing intermediates. Finally, intermediates that mature into Holliday structures are disjoined by either nucleolytic resolution or topological dissolution. PMID:26525148

  9. 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.

  10. DNA Microarrays

    NASA Astrophysics Data System (ADS)

    Nguyen, C.; Gidrol, X.

    Genomics has revolutionised biological and biomedical research. This revolution was predictable on the basis of its two driving forces: the ever increasing availability of genome sequences and the development of new technology able to exploit them. Up until now, technical limitations meant that molecular biology could only analyse one or two parameters per experiment, providing relatively little information compared with the great complexity of the systems under investigation. This gene by gene approach is inadequate to understand biological systems containing several thousand genes. It is essential to have an overall view of the DNA, RNA, and relevant proteins. A simple inventory of the genome is not sufficient to understand the functions of the genes, or indeed the way that cells and organisms work. For this purpose, functional studies based on whole genomes are needed. Among these new large-scale methods of molecular analysis, DNA microarrays provide a way of studying the genome and the transcriptome. The idea of integrating a large amount of data derived from a support with very small area has led biologists to call these chips, borrowing the term from the microelectronics industry. At the beginning of the 1990s, the development of DNA chips on nylon membranes [1, 2], then on glass [3] and silicon [4] supports, made it possible for the first time to carry out simultaneous measurements of the equilibrium concentration of all the messenger RNA (mRNA) or transcribed RNA in a cell. These microarrays offer a wide range of applications, in both fundamental and clinical research, providing a method for genome-wide characterisation of changes occurring within a cell or tissue, as for example in polymorphism studies, detection of mutations, and quantitative assays of gene copies. With regard to the transcriptome, it provides a way of characterising differentially expressed genes, profiling given biological states, and identifying regulatory channels.

  11. Mechanical unfolding of human telomere G-quadruplex DNA probed by integrated fluorescence and magnetic tweezers spectroscopy.

    PubMed

    Long, Xi; Parks, Joseph W; Bagshaw, Clive R; Stone, Michael D

    2013-02-01

    Single-molecule techniques facilitate analysis of mechanical transitions within nucleic acids and proteins. Here, we describe an integrated fluorescence and magnetic tweezers instrument that permits detection of nanometer-scale DNA structural rearrangements together with the application of a wide range of stretching forces to individual DNA molecules. We have analyzed the force-dependent equilibrium and rate constants for telomere DNA G-quadruplex (GQ) folding and unfolding, and have determined the location of the transition state barrier along the well-defined DNA-stretching reaction coordinate. Our results reveal the mechanical unfolding pathway of the telomere DNA GQ is characterized by a short distance (<1 nm) to the transition state for the unfolding reaction. This mechanical unfolding response reflects a critical contribution of long-range interactions to the global stability of the GQ fold, and suggests that telomere-associated proteins need only disrupt a few base pairs to destabilize GQ structures. Comparison of the GQ unfolded state with a single-stranded polyT DNA revealed the unfolded GQ exhibits a compacted non-native conformation reminiscent of the protein molten globule. We expect the capacity to interrogate macromolecular structural transitions with high spatial resolution under conditions of low forces will have broad application in analyses of nucleic acid and protein folding. PMID:23303789

  12. 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

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

    PubMed

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

    2014-05-20

    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

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

    PubMed Central

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

    2015-01-01

    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. PMID:26569308

  15. Photographic copy of undated drawing. Printed copy located in plan ...

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

    Photographic copy of undated drawing. Printed copy located in plan files at the New Orleans Public Belt Railroad Administration Office at 5100 Jefferson Highway, Jefferson, Louisiana 70123. Draftsman Unknown. DRAWING OF ENTIRE BRIDGE PLAN AND PARTIAL NORTHEAST ELEVATION. PROPERTY OF NEW ORLEANS PUBLIC BELT RAILROAD. - Huey P. Long Bridge, Spanning Mississippi River approximately midway between nine & twelve mile points upstream from & west of New Orleans, Jefferson, Jefferson Parish, LA

  16. 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.

  17. 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

    2013-04-01

    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

  18. 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

  19. Principles and concepts of DNA replication in bacteria, archaea, and eukarya.

    PubMed

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

    2013-07-01

    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

  20. 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

  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. Agrobacterium T-DNA integration into the plant genome can occur without the activity of key non-homologous end-joining proteins.

    PubMed

    Park, So-Yon; Vaghchhipawala, Zarir; Vasudevan, Balaji; Lee, Lan-Ying; Shen, Yunjia; Singer, Kamy; Waterworth, Wanda M; Zhang, Zhanyuan J; West, Christopher E; Mysore, Kirankumar S; Gelvin, Stanton B

    2015-03-01

    Non-homologous end joining (NHEJ) is the major model proposed for Agrobacterium T-DNA integration into the plant genome. In animal cells, several proteins, including KU70, KU80, ARTEMIS, DNA-PKcs, DNA ligase IV (LIG4), Ataxia telangiectasia mutated (ATM), and ATM- and Rad3-related (ATR), play an important role in 'classical' (c)NHEJ. Other proteins, including histone H1 (HON1), XRCC1, and PARP1, participate in a 'backup' (b)NHEJ process. We examined transient and stable transformation frequencies of Arabidopsis thaliana roots mutant for numerous NHEJ and other related genes. Mutants of KU70, KU80, and the plant-specific DNA Ligase VI (LIG6) showed increased stable transformation susceptibility. However, these mutants showed transient transformation susceptibility similar to that of wild-type plants, suggesting enhanced T-DNA integration in these mutants. These results were confirmed using a promoter-trap transformation vector that requires T-DNA integration into the plant genome to activate a promoterless gusA (uidA) gene, by virus-induced gene silencing (VIGS) of Nicotiana benthamiana NHEJ genes, and by biochemical assays for T-DNA integration. No alteration in transient or stable transformation frequencies was detected with atm, atr, lig4, xrcc1, or parp1 mutants. However, mutation of parp1 caused high levels of T-DNA integration and transgene methylation. A double mutant (ku80/parp1), knocking out components of both NHEJ pathways, did not show any decrease in stable transformation or T-DNA integration. Thus, T-DNA integration does not require known NHEJ proteins, suggesting an alternative route for integration. PMID:25641249

  3. Does Ataxia Telangiectasia Mutated (ATM) protect testicular and germ cell DNA integrity by regulating the redox status?

    PubMed

    Godschalk, Roger W L; Vanhees, Kimberly; Maas, Lou; Drittij, Marie-Jose; Pachen, Daniëlle; van Doorn-Khosrovani, Sahar van Waalwijk; van Schooten, Frederik J; Haenen, Guido R M M

    2016-08-01

    A balanced redox homeostasis in the testis is essential for genetic integrity of sperm. Reactive oxygen species can disturb this balance by oxidation of glutathione, which is regenerated using NADPH, formed by glucose-6-phosphate dehydrogenase (G6PDH). G6PDH is regulated by the Ataxia Telangiectasia Mutated (Atm) protein. Therefore, we studied the redox status and DNA damage in testes and sperm of mice that carried a deletion in Atm. The redox status in heterozygote mice, reflected by glutathione levels and antioxidant capacity, was lower than in wild type mice, and in homozygotes the redox status was even lower. The redox status correlated with oxidative DNA damage that was highest in mice that carried Atm deletions. Surprisingly, G6PDH activity was highest in homozygotes carrying the deletion. These data indicate that defective Atm reduces the redox homeostasis of the testis and genetic integrity of sperm by regulating glutathione levels independently from G6PDH activity. PMID:27318254

  4. RNF138 interacts with RAD51D and is required for DNA interstrand crosslink repair and maintaining chromosome integrity.

    PubMed

    Yard, Brian D; Reilly, Nicole M; Bedenbaugh, Michael K; Pittman, Douglas L

    2016-06-01

    The RAD51 family is integral for homologous recombination (HR) mediated DNA repair and maintaining chromosome integrity. RAD51D, the fourth member of the family, is a known ovarian cancer susceptibility gene and required for the repair of interstrand crosslink DNA damage and preserving chromosomal stability. In this report, we describe the RNF138 E3 ubiquitin ligase that interacts with and ubiquitinates the RAD51D HR protein. RNF138 is a member of an E3 ligase family that contains an amino-terminal RING finger domain and a putative carboxyl-terminal ubiquitin interaction motif. In mammalian cells, depletion of RNF138 increased the stability of the RAD51D protein, suggesting that RNF138 governs ubiquitin-proteasome-mediated degradation of RAD51D. However, RNF138 depletion conferred sensitivity to DNA damaging agents, reduced RAD51 focus formation, and increased chromosomal instability. Site-specific mutagenesis of the RNF138 RING finger domain demonstrated that it was necessary for RAD51D ubiquitination. Presence of RNF138 also enhanced the interaction between RAD51D and a known interacting RAD51 family member XRCC2 in a yeast three-hybrid assay. Therefore, RNF138 is a newly identified regulatory component of the HR mediated DNA repair pathway that has implications toward understanding how ubiquitination modifies the functions of the RAD51 paralog protein complex. PMID:27161866

  5. Effect of vitamin E on sperm parameters and DNA integrity in sodium arsenite-treated rats

    PubMed Central

    Momeni, Hamid Reza; Eskandari, Najmeh

    2012-01-01

    Background: Arsenic as an environmental toxicant is able to exert malformations in male reproductive system by inducing oxidative stress. Vitamin E (Vit.E) is known as antioxidant vitamin. Objective: The aim of this study was to investigate the harmful effects of sodium arsenite on sperm parameters and the antioxidant effects of Vit.E on sperm anomalies in sodium arsenite treated rats. Materials and Methods: Adult male rats were divided into 4 groups: control, sodium arsenite (8 mg/kg/day), Vit.E (100 mg/kg/day) and sodium arsenite+Vit.E. Oral treatments were performed till 8 weeks. Body and left testis weight were recorded and then left caudal epididymis was cut in Ham's F10. Released spermatozoa were used to analyze number, motility, viability and abnormalities of the sperm. Sperm chromatin quality was assessed by nuclear staining using acridine orange and aniline blue. Results: Body and testis weight showed no significant change in 4 groups (p>0.05). A significant decrease in the number, motility, viability and normal sperm morphology was found in sodium arsenite-treated rats compared to the control (p<0.001). Sodium arsenite had no effect on sperm DNA integrity and histon-protamine replacement (p>0.05). In sodium arsenite+Vit.E group, Vit.E could significantly compensate the harmful effects of sodium arsenite on sperm number, motility, viability and morphology compared to sodium arsenite group. In addition, sperm viability and motility was significantly increased in rats treated with Vit.E alone compared to the control and sodium arsenite+Vit.E group. Conclusion: Vitamin E could compensate the adverse effects of sodium arsenite on sperm parameters in adult rats. PMID:25243001

  6. A new large-DNA-fragment delivery system based on integrase activity from an integrative and conjugative element.

    PubMed

    Miyazaki, Ryo; van der Meer, Jan Roelof

    2013-07-01

    During the past few decades, numerous plasmid vectors have been developed for cloning, gene expression analysis, and genetic engineering. Cloning procedures typically rely on PCR amplification, DNA fragment restriction digestion, recovery, and ligation, but increasingly, procedures are being developed to assemble large synthetic DNAs. In this study, we developed a new gene delivery system using the integrase activity of an integrative and conjugative element (ICE). The advantage of the integrase-based delivery is that it can stably introduce a large DNA fragment (at least 75 kb) into one or more specific sites (the gene for glycine-accepting tRNA) on a target chromosome. Integrase recombination activity in Escherichia coli is kept low by using a synthetic hybrid promoter, which, however, is unleashed in the final target host, forcing the integration of the construct. Upon integration, the system is again silenced. Two variants with different genetic features were produced, one in the form of a cloning vector in E. coli and the other as a mini-transposable element by which large DNA constructs assembled in E. coli can be tagged with the integrase gene. We confirmed that the system could successfully introduce cosmid and bacterial artificial chromosome (BAC) DNAs from E. coli into the chromosome of Pseudomonas putida in a site-specific manner. The integrase delivery system works in concert with existing vector systems and could thus be a powerful tool for synthetic constructions of new metabolic pathways in a variety of host bacteria. PMID:23686268

  7. Genetic analysis of the virD operon of Agrobacterium tumefaciens: a search for functions involved in transport of T-DNA into the plant cell nucleus and in T-DNA integration.

    PubMed Central

    Koukolíková-Nicola, Z; Raineri, D; Stephens, K; Ramos, C; Tinland, B; Nester, E W; Hohn, B

    1993-01-01

    The transferred DNA (T-DNA) is transported from Agrobacterium tumefaciens to the nucleus and is stably integrated into the genome of many plant species. It has been proposed that the VirD2 protein, tightly attached to the T-DNA, pilots the T-DNA into the plant cell nucleus and that it is involved in integration. Using agroinfection and beta-glucuronidase expression as two different very sensitive transient assays for T-DNA transfer, together with assays for stable integration, we have shown that the C-terminal half of the VirD2 protein and the VirD3 protein are not involved in T-DNA integration. However, the bipartite nuclear localization signal, which is located within the C terminus of the VirD2 protein and which has previously been shown to be able to target a foreign protein into the plant cell nucleus, was shown to be required for efficient T-DNA transfer. virD4 mutants were shown by agroinfection to be completely inactive in T-DNA transfer. Images PMID:8380800

  8. 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...

  9. Inhibition of recombinase polymerase amplification by background DNA: a lateral flow-based method for enriching target DNA.

    PubMed

    Rohrman, Brittany; Richards-Kortum, Rebecca

    2015-02-01

    Recombinase polymerase amplification (RPA) may be used to detect a variety of pathogens, often after minimal sample preparation. However, previous work has shown that whole blood inhibits RPA. In this paper, we show that the concentrations of background DNA found in whole blood prevent the amplification of target DNA by RPA. First, using an HIV-1 RPA assay with known concentrations of nonspecific background DNA, we show that RPA tolerates more background DNA when higher HIV-1 target concentrations are present. Then, using three additional assays, we demonstrate that the maximum amount of background DNA that may be tolerated in RPA reactions depends on the DNA sequences used in the assay. We also show that changing the RPA reaction conditions, such as incubation time and primer concentration, has little effect on the ability of RPA to function when high concentrations of background DNA are present. Finally, we develop and characterize a lateral flow-based method for enriching the target DNA concentration relative to the background DNA concentration. This sample processing method enables RPA of 10(4) copies of HIV-1 DNA in a background of 0-14 μg of background DNA. Without lateral flow sample enrichment, the maximum amount of background DNA tolerated is 2 μg when 10(6) copies of HIV-1 DNA are present. This method requires no heating or other external equipment, may be integrated with upstream DNA extraction and purification processes, is compatible with the components of lysed blood, and has the potential to detect HIV-1 DNA in infant whole blood with high proviral loads. PMID:25560368

  10. Mycobacterium avium Possesses Extracellular DNA that Contributes to Biofilm Formation, Structural Integrity, and Tolerance to Antibiotics

    PubMed Central

    Rose, Sasha J.; Babrak, Lmar M.; Bermudez, Luiz E.

    2015-01-01

    Mycobacterium avium subsp. hominissuis is an opportunistic pathogen that is associated with biofilm-related infections of the respiratory tract and is difficult to treat. In recent years, extracellular DNA (eDNA) has been found to be a major component of bacterial biofilms, including many pathogens involved in biofilm-associated infections. To date, eDNA has not been described as a component of mycobacterial biofilms. In this study, we identified and characterized eDNA in a high biofilm-producing strain of Mycobacterium avium subsp. hominissuis (MAH). In addition, we surveyed for presence of eDNA in various MAH strains and other nontuberculous mycobacteria. Biofilms of MAH A5 (high biofilm-producing strain) and MAH 104 (reference strain) were established at 22°C and 37°C on abiotic surfaces. Acellular biofilm matrix and supernatant from MAH A5 7 day-old biofilms both possess abundant eDNA, however very little eDNA was found in MAH 104 biofilms. A survey of MAH clinical isolates and other clinically relevant nontuberculous mycobacterial species revealed many species and strains that also produce eDNA. RAPD analysis demonstrated that eDNA resembles genomic DNA. Treatment with DNase I reduced the biomass of MAH A5 biofilms when added upon biofilm formation or to an already established biofilm both on abiotic surfaces and on top of human pharyngeal epithelial cells. Furthermore, co-treatment of an established biofilm with DNase 1 and either moxifloxacin or clarithromycin significantly increased the susceptibility of the bacteria within the biofilm to these clinically used antimicrobials. Collectively, our results describe an additional matrix component of mycobacterial biofilms and a potential new target to help treat biofilm-associated nontuberculous mycobacterial infections. PMID:26010725

  11. Human Papilloma Viral DNA Replicates as a Stable Episome in Cultured Epidermal Keratinocytes

    NASA Astrophysics Data System (ADS)

    Laporta, Robert F.; Taichman, Lorne B.

    1982-06-01

    Human papilloma virus (HPV) is poorly understood because systems for its growth in tissue culture have not been developed. We report here that cultured human epidermal keratinocytes could be infected with HPV from plantar warts and that the viral DNA persisted and replicated as a stable episome. There were 50-200 copies of viral DNA per cell and there was no evidence to indicate integration of viral DNA into the cellular genome. There was also no evidence to suggest that viral DNA underwent productive replication. We conclude that cultured human epidermal keratinocytes may be a model for the study of certain aspects of HPV biology.

  12. Plasmid Copy Number Determination by Quantitative Polymerase Chain Reaction.

    PubMed

    Anindyajati; Artarini, A Anita; Riani, Catur; Retnoningrum, Debbie S

    2016-01-01

    that both primer pairs were acceptable and were predicted to have good performance. Those predictions were in agreement with the in vitro test that gave a single band in the PCR product's electropherogram and a single peak in DNA amplicon's melting curve with a Tm value of 79.01 ± 0.11°C for the tdk primer and 81.53 ± 0.29°C for the ori primer. The efficiency of each primer was 1.95 and 1.97, respectively. The calculation result of pCAD's copy number was 13.1 ± 0.3 copies/cell, showing that pCAD's low copy number has been determined and confirmed. Meanwhile, it was 576.3 ± 91.9 copies/cell for pJExpress414-sod, in accordance with the hypothesis that pUC ori regulates the high copy number plasmid. In conclusion, the designed primers and qPCR conditions used in this study can be used to determine plasmid copy number for plasmids with pBR322 and pUC ori. The method should be tested further on plasmids harboring other type of ori. PMID:27110501

  13. The role of mutation in genetic copy number variation

    NASA Astrophysics Data System (ADS)

    Clark, B. K.; Weidner, Jacob; Wabick, Kevin

    2010-03-01

    Until very recently, the standard model of DNA included two genes for each trait. This dated model has given way to a model that includes copies of some genes well in excess of the canonical two. Copy number variations in the human genome play critical roles in causing or aggravating a number of syndromes and diseases while providing increased resistance to others. We explore the role of mutation, crossover, inversion, and reproduction in determining copy number variations in a numerical simulation of a population. The numerical model consists of a population of individuals, where each individual is represented by a single strand of DNA with the same number of genes. Each gene is initially assigned to one of two traits. Fitness of the individual is determined by the two most fit genes for trait one, and trait two genetic material is treated as a reservoir of junk DNA. After a sufficient number of generations, during which the genetic distribution is allowed to reach a steady-state, the mean number of genes per trait and the copy number variation are recorded. Here, we focus on the role of mutation and compare simulation results to theory.

  14. Sperm DNA integrity in frozen-thawed semen from Italian Mediterranean Buffalo bulls and its relationship to in vivo fertility.

    PubMed

    Serafini, Rosanna; Love, Charles C; Coletta, Angelo; Mari, Gaetano; Mislei, Beatrice; Caso, Chiara; Di Palo, Rossella

    2016-09-01

    The relationship among sperm attributes of DNA integrity, sperm motility, morphology, viability, acrosome integrity and in vivo fertility of frozen-thawed Italian Mediterranean Buffalo (IMB) sperm has not been reported. Straws of frozen-thawed semen samples from three bulls were examined. Sperm DNA assays (i.e., neutral Comet assay, Sperm Bos Halomax-SBH and Sperm Chromatin Structure Assay-SCSA) were not correlated to each other (P>0.05). Many neutral Comet assay measures were correlated to total sperm motility-TMOT (% head-H-DNA, r=0.74; Olive moment, r=-0.76; P<0.05) and coiled tails (r-values ranged from% H-DNA, r=-0.80 to tail length, r=-0.71; P<0.05). The COMP-αt was negatively correlated to viable acrosome intact (VAI) sperm, and distal droplets (r=-0.60 and -0.61; P<0.05), whereas Mean-αt and Mode-αt were positively correlated to bent midpieces (r=0.63 and 0.61; P<0.05). The SBH assay was positively correlated to non-viable acrosome damaged (NVAD) sperm (r=0.60; P<0.05) and negatively correlated to viable acrosome damaged (VAD) sperm (r=-0.63; P<0.05). The overall pregnancy rate (PR-at 30 and 45d post artificial insemination-AI) and the calving rate were 57%, 55% and 45%, respectively. Among sperm features analyzed the area under the Receiver Operating Characteristic (ROC) Curve was significant (P<0.05) for TMOT, NVAD, Standard Deviation-αt (SD-αt) and neutral comet measures (Olive tail moment and tail moment, % H- DNA and tail area) in estimating pregnancy. PMID:27421229

  15. Performance of Molecular Inversion Probes (MIP) in Allele CopyNumber Determination

    SciTech Connect

    Wang, Yuker; Moorhead, Martin; Karlin-Neumann, George; Wang,Nicolas J.; Ireland, James; Lin, Steven; Chen, Chunnuan; Heiser, LauraM.; Chin, Koei; Esserman, Laura; Gray, Joe W.; Spellman, Paul T.; Faham,Malek

    2007-05-14

    We have developed a new protocol for using MolecularInversion Probes (MIP) to accurately and specifically measure allele copynumber (ACN). The new protocol provides for significant improvementsincluding the reduction of input DNA (from 2?g) by more than 25 fold (to75ng total genomic DNA), higher overall precision resulting in one orderof magnitude lower false positive rate, and greater dynamic range withaccurate absolute copy number up to 60 copies.

  16. Overproduction of Bacillus amyloliquefaciens extracellular glutamyl-endopeptidase as a result of ectopic multi-copy insertion of an efficiently-expressed mpr gene into the Bacillus subtilis chromosome

    PubMed Central

    2011-01-01

    Background Plasmid-less, engineered Bacillus strains have several advantages over plasmid-carrier variants. Specifically, their stability and potential ecological safety make them of use in industrial applications. As a rule, however, it is necessary to incorporate many copies of a key gene into a chromosome to achieve strain performance that is comparable to that of cells carrying multiple copies of a recombinant plasmid. Results A plasmid-less B. subtilis JE852-based strain secreting glutamyl-specific protease (GSP-the protein product of the mpr gene from B. amyloliquefaciens) was constructed that exhibits decreased levels of other extracellular proteases. Ten copies of an mprB.amy cassette in which the GSP gene was placed between the promoter of the B. amyloliquefaciens rplU-rpmA genes and the Rho-independent transcription terminator were ectopically inserted into designated (3 copies) and random (7 copies) points in the recipient chromosome. The resulting strain produced approximately 0.5 g/L of secreted GSP after bacterial cultivation in flasks with starch-containing media, and its performance was comparable to an analogous strain in which the mprB.amy cassette was carried on a multi-copy plasmid. Conclusion A novel strategy for ectopically integrating a cassette into multiple random locations in the B. subtilis chromosome was developed. This new method is based on the construction of DNA fragments in which the desired gene, marked by antibiotic resistance, is sandwiched between "front" and "back" portions of random chromosomal DNA restriction fragments. These fragments were subsequently inserted into the targeted sites of the chromosome using double-cross recombination. The construction of a marker-free strain was achieved by gene conversion between the integrated marked gene and a marker-less variant carried by plasmid DNA, which was later removed from the cells. PMID:21819557

  17. Integrated analysis of DNA methylation profiles and gene expression profiles to identify genes associated with pilocytic astrocytomas.

    PubMed

    Zhou, Ruigang; Man, Yigang

    2016-04-01

    The present study performed an integral analysis of the gene expression and DNA methylation profile of pilocytic astrocytomas (PAs). Weighted gene co-expression network analysis (WGCNA) was also performed to examine and identify the genes correlated to PAs, to identify candidate therapeutic targets for the treatment of PAs. The DNA methylation profile and gene expression profile were downloaded from the Gene Expression Omnibus database. Following screening of the differentially expressed genes (DEGs) and differentially methylated regions (DMRs), respectively, integrated analysis of the DEGs and DMRs was performed to detect their correlation. Subsequently, the WGCNA algorithm was applied to identify the significant modules and construct the co‑expression network associated with PAs. Furthermore, Gene Ontology enrichment analysis of the associated genes was performed using the Database for Annotation, Visualization and Integrated Discovery. A total number of 2,259 DEGs and 235 DMRs were screened out. Integrated analysis revealed that 30 DEGs were DMRs with prominent negative correlation (cor=‑0.82; P=0.02). Based on the DEGs, the gene co‑expression network was constructed, and nine network modules associated with PAs were identified. The functional analysis results showed that genes relevant to PAs were closely associated with cell differentiation modulation. The screened PA-associated genes were significantly different at the expression and methylation levels. These genes may be used as reliable candidate target genes for the treatment of PAs. PMID:26934913

  18. Integrated analysis of DNA methylation profiles and gene expression profiles to identify genes associated with pilocytic astrocytomas

    PubMed Central

    ZHOU, RUIGANG; MAN, YIGANG

    2016-01-01

    The present study performed an integral analysis of the gene expression and DNA methylation profile of pilocytic astrocytomas (PAs). Weighted gene co-expression network analysis (WGCNA) was also performed to examine and identify the genes correlated to PAs, to identify candidate therapeutic targets for the treatment of PAs. The DNA methylation profile and gene expression profile were downloaded from the Gene Expression Omnibus database. Following screening of the differentially expressed genes (DEGs) and differentially methylated regions (DMRs), respectively, integrated analysis of the DEGs and DMRs was performed to detect their correlation. Subsequently, the WGCNA algorithm was applied to identify the significant modules and construct the co-expression network associated with PAs. Furthermore, Gene Ontology enrichment analysis of the associated genes was performed using the Database for Annotation, Visualization and Integrated Discovery. A total number of 2,259 DEGs and 235 DMRs were screened out. Integrated analysis revealed that 30 DEGs were DMRs with prominent negative correlation (cor=−0.82; P=0.02). Based on the DEGs, the gene co-expression network was constructed, and nine network modules associated with PAs were identified. The functional analysis results showed that genes relevant to PAs were closely associated with cell differentiation modulation. The screened PA-associated genes were significantly different at the expression and methylation levels. These genes may be used as reliable candidate target genes for the treatment of PAs. PMID:26934913

  19. Retrotransposon mdg3 of Drosophila: General structure and functional domains of the full-length copy

    SciTech Connect

    Avedisov, S.N.; Ilyin, Yu.V.

    1995-09-01

    A full-length copy of the transposable element mdg3 from the plasmid clone Dm38 of Drosophila melanogaster was obtained by screening the DNA library of the cell culture 67J25D. Previous work demonstrated that only full-length copies of mdg3 (5.5 kb) are amplified in this culture, whereas the number of deleted copies probably has not changed since the cell line was established. We sequenced the full-length copy of mdg3 from cDm38 by the method described by Sanger. 10 refs., 2 figs., 2 tabs.

  20. EVALUATION OF CHROMOSOME BREAKAGE AND DNA INTEGRITY IN SPERM: AN INVESTIGATION OF REMOTE SEMEN COLLECTION CONDITIONS

    EPA Science Inventory

    Home collection of ejaculated semen would facilitate participation rates and geographic diversity in reproductive epidemiology studies. Our study addressed concerns that home collection and overnight mail return might induce chromosome/DNA damage. We collected semen from 10 hea...

  1. An integrated microfluidic device for rapid cell lysis and DNA purification of epithelial cell samples.

    PubMed

    Ha, Seung-Mo; Cho, Woong; Ahn, Yoomin; Hwang, Seung Yong

    2011-05-01

    In this paper, we describe the design and fabrication of a microfluidic device for cell lysis and DNA purification, and the results of device tests using a real sample of buccal cells. Cell lysis was thermally executed for two minutes at 80 degrees C in a serpentine type microreactor (20 microL) using an Au microheater with a microsensor. The DNA was then mixed with other residual products and purified by a new filtration process involving micropillars and 50-80 microm microbeads. The entire process of sample loading, cell lysis, DNA purification, and sample extraction was successfully completed in the microchip within five minutes. Sample preparation within the microchip was verified by performing a SY158 gene PCR analysis and gel electrophoresis on the products obtained from the chip. The new purification method enhanced DNA purity from 0.93 to 1.62 after purification. PMID:21780436

  2. DNA Integrity and Shock Wave Transformation Efficiency of Bacteria and Fungi

    NASA Astrophysics Data System (ADS)

    Loske, Achim M.; Campos-Guillén, Juan; Fernández, Francisco; Pastrana, Xóchitl; Magaña-Ortíz, Denis; Coconi-Linares, Nancy; Ortíz-Vázquez, Elizabeth; Gómez-Lim, Miguel

    Delivery of DNA into bacteria and fungi is essential in medicine and biotechnology to produce metabolites, enzymes, antibiotics and proteins. So far, protocols to genetically transform bacteria and fungi are inefficient and have low reproducibility.

  3. Copy number variations alter methylation and parallel IGF2 overexpression in adrenal tumors

    PubMed Central

    Nielsen, Helene Myrtue; How-Kit, Alexandre; Guerin, Carole; Castinetti, Frederic; Vollan, Hans Kristian Moen; De Micco, Catherine; Daunay, Antoine; Taieb, David; Van Loo, Peter; Besse, Celine; Kristensen, Vessela N; Hansen, Lise Lotte; Barlier, Anne; Sebag, Frederic; Tost, Jörg

    2015-01-01

    Overexpression of insulin growth factor 2 (IGF2) is a hallmark of adrenocortical carcinomas and pheochromocytomas. Previous studies investigating the IGF2/H19 locus have mainly focused on a single molecular level such as genomic alterations or altered DNA methylation levels and the causal changes underlying IGF2 overexpression are still not fully established. In the current study, we analyzed 62 tumors of the adrenal gland from patients with Conn's adenoma (CA, n=12), pheochromocytomas (PCC, n=10), adrenocortical benign tumors (ACBT, n=20), and adrenocortical carcinomas (ACC, n=20). Gene expression, somatic copy number variation of chr11p15.5, and DNA methylation status of three differential methylated regions of the IGF2/H19 locus including the H19 imprinting control region were integratively analyzed. IGF2 overexpression was found in 85% of the ACCs and 100% of the PCCs compared to 23% observed in CAs and ACBTs. Copy number aberrations of chr11p15.5 were abundant in both PCCs and ACCs but while PCCs retained a diploid state, ACCs were frequently tetraploid (7/19). Loss of either a single allele or loss of two alleles of the same parental origin in tetraploid samples resulted in a uniparental disomy-like genotype. These copy number changes correlated with hypermethylation of the H19 ICR suggesting that the lost alleles were the unmethylated maternal alleles. Our data provide conclusive evidence that loss of the maternal allele correlates with IGF2 overexpression in adrenal tumors and that hypermethylation of the H19 ICR is a consequence thereof. PMID:26400872

  4. Copy number variations alter methylation and parallel IGF2 overexpression in adrenal tumors.

    PubMed

    Nielsen, Helene Myrtue; How-Kit, Alexandre; Guerin, Carole; Castinetti, Frederic; Vollan, Hans Kristian Moen; De Micco, Catherine; Daunay, Antoine; Taieb, David; Van Loo, Peter; Besse, Celine; Kristensen, Vessela N; Hansen, Lise Lotte; Barlier, Anne; Sebag, Frederic; Tost, Jörg

    2015-12-01

    Overexpression of insulin growth factor 2 (IGF2) is a hallmark of adrenocortical carcinomas and pheochromocytomas. Previous studies investigating the IGF2/H19 locus have mainly focused on a single molecular level such as genomic alterations or altered DNA methylation levels and the causal changes underlying IGF2 overexpression are still not fully established. In the current study, we analyzed 62 tumors of the adrenal gland from patients with Conn's adenoma (CA, n=12), pheochromocytomas (PCC, n=10), adrenocortical benign tumors (ACBT, n=20), and adrenocortical carcinomas (ACC, n=20). Gene expression, somatic copy number variation of chr11p15.5, and DNA methylation status of three differential methylated regions of the IGF2/H19 locus including the H19 imprinting control region were integratively analyzed. IGF2 overexpression was found in 85% of the ACCs and 100% of the PCCs compared to 23% observed in CAs and ACBTs. Copy number aberrations of chr11p15.5 were abundant in both PCCs and ACCs but while PCCs retained a diploid state, ACCs were frequently tetraploid (7/19). Loss of either a single allele or loss of two alleles of the same parental origin in tetraploid samples resulted in a uniparental disomy-like genotype. These copy number changes correlated with hypermethylation of the H19 ICR suggesting that the lost alleles were the unmethylated maternal alleles. Our data provide conclusive evidence that loss of the maternal allele correlates with IGF2 overexpression in adrenal tumors and that hypermethylation of the H19 ICR is a consequence thereof. PMID:26400872

  5. Mapping copy number variation by population-scale genome sequencing.

    PubMed

    Mills, Ryan E; Walter, Klaudia; Stewart, Chip; Handsaker, Robert E; Chen, Ken; Alkan, Can; Abyzov, Alexej; Yoon, Seungtai Chris; Ye, Kai; Cheetham, R Keira; Chinwalla, Asif; Conrad, Donald F; Fu, Yutao; Grubert, Fabian; Hajirasouliha, Iman; Hormozdiari, Fereydoun; Iakoucheva, Lilia M; Iqbal, Zamin; Kang, Shuli; Kidd, Jeffrey M; Konkel, Miriam K; Korn, Joshua; Khurana, Ekta; Kural, Deniz; Lam, Hugo Y K; Leng, Jing; Li, Ruiqiang; Li, Yingrui; Lin, Chang-Yun; Luo, Ruibang; Mu, Xinmeng Jasmine; Nemesh, James; Peckham, Heather E; Rausch, Tobias; Scally, Aylwyn; Shi, Xinghua; Stromberg, Michael P; Stütz, Adrian M; Urban, Alexander Eckehart; Walker, Jerilyn A; Wu, Jiantao; Zhang, Yujun; Zhang, Zhengdong D; Batzer, Mark A; Ding, Li; Marth, Gabor T; McVean, Gil; Sebat, Jonathan; Snyder, Michael; Wang, Jun; Ye, Kenny; Eichler, Evan E; Gerstein, Mark B; Hurles, Matthew E; Lee, Charles; McCarroll, Steven A; Korbel, Jan O

    2011-02-01

    Genomic structural variants (SVs) are abundant in humans, differing from other forms of variation in extent, origin and functional impact. Despite progress in SV characterization, the nucleotide resolution architecture of most SVs remains unknown. We constructed a map of unbalanced SVs (that is, copy number variants) based on whole genome DNA sequencing data from 185 human genomes, integrating evidence from complementary SV discovery approaches with extensive experimental validations. Our map encompassed 22,025 deletions and 6,000 additional SVs, including insertions and tandem duplications. Most SVs (53%) were mapped to nucleotide resolution, which facilitated analysing their origin and functional impact. We examined numerous whole and partial gene deletions with a genotyping approach and observed a depletion of gene disruptions amongst high frequency deletions. Furthermore, we observed differences in the size spectra of SVs originating from distinct formation mechanisms, and constructed a map of SV hotspots formed by common mechanisms. Our analytical framework and SV map serves as a resource for sequencing-based association studies. PMID:21293372

  6. Integrated Stochastic Model of DNA Damage Repair by Non-homologous End Joining and p53/p21- Mediated Early Senescence Signalling

    PubMed Central

    Nelson, Glyn; Hall, Philip; Miwa, Satomi; Kirkwood, Thomas B. L.; Shanley, Daryl P.

    2015-01-01

    Unrepaired or inaccurately repaired DNA damage can lead to a range of cell fates, such as apoptosis, cellular senescence or cancer, depending on the efficiency and accuracy of DNA damage repair and on the downstream DNA damage signalling. DNA damage repair and signalling have been studied and modelled in detail separately, but it is not yet clear how they integrate with one another to control cell fate. In this study, we have created an integrated stochastic model of DNA damage repair by non-homologous end joining and of gamma irradiation-induced cellular senescence in human cells that are not apoptosis-prone. The integrated model successfully explains the changes that occur in the dynamics of DNA damage repair after irradiation. Simulations of p53/p21 dynamics after irradiation agree well with previously published experimental studies, further validating the model. Additionally, the model predicts, and we offer some experimental support, that low-dose fractionated irradiation of cells leads to temporal patterns in p53/p21 that lead to significant cellular senescence. The integrated model is valuable for studying the processes of DNA damage induced cell fate and predicting the effectiveness of DNA damage related medical interventions at the cellular level. PMID:26020242

  7. Drosophila Uri, a PP1α binding protein, is essential for viability, maintenance of DNA integrity and normal transcriptional activity

    PubMed Central

    Kirchner, Jasmin; Vissi, Emese; Gross, Sascha; Szoor, Balazs; Rudenko, Andrey; Alphey, Luke; White-Cooper, Helen

    2008-01-01

    Background Protein phosphatase 1 (PP1) is involved in diverse cellular processes, and is targeted to substrates via interaction with many different protein binding partners. PP1 catalytic subunits (PP1c) fall into PP1α and PP1β subfamilies based on sequence analysis, however very few PP1c binding proteins have been demonstrated to discriminate between PP1α and PP1β. Results URI (unconventional prefoldin RPB5 interactor) is a conserved molecular chaperone implicated in a variety of cellular processes, including the transcriptional response to nutrient signalling and maintenance of DNA integrity. We show that Drosophila Uri binds PP1α with much higher affinity than PP1β, and that this ability to discriminate between PP1c forms is conserved to humans. Most Uri is cytoplasmic, however we found some protein associated with active RNAPII on chromatin. We generated a uri loss of function allele, and show that uri is essential for viability in Drosophila. uri mutants have transcriptional defects, reduced cell viability and differentiation in the germline, and accumulate DNA damage in their nuclei. Conclusion Uri is the first PP1α specific binding protein to be described in Drosophila. Uri protein plays a role in transcriptional regulation. Activity of uri is required to maintain DNA integrity and cell survival in normal development. PMID:18412953

  8. 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.

  9. Repetitive, marker-free, site-specific integration as a novel tool for multiple chromosomal integration of DNA.

    PubMed

    Petersen, Kia Vest; Martinussen, Jan; Jensen, Peter Ruhdal; Solem, Christian

    2013-06-01

    We present a tool for repetitive, marker-free, site-specific integration in Lactococcus lactis, in which a nonreplicating plasmid vector (pKV6) carrying a phage attachment site (attP) can be integrated into a bacterial attachment site (attB). The novelty of the tool described here is the inclusion of a minimal bacterial attachment site (attB(min)), two mutated loxP sequences (lox66 and lox71) allowing for removal of undesirable vector elements (antibiotic resistance marker), and a counterselection marker (oroP) for selection of loxP recombination on the pKV6 vector. When transformed into L. lactis expressing the phage TP901-1 integrase, pKV6 integrates with high frequency into the chromosome, where it is flanked by attL and attR hybrid attachment sites. After expression of Cre recombinase from a plasmid that is not able to replicate in L. lactis, loxP recombinants can be selected for by using 5-fluoroorotic acid. The introduced attB(min) site can subsequently be used for a second round of integration. To examine if attP recombination was specific to the attB site, integration was performed in strains containing the attB, attL, and attR sites or the attL and attR sites only. Only attP-attB recombination was observed when all three sites were present. In the absence of the attB site, a low frequency of attP-attL recombination was observed. To demonstrate the functionality of the system, the xylose utilization genes (xylABR and xylT) from L. lactis strain KF147 were integrated into the chromosome of L. lactis strain MG1363 in two steps. PMID:23542630

  10. Tight regulation of ubiquitin-mediated DNA damage response by USP3 preserves the functional integrity of hematopoietic stem cells

    PubMed Central

    Lancini, Cesare; van den Berk, Paul C.M.; Vissers, Joseph H.A.; Gargiulo, Gaetano; Song, Ji-Ying; Hulsman, Danielle; Serresi, Michela; Tanger, Ellen; Blom, Marleen; Vens, Conchita; van Lohuizen, Maarten; Jacobs, Heinz

    2014-01-01

    Histone ubiquitination at DNA breaks is required for activation of the DNA damage response (DDR) and DNA repair. How the dynamic removal of this modification by deubiquitinating enzymes (DUBs) impacts genome maintenance in vivo is largely unknown. To address this question, we generated mice deficient for Ub-specific protease 3 (USP3; Usp3Δ/Δ), a histone H2A DUB which negatively regulates ubiquitin-dependent DDR signaling. Notably, USP3 deletion increased the levels of histone ubiquitination in adult tissues, reduced the hematopoietic stem cell (HSC) reserves over time, and shortened animal life span. Mechanistically, our data show that USP3 is important in HSC homeostasis, preserving HSC self-renewal, and repopulation potential in vivo and proliferation in vitro. A defective DDR and unresolved spontaneous DNA damage contribute to cell cycle restriction of Usp3Δ/Δ HSCs. Beyond the hematopoietic system, Usp3Δ/Δ animals spontaneously developed tumors, and primary Usp3Δ/Δ cells failed to preserve chromosomal integrity. These findings broadly support the regulation of chromatin ubiquitination as a key pathway in preserving tissue function through modulation of the response to genotoxic stress. PMID:25113974

  11. Incidental copy-number variants identified by routine genome testing in a clinical population

    PubMed Central

    Boone, Philip M.; Soens, Zachry T.; Campbell, Ian M.; Stankiewicz, Pawel; Cheung, Sau Wai; Patel, Ankita; Beaudet, Arthur L.; Plon, Sharon E.; Shaw, Chad A.; McGuire, Amy L.; Lupski, James R.

    2013-01-01

    Purpose Mutational load of susceptibility variants has not been studied on a genomic scale in a clinical population, nor has the potential to identify these mutations as incidental findings during clinical testing been systematically ascertained. Methods Array comparative genomic hybridization, a method for genome-wide detection of DNA copy-number variants, was performed clinically on DNA from 9,005 individuals. Copy-number variants encompassing or disrupting single genes were identified and analyzed for their potential to confer predisposition to dominant, adult-onset disease. Multigene copy-number variants affecting dominant, adult-onset cancer syndrome genes were also assessed. Results In our cohort, 83 single-gene copy-number variants affected 40 unique genes associated with dominant, adult-onset disorders and unrelated to the patients’ referring diagnoses (i.e., incidental) were found. Fourteen of these copy-number variants are likely disease-predisposing, 25 are likely benign, and 44 are of unknown clinical consequence. When incidental copy-number variants spanning up to 20 genes were considered, 27 copy-number variants affected 17 unique genes associated with dominant, adult-onset cancer predisposition. Conclusion Copy-number variants potentially conferring susceptibility to adult-onset disease can be identified as incidental findings during routine genome-wide testing. Some of these mutations may be medically actionable, enabling disease surveillance or prevention; however, most incidentally observed single-gene copy-number variants are currently of unclear significance to the patient. PMID:22878507

  12. 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 through the Integrative Studies Element of the former NABIR Program (now the Environmental Remediation Sciences Program) within the Office of Biological and Environmental Research. Dr. Darrell Chandler (originally at Argonne National Laboratory, now with Akonni Biosystems) was the overall PI/PD for the project. The overall project goals were to (1) apply a model iron-reducer and sulfate-reducer microarray and instrumentation systems to sediment and groundwater samples from the Scheibe et al. FRC Area 2 field site, UMTRA sediments, and other DOE contaminated sites; (2) continue development and expansion of a 16S rRNA/rDNA¬-targeted probe suite for microbial community dynamics as new sequences are obtained from DOE-relevant sites; and (3) address the fundamental molecular biology and analytical chemistry associated with the extraction, purification and analysis of functional genes and mRNA in environmental samples. Work on the UW subproject focused on conducting detailed batch and semicontinuous culture reactor experiments with uranium-contaminated FRC Area 2 sediment. The reactor experiments were designed to provide coherent geochemical and microbiological data in support of microarray analyses of microbial communities in Area 2 sediments undergoing biostimulation with ethanol. A total of four major experiments were conducted (one batch and three semicontinuous culture), three of which (the batch and two semicontinuous culture) provided samples for DNA microarray analysis. A variety of other molecular analyses (clone libraries, 16S PhyloChip, RT-PCR, and T-RFLP) were conducted on parallel samples from the various experiments in order to provide independent information on microbial community response to biostimulation.

  13. Trans-activation function of a 3 prime truncated X gene-cell fusion product from integrated hepatitis B virus DNA in chronic hepatitis tissues

    SciTech Connect

    Takada, Shinako; Koike, Katsuro )

    1990-08-01

    To investigate the expression and transactivation function of the X gene in integrated hepatitis B virus (HBV) DNA from chronic hepatitis tissues, a series of transfectants containing cloned integrated HBV DNAs was made and analyzed for X mRNA expression and trans-activation activity by using a chloramphenicol acetyltransferase assay. Most of the integrated HBV DNAs expressed X mRNA and encoded a product with trans-activation activity in spite of the loss of the 3{prime} end region of the X gene due to integration. From cDNA cloning and sequence analysis of X mRNA transcribed from native or integrated HBV DNA, the X protein was found to be translated from the X open reading frame without splicing. For integrated HBV DNA, transcription was extended to a cellular flanking DNA and an X gene-cell fusion transcript was terminated by using a cellular poly(A) signal. The amino acid sequence deduced from an X-cell fusion transcript indicated truncation of the carboxyl-terminal five amino acids, but the upstream region of seven amino acids conserved among hepadnaviruses was retained in the integrated HBV DNA, suggesting that this conserved region is essential for the transactivation function of the X protein. These findings support the following explanation for hepatocarcinogenesis by HBV DNA integration: the expression of a cellular oncogene(s) is transactivated at the time of chronic infection by the increasing amounts of the integrated HBV gene product(s), such as the X-cell fusion product.

  14. Copy variations in schizophrenia and bipolar disorder

    PubMed Central

    Lachman, H.M.

    2009-01-01

    The analysis of copy number variations (CNVs) is an emerging tool for identifying genetic factors underlying complex traits. In this chapter I will review studies that have been carried out showing that CNVs play a role in the development of two such complex traits; schizophrenia (SZ) and bipolar disorder (BD). There are two aspects to consider regarding the role of copy variations in these conditions. One is gene discovery in which DNA from patients is analyzed for the purpose of identifying rare, patient-specific CNVs that may be informative to a larger population of affected individuals. The model for this concept is based on the emergence of DISC1 as a SZ candidate gene, which was discovered in a single informative family with a rare chromosomal translocation. Another aspect revolves around the idea that polymorphic CNVs found in the general population, many of which appear to disrupt previously identified SZ and BD candidate genes, contribute to disease pathogenesis. Here, gene-disrupting CNVs are viewed in the same manner as functional SNPs and analyzed for involvement in disease susceptibility using genetic association. Although the analysis of CNVs in patients with psychiatric disorders is in its infancy, informative new findings have already been made, suggesting that this is a very promising line of research. PMID:19287136

  15. RIP1 maintains DNA integrity and cell proliferation by regulating PGC-1α-mediated mitochondrial oxidative phosphorylation and glycolysis

    PubMed Central

    Chen, W; Wang, Q; Bai, L; Chen, W; Wang, X; Tellez, C S; Leng, S; Padilla, M T; Nyunoya, T; Belinsky, S A; Lin, Y

    2014-01-01

    Aerobic glycolysis or the Warburg effect contributes to cancer cell proliferation; however, how this glucose metabolism pathway is precisely regulated remains elusive. Here we show that receptor-interacting protein 1 (RIP1), a cell death and survival signaling factor, regulates mitochondrial oxidative phosphorylation and aerobic glycolysis. Loss of RIP1 in lung cancer cells suppressed peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) expression, impairing mitochondrial oxidative phosphorylation and accelerating glycolysis, resulting in spontaneous DNA damage and p53-mediated cell proliferation inhibition. Thus, although aerobic glycolysis within a certain range favors cancer cell proliferation, excessive glycolysis causes cytostasis. Our data suggest that maintenance of glycolysis by RIP1 is pivotal to cancer cell energy homeostasis and DNA integrity and may be exploited for use in anticancer therapy. PMID:24583643

  16. Effects of a petunia scaffold/matrix attachment region on copy number dependency and stability of transgene expression in Nicotiana tabacum.

    PubMed

    Dietz-Pfeilstetter, Antje; Arndt, Nicola; Manske, Ulrike

    2016-04-01

    Transgenes in genetically modified plants are often not reliably expressed during development or in subsequent generations. Transcriptional gene silencing (TGS) as well as post-transcriptional gene silencing (PTGS) have been shown to occur in transgenic plants depending on integration pattern, copy number and integration site. In an effort to reduce position effects, to prevent read-through transcription and to provide a more accessible chromatin structure, a P35S-ß-glucuronidase (P35S-gus) transgene flanked by a scaffold/matrix attachment region from petunia (Petun-SAR), was introduced in Nicotiana tabacum plants by Agrobacterium tumefaciens mediated transformation. It was found that Petun-SAR mediates enhanced expression and copy number dependency up to 2 gene copies, but did not prevent gene silencing in transformants with multiple and rearranged gene copies. However, in contrast to the non-SAR transformants where silencing was irreversible and proceeded during long-term vegetative propagation and in progeny plants, gus expression in Petun-SAR plants was re-established in the course of development. Gene silencing was not necessarily accompanied by DNA methylation, while the gus transgene could still be expressed despite considerable CG methylation within the coding region. PMID:26732611

  17. FSHD: copy number variations on the theme of muscular dystrophy

    PubMed Central

    Cabianca, Daphne Selvaggia

    2010-01-01

    In humans, copy number variations (CNVs) are a common source of phenotypic diversity and disease susceptibility. Facioscapulohumeral muscular dystrophy (FSHD) is an important genetic disease caused by CNVs. It is an autosomal-dominant myopathy caused by a reduction in the copy number of the D4Z4 macrosatellite repeat located at chromosome 4q35. Interestingly, the reduction of D4Z4 copy number is not sufficient by itself to cause FSHD. A number of epigenetic events appear to affect the severity of the disease, its rate of progression, and the distribution of muscle weakness. Indeed, recent findings suggest that virtually all levels of epigenetic regulation, from DNA methylation to higher order chromosomal architecture, are altered at the disease locus, causing the de-regulation of 4q35 gene expression and ultimately FSHD. PMID:21149563

  18. Genome Architecture and Its Roles in Human Copy Number Variation

    PubMed Central

    Chen, Lu; Zhou, Weichen; Zhang, Ling

    2014-01-01

    Besides single-nucleotide variants in the human genome, large-scale genomic variants, such as copy number variations (CNVs), are being increasingly discovered as a genetic source of human diversity and the pathogenic factors of diseases. Recent experimental findings have shed light on the links between different genome architectures and CNV mutagenesis. In this review, we summarize various genomic features and discuss their contributions to CNV formation. Genomic repeats, including both low-copy and high-copy repeats, play important roles in CNV instability, which was initially known as DNA recombination events. Furthermore, it has been found that human genomic repeats can also induce DNA replication errors and consequently result in CNV mutations. Some recent studies showed that DNA replication timing, which reflects the high-order information of genomic organization, is involved in human CNV mutations. Our review highlights that genome architecture, from DNA sequence to high-order genomic organization, is an important molecular factor in CNV mutagenesis and human genomic instability. PMID:25705150

  19. Integrating prior knowledge in multiple testing under dependence with applications to detecting differential DNA methylation.

    PubMed

    Kuan, Pei Fen; Chiang, Derek Y

    2012-09-01

    DNA methylation has emerged as an important hallmark of epigenetics. Numerous platforms including tiling arrays and next generation sequencing, and experimental protocols are available for profiling DNA methylation. Similar to other tiling array data, DNA methylation data shares the characteristics of inherent correlation structure among nearby probes. However, unlike gene expression or protein DNA binding data, the varying CpG density which gives rise to CpG island, shore and shelf definition provides exogenous information in detecting differential methylation. This article aims to introduce a robust testing and probe ranking procedure based on a nonhomogeneous hidden Markov model that incorporates the above-mentioned features for detecting differential methylation. We revisit the seminal work of Sun and Cai (2009, Journal of the Royal Statistical Society: Series B (Statistical Methodology)71, 393-424) and propose modeling the nonnull using a nonparametric symmetric distribution in two-sided hypothesis testing. We show that this model improves probe ranking and is robust to model misspecification based on extensive simulation studies. We further illustrate that our proposed framework achieves good operating characteristics as compared to commonly used methods in real DNA methylation data that aims to detect differential methylation sites. PMID:22260651

  20. PRMT5 Protects Genomic Integrity during Global DNA Demethylation in Primordial Germ Cells and Preimplantation Embryos

    PubMed Central

    Kim, Shinseog; Günesdogan, Ufuk; Zylicz, Jan J.; Hackett, Jamie A.; Cougot, Delphine; Bao, Siqin; Lee, Caroline; Dietmann, Sabine; Allen, George E.; Sengupta, Roopsha; Surani, M. Azim

    2014-01-01

    Summary Primordial germ cells (PGCs) and preimplantation embryos undergo epigenetic reprogramming, which includes comprehensive DNA demethylation. We found that PRMT5, an arginine methyltransferase, translocates from the cytoplasm to the nucleus during this process. Here we show that conditional loss of PRMT5 in early PGCs causes complete male and female sterility, preceded by the upregulation of LINE1 and IAP transposons as well as activation of a DNA damage response. Similarly, loss of maternal-zygotic PRMT5 also leads to IAP upregulation. PRMT5 is necessary for the repressive H2A/H4R3me2s chromatin modification on LINE1 and IAP transposons in PGCs, directly implicating this modification in transposon silencing during DNA hypomethylation. PRMT5 translocates back to the cytoplasm subsequently, to participate in the previously described PIWI-interacting RNA (piRNA) pathway that promotes transposon silencing via de novo DNA remethylation. Thus, PRMT5 is directly involved in genome defense during preimplantation development and in PGCs at the time of global DNA demethylation. PMID:25457166

  1. An integrated multiple capillary array electrophoresis system for high-throughput DNA sequencing

    SciTech Connect

    Lu, X.

    1998-03-27

    A capillary array electrophoresis system was chosen to perform DNA sequencing because of several advantages such as rapid heat dissipation, multiplexing capabilities, gel matrix filling simplicity, and the mature nature of the associated manufacturing technologies. There are two major concerns for the multiple capillary systems. One concern is inter-capillary cross-talk, and the other concern is excitation and detection efficiency. Cross-talk is eliminated through proper optical coupling, good focusing and immersing capillary array into index matching fluid. A side-entry excitation scheme with orthogonal detection was established for large capillary array. Two 100 capillary array formats were used for DNA sequencing. One format is cylindrical capillary with 150 {micro}m o.d., 75 {micro}m i.d and the other format is square capillary with 300 {micro}m out edge and 75 {micro}m inner edge. This project is focused on the development of excitation and detection of DNA as well as performing DNA sequencing. The DNA injection schemes are discussed for the cases of single and bundled capillaries. An individual sampling device was designed. The base-calling was performed for a capillary from the capillary array with the accuracy of 98%.

  2. To Copy-Protect or Not to Copy-Protect?

    ERIC Educational Resources Information Center

    Sacks, Jonathan

    1985-01-01

    Discusses the issues of software piracy, why people illegally copy software, protection afforded software developers by copyright laws, and current and future methods of disk-based protection built into software by developers and the problems these methods have created. (MBR)

  3. The effects of pyridaben pesticide on the DNA integrity of sperms and early in vitro embryonic development in mice

    PubMed Central

    Ebadi Manas, Ghodrat; Hasanzadeh, Shapour; Najafi, Golamreza; Parivar, Kazem; Yaghmaei, Parichehr

    2013-01-01

    Background: Pyridaben, a pyridazinone derivative, is a new acaricide and insecticide for control of mites and some insects such as white flies, aphids and thrips. Objective: This study was designed to elucidate how pyridaben can affect the sperms' morphological parameters, its DNA integrity, and to estimate the effect of various quantities of pyridaben on in vitro fertilization rate. Materials and Methods: In this study, 80 adult male Balb/C strain mice were used. Animals were divided into control and two test groups. Control group received distilled water. The test group was divided into two subgroups, viz, high dose (212 mg/kg/day) and low dose (53 mg/kg/day) and they received the pyridaben, orally for duration of 45 days. The spermatozoa were obtained from caudae epididymides on day 45 in all groups. Sperm viability, protamin compression (nuclear maturity), DNA double-strand breaks, and in vitro fertilizing (IVF) ability were examined. Results: The pyridaben treatment provoked a significant decrease in sperm population and viability in epididymides. The data obtained from this experiment revealed that, the pyridaben brings about negative impact on the sperm maturation and DNA integrity in a time-dependent manner, which consequently caused a significant (p<0.05) reduction in IVF capability. Embryo developing arrest was significantly (p<0.05) higher in treated than the control group. Conclusion: Theses results confirmed that, the pyridaben is able to induce DNA damage and chromatin abnormalities in spermatozoa which were evident by low IVF rate. This article extracted from Ph.D. thesis. (Ghodrat Ebadi Mans) PMID:24639796

  4. Arrest of Viral Proliferation by Ectopic Copies of Its Cognate Replication Origin.

    PubMed

    Valenzuela, Manuel S; Sharan, Chakradhari

    2015-01-01

    The initiation step of DNA replication is the crucial determinant of proliferation in all organisms. This step depends on the specific interaction of DNA sequences present at origins of DNA replication and their cognate activators. We wished to explore the hypothesis that the presence of ectopic origin copies may interfere with proper genome duplication. Bacteriophage λ was used as a model system. To this end, the outcome of an infection of an E. coli strain harboring ectopic copies of the λ origin region was analyzed. By measuring the effect on the host growth, viral production, and electro-microscopic visualization of the resulting λ replicative intermediates, we concluded that the ectopic copies had prevented the normal initiation step of λ DNA replication. These results suggest that DNA decoys encoding viral origins could constitute effective tools to specifically arrest viral proliferation. PMID:26110319

  5. BRIT1 regulates early DNA damage response, chromosomal integrity,and cancer

    SciTech Connect

    Rai, Rekha; Dai, Hui; Multani, Asha S.; Li, Kaiyi; Chin, Koei; Gray, Joe; Lahad, John P.; Liang, Jiyong; Mills, Gordon B.; Meric-Bernstam, Funda; Lin, Shiaw-Yih

    2006-08-24

    BRIT1, initially identified as an hTERT repressor, hasadditional functions at DNA damage checkpoints. Here, we demonstratedthat BRIT1 formed nuclear foci minutes after irradiation. The foci ofBRIT1 co-localized with 53BP1, MDC1, NBS1, ATM, RPA, and ATR. BRIT1 wasrequired for activation of these elements, indicating that BRIT1 is aproximal factor in the DNA damage response pathway. Depletion of BRIT1increased the accumulation of chromosomal aberrations. In addition,decreased levels of BRIT1 were detected in several types of human cancerwith BRIT1 expression being inversely correlated with genomic instabilityand metastasis. These results identify BRIT1 as a crucial DNA damageregulator in the ATM/ATR pathways and suggest that it functions as atumor suppressor gene.

  6. Effect of bile salts on the DNA and membrane integrity of enteric bacteria.

    PubMed

    Merritt, Megan E; Donaldson, Janet R

    2009-12-01

    Enteric bacteria are able to resist the high concentrations of bile encountered throughout the gastrointestinal tract. Here we review the current mechanisms identified in the enteric bacteria Salmonella, Escherichia coli, Bacillus cereus and Listeria monocytogenes to resist the dangerous effects of bile. We describe the role of membrane transport systems, and their connection with DNA repair pathways, in conferring bile resistance to these enterics. We discuss the findings from recent investigations that indicate bile tolerance is dependent upon being able to resist the detergent properties of bile at both the membrane and DNA level. PMID:19762477

  7. Endogenous RNA interference is driven by copy number

    PubMed Central

    Cruz, Cristina; Houseley, Jonathan

    2014-01-01

    A plethora of non-protein coding RNAs are produced throughout eukaryotic genomes, many of which are transcribed antisense to protein-coding genes and could potentially instigate RNA interference (RNAi) responses. Here we have used a synthetic RNAi system to show that gene copy number is a key factor controlling RNAi for transcripts from endogenous loci, since transcripts from multi-copy loci form double stranded RNA more efficiently than transcripts from equivalently expressed single-copy loci. Selectivity towards transcripts from high-copy DNA is therefore an emergent property of a minimal RNAi system. The ability of RNAi to selectively degrade transcripts from high-copy loci would allow suppression of newly emerging transposable elements, but such a surveillance system requires transcription. We show that low-level genome-wide pervasive transcription is sufficient to instigate RNAi, and propose that pervasive transcription is part of a defense mechanism capable of directing a sequence-independent RNAi response against transposable elements amplifying within the genome. DOI: http://dx.doi.org/10.7554/eLife.01581.001 PMID:24520161

  8. Copying Services and Copyright Infringement

    ERIC Educational Resources Information Center

    College Store Journal (Sec 1), 1977

    1977-01-01

    The impact of the Copyright Revision Act of 1976 on copying services supplied by many college stores is assessed. It is suggested that even unsupervised photocopying of copyrighted books and periodicals may constitute vicarious infringement by the store, but that in an academic environment, photocopying may constitute defensible "fair use." (LBH)

  9. USF-1 Is Critical for Maintaining Genome Integrity in Response to UV-Induced DNA Photolesions

    PubMed Central

    Mouchet, Nicolas; Vaulont, Sophie; Prince, Sharon; Galibert, Marie-Dominique

    2012-01-01

    An important function of all organisms is to ensure that their genetic material remains intact and unaltered through generations. This is an extremely challenging task since the cell's DNA is constantly under assault by endogenous and environmental agents. To protect aga