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Sample records for gene specific repair

  1. FISH comets show that the salvage enzyme TK1 contributes to gene-specific DNA repair

    Directory of Open Access Journals (Sweden)

    Stephen eDownes

    2014-08-01

    Full Text Available Thymidine kinase 1 (TK1 is a salvage enzyme that phosphorylates thymidine, imported from surrounding fluids, to create dTMP, which is further phosphorylated to the DNA precursor dTTP. TK1 deficiency has for a long time been known to cause increased cellular sensitivity to DNA damage. We have examined preferential strand break repair of DNA domains in TK1+ and TK1- clones of the Raji cell line, by the Comet-FISH technique, in bulk DNA and in the actively transcribed tumour suppressor (TP53 and human telomerase reverse transcriptase (hTERT gene regions, over 1 hour after 5Gy γ-irradiation. Results showed that repair of the TP53 and hTERT gene regions was more efficient in TK1+ compared to TK1- cells, while levels of genomic DNA repair were consistant between the two cell-lines. The targeted gene-specific repair in TK+ cells occurred rapidly, mainly over the first 15 minute repair-period. Therefore, TK1 is needed for preferential repair of actively transcribed regions, through a previously unsuspected mechanism. In principle, TK1 could exert its protective effects through supply of a supplementary dTTP pool for accurate repair of damaged genes; but Raji TK1+ cells in thymidine free media still show preferential repair of transcribed regions. TK1 therefore does not exert its protective effects through dTTP pools, but through another unidentified mechanism, which affects sensitivity to and mutagenicity by DNA damaging agents.

  2. FISH comets show that the salvage enzyme TK1 contributes to gene-specific DNA repair

    Science.gov (United States)

    McAllister, Katherine A.; Yasseen, Akeel A.; McKerr, George; Downes, C. S.; McKelvey-Martin, Valerie J.

    2014-01-01

    Thymidine kinase 1 (TK1) is a salvage enzyme that phosphorylates thymidine, imported from surrounding fluids, to create dTMP, which is further phosphorylated to the DNA precursor dTTP. TK1 deficiency has for a long time been known to cause increased cellular sensitivity to DNA damage. We have examined preferential strand break repair of DNA domains in TK1+ and TK1- clones of the Raji cell line, by the Comet-FISH technique, in bulk DNA and in the actively transcribed tumor suppressor (TP53) and human telomerase reverse transcriptase (hTERT) gene regions, over 1 h after 5Gy γ-irradiation. Results showed that repair of the TP53 and hTERT gene regions was more efficient in TK1+ compared to TK1- cells, a trend also reflected to a lesser degree in genomic DNA repair between the cell-lines. The targeted gene-specific repair in TK+ cells occurred rapidly, mainly over the first 15 min repair-period. Therefore, TK1 is needed for preferential repair of actively transcribed regions, through a previously unsuspected mechanism. In principle, TK1 could exert its protective effects through supply of a supplementary dTTP pool for accurate repair of damaged genes; but Raji TK1+ cells in thymidine free media still show preferential repair of transcribed regions. TK1 therefore does not exert its protective effects through dTTP pools, but through another unidentified mechanism, which affects sensitivity to and mutagenicity by DNA damaging agents. PMID:25152750

  3. The democratization of gene editing: Insights from site-specific cleavage and double-strand break repair.

    Science.gov (United States)

    Jasin, Maria; Haber, James E

    2016-08-01

    DNA double-strand breaks (DSBs) are dangerous lesions that if not properly repaired can lead to genomic change or cell death. Organisms have developed several pathways and have many factors devoted to repairing DSBs, which broadly occurs by homologous recombination, which relies on an identical or homologous sequence to template repair, or nonhomologous end-joining. Much of our understanding of these repair mechanisms has come from the study of induced DNA cleavage by site-specific endonucleases. In addition to their biological role, these cellular pathways can be co-opted for gene editing to study gene function or for gene therapy or other applications. While the first gene editing experiments were done more than 20 years ago, the recent discovery of RNA-guided endonucleases has simplified approaches developed over the years to make gene editing an approach that is available to the entire biomedical research community. Here, we review DSB repair mechanisms and site-specific cleavage systems that have provided insight into these mechanisms and led to the current gene editing revolution.

  4. Specific targeted gene repair using single-stranded DNA oligonucleotides at an endogenous locus in mammalian cells uses homologous recombination.

    Science.gov (United States)

    McLachlan, Jennifer; Fernandez, Serena; Helleday, Thomas; Bryant, Helen E

    2009-12-03

    The feasibility of introducing point mutations in vivo using single-stranded DNA oligonucleotides (ssON) has been demonstrated but the efficiency and mechanism remain elusive and potential side effects have not been fully evaluated. Understanding the mechanism behind this potential therapy may help its development. Here, we demonstrate the specific repair of an endogenous non-functional hprt gene by a ssON in mammalian cells, and show that the frequency of such an event is enhanced when cells are in S-phase of the cell cycle. A potential barrier in using ssONs as gene therapy could be non-targeted mutations or gene rearrangements triggered by the ssON. Both the non-specific mutation frequencies and the frequency of gene rearrangements were largely unaffected by ssONs. Furthermore, we find that the introduction of a mutation causing the loss of a functional endogenous hprt gene by a ssON occurred at a similarly low but statistically significant frequency in wild type cells and in cells deficient in single strand break repair, nucleotide excision repair and mismatch repair. However, this mutation was not induced in XRCC3 mutant cells deficient in homologous recombination. Thus, our data suggest ssON-mediated targeted gene repair is more efficient in S-phase and involves homologous recombination.

  5. A small interfering RNA screen of genes involved in DNA repair identifies tumor-specific radiosensitization by POLQ knockdown

    DEFF Research Database (Denmark)

    Higgins, Geoff S; Prevo, Remko; Lee, Yin-Fai

    2010-01-01

    radiosensitivity are largely unknown. We have conducted a small interfering RNA (siRNA) screen of 200 genes involved in DNA damage repair aimed at identifying genes whose knockdown increased tumor radiosensitivity. Parallel siRNA screens were conducted in irradiated and unirradiated tumor cells (SQ20B...... polymerase ) as a potential tumor-specific target. Subsequent investigations showed that POLQ knockdown resulted in radiosensitization of a panel of tumor cell lines from different primary sites while having little or no effect on normal tissue cell lines. These findings raise the possibility that POLQ...

  6. Three-dimensionally Specific Inhibition of DNA Repair-Related Genes by Activated KRAS in Colon Crypt Model

    Directory of Open Access Journals (Sweden)

    Toshiyuki Tsunoda

    2010-05-01

    Full Text Available Growth and differentiation of colonic epithelium are regulated in the three-dimensional (3D physiological architecture, colonic crypt, and deregulation of 3D interactions is involved in tumorigenesis. Cell-based 3D culture systems provide a suitable approach bridging the gap between two-dimensional (2D culture and animal models. KRAS mutations are found at high frequencies in human colorectal cancer (CRC; however, KRAS-targeted cancer therapy has not been developed. Here, we have established a 3D cell culture model resembling the colonic crypt by use of HKe3 cells, human CRC HCT116 cells disrupted at activated KRAS. In this 3D colonic crypt model, HKe3 cells showed the features of time course-dependent transit-amplifying and terminal-differentiated stages, which are characteristic of normal colonic crypt. On the basis of the features of HCT116 cells, activated KRAS inhibited normal cell polarity and apoptosis in 3D culture. The expression of DNA repair-related tumor suppressor genes including TP53, BRCA1, BRCA2, and EXO-1 was markedly suppressed by activated KRAS in 3D culture but not in 2D culture. These results together suggest that activated KRAS plays critical roles in the accumulation of genetic alterations through inhibition of DNA repair genes and apoptosis and that this 3D culture model will provide a useful tool for investigating the molecular mechanisms of CRC development.

  7. Macrophage-specific apoE gene repair reduces diet-induced hyperlipidemia and atherosclerosis in hypomorphic Apoe mice.

    Directory of Open Access Journals (Sweden)

    Nathalie Gaudreault

    Full Text Available BACKGROUND: Apolipoprotein (apo E is best known for its ability to lower plasma cholesterol and protect against atherosclerosis. Although the liver is the major source of plasma apoE, extra-hepatic sources of apoE, including from macrophages, account for up to 10% of plasma apoE levels. This study examined the contribution of macrophage-derived apoE expression levels in diet-induced hyperlipidemia and atherosclerosis. METHODOLOGY/PRINCIPAL FINDINGS: Hypomorphic apoE (Apoe(h/h mice expressing wildtype mouse apoE at ∼2-5% of physiological levels in all tissues were derived by gene targeting in embryonic stem cells. Cre-mediated gene repair of the Apoe(h/h allele in Apoe(h/hLysM-Cre mice raised apoE expression levels by 26 fold in freshly isolated peritoneal macrophages, restoring it to 37% of levels seen in wildtype mice. Chow-fed Apoe(h/hLysM-Cre and Apoe(h/h mice displayed similar plasma apoE and cholesterol levels (55.53±2.90 mg/dl versus 62.70±2.77 mg/dl, n = 12. When fed a high-cholesterol diet (HCD for 16 weeks, Apoe(h/hLysM-Cre mice displayed a 3-fold increase in plasma apoE and a concomitant 32% decrease in plasma cholesterol when compared to Apoe(h/h mice (602.20±22.30 mg/dl versus 888.80±24.99 mg/dl, n = 7. On HCD, Apoe(h/hLysM-Cre mice showed increased apoE immunoreactivity in lesional macrophages and liver-associated Kupffer cells but not hepatocytes. In addition, Apoe(h/hLysM-Cre mice developed 35% less atherosclerotic lesions in the aortic root than Apoe(h/h mice (167×10(3±16×10(3 µm(2 versus 259×10(3±56×10(3 µm(2, n = 7. This difference in atherosclerosis lesions size was proportional to the observed reduction in plasma cholesterol. CONCLUSIONS/SIGNIFICANCE: Macrophage-derived apoE raises plasma apoE levels in response to diet-induced hyperlipidemia and by such reduces atherosclerosis proportionally to the extent to which it lowers plasma cholesterol levels.

  8. Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis in Aedes aegypti.

    Science.gov (United States)

    Basu, Sanjay; Aryan, Azadeh; Overcash, Justin M; Samuel, Glady Hazitha; Anderson, Michelle A E; Dahlem, Timothy J; Myles, Kevin M; Adelman, Zach N

    2015-03-31

    Conventional control strategies for mosquito-borne pathogens such as malaria and dengue are now being complemented by the development of transgenic mosquito strains reprogrammed to generate beneficial phenotypes such as conditional sterility or pathogen resistance. The widespread success of site-specific nucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 in model organisms also suggests that reprogrammable gene drive systems based on these nucleases may be capable of spreading such beneficial phenotypes in wild mosquito populations. Using the mosquito Aedes aegypti, we determined that mutations in the FokI domain used in TALENs to generate obligate heterodimeric complexes substantially and significantly reduce gene editing rates. We found that CRISPR/Cas9-based editing in the mosquito Ae. aegypti is also highly variable, with the majority of guide RNAs unable to generate detectable editing. By first evaluating candidate guide RNAs using a transient embryo assay, we were able to rapidly identify highly effective guide RNAs; focusing germ line-based experiments only on this cohort resulted in consistently high editing rates of 24-90%. Microinjection of double-stranded RNAs targeting ku70 or lig4, both essential components of the end-joining response, increased recombination-based repair in early embryos as determined by plasmid-based reporters. RNAi-based suppression of Ku70 concurrent with embryonic microinjection of site-specific nucleases yielded consistent gene insertion frequencies of 2-3%, similar to traditional transposon- or ΦC31-based integration methods but without the requirement for an initial docking step. These studies should greatly accelerate investigations into mosquito biology, streamline development of transgenic strains for field releases, and simplify the evaluation of novel Cas9-based gene drive systems.

  9. Targeted gene repair – in the arena

    OpenAIRE

    2003-01-01

    The development of targeted gene repair is under way and, despite some setbacks, shows promise as an alternative form of gene therapy. This approach uses synthetic DNA molecules to activate and direct the cell’s inherent DNA repair systems to correct inborn errors. The progress of this technique and its therapeutic potential are discussed in relation to the treatment of genetic diseases.

  10. Epigenetic changes of DNA repair genes in cancer

    Institute of Scientific and Technical Information of China (English)

    Christoph Lahtz; Gerd P. Pfeifer

    2011-01-01

    'Every Hour Hurts, The Last One Kills'. That is an old saying about getting old. Every day, thousands of DNA damaging events take place in each cell of our body, but efficient DNA repair systems have evolved to prevent that. However, our DNA repair system and that of most other organisms are not as perfect as that of Deinococcus radiodurans, for example, which is able to repair massive amounts of DNA damage at one time. In many instances, accumulation of DNA damage has been linked to cancer, and genetic deficiencies in specific DNA repair genes are associated with tumor-prone phenotypes. In addition to mutations, which can be either inherited or somatically acquired, epigenetic silencing of DNA repair genes may promote tumorigenesis. This review will summarize current knowledge of the epigenetic inactivation of different DNA repair components in human cancer.

  11. DNA repair genes in the Megavirales pangenome.

    Science.gov (United States)

    Blanc-Mathieu, Romain; Ogata, Hiroyuki

    2016-06-01

    The order 'Megavirales' represents a group of eukaryotic viruses with a large genome encoding a few hundred up to two thousand five hundred genes. Several members of Megavirales possess genes involved in major DNA repair pathways. Some of these genes were likely inherited from an ancient virus world and some others were derived from the genomes of their hosts. Here we examine molecular phylogenies of key DNA repair enzymes in light of recent hypotheses on the origin of Megavirales, and propose that the last common ancestors of the individual families of the order Megavirales already possessed DNA repair functions to achieve and maintain a moderately large genome and that this repair capacity gradually increased, in a family-dependent manner, during their recent evolution.

  12. Transactivation of repair genes by BRCA1.

    Science.gov (United States)

    El-Deiry, Wafik S

    2002-01-01

    Recent studies have identified a link between the BRCA1 tumor suppressor and transcriptional regulation of a group of genes involved in nucleotide excision repair. There is some controversy regarding the precise mechanism of upregulation of XPE DDB2 or XPC by BRCA1, with some evidence suggesting that p53 is involved in their regulation. Some evidence suggests BRCA1 may stabilize p53 and direct regulation of DNA repair genes, although how BRCA1 stabilizes p53 remains unclear and whether BRCA1 can upregulate DNA repair genes in a p53-independent manner remains a possibility. A transcriptional component to the action of BRCA1 and involvement of XP genes brings up new and interesting questions about breast cancer development and therapy.

  13. Targeted gene repair: the ups and downs of a promising gene therapy approach.

    Science.gov (United States)

    de Semir, David; Aran, Josep M

    2006-08-01

    As a novel form of molecular medicine based on direct actions over the genes, targeted gene repair has raised consideration recently above classical gene therapy strategies based on genetic augmentation or complementation. Targeted gene repair relies on the local induction of the cell's endogenous DNA repair mechanisms to attain a therapeutic gene conversion event within the genome of the diseased cell. Successful repair has been achieved both in vitro and in vivo with a variety of corrective molecules ranging from oligonucleotides (chimeraplasts, modified single-stranded oligonucleotides, triplex-forming oligonucleotides), to small DNA fragments (small fragment homologous replacement (SFHR)), and even viral vectors (AAV-based). However, controversy on the consistency and lack of reproducibility of early experiments regarding frequencies and persistence of targeted gene repair, particularly for chimeraplasty, has flecked the field. Nevertheless, several hurdles such as inefficient nuclear uptake of the corrective molecules, and misleading assessment of targeted repair frequencies have been identified and are being addressed. One of the key bottlenecks for exploiting the overall potential of the different targeted gene repair modalities is the lack of a detailed knowledge of their mechanisms of action at the molecular level. Several studies are now focusing on the assessment of the specific repair pathway(s) involved (homologous recombination, mismatch repair, etc.), devising additional strategies to increase their activity (using chemotherapeutic drugs, chimeric nucleases, etc.), and assessing the influence of the cell cycle in the regulation of the repair process. Until therapeutic correction frequencies for single gene disorders are reached both in cellular and animal models, precision and undesired side effects of this promising gene therapy approach will not be thoroughly evaluated.

  14. Gene Therapy for Fracture Repair

    Science.gov (United States)

    2007-05-01

    case, the external catheter hub is visible (D), though the internal tubing cannot be visualized by X-Ray. 11 MLV-based vector with BMP-2/4...catheter) injection. Top: A fluoroscope was used to visualize a radio- opaque contrast dye during a percutaneous injection from the lateral aspect...analysis was performed using ImaGene software (BioDiscovery, El Segundo, CA), that used an internal statistical analysis of the signal intensity of

  15. Specification guidelines for surface preparation of concrete prior to repair

    OpenAIRE

    Courard, Luc; Bissonnette, Benoît; Garbacz, Andrzej

    2017-01-01

    The repair of concrete requests specific preparation operations needed for guaranteeing compatibility between substrate and new materials as well as the development of adhesion properties. These specification guidelines contain design and construction recommendations for surface preparation of concrete for repair and overlay. The paper summarizes current knowledge, best practices and results of the research concerning the surface preparation of concrete prior to application of repair/overlay ...

  16. SELECTIVE-INHIBITION OF REPAIR OF ACTIVE GENES BY HYPERTHERMIA IS DUE TO INHIBITION OF GLOBAL AND TRANSCRIPTION COUPLED REPAIR PATHWAYS

    NARCIS (Netherlands)

    SAKKERS, RJ; FILON, AR; BRUNSTING, JF; KAMPINGA, HH; KONINGS, AWT; MULLENDERS, LHF

    1995-01-01

    Hyperthermia specifically inhibits the repair of UV-induced DNA photolesions in transcriptionally active genes, To define more precisely which mechanisms underlie the heat-induced inhibition of repair of active genes, removal of cyclobutane pyrimidine dimers (CPDs) was studied in human fibroblasts w

  17. Application of a 5-tiered scheme for standardized classification of 2,360 unique mismatch repair gene variants in the InSiGHT locus-specific database

    NARCIS (Netherlands)

    Thompson, Bryony A.; Spurdle, Amanda B.; Plazzer, John-Paul; Greenblatt, Marc S.; Akagi, Kiwamu; Al-Mulla, Fahd; Bapat, Bharati; Bernstein, Inge; Capella, Gabriel; den Dunnen, Johan T.; du Sart, Desiree; Fabre, Aurelie; Farrell, Michael P.; Farrington, Susan M.; Frayling, Ian M.; Frebourg, Thierry; Goldgar, David E.; Heinen, Christopher D.; Holinski-Feder, Elke; Kohonen-Corish, Maija; Robinson, Kristina Lagerstedt; Leung, Suet Yi; Martins, Alexandra; Moller, Pal; Morak, Monika; Nystrom, Minna; Peltomaki, Paivi; Pineda, Marta; Qi, Ming; Ramesar, Rajkumar; Rasmussen, Lene Juel; Royer-Pokora, Brigitte; Scott, Rodney J.; Sijmons, Rolf; Tavtigian, Sean V.; Tops, Carli M.; Weber, Thomas; Wijnen, Juul; Woods, Michael O.; Macrae, Finlay; Genuardi, Maurizio

    2014-01-01

    The clinical classification of hereditary sequence variants identified in disease-related genes directly affects clinical management of patients and their relatives. The International Society for Gastrointestinal Hereditary Tumours (InSiGHT) undertook a collaborative effort to develop, test and appl

  18. Application of a 5-tiered scheme for standardized classification of 2,360 unique mismatch repair gene variants in the InSiGHT locus-specific database

    DEFF Research Database (Denmark)

    Thompson, Bryony A; Spurdle, Amanda B; Plazzer, John-Paul

    2014-01-01

    The clinical classification of hereditary sequence variants identified in disease-related genes directly affects clinical management of patients and their relatives. The International Society for Gastrointestinal Hereditary Tumours (InSiGHT) undertook a collaborative effort to develop, test and a...

  19. Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis in Aedes aegypti

    OpenAIRE

    Basu, Sanjay; Aryan, Azadeh; Overcash, Justin M.; Samuel, Glady Hazitha; Anderson, Michelle A.E.; Dahlem, Timothy J.; Myles, Kevin M.; Adelman, Zach N.

    2015-01-01

    Mosquitoes are vectors of both parasites and viruses responsible for high-impact diseases including malaria, dengue, and chikungunya. Novel interventions based on genetic modification of the mosquito genome are currently being developed and implemented. To comprehensively exploit such interventions, detailed knowledge of mosquito physiology, genetics, and genome engineering are required. We developed and validated a two-step process for performing high-efficiency site-specific insertion of ge...

  20. Device-specific outcomes after endovascular abdominal aortic aneurysm repair

    NARCIS (Netherlands)

    F.B. Gonçalves (Frederico Bastos); E.V. Rouwet (Ellen); R. Metz (Roderik); J.M. Hendriks (Joke); M.P.F.V. Peeters; B.E. Muhs (Bart); H.J.M. Verhagen (Hence)

    2010-01-01

    textabstractOver the last decade, endovascular aneurysm repair (EVAR) has been used extensively for the elective treatment of infra-renal abdominal aneurysms. However, it remains unclear how specific devices perform and how they compare to others. We provide an overview of currently used endografts,

  1. Dynamic regulation of cerebral DNA repair genes by psychological stress

    DEFF Research Database (Denmark)

    Forsberg, Kristin; Aalling, Nadia; Wörtwein, Gitta

    2015-01-01

    for maintaining genomic integrity. The aim of the present study was to characterize the pattern of cerebral DNA repair enzyme regulation after stress through the quantification of a targeted range of gene products involved in different types of DNA repair. 72 male Sprague-Dawley rats were subjected to either...... was seen in HC, but with overall smaller effects and without the induction after acute stress. Nuclear DNA damage from oxidation as measured by the comet assay was unaffected by stress in both regions. We conclude that psychological stress have a dynamic influence on brain DNA repair gene expression...

  2. Genomic survey and expression analysis of DNA repair genes in the genus Leptospira.

    Science.gov (United States)

    Martins-Pinheiro, Marinalva; Schons-Fonseca, Luciane; da Silva, Josefa B; Domingos, Renan H; Momo, Leonardo Hiroyuki Santos; Simões, Ana Carolina Quirino; Ho, Paulo Lee; da Costa, Renata M A

    2016-04-01

    Leptospirosis is an emerging zoonosis with important economic and public health consequences and is caused by pathogenic leptospires. The genus Leptospira belongs to the order Spirochaetales and comprises saprophytic (L. biflexa), pathogenic (L. interrogans) and host-dependent (L. borgpetersenii) members. Here, we present an in silico search for DNA repair pathways in Leptospira spp. The relevance of such DNA repair pathways was assessed through the identification of mRNA levels of some genes during infection in animal model and after exposition to spleen cells. The search was performed by comparison of available Leptospira spp. genomes in public databases with known DNA repair-related genes. Leptospires exhibit some distinct and unexpected characteristics, for instance the existence of a redundant mechanism for repairing a chemically diverse spectrum of alkylated nucleobases, a new mutS-like gene and a new shorter version of uvrD. Leptospira spp. shares some characteristics from Gram-positive, as the presence of PcrA, two RecQ paralogs and two SSB proteins; the latter is considered a feature shared by naturally competent bacteria. We did not find a significant reduction in the number of DNA repair-related genes in both pathogenic and host-dependent species. Pathogenic leptospires were enriched for genes dedicated to base excision repair and non-homologous end joining. Their evolutionary history reveals a remarkable importance of lateral gene transfer events for the evolution of the genus. Up-regulation of specific DNA repair genes, including components of SOS regulon, during infection in animal model validates the critical role of DNA repair mechanisms for the complex interplay between host/pathogen.

  3. Control of gene editing by manipulation of DNA repair mechanisms.

    Science.gov (United States)

    Danner, Eric; Bashir, Sanum; Yumlu, Saniye; Wurst, Wolfgang; Wefers, Benedikt; Kühn, Ralf

    2017-04-03

    DNA double-strand breaks (DSBs) are produced intentionally by RNA-guided nucleases to achieve genome editing through DSB repair. These breaks are repaired by one of two main repair pathways, classic non-homologous end joining (c-NHEJ) and homology-directed repair (HDR), the latter being restricted to the S/G2 phases of the cell cycle and notably less frequent. Precise genome editing applications rely on HDR, with the abundant c-NHEJ formed mutations presenting a barrier to achieving high rates of precise sequence modifications. Here, we give an overview of HDR- and c-NHEJ-mediated DSB repair in gene editing and summarize the current efforts to promote HDR over c-NHEJ.

  4. Suppressed expression of non-DSB repair genes inhibits gamma-radiation-induced cytogenetic repair and cell cycle arrest.

    Science.gov (United States)

    Zhang, Ye; Rohde, Larry H; Emami, Kamal; Hammond, Dianne; Casey, Rachael; Mehta, Satish K; Jeevarajan, Antony S; Pierson, Duane L; Wu, Honglu

    2008-11-01

    Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in double-strand break (DSB) repair, and its impact on cytogenetic responses has not been well studied. The purpose of this study is to identify new roles of IR inducible genes in regulating DSB repair and cell cycle progression. In this study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by small interfering RNA in human fibroblast cells. Frequency of micronuclei (MN) formation and chromosome aberrations were measured to determine efficiency of cytogenetic repair, especially DSB repair. In response to IR, the formation of MN was significantly increased by suppressed expression of five genes: Ku70 (DSB repair pathway), XPA (nucleotide excision repair pathway), RPA1 (mismatch repair pathway), RAD17 and RBBP8 (cell cycle control). Knocked-down expression of four genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Moreover, decreased XPA, p21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Nine of these eleven genes, whose knock-down expression affected cytogenetic repair, were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate IR-induced biological consequences. Furthermore, eight non-DBS repair genes showed involvement in regulating DSB repair, indicating that

  5. A multistep genomic screen identifies new genes required for repair of DNA double-strand breaks in Saccharomyces cerevisiae.

    Science.gov (United States)

    McKinney, Jennifer Summers; Sethi, Sunaina; Tripp, Jennifer DeMars; Nguyen, Thuy N; Sanderson, Brian A; Westmoreland, James W; Resnick, Michael A; Lewis, L Kevin

    2013-04-15

    Efficient mechanisms for rejoining of DNA double-strand breaks (DSBs) are vital because misrepair of such lesions leads to mutation, aneuploidy and loss of cell viability. DSB repair is mediated by proteins acting in two major pathways, called homologous recombination and nonhomologous end-joining. Repair efficiency is also modulated by other processes such as sister chromatid cohesion, nucleosome remodeling and DNA damage checkpoints. The total number of genes influencing DSB repair efficiency is unknown. To identify new yeast genes affecting DSB repair, genes linked to gamma radiation resistance in previous genome-wide surveys were tested for their impact on repair of site-specific DSBs generated by in vivo expression of EcoRI endonuclease. Eight members of the RAD52 group of DNA repair genes (RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, MRE11 and XRS2) and 73 additional genes were found to be required for efficient repair of EcoRI-induced DSBs in screens utilizing both MATa and MATα deletion strain libraries. Most mutants were also sensitive to the clastogenic chemicals MMS and bleomycin. Several of the non-RAD52 group genes have previously been linked to DNA repair and over half of the genes affect nuclear processes. Many proteins encoded by the protective genes have previously been shown to associate physically with each other and with known DNA repair proteins in high-throughput proteomics studies. A majority of the proteins (64%) share sequence similarity with human proteins, suggesting that they serve similar functions. We have used a genetic screening approach to detect new genes required for efficient repair of DSBs in Saccharomyces cerevisiae. The findings have spotlighted new genes that are critical for maintenance of genome integrity and are therefore of greatest concern for their potential impact when the corresponding gene orthologs and homologs are inactivated or polymorphic in human cells.

  6. Molecular cloning of the human excision repair gene ERCC-6.

    NARCIS (Netherlands)

    C. Troelstra (Christine); H. Odijk (Hanny); J. de Wit (Jan); A. Westerveld (Andries); L.H. Thompson; D. Bootsma (Dirk); J.H.J. Hoeijmakers (Jan)

    1990-01-01

    textabstractThe UV-sensitive, nucleotide excision repair-deficient Chinese hamster mutant cell line UV61 was used to identify and clone a correcting human gene, ERCC-6. UV61, belonging to rodent complementation group 6, is only moderately UV sensitive in comparison with mutant lines in groups 1 to 5

  7. DNA damage and gene therapy of xeroderma pigmentosum, a human DNA repair-deficient disease

    Energy Technology Data Exchange (ETDEWEB)

    Dupuy, Aurélie [Laboratory of Genetic Instability and Oncogenesis UMR8200CNRS, Institut Gustave Roussy and University Paris-Sud, Villejuif (France); Sarasin, Alain, E-mail: alain.sarasin@gustaveroussy.fr [Laboratory of Genetic Instability and Oncogenesis UMR8200CNRS, Institut Gustave Roussy and University Paris-Sud, Villejuif (France); Service de Génétique, Institut Gustave Roussy (France)

    2015-06-15

    Graphical abstract: - Highlights: • Full correction of mutation in the XPC gene by engineered nucleases. • Meganucleases and TALENs are inhibited by 5-MeC for inducing double strand breaks. • Gene therapy of XP cells is possible using homologous recombination for DSB repair. - Abstract: Xeroderma pigmentosum (XP) is a genetic disease characterized by hypersensitivity to ultra-violet and a very high risk of skin cancer induction on exposed body sites. This syndrome is caused by germinal mutations on nucleotide excision repair genes. No cure is available for these patients except a complete protection from all types of UV radiations. We reviewed the various techniques to complement or to correct the genetic defect in XP cells. We, particularly, developed the correction of XP-C skin cells using the fidelity of the homologous recombination pathway during repair of double-strand break (DSB) in the presence of XPC wild type sequences. We used engineered nucleases (meganuclease or TALE nuclease) to induce a DSB located at 90 bp of the mutation to be corrected. Expression of specific TALE nuclease in the presence of a repair matrix containing a long stretch of homologous wild type XPC sequences allowed us a successful gene correction of the original TG deletion found in numerous North African XP patients. Some engineered nucleases are sensitive to epigenetic modifications, such as cytosine methylation. In case of methylated sequences to be corrected, modified nucleases or demethylation of the whole genome should be envisaged. Overall, we showed that specifically-designed TALE-nuclease allowed us to correct a 2 bp deletion in the XPC gene leading to patient's cells proficient for DNA repair and showing normal UV-sensitivity. The corrected gene is still in the same position in the human genome and under the regulation of its physiological promoter. This result is a first step toward gene therapy in XP patients.

  8. Mismatch-mediated error prone repair at the immunoglobulin genes.

    Science.gov (United States)

    Chahwan, Richard; Edelmann, Winfried; Scharff, Matthew D; Roa, Sergio

    2011-12-01

    The generation of effective antibodies depends upon somatic hypermutation (SHM) and class-switch recombination (CSR) of antibody genes by activation induced cytidine deaminase (AID) and the subsequent recruitment of error prone base excision and mismatch repair. While AID initiates and is required for SHM, more than half of the base changes that accumulate in V regions are not due to the direct deamination of dC to dU by AID, but rather arise through the recruitment of the mismatch repair complex (MMR) to the U:G mismatch created by AID and the subsequent perversion of mismatch repair from a high fidelity process to one that is very error prone. In addition, the generation of double-strand breaks (DSBs) is essential during CSR, and the resolution of AID-generated mismatches by MMR to promote such DSBs is critical for the efficiency of the process. While a great deal has been learned about how AID and MMR cause hypermutations and DSBs, it is still unclear how the error prone aspect of these processes is largely restricted to antibody genes. The use of knockout models and mice expressing mismatch repair proteins with separation-of-function point mutations have been decisive in gaining a better understanding of the roles of each of the major MMR proteins and providing further insight into how mutation and repair are coordinated. Here, we review the cascade of MMR factors and repair signals that are diverted from their canonical error free role and hijacked by B cells to promote genetic diversification of the Ig locus. This error prone process involves AID as the inducer of enzymatically-mediated DNA mismatches, and a plethora of downstream MMR factors acting as sensors, adaptors and effectors of a complex and tightly regulated process from much of which is not yet well understood.

  9. DNA repair and gene therapy: implications for translational uses.

    Science.gov (United States)

    Limp-Foster, M; Kelley, M R

    2000-01-01

    Gene therapy has been proposed to have implications in the treatment of cancer. By genetically manipulating the hematopoietic stem cell compartment with genes that confer resistance to chemotherapeutic agents, the dose escalation that is necessary to effectively treat the cancers could potentially be achieved. DNA repair genes are some of the potential candidates to confer increased resistance to chemotherapeutic agents. Although initial focus in this area has been on the direct reversal protein (MGMT), its protective ability is limited to those agents that produce O(6)-methylGuanine cross-links-agents that are not extensively used clinically (e.g., nitrosoureas). Furthermore, most alkylating agents attack more sites in DNA other than O(6)-methylGuanine, such that the protections afforded by MGMT may prevent the initial cytotoxicity, but at a price of increased mutational burden and potential secondary leukemias. Therefore, some of the genes that are being tested as candidates for gene transfer are base excision repair (BER) genes. We and others have found that overexpression of selective BER genes confers resistance to chemotherapeutic agents such as thiotepa, ionizing radiation, bleomycin, and other agents. As these "proof of concept" analyses mature, many more clinically relevant chemotherapeutic agents can be tested for BER protective ability.

  10. 49 CFR 180.413 - Repair, modification, stretching, rebarrelling, or mounting of specification cargo tanks.

    Science.gov (United States)

    2010-10-01

    ... 49 Transportation 2 2010-10-01 2010-10-01 false Repair, modification, stretching, rebarrelling, or... of Cargo Tanks § 180.413 Repair, modification, stretching, rebarrelling, or mounting of specification cargo tanks. (a) General. Any repair, modification, stretching, rebarrelling, or mounting of a cargo...

  11. Gene therapy and peripheral nerve repair: a perspective

    Directory of Open Access Journals (Sweden)

    Stefan A. Hoyng

    2015-07-01

    Full Text Available Clinical phase I/II studies have demonstrated the safety of gene therapy for a variety of central nervous system disorders, including Canavan’s, Parkinson’s and Alzheimer’s disease, retinal diseases and pain. The majority of gene therapy studies in the CNS have used adeno-associated viral vectors (AAV and the first AAV-based therapeutic, a vector encoding lipoprotein lipase, is now marketed in Europe under the name Glybera. These remarkable advances may become relevant to translational research on gene therapy to promote peripheral nervous system (PNS repair. This short review first summarizes the results of gene therapy in animal models for peripheral nerve repair. Secondly, we identify key areas of future research in the domain of PNS-gene therapy. Finally, a perspective is provided on the path to clinical translation of PNS gene therapy for traumatic nerve injuries. In the latter section we discuss the route and mode of delivery of the vector to human patients, the efficacy and safety of the vector, and the choice of the patient population for a first possible proof-of-concept clinical study.

  12. Enhanced gene repair mediated by methyl-CpG-modified single-stranded oligonucleotides

    Science.gov (United States)

    Bertoni, Carmen; Rustagi, Arjun; Rando, Thomas A.

    2009-01-01

    Gene editing mediated by oligonucleotides has been shown to induce stable single base alterations in genomic DNA in both prokaryotic and eukaryotic organisms. However, the low frequencies of gene repair have limited its applicability for both basic manipulation of genomic sequences and for the development of therapeutic approaches for genetic disorders. Here, we show that single-stranded oligodeoxynucleotides (ssODNs) containing a methyl-CpG modification and capable of binding to the methyl-CpG binding domain protein 4 (MBD4) are able to induce >10-fold higher levels of gene correction than ssODNs lacking the specific modification. Correction was stably inherited through cell division and was confirmed at the protein, transcript and genomic levels. Downregulation of MBD4 expression using RNAi prevented the enhancement of gene correction efficacy obtained using the methyl-CpG-modified ssODN, demonstrating the specificity of the repair mechanism being recruited. Our data demonstrate that efficient manipulation of genomic targets can be achieved and controlled by the type of ssODN used and by modulation of the repair mechanism involved in the correction process. This new generation of ssODNs represents an important technological advance that is likely to have an impact on multiple applications, especially for gene therapy where permanent correction of the genetic defect has clear advantages over viral and other nonviral approaches currently being tested. PMID:19854937

  13. Induction of a mutant phenotype in human repair proficient cells after overexpression of a mutated human DNA repair gene.

    NARCIS (Netherlands)

    P.B.G.M. Belt; M.F. van Oostenrijk; H. Odijk (Hanny); J.H.J. Hoeijmakers (Jan); C.M.P. Backendorf (Claude)

    1991-01-01

    textabstractAntisense and mutated cDNA of the human excision repair gene ERCC-1 were overexpressed in repair efficient HeLa cells by means of an Epstein-Barr-virus derived CDNA expression vector. Whereas antisense RNA did not influence the survival of the transfected cells, a mutated cDNA generating

  14. Loss of transcription factor early growth response gene 1 results in impaired endochondral bone repair.

    Science.gov (United States)

    Reumann, Marie K; Strachna, Olga; Yagerman, Sarah; Torrecilla, Daniel; Kim, Jihye; Doty, Stephen B; Lukashova, Lyudmila; Boskey, Adele L; Mayer-Kuckuk, Philipp

    2011-10-01

    Transcription factors that play a role in ossification during development are expected to participate in postnatal fracture repair since the endochondral bone formation that occurs in embryos is recapitulated during fracture repair. However, inherent differences exist between bone development and fracture repair, including a sudden disruption of tissue integrity followed by an inflammatory response. This raises the possibility that repair-specific transcription factors participate in bone healing. Here, we assessed the consequence of loss of early growth response gene 1 (EGR-1) on endochondral bone healing because this transcription factor has been shown to modulate repair in vascularized tissues. Model fractures were created in ribs of wild type (wt) and EGR-1(-/-) mice. Differences in tissue morphology and composition between these two animal groups were followed over 28 post fracture days (PFDs). In wt mice, bone healing occurred in healing phases characteristic of endochondral bone repair. A similar healing sequence was observed in EGR-1(-/-) mice but was impaired by alterations. A persistent accumulation of fibrin between the disconnected bones was observed on PFD7 and remained pronounced in the callus on PFD14. Additionally, the PFD14 callus was abnormally enlarged and showed increased deposition of mineralized tissue. Cartilage ossification in the callus was associated with hyper-vascularity and -proliferation. Moreover, cell deposits located in proximity to the callus within skeletal muscle were detected on PFD14. Despite these impairments, repair in EGR-1(-/-) callus advanced on PFD28, suggesting EGR-1 is not essential for healing. Together, this study provides genetic evidence that EGR-1 is a pleiotropic regulator of endochondral fracture repair. Copyright © 2011 Elsevier Inc. All rights reserved.

  15. Non-DBS DNA Repair Genes Regulate Radiation-induced Cytogenetic Damage Repair and Cell Cycle Progression

    Science.gov (United States)

    Zhang, Ye; Rohde, Larry H.; Emami, Kamal; Casey, Rachael; Wu, Honglu

    2008-01-01

    Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in DSB repair, and its impact on cytogenetic responses has not been systematically studied. In the present study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by transfection with small interfering RNA in human fibroblast cells. The purpose of this study is to identify new roles of these selected genes on regulating DSB repair and cell cycle progression , as measured in the micronuclei formation and chromosome aberration. In response to IR, the formation of MN was significantly increased by suppressed expression of 5 genes: Ku70 in the DSB repair pathway, XPA in the NER pathway, RPA1 in the MMR pathway, and RAD17 and RBBP8 in cell cycle control. Knocked-down expression of 4 genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Furthermore, loss of XPA, P21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Most of the 11 genes that affected cytogenetic responses are not known to have clear roles influencing DBS repair. Nine of these 11 genes were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate the biological consequences after IR.

  16. Non-DBS DNA Repair Genes Regulate Radiation-induced Cytogenetic Damage Repair and Cell Cycle Progression

    Science.gov (United States)

    Zhang, Ye; Rohde, Larry H.; Emami, Kamal; Casey, Rachael; Wu, Honglu

    2008-01-01

    Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in DSB repair, and its impact on cytogenetic responses has not been systematically studied. In the present study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by transfection with small interfering RNA in human fibroblast cells. The purpose of this study is to identify new roles of these selected genes on regulating DSB repair and cell cycle progression , as measured in the micronuclei formation and chromosome aberration. In response to IR, the formation of MN was significantly increased by suppressed expression of 5 genes: Ku70 in the DSB repair pathway, XPA in the NER pathway, RPA1 in the MMR pathway, and RAD17 and RBBP8 in cell cycle control. Knocked-down expression of 4 genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Furthermore, loss of XPA, P21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Most of the 11 genes that affected cytogenetic responses are not known to have clear roles influencing DBS repair. Nine of these 11 genes were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate the biological consequences after IR.

  17. Sister chromatid gene conversion is a prominent double-strand break repair pathway in mammalian cells

    OpenAIRE

    Johnson, Roger D.; Jasin, Maria

    2000-01-01

    In mammalian cells, repair of DNA double-strand breaks (DSBs) occurs by both homologous and non-homologous mechanisms. By definition, homologous recombination requires a template with sufficient sequence identity to the damaged molecule in order to direct repair. We now show that the sister chromatid acts as a repair template in a substantial proportion of DSB repair events. The outcome of sister chromatid repair is primarily gene conversion unassociated with reciprocal exchange. This contras...

  18. Cell type-specific properties and environment shape tissue specificity of cancer genes.

    Science.gov (United States)

    Schaefer, Martin H; Serrano, Luis

    2016-02-09

    One of the biggest mysteries in cancer research remains why mutations in certain genes cause cancer only at specific sites in the human body. The poor correlation between the expression level of a cancer gene and the tissues in which it causes malignant transformations raises the question of which factors determine the tissue-specific effects of a mutation. Here, we explore why some cancer genes are associated only with few different cancer types (i.e., are specific), while others are found mutated in a large number of different types of cancer (i.e., are general). We do so by contrasting cellular functions of specific-cancer genes with those of general ones to identify properties that determine where in the body a gene mutation is causing malignant transformations. We identified different groups of cancer genes that did not behave as expected (i.e., DNA repair genes being tissue specific, immune response genes showing a bimodal specificity function or strong association of generally expressed genes to particular cancers). Analysis of these three groups demonstrates the importance of environmental impact for understanding why certain cancer genes are only involved in the development of some cancer types but are rarely found mutated in other types of cancer.

  19. DNA damage and gene therapy of xeroderma pigmentosum, a human DNA repair-deficient disease.

    Science.gov (United States)

    Dupuy, Aurélie; Sarasin, Alain

    2015-06-01

    Xeroderma pigmentosum (XP) is a genetic disease characterized by hypersensitivity to ultra-violet and a very high risk of skin cancer induction on exposed body sites. This syndrome is caused by germinal mutations on nucleotide excision repair genes. No cure is available for these patients except a complete protection from all types of UV radiations. We reviewed the various techniques to complement or to correct the genetic defect in XP cells. We, particularly, developed the correction of XP-C skin cells using the fidelity of the homologous recombination pathway during repair of double-strand break (DSB) in the presence of XPC wild type sequences. We used engineered nucleases (meganuclease or TALE nuclease) to induce a DSB located at 90 bp of the mutation to be corrected. Expression of specific TALE nuclease in the presence of a repair matrix containing a long stretch of homologous wild type XPC sequences allowed us a successful gene correction of the original TG deletion found in numerous North African XP patients. Some engineered nucleases are sensitive to epigenetic modifications, such as cytosine methylation. In case of methylated sequences to be corrected, modified nucleases or demethylation of the whole genome should be envisaged. Overall, we showed that specifically-designed TALE-nuclease allowed us to correct a 2 bp deletion in the XPC gene leading to patient's cells proficient for DNA repair and showing normal UV-sensitivity. The corrected gene is still in the same position in the human genome and under the regulation of its physiological promoter. This result is a first step toward gene therapy in XP patients.

  20. Xeroderma pigmentosum group F caused by a defect in a structure-specific DNA repair endonuclease.

    NARCIS (Netherlands)

    A.M. Sijbers (Anneke); W.L. de Laat (Wouter); R.A. Ariza (Rafael); M. Biggerstaff (Maureen); Y-F. Wei; J.G. Moggs (Jonathan); K.C. Carter (Kenneth); B.K. Shell (Brenda); E. Evans (Elizabeth); M.C. de Jong (Mariska); S. Rademakers (Suzanne); J.D. de Rooij (Johan); N.G.J. Jaspers (Nicolaas); J.H.J. Hoeijmakers (Jan); R.D. Wood (Richard)

    1996-01-01

    textabstractNucleotide excision repair, which is defective in xeroderma pigmentosum (XP), involves incision of a DNA strand on each side of a lesion. We isolated a human gene homologous to yeast Rad1 and found that it corrects the repair defects of XP group F as well as rodent groups 4 and 11. Causa

  1. Regulation of the Saccharomyces cerevisiae DNA repair gene RAD16.

    Science.gov (United States)

    Bang, D D; Timmermans, V; Verhage, R; Zeeman, A M; van de Putte, P; Brouwer, J

    1995-05-25

    The RAD16 gene product has been shown to be essential for the repair of the silenced mating type loci [Bang et al. (1992) Nucleic Acids Res. 20, 3925-3931]. More recently we demonstrated that the RAD16 and RAD7 proteins are also required for repair of non-transcribed strands of active genes in Saccharomyces cerevisiae [Waters et al. (1993) Mol. Gen. Genet. 239, 28-32]. We have studied the regulation of the RAD16 gene and found that the RAD16 transcript levels increased up to 7-fold upon UV irradiation. Heat shock at 42 degrees C also results in elevated levels of RAD16 mRNA. In sporulating MAT alpha/MATa diploid cells RAD16 mRNA is also induced. The basal level of the RAD16 transcript is constant during the mitotic cell cycle. G1-arrested cells show normal induction of RAD16 mRNA upon UV irradiation demonstrating that the induction is not a secondary consequence of G2 cell cycle arrest following UV irradiation. However, in cells arrested in G1 the induction of RAD16 mRNA after UV irradiation is not followed by a rapid decline as occurs in normal growing cells suggesting that the down regulation of RAD16 transcription is dependent on progression into the cell cycle.

  2. Double-strand break damage and associated DNA repair genes predispose smokers to gene methylation

    OpenAIRE

    Leng, Shuguang; Stidley, Christine A.; Willink, Randy; Bernauer, Amanda; Do, Kieu; Picchi, Maria A.; Sheng, Xin; Frasco, Melissa, A.; Berg, David Van Den; Gilliland, Frank D.; Zima, Christopher; Crowell, Richard E.; Belinsky, Steven A.

    2008-01-01

    Gene promoter hypermethylation in sputum is a promising biomarker for predicting lung cancer. Identifying factors that predispose smokers to methylation of multiple gene promoters in the lung could impact strategies for early detection and chemoprevention. This study evaluated the hypothesis that double-strand break repair capacity and sequence variation in genes in this pathway are associated with a high methylation index in a cohort of current and former cancer-free smokers. A 50% reduction...

  3. Reduced Activity of Double-Strand Break Repair Genes in Prostate Cancer Patients With Late Normal Tissue Radiation Toxicity

    Energy Technology Data Exchange (ETDEWEB)

    Oorschot, Bregje van, E-mail: b.vanoorschot@amc.uva.nl [Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Molecular Medicine (CEMM), Department of Radiation Oncology, Academic Medical Center, University of Amsterdam, Amsterdam (Netherlands); Hovingh, Suzanne E. [Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Molecular Medicine (CEMM), Department of Radiation Oncology, Academic Medical Center, University of Amsterdam, Amsterdam (Netherlands); Moerland, Perry D. [Bioinformatics Laboratory, Department of Clinical Epidemiology, Biostatistics and Bioinformatics, Academic Medical Center, University of Amsterdam, Amsterdam (Netherlands); Medema, Jan Paul; Stalpers, Lukas J.A. [Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Molecular Medicine (CEMM), Department of Radiation Oncology, Academic Medical Center, University of Amsterdam, Amsterdam (Netherlands); Vrieling, Harry [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Franken, Nicolaas A.P. [Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Molecular Medicine (CEMM), Department of Radiation Oncology, Academic Medical Center, University of Amsterdam, Amsterdam (Netherlands)

    2014-03-01

    Purpose: To investigate clinical parameters and DNA damage response as possible risk factors for radiation toxicity in the setting of prostate cancer. Methods and Materials: Clinical parameters of 61 prostate cancer patients, 34 with (overresponding, OR) and 27 without (non-responding, NR) severe late radiation toxicity were assembled. In addition, for a matched subset the DNA damage repair kinetics (γ-H2AX assay) and expression profiles of DNA repair genes were determined in ex vivo irradiated lymphocytes. Results: Examination of clinical data indicated none of the considered clinical parameters to be correlated with the susceptibility of patients to develop late radiation toxicity. Although frequencies of γ-H2AX foci induced immediately after irradiation were similar (P=.32), significantly higher numbers of γ-H2AX foci were found 24 hours after irradiation in OR compared with NR patients (P=.03). Patient-specific γ-H2AX foci decay ratios were significantly higher in NR patients than in OR patients (P<.0001). Consequently, NR patients seem to repair DNA double-strand breaks (DSBs) more efficiently than OR patients. Moreover, gene expression analysis indicated several genes of the homologous recombination pathway to be stronger induced in NR compared with OR patients (P<.05). A similar trend was observed in genes of the nonhomologous end-joining repair pathway (P=.09). This is congruent with more proficient repair of DNA DSBs in patients without late radiation toxicity. Conclusions: Both gene expression profiling and DNA DSB repair kinetics data imply that less-efficient repair of radiation-induced DSBs may contribute to the development of late normal tissue damage. Induction levels of DSB repair genes (eg, RAD51) may potentially be used to assess the risk for late radiation toxicity.

  4. Germline mutations in DNA repair genes predispose asbestos-exposed patients to malignant pleural mesothelioma.

    Science.gov (United States)

    Betti, Marta; Casalone, Elisabetta; Ferrante, Daniela; Aspesi, Anna; Morleo, Giulia; Biasi, Alessandra; Sculco, Marika; Mancuso, Giuseppe; Guarrera, Simonetta; Righi, Luisella; Grosso, Federica; Libener, Roberta; Pavesi, Mansueto; Mariani, Narciso; Casadio, Caterina; Boldorini, Renzo; Mirabelli, Dario; Pasini, Barbara; Magnani, Corrado; Matullo, Giuseppe; Dianzani, Irma

    2017-10-01

    Malignant pleural mesothelioma (MPM) is a rare, aggressive cancer caused by asbestos exposure. An inherited predisposition has been suggested to explain multiple cases in the same family and the observation that not all individuals highly exposed to asbestos develop the tumor. Germline mutations in BAP1 are responsible for a rare cancer predisposition syndrome that includes predisposition to mesothelioma. We hypothesized that other genes involved in hereditary cancer syndromes could be responsible for the inherited mesothelioma predisposition. We investigated the prevalence of germline variants in 94 cancer-predisposing genes in 93 MPM patients with a quantified asbestos exposure. Ten pathogenic truncating variants (PTVs) were identified in PALB2, BRCA1, FANCI, ATM, SLX4, BRCA2, FANCC, FANCF, PMS1 and XPC. All these genes are involved in DNA repair pathways, mostly in homologous recombination repair. Patients carrying PTVs represented 9.7% of the panel and showed lower asbestos exposure than did all the other patients (p = 0.0015). This suggests that they did not efficiently repair the DNA damage induced by asbestos and leading to carcinogenesis. This study shows that germline variants in several genes may increase MPM susceptibility in the presence of asbestos exposure and may be important for specific treatment. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  5. Tumor site- and stage-specific associations between allelic variants of glutathione S-transferase and DNA-repair genes and overall survival in colorectal cancer patients receiving 5-fluorouracil-based chemotherapy.

    Directory of Open Access Journals (Sweden)

    Ching-Yu Lai

    Full Text Available INTRODUCTION: Our retrospective cohort study investigated the effect of tumor site and stage on the associations between the allelic variants of glutathione S-transferase (GST and DNA-repair genes and overall survival (OS in CRC patients treated with 5-fluorouracil (5-FU-based adjuvant chemotherapy. MATERIAL AND METHODS: We genotyped GSTM1, GSTT1, GSTP1 Ile105Val, XRCC1 Arg399Gln, XRCC3 Thr241Met, and XPD Lys751Gln in 491 CRC patients between 1995 and 2001. A Cox proportional-hazards model was used to calculate the hazard ratios (HRs and 95% confidence intervals (CIs for the relationships between the allelic variants and OS. Survival analyses were performed for each allelic variant by using the log-rank test and Kaplan-Meier analysis. RESULTS: The CRC patients with the XPD Gln allelic variants had poorer survival than patients with the Lys/Lys genotype (HR  =1.38, 95% CI  =1.02-1.87, and rectal cancer patients had the poorest survival among them (HR  =1.87, 95% CI  =1.18-2.95. A significantly shorter OS was observed among stage II/III colon cancer patients with the XRCC1 Gln allelic variants (HR  =1.69, 95% CI  =1.06-2.71, compared to those with XRCC1 Arg/Arg genotype. In the combined analysis of the XRCC1 and XPD genes patients with stage II/III tumors, the poorest OS occurred in colon cancer patients with the XRCC1 Gln and XPD Gln allelic variants (HR  =2.60, 95% CI  =1.19-5.71 and rectal cancer patients with the XRCC1 Arg/Arg and XPD Gln allelic variants (HR  =2.77, 95% CI  =1.25-6.17. CONCLUSION: The XPD and XRCC1 allelic variants may be prognostic markers for CRC patients receiving 5-FU based chemotherapy. The contributions of the XPD and XRCC1 allelic variants to OS are tumor site- and/or stage-dependent.

  6. Simulated microgravity influenced the expression of DNA damage repair genes

    Science.gov (United States)

    Zhang, Meng; Sun, Yeqing; Jiawei, Liu; Wang, Ting

    2016-07-01

    Ionizing radiation and microgravity were considered to be the most important stress factors of space environmental the respective study of the biological effects of the radiation and microgravity carried out earlier, but the interaction of the effects of radiation with microgravity started later, and due to difference of the materials and methods the result of this experiment were not consistent. To further investigate the influence of microgravity on the expression of the radiation damage repair genes, the seed of Arabidopsis (Col) and its gravity-insensitive mutant (PIN2) were exposed to 0.1Gy of the dose of energetic carbon-ion beam radiation (LET = 30KeV / μm), and the germinated seed were than fixed in the 3D random positioning apparatus immediately for a 10-day simulated microgravity. By measuring the deflection angle of root tip and the changes of the expression of Ku70 and RAD51 protein, we investigated the impact of microgravity effect on radiation damage repair systems. The results shown that radiation, microgravity and microgravity with radiation could increase the angle of the root of the Col significantly, but no obvious effect on PIN2 type. The radiation could increase the expression of Ku70 significantly in both Col and PIN2, microgravity does not affect the expression, but the microgravity with radiation could decrease the expression of Ku70. This result shown that the microgravity could influence the radiation damage repair systems in molecular level. Moreover, our findings were important to understand the molecular mechanism of the impact of microgravity effect on radiation damage repair systems in vivo.

  7. Polymorphism of the DNA Base Excision Repair Genes in Keratoconus

    Directory of Open Access Journals (Sweden)

    Katarzyna A. Wojcik

    2014-10-01

    Full Text Available Keratoconus (KC is a degenerative corneal disorder for which the exact pathogenesis is not yet known. Oxidative stress is reported to be associated with this disease. The stress may damage corneal biomolecules, including DNA, and such damage is primarily removed by base excision repair (BER. Variation in genes encoding BER components may influence the effectiveness of corneal cells to cope with oxidative stress. In the present work we genotyped 5 polymorphisms of 4 BER genes in 284 patients and 353 controls. The A/A genotype of the c.–1370T>A polymorphism of the DNA polymerase γ (POLG gene was associated with increased occurrence of KC, while the A/T genotype was associated with decreased occurrence of KC. The A/G genotype and the A allele of the c.1196A>G polymorphism of the X-ray repair cross-complementing group 1 (XRCC1 were associated with increased, and the G/G genotype and the G allele, with decreased KC occurrence. Also, the C/T and T as well as C/C genotypes and alleles of the c.580C>T polymorphism of the same gene displayed relationship with KC occurrence. Neither the g.46438521G>C polymorphism of the Nei endonuclease VIII-like 1 (NEIL1 nor the c.2285T>C polymorphism of the poly(ADP-ribose polymerase-1 (PARP-1 was associated with KC. In conclusion, the variability of the XRCC1 and POLG genes may play a role in KC pathogenesis and determine the risk of this disease.

  8. Evolution and mutagenesis of the mammalian excision repair gene ERCC-1

    NARCIS (Netherlands)

    M. van Duin (Mark); J. van den Tol; P. Warmerdam (Peter); H. Odijk (Hanny); D.N. Meijer (Dies); A. Westerveld (Andries); D. Bootsma (Dirk); J.H.J. Hoeijmakers (Jan)

    1988-01-01

    textabstractThe human DNA excision repair protein ERCC-1 exhibits homology to the yeast RADIO repair protein and its longer C-terminus displays similarity to parts of the E.coli repair proteins uvrA and uvrC. To study the evolution of this 'mosaic' ERCC-1 gene we have isolated the mouse homologue.

  9. A sensitive test for the detection of specific DSB repair defects in primary cells from breast cancer specimens.

    Science.gov (United States)

    Keimling, Marlen; Kaur, Jatinder; Bagadi, Sarangadhara Appala Raju; Kreienberg, Rolf; Wiesmüller, Lisa; Ralhan, Ranju

    2008-08-01

    Increasing evidence indicates that breast cancer pathogenesis is linked with DNA double-strand break (DSB) repair dysfunction. This conclusion is based on advances in the study of functions of breast cancer susceptibility genes such as BRCA1 and BRCA2, on the identification of breast cancer-associated changes regarding the genetics, expression, and localization of multiple DSB repair factors, and on observations indicating enhanced radiation-induced chromosomal damage in cells from predisposed individuals and sporadic breast cancer patients. In this pilot study, we describe a sensitive method for the analysis of DSB repair functions in mammary carcinomas. Using this method we firstly document alterations in pathway-specific DSB repair activities in primary cells originating from familial as well as sporadic breast cancer. In particular, we identified increases in the mutagenic nonhomologous end joining and single-strand annealing mechanisms in sporadic breast cancers with wild-type BRCA1 and BRCA2, and, thus, similar phenotypes to tumors with mutant alleles of BRCA1 and BRCA2. This suggests that detection of error-prone DSB repair activities may be useful to extend the limits of genotypic characterization of high-risk susceptibility genes. This method may, therefore, serve as a marker for breast cancer risk assessment and, even more importantly, for the prediction of responsiveness to targeted therapies such as to inhibitors of poly(ADP-ribose)polymerase (PARP1).

  10. Rearrangement of Rag-1 recombinase gene in DNA-repair deficient/immunodeficient ``wasted`` mice

    Energy Technology Data Exchange (ETDEWEB)

    Woloschak, G.E.; Weaver, P.; Churchill, M.; Chang-Liu, C-M. [Argonne National Lab., IL (United States); Libertin, C.R. [Loyola Univ., Maywood, IL (United States)

    1992-11-01

    Mice recessive for the autosomal gene ``wasted`` (wst) display a disease pattern which includes increased sensitivity to the killing effects of ionizing radiation, immunodeficiency, and neurologic dysfunction. The recent cloning and characterization of recombinase genes (Rag-l/Rag-2) expressed in lymphoid and possibly central nervous system tissues prompted us to examine expression of these genes in DNA repair-deficient/immunodeficient wasted mice. Our results revealed that in thymus tissue, a small Rag-I transcript (1.0 kb) was detected in wst/wst mice that was not evident in thymus from control mice. In wst/{sm_bullet} mice, a two-fold increase in Rag-1 mRNA was evident in thymus tissue. Rag-2 mRNA could only be detected in thymus tissue from wst/{sm_bullet} and not from wst/wst or parental control BCF, mice. Southern blots revealed a rearrangement or deletion within the Rag-1 gene of affected wasted mice that was not evident in known strain-specific parental or littermate controls. These results support the idea that the Rag-1 gene may map at or near the locus for the wasted mutation. In addition, they suggest the importance of recombinase function in normal immune and central nervous system development as well as the potential contribution of this gene family to the normal repair of radiation-induced DNA damage.

  11. Interaction of DNA repair gene polymorphisms and aflatoxin B1 in the risk of hepatocellular carcinoma

    OpenAIRE

    2014-01-01

    Aflatoxin B1 (AFB1) is an important environmental carcinogen and can induce DNA damage and involve in the carcinogenesis of hepatocellular carcinoma (HCC). The deficiency of DNA repair capacity related to the polymorphisms of DNA repair genes might play a central role in the process of HCC tumorigenesis. However, the interaction of DNA repair gene polymorphisms and AFB1 in the risk of hepatocellular carcinoma has not been elucidated. In this study, we investigated whether six polymorphisms (i...

  12. Molecular characterization of the human excision repair gene ERCC-1: cDNA cloning and aminoacid homology with the yeast DNA repair gene RAD10.

    NARCIS (Netherlands)

    M. van Duin (Mark); J. de Wit (Jan); H. Odijk (Hanny); A. Westerveld (Andries); A. Yasui (Akira); M.H.M. Koken (Marcel); J.H.J. Hoeijmakers (Jan); D. Bootsma (Dirk)

    1986-01-01

    textabstractThe human excision repair gene ERCC-7 was cloned after DNA mediated gene transfer to the CHO mutant 43-38, which is sensitive to ultraviolet light and mitomycin-C. We describe the cloning and sequence analysis of the ERCC-7 cDNA and partial characterization of the gene. ERCC.1 has a size

  13. Assessment by Southern blot analysis of UV-induced damage and repair in human immunoglobulin genes.

    Science.gov (United States)

    Bianchi, M S; Bianchi, N O; de la Chapelle, A

    1990-09-01

    Irradiation of DNA with UV light induces pyrimidine dimers and (6-4) photoproducts. The presence of one of these photolesions in the restriction site of a given endonuclease inhibits DNA cleavage and induces the formation of fragments by incomplete DNA digestion which appear as additional, facultative bands in Southern hybridization autoradiograms. The number and size of these fragments show a positive correlation with the UV dose. The response to UV light of immunoglobulin light-chain constant kappa and heavy-chain constant mu genes was analyzed with 2 specific probes. Constant kappa and mu genes when irradiated as part of the chromatin of living lymphocytes showed a UV sensitivity similar to that of naked DNA. The same genes from granulocytes had 50-60 times lower UV sensitivity. When cells were allowed to repair photolesions for 24 h the facultative bands from granulocytes disappeared indicating that these cells were able to remove photolesions from constant kappa and mu genes. Facultative bands from lymphocytes showed a smaller decrease of density after 24 h repair. This suggests that lymphocytes are less efficient than granulocytes in removing UV damage from constant kappa and mu genes.

  14. Analysis of DNA repair gene polymorphisms and survival in low-grade and anaplastic gliomas

    DEFF Research Database (Denmark)

    Berntsson, Shala Ghaderi; Wibom, Carl; Sjöström, Sara;

    2011-01-01

    The purpose of this study was to explore the variation in DNA repair genes in adults with WHO grade II and III gliomas and their relationship to patient survival. We analysed a total of 1,458 tagging single-nucleotide polymorphisms (SNPs) that were selected to cover DNA repair genes, in 81 grade ...

  15. p53 Gene repair with zinc finger nucleases optimised by yeast 1-hybrid and validated by Solexa sequencing.

    Directory of Open Access Journals (Sweden)

    Frank Herrmann

    Full Text Available The tumor suppressor gene p53 is mutated or deleted in over 50% of human tumors. As functional p53 plays a pivotal role in protecting against cancer development, several strategies for restoring wild-type (wt p53 function have been investigated. In this study, we applied an approach using gene repair with zinc finger nucleases (ZFNs. We adapted a commercially-available yeast one-hybrid (Y1H selection kit to allow rapid building and optimization of 4-finger constructs from randomized PCR libraries. We thus generated novel functional zinc finger nucleases against two DNA sites in the human p53 gene, near cancer mutation 'hotspots'. The ZFNs were first validated using in vitro cleavage assays and in vivo episomal gene repair assays in HEK293T cells. Subsequently, the ZFNs were used to restore wt-p53 status in the SF268 human cancer cell line, via ZFN-induced homologous recombination. The frequency of gene repair and mutation by non-homologous end-joining was then ascertained in several cancer cell lines, using a deep sequencing strategy. Our Y1H system facilitates the generation and optimisation of novel, sequence-specific four- to six-finger peptides, and the p53-specific ZFN described here can be used to mutate or repair p53 in genomic loci.

  16. p53 Gene Repair with Zinc Finger Nucleases Optimised by Yeast 1-Hybrid and Validated by Solexa Sequencing

    Science.gov (United States)

    Herrmann, Frank; Garriga-Canut, Mireia; Baumstark, Rebecca; Fajardo-Sanchez, Emmanuel; Cotterell, James; Minoche, André; Himmelbauer, Heinz; Isalan, Mark

    2011-01-01

    The tumor suppressor gene p53 is mutated or deleted in over 50% of human tumors. As functional p53 plays a pivotal role in protecting against cancer development, several strategies for restoring wild-type (wt) p53 function have been investigated. In this study, we applied an approach using gene repair with zinc finger nucleases (ZFNs). We adapted a commercially-available yeast one-hybrid (Y1H) selection kit to allow rapid building and optimization of 4-finger constructs from randomized PCR libraries. We thus generated novel functional zinc finger nucleases against two DNA sites in the human p53 gene, near cancer mutation ‘hotspots’. The ZFNs were first validated using in vitro cleavage assays and in vivo episomal gene repair assays in HEK293T cells. Subsequently, the ZFNs were used to restore wt-p53 status in the SF268 human cancer cell line, via ZFN-induced homologous recombination. The frequency of gene repair and mutation by non-homologous end-joining was then ascertained in several cancer cell lines, using a deep sequencing strategy. Our Y1H system facilitates the generation and optimisation of novel, sequence-specific four- to six-finger peptides, and the p53-specific ZFN described here can be used to mutate or repair p53 in genomic loci. PMID:21695267

  17. Mating-type suppression of the DNA-repair defect of the yeast rad6 delta mutation requires the activity of genes in the RAD52 epistasis group.

    Science.gov (United States)

    Yan, Y X; Schiestl, R H; Prakash, L

    1995-06-01

    The RAD6 gene of Saccharomyces cerevisiae is required for post-replication repair of UV-damaged DNA, UV mutagenesis, and sporulation. Here, we show that the radiation sensitivity of a MATa rad6 delta strain can be suppressed by the MAT alpha 2 gene carried on a multicopy plasmid. The a1-alpha 2 suppression is specific to the RAD6 pathway, as mutations in genes required for nucleotide excision repair or for recombinational repair do not show such mating-type suppression. The a1-alpha 2 suppression of the rad6 delta mutation requires the activity of the RAD52 group of genes, suggesting that suppression occurs by channelling of post-replication gaps present in the rad6 delta mutant into the RAD52 recombinational repair pathway. The a1-alpha 2 repressor could mediate this suppression via an enhancement in the expression, or the activity, of recombination genes.

  18. Transfection of the cloned human excision repair gene ERCC-1 to UV-sensitive CHO mutants only corrects the repair defect in complementation group 2 mutants.

    NARCIS (Netherlands)

    M. van Duin (Mark); J.H. Janssen; J. de Wit (Jan); J.H.J. Hoeijmakers (Jan); L.H. Thompson; D. Bootsma (Dirk); A. Westerveld (Andries)

    1988-01-01

    textabstractThe human DNA-excision repair gene ERCC-1 is cloned by its ability to correct the excision-repair defect of the ultraviolet light- and mitomycin-C-sensitive CHO mutant cell line 43-3B. This mutant is assigned to complementation group 2 of the excision-repair-deficient CHO mutants. In ord

  19. Tumor-specific gene expression patterns with gene expression profiles

    Institute of Scientific and Technical Information of China (English)

    RUAN Xiaogang; LI Yingxin; LI Jiangeng; GONG Daoxiong; WANG Jinlian

    2006-01-01

    Gene expression profiles of 14 common tumors and their counterpart normal tissues were analyzed with machine learning methods to address the problem of selection of tumor-specific genes and analysis of their differential expressions in tumor tissues. First, a variation of the Relief algorithm, "RFE_Relief algorithm" was proposed to learn the relations between genes and tissue types. Then, a support vector machine was employed to find the gene subset with the best classification performance for distinguishing cancerous tissues and their counterparts. After tissue-specific genes were removed, cross validation experiments were employed to demonstrate the common deregulated expressions of the selected gene in tumor tissues. The results indicate the existence of a specific expression fingerprint of these genes that is shared in different tumor tissues, and the hallmarks of the expression patterns of these genes in cancerous tissues are summarized at the end of this paper.

  20. The Gene Targeting Approach of Small Fragment Homologous Replacement (SFHR Alters the Expression Patterns of DNA Repair and Cell Cycle Control Genes

    Directory of Open Access Journals (Sweden)

    Silvia Pierandrei

    2016-01-01

    Full Text Available Cellular responses and molecular mechanisms activated by exogenous DNA that invades cells are only partially understood. This limits the practical use of gene targeting strategies. Small fragment homologous replacement (SFHR uses a small exogenous wild-type DNA fragment to restore the endogenous wild-type sequence; unfortunately, this mechanism has a low frequency of correction. In this study, we used a mouse embryonic fibroblast cell line with a stably integrated mutated gene for enhanced green fluorescence protein. The restoration of a wild-type sequence can be detected by flow cytometry analysis. We quantitatively analyzed the expression of 84 DNA repair genes and 84 cell cycle control genes. Peculiar temporal gene expression patterns were observed for both pathways. Different DNA repair pathways, not only homologous recombination, as well as the three main cell cycle checkpoints appeared to mediate the cellular response. Eighteen genes were selected as highly significant target/effectors of SFHR. We identified a wide interconnection between SFHR, DNA repair, and cell cycle control. Our results increase the knowledge of the molecular mechanisms involved in cell invasion by exogenous DNA and SFHR. Specific molecular targets of both the cell cycle and DNA repair machineries were selected for manipulation to enhance the practical application of SFHR.

  1. Gene expression of the mismatch repair gene MSH2 in primary colorectal cancer

    DEFF Research Database (Denmark)

    Jensen, Lars Henrik; Kuramochi, Hidekazu; Crüger, Dorthe Gylling

    2011-01-01

    Microsatellite instability (MSI) is caused by defective mismatch repair (MMR) and is one of the very few molecular markers with proven clinical importance in colorectal cancer with respect to heredity, prognosis, and treatment effect. The gene expression of the MMR gene MSH2 may be a quantitative...... marker for the level of MMR and a potential molecular marker with clinical relevance. The aim was to investigate the gene expression of MSH2 in primary operable colorectal cancer in correlation with MSI, protein expression, and promoter hypermethylation. In a cohort of 210 patients, the primary tumor...... and lymphnode metastases were analyzed with immunohistochemistry, methylation and MSI analyses, and quantitative polymerase chain reaction (PCR). The median gene expression of MSH2 was 1.00 (range 0.16-11.2, quartiles 0.70-1.51) and there was good agreement between the gene expression in primary tumor and lymph...

  2. Ndrg3 gene regulates DSB repair during meiosis through modulation the ERK signal pathway in the male germ cells

    Science.gov (United States)

    Pan, Hongjie; Zhang, Xuan; Jiang, Hanwei; Jiang, Xiaohua; Wang, Liu; Qi, Qi; Bi, Yuan; Wang, Jian; Shi, Qinghua; Li, Runsheng

    2017-01-01

    The N-myc downstream regulated gene (NDRG) family consists of 4 members, NDRG-1, -2, -3, -4. Physiologically, we found Ndrg3, a critical gene which led to homologous lethality in the early embryo development, regulated the male meiosis in mouse. The expression of Ndrg3 was enhanced specifically in germ cells, and reached its peak level in the pachytene stage spermatocyte. Haplo-insufficiency of Ndrg3 gene led to sub-infertility during the male early maturation. In the Ndrg3+/− germ cells, some meiosis events such as DSB repair and synaptonemal complex formation were impaired. Disturbances on meiotic prophase progression and spermatogenesis were observed. In mechanism, the attenuation of pERK1/2 signaling was detected in the heterozygous testis. With our primary spermatocyte culture system, we found that lactate promoted DSB repair via ERK1/2 signaling in the male mouse germ cells in vitro. Deficiency of Ndrg3 gene attenuated the activation of ERK which further led to the aberrancy of DSB repair in the male germ cells in mouse. Taken together, we reported that Ndrg3 gene modulated the lactate induced ERK pathway to facilitate DSB repair in male germ cells, which further regulated meiosis and subsequently fertility in male mouse. PMID:28290521

  3. Ndrg3 gene regulates DSB repair during meiosis through modulation the ERK signal pathway in the male germ cells.

    Science.gov (United States)

    Pan, Hongjie; Zhang, Xuan; Jiang, Hanwei; Jiang, Xiaohua; Wang, Liu; Qi, Qi; Bi, Yuan; Wang, Jian; Shi, Qinghua; Li, Runsheng

    2017-03-14

    The N-myc downstream regulated gene (NDRG) family consists of 4 members, NDRG-1, -2, -3, -4. Physiologically, we found Ndrg3, a critical gene which led to homologous lethality in the early embryo development, regulated the male meiosis in mouse. The expression of Ndrg3 was enhanced specifically in germ cells, and reached its peak level in the pachytene stage spermatocyte. Haplo-insufficiency of Ndrg3 gene led to sub-infertility during the male early maturation. In the Ndrg3(+/-) germ cells, some meiosis events such as DSB repair and synaptonemal complex formation were impaired. Disturbances on meiotic prophase progression and spermatogenesis were observed. In mechanism, the attenuation of pERK1/2 signaling was detected in the heterozygous testis. With our primary spermatocyte culture system, we found that lactate promoted DSB repair via ERK1/2 signaling in the male mouse germ cells in vitro. Deficiency of Ndrg3 gene attenuated the activation of ERK which further led to the aberrancy of DSB repair in the male germ cells in mouse. Taken together, we reported that Ndrg3 gene modulated the lactate induced ERK pathway to facilitate DSB repair in male germ cells, which further regulated meiosis and subsequently fertility in male mouse.

  4. Use of the comet-FISH assay to compare DNA damage and repair in p53 and hTERT genes following ionizing radiation.

    Directory of Open Access Journals (Sweden)

    Declan J McKenna

    Full Text Available The alkaline single cell gel electrophoresis (comet assay can be combined with fluorescent in situ hybridisation (FISH methodology in order to investigate the localisation of specific gene domains within an individual cell. The number and position of the fluorescent signal(s provides information about the relative damage and subsequent repair that is occurring in the targeted gene domain(s. In this study, we have optimised the comet-FISH assay to detect and compare DNA damage and repair in the p53 and hTERT gene regions of bladder cancer cell-lines RT4 and RT112, normal fibroblasts and Cockayne Syndrome (CS fibroblasts following γ-radiation. Cells were exposed to 5Gy γ-radiation and repair followed for up to 60 minutes. At each repair time-point, the number and location of p53 and hTERT hybridisation spots was recorded in addition to standard comet measurements. In bladder cancer cell-lines and normal fibroblasts, the p53 gene region was found to be rapidly repaired relative to the hTERT gene region and the overall genome, a phenomenon that appeared to be independent of hTERT transcriptional activity. However, in the CS fibroblasts, which are defective in transcription coupled repair (TCR, this rapid repair of the p53 gene region was not observed when compared to both the hTERT gene region and the overall genome, proving the assay can detect variations in DNA repair in the same gene. In conclusion, we propose that the comet-FISH assay is a sensitive and rapid method for detecting differences in DNA damage and repair between different gene regions in individual cells in response to radiation. We suggest this increases its potential for measuring radiosensitivity in cells and may therefore have value in a clinical setting.

  5. A novel method for monitoring functional lesion-specific recruitment of repair proteins in live cells

    Energy Technology Data Exchange (ETDEWEB)

    Woodrick, Jordan; Gupta, Suhani; Khatkar, Pooja; Dave, Kalpana; Levashova, Darya; Choudhury, Sujata; Elias, Hadi; Saha, Tapas; Mueller, Susette; Roy, Rabindra, E-mail: rr228@georgetown.edu

    2015-05-15

    Highlights: • A method of monitoring lesion-specific recruitment of proteins in vivo is described. • Recruitment of repair enzymes to abasic sites is monitored by co-localization. • Repair protein recruitment is consistent with known protein–protein relationships. • Cells demonstrated complete repair of abasic sites by 90 min. - Abstract: DNA–protein relationships have been studied by numerous methods, but a particular gap in methodology lies in the study of DNA adduct-specific interactions with proteins in vivo, which particularly affects the field of DNA repair. Using the repair of a well-characterized and ubiquitous adduct, the abasic (AP) site, as a model, we have developed a comprehensive method of monitoring DNA lesion-specific recruitment of proteins in vivo over time. We utilized a surrogate system in which a Cy3-labeled plasmid containing a single AP-site was transfected into cells, and the interaction of the labeled DNA with BER enzymes, including APE1, Polβ, LIG1, and FEN1, was monitored by immunofluorescent staining of the enzymes by Alexafluor-488-conjugated secondary antibody. The recruitment of enzymes was characterized by quantification of Cy3-Alexafluor-488 co-localization. To validate the microscopy-based method, repair of the transfected AP-site DNA was also quantified at various time points post-transfection using a real time PCR-based method. Notably, the recruitment time kinetics for each enzyme were consistent with AP-site repair time kinetics. This microscopy-based methodology is reliable in detecting the recruitment of proteins to specific DNA substrates and can be extended to study other in vivo DNA–protein relationships in any DNA sequence and in the context of any DNA structure in transfectable proliferating or quiescent cells. The method may be applied to a variety of disciplines of nucleic acid transaction pathways, including repair, replication, transcription, and recombination.

  6. Are SNP-Smoking Association Studies Needed in Controls? DNA Repair Gene Polymorphisms and Smoking Intensity.

    Science.gov (United States)

    Verde, Zoraida; Reinoso, Luis; Chicharro, Luis Miguel; Resano, Pilar; Sánchez-Hernández, Ignacio; Rodríguez González-Moro, Jose Miguel; Bandrés, Fernando; Gómez-Gallego, Félix; Santiago, Catalina

    2015-01-01

    Variations in tobacco-related cancers, incidence and prevalence reflect differences in tobacco consumption in addition to genetic factors. Besides, genes related to lung cancer risk could be related to smoking behavior. Polymorphisms altering DNA repair capacity may lead to synergistic effects with tobacco carcinogen-induced lung cancer risk. Common problems in genetic association studies, such as presence of gene-by-environment (G x E) correlation in the population, may reduce the validity of these designs. The main purpose of this study was to evaluate the independence assumption for selected SNPs and smoking behaviour in a cohort of 320 healthy Spanish smokers. We found an association between the wild type alleles of XRCC3 Thr241Met or KLC3 Lys751Gln and greater smoking intensity (OR = 12.98, 95% CI = 2.86-58.82 and OR=16.90, 95% CI=2.09-142.8; respectively). Although preliminary, the results of our study provide evidence that genetic variations in DNA-repair genes may influence both smoking habits and the development of lung cancer. Population-specific G x E studies should be carried out when genetic and environmental factors interact to cause the disease.

  7. Are SNP-Smoking Association Studies Needed in Controls? DNA Repair Gene Polymorphisms and Smoking Intensity.

    Directory of Open Access Journals (Sweden)

    Zoraida Verde

    Full Text Available Variations in tobacco-related cancers, incidence and prevalence reflect differences in tobacco consumption in addition to genetic factors. Besides, genes related to lung cancer risk could be related to smoking behavior. Polymorphisms altering DNA repair capacity may lead to synergistic effects with tobacco carcinogen-induced lung cancer risk. Common problems in genetic association studies, such as presence of gene-by-environment (G x E correlation in the population, may reduce the validity of these designs. The main purpose of this study was to evaluate the independence assumption for selected SNPs and smoking behaviour in a cohort of 320 healthy Spanish smokers. We found an association between the wild type alleles of XRCC3 Thr241Met or KLC3 Lys751Gln and greater smoking intensity (OR = 12.98, 95% CI = 2.86-58.82 and OR=16.90, 95% CI=2.09-142.8; respectively. Although preliminary, the results of our study provide evidence that genetic variations in DNA-repair genes may influence both smoking habits and the development of lung cancer. Population-specific G x E studies should be carried out when genetic and environmental factors interact to cause the disease.

  8. Triple Negative Breast Cancers Have a Reduced Expression of DNA Repair Genes

    Science.gov (United States)

    Andreis, Daniele; Bertoni, Ramona; Giardini, Roberto; Fox, Stephen B.; Broggini, Massimo; Bottini, Alberto; Zanoni, Vanessa; Bazzola, Letizia; Foroni, Chiara; Generali, Daniele; Damia, Giovanna

    2013-01-01

    DNA repair is a key determinant in the cellular response to therapy and tumor repair status could play an important role in tailoring patient therapy. Our goal was to evaluate the mRNA of 13 genes involved in different DNA repair pathways (base excision, nucleotide excision, homologous recombination, and Fanconi anemia) in paraffin embedded samples of triple negative breast cancer (TNBC) compared to luminal A breast cancer (LABC). Most of the genes involved in nucleotide excision repair and Fanconi Anemia pathways, and CHK1 gene were significantly less expressed in TNBC than in LABC. PARP1 levels were higher in TNBC than in LABC. In univariate analysis high level of FANCA correlated with an increased overall survival and event free survival in TNBC; however multivariate analyses using Cox regression did not confirm FANCA as independent prognostic factor. These data support the evidence that TNBCs compared to LABCs harbour DNA repair defects. PMID:23825533

  9. Polymorphisms of the DNA repair genes XRCC1 and XRCC3 in a Brazilian population

    Directory of Open Access Journals (Sweden)

    Duarte Márcia Cristina

    2005-01-01

    Full Text Available In several DNA repair genes, polymorphisms may result in reduced repair capacity, which has been implicated as a risk factor for various types of cancer. The frequency of the polymorphic alleles varies among populations, suggesting an ethnic distribution of genotypes. We genotyped 300 healthy Southeastern Brazilian individuals (262 of European ancestry and 38 of African ancestry for polymorphisms of codons 194 and 399 of the XRCC1 base excision repair pathway gene and of codon 241 of the XRCC3 homologous recombination repair pathway gene. The allele frequencies were 0.07 for the Arg194Trp and 0.33 for the Arg399Gln codons of the XRCC1 gene and 0.35 for the Thr241Met codon of the XRCC3 gene. The genotypic frequencies were within Hardy-Weinberg equilibrium. These frequencies showed ethnic variability when compared with those obtained for different populations from several countries.

  10. Rearrangement of RAG-1 recombinase gene in DNA-repair deficient ``wasted`` mice

    Energy Technology Data Exchange (ETDEWEB)

    Woloschak, G.E.; Libertin, C.R.; Weaver, P. [Loyola Univ., Chicago, IL (United States); Churchill, M.; Chang-Liu, C.M. [Argonne National Lab., IL (United States)

    1993-11-01

    Mice recessive for the autosomal gene ``wasted`` wst display a disease pattern which includes increased sensitivity to the killing effects of ionizing radiation, immunodeficiency, and neurologic dysfunction. The recent cloning and characterization of recombinase genes (RAG-l/RAG-2) expressed in lymphoid and possibly central nervous system tissues prompted us to examine expression of these genes in DNA repair-deficient/immunodeficient wasted mice. Our results revealed expression of RAG-1 mRNA in spinal cord (but not brain) of control mice; no expression of RAG-1 mRNA was detected in spinal cord or brain from wst/wst mice or their normal littermates (wst/{center_dot}mice). In thymus tissue, a small RAG-1 transcript (1.0 kb) was detected in wst/wst mice that was not evident in thymus from control mice. In wst/{center_dot}mice, a two-fold increase in RAG-1 mRNA was evident in thymus tissue. RAG-2 mRNA could only be detected in thymus tissue from wst/{center_dot} and not from wst/wst or parental control BCF{sub 1} mice. Southern blots revealed a rearrangement/deletion within the RAG-1 gene of affected wasted mice, not evident in known strain-specific parental or littermate controls. These results support the idea that the RAG-1 gene may map at or near the locus for the wasted mutation. In addition, they suggest the importance of recombinase function in normal immune and central nervous system development as well as the potential contribution of this gene family to the normal repair of radiation-induced DNA damage.

  11. DNA repair in human cells: from genetic complementation to isolation of genes.

    NARCIS (Netherlands)

    D. Bootsma (Dirk); A. Westerveld (Andries); J.H.J. Hoeijmakers (Jan)

    1988-01-01

    textabstractThe genetic disease xeroderma pigmentosum (XP) demonstrates the association between defective repair of DNA lesions and cancer. Complementation analysis performed on XP cell strains and on repair deficient rodent cell lines has revealed that at least nine and possibly more than 13 genes

  12. Cloning and characterization of the human DNA-excision repair gene ERCC-1

    NARCIS (Netherlands)

    M. van Duin (Michel)

    1988-01-01

    textabstractIt is the aim of the work described in this thesis to isolate and characterize human genes involved DNA excision repair. This will facilitate the understanding of the mechanism of this repair process whereas it also provides an important step to better understand the relationship

  13. Cloning and characterization of the human DNA-excision repair gene ERCC-1

    NARCIS (Netherlands)

    M. van Duin (Michel)

    1988-01-01

    textabstractIt is the aim of the work described in this thesis to isolate and characterize human genes involved DNA excision repair. This will facilitate the understanding of the mechanism of this repair process whereas it also provides an important step to better understand the relationship between

  14. DNA repair in human cells: from genetic complementation to isolation of genes.

    NARCIS (Netherlands)

    D. Bootsma (Dirk); A. Westerveld (Andries); J.H.J. Hoeijmakers (Jan)

    1988-01-01

    textabstractThe genetic disease xeroderma pigmentosum (XP) demonstrates the association between defective repair of DNA lesions and cancer. Complementation analysis performed on XP cell strains and on repair deficient rodent cell lines has revealed that at least nine and possibly more than 13 genes

  15. Symptomatic and quality of life outcomes after site-specific fascial reattachment for pelvic organ prolapse repair.

    Science.gov (United States)

    Fayyad, Abdalla M; Redhead, Emma; Awan, Noveen; Kyrgiou, Maria; Prashar, Sanjeev; Hill, Simon R

    2008-02-01

    The aim of this study was to assess symptomatic and quality of life outcome scores following site specific fascial reattachment surgery for pelvic organ prolapse using the validated Prolapse Quality of Life (P-QOL) questionnaires. One hundred and ninety two women underwent surgery for pelvic organ prolapse; ninety four underwent anterior repair (thirty four of them had vaginal hysterectomy), and ninety eight had posterior repair. Patients filled P-QOL questionnaires 24 hours prior to surgery and a postal P-QOL questionnaire six months post operatively. Pre and post operative questionnaires were paired. Quality of life and symptoms scores were calculated using Wilcoxon signed rank test. One hundred and one women returned their questionnaires and were suitable to include in the study. Forty nine underwent anterior repair (fifteen had vaginal hysterectomy) and 52 underwent posterior repair. Quality of life scores showed significant improvement in the anterior and posterior repair groups with the exception of general health in the anterior repair group and general health and prolapse impact in the posterior repair group. Anterior repair significantly improved urinary voiding and storage symptoms. Posterior repair group showed significant improvement in defecatory symptoms. Both groups showed improvement in sexual function and general prolapse symptoms. Prolapse repair with site specific fascial reattachment results in significant improvement in quality of life scores six months after surgery. Anterior repair improves urinary voiding and storage symptoms and posterior repair improves defecatory dysfunction and urinary voiding. Sexual function improves following prolapse repair with site specific fascial reattachment.

  16. Practices of Other-Initiated Repair in the Classrooms of Children with Specific Speech and Language Difficulties

    Science.gov (United States)

    Radford, Julie

    2010-01-01

    Repair practices used by teachers who work with children with specific speech and language difficulties (SSLDs) have hitherto remained largely unexplored. Such classrooms therefore offer a new context for researching repairs and considering how they compare with non-SSLD interactions. Repair trajectories are of interest because they are dialogic…

  17. Differential expression of tissue repair genes in the pathogenesis of chronic obstructive pulmonary disease

    National Research Council Canada - National Science Library

    Gosselink, John V; Hayashi, Shizu; Elliott, W Mark; Xing, Li; Chan, Becky; Yang, Luojia; Wright, Claire; Sin, Don; Paré, Peter D; Pierce, John A; Pierce, Richard A; Patterson, Alex; Cooper, Joel; Hogg, James C

    2010-01-01

    .... The expression of 54 genes associated with repair of repetitively damaged tissue was measured in 136 paired samples of small bronchioles and surrounding lung tissue separated by laser capture microdissection...

  18. Specific sizes of hyaluronan oligosaccharides stimulate fibroblast migration and excisional wound repair.

    Directory of Open Access Journals (Sweden)

    Cornelia Tolg

    Full Text Available The extracellular matrix polysaccharide hyaluronan (HA plays a key role in both fibrotic and regenerative tissue repair. Accumulation of high molecular weight HA is typical of regenerative repair, which is associated with minimal inflammation and fibrosis, while fragmentation of HA is typical of postnatal wounds, which heal in the presence of inflammation and transient fibrosis. It is generally considered that HA oligosaccharides and fragments of a wide size range support these processes of adult, fibrotic wound repair yet the consequences of sized HA fragments/oligosaccharides to each repair stage is not well characterized. Here, we compared the effects of native HA, HA oligosaccharide mixtures and individual sizes (4-10 mer oligosaccharides, 5 and, 40 kDa of HA oligosaccharides and fragments, on fibroblast migration in scratch wound assays and on excisional skin wound repair in vivo. We confirm that 4-10 mer mixtures significantly stimulated scratch wound repair and further report that only the 6 and 8 mer oligosaccharides in this mixture are responsible for this effect. The HA 6 mer promoted wound closure, accumulation of wound M1 and M2 macrophages and the M2 cytokine TGFβ1, but did not increase myofibroblast differentiation. The effect of 6 mer HA on wound closure required both RHAMM and CD44 expression. In contrast, The 40 kDa HA fragment inhibited wound closure, increased the number of wound macrophages but had no effect on TGFβ1 accumulation or subsequent fibrosis. These results show that specific sizes of HA polymer have unique effects on postnatal wound repair. The ability of 6 mer HA to promote wound closure and inflammation resolution without increased myofibroblast differentiation suggests that this HA oligosaccharide could be useful for treatment of delayed or inefficient wound repair where minimal fibrosis is advantageous.

  19. Transcript RNA supports precise repair of its own DNA gene.

    Science.gov (United States)

    Keskin, Havva; Meers, Chance; Storici, Francesca

    2016-01-01

    The transfer of genetic information from RNA to DNA is considered an extraordinary process in molecular biology. Despite the fact that cells transcribe abundant amount of RNA with a wide range of functions, it has been difficult to uncover whether RNA can serve as a template for DNA repair and recombination. An increasing number of experimental evidences suggest a direct role of RNA in DNA modification. Recently, we demonstrated that endogenous transcript RNA can serve as a template to repair a DNA double-strand break (DSB), the most harmful DNA lesion, not only indirectly via formation of a DNA copy (cDNA) intermediate, but also directly in a homology driven mechanism in budding yeast. These results point out that the transfer of genetic information from RNA to DNA is more general than previously thought. We found that transcript RNA is more efficient in repairing a DSB in its own DNA (in cis) than in a homologous but ectopic locus (in trans). Here, we summarize current knowledge about the process of RNA-driven DNA repair and recombination, and provide further data in support of our model of DSB repair by transcript RNA in cis. We show that a DSB is precisely repaired predominately by transcript RNA and not by residual cDNA in conditions in which formation of cDNA by reverse transcription is inhibited. Additionally, we demonstrate that defects in ribonuclease (RNase) H stimulate precise DSB repair by homologous RNA or cDNA sequence, and not by homologous DNA sequence carried on a plasmid. These results highlight an antagonistic role of RNase H in RNA-DNA recombination. Ultimately, we discuss several questions that should be addressed to better understand mechanisms and implications of RNA-templated DNA repair and recombination.

  20. Manipulation of cell cycle progression can counteract the apparent loss of correction frequency following oligonucleotide-directed gene repair

    Directory of Open Access Journals (Sweden)

    Kmiec Eric B

    2007-02-01

    Full Text Available Abstract Background Single-stranded oligonucleotides (ssODN are used routinely to direct specific base alterations within mammalian genomes that result in the restoration of a functional gene. Despite success with the technique, recent studies have revealed that following repair events, correction frequencies decrease as a function of time, possibly due to a sustained activation of damage response signals in corrected cells that lead to a selective stalling. In this study, we use thymidine to slow down the replication rate to enhance repair frequency and to maintain substantial levels of correction over time. Results First, we utilized thymidine to arrest cells in G1 and released the cells into S phase, at which point specific ssODNs direct the highest level of correction. Next, we devised a protocol in which cells are maintained in thymidine following the repair reaction, in which the replication is slowed in both corrected and non-corrected cells and the initial correction frequency is retained. We also present evidence that cells enter a senescence state upon prolonged treatment with thymidine but this passage can be avoided by removing thymidine at 48 hours. Conclusion Taken together, we believe that thymidine may be used in a therapeutic fashion to enable the maintenance of high levels of treated cells bearing repaired genes.

  1. DNA repair gene polymorphisms in relation to chromosome aberration frequencies in retired radiation workers

    Energy Technology Data Exchange (ETDEWEB)

    Wilding, Craig S. [Genetics Department, Westlakes Research Institute, Westlakes Science and Technology Park, Moor Row, Cumbria CA24 3JY (United Kingdom)]. E-mail: craig.wilding@westlakes.ac.uk; Relton, Caroline L. [Genetics Department, Westlakes Research Institute, Westlakes Science and Technology Park, Moor Row, Cumbria CA24 3JY (United Kingdom); Paediatric and Lifecourse Epidemiology Research Group, School of Clinical Medical Sciences (Child Health), Newcastle University, Sir James Spence Institute, Royal Victoria Infirmary, Newcastle-upon-Tyne NE1 4LP (United Kingdom); Rees, Gwen S. [Genetics Department, Westlakes Research Institute, Westlakes Science and Technology Park, Moor Row, Cumbria CA24 3JY (United Kingdom); Tarone, Robert E. [International Epidemiology Institute, 1455 Research Boulevard, Suite 550, Rockville, MD 20850 (United States); Whitehouse, Caroline A. [Genetics Department, Westlakes Research Institute, Westlakes Science and Technology Park, Moor Row, Cumbria CA24 3JY (United Kingdom); Tawn, E. Janet [Genetics Department, Westlakes Research Institute, Westlakes Science and Technology Park, Moor Row, Cumbria CA24 3JY (United Kingdom)

    2005-02-15

    Polymorphic variation in DNA repair genes was examined in a group of retired workers from the British Nuclear Fuels plc facility at Sellafield in relation to previously determined translocation frequencies in peripheral blood lymphocytes. Variation at seven polymorphisms in four genes involved in the base excision repair (XRCC1 R194W, R399Q and a [AC]{sub n} microsatellite in the 3' UTR) and double strand break repair (XRCC3 T241M and a [AC]{sub n} microsatellite in intron 3 of XRCC3, XRCC4 I134T, and a GACTAn microsatellite located 120kb 5' of XRCC5) pathways was determined for 291 retired radiation workers who had received cumulative occupational external radiation doses of between 0 and 1873mSv. When the interaction between radiation dose and each DNA repair gene polymorphism was examined in relation to translocation frequency there was no evidence for any of the polymorphisms studied influencing the response to occupational exposure. A positive interaction observed between genotype (individuals with at least one allele >=20 repeat units) at a microsatellite locus in the XRCC3 gene and smoking status should be interpreted cautiously because interactions were investigated for seven polymorphisms and two exposures. Nonetheless, further research is warranted to examine whether this DNA repair gene variant might be associated with a sub-optimal repair response to smoking-induced DNA damage and hence an increased frequency of translocations.

  2. Chromosomal Aberrations and DNA Repair Gene Variants in a Radon-exposed Population

    Energy Technology Data Exchange (ETDEWEB)

    Kiuru, A.; Lindholm, C.; Koivistoinen, A.; Salomaa, S.

    2004-07-01

    Polymorphisms of XRCC1 (X-ray repair cross-complementing group 1), XRCC3 (X-ray repair cross-complementing group 3), and hOGG1 (the human homologue of the yeast OGG1 gene) DNA repair genes have been associated with altered DNA repair capacity and risk of various cancers. In the present study our goal was to clarify the influence of various DNA repair gene variants on the frequency of chromosomal aberrations (CA) in subjects exposed to residential radon. The study group of 84 non-smoking, healthy individuals exposed to domestic radon were analysed using the fluorescence in-situ hybridization (FISH) technique. No association between radon concentration and CA frequencies was observed. However, a significant increase with age was shown as well as a large variability in translocation frequencies between individuals within the same age group. In order to investigate the role of individual susceptibility to this variation genotypes of DNA repair genes XRCC1 (codons 194, 280 and 399), XRCC3 (codon 241) and hOGG1 (codon 326) were determined from leukocyte DNA using methods based on polymerase chain reaction. Multiple regression analysis was applied to evaluate the effect of the polymorphisms and the other confounding factors (age, exposure to randon etc) to the frequency of CA. The preliminary statistical analyses showed that the different gene appeared not to be related to a pronounced increase in chromosome aberration frequencies observed by FISH painting. However, the analysis indicated that the homozygous variant of XRCC3 codon 241 was associated (P<0.05) with two-ways translocations in conjunction with age. Larger studies, both with regard to the cohort and the number of gene variants are needed to elucidate the influence of other DNA repair variants to the yield of chromosomal aberrations. The results indicate that the chromosomal translocations accumulated by age (spontaneous background) may be partly explained by defects in homologous recombination repair. (Author

  3. Genome analysis of DNA repair genes in the alpha proteobacterium Caulobacter crescentus

    Directory of Open Access Journals (Sweden)

    Menck Carlos FM

    2007-03-01

    Full Text Available Abstract Background The integrity of DNA molecules is fundamental for maintaining life. The DNA repair proteins protect organisms against genetic damage, by removal of DNA lesions or helping to tolerate them. DNA repair genes are best known from the gamma-proteobacterium Escherichia coli, which is the most understood bacterial model. However, genome sequencing raises questions regarding uniformity and ubiquity of these DNA repair genes and pathways, reinforcing the need for identifying genes and proteins, which may respond to DNA damage in other bacteria. Results In this study, we employed a bioinformatic approach, to analyse and describe the open reading frames potentially related to DNA repair from the genome of the alpha-proteobacterium Caulobacter crescentus. This was performed by comparison with known DNA repair related genes found in public databases. As expected, although C. crescentus and E. coli bacteria belong to separate phylogenetic groups, many of their DNA repair genes are very similar. However, some important DNA repair genes are absent in the C. crescentus genome and other interesting functionally related gene duplications are present, which do not occur in E. coli. These include DNA ligases, exonuclease III (xthA, endonuclease III (nth, O6-methylguanine-DNA methyltransferase (ada gene, photolyase-like genes, and uracil-DNA-glycosylases. On the other hand, the genes imuA and imuB, which are involved in DNA damage induced mutagenesis, have recently been described in C. crescentus, but are absent in E. coli. Particularly interesting are the potential atypical phylogeny of one of the photolyase genes in alpha-proteobacteria, indicating an origin by horizontal transfer, and the duplication of the Ada orthologs, which have diverse structural configurations, including one that is still unique for C. crescentus. Conclusion The absence and the presence of certain genes are discussed and predictions are made considering the particular

  4. Expression Silence of DNA Repair Gene hMGMT Induced by RNA Interference

    Institute of Scientific and Technical Information of China (English)

    LI Xiu-ying; LAI Yan-dong

    2007-01-01

    Objective: MGMT protein expression has been associated with tumor resistance to alkylating agents. The objective of this paper is to construct the RNA interference vector which can specifically induce the expression silence of human DNA repair gene hMGMT. Methods: The hMGMT specific siRNA expression cassette was made by two steps PCR, linked with pUC19 to get pU6-MGMTi, co-transfected with pEGFP-C1 into 16HBE and screened by G418. The MGMT mRNA and protein levels were detected by RT-PCR and Western Blot respectively. Results: hMGMT specific RNA interfere vector pU6-MGMTi was constructed successfully. In transfected 16HBE cells MGMT mRNA level could hardly be detected and the protein level was only 10% of control. Conclusion: MGMT specific RNAi expression cassette can effectively inhibit MGMT expression. MGMT silence cell line was built by co-transfection technology, which offered condition for studying the gene function of MGMT.

  5. Assessment of single nucleotide polymorphisms in screening 52 DNA repair and cell cycle control genes in Fanconi anemia patients

    Directory of Open Access Journals (Sweden)

    Petrović Sandra

    2015-01-01

    Full Text Available Fanconi anemia (FA is a rare genetically heterogeneous disorder associated with bone marrow failure, birth defects and cancer susceptibility. Apart from the disease- causing mutations in FANC genes, the identification of specific DNA variations, such as single nucleotide polymorphisms (SNPs, in other candidate genes may lead to a better clinical description of this condition enabling individualized treatment with improvement of the prognosis. In this study, we have assessed 95 SNPs located in 52 key genes involved in base excision repair (BER, nucleotide excision repair (NER, mismatch repair (MMR, double strand break (DSB repair and cell cycle control using a DNA repair chip (Asper Biotech, Estonia which includes most of the common variants for the candidate genes. The SNP genotyping was performed in five FA-D2 patients and in one FA-A patient. The polymorphisms studied were synonymous (n=10, nonsynonymous (missense (n=52 and in non-coding regions of the genome (introns and 5 ‘and 3’ untranslated regions (UTR (n=33. Polymorphisms found at the homozygous state are selected for further analysis. Our results have shown a significant inter-individual variability among patients in the type and the frequency of SNPs and also elucidate the need for further studies of polymorphisms located in ATM, APEX APE 1, XRCC1, ERCC2, MSH3, PARP4, NBS1, BARD1, CDKN1B, TP53 and TP53BP1 which may be of great importance for better clinical description of FA. In addition, the present report recommends the use of SNPs as predictive and prognostic genetic markers to individualize therapy of FA patients. [Projekat Ministarstva nauke Republike Srbije, br. 173046

  6. Preferential repair of DNA double-strand break at the active gene in vivo.

    Science.gov (United States)

    Chaurasia, Priyasri; Sen, Rwik; Pandita, Tej K; Bhaumik, Sukesh R

    2012-10-19

    Previous studies have demonstrated transcription-coupled nucleotide/base excision repair. We report here for the first time that DNA double-strand break (DSB) repair is also coupled to transcription. We generated a yeast strain by introducing a homing (Ho) endonuclease cut site followed by a nucleotide sequence for multiple Myc epitopes at the 3' end of the coding sequence of a highly active gene, ADH1. This yeast strain also contains the Ho cut site at the nearly silent or poorly active mating type α (MATα) locus and expresses Ho endonuclease under the galactose-inducible GAL1 promoter. Using this strain, DSBs were generated at the ADH1 and MATα loci in galactose-containing growth medium that induced HO expression. Subsequently, yeast cells were transferred to dextrose-containing growth medium to stop HO expression, and the DSB repair was monitored at the ADH1 and MATα loci by PCR, using the primer pairs flanking the Ho cut sites. Our results revealed a faster DSB repair at the highly active ADH1 than that at the nearly silent MATα locus, hence implicating a transcription-coupled DSB repair at the active gene in vivo. Subsequently, we extended this study to another gene, PHO5 (carrying the Ho cut site at its coding sequence), under transcriptionally active and inactive growth conditions. We found a fast DSB repair at the active PHO5 gene in comparison to its inactive state. Collectively, our results demonstrate a preferential DSB repair at the active gene, thus supporting transcription-coupled DSB repair in living cells.

  7. Polymorphisms in human DNA repair genes and head and neck squamous cell carcinoma

    Indian Academy of Sciences (India)

    Rim Khlifi; Ahmed Rebai; Amel Hamza-Chaffai

    2012-12-01

    Genetic polymorphisms in some DNA repair proteins are associated with a number of malignant transformations like head and neck squamous cell carcinoma (HNSCC). Xeroderma pigmentosum group D (XPD) and X-ray repair cross-complementing proteins 1 (XRCC1) and 3 (XRCC3) genes are involved in DNA repair and were found to be associated with HNSCC in numerous studies. To establish our overall understanding of possible relationships between DNA repair gene polymorphisms and development of HNSCC, we surveyed the literature on epidemiological studies that assessed potential associations with HNSCC risk in terms of gene–environment interactions, genotype-induced functional defects in enzyme activity and/or protein expression, and the influence of ethnic origin on these associations.We conclude that large, well-designed studies of common polymorphisms in DNA repair genes are needed. Such studies may benefit from analysis of multiple genes or polymorphisms and from the consideration of relevant exposures that may influence the likelihood of HNSCC when DNA repair capacity is reduced.

  8. Approaches to diagnose DNA mismatch repair gene defects in cancer

    DEFF Research Database (Denmark)

    Peña-Diaz, Javier; Rasmussen, Lene Juel

    2016-01-01

    The DNA repair pathway mismatch repair (MMR) is responsible for the recognition and correction of DNA biosynthetic errors caused by inaccurate nucleotide incorporation during replication. Faulty MMR leads to failure to address the mispairs or insertion deletion loops (IDLs) left behind...... already been well defined and their pathogenicity assessed. Despite this substantial wealth of knowledge, the effects of a large number of alterations remain uncharacterized (variants of uncertain significance, VUSs). The advent of personalized genomics is likely to increase the list of VUSs found in MMR...

  9. Targeted Modification of Gene Function Exploiting Homology-Directed Repair of TALEN-Mediated Double-Strand Breaks in Barley.

    Science.gov (United States)

    Budhagatapalli, Nagaveni; Rutten, Twan; Gurushidze, Maia; Kumlehn, Jochen; Hensel, Goetz

    2015-07-06

    Transcription activator-like effector nucleases open up new opportunities for targeted mutagenesis in eukaryotic genomes. Similar to zinc-finger nucleases, sequence-specific DNA-binding domains can be fused with effector domains like the nucleolytically active part of FokI to induce double-strand breaks and thereby modify the host genome on a predefined target site via nonhomologous end joining. More sophisticated applications of programmable endonucleases involve the use of a DNA repair template facilitating homology-directed repair (HDR) so as to create predefined rather than random DNA sequence modifications. The aim of this study was to demonstrate the feasibility of editing the barley genome by precisely modifying a defined target DNA sequence resulting in a predicted alteration of gene function. We used gfp-specific transcription activator-like effector nucleases along with a repair template that, via HDR, facilitates conversion of gfp into yfp, which is associated with a single amino acid exchange in the gene product. As a result of co-bombardment of leaf epidermis, we detected yellow fluorescent protein accumulation in about three of 100 mutated cells. The creation of a functional yfp gene via HDR was unambiguously confirmed by sequencing of the respective genomic site. In addition to the allele conversion accomplished in planta, a readily screenable marker system is introduced that might be useful for optimization approaches in the field of genome editing.

  10. Assignment of ten DNA repair genes from Schizosaccharomyces pombe to chromosomal NotI restriction fragments

    NARCIS (Netherlands)

    B.C. Broughton; N.C. Barbet; J. Murray (Johanne); F.Z. Watts (Felicity); M.H.M. Koken (Marcel); A.R. Lehmann (Alan); A.M. Carr (Anthony)

    1991-01-01

    textabstractTen DNA repair (rad) genes from the fission yeast, Schizosaccharomyces pombe were mapped to the 17 NotI fragments of the three chromosomes. Nine of the genes map to chromosome I, but there is no evidence for significant clustering.

  11. 'Hide-then-hit' to explain the importance of genotypic polymorphism of DNA repair genes in determining susceptibility to cancer

    Institute of Scientific and Technical Information of China (English)

    Pei-Ei Wu; Chen-Yang Shen

    2011-01-01

    Interindividual variations in DNA repair capacity/efficiency linked to the presence of polymorphisms in DNA repair-related genes have been suggested to account for different risk of developing cancers. In this review article, on the basis of breast cancer formation as a model, we propose a 'hide-then-hit' hypothesis indicating the importance of escaping checkpoint surveillance for sub-optimal DNA repair variants to cause cancer. Therefore, only cells with subtle defects in repair capacity arising from low-penetrance variants of DNA repair genes would have the opportunity to grow and accumulate the genetic changes needed for cancer formation, without triggering cell-cycle checkpoint surveillance. Furthermore, distinct from high-penetrance alleles, these polymorphic alleles of DNA repair genes would predispose carriers to a higher risk of developing cancer but would not necessarily cause cancer. To examine this,we simultaneously genotyped multiple SNPs of cell-cycle checkpoint genes and the DNA repair genes. Support for the hypothesis came from observations that breast cancer risk associated with variant genotypes of DNA repair genes became more significant in be confirmed by biological evidence in which a cause-effect relationship has to be established. However, based on this, possible gene-gene interaction is considered to play an important role in modifying the cancer risk associated with genotypic polymorphism of DNA repair gene in different study populations.

  12. Blockage of Src by Specific siRNA as a Novel Therapeutic Strategy to Prevent Destructive Repair in Steroid-Associated Osteonecrosis in Rabbits.

    Science.gov (United States)

    Zheng, Li-zhen; Cao, Hui-juan; Chen, Shi-hui; Tang, Tao; Fu, Wei-min; Huang, Le; Chow, Dick Ho Kiu; Wang, Yi-xiang; Griffith, James Francis; He, Wei; Zhou, Hong; Zhao, De-wei; Zhang, Ge; Wang, Xin-luan; Qin, Ling

    2015-11-01

    Vascular hyperpermeability and highly upregulated bone resorption in the destructive repair progress of steroid-associated osteonecrosis (SAON) are associated with a high expression of VEGF and high Src activity (Src is encoded by the cellular sarcoma [c-src] gene). This study was designed to prove our hypothesis that blocking the VEGF-Src signaling pathway by specific Src siRNA is able to prevent destructive repair in a SAON rabbit model. Destructive repair in SAON was induced in rabbits. At 2, 4, and 6 weeks after SAON induction, VEGF, anti-VEGF, Src siRNA, Src siRNA+VEGF, control siRNA, and saline were introduced via intramedullary injection into proximal femora for each group, respectively. Vascularization and permeability were quantified by dynamic contrast-enhanced (DCE) MRI. At week 6 after SAON induction, proximal femurs were dissected for micro-computed tomography (μCT)-based trabecular architecture with finite element analysis (FEA), μCT-based angiography, and histological analysis. Histological evaluation revealed that VEGF enhanced destructive repair, whereas anti-VEGF prevented destructive repair and Src siRNA and Src siRNA+VEGF prevented destructive repair and enhanced reparative osteogenesis. Findings of angiography and histomorphometry were consistent with those determined by DCE MRI. Src siRNA inhibited VEGF-mediated vascular hyperpermeability but preserved VEGF-induced neovascularization. Bone resorption was enhanced in the VEGF group and inhibited in the anti-VEGF, Src siRNA, Src siRNA+VEGF groups as determined by both 3D μCT and 2D histomorphometry. FEA showed higher estimated failure load in the Src siRNA and Src siRNA+VEGF groups when compared to the vehicle control group. Blockage of VEGF-Src signaling pathway by specific Src siRNA was able to prevent steroid-associated destructive repair while improving reconstructive repair in SAON, which might become a novel therapeutic strategy.

  13. Genetic variants of the DNA repair genes from Exome Aggregation Consortium (EXAC) database: significance in cancer.

    Science.gov (United States)

    Das, Raima; Ghosh, Sankar Kumar

    2017-04-01

    DNA repair pathway is a primary defense system that eliminates wide varieties of DNA damage. Any deficiencies in them are likely to cause the chromosomal instability that leads to cell malfunctioning and tumorigenesis. Genetic polymorphisms in DNA repair genes have demonstrated a significant association with cancer risk. Our study attempts to give a glimpse of the overall scenario of the germline polymorphisms in the DNA repair genes by taking into account of the Exome Aggregation Consortium (ExAC) database as well as the Human Gene Mutation Database (HGMD) for evaluating the disease link, particularly in cancer. It has been found that ExAC DNA repair dataset (which consists of 228 DNA repair genes) comprises 30.4% missense, 12.5% dbSNP reported and 3.2% ClinVar significant variants. 27% of all the missense variants has the deleterious SIFT score of 0.00 and 6% variants carrying the most damaging Polyphen-2 score of 1.00, thus affecting the protein structure and function. However, as per HGMD, only a fraction (1.2%) of ExAC DNA repair variants was found to be cancer-related, indicating remaining variants reported in both the databases to be further analyzed. This, in turn, may provide an increased spectrum of the reported cancer linked variants in the DNA repair genes present in ExAC database. Moreover, further in silico functional assay of the identified vital cancer-associated variants, which is essential to get their actual biological significance, may shed some lights in the field of targeted drug development in near future. Copyright © 2017. Published by Elsevier B.V.

  14. Rearrangement of Rag-1 recombinase gene in DNA-repair deficient/immunodeficient wasted'' mice

    Energy Technology Data Exchange (ETDEWEB)

    Woloschak, G.E.; Weaver, P.; Churchill, M.; Chang-Liu, C-M. (Argonne National Lab., IL (United States)); Libertin, C.R. (Loyola Univ., Maywood, IL (United States))

    1992-01-01

    Mice recessive for the autosomal gene wasted'' (wst) display a disease pattern which includes increased sensitivity to the killing effects of ionizing radiation, immunodeficiency, and neurologic dysfunction. The recent cloning and characterization of recombinase genes (Rag-l/Rag-2) expressed in lymphoid and possibly central nervous system tissues prompted us to examine expression of these genes in DNA repair-deficient/immunodeficient wasted mice. Our results revealed that in thymus tissue, a small Rag-I transcript (1.0 kb) was detected in wst/wst mice that was not evident in thymus from control mice. In wst/[sm bullet] mice, a two-fold increase in Rag-1 mRNA was evident in thymus tissue. Rag-2 mRNA could only be detected in thymus tissue from wst/[sm bullet] and not from wst/wst or parental control BCF, mice. Southern blots revealed a rearrangement or deletion within the Rag-1 gene of affected wasted mice that was not evident in known strain-specific parental or littermate controls. These results support the idea that the Rag-1 gene may map at or near the locus for the wasted mutation. In addition, they suggest the importance of recombinase function in normal immune and central nervous system development as well as the potential contribution of this gene family to the normal repair of radiation-induced DNA damage.

  15. Repairing the sickle cell mutation. I. Specific covalent binding of a photoreactive third strand to the mutated base pair.

    Science.gov (United States)

    Broitman, S; Amosova, O; Dolinnaya, N G; Fresco, J R

    1999-07-30

    A DNA third strand with a 3'-psoralen substituent was designed to form a triplex with the sequence downstream of the T.A mutant base pair of the human sickle cell beta-globin gene. Triplex-mediated psoralen modification of the mutant T residue was sought as an approach to gene repair. The 24-nucleotide purine-rich target sequence switches from one strand to the other and has four pyrimidine interruptions. Therefore, a third strand sequence favorable to two triplex motifs was used, one parallel and the other antiparallel to it. To cope with the pyrimidine interruptions, which weaken third strand binding, 5-methylcytosine and 5-propynyluracil were used in the third strand. Further, a six residue "hook" complementary to an overhang of a linear duplex target was added to the 5'-end of the third strand via a T(4) linker. In binding to the overhang by Watson-Crick pairing, the hook facilitates triplex formation. This third strand also binds specifically to the target within a supercoiled plasmid. The psoralen moiety at the 3'-end of the third strand forms photoadducts to the targeted T with high efficiency. Such monoadducts are known to preferentially trigger reversion of the mutation by DNA repair enzymes.

  16. Gene Expression in Experimental Aortic Coarctation and Repair: Candidate Genes for Therapeutic Intervention?

    Science.gov (United States)

    LaDisa, John F; Bozdag, Serdar; Olson, Jessica; Ramchandran, Ramani; Kersten, Judy R; Eddinger, Thomas J

    2015-01-01

    Coarctation of the aorta (CoA) is a constriction of the proximal descending thoracic aorta and is one of the most common congenital cardiovascular defects. Treatments for CoA improve life expectancy, but morbidity persists, particularly due to the development of chronic hypertension (HTN). Identifying the mechanisms of morbidity is difficult in humans due to confounding variables such as age at repair, follow-up duration, coarctation severity and concurrent anomalies. We previously developed an experimental model that replicates aortic pathology in humans with CoA without these confounding variables, and mimics correction at various times using dissolvable suture. Here we present the most comprehensive description of differentially expressed genes (DEGs) to date from the pathology of CoA, which were obtained using this model. Aortic samples (n=4/group) from the ascending aorta that experiences elevated blood pressure (BP) from induction of CoA, and restoration of normal BP after its correction, were analyzed by gene expression microarray, and enriched genes were converted to human orthologues. 51 DEGs with >6 fold-change (FC) were used to determine enriched Gene Ontology terms, altered pathways, and association with National Library of Medicine Medical Subject Headers (MeSH) IDs for HTN, cardiovascular disease (CVD) and CoA. The results generated 18 pathways, 4 of which (cell cycle, immune system, hemostasis and metabolism) were shared with MeSH ID's for HTN and CVD, and individual genes were associated with the CoA MeSH ID. A thorough literature search further uncovered association with contractile, cytoskeletal and regulatory proteins related to excitation-contraction coupling and metabolism that may explain the structural and functional changes observed in our experimental model, and ultimately help to unravel the mechanisms responsible for persistent morbidity after treatment for CoA.

  17. Polyandry and sex-specific gene expression.

    Science.gov (United States)

    Mank, Judith E; Wedell, Nina; Hosken, David J

    2013-03-05

    Polyandry is widespread in nature, and has important evolutionary consequences for the evolution of sexual dimorphism and sexual conflict. Although many of the phenotypic consequences of polyandry have been elucidated, our understanding of the impacts of polyandry and mating systems on the genome is in its infancy. Polyandry can intensify selection on sexual characters and generate more intense sexual conflict. This has consequences for sequence evolution, but also for sex-biased gene expression, which acts as a link between mating systems, sex-specific selection and the evolution of sexual dimorphism. We discuss this and the remarkable confluence of sexual-conflict theory and patterns of gene expression, while also making predictions about transcription patterns, mating systems and sexual conflict. Gene expression is a key link in the genotype-phenotype chain, and although in its early stages, understanding the sexual selection-transcription relationship will provide significant insights into this critical association.

  18. The plant-specific CDKB1-CYCB1 complex mediates homologous recombination repair in Arabidopsis.

    Science.gov (United States)

    Weimer, Annika K; Biedermann, Sascha; Harashima, Hirofumi; Roodbarkelari, Farshad; Takahashi, Naoki; Foreman, Julia; Guan, Yonsheng; Pochon, Gaëtan; Heese, Maren; Van Damme, Daniël; Sugimoto, Keiko; Koncz, Csaba; Doerner, Peter; Umeda, Masaaki; Schnittger, Arp

    2016-10-04

    Upon DNA damage, cyclin-dependent kinases (CDKs) are typically inhibited to block cell division. In many organisms, however, it has been found that CDK activity is required for DNA repair, especially for homology-dependent repair (HR), resulting in the conundrum how mitotic arrest and repair can be reconciled. Here, we show that Arabidopsis thaliana solves this dilemma by a division of labor strategy. We identify the plant-specific B1-type CDKs (CDKB1s) and the class of B1-type cyclins (CYCB1s) as major regulators of HR in plants. We find that RADIATION SENSITIVE 51 (RAD51), a core mediator of HR, is a substrate of CDKB1-CYCB1 complexes. Conversely, mutants in CDKB1 and CYCB1 fail to recruit RAD51 to damaged DNA CYCB1;1 is specifically activated after DNA damage and we show that this activation is directly controlled by SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1), a transcription factor that acts similarly to p53 in animals. Thus, while the major mitotic cell-cycle activity is blocked after DNA damage, CDKB1-CYCB1 complexes are specifically activated to mediate HR. © 2016 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

  19. [Advances in lineage-specific genes].

    Science.gov (United States)

    Huanping, Zhang; Tongming, Yin

    2015-06-01

    Lineage-specific genes (LSGs) are defined as genes found in one particular taxonomic group but have no significant sequence similarity with genes from other lineages, which compose about 10%?20% of the total genes in the genome of a focal organism. LSGs were first uncovered in the yeast genome in 1996. The development of the whole genome sequencing leads to the emergence of studies on LSGs as a hot topic in comparative genomics. LSGs have been extensively studied on microbial species, lower marine organisms, plant (such as Arabidopsis thaliana, Oryza sativa, Populus), insects, primate, etc; the biological functions of LSGs are important to clarify the evolution and adaptability of a species. In this review, we summarize the progress of LSGs studies, including LSGs identification, gene characterization, origin and evolution of LSGs, biological function, and expression analysis of LSGs. In addition, we discuss the existing problems and future directions for studies in this area. Our purpose is to provide some unique insights into the researches of LSGs.

  20. 49 CFR 180.212 - Repair of seamless DOT 3-series specification cylinders and seamless UN pressure receptacles.

    Science.gov (United States)

    2010-10-01

    ... TRANSPORTATION HAZARDOUS MATERIALS REGULATIONS CONTINUING QUALIFICATION AND MAINTENANCE OF PACKAGINGS Qualification, Maintenance and Use of Cylinders § 180.212 Repair of seamless DOT 3-series specification... pressure receptacle unless— (i) The repair facility holds an approval issued under the provisions in §...

  1. The Saccharomyces cerevisiae RAD7 and RAD16 genes are required for inducible excision of endonuclease III sensitive-sites, yet are not needed for the repair of these lesions following a single UV dose.

    Science.gov (United States)

    Scott, A D; Waters, R

    1997-01-31

    The RAD7 and RAD16 genes of Saccharomyces cerevisiae have roles in the repair of UV induced CPDs in nontranscribed genes [1], and in the repair of CPDs in the nontranscribed strand of transcribed genes [2]. Previously, we identified an inducible component to nucleotide excision repair (NER), which is absent in a rad16 delta strain [3]. We have examined the repair of UV induced endonuclease III sensitive-sites (EIIISS), and have shown repair of these lesions to proceed by NER but their removal from nontranscribed regions is independent of RAD7 and RAD16. Furthermore, EIIISS are repaired with equal efficiency from both transcribed and nontranscribed genes [4]. In order to dissect the roles of RAD7 and RAD16 in the above processes we examined the repair of EIIISS in the MAT alpha and HML alpha loci, which are, respectively, transcriptionally active and inactive in alpha haploid cells. These loci have elevated levels of these lesions after UV (in genomic DNA EIIISS constitute about 10% of total lesions, whereas CPDs are about 70% of total lesions). We have shown that excision of UV induced EIIISS is enhanced following a prior UV irradiation. No enhancement of repair was detected in either the rad7 delta or the rad16 delta mutant. The fact that RAD7 and RAD16 are not required for the repair of EIIISS per se yet are required for the enhanced excision of these lesions from MAT alpha and HML alpha suggests two possibilities. These genes have two roles in NER, namely in the repair of CPDs from nontranscribed sequences, and in enhancing NER itself regardless of whether these genes' products are required for the excision of the specific lesion being repaired. In the latter case, the induction of RAD7 and RAD16 may increase the turnover of complexes stalled in nontranscribed DNA so as to increase the availability of NER proteins for the repair of CPDs and EIIISS in all regions of the genome.

  2. Polymorphisms in base excision repair genes: Breast cancer risk and individual radiosensitivity

    Science.gov (United States)

    Patrono, Clarice; Sterpone, Silvia; Testa, Antonella; Cozzi, Renata

    2014-01-01

    Breast cancer (BC) is the most common cancer among women worldwide. The aetiology and carcinogenesis of BC are not clearly defined, although genetic, hormonal, lifestyle and environmental risk factors have been established. The most common treatment for BC includes breast-conserving surgery followed by a standard radiotherapy (RT) regimen. However, radiation hypersensitivity and the occurrence of RT-induced toxicity in normal tissue may affect patients’ treatment. The role of DNA repair in cancer has been extensively investigated, and an impaired DNA damage response may increase the risk of BC and individual radiosensitivity. Single nucleotide polymorphisms (SNPs) in DNA repair genes may alter protein function and modulate DNA repair efficiency, influencing the development of various cancers, including BC. SNPs in DNA repair genes have also been studied as potential predictive factors for the risk of RT-induced side effects. Here, we review the literature on the association between SNPs in base excision repair (BER) genes and BC risk. We focused on X-ray repair cross complementing group 1 (XRCC1), which plays a key role in BER, and on 8-oxoguanine DNA glycosylase 1, apurinic/apyrimidinic endonuclease 1 and poly (ADP-ribose) polymerase-1, which encode three important BER enzymes that interact with XRCC1. Although no association between SNPs and radiation toxicity has been validated thus far, we also report published studies on XRCC1 SNPs and variants in other BER genes and RT-induced side effects in BC patients, emphasising that large well-designed studies are needed to determine the genetic components of individual radiosensitivity. PMID:25493225

  3. Gene therapy and peripheral nerve repair : a perspective

    NARCIS (Netherlands)

    Hoyng, Stefan A; de Winter, Fred; Tannemaat, Martijn R; Blits, Bas; Malessy, Martijn J A; Verhaagen, J.

    2015-01-01

    Clinical phase I/II studies have demonstrated the safety of gene therapy for a variety of central nervous system disorders, including Canavan's, Parkinson's (PD) and Alzheimer's disease (AD), retinal diseases and pain. The majority of gene therapy studies in the CNS have used adeno-associated viral

  4. Gene therapy and peripheral nerve repair : a perspective

    NARCIS (Netherlands)

    Hoyng, Stefan A; de Winter, Fred; Tannemaat, Martijn R; Blits, Bas; Malessy, Martijn J A; Verhaagen, J.

    2015-01-01

    Clinical phase I/II studies have demonstrated the safety of gene therapy for a variety of central nervous system disorders, including Canavan's, Parkinson's (PD) and Alzheimer's disease (AD), retinal diseases and pain. The majority of gene therapy studies in the CNS have used adeno-associated viral

  5. Cloning of human and mouse genes homologous to RAD52, a yeast gene involved in DNA repair and recombination.

    NARCIS (Netherlands)

    D.F.R. Muris; O.Y. Bezzubova (Olga); J-M. Buerstedde; K. Vreeken; A.S. Balajee; C.J. Osgood; C. Troelstra (Christine); J.H.J. Hoeijmakers (Jan); K. Ostermann; H. Schmidt (Henning); A.T. Natarajan; J.C.J. Eeken; P.H.M. Lohmann (Paul); A. Pastink (Albert)

    1994-01-01

    textabstractThe RAD52 gene of Saccharomyces cerevisiae is required for recombinational repair of double-strand breaks. Using degenerate oligonucleotides based on conserved amino acid sequences of RAD52 and rad22, its counterpart from Schizosaccharomyces pombe, RAD52 homologs from man and mouse were

  6. Chromosomal localization of three repair genes: the xeroderma pigmentosum group C gene and two human homologs of yeast RAD23.

    NARCIS (Netherlands)

    P.J. van der Spek (Peter); E.M.E. Smit (Elisabeth); H.B. Beverloo (Berna); K. Sugasawa (Kaoru); C. Matsutani; F. Hanaoka (Fumio); J.H.J. Hoeijmakers (Jan); A. Hagemeier

    1994-01-01

    textabstractThe nucleotide excision repair (NER) disorder xeroderma pigmentosum (XP) is characterized by sun (UV) sensitivity, predisposition to skin cancer, and extensive genetic heterogeneity. Recently, we reported the cloning and analysis of three human NER genes, XPC, HHR23A, and HHR23B. The

  7. Structural and functional conservation of two human homologs of the yeast DNA repair gene RAD6.

    NARCIS (Netherlands)

    M.H.M. Koken (Marcel); P. Reynolds (Paul); I. Jaspers-Dekker (Iris); L. Prakash; S. Prakash; D. Bootsma (Dirk); J.H.J. Hoeijmakers (Jan)

    1991-01-01

    textabstractThe RAD6 gene of Saccharomyces cerevisiae encodes a ubiquitin-conjugating enzyme (E2) that is required for DNA repair, damage-induced mutagenesis, and sporulation. We have cloned the two human RAD6 homologs, designated HHR6A and HHR6B. The two 152-amino acid human proteins share 95% sequ

  8. Comprehensive analysis of DNA repair gene variants and risk of meningioma

    DEFF Research Database (Denmark)

    Bethke, L.; Murray, A.; Webb, E.

    2008-01-01

    of meningioma and exposure to ionizing radiation is also well known and led us to examine whether variants in DNA repair genes contribute to disease susceptibility. METHODS: We analyzed 1127 tagging single-nucleotide polymorphisms (SNPs) that were selected to capture most of the common variation in 136 DNA...

  9. Semiconservative replication, genetic repair, and many-gened genomes: Extending the quasispecies paradigm to living systems

    Science.gov (United States)

    Tannenbaum, Emmanuel; Shakhnovich, Eugene I.

    2005-12-01

    Quasispecies theory has emerged as an important tool for modeling the evolutionary dynamics of biological systems. We review recent advances in the field, with an emphasis on the quasispecies dynamics of semiconservatively replicating genomes. Applications to cancer and adult stem cell growth are discussed. Additional topics, such as genetic repair and many-gene genomes, are covered as well.

  10. A Database to Support the Interpretation of Human Mismatch Repair Gene Variants

    NARCIS (Netherlands)

    Ou, Jianghua; Niessen, Renee C.; Vonk, Jan; Westers, Helga; Hofstra, Robert M. W.; Sijmons, Rolf H.

    2008-01-01

    Germline mutations in the mismatch repair (MMR) genes MLH1, MSH2, MSH6, or PMS2 can cause Lynch syndrome. This syndrome, also known as hereditary nonpolyposis colorectal cancer (HNPCC), is an autosomal dominantly-inherited disorder predominantly characterized by colorectal and endometrial cancer. Tr

  11. Polymorphisms in DNA Repair Genes and MDR1 and the Risk for Non-Hodgkin Lymphoma

    Directory of Open Access Journals (Sweden)

    Hee Nam Kim

    2014-04-01

    Full Text Available The damage caused by oxidative stress and exposure to cigarette smoke and alcohol necessitate DNA damage repair and transport by multidrug resistance-1 (MDR1. To explore the association between polymorphisms in these genes and non-Hodgkin lymphoma risk, we analyzed 15 polymorphisms of 12 genes in a population-based study in Korea (694 cases and 1700 controls. Four genotypes of DNA repair pathway genes (XRCC1 399 GA, OGG1 326 GG, BRCA1 871 TT, and WRN 787 TT were associated with a decreased risk for NHL [odds ratio (ORXRCC1 GA = 0.80, p = 0.02; OROGG1 GG = 0.70, p = 0.008; ORBRCA1 TT = 0.71, p = 0.048; ORWRN TT = 0.68, p = 0.01]. Conversely, the MGMT 115 CT genotype was associated with an increased risk for NHL (OR = 1.25, p = 0.04. In the MDR1 gene, the 1236 CC genotype was associated with a decreased risk for NHL (OR = 0.74, p = 0.04, and the 3435 CT and TT genotypes were associated with an increased risk (OR3435CT = 1.50, p < 0.0001; OR3435TT = 1.43, p = 0.02. These results suggest that polymorphisms in the DNA repair genes XRCC1, OGG1, BRCA1, WRN1, and MGMT and in the MDR1 gene may affect the risk for NHL in Korean patients.

  12. Cytogenetic Response to Ionizing Radiation Exposure in Human Fibroblasts with Suppressed Expression of Non-DSB Repair Genes

    Science.gov (United States)

    Zhang, Ye; Rohde, Larry H.; Emami, Kamal; Hammond, Dianne; Mehta, Satish K.; Jeevarajan, Antony S.; Pierson, Duane L.; Wu, Honglu

    2009-01-01

    Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in double-strand break (DSB) repair, and its impact on cytogenetic responses has not been well studied. The purpose of this study is to identify new roles of IR inducible genes in radiation-induced chromosome aberrations and micronuclei formation. In the study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by small interfering RNA in human fibroblast cells. Frequencies of micronuclei (MN) formation and chromosome aberrations were measured to determine the efficiency of cytogenetic repair, and the fraction of bi-nucleated cells in the MN analysis was used as a marker for cell cycle progression. In response to gamma radiation, the formation of MN was significantly increased by suppressed expression of five genes: Ku70 (DSB repair pathway), XPA (nucleotide excision repair pathway), RPA1 (mismatch repair pathway), RAD17 and RBBP8 (cell cycle control). Knocked-down expression of four genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Moreover, decreased XPA, p21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Nine of these eleven genes, whose knock-down expression affected cytogenetic repair, were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate IR

  13. Cytogenetic Response to Ionizing Radiation Exposure in Human Fibroblasts with Suppressed Expression of Non-DSB Repair Genes

    Science.gov (United States)

    Zhang, Ye; Rohde, Larry H.; Emami, Kamal; Hammond, Dianne; Mehta, Satish K.; Jeevarajan, Antony S.; Pierson, Duane L.; Wu, Honglu

    2009-01-01

    Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in double-strand break (DSB) repair, and its impact on cytogenetic responses has not been well studied. The purpose of this study is to identify new roles of IR inducible genes in radiation-induced chromosome aberrations and micronuclei formation. In the study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by small interfering RNA in human fibroblast cells. Frequencies of micronuclei (MN) formation and chromosome aberrations were measured to determine the efficiency of cytogenetic repair, and the fraction of bi-nucleated cells in the MN analysis was used as a marker for cell cycle progression. In response to gamma radiation, the formation of MN was significantly increased by suppressed expression of five genes: Ku70 (DSB repair pathway), XPA (nucleotide excision repair pathway), RPA1 (mismatch repair pathway), RAD17 and RBBP8 (cell cycle control). Knocked-down expression of four genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Moreover, decreased XPA, p21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Nine of these eleven genes, whose knock-down expression affected cytogenetic repair, were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate IR

  14. Redox regulation of genome stability by effects on gene expression, epigenetic pathways and DNA damage/repair.

    Science.gov (United States)

    Mikhed, Yuliya; Görlach, Agnes; Knaus, Ulla G; Daiber, Andreas

    2015-08-01

    Reactive oxygen and nitrogen species (e.g. H2O2, nitric oxide) confer redox regulation of essential cellular signaling pathways such as cell differentiation, proliferation, migration and apoptosis. In addition, classical regulation of gene expression or activity, including gene transcription to RNA followed by translation to the protein level, by transcription factors (e.g. NF-κB, HIF-1α) and mRNA binding proteins (e.g. GAPDH, HuR) is subject to redox regulation. This review will give an update of recent discoveries in this field, and specifically highlight the impact of reactive oxygen and nitrogen species on DNA repair systems that contribute to genomic stability. Emphasis will be placed on the emerging role of redox mechanisms regulating epigenetic pathways (e.g. miRNA, DNA methylation and histone modifications). By providing clinical correlations we discuss how oxidative stress can impact on gene regulation/activity and vise versa, how epigenetic processes, other gene regulatory mechanisms and DNA repair can influence the cellular redox state and contribute or prevent development or progression of disease.

  15. Redox regulation of genome stability by effects on gene expression, epigenetic pathways and DNA damage/repair

    Directory of Open Access Journals (Sweden)

    Yuliya Mikhed

    2015-08-01

    Full Text Available Reactive oxygen and nitrogen species (e.g. H2O2, nitric oxide confer redox regulation of essential cellular signaling pathways such as cell differentiation, proliferation, migration and apoptosis. In addition, classical regulation of gene expression or activity, including gene transcription to RNA followed by translation to the protein level, by transcription factors (e.g. NF-κB, HIF-1α and mRNA binding proteins (e.g. GAPDH, HuR is subject to redox regulation. This review will give an update of recent discoveries in this field, and specifically highlight the impact of reactive oxygen and nitrogen species on DNA repair systems that contribute to genomic stability. Emphasis will be placed on the emerging role of redox mechanisms regulating epigenetic pathways (e.g. miRNA, DNA methylation and histone modifications. By providing clinical correlations we discuss how oxidative stress can impact on gene regulation/activity and vise versa, how epigenetic processes, other gene regulatory mechanisms and DNA repair can influence the cellular redox state and contribute or prevent development or progression of disease.

  16. Site-specific rectocele repair with dermal graft augmentation: comparison of porcine dermal xenograft (Pelvicol) and human dermal allograft.

    Science.gov (United States)

    Biehl, Roger C; Moore, Robert D; Miklos, John R; Kohli, Neeraj; Anand, Indu S; Mattox, T Fleming

    2008-01-01

    This study is a retrospective chart review comparing 195 women who underwent rectocele repair with either a porcine dermal xenograft or human allogenic cadaveric dermal graft augmentation over a two year period. A site-specific defect repair was completed prior to augmentation with the graft. Examinations were performed preoperatively and postoperatively using the pelvic organ prolapse quantification system. Questionnaires were used to assess constipation and dyspareunia. De novo dyspareunia and cure rates for constipation and dyspareunia were not statistically different between the two groups. Site-specific fascial rectocele repairs with xenograft or allograft augmentation were found to have similar complication rates as well as objective and subjective cure rates.

  17. Human longevity and variation in DNA damage response and repair: study of the contribution of sub-processes using competitive gene-set analysis.

    Science.gov (United States)

    Debrabant, Birgit; Soerensen, Mette; Flachsbart, Friederike; Dato, Serena; Mengel-From, Jonas; Stevnsner, Tinna; Bohr, Vilhelm A; Kruse, Torben A; Schreiber, Stefan; Nebel, Almut; Christensen, Kaare; Tan, Qihua; Christiansen, Lene

    2014-09-01

    DNA-damage response and repair are crucial to maintain genetic stability, and are consequently considered central to aging and longevity. Here, we investigate whether this pathway overall associates to longevity, and whether specific sub-processes are more strongly associated with longevity than others. Data were applied on 592 SNPs from 77 genes involved in nine sub-processes: DNA-damage response, base excision repair (BER), nucleotide excision repair, mismatch repair, non-homologous end-joining, homologous recombinational repair (HRR), RecQ helicase activities (RECQ), telomere functioning and mitochondrial DNA processes. The study population was 1089 long-lived and 736 middle-aged Danes. A self-contained set-based test of all SNPs displayed association with longevity (P-value=9.9 × 10(-5)), supporting that the overall pathway could affect longevity. Investigation of the nine sub-processes using the competitive gene-set analysis by Wang et al indicated that BER, HRR and RECQ associated stronger with longevity than the respective remaining genes of the pathway (P-values=0.004-0.048). For HRR and RECQ, only one gene contributed to the significance, whereas for BER several genes contributed. These associations did, however, generally not pass correction for multiple testing. Still, these findings indicate that, of the entire pathway, variation in BER might influence longevity the most. These modest sized P-values were not replicated in a German sample. This might, though, be due to differences in genotyping procedures and investigated SNPs, potentially inducing differences in the coverage of gene regions. Specifically, five genes were not covered at all in the German data. Therefore, investigations in additional study populations are needed before final conclusion can be drawn.

  18. Mutagen sensitivity and DNA repair of the EGFR gene in oropharyngeal cancer.

    Science.gov (United States)

    Reiter, Maximilian; Welz, Christian; Baumeister, Philipp; Schwenk-Zieger, Sabina; Harréus, Ulrich

    2010-07-01

    Epidermal growth factor receptor (EGFR) is overexpressed in several epithelial malignancies, including head and neck squamous cell cancer. Up to 90% of the tumour cases in this area exhibit EGFR overexpression. The reasons for overexpression are still not clear. Mutagen sensitivity, pre-existing conditions for genotoxic damage, gene amplification, and reduced DNA repair of the EGFR gene are possible causes for EGFR protein overexpression. DNA damage in macroscopically healthy pharyngeal mucosal tissue of 30 patients with (15) and without cancer (15) of the oropharynx was evaluated after incubation with Benz[a]pyren-7,8-diol-9,10-epoxid (BPDE), a tobacco-associated carcinogen. Emerging DNA fragmentation of the EGFR gene located on chromosome 7 was evaluated. The centromere of the chromosome served as a reference gene. Comet FISH was applied to assess the mutagen sensitivity in these regions. The extent of DNA repair was evaluated in the same samples after a 24-h repair-period. Differences in gene amplification and protein expression between the two groups were analysed by Interphase-FISH (I-FISH) and immunohistochemistry (IHC), respectively. BPDE caused significant DNA damage compared to the negative control in oropharyngeal mucosa cells of patients with- and without carcinoma. DNA fragmentation of the EGFR gene in the two groups was comparable. Mutagen sensitivity was significantly higher in the EGFR gene than in the reference gene, but fragmentation of the EGFR gene was not enhanced compared to the DNA damage of the entire DNA. The DNA repair period led to a significant reduction in DNA damage levels in all groups, without preference for any of the groups or genes. EGFR amplification was found in 7.7% of the tumour patients but not in control patients. Of the patients with oropharyngeal carcinoma, 66.6% showed enhanced expression of EGFR protein (grades 2 and 3), whereas only 13% of tumour-free patients showed such protein expression. No significant differences in

  19. Evaluating the effects of genetic variants of DNA repair genes using cytogenetic mutagen sensitivity approaches.

    Science.gov (United States)

    Abdel-Rahman, Sherif Z; El-Zein, Randa A

    2011-08-01

    Mutagen sensitivity, measured in short-term cultures of peripheral blood lymphocytes by cytogenetic endpoints, is an indirect measure for DNA repair capacity and has been used for many years as a biomarker for intrinsic susceptibility for cancer. In this article, we briefly give an overview of the different cytogenetic mutagen sensitivity approaches that have been used successfully to evaluate the biological effects of polymorphisms in DNA repair genes based on a current review of the literature and based on the need for biomarkers that would allow the characterization of the biological and functional significance of such polymorphisms. We also address some of the future challenges facing this emerging area of research.

  20. Determining the functional significance of mismatch repair gene missense variants using biochemical and cellular assays

    DEFF Research Database (Denmark)

    Heinen, Christopher D; Juel Rasmussen, Lene

    2012-01-01

    provided an important experimental tool for studying the functional consequences of VUS. However, beyond this repair assay, a number of other experimental methods have been developed that allow us to test the effect of a VUS on discrete biochemical steps or other aspects of MMR function. Here, we describe......ABSTRACT: With the discovery that the hereditary cancer susceptibility disease Lynch syndrome (LS) is caused by deleterious germline mutations in the DNA mismatch repair (MMR) genes nearly 20 years ago, genetic testing can now be used to diagnose this disorder in patients. A definitive diagnosis...

  1. Low-level infrared laser modulates muscle repair and chromosome stabilization genes in myoblasts.

    Science.gov (United States)

    da Silva Neto Trajano, Larissa Alexsandra; Stumbo, Ana Carolina; da Silva, Camila Luna; Mencalha, Andre Luiz; Fonseca, Adenilson S

    2016-08-01

    Infrared laser therapy is used for skeletal muscle repair based on its biostimulative effect on satellite cells. However, shortening of telomere length limits regenerative potential in satellite cells, which occurs after each cell division cycle. Also, laser therapy could be more effective on non-physiologic tissues. This study evaluated low-level infrared laser exposure effects on mRNA expression from muscle injury repair and telomere stabilization genes in myoblasts in normal and stressful conditions. Laser fluences were those used in clinical protocols. C2C12 myoblast cultures were exposed to low-level infrared laser (10, 35, and 70 J/cm(2)) in standard or normal (10 %) and reduced (2 %) fetal bovine serum concentrations; total RNA was extracted for mRNA expression evaluation from muscle injury repair (MyoD and Pax7) and chromosome stabilization (TRF1 and TRF2) genes by real time quantitative polymerization chain reaction. Data show that low-level infrared laser increases the expression of MyoD and Pax7 in 10 J/cm(2) fluence, TRF1 expression in all fluences, and TRF2 expression in 70 J/cm(2) fluence in both 10 and 2 % fetal bovine serum. Low-level infrared laser increases mRNA expression from genes related to muscle repair and telomere stabilization in myoblasts in standard or normal and stressful conditions.

  2. Stem/progenitor cells: a potential source of retina-specific cells for retinal repair.

    Science.gov (United States)

    Bi, Yong-Yan; Feng, Dong-Fu; Pan, Dong-Chao

    2009-11-01

    Retinal injury generally results in permanent visual disturbance or even blindness. Any effort to restore vision in such condition would require replacement of the highly specialized retinal cells. Stem/progenitor cells have been proposed as a potential source of new retina-specific cells to replace those lost due to retina injury. Evidence to date suggests that continued development of stem cell therapies may ultimately lead to viable treatment options for retina injury. A wide range of stem/progenitor cells from various sources is currently being investigated for the treatment of retinal injury. This article reviews the recent achievements about stem/progenitor cell source for retinal repair.

  3. DNA-repair gene variants are associated with glioblastoma survival

    DEFF Research Database (Denmark)

    Wibom, Carl; Sjöström, Sara; Henriksson, Roger

    2012-01-01

    genes, in 138 glioblastoma samples from Sweden and Denmark. We confirmed our findings in an independent cohort of 121 glioblastoma patients from the UK. Our analysis revealed nine SNPs annotating MSH2, RAD51L1 and RECQL4 that were significantly (p

  4. Gene therapy on demand: site specific regulation of gene therapy.

    Science.gov (United States)

    Jazwa, Agnieszka; Florczyk, Urszula; Jozkowicz, Alicja; Dulak, Jozef

    2013-08-10

    Since 1990 when the first clinical gene therapy trial was conducted, much attention and considerable promise have been given to this form of treatment. Gene therapy has been used with success in patients suffering from severe combined immunodeficiency syndromes (X-SCID and ADA-deficiency), Leber's congenital amaurosis, hemophilia, β-thalassemia and adrenoleukodystrophy. Last year, the first therapeutic vector (Glybera) for treatment of lipoprotein lipase deficiency has been registered in the European Union. Nevertheless, there are still several numerous issues that need to be improved to make this technique more safe, effective and easily accessible for patients. Introduction of the therapeutic gene to the given cells should provide the level of expression which will restore the production of therapeutic protein to normal values or will provide therapeutic efficacy despite not fully physiological expression. However, in numerous diseases the expression of therapeutic genes has to be kept at certain level for some time, and then might be required to be switched off to be activated again when worsening of the symptoms may aggravate the risk of disease relapse. In such cases the promoters which are regulated by local conditions may be more required. In this article the special emphasis is to discuss the strategies of regulation of gene expression by endogenous stimuli. Particularly, the hypoxia- or miRNA-regulated vectors offer the possibilities of tight but, at the same time, condition-dependent and cell-specific expression. Such means have been already tested in certain pathophysiological conditions. This creates the chance for the translational approaches required for development of effective treatments of so far incurable diseases.

  5. Evaluation of cell proliferation, apoptosis, and dna-repair genes as potential biomarkers for ethanol-induced cns alterations

    Directory of Open Access Journals (Sweden)

    Hicks Steven D

    2012-10-01

    Full Text Available Abstract Background Alcohol use disorders (AUDs lead to alterations in central nervous system (CNS architecture along with impaired learning and memory. Previous work from our group and that of others suggests that one mechanism underlying these changes is alteration of cell proliferation, apoptosis, and DNA-repair in neural stem cells (NSCs produced as a consequence of ethanol-induced effects on the expression of genes related to p53-signaling. This study tests the hypothesis that changes in the expression of p53-signaling genes represent biomarkers of ethanol abuse which can be identified in the peripheral blood of rat drinking models and human AUD subjects and posits that specific changes may be correlated with differences in neuropsychological measures and CNS structure. Results Remarkably, microarray analysis of 350 genes related to p53-signaling in peripheral blood leukocytes (PBLs of binge-drinking rats revealed 190 genes that were significantly altered after correcting for multiple testing. Moreover, 40 of these genes overlapped with those that we had previously observed to be changed in ethanol-exposed mouse NSCs. Expression changes in nine of these genes were tested for independent confirmation by a custom QuantiGene Plex (QGP assay for a subset of p53-signaling genes, where a consistent trend for decreased expression of mitosis-related genes was observed. One mitosis-related gene (Pttg1 was also changed in human lymphoblasts cultured with ethanol. In PBLs of human AUD subjects seven p53-signaling genes were changed compared with non-drinking controls. Correlation and principal components analysis were then used to identify significant relationships between the expression of these seven genes and a set of medical, demographic, neuropsychological and neuroimaging measures that distinguished AUD and control subjects. Two genes (Ercc1 and Mcm5 showed a highly significant correlation with AUD-induced decreases in the volume of the left

  6. Evaluation of cell proliferation, apoptosis, and DNA-repair genes as potential biomarkers for ethanol-induced CNS alterations.

    Science.gov (United States)

    Hicks, Steven D; Lewis, Lambert; Ritchie, Julie; Burke, Patrick; Abdul-Malak, Ynesse; Adackapara, Nyssa; Canfield, Kelly; Shwarts, Erik; Gentile, Karen; Meszaros, Zsuzsa Szombathyne; Middleton, Frank A

    2012-10-25

    Alcohol use disorders (AUDs) lead to alterations in central nervous system (CNS) architecture along with impaired learning and memory. Previous work from our group and that of others suggests that one mechanism underlying these changes is alteration of cell proliferation, apoptosis, and DNA-repair in neural stem cells (NSCs) produced as a consequence of ethanol-induced effects on the expression of genes related to p53-signaling. This study tests the hypothesis that changes in the expression of p53-signaling genes represent biomarkers of ethanol abuse which can be identified in the peripheral blood of rat drinking models and human AUD subjects and posits that specific changes may be correlated with differences in neuropsychological measures and CNS structure. Remarkably, microarray analysis of 350 genes related to p53-signaling in peripheral blood leukocytes (PBLs) of binge-drinking rats revealed 190 genes that were significantly altered after correcting for multiple testing. Moreover, 40 of these genes overlapped with those that we had previously observed to be changed in ethanol-exposed mouse NSCs. Expression changes in nine of these genes were tested for independent confirmation by a custom QuantiGene Plex (QGP) assay for a subset of p53-signaling genes, where a consistent trend for decreased expression of mitosis-related genes was observed. One mitosis-related gene (Pttg1) was also changed in human lymphoblasts cultured with ethanol. In PBLs of human AUD subjects seven p53-signaling genes were changed compared with non-drinking controls. Correlation and principal components analysis were then used to identify significant relationships between the expression of these seven genes and a set of medical, demographic, neuropsychological and neuroimaging measures that distinguished AUD and control subjects. Two genes (Ercc1 and Mcm5) showed a highly significant correlation with AUD-induced decreases in the volume of the left parietal supramarginal gyrus and

  7. Chromosomal localization of three repair genes: The xeroderma pigmentosum group C gene and two human homologs of yeast RAD23

    Energy Technology Data Exchange (ETDEWEB)

    Spek, P.J. van der; Smit, E.M.E.; Beverloo, H.B. [Erasmus Univ., Rotterdam (Netherlands)] [and others

    1994-10-01

    The nucleotide excision repair (NER) disorder xeroderma pigmentosum (XP) is characterized by sun (UV) sensitivity, predisposition to skin cancer, and extensive genetic heterogeneity. Recently, we reported the cloning and analysis of three human NER genes, XPC, HHR23A, and HHR23B. The previously cloned XPC gene is involved in the common XP complementation group C, which is defective in excision repair of nontranscribed sequences in the genome. The XPC protein was found to be complexed with the product of HHR23B, one of the two human homologs of the Saccharomyes cerevisiae NER gene RAD23. Here we present the chromosomal localization by in situ hybridization using haptenized probes of all three genes. The HHR23A gene was assigned to chromosome 19p13.2. Interestingly, the HHR23B and XPC genes, the product of which forms a tight complex, were found to colocalize on band 3p25.1. Pulsed-field gel electrophoresis revealed that the HHR23B and XPC genes possibly share a MluI restriction fragment of about 625 kb. Potential involvement of the HHR23 genes in human genetic disorders is discussed. 53 refs., 4 figs., 2 tabs.

  8. Gene Expression Profiling in the Injured Spinal Cord of Trachemys scripta elegans: An Amniote with Self-Repair Capabilities

    Science.gov (United States)

    Valentin-Kahan, Adrián; García-Tejedor, Gabriela B.; Robello, Carlos; Trujillo-Cenóz, Omar; Russo, Raúl E.; Alvarez-Valin, Fernando

    2017-01-01

    Slider turtles are the only known amniotes with self-repair mechanisms of the spinal cord that lead to substantial functional recovery. Their strategic phylogenetic position makes them a relevant model to investigate the peculiar genetic programs that allow anatomical reconnection in some vertebrate groups but are absent in others. Here, we analyze the gene expression profile of the response to spinal cord injury (SCI) in the turtle Trachemys scripta elegans. We found that this response comprises more than 1000 genes affecting diverse functions: reaction to ischemic insult, extracellular matrix re-organization, cell proliferation and death, immune response, and inflammation. Genes related to synapses and cholesterol biosynthesis are down-regulated. The analysis of the evolutionary distribution of these genes shows that almost all are present in most vertebrates. Additionally, we failed to find genes that were exclusive of regenerating taxa. The comparison of expression patterns among species shows that the response to SCI in the turtle is more similar to that of mice and non-regenerative Xenopus than to Xenopus during its regenerative stage. This observation, along with the lack of conserved “regeneration genes” and the current accepted phylogenetic placement of turtles (sister group of crocodilians and birds), indicates that the ability of spinal cord self-repair of turtles does not represent the retention of an ancestral vertebrate character. Instead, our results suggest that turtles developed this capability from a non-regenerative ancestor (i.e., a lineage specific innovation) that was achieved by re-organizing gene expression patterns on an essentially non-regenerative genetic background. Among the genes activated by SCI exclusively in turtles, those related to anoxia tolerance, extracellular matrix remodeling, and axonal regrowth are good candidates to underlie functional recovery. PMID:28223917

  9. Transcriptional and post-transcriptional regulation of nucleotide excision repair genes in human cells

    Energy Technology Data Exchange (ETDEWEB)

    Lefkofsky, Hailey B. [Translational Oncology Program, University of Michigan Medical School, Ann Arbor, MI (United States); Veloso, Artur [Translational Oncology Program, University of Michigan Medical School, Ann Arbor, MI (United States); Department of Radiation Oncology, University of Michigan Medical School, Ann Arbor, MI (United States); Bioinformatics Program, Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI (United States); Ljungman, Mats, E-mail: ljungman@umich.edu [Translational Oncology Program, University of Michigan Medical School, Ann Arbor, MI (United States); Department of Radiation Oncology, University of Michigan Medical School, Ann Arbor, MI (United States); Department of Environmental Health Sciences, School of Public Health, University of Michigan, Ann Arbor, MI (United States)

    2015-06-15

    Nucleotide excision repair (NER) removes DNA helix-distorting lesions induced by UV light and various chemotherapeutic agents such as cisplatin. These lesions efficiently block the elongation of transcription and need to be rapidly removed by transcription-coupled NER (TC-NER) to avoid the induction of apoptosis. Twenty-nine genes have been classified to code for proteins participating in nucleotide excision repair (NER) in human cells. Here we explored the transcriptional and post-transcriptional regulation of these NER genes across 13 human cell lines using Bru-seq and BruChase-seq, respectively. Many NER genes are relatively large in size and therefore will be easily inactivated by UV-induced transcription-blocking lesions. Furthermore, many of these genes produce transcripts that are rather unstable. Thus, these genes are expected to rapidly lose expression leading to a diminished function of NER. One such gene is ERCC6 that codes for the CSB protein critical for TC-NER. Due to its large gene size and high RNA turnover rate, the ERCC6 gene may act as dosimeter of DNA damage so that at high levels of damage, ERCC6 RNA levels would be diminished leading to the loss of CSB expression, inhibition of TC-NER and the promotion of cell death.

  10. A data mining approach for classifying DNA repair genes into ageing-related or non-ageing-related

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    Vasieva Olga

    2011-01-01

    Full Text Available Abstract Background The ageing of the worldwide population means there is a growing need for research on the biology of ageing. DNA damage is likely a key contributor to the ageing process and elucidating the role of different DNA repair systems in ageing is of great interest. In this paper we propose a data mining approach, based on classification methods (decision trees and Naive Bayes, for analysing data about human DNA repair genes. The goal is to build classification models that allow us to discriminate between ageing-related and non-ageing-related DNA repair genes, in order to better understand their different properties. Results The main patterns discovered by the classification methods are as follows: (a the number of protein-protein interactions was a predictor of DNA repair proteins being ageing-related; (b the use of predictor attributes based on protein-protein interactions considerably increased predictive accuracy of attributes based on Gene Ontology (GO annotations; (c GO terms related to "response to stimulus" seem reasonably good predictors of ageing-relatedness for DNA repair genes; (d interaction with the XRCC5 (Ku80 protein is a strong predictor of ageing-relatedness for DNA repair genes; and (e DNA repair genes with a high expression in T lymphocytes are more likely to be ageing-related. Conclusions The above patterns are broadly integrated in an analysis discussing relations between Ku, the non-homologous end joining DNA repair pathway, ageing and lymphocyte development. These patterns and their analysis support non-homologous end joining double strand break repair as central to the ageing-relatedness of DNA repair genes. Our work also showcases the use of protein interaction partners to improve accuracy in data mining methods and our approach could be applied to other ageing-related pathways.

  11. DNA Repair Gene Polymorphisms in Relation to Non-Small Cell Lung Cancer Survival

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    Yuliang Su

    2015-07-01

    Full Text Available Background: Single nucleotide polymorphisms (SNPs in the DNA repair genes are suspected to be related to the survival of lung cancer patients due to their possible influence on DNA repair capacity (DRC. However, the study results are inconsistent. Methods: A follow-up study of 610 non-small cell lung cancer (NSCLC patients was conducted to investigate genetic polymorphisms associated with the DNA repair genes in relation to NSCLC survival; 6 SNPs were genotyped, including XRCC1 (rs25487 G>A, hOGG1 (rs1052133 C>G, MUTYH (rs3219489 G>C, XPA (rs1800975 G>A, ERCC2 (rs1799793 G>A and XRCC3 (rs861539 C>T. Kaplan-Meier survival curve and Cox proportional hazards regression analyses were performed. SNP-SNP interaction was also examined using the survival tree analysis. Results: Advanced disease stage and older age at diagnosis were associated with poor prognosis of NSCLC. Patients with the variant ‘G' allele of hOGG1 rs1052133 had poor overall survival compared with those with the homozygous wild ‘CC' genotype, especially in female patients, adenocarcinoma histology, early stage, light smokers and without family history of cancer. For never smoking female lung cancer patients, individuals carrying homozygous variant ‘AA' genotype of XPA had shorter survival time compared to those with wild ‘G' alleles. Furthermore, females carrying homozygous variant XPA and hOGG1 genotypes simultaneously had 2.78-fold increased risk for death. Among all 6 polymorphisms, the homozygous variant ‘AA' of XPA carriers had poor prognosis compared to the carriers of wild ‘G' alleles of XPA together with other base excision repair (BER polymorphisms. Conclusions: Besides disease stage and age, the study found DNA repair gene polymorphisms were associated with lung cancer survival.

  12. Low intensity infrared laser affects expression of oxidative DNA repair genes in mitochondria and nucleus

    Science.gov (United States)

    Fonseca, A. S.; Magalhães, L. A. G.; Mencalha, A. L.; Geller, M.; Paoli, F.

    2014-11-01

    Practical properties and physical characteristics of low intensity lasers have made possible their application to treat soft tissue diseases. Excitation of intracellular chromophores by red and infrared radiation at low energy fluences with increase of mitochondrial metabolism is the basis of the biostimulation effect but free radicals can be produced. DNA lesions induced by free radicals are repaired by the base excision repair pathway. In this work, we evaluate the expression of POLγ and APEX2 genes related to repair of mitochondrial and nuclear DNA, respectively. Skin and muscle tissue of Wistar rats were exposed to low intensity infrared laser at different fluences. One hour and 24 hours after laser exposure, tissue samples were withdrawn for total RNA extraction, cDNA synthesis, and evaluation of POLγ and APEX2 mRNA expression by real time quantitative polymerase chain reaction. Skin and muscle tissue of Wistar rats exposed to laser radiation show different expression of POLγ and APEX2 mRNA depending of the fluence and time after exposure. Our study suggests that a low intensity infrared laser affects expression of genes involved in repair of oxidative lesions in mitochondrial and nuclear DNA.

  13. Beryllium chloride-induced oxidative DNA damage and alteration in the expression patterns of DNA repair-related genes.

    Science.gov (United States)

    Attia, Sabry M; Harisa, Gamaleldin I; Hassan, Memy H; Bakheet, Saleh A

    2013-09-01

    Beryllium metal has physical properties that make its use essential for very specific applications, such as medical diagnostics, nuclear/fusion reactors and aerospace applications. Because of the widespread human exposure to beryllium metals and the discrepancy of the genotoxic results in the reported literature, detail assessments of the genetic damage of beryllium are warranted. Mice exposed to beryllium chloride at an oral dose of 23mg/kg for seven consecutive days exhibited a significant increase in the level of DNA-strand breaking and micronuclei formation as detected by a bone marrow standard comet assay and micronucleus test. Whereas slight beryllium chloride-induced oxidative DNA damage was detected following formamidopyrimidine DNA glycosylase digestion, digestion with endonuclease III resulted in considerable increases in oxidative DNA damage after the 11.5 and 23mg/kg/day treatment as detected by enzyme-modified comet assays. Increased 8-hydroxydeoxyguanosine was also directly correlated with increased bone marrow micronuclei formation and DNA strand breaks, which further confirm the involvement of oxidative stress in the induction of bone marrow genetic damage after exposure to beryllium chloride. Gene expression analysis on the bone marrow cells from beryllium chloride-exposed mice showed significant alterations in genes associated with DNA damage repair. Therefore, beryllium chloride may cause genetic damage to bone marrow cells due to the oxidative stress and the induced unrepaired DNA damage is probably due to the down-regulation in the expression of DNA repair genes, which may lead to genotoxicity and eventually cause carcinogenicity.

  14. DNA repair gene XRCC3 241Met variant and breast cancer susceptibility of Azeri population in Iranian

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    Gohari-Lasaki Sahar

    2015-01-01

    Full Text Available DNA-repair systems are essential for repairing damage that occurs when there is recombination between homologous chromosomes. The gene XRCC3 (X-ray cross complementing group 3 encodes a member of the RecA/Rad51-related protein family that participates in homologous recombination to maintain chromosome stability and repair DNA damage. The Thr241Met XRCC3-18067C>T, rs861539 substitution, a C to T transition at codon 241 in exon7, thus plays critical roles in cancer development. The aim of this study was association between XRCC3 Thr241Met polymorphism and risk of sporadic breast cancer in Azari population. We analysed DNA samples from 100 sporadic breast cancer patients and 100 healthy women, for XRCC3 Thr241Met polymorphism using PCR-RFLP. Genotype specific risks were tested using chi-test with 95% confident intervals. Frequency of Thr/Thr at codon 241was 69% in controls and 70% in patients, Thr/Met frequency was 22% in controls and 13 % in patients, the Met/Met genotype was 9% incontrols and 17% in patients. No correlation between the genotype and allele distribution and increased susceptibility for breast Cancer. Our results suggested that in pre-menopausal women, breast cancer riskis not significantly associated with rs861539 in Azari population.

  15. Comet-FISH with strand-specific probes reveals transcription-coupled repair of 8-oxoGuanine in human cells.

    Science.gov (United States)

    Guo, Jia; Hanawalt, Philip C; Spivak, Graciela

    2013-09-01

    Oxidized bases in DNA have been implicated in cancer, aging and neurodegenerative disease. We have developed an approach combining single-cell gel electrophoresis (comet) with fluorescence in situ hybridization (FISH) that enables the comparative quantification of low, physiologically relevant levels of DNA lesions in the respective strands of defined nucleotide sequences and in the genome overall. We have synthesized single-stranded probes targeting the termini of DNA segments of interest using a polymerase chain reaction-based method. These probes facilitate detection of damage at the single-molecule level, as the lesions are converted to DNA strand breaks by lesion-specific endonucleases or glycosylases. To validate our method, we have documented transcription-coupled repair of cyclobutane pyrimidine dimers in the ataxia telangiectasia-mutated (ATM) gene in human fibroblasts irradiated with 254 nm ultraviolet at 0.1 J/m2, a dose ∼100-fold lower than those typically used. The high specificity and sensitivity of our approach revealed that 7,8-dihydro-8-oxoguanine (8-oxoG) at an incidence of approximately three lesions per megabase is preferentially repaired in the transcribed strand of the ATM gene. We have also demonstrated that the hOGG1, XPA, CSB and UVSSA proteins, as well as actively elongating RNA polymerase II, are required for this process, suggesting cross-talk between DNA repair pathways.

  16. DNA repair gene ERCC2 polymorphisms and associations with breast and ovarian cancer risk

    Directory of Open Access Journals (Sweden)

    Rabiau Nadège

    2008-05-01

    Full Text Available Abstract Breast and ovarian cancers increased in the last decades. Except rare cases with a genetic predisposition and high penetrance, these pathologies are viewed as a polygenic disease. In this concept, association studies look for genetic variations such as polymorphisms in low penetrance genes, i.e. genes in interaction with environmental factors. DNA repair systems that protect the genome from deleterious endogenous and exogenous damages have been shown to have significantly reduced. In particular, enzymes of the nucleotide excision repair pathway are suspected to be implicated in cancer. In this study, 2 functional polymorphisms in a DNA repair gene ERCC2 were analyzed. The population included 911 breast cancer cases, 51 ovarian cancer cases and 1000 controls. The genotyping of 2 SNP (Single Nucleotide Polymorphism was carried out on the population with the MGB (Minor Groove Binder probe technique which consists of the use of the allelic discrimination with the Taqman® method. This study enabled us to show an increase in risk of breast cancer with no oral contraceptive users and with women exhibiting a waist-to-hip ratio (WHR > 0.85 for Asn homozygous for ERCC2 312.

  17. Mutation mismatch repair gene deletions in diffuse large B-cell lymphoma.

    Science.gov (United States)

    Couronné, Lucile; Ruminy, Philippe; Waultier-Rascalou, Agathe; Rainville, Vinciane; Cornic, Marie; Picquenot, Jean-Michel; Figeac, Martin; Bastard, Christian; Tilly, Hervé; Jardin, Fabrice

    2013-05-01

    To further unravel the molecular pathogenesis of diffuse large B-cell lymphoma (DLBCL), we performed high-resolution comparative genomic hybridization on lymph node biopsies from 70 patients. With this strategy, we identified microdeletions of genes involved in the mutation mismatch repair (MMR) pathway in two samples. The first patient presented with a homozygous deletion of MSH2-MSH6 due to duplication of an unbalanced pericentric inversion of chromosome 2. The other case showed a PMS2 heterozygous deletion. PMS2 and MSH2-MSH6 abnormalities, respectively, resulted in a decrease and complete loss of gene expression. However, unlike tumors associated with the hereditary non-polyposis colorectal cancer syndrome or immunodeficiency-related lymphomas, no microsatellite instability was detected. Mutational profiles revealed especially in one patient an aberrant hypermutation without a clear activation-induced cytidine deaminase signature, indicating a breakdown of the high-fidelity repair in favor of the error-prone repair pathway. Our findings suggest that in a rare subset of patients, inactivation of the genes of the MMR pathway is likely an important step in the molecular pathogenesis of DLBCL and does not involve the same molecular mechanisms as other common neoplasms with MMR deficiency.

  18. Site-specific analysis of UV-induced cyclobutane pyrimidine dimers in nucleotide excision repair-proficient and -deficient hamster cells: Lack of correlation with mutational spectra

    Energy Technology Data Exchange (ETDEWEB)

    Vreeswijk, Maaike P.G., E-mail: vreeswijk@lumc.nl [Department of Toxicogenetics, Leiden University Medical Center, Einthovenweg 20, P.O. Box 9600, Postzone S4-P, 2300 RC Leiden (Netherlands); Department of Human Genetics, Center for Human and Clinical Genetics, Leiden University Medical Center, Building 2, Postzone S-04, P.O. Box 9600, 2300 RC Leiden (Netherlands); Meijers, Caro M.; Giphart-Gassler, Micheline; Vrieling, Harry; Zeeland, Albert A. van; Mullenders, Leon H.F.; Loenen, Wil A.M. [Department of Toxicogenetics, Leiden University Medical Center, Einthovenweg 20, P.O. Box 9600, Postzone S4-P, 2300 RC Leiden (Netherlands)

    2009-04-26

    Irradiation of cells with UVC light induces two types of mutagenic DNA photoproducts, i.e. cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6-4PP). To investigate the relationship between the frequency of UV-induced photolesions at specific sites and their ability to induce mutations, we quantified CPD formation at the nucleotide level along exons 3 and 8 of the hprt gene using ligation-mediated PCR, and determined the mutational spectrum of 132 UV-induced hprt mutants in the AA8 hamster cell line and of 165 mutants in its nucleotide excision repair-defective derivative UV5. In AA8 cells, transversions predominated with a strong strand bias towards thymine-containing photolesions in the non-transcribed strand. As hamster AA8 cells are proficient in global genome repair of 6-4PP but selectively repair CPD from the transcribed strand of active genes, most mutations probably resulted from erroneous bypass of CPD in the non-transcribed strand. However, the relative incidence of CPD and the positions where mutations most frequently arose do not correlate. In fact some major damage sites hardly gave rise to the formation of mutations. In the repair-defective UV5 cells, mutations were almost exclusively C > T transitions caused by photoproducts at PyC sites in the transcribed strand. Even though CPD were formed at high frequencies at some TT sites in UV5, these photoproducts did not contribute to mutation induction at all. We conclude that, even in the absence of repair, large variations in the level of induction of CPD at different sites throughout the two exons do not correspond to frequencies of mutation induction.

  19. Site-specific analysis of UV-induced cyclobutane pyrimidine dimers in nucleotide excision repair-proficient and -deficient hamster cells: Lack of correlation with mutational spectra.

    Science.gov (United States)

    Vreeswijk, Maaike P G; Meijers, Caro M; Giphart-Gassler, Micheline; Vrieling, Harry; van Zeeland, Albert A; Mullenders, Leon H F; Loenen, Wil A M

    2009-04-26

    Irradiation of cells with UVC light induces two types of mutagenic DNA photoproducts, i.e. cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6-4 PP). To investigate the relationship between the frequency of UV-induced photolesions at specific sites and their ability to induce mutations, we quantified CPD formation at the nucleotide level along exons 3 and 8 of the hprt gene using ligation-mediated PCR, and determined the mutational spectrum of 132 UV-induced hprt mutants in the AA8 hamster cell line and of 165 mutants in its nucleotide excision repair-defective derivative UV5. In AA8 cells, transversions predominated with a strong strand bias towards thymine-containing photolesions in the non-transcribed strand. As hamster AA8 cells are proficient in global genome repair of 6-4 PP but selectively repair CPD from the transcribed strand of active genes, most mutations probably resulted from erroneous bypass of CPD in the non-transcribed strand. However, the relative incidence of CPD and the positions where mutations most frequently arose do not correlate. In fact some major damage sites hardly gave rise to the formation of mutations. In the repair-defective UV5 cells, mutations were almost exclusively C>T transitions caused by photoproducts at PyC sites in the transcribed strand. Even though CPD were formed at high frequencies at some TT sites in UV5, these photoproducts did not contribute to mutation induction at all. We conclude that, even in the absence of repair, large variations in the level of induction of CPD at different sites throughout the two exons do not correspond to frequencies of mutation induction.

  20. Immunoglobulin genes undergo legitimate repair in human B cells not only after cis- but also frequent trans-class switch recombination.

    Science.gov (United States)

    Laffleur, B; Bardet, S M; Garot, A; Brousse, M; Baylet, A; Cogné, M

    2014-01-01

    Immunoglobulin (Ig) genes specifically recruit activation-induced deaminase (AID) for 'on-target' DNA deamination, initiating either variable (V) region somatic hypermutation, or double-strand break intermediates of class switch recombination (CSR). Such breaks overwhelmingly undergo legitimate intra-Ig repair rather than rare illegitimate and potentially oncogenic junctions outside of Ig loci. We show that in human B cells, legitimate synapsis and repair efficiently join Ig genes whether physically linked on one chromosome or located apart on both alleles. This indicates mechanisms faithfully recognizing and/or pairing loci with homology in structure and accessibility, thus licensing interchromosomal trans-CSR junctions while usually preventing illegitimate interchromosomal recombination with AID off-target genes. Physical linkage of IgH genes in cis on the same allele just increases the likelihood of legitimate repair by another fourfold. The strongest force driving CSR might thus be recognition of legitimate target genes. Formation of IgH intra-allelic loops along this process would then constitute a consequence rather than a pre-requisite of this gene-pairing process.

  1. A comparison of synthetic oligodeoxynucleotides, DNA fragments and AAV-1 for targeted episomal and chromosomal gene repair

    Directory of Open Access Journals (Sweden)

    Leclerc Xavier

    2009-04-01

    Full Text Available Abstract Background Current strategies for gene therapy of inherited diseases consist in adding functional copies of the gene that is defective. An attractive alternative to these approaches would be to correct the endogenous mutated gene in the affected individual. This study presents a quantitative comparison of the repair efficiency using different forms of donor nucleic acids, including synthetic DNA oligonucleotides, double stranded DNA fragments with sizes ranging from 200 to 2200 bp and sequences carried by a recombinant adeno-associated virus (rAAV-1. Evaluation of each gene repair strategy was carried out using two different reporter systems, a mutated eGFP gene or a dual construct with a functional eGFP and an inactive luciferase gene, in several different cell systems. Gene targeting events were scored either following transient co-transfection of reporter plasmids and donor DNAs, or in a system where a reporter construct was stably integrated into the chromosome. Results In both episomal and chromosomal assays, DNA fragments were more efficient at gene repair than oligonucleotides or rAAV-1. Furthermore, the gene targeting frequency could be significantly increased by using DNA repair stimulating drugs such as doxorubicin and phleomycin. Conclusion Our results show that it is possible to obtain repair frequencies of 1% of the transfected cell population under optimized transfection protocols when cells were pretreated with phleomycin using rAAV-1 and dsDNA fragments.

  2. A study of molecular signals deregulating mismatch repair genes in prostate cancer compared to benign prostatic hyperplasia.

    Science.gov (United States)

    Basu, Sanmitra; Majumder, Subhadipa; Bhowal, Ankur; Ghosh, Alip; Naskar, Sukla; Nandy, Sumit; Mukherjee, Subhabrata; Sinha, Rajan Kumar; Basu, Keya; Karmakar, Dilip; Banerjee, Soma; Sengupta, Sanghamitra

    2015-01-01

    Prostate cancer is one of the leading causes of mortality among aging males. There is an unmet requirement of clinically useful biomarkers for early detection of prostate cancer to reduce the liabilities of overtreatment and accompanying morbidity. The present population-based study investigates the factors disrupting expression of multiple functionally related genes of DNA mismatch repair pathway in prostate cancer patients to identify molecular attributes distinguishing adenocarcinoma from benign hyperplasia of prostate. Gene expression was compared between tissue samples from prostate cancer and benign prostatic hyperplasia using real-time-PCR, western blot and immunohistochemistry. Assessment of genotypes of seven single-nucleotide-polymorphisms of three MMR genes was conducted using PCR-coupled RFLP and sequencing. Promoter methylation was interrogated by methylation-specific-PCR and bisulfite-sequencing. Interaction between microRNAs and MMR genes was verified by 3'UTR-based dual luciferase assays. Concurrent reduction of three MMR genes namely hMLH1, hMSH6 and hMSH2 (34-85%, Pprostate cancer tissues. hMSH6 polymorphism rs1800932(Pro92Pro) conferred a borderline protection in cancer patients (OR = 0.33, 95% CI = 0.15-0.75). Relative transcript level of hMLH1 was inversely related (r = -0.59, Pprostate cancer. This comparative study reflects that microRNA expression level, particularly hsa-miR-155, exhibits predictive signature of prostate adenocarcinoma.

  3. Tissue repair genes: the TiRe database and its implication for skin wound healing.

    Science.gov (United States)

    Yanai, Hagai; Budovsky, Arie; Tacutu, Robi; Barzilay, Thomer; Abramovich, Amir; Ziesche, Rolf; Fraifeld, Vadim E

    2016-04-19

    Wound healing is an inherent feature of any multicellular organism and recent years have brought about a huge amount of data regarding regular and abnormal tissue repair. Despite the accumulated knowledge, modulation of wound healing is still a major biomedical challenge, especially in advanced ages. In order to collect and systematically organize what we know about the key players in wound healing, we created the TiRe (Tissue Repair) database, an online collection of genes and proteins that were shown to directly affect skin wound healing. To date, TiRe contains 397 entries for four organisms: Mus musculus, Rattus norvegicus, Sus domesticus, and Homo sapiens. Analysis of the TiRe dataset of skin wound healing-associated genes showed that skin wound healing genes are (i) over-conserved among vertebrates, but are under-conserved in invertebrates; (ii) enriched in extracellular and immuno-inflammatory genes; and display (iii) high interconnectivity and connectivity to other proteins. The latter may provide potential therapeutic targets. In addition, a slower or faster skin wound healing is indicative of an aging or longevity phenotype only when assessed in advanced ages, but not in the young. In the long run, we aim for TiRe to be a one-station resource that provides researchers and clinicians with the essential data needed for a better understanding of the mechanisms of wound healing, designing new experiments, and the development of new therapeutic strategies. TiRe is freely available online at http://www.tiredb.org.

  4. Altered Gene Expressions and Cytogenetic Repair Efficiency in Cells with Suppressed Expression of XPA after Proton Exposure

    Science.gov (United States)

    Zhang, Ye; Rohde, Larry H.; Gridley, Daila S.; Mehta, Satish K.; Pierson, Duane L.; Wu, Honglu

    2009-01-01

    Cellular responses to damages from ionizing radiation (IR) exposure are influenced not only by the genes involved in DNA double strand break (DSB) repair, but also by non- DSB repair genes. We demonstrated previously that suppressed expression of several non-DSB repair genes, such as XPA, elevated IR-induced cytogenetic damages. In the present study, we exposed human fibroblasts that were treated with control or XPA targeting siRNA to 250 MeV protons (0 to 4 Gy), and analyzed chromosome aberrations and expressions of genes involved in DNA repair. As expected, after proton irradiation, cells with suppressed expression of XPA showed a significantly elevated frequency of chromosome aberrations compared with control siRNA treated (CS) cells. Protons caused more severe DNA damages in XPA knock-down cells, as 36% cells contained multiple aberrations compared to 25% in CS cells after 4Gy proton irradiation. Comparison of gene expressions using the real-time PCR array technique revealed that expressions of p53 and its regulated genes in irradiated XPA suppressed cells were altered similarly as in CS cells, suggesting that the impairment of IR induced DNA repair in XPA suppressed cells is p53-independent. Except for XPA, which was more than 2 fold down regulated in XPA suppressed cells, several other DNA damage sensing and repair genes (GTSE1, RBBP8, RAD51, UNG and XRCC2) were shown a more than 1.5 fold difference between XPA knock-down cells and CS cells after proton exposure. The possible involvement of these genes in the impairment of DNA repair in XPA suppressed cells will be further investigated.

  5. Repair of full-thickness articular cartilage defects by cultured mesenchymal stem cells transfected with the transforming growth factor {beta}{sub 1} gene

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    Guo Xiaodong [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Zheng Qixin [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Yang Shuhua [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Shao Zengwu [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Yuan Quan [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Pan Zhengqi [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Tang Shuo [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Liu Kai [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Quan Daping [Institute of Polymer Science, School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275 (China)

    2006-12-15

    Articular cartilage repair remains a clinical and scientific challenge with increasing interest focused on the combined techniques of gene transfer and tissue engineering. Transforming growth factor beta 1 (TGF-{beta}{sub 1}) is a multifunctional molecule that plays a central role in promotion of cartilage repair, and inhibition of inflammatory and alloreactive immune response. Cell mediated gene therapy can allow a sustained expression of TGF-{beta}{sub 1} that may circumvent difficulties associated with growth factor delivery. The objective of this study was to investigate whether TGF-{beta}{sub 1} gene modified mesenchymal stem cells (MSCs) could enhance the repair of full-thickness articular cartilage defects in allogeneic rabbits. The pcDNA{sub 3}-TGF-{beta}{sub 1} gene transfected MSCs were seeded onto biodegradable poly-L-lysine coated polylactide (PLA) biomimetic scaffolds in vitro and allografted into full-thickness articular cartilage defects in 18 New Zealand rabbits. The pcDNA{sub 3} gene transfected MSCs/biomimetic scaffold composites and the cell-free scaffolds were taken as control groups I and II, respectively. The follow-up times were 2, 4, 12 and 24 weeks. Macroscopical, histological and ultrastructural studies were performed. In vitro SEM studies found that abundant cartilaginous matrices were generated and completely covered the interconnected pores of the scaffolds two weeks post-seeding in the experimental groups. In vivo, the quality of regenerated tissue improved over time with hyaline cartilage filling the chondral region and a mixture of trabecular and compact bone filling the subchondral region at 24 weeks post-implantation. Joint repair in the experimental groups was better than that of either control group I or II, with respect to: (1) synthesis of hyaline cartilage specific extracellular matrix at the upper portion of the defect; (2) reconstitution of the subchondral bone at the lower portion of the defect and (3) inhibition of

  6. Proteasome inhibition enhances resistance to DNA damage via upregulation of Rpn4-dependent DNA repair genes.

    Science.gov (United States)

    Karpov, Dmitry S; Spasskaya, Daria S; Tutyaeva, Vera V; Mironov, Alexander S; Karpov, Vadim L

    2013-09-17

    The 26S proteasome is an ATP-dependent multi-subunit protease complex and the major regulator of intracellular protein turnover and quality control. However, its role in the DNA damage response is controversial. We addressed this question in yeast by disrupting the transcriptional regulation of the PRE1 proteasomal gene. The mutant strain has decreased proteasome activity and is hyper-resistant to various DNA-damaging agents. We found that Rpn4-target genes MAG1, RAD23, and RAD52 are overexpressed in this strain due to Rpn4 stabilisation. These genes represent three different pathways of base excision, nucleotide excision and double strand break repair by homologous recombination (DSB-HR). Consistently, the proteasome mutant displays increased DSB-HR activity. Our data imply that the proteasome may have a negative role in DNA damage response.

  7. Rapid assessment of repair of ultraviolet DNA damage with a modified host-cell reactivation assay using a luciferase reporter gene and correlation with polymorphisms of DNA repair genes in normal human lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Qiao Yawei; Spitz, Margaret R.; Guo Zhaozheng; Hadeyati, Mohammad; Grossman, Lawrence; Kraemer, Kenneth H.; Wei Qingyi

    2002-11-30

    As DNA repair plays an important role in genetic susceptibility to cancer, assessment of the DNA repair phenotype is critical for molecular epidemiological studies of cancer. In this report, we compared use of the luciferase (luc) reporter gene in a host-cell reactivation (HCR) (LUC) assay of repair of ultraviolet (UV) damage to DNA to use of the chloramphenicol (cat) gene-based HCR (CAT) assay we used previously for case-control studies. We performed both the assays on cryopreserved lymphocytes from 102 healthy non-Hispanic white subjects. There was a close correlation between DNA repair capacity (DRC) as measured by the LUC and CAT assays. Although these two assays had similar variation, the LUC assay was faster and more sensitive. We also analyzed the relationship between DRC and the subjects' previously determined genotypes for four polymorphisms of two nucleotide-excision repair (NER) genes (in intron 9 of xeroderma pigmentosum (XP) C and exons 6, 10 and 23 of XPD) and one polymorphism of a base-excision repair gene in exon 10 of X-ray complementing group 1 (XRCC1). The DRC was significantly lower in subjects homozygous for one or more polymorphisms of the two NER genes than in subjects with other genotypes (P=0.010). In contrast, the polymorphic XRCC1 allele had no significant effect on DRC. These results suggest that the post-UV LUC assay measures NER phenotype and that polymorphisms of XPC and XPD genes modulate DRC. For population studies of the DNA repair phenotype, many samples need to be evaluated, and so the LUC assay has several advantages over the CAT assay: the LUC assay was more sensitive, had less variation, was not radioactive, was easier to perform, and required fewer cryopreserved cells. These features make the LUC-based HCR assay suitable for molecular epidemiological studies.

  8. Paradoxical DNA repair and peroxide resistance gene conservation in Bacillus pumilus SAFR-032.

    Directory of Open Access Journals (Sweden)

    Jason Gioia

    Full Text Available BACKGROUND: Bacillus spores are notoriously resistant to unfavorable conditions such as UV radiation, gamma-radiation, H2O2, desiccation, chemical disinfection, or starvation. Bacillus pumilus SAFR-032 survives standard decontamination procedures of the Jet Propulsion Lab spacecraft assembly facility, and both spores and vegetative cells of this strain exhibit elevated resistance to UV radiation and H2O2 compared to other Bacillus species. PRINCIPAL FINDINGS: The genome of B. pumilus SAFR-032 was sequenced and annotated. Lists of genes relevant to DNA repair and the oxidative stress response were generated and compared to B. subtilis and B. licheniformis. Differences in conservation of genes, gene order, and protein sequences are highlighted because they potentially explain the extreme resistance phenotype of B. pumilus. The B. pumilus genome includes genes not found in B. subtilis or B. licheniformis and conserved genes with sequence divergence, but paradoxically lacks several genes that function in UV or H2O2 resistance in other Bacillus species. SIGNIFICANCE: This study identifies several candidate genes for further research into UV and H2O2 resistance. These findings will help explain the resistance of B. pumilus and are applicable to understanding sterilization survival strategies of microbes.

  9. DNA repair genes implicated in triple negative familial non-BRCA1/2 breast cancer predisposition.

    Science.gov (United States)

    Ollier, Marie; Radosevic-Robin, Nina; Kwiatkowski, Fabrice; Ponelle, Flora; Viala, Sandrine; Privat, Maud; Uhrhammer, Nancy; Bernard-Gallon, Dominique; Penault-Llorca, Frédérique; Bignon, Yves-Jean; Bidet, Yannick

    2015-01-01

    Among breast cancers, 10 to 15% of cases would be due to hereditary risk. In these familial cases, mutations in BRCA1 and BRCA2 are found in only 15% to 20%, meaning that new susceptibility genes remain to be found. Triple-negative breast cancers represent 15% of all breast cancers, and are generally aggressive tumours without targeted therapies available. Our hypothesis is that some patients with triple negative breast cancer could share a genetic susceptibility different from other types of breast cancers. We screened 36 candidate genes, using pyrosequencing, in all the 50 triple negative breast cancer patients with familial history of cancer but no BRCA1 or BRCA2 mutation of a population of 3000 families who had consulted for a familial breast cancer between 2005 and 2013. Any mutations were also sequenced in available relatives of cases. Protein expression and loss of heterozygosity were explored in tumours. Seven deleterious mutations in 6 different genes (RAD51D, MRE11A, CHEK2, MLH1, MSH6, PALB2) were observed in one patient each, except the RAD51D mutation found in two cases. Loss of heterozygosity in the tumour was found for 2 of the 7 mutations. Protein expression was absent in tumour tissue for 5 mutations. Taking into consideration a specific subtype of tumour has revealed susceptibility genes, most of them in the homologous recombination DNA repair pathway. This may provide new possibilities for targeted therapies, along with better screening and care of patients.

  10. Expression of DNA repair genes in burned skin exposed to low-level red laser.

    Science.gov (United States)

    Trajano, Eduardo Tavares Lima; Mencalha, Andre Luiz; Monte-Alto-Costa, Andréa; Pôrto, Luís Cristóvão; de Souza da Fonseca, Adenilson

    2014-11-01

    Although red laser lights lie in the region of non-ionizing radiations in the electromagnetic spectrum, there are doubts whether absorption of these radiations causes lesions in the DNA molecule. Our aim was to investigate the expression of the genes involved with base excision and nucleotide excision repair pathways in skin tissue submitted to burn injury and exposed to low-level red laser. Wistar rats were divided as follows: control group-rats burned and not irradiated, laser group-rats burned and irradiated 1 day after injury for five consecutive days, and later laser group-rats injured and treated 4 days after injury for five consecutive days. Irradiation was performed according to a clinical protocol (20 J/cm(2), 100 mW, continuous wave emission mode). The animals were sacrificed on day 10, and scarred tissue samples were withdrawn for total RNA extraction, complementary DNA (cDNA) synthesis, and evaluation of gene expression by quantitative polymerase chain reaction. Low-level red laser exposure (1) reduces the expression of APE1 messenger (mRNA), (2) increases the expression of OGG1 mRNA, (3) reduces the expression of XPC mRNA, and (4) increases the expression of XPA mRNA both in laser and later laser groups. Red laser exposure at therapeutic fluences alters the expression of genes related to base excision and nucleotide excision pathways of DNA repair during wound healing of burned skin.

  11. A systematic gene-gene and gene-environment interaction analysis of DNA repair genes XRCC1, XRCC2, XRCC3, XRCC4, and oral cancer risk.

    Science.gov (United States)

    Yang, Cheng-Hong; Lin, Yu-Da; Yen, Ching-Yui; Chuang, Li-Yeh; Chang, Hsueh-Wei

    2015-04-01

    Oral cancer is the sixth most common cancer worldwide with a high mortality rate. Biomarkers that anticipate susceptibility, prognosis, or response to treatments are much needed. Oral cancer is a polygenic disease involving complex interactions among genetic and environmental factors, which require multifaceted analyses. Here, we examined in a dataset of 103 oral cancer cases and 98 controls from Taiwan the association between oral cancer risk and the DNA repair genes X-ray repair cross-complementing group (XRCCs) 1-4, and the environmental factors of smoking, alcohol drinking, and betel quid (BQ) chewing. We employed logistic regression, multifactor dimensionality reduction (MDR), and hierarchical interaction graphs for analyzing gene-gene (G×G) and gene-environment (G×E) interactions. We identified a significantly elevated risk of the XRCC2 rs2040639 heterozygous variant among smokers [adjusted odds ratio (OR) 3.7, 95% confidence interval (CI)=1.1-12.1] and alcohol drinkers [adjusted OR=5.7, 95% CI=1.4-23.2]. The best two-factor based G×G interaction of oral cancer included the XRCC1 rs1799782 and XRCC2 rs2040639 [OR=3.13, 95% CI=1.66-6.13]. For the G×E interaction, the estimated OR of oral cancer for two (drinking-BQ chewing), three (XRCC1-XRCC2-BQ chewing), four (XRCC1-XRCC2-age-BQ chewing), and five factors (XRCC1-XRCC2-age-drinking-BQ chewing) were 32.9 [95% CI=14.1-76.9], 31.0 [95% CI=14.0-64.7], 49.8 [95% CI=21.0-117.7] and 82.9 [95% CI=31.0-221.5], respectively. Taken together, the genotypes of XRCC1 rs1799782 and XRCC2 rs2040639 DNA repair genes appear to be significantly associated with oral cancer. These were enhanced by exposure to certain environmental factors. The observations presented here warrant further research in larger study samples to examine their relevance for routine clinical care in oncology.

  12. Structure-specific endonucleases Xpf and Mus81 play overlapping but essential roles in DNA repair by homologous recombination

    NARCIS (Netherlands)

    K. Kikuchi (Koji); T. Narita (Takeo); V.C. Pham (Van Ca); J. Iijima (Junko); T. Hirota (Tomomitsu); I.S. Keka (Islam Shamima); Mohiuddin; K. Okawa (Katsuya); T. Hori (Toshiyuki); T. Fukagawa (Tatsuo); J. Essers (Jeroen); R. Kanaar (Roland); M.C. Whitby (Matthew); K. Sugasawa (Kaoru); Y. Taniguchi (Yoshihito); K. Kitagawa; S. Takeda (Shiunichi)

    2013-01-01

    textabstractDNA double-strand breaks (DSB) occur frequently during replication in sister chromatids and are dramatically increased when cells are exposed to chemotherapeutic agents including camptothecin. Such DSBs are efficiently repaired specifically by homologous recombination (HR) with the intac

  13. Structure-specific endonucleases Xpf and Mus81 play overlapping but essential roles in DNA repair by homologous recombination

    NARCIS (Netherlands)

    K. Kikuchi (Koji); T. Narita (Takeo); V.C. Pham (Van Ca); J. Iijima (Junko); T. Hirota (Tomomitsu); I.S. Keka (Islam Shamima); Mohiuddin; K. Okawa (Katsuya); T. Hori (Toshiyuki); T. Fukagawa (Tatsuo); J. Essers (Jeroen); R. Kanaar (Roland); M.C. Whitby (Matthew); K. Sugasawa (Kaoru); Y. Taniguchi (Yoshihito); K. Kitagawa; S. Takeda (Shiunichi)

    2013-01-01

    textabstractDNA double-strand breaks (DSB) occur frequently during replication in sister chromatids and are dramatically increased when cells are exposed to chemotherapeutic agents including camptothecin. Such DSBs are efficiently repaired specifically by homologous recombination (HR) with the intac

  14. Dietary Berries and Ellagic Acid Prevent Oxidative DNA Damage and Modulate Expression of DNA Repair Genes

    Directory of Open Access Journals (Sweden)

    Ramesh C. Gupta

    2008-03-01

    Full Text Available DNA damage is a pre-requisite for the initiation of cancer and agents that reduce this damage are useful in cancer prevention. In this study, we evaluated the ability of whole berries and berry phytochemical, ellagic acid to reduce endogenous oxidative DNA damage. Ellagic acid was selected based on > 95% inhibition of 8-oxodeoxyguosine (8-oxodG and other unidentified oxidative DNA adducts induced by 4-hydroxy-17B;-estradiol and CuCl2 in vitro. Inhibition of the latter occurred at lower concentrations (10 u(microM than that for 8-oxodG (100 u(microM. In the in vivo study, female CD-1 mice (n=6 were fed either a control diet or diet supplemented with ellagic acid (400 ppm and dehydrated berries (5% w/w with varying ellagic acid contents -- blueberry (low, strawberry (medium and red raspberry (high, for 3 weeks. Blueberry and strawberry diets showed moderate reductions in endogenous DNA adducts (25%. However, both red raspberry and ellagic acid diets showed a significant reduction of 59% (p < 0.001 and 48% (p < 0.01, respectively. Both diets also resulted in a 3-8 fold over-expression of genes involved in DNA repair such as xeroderma pigmentosum group A complementing protein (XPA, DNA excision repair protein (ERCC5 and DNA ligase III (DNL3. These results suggest that red raspberry and ellagic acid reduce endogenous oxidative DNA damage by mechanisms which may involve increase in DNA repair.

  15. Nucleotide Excision Repair in Cellular Chromatin: Studies with Yeast from Nucleotide to Gene to Genome

    Directory of Open Access Journals (Sweden)

    Simon Reed

    2012-09-01

    Full Text Available Here we review our development of, and results with, high resolution studies on global genome nucleotide excision repair (GGNER in Saccharomyces cerevisiae. We have focused on how GGNER relates to histone acetylation for its functioning and we have identified the histone acetyl tranferase Gcn5 and acetylation at lysines 9/14 of histone H3 as a major factor in enabling efficient repair. We consider results employing primarily MFA2 as a model gene, but also those with URA3 located at subtelomeric sequences. In the latter case we also see a role for acetylation at histone H4. We then go on to outline the development of a high resolution genome-wide approach that enables one to examine correlations between histone modifications and the nucleotide excision repair (NER of UV-induced cyclobutane pyrimidine dimers throughout entire genomes. This is an approach that will enable rapid advances in understanding the complexities of how compacted chromatin in chromosomes is processed to access DNA damage and then returned to its pre-damaged status to maintain epigenetic codes.

  16. Strategies for psbA gene expression in cyanobacteria, green algae and higher plants: from transcription to PSII repair.

    Science.gov (United States)

    Mulo, Paula; Sakurai, Isamu; Aro, Eva-Mari

    2012-01-01

    The Photosystem (PS) II of cyanobacteria, green algae and higher plants is prone to light-induced inactivation, the D1 protein being the primary target of such damage. As a consequence, the D1 protein, encoded by the psbA gene, is degraded and re-synthesized in a multistep process called PSII repair cycle. In cyanobacteria, a small gene family codes for the various, functionally distinct D1 isoforms. In these organisms, the regulation of the psbA gene expression occurs mainly at the level of transcription, but the expression is fine-tuned by regulation of translation elongation. In plants and green algae, the D1 protein is encoded by a single psbA gene located in the chloroplast genome. In chloroplasts of Chlamydomonas reinhardtii the psbA gene expression is strongly regulated by mRNA processing, and particularly at the level of translation initiation. In chloroplasts of higher plants, translation elongation is the prevalent mechanism for regulation of the psbA gene expression. The pre-existing pool of psbA transcripts forms translation initiation complexes in plant chloroplasts even in darkness, while the D1 synthesis can be completed only in the light. Replacement of damaged D1 protein requires also the assistance by a number of auxiliary proteins, which are encoded by the nuclear genome in green algae and higher plants. Nevertheless, many of these chaperones are conserved between prokaryotes and eukaryotes. Here, we describe the specific features and fundamental differences of the psbA gene expression and the regeneration of the PSII reaction center protein D1 in cyanobacteria, green algae and higher plants. This article is part of a Special Issue entitled Photosystem II.

  17. Polymorphisms in nucleotide excision repair genes, smoking and intake of fruit and vegetables in relation to lung cancer

    DEFF Research Database (Denmark)

    Raaschou-Nielsen, Ole; Sørensen, Mette; Overvad, Kim

    2007-01-01

    Polymorphisms in nucleotide excision repair genes have been associated with risk for lung cancer. We examined gene-environment interactions in relation to lung cancer in 430 cases and 790 comparison persons identified within a prospective cohort of 57,053 persons. We included polymorphisms...... in the XPC, XPA and XPD genes involved in the nucleotide excision DNA repair pathway and analysed possible interactions with smoking and dietary intake of fruit and vegetables in relation to risk for lung cancer. We found that intake of fruit was associated with lower risk for lung cancer only among carriers...

  18. Early passage bone marrow stromal cells express genes involved in nervous system development supporting their relevance for neural repair

    NARCIS (Netherlands)

    Nandoe, R.D.S.; Bossers, K.; Ritfeld, G.J.; Blits, B.; Grotenhuis, J.A.; Verhaagen, J.; Oudega, M.

    2011-01-01

    PURPOSE: The assessment of the capacity of bone marrow stromal cells (BMSC) to repair the nervous system using gene expression profiling. The evaluation of effects of long-term culturing on the gene expression profile of BMSC. METHODS: Fourty four k whole genome rat microarrays were used to study

  19. Discovering Study-Specific Gene Regulatory Networks

    OpenAIRE

    2014-01-01

    This article has been made available through the Brunel Open Access Publishing Fund. This article has been made available through the Brunel Open Access Publishing Fund. Microarrays are commonly used in biology because of their ability to simultaneously measure thousands of genes under different conditions. Due to their structure, typically containing a high amount of variables but far fewer samples, scalable network analysis techniques are often employed. In particular, consensus appro...

  20. Ubiquitin-specific protease 5 is required for the efficient repair of DNA double-strand breaks.

    Directory of Open Access Journals (Sweden)

    Satoshi Nakajima

    Full Text Available During the DNA damage response (DDR, ubiquitination plays an important role in the recruitment and regulation of repair proteins. However, little is known about elimination of the ubiquitination signal after repair is completed. Here we show that the ubiquitin-specific protease 5 (USP5, a deubiquitinating enzyme, is involved in the elimination of the ubiquitin signal from damaged sites and is required for efficient DNA double-strand break (DSB repair. Depletion of USP5 sensitizes cells to DNA damaging agents, produces DSBs, causes delayed disappearance of γH2AX foci after Bleocin treatment, and influences DSB repair efficiency in the homologous recombination pathway but not in the non-homologous end joining pathway. USP5 co-localizes to DSBs induced by laser micro-irradiation in a RAD18-dependent manner. Importantly, polyubiquitin chains at sites of DNA damage remained for longer periods in USP5-depleted cells. Our results show that disassembly of polyubiquitin chains by USP5 at sites of damage is important for efficient DSB repair.

  1. Dissecting specific and global transcriptional regulation of bacterial gene expression

    NARCIS (Netherlands)

    Gerosa, Luca; Kochanowski, Karl; Heinemann, Matthias; Sauer, Uwe

    2013-01-01

    Gene expression is regulated by specific transcriptional circuits but also by the global expression machinery as a function of growth. Simultaneous specific and global regulation thus constitutes an additional-but often neglected-layer of complexity in gene expression. Here, we develop an experiment

  2. Synergistic interactions between RAD5, RAD16, and RAD54, three partially homologous yeast DNA repair genes each in a different repair pathway

    Energy Technology Data Exchange (ETDEWEB)

    Glassner, B.J. [Univ. of California, Berkeley, CA (United States); Mortimer, R.K. [Univ. of California, Berkeley, CA (United States)]|[Lawrence Berkeley Laboratory, Berkeley, CA (United States)

    1994-07-01

    Considerable homology has recently been noted between the proteins encoded by the RAD5, RAD16 and RAD54 genes of Saccharomyces cerevisiae. These genes are members of the RAD6, RAD3 and RAD50 epistasis groups, respectively, which correspond to the three major DNA repair pathways in yeast. These proteins also share homology with other eucaryotic proteins, including those encoded by SNF2 and MO1 of yeast, brahma and lodestar of Drosophila and the human ERCC6 gene. The homology shares features with known helicases, suggesting a newly identified helicase subfamily. We have constructed a series of congenic single-, double- and triple-deletion mutants involving RAD5, RAD16 and RAD54 to examine the interactions between these genes. Each deletion mutation alone has only a moderate effect on survival after exposure to UV radiation. Each pairwise-double mutant exhibits marked synergism. The triple-deletion mutant displays further synergism. These results confirm the assignment of the RAD54 gene to the RAD50 epistasis group and suggest that the RAD16 gene plays a larger role in DNA repair after exposure to UV radiation than has been suggested previously. Additionally, the proteins encoded by RAD5, RAD16, and RAD54 may compete for the same substrate after damage induced by UV radiation, possibly at an early step in their respective pathways. 49 refs., 6 figs., 2 tabs.

  3. Shoulder sport-specific impairments after arthroscopic Bankart repair: a prospective longitudinal assessment.

    Science.gov (United States)

    Stein, Thomas; Linke, Ralf Dieter; Buckup, Johannes; Efe, Turgay; von Eisenhart-Rothe, Rudiger; Hoffmann, Reinhard; Jäger, Alwin; Welsch, Frederic

    2011-11-01

    Reports of return to shoulder-dependent sport after surgical stabilization previously underestimated impairments, which were not reflected in the score systems used. Return to shoulder-dependent sport depends on the type of sport performed. Case series; Level of evidence, 4. Forty-seven athletes (26.9 years of age at surgery) who underwent isolated arthroscopic Bankart repair were longitudinally monitored by shoulder-dependent sport-specific activity (Shoulder Sport Activity Score [SSAS]) and ability (Athletic Shoulder Outcome Scoring System [ASOSS]) scores and visual analog scales for reachieved proficiency level, sport-specific shoulder pain, and functional deficits. Data were assessed at 4 points of treatment: preoperatively, and postoperatively after 6, 16, and 32 months (P0-P3). Athletes were analyzed separately according to shoulder sport: noncollision/nonoverhead (G1), collision (G2), overhead (G3), and martial arts (G4). The G1 and G2 athletes had re-achieved the preinjury sport activity and sport proficiency status and excellent ASOSS scores after 32 months (SSAS(G1) = 7.2, SSAS(G2) = 8.1, ASOSS(G1) = 94.4, ASOSS(G2) = 95.2), whereas G3 and G4 athletes remained at an inferior activity level (SSAS(G3) = 8.0, SSAS(G4) = 8.3) and proficiency level. The ASOSS documented a prolonged period of shoulder rehabilitation for G3 and G4 athletes to reach a good shoulder-dependent sport ability outcome after 32 months (ASOSS(G3) = 89.0, ASOSS(G4) = 93.1). All groups recorded persisting limitations in visual analog scales for sport-specific shoulder function and pain. The established scores (Rowe = 95.9, Walch-Duplay = 93.3, Constant = 94.0) did not reflect these sport-specific impairments. Athletes with 5 or more preoperative dislocations had significantly longer surgery-to-sport resumption intervals with a prolonged proficiency recovery. The athletes' shoulder stabilization resulted in a prolonged rehabilitation depending on the functional demand of the performed

  4. Haplotype analyses of DNA repair gene polymorphisms and their role in ulcerative colitis.

    Directory of Open Access Journals (Sweden)

    Avinash Bardia

    Full Text Available Ulcerative colitis (UC is a major clinical form of inflammatory bowel disease. UC is characterized by mucosal inflammation limited to the colon, always involving the rectum and a variable extent of the more proximal colon in a continuous manner. Genetic variations in DNA repair genes may influence the extent of repair functions, DNA damage, and thus the manifestations of UC. This study thus evaluated the role of polymorphisms of the genes involved in DNA repair mechanisms. A total of 171 patients and 213 controls were included. Genotyping was carried out by ARMS PCR and PCR-RFLP analyses for RAD51, XRCC3 and hMSH2 gene polymorphisms. Allelic and genotypic frequencies were computed in both control & patient groups and data was analyzed using appropriate statistical tests. The frequency of 'A' allele of hMSH2 in the UC group caused statistically significant increased risk for UC compared to controls (OR 1.64, 95% CI 1.16-2.31, p = 0.004. Similarly, the CT genotype of XRCC3 gene was predominant in the UC group and increased the risk for UC by 1.75 fold compared to controls (OR 1.75, 95% CI 1.15-2.67, p = 0.03, further confirming the risk of 'T' allele in UC. The GC genotype frequency of RAD51 gene was significantly increased (p = 0.02 in the UC group (50.3% compared to controls (38%. The GC genotype significantly increased the risk for UC compared to GG genotype by 1.73 fold (OR 1.73, 95% CI 1.14-2.62, p = 0.02 confirming the strong association of 'C' allele with UC. Among the controls, the SNP loci combination of hMSH2:XRCC3 were in perfect linkage. The GTC and ACC haplotypes were found to be predominant in UC than controls with a 2.28 and 2.93 fold significant increase risk of UC.

  5. Polymorphisms in genes controlling inflammation and tissue repair in rheumatoid arthritis: a case control study

    Directory of Open Access Journals (Sweden)

    de Vogel Lisette

    2011-03-01

    Full Text Available Abstract Background Various cytokines and inflammatory mediators are known to be involved in the pathogenesis of rheumatoid arthritis (RA. We hypothesized that polymorphisms in selected inflammatory response and tissue repair genes contribute to the susceptibility to and severity of RA. Methods Polymorphisms in TNFA, IL1B, IL4, IL6, IL8, IL10, PAI1, NOS2a, C1INH, PARP, TLR2 and TLR4 were genotyped in 376 Caucasian RA patients and 463 healthy Caucasian controls using single base extension. Genotype distributions in patients were compared with those in controls. In addition, the association of polymorphisms with the need for anti-TNF-α treatment as a marker of RA severity was assessed. Results The IL8 781 CC genotype was associated with early onset of disease. The TNFA -238 G/A polymorphism was differentially distributed between RA patients and controls, but only when not corrected for age and gender. None of the polymorphisms was associated with disease severity. Conclusions We here report an association between IL8 781 C/T polymorphism and age of onset of RA. Our findings indicate that there might be a role for variations in genes involved in the immune response and in tissue repair in RA pathogenesis. Nevertheless, additional larger genomic and functional studies are required to further define their role in RA.

  6. Repair of spinal cord injury by neural stem cells modified with BDNF gene in rats

    Institute of Scientific and Technical Information of China (English)

    Wei LI; Wen-Qin CAI; Cheng-Ren LI

    2006-01-01

    Objective To explore repair of spinal cord injury by neural stem cells (NSCs) modified with brain derived neurotrophic factor (BDNF) gene (BDNF-NSCs) in rats. Methods Neural stem cells modified with BDNF gene were transplanted into the complete transection site of spinal cord at the lumbar 4 (L4) level in rats. Motor function of rats'hind limbs was observed and HE and X-gal immunocytochemical staining, in situ hybridization, and retrograde HRP tracing were also performed. Results BDNF-NSCs survived and integrated well with host spinal cord. In the transplant group, some X-gal positive, NF-200 positive, GFAP positive, BDNF positive, and BDNF mRNA positive cells, and many NF-200 positive nerve fibers were observed in the injury site. Retrograde HRP tracing through sciatic nerve showed some HRP positive cells and nerve fibers near the rostral side of the injury one month after transplant and with time, they increased in number. Examinations on rats' motor function and behavior demonstrated that motor function of rats' hind limbs improved better in the transplant group than the injury group. Conclusion BDNF-NSCs can survive, differentiate,and partially integrate with host spinal cord, and they significantly ameliorate rats ' motor function of hind limbs, indicating their promising role in repairing spinal cord injury.

  7. Role of APC and DNA mismatch repair genes in the development of colorectal cancers

    Directory of Open Access Journals (Sweden)

    Roy Deodutta

    2003-12-01

    Full Text Available Abstract Colorectal cancer is the third most common cause of cancer-related death in both men and women in the western hemisphere. According to the American Cancer Society, an estimated 105,500 new cases of colon cancer with 57,100 deaths will occur in the U.S. in 2003, accounting for about 10% of cancer deaths. Among the colon cancer patients, hereditary risk contributes approximately 20%. The main inherited colorectal cancers are the familial adenomatous polyposis (FAP and the hereditary nonpolyposis colorectal cancers (HNPCC. The FAP and HNPCC are caused due to mutations in the adenomatous polyposis coli (APC and DNA mismatch repair (MMR genes. The focus of this review is to summarize the functions of APC and MMR gene products in the development of colorectal cancers.

  8. Repairing DNA damage in xeroderma pigmentosum: T4N5 lotion and gene therapy.

    Science.gov (United States)

    Zahid, Sarwar; Brownell, Isaac

    2008-04-01

    Patients with xeroderma pigmentosum (XP) have defective DNA repair and are at a high risk for cutaneous malignancies. Standard treatments for XP are limited in scope and effectiveness. Understanding the molecular etiology of XP has led to the development of novel therapeutic approaches, including enzyme and gene therapies. One new topical treatment utilizing bacteriophage T4 endonuclease 5 (T4N5) in a liposomal lotion is currently in clinical trials and has received a Fast Track designation from the FDA. Gene therapy for XP, while making leaps in preclinical studies, has been slower to develop due to tactical hurdles, but seems to have much potential for future treatment. If these treatments prove effective in lowering the risk of cancer in patients with XP, they may also be found useful in reducing skin cancers in other at-risk patient populations.

  9. Insertional Mutagenesis by CRISPR/Cas9 Ribonucleoprotein Gene Editing in Cells Targeted for Point Mutation Repair Directed by Short Single-Stranded DNA Oligonucleotides.

    Science.gov (United States)

    Rivera-Torres, Natalia; Banas, Kelly; Bialk, Pawel; Bloh, Kevin M; Kmiec, Eric B

    2017-01-01

    CRISPR/Cas9 and single-stranded DNA oligonucleotides (ssODNs) have been used to direct the repair of a single base mutation in human genes. Here, we examine a method designed to increase the precision of RNA guided genome editing in human cells by utilizing a CRISPR/Cas9 ribonucleoprotein (RNP) complex to initiate DNA cleavage. The RNP is assembled in vitro and induces a double stranded break at a specific site surrounding the mutant base designated for correction by the ssODN. We use an integrated mutant eGFP gene, bearing a single base change rendering the expressed protein nonfunctional, as a single copy target in HCT 116 cells. We observe significant gene correction activity of the mutant base, promoted by the RNP and single-stranded DNA oligonucleotide with validation through genotypic and phenotypic readout. We demonstrate that all individual components must be present to obtain successful gene editing. Importantly, we examine the genotype of individually sorted corrected and uncorrected clonally expanded cell populations for the mutagenic footprint left by the action of these gene editing tools. While the DNA sequence of the corrected population is exact with no adjacent sequence modification, the uncorrected population exhibits heterogeneous mutagenicity with a wide variety of deletions and insertions surrounding the target site. We designate this type of DNA aberration as on-site mutagenicity. Analyses of two clonal populations bearing specific DNA insertions surrounding the target site, indicate that point mutation repair has occurred at the level of the gene. The phenotype, however, is not rescued because a section of the single-stranded oligonucleotide has been inserted altering the reading frame and generating truncated proteins. These data illustrate the importance of analysing mutagenicity in uncorrected cells. Our results also form the basis of a simple model for point mutation repair directed by a short single-stranded DNA oligonucleotides and

  10. Insertional Mutagenesis by CRISPR/Cas9 Ribonucleoprotein Gene Editing in Cells Targeted for Point Mutation Repair Directed by Short Single-Stranded DNA Oligonucleotides

    Science.gov (United States)

    Rivera-Torres, Natalia; Bialk, Pawel; Bloh, Kevin M.; Kmiec, Eric B.

    2017-01-01

    CRISPR/Cas9 and single-stranded DNA oligonucleotides (ssODNs) have been used to direct the repair of a single base mutation in human genes. Here, we examine a method designed to increase the precision of RNA guided genome editing in human cells by utilizing a CRISPR/Cas9 ribonucleoprotein (RNP) complex to initiate DNA cleavage. The RNP is assembled in vitro and induces a double stranded break at a specific site surrounding the mutant base designated for correction by the ssODN. We use an integrated mutant eGFP gene, bearing a single base change rendering the expressed protein nonfunctional, as a single copy target in HCT 116 cells. We observe significant gene correction activity of the mutant base, promoted by the RNP and single-stranded DNA oligonucleotide with validation through genotypic and phenotypic readout. We demonstrate that all individual components must be present to obtain successful gene editing. Importantly, we examine the genotype of individually sorted corrected and uncorrected clonally expanded cell populations for the mutagenic footprint left by the action of these gene editing tools. While the DNA sequence of the corrected population is exact with no adjacent sequence modification, the uncorrected population exhibits heterogeneous mutagenicity with a wide variety of deletions and insertions surrounding the target site. We designate this type of DNA aberration as on-site mutagenicity. Analyses of two clonal populations bearing specific DNA insertions surrounding the target site, indicate that point mutation repair has occurred at the level of the gene. The phenotype, however, is not rescued because a section of the single-stranded oligonucleotide has been inserted altering the reading frame and generating truncated proteins. These data illustrate the importance of analysing mutagenicity in uncorrected cells. Our results also form the basis of a simple model for point mutation repair directed by a short single-stranded DNA oligonucleotides and

  11. Mutation screening of mismatch repair gene Mlh3 in familial esophageal cancer

    Institute of Scientific and Technical Information of China (English)

    Hong-Xu Liu; Yu Li; Xue-Dong Jiang; Hong-Nian Yin; Lin Zhang; Yu Wang; Jun Yang

    2006-01-01

    AIM: To shed light on the possible role of mismatch repair gene Mlh3 in familial esophageal cancer (FEC).METHODS: A total of 66 members from 10 families suggestive of a genetic predisposition to hereditary esophageal cancer were screened for germline mutations in Mlh3 with denaturing high performance liquid chromatography (DHPLC), a newly developed method of comparative sequencing based on heteroduplex detection. For all samples exhibiting abnormal DHPLC profiles,sequence changes were evaluated by cycle sequencing.For any mutation in family members, we conducted a segregation study to compare its prevalence in sporadic esophageal cancer patients and normal controls.RESULTS: Exons of Mlh3 in all samples were successfully examined. Overall, 4 missense mutations and 3 polymorphisms were identified in 4 families. Mlh3 missense mutations in families 9 and 10 might be pathogenic, but had a reduced penetrance. While in families 1 and 7,there was no sufficient evidence supporting the monogenic explanations of esophageal cancers in families.The mutations were found in 33% of high-risk families and 50% of low-risk families.CONCLUSION: Mlh3 is a high risk gene with a reduced penetrance in some families. However, it acts as a low risk gene for esophageal cancer in most families. Mutations of Mlh3 may work together with other genes in an accumulated manner and result in an increased risk of esophageal tumor. DHPLC is a robust and sensitive technique for screening gene mutations.

  12. Tissue-engineering strategies to repair joint tissue in osteoarthritis: nonviral gene-transfer approaches.

    Science.gov (United States)

    Madry, Henning; Cucchiarini, Magali

    2014-10-01

    Loss of articular cartilage is a common clinical consequence of osteoarthritis (OA). In the past decade, substantial progress in tissue engineering, nonviral gene transfer, and cell transplantation have provided the scientific foundation for generating cartilaginous constructs from genetically modified cells. Combining tissue engineering with overexpression of therapeutic genes enables immediate filling of a cartilage defect with an engineered construct that actively supports chondrogenesis. Several pioneering studies have proved that spatially defined nonviral overexpression of growth-factor genes in constructs of solid biomaterials or hydrogels is advantageous compared with gene transfer or scaffold alone, both in vitro and in vivo. Notably, these investigations were performed in models of focal cartilage defects, because advanced cartilage-repair strategies based on the principles of tissue engineering have not advanced sufficiently to enable resurfacing of extensively degraded cartilage as therapy for OA. These studies serve as prototypes for future technological developments, because they raise the possibility that cartilage constructs engineered from genetically modified chondrocytes providing autocrine and paracrine stimuli could similarly compensate for the loss of articular cartilage in OA. Because cartilage-tissue-engineering strategies are already used in the clinic, combining tissue engineering and nonviral gene transfer could prove a powerful approach to treat OA.

  13. Precise Genome Modification via Sequence-Specific Nucleases-Mediated Gene Targeting for Crop Improvement.

    Science.gov (United States)

    Sun, Yongwei; Li, Jingying; Xia, Lanqin

    2016-01-01

    Genome editing technologies enable precise modifications of DNA sequences in vivo and offer a great promise for harnessing plant genes in crop improvement. The precise manipulation of plant genomes relies on the induction of DNA double-strand breaks by sequence-specific nucleases (SSNs) to initiate DNA repair reactions that are based on either non-homologous end joining (NHEJ) or homology-directed repair (HDR). While complete knock-outs and loss-of-function mutations generated by NHEJ are very valuable in defining gene functions, their applications in crop improvement are somewhat limited because many agriculturally important traits are conferred by random point mutations or indels at specific loci in either the genes' encoding or promoter regions. Therefore, genome modification through SSNs-mediated HDR for gene targeting (GT) that enables either gene replacement or knock-in will provide an unprecedented ability to facilitate plant breeding by allowing introduction of precise point mutations and new gene functions, or integration of foreign genes at specific and desired "safe" harbor in a predefined manner. The emergence of three programmable SSNs, such as zinc finger nucleases, transcriptional activator-like effector nucleases, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems has revolutionized genome modification in plants in a more controlled manner. However, while targeted mutagenesis is becoming routine in plants, the potential of GT technology has not been well realized for traits improvement in crops, mainly due to the fact that NHEJ predominates DNA repair process in somatic cells and competes with the HDR pathway, and thus HDR-mediated GT is a relative rare event in plants. Here, we review recent research findings mainly focusing on development and applications of precise GT in plants using three SSNs systems described above, and the potential mechanisms underlying HDR events in plant

  14. Integration-defective lentiviral vector mediates efficient gene editing through homology-directed repair in human embryonic stem cells.

    Science.gov (United States)

    Wang, Yebo; Wang, Yingjia; Chang, Tammy; Huang, He; Yee, Jiing-Kuan

    2016-11-28

    Human embryonic stem cells (hESCs) are used as platforms for disease study, drug screening and cell-based therapy. To facilitate these applications, it is frequently necessary to genetically manipulate the hESC genome. Gene editing with engineered nucleases enables site-specific genetic modification of the human genome through homology-directed repair (HDR). However, the frequency of HDR remains low in hESCs. We combined efficient expression of engineered nucleases and integration-defective lentiviral vector (IDLV) transduction for donor template delivery to mediate HDR in hESC line WA09. This strategy led to highly efficient HDR with more than 80% of the selected WA09 clones harboring the transgene inserted at the targeted genomic locus. However, certain portions of the HDR clones contained the concatemeric IDLV genomic structure at the target site, probably resulted from recombination of the IDLV genomic input before HDR with the target. We found that the integrase protein of IDLV mediated the highly efficient HDR through the recruitment of a cellular protein, LEDGF/p75. This study demonstrates that IDLV-mediated HDR is a powerful and broadly applicable technology to carry out site-specific gene modification in hESCs.

  15. Fibrin patch-based insulin-like growth factor-1 gene-modified stem cell transplantation repairs ischemic myocardium

    OpenAIRE

    Li, Jun; Zhu, Kai; Yang, Shan; WANG, YULIN; Guo, Changfa; Yin, Kanhua; Wang, Chunsheng; Lai, Hao

    2015-01-01

    Bone marrow mesenchymal stem cells (BMSCs), tissue-engineered cardiac patch, and therapeutic gene have all been proposed as promising therapy strategies for cardiac repair after myocardial infarction. In our study, BMSCs were modified with insulin-like growth factor-1 (IGF-1) gene, loaded into a fibrin patch, and then transplanted into a porcine model of ischemia/reperfusion (I/R) myocardium injury. The results demonstrated that IGF-1 gene overexpression could promote proliferation of endothe...

  16. Horizontal gene transfer regulation in bacteria as a "spandrel" of DNA repair mechanisms.

    Directory of Open Access Journals (Sweden)

    Saliou Fall

    Full Text Available Horizontal gene transfer (HGT is recognized as the major force for bacterial genome evolution. Yet, numerous questions remain about the transferred genes, their function, quantity and frequency. The extent to which genetic transformation by exogenous DNA has occurred over evolutionary time was initially addressed by an in silico approach using the complete genome sequence of the Ralstonia solanacearum GMI1000 strain. Methods based on phylogenetic reconstruction of prokaryote homologous genes families detected 151 genes (13.3% of foreign origin in the R. solanacearum genome and tentatively identified their bacterial origin. These putative transfers were analyzed in comparison to experimental transformation tests involving 18 different genomic DNA positions in the genome as sites for homologous or homeologous recombination. Significant transformation frequency differences were observed among these positions tested regardless of the overall genomic divergence of the R. solanacearum strains tested as recipients. The genomic positions containing the putative exogenous DNA were not systematically transformed at the highest frequencies. The two genomic "hot spots", which contain recA and mutS genes, exhibited transformation frequencies from 2 to more than 4 orders of magnitude higher than positions associated with other genes depending on the recipient strain. These results support the notion that the bacterial cell is equipped with active mechanisms to modulate acquisition of new DNA in different genomic positions. Bio-informatics study correlated recombination "hot-spots" to the presence of Chi-like signature sequences with which recombination might be preferentially initiated. The fundamental role of HGT is certainly not limited to the critical impact that the very rare foreign genes acquired mainly by chance can have on the bacterial adaptation potential. The frequency to which HGT with homologous and homeologous DNA happens in the environment

  17. Stimulation of proteoglycan synthesis by glucuronosyltransferase-I gene delivery: a strategy to promote cartilage repair.

    Science.gov (United States)

    Venkatesan, N; Barré, L; Benani, A; Netter, P; Magdalou, J; Fournel-Gigleux, S; Ouzzine, M

    2004-12-28

    Osteoarthritis is a degenerative joint disease characterized by a progressive loss of articular cartilage components, mainly proteoglycans (PGs), leading to destruction of the tissue. We investigate a therapeutic strategy based on stimulation of PG synthesis by gene transfer of the glycosaminoglycan (GAG)-synthesizing enzyme, beta1,3-glucuronosyltransferase-I (GlcAT-I) to promote cartilage repair. We previously reported that IL-1beta down-regulated the expression and activity of GlcAT-I in primary rat chondrocytes. Here, by using antisense oligonucleotides, we demonstrate that GlcAT-I inhibition impaired PG synthesis and deposition in articular cartilage explants, emphasizing the crucial role of this enzyme in PG anabolism. Thus, primary chondrocytes and cartilage explants were engineered by lipid-mediated gene delivery to efficiently overexpress a human GlcAT-I cDNA. Interestingly, GlcAT-I overexpression significantly enhanced GAG synthesis and deposition as evidenced by (35)S-sulfate incorporation, histology, estimation of GAG content, and fluorophore-assisted carbohydrate electrophoresis analysis. Metabolic labeling and Western blot analyses further suggested that GlcAT-I expression led to an increase in the abundance rather than in the length of GAG chains. Importantly, GlcAT-I delivery was able to overcome IL-1beta-induced PG depletion and maintain the anabolic activity of chondrocytes. Moreover, GlcAT-I also restored PG synthesis to a normal level in cartilage explants previously depleted from endogenous PGs by IL-1beta-treatment. In concert, our investigations strongly indicated that GlcAT-I was able to control and reverse articular cartilage defects in terms of PG anabolism and GAG content associated with IL-1beta. This study provides a basis for a gene therapy approach to promote cartilage repair in degenerative joint diseases.

  18. Gene mutation-based and specific therapies in precision medicine.

    Science.gov (United States)

    Wang, Xiangdong

    2016-04-01

    Precision medicine has been initiated and gains more and more attention from preclinical and clinical scientists. A number of key elements or critical parts in precision medicine have been described and emphasized to establish a systems understanding of precision medicine. The principle of precision medicine is to treat patients on the basis of genetic alterations after gene mutations are identified, although questions and challenges still remain before clinical application. Therapeutic strategies of precision medicine should be considered according to gene mutation, after biological and functional mechanisms of mutated gene expression or epigenetics, or the correspondent protein, are clearly validated. It is time to explore and develop a strategy to target and correct mutated genes by direct elimination, restoration, correction or repair of mutated sequences/genes. Nevertheless, there are still numerous challenges to integrating widespread genomic testing into individual cancer therapies and into decision making for one or another treatment. There are wide-ranging and complex issues to be solved before precision medicine becomes clinical reality. Thus, the precision medicine can be considered as an extension and part of clinical and translational medicine, a new alternative of clinical therapies and strategies, and have an important impact on disease cures and patient prognoses.

  19. Precise Genome Modification via Sequence-Specific Nucleases-Mediated Gene Targeting for Crop Improvement

    Science.gov (United States)

    Sun, Yongwei; Li, Jingying; Xia, Lanqin

    2016-01-01

    Genome editing technologies enable precise modifications of DNA sequences in vivo and offer a great promise for harnessing plant genes in crop improvement. The precise manipulation of plant genomes relies on the induction of DNA double-strand breaks by sequence-specific nucleases (SSNs) to initiate DNA repair reactions that are based on either non-homologous end joining (NHEJ) or homology-directed repair (HDR). While complete knock-outs and loss-of-function mutations generated by NHEJ are very valuable in defining gene functions, their applications in crop improvement are somewhat limited because many agriculturally important traits are conferred by random point mutations or indels at specific loci in either the genes’ encoding or promoter regions. Therefore, genome modification through SSNs-mediated HDR for gene targeting (GT) that enables either gene replacement or knock-in will provide an unprecedented ability to facilitate plant breeding by allowing introduction of precise point mutations and new gene functions, or integration of foreign genes at specific and desired “safe” harbor in a predefined manner. The emergence of three programmable SSNs, such as zinc finger nucleases, transcriptional activator-like effector nucleases, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems has revolutionized genome modification in plants in a more controlled manner. However, while targeted mutagenesis is becoming routine in plants, the potential of GT technology has not been well realized for traits improvement in crops, mainly due to the fact that NHEJ predominates DNA repair process in somatic cells and competes with the HDR pathway, and thus HDR-mediated GT is a relative rare event in plants. Here, we review recent research findings mainly focusing on development and applications of precise GT in plants using three SSNs systems described above, and the potential mechanisms underlying HDR events in

  20. Precise genome modification via sequence-specific nucleases-mediated gene targeting for crop improvement

    Directory of Open Access Journals (Sweden)

    Yongwei Sun

    2016-12-01

    Full Text Available Genome editing technologies enable precise modifications of DNA sequences in vivo and offer a great promise for harnessing plant genes in crop improvement. The precise manipulation of plant genomes relies on the induction of DNA double-strand breaks (DSBs by sequence-specific nucleases (SSNs to initiate DNA repair reactions that are based on either non-homologous end joining (NHEJ or homology-directed repair (HDR. While complete knock-outs and loss-of-function mutations generated by NHEJ are very valuable in defining gene functions, their applications in crop improvement are somewhat limited because many agriculturally important traits are conferred by random point mutations or indels at specific loci in either the genes’ encoding or promoter regions. Therefore, genome modification through SSNs-mediated HDR for gene targeting (GT that enables either gene replacement or knock-in will provide an unprecedented ability to facilitate plant breeding by allowing introduction of precise point mutations and new gene functions, or integration of foreign genes at specific and desired ‘safe’ harbor in a predefined manner. The emergence of three programmable SSNs such as zinc finger nucleases (ZFNs, transcriptional activator-like effector nucleases (TALENs, and the clustered regularly interspaced short palindromic repeat (CRISPR/CRISPR-associated protein 9 (Cas9 systems has revolutionized genome modification in plants in a more controlled manner. However, while targeted mutagenesis is becoming routine in plants, the potential of GT technology has not been well realized for traits improvement in crops, mainly due to the fact that NHEJ predominates DNA repair process in somatic cells and competes with the HDR pathway, and thus HDR-mediated GT is a relative rare event in plants. Here, we review recent research findings mainly focusing on development and applications of precise GT in plants using three SSNs systems described above, and the potential

  1. Polymorphisms in DNA Repair Genes and Susceptibility to Glioma in a Chinese Population

    Directory of Open Access Journals (Sweden)

    Jun-Hong Guan

    2013-02-01

    Full Text Available The excision repair cross-complementing rodent repair deficiency complementation group 1 (ERCC1, and X-ray repair cross-complementing group 1 (XRCC1 genes appear to protect mammalian cells from the harmful effects of ionizing radiation. We conducted a large case-control study to investigate the association of polymorphisms in ERCC1 C118T, ERCC1 C8092A, XRCC1 A194T, XRCC1 A194T, and XRCC3 C241T, with glioma risk in a Chinese population. Five single nucleotide polymorphisms (SNPs were genotyped, using the MassARRAY IPLEX platform, in 443 glioma cases and 443 controls. Association analyses based on an χ2 test and binary logistic regression were performed to determine the odds ratio (OR and a 95% confidence interval (95% CI for each SNP. For XRCC1 Arg194Trp, the variant genotype T/T was strongly associated with a lower risk of glioma cancer when compared with the wild type C/C (OR = 2.45, 95% CI = 1.43–4.45. Individuals carrying the XRCC1 399A allele had an increased risk of glioma (OR = 1.33, 95% CI = 1.02–1.64. The XRCC3 241T/T genotype was associated with a strong increased glioma risk (OR = 3.78, 95% CI = 1.86–9.06. Further analysis of the interactions of two susceptibility-associated SNPs, XRCC1 Arg194Trp and XRCC3 Thr241Met, showed that the combination of the XRCC1 194T and XRCC3 241T alleles brought a large increase in glioma risk (OR = 2.75, 95% CI = 1.54–4.04. XRCC1 Arg194Trp, XRCC1 Arg399Gln, and XRCC3 C241T, appear to be associated with susceptibility to glioma in a Chinese population.

  2. Assessing somatic hypermutation in Ramos B cells after overexpression or knockdown of specific genes.

    Science.gov (United States)

    Upton, Dana C; Unniraman, Shyam

    2011-11-01

    B cells start their life with low affinity antibodies generated by V(D)J recombination. However, upon detecting a pathogen, the variable (V) region of an immunoglobulin (Ig) gene is mutated approximately 100,000-fold more than the rest of the genome through somatic hypermutation (SHM), resulting in high affinity antibodies. In addition, class switch recombination (CSR) produces antibodies with different effector functions depending on the kind of immune response that is needed for a particular pathogen. Both CSR and SHM are initiated by activation-induced cytidine deaminase (AID), which deaminates cytosine residues in DNA to produce uracils. These uracils are processed by error-prone forms of repair pathways, eventually leading to mutations and recombination. Our current understanding of the molecular details of SHM and CSR come from a combination of studies in mice, primary cells, cell lines, and cell-free experiments. Mouse models remain the gold standard with genetic knockouts showing critical roles for many repair factors (e.g. Ung, Msh2, Msh6, Exo1, and polymerase η). However, not all genes are amenable for knockout studies. For example, knockouts of several double-strand break repair proteins are embryonically lethal or impair B-cell development. Moreover, sometimes the specific function of a protein in SHM or CSR may be masked by more global defects caused by the knockout. In addition, since experiments in mice can be lengthy, altering expression of individual genes in cell lines has become an increasingly popular first step to identifying and characterizing candidate genes. Ramos - a Burkitt lymphoma cell line that constitutively undergoes SHM - has been a popular cell-line model to study SHM. One advantage of Ramos cells is that they have a built-in convenient semi-quantitative measure of SHM. Wild type cells express IgM and, as they pick up mutations, some of the mutations knock out IgM expression. Therefore, assaying IgM loss by fluorescence

  3. Fibrin patch-based insulin-like growth factor-1 gene-modified stem cell transplantation repairs ischemic myocardium

    Science.gov (United States)

    Li, Jun; Zhu, Kai; Yang, Shan; Wang, Yulin; Guo, Changfa; Yin, Kanhua; Wang, Chunsheng

    2015-01-01

    Bone marrow mesenchymal stem cells (BMSCs), tissue-engineered cardiac patch, and therapeutic gene have all been proposed as promising therapy strategies for cardiac repair after myocardial infarction. In our study, BMSCs were modified with insulin-like growth factor-1 (IGF-1) gene, loaded into a fibrin patch, and then transplanted into a porcine model of ischemia/reperfusion (I/R) myocardium injury. The results demonstrated that IGF-1 gene overexpression could promote proliferation of endothelial cells and cardiomyocyte-like differentiation of BMSCs in vitro. Four weeks after transplantation of fibrin patch loaded with gene-modified BMSCs, IGF-1 overexpression could successfully promote angiogenesis, inhibit remodeling, increase grafted cell survival and reduce apoptosis. In conclusion, the integrated strategy, which combined fibrin patch with IGF-1 gene modified BMSCs, could promote the histological cardiac repair for a clinically relevant porcine model of I/R myocardium injury. PMID:25767192

  4. Vascular endothelial growth factor gene transfection to enhance the repair of avascular necrosis of the femoral head of rabbit

    Institute of Scientific and Technical Information of China (English)

    杨操; 杨述华; 杜靖远; 李进; 许伟华; 熊宇芳

    2003-01-01

    Objective To explore a new method for the therapy of avascular necrosis of the femoral head.Methods The recombinant plasmid pCD-hVEGF165 was mixed with collagen and was implanted in the necrotic femoral head. The expression of vascular endothelial growth factor (VEGF) was examined by RNA dot hybridization and immunohistochemical techniques. Repair of the femoral head was observed by histological and histomorphometric analysis.Results The expression of VEGF was detected in the femoral head transfected with the VEGF gene. The femoral head transfected with the VEGF gene showed a significant increase in angiogenesis 2 and 4 weeks after gene transfection and a significant increase in bone formation 6 and 8 weeks after gene transfection on histomorphometric analysis (P<0.01).Conclusions Transfection of the VEGF gene enhances bone tissue angiogenesis. Repair of osteonecrosis could be accelerated accordingly, thus providing a potential method for therapy of osteonecrosis.

  5. Genes2GO: A web application for querying gene sets for specific GO terms.

    Science.gov (United States)

    Chawla, Konika; Kuiper, Martin

    2016-01-01

    Gene ontology annotations have become an essential resource for biological interpretations of experimental findings. The process of gathering basic annotation information in tables that link gene sets with specific gene ontology terms can be cumbersome, in particular if it requires above average computer skills or bioinformatics expertise. We have therefore developed Genes2GO, an intuitive R-based web application. Genes2GO uses the biomaRt package of Bioconductor in order to retrieve custom sets of gene ontology annotations for any list of genes from organisms covered by the Ensembl database. Genes2GO produces a binary matrix file, indicating for each gene the presence or absence of specific annotations for a gene. It should be noted that other GO tools do not offer this user-friendly access to annotations. Genes2GO is freely available and listed under http://www.semantic-systems-biology.org/tools/externaltools/.

  6. Enzymatic repair of selected cross-linked homoduplex molecules enhances nuclear gene rescue from Pompeii and Herculaneum remains.

    Science.gov (United States)

    Di Bernardo, Giovanni; Del Gaudio, Stefania; Cammarota, Marcella; Galderisi, Umberto; Cascino, Antonino; Cipollaro, Marilena

    2002-02-15

    Ancient DNA (aDNA) samples extracted from the bone remains of six equids buried by the Vesuvius eruption in 79 AD were investigated to test pre-amplification and enzymatic repair procedures designed to enhance the rescue of nuclear genes. The extracts, which proved all positive for Equidae mtDNA amplification, proved positive only four times out of 18 when tested for single-copy Equidae nuclear genes (epsilon globin, p53 and gamma interferon). Pre-amplification did not change the number of retrieved aDNA sequences but 10 times out of 14 enzymatic repair restored the amplifiability of the genes analysed, proving that repair increases the rate of successful rescue from 22 to alpha(lambda)mu(omicron)sigma(tau) 80%. These findings support the hypothesis that some of these cross-linked aDNA molecules, which are not completely separated when DNA is extracted under denaturing conditions, become homoduplex substrates for Pol I and/or T4 ligase action upon renaturation. aDNA authenticity is proved by the homology of the nucleotide sequences of loci tested to the corresponding modern Equidae sequences. Data also indicate that cross-linked homoduplex molecules selected by denaturation of the extract are repaired without any chimera formation. The general features of aDNA amplification with and without denaturation and enzymatic repair are discussed.

  7. Recombination-dependent deletion formation in mammalian cells deficient in the nucleotide excision repair gene ERCC1.

    Science.gov (United States)

    Sargent, R G; Rolig, R L; Kilburn, A E; Adair, G M; Wilson, J H; Nairn, R S

    1997-11-25

    Nucleotide excision repair proteins have been implicated in genetic recombination by experiments in Saccharomyces cerevisiae and Drosophila melanogaster, but their role, if any, in mammalian cells is undefined. To investigate the role of the nucleotide excision repair gene ERCC1, the hamster homologue to the S. cerevisiae RADIO gene, we disabled the gene by targeted knockout. Partial tandem duplications of the adenine phosphoribosyltransferase (APRT) gene then were constructed at the endogenous APRT locus in ERCC1- and ERCC1+ cells. To detect the full spectrum of gene-altering events, we used a loss-of-function assay in which the parental APRT+ tandem duplication could give rise to APRT- cells by homologous recombination, gene rearrangement, or point mutation. Measurement of rates and analysis of individual APRT- products indicated that gene rearrangements (principally deletions) were increased at least 50-fold, whereas homologous recombination was affected little. The formation of deletions is not caused by a general effect of the ERCC1 deficiency on gene stability, because ERCC1- cell lines with a single wild-type copy of the APRT gene yielded no increase in deletions. Thus, deletion formation is dependent on the tandem duplication, and presumably the process of homologous recombination. Recombination-dependent deletion formation in ERCC1- cells is supported by a significant decrease in a particular class of crossover products that are thought to arise by repair of a heteroduplex intermediate in recombination. We suggest that the ERCC1 gene product in mammalian cells is involved in the processing of heteroduplex intermediates in recombination and that the misprocessed intermediates in ERCC1- cells are repaired by illegitimate recombination.

  8. Dynamic Gene Regulatory Networks Drive Hematopoietic Specification and Differentiation

    Science.gov (United States)

    Goode, Debbie K.; Obier, Nadine; Vijayabaskar, M.S.; Lie-A-Ling, Michael; Lilly, Andrew J.; Hannah, Rebecca; Lichtinger, Monika; Batta, Kiran; Florkowska, Magdalena; Patel, Rahima; Challinor, Mairi; Wallace, Kirstie; Gilmour, Jane; Assi, Salam A.; Cauchy, Pierre; Hoogenkamp, Maarten; Westhead, David R.; Lacaud, Georges; Kouskoff, Valerie; Göttgens, Berthold; Bonifer, Constanze

    2016-01-01

    Summary Metazoan development involves the successive activation and silencing of specific gene expression programs and is driven by tissue-specific transcription factors programming the chromatin landscape. To understand how this process executes an entire developmental pathway, we generated global gene expression, chromatin accessibility, histone modification, and transcription factor binding data from purified embryonic stem cell-derived cells representing six sequential stages of hematopoietic specification and differentiation. Our data reveal the nature of regulatory elements driving differential gene expression and inform how transcription factor binding impacts on promoter activity. We present a dynamic core regulatory network model for hematopoietic specification and demonstrate its utility for the design of reprogramming experiments. Functional studies motivated by our genome-wide data uncovered a stage-specific role for TEAD/YAP factors in mammalian hematopoietic specification. Our study presents a powerful resource for studying hematopoiesis and demonstrates how such data advance our understanding of mammalian development. PMID:26923725

  9. Gene specific actions of thyroid hormone receptor subtypes.

    Directory of Open Access Journals (Sweden)

    Jean Z Lin

    Full Text Available There are two homologous thyroid hormone (TH receptors (TRs α and β, which are members of the nuclear hormone receptor (NR family. While TRs regulate different processes in vivo and other highly related NRs regulate distinct gene sets, initial studies of TR action revealed near complete overlaps in their actions at the level of individual genes. Here, we assessed the extent that TRα and TRβ differ in target gene regulation by comparing effects of equal levels of stably expressed exogenous TRs +/- T(3 in two cell backgrounds (HepG2 and HeLa. We find that hundreds of genes respond to T(3 or to unliganded TRs in both cell types, but were not able to detect verifiable examples of completely TR subtype-specific gene regulation. TR actions are, however, far from identical and we detect TR subtype-specific effects on global T(3 response kinetics in HepG2 cells and many examples of TR subtype specificity at the level of individual genes, including effects on magnitude of response to TR +/- T(3, TR regulation patterns and T(3 dose response. Cycloheximide (CHX treatment confirms that at least some differential effects involve verifiable direct TR target genes. TR subtype/gene-specific effects emerge in the context of widespread variation in target gene response and we suggest that gene-selective effects on mechanism of TR action highlight differences in TR subtype function that emerge in the environment of specific genes. We propose that differential TR actions could influence physiologic and pharmacologic responses to THs and selective TR modulators (STRMs.

  10. Systematic design of mouse Vh gene family-specific oligonucleotides

    NARCIS (Netherlands)

    Seijen, AM; Seijen, HG; Bos, NA

    2001-01-01

    Kabat's database has often been used to design mouse Vh gene-specific 5 ' primers. The emphasis was mostly on constructing a universal (degenerate) 5 ' primer or 5 ' primer set, which would be able to match every mouse Vh gene. We were interested in finding oligonucleotides that could be used as pri

  11. Systematic design of mouse Vh gene family-specific oligonucleotides

    NARCIS (Netherlands)

    Seijen, AM; Seijen, HG; Bos, NA

    2001-01-01

    Kabat's database has often been used to design mouse Vh gene-specific 5 ' primers. The emphasis was mostly on constructing a universal (degenerate) 5 ' primer or 5 ' primer set, which would be able to match every mouse Vh gene. We were interested in finding oligonucleotides that could be used as pri

  12. A peek into the possible future of management of articular cartilage injuries: gene therapy and scaffolds for cartilage repair.

    Science.gov (United States)

    Kim, Hubert T; Zaffagnini, Stefano; Mizuno, Shuichi; Abelow, Stephen; Safran, Marc R

    2006-10-01

    Two rapidly progressing areas of research will likely contribute to cartilage repair procedures in the foreseeable future: gene therapy and synthetic scaffolds. Gene therapy refers to the transfer of new genetic information to cells that contribute to the cartilage repair process. This approach allows for manipulation of cartilage repair at the cellular and molecular level. Scaffolds are the core technology for the next generation of autologous cartilage implantation procedures in which synthetic matrices are used in conjunction with chondrocytes. This approach can be improved further using bioreactor technologies to enhance the production of extracellular matrix proteins by chondrocytes seeded onto a scaffold. The resulting "neo-cartilage implant" matures within the bioreactor, and can then be used to fill cartilage defects.

  13. Phenotypic Heterogeneity by Germline Mismatch Repair Gene Defect in Lynch Syndrome Patients.

    Science.gov (United States)

    Hernâni-Eusébio, Jorge; Barbosa, Elisabete

    2016-10-01

    Introdução: A síndrome de Lynch é a forma hereditária mais comum de cancro colo-rectal, sendo também responsável por cancro do endométrio e de outros tipos. Associa-se a mutações germinativas nos genes de mismatch repair do ADN e a instabilidade de microssatélites. As mutações MLH1 e MSH2 têm um fenótipo de síndrome de Lynch ‘clássico’, sendo o MSH2 mais associado a cancro extra-cólico. Mutações do MSH6 e PMS2 têm um fenótipo atípico. A expressão clínica é heterogénea, existindo uma correlação entre o gene mismatch repair mutado e o padrão fenotípico. Material e Métodos: Análise retrospetiva dos dados clínicos de doentes que cumpriam os critérios de Amesterdão ou que tinha mutações nos genes mismatch repair, entre setembro de 2012 e outubro de 2015. Resultados: Identificámos 28 doentes. Dezassete tinham cancro colo-rectal sendo a localização no cólon direito predominante. Cinco tiveram cancro do endométrio (mediana da idade de diagnóstico – 53), sem qualquer mutação no MSH6. Cinco desenvolveram outros cancros. Todos os casos com mutações mismatch repair estudados tinham instabilidade de microssatélites. Discussão: Na maioria dos casos foi encontrada mutação no MSH2 apesar de o MLH1 ser descrito na literatura como o gene mais frequentemente mutado. Interessa dizer que os doentes com cancro colo-rectal não evidenciam uma tendência para ter muito infiltrado inflamatório. Na maioria dos casos foi realizada colectomia parcial apesar da incidência elevada de lesões síncronas e metácronas associadas. Histerectomia e anexectomia profilática foi realizada em doentes em menopausa/perimenopausa. Conclusão: O registo standardizado dos dados dos doentes poderá levar a um melhor acompanhamento e conhecimento desta síndrome. O uso das Guidelines de Bethesda poderá identificar novos casos que escapam aos critérios de Amesterdão. A pesquisa de instabilidade de microssatélites deve ser feita em muito maior n

  14. Specific patient-related prognostic factors for rotator cuff repair : a systematic review

    NARCIS (Netherlands)

    Heerspink, Frederik O. Lambers; Dorrestijn, Oscar; van Raay, Jos J. A. M.; Diercks, Ron L.

    2014-01-01

    Background: Many studies that describe factors affecting outcome in primary rotator cuff repair (RCR) have been published, but so far there is no review that summarizes them. This systematic review was conducted to identify prognostic factors influencing functional (clinical) outcome and radiologica

  15. Specific patient-related prognostic factors for rotator cuff repair : a systematic review

    NARCIS (Netherlands)

    Heerspink, Frederik O. Lambers; Dorrestijn, Oscar; van Raay, Jos J. A. M.; Diercks, Ron L.

    Background: Many studies that describe factors affecting outcome in primary rotator cuff repair (RCR) have been published, but so far there is no review that summarizes them. This systematic review was conducted to identify prognostic factors influencing functional (clinical) outcome and

  16. A study of molecular signals deregulating mismatch repair genes in prostate cancer compared to benign prostatic hyperplasia.

    Directory of Open Access Journals (Sweden)

    Sanmitra Basu

    Full Text Available Prostate cancer is one of the leading causes of mortality among aging males. There is an unmet requirement of clinically useful biomarkers for early detection of prostate cancer to reduce the liabilities of overtreatment and accompanying morbidity. The present population-based study investigates the factors disrupting expression of multiple functionally related genes of DNA mismatch repair pathway in prostate cancer patients to identify molecular attributes distinguishing adenocarcinoma from benign hyperplasia of prostate. Gene expression was compared between tissue samples from prostate cancer and benign prostatic hyperplasia using real-time-PCR, western blot and immunohistochemistry. Assessment of genotypes of seven single-nucleotide-polymorphisms of three MMR genes was conducted using PCR-coupled RFLP and sequencing. Promoter methylation was interrogated by methylation-specific-PCR and bisulfite-sequencing. Interaction between microRNAs and MMR genes was verified by 3'UTR-based dual luciferase assays. Concurrent reduction of three MMR genes namely hMLH1, hMSH6 and hMSH2 (34-85%, P<0.05 was observed in prostate cancer tissues. hMSH6 polymorphism rs1800932(Pro92Pro conferred a borderline protection in cancer patients (OR = 0.33, 95% CI = 0.15-0.75. Relative transcript level of hMLH1 was inversely related (r = -0.59, P<0.05 with methylation quotient of its promoter which showed a significantly higher methylation density (P = 0.008, Z = -2.649 in cancer patients. hsa-miR-155, hsa-miR-141 and hsa-miR-21 gene expressions were significantly elevated (66-85%, P<0.05 in tumor specimens and negatively correlated (r = -0.602 to -0.527, P<0.05 with that of MMR genes. hsa-miR-155 & hsa-miR-141 and hsa-miR-155 & hsa-miR-21 were demonstrated to bind to their putative seed sequences in hMLH1 and hMSH6 3'UTRs respectively. Relatively higher expression of DNA methyl-transferases (DNMT1 and DNMT3b and HIF-1α genes (34-50%, P<0.05 were also detected in tumor

  17. Housekeeping and tissue-specific genes in mouse tissues

    Directory of Open Access Journals (Sweden)

    St-Amand Jonny

    2007-05-01

    Full Text Available Abstract Background This study aims to characterize the housekeeping and tissue-specific genes in 15 mouse tissues by using the serial analysis of gene expression (SAGE strategy which indicates the relative level of expression for each transcript matched to the tag. Results Here, we identified constantly expressed housekeeping genes, such as eukaryotic translation elongation factor 2, which is expressed in all tissues without significant difference in expression levels. Moreover, most of these genes were not regulated by experimental conditions such as steroid hormones, adrenalectomy and gonadectomy. In addition, we report previously postulated housekeeping genes such as peptidyl-prolyl cis-trans isomerase A, glyceraldehyde-3-phosphate dehydrogenase and beta-actin, which are expressed in all the tissues, but with significant difference in their expression levels. We have also identified genes uniquely detected in each of the 15 tissues and other tissues from public databases. Conclusion These identified housekeeping genes could represent appropriate controls for RT-PCR and northern blot when comparing the expression levels of genes in several tissues. The results reveal several tissue-specific genes highly expressed in testis and pituitary gland. Furthermore, the main function of tissue-specific genes expressed in liver, lung and bone is the cell defence, whereas several keratins involved in cell structure function are exclusively detected in skin and vagina. The results from this study can be used for example to target a tissue for agent delivering by using the promoter of tissue-specific genes. Moreover, this study could be used as basis for further researches on physiology and pathology of these tissues.

  18. The structure-specific endonuclease Ercc1–Xpf is required for targeted gene replacement in embryonic stem cells

    Science.gov (United States)

    Niedernhofer, Laura J.; Essers, Jeroen; Weeda, Geert; Beverloo, Berna; de Wit, Jan; Muijtjens, Manja; Odijk, Hanny; Hoeijmakers, Jan H.J.; Kanaar, Roland

    2001-01-01

    The Ercc1–Xpf heterodimer, a highly conserved structure-specific endonuclease, functions in multiple DNA repair pathways that are pivotal for maintaining genome stability, including nucleotide excision repair, interstrand crosslink repair and homologous recombination. Ercc1–Xpf incises double-stranded DNA at double-strand/single-strand junctions, making it an ideal enzyme for processing DNA structures that contain partially unwound strands. Here we demonstrate that although Ercc1 is dispensable for recombination between sister chromatids, it is essential for targeted gene replacement in mouse embryonic stem cells. Surprisingly, the role of Ercc1–Xpf in gene targeting is distinct from its previously identified role in removing nonhomologous termini from recombination intermediates because it was required irrespective of whether the ends of the DNA targeting constructs were heterologous or homologous to the genomic locus. Our observations have implications for the mechanism of gene targeting in mammalian cells and define a new role for Ercc1–Xpf in mammalian homologous recombination. We propose a model for the mechanism of targeted gene replacement that invokes a role for Ercc1–Xpf in making the recipient genomic locus receptive for gene replacement. PMID:11707424

  19. Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway

    DEFF Research Database (Denmark)

    Lee, Jae Seong; Beuchert Kallehauge, Thomas; Pedersen, Lasse Ebdrup

    2015-01-01

    gene integration into site-specific loci in CHO cells using CRISPR/Cas9 genome editing system and compatible donor plasmid harboring a gene of interest (GOI) and short homology arms. This strategy has enabled precise insertion of a 3.7-kb gene expression cassette at defined loci in CHO cells following...

  20. Identification of the two rotavirus genes determining neutralization specificities

    Energy Technology Data Exchange (ETDEWEB)

    Offit, P.A.; Blavat, G.

    1986-01-01

    Bovine rotavirus NCDV and simian rotavirus SA-11 represent two distinct rotavirus serotypes. A genetic approach was used to determine which viral gene segments segregated with serotype-specific viral neutralization. There were 16 reassortant rotarviruses derived by coinfection of MA-104 cells in vitro with the SA-11 and NCDV strains. The parental origin of reassortant rotavirus double-stranded RNA segments was determined by gene segment mobility in polyacrylamide gels and by hybridization with radioactively labeled parental viral transcripts. The authors found that two rotavirus gene segments found previously to code for outer capsid proteins vp3 and vp7 cosegreated with virus neutralization specificities.

  1. XRCC1 and XPD DNA repair gene polymorphisms: a potential risk factor for glaucoma in the Pakistani population

    NARCIS (Netherlands)

    Yousaf, S.; Khan, M.I.; Micheal, S.; Akhtar, F.; Ali, S.H.; Riaz, M.; Ali, M.; Lall, P.; Waheed, N.K.; Hollander, A.I. den; Ahmed, A.; Qamar, R.

    2011-01-01

    PURPOSE: The present study was designed to determine the association of polymorphisms of the DNA repair genes X-ray cross-complementing group 1 (XRCC1) (c.1316G>A [rs25487]) and xeroderma pigmentosum complementation group D (XPD) (c.2298A>C [rs13181]) with primary open-angle glaucoma (POAG) an

  2. Cloning, comparative mapping, and RNA expression of the mouse homologues of the Saccharomyces cerevisiae nucleotide excision repair gene RAD23.

    NARCIS (Netherlands)

    P.J. van der Spek (Peter); C.E. Visser (Cécile); F. Hanaoka (Fumio); B. Smit (Bep); A. Hagemeijer (Anne); D. Bootsma (Dirk); J.H.J. Hoeijmakers (Jan)

    1996-01-01

    textabstractThe Saccharomyces cerevisiae RAD23 gene is involved in nucleotide excision repair (NER). Two human homologs of RAD23, HHR23A and HHR23B (HGMW-approved symbols RAD23A and RAD23B), were previously isolated. The HHR23B protein is complexed with the protein defective in the cancer-prone

  3. Conserved pattern of antisense overlapping transcription in the homologous ERCC-1 and yeast RAD10 DNA repair gene regions.

    NARCIS (Netherlands)

    M. van Duin (Mark); J. van den Tol; J.H.J. Hoeijmakers (Jan); D. Bootsma (Dirk); I.P. Rupp; P. Reynolds (Paul); L. Prakash; S. Prakash

    1989-01-01

    textabstractWe report that the genes for the homologous Saccharomyces cerevisiae RAD10 and human ERCC-1 DNA excision repair proteins harbor overlapping antisense transcription units in their 3' regions. Since naturally occurring antisense transcription is rare in S. cerevisiae and humans (this is

  4. Genetic Variation in Base Excision Repair Pathway Genes, Pesticide Exposure, and Prostate Cancer Risk

    National Research Council Canada - National Science Library

    Kathryn Hughes Barry; Stella Koutros; Sonja I. Berndt; Gabriella Andreotti; Jane A. Hoppin; Dale P. Sandler; Laurie A. Burdette; Meredith Yeager; Laura E. Beane Freeman; Jay H. Lubin; Xiaomei Ma; Tongzhang Zheng; Michael C. R. Alavanja

    2011-01-01

    .... OBJECTIVES: Because base excision repair (BER) is the predominant pathway involved in repairing oxidative damage, we evaluated interactions between 39 pesticides and 394 tag single-nucleotide polymorphisms (SNPs...

  5. H. pylori-Induced DNA Strand Breaks Are Introduced by Nucleotide Excision Repair Endonucleases and Promote NF-κB Target Gene Expression

    Directory of Open Access Journals (Sweden)

    Mara L. Hartung

    2015-10-01

    Full Text Available The human bacterial pathogen Helicobacter pylori exhibits genotoxic properties that promote gastric carcinogenesis. H. pylori introduces DNA double strand breaks (DSBs in epithelial cells that trigger host cell DNA repair efforts. Here, we show that H. pylori-induced DSBs are repaired via error-prone, potentially mutagenic non-homologous end-joining. A genome-wide screen for factors contributing to DSB induction revealed a critical role for the H. pylori type IV secretion system (T4SS. Inhibition of transcription, as well as NF-κB/RelA-specific RNAi, abrogates DSB formation. DSB induction further requires β1-integrin signaling. DSBs are introduced by the nucleotide excision repair endonucleases XPF and XPG, which, together with RelA, are recruited to chromatin in a highly coordinated, T4SS-dependent manner. Interestingly, XPF/XPG-mediated DNA DSBs promote NF-κB target gene transactivation and host cell survival. In summary, H. pylori induces XPF/XPG-mediated DNA damage through activation of the T4SS/β1-integrin signaling axis, which promotes NF-κB target gene expression and host cell survival.

  6. Mice with DNA repair gene Ercc1 deficiency in a neural crest lineage are a model for late-onset Hirschsprung disease.

    Science.gov (United States)

    Selfridge, Jim; Song, Liang; Brownstein, David G; Melton, David W

    2010-06-04

    The Ercc1 gene is essential for nucleotide excision repair and is also important in recombination repair and the repair of interstrand crosslinks. We have previously used a floxed Ercc1 allele with a keratinocyte-specific Cre recombinase transgene to inactivate Ercc1 in the epidermal layer of the skin and so generate a mouse model for UV-induced non-melanoma skin cancer. Now, in an attempt to generate a model for UV-induced melanoma, we have used the floxed Ercc1 allele in combination with a Cre transgene under the control of the tyrosinase gene promoter to produce mice with Ercc1-deficient melanocytes that are hypersensitive to UV irradiation. These animals developed normally, but died when 4-6 months old with severe colonic obstruction. Melanocytes are derived from the neural crest and the tyrosinase promoter is also expressed in additional neural crest-derived lineages, including the progenitors of the parasympathetic nervous system that innervates the gastrointestinal tract and controls gut peristalsis. A functional enteric nervous system developed in floxed Ercc1 mice with the tyrosinase Cre transgene, but was found to have degenerated in the colons of affected mice. We suggest that accumulating unrepaired endogenous DNA damage in the Ercc1-deficient colonic parasympathetic ganglia leads to the degeneration of this network and results in a colonic obstructive disorder that resembles late-onset Hirschsprung disease in man.

  7. Cancer specificity of promoters of the genes controlling cell proliferation.

    Science.gov (United States)

    Kashkin, Kirill; Chernov, Igor; Stukacheva, Elena; Monastyrskaya, Galina; Uspenskaya, Natalya; Kopantzev, Eugene; Sverdlov, Eugene

    2015-02-01

    Violation of proliferation control is a common feature of cancer cells. We put forward the hypothesis that promoters of genes involved in the control of cell proliferation should possess intrinsic cancer specific activity. We cloned promoter regions of CDC6, POLD1, CKS1B, MCM2, and PLK1 genes into pGL3 reporter vector and studied their ability to drive heterologous gene expression in transfected cancer cells of different origin and in normal human fibroblasts. Each promoter was cloned in short (335-800 bp) and long (up to 2.3 kb) variants to cover probable location of core and whole promoter regulatory elements. Cloned promoters were significantly more active in cancer cells than in normal fibroblasts that may indicate their cancer specificity. Both versions of CDC6 promoters were shown to be most active while the activities of others were close to that of BIRC5 gene (survivin) gene promoter. Long and short variants of each cloned promoter demonstrated very similar cancer specificity with the exception of PLK1-long promoter that was substantially more specific than its short variant and other promoters under study. The data indicate that most of the important cis-regulatory transcription elements responsible for intrinsic cancer specificity are located in short variants of the promoters under study. CDC6 short promoter may serve as a promising candidate for transcription targeted cancer gene therapy.

  8. Nucleus- and cell-specific gene expression in monkey thalamus.

    Science.gov (United States)

    Murray, Karl D; Choudary, Prabhakara V; Jones, Edward G

    2007-02-06

    Nuclei of the mammalian thalamus are aggregations of neurons with unique architectures and input-output connections, yet the molecular determinants of their organizational specificity remain unknown. By comparing expression profiles of thalamus and cerebral cortex in adult rhesus monkeys, we identified transcripts that are unique to dorsal thalamus or to individual nuclei within it. Real-time quantitative PCR and in situ hybridization analyses confirmed the findings. Expression profiling of individual nuclei microdissected from the dorsal thalamus revealed additional subsets of nucleus-specific genes. Functional annotation using Gene Ontology (GO) vocabulary and Ingenuity Pathways Analysis revealed overrepresentation of GO categories related to development, morphogenesis, cell-cell interactions, and extracellular matrix within the thalamus- and nucleus-specific genes, many involved in the Wnt signaling pathway. Examples included the transcription factor TCF7L2, localized exclusively to excitatory neurons; a calmodulin-binding protein PCP4; the bone extracellular matrix molecules SPP1 and SPARC; and other genes involved in axon outgrowth and cell matrix interactions. Other nucleus-specific genes such as CBLN1 are involved in synaptogenesis. The genes identified likely underlie nuclear specification, cell phenotype, and connectivity during development and their maintenance in the adult thalamus.

  9. SNPs in DNA repair or oxidative stress genes and late subcutaneous fibrosis in patients following single shot partial breast irradiation

    OpenAIRE

    2012-01-01

    Abstract Background The aim of this study was to evaluate the potential association between single nucleotide polymorphisms related response to radiotherapy injury, such as genes related to DNA repair or enzymes involved in anti-oxidative activities. The paper aims to identify marker genes able to predict an increased risk of late toxicity studying our group of patients who underwent a Single Shot 3D-CRT PBI (SSPBI) after BCS (breast conserving surgery). Methods A total of 57 breast cancer pa...

  10. DNA Damage/Repair and Polymorphism of the hOGG1 Gene in Lymphocytes of AMD Patients

    Directory of Open Access Journals (Sweden)

    Katarzyna Wozniak

    2009-01-01

    Full Text Available Oxidative stress is thought to play a role in the pathogenesis of age-related macular degeneration (AMD. We determined the extent of oxidative DNA damage and the kinetics of its removal as well as the genotypes of the Ser326Cys polymorphism of the hOGG1 gene in lymphocytes of 30 wet AMD patients and 30 controls. Oxidative DNA damage induced by hydrogen peroxide and its repair were evaluated by the comet assay and DNA repair enzymes. We observed a higher extent of endogenous oxidative DNA damage and a lower efficacy of its repair in AMD patients as compared with the controls. We did not find any correlation between the extent of DNA damage and efficacy of DNA repair with genotypes of the Ser326Cys polymorphism. The results obtained suggest that oxidative DNA damage and inefficient DNA repair can be associated with AMD and the variability of the hOOG1 gene may not contribute to this association.

  11. Integrative characterization of germ cell-specific genes from mouse spermatocyte UniGene library

    Directory of Open Access Journals (Sweden)

    Eddy Edward M

    2007-07-01

    Full Text Available Abstract Background The primary regulator of spermatogenesis, a highly ordered and tightly regulated developmental process, is an intrinsic genetic program involving male germ cell-specific genes. Results We analyzed the mouse spermatocyte UniGene library containing 2155 gene-oriented transcript clusters. We predict that 11% of these genes are testis-specific and systematically identified 24 authentic genes specifically and abundantly expressed in the testis via in silico and in vitro approaches. Northern blot analysis disclosed various transcript characteristics, such as expression level, size and the presence of isoform. Expression analysis revealed developmentally regulated and stage-specific expression patterns in all of the genes. We further analyzed the genes at the protein and cellular levels. Transfection assays performed using GC-2 cells provided information on the cellular characteristics of the gene products. In addition, antibodies were generated against proteins encoded by some of the genes to facilitate their identification and characterization in spermatogenic cells and sperm. Our data suggest that a number of the gene products are implicated in transcriptional regulation, nuclear integrity, sperm structure and motility, and fertilization. In particular, we found for the first time that Mm.333010, predicted to contain a trypsin-like serine protease domain, is a sperm acrosomal protein. Conclusion We identify 24 authentic genes with spermatogenic cell-specific expression, and provide comprehensive information about the genes. Our findings establish a new basis for future investigation into molecular mechanisms underlying male reproduction.

  12. Improved generation of rat gene knockouts by target-selected mutagenesis in mismatch repair-deficient animals

    Directory of Open Access Journals (Sweden)

    van Roekel Henk S

    2008-10-01

    Full Text Available Abstract Background The laboratory rat (Rattus norvegicus is one of the preferred model organisms in physiological and pharmacological research, although the availability of specific genetic models, especially gene knockouts, is limited. N-ethyl-N-nitrosourea (ENU-driven target-selected mutagenesis is currently the most successful method in rats, although it is still very laborious and expensive. Results As ENU-induced DNA damage is normally recognized by the mismatch repair (MMR system, we hypothesized that the effectiveness of the target-selected mutagenesis approach could be improved by using a MMR-deficient genetic background. Indeed, Msh6 knockout rats were found to be more sensitive to ENU treatment and the germ line mutation rate was boosted more than two-fold to 1 mutation per 585 kb. In addition, the molecular mutation spectrum was found to be changed in favor of generating knockout-type alleles by ~20%, resulting in an overall increase in efficiency of ~2.5 fold. The improved effectiveness was demonstrated by high throughput mutation discovery in 70 Mb of sequence in a set of only 310 mutant F1 rats. This resulted in the identification of 89 mutations of which four introduced a premature stopcodon and 64 resulted in amino acid changes. Conclusion Taken together, we show that the use of a MMR-deficient background considerably improves ENU-driven target-selected mutagenesis in the rat, thereby reducing animal use as well as screening costs. The use of a mismatch repair-deficient genetic background for improving mutagenesis and target-selected knockout efficiency is in principle applicable to any organism of interest.

  13. Improved generation of rat gene knockouts by target-selected mutagenesis in mismatch repair-deficient animals

    Science.gov (United States)

    van Boxtel, Ruben; Toonen, Pim W; Verheul, Mark; van Roekel, Henk S; Nijman, Isaac J; Guryev, Victor; Cuppen, Edwin

    2008-01-01

    Background The laboratory rat (Rattus norvegicus) is one of the preferred model organisms in physiological and pharmacological research, although the availability of specific genetic models, especially gene knockouts, is limited. N-ethyl-N-nitrosourea (ENU)-driven target-selected mutagenesis is currently the most successful method in rats, although it is still very laborious and expensive. Results As ENU-induced DNA damage is normally recognized by the mismatch repair (MMR) system, we hypothesized that the effectiveness of the target-selected mutagenesis approach could be improved by using a MMR-deficient genetic background. Indeed, Msh6 knockout rats were found to be more sensitive to ENU treatment and the germ line mutation rate was boosted more than two-fold to 1 mutation per 585 kb. In addition, the molecular mutation spectrum was found to be changed in favor of generating knockout-type alleles by ~20%, resulting in an overall increase in efficiency of ~2.5 fold. The improved effectiveness was demonstrated by high throughput mutation discovery in 70 Mb of sequence in a set of only 310 mutant F1 rats. This resulted in the identification of 89 mutations of which four introduced a premature stopcodon and 64 resulted in amino acid changes. Conclusion Taken together, we show that the use of a MMR-deficient background considerably improves ENU-driven target-selected mutagenesis in the rat, thereby reducing animal use as well as screening costs. The use of a mismatch repair-deficient genetic background for improving mutagenesis and target-selected knockout efficiency is in principle applicable to any organism of interest. PMID:18840264

  14. DNA repair and gene targeting in plant end-joining mutants

    NARCIS (Netherlands)

    Jia, Qi

    2011-01-01

    DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR) or by non-homologous end joining (NHEJ). The latter mechanism is the major route for DSB repair in the somatic cells of higher eukaryotes, including plants. If we could manipulate the balance of the DSB repair pathways

  15. DNA repair and gene targeting in plant end-joining mutants

    NARCIS (Netherlands)

    Jia, Qi

    2011-01-01

    DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR) or by non-homologous end joining (NHEJ). The latter mechanism is the major route for DSB repair in the somatic cells of higher eukaryotes, including plants. If we could manipulate the balance of the DSB repair pathways

  16. Mismatch repair genes of Streptococcus pneumoniae: HexA confers a mutator phenotype in Escherichia coli by negative complementation.

    Science.gov (United States)

    Prudhomme, M; Méjean, V; Martin, B; Claverys, J P

    1991-11-01

    DNA repair systems able to correct base pair mismatches within newly replicated DNA or within heteroduplex molecules produced during recombination are widespread among living organisms. Evidence that such generalized mismatch repair systems evolved from a common ancestor is particularly strong for two of them, the Hex system of the gram-positive Streptococcus pneumoniae and the Mut system of the gram-negative Escherichia coli and Salmonella typhimurium. The homology existing between HexA and MutS and between HexB and MutL prompted us to investigate the effect of expressing hex genes in E. coli. Complementation of mutS or mutL mutations, which confer a mutator phenotype, was assayed by introducing on a multicopy plasmid the hexA and hexB genes, under the control of an inducible promoter, either individually or together in E. coli strains. No decrease in mutation rate was conferred by either hexA or hexB gene expression. However, a negative complementation effect was observed in wild-type E. coli cells: expression of hexA resulted in a typical Mut- mutator phenotype. hexB gene expression did not increase the mutation rate either individually or in conjunction with hexA. Since expression of hexA did not affect the mutation rate in mutS mutant cells and the hexA-induced mutator effect was recA independent, it is concluded that this effect results from inhibition of the Mut system. We suggest that HexA, like its homolog MutS, binds to mismatches resulting from replication errors, but in doing so it protects them from repair by the Mut system. In agreement with this hypothesis, an increase in mutS gene copy number abolished the hexA-induced mutator phenotype. HexA protein could prevent repair either by being unable to interact with Mut proteins or by producing nonfunctional repair complexes.

  17. Site-specific regulation of cell cycle and DNA repair in post-mitotic GABA cells in schizophrenic versus bipolars.

    Science.gov (United States)

    Benes, Francine M; Lim, Benjamin; Subburaju, Sivan

    2009-07-14

    GABA cell dysfunction in both schizophrenia (SZ) and bipolar disorder (BD) involves decreased GAD(67) expression, although this change involves fundamentally different networks of genes in the 2 disorders. One gene that is common to these 2 networks is cyclin D2, a key component of cell cycle regulation that shows increased expression in SZ, but decreased expression in BD. Because of the importance of cell cycle regulation in maintaining functional differentiation and DNA repair, the current study has examined the genes involved in the G(1) and G(2) checkpoints to generate new hypotheses regarding the regulation of the GABA cell phenotype in the hippocampus of SZ and BD. The results have demonstrated significant changes in cell cycle regulation in both SZ and BD and these changes include the transcriptional complex (TC) that controls the expression of E2F/DP-1 target genes critical for progression to G(2)/M. The methyl-CpG binding domain protein (MBD4) that is pivotal for DNA repair, is significantly up-regulated in the stratum oriens (SO) of CA3/2 and CA1 in SZs and BDs. However, other genes associated with the TC, and the G(1) and G(2) checkpoints, show complex changes in expression in the SO of CA3/2 and CA1 of both SZs and BDS. Overall, the patterns of expression observed have suggested that the regulation of functional differentiation and/or genomic integrity of hippocampal GABA cells varies according to diagnosis and their location within the trisynaptic pathway.

  18. Molecular basis for gene-specific transactivation by nuclear receptors

    DEFF Research Database (Denmark)

    Jørgensen, Mads Aagaard; Siersbæk, Rasmus; Mandrup, Susanne

    2010-01-01

    most likely be accounted for by mechanisms involving receptor-specific interactions with DNA as well as receptor-specific interactions with protein complexes binding to adjacent and distant DNA sequences. Here, we review key molecular aspects of transactivation by NRs with special emphasis......Nuclear receptors (NRs) are key transcriptional regulators of metazoan physiology and metabolism. Different NRs bind to similar or even identical core response elements; however, they regulate transcription in a highly receptor- and gene-specific manner. These differences in gene activation can...... on the recent advances in the molecular mechanisms responsible for receptor- and gene-specific transcriptional activation. This article is part of a Special Issue entitled: Translating nuclear receptors from health to disease....

  19. Lineage-specific expansion of IFIT gene family: an insight into coevolution with IFN gene family.

    Directory of Open Access Journals (Sweden)

    Ying Liu

    Full Text Available In mammals, IFIT (Interferon [IFN]-induced proteins with Tetratricopeptide Repeat [TPR] motifs family genes are involved in many cellular and viral processes, which are tightly related to mammalian IFN response. However, little is known about non-mammalian IFIT genes. In the present study, IFIT genes are identified in the genome databases from the jawed vertebrates including the cartilaginous elephant shark but not from non-vertebrates such as lancelet, sea squirt and acorn worm, suggesting that IFIT gene family originates from a vertebrate ancestor about 450 million years ago. IFIT family genes show conserved gene structure and gene arrangements. Phylogenetic analyses reveal that this gene family has expanded through lineage-specific and species-specific gene duplication. Interestingly, IFN gene family seem to share a common ancestor and a similar evolutionary mechanism; the function link of IFIT genes to IFN response is present early since the origin of both gene families, as evidenced by the finding that zebrafish IFIT genes are upregulated by fish IFNs, poly(I:C and two transcription factors IRF3/IRF7, likely via the IFN-stimulated response elements (ISRE within the promoters of vertebrate IFIT family genes. These coevolution features creates functional association of both family genes to fulfill a common biological process, which is likely selected by viral infection during evolution of vertebrates. Our results are helpful for understanding of evolution of vertebrate IFN system.

  20. Basic fibroblast growth factor gene transfection in repair of internal carotid artery aneurysm wall

    Institute of Scientific and Technical Information of China (English)

    Lei Jiao; Ming Jiang; Jinghai Fang; Yinsheng Deng; Zejun Chen; Min Wu

    2012-01-01

    Surgery or interventional therapy has some risks in the treatment of cerebral aneurysm. We established an internal carotid artery aneurysm model by dripping elastase in the crotch of the right internal and external carotid arteries of New Zealand rabbits. Following model induction, lentivirus carrying basic fibroblast growth factor was injected through the ear vein. We found that the longer the action time of the lentivirus, the smaller the aneurysm volume. Moreover, platelet-derived growth factor expression in the aneurysm increased, but smooth muscle 22 alpha and hypertension-related gene 1 mRNA expression decreased. At 1, 2, 3, and 4 weeks following model establishment, following 1 week of injection of lentivirus carrying basic fibroblast growth factor, the later the intervention time, the more severe the blood vessel damage, and the bigger the aneurysm volume, the lower the smooth muscle 22 alpha and hypertension-related gene 1 mRNA expression. Simultaneously, platelet-derived growth factor expression decreased. These data suggest that recombinant lentivirus carrying basic fibroblast growth factor can repair damaged cells in the aneurysmal wall and inhibit aneurysm dynamic growth, and that the effect is dependent on therapeutic duration.

  1. Cell and gene therapy for arrhythmias: Repair of cardiac conduction damage

    Institute of Scientific and Technical Information of China (English)

    Yong-Fu Xiao

    2011-01-01

    Action potentials generated in the sinoatrial node(SAN)dominate the rhythm and rate of a healthy human heart.Subsequently,these action potentials propagate to the whole heart via its conduction system .Abnormalities of impulse generation and/or propagation in a heart can cause arrhythmias.For example,SAN dysfunction or conduction block of the atrioventricular node can lead to serious bradycardia which is currently treated with an implanted electronic pacemaker.On the other hand conduction damage may cause reentrant tachyarrhythmias which are primarily treated pharmacologically or by medical device-based therapies,including defibrillation and tissue ablation.However,drug therapies sometimes may not be effective or are associated with serious side effects.Device-based therapies for cardiac arrhythmias,even with well developed technology,still face inadequacies,limitations,hardware complications,and other challenges.Therefore,scientists are actively seeking other alternatives for antiarrhythmic therapy.In particular,cells and genes used for repairing cardiac conduction damage/defect have been investigated in various studies both in vitro and in vivo.Despite the complexities of the excitation and conduction systems of the heart,cell and gene-based strategies provide novel alternatives for treatment or cure of cardiac anhythmias.This review summarizes some highlights of recent research progress in this field.

  2. Characterisation of the promoter region of the human DNA-repair gene Rad51.

    Science.gov (United States)

    Hasselbach, L; Haase, S; Fischer, D; Kolberg, H C; Stürzbecher, H W

    2005-01-01

    Regulatory elements of the 5'-flanking region of the DNA-repair gene Rad51 were analysed to characterise pathological alterations of Rad51 mRNA expression during tumour development. Various fragments of the Rad51 promoter were cloned into the pGL3 reporter vector and the respective promoter activity was determined by luciferase assays in transfected U2-OS cells. Transcription factor binding was identified using Protein/DNA arrays. The region encompassing base pairs -204 to -58 was identified as crucial for Rad51 gene transcription. Down regulator sequences are present upstream (-305 to -204) and downstream (-48 and +204) of this core promoter element. Promoter activity is significantly enhanced by substituting G at the polymorphic positions +135 and +172 for C and T, respectively. Transcription factors Ets1/PEA3, E2F1, p53, EGR1, and Stat5 were identified as relevant for regulating expression of Rad51. We identified three separate cis-sequence elements within the Rad51 transcriptional promoter, one ensuring basal levels of expression and two elements limiting expression to relatively low levels. The characterisation of transcription factor binding might help to explain high-level expression of Rad51 in a variety of solid tumours. The polymorphic sites appear important for the increased risk of breast and/or ovarian cancer for BRCA2 mutation carriers.

  3. The effect of a DNA repair gene on cellular invasiveness: XRCC3 over-expression in breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Veronica L Martinez-Marignac

    Full Text Available Over-expression of DNA repair genes has been associated with resistance to radiation and DNA-damage induced by chemotherapeutic agents such as cisplatin. More recently, based on the analysis of genome expression profiling, it was proposed that over-expression of DNA repair genes enhances the invasive behaviour of tumour cells. In this study we present experimental evidence utilizing functional assays to test this hypothesis. We assessed the effect of the DNA repair proteins known as X-ray complementing protein 3 (XRCC3 and RAD51, to the invasive behavior of the MCF-7 luminal epithelial-like and BT20 basal-like triple negative human breast cancer cell lines. We report that stable or transient over-expression of XRCC3 but not RAD51 increased invasiveness in both cell lines in vitro. Moreover, XRCC3 over-expressing MCF-7 cells also showed a higher tumorigenesis in vivo and this phenotype was associated with increased activity of the metalloproteinase MMP-9 and the expression of known modulators of cell-cell adhesion and metastasis such as CD44, ID-1, DDR1 and TFF1. Our results suggest that in addition to its' role in facilitating repair of DNA damage, XRCC3 affects invasiveness of breast cancer cell lines and the expression of genes associated with cell adhesion and invasion.

  4. Risk of colorectal cancer for people with a mutation in both a MUTYH and a DNA mismatch repair gene

    Science.gov (United States)

    Win, Aung Ko; Reece, Jeanette C.; Buchanan, Daniel D.; Clendenning, Mark; Young, Joanne P.; Cleary, Sean P.; Kim, Hyeja; Cotterchio, Michelle; Dowty, James G.; MacInnis, Robert J.; Tucker, Katherine M.; Winship, Ingrid M.; Macrae, Finlay A.; Burnett, Terrilea; Le Marchand, Loïc; Casey, Graham; Haile, Robert W.; Newcomb, Polly A.; Thibodeau, Stephen N.; Lindor, Noralane M.; Hopper, John L.; Gallinger, Steven; Jenkins, Mark A.

    2015-01-01

    The base excision repair protein, MUTYH, functionally interacts with the DNA mismatch repair (MMR) system. As genetic testing moves from testing one gene at a time, to gene panel and whole exome next generation sequencing approaches, understanding the risk associated with co-existence of germline mutations in these genes will be important for clinical interpretation and management. From the Colon Cancer Family Registry, we identified 10 carriers who had both a MUTYH mutation (6 with c.1187G>A p.(Gly396Asp), 3 with c.821G>A p.(Arg274Gln), and 1 with c.536A>G p.(Tyr179Cys)) and a MMR gene mutation (3 in MLH1, 6 in MSH2, and 1 in PMS2), 375 carriers of a single (monoallelic) MUTYH mutation alone, and 469 carriers of a MMR gene mutation alone. Of the 10 carriers of both gene mutations, 8 were diagnosed with colorectal cancer. Using a weighted cohort analysis, we estimated that risk of colorectal cancer for carriers of both a MUTYH and a MMR gene mutation was substantially higher than that for carriers of a MUTYH mutation alone [hazard ratio (HR) 21.5, 95 % confidence interval (CI) 9.19–50.1; p colorectal cancer for carriers of a MMR gene mutation alone. Our finding suggests MUTYH mutation testing in MMR gene mutation carriers is not clinically informative. PMID:26202870

  5. Gene and pathway level analyses of germline DNA-repair gene variants and prostate cancer susceptibility using the iCOGS-genotyping array

    DEFF Research Database (Denmark)

    Saunders, Edward J; Dadaev, Tokhir; Leongamornlert, Daniel A

    2016-01-01

    BACKGROUND: Germline mutations within DNA-repair genes are implicated in susceptibility to multiple forms of cancer. For prostate cancer (PrCa), rare mutations in BRCA2 and BRCA1 give rise to moderately elevated risk, whereas two of B100 common, low-penetrance PrCa susceptibility variants identif...

  6. Gene-Specific Assessment of Guanine Oxidation as an Epigenetic Modulator for Cardiac Specification of Mouse Embryonic Stem Cells.

    Directory of Open Access Journals (Sweden)

    Joonghoon Park

    Full Text Available Epigenetics have essential roles in development and human diseases. Compared to the complex histone modifications, epigenetic changes on mammalian DNA are as simple as methylation on cytosine. Guanine, however, can be oxidized as an epigenetic change which can undergo base-pair transversion, causing a genetic difference. Accumulating evidence indicates that reactive oxygen species (ROS are important signaling molecules for embryonic stem cell (ESC differentiation, possibly through transient changes on genomic DNA such as 7,8-dihydro-8-oxoguanine (8-oxoG. Technical limitations on detecting such DNA modifications, however, restrict the investigation of the role of 8-oxoG in ESC differentiation. Here, we developed a Hoogsteen base pairing-mediated PCR-sequencing assay to detect 8-oxoG lesions that can subsequently cause G to T transversions during PCR. We then used this assay to assess the epigenetic and transient 8-oxoG formation in the Tbx5 gene of R1 mouse ESCs subjected to oxidative stress by removing 2-mercaptoethanol (2ME from the culture media. To our surprise, significantly higher numbers of 8-oxoG-mediated G∙C to C∙G transversion, not G∙C to T∙A, were detected at 7th and 9th base position from the transcription start site of exon 1 of Tbx5 in ESCs in the (-2ME than (+2ME group (p < 0.05. This was consistent with the decrease in the amount of amplifiable of DNA harboring the 8-oxoG lesions at the Tbx5 promoter region in the oxidative stressed ESCs. The ESCs responded to oxidative stress, possibly through the epigenetic effects of guanine oxidation with decreased proliferation (p < 0.05 and increased formation of beating embryoid bodies (EBs; p < 0.001. Additionally, the epigenetic changes of guanine induced up-regulation of Ogg1 and PolB, two base excision repairing genes for 8-oxoG, in ESCs treated with (-2ME (p < 0.01. Together, we developed a gene-specific and direct quantification assay for guanine oxidation. Using oxidative

  7. Lab-specific gene expression signatures in pluripotent stem cells.

    Science.gov (United States)

    Newman, Aaron M; Cooper, James B

    2010-08-06

    Pluripotent stem cells derived from both embryonic and reprogrammed somatic cells have significant potential for human regenerative medicine. Despite similarities in developmental potential, however, several groups have found fundamental differences between embryonic stem cell (ESC) and induced-pluripotent stem cell (iPSC) lines that may have important implications for iPSC-based medical therapies. Using an unsupervised clustering algorithm, we further studied the genetic homogeneity of iPSC and ESC lines by reanalyzing microarray gene expression data from seven different laboratories. Unexpectedly, this analysis revealed a strong correlation between gene expression signatures and specific laboratories in both ESC and iPSC lines. Nearly one-third of the genes with lab-specific expression signatures are also differentially expressed between ESCs and iPSCs. These data are consistent with the hypothesis that in vitro microenvironmental context differentially impacts the gene expression signatures of both iPSCs and ESCs.

  8. Downregulation of homologous recombination DNA repair genes by HDAC inhibition in prostate cancer is mediated through the E2F1 transcription factor.

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    Sushant K Kachhap

    Full Text Available BACKGROUND: Histone deacetylase inhibitors (HDACis re-express silenced tumor suppressor genes and are currently undergoing clinical trials. Although HDACis have been known to induce gene expression, an equal number of genes are downregulated upon HDAC inhibition. The mechanism behind this downregulation remains unclear. Here we provide evidence that several DNA repair genes are downregulated by HDAC inhibition and provide a mechanism involving the E2F1 transcription factor in the process. METHODOLOGY/PRINCIPAL FINDINGS: Applying Analysis of Functional Annotation (AFA on microarray data of prostate cancer cells treated with HDACis, we found a number of genes of the DNA damage response and repair pathways are downregulated by HDACis. AFA revealed enrichment of homologous recombination (HR DNA repair genes of the BRCA1 pathway, as well as genes regulated by the E2F1 transcription factor. Prostate cancer cells demonstrated a decreased DNA repair capacity and an increased sensitization to chemical- and radio-DNA damaging agents upon HDAC inhibition. Recruitment of key HR repair proteins to the site of DNA damage, as well as HR repair capacity was compromised upon HDACi treatment. Based on our AFA data, we hypothesized that the E2F transcription factors may play a role in the downregulation of key repair genes upon HDAC inhibition in prostate cancer cells. ChIP analysis and luciferase assays reveal that the downregulation of key repair genes is mediated through decreased recruitment of the E2F1 transcription factor and not through active repression by repressive E2Fs. CONCLUSIONS/SIGNIFICANCE: Our study indicates that several genes in the DNA repair pathway are affected upon HDAC inhibition. Downregulation of the repair genes is on account of a decrease in amount and promoter recruitment of the E2F1 transcription factor. Since HDAC inhibition affects several pathways that could potentially have an impact on DNA repair, compromised DNA repair upon HDAC

  9. Freedom of expression: cell-type-specific gene profiling.

    Science.gov (United States)

    Otsuki, Leo; Cheetham, Seth W; Brand, Andrea H

    2014-01-01

    Cell fate and behavior are results of differential gene regulation, making techniques to profile gene expression in specific cell types highly desirable. Many methods now enable investigation at the DNA, RNA and protein level. This review introduces the most recent and popular techniques, and discusses key issues influencing the choice between these such as ease, cost and applicability of information gained. Interdisciplinary collaborations will no doubt contribute further advances, including not just in single cell type but single-cell expression profiling.

  10. Injury, inflammation and the emergence of human specific genes

    Science.gov (United States)

    2016-07-12

    indistinguishable.6 Interestingly, just as we noted the expression of human -specific genes in human immune cells (Table 1), Long and colleagues noted the wide...nervous system, it presumably alters a7AChR activities on human cognition and memory . In other examples, the human antimicrobial defensins are highly...genes in circulating and resident human immune cells can be studied in mice after the transplantation and engraft- ment of human hemato-lymphoid immune

  11. Cell-specific DNA methylation patterns of retina-specific genes.

    Directory of Open Access Journals (Sweden)

    Shannath L Merbs

    Full Text Available Many studies have demonstrated that epigenetic mechanisms are important in the regulation of gene expression during embryogenesis, gametogenesis, and other forms of tissue-specific gene regulation. We sought to explore the possible role of epigenetics, specifically DNA methylation, in the establishment and maintenance of cell type-restricted gene expression in the retina. To assess the relationship between DNA methylation status and expression level of retinal genes, bisulfite sequence analysis of the 1000 bp region around the transcription start sites (TSS of representative rod and cone photoreceptor-specific genes and gene expression analysis were performed in the WERI and Y79 human retinoblastoma cell lines. Next, the homologous genes in mouse were bisulfite sequenced in the retina and in non-expressing tissues. Finally, bisulfite sequencing was performed on isolated photoreceptor and non-photoreceptor retinal cells isolated by laser capture microdissection. Differential methylation of rhodopsin (RHO, retinal binding protein 3 (RBP3, IRBP cone opsin, short-wave-sensitive (OPN1SW, cone opsin, middle-wave-sensitive (OPN1MW, and cone opsin, long-wave-sensitive (OPN1LW was found in the retinoblastoma cell lines that inversely correlated with gene expression levels. Similarly, we found tissue-specific hypomethylation of the promoter region of Rho and Rbp3 in mouse retina as compared to non-expressing tissues, and also observed hypomethylation of retinal-expressed microRNAs. The Rho and Rbp3 promoter regions were unmethylated in expressing photoreceptor cells and methylated in non-expressing, non-photoreceptor cells from the inner nuclear layer. A third regional hypomethylation pattern of photoreceptor-specific genes was seen in a subpopulation of non-expressing photoreceptors (Rho in cones from the Nrl -/- mouse and Opn1sw in rods. These results demonstrate that a number of photoreceptor-specific genes have cell-specific differential DNA

  12. Heritable and lineage-specific gene knockdown in zebrafish embryo.

    Directory of Open Access Journals (Sweden)

    Mei Dong

    Full Text Available BACKGROUND: Reduced expression of developmentally important genes and tumor suppressors due to haploinsufficiency or epigenetic suppression has been shown to contribute to the pathogenesis of various malignancies. However, methodology that allows spatio-temporally knockdown of gene expression in various model organisms such as zebrafish has not been well established, which largely limits the potential of zebrafish as a vertebrate model of human malignant disorders. PRINCIPAL FINDING: Here, we report that multiple copies of small hairpin RNA (shRNA are expressed from a single transcript that mimics the natural microRNA-30e precursor (mir-shRNA. The mir-shRNA, when microinjected into zebrafish embryos, induced an efficient knockdown of two developmentally essential genes chordin and alpha-catenin in a dose-controllable fashion. Furthermore, we designed a novel cassette vector to simultaneously express an intronic mir-shRNA and a chimeric red fluorescent protein driven by lineage-specific promoter, which efficiently reduced the expression of a chromosomally integrated reporter gene and an endogenously expressed gata-1 gene in the developing erythroid progenitors and hemangioblasts, respectively. SIGNIFICANCE: This methodology provides an invaluable tool to knockdown developmental important genes in a tissue-specific manner or to establish animal models, in which the gene dosage is critically important in the pathogenesis of human disorders. The strategy should be also applicable to other model organisms.

  13. Global Identification of Genes Specific for Rice Meiosis.

    Science.gov (United States)

    Zhang, Bingwei; Xu, Meng; Bian, Shiquan; Hou, Lili; Tang, Ding; Li, Yafei; Gu, Minghong; Cheng, Zhukuan; Yu, Hengxiu

    2015-01-01

    The leptotene-zygotene transition is a major step in meiotic progression during which pairing between homologous chromosomes is initiated and double strand breaks occur. OsAM1, a homologue of maize AM1 and Arabidopsis SWI1, encodes a protein with a coiled-coil domain in its central region that is required for the leptotene-zygotene transition during rice meiosis. To gain more insight into the role of OsAM1 in rice meiosis and identify additional meiosis-specific genes, we characterized the transcriptomes of young panicles of Osam1 mutant and wild-type rice plants using RNA-Seq combined with bioinformatic and statistical analyses. As a result, a total of 25,750 and 28,455 genes were expressed in young panicles of wild-type and Osam1 mutant plants, respectively, and 4,400 differentially expressed genes (DEGs; log2 Ratio ≥ 1, FDR ≤ 0.05) were identified. Of these DEGs, four known rice meiosis-specific genes were detected, and 22 new putative meiosis-related genes were found by mapping these DEGs to reference biological pathways in the KEGG database. We identified eight additional well-conserved OsAM1-responsive rice meiotic genes by comparing our RNA-Seq data with known meiotic genes in Arabidopsis and fission yeast.

  14. Gene polymorphisms of the DNA Repairing Genes APE1 and XRCC1 among Smoking Lung Cancer Egyptians

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    Rezk Ahmed Abd-ellateef Elbaz, Salim Abd-elhady Habib, Maha Ebraheem Esmael Ebraheem, Gamal Kamel EL-Ebidy, Lamiaa Mohamed Mahmoud Ramadan and Ahmed Settin

    2012-04-01

    Full Text Available Lung cancer is the leading cause of cancer-related mortality worldwide and is thus a major public health problem. DNA base damage or losses caused by endogenous and exogenous agents occur constantly at a high frequency in human cells. The removal or repair of damaged bases is an important mechanism in protecting the integrity of the genome. APE1 (Apurinic/Apyrimidinic Endonuclease 1 and XRCC1 (X-ray cross-complementing group1 are DNA repair proteins that play important roles in the base excision repair (BER pathway. The focus of this work is limited to the association between polymorphisms in the DNA repair genes, (APE1 Asp148Glu (2197 T→G and XRCC1 Arg399Gln (28152 G→A genotypes, cigarette smoking and lung cancer. This study has included 131 cases affected with lung cancers include; 33cases with small cell carcinoma (25.2% and 98 cases with non-small cell carcinoma (74.8%. They were recruited from oncology Center, Mansoura University, Egypt; in the period between April 2008 to March 2010. For comparison, a negative control group including 150 healthy individuals randomly selected from blood donors. Controls were selected by random sampling cancer-free individuals without a past history of cancer, who visited Mansoura University hospitals and provided peripheral blood between April 2008 and March 2010. DNA was extracted from the whole peripheral blood using generation DNA purification capture column kit (Gentra system, USA and genotyping for APE1 Glu148Asp and XRCC1 Arg399Gln polymorphisms was performed by a PCR--CTPP (PCR with confronting two-pair primers method. The collected data were organized and statistically analyzed using SPSS statistical computer package version 10 software. we observed that, There were no significant differences in the frequencies of the APE1 Asp148Glu (2197 T→G polymorphism of all genotypes and alleles in all lung cancer cases compared to all healthy controls. Also, there were no significant differences in the

  15. Association of common variants in mismatch repair genes and breast cancer susceptibility: a multigene study

    Directory of Open Access Journals (Sweden)

    Pina Julieta

    2009-09-01

    Full Text Available Abstract Background MMR is responsible for the repair of base-base mismatches and insertion/deletion loops. Besides this, MMR is also associated with an anti-recombination function, suppressing homologous recombination. Losses of heterozygosity and/or microsatellite instability have been detected in a large number of skin samples from breast cancer patients, suggesting a potential role of MMR in breast cancer susceptibility. Methods We carried out a hospital-based case-control study in a Caucasian Portuguese population (287 cases and 547 controls to estimate the susceptibility to non-familial breast cancer associated with some polymorphisms in mismatch repair genes (MSH3, MSH4, MSH6, MLH1, MLH3, PMS1 and MUTYH. Results Using unconditional logistic regression we found that MLH3 (L844P, G>A polymorphism GA (Leu/Pro and AA (Pro/Pro genotypes were associated with a decreased risk: OR = 0.65 (0.45-0.95 (p = 0.03 and OR = 0.62 (0.41-0.94 (p = 0.03, respectively. Analysis of two-way SNP interaction effects on breast cancer revealed two potential associations to breast cancer susceptibility: MSH3 Ala1045Thr/MSH6 Gly39Glu - AA/TC [OR = 0.43 (0.21-0.83, p = 0.01] associated with a decreased risk; and MSH4 Ala97Thr/MLH3 Leu844Pro - AG/AA [OR = 2.35 (1.23-4.49, p = 0.01], GG/AA [OR = 2.11 (1.12-3,98, p = 0.02], and GG/AG [adjusted OR = 1.88 (1.12-3.15, p = 0.02] all associated with an increased risk for breast cancer. Conclusion It is possible that some of these common variants in MMR genes contribute significantly to breast cancer susceptibility. However, further studies with a large sample size will be needed to support our results.

  16. Cloning of the hexA mismatch-repair gene of Streptococcus pneumoniae and identification of the product.

    Science.gov (United States)

    Martin, B; Prats, H; Claverys, J P

    1985-01-01

    The hexA mismatch repair gene of Streptococcus pneumoniae has been cloned into multicopy plasmid vectors. The cloned hexA gene is expressed as judged from its ability to complement various chromosomal hexA- alleles. Its direction of transcription was defined and the functional limits were localized by original methods relying on homology-dependent integration of nonautonomous chimeric plasmids carrying chromosomal inserts into the chromosome. Comparison of the proteins encoded by recombinant plasmids and by restriction fragments allowed us to identify an Mr 94 000 protein as the probable product of the hexA gene.

  17. Polymorphisms in XPC, XPD, XRCC1, and XRCC3 DNA repair genes and lung cancer risk in a population of Northern Spain

    Directory of Open Access Journals (Sweden)

    Tardón Adonina

    2007-08-01

    Full Text Available Abstract Background Polymorphisms in DNA repair genes have been associated to repair DNA lesions, and might contribute to the individual susceptibility to develop different types of cancer. Nucleotide excision repair (NER, base excision repair (BER, and double-strand break repair (DSBR are the main DNA repair pathways. We investigated the relationship between polymorphisms in two NER genes, XPC (poly (AT insertion/deletion: PAT-/+ and XPD (Asp312Asn and Lys751Gln, the BER gene XRCC1 (Arg399Gln, and the DSBR gene XRCC3 (Thr241Met and the risk of developing lung cancer. Methods A hospital-based case-control study was designed with 516 lung cancer patients and 533 control subjects, matched on ethnicity, age, and gender. Genotypes were determined by PCR-RFLP and the results were analysed using multivariate unconditional logistic regression, adjusting for age, gender and pack-years. Results Borderline association was found for XPC and XPD NER genes polymorphisms, while no association was observed for polymorphisms in BER and DSBR genes. XPC PAT+/+ genotype was associated with no statistically significant increased risk among ever smokers (OR = 1.40; 95%CI = 0.94–2.08, squamous cell carcinoma (OR = 1.44; 95%CI = 0.85–2.44, and adenocarcinoma (OR = 1.72; 95%CI = 0.97–3.04. XPD variant genotypes (312Asn/Asn and 751Gln/Gln presented a not statistically significant risk of developing lung cancer (OR = 1.52; 95%CI = 0.91–2.51; OR = 1.38; 95%CI = 0.85–2.25, respectively, especially among ever smokers (OR = 1.58; 95%CI = 0.96–2.60, heavy smokers (OR = 2.07; 95%CI = 0.74–5.75, and adenocarcinoma (OR = 1.88; 95%CI = 0.97–3.63. On the other hand, individuals homozygous for the XRCC1 399Gln allele presented no risk of developing lung cancer (OR = 0.87; 95%CI = 0.57–1.31 except for individuals carriers of 399Gln/Gln genotype and without family history of cancer (OR = 0.57; 95%CI = 0.33–0.98 and no association was found between XRCC3 Thr241Met

  18. The human Bloom syndrome gene suppresses the DNA replication and repair defects of yeast dna2 mutants.

    Science.gov (United States)

    Imamura, Osamu; Campbell, Judith L

    2003-07-08

    Bloom syndrome is a disorder of profound and early cancer predisposition in which cells become hypermutable, exhibit high frequency of sister chromatid exchanges, and show increased micronuclei. BLM, the gene mutated in Bloom syndrome, has been cloned previously, and the BLM protein is a member of the RecQ family of DNA helicases. Many lines of evidence suggest that BLM is involved either directly in DNA replication or in surveillance during DNA replication, but its specific roles remain unknown. Here we show that hBLM can suppress both the temperature-sensitive growth defect and the DNA damage sensitivity of the yeast DNA replication mutant dna2-1. The dna2-1 mutant is defective in a helicase-nuclease that is required either to coordinate with the crucial Saccharomyces cerevisiae (sc) FEN1 nuclease in Okazaki fragment maturation or to compensate for scFEN1 when its activity is impaired. We show that human BLM interacts with both scDna2 and scFEN1 by using coimmunoprecipitation from yeast extracts, suggesting that human BLM participates in the same steps of DNA replication or repair as scFEN1 and scDna2.

  19. Stimulation of DNA repair and increased light output in response to UV irradiation in Escherichia coli expressing lux genes.

    Science.gov (United States)

    Cutter, Kerry L; Alloush, Habib M; Salisbury, Vyv C

    2007-01-01

    It has previously been suggested that the evolutionary drive of bacterial bioluminescence is a mechanism of DNA repair. By assessing the UV sensitivity of Escherichia coli, it is shown that the survival of UV-irradiated E. coli constitutively expressing luxABCDE in the dark is significantly better than either a strain with no lux gene expression or the same strain expressing only luciferase (luxAB) genes. This shows that UV resistance is dependent on light output, and not merely on luciferase production. Also, bacterial survival was found to be dependent on the conditions following UV irradiation, as bioluminescence-mediated repair was not as efficient as repair in visible light. Moreover, photon emission revealed a dose-dependent increase in light output per cell after UV exposure, suggesting that increased lux gene expression correlates with UV-induced DNA damage. This phenomenon has been previously documented in organisms where the lux genes are under their natural luxR regulation but has not previously been demonstrated under the regulation of a constitutive promoter.

  20. c-Myc directly regulates the transcription of the NBS1 gene involved in DNA double-strand break repair.

    Science.gov (United States)

    Chiang, Yu-Chi; Teng, Shu-Chun; Su, Yi-Ning; Hsieh, Fon-Jou; Wu, Kou-Juey

    2003-05-23

    The c-myc proto-oncogene encodes a ubiquitous transcription factor involved in the control of cell growth and implicated in inducing tumorigenesis. Understanding the function of c-Myc and its role in cancer depends upon the identification of c-Myc target genes. Nijmegen breakage syndrome (NBS) is a chromosomal-instability syndrome associated with cancer predisposition, radiosensitivity, and chromosomal instability. The NBS gene product, NBS1 (p95 or nibrin), is a part of the hMre11 complex, a central player associated with double-strand break (DSB) repair. NBS1 contains domains characteristic for proteins involved in DNA repair, recombination, and replication. Here we show that c-Myc directly activates NBS1. c-Myc-mediated induction of NBS1 gene transcription occurs in different tissues, is independent of cell proliferation, and is mediated by a c-Myc binding site in the intron 1 region of NBS1 gene. Overexpression of NBS1 in Rat1a cells increased cell proliferation. These results indicate that NBS1 is a direct transcriptional target of c-Myc and links the function of c-Myc to the regulation of DNA DSB repair pathway operating during DNA replication.

  1. Gypenosides causes DNA damage and inhibits expression of DNA repair genes of human oral cancer SAS cells.

    Science.gov (United States)

    Lu, Kung-Wen; Chen, Jung-Chou; Lai, Tung-Yuan; Yang, Jai-Sing; Weng, Shu-Wen; Ma, Yi-Shih; Tang, Nou-Ying; Lu, Pei-Jung; Weng, Jing-Ru; Chung, Jing-Gung

    2010-01-01

    Gypenosides (Gyp) are the major components of Gynostemma pentaphyllum Makino, a Chinese medical plant. Recently, Gyp has been shown to induce cell cycle arrest and apoptosis in many human cancer cell lines. However, there is no available information to address the effects of Gyp on DNA damage and DNA repair-associated gene expression in human oral cancer cells. Therefore, we investigated whether Gyp induced DNA damage and DNA repair gene expression in human oral cancer SAS cells. The results from flow cytometric assay indicated that Gyp-induced cytotoxic effects led to a decrease in the percentage of viable SAS cells. The results from comet assay revealed that the incubation of SAS cells with Gyp led to a longer DNA migration smear (comet tail) when compared with control and this effect was dose-dependent. The results from real-time PCR analysis indicated that treatment of SAS cells with 180 mug/ml of Gyp for 24 h led to a decrease in 14-3-3sigma, DNA-dependent serine/threonine protein kinase (DNAPK), p53, ataxia telangiectasia mutated (ATM), ataxia-telangiectasia and Rad3-related (ATR) and breast cancer gene 1 (BRCA1) mRNA expression. These observations may explain the cell death caused by Gyp in SAS cells. Taken together, Gyp induced DNA damage and inhibited DNA repair-associated gene expressions in human oral cancer SAS cells in vitro.

  2. Contribution of DNA double-strand break repair gene XRCC3 genotypes to oral cancer susceptibility in Taiwan.

    Science.gov (United States)

    Tsai, Chia-Wen; Chang, Wen-Shin; Liu, Juhn-Cherng; Tsai, Ming-Hsui; Lin, Cheng-Chieh; Bau, Da-Tian

    2014-06-01

    The DNA repair gene X-ray repair cross complementing protein 3 (XRCC3) is thought to play a major role in double-strand break repair and in maintaining genomic stability. Very possibly, defective double-strand break repair of cells can lead to carcinogenesis. Therefore, a case-control study was performed to reveal the contribution of XRCC3 genotypes to individual oral cancer susceptibility. In this hospital-based research, the association of XRCC3 rs1799794, rs45603942, rs861530, rs3212057, rs1799796, rs861539, rs28903081 genotypes with oral cancer risk in a Taiwanese population was investigated. In total, 788 patients with oral cancer and 956 age- and gender-matched healthy controls were genotyped. The results showed that there was significant differential distribution among oral cancer and controls in the genotypic (p=0.001428) and allelic (p=0.0013) frequencies of XRCC3 rs861539. As for the other polymorphisms, there was no difference between case and control groups. In gene-lifestyle interaction analysis, we have provided the first evidence showing that there is an obvious joint effect of XRCC3 rs861539 genotype with individual areca chewing habits on oral cancer risk. In conclusion, the T allele of XRCC3 rs861539, which has an interaction with areca chewing habit in oral carcinogenesis, may be an early marker for oral cancer in Taiwanese.

  3. Identification and Analysis of MS5(d): A Gene That Affects Double-Strand Break (DSB) Repair during Meiosis I in Brassica napus Microsporocytes.

    Science.gov (United States)

    Zeng, Xinhua; Yan, Xiaohong; Yuan, Rong; Li, Keqi; Wu, Yuhua; Liu, Fang; Luo, Junling; Li, Jun; Wu, Gang

    2016-01-01

    Here, we report the identification of the Brassica-specific gene MS5(d), which is responsible for male sterility in Brassica napus. The MS5(d) gene is highly expressed in the microsporocyte and encodes a protein that localizes to the nucleus. Light microscopy analyses have demonstrated that the MS5(d) gene affects microsporocyte meiosis in the thermosensitive genic male sterility line TE5A. Sequence comparisons and genetic complementation revealed a C-to-T transition in MS5(d), encoding a Leu-to-Phe (L281F) substitution and causing abnormal male meiosis in TE5A. These findings suggest arrested meiotic chromosome dynamics at pachytene. Furthermore, immunofluorescence analyses showed that double-strand break (DSB) formation and axial elements were normal but that DSB repair and spindle behavior were aberrant in TE5A meiocytes. Collectively, our results indicate that MS5(d) likely encodes a protein required for chromosomal DSB repair at early stages of meiosis in B. napus.

  4. Escherichia coli radD (yejH) gene: a novel function involved in radiation resistance and double-strand break repair

    OpenAIRE

    Chen, Stefanie H.; Byrne, Rose T.; Wood, Elizabeth A; Cox, Michael M.

    2015-01-01

    A transposon insertion screen implicated the yejH gene in the repair of ionizing radiation-induced damage. The yejH gene, which exhibits significant homology to the human transcription-coupled DNA repair gene XPB, is involved in the repair of double strand DNA breaks. Deletion of yejH significantly sensitized cells to agents that cause double strand breaks (ionizing radiation, UV radiation, ciprofloxacin). In addition, deletion of both yejH and radA hypersensitized the cells to ionizing radia...

  5. Deletion of the nucleotide excision repair gene Ercc1 reduces immunoglobulin class switching and alters mutations near switch recombination junctions

    NARCIS (Netherlands)

    C.E. Schrader; J. Vardo; E. Linehan; M.Z. Twarog; L.J. Niedernhofer (Laura); J. Stavnezer; J.H.J. Hoeijmakers (Jan)

    2004-01-01

    textabstractThe structure-specific endonuclease ERCC1-XPF is an essential component of the nucleotide excision DNA repair pathway. ERCC1-XPF nicks double-stranded DNA immediately adjacent to 3' single-strand regions. Substrates include DNA bubbles and flaps. Furthermore, ERCC1 interacts with Msh2, a

  6. Exome sequencing identifies rare deleterious mutations in DNA repair genes FANCC and BLM as potential breast cancer susceptibility alleles.

    Directory of Open Access Journals (Sweden)

    Ella R Thompson

    2012-09-01

    Full Text Available Despite intensive efforts using linkage and candidate gene approaches, the genetic etiology for the majority of families with a multi-generational breast cancer predisposition is unknown. In this study, we used whole-exome sequencing of thirty-three individuals from 15 breast cancer families to identify potential predisposing genes. Our analysis identified families with heterozygous, deleterious mutations in the DNA repair genes FANCC and BLM, which are responsible for the autosomal recessive disorders Fanconi Anemia and Bloom syndrome. In total, screening of all exons in these genes in 438 breast cancer families identified three with truncating mutations in FANCC and two with truncating mutations in BLM. Additional screening of FANCC mutation hotspot exons identified one pathogenic mutation among an additional 957 breast cancer families. Importantly, none of the deleterious mutations were identified among 464 healthy controls and are not reported in the 1,000 Genomes data. Given the rarity of Fanconi Anemia and Bloom syndrome disorders among Caucasian populations, the finding of multiple deleterious mutations in these critical DNA repair genes among high-risk breast cancer families is intriguing and suggestive of a predisposing role. Our data demonstrate the utility of intra-family exome-sequencing approaches to uncover cancer predisposition genes, but highlight the major challenge of definitively validating candidates where the incidence of sporadic disease is high, germline mutations are not fully penetrant, and individual predisposition genes may only account for a tiny proportion of breast cancer families.

  7. Mismatch repair genes Mlh1 and Mlh3 modify CAG instability in Huntington's disease mice: genome-wide and candidate approaches.

    Directory of Open Access Journals (Sweden)

    Ricardo Mouro Pinto

    2013-10-01

    Full Text Available The Huntington's disease gene (HTT CAG repeat mutation undergoes somatic expansion that correlates with pathogenesis. Modifiers of somatic expansion may therefore provide routes for therapies targeting the underlying mutation, an approach that is likely applicable to other trinucleotide repeat diseases. Huntington's disease Hdh(Q111 mice exhibit higher levels of somatic HTT CAG expansion on a C57BL/6 genetic background (B6.Hdh(Q111 than on a 129 background (129.Hdh(Q111 . Linkage mapping in (B6x129.Hdh(Q111 F2 intercross animals identified a single quantitative trait locus underlying the strain-specific difference in expansion in the striatum, implicating mismatch repair (MMR gene Mlh1 as the most likely candidate modifier. Crossing B6.Hdh(Q111 mice onto an Mlh1 null background demonstrated that Mlh1 is essential for somatic CAG expansions and that it is an enhancer of nuclear huntingtin accumulation in striatal neurons. Hdh(Q111 somatic expansion was also abolished in mice deficient in the Mlh3 gene, implicating MutLγ (MLH1-MLH3 complex as a key driver of somatic expansion. Strikingly, Mlh1 and Mlh3 genes encoding MMR effector proteins were as critical to somatic expansion as Msh2 and Msh3 genes encoding DNA mismatch recognition complex MutSβ (MSH2-MSH3. The Mlh1 locus is highly polymorphic between B6 and 129 strains. While we were unable to detect any difference in base-base mismatch or short slipped-repeat repair activity between B6 and 129 MLH1 variants, repair efficiency was MLH1 dose-dependent. MLH1 mRNA and protein levels were significantly decreased in 129 mice compared to B6 mice, consistent with a dose-sensitive MLH1-dependent DNA repair mechanism underlying the somatic expansion difference between these strains. Together, these data identify Mlh1 and Mlh3 as novel critical genetic modifiers of HTT CAG instability, point to Mlh1 genetic variation as the likely source of the instability difference in B6 and 129 strains and suggest

  8. Mismatch repair genes Mlh1 and Mlh3 modify CAG instability in Huntington's disease mice: genome-wide and candidate approaches.

    Science.gov (United States)

    Pinto, Ricardo Mouro; Dragileva, Ella; Kirby, Andrew; Lloret, Alejandro; Lopez, Edith; St Claire, Jason; Panigrahi, Gagan B; Hou, Caixia; Holloway, Kim; Gillis, Tammy; Guide, Jolene R; Cohen, Paula E; Li, Guo-Min; Pearson, Christopher E; Daly, Mark J; Wheeler, Vanessa C

    2013-10-01

    The Huntington's disease gene (HTT) CAG repeat mutation undergoes somatic expansion that correlates with pathogenesis. Modifiers of somatic expansion may therefore provide routes for therapies targeting the underlying mutation, an approach that is likely applicable to other trinucleotide repeat diseases. Huntington's disease Hdh(Q111) mice exhibit higher levels of somatic HTT CAG expansion on a C57BL/6 genetic background (B6.Hdh(Q111) ) than on a 129 background (129.Hdh(Q111) ). Linkage mapping in (B6x129).Hdh(Q111) F2 intercross animals identified a single quantitative trait locus underlying the strain-specific difference in expansion in the striatum, implicating mismatch repair (MMR) gene Mlh1 as the most likely candidate modifier. Crossing B6.Hdh(Q111) mice onto an Mlh1 null background demonstrated that Mlh1 is essential for somatic CAG expansions and that it is an enhancer of nuclear huntingtin accumulation in striatal neurons. Hdh(Q111) somatic expansion was also abolished in mice deficient in the Mlh3 gene, implicating MutLγ (MLH1-MLH3) complex as a key driver of somatic expansion. Strikingly, Mlh1 and Mlh3 genes encoding MMR effector proteins were as critical to somatic expansion as Msh2 and Msh3 genes encoding DNA mismatch recognition complex MutSβ (MSH2-MSH3). The Mlh1 locus is highly polymorphic between B6 and 129 strains. While we were unable to detect any difference in base-base mismatch or short slipped-repeat repair activity between B6 and 129 MLH1 variants, repair efficiency was MLH1 dose-dependent. MLH1 mRNA and protein levels were significantly decreased in 129 mice compared to B6 mice, consistent with a dose-sensitive MLH1-dependent DNA repair mechanism underlying the somatic expansion difference between these strains. Together, these data identify Mlh1 and Mlh3 as novel critical genetic modifiers of HTT CAG instability, point to Mlh1 genetic variation as the likely source of the instability difference in B6 and 129 strains and suggest that MLH1

  9. Polymorphism of the DNA repair gene XPA and susceptibility to lung cancer

    Institute of Scientific and Technical Information of China (English)

    Jinfu Zhu; Zhibin Hu; Hongxia Ma; Xiang Huo; Lin Xu; Jiannong Zhou; Hongbing Shen; Yijiang Chen

    2005-01-01

    Objective: To study the relationship between one polymorphism in the promoter of the DNA repair gene XPA and the susceptibility to lung cancer. Methods: Genotypes were determined by the PCR-restriction fragment length polymorphism (PCR-RFLP)method in 310 histologically-confirmed lung cancer cases and 341 age and sex frequency-matched cancer-free controls. Results: The XPA A23G genotype frequencies were 27.1% (AA), 42.9% (AG), and 30.0% (GG) in case patients and 21.1% (AA), 52.8% (AG),and 26.1% (GG) in control subjects. Multivariate logistic regression analysis revealed that individuals carrying at least one 23G variant allele (AG + GG genotypes) had a significantly decreased risk for lung cancer (adjusted OR = 0.66; 95% CI = 0.44- 0.98) compared with the wild-type genotype (23AA). Stratified analysis showed that the protective effect was more evident in subjects with a family history of cancer. Conclusion: These results suggest that the XPA A23G polymorphism may have a role in lung cancer susceptibility in this study population.

  10. Global and gene specific DNA methylation changes during zebrafish development

    Science.gov (United States)

    DNA methylation is dynamic through the life of an organism. In this study, we measured the global and gene specific DNA methylation changes in zebrafish at different developmental stages. We found that the methylation percentage of cytosines was 11.75 ± 0.96% in 3.3 hour post fertilization (hpf) zeb...

  11. Human INO80 chromatin-remodelling complex contributes to DNA double-strand break repair via the expression of Rad54B and XRCC3 genes.

    Science.gov (United States)

    Park, Eun-Jung; Hur, Shin-Kyoung; Kwon, Jongbum

    2010-10-15

    Recent studies have shown that the SWI/SNF family of ATP-dependent chromatin-remodelling complexes play important roles in DNA repair as well as in transcription. The INO80 complex, the most recently described member of this family, has been shown in yeast to play direct role in DNA DSB (double-strand break) repair without affecting the expression of the genes involved in this process. However, whether this function of the INO80 complex is conserved in higher eukaryotes has not been investigated. In the present study, we found that knockdown of hINO80 (human INO80) confers DNA-damage hypersensitivity and inefficient DSB repair. Microarray analysis and other experiments have identified the Rad54B and XRCC3 (X-ray repair complementing defective repair in Chinese-hamster cells 3) genes, implicated in DSB repair, to be repressed by hINO80 deficiency. Chromatin immunoprecipitation studies have shown that hINO80 binds to the promoters of the Rad54B and XRCC3 genes. Re-expression of the Rad54B and XRCC3 genes rescues the DSB repair defect in hINO80-deficient cells. These results suggest that hINO80 assists DSB repair by positively regulating the expression of the Rad54B and XRCC3 genes. Therefore, unlike yeast INO80, hINO80 can contribute to DSB repair indirectly via gene expression, suggesting that the mechanistic role of this chromatin remodeller in DSB repair is evolutionarily diversified.

  12. Promoter Hypermethylation of DNA Repair Gene MGMT in Laryngeal Squamous Cell Carcinoma

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    The relationship between hypermethylation of CpG islands in the promoter regions of O6methylguanine DNA methyltransferase (MGMT)genes and laryngeal squamous cell carcinoma was explored. Methylation-specific PCR and semi-quantitative RT-PCR were used to study the promoter methylation and mRNA expression of the MGMT gene in laryngeal carcinoma tissues, t issues adjacent to the tumor and normal laryngeal tissues. Hypermethylation of MGMT gene was detected in 16 samples of 46 (34.8 %) laryngeal squamous cell carcinoma samples. However, the MGMT hypermethylation was not detected in all tissues adjacent to the tumors and normal tissues. No significant difference in MGMT gene hypermethylation was found in samples with different histological grades (x2= 3. 130, P=0. 077) or in samples from patients with different TNM status (x2=3. 957, P=0. 138). No expression of MGMT mRNA was detected in all hypermethylated laryngeal carcinoma tissues. The expression of MGMT mRNA was detected in all unmethylated laryngeal carcinoma tissues, tissues adjacent to the tumors and normal tissues. It suggests that MGMT gene promoter hypermethylation is associated with MGMT gene transcription loss in laryngeal carcinoma tissues and possibly plays an important role in carcinogenesis of laryngeal tissues.

  13. Hypertension and genetic variation in endothelial-specific genes.

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    Erik Larsson

    Full Text Available Genome-wide association (GWA studies usually detect common genetic variants with low-to-medium effect sizes. Many contributing variants are not revealed, since they fail to reach significance after strong correction for multiple comparisons. The WTCCC study for hypertension, for example, failed to identify genome-wide significant associations. We hypothesized that genetic variation in genes expressed specifically in the endothelium may be important for hypertension development. Results from the WTCCC study were combined with previously published gene expression data from mice to specifically investigate SNPs located within endothelial-specific genes, bypassing the requirement for genome-wide significance. Six SNPs from the WTCCC study were selected for independent replication in 5205 hypertensive patients and 5320 population-based controls, and successively in a cohort of 16,537 individuals. A common variant (rs10860812 in the DRAM (damage-regulated autophagy modulator locus showed association with hypertension (P = 0.008 in the replication study. The minor allele (A had a protective effect (OR = 0.93; 95% CI 0.88-0.98 per A-allele, which replicates the association in the WTCCC GWA study. However, a second follow-up, in the larger cohort, failed to reveal an association with blood pressure. We further tested the endothelial-specific genes for co-localization with a panel of newly discovered SNPs from large meta-GWAS on hypertension or blood pressure. There was no significant overlap between those genes and hypertension or blood pressure loci. The result does not support the hypothesis that genetic variation in genes expressed in endothelium plays an important role for hypertension development. Moreover, the discordant association of rs10860812 with blood pressure in the case control study versus the larger Malmö Preventive Project-study highlights the importance of rigorous replication in multiple large independent studies.

  14. Clinical features and mismatch repair gene mutation screening in Chinese patients with hereditary nonpolyposis colorectal carcinoma

    Institute of Scientific and Technical Information of China (English)

    Shan-Run Liu; Bo Zhao; Zhen-Jun Wang; Yuan-Lian Wan; Yan-Ting Huang

    2004-01-01

    AIM: Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominantly- inherited cancer-susceptibility syndrome that confers an increased risk for colorectal cancer and a variety of other tumors at a young age. It has been associated with germline mutations in five mismatch repair (MMR) genes (hMSH2, hMLH1, hPMS1, hPMS2, and hMSH6/GTBP). The great majority of germline mutations were found in hMSH2 and hMLH1. The purpose of this study was to analyze the clinical features of Chinese HNPCC patients and to screen hMSH2 and hMLH1 gene mutations. METHODS: Twenty-eight independent Chinese families were collected, of which 15 met Amsterdam criteria I and 13 met the Japanese clinical diagnosis criteria. The data were recorded including sex, site of colorectal cancer (CRC),age of diagnosis, history of synchronous and/or metachronous CRC, instance of extracolonic cancers, and histopathology of tumors. Peripheral blood samples were collected from all pedigrees after formal written consents were signed. PCR and denaturing high-performance liquid chromatography (DHPLC) were used to screen the coding regions of hMSH2 and hMLH1 genes. The samples showing abnormal DHPLC profiles were sequenced by a 377 DNA sequencer.RESULTS: One hundred and seventy malignant neoplasms were found in one hundred and twenty-six patients (multiple cancer in twenty-three), including one hundred and twentyseven CRCs, fifteen gastric, seven endometrial, and five esophageal cancers. Seventy-seven point eight percent of the patients had CRCs, sharing the features of early occurrence (average age of onset, 45.9 years) and of the right-sided predominance reported in the literature. In Chinese HNPCC patients, gastric cancer occurred more frequently, accounting for 11.9% of all cancers patients and ranking second in the spectrum of HNPCC predisposing cancers. Synchronous CRCs occurred less frequently, only accounting for 3.1% of the total CRCs. Twenty percent of the colorectal patients had

  15. A maize-specifically expressed gene cluster in Ustilago maydis.

    Science.gov (United States)

    Basse, Christoph W; Kolb, Sebastian; Kahmann, Regine

    2002-01-01

    The corn pathogen Ustilago maydis requires its host plant maize for development and completion of its sexual cycle. We have identified the fungal mig2-1 gene as being specifically expressed during this biotrophic stage. Intriguingly, mig2-1 is part of a gene cluster comprising five highly homologous and similarly regulated genes designated mig2-1 to mig2-5. Deletion analysis of the mig2-1 promoter provides evidence for negative and positive regulation. The predicted polypeptides of all five genes lack significant homologies to known genes but have characteristic N-terminal secretion sequences. The secretion signals of mig2-1 and mig2-5 were shown to be functional, and secretion of a full length Mig2-1-eGFP fusion protein to the extracellular space was demonstrated. The central domains of the Mig2 proteins are highly variable whereas the C-termini are strongly conserved and share a characteristic pattern of eight cysteine residues. The mig2 gene cluster was conserved in a wide collection of U. maydis strains. Interestingly, some U. maydis isolates from South America had lost the mig2-4 gene as a result of a homologous recombination event. Furthermore, the related Ustilago scitaminea strain, which is pathogenic on sugar cane, appears to lack the mig2 cluster. We describe a model of how the mig2 cluster might have evolved and discuss its possible role in governing host interaction.

  16. Multiple lineage specific expansions within the guanylyl cyclase gene family

    Directory of Open Access Journals (Sweden)

    O'Halloran Damien M

    2006-03-01

    Full Text Available Abstract Background Guanylyl cyclases (GCs are responsible for the production of the secondary messenger cyclic guanosine monophosphate, which plays important roles in a variety of physiological responses such as vision, olfaction, muscle contraction, homeostatic regulation, cardiovascular and nervous function. There are two types of GCs in animals, soluble (sGCs which are found ubiquitously in cell cytoplasm, and receptor (rGC forms which span cell membranes. The complete genomes of several vertebrate and invertebrate species are now available. These data provide a platform to investigate the evolution of GCs across a diverse range of animal phyla. Results In this analysis we located GC genes from a broad spectrum of vertebrate and invertebrate animals and reconstructed molecular phylogenies for both sGC and rGC proteins. The most notable features of the resulting phylogenies are the number of lineage specific rGC and sGC expansions that have occurred during metazoan evolution. Among these expansions is a large nematode specific rGC clade comprising 21 genes in C. elegans alone; a vertebrate specific expansion in the natriuretic receptors GC-A and GC-B; a vertebrate specific expansion in the guanylyl GC-C receptors, an echinoderm specific expansion in the sperm rGC genes and a nematode specific sGC clade. Our phylogenetic reconstruction also shows the existence of a basal group of nitric oxide (NO insensitive insect and nematode sGCs which are regulated by O2. This suggests that the primordial eukaryotes probably utilized sGC as an O2 sensor, with the ligand specificity of sGC later switching to NO which provides a very effective local cell-to-cell signalling system. Phylogenetic analysis of the sGC and bacterial heme nitric oxide/oxygen binding protein domain supports the hypothesis that this domain originated from a cyanobacterial source. Conclusion The most salient feature of our phylogenies is the number of lineage specific expansions

  17. Kidney-specific transposon-mediated gene transfer in vivo

    Science.gov (United States)

    Woodard, Lauren E.; Cheng, Jizhong; Welch, Richard C.; Williams, Felisha M.; Luo, Wentian; Gewin, Leslie S.; Wilson, Matthew H.

    2017-01-01

    Methods enabling kidney-specific gene transfer in adult mice are needed to develop new therapies for kidney disease. We attempted kidney-specific gene transfer following hydrodynamic tail vein injection using the kidney-specific podocin and gamma-glutamyl transferase promoters, but found expression primarily in the liver. In order to achieve kidney-specific transgene expression, we tested direct hydrodynamic injection of a DNA solution into the renal pelvis and found that luciferase expression was strong in the kidney and absent from extra-renal tissues. We observed heterogeneous, low-level transfection of the collecting duct, proximal tubule, distal tubule, interstitial cells, and rarely glomerular cells following injection. To assess renal injury, we performed the renal pelvis injections on uninephrectomised mice and found that their blood urea nitrogen was elevated at two days post-transfer but resolved within two weeks. Although luciferase expression quickly decreased following renal pelvis injection, the use of the piggyBac transposon system improved long-term expression. Immunosuppression with cyclophosphamide stabilised luciferase expression, suggesting immune clearance of the transfected cells occurs in immunocompetent animals. Injection of a transposon expressing erythropoietin raised the haematocrit, indicating that the developed injection technique can elicit a biologic effect in vivo. Hydrodynamic renal pelvis injection enables transposon mediated-kidney specific gene transfer in adult mice. PMID:28317878

  18. Association between DNA damage response and repair genes and risk of invasive serous ovarian cancer.

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    Joellen M Schildkraut

    Full Text Available BACKGROUND: We analyzed the association between 53 genes related to DNA repair and p53-mediated damage response and serous ovarian cancer risk using case-control data from the North Carolina Ovarian Cancer Study (NCOCS, a population-based, case-control study. METHODS/PRINCIPAL FINDINGS: The analysis was restricted to 364 invasive serous ovarian cancer cases and 761 controls of white, non-Hispanic race. Statistical analysis was two staged: a screen using marginal Bayes factors (BFs for 484 SNPs and a modeling stage in which we calculated multivariate adjusted posterior probabilities of association for 77 SNPs that passed the screen. These probabilities were conditional on subject age at diagnosis/interview, batch, a DNA quality metric and genotypes of other SNPs and allowed for uncertainty in the genetic parameterizations of the SNPs and number of associated SNPs. Six SNPs had Bayes factors greater than 10 in favor of an association with invasive serous ovarian cancer. These included rs5762746 (median OR(odds ratio(per allele = 0.66; 95% credible interval (CI = 0.44-1.00 and rs6005835 (median OR(per allele = 0.69; 95% CI = 0.53-0.91 in CHEK2, rs2078486 (median OR(per allele = 1.65; 95% CI = 1.21-2.25 and rs12951053 (median OR(per allele = 1.65; 95% CI = 1.20-2.26 in TP53, rs411697 (median OR (rare homozygote = 0.53; 95% CI = 0.35 - 0.79 in BACH1 and rs10131 (median OR( rare homozygote = not estimable in LIG4. The six most highly associated SNPs are either predicted to be functionally significant or are in LD with such a variant. The variants in TP53 were confirmed to be associated in a large follow-up study. CONCLUSIONS/SIGNIFICANCE: Based on our findings, further follow-up of the DNA repair and response pathways in a larger dataset is warranted to confirm these results.

  19. Transcript levels of the Saccharomyes cerevisiae DNA repair gene RAD23 increase in response to UV light and in meiosis but remain constant in the mitotic cell cycle.

    Science.gov (United States)

    Madura, K; Prakash, S

    1990-08-25

    The RAD23 gene of Saccharomyces cerevisiae is required for excision-repair of UV damaged DNA. In this paper, we determine the location of the RAD23 gene in a cloned DNA fragment, identify the 1.6 kb RAD23 transcript, and examine RAD23 transcript levels in UV damaged cells, during the mitotic cell cycle, and in meiosis. The RAD23 mRNA levels are elevated 5-fold between 30 to 60 min after 37 J/m2 of UV light. RAD23 mRNA levels rise over 6-fold during meiosis at a stage coincident with high levels of genetic recombination. This response is specific to sporulation competent MATa/MAT alpha diploid cells, and is not observed in asporogenous MATa/MATa diploids. RAD23 mRNA levels, however, remain constant during the mitotic cell cycle.

  20. FRAGILE HISTIDINE TRIAD GENE EXPRESSION AND ITS CORRALATION WITH MISMATCH REPAIR PROTEIN IN HUMAN SPORADIC COLORECTAL CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    姚成才; 林从尧

    2004-01-01

    Objective: To investigate the expression of fragile histidine triad (FHIT) gene and its correlation with clinicopathological features and correlation with mismatch repair protein (mainly MLH1 and MSH2) in human sporadic colorectal carcinoma (SCC). Methods:Immunohistochemistry SP method was used to determine the expression of FHIT, MLH1 and MSH2 protein in surgically resected specimens of 84 human SCC. Results:The positive rates of FHIT, MLH1 and MSH2 protein expression were 48.81%, 92.86% and 100% respectively.Loss or reduced expression of FHIT protein was not related with tumors clinicopathological features such as age, gender,tumors site and histological type (P>0.05), but was correlated with tumors invade depth, degree of the differentiation, Ducks' stage and metastasis (P<0.05). There was no relationship between FHIT gene expression and MLH1 protein (r=0.0991, P>0.05) and MSH2 protein (r=0.0000, P=l.00) expression in human SCC. Conclusion:Absent or reduction of FHIT gene expression consists of high proportion and is a frequent event in SCC. FHIT gene is involved in the development and progression of human SCC and may be a candidate tumors suppressor gene. The relationship between alteration of FHIT gene expression and mismatch repair protein (mainly MLH1 and MSH2)deserved further study in human SCC.

  1. Polymorphisms in DNA repair genes, smoking, and bladder cancer risk: findings from the International Consortium of Bladder Cancer

    Science.gov (United States)

    Stern, Mariana C.; Lin, Jie; Figueroa, Jonine D.; Kelsey, Karl T.; Kiltie, Anne E.; Yuan, Jian-Min; Matullo, Giuseppe; Fletcher, Tony; Benhamou, Simone; Taylor, Jack A.; Placidi, Donatella; Zhang, Zuo-Feng; Steineck, Gunnar; Rothman, Nathaniel; Kogevinas, Manolis; Silverman, Debra; Malats, Nuria; Chanock, Stephen; Wu, Xifeng; Karagas, Margaret R.; Andrew, Angeline S.; Nelson, Heather H.; Bishop, D. Timothy; Sak, Sei Chung; Choudhury, Ananya; Barrett, Jennifer H; Elliot, Faye; Corral, Román; Joshi, Amit D.; Gago-Dominguez, Manuela; Cortessis, Victoria K.; Xiang, Yong-Bing; Vineis, Paolo; Sacerdote, Carlotta; Guarrera, Simonetta; Polidoro, Silvia; Allione, Alessandra; Gurzau, Eugen; Koppova, Kvetoslava; Kumar, Rajiv; Rudnai, Peter; Porru, Stefano; Carta, Angela; Campagna, Marcello; Arici, Cecilia; Park, SungShim Lani; Garcia-Closas, Montserrat

    2009-01-01

    Tobacco smoking is the most important and well-established bladder cancer risk factor, and a rich source of chemical carcinogens and reactive oxygen species that can induce damage to DNA in urothelial cells. Therefore, common variation in DNA repair genes might modify bladder cancer risk. In this study we present results from meta- and pooled analyses conducted as part of the International Consortium of Bladder Cancer. We included data on 10 single nucleotide polymorphisms corresponding to 7 DNA repair genes from 13 studies. Pooled- and meta-analyses included 5,282 cases and 5,954 controls of non-Latino white origin. We found evidence for weak but consistent associations with ERCC2 D312N (rs1799793) (per allele OR = 1.10; 95% CI = 1.01–1.19; p = 0.021), NBN E185Q (rs1805794) (per allele OR = 1.09; 95% CI = 1.01–1.18; p = 0.028), and XPC A499V (rs2228000) (per allele OR = 1.10; 95% CI = 1.00–1.21, p = 0.044). The association with NBN E185Q was limited to ever smokers (interaction p = 0.002), and was strongest for the highest levels of smoking dose and smoking duration. Overall, our study provides the strongest evidence to date for a role of common variants in DNA repair genes in bladder carcinogenesis. PMID:19706757

  2. The Mutyh base excision repair gene influences the inflammatory response in a mouse model of ulcerative colitis.

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    Ida Casorelli

    Full Text Available BACKGROUND: The Mutyh DNA glycosylase is involved in the repair of oxidized DNA bases. Mutations in the human MUTYH gene are responsible for colorectal cancer in familial adenomatous polyposis. Since defective DNA repair genes might contribute to the increased cancer risk associated with inflammatory bowel diseases, we compared the inflammatory response of wild-type and Mutyh(-/- mice to oxidative stress. METHODOLOGY/PRINCIPAL FINDINGS: The severity of colitis, changes in expression of genes involved in DNA repair and inflammation, DNA 8-oxoguanine levels and microsatellite instability were analysed in colon of mice treated with dextran sulfate sodium (DSS. The Mutyh(-/- phenotype was associated with a significant accumulation of 8-oxoguanine in colon DNA of treated mice. A single DSS cycle induced severe acute ulcerative colitis in wild-type mice, whereas lesions were modest in Mutyh(-/- mice, and this was associated with moderate variations in the expression of several cytokines. Eight DSS cycles caused chronic colitis in both wild-type and Mutyh(-/- mice. Lymphoid hyperplasia and a significant reduction in Foxp3(+ regulatory T cells were observed only in Mutyh(-/- mice. CONCLUSIONS: The findings indicate that, in this model of ulcerative colitis, Mutyh plays a major role in maintaining intestinal integrity by affecting the inflammatory response.

  3. New single nucleotide polymorphisms (SNPs) in homologous recombination repair genes detected by microarray analysis in Polish breast cancer patients.

    Science.gov (United States)

    Romanowicz, Hanna; Strapagiel, Dominik; Słomka, Marcin; Sobalska-Kwapis, Marta; Kępka, Ewa; Siewierska-Górska, Anna; Zadrożny, Marek; Bieńkiewicz, Jan; Smolarz, Beata

    2016-11-30

    Breast cancer is the most common cause of malignancy and mortality in women worldwide. This study aimed at localising homologous recombination repair (HR) genes and their chromosomal loci and correlating their nucleotide variants with susceptibility to breast cancer. In this study, authors analysed the association between single nucleotide polymorphisms (SNPs) in homologous recombination repair genes and the incidence of breast cancer in the population of Polish women. Blood samples from 94 breast cancer patients were analysed as test group. Individuals were recruited into the study at the Department of Oncological Surgery and Breast Diseases of the Institute of the Polish Mother's Memorial Hospital in Lodz, Poland. Healthy controls (n = 500) were obtained from the Biobank Laboratory, Department of Molecular Biophysics, University of Lodz. Then, DNA of breast cancer patients was compared with one of the disease-free women. The test was supported by microarray analysis. Statistically significant correlations were identified between breast cancer and 3 not described previously SNPs of homologous recombination repair genes BRCA1 and BRCA2: rs59004709, rs4986852 and rs1799950. Further studies on larger groups are warranted to support the hypothesis of correlation between the abovementioned genetic variants and breast cancer risk.

  4. Study on the Specific Gene Expression during Spermatogenesis of Rat

    Institute of Scientific and Technical Information of China (English)

    吴燕婉; 王春梅; 黄海燕; 石心泉; 邢志军; 刘德瑜

    2001-01-01

    Objective To explore specific gene expression for regulating meiosis of germ cells during spermatogenesis of rat testis Materials & Methods Male SD rats, aged 1, 3 and 8 weeks, were observed in this study. The methods of morphological observation on testicular tissues embedded by resin and mRNA differential display (DDRT-PCR) were combined to obtain specific mRNA expression gene fragments during the testicular development. Reverse dot blot hybridization was operated to further screen the positive differential DNA fragments.The positive DNA segments were sub-cloned in pGEM-T-Easy vector and transformed into the competent E-coli 109 straint. Northern blot analysis and in situ hybridization were also carried out for identifying tissue-specific expression as well as cell-specific expression DNA fragments. To screen λ-ZAP-II rat testicular gene library was searched for the original gene.Results Eighty two differential cDNA fragments were obtained through primary DDRT-PCR, among which 40 differential cDNA fragments were selected for further screening with reverse dot blot hybridization. After the reverse dot blot hybridization,12 primary differential DNA fragments were obtained. The size of DNA fragments ranged from 250 to 500 bp. The in situ hybridization of the testicular tissue showed that a specific DNA fragment derived from 8-week old rat testis, named CG14, was hybridized in adult rat testicular section, in which the positive nucleic acid signals were distributed specifically in the primary spermatocytes. Another DNA fragment derived from 1-week old rat testis, named AA 1 1, was hybridized specifically in Sertoli cell of 1-week old rat testis. Northern blot hybridization with [α-32P] dCTP labeled CG 1 4 probe, including cardiac, liver, kidney, brain, testis, and epididymis tissue mRNAs of rat, showed that an mRNA specific hybridization band, size of 1. 258 kb, was found in testis tissue and size of 1. 531 kb of another hybridization band present in epididymis

  5. A gene regulatory network armature for T-lymphocyte specification

    Energy Technology Data Exchange (ETDEWEB)

    Fung, Elizabeth-sharon [Los Alamos National Laboratory

    2008-01-01

    Choice of a T-lymphoid fate by hematopoietic progenitor cells depends on sustained Notch-Delta signaling combined with tightly-regulated activities of multiple transcription factors. To dissect the regulatory network connections that mediate this process, we have used high-resolution analysis of regulatory gene expression trajectories from the beginning to the end of specification; tests of the short-term Notchdependence of these gene expression changes; and perturbation analyses of the effects of overexpression of two essential transcription factors, namely PU.l and GATA-3. Quantitative expression measurements of >50 transcription factor and marker genes have been used to derive the principal components of regulatory change through which T-cell precursors progress from primitive multipotency to T-lineage commitment. Distinct parts of the path reveal separate contributions of Notch signaling, GATA-3 activity, and downregulation of PU.l. Using BioTapestry, the results have been assembled into a draft gene regulatory network for the specification of T-cell precursors and the choice of T as opposed to myeloid dendritic or mast-cell fates. This network also accommodates effects of E proteins and mutual repression circuits of Gfil against Egr-2 and of TCF-l against PU.l as proposed elsewhere, but requires additional functions that remain unidentified. Distinctive features of this network structure include the intense dose-dependence of GATA-3 effects; the gene-specific modulation of PU.l activity based on Notch activity; the lack of direct opposition between PU.l and GATA-3; and the need for a distinct, late-acting repressive function or functions to extinguish stem and progenitor-derived regulatory gene expression.

  6. Gene recovery microdissection (GRM) a process for producing chromosome region-specific libraries of expressed genes

    Energy Technology Data Exchange (ETDEWEB)

    Christian, A T; Coleman, M A; Tucker, J D

    2001-02-08

    Gene Recovery Microdissection (GRM) is a unique and cost-effective process for producing chromosome region-specific libraries of expressed genes. It accelerates the pace, reduces the cost, and extends the capabilities of functional genomic research, the means by which scientists will put to life-saving, life-enhancing use their knowledge of any plant or animal genome.

  7. Transplantation of erythropoietin gene-modified neural stem cells improves the repair of injured spinal cord

    Directory of Open Access Journals (Sweden)

    Min-fei Wu

    2015-01-01

    Full Text Available The protective effects of erythropoietin on spinal cord injury have not been well described. Here, the eukaryotic expression plasmid pcDNA3.1 human erythropoietin was transfected into rat neural stem cells cultured in vitro. A rat model of spinal cord injury was established using a free falling object. In the human erythropoietin-neural stem cells group, transfected neural stem cells were injected into the rat subarachnoid cavity, while the neural stem cells group was injected with non-transfected neural stem cells. Dulbecco′s modified Eagle′s medium/F12 medium was injected into the rats in the spinal cord injury group as a control. At 1-4 weeks post injury, the motor function in the rat lower limbs was best in the human erythropoietin-neural stem cells group, followed by the neural stem cells group, and lastly the spinal cord injury group. At 72 hours, compared with the spinal cord injury group, the apoptotic index and Caspase-3 gene and protein expressions were apparently decreased, and the bcl-2 gene and protein expressions were noticeably increased, in the tissues surrounding the injured region in the human erythropoietin-neural stem cells group. At 4 weeks, the cavities were clearly smaller and the motor and somatosensory evoked potential latencies were remarkably shorter in the human erythropoietin-neural stem cells group and neural stem cells group than those in the spinal cord injury group. These differences were particularly obvious in the human erythropoietin-neural stem cells group. More CM-Dil-positive cells and horseradish peroxidase-positive nerve fibers and larger amplitude motor and somatosensory evoked potentials were found in the human erythropoietin-neural stem cells group and neural stem cells group than in the spinal cord injury group. Again, these differences were particularly obvious in the human erythropoietin-neural stem cells group. These data indicate that transplantation of erythropoietin gene-modified neural stem

  8. Transplantation of erythropoietin gene-modified neural stem cells improves the repair of injured spinal cord.

    Science.gov (United States)

    Wu, Min-Fei; Zhang, Shu-Quan; Gu, Rui; Liu, Jia-Bei; Li, Ye; Zhu, Qing-San

    2015-09-01

    The protective effects of erythropoietin on spinal cord injury have not been well described. Here, the eukaryotic expression plasmid pcDNA3.1 human erythropoietin was transfected into rat neural stem cells cultured in vitro. A rat model of spinal cord injury was established using a free falling object. In the human erythropoietin-neural stem cells group, transfected neural stem cells were injected into the rat subarachnoid cavity, while the neural stem cells group was injected with non-transfected neural stem cells. Dulbecco's modified Eagle's medium/F12 medium was injected into the rats in the spinal cord injury group as a control. At 1-4 weeks post injury, the motor function in the rat lower limbs was best in the human erythropoietin-neural stem cells group, followed by the neural stem cells group, and lastly the spinal cord injury group. At 72 hours, compared with the spinal cord injury group, the apoptotic index and Caspase-3 gene and protein expressions were apparently decreased, and the bcl-2 gene and protein expressions were noticeably increased, in the tissues surrounding the injured region in the human erythropoietin-neural stem cells group. At 4 weeks, the cavities were clearly smaller and the motor and somatosensory evoked potential latencies were remarkably shorter in the human erythropoietin-neural stem cells group and neural stem cells group than those in the spinal cord injury group. These differences were particularly obvious in the human erythropoietin-neural stem cells group. More CM-Dil-positive cells and horseradish peroxidase-positive nerve fibers and larger amplitude motor and somatosensory evoked potentials were found in the human erythropoietin-neural stem cells group and neural stem cells group than in the spinal cord injury group. Again, these differences were particularly obvious in the human erythropoietin-neural stem cells group. These data indicate that transplantation of erythropoietin gene-modified neural stem cells into the

  9. Transplantation of erythropoietin gene-modiifed neural stem cells improves the repair of injured spinal cord

    Institute of Scientific and Technical Information of China (English)

    Min-fei Wu; Shu-quan Zhang; Rui Gu; Jia-bei Liu; Ye Li; Qing-san Zhu

    2015-01-01

    The protective effects of erythropoietin on spinal cord injury have not been well described. Here, the eukaryotic expression plasmid pcDNA3.1 human erythropoietin was transfected into rat neural stem cells culturedin vitro. A rat model of spinal cord injury was established using a free falling object. In the human erythropoietin-neural stem cells group, transfected neural stem cells were injected into the rat subarachnoid cavity, while the neural stem cells group was inject-ed with non-transfected neural stem cells. Dulbecco’s modified Eagle’s medium/F12 medium was injected into the rats in the spinal cord injury group as a control. At 1–4 weeks post injury, the motor function in the rat lower limbs was best in the human erythropoietin-neural stem cells group, followed by the neural stem cells group, and lastly the spinal cord injury group. At 72 hours, compared with the spinal cord injury group, the apoptotic index and Caspase-3 gene and protein expressions were apparently decreased, and the bcl-2 gene and protein expressions were noticeably increased, in the tissues surrounding the injured region in the human erythro-poietin-neural stem cells group. At 4 weeks, the cavities were clearly smaller and the motor and somatosensory evoked potential latencies were remarkably shorter in the human erythropoi-etin-neural stem cells group and neural stem cells group than those in the spinal cord injury group. These differences were particularly obvious in the human erythropoietin-neural stem cells group. More CM-Dil-positive cells and horseradish peroxidase-positive nerve fibers and larger amplitude motor and somatosensory evoked potentials were found in the human erythro-poietin-neural stem cells group and neural stem cells group than in the spinal cord injury group. Again, these differences were particularly obvious in the human erythropoietin-neural stem cells group. These data indicate that transplantation of erythropoietin gene-modified neural stem cells into the

  10. Alcohol Consumption and the Risk of Colorectal Cancer for Mismatch Repair Gene Mutation Carriers.

    Science.gov (United States)

    Dashti, S Ghazaleh; Buchanan, Daniel D; Jayasekara, Harindra; Ait Ouakrim, Driss; Clendenning, Mark; Rosty, Christophe; Winship, Ingrid M; Macrae, Finlay A; Giles, Graham G; Parry, Susan; Casey, Graham; Haile, Robert W; Gallinger, Steven; Le Marchand, Loïc; Thibodeau, Stephen N; Lindor, Noralane M; Newcomb, Polly A; Potter, John D; Baron, John A; Hopper, John L; Jenkins, Mark A; Win, Aung Ko

    2017-03-01

    Background: People with germline mutation in one of the DNA mismatch repair (MMR) genes have increased colorectal cancer risk. For these high-risk people, study findings of the relationship between alcohol consumption and colorectal cancer risk have been inconclusive.Methods: 1,925 MMR gene mutations carriers recruited into the Colon Cancer Family Registry who had completed a questionnaire on lifestyle factors were included. Weighted Cox proportional hazard regression models were used to estimate hazard ratios (HR) and 95% confidence intervals (CI) for the association between alcohol consumption and colorectal cancer.Results: Colorectal cancer was diagnosed in 769 carriers (40%) at a mean (SD) age of 42.6 (10.3) years. Compared with abstention, ethanol consumption from any alcoholic beverage up to 14 g/day and >28 g/day was associated with increased colorectal cancer risk (HR, 1.50; 95% CI, 1.09-2.07 and 1.69; 95% CI, 1.07-2.65, respectively; Ptrend = 0.05), and colon cancer risk (HR, 1.78; 95% CI, 1.27-2.49 and 1.94; 95% CI, 1.19-3.18, respectively; Ptrend = 0.02). However, there was no clear evidence for an association with rectal cancer risk. Also, there was no evidence for associations between consumption of individual alcoholic beverage types (beer, wine, spirits) and colorectal, colon, or rectal cancer risk.Conclusions: Our data suggest that alcohol consumption, particularly more than 28 g/day of ethanol (∼2 standard drinks of alcohol in the United States), is associated with increased colorectal cancer risk for MMR gene mutation carriers.Impact: Although these data suggested that alcohol consumption in MMR carriers was associated with increased colorectal cancer risk, there was no evidence of a dose-response, and not all types of alcohol consumption were associated with increased risk. Cancer Epidemiol Biomarkers Prev; 26(3); 366-75. ©2016 AACR. ©2016 American Association for Cancer Research.

  11. Laminin Mediates Tissue-specific Gene Expression in Mammary Epithelia

    Energy Technology Data Exchange (ETDEWEB)

    Streuli, Charles H; Schmidhauser, Christian; Bailey, Nina; Yurchenco, Peter; Skubitz, Amy P. N.; Roskelley, Calvin; Bissell, Mina J

    1995-04-01

    Tissue-specific gene expression in mammary epithelium is dependent on the extracellular matrix as well as hormones. There is good evidence that the basement membrane provides signals for regulating beta-casein expression, and that integrins are involved in this process. Here, we demonstrate that in the presence of lactogenic hormones, laminin can direct expression of the beta-casein gene. Mouse mammary epithelial cells plated on gels of native laminin or laminin-entactin undergo functional differentiation. On tissue culture plastic, mammary cells respond to soluble basement membrane or purified laminin, but not other extracellular matrix components, by synthesizing beta-casein. In mammary cells transfected with chloramphenicol acetyl transferase reporter constructs, laminin activates transcription from the beta-casein promoter through a specific enhancer element. The inductive effect of laminin on casein expression was specifically blocked by the E3 fragment of the carboxy terminal region of the alpha 1 chain of laminin, by antisera raised against the E3 fragment, and by a peptide corresponding to a sequence within this region. Our results demonstrate that laminin can direct tissue-specific gene expression in epithelial cells through its globular domain.

  12. Heteroduplex DNA mismatch repair system of Streptococcus pneumoniae: cloning and expression of the hexA gene.

    OpenAIRE

    Balganesh, T S; Lacks, S A

    1985-01-01

    Mutations affecting heteroduplex DNA mismatch repair in Streptococcus pneumoniae were localized in two genes, hexA and hexB, by fractionation of restriction fragments carrying mutant alleles. A fragment containing the hexA4 allele was cloned in the S. pneumoniae cloning system, and the hexA+ allele was introduced into the recombinant plasmid by chromosomal facilitation of plasmid transfer. Subcloning localized the functional hexA gene to a 3.5-kilobase segment of the cloned pneumococcal DNA. ...

  13. Topoisomerase-1 and -2A gene copy numbers are elevated in mismatch repair-proficient colorectal cancers

    DEFF Research Database (Denmark)

    Sønderstrup, Ida Marie Heeholm; Nygård, Sune Boris; Poulsen, Tim Svenstrup

    2015-01-01

    (MMR) subtypes of CRC have been associated with benefit from adjuvant chemotherapy of primary CRC. Given the involvement of the topoisomerase enzymes in DNA replication and repair, we raised the hypothesis that an association may exist between TOP gene copy numbers and MMR proficiency/deficiency in CRC...... patients with deficient MMR (dMMR) CRC. TOP1 and TOP2A gene copy numbers and their ratios per nucleus were correlated with MMR status using the Mann-Whitney test. Validation cohort: FFPE samples from 154 patients with primary stage III CRC (originally included in the RANX05 study) were classified according...

  14. Two DNA repair and recombination genes in Saccharomyces cerevisiae, RAD52 and RAD54, are induced during meiosis

    Energy Technology Data Exchange (ETDEWEB)

    Cole, G.M.; Mortimer, R.K. (Univ. of California, Berkeley (United States)); Schild, D. (Lawrence Berkeley Lab., CA (United States))

    1989-07-01

    The DNA repair and recombination genes of Saccharomyces cerevisiae, RAD52 and RAD54, were transcriptionally induced approximately 10- to 15-fold in sporulating MATa/{alpha} cells. Congenic MATa/a cells, which did not sporulate, did not show similar increases. Assays of {beta}-galactosidase activity in strains harboring either a RAD52- or RAD54-lacZ gene fusion indicated that this induction occurred at a time concomitant with a commitment to meiotic recombination, as measured by prototroph formation from his1 heteroalleles.

  15. RAD1 and RAD10, but not other excision repair genes, are required for double-strand break-induced recombination in Saccharomyces cerevisiae.

    Science.gov (United States)

    Ivanov, E L; Haber, J E

    1995-04-01

    HO endonuclease-induced double-strand breaks (DSBs) in the yeast Saccharomyces cerevisiae can be repaired by the process of gap repair or, alternatively, by single-strand annealing if the site of the break is flanked by directly repeated homologous sequences. We have shown previously (J. Fishman-Lobell and J. E. Haber, Science 258:480-484, 1992) that during the repair of an HO-induced DSB, the excision repair gene RAD1 is needed to remove regions of nonhomology from the DSB ends. In this report, we present evidence that among nine genes involved in nucleotide excision repair, only RAD1 and RAD10 are required for removal of nonhomologous sequences from the DSB ends. rad1 delta and rad10 delta mutants displayed a 20-fold reduction in the ability to execute both gap repair and single-strand annealing pathways of HO-induced recombination. Mutations in RAD2, RAD3, and RAD14 reduced HO-induced recombination by about twofold. We also show that RAD7 and RAD16, which are required to remove UV photodamage from the silent HML, locus, are not required for MAT switching with HML or HMR as a donor. Our results provide a molecular basis for understanding the role of yeast nucleotide excision repair gene and their human homologs in DSB-induced recombination and repair.

  16. Human DJ-1-specific Transcriptional Activation of Tyrosine Hydroxylase Gene*

    Science.gov (United States)

    Ishikawa, Shizuma; Taira, Takahiro; Takahashi-Niki, Kazuko; Niki, Takeshi; Ariga, Hiroyoshi; Iguchi-Ariga, Sanae M. M.

    2010-01-01

    Loss-of-function mutation in the DJ-1 gene causes a subset of familial Parkinson disease. The mechanism underlying DJ-1-related selective vulnerability in the dopaminergic pathway is, however, not known. DJ-1 has multiple functions, including transcriptional regulation, and one of transcriptional target genes for DJ-1 is the tyrosine hydroxylase (TH) gene, the product of which is a key enzyme for dopamine biosynthesis. It has been reported that DJ-1 is a neuroprotective transcriptional co-activator that sequesters a transcriptional co-repressor polypyrimidine tract-binding protein-associated splicing factor (PSF) from the TH gene promoter. In this study, we found that knockdown of human DJ-1 by small interference RNA in human dopaminergic cell lines attenuated TH gene expression and 4-dihydroxy-l-phenylalanine production but that knockdown or knock-out of mouse DJ-1 in mouse cell lines or in mice did not affect such expression and TH activity. In reporter assays using the human TH gene promoter linked to the luciferase gene, stimulation of TH promoter activity was observed in human cells, but not mouse cells, that had been transfected with DJ-1. Although human DJ-1 and mouse DJ-1 were associated either with human or with mouse PSF, TH promoter activity inhibited by PSF was restored by human DJ-1 but not by mouse DJ-1. Chromatin immunoprecipitation assays revealed that the complex of PSF with DJ-1 bound to the human but not the mouse TH gene promoter. These results suggest a novel species-specific transcriptional regulation of the TH promoter by DJ-1 and one of the mechanisms for no reduction of TH in DJ-1-knock-out mice. PMID:20938049

  17. Universal and specific quantitative detection of botulinum neurotoxin genes

    Directory of Open Access Journals (Sweden)

    Arnon Stephen S

    2010-10-01

    Full Text Available Abstract Background Clostridium botulinum, an obligate anaerobic spore-forming bacterium, produces seven antigenic variants of botulinum toxin that are distinguished serologically and termed "serotypes". Botulinum toxin blocks the release of acetylcholine at neuromuscular junctions resulting in flaccid paralysis. The potential lethality of the disease warrants a fast and accurate means of diagnosing suspected instances of food contamination or human intoxication. Currently, the Food and Drug Administration (FDA-accepted assay to detect and type botulinum neurotoxins (BoNTs is the mouse protection bioassay. While specific and sensitive, this assay requires the use of laboratory animals, may take up to four days to achieve a diagnosis, and is unsuitable for high-throughput analysis. We report here a two-step PCR assay that identifies all toxin types, that achieves the specificity of the mouse bioassay while surpassing it in equivalent sensitivity, that has capability for high-throughput analysis, and that provides quantitative results within hours. The first step of our assay consists of a conventional PCR that detects the presence of C. botulinum regardless of the neurotoxin type. The second step uses quantitative PCR (qPCR technology to determine the specific serotype of the neurotoxin. Results We assayed purified C. botulinum DNA and crude toxin preparations, as well as food and stool from healthy individuals spiked with purified BoNT DNA, and one stool sample from a case of infant botulism for the presence of the NTNH gene, which is part of the BoNT gene cluster, and for the presence of serotype-specific BoNT genes. The PCR surpassed the mouse bioassay both in specificity and sensitivity, detecting positive signals in BoNT preparations containing well below the 1 LD50 required for detection via the mouse bioassay. These results were type-specific and we were reliably able to quantify as few as 10 genomic copies. Conclusions While other studies

  18. Repair genes expression profile of MLH1, MSH2 and ATM in the normal oral mucosa of chronic smokers.

    Science.gov (United States)

    Alves, Mônica Ghislaine Oliveira; Carta, Celina Faig Lima; de Barros, Patrícia Pimentel; Issa, Jaqueline Scholz; Nunes, Fábio Daumas; Almeida, Janete Dias

    2017-01-01

    The aim of this study was to evaluate the effect of chronic smoking on the expression profile of the repair genes MLH1, MSH2 and ATM in the normal oral mucosa of chronic smokers and never smokers. The sample consisted of thirty exfoliative cytology smears per group obtained from Smokers and Never Smokers. Total RNA was extracted and expression of the MLH1, MSH2 and ATM genes were evaluated by quantitative real-time and immunocytochemistry. The gene and protein expression data were correlated to the clinical data. Gene expression was analyzed statistically using the Student t-test and Pearson's correlation coefficient, with pexpression of MLH1, MSH2, ATM and age, number of cigarettes consumed per day, time of smoking during life, smoking history or levels of CO in expired air. The expression of genes and proteins related to DNA repair mechanism MLH1, MSH2 and ATM in the normal oral mucosa of chronic smokers was reduced. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Repressor-mediated tissue-specific gene expression in plants

    Science.gov (United States)

    Meagher, Richard B.; Balish, Rebecca S.; Tehryung, Kim; McKinney, Elizabeth C.

    2009-02-17

    Plant tissue specific gene expression by way of repressor-operator complexes, has enabled outcomes including, without limitation, male sterility and engineered plants having root-specific gene expression of relevant proteins to clean environmental pollutants from soil and water. A mercury hyperaccumulation strategy requires that mercuric ion reductase coding sequence is strongly expressed. The actin promoter vector, A2pot, engineered to contain bacterial lac operator sequences, directed strong expression in all plant vegetative organs and tissues. In contrast, the expression from the A2pot construct was restricted primarily to root tissues when a modified bacterial repressor (LacIn) was coexpressed from the light-regulated rubisco small subunit promoter in above-ground tissues. Also provided are analogous repressor operator complexes for selective expression in other plant tissues, for example, to produce male sterile plants.

  20. DNA repair and damage pathway related cancer suppressor genes in low-dose-rate irradiated AKR/J an IR mice

    Energy Technology Data Exchange (ETDEWEB)

    Bang, Hyun Soon; Bong, Jin Jong; Kang, Yumi; Choi, Moo Hyun; Lee, Hae Un; Yoo, Jae Young; Choi, Seung Jin; Kim, Hee Sun [Radiation Health Research Institute, Korea Hydro and Nuclear Power Co., Ltd, Gyeongju (Korea, Republic of); Lee, Kyung Mi [Global Research Lab, BAERI Institute, Dept. of Biochemistry and Molecular Biology, Korea University College of Medicine, Seoul (Korea, Republic of)

    2012-11-15

    It has been reported that low-dose-rate radiation stimulates the immune response, prolongs life span and inhibits carcinogenesis. The high dose-rate radiation influences the expression of DNA repair and damage-related genes. In contrast, DNA repair and damage signaling triggered by low-dose-rate irradiation remain unclear. In the present study, we investigated the differential expression of DNA repair and damage pathway related genes in the thymus of AKR/J and ICR mice after 100th day low-dose-rate irradiation. Our findings demonstrated that low-dose-rate γ -radiation suppressed tumorigenesis.

  1. Prognostic impact of mismatch repair genes germline defects in colorectal cancer patients: are all mutations equal?

    Science.gov (United States)

    Maccaroni, Elena; Bracci, Raffaella; Giampieri, Riccardo; Bianchi, Francesca; Belvederesi, Laura; Brugiati, Cristiana; Pagliaretta, Silvia; Del Prete, Michela; Scartozzi, Mario; Cascinu, Stefano

    2015-01-01

    Background Lynch syndrome (LS) is the most common hereditary colorectal cancer (CRC) syndrome, caused by germline mutations in MisMatch Repair (MMR) genes, particularly in MLH1, MSH2 and MSH6. Patients with LS seem to have a more favourable prognosis than those with sporadic CRC, although the prognostic impact of different mutation types is unknown. Aim of our study is to compare survival outcomes of different types of MMR mutations in patients with LS-related CRC. Methods 302 CRC patients were prospectively selected on the basis of Amsterdam or Revised Bethesda criteria to undergo genetic testing: direct sequencing of DNA and MLPA were used to examine the entire MLH1, MSH2 and MSH6 coding sequence. Patients were classified as mutation-positive or negative according to the genetic testing result. Results A deleterious MMR mutation was found in 38/302 patients. Median overall survival (OS) was significantly higher in mutation-positive vs mutation-negative patients (102.6 vs 77.7 months, HR:0.63, 95%CI:0.46–0.89, p = 0.0083). Different types of mutation were significantly related with OS: missense or splicing-site mutations were associated with better OS compared with rearrangement, frameshift or non-sense mutations (132.5 vs 82.5 months, HR:0.46, 95%CI:0.16–0.82, p = 0.0153). Conclusions Our study confirms improved OS for LS-patients compared with mutation-negative CRC patients. In addition, not all mutations could be considered equal: the better prognosis in CRC patients with MMR pathogenic missense or splicing site mutation could be due to different functional activity of the encoded MMR protein. This matter should be investigated by use of functional assays in the future. PMID:26485756

  2. Important aspects of placental-specific gene transfer.

    Science.gov (United States)

    Kaufman, Melissa R; Albers, Renee E; Keoni, Chanel; Kulkarni-Datar, Kashmira; Natale, David R; Brown, Thomas L

    2014-10-15

    The placenta is a unique and highly complex organ that develops only during pregnancy and is essential for growth and survival of the developing fetus. The placenta provides the vital exchange of gases and wastes, the necessary nutrients for fetal development, acts as immune barrier that protects against maternal rejection, and produces numerous hormones and growth factors that promote fetal maturity to regulate pregnancy until parturition. Abnormal placental development is a major underlying cause of pregnancy-associated disorders that often result in preterm birth. Defects in placental stem cell propagation, growth, and differentiation are the major factors that affect embryonic and fetal well-being and dramatically increase the risk of pregnancy complications. Understanding the processes that regulate placentation is important in determining the underlying factors behind abnormal placental development. The ability to manipulate genes in a placenta-specific manner provides a unique tool to analyze development and eliminates potentially confounding results that can occur with traditional gene knockouts. Trophoblast stem cells and mouse embryos are not overly amenable to traditional gene transfer techniques. Most viral vectors, however, have a low infection rate and often lead to mosaic transgenesis. Although the traditional method of embryo transfer is intrauterine surgical implantation, the methodology reported here, combining lentiviral blastocyst infection and nonsurgical embryo transfer, leads to highly efficient and placental-specific gene transfer. Numerous advantages of our optimized procedures include increased investigator safety, a reduction in animal stress, rapid and noninvasive embryo transfer, and higher a rate of pregnancy and live birth.

  3. Candidate driver genes involved in genome maintenance and DNA repair in Sézary syndrome.

    Science.gov (United States)

    Woollard, Wesley J; Pullabhatla, Venu; Lorenc, Anna; Patel, Varsha M; Butler, Rosie M; Bayega, Anthony; Begum, Nelema; Bakr, Farrah; Dedhia, Kiran; Fisher, Joshua; Aguilar-Duran, Silvia; Flanagan, Charlotte; Ghasemi, Aria A; Hoffmann, Ricarda M; Castillo-Mosquera, Nubia; Nuttall, Elisabeth A; Paul, Arisa; Roberts, Ceri A; Solomonidis, Emmanouil G; Tarrant, Rebecca; Yoxall, Antoinette; Beyers, Carl Z; Ferreira, Silvia; Tosi, Isabella; Simpson, Michael A; de Rinaldis, Emanuele; Mitchell, Tracey J; Whittaker, Sean J

    2016-06-30

    Sézary syndrome (SS) is a leukemic variant of cutaneous T-cell lymphoma (CTCL) and represents an ideal model for study of T-cell transformation. We describe whole-exome and single-nucleotide polymorphism array-based copy number analyses of CD4(+) tumor cells from untreated patients at diagnosis and targeted resequencing of 101 SS cases. A total of 824 somatic nonsynonymous gene variants were identified including indels, stop-gain/loss, splice variants, and recurrent gene variants indicative of considerable molecular heterogeneity. Driver genes identified using MutSigCV include POT1, which has not been previously reported in CTCL; and TP53 and DNMT3A, which were also identified consistent with previous reports. Mutations in PLCG1 were detected in 11% of tumors including novel variants not previously described in SS. This study is also the first to show BRCA2 defects in a significant proportion (14%) of SS tumors. Aberrations in PRKCQ were found to occur in 20% of tumors highlighting selection for activation of T-cell receptor/NF-κB signaling. A complex but consistent pattern of copy number variants (CNVs) was detected and many CNVs involved genes identified as putative drivers. Frequent defects involving the POT1 and ATM genes responsible for telomere maintenance were detected and may contribute to genomic instability in SS. Genomic aberrations identified were enriched for genes implicated in cell survival and fate, specifically PDGFR, ERK, JAK STAT, MAPK, and TCR/NF-κB signaling; epigenetic regulation (DNMT3A, ASLX3, TET1-3); and homologous recombination (RAD51C, BRCA2, POLD1). This study now provides the basis for a detailed functional analysis of malignant transformation of mature T cells and improved patient stratification and treatment.

  4. Activation of GATA4 gene expression at the early stage of cardiac specification

    Directory of Open Access Journals (Sweden)

    Ayse eYilbas

    2014-03-01

    Full Text Available Currently, there are no effective treatments to directly repair damaged heart tissue after cardiac injury since existing therapies focus on rescuing or preserving reversibly damaged tissue. Cell-based therapies using cardiomyocytes generated from stem cells present a promising therapeutic approach to directly replace damaged myocardium with new healthy tissue. However, the molecular mechanisms underlying the commitment of stem cells into cardiomyocytes are not fully understood and will be critical to guide this new technology into the clinic. Since GATA4 is a critical regulator of cardiac differentiation, we examined the molecular basis underlying the early activation of GATA4 gene expression during cardiac differentiation of pluripotent stem cells. Our studies demonstrate the direct involvement of histone acetylation and transcriptional coactivator p300 in the regulation of GATA4 gene expression. More importantly, we show that histone acetyltransferase (HAT activity is important for GATA4 gene expression with the use of curcumin, a HAT inhibitor. In addition, the widely used histone deacetylase inhibitor valproic acid enhances both histone acetylation and cardiac specification.

  5. Activation of GATA4 gene expression at the early stage of cardiac specification

    Science.gov (United States)

    Yilbas, Ayse; Hamilton, Alison; Wang, Yingjian; Mach, Hymn; Lacroix, Natascha; Davis, Darryl; Chen, Jihong; LI, Qiao

    2014-03-01

    Currently, there are no effective treatments to directly repair damaged heart tissue after cardiac injury since existing therapies focus on rescuing or preserving reversibly damaged tissue. Cell-based therapies using cardiomyocytes generated from stem cells present a promising therapeutic approach to directly replace damaged myocardium with new healthy tissue. However, the molecular mechanisms underlying the commitment of stem cells into cardiomyocytes are not fully understood and will be critical to guide this new technology into the clinic. Since GATA4 is a critical regulator of cardiac differentiation, we examined the molecular basis underlying the early activation of GATA4 gene expression during cardiac differentiation of pluripotent stem cells. Our studies demonstrate the direct involvement of histone acetylation and transcriptional coactivator p300 in the regulation of GATA4 gene expression. More importantly, we show that histone acetyltransferase (HAT) activity is important for GATA4 gene expression with the use of curcumin, a HAT inhibitor. In addition, the widely used histone deacetylase inhibitor valproic acid enhances both histone acetylation and cardiac specification.

  6. Polymorphisms in DNA repair genes XRCC2 and XRCC3 risk of gastric cancer in Turkey

    Directory of Open Access Journals (Sweden)

    İlhami Gok

    2014-09-01

    Full Text Available We studied the prevalence of polymorphisms in genes XRCC2 and XRCC3 in stomach cancer patients who lived in North Eastern Turkey. A total of 61 cancer patients and 78 controls were included in this study. Single nucleotide changes were studied in XRCC2 and XRCC3 genes at locus Arg188His and Thr241Met. Blood samples were taken from the patients and controls, and DNA was isolated. The regions of interest were amplified using a polymerase chain reaction method. After amplification, we used restriction enzymes (HphI and NcoI to digest the amplified product. Digested product was then run through gel electrophoresis. We identified changes in the nucleotides in these specific regions. It was found that the Arg188His polymorphism of the XRCC2 gene was about 39% (24 out of the 61 among cancer patients. However, only 15% (12 out of 78 of the control group indicated this polymorphism. We also observed that 18 of the 61 cancer patients (29% carried the Thr241Met polymorphism of the XRCC3 gene whereas 11 of the 78 (14% individuals in the control group had the polymorphism. Our results showed a significant difference in polymorphism ratios between the cancer patients and health control group for the regions of interest. This result clearly showed that these polymorphisms increase the risk of stomach cancer and might be a strong marker for early diagnosis of gastric cancer.

  7. Failure to induce a DNA repair gene, RAD54, in Saccharomyces cerevisiae does not affect DNA repair or recombination phenotypes

    Energy Technology Data Exchange (ETDEWEB)

    Cole, G.M.; Mortimer, R.K. (Univ. of California, Berkeley (USA))

    1989-08-01

    The Saccharomyces cerevisiae RAD54 gene is transcriptionally regulated by a broad spectrum of DNA-damaging agents. Induction of RAD54 by DNA-damaging agents is under positive control. Sequences responsible for DNA damage induction (the DRS element) lie within a 29-base-pair region from -99 to -70 from the most proximal transcription start site. This inducible promoter element is functionally separable from a poly(dA-dT) region immediately downstream which is required for constitutive expression. Deletions which eliminate induction of RAD54 transcription by DNA damage but do not affect constitutive expression have no effect on growth or survival of noninducible strains relative to wild-type strains in the presence of DNA-damaging agents. The DRS element is also not required for homothallic mating type switching, transcriptional induction of RAD54 during meiosis, meiotic recombination, or spontaneous or X-ray-induced mitotic recombination. We find no phenotype for a lack of induction of RAD54 message via the damage-inducible DRS, which raises significant questions about the physiology of DNA damage induction in S. cerevisiae.

  8. Homology Requirements and Competition between Gene Conversion and Break-Induced Replication during Double-Strand Break Repair.

    Science.gov (United States)

    Mehta, Anuja; Beach, Annette; Haber, James E

    2017-02-02

    Saccharomyces cerevisiae mating-type switching is initiated by a double-strand break (DSB) at MATa, leaving one cut end perfectly homologous to the HMLα donor, while the second end must be processed to remove a non-homologous tail before completing repair by gene conversion (GC). When homology at the matched end is ≤150 bp, efficient repair depends on the recombination enhancer, which tethers HMLα near the DSB. Thus, homology shorter than an apparent minimum efficient processing segment can be rescued by tethering the donor near the break. When homology at the second end is ≤150 bp, second-end capture becomes inefficient and repair shifts from GC to break-induced replication (BIR). But when pol32 or pif1 mutants block BIR, GC increases 3-fold, indicating that the steps blocked by these mutations are reversible. With short second-end homology, absence of the RecQ helicase Sgs1 promotes gene conversion, whereas deletion of the FANCM-related Mph1 helicase promotes BIR. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Complex oncogenic translocations with gene amplification are initiated by specific DNA breaks in lymphocytes.

    Science.gov (United States)

    Wright, Sarah M; Woo, Yong H; Alley, Travis L; Shirley, Bobbi-Jo; Akeson, Ellen C; Snow, Kathy J; Maas, Sarah A; Elwell, Rachel L; Foreman, Oded; Mills, Kevin D

    2009-05-15

    Chromosomal instability is a hallmark of many tumor types. Complex chromosomal rearrangements with associated gene amplification, known as complicons, characterize many hematologic and solid cancers. Whereas chromosomal aberrations, including complicons, are useful diagnostic and prognostic cancer markers, their molecular origins are not known. Although accumulating evidence has implicated DNA double-strand break repair in suppression of oncogenic genome instability, the genomic elements required for chromosome rearrangements, especially complex lesions, have not been elucidated. Using a mouse model of B-lineage lymphoma, characterized by complicon formation involving the immunoglobulin heavy chain (Igh) locus and the c-myc oncogene, we have now investigated the requirement for specific genomic segments as donors for complex rearrangements. We now show that specific DNA double-strand breaks, occurring within a narrow segment of Igh, are necessary to initiate complicon formation. By contrast, neither specific DNA breaks nor the powerful intronic enhancer Emu are required for complicon-independent oncogenesis. This study is the first to delineate mechanisms of complex versus simple instability and the first to identify specific chromosomal elements required for complex chromosomal aberrations. These findings will illuminate genomic cancer susceptibility and risk factors.

  10. The CREB Coactivator CRTC2 is a Lymphoma Tumor Suppressor that Preserves Genome Integrity Through Transcription of DNA Mismatch Repair Genes

    Science.gov (United States)

    Fang, Minggang; Pak, Magnolia L.; Chamberlain, Lynn; Xing, Wei; Yu, Hongbo; Green, Michael R.

    2015-01-01

    SUMMARY The CREB-regulated transcription coactivator CRTC2 stimulates CREB target gene expression and has a well-established role in modulating glucose and lipid metabolism. Here we find, unexpectedly, that loss of CRTC2, as well as CREB1 and its coactivator CREB-binding protein (CBP), results in a deficiency in DNA mismatch repair (MMR) and a resultant increased mutation frequency. We show that CRTC2, CREB1 and CBP are transcriptional activators of well-established MMR genes, including EXO1, MSH6, PMS1 and POLD2. Mining of expression profiling databases and analysis of patient samples reveal that CRTC2 and its target MMR genes are down-regulated in specific T-cell lymphoma subtypes, which are microsatellite unstable. The levels of acetylated histone H3 on the CRTC2 promoter are significantly reduced in lymphoma compared to normal tissue, explaining the decreased CRTC2 expression. Our results establish a role for CRTC2 as a lymphoma tumor suppressor gene that preserves genome integrity by stimulating transcription of MMR genes. PMID:26004186

  11. Epstein-Barr virus-specific methylation of human genes in gastric cancer cells

    Directory of Open Access Journals (Sweden)

    Coleman William B

    2010-12-01

    Full Text Available Abstract Background Epstein-Barr Virus (EBV is found in 10% of all gastric adenocarcinomas but its role in tumor development and maintenance remains unclear. The objective of this study was to examine EBV-mediated dysregulation of cellular factors implicated in gastric carcinogenesis. Methods Gene expression patterns were examined in EBV-negative and EBV-positive AGS gastric epithelial cells using a low density microarray, reverse transcription PCR, histochemical stains, and methylation-specific DNA sequencing. Expression of PTGS2 (COX2 was measured in AGS cells and in primary gastric adenocarcinoma tissues. Results In array studies, nearly half of the 96 human genes tested, representing 15 different cancer-related signal transduction pathways, were dysregulated after EBV infection. Reverse transcription PCR confirmed significant impact on factors having diverse functions such as cell cycle regulation (IGFBP3, CDKN2A, CCND1, HSP70, ID2, ID4, DNA repair (BRCA1, TFF1, cell adhesion (ICAM1, inflammation (COX2, and angiogenesis (HIF1A. Demethylation using 5-aza-2'-deoxycytidine reversed the EBV-mediated dysregulation for all 11 genes listed here. For some promoter sequences, CpG island methylation and demethylation occurred in an EBV-specific pattern as shown by bisulfite DNA sequencing. Immunohistochemistry was less sensitive than was western blot for detecting downregulation of COX2 upon EBV infection. Virus-related dysregulation of COX2 levels in vitro was not recapitulated in vivo among naturally infected gastric cancer tissues. Conclusions EBV alters human gene expression in ways that could contribute to the unique pathobiology of virus-associated cancer. Furthermore, the frequency and reversability of methylation-related transcriptional alterations suggest that demethylating agents have therapeutic potential for managing EBV-related carcinoma.

  12. Comparative analysis of meiotic progression in female mice bearing mutations in genes of the DNA mismatch repair pathway.

    Science.gov (United States)

    Kan, Rui; Sun, Xianfei; Kolas, Nadine K; Avdievich, Elena; Kneitz, Burkhard; Edelmann, Winfried; Cohen, Paula E

    2008-03-01

    The DNA mismatch repair (MMR) family functions in a variety of contexts to preserve genome integrity in most eukaryotes. In particular, members of the MMR family are involved in the process of meiotic recombination in germ cells. MMR gene mutations in mice result in meiotic disruption during prophase I, but the extent of this disruption often differs between male and female meiocytes. To address the role of MMR proteins specifically in female meiosis, we explored the progression of oocytes through prophase I and the meiotic divisions in mice harboring deletions in members of the MMR pathway (Mlh1, Mlh3, Exo1, and an ATPase-deficient variant of Mlh1, Mlh1(G67R)). The colocalization of MLH1 and MLH3, key proteins involved in stabilization of nascent crossovers, was dependent on intact heterodimer formation and was highly correlated with the ability of oocytes to progress through to metaphase II. The exception was Exo1(-/-) oocytes, in which normal MLH1/MLH3 localization was observed followed by failure to proceed to metaphase II. All mutant oocytes were able to resume meiosis after dictyate arrest, but they showed a dramatic decline in chiasmata (to less than 25% of normal), accompanied by varied progression through metaphase I. Taken together, these results demonstrate that MMR function is required for the formation and stabilization of crossovers in mammalian oocytes and that, in the absence of a functional MMR system, the failure to maintain chiasmata results in a reduced ability to proceed normally through the first and second meiotic divisions, despite near-normal levels of meiotic resumption after dictyate arrest.

  13. Deficiency in nucleotide excision repair family gene activity, especially ERCC3, is associated with non-pigmented hair fiber growth.

    Directory of Open Access Journals (Sweden)

    Mei Yu

    Full Text Available We conducted a microarray study to discover gene expression patterns associated with a lack of melanogenesis in non-pigmented hair follicles (HF by microarray. Pigmented and non-pigmented HFs were collected and micro-dissected into the hair bulb (HB and the upper hair sheaths (HS including the bulge region. In comparison to pigmented HS and HBs, nucleotide excision repair (NER family genes ERCC1, ERCC2, ERCC3, ERCC4, ERCC5, ERCC6, XPA, NTPBP, HCNP, DDB2 and POLH exhibited statistically significantly lower expression in non- pigmented HS and HBs. Quantitative PCR verified microarray data and identified ERCC3 as highly differentially expressed. Immunohistochemistry confirmed ERCC3 expression in HF melanocytes. A reduction in ERCC3 by siRNA interference in human melanocytes in vitro reduced their tyrosinase production ability. Our results suggest that loss of NER gene function is associated with a loss of melanin production capacity. This may be due to reduced gene transcription and/or reduced DNA repair in melanocytes which may eventually lead to cell death. These results provide novel information with regard to melanogenesis and its regulation.

  14. DNA Repair Gene Polymorphisms in the Nucleotide Excision Repair Pathway and Lung Cancer Risk: A Meta-analysis

    Institute of Scientific and Technical Information of China (English)

    Chao-rong Mei; Meng Luo; Hong-mei Li; Wen-jun Deng; Qing-hua Zhou

    2011-01-01

    Objective: A number of studies have reported the association of “XPA”, “XPC”, “XPD/ERCC2” gene polymorphisms with lung cancer risk. However, the results were conflict. To clarify the impact of polymorphisms of “XPA”, “XPC”; “XPD/ERCC2”, on lung cancer risk, a meta-analysis was performed in this study. Methods: The electronic databases PubMed and Embase were retrieved for studies included in this meta-analysis by “XPA”; “XPC”, “XPD/ERCC2”, “lung”, “cancer/neoplasm/tumor/carcinoma”, “polymorphism” (An upper date limit of October, 31, 2009). A meta-analysis was performed to evaluate the relationship among XPA, XPC and XPD polymorphism and lung cancer risks. Results: A total of 31 publications retrieved from Pubmed and Embase included in this study. XPC A939C CC genotype increased lung cancer risk in total population (recessive genetic model: OR=1.23, 95% Cl:1.05-1.44;homozygote comparison: OR=1.21,95%Cl:1.02-1.43and CC vs. CA contrast: OR=1.25,95%Cl:1.06-1.48), except in Asians. XPD A751C, 751C allele and CC genotype also increased lung cancer risk in total population and in Caucasians (recessive genetic model: Total population: OR=1.20, 95%Cl:1.07-1.35). No significant correlation was found between XPD A751C and lung cancer risk in Asians and African Americans. XPD G312A AA genotype increased lung cancer risk in total population, in Asians and Caucasians(recessive genetic model: Total population: OR=1.20, 95%Cl:1.06-1.36). No significant association was found between XPA G23A, XPC C499T, XPD C156A and lung cancer risk. Conclusion: Our results suggest that the polymorphisms in XPC and XPD involve in lung cancer risks. XPA polymorphisms is less related to lung cancer risk.

  15. The imaging assessment and specific endograft design for the endovascular repair of ascending aortic dissection

    Directory of Open Access Journals (Sweden)

    Zhang Y

    2016-07-01

    (10.4%, 22 (45.9%, 13 (27.1%, six (12.5%, and two (4.2% patients, respectively. Conclusion: In this selected number of Chinese patients, the suitability of endovascular repair has been demonstrated based on the CT imaging. Shorter, larger, and bare spring-free conical endografts were preferred in the ascending aortic pathology. Keywords: type A dissection, endovascular, endograft, design

  16. Association Between Polymorphisms of DNA Repair Gene XRCC1 and DNA Damage in Asbestos-Exposed Workers

    Institute of Scientific and Technical Information of China (English)

    XIAO-HONG ZHAO; GUANG JIA; YONG-QUAN LIU; SHAO-WEI LIU; LEI YAN; YU JIN; NIAN LIU

    2006-01-01

    Objective To compare the asbestos-induced DNA damage and repair capacities of DNA damage between 104 asbestos exposed workers and 101 control workers in Qingdao City of China and to investigate the possible association between polymorphisms in codon 399 of XRCC1 and susceptibility to asbestosis. Methods DNA damage levels in peripheral bloodlymphocytes were determined by comet assay, and XRCC 1 genetic polymorphisms of DNA samples from 51 asbestosis cases and 53 non-asbestosis workers with a similar asbestos exposure history were analyzed by PCR/RFLP. Results The basal comet scores (3.95±2.95) were significantly higher in asbestos-exposed workers than in control workers (0.10±0.28). After 1 h H2O2 stimulation, DNA damage of lymphocytes exhibited different increases. After a 4 h repair period, the comet scores were 50.98±19.53 in asbestos-exposed workers and 18.32±12.04 in controls. The residual DNA damage (RD) was significantly greater (P<0.01) in asbestos-exposed workers (35.62%) than in controls (27.75%). XRCC1 genetic polymorphism in 104 asbestos-exposed workers was not associated with increased risk of asbestosis. But compared with polymorphisms in the DNA repair gene XRCC1 (polymorphisms in codon 399) and the DNA damage induced by asbestos, the comet scores in asbestosis cases with Gln/Gln, Gln/Arg, and Arg/Arg were 40.26±18.94, 38.03±28.22, and 32.01±11.65, respectively, which were higher than those in non-asbestosis workers with the same genotypes (25.58±11.08, 37.08±14.74, and 29.38±10.15). There were significant differences in the comet scores between asbestosis cases and non-asbestosis workers with Gln/Gln by Student's t-test (P<0.05 or 0.01). The comet scores were higher in asbestosis workers with Gln/Gln than in those with Arg/Arg and in non-asbestosis workers exposed to asbestos, but without statistically significant difference. Conclusions Exposure to asbestos may be related to DNA damage or the capacity of cells to repair H2O2-induced

  17. Gene expression profiles of hepatic cell-type specific marker genes in progression of liver fibrosis

    Institute of Scientific and Technical Information of China (English)

    Yoshiyuki Takahara; Mitsuo Takahashi; Hiroki Wagatsuma; Fumihiko Yokoya; Qing-Wei Zhang; Mutsuyo Yamaguchi; Hiroyuki Aburatani; Norifumi Kawada

    2006-01-01

    AIM: To determine the gene expression profile data for the whole liver during development of dimethylnitrosamine (DMN)-induced hepatic fibrosis.METHODS: Marker genes were identified for different types of hepatic cells, including hepatic stellate cells (HSCs), Kupffer cells (including other inflammatory cells),and hepatocytes, using independent temporal DNA microarray data obtained from isolated hepatic cells.RESULTS: The cell-type analysis of gene expression gave several key results and led to formation of three hypotheses: (1) changes in the expression of HSCspecific marker genes during fibrosis were similar to gene expression data in in vitro cultured HSCs, suggesting a major role of the self-activating characteristics of HSCs in formation of fibrosis; (2) expression of mast cell-specific marker genes reached a peak during liver fibrosis,suggesting a possible role of mast cells in formation of fibrosis; and (3) abnormal expression of hepatocytespecific marker genes was found across several metabolic pathways during fibrosis, including sulfur-containing amino acid metabolism, fatty acid metabolism, and drug metabolism, suggesting a mechanistic relationship between these abnormalities and symptoms of liver fibrosis.CONCLUSION: Analysis of marker genes for specific hepatic cell types can identify the key aspects of fibrogenesis. Sequential activation of inflammatory cells and the self-supporting properties of HSCs play an important role in development of fibrosis.

  18. Site-specific gene expression patterns in oral cancer.

    Science.gov (United States)

    Frohwitter, Gesche; Buerger, Horst; Korsching, Eberhard; van Diest, Paul J; Kleinheinz, Johannes; Fillies, Thomas

    2017-05-10

    Squamous cell carcinomas (SCCs) are the most prevalent malignant tumours within the head and neck. Evidence exists that distinct genes are differentially regulated in SCCs of the oral cavity compared to other head and neck regions. Given this background, the aim of this study was to investigate whether such tumour site-specific gene expression can also be observed in different localizations within the oral cavity. Using tissue microarrays (TMAs), we investigated 76 SCCs of the floor of the mouth, 49 SCCs of the tongue and 68 SCCs of other anatomic regions within the oral cavity. The expression of 17 genes involved in cell cycle and growth control (p16, p21, p27, p53, cyclin D1, EGFR, c-kit, bcl-6), cell adhesion (alpha-, beta-, and gamma-catenin), and apoptosis/stress response genes (Hif-1-alpha, Glut 1, CA IX, caspase, hsp70, XIAP) were investigated by means of immunohistochemistry. The data were subjected to chi(2), interdependency and Kaplan-Meier analysis. Our study suggests a remote difference in the site-specific gene expression patterns of oral cancer. X-linked inhibitor of apoptosis (XIAP) showed a significantly higher expression (p oral cavity. The increased XIAP expression was further associated with significantly decreased overall survival in all cases of SCCs of the oral cavity (p Expression levels of p53, CA IX, beta-catenin, Hif-1-alpha, and c-kit were also observed to be inversely related between SCCs of the floor of the mouth and those of the tongue respectively, although these differences did not reach statistical significance. Overall and event-free survival did not differ in patients with T1/T2/N0 SCCs according to tumour localization. In summary, the protein expression patterns of SCCs of the oral cavity suggest the existence of a molecular and morphological spectrum of SCCs in the oral cavity. In particular the expression pattern of XIAP indicates distinct gene expression patterns between carcinomas of the floor of the mouth and oral tongue

  19. The DNA repair-ubiquitin-associated HR23 proteins are constituents of neuronal inclusions in specific neurodegenerative disorders without hampering DNA repair

    NARCIS (Netherlands)

    Bergink, Steven; Severijnen, Lies-Anne; Wijgers, Nils; Sugasawa, Kaoru; Yousaf, Humaira; Kros, Johan M.; van Swieten, John; Oostra, Ben A.; Hoeijmakers, Jan H.; Vermeulen, Wim; Willemsen, Rob

    2006-01-01

    Intracellular inclusions play a profound role in many neurodegenerative diseases. Here, we report that HR23B and HR23A, proteins that are involved in both DNA repair and shuttling proteins to the 26S proteasome for degradation, accumulate in neuronal inclusions in brain from a mouse model for FXTAS,

  20. A mutation in the XPB/ERCC3 DNA repair transcription gene, associated with trichothiodystrophy

    Energy Technology Data Exchange (ETDEWEB)

    Weeda, G.; Donker, I.; Vermeulen, W. [Erasmus Univ., Rotterdam (Netherlands)] [and others

    1997-02-01

    Trichothiodystrophy (TTD) is a rare, autosomal recessive disorder characterized by sulfur-deficient brittle hair and nails, mental retardation, impaired sexual development, and ichthyosis. Photosensitivity has been reported in {approximately}50% of the cases, but no skin cancer is associated with TTD. Virtually all photosensitive TTD patients have a deficiency in the nucleotide excision repair (NER) of UV-induced DNA damage that is indistinguishable from that of xeroderma pigmentosum (XP) complementation group D (XP-D) patients. DNA repair defects in XP-D are associated with two additional, quite different diseases; XP, a sun-sensitive and cancer-prone repair disorder, and Cockayne syndrome (CS), a photosensitive condition characterized by physical and mental retardation and wizened facial appearance. One photosensitive TTD case constitutes a new repair-deficient complementation group, TTD-A. Remarkably, both TTD-A and XP-D defects are associated with subunits of TFIIH, a basal transcription factor with a second function in DNA repair. Thus, mutations in TFIIH components may, on top of a repair defect, also cause transcriptional insufficiency, which may explain part of the non-XP clinical features of TTD. To date, three patients with the remarkable conjunction of XP and CS but not TM have been assigned to XP complementation group B (XP-B). Here we present the characterization of the NER defect in two mild TTD patients (TTD6VI and TTD4VI) and confirm the assignment to X-PB. The causative mutation was found to be a single base substitution resulting in a missense mutation (T119P) in a region of the XPB protein. These findings define a third TTD complementation group, extend the clinical heterogeneity associated with XP-B, stress the exclusive relationship between TTD and mutations in subunits of repair/transcription factor TFIIH, and strongly support the concept of {open_quotes}transcription syndromes.{close_quotes} 46 refs., 6 figs., 2 tabs.

  1. Variations in CCL3L gene cluster sequence and non-specific gene copy numbers

    Directory of Open Access Journals (Sweden)

    Edberg Jeffrey C

    2010-03-01

    Full Text Available Abstract Background Copy number variations (CNVs of the gene CC chemokine ligand 3-like1 (CCL3L1 have been implicated in HIV-1 susceptibility, but the association has been inconsistent. CCL3L1 shares homology with a cluster of genes localized to chromosome 17q12, namely CCL3, CCL3L2, and, CCL3L3. These genes are involved in host defense and inflammatory processes. Several CNV assays have been developed for the CCL3L1 gene. Findings Through pairwise and multiple alignments of these genes, we have shown that the homology between these genes ranges from 50% to 99% in complete gene sequences and from 70-100% in the exonic regions, with CCL3L1 and CCL3L3 being identical. By use of MEGA 4 and BioEdit, we aligned sense primers, anti-sense primers, and probes used in several previously described assays against pre-multiple alignments of all four chemokine genes. Each set of probes and primers aligned and matched with overlapping sequences in at least two of the four genes, indicating that previously utilized RT-PCR based CNV assays are not specific for only CCL3L1. The four available assays measured median copies of 2 and 3-4 in European and African American, respectively. The concordance between the assays ranged from 0.44-0.83 suggesting individual discordant calls and inconsistencies with the assays from the expected gene coverage from the known sequence. Conclusions This indicates that some of the inconsistencies in the association studies could be due to assays that provide heterogenous results. Sequence information to determine CNV of the three genes separately would allow to test whether their association with the pathogenesis of a human disease or phenotype is affected by an individual gene or by a combination of these genes.

  2. Phylogenetics of Lophotrochozoan bHLH Genes and the Evolution of Lineage-Specific Gene Duplicates

    Science.gov (United States)

    Bao, Yongbo

    2017-01-01

    The gain and loss of genes encoding transcription factors is of importance to understanding the evolution of gene regulatory complexity. The basic helix–loop–helix (bHLH) genes encode a large superfamily of transcription factors. We systematically classify the bHLH genes from five mollusc, two annelid and one brachiopod genomes, tracing the pattern of bHLH gene evolution across these poorly studied Phyla. In total, 56–88 bHLH genes were identified in each genome, with most identifiable as members of previously described bilaterian families, or of new families we define. Of such families only one, Mesp, appears lost by all these species. Additional duplications have also played a role in the evolution of the bHLH gene repertoire, with many new lophotrochozoan-, mollusc-, bivalve-, or gastropod-specific genes defined. Using a combination of transcriptome mining, RT-PCR, and in situ hybridization we compared the expression of several of these novel genes in tissues and embryos of the molluscs Crassostrea gigas and Patella vulgata, finding both conserved expression and evidence for neofunctionalization. We also map the positions of the genes across these genomes, identifying numerous gene linkages. Some reflect recent paralog divergence by tandem duplication, others are remnants of ancient tandem duplications dating to the lophotrochozoan or bilaterian common ancestors. These data are built into a model of the evolution of bHLH genes in molluscs, showing formidable evolutionary stasis at the family level but considerable within-family diversification by tandem gene duplication. PMID:28338988

  3. Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases.

    Science.gov (United States)

    Remy, Séverine; Tesson, Laurent; Menoret, Séverine; Usal, Claire; De Cian, Anne; Thepenier, Virginie; Thinard, Reynald; Baron, Daniel; Charpentier, Marine; Renaud, Jean-Baptiste; Buelow, Roland; Cost, Gregory J; Giovannangeli, Carine; Fraichard, Alexandre; Concordet, Jean-Paul; Anegon, Ignacio

    2014-08-01

    The generation of genetically modified animals is important for both research and commercial purposes. The rat is an important model organism that until recently lacked efficient genetic engineering tools. Sequence-specific nucleases, such as ZFNs, TALE nucleases, and CRISPR/Cas9 have allowed the creation of rat knockout models. Genetic engineering by homology-directed repair (HDR) is utilized to create animals expressing transgenes in a controlled way and to introduce precise genetic modifications. We applied TALE nucleases and donor DNA microinjection into zygotes to generate HDR-modified rats with large new sequences introduced into three different loci with high efficiency (0.62%-5.13% of microinjected zygotes). Two of these loci (Rosa26 and Hprt1) are known to allow robust and reproducible transgene expression and were targeted for integration of a GFP expression cassette driven by the CAG promoter. GFP-expressing embryos and four Rosa26 GFP rat lines analyzed showed strong and widespread GFP expression in most cells of all analyzed tissues. The third targeted locus was Ighm, where we performed successful exon exchange of rat exon 2 for the human one. At all three loci we observed HDR only when using linear and not circular donor DNA. Mild hypothermic (30°C) culture of zygotes after microinjection increased HDR efficiency for some loci. Our study demonstrates that TALE nuclease and donor DNA microinjection into rat zygotes results in efficient and reproducible targeted donor integration by HDR. This allowed creation of genetically modified rats in a work-, cost-, and time-effective manner.

  4. PAH-DNA adducts in environmentally exposed population in relation to metabolic and DNA repair gene polymorphisms

    Energy Technology Data Exchange (ETDEWEB)

    Binkova, Blanka [Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Videnska 1083, 14220 Prague (Czech Republic); Chvatalova, Irena [Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Videnska 1083, 14220 Prague (Czech Republic); Lnenickova, Zdena [Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Videnska 1083, 14220 Prague (Czech Republic); Milcova, Alena [Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Videnska 1083, 14220 Prague (Czech Republic); Tulupova, Elena [Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Videnska 1083, 14220 Prague (Czech Republic); Cancer Biomarkers and Prevention Group, Biocentre, University of Leicester (United Kingdom); Farmer, Peter B. [Cancer Biomarkers and Prevention Group, Biocentre, University of Leicester (United Kingdom); Sram, Radim J. [Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Videnska 1083, 14220 Prague (Czech Republic)]. E-mail: sram@biomed.cas.cz

    2007-07-01

    Epidemiologic studies indicate that prolonged exposure to particulate air pollution may be associated with increased risk of cardiovascular diseases and cancer in general population. These effects may be attributable to polycyclic aromatic hydrocarbons (PAHs) adsorbed to respirable air particles. It is expected that metabolic and DNA repair gene polymorphisms may modulate individual susceptibility to PAH exposure. This study investigates relationships between exposure to PAHs, polymorphisms of these genes and DNA adducts in group of occupationally exposed policemen (EXP, N = 53, males, aged 22-50 years) working outdoors in the downtown area of Prague and in matched 'unexposed' controls (CON, N = 52). Personal exposure to eight carcinogenic PAHs (c-PAHs) was evaluated by personal samplers during working shift prior to collection of biological samples. Bulky-aromatic DNA adducts were analyzed in lymphocytes by {sup 32}P-postlabeling assay. Polymorphisms of metabolizing (GSTM1, GSTP1, GSTT1, EPHX1, CYP1A1-MspI) and DNA repair (XRCC1, XPD) genes were determined by PCR-based RFLP assays. As potential modifiers and/or cofounders, urinary cotinine levels were analyzed by radioimmunoassay, plasma levels of vitamins A, C, E and folates by HPLC, cholesterol and triglycerides using commercial kits. During the sampling period ambient particulate air pollution was as follows: PM10 32-55 {mu}g/m{sup 3}, PM2.5 27-38 {mu}g/m{sup 3}, c-PAHs 18-22 ng/m{sup 3}; personal exposure to c-PAHs: 9.7 ng/m{sup 3} versus 5.8 ng/m{sup 3} (P < 0.01) for EXP and CON groups, respectively. The total DNA adduct levels did not significantly differ between EXP and CON groups (0.92 {+-} 0.28 adducts/10{sup 8} nucleotides versus 0.82 {+-} 0.23 adducts/10{sup 8} nucleotides, P = 0.065), whereas the level of the B[a]P-'like' adduct was significantly higher in exposed group (0.122 {+-} 0.036 adducts/10{sup 8} nucleotides versus 0.099 {+-} 0.035 adducts/10{sup 8} nucleotides, P = 0

  5. Methylation Analysis of DNA Mismatch Repair Genes Using DNA Derived from the Peripheral Blood of Patients with Endometrial Cancer: Epimutation in Endometrial Carcinogenesis.

    Science.gov (United States)

    Takeda, Takashi; Banno, Kouji; Yanokura, Megumi; Adachi, Masataka; Iijima, Moito; Kunitomi, Haruko; Nakamura, Kanako; Iida, Miho; Nogami, Yuya; Umene, Kiyoko; Masuda, Kenta; Kobayashi, Yusuke; Yamagami, Wataru; Hirasawa, Akira; Tominaga, Eiichiro; Susumu, Nobuyuki; Aoki, Daisuke

    2016-10-14

    Germline mutation of DNA mismatch repair (MMR) genes is a cause of Lynch syndrome. Methylation of MutL homolog 1 (MLH1) and MutS homolog 2 (MSH2) has been detected in peripheral blood cells of patients with colorectal cancer. This methylation is referred to as epimutation. Methylation of these genes has not been studied in an unselected series of endometrial cancer cases. Therefore, we examined methylation of MLH1, MSH2, and MSH6 promoter regions of peripheral blood cells in 206 patients with endometrial cancer using a methylation-specific polymerase chain reaction (MSP). Germline mutation of MMR genes, microsatellite instability (MSI), and immunohistochemistry (IHC) were also analyzed in each case with epimutation. MLH1 epimutation was detected in a single patient out of a total of 206 (0.49%)-1 out of 58 (1.72%) with an onset age of less than 50 years. The patient with MLH1 epimutation showed high level MSI (MSI-H), loss of MLH1 expression and had developed endometrial cancer at 46 years old, complicated with colorectal cancer. No case had epimutation of MSH2 or MSH6. The MLH1 epimutation detected in a patient with endometrial cancer may be a cause of endometrial carcinogenesis. This result indicates that it is important to check epimutation in patients with endometrial cancer without a germline mutation of MMR genes.

  6. Specific regulatory motifs predict glucocorticoid responsiveness of hippocampal gene expression.

    Science.gov (United States)

    Datson, N A; Polman, J A E; de Jonge, R T; van Boheemen, P T M; van Maanen, E M T; Welten, J; McEwen, B S; Meiland, H C; Meijer, O C

    2011-10-01

    The glucocorticoid receptor (GR) is an ubiquitously expressed ligand-activated transcription factor that mediates effects of cortisol in relation to adaptation to stress. In the brain, GR affects the hippocampus to modulate memory processes through direct binding to glucocorticoid response elements (GREs) in the DNA. However, its effects are to a high degree cell specific, and its target genes in different cell types as well as the mechanisms conferring this specificity are largely unknown. To gain insight in hippocampal GR signaling, we characterized to which GRE GR binds in the rat hippocampus. Using a position-specific scoring matrix, we identified evolutionary-conserved putative GREs from a microarray based set of hippocampal target genes. Using chromatin immunoprecipitation, we were able to confirm GR binding to 15 out of a selection of 32 predicted sites (47%). The majority of these 15 GREs are previously undescribed and thus represent novel GREs that bind GR and therefore may be functional in the rat hippocampus. GRE nucleotide composition was not predictive for binding of GR to a GRE. A search for conserved flanking sequences that may predict GR-GRE interaction resulted in the identification of GC-box associated motifs, such as Myc-associated zinc finger protein 1, within 2 kb of GREs with GR binding in the hippocampus. This enrichment was not present around nonbinding GRE sequences nor around proven GR-binding sites from a mesenchymal stem-like cell dataset that we analyzed. GC-binding transcription factors therefore may be unique partners for DNA-bound GR and may in part explain cell-specific transcriptional regulation by glucocorticoids in the context of the hippocampus.

  7. Gametogenesis in the Pacific Oyster Crassostrea gigas: A Microarrays-Based Analysis Identifies Sex and Stage Specific Genes

    Science.gov (United States)

    Dheilly, Nolwenn M.; Lelong, Christophe; Huvet, Arnaud; Kellner, Kristell; Dubos, Marie-Pierre; Riviere, Guillaume; Boudry, Pierre; Favrel, Pascal

    2012-01-01

    Background The Pacific oyster Crassostrea gigas (Mollusca, Lophotrochozoa) is an alternative and irregular protandrous hermaphrodite: most individuals mature first as males and then change sex several times. Little is known about genetic and phenotypic basis of sex differentiation in oysters, and little more about the molecular pathways regulating reproduction. We have recently developed and validated a microarray containing 31,918 oligomers (Dheilly et al., 2011) representing the oyster transcriptome. The application of this microarray to the study of mollusk gametogenesis should provide a better understanding of the key factors involved in sex differentiation and the regulation of oyster reproduction. Methodology/Principal Findings Gene expression was studied in gonads of oysters cultured over a yearly reproductive cycle. Principal component analysis and hierarchical clustering showed a significant divergence in gene expression patterns of males and females coinciding with the start of gonial mitosis. ANOVA analysis of the data revealed 2,482 genes differentially expressed during the course of males and/or females gametogenesis. The expression of 434 genes could be localized in either germ cells or somatic cells of the gonad by comparing the transcriptome of female gonads to the transcriptome of stripped oocytes and somatic tissues. Analysis of the annotated genes revealed conserved molecular mechanisms between mollusks and mammals: genes involved in chromatin condensation, DNA replication and repair, mitosis and meiosis regulation, transcription, translation and apoptosis were expressed in both male and female gonads. Most interestingly, early expressed male-specific genes included bindin and a dpy-30 homolog and female-specific genes included foxL2, nanos homolog 3, a pancreatic lipase related protein, cd63 and vitellogenin. Further functional analyses are now required in order to investigate their role in sex differentiation in oysters. Conclusions

  8. Transcription-coupled repair and apoptosis provide specific protection against transcription-associated mutagenesis by ultraviolet light.

    Science.gov (United States)

    Hendriks, Giel; Jansen, Jacob G; Mullenders, Leon H F; de Wind, Niels

    2010-01-01

    Recent data reveal that gene transcription affects genome stability in mammalian cells. For example, transcription of DNA that is damaged by the most prevalent exogenous genotoxin, UV light, induces nucleotide substitutions and chromosomal instability, collectively called UV-induced transcription-associated mutations (UV-TAM). An important class of UV-TAM consists of nucleotide transitions that are caused by deamination of cytosine-containing photolesions to uracil, presumably occurring at stalled transcription complexes. Transcription-associated deletions and recombinational events after UV exposure may be triggered by collisions of replication forks with stalled transcription complexes. In this Point-of-View we propose that mammalian cells possess two tailored mechanisms to prevent UV-TAM in dermal stem cells. First, the transcription-coupled nucleotide excision repair (TCR) pathway removes lesions at transcribed DNA strands, forming the primary barrier against the mutagenic consequences of transcription at a damaged template. Second, when TCR is absent or when the capacity of TCR is exceeded, persistently stalled transcription complexes induce apoptosis, averting the generation of mutant cells following replication. We hypothesize that TCR and the apoptotic response in conjunction reduce the risk of skin carcinogenesis.

  9. Specificity of reinnervation and motor recovery after interposition of an artificial barrier between transected and repaired nerves in adjacency--an experimental study in the rat.

    Science.gov (United States)

    Lutz, B S; Ma, S F; Chuang, D C; Lidman, D; Wei, F C

    2001-01-01

    Non-specific re-innervation of target organs caused by misdirected axonal growth at the repair site is regarded as one reason for a poor functional outcome after peripheral nerve transsection and repair. This study investigates the rate of aberrant re-innervation and its influence on motor recovery in the rat sciatic nerve using artificial sheets as barrier between tibial and peroneal nerves. The sciatic nerve was transsected and repaired as follows: epineural sutures (A x 6), fascicular repair of tibial and peroneal nerves respectively (B x 8), and the same as in group B, but separating both nerves using an Integra-sheet with silicone (C x 8), or Integra without silicone (D x 8). As control, solely the tibial nerve was transsected and repaired (E x 5). Final investigations after 4 months revealed that in group C, 50% of the Integra-silicone sheets were dislocated. No dislocation was found in group D. Muscle contraction force of the gastrocnemius muscle was significantly higher in group E as compared to all other groups. However although not significant, group D showed a consistently higher muscle contraction force than groups A, B, and C. Histology in groups A, B, and C with dislocated sheets demonstrated multiple axons growing from the tibial to the peroneal nerve and vice versa. In groups D and E, no such axonal growth was visible. These findings were confirmed by a significantly higher rate of specific reinnervation of the soleus muscle using sequential retrograde double labelling technique. Results of this study suggest that an artificial sheet such as Integra bears the potential of preventing aberrant re-innervation between repaired adjacent nerves resulting in improved motor recovery. Clinically, this technique may be of importance for brachial plexus, sciatic nerve, and facial nerve repair.

  10. Specific expression of bioluminescence reporter gene in cardiomyocyte regulated by tissue specific promoter

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, Vu Hong; Tae, Seong Ho; Le, Nguyen Uyen Chi; Min, Jung Joon [Chonnam National University Medical School, Gwangju (Korea, Republic of)

    2007-07-01

    As the human heart is not capable of regenerating the great numbers of cardiac cells that are lost after myocardial infarction, impaired cardiac function is the inevitable result of ischemic disease. Recently, human embryonic stem cells (hESCs) have gained popularity as a potentially ideal cell candidate for tissue regeneration. In particular, hESCs are capable of cardiac lineage-specific differentiation and confer improvement of cardiac function following transplantation into animal models. Although such data are encouraging, the specific strategy for in vivo and non-invasive detection of differentiated cardiac lineage is still limited. Therefore, in the present study, we established the gene construction in which the optical reporter gene Firefly luciferase was controlled by Myosin Heavy Chain promoter for specific expressing in heart cells. The vector consisting of - MHC promoter and a firefly luciferase coding sequence flanked by full-length bovine growth hormone (BGH) 3'-polyadenylation sequence based on pcDNA3.1- vector backbone. To test the specific transcription of this promoter in g of MHC-Fluc or CMV-Flue (for control) plasmid DNA in myocardial tissue, 20 phosphate-buffered saline was directly injected into mouse myocardium through a midline sternotomy and liver. After 1 week of injection, MHC-Fluc expression was detected from heart region which was observed under cooled CCD camera of in vivo imaging system but not from liver. In control group injected with CMV-Flue, the bioluminescence was detected from all these organs. The expression of Flue under control of Myosin Heavy Chain promoter may become a suitable optical reporter gene for stem cell-derived cardiac lineage differentiation study.

  11. Mouse BAZ1A (ACF1 is dispensable for double-strand break repair but is essential for averting improper gene expression during spermatogenesis.

    Directory of Open Access Journals (Sweden)

    James A Dowdle

    2013-11-01

    Full Text Available ATP-dependent chromatin remodelers control DNA access for transcription, recombination, and other processes. Acf1 (also known as BAZ1A in mammals is a defining subunit of the conserved ISWI-family chromatin remodelers ACF and CHRAC, first purified over 15 years ago from Drosophila melanogaster embryos. Much is known about biochemical properties of ACF and CHRAC, which move nucleosomes in vitro and in vivo to establish ordered chromatin arrays. Genetic studies in yeast, flies and cultured human cells clearly implicate these complexes in transcriptional repression via control of chromatin structures. RNAi experiments in transformed mammalian cells in culture also implicate ACF and CHRAC in DNA damage checkpoints and double-strand break repair. However, their essential in vivo roles in mammals are unknown. Here, we show that Baz1a-knockout mice are viable and able to repair developmentally programmed DNA double-strand breaks in the immune system and germ line, I-SceI endonuclease-induced breaks in primary fibroblasts via homologous recombination, and DNA damage from mitomycin C exposure in vivo. However, Baz1a deficiency causes male-specific sterility in accord with its high expression in male germ cells, where it displays dynamic, stage-specific patterns of chromosomal localization. Sterility is caused by pronounced defects in sperm development, most likely a consequence of massively perturbed gene expression in spermatocytes and round spermatids in the absence of BAZ1A: the normal spermiogenic transcription program is largely intact but more than 900 other genes are mis-regulated, primarily reflecting inappropriate up-regulation. We propose that large-scale changes in chromatin composition that occur during spermatogenesis create a window of vulnerability to promiscuous transcription changes, with an essential function of ACF and/or CHRAC chromatin remodeling activities being to safeguard against these alterations.

  12. Complex Oncogenic Translocations with Gene Amplification are Initiated by Specific DNA Breaks in Lymphocytes

    OpenAIRE

    2009-01-01

    Chromosomal instability is a hallmark of many tumor types. Complex chromosomal rearrangements with associated gene amplification, known as complicons, characterize many hematologic and solid cancers. While chromosomal aberrations, including complicons, are useful diagnostic and prognostic cancer markers, their molecular origins are not known. Although accumulating evidence has implicated DNA double strand break repair in suppression of oncogenic genome instability, the genomic elements requir...

  13. The immunoglobulin genes: structure and specificity in chronic lymphocytic leukemia.

    Science.gov (United States)

    Tobin, Gerard

    2007-06-01

    The rearrangement of the immunoglobulin genes (IG) provides a large diversity of B-cell receptors conformations and allows the immune system to respond differently to foreign antigens. In chronic lymphocytic leukemia (CLL), there are a restricted number of stereotyped B-cell receptors rearranged by the tumor B-cells between CLL patients. These subsets with stereotyped receptors appear to have clinical implications, for example cases that rearrange the IGHV3-21 gene display poor clinical prognosis. The number of subsets with stereotyped receptors has been reported at a frequency of over 20% of CLL cases; however, the specificities of these receptors are still not clearly defined. Reactivity to epitopes from bacterial antigen, cytoskeleton components such as vimentin, and antigens on viable and apoptotic T-cell have been proposed. The role of antigen in CLL development is currently being more clearly defined with identification of stereotyped receptors, and their antigen specificity and the continued role antigen stimulation plays in CLL disease will be an important question in the future.

  14. Zebrafish with mutations in mismatch repair genes develop neurofibromas and other tumors.

    NARCIS (Netherlands)

    Feitsma, H.; Kuiper, R.V.; Korving, J.; Nijman, I.J.; Cuppen, E.

    2008-01-01

    Defective mismatch repair (MMR) in humans causes hereditary nonpolyposis colorectal cancer. This genetic predisposition to colon cancer is linked to heterozygous familial mutations, and loss-of-heterozygosity is necessary for tumor development. In contrast, the rare cases with biallelic MMR

  15. Impaired Cytogenetic Damage Repair and Cell Cycle Regulation in Response to Ionizing Radiation in Human Fibroblast Cells with Individual Knock-down of 25 Genes

    Science.gov (United States)

    Zhang, Ye; Rohde, Larry; Emami, Kamal; Hammond, Dianne; Casey, Rachael; Mehta, Satish; Jeevarajan, Antony; Pierson, Duane; Wu, Honglu

    2008-01-01

    Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have demonstrated that genes with upregulated expression induced by IR may play important roles in DNA damage sensing, cell cycle checkpoint and chromosomal repair, the relationship between the regulation of gene expression by IR and its impact on cytogenetic responses to ionizing radiation has not been systematically studied. In our present study, the expression of 25 genes selected based on their transcriptional changes in response to IR or from their known DNA repair roles were individually knocked down by siRNA transfection in human fibroblast cells. Chromosome aberrations (CA) and micronuclei (MN) formation were measured as the cytogenetic endpoints. Our results showed that the yield of MN and/or CA formation were significantly increased by suppressed expression of 5 genes that included Ku70 in the DSB repair pathway; XPA in the NER pathway; RPA1 in the MMR pathway; RAD17 and RBBP8 in cell cycle control. Knocked-down expression of 4 genes including MRE11A, RAD51 in the DSB pathway, and SESN1 and SUMO1 showed significant inhibition of cell cycle progression, possibly because of severe impairment of DNA damage repair. Furthermore, loss of XPA, p21 and MLH1 expression resulted in both enhanced cell cycle progression and significantly higher yield of cytogenetic damage, indicating the involvement of these gene products in both cell cycle control and DNA damage repair. Of these 11 genes that affected the cytogenetic response, 9 were up-regulated in the cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulating the biological consequences after IR. Failure to express these IR-responsive genes, such as by gene mutation, could seriously change the outcome of the post IR scenario and lead to carcinogenesis.

  16. Shoulder-specific outcomes 1 year after nontraumatic full-thickness rotator cuff repair: a systematic literature review and meta-analysis.

    Science.gov (United States)

    Gurnani, Navin; van Deurzen, Derek F P; van den Bekerom, Michel P J

    2017-10-01

    Nontraumatic full-thickness rotator cuff tears are commonly initially treated conservatively. If conservative treatment fails, rotator cuff repair is a viable subsequent option. The objective of the present meta-analysis is to evaluate the shoulder-specific outcomes one year after arthroscopic or mini-open rotator cuff repair of nontraumatic rotator cuff tears. A literature search was conducted in PubMed and EMBASE within the period January 2000 to January 2017. All studies measuring the clinical outcome at 12 months after nontraumatic rotator cuff repair of full-thickness rotator cuff tears were listed. We included 16 randomized controlled trials that met our inclusion criteria with a total of 1.221 shoulders. At 12 months after rotator cuff repair, the mean Constant score had increased 29.5 points; the mean American Shoulder and Elbow Score score increased by 38.6 points; mean Simple Shoulder Test score was 5.6 points; mean University of California Los Angeles score improved by 13.0 points; and finally, mean Visual Analogue Scale score decreased by 4.1 points. Based on this meta-analysis, significant improvements in the shoulder-specific indices are observed 12 months after nontraumatic arthroscopic or mini-open rotator cuff repair.

  17. A hierarchy of ECM-mediated signalling tissue-specific gene expression regulates tissue-specific gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Roskelley, Calvin D; Srebrow, Anabella; Bissell, Mina J

    1995-10-07

    A dynamic and reciprocal flow of information between cells and the extracellular matrix contributes significantly to the regulation of form and function in developing systems. Signals generated by the extracellular matrix do not act in isolation. Instead, they are processed within the context of global signalling hierarchies whose constituent inputs and outputs are constantly modulated by all the factors present in the cell's surrounding microenvironment. This is particularly evident in the mammary gland, where the construction and subsequent destruction of such a hierarchy regulates changes in tissue-specific gene expression, morphogenesis and apoptosis during each developmental cycle of pregnancy, lactation and involution.

  18. Syntenator: Multiple gene order alignments with a gene-specific scoring function

    Directory of Open Access Journals (Sweden)

    Dieterich Christoph

    2008-11-01

    Full Text Available Abstract Background Identification of homologous regions or conserved syntenies across genomes is one crucial step in comparative genomics. This task is usually performed by genome alignment softwares like WABA or blastz. In case of conserved syntenies, such regions are defined as conserved gene orders. On the gene order level, homologous regions can even be found between distantly related genomes, which do not align on the nucleotide sequence level. Results We present a novel approach to identify regions of conserved synteny across multiple genomes. Syntenator represents genomes and alignments thereof as partial order graphs (POGs. These POGs are aligned by a dynamic programming approach employing a gene-specific scoring function. The scoring function reflects the level of protein sequence similarity for each possible gene pair. Our method consistently defines larger homologous regions in pairwise gene order alignments than nucleotide-level comparisons. Our method is superior to methods that work on predefined homology gene sets (as implemented in Blockfinder. Syntenator successfully reproduces 80% of the EnsEMBL man-mouse conserved syntenic blocks. The full potential of our method becomes visible by comparing remotely related genomes and multiple genomes. Gene order alignments potentially resolve up to 75% of the EnsEMBL 1:many orthology relations and 27% of the many:many orthology relations. Conclusion We propose Syntenator as a software solution to reliably infer conserved syntenies among distantly related genomes. The software is available from http://www2.tuebingen.mpg.de/abt4/plone.

  19. Expression Patterns of Glucose Transporter-1 Gene and Thyroid Specific Genes in Human Papillary Thyroid Carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sungeun; Chung, Junekey; Min Haesook and others

    2014-06-15

    The expression of glucose transporter-1 (Glut-1) gene and those of major thyroid-specific genes were examined in papillary carcinoma tissues, and the expressions of these genes were compared with cancer differentiation grades. Twenty-four human papillary carcinoma tissues were included in this study. The expressions of Glut-1- and thyroid-specific genes [sodium/iodide symporter (NIS), thyroid peroxidase, thyroglobulin, TSH receptor and pendrin] were analyzed by RT-PCR. Expression levels were expressed as ratios versus the expression of beta-actin. Pathologic differentiation of papillary carcinoma was classified into a relatively well-differentiated group (n=13) and relatively less differentiated group (n=11). Glut-1 gene expression was significantly higher in the less differentiated group (0.66±0.04) than in the well-differentiated group (0.59±0.07). The expression levels of the NIS, PD and TG genes were significantly higher in the well-differentiated group (NIS: 0.67±0.20, PD: 0.65±0.21, TG: 0.74±0.16) than in the less differentiated group (NIS: 0.36±0.05, PD: 0.49±0.08, TG: 0.60±0.11), respectively. A significant negative correlation was found between Glut-1 and NIS expression, and positive correlations were found between NIS and TG, and between NIS and PD. The NIS, PD and TG genes were highly expressed in well-differentiated thyroid carcinomas, whereas the Glut-1 gene was highly expressed in less differentiated thyroid carcinomas. These findings provide a molecular rationale for the management of papillary carcinoma, especially in the selection of FDG PET or radioiodine whole-body scan and I-131-based therapy.

  20. Efficacy of DNA double-strand breaks repair in breast cancer is decreased in carriers of the variant allele of the UBC9 gene c.73G>A polymorphism

    Energy Technology Data Exchange (ETDEWEB)

    Synowiec, Ewelina [Department of Molecular Genetics, University of Lodz, Lodz (Poland); Krupa, Renata [Laboratory of DNA Repair, Department of Molecular Genetics, University of Lodz, Banacha 12/16, Lodz (Poland); Morawiec, Zbigniew; Wasylecka, Maja [Department of Surgical Oncology, N. Copernicus Hospital, Lodz (Poland); Dziki, Lukasz; Morawiec, Jan [Department of General and Colorectal Surgery, Medical University of Lodz, Lodz (Poland); Blasiak, Janusz [Department of Molecular Genetics, University of Lodz, Lodz (Poland); Wozniak, Katarzyna, E-mail: wozniak@biol.uni.lodz.pl [Laboratory of DNA Repair, Department of Molecular Genetics, University of Lodz, Banacha 12/16, Lodz (Poland)

    2010-12-10

    UBC9 (E2) SUMO conjugating enzyme plays an important role in the maintenance of genome stability and integrity. In the present work we examined the association between the c.73G>A (Val25Met) polymorphism of the UBC9 gene (rs11553473) and efficacy of DNA double-strand breaks (DSBs) repair (DRE) in breast cancer patients. We determined the level of endogenous (basal) and exogenous (induced by {gamma}-irradiation) DSBs and efficacy of their repair in peripheral blood lymphocytes of 57 breast cancer patients and 70 healthy individuals. DNA damage and repair were studied by neutral comet assay. Genotypes were determined in DNA from peripheral blood lymphocytes by allele-specific PCR (ASO-PCR). We also correlated genotypes with the clinical characteristics of breast cancer patients. We observed a strong association between breast cancer occurrence and the variant allele carried genotypes in patients with elevated level of basal as well as induced DNA damage (OR 6.74, 95% CI 2.27-20.0 and OR 5.33, 95% CI 1.81-15.7, respectively). We also found statistically significant (p < 0.05) difference in DRE related to the c.73G>A polymorphism of the UBC9 gene in breast cancer patients. Carriers of variant allele have decreased DNA DRE as compared to wild type genotype carriers. We did not find any association with the UBC9 gene polymorphism and estrogen and progesterone receptor status. The variant allele of the UBC9 gene polymorphism was strongly inversely related to HER negative breast cancer patients (OR 0.03, 95% CI 0.00-0.23). Our results suggest that the c.73G>A polymorphism of the UBC9 gene may affect DNA DSBs repair efficacy in breast cancer patients.

  1. Antioxidative Dietary Compounds Modulate Gene Expression Associated with Apoptosis, DNA Repair, Inhibition of Cell Proliferation and Migration

    Directory of Open Access Journals (Sweden)

    Likui Wang

    2014-09-01

    Full Text Available Many dietary compounds are known to have health benefits owing to their antioxidative and anti-inflammatory properties. To determine the molecular mechanism of these food-derived compounds, we analyzed their effect on various genes related to cell apoptosis, DNA damage and repair, oxidation and inflammation using in vitro cell culture assays. This review further tests the hypothesis proposed previously that downstream products of COX-2 (cyclooxygenase-2 called electrophilic oxo-derivatives induce antioxidant responsive elements (ARE, which leads to cell proliferation under antioxidative conditions. Our findings support this hypothesis and show that cell proliferation was inhibited when COX-2 was down-regulated by polyphenols and polysaccharides. Flattened macrophage morphology was also observed following the induction of cytokine production by polysaccharides extracted from viili, a traditional Nordic fermented dairy product. Coix lacryma-jobi (coix polysaccharides were found to reduce mitochondrial membrane potential and induce caspase-3- and 9-mediated apoptosis. In contrast, polyphenols from blueberries were involved in the ultraviolet-activated p53/Gadd45/MDM2 DNA repair system by restoring the cell membrane potential. Inhibition of hypoxia-inducible factor-1 by saponin extracts of ginsenoside (Ginsen and Gynostemma and inhibition of S100A4 by coix polysaccharides inhibited cancer cell migration and invasion. These observations suggest that antioxidants and changes in cell membrane potential are the major driving forces that transfer signals through the cell membrane into the cytosol and nucleus, triggering gene expression, changes in cell proliferation and the induction of apoptosis or DNA repair.

  2. SNPs in DNA repair or oxidative stress genes and late subcutaneous fibrosis in patients following single shot partial breast irradiation

    Directory of Open Access Journals (Sweden)

    Falvo Elisabetta

    2012-01-01

    Full Text Available Abstract Background The aim of this study was to evaluate the potential association between single nucleotide polymorphisms related response to radiotherapy injury, such as genes related to DNA repair or enzymes involved in anti-oxidative activities. The paper aims to identify marker genes able to predict an increased risk of late toxicity studying our group of patients who underwent a Single Shot 3D-CRT PBI (SSPBI after BCS (breast conserving surgery. Methods A total of 57 breast cancer patients who underwent SSPBI were genotyped for SNPs (single nucleotide polymorphisms in XRCC1, XRCC3, GST and RAD51 by Pyrosequencing technology. Univariate analysis (ORs and 95% CI was performed to correlate SNPs with the risk of developing ≥ G2 fibrosis or fat necrosis. Results A higher significant risk of developing ≥ G2 fibrosis or fat necrosis in patients with: polymorphic variant GSTP1 (Ile105Val (OR = 2.9; 95%CI, 0.88-10.14, p = 0.047. Conclusions The presence of some SNPs involved in DNA repair or response to oxidative stress seem to be able to predict late toxicity. Trial Registration ClinicalTrials.gov: NCT01316328

  3. Trichostatin A enhances vascular repair by injected human endothelial progenitors through increasing the expression of TAL1-dependent genes.

    Science.gov (United States)

    Palii, Carmen G; Vulesevic, Branka; Fraineau, Sylvain; Pranckeviciene, Erinija; Griffith, Alexander J; Chu, Alphonse; Faralli, Hervé; Li, Yuhua; McNeill, Brian; Sun, Jie; Perkins, Theodore J; Dilworth, F Jeffrey; Perez-Iratxeta, Carol; Suuronen, Erik J; Allan, David S; Brand, Marjorie

    2014-05-01

    A major goal of cell therapy for vascular diseases is to promote revascularization through the injection of endothelial stem/progenitor cells. The gene regulatory mechanisms that underlie endothelial progenitor-mediated vascular repair, however, remain elusive. Here, we identify the transcription factor TAL1/SCL as a key mediator of the vascular repair function of primary human endothelial colony-forming cells (ECFCs). Genome-wide analyses in ECFCs demonstrate that TAL1 activates a transcriptional program that promotes cell adhesion and migration. At the mechanistic level, we show that TAL1 upregulates the expression of migratory and adhesion genes through recruitment of the histone acetyltransferase p300. Based on these findings, we establish a strategy that enhances the revascularization efficiency of ECFCs after ischemia through ex vivo priming with the histone deacetylase inhibitor TSA. Thus, small molecule epigenetics drugs are effective tools for modifying the epigenome of stem/progenitor cells prior to transplantation as a means to enhance their therapeutic potential.

  4. Regulated expression of the Saccharomyces cerevisiae DNA repair gene RAD7 in response to DNA damage and during sporulation.

    Science.gov (United States)

    Jones, J S; Prakash, L; Prakash, S

    1990-06-11

    The RAD7 gene of Saccharomyces cerevisiae affects the proficiency of excision repair of DNA damaged by UV light. Here, we report our studies on the regulation of the RAD7 gene in response to UV irradiation and during sporulation. RAD7 transcript levels increased 6-fold within 40 min of exposure of cells to 37 J/m2 of UV light. Higher UV doses also elicited rapid increases in the level of RAD7 mRNA. RAD7 mRNA levels increased in sporulating MATa/MAT alpha diploid cells, but not in the asporogenous MATa/MATa strain exposed to sporulation conditions. The increase in RAD7 mRNA level in MATa/MAT alpha cells was 15-fold after 6 h and 9-fold after 7 h in sporulation medium; thereafter, RAD7 mRNA levels declined. Periodic transcription of RAD7 during sporulation suggests a role for RAD7 in this process.

  5. Sequence analysis of the DNA-repair gene rad51 in the tardigrades Milnesium cf. tardigradum, Hypsibius dujardini and Macrobiotus cf. harmsworthi

    Directory of Open Access Journals (Sweden)

    Eliana A. Beltrán-Pardo

    2013-05-01

    Full Text Available Tardigrades are known for being resistant to extreme conditions, including tolerance to ionising and UV radiation in both the hydrated and the dehydrated state. It is known that these factors may cause damage to DNA. It has recently been shown that single and double DNA strand breaks occur when tardigrades are maintained for a long time in the anhydrobiotic state. This may suggest that perhaps tardigrades rely on efficient DNA repair mechanisms. Among all proteins that comprise the DNA repair system, recombinases such as RecA or Rad51 have a very important function: DNA exchange activity. This enzyme is used in the homologous recombination and allows repair of the damaged strand using homologous non-damaged strands as a template. In this study, Rad51 induction was evaluated by western blot in Milnesium cf. tardigradum, after exposure to gamma radiation. The Rad51 protein was highly induced by radiation, when compared to the control. The rad51 genes were searched in three tardigrades: Milnesium cf. tardigradum, Hypsibius dujardini and Macrobiotus cf. harmsworthi. The gene sequences were obtained by preparing and sequencing transcriptome libraries for H. dujardini and M. cf. harmsworthi and designing rad51 degenerate primers specific for M. cf. tardigradum. Comparison of Rad51 putative proteins from tardigrades with other organisms showed that they are highly similar to the corresponding sequence from the nematode Trichinella spiralis. A structure-based sequence alignment from tardigrades and other organisms revealed that putative Rad51 predicted proteins from tardigrades contain the expected motifs for these important recombinases. In a cladogram tree based on this alignment, tardigrades tend to cluster together suggesting that they have selective differences in these genes that make them diverge between species. Predicted Rad51 structures from tardigrades were also compared with crystalline structure of Rad51 in Saccharomyces cerevisiae. These

  6. The SLEEPER genes: a transposase-derived angiosperm-specific gene family

    Directory of Open Access Journals (Sweden)

    Knip Marijn

    2012-10-01

    Full Text Available Abstract Background DAYSLEEPER encodes a domesticated transposase from the hAT-superfamily, which is essential for development in Arabidopsis thaliana. Little is known about the presence of DAYSLEEPER orthologs in other species, or how and when it was domesticated. We studied the presence of DAYSLEEPER orthologs in plants and propose a model for the domestication of the ancestral DAYSLEEPER gene in angiosperms. Results Using specific BLAST searches in genomic and EST libraries, we found that DAYSLEEPER-like genes (hereafter called SLEEPER genes are unique to angiosperms. Basal angiosperms as well as grasses (Poaceae and dicotyledonous plants possess such putative orthologous genes, but SLEEPER-family genes were not found in gymnosperms, mosses and algae. Most species contain more than one SLEEPER gene. All SLEEPERs contain a C2H2 type BED-zinc finger domain and a hATC dimerization domain. We designated 3 motifs, partly overlapping the BED-zinc finger and dimerization domain, which are hallmark features in the SLEEPER family. Although SLEEPER genes are structurally conserved between species, constructs with SLEEPER genes from grapevine and rice did not complement the daysleeper phenotype in Arabidopsis, when expressed under control of the DAYSLEEPER promoter. However these constructs did cause a dominant phenotype when expressed in Arabidopsis. Rice plant lines with an insertion in the RICESLEEPER1 or 2 locus displayed phenotypic abnormalities, indicating that these genes are functional and important for normal development in rice. We suggest a model in which we hypothesize that an ancestral hAT transposase was retrocopied and stably integrated in the genome during early angiosperm evolution. Evidence is also presented for more recent retroposition events of SLEEPER genes, such as an event in the rice genome, which gave rise to the RICESLEEPER1 and 2 genes. Conclusions We propose the ancestral SLEEPER gene was formed after a process of retro

  7. NANOPARTICLE AS A NEW GENE TRANSFERRING VECTOR IN SPECIFIC EXPRESSION GENE

    Institute of Scientific and Technical Information of China (English)

    管珩; 李拥军; 郑曰宏; 刘昌伟; 杨菁; 宋存先; 王彭延; 赵三妹; 王宗立; 佘铭鹏

    2002-01-01

    Objective. To evaluate the possibility and efficiency of nanoparticle as a new vector in specific gene transference.Methods. Nanoparticle-DNA complex was prepared with Poly- dl-lactic-co-glycolic acid (PLGA) beating antisense monocyte chemotactic protein-1 (A-MCP-1), a specific expression gene, and the package efficiency, release progress in vitro, and the size of the complex were determined. The possibility of the new vector was evaluated with genomic DNA PCR by transferring gene into cultured smooth muscle cells (SMC), cationic lipids as a control. For study in vivo, jugular vein-to-artery bypass grafting procedures were performed on 20 New Zealand white rabbits, of which 6 grafts were transferred with nanoparticle-A-MCP-1 (200 μg), 6 with A - MCP - 1(200 μ g) by cationic liposome, 4 with LNCX plasmid, and 4 as control. Fourteen days after the grafts were harvested, the expression of A-MCP-1 and its effect on MCP-1 in vein grafts were detected by dot blot, and the morphologic evaluation of grafts was performed.Results. The package efficiency of the nanoparticle-DNA complex was 0. 9%, release progress in vitro lasted 2 weeks, and the size ranged from 150 to 300nm. SMC genomic DNA PCR showed that A-MCP-1 gene could be successfully transfected into cells by nanoparticle. The study in vivo indicated that A-MCP-1 mRNA was expressed in both local gene delivery groups, nanoparticle and liposome, meanwhile, MCP-1 expression in vein grafts was significantly inhibited and neointimal hyperplasia was notably reduced.Conclusion. Nanoparticle can act as a vector to transfect specific gene.

  8. 14-3-3 checkpoint regulatory proteins interact specifically with DNA repair protein human exonuclease 1 (hEXO1) via a semi-conserved motif

    DEFF Research Database (Denmark)

    Andersen, Sofie Dabros; Keijzers, Guido; Rampakakis, Emmanouil

    2012-01-01

    Human exonuclease 1 (hEXO1) acts directly in diverse DNA processing events, including replication, mismatch repair (MMR), and double strand break repair (DSBR), and it was also recently described to function as damage sensor and apoptosis inducer following DNA damage. In contrast, 14-3-3 proteins...... are regulatory phosphorserine/threonine binding proteins involved in the control of diverse cellular events, including cell cycle checkpoint and apoptosis signaling. hEXO1 is regulated by post-translation Ser/Thr phosphorylation in a yet not fully clarified manner, but evidently three phosphorylation sites...... are specifically induced by replication inhibition leading to protein ubiquitination and degradation. We demonstrate direct and robust interaction between hEXO1 and six of the seven 14-3-3 isoforms in vitro, suggestive of a novel protein interaction network between DNA repair and cell cycle control. Binding...

  9. Transcription-coupled DNA repair in prokaryotes.

    Science.gov (United States)

    Ganesan, Ann; Spivak, Graciela; Hanawalt, Philip C

    2012-01-01

    Transcription-coupled repair (TCR) is a subpathway of nucleotide excision repair (NER) that acts specifically on lesions in the transcribed strand of expressed genes. First reported in mammalian cells, TCR was then documented in Escherichia coli. In this organism, an RNA polymerase arrested at a lesion is displaced by the transcription repair coupling factor, Mfd. This protein recruits the NER lesion-recognition factor UvrA, and then dissociates from the DNA. UvrA binds UvrB, and the assembled UvrAB* complex initiates repair. In mutants lacking active Mfd, TCR is absent. A gene transcribed by the bacteriophage T7 RNA polymerase in E. coli also requires Mfd for TCR. The CSB protein (missing or defective in cells of patients with Cockayne syndrome, complementation group B) is essential for TCR in humans. CSB and its homologs in higher eukaryotes are likely functional equivalents of Mfd. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. Generation of healthy mice from gene-corrected disease-specific induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Guangming Wu

    2011-07-01

    Full Text Available Using the murine model of tyrosinemia type 1 (fumarylacetoacetate hydrolase [FAH] deficiency; FAH⁻/⁻ mice as a paradigm for orphan disorders, such as hereditary metabolic liver diseases, we evaluated fibroblast-derived FAH⁻/⁻-induced pluripotent stem cells (iPS cells as targets for gene correction in combination with the tetraploid embryo complementation method. First, after characterizing the FAH⁻/⁻ iPS cell lines, we aggregated FAH⁻/⁻-iPS cells with tetraploid embryos and obtained entirely FAH⁻/⁻-iPS cell-derived mice that were viable and exhibited the phenotype of the founding FAH⁻/⁻ mice. Then, we transduced FAH cDNA into the FAH⁻/⁻-iPS cells using a third-generation lentiviral vector to generate gene-corrected iPS cells. We could not detect any chromosomal alterations in these cells by high-resolution array CGH analysis, and after their aggregation with tetraploid embryos, we obtained fully iPS cell-derived healthy mice with an astonishing high efficiency for full-term development of up to 63.3%. The gene correction was validated functionally by the long-term survival and expansion of FAH-positive cells of these mice after withdrawal of the rescuing drug NTBC (2-(2-nitro-4-fluoromethylbenzoyl-1,3-cyclohexanedione. Furthermore, our results demonstrate that both a liver-specific promoter (transthyretin, TTR-driven FAH transgene and a strong viral promoter (from spleen focus-forming virus, SFFV-driven FAH transgene rescued the FAH-deficiency phenotypes in the mice derived from the respective gene-corrected iPS cells. In conclusion, our data demonstrate that a lentiviral gene repair strategy does not abrogate the full pluripotent potential of fibroblast-derived iPS cells, and genetic manipulation of iPS cells in combination with tetraploid embryo aggregation provides a practical and rapid approach to evaluate the efficacy of gene correction of human diseases in mouse models.

  11. Gene-specific function prediction for non-synonymous mutations in monogenic diabetes genes.

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    Quan Li

    Full Text Available The rapid progress of genomic technologies has been providing new opportunities to address the need of maturity-onset diabetes of the young (MODY molecular diagnosis. However, whether a new mutation causes MODY can be questionable. A number of in silico methods have been developed to predict functional effects of rare human mutations. The purpose of this study is to compare the performance of different bioinformatics methods in the functional prediction of nonsynonymous mutations in each MODY gene, and provides reference matrices to assist the molecular diagnosis of MODY. Our study showed that the prediction scores by different methods of the diabetes mutations were highly correlated, but were more complimentary than replacement to each other. The available in silico methods for the prediction of diabetes mutations had varied performances across different genes. Applying gene-specific thresholds defined by this study may be able to increase the performance of in silico prediction of disease-causing mutations.

  12. The GH/IGF-1 axis in a critical period early in life determines cellular DNA repair capacity by altering transcriptional regulation of DNA repair-related genes: implications for the developmental origins of cancer.

    Science.gov (United States)

    Podlutsky, Andrej; Valcarcel-Ares, Marta Noa; Yancey, Krysta; Podlutskaya, Viktorija; Nagykaldi, Eszter; Gautam, Tripti; Miller, Richard A; Sonntag, William E; Csiszar, Anna; Ungvari, Zoltan

    2017-02-23

    Experimental, clinical, and epidemiological findings support the concept of developmental origins of health and disease (DOHAD), suggesting that early-life hormonal influences during a sensitive period around adolescence have a powerful impact on cancer morbidity later in life. The endocrine changes that occur during puberty are highly conserved across mammalian species and include dramatic increases in circulating GH and IGF-1 levels. Importantly, patients with developmental IGF-1 deficiency due to GH insensitivity (Laron syndrome) do not develop cancer during aging. Rodents with developmental GH/IGF-1 deficiency also exhibit significantly decreased cancer incidence at old age, marked resistance to chemically induced carcinogenesis, and cellular resistance to genotoxic stressors. Early-life treatment of GH/IGF-1-deficient mice and rats with GH reverses the cancer resistance phenotype; however, the underlying molecular mechanisms remain elusive. The present study was designed to test the hypothesis that developmental GH/IGF-1 status impacts cellular DNA repair mechanisms. To achieve that goal, we assessed repair of γ-irradiation-induced DNA damage (single-cell gel electrophoresis/comet assay) and basal and post-irradiation expression of DNA repair-related genes (qPCR) in primary fibroblasts derived from control rats, Lewis dwarf rats (a model of developmental GH/IGF-1 deficiency), and GH-replete dwarf rats (GH administered beginning at 5 weeks of age, for 30 days). We found that developmental GH/IGF-1 deficiency resulted in persisting increases in cellular DNA repair capacity and upregulation of several DNA repair-related genes (e.g., Gadd45a, Bbc3). Peripubertal GH treatment reversed the radiation resistance phenotype. Fibroblasts of GH/IGF-1-deficient Snell dwarf mice also exhibited improved DNA repair capacity, showing that the persisting influence of peripubertal GH/IGF-1 status is not species-dependent. Collectively, GH/IGF-1 levels during a critical period

  13. iFish: predicting the pathogenicity of human nonsynonymous variants using gene-specific/family-specific attributes and classifiers.

    Science.gov (United States)

    Wang, Meng; Wei, Liping

    2016-08-16

    Accurate prediction of the pathogenicity of genomic variants, especially nonsynonymous single nucleotide variants (nsSNVs), is essential in biomedical research and clinical genetics. Most current prediction methods build a generic classifier for all genes. However, different genes and gene families have different features. We investigated whether gene-specific and family-specific customized classifiers could improve prediction accuracy. Customized gene-specific and family-specific attributes were selected with AIC, BIC, and LASSO, and Support Vector Machine classifiers were generated for 254 genes and 152 gene families, covering a total of 5,985 genes. Our results showed that the customized attributes reflected key features of the genes and gene families, and the customized classifiers achieved higher prediction accuracy than the generic classifier. The customized classifiers and the generic classifier for other genes and families were integrated into a new tool named iFish (integrated Functional inference of SNVs in human, http://ifish.cbi.pku.edu.cn). iFish outperformed other methods on benchmark datasets as well as on prioritization of candidate causal variants from whole exome sequencing. iFish provides a user-friendly web-based interface and supports other functionalities such as integration of genetic evidence. iFish would facilitate high-throughput evaluation and prioritization of nsSNVs in human genetics research.

  14. Exploring the potential relevance of human-specific genes to complex disease

    Directory of Open Access Journals (Sweden)

    Cooper David N

    2011-01-01

    Full Text Available Abstract Although human disease genes generally tend to be evolutionarily more ancient than non-disease genes, complex disease genes appear to be represented more frequently than Mendelian disease genes among genes of more recent evolutionary origin. It is therefore proposed that the analysis of human-specific genes might provide new insights into the genetics of complex disease. Cross-comparison with the Human Gene Mutation Database (http://www.hgmd.org revealed a number of examples of disease-causing and disease-associated mutations in putatively human-specific genes. A sizeable proportion of these were missense polymorphisms associated with complex disease. Since both human-specific genes and genes associated with complex disease have often experienced particularly rapid rates of evolutionary change, either due to weaker purifying selection or positive selection, it is proposed that a significant number of human-specific genes may play a role in complex disease.

  15. Polysaccharides of Aloe vera induce MMP-3 and TIMP-2 gene expression during the skin wound repair of rat.

    Science.gov (United States)

    Tabandeh, Mohammad Reza; Oryan, Ahmad; Mohammadalipour, Adel

    2014-04-01

    Polysaccharides are the main macromolecules of Aloe vera gel but no data about their effect on extracellular matrix (ECM) elements are available. Here, mannose rich Aloe vera polysaccharides (AVP) with molecular weight between 50 and 250 kDa were isolated and characterized. Open cutaneous wounds on the back of 45 rats (control and treated) were daily treated with 25mg (n=15) and 50 mg (n=15) AVP for 30 days. The levels of MMP-3 and TIMP-2 gene expression were analyzed using real time PCR. The levels of n-acetyl glucosamine (NAGA), n-acetyl galactosamine (NAGLA) and collagen contents were also measured using standard biochemical methods. Faster wound closure was observed at day 15 post wounding in AVP treated animals in comparison with untreated group. At day 10 post wounding, AVP inhibited MMP-3 gene expression, while afterwards MMP-3 gene expression was upregulated. AVP enhanced TIMP-2 gene expression, collagen, NAGLA and NAGA synthesis in relation to untreated wounds. Our results suggest that AVP has positive effects on the regulation of ECM factor synthesis, which open up new perspectives for the wound repair activity of Aloe vera polysaccharide at molecular level.

  16. Genomic structure and characterization of the Drosophila S3 ribosomal/DNA repair gene and mutant alleles.

    Science.gov (United States)

    Kelley, M R; Xu, Y; Wilson, D M; Deutsch, W A

    2000-03-01

    The Drosophila S3 protein is known to be associated with ribosomes, where it is thought to play a role in the initiation of protein translation. The S3 protein also contains a DNA repair activity, efficiently processing 8-oxoguanine residues in DNA via an N-glycosylase/apurinic-apyrimidinic (AP) lyase activity. The gene that encodes S3 has previously been localized to one of the Minute loci on chromosome 3 in Drosophila. This study focused on the genomic organization of S3 at M(3)95A, initial promoter characterization, and analysis of three mutant alleles at this locus. The S3 gene was found to be a single-copy gene 2 to 3 kb in length and containing a single intron. The upstream 1.6-kb region was analyzed for promoter activity, identifying a presumptive regulatory domain containing potential enhancer and suppressor elements. This finding is of interest, as the S3 gene is constitutively expressed throughout development and mRNA is most likely maternally inherited. Lastly, three Minute alleles from the same locus were sequenced and two alleles found to contain a 22-bp deletion in exon 2, resulting in a truncated S3 protein, although wildtype levels of S3 mRNA and protein were detected in the viable heterozygous Minute alleles, possibly reflecting dosage compensation.

  17. Repairing the Sickle Cell mutation. III. Effect of irradiation wavelength on the specificity and type of photoproduct formed by a 3′-terminal psoralen on a third strand directed to the mutant base pair

    Science.gov (United States)

    Broitman, Steven L.; Amosova, Olga; Fresco, Jacques R.

    2003-01-01

    Using a psoralen delivery system mediated by a DNA third strand that binds selectively to linear target duplexes immediately downstream from the Sickle Cell β-globin gene mutation and the comparable wild-type β-globin gene sequence, the kinetics of formation and yield of psoralen monoadducts and crosslinks with pyrimidine residues at and near the mutant base pair site and its wild-type counterpart were determined. By exploiting irradiation specificities at 300, 365 and 419 nm, it was possible to evaluate the orientation equilibrium of 3′-linked intercalated psoralen and to develop conditions that lead to preferential formation of each type of photoproduct in both the mutant and wild-type sequences. This makes possible the preparation of each type of photoproduct for use as a substrate for DNA repair. In this way, the base pair change(s) that each generates can be established. PMID:12907707

  18. Repairing the Sickle Cell mutation. III. Effect of irradiation wavelength on the specificity and type of photoproduct formed by a 3'-terminal psoralen on a third strand directed to the mutant base pair.

    Science.gov (United States)

    Broitman, Steven L; Amosova, Olga; Fresco, Jacques R

    2003-08-15

    Using a psoralen delivery system mediated by a DNA third strand that binds selectively to linear target duplexes immediately downstream from the Sickle Cell beta-globin gene mutation and the comparable wild-type beta-globin gene sequence, the kinetics of formation and yield of psoralen monoadducts and crosslinks with pyrimidine residues at and near the mutant base pair site and its wild-type counterpart were determined. By exploiting irradiation specificities at 300, 365 and 419 nm, it was possible to evaluate the orientation equilibrium of 3'-linked intercalated psoralen and to develop conditions that lead to preferential formation of each type of photoproduct in both the mutant and wild-type sequences. This makes possible the preparation of each type of photoproduct for use as a substrate for DNA repair. In this way, the base pair change(s) that each generates can be established.

  19. RAD25 (SSL2), the yeast homolog of the human xeroderma pigmentosum group B DNA repair gene, is essential for viability

    Energy Technology Data Exchange (ETDEWEB)

    Park, E.; Prakash, L. (Univ. of Rochester School of Medicine, NY (United States)); Guzder, S.N.; Prakash, S. (Univ. of Rochester, NY (United States)); Koken, M.H.M.; Jaspers-Dekker, I.; Weeda, G.; Hoeijmakers, H.J. (Erasmus Univ., Rotterdam (Netherlands))

    1992-12-01

    Xeroderma pigmentosum (XP) patients are extremely sensitive to ultraviolet (UV) light and suffer from a high incidence of skin cancers, due to a defect in nucleotide excision repair. The disease is genetically heterogeneous, and seven complementation groups, A-G, have been identified. Homologs of human excision repair genes ERCC1, XPDC/ERCC2, and XPAC have been identified in the yeast Saccharomyces cerevisiae. Since no homolog of human XPBC/ERCC3 existed among the known yeast genes, we cloned the yeast homolog by using XPBC cDNA as a hybridization probe. The yeast homolog, RAD25 (SSL2), encodes a protein of 843 amino acids (M[sub r] 95,356). The RAD25 (SSL2)- and XPCX-encoded proteins share 55% identical and 72% conserved amino acid residues, and the two proteins resemble one another in containing the conserved DNA helicase sequence motifs. A nonsense mutation at codon 799 that deletes the 45 C-terminal amino acid residues in RAD25 (SSL2) confers UV sensitivity. This mutation shows epistasis with genes in the excision repair group, whereas a synergistic increase in UN sensitivity occurs when it is combined with mutations in genes in other DNA repair pathways, indicating that RAD25 (SSL2) functions in excision repair but not in other repair pathways. We also show that RAD25 (SSL2) is an essential gene. A mutation of the Lys[sup 392] residue to arginine in the conserved Walker type A nucleotide-binding motif is lethal, suggesting an essential role of the putative RAD 25 (SSL2) ATPase/DNA helicase activity in viability. 40 refs., 3 figs., 1 tab.

  20. Low-level laser irradiation alters mRNA expression from genes involved in DNA repair and genomic stabilization in myoblasts

    Science.gov (United States)

    Trajano, L. A. S. N.; Sergio, L. P. S.; Silva, C. L.; Carvalho, L.; Mencalha, A. L.; Stumbo, A. C.; Fonseca, A. S.

    2016-07-01

    Low-level lasers are used for the treatment of diseases in soft and bone tissues, but few data are available regarding their effects on genomic stability. In this study, we investigated mRNA expression from genes involved in DNA repair and genomic stabilization in myoblasts exposed to low-level infrared laser. C2C12 myoblast cultures in different fetal bovine serum concentrations were exposed to low-level infrared laser (10, 35 and 70 J cm-2), and collected for the evaluation of DNA repair gene expression. Laser exposure increased gene expression related to base excision repair (8-oxoguanine DNA glycosylase and apurinic/apyrimidinic endonuclease 1), nucleotide excision repair (excision repair cross-complementation group 1 and xeroderma pigmentosum C protein) and genomic stabilization (ATM serine/threonine kinase and tumor protein p53) in normal and low fetal bovine serum concentrations. Results suggest that genomic stability could be part of a biostimulation effect of low-level laser therapy in injured muscles.

  1. Many amino acid substitution variants identified in DNA repair genes during human population screenings are predicted to impact protein function

    Energy Technology Data Exchange (ETDEWEB)

    Xi, T; Jones, I M; Mohrenweiser, H W

    2003-11-03

    Over 520 different amino acid substitution variants have been previously identified in the systematic screening of 91 human DNA repair genes for sequence variation. Two algorithms were employed to predict the impact of these amino acid substitutions on protein activity. Sorting Intolerant From Tolerant (SIFT) classified 226 of 508 variants (44%) as ''Intolerant''. Polymorphism Phenotyping (PolyPhen) classed 165 of 489 amino acid substitutions (34%) as ''Probably or Possibly Damaging''. Another 9-15% of the variants were classed as ''Potentially Intolerant or Damaging''. The results from the two algorithms are highly associated, with concordance in predicted impact observed for {approx}62% of the variants. Twenty one to thirty one percent of the variant proteins are predicted to exhibit reduced activity by both algorithms. These variants occur at slightly lower individual allele frequency than do the variants classified as ''Tolerant'' or ''Benign''. Both algorithms correctly predicted the impact of 26 functionally characterized amino acid substitutions in the APE1 protein on biochemical activity, with one exception. It is concluded that a substantial fraction of the missense variants observed in the general human population are functionally relevant. These variants are expected to be the molecular genetic and biochemical basis for the associations of reduced DNA repair capacity phenotypes with elevated cancer risk.

  2. Heritable and Lineage-Specific Gene Knockdown in Zebrafish Embryo

    NARCIS (Netherlands)

    Dong, Mei; Fu, Yan-Fang; Du, Ting-Ting; Jing, Chang-Bin; Fu, Chun-Tang; Chen, Yi; Jin, Yi; Deng, Min; Liu, Ting Xi

    2009-01-01

    Background: Reduced expression of developmentally important genes and tumor suppressors due to haploinsufficiency or epigenetic suppression has been shown to contribute to the pathogenesis of various malignancies. However, methodology that allows spatio-temporally knockdown of gene expression in var

  3. Automatic, context-specific generation of Gene Ontology slims

    Directory of Open Access Journals (Sweden)

    Sehgal Muhammad

    2010-10-01

    Full Text Available Abstract Background The use of ontologies to control vocabulary and structure annotation has added value to genome-scale data, and contributed to the capture and re-use of knowledge across research domains. Gene Ontology (GO is widely used to capture detailed expert knowledge in genomic-scale datasets and as a consequence has grown to contain many terms, making it unwieldy for many applications. To increase its ease of manipulation and efficiency of use, subsets called GO slims are often created by collapsing terms upward into more general, high-level terms relevant to a particular context. Creation of a GO slim currently requires manipulation and editing of GO by an expert (or community familiar with both the ontology and the biological context. Decisions about which terms to include are necessarily subjective, and the creation process itself and subsequent curation are time-consuming and largely manual. Results Here we present an objective framework for generating customised ontology slims for specific annotated datasets, exploiting information latent in the structure of the ontology graph and in the annotation data. This framework combines ontology engineering approaches, and a data-driven algorithm that draws on graph and information theory. We illustrate this method by application to GO, generating GO slims at different information thresholds, characterising their depth of semantics and demonstrating the resulting gains in statistical power. Conclusions Our GO slim creation pipeline is available for use in conjunction with any GO-annotated dataset, and creates dataset-specific, objectively defined slims. This method is fast and scalable for application to other biomedical ontologies.

  4. Associations of polymorphisms in DNA repair genes and MDR1 gene with chemotherapy response and survival of non-small cell lung cancer.

    Directory of Open Access Journals (Sweden)

    Yan Du

    Full Text Available OBJECTIVES: We aimed to determine the associations of genetic polymorphisms of excision repair cross-complementation group 1 (ERCC1 rs11615, xeroderma pigmentosum group D (XPD/ERCC2 rs13181, X-ray repair cross complementing group 1 (XRCC1 rs25487, XRCC3 rs1799794, and breast cancer susceptibility gene 1 (BRCA1 rs1799966 from the DNA repair pathway and multiple drug resistance 1 (MDR1/ABCB1 rs1045642 with response to chemotherapy and survival of non-small cell lung cancer (NSCLC in a Chinese population. MATERIALS AND METHODS: A total of 352 NSCLC patients were enrolled to evaluate the associations of the six SNPs with response to chemotherapy and overall survival. Logistic regressions were applied to test the associations of genetic polymorphisms with response to chemotherapy in 161 advanced NSCLC patients. Overall survival was analyzed in 161 advanced and 156 early stage NSCLC patients using the Kaplan-Meier method with log-rank test, respectively. Multivariate Cox proportional hazards model was performed to determine the factors independently associated with NSCLC prognosis. RESULTS: BRCA1 rs1799966 minor allele C (TC+CC vs. TT, OR = 0.402, 95% CI = 0.204-0.794, p = 0.008 and MDR1/ABCB1 rs1045642 minor allele A (GA +AA vs. GG, OR = 0.478, 95% CI = 0.244-0.934, p = 0.030 were associated with a better response to chemotherapy in advanced NSCLC patients. Survival analyses indicated that BRCA1 rs1799966 TC+CC genotypes were associated with a decreased risk of death (HR = 0.617, 95% CI = 0.402-0.948, p = 0.028 in advanced NSCLC patients, and the association was still significant after the adjustment for covariates. Multivariate Cox regression analysis showed that ERCC1 rs11615 AA genotype (P = 0.020 and smoking (p = 0.037 were associated with increased risks of death in early stage NSCLC patients after surgery. CONCLUSIONS: Polymorphisms of genes in DNA repair pathway and MDR1 could contribute to chemotherapy response and survival of patients with

  5. Adaptations to endosymbiosis in a cnidarian-dinoflagellate association: differential gene expression and specific gene duplications.

    Directory of Open Access Journals (Sweden)

    Philippe Ganot

    2011-07-01

    Full Text Available Trophic endosymbiosis between anthozoans and photosynthetic dinoflagellates forms the key foundation of reef ecosystems. Dysfunction and collapse of symbiosis lead to bleaching (symbiont expulsion, which is responsible for the severe worldwide decline of coral reefs. Molecular signals are central to the stability of this partnership and are therefore closely related to coral health. To decipher inter-partner signaling, we developed genomic resources (cDNA library and microarrays from the symbiotic sea anemone Anemonia viridis. Here we describe differential expression between symbiotic (also called zooxanthellate anemones or aposymbiotic (also called bleached A. viridis specimens, using microarray hybridizations and qPCR experiments. We mapped, for the first time, transcript abundance separately in the epidermal cell layer and the gastrodermal cells that host photosynthetic symbionts. Transcriptomic profiles showed large inter-individual variability, indicating that aposymbiosis could be induced by different pathways. We defined a restricted subset of 39 common genes that are characteristic of the symbiotic or aposymbiotic states. We demonstrated that transcription of many genes belonging to this set is specifically enhanced in the symbiotic cells (gastroderm. A model is proposed where the aposymbiotic and therefore heterotrophic state triggers vesicular trafficking, whereas the symbiotic and therefore autotrophic state favors metabolic exchanges between host and symbiont. Several genetic pathways were investigated in more detail: i a key vitamin K-dependant process involved in the dinoflagellate-cnidarian recognition; ii two cnidarian tissue-specific carbonic anhydrases involved in the carbon transfer from the environment to the intracellular symbionts; iii host collagen synthesis, mostly supported by the symbiotic tissue. Further, we identified specific gene duplications and showed that the cnidarian-specific isoform was also up-regulated both

  6. Cell-Specific Promoters Enable Lipid-Based Nanoparticles to Deliver Genes to Specific Cells of the Retina In Vivo.

    Science.gov (United States)

    Wang, Yuhong; Rajala, Ammaji; Cao, Binrui; Ranjo-Bishop, Michelle; Agbaga, Martin-Paul; Mao, Chuanbin; Rajala, Raju V S

    2016-01-01

    Non-viral vectors, such as lipid-based nanoparticles (liposome-protamine-DNA complex [LPD]), could be used to deliver a functional gene to the retina to correct visual function and treat blindness. However, one of the limitations of LPD is the lack of cell specificity, as the retina is composed of seven types of cells. If the same gene is expressed in multiple cell types or is absent from one desired cell type, LPD-mediated gene delivery to every cell may have off-target effects. To circumvent this problem, we have tested LPD-mediated gene delivery using various generalized, modified, and retinal cell-specific promoters. We achieved retinal pigment epithelium cell specificity with vitelliform macular dystrophy (VMD2), rod cell specificity with mouse rhodopsin, cone cell specificity with red/green opsin, and ganglion cell specificity with thymocyte antigen promoters. Here we show for the first time that cell-specific promoters enable lipid-based nanoparticles to deliver genes to specific cells of the retina in vivo. This work will inspire investigators in the field of lipid nanotechnology to couple cell-specific promoters to drive expression in a cell- and tissue-specific manner.

  7. Intergenerational and striatal CAG repeat instability in Huntington's disease knock-in mice involve different DNA repair genes.

    Science.gov (United States)

    Dragileva, Ella; Hendricks, Audrey; Teed, Allison; Gillis, Tammy; Lopez, Edith T; Friedberg, Errol C; Kucherlapati, Raju; Edelmann, Winfried; Lunetta, Kathryn L; MacDonald, Marcy E; Wheeler, Vanessa C

    2009-01-01

    Modifying the length of the Huntington's disease (HD) CAG repeat, the major determinant of age of disease onset, is an attractive therapeutic approach. To explore this we are investigating mechanisms of intergenerational and somatic HD CAG repeat instability. Here, we have crossed HD CAG knock-in mice onto backgrounds deficient in mismatch repair genes, Msh3 and Msh6, to discern the effects on CAG repeat size and disease pathogenesis. We find that different mechanisms predominate in inherited and somatic instability, with Msh6 protecting against intergenerational contractions and Msh3 required both for increasing CAG length and for enhancing an early disease phenotype in striatum. Therefore, attempts to decrease inherited repeat size may entail a full understanding of Msh6 complexes, while attempts to block the age-dependent increases in CAG size in striatal neurons and to slow the disease process will require a full elucidation of Msh3 complexes and their function in CAG repeat instability.

  8. Exposure to Hycanthone alters chromatin structure around specific gene functions and specific repeats in Schistosoma mansoni

    Directory of Open Access Journals (Sweden)

    David eRoquis

    2014-07-01

    Full Text Available Schistosoma mansoni is a parasitic plathyhelminth responsible for intestinal schistosomiasis (or bilharziasis, a disease affecting 67 million people worldwide and causing an important economic burden. The schistosomicides hycanthone, and its later proxy oxamniquine, were widely used for treatments in endemic areas during the 20th century. Recently, the mechanism of action, as well as the genetic origin of a stably and Mendelian inherited resistance for both drugs was elucidated in two strains. However, several observations suggested early on that alternative mechanisms might exist, by which resistance could be induced for these two drugs in sensitive lines of schistosomes. This induced resistance appeared rapidly, within the first generation, but was metastable (not stably inherited. Epigenetic inheritance could explain such a phenomenon and we therefore re-analyzed the historical data with our current knowledge of epigenetics. In addition, we performed new experiments such as ChIP-seq on hycanthone treated worms. We found distinct chromatin structure changes between sensitive worms and induced resistant worms from the same strain. No specific pathway was discovered, but genes in which chromatin structure modification were observed are mostly associated with transport and catabolism, which makes sense in the context of the elimination of the drug. Specific differences were observed in the repetitive compartment of the genome. We finally describe what types of experiments are needed to understand the complexity of heritability that can be based on genetic and/or epigenetic mechanisms for drug resistance in schistosomes.

  9. Tissue Specificity and Sex-Specific Regulatory Variation Permit the Evolution of Sex-Biased Gene Expression.

    Science.gov (United States)

    Dean, Rebecca; Mank, Judith E

    2016-09-01

    Genetic correlations between males and females are often thought to constrain the evolution of sexual dimorphism. However, sexually dimorphic traits and the underlying sexually dimorphic gene expression patterns are often rapidly evolving. We explore this apparent paradox by measuring the genetic correlation in gene expression between males and females (Cmf) across broad evolutionary timescales, using two RNA-sequencing data sets spanning multiple populations and multiple species. We find that unbiased genes have higher Cmf than sex-biased genes, consistent with intersexual genetic correlations constraining the evolution of sexual dimorphism. However, we found that highly sex-biased genes (both male and female biased) also had higher tissue specificity, and unbiased genes had greater expression breadth, suggesting that pleiotropy may constrain the breakdown of intersexual genetic correlations. Finally, we show that genes with high Cmf showed some degree of sex-specific changes in gene expression in males and females. Together, our results suggest that genetic correlations between males and females may be less important in constraining the evolution of sex-biased gene expression than pleiotropy. Sex-specific regulatory variation and tissue specificity may resolve the paradox of widespread sex bias within a largely shared genome.

  10. Deproteinized bone with VEGF gene transfer to facilitate the repair of early avascular necrosis of femoral head of rabbit

    Institute of Scientific and Technical Information of China (English)

    CAO Kai; HUANG Wei; AN Hong; JIANG Dian-ming; SHU Yong; HAN Zhi-min

    2009-01-01

    Objective: To explore a new method for early avascular necrosis of femoral head (AVNFH) therapy.Methods: Sixty-nine AVNFH New Zealand adult rabbits were randomly divided into three groups with equal number. In Group A, deproteinized bone (DPB) that absorbed with recombinant plasmid pcDNA3.1-hVEGF165 was implanted into the drilled tunnel of necrotic femoral head. In Group B, only DPB was implanted. In Group C, only tunnel was drilled without DPB or plasmid implanted. Femoral head specimens were obtained at postoperative 1, 2, 4, 8, 16 weeks. The expression of VEGF165 and collagen I was detected by immunohistochemistry. Bone formation was detected generally by X-ray. Angiogenesis and the repair of the femoral head were observed histologically.Results: The expression of VEGF 165 could be detected 2 weeks after implantation in Group A, but it was not observed in other groups. The result of collagen I expression had a significantly difference 2, 4 and 8 weeks after operation in Group A from those in other groups (P<0.01).X-ray results indicated that there was more bone formation in Group A than in other groups. The regenerated capillary vessels staining result of necrotic femoral head in Group A was significantly different from those in other groups at postoperative 2 and 4 weeks (P<0.01).Conclusions: Transfection ofhVEGF165 gene enhances local angiogenesis and DPB-VEGF compound improves the repair of necrotic femoral head. Deproteinized bone grafting with VEGF gene transfer provides a potential method for the treatment of osteonecrosis.

  11. Sequence and stress-response analyses of the DNA mismatch repair gene hexA in Lactococcus lactis.

    Science.gov (United States)

    Ren, J; Park, J H; Dunn, N W; Kim, W S

    2001-10-01

    The DNA mismatch repair gene hexA was identified in Lactococcus lactis by PCR amplification by using a pair of primers homologous to the DNA-binding Dps protein. The gene in its entirety, including the regulatory regions, was sequenced, by using a strategy of chromosomal walking based on two PCR protocols. The open reading frame of 2526 bp was preceded by a strong ribosome-binding site (AGGAAG) and was followed by a potential transcription terminator (hairpin loop structure). The 5' terminus of the hexA mRNA was located 135 bp upstream of the start codon, and putative -10 and -35 regions were identified. The deduced amino acid sequence revealed two motifs, the ATP/GTP-binding site (P-loop) and the "MutS family signature". The hexA promoter was cloned into pMU1327, which contained a promoter-less CAT reporter gene, and the promoter activity was examined under oxidative-stress conditions. It appears that the promoter activity is down-shifted by H2O2 at 4 mM.

  12. A New Powerful Method for Site-Specific Transgene Stabilization Based on Chromosomal Double-Strand Break Repair

    Science.gov (United States)

    Kravchuk, Oksana; Savitsky, Mikhail

    2011-01-01

    Transgenic insects are a promising tool in sterile insect techniques and population replacement strategies. Such transgenic insects can be created using nonautonomous transposons, which cannot be transferred without a transposase source. In biocontrol procedures where large numbers of insects are released, there is increased risk of transgene remobilization caused by external transposase sources that can alter the characteristics of the transgenic organisms lead horizontal transgene transfer to other species. Here we describe a novel, effective method for transgene stabilization based on the introduction of directed double-strand breaks (DSB) into a genome-integrated sequence and their subsequent repair by the single-strand annealing (SSA) pathway. Due to the construct's organization, the repair pathway is predictable, such that all transposon and marker sequences can be deleted, while preserving integration of exogenous DNA in the genome. The exceptional conservation of DNA repair pathways makes this method suitable for a broad range of organisms. PMID:22022613

  13. A new powerful method for site-specific transgene stabilization based on chromosomal double-strand break repair.

    Directory of Open Access Journals (Sweden)

    Artem Tkachuk

    Full Text Available Transgenic insects are a promising tool in sterile insect techniques and population replacement strategies. Such transgenic insects can be created using nonautonomous transposons, which cannot be transferred without a transposase source. In biocontrol procedures where large numbers of insects are released, there is increased risk of transgene remobilization caused by external transposase sources that can alter the characteristics of the transgenic organisms lead horizontal transgene transfer to other species. Here we describe a novel, effective method for transgene stabilization based on the introduction of directed double-strand breaks (DSB into a genome-integrated sequence and their subsequent repair by the single-strand annealing (SSA pathway. Due to the construct's organization, the repair pathway is predictable, such that all transposon and marker sequences can be deleted, while preserving integration of exogenous DNA in the genome. The exceptional conservation of DNA repair pathways makes this method suitable for a broad range of organisms.

  14. Rapid evolution and gene-specific patterns of selection for three genes of spermatogenesis in Drosophila.

    Science.gov (United States)

    Civetta, Alberto; Rajakumar, Sujeetha A; Brouwers, Barb; Bacik, John P

    2006-03-01

    Hybrid males resulting from crosses between closely related species of Drosophila are sterile. The F1 hybrid sterility phenotype is mainly due to defects occurring during late stages of development that relate to sperm individualization, and so genes controlling sperm development may have been subjected to selective diversification between species. It is also possible that genes of spermatogenesis experience selective constraints given their role in a developmental pathway. We analyzed the molecular evolution of three genes playing a role during the sperm developmental pathway in Drosophila at an early (bam), a mid (aly), and a late (dj) stage. The complete coding region of these genes was sequenced in different strains of Drosophila melanogaster and Drosophila simulans. All three genes showed rapid divergence between species, with larger numbers of nonsynonymous to synonymous differences between species than polymorphisms. Although this could be interpreted as evidence for positive selection at all three genes, formal tests of selection do not support such a conclusion. Departures from neutrality were detected only for dj and bam but not aly. The role played by selection is unique and determined by gene-specific characteristics rather than site of expression. In dj, the departure was due to a high proportion of neutral synonymous polymorphisms in D. simulans, and there was evidence of purifying selection maintaining a high lysine amino acid protein content that is characteristic of other DNA-binding proteins. The earliest spermatogenesis gene surveyed, which plays a role in both male and female gametogenesis, was bam, and its significant departure from neutrality was due to an excess of nonsynonymous substitutions between species. Bam is degraded at the end of mitosis, and rapid evolutionary changes among species might be a characteristic shared with other degradable transient proteins. However, the large number of nonsynonymous changes between D. melanogaster and

  15. Ultraviolet B retards growth, induces oxidative stress, and modulates DNA repair-related gene and heat shock protein gene expression in the monogonont rotifer, Brachionus sp

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Ryeo-Ok [Department of Chemistry, and Research Institute for Natural Sciences, College of Natural Sciences, Hanyang University, Seoul 133-791 (Korea, Republic of); Rhee, Jae-Sung [Department of Molecular and Environmental Bioscience, Graduate School, Hanyang University, Seoul 133-791 (Korea, Republic of); Won, Eun-Ji [Department of Environmental Marine Sciences, College of Science and Technology, Hanyang University, Ansan 426-791 (Korea, Republic of); Lee, Kyun-Woo [Department of Chemistry, and Research Institute for Natural Sciences, College of Natural Sciences, Hanyang University, Seoul 133-791 (Korea, Republic of); Kang, Chang-Mo [Laboratory of Cytogenetics and Tissue Regeneration, Korea Institute of Radiological and Medical Science, Seoul 139-709 (Korea, Republic of); Lee, Young-Mi [Department of Green Life Science, College of Convergence, Sangmyung University, Seoul 110-743 (Korea, Republic of); Lee, Jae-Seong, E-mail: jslee2@hanyang.ac.kr [Department of Chemistry, and Research Institute for Natural Sciences, College of Natural Sciences, Hanyang University, Seoul 133-791 (Korea, Republic of); Department of Molecular and Environmental Bioscience, Graduate School, Hanyang University, Seoul 133-791 (Korea, Republic of)

    2011-02-15

    Ultraviolet B (UV-B) radiation causes direct cellular damage by breakage of DNA strands and oxidative stress induction in aquatic organisms. To understand the effect of UV-B radiation on the rotifer, Brachionus sp., several parameters including 24-h survival rate, population growth rate, and ROS level were measured after exposure to a wide range of UV-B doses. To check the expression of other important inducible genes such as replication protein A (RPA), DNA-dependent protein kinase (DNA-PK), Ku70, Ku80, and heat shock proteins (hsps) after UV-B radiation, we observed dose- and time-dependency at 2 kJ/m{sup 2}. We also examined 13 hsp genes for their roles in the UV-B damaged rotifer. Results showed that UV-B remarkably inhibited the population growth of Brachionus sp. The level of intracellular reactive oxygen species (ROS) was high at 2 kJ/m{sup 2}, suggesting that 2 kJ/m{sup 2} would already be toxic. This result was supported by other enzymatic activities, such as GSH levels, glutathione peroxidase, glutathione S-transferase, and glutathione reductase. For dose dependency, low doses of UV-B radiation (2, 4, and 6 kJ/m{sup 2}) significantly up-regulated the examined genes (e.g. RPA, DNA-PK, Ku70, and Ku80). For the time course study, RPA genes showed immediate up-regulation but returned to basal or lower expression levels compared to the control 3 h after UV-B exposure. The DNA-PK and Ku70/80 genes significantly increased, indicating that they may be involved in repairing processes against a low dose of UV-B exposure (2 kJ/m{sup 2}). At the basal level, the hsp90{alpha}1 gene showed the highest expression, and followed by hsp10, hsp30, hsp60, and hsc70, and hsp90{beta} in adults (w/o egg). In eggs, the hsp10 gene was expressed the highest, and followed by hsp30, hsp27, hsp90{alpha}1, and hsp60 genes. In real-time RT-PCR array on rotifer hsp genes, low doses of UV-B radiation (2 and 4 kJ/m{sup 2}) showed up-regulation of several hsp genes but most of the hsp

  16. Identification of mismatch repair gene mutations in young patients with colorectal cancer and in patients with multiple tumours associated with hereditary non-polyposis colorectal cancer

    NARCIS (Netherlands)

    Niessen, R C; Berends, M J W; Wu, Y; Sijmons, R H; Hollema, H; Ligtenberg, M J L; de Walle, H E K; de Vries, E G E; Karrenbeld, A; Buys, C H C M; van der Zee, A G J; Hofstra, R M W; Kleibeuker, J H

    2006-01-01

    Background: Patients with early-onset colorectal cancer (CRC) or those with multiple tumours associated with hereditary non-polyposis colorectal cancer (HNPCC) raise suspicion of the presence of germline DNA mismatch repair (MMR) gene mutations. Aim: To analyse the value of family history,

  17. Identification of mismatch repair gene mutations in young patients with colorectal cancer and in patients with multiple tumours associated with hereditary non-polyposis colorectal cancer.

    NARCIS (Netherlands)

    Niessen, R.C.; Berends, M.J.; Wu, Y.; Sijmons, R.H.; Hollema, H.; Ligtenberg, M.J.L.; Walle, H.E. de; Vries, E.G.F. de; Karrenbeld, A.; Buys, C.H.C.M.; Zee, A.G. van der; Hofstra, R.M.; Kleibeuker, J.H.

    2006-01-01

    BACKGROUND: Patients with early-onset colorectal cancer (CRC) or those with multiple tumours associated with hereditary non-polyposis colorectal cancer (HNPCC) raise suspicion of the presence of germline DNA mismatch repair (MMR) gene mutations. AIM: To analyse the value of family history,

  18. Tissue-specific transplantation antigen P35B (TSTA3) immune response-mediated metabolism coupling cell cycle to postreplication repair network in no-tumor hepatitis/cirrhotic tissues (HBV or HCV infection) by biocomputation.

    Science.gov (United States)

    Wang, Lin; Huang, Juxiang; Jiang, Minghu; Lin, Hong

    2012-06-01

    We constructed the low-expression tissue-specific transplantation antigen P35B (TSTA3) immune response-mediated metabolism coupling cell cycle to postreplication repair network in no-tumor hepatitis/cirrhotic tissues (HBV or HCV infection) compared with high-expression (fold change ≥ 2) human hepatocellular carcinoma in GEO data set, by using integration of gene regulatory network inference method with gene ontology analysis of TSTA3-activated up- and downstream networks. Our results showed TSTA3 upstream-activated CCNB2, CKS1B, ELAVL3, GAS7, NQO1, NTN1, OCRL, PLA2G1B, REG3A, SSTR5, etc. and TSTA3 downstream-activated BAP1, BRCA1, CCL20, MCM2, MS4A2, NTN1, REG1A, TP53I11, VCAN, SLC16A3, etc. in no-tumor hepatitis/cirrhotic tissues. TSTA3-activated network enhanced the regulation of apoptosis, cyclin-dependent protein kinase activity, cell migration, insulin secretion, transcription, cell division, cell proliferation, DNA replication, postreplication repair, cell differentiation, T-cell homeostasis, neutrophil-mediated immunity, neutrophil chemotaxis, interleukin-8 production, inflammatory response, immune response, B-cell activation, humoral immune response, actin filament organization, xenobiotic metabolism, lipid metabolism, phospholipid metabolism, leukotriene biosynthesis, organismal lipid catabolism, phosphatidylcholine metabolism, arachidonic acid secretion, activation of phospholipase A2, deoxyribonucleotide biosynthesis, heterophilic cell adhesion, activation of MAPK activity, signal transduction by p53 class mediator resulting in transcription of p21 class mediator, G-protein-coupled receptor protein signaling pathway, response to toxin, acute-phase response, DNA damage response, intercellular junction assembly, cell communication, and cell recognition, as a result of inducing immune response-mediated metabolism coupling cell cycle to postreplication repair in no-tumor hepatitis/cirrhotic tissues.

  19. Genome-scale analysis of positional clustering of mouse testis-specific genes

    Directory of Open Access Journals (Sweden)

    Lee Bernett TK

    2005-01-01

    Full Text Available Abstract Background Genes are not randomly distributed on a chromosome as they were thought even after removal of tandem repeats. The positional clustering of co-expressed genes is known in prokaryotes and recently reported in several eukaryotic organisms such as Caenorhabditis elegans, Drosophila melanogaster, and Homo sapiens. In order to further investigate the mode of tissue-specific gene clustering in higher eukaryotes, we have performed a genome-scale analysis of positional clustering of the mouse testis-specific genes. Results Our computational analysis shows that a large proportion of testis-specific genes are clustered in groups of 2 to 5 genes in the mouse genome. The number of clusters is much higher than expected by chance even after removal of tandem repeats. Conclusion Our result suggests that testis-specific genes tend to cluster on the mouse chromosomes. This provides another piece of evidence for the hypothesis that clusters of tissue-specific genes do exist.

  20. Gene-Specific Demethylation as Targeted Therapy in MDS

    Science.gov (United States)

    2016-07-01

    methylation is considered a dominant mechanism for Tumor Suppressor Genes silencing during MDS evolution to AML, but the causes leading to aberrant DNA...DNA methylation is considered a dominant mechanism for Tumor Suppressor Genes silencing during MDS evolution to AML, but the causes leading to...study two tumor suppressor genes chromosomally linked and frequently methylated in cancer: the CDKN2A (aka P16 ) and CDKN2B (aka: P15). While P16 is

  1. Locus-specific ribosomal RNA gene silencing in nucleolar dominance.

    Directory of Open Access Journals (Sweden)

    Michelle S Lewis

    Full Text Available The silencing of one parental set of rRNA genes in a genetic hybrid is an epigenetic phenomenon known as nucleolar dominance. We showed previously that silencing is restricted to the nucleolus organizer regions (NORs, the loci where rRNA genes are tandemly arrayed, and does not spread to or from neighboring protein-coding genes. One hypothesis is that nucleolar dominance is the net result of hundreds of silencing events acting one rRNA gene at a time. A prediction of this hypothesis is that rRNA gene silencing should occur independent of chromosomal location. An alternative hypothesis is that the regulatory unit in nucleolar dominance is the NOR, rather than each individual rRNA gene, in which case NOR localization may be essential for rRNA gene silencing. To test these alternative hypotheses, we examined the fates of rRNA transgenes integrated at ectopic locations. The transgenes were accurately transcribed in all independent transgenic Arabidopsis thaliana lines tested, indicating that NOR localization is not required for rRNA gene expression. Upon crossing the transgenic A. thaliana lines as ovule parents with A. lyrata to form F1 hybrids, a new system for the study of nucleolar dominance, the endogenous rRNA genes located within the A. thaliana NORs are silenced. However, rRNA transgenes escaped silencing in multiple independent hybrids. Collectively, our data suggest that rRNA gene activation can occur in a gene-autonomous fashion, independent of chromosomal location, whereas rRNA gene silencing in nucleolar dominance is locus-dependent.

  2. Genetic variations in DNA repair genes, radiosensitivity to cancer and susceptibility to acute tissue reactions in radiotherapy-treated cancer patients

    Energy Technology Data Exchange (ETDEWEB)

    Chistiakov, Dimitry A. (Dept. of Pathology, Univ. of Pittsburgh, Pittsburgh (US)); Voronova, Natalia V. (Dept. of Molecular Diagnostics, National Research Center GosNIIgenetika, Moscow (RU)); Chistiakov, Pavel A. (Dept. of Radiology, Cancer Research Center, Moscow (RU))

    2008-06-15

    Ionizing radiation is a well established carcinogen for human cells. At low doses, radiation exposure mainly results in generation of double strand breaks (DSBs). Radiation-related DSBs could be directly linked to the formation of chromosomal rearrangements as has been proven for radiation-induced thyroid tumors. Repair of DSBs presumably involves two main pathways, non-homologous end joining (NHEJ) and homologous recombination (HR). A number of known inherited syndromes, such as ataxia telangiectasia, ataxia-telangiectasia like-disorder, radiosensitive severe combined immunodeficiency, Nijmegen breakage syndrome, and LIG4 deficiency are associated with increased radiosensitivity and/or cancer risk. Many of them are caused by mutations in DNA repair genes. Recent studies also suggest that variations in the DNA repair capacity in the general population may influence cancer susceptibility. In this paper, we summarize the current status of DNA repair proteins as potential targets for radiation-induced cancer risk. We will focus on genetic alterations in genes involved in HR- and NHEJ-mediated repair of DSBs, which could influence predisposition to radiation-related cancer and thereby explain interindividual differences in radiosensitivity or radioresistance in a general population

  3. Large Scale Gene Expression Meta-Analysis Reveals Tissue-Specific, Sex-Biased Gene Expression in Humans

    Science.gov (United States)

    Mayne, Benjamin T.; Bianco-Miotto, Tina; Buckberry, Sam; Breen, James; Clifton, Vicki; Shoubridge, Cheryl; Roberts, Claire T.

    2016-01-01

    The severity and prevalence of many diseases are known to differ between the sexes. Organ specific sex-biased gene expression may underpin these and other sexually dimorphic traits. To further our understanding of sex differences in transcriptional regulation, we performed meta-analyses of sex biased gene expression in multiple human tissues. We analyzed 22 publicly available human gene expression microarray data sets including over 2500 samples from 15 different tissues and 9 different organs. Briefly, by using an inverse-variance method we determined the effect size difference of gene expression between males and females. We found the greatest sex differences in gene expression in the brain, specifically in the anterior cingulate cortex, (1818 genes), followed by the heart (375 genes), kidney (224 genes), colon (218 genes), and thyroid (163 genes). More interestingly, we found different parts of the brain with varying numbers and identity of sex-biased genes, indicating that specific cortical regions may influence sexually dimorphic traits. The majority of sex-biased genes in other tissues such as the bladder, liver, lungs, and pancreas were on the sex chromosomes or involved in sex hormone production. On average in each tissue, 32% of autosomal genes that were expressed in a sex-biased fashion contained androgen or estrogen hormone response elements. Interestingly, across all tissues, we found approximately two-thirds of autosomal genes that were sex-biased were not under direct influence of sex hormones. To our knowledge this is the largest analysis of sex-biased gene expression in human tissues to date. We identified many sex-biased genes that were not under the direct influence of sex chromosome genes or sex hormones. These may provide targets for future development of sex-specific treatments for diseases.

  4. Large scale gene expression meta-analysis reveals tissue-specific, sex-biased gene expression in humans

    Directory of Open Access Journals (Sweden)

    Benjamin Mayne

    2016-10-01

    Full Text Available The severity and prevalence of many diseases are known to differ between the sexes. Organ specific sex-biased gene expression may underpin these and other sexually dimorphic traits. To further our understanding of sex differences in transcriptional regulation, we performed meta-analyses of sex biased gene expression in multiple human tissues. We analysed 22 publicly available human gene expression microarray data sets including over 2500 samples from 15 different tissues and 9 different organs. Briefly, by using an inverse-variance method we determined the effect size difference of gene expression between males and females. We found the greatest sex differences in gene expression in the brain, specifically in the anterior cingulate cortex, (1818 genes, followed by the heart (375 genes, kidney (224 genes, colon (218 genes and thyroid (163 genes. More interestingly, we found different parts of the brain with varying numbers and identity of sex-biased genes, indicating that specific cortical regions may influence sexually dimorphic traits. The majority of sex-biased genes in other tissues such as the bladder, liver, lungs and pancreas were on the sex chromosomes or involved in sex hormone production. On average in each tissue, 32% of autosomal genes that were expressed in a sex-biased fashion contained androgen or estrogen hormone response elements. Interestingly, across all tissues, we found approximately two-thirds of autosomal genes that were sex-biased were not under direct influence of sex hormones. To our knowledge this is the largest analysis of sex-biased gene expression in human tissues to date. We identified many sex-biased genes that were not under the direct influence of sex chromosome genes or sex hormones. These may provide targets for future development of sex-specific treatments for diseases.

  5. Genetic variants in DNA double-strand break repair genes and risk of salivary gland carcinoma: a case-control study.

    Directory of Open Access Journals (Sweden)

    Li Xu

    Full Text Available DNA double strand break (DSB repair is the primary defense mechanism against ionizing radiation-induced DNA damage. Ionizing radiation is the only established risk factor for salivary gland carcinoma (SGC. We hypothesized that genetic variants in DSB repair genes contribute to individual variation in susceptibility to SGC. To test this hypothesis, we conducted a case-control study in which we analyzed 415 single nucleotide polymorphisms (SNPs in 45 DSB repair genes in 352 SGC cases and 598 controls. Multivariate logistic regression analysis was performed to calculate odds ratios (ORs and 95% confidence intervals (CIs. Rs3748522 in RAD52 and rs13180356 in XRCC4 were significantly associated with SGC after Bonferroni adjustment; ORs (95% CIs for the variant alleles of these SNPs were 1.71 (1.40-2.09, P = 1.70 × 10(-7 and 0.58 (0.45-0.74, P = 2.00 × 10(-5 respectively. The genetic effects were modulated by histological subtype. The association of RAD52-rs3748522 with SGC was strongest for mucoepidermoid carcinoma (OR = 2.21, 95% CI: 1.55-3.15, P = 1.25 × 10(-5, n = 74, and the association of XRCC4-rs13180356 with SGC was strongest for adenoid cystic carcinoma (OR = 0.60, 95% CI: 0.42-0.87, P = 6.91 × 10(-3, n = 123. Gene-level association analysis revealed one gene, PRKDC, with a marginally significant association with SGC risk in non-Hispanic whites. To our knowledge, this study is the first to comprehensively evaluate the genetic effect of DSB repair genes on SGC risk. Our results indicate that genetic variants in the DSB repair pathways contribute to inter-individual differences in susceptibility to SGC and show that the impact of genetic variants differs by histological subtype. Independent studies are warranted to confirm these findings.

  6. Loss of genes related to Nucleotide Excision Repair (NER) and implications for reductive genome evolution in symbionts of deep-sea vesicomyid clams

    Science.gov (United States)

    Shimamura, Shigeru; Kaneko, Takashi; Ozawa, Genki; Matsumoto, Mamiko Nishino; Koshiishi, Takeru; Takaki, Yoshihiro; Kato, Chiaki; Takai, Ken; Yoshida, Takao; Fujikura, Katsunori; Barry, James P.

    2017-01-01

    Intracellular thioautotrophic symbionts of deep-sea vesicomyid clams lack some DNA repair genes and are thought to be undergoing reductive genome evolution (RGE). In this study, we addressed two questions, 1) how these symbionts lost their DNA repair genes and 2) how such losses affect RGE. For the first question, we examined genes associated with nucleotide excision repair (NER; uvrA, uvrB, uvrC, uvrD, uvrD paralog [uvrDp] and mfd) in 12 symbionts of vesicomyid clams belonging to two clades (5 clade I and 7 clade II symbionts). While uvrA, uvrDp and mfd were conserved in all symbionts, uvrB and uvrC were degraded in all clade I symbionts but were apparently intact in clade II symbionts. UvrD was disrupted in two clade II symbionts. Among the intact genes in Ca. Vesicomyosocius okutanii (clade I), expressions of uvrD and mfd were detected by reverse transcription-polymerase chain reaction (RT-PCR), but those of uvrA and uvrDp were not. In contrast, all intact genes were expressed in the symbiont of Calyptogena pacifica (clade II). To assess how gene losses affect RGE (question 2), genetic distances of the examined genes in symbionts from Bathymodiolus septemdierum were shown to be larger in clade I than clade II symbionts. In addition, these genes had lower guanine+cytosine (GC) content and higher repeat sequence densities in clade I than measured in clade II. Our results suggest that NER genes are currently being lost from the extant lineages of vesicomyid clam symbionts. The loss of NER genes and mutY in these symbionts is likely to promote increases in genetic distance and repeat sequence density as well as reduced GC content in genomic genes, and may have facilitated reductive evolution of the genome. PMID:28199404

  7. Tissue Non-Specific Genes and Pathways Associated with Diabetes: An Expression Meta-Analysis.

    Science.gov (United States)

    Mei, Hao; Li, Lianna; Liu, Shijian; Jiang, Fan; Griswold, Michael; Mosley, Thomas

    2017-01-21

    We performed expression studies to identify tissue non-specific genes and pathways of diabetes by meta-analysis. We searched curated datasets of the Gene Expression Omnibus (GEO) database and identified 13 and five expression studies of diabetes and insulin responses at various tissues, respectively. We tested differential gene expression by empirical Bayes-based linear method and investigated gene set expression association by knowledge-based enrichment analysis. Meta-analysis by different methods was applied to identify tissue non-specific genes and gene sets. We also proposed pathway mapping analysis to infer functions of the identified gene sets, and correlation and independent analysis to evaluate expression association profile of genes and gene sets between studies and tissues. Our analysis showed that PGRMC1 and HADH genes were significant over diabetes studies, while IRS1 and MPST genes were significant over insulin response studies, and joint analysis showed that HADH and MPST genes were significant over all combined data sets. The pathway analysis identified six significant gene sets over all studies. The KEGG pathway mapping indicated that the significant gene sets are related to diabetes pathogenesis. The results also presented that 12.8% and 59.0% pairwise studies had significantly correlated expression association for genes and gene sets, respectively; moreover, 12.8% pairwise studies had independent expression association for genes, but no studies were observed significantly different for expression association of gene sets. Our analysis indicated that there are both tissue specific and non-specific genes and pathways associated with diabetes pathogenesis. Compared to the gene expression, pathway association tends to be tissue non-specific, and a common pathway influencing diabetes development is activated through different genes at different tissues.

  8. Gene program-specific regulation of PGC-1{alpha} activity

    DEFF Research Database (Denmark)

    Schmidt, Søren F; Mandrup, Susanne

    2011-01-01

    Peroxisome proliferator-activated receptor γ (PPARγ) coactivator 1 α (PGC-1α) activation coordinates induction of the hepatic fasting response through coactivation of numerous transcription factors and gene programs. In the June 15, 2011, issue of Genes & Development, Lustig and colleagues (pp...

  9. Phylogeny of Mycobacterium tuberculosis Beijing strains constructed from polymorphisms in genes involved in DNA replication, recombination and repair.

    Directory of Open Access Journals (Sweden)

    Olga Mestre

    Full Text Available BACKGROUND: The Beijing family is a successful group of M. tuberculosis strains, often associated with drug resistance and widely distributed throughout the world. Polymorphic genetic markers have been used to type particular M. tuberculosis strains. We recently identified a group of polymorphic DNA repair replication and recombination (3R genes. It was shown that evolution of M. tuberculosis complex strains can be studied using 3R SNPs and a high-resolution tool for strain discrimination was developed. Here we investigated the genetic diversity and propose a phylogeny for Beijing strains by analyzing polymorphisms in 3R genes. METHODOLOGY/PRINCIPAL FINDINGS: A group of 3R genes was sequenced in a collection of Beijing strains from different geographic origins. Sequence analysis and comparison with the ones of non-Beijing strains identified several SNPs. These SNPs were used to type a larger collection of Beijing strains and allowed identification of 26 different sequence types for which a phylogeny was constructed. Phylogenetic relationships established by sequence types were in agreement with evolutionary pathways suggested by other genetic markers, such as Large Sequence Polymorphisms (LSPs. A recent Beijing genotype (Bmyc10, which included 60% of strains from distinct parts of the world, appeared to be predominant. CONCLUSIONS/SIGNIFICANCE: We found SNPs in 3R genes associated with the Beijing family, which enabled discrimination of different groups and the proposal of a phylogeny. The Beijing family can be divided into different groups characterized by particular genetic polymorphisms that may reflect pathogenic features. These SNPs are new, potential genetic markers that may contribute to better understand the success of the Beijing family.

  10. Repair of endonuclease-induced double-strand breaks in Saccharomyces cerevisiae: essential role for genes associated with nonhomologous end-joining.

    OpenAIRE

    Lewis, L K; Westmoreland, J W; Resnick, M A

    1999-01-01

    Repair of double-strand breaks (DSBs) in chromosomal DNA by nonhomologous end-joining (NHEJ) is not well characterized in the yeast Saccharomyces cerevisiae. Here we demonstrate that several genes associated with NHEJ perform essential functions in the repair of endonuclease-induced DSBs in vivo. Galactose-induced expression of EcoRI endonuclease in rad50, mre11, or xrs2 mutants, which are deficient in plasmid DSB end-joining and some forms of recombination, resulted in G2 arrest and rapid ce...

  11. Cobalt-induced genotoxicity in male zebrafish (Danio rerio), with implications for reproduction and expression of DNA repair genes

    Energy Technology Data Exchange (ETDEWEB)

    Reinardy, Helena C.; Syrett, James R. [School of Biomedical and Biological Sciences, University of Plymouth (United Kingdom); Jeffree, Ross A. [Faculty of Science, University of Technology, Sydney, PO Box 123, Broadway, NSW 2007 (Australia); Henry, Theodore B., E-mail: ted.henry@plymouth.ac.uk [School of Biomedical and Biological Sciences, University of Plymouth (United Kingdom); Center for Environmental Biotechnology, University of Tennessee, Knoxville, TN 37996 (United States); Department of Forestry, Wildlife and Fisheries, University of Tennessee, Knoxville, TN 37996. USA (United States); Jha, Awadhesh N. [School of Biomedical and Biological Sciences, The University of Plymouth (United Kingdom)

    2013-01-15

    Although cobalt (Co) is an environmental contaminant of surface waters in both radioactive (e.g. {sup 60}Co) and non-radioactive forms, there is relatively little information about Co toxicity in fishes. The objective of this study was to investigate acute and chronic toxicity of Co in zebrafish, with emphasis on male genotoxicity and implications for reproductive success. The lethal concentration for 50% mortality (LC{sub 50}) in larval zebrafish exposed (96 h) to 0-50 mg l{sup -1} Co was 35.3 {+-} 1.1 (95% C.I.) mg l{sup -1} Co. Adult zebrafish were exposed (13 d) to sub-lethal (0-25 mg l{sup -1}) Co and allowed to spawn every 4 d and embryos were collected. After 12-d exposure, fertilisation rate was reduced (6% total eggs fertilised, 25 mg l{sup -1}) and embryo survival to hatching decreased (60% fertilised eggs survived, 25 mg l{sup -1}). A concentration-dependent increase in DNA strand breaks was detected in sperm from males exposed (13 d) to Co, and DNA damage in sperm returned to control levels after males recovered for 6 d in clean water. Induction of DNA repair genes (rad51, xrcc5, and xrcc6) in testes was complex and not directly related to Co concentration, although there was significant induction in fish exposed to 15 and 25 mg l{sup -1} Co relative to controls. Induction of 4.0 {+-} 0.9, 2.5 {+-} 0.7, and 3.1 {+-} 0.7-fold change (mean {+-} S.E.M. for rad51, xrcc5, and xrcc6, respectively) was observed in testes at the highest Co concentration (25 mg l{sup -1}). Expression of these genes was not altered in offspring (larvae) spawned after 12-d exposure. Chronic exposure to Co resulted in DNA damage in sperm, induction of DNA repair genes in testes, and indications of reduced reproductive success.

  12. Saliva microbiota carry caries-specific functional gene signatures.

    Directory of Open Access Journals (Sweden)

    Fang Yang

    Full Text Available Human saliva microbiota is phylogenetically divergent among host individuals yet their roles in health and disease are poorly appreciated. We employed a microbial functional gene microarray, HuMiChip 1.0, to reconstruct the global functional profiles of human saliva microbiota from ten healthy and ten caries-active adults. Saliva microbiota in the pilot population featured a vast diversity of functional genes. No significant distinction in gene number or diversity indices was observed between healthy and caries-active microbiota. However, co-presence network analysis of functional genes revealed that caries-active microbiota was more divergent in non-core genes than healthy microbiota, despite both groups exhibited a similar degree of conservation at their respective core genes. Furthermore, functional gene structure of saliva microbiota could potentially distinguish caries-active patients from healthy hosts. Microbial functions such as Diaminopimelate epimerase, Prephenate dehydrogenase, Pyruvate-formate lyase and N-acetylmuramoyl-L-alanine amidase were significantly linked to caries. Therefore, saliva microbiota carried disease-associated functional signatures, which could be potentially exploited for caries diagnosis.

  13. Meiosis-specific gene discovery in plants: RNA-Seq applied to isolated Arabidopsis male meiocytes

    Directory of Open Access Journals (Sweden)

    May Gregory D

    2010-12-01

    Full Text Available Abstract Background Meiosis is a critical process in the reproduction and life cycle of flowering plants in which homologous chromosomes pair, synapse, recombine and segregate. Understanding meiosis will not only advance our knowledge of the mechanisms of genetic recombination, but also has substantial applications in crop improvement. Despite the tremendous progress in the past decade in other model organisms (e.g., Saccharomyces cerevisiae and Drosophila melanogaster, the global identification of meiotic genes in flowering plants has remained a challenge due to the lack of efficient methods to collect pure meiocytes for analyzing the temporal and spatial gene expression patterns during meiosis, and for the sensitive identification and quantitation of novel genes. Results A high-throughput approach to identify meiosis-specific genes by combining isolated meiocytes, RNA-Seq, bioinformatic and statistical analysis pipelines was developed. By analyzing the studied genes that have a meiosis function, a pipeline for identifying meiosis-specific genes has been defined. More than 1,000 genes that are specifically or preferentially expressed in meiocytes have been identified as candidate meiosis-specific genes. A group of 55 genes that have mitochondrial genome origins and a significant number of transposable element (TE genes (1,036 were also found to have up-regulated expression levels in meiocytes. Conclusion These findings advance our understanding of meiotic genes, gene expression and regulation, especially the transcript profiles of MGI genes and TE genes, and provide a framework for functional analysis of genes in meiosis.

  14. Methylation and protein expression of DNA repair genes: association with chemotherapy exposure and survival in sporadic ovarian and peritoneal carcinomas

    Directory of Open Access Journals (Sweden)

    Walsh Tom

    2009-07-01

    Full Text Available Abstract Background DNA repair genes critically regulate the cellular response to chemotherapy and epigenetic regulation of these genes may be influenced by chemotherapy exposure. Restoration of BRCA1 and BRCA2 mediates resistance to platinum chemotherapy in recurrent BRCA1 and BRCA2 mutated hereditary ovarian carcinomas. We evaluated BRCA1, BRCA2, and MLH1 protein expression in 115 sporadic primary ovarian carcinomas, of which 31 had paired recurrent neoplasms collected after chemotherapy. Additionally, we assessed whether promoter methylation of BRCA1, MLH1 or FANCF influenced response to chemotherapy or explained alterations in protein expression after chemotherapy exposure. Results Of 115 primary sporadic ovarian carcinomas, 39 (34% had low BRCA1 protein and 49 (42% had low BRCA2 expression. BRCA1 and BRCA2 protein expression were highly concordant (p Conclusion Low BRCA1 expression in primary sporadic ovarian carcinoma is associated with prolonged survival. Recurrent ovarian carcinomas commonly have increased BRCA1 and/or BRCA2 protein expression post chemotherapy exposure which could mediate resistance to platinum based therapies. However, alterations in expression of these proteins after chemotherapy are not commonly mediated by promoter methylation, and other regulatory mechanisms are likely to contribute to these alterations.

  15. Defects in the DNA repair and transcription gene ERCC2(XPD) in trichothiodystrophy

    Energy Technology Data Exchange (ETDEWEB)

    Takayama, K.; Salazar, E.P.; Thompson, L.H. [Lawrence Livermore National Lab., CA (United States)] [and others

    1996-02-01

    Trichothiodystrophy (TTD) is a rare autosomal recessive disorder characterized by brittle hair with reduced sulfur content, ichthyosis, peculiar face, and mental and growth retardation. Clinical photosensitivity is present in {approximately}50% of TTD patients but is not associated with an elevated frequency of cancers. Previous complementation studies show that the photosensitivity in nearly all of the studied patients is due to a defect in the same genetic locus that underlies the cancer-prone genetic disorder xeroderma pigmentosum group D (XP-D). Nucleotide-sequence analysis of the ERCC2 cDNA from three TTD cell strains (TTD1VI, TTD3VI, and TTD1RO) revealed mutations within the region from amino acid 713-730 and within previously identified helicase functional domains. The various clinical presentations and DNA repair characteristics of the cell strains can be correlated with the particular mutations found in the ERCC2 locus. Mutations of Arg658 to either His or Cys correlate with TTD cell strains with intermediate UV-sensitivity, mutation of Arg722 to Trp correlates with highly UV-sensitive TTD cell strains, and mutation of Arg683 to Trp correlates with XP-D. Alleles with mutation of Arg616 to Pro or with the combined mutation of Leu461 to Val and deletion of 716-730 are found in both XP-D and TTD cell strains. 39 refs., 2 figs., 3 tabs.

  16. The first determination of DNA sequence of a specific gene.

    Science.gov (United States)

    Inouye, Masayori

    2016-05-10

    How and when the first DNA sequence of a gene was determined? In 1977, F. Sanger came up with an innovative technology to sequence DNA by using chain terminators, and determined the entire DNA sequence of the 5375-base genome of bacteriophage φX 174 (Sanger et al., 1977). While this Sanger's achievement has been recognized as the first DNA sequencing of genes, we had determined DNA sequence of a gene, albeit a partial sequence, 11 years before the Sanger's DNA sequence (Okada et al., 1966).

  17. Site-Specific Gene Targeting Using Transcription Activator-Like Effector (TALE)-Based Nuclease in Brassica oleracea

    Institute of Scientific and Technical Information of China (English)

    Zijian Sun; Nianzu Li; Guodong Huang; Junqiang Xu; Yu Pan; Zhimin Wang; Qinglin Tang; Ming Song; Xiaojia Wang

    2013-01-01

    Site-specific recognition modules with DNA nuclease have tremendous potential as molecular tools for genome targeting. The type III transcription activator-like effectors (TALEs) contain a DNA binding domain consisting of tandem repeats that can be engineered to bind user-defined specific DNA sequences. We demonstrated that customized TALE-based nucleases (TALENs), constructed using a method called“unit assembly”, specifically target the endogenous FRIGIDA gene in Brassica oleracea L. var. capitata L. The results indicate that the TALENs bound to the target site and cleaved double-strand DNA in vitro and in vivo, whereas the effector binding elements have a 23 bp spacer. The T7 endonuclease I assay and sequencing data show that TALENs made double-strand breaks, which were repaired by a non-homologous end-joining pathway within the target sequence. These data show the feasibility of applying customized TALENs to target and modify the genome with deletions in those organisms that are still in lacking gene target methods to provide germplasms in breeding improvement.

  18. Site-specific gene targeting using transcription activator-like effector (TALE)-based nuclease in Brassica oleracea.

    Science.gov (United States)

    Sun, Zijian; Li, Nianzu; Huang, Guodong; Xu, Junqiang; Pan, Yu; Wang, Zhimin; Tang, Qinglin; Song, Ming; Wang, Xiaojia

    2013-11-01

    Site-specific recognition modules with DNA nuclease have tremendous potential as molecular tools for genome targeting. The type III transcription activator-like effectors (TALEs) contain a DNA binding domain consisting of tandem repeats that can be engineered to bind user-defined specific DNA sequences. We demonstrated that customized TALE-based nucleases (TALENs), constructed using a method called "unit assembly", specifically target the endogenous FRIGIDA gene in Brassica oleracea L. var. capitata L. The results indicate that the TALENs bound to the target site and cleaved double-strand DNA in vitro and in vivo, whereas the effector binding elements have a 23 bp spacer. The T7 endonuclease I assay and sequencing data show that TALENs made double-strand breaks, which were repaired by a non-homologous end-joining pathway within the target sequence. These data show the feasibility of applying customized TALENs to target and modify the genome with deletions in those organisms that are still in lacking gene target methods to provide germplasms in breeding improvement.

  19. Gametogenesis in the Pacific oyster Crassostrea gigas: a microarrays-based analysis identifies sex and stage specific genes.

    Directory of Open Access Journals (Sweden)

    Nolwenn M Dheilly

    Full Text Available BACKGROUND: The Pacific oyster Crassostrea gigas (Mollusca, Lophotrochozoa is an alternative and irregular protandrous hermaphrodite: most individuals mature first as males and then change sex several times. Little is known about genetic and phenotypic basis of sex differentiation in oysters, and little more about the molecular pathways regulating reproduction. We have recently developed and validated a microarray containing 31,918 oligomers (Dheilly et al., 2011 representing the oyster transcriptome. The application of this microarray to the study of mollusk gametogenesis should provide a better understanding of the key factors involved in sex differentiation and the regulation of oyster reproduction. METHODOLOGY/PRINCIPAL FINDINGS: Gene expression was studied in gonads of oysters cultured over a yearly reproductive cycle. Principal component analysis and hierarchical clustering showed a significant divergence in gene expression patterns of males and females coinciding with the start of gonial mitosis. ANOVA analysis of the data revealed 2,482 genes differentially expressed during the course of males and/or females gametogenesis. The expression of 434 genes could be localized in either germ cells or somatic cells of the gonad by comparing the transcriptome of female gonads to the transcriptome of stripped oocytes and somatic tissues. Analysis of the annotated genes revealed conserved molecular mechanisms between mollusks and mammals: genes involved in chromatin condensation, DNA replication and repair, mitosis and meiosis regulation, transcription, translation and apoptosis were expressed in both male and female gonads. Most interestingly, early expressed male-specific genes included bindin and a dpy-30 homolog and female-specific genes included foxL2, nanos homolog 3, a pancreatic lipase related protein, cd63 and vitellogenin. Further functional analyses are now required in order to investigate their role in sex differentiation in oysters

  20. Relationships between chromatin remodeling and DNA damage repair induced by 8-methoxypsoralen and UVA in yeast Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Lavínia Almeida Cruz

    2012-01-01

    Full Text Available Eukaryotic cells have developed mechanisms to prevent genomic instability, such as DNA damage detection and repair, control of cell cycle progression and cell death induction. The bifunctional compound furocumarin 8-methoxy-psoralen (8-MOP is widely used in the treatment of various inflammatory skin diseases. In this review, we summarize recent data about the role of chromatin remodeling in the repair of DNA damage induced by treatment with 8-methoxypsoralen plus UVA (8-MOP+UVA, focusing on repair proteins in budding yeast Saccharomyces cerevisiae, an established model system for studying DNA repair pathways. The interstrand crosslinks (ICL formed by the 8-MOP+UVA treatment are detrimental lesions that can block transcription and replication, leading to cell death if not repaired. Current data show the involvement of different pathways in ICL processing, such as nucleotide excision repair (NER, base excision repair (BER, translesion repair (TLS and double-strand break repair. 8-MOP+UVA treatment in yeast enhances the expression of genes involved in the DNA damage response, double strand break repair by homologous replication, as well as genes related to cell cycle regulation. Moreover, alterations in the expression of subtelomeric genes and genes related to chromatin remodeling are consistent with structural modifications of chromatin relevant to DNA repair. Taken together, these findings indicate a specific profile in 8-MOP+UVA responses related to chromatin remodeling and DNA repair.

  1. Initial description of primate-specific cystine-knot Prometheus genes and differential gene expansions of D-dopachrome tautomerase genes.

    Science.gov (United States)

    Premzl, Marko

    2015-06-01

    Using eutherian comparative genomic analysis protocol and public genomic sequence data sets, the present work attempted to update and revise two gene data sets. The most comprehensive third party annotation gene data sets of eutherian adenohypophysis cystine-knot genes (128 complete coding sequences), and d-dopachrome tautomerases and macrophage migration inhibitory factor genes (30 complete coding sequences) were annotated. For example, the present study first described primate-specific cystine-knot Prometheus genes, as well as differential gene expansions of D-dopachrome tautomerase genes. Furthermore, new frameworks of future experiments of two eutherian gene data sets were proposed.

  2. Percutaneous mitral valve repair.

    Science.gov (United States)

    Gillinov, A Marc; Liddicoat, John R

    2006-01-01

    Surgical mitral valve repair is the procedure of choice to treat mitral regurgitation of all etiologies. Whereas annuloplasty is the cornerstone of mitral valve repair, a variety of other surgical techniques are utilized to correct dysfunction of the leaflets and subvalvular apparatus; in most cases, surgical repair entails application of multiple repair techniques in each patient. Preclinical studies and early human experience have demonstrated that some of these surgical repair techniques can be performed using percutaneous approaches. Specifically, there has been great progress in the development of novel technology to facilitate percutaneous annuloplasty and percutaneous edge-to-edge repair. The objectives of this report were to (1) discuss the surgical foundations for these percutaneous approaches; (2) review device design and experimental and clinical results of percutaneous valve repair; and (3) address future directions, including the key challenges of patient selection and clinical trial design.

  3. Tissue Specific and Hormonal Regulation of Gene Expression

    Science.gov (United States)

    1998-07-01

    cAMP responsive region located at -200 to -99 bp in CRH. 14. SUBJECT TERMS 15. NUMfER OF PAGES Breast Cancer gene regulation, transcription, placenta...known mediators of labor, and it may also the stress response. The peptide sequence and expression of potentiate the effect of oxytocin on uterine...regulation of other rodent trophoblast genes has 220 not yet been investigated. 2. Robinson BG, Arbiser JL, Emanuel RL, Majzoub JA 1989 Species- 3008

  4. Prevalence of Germline Mutations in Genes Engaged in DNA Damage Repair by Homologous Recombination in Patients with Triple-Negative and Hereditary Non-Triple-Negative Breast Cancers.

    Directory of Open Access Journals (Sweden)

    Pawel Domagala

    Full Text Available This study sought to assess the prevalence of common germline mutations in several genes engaged in the repair of DNA double-strand break by homologous recombination in patients with triple-negative breast cancers and hereditary non-triple-negative breast cancers. Tumors deficient in this type of DNA damage repair are known to be especially sensitive to DNA cross-linking agents (e.g., platinum drugs and to poly(ADP-ribose polymerase (PARP inhibitors.Genetic testing was performed for 36 common germline mutations in genes engaged in the repair of DNA by homologous recombination, i.e., BRCA1, BRCA2, CHEK2, NBN, ATM, PALB2, BARD1, and RAD51D, in 202 consecutive patients with triple-negative breast cancers and hereditary non-triple-negative breast cancers.Thirty five (22.2% of 158 patients in the triple-negative group carried mutations in genes involved in DNA repair by homologous recombination, while 10 (22.7% of the 44 patients in the hereditary non-triple-negative group carried such mutations. Mutations in BRCA1 were most frequent in patients with triple-negative breast cancer (18.4%, and mutations in CHEK2 were most frequent in patients with hereditary non-triple-negative breast cancers (15.9%. In addition, in the triple-negative group, mutations in CHEK2, NBN, and ATM (3.8% combined were found, while mutations in BRCA1, NBN, and PALB2 (6.8% combined were identified in the hereditary non-triple-negative group.Identifying mutations in genes engaged in DNA damage repair by homologous recombination other than BRCA1/2 can substantially increase the proportion of patients with triple-negative breast cancer and hereditary non-triple-negative breast cancer who may be eligible for therapy using PARP inhibitors and platinum drugs.

  5. Strain Specific Factors Control Effector Gene Silencing in Phytophthora sojae.

    Directory of Open Access Journals (Sweden)

    Sirjana Devi Shrestha

    Full Text Available The Phytophthora sojae avirulence gene Avr3a encodes an effector that is capable of triggering immunity on soybean plants carrying the resistance gene Rps3a. P. sojae strains that express Avr3a are avirulent to Rps3a plants, while strains that do not are virulent. To study the inheritance of Avr3a expression and virulence towards Rps3a, genetic crosses and self-fertilizations were performed. A cross between P. sojae strains ACR10 X P7076 causes transgenerational gene silencing of Avr3a allele, and this effect is meiotically stable up to the F5 generation. However, test-crosses of F1 progeny (ACR10 X P7076 with strain P6497 result in the release of silencing of Avr3a. Expression of Avr3a in the progeny is variable and correlates with the phenotypic penetrance of the avirulence trait. The F1 progeny from a direct cross of P6497 X ACR10 segregate for inheritance for Avr3a expression, a result that could not be explained by parental imprinting or heterozygosity. Analysis of small RNA arising from the Avr3a gene sequence in the parental strains and hybrid progeny suggests that the presence of small RNA is necessary but not sufficient for gene silencing. Overall, we conclude that inheritance of the Avr3a gene silenced phenotype relies on factors that are variable among P. sojae strains.

  6. The genetics of alcoholism: identifying specific genes through family studies.

    Science.gov (United States)

    Edenberg, Howard J; Foroud, Tatiana

    2006-09-01

    Alcoholism is a complex disorder with both genetic and environmental risk factors. Studies in humans have begun to elucidate the genetic underpinnings of the risk for alcoholism. Here we briefly review strategies for identifying individual genes in which variations affect the risk for alcoholism and related phenotypes, in the context of one large study that has successfully identified such genes. The Collaborative Study on the Genetics of Alcoholism (COGA) is a family-based study that has collected detailed phenotypic data on individuals in families with multiple alcoholic members. A genome-wide linkage approach led to the identification of chromosomal regions containing genes that influenced alcoholism risk and related phenotypes. Subsequently, single nucleotide polymorphisms (SNPs) were genotyped in positional candidate genes located within the linked chromosomal regions, and analyzed for association with these phenotypes. Using this sequential approach, COGA has detected association with GABRA2, CHRM2 and ADH4; these associations have all been replicated by other researchers. COGA has detected association to additional genes including GABRG3, TAS2R16, SNCA, OPRK1 and PDYN, results that are awaiting confirmation. These successes demonstrate that genes contributing to the risk for alcoholism can be reliably identified using human subjects.

  7. Strain Specific Factors Control Effector Gene Silencing in Phytophthora sojae.

    Science.gov (United States)

    Shrestha, Sirjana Devi; Chapman, Patrick; Zhang, Yun; Gijzen, Mark

    2016-01-01

    The Phytophthora sojae avirulence gene Avr3a encodes an effector that is capable of triggering immunity on soybean plants carrying the resistance gene Rps3a. P. sojae strains that express Avr3a are avirulent to Rps3a plants, while strains that do not are virulent. To study the inheritance of Avr3a expression and virulence towards Rps3a, genetic crosses and self-fertilizations were performed. A cross between P. sojae strains ACR10 X P7076 causes transgenerational gene silencing of Avr3a allele, and this effect is meiotically stable up to the F5 generation. However, test-crosses of F1 progeny (ACR10 X P7076) with strain P6497 result in the release of silencing of Avr3a. Expression of Avr3a in the progeny is variable and correlates with the phenotypic penetrance of the avirulence trait. The F1 progeny from a direct cross of P6497 X ACR10 segregate for inheritance for Avr3a expression, a result that could not be explained by parental imprinting or heterozygosity. Analysis of small RNA arising from the Avr3a gene sequence in the parental strains and hybrid progeny suggests that the presence of small RNA is necessary but not sufficient for gene silencing. Overall, we conclude that inheritance of the Avr3a gene silenced phenotype relies on factors that are variable among P. sojae strains.

  8. Symbiont modulates expression of specific gene categories in Angomonas deanei

    Directory of Open Access Journals (Sweden)

    Luciana Loureiro Penha

    Full Text Available Trypanosomatids are parasites that cause disease in humans, animals, and plants. Most are non-pathogenic and some harbor a symbiotic bacterium. Endosymbiosis is part of the evolutionary process of vital cell functions such as respiration and photosynthesis. Angomonas deanei is an example of a symbiont-containing trypanosomatid. In this paper, we sought to investigate how symbionts influence host cells by characterising and comparing the transcriptomes of the symbiont-containing A. deanei (wild type and the symbiont-free aposymbiotic strains. The comparison revealed that the presence of the symbiont modulates several differentially expressed genes. Empirical analysis of differential gene expression showed that 216 of the 7625 modulated genes were significantly changed. Finally, gene set enrichment analysis revealed that the largest categories of genes that downregulated in the absence of the symbiont were those involved in oxidation-reduction process, ATP hydrolysis coupled proton transport and glycolysis. In contrast, among the upregulated gene categories were those involved in proteolysis, microtubule-based movement, and cellular metabolic process. Our results provide valuable information for dissecting the mechanism of endosymbiosis in A. deanei.

  9. DNA mismatch repair gene MSH6 implicated in determining age at natural menopause

    DEFF Research Database (Denmark)

    Perry, John R B; Hsu, Yi-Hsiang; Chasman, Daniel I

    2014-01-01

    The length of female reproductive lifespan is associated with multiple adverse outcomes, including breast cancer, cardiovascular disease and infertility. The biological processes that govern the timing of the beginning and end of reproductive life are not well understood. Genetic variants are kno...... to contribute to ∼50% of the variation in both age at menarche and menopause, but to date the known genes explain...

  10. Lineage-Specific Genes Are Prominent DNA Damage Hotspots during Leukemic Transformation of B Cell Precursors

    Directory of Open Access Journals (Sweden)

    Bryant Boulianne

    2017-02-01

    Full Text Available In human leukemia, lineage-specific genes represent predominant targets of deletion, with lymphoid-specific genes frequently affected in lymphoid leukemia and myeloid-specific genes in myeloid leukemia. To investigate the basis of lineage-specific alterations, we analyzed global DNA damage in primary B cell precursors expressing leukemia-inducing oncogenes by ChIP-seq. We identified more than 1,000 sensitive regions, of which B lineage-specific genes constitute the most prominent targets. Identified hotspots at B lineage genes relate to DNA-DSBs, affect genes that harbor genomic lesions in human leukemia, and associate with ectopic deletion in successfully transformed cells. Furthermore, we show that most identified regions overlap with gene bodies of highly expressed genes and that induction of a myeloid lineage phenotype in transformed B cell precursors promotes de novo DNA damage at myeloid loci. Hence, we demonstrate that lineage-specific transcription predisposes lineage-specific genes in transformed B cell precursors to DNA damage, which is likely to promote the frequent alteration of lineage-specific genes in human leukemia.

  11. Classical and alternative end-joining pathways for repair of lymphocyte-specific and general DNA double-strand breaks.

    Science.gov (United States)

    Boboila, Cristian; Alt, Frederick W; Schwer, Bjoern

    2012-01-01

    Classical nonhomologous end joining (C-NHEJ) is one of the two major known pathways for the repair of DNA double-strand breaks (DSBs) in mammalian cells. Our understanding of C-NHEJ has been derived, in significant part, through studies of programmed physiologic DNA DSBs formed during V(D)J recombination in the developing immune system. Studies of immunoglobulin heavy-chain (IgH) class-switch recombination (CSR) also have revealed that there is an "alternative" end-joining process (A-EJ) that can function, relatively robustly, in the repair of DSBs in activated mature B lymphocytes. This A-EJ process has also been implicated in the formation of oncogenic translocations found in lymphoid tumors. In this review, we discuss our current understanding of C-NHEJ and A-EJ in the context of V(D)J recombination, CSR, and the formation of chromosomal translocations. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Community specificity: life and afterlife effects of genes.

    Science.gov (United States)

    Whitham, Thomas G; Gehring, Catherine A; Lamit, Louis J; Wojtowicz, Todd; Evans, Luke M; Keith, Arthur R; Smith, David Solance

    2012-05-01

    Community-level genetic specificity results when individual genotypes or populations of the same species support different communities. Our review of the literature shows that genetic specificity exhibits both life and afterlife effects; it is a widespread phenomenon occurring in diverse taxonomic groups, aquatic to terrestrial ecosystems, and species-poor to species-rich systems. Such specificity affects species interactions, evolution, ecosystem processes and leads to community feedbacks on the performance of the individuals expressing the traits. Thus, genetic specificity by communities appears to be fundamentally important, suggesting that specificity is a major driver of the biodiversity and stability of the world's ecosystems.

  13. The Effect of Polymorphisms in DNA Repair Genes and Carcinogen Metabolizers on Leukocyte Telomere Length: A Cohort of Healthy Spanish Smokers.

    Science.gov (United States)

    Verde, Zoraida; Reinoso-Barbero, Luis; Chicharro, Luis; Resano, Pilar; Sánchez-Hernández, Ignacio; Rodríguez González-Moro, Jose Miguel; Bandrés, Fernando; Gómez-Gallego, Félix; Santiago, Catalina

    2016-04-01

    Smoking implies exposure to carcinogenic agents that causes DNA damage, which could be suspected to enhance telomere attrition. To protect and deal with DNA damage, cells possess mechanisms that repair and neutralize harmful substances. Polymorphisms altering DNA repair capacity or carcinogen metabolism may lead to synergistic effects with tobacco carcinogen-induced shorter telomere length independently of cancer interaction. The aim of this study was to explore the association between leukocyte telomere length (LTL) and several genetic polymorphisms in DNA repair genes and carcinogen metabolizers in a cohort of healthy smokers. We evaluated the effect of six genetic polymorphisms in cytochrome P1A1 (Ile462Val), XRCC1 (Arg399Gln), APEX1 (Asp148Glu), XRCC3 (Thr241Met), and XPD (Asp312Asn; Lys751Gln) on LTL in a cohort of 145 healthy smokers in addition to smoking habits. Logistic regression analysis showed an association between XRCC1 399Gln allele and shorter telomere length (OR = 5.03, 95% CI = 1.08% to 23.36%). There were not association between the rest of polymorphisms analyzed and LTL. Continuous exposure to tobacco could overwhelm the DNA repair machinery, making the effect of the polymorphisms that reduce repair capacity more pronounced. Analyzing the function of smoking-induced DNA-repair genes and LTL is an important goal in order to identify therapeutic targets to treat smoking-induced diseases. © The Author 2015. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  14. Improved generation of rat gene knockouts by target-selected mutagenesis in mismatch repair-deficient animals.

    NARCIS (Netherlands)

    van Boxtel, R.; Toonen, P.W.; Verheul, M.; van Roekel, H.S.; Nijman, I.J.; Guryev, V.; Cuppen, E.

    2008-01-01

    BACKGROUND: The laboratory rat (Rattus norvegicus) is one of the preferred model organisms in physiological and pharmacological research, although the availability of specific genetic models, especially gene knockouts, is limited. N-ethyl-N-nitrosourea (ENU)-driven target-selected mutagenesis is cur

  15. Improved generation of rat gene knockouts by target-selected mutagenesis in mismatch repair-deficient animals

    NARCIS (Netherlands)

    van Boxtel, Ruben; Toonen, Pim W; Verheul, Mark; van Roekel, Henk S; Nijman, Isaac J; Guryev, Victor; Cuppen, Edwin

    2008-01-01

    BACKGROUND: The laboratory rat (Rattus norvegicus) is one of the preferred model organisms in physiological and pharmacological research, although the availability of specific genetic models, especially gene knockouts, is limited. N-ethyl-N-nitrosourea (ENU)-driven target-selected mutagenesis is cur

  16. Sequence-specific "gene signatures" can be obtained by PCR with single specific primers at low stringency.

    OpenAIRE

    Pena, S D; Barreto, G.; Vago, A. R.; De Marco, L; Reinach,F. C.; Dias Neto, E; Simpson, A J

    1994-01-01

    Low-stringency single specific primer PCR (LSSP-PCR) is an extremely simple PCR-based technique that detects single or multiple mutations in gene-sized DNA fragments. A purified DNA fragment is subjected to PCR using high concentrations of a single specific oligonucleotide primer, large amounts of Taq polymerase, and a very low annealing temperature. Under these conditions the primer hybridizes specifically to its complementary region and nonspecifically to multiple sites wi...

  17. Gene expression and DNA repair in progeroid syndromes and human aging.

    Science.gov (United States)

    Kyng, Kasper J; Bohr, Vilhelm A

    2005-11-01

    Human progeroid syndromes are caused by mutations in single genes accelerating some but not all features of normal aging. Most progeroid disorders are linked to defects in genome maintenance, and while it remains unknown if similar processes underlie normal and premature aging, they provide useful models for the study of aging. Altered transcription is speculated to play a causative role in aging, and is involved in the pathology of most if not all progeroid syndromes. Previous studies demonstrate that there is a similar pattern of gene expression changes in primary cells from old and Werner syndrome compared to young suggesting a presence of common cellular aging mechanisms in old and progeria. Here we review the role of transcription in progeroid syndromes and discuss the implications of similar transcription aberrations in normal and premature aging.

  18. Specifically targeted gene therapy for small-cell lung cancer

    DEFF Research Database (Denmark)

    Christensen, C.L.; Zandi, R.; Gjetting, T.

    2009-01-01

    Small-cell lung cancer (SCLC) is a highly malignant disease with poor prognosis. Hence, there is great demand for new therapies that can replace or supplement the current available treatment regimes. Gene therapy constitutes a promising strategy and relies on the principle of introducing exogenous....... This review describes and discusses the current status of the application of gene therapy in relation to SCLC Udgivelsesdato: 2009/4...... DNA into malignant cells causing them to die. Since SCLC is a highly disseminated malignancy, the gene therapeutic agent must be administered systemically, obligating a high level of targeting of tumor tissue and the use of delivery vehicles designed for systemic circulation of the therapeutic DNA...

  19. MGFM: a novel tool for detection of tissue and cell specific marker genes from microarray gene expression data.

    Science.gov (United States)

    El Amrani, Khadija; Stachelscheid, Harald; Lekschas, Fritz; Kurtz, Andreas; Andrade-Navarro, Miguel A

    2015-08-28

    Identification of marker genes associated with a specific tissue/cell type is a fundamental challenge in genetic and cell research. Marker genes are of great importance for determining cell identity, and for understanding tissue specific gene function and the molecular mechanisms underlying complex diseases. We have developed a new bioinformatics tool called MGFM (Marker Gene Finder in Microarray data) to predict marker genes from microarray gene expression data. Marker genes are identified through the grouping of samples of the same type with similar marker gene expression levels. We verified our approach using two microarray data sets from the NCBI's Gene Expression Omnibus public repository encompassing samples for similar sets of five human tissues (brain, heart, kidney, liver, and lung). Comparison with another tool for tissue-specific gene identification and validation with literature-derived established tissue markers established functionality, accuracy and simplicity of our tool. Furthermore, top ranked marker genes were experimentally validated by reverse transcriptase-polymerase chain reaction (RT-PCR). The sets of predicted marker genes associated with the five selected tissues comprised well-known genes of particular importance in these tissues. The tool is freely available from the Bioconductor web site, and it is also provided as an online application integrated into the CellFinder platform ( http://cellfinder.org/analysis/marker ). MGFM is a useful tool to predict tissue/cell type marker genes using microarray gene expression data. The implementation of the tool as an R-package as well as an application within CellFinder facilitates its use.

  20. The Evaluation of Nerve Growth Factor Over Expression on Neural Lineage Specific Genes in Human Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Mortazavi Yousef

    2016-07-01

    Full Text Available Objective Treatment and repair of neurodegenerative diseases such as brain tumors, spinal cord injuries, and functional disorders, including Alzheimer’s disease, are challenging problems. A common treatment approach for such disorders involves the use of mesenchymal stem cells (MSCs as an alternative cell source to replace injured cells. However, use of these cells in hosts may potentially cause adverse outcomes such as tumorigenesis and uncontrolled differentiation. In attempt to generate mesenchymal derived neural cells, we have infected MSCs with recombinant lentiviruses that expressed nerve growth factor (NGF and assessed their neural lineage genes. Materials and Methods In this experimental study, we cloned the NGF gene sequence into a helper dependent lentiviral vector that contained the green fluorescent protein (GFP gene. The recombinant vector was amplified in DH5 bacterial cells. Recombinant viruses were generated in the human embryonic kidney 293 (HEK-293 packaging cell line with the helper vectors and analyzed under fluorescent microscopy. Bone marrow mesenchymal cells were infected by recombinant viruses for three days followed by assessment of neural differentiation. We evaluated expression of NGF through measurement of the NGF protein in culture medium by ELISA; neural specific genes were quantified by real-time polymerase chain reaction (PCR. Results We observed neural morphological changes after three days. Quantitative PCR showed that expressions of NESTIN, glial derived neurotrophic factor (GDNF, glial fibrillary acidic protein (GFAP and Microtubule-associated protein 2 (MAP2 genes increased following induction of NGF overexpression, whereas expressions of endogenous NGF and brain derived neural growth factor (BDNF genes reduced. Conclusion Ectopic expression of NGF can induce neurogenesis in MSCs. Direct injection of MSCs may cause tumorigenesis and an undesirable outcome. Therefore an alternative choice to overcome this

  1. Gene targeting in melanoma therapy: exploiting of surface markers and specific promoters

    Directory of Open Access Journals (Sweden)

    Sverdlov E. D.

    2012-01-01

    Full Text Available One of the problems of gene therapy of melanoma is effective expression of therapeutic gene in tumor cells and their metastases but not in normal cells. In this review, we will consider a two-step approach to a highly specific gene therapy. At the first step, therapeutic genes are delivered specifically to tumor cells using cell surface markers of melanoma cells as targets. At the second step, a specific expression of the therapeutic genes in tumor cells is ensured. Surface markers of melanoma cells were analyzed as potential targets for therapeutic treatment. Criteria for choosing the most promising targets are proposed. The use of specific melanoma promoters allows to further increase the specificity of treatment via transcriptional control of therapeutic gene expression in melanoma cells.

  2. Genome-wide identification of lineage-specific genes within Caenorhabditis elegans.

    Science.gov (United States)

    Zhou, Kun; Huang, Beibei; Zou, Ming; Lu, Dandan; He, Shunping; Wang, Guoxiu

    2015-10-01

    With the rapid growth of sequencing technology, a number of genomes and transcriptomes of various species have been sequenced, contributing to the study of lineage-specific genes (LSGs). We identified two sets of LSGs using BLAST: one included Caenorhabditis elegans species-specific genes (1423, SSGs), and the other consisted of Caenorhabditis genus-specific genes (4539, GSGs). The subsequent characterization and analysis of the SSGs and GSGs showed that they have significant differences in evolution and that most LSGs were generated by gene duplication and integration of transposable elements (TEs). We then performed temporal expression profiling and protein function prediction and observed that many SSGs and GSGs are expressed and that genes involved with sex determination, specific stress, immune response, and morphogenesis are over-represented, suggesting that these specific genes may be related to the Caenorhabditis nematodes' special ability to survive in severe and extreme environments.

  3. Age-Specific Gene Expression Profiles of Rhesus Monkey Ovaries Detected by Microarray Analysis

    Directory of Open Access Journals (Sweden)

    Hengxi Wei

    2015-01-01

    Full Text Available The biological function of human ovaries declines with age. To identify the potential molecular changes in ovarian aging, we performed genome-wide gene expression analysis by microarray of ovaries from young, middle-aged, and old rhesus monkeys. Microarray data was validated by quantitative real-time PCR. Results showed that a total of 503 (60 upregulated, 443 downregulated and 84 (downregulated genes were differentially expressed in old ovaries compared to young and middle-aged groups, respectively. No difference in gene expression was found between middle-aged and young groups. Differentially expressed genes were mainly enriched in cell and organelle, cellular and physiological process, binding, and catalytic activity. These genes were primarily associated with KEGG pathways of cell cycle, DNA replication and repair, oocyte meiosis and maturation, MAPK, TGF-beta, and p53 signaling pathway. Genes upregulated were involved in aging, defense response, oxidation reduction, and negative regulation of cellular process; genes downregulated have functions in reproduction, cell cycle, DNA and RNA process, macromolecular complex assembly, and positive regulation of macromolecule metabolic process. These findings show that monkey ovary undergoes substantial change in global transcription with age. Gene expression profiles are useful in understanding the mechanisms underlying ovarian aging and age-associated infertility in primates.

  4. Inferring gene dependency network specific to phenotypic alteration based on gene expression data and clinical information of breast cancer.

    Directory of Open Access Journals (Sweden)

    Xionghui Zhou

    Full Text Available Although many methods have been proposed to reconstruct gene regulatory network, most of them, when applied in the sample-based data, can not reveal the gene regulatory relations underlying the phenotypic change (e.g. normal versus cancer. In this paper, we adopt phenotype as a variable when constructing the gene regulatory network, while former researches either neglected it or only used it to select the differentially expressed genes as the inputs to construct the gene regulatory network. To be specific, we integrate phenotype information with gene expression data to identify the gene dependency pairs by using the method of conditional mutual information. A gene dependency pair (A,B means that the influence of gene A on the phenotype depends on gene B. All identified gene dependency pairs constitute a directed network underlying the phenotype, namely gene dependency network. By this way, we have constructed gene dependency network of breast cancer from gene expression data along with two different phenotype states (metastasis and non-metastasis. Moreover, we have found the network scale free, indicating that its hub genes with high out-degrees may play critical roles in the network. After functional investigation, these hub genes are found to be biologically significant and specially related to breast cancer, which suggests that our gene dependency network is meaningful. The validity has also been justified by literature investigation. From the network, we have selected 43 discriminative hubs as signature to build the classification model for distinguishing the distant metastasis risks of breast cancer patients, and the result outperforms those classification models with published signatures. In conclusion, we have proposed a promising way to construct the gene regulatory network by using sample-based data, which has been shown to be effective and accurate in uncovering the hidden mechanism of the biological process and identifying the gene

  5. Interactive effects of ultraviolet-B radiation and pesticide exposure on DNA photo-adduct accumulation and expression of DNA damage and repair genes in Xenopus laevis embryos.

    Science.gov (United States)

    Yu, Shuangying; Tang, Song; Mayer, Gregory D; Cobb, George P; Maul, Jonathan D

    2015-02-01

    Pesticide use and ultraviolet-B (UVB) radiation have both been suggested to adversely affect amphibians; however, little is known about their interactive effects. One potential adverse interaction could involve pesticide-induced dysregulation of DNA repair pathways, resulting in greater numbers of DNA photo-adducts from UVB exposure. In the present study, we investigated the interactive effects of UVB radiation and two common pesticides (endosulfan and α-cypermethrin) on induction of DNA photo-adducts and expression of DNA damage and repair related genes in African clawed frog (Xenopus laevis) embryos. We examined 13 genes that are, collectively, involved in stress defense, cell cycle arrest, nucleotide excision repair (NER), base excision repair, mismatch repair, DNA repair regulation, and apoptosis. We exposed X. laevis embryos to 0, 25, and 50 μg/L endosulfan or 0, 2.5, and 5.0 μg/L α-cypermethrin for 96 h, with environmentally relevant exposures of UVB radiation during the last 7 h of the 96 h exposure. We measured the amount of cyclobutane pyrimidine dimers (CPDs) and mRNA abundance of the 13 genes among treatments including control, pesticide only, UVB only, and UVB and pesticide co-exposures. Each of the co-exposure scenarios resulted in elevated CPD levels compared to UVB exposure alone, suggesting an inhibitory effect of endosulfan and α-cypermethrin on CPD repair. This is attributed to results indicating that α-cypermethrin and endosulfan reduced mRNA abundance of XPA and HR23B, respectively, to levels that may affect the initial recognition of DNA lesions. In contrast, both pesticides increased transcript abundance of CSA and MUTL. In addition, mRNA abundance of HSP70 and GADD45α were increased by endosulfan and mRNA abundance of XPG was increased by α-cypermethrin. XPC, HR23B, XPG, and GADD45α exhibited elevated mRNA concentrations whereas there was a reduction in MUTL transcript concentrations in UVB-alone treatments. It appeared that even

  6. Participation of different genes in the ruptures repair of double chain in Escherichia coli stumps exposed to gamma radiation; Participacion de diferentes genes en la reparacion de rupturas de doble cadena en cepas de Escherichia coli expuestas a radiacion gamma

    Energy Technology Data Exchange (ETDEWEB)

    Serment G, J. H.; Martinez M, E.; Alcantara D, D., E-mail: jorge.serment@inin.gob.mx [ININ, Departamento de Biologia, Carretera Mexico-Toluca s/n, 52750 Ocoyoacac, Estado de Mexico (Mexico)

    2013-05-01

    All living organisms are naturally exposed to radiation from different sources. Ionizing radiation produces a plethora of lesions upon DNA that can be categorized as single and double strand breaks and base damage. Among them, unrepaired double strand breaks (Dbs) have the greatest biological significance, since they are responsible of cell death. In Escherichia coli this kind of lesions are repaired mostly by homologous recombination. In this work the participation of some recombination genes in the repair of Dbs is evaluated. Escherichia coli defective strains were exposed to gamma radiation and incubated for different periods in ideal conditions. Both micro electrophoresis and pulse field gel electrophoresis techniques were used to evaluate the kinetics of repair of such lesions, reflecting the importance of each defective gene in the process. (Author)

  7. Consistent and contrasting properties of lineage-specific genes in the apicomplexan parasites Plasmodium and Theileria

    Directory of Open Access Journals (Sweden)

    Kissinger Jessica C

    2008-04-01

    Full Text Available Abstract Background Lineage-specific genes, the genes that are restricted to a limited subset of related organisms, may be important in adaptation. In parasitic organisms, lineage-specific gene products are possible targets for vaccine development or therapeutics when these genes are absent from the host genome. Results In this study, we utilized comparative approaches based on a phylogenetic framework to characterize lineage-specific genes in the parasitic protozoan phylum Apicomplexa. Genes from species in two major apicomplexan genera, Plasmodium and Theileria, were categorized into six levels of lineage specificity based on a nine-species phylogeny. In both genera, lineage-specific genes tend to have a higher level of sequence divergence among sister species. In addition, species-specific genes possess a strong codon usage bias compared to other genes in the genome. We found that a large number of genus- or species-specific genes are putative surface antigens that may be involved in host-parasite interactions. Interestingly, the two parasite lineages exhibit several notable differences. In Plasmodium, the (G + C content at the third codon position increases with lineage specificity while Theileria shows the opposite trend. Surface antigens in Plasmodium are species-specific and mainly located in sub-telomeric regions. In contrast, surface antigens in Theileria are conserved at the genus level and distributed across the entire lengths of chromosomes. Conclusion Our results provide further support for the model that gene duplication followed by rapid divergence is a major mechanism for generating lineage-specific genes. The result that many lineage-specific genes are putative surface antigens supports the hypothesis that lineage-specific genes could be important in parasite adaptation. The contrasting properties between the lineage-specific genes in two major apicomplexan genera indicate that the mechanisms of generating lineage-specific genes

  8. Comparative analysis of chromatin landscape in regulatory regions of human housekeeping and tissue specific genes

    Directory of Open Access Journals (Sweden)

    Dasgupta Dipayan

    2005-05-01

    Full Text Available Abstract Background Global regulatory mechanisms involving chromatin assembly and remodelling in the promoter regions of genes is implicated in eukaryotic transcription control especially for genes subjected to spatial and temporal regulation. The potential to utilise global regulatory mechanisms for controlling gene expression might depend upon the architecture of the chromatin in and around the gene. In-silico analysis can yield important insights into this aspect, facilitating comparison of two or more classes of genes comprising of a large number of genes within each group. Results In the present study, we carried out a comparative analysis of chromatin characteristics in terms of the scaffold/matrix attachment regions, nucleosome formation potential and the occurrence of repetitive sequences, in the upstream regulatory regions of housekeeping and tissue specific genes. Our data show that putative scaffold/matrix attachment regions are more abundant and nucleosome formation potential is higher in the 5' regions of tissue specific genes as compared to the housekeeping genes. Conclusion The differences in the chromatin features between the two groups of genes indicate the involvement of chromatin organisation in the control of gene expression. The presence of global regulatory mechanisms mediated through chromatin organisation can decrease the burden of invoking gene specific regulators for maintenance of the active/silenced state of gene expression. This could partially explain the lower number of genes estimated in the human genome.

  9. The novel quinolone CHM-1 induces DNA damage and inhibits DNA repair gene expressions in a human osterogenic sarcoma cell line.

    Science.gov (United States)

    Chen, Hung-Yi; Lu, Hsu-Feng; Yang, Jai-Sing; Kuo, Sheng-Chu; Lo, Chyi; Yang, Mei-Due; Chiu, Tsan-Hung; Chueh, Fu-Shin; Ho, Heng-Chien; Ko, Yang-Ching; Chung, Jing-Gung

    2010-10-01

    20-Fluoro-6,7-methylenedioxy-2-phenyl-4-quino-lone (CHM-1) has been reported to induce cell cycle arrest and apoptosis in many types of cancer cells. However, there is no available information to show CHM-1 affecting DNA damage and expression of associated repair genes. Herein, we investigated whether or not CHM-1 induced DNA damage and affected DNA repair gene expression in U-2 OS human osterogenic sarcoma cells. The comet assay showed that incubation of U-2 OS cells with 0, 0.75, 1.5, 3 and 6 μM of CHM-1 led to a longer DNA migration smear (comet tail). DNA gel electrophoresis showed that 3 μM of CHM-1 for 24 and 48 h treatment induced DNA fragmentation in U-2 OS cells. Real-time PCR analysis showed that treatment with 3 μM of CHM-1 for 24 h reduced the mRNA expression levels of ataxia telangiectasia mutated (ATM), ataxia-telangiectasia and Rad3-related (ATR), breast cancer 1, early onset (BRCA1), 14-3-3sigma (14-3-3σ), DNA-dependent serine/threonine protein kinase (DNA-PK) and O(6)-methylguanine-DNA methyltransferase (MGMT) genes in a time-dependent manner. Taken together, the results indicate that CHM-1 caused DNA damage and reduced DNA repair genes in U-2 OS cells, which may be the mechanism for CHM-1-inhibited cell growth and induction of apoptosis.

  10. Characterization of the mouse homolog of the XPBC/ERCC-3 gene implicated in xeroderma pigmentosum and Cockayne's Syndrome.

    NARCIS (Netherlands)

    G. Weeda (Geert); L. Ma (Libin); R.C.A. van Ham; D. Bootsma (Dirk); A.J. van der Eb; J.H.J. Hoeijmakers (Jan)

    1991-01-01

    textabstractThe human XPBC/ERCC-3 DNA repair gene specifically corrects the repair defect of xeroderma pigmentosum (XP) complementation group B and rodent repair mutant cell lines of group 3. The gene encodes a presumed DNA- and chromatin-binding helicase involved in early steps of the excision

  11. New frontier in regenerative medicine: site-specific gene correction in patient-specific induced pluripotent stem cells.

    Science.gov (United States)

    Garate, Zita; Davis, Brian R; Quintana-Bustamante, Oscar; Segovia, Jose C

    2013-06-01

    Advances in cell and gene therapy are opening up new avenues for regenerative medicine. Because of their acquired pluripotency, human induced pluripotent stem cells (hiPSCs) are a promising source of autologous cells for regenerative medicine. They show unlimited self-renewal while retaining the ability, in principle, to differentiate into any cell type of the human body. Since Yamanaka and colleagues first reported the generation of hiPSCs in 2007, significant efforts have been made to understand the reprogramming process and to generate hiPSCs with potential for clinical use. On the other hand, the development of gene-editing platforms to increase homologous recombination efficiency, namely DNA nucleases (zinc finger nucleases, TAL effector nucleases, and meganucleases), is making the application of locus-specific gene therapy in human cells an achievable goal. The generation of patient-specific hiPSC, together with gene correction by homologous recombination, will potentially allow for their clinical application in the near future. In fact, reports have shown targeted gene correction through DNA-Nucleases in patient-specific hiPSCs. Various technologies have been described to reprogram patient cells and to correct these patient hiPSCs. However, no approach has been clearly more efficient and safer than the others. In addition, there are still significant challenges for the clinical application of these technologies, such as inefficient differentiation protocols, genetic instability resulting from the reprogramming process and hiPSC culture itself, the efficacy and specificity of the engineered DNA nucleases, and the overall homologous recombination efficiency. To summarize advances in the generation of gene corrected patient-specific hiPSCs, this review focuses on the available technological platforms, including their strengths and limitations regarding future therapeutic use of gene-corrected hiPSCs.

  12. Organ-specific gene expression: the bHLH protein Sage provides tissue specificity to Drosophila FoxA.

    Science.gov (United States)

    Fox, Rebecca M; Vaishnavi, Aria; Maruyama, Rika; Andrew, Deborah J

    2013-05-01

    FoxA transcription factors play major roles in organ-specific gene expression, regulating, for example, glucagon expression in the pancreas, GLUT2 expression in the liver, and tyrosine hydroxylase expression in dopaminergic neurons. Organ-specific gene regulation by FoxA proteins is achieved through cooperative regulation with a broad array of transcription factors with more limited expression domains. Fork head (Fkh), the sole Drosophila FoxA family member, is required for the development of multiple distinct organs, yet little is known regarding how Fkh regulates tissue-specific gene expression. Here, we characterize Sage, a bHLH transcription factor expressed exclusively in the Drosophila salivary gland (SG). We show that Sage is required for late SG survival and normal tube morphology. We find that many Sage targets, identified by microarray analysis, encode SG-specific secreted cargo, transmembrane proteins, and the enzymes that modify these proteins. We show that both Sage and Fkh are required for the expression of Sage target genes, and that co-expression of Sage and Fkh is sufficient to drive target gene expression in multiple cell types. Sage and Fkh drive expression of the bZip transcription factor Senseless (Sens), which boosts expression of Sage-Fkh targets, and Sage, Fkh and Sens colocalize on SG chromosomes. Importantly, expression of Sage-Fkh target genes appears to simply add to the tissue-specific gene expression programs already established in other cell types, and Sage and Fkh cannot alter the fate of most embryonic cell types even when expressed early and continuously.

  13. Caste-Specific and Sex-Specific Expression of Chemoreceptor Genes in a Termite.

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    Yuki Mitaka

    Full Text Available The sophisticated colony organization of eusocial insects is primarily maintained through the utilization of pheromones. The regulation of these complex social interactions requires intricate chemoreception systems. The recent publication of the genome of Zootermopsis nevadensis opened a new avenue to study molecular basis of termite caste systems. Although there has been a growing interest in the termite chemoreception system that regulates their sophisticated caste system, the relationship between division of labor and expression of chemoreceptor genes remains to be explored. Using high-throughput mRNA sequencing (RNA-seq, we found several chemoreceptors that are differentially expressed among castes and between sexes in a subterranean termite Reticulitermes speratus. In total, 53 chemoreception-related genes were annotated, including 22 odorant receptors, 7 gustatory receptors, 12 ionotropic receptors, 9 odorant-binding proteins, and 3 chemosensory proteins. Most of the chemoreception-related genes had caste-related and sex-related expression patterns; in particular, some chemoreception genes showed king-biased or queen-biased expression patterns. Moreover, more than half of the genes showed significant age-dependent differences in their expression in female and/or male reproductives. These results reveal a strong relationship between the evolution of the division of labor and the regulation of chemoreceptor gene expression, thereby demonstrating the chemical communication and underlining chemoreception mechanism in social insects.

  14. Caste-Specific and Sex-Specific Expression of Chemoreceptor Genes in a Termite.

    Science.gov (United States)

    Mitaka, Yuki; Kobayashi, Kazuya; Mikheyev, Alexander; Tin, Mandy M Y; Watanabe, Yutaka; Matsuura, Kenji

    2016-01-01

    The sophisticated colony organization of eusocial insects is primarily maintained through the utilization of pheromones. The regulation of these complex social interactions requires intricate chemoreception systems. The recent publication of the genome of Zootermopsis nevadensis opened a new avenue to study molecular basis of termite caste systems. Although there has been a growing interest in the termite chemoreception system that regulates their sophisticated caste system, the relationship between division of labor and expression of chemoreceptor genes remains to be explored. Using high-throughput mRNA sequencing (RNA-seq), we found several chemoreceptors that are differentially expressed among castes and between sexes in a subterranean termite Reticulitermes speratus. In total, 53 chemoreception-related genes were annotated, including 22 odorant receptors, 7 gustatory receptors, 12 ionotropic receptors, 9 odorant-binding proteins, and 3 chemosensory proteins. Most of the chemoreception-related genes had caste-related and sex-related expression patterns; in particular, some chemoreception genes showed king-biased or queen-biased expression patterns. Moreover, more than half of the genes showed significant age-dependent differences in their expression in female and/or male reproductives. These results reveal a strong relationship between the evolution of the division of labor and the regulation of chemoreceptor gene expression, thereby demonstrating the chemical communication and underlining chemoreception mechanism in social insects.

  15. Quantitative analysis of cell-type specific gene expression in the green alga Volvox carteri

    Directory of Open Access Journals (Sweden)

    Hallmann Armin

    2006-12-01

    Full Text Available Abstract Background The multicellular alga Volvox carteri possesses only two cell types: mortal, motile somatic cells and potentially immortal, immotile reproductive cells. It is therefore an attractive model system for studying how cell-autonomous cytodifferentiation is programmed within a genome. Moreover, there are ongoing genome projects both in Volvox carteri and in the closely related unicellular alga Chlamydomonas reinhardtii. However, gene sequencing is only the beginning. To identify cell-type specific expression and to determine relative expression rates, we evaluate the potential of real-time RT-PCR for quantifying gene transcript levels. Results Here we analyze a diversified pool of 39 target genes by real-time RT-PCR for each cell type. This gene pool contains previously known genes with unknown localization of cellular expression, 28 novel genes which are described in this study for the first time, and a few known, cell-type specific genes as a control. The respective gene products are, for instance, part of photosynthesis, cellular regulation, stress response, or transport processes. We provide expression data for all these genes. Conclusion The results show that quantitative real-time RT-PCR is a favorable approach to analyze cell-type specific gene expression in Volvox, which can be extended to a much larger number of genes or to developmental or metabolic mutants. Our expression data also provide a basis for a detailed analysis of individual, previously unknown, cell-type specifically expressed genes.

  16. Inactivation of RAD52 and HDF1 DNA repair genes leads to premature chronological aging and cellular instability

    Indian Academy of Sciences (India)

    SILVIA MERCADO-SÁENZ; BEATRIZ LÓPEZ-DÍAZ; FRANCISCO SENDRA-PORTERO; MANUEL MARTÍNEZ-MORILLO; MIGUEL J RUIZ-GÓMEZ

    2017-06-01

    The present study aims to investigate the role of radiation sensitive 52 (RAD52) and high-affinity DNA binding factor1 (HDF1) DNA repair genes on the life span of budding yeasts during chronological aging. Wild type (wt) and rad52,hdf1, and rad52 hdf1 mutant Saccharomyces cerevisiae strains were used. Chronological aging and survival assayswere studied by clonogenic assay and drop test. DNA damage was analyzed by electrophoresis after phenol extraction.Mutant analysis, colony forming units and the index of respiratory competence were studied by growing on dextroseand glycerol plates as a carbon source. Rad52 and rad52 hdf1 mutants showed a gradual decrease in surviving fractionin relation to wt and hdf1 mutant during aging. Genomic DNA was spontaneously more degraded during aging,mainly in rad52 mutants. This strain showed an increased percentage of revertant colonies. Moreover, all mutantsshowed a decrease in the index of respiratory competence during aging. The inactivation of RAD52 leads to prematurechronological aging with an increase in DNA degradation and mutation frequency. In addition, RAD52 and HDF1contribute to maintain the metabolic state, in a different way, during chronological aging. The results obtained couldhave important implications in the chronobiology of aging.

  17. Distinctive adaptive response to repeated exposure to hydrogen peroxide associated with upregulation of DNA repair genes and cell cycle arrest

    Directory of Open Access Journals (Sweden)

    Gloria A. Santa-Gonzalez

    2016-10-01

    Full Text Available Many environmental and physiological stresses are chronic. Thus, cells are constantly exposed to diverse types of genotoxic insults that challenge genome stability, including those that induce oxidative DNA damage. However, most in vitro studies that model cellular response to oxidative stressors employ short exposures and/or acute stress models. In this study, we tested the hypothesis that chronic and repeated exposure to a micromolar concentration of hydrogen peroxide (H2O2 could activate DNA damage responses, resulting in cellular adaptations. For this purpose, we developed an in vitro model in which we incubated mouse myoblast cells with a steady concentration of ~50 μM H2O2 for one hour daily for seven days, followed by a final challenge of a 10 or 20X higher dose of H2O2 (0.5 or 1 mM. We report that intermittent long-term exposure to this oxidative stimulus nearly eliminated cell toxicity and significantly decreased genotoxicity (in particular, a >5-fold decreased in double-strand breaks resulting from subsequent acute exposure to oxidative stress. This protection was associated with cell cycle arrest in G2/M and induction of expression of nine DNA repair genes. Together, this evidence supports an adaptive response to chronic, low-level oxidative stress that results in genomic protection and up-regulated maintenance of cellular homeostasis.

  18. Evolution of DNA Double-Strand Break Repair by Gene Conversion: Coevolution Between a Phage and a Restriction-Modification System

    Science.gov (United States)

    Yahara, Koji; Horie, Ryota; Kobayashi, Ichizo; Sasaki, Akira

    2007-01-01

    The necessity to repair genome damage has been considered to be an immediate factor responsible for the origin of sex. Indeed, attack by a cellular restriction enzyme of invading DNA from several bacteriophages initiates recombinational repair by gene conversion if there is homologous DNA. In this work, we modeled the interaction between a bacteriophage and a bacterium carrying a restriction enzyme as antagonistic coevolution. We assume a locus on the bacteriophage genome has either a restriction-sensitive or a restriction-resistant allele, and another locus determines whether it is recombination/repair proficient or defective. A restriction break can be repaired by a co-infecting phage genome if one of them is recombination/repair proficient. We define the fitness of phage (resistant/sensitive and repair-positive/-negative) genotypes and bacterial (restriction-positive/-negative) genotypes by assuming random encounter of the genotypes, with given probabilities of single and double infections, and the costs of resistance, repair, and restriction. Our results show the evolution of the repair allele depends on \\documentclass[10pt]{article} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{pmc} \\pagestyle{empty} \\oddsidemargin -1.0in \\begin{document} \\begin{equation*}b_{1}/b_{0},\\end{equation*}\\end{document} the ratio of the burst size \\documentclass[10pt]{article} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{pmc} \\pagestyle{empty} \\oddsidemargin -1.0in \\begin{document} \\begin{equation*}b_{1}\\end{equation*}\\end{document} under damage to host cell physiology induced by an unrepaired double-strand break to the default burst size \\documentclass[10pt]{article} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage

  19. Micronuclei in humans induced by exposure to low level of ionizing radiation: influence of polymorphisms in DNA repair genes

    Energy Technology Data Exchange (ETDEWEB)

    Angelini, Sabrina [Department of Pharmacology, University of Bologna, Via Irnerio 48, Bologna 40126 (Italy) and Department of Biosciences, Karolinska Institute, Novum, Huddinge 141 57 (Sweden)]. E-mail: angelini@biocfarm.unibo.it; Kumar, Rajiv [Department of Biosciences, Karolinska Institute, Novum, Huddinge 141 57 (Sweden); Division of Molecular Genetic Epidemiology, German Cancer Research Center (DKFZ), Heidelberg (Germany); Carbone, Fabio [Department of Pharmacology, University of Bologna, Via Irnerio 48, Bologna 40126 (Italy); Maffei, Francesca [Department of Pharmacology, University of Bologna, Via Irnerio 48, Bologna 40126 (Italy); Forti, Giorgio Cantelli [Department of Pharmacology, University of Bologna, Via Irnerio 48, Bologna 40126 (Italy); Violante, Francesco Saverio [Department of Biosciences, Karolinska Institute, Novum, Huddinge 141 57 (Sweden); Occupational Medicine Unit, S. Orsola-Malpighi Hospital, Via Pelagi 9, Bologna 40100 (Italy); Lodi, Vittorio [Department of Biosciences, Karolinska Institute, Novum, Huddinge 141 57 (Sweden); Curti, Stefania [Department of Biosciences, Karolinska Institute, Novum, Huddinge 141 57 (Sweden); Hemminki, Kari [Department of Biosciences, Karolinska Institute, Novum, Huddinge 141 57 (Sweden); Hrelia, Patrizia [Department of Pharmacology, University of Bologna, Via Irnerio 48, Bologna 40126 (Italy)

    2005-02-15

    Understanding the risks deriving from protracted exposure to low doses of ionizing radiation has remarkable societal importance in view of the large number of work settings in which sources of IR are encountered. To address this question, we studied the frequency of micronuclei (MN), which is an indicator of DNA damage, in a population exposed to low levels of ionizing radiation and in matched controls. In both exposed population and controls, the possible influence of single nucleotide polymorphisms in XRCC1, XRCC3 and XPD genes on the frequency of micronuclei was also evaluated. We also considered the effects of confounding factors, like smoking status, age and gender. The results indicated that MN frequency was significantly higher in the exposed workers than in the controls [8.62 {+-} 2.80 versus 6.86 {+-} 2.65; P = 0.019]. Radiological workers with variant alleles for XRCC1 or XRCC3 polymorphisms or wild-type alleles for XPD exon 23 or 10 polymorphisms showed a significantly higher MN frequency than controls with the same genotypes. Smoking status did not affect micronuclei frequency either in exposed workers or controls, while age was associated with increased MN frequency in the exposed only. In the combined population, gender but not age exerted an influence on the yield of MN, being higher in females than in males. Even though there is a limitation in this study due to the small number of subjects, these results suggest that even exposures to low level of ionizing radiation could have genotoxic effects and that XRCC3, XRCC1 and XPD polymorphisms might contribute to the increased genetic damage in susceptible individuals occupationally exposed to chronic low levels of ionizing radiation. For a clear conclusion on the induction of DNA damage caused by protracted exposure to low doses of ionizing radiation and the possible influence of genetic polymorphism in DNA repair genes larger studies are needed.

  20. Assessing SNP-SNP interactions among DNA repair, modification and metabolism related pathway genes in breast cancer susceptibility.

    Directory of Open Access Journals (Sweden)

    Yadav Sapkota

    Full Text Available Genome-wide association studies (GWASs have identified low-penetrance common variants (i.e., single nucleotide polymorphisms, SNPs associated with breast cancer susceptibility. Although GWASs are primarily focused on single-locus effects, gene-gene interactions (i.e., epistasis are also assumed to contribute to the genetic risks for complex diseases including breast cancer. While it has been hypothesized that moderately ranked (P value based weak single-locus effects in GWASs could potentially harbor valuable information for evaluating epistasis, we lack systematic efforts to investigate SNPs showing consistent associations with weak statistical significance across independent discovery and replication stages. The objectives of this study were i to select SNPs showing single-locus effects with weak statistical significance for breast cancer in a GWAS and/or candidate-gene studies; ii to replicate these SNPs in an independent set of breast cancer cases and controls; and iii to explore their potential SNP-SNP interactions contributing to breast cancer susceptibility. A total of 17 SNPs related to DNA repair, modification and metabolism pathway genes were selected since these pathways offer a priori knowledge for potential epistatic interactions and an overall role in breast carcinogenesis. The study design included predominantly Caucasian women (2,795 cases and 4,505 controls from Alberta, Canada. We observed two two-way SNP-SNP interactions (APEX1-rs1130409 and RPAP1-rs2297381; MLH1-rs1799977 and MDM2-rs769412 in logistic regression that conferred elevated risks for breast cancer (P(interaction<7.3 × 10(-3. Logic regression identified an interaction involving four SNPs (MBD2-rs4041245, MLH1-rs1799977, MDM2-rs769412, BRCA2-rs1799943 (P(permutation = 2.4 × 10(-3. SNPs involved in SNP-SNP interactions also showed single-locus effects with weak statistical significance, while BRCA2-rs1799943 showed stronger statistical significance (P

  1. Interactive effects of ultraviolet-B radiation and pesticide exposure on DNA photo-adduct accumulation and expression of DNA damage and repair genes in Xenopus laevis embryos

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Shuangying, E-mail: shuangying.yu@ttu.edu [Department of Environmental Toxicology, The Institute of Environmental and Human Health, Texas Tech University, 1207 S. Gilbert Dr., Lubbock, TX 79416 (United States); Tang, Song, E-mail: song.tang@usask.ca [Department of Environmental Toxicology, The Institute of Environmental and Human Health, Texas Tech University, 1207 S. Gilbert Dr., Lubbock, TX 79416 (United States); Mayer, Gregory D., E-mail: greg.mayer@ttu.edu [Department of Environmental Toxicology, The Institute of Environmental and Human Health, Texas Tech University, 1207 S. Gilbert Dr., Lubbock, TX 79416 (United States); Cobb, George P., E-mail: george_cobb@baylor.edu [Department of Environmental Science, Baylor University, One Bear Place #97266, Waco, TX 76798 (United States); Maul, Jonathan D., E-mail: jonathan.maul@ttu.edu [Department of Environmental Toxicology, The Institute of Environmental and Human Health, Texas Tech University, 1207 S. Gilbert Dr., Lubbock, TX 79416 (United States)

    2015-02-15

    Highlights: • Interactive effects of UVB radiation-pesticide co-exposures were examined in frogs. • Responses included induction of DNA photo-adducts and DNA damage and repair genes. • Elevated DNA adduct levels occurred for co-exposures compared to UVB alone. • One mechanism is that pesticides may alter nuclear excision repair gene expression. - Abstract: Pesticide use and ultraviolet-B (UVB) radiation have both been suggested to adversely affect amphibians; however, little is known about their interactive effects. One potential adverse interaction could involve pesticide-induced dysregulation of DNA repair pathways, resulting in greater numbers of DNA photo-adducts from UVB exposure. In the present study, we investigated the interactive effects of UVB radiation and two common pesticides (endosulfan and α-cypermethrin) on induction of DNA photo-adducts and expression of DNA damage and repair related genes in African clawed frog (Xenopus laevis) embryos. We examined 13 genes that are, collectively, involved in stress defense, cell cycle arrest, nucleotide excision repair (NER), base excision repair, mismatch repair, DNA repair regulation, and apoptosis. We exposed X. laevis embryos to 0, 25, and 50 μg/L endosulfan or 0, 2.5, and 5.0 μg/L α-cypermethrin for 96 h, with environmentally relevant exposures of UVB radiation during the last 7 h of the 96 h exposure. We measured the amount of cyclobutane pyrimidine dimers (CPDs) and mRNA abundance of the 13 genes among treatments including control, pesticide only, UVB only, and UVB and pesticide co-exposures. Each of the co-exposure scenarios resulted in elevated CPD levels compared to UVB exposure alone, suggesting an inhibitory effect of endosulfan and α-cypermethrin on CPD repair. This is attributed to results indicating that α-cypermethrin and endosulfan reduced mRNA abundance of XPA and HR23B, respectively, to levels that may affect the initial recognition of DNA lesions. In contrast, both pesticides

  2. An approach for the identification of targets specific to bone metastasis using cancer genes interactome and gene ontology analysis.

    Science.gov (United States)

    Vashisht, Shikha; Bagler, Ganesh

    2012-01-01

    Metastasis is one of the most enigmatic aspects of cancer pathogenesis and is a major cause of cancer-associated mortality. Secondary bone cancer (SBC) is a complex disease caused by metastasis of tumor cells from their primary site and is characterized by intricate interplay of molecular interactions. Identification of targets for multifactorial diseases such as SBC, the most frequent complication of breast and prostate cancers, is a challenge. Towards achieving our aim of identification of targets specific to SBC, we constructed a 'Cancer Genes Network', a representative protein interactome of cancer genes. Using graph theoretical methods, we obtained a set of key genes that are relevant for generic mechanisms of cancers and have a role in biological essentiality. We also compiled a curated dataset of 391 SBC genes from published literature which serves as a basis of ontological correlates of secondary bone cancer. Building on these results, we implement a strategy based on generic cancer genes, SBC genes and gene ontology enrichment method, to obtain a set of targets that are specific to bone metastasis. Through this study, we present an approach for probing one of the major complications in cancers, namely, metastasis. The results on genes that play generic roles in cancer phenotype, obtained by network analysis of 'Cancer Genes Network', have broader implications in understanding the role of molecular regulators in mechanisms of cancers. Specifically, our study provides a set of potential targets that are of ontological and regulatory relevance to secondary bone cancer.

  3. An approach for the identification of targets specific to bone metastasis using cancer genes interactome and gene ontology analysis.

    Directory of Open Access Journals (Sweden)

    Shikha Vashisht

    Full Text Available Metastasis is one of the most enigmatic aspects of cancer pathogenesis and is a major cause of cancer-associated mortality. Secondary bone cancer (SBC is a complex disease caused by metastasis of tumor cells from their primary site and is characterized by intricate interplay of molecular interactions. Identification of targets for multifactorial diseases such as SBC, the most frequent complication of breast and prostate cancers, is a challenge. Towards achieving our aim of identification of targets specific to SBC, we constructed a 'Cancer Genes Network', a representative protein interactome of cancer genes. Using graph theoretical methods, we obtained a set of key genes that are relevant for generic mechanisms of cancers and have a role in biological essentiality. We also compiled a curated dataset of 391 SBC genes from published literature which serves as a basis of ontological correlates of secondary bone cancer. Building on these results, we implement a strategy based on generic cancer genes, SBC genes and gene ontology enrichment method, to obtain a set of targets that are specific to bone metastasis. Through this study, we present an approach for probing one of the major complications in cancers, namely, metastasis. The results on genes that play generic roles in cancer phenotype, obtained by network analysis of 'Cancer Genes Network', have broader implications in understanding the role of molecular regulators in mechanisms of cancers. Specifically, our study provides a set of potential targets that are of ontological and regulatory relevance to secondary bone cancer.

  4. Genome-wide identification of lineage-specific genes in Arabidopsis, Oryza and Populus

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Xiaohan [ORNL; Jawdy, Sara [ORNL; Tschaplinski, Timothy J [ORNL; Tuskan, Gerald A [ORNL

    2009-01-01

    Protein sequences were compared among Arabidopsis, Oryza and Populus to identify differential gene (DG) sets that are in one but not the other two genomes. The DG sets were screened against a plant transcript database, the NR protein database and six newly-sequenced genomes (Carica, Glycine, Medicago, Sorghum, Vitis and Zea) to identify a set of species-specific genes (SS). Gene expression, protein motif and intron number were examined. 192, 641 and 109 SS genes were identified in Arabidopsis, Oryza and Populus, respectively. Some SS genes were preferentially expressed in flowers, roots, xylem and cambium or up-regulated by stress. Six conserved motifs in Arabidopsis and Oryza SS proteins were found in other distant lineages. The SS gene sets were enriched with intronless genes. The results reflect functional and/or anatomical differences between monocots and eudicots or between herbaceous and woody plants. The Populus-specific genes are candidates for carbon sequestration and biofuel research.

  5. High-throughput analysis of candidate imprinted genes and allele-specific gene expression in the human term placenta

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    Clark Taane G

    2010-04-01

    Full Text Available Abstract Background Imprinted genes show expression from one parental allele only and are important for development and behaviour. This extreme mode of allelic imbalance has been described for approximately 56 human genes. Imprinting status is often disrupted in cancer and dysmorphic syndromes. More subtle variation of gene expression, that is not parent-of-origin specific, termed 'allele-specific gene expression' (ASE is more common and may give rise to milder phenotypic differences. Using two allele-specific high-throughput technologies alongside bioinformatics predictions, normal term human placenta was screened to find new imprinted genes and to ascertain the extent of ASE in this tissue. Results Twenty-three family trios of placental cDNA, placental genomic DNA (gDNA and gDNA from both parents were tested for 130 candidate genes with the Sequenom MassArray system. Six genes were found differentially expressed but none imprinted. The Illumina ASE BeadArray platform was then used to test 1536 SNPs in 932 genes. The array was enriched for the human orthologues of 124 mouse candidate genes from bioinformatics predictions and 10 human candidate imprinted genes from EST database mining. After quality control pruning, a total of 261 informative SNPs (214 genes remained for analysis. Imprinting with maternal expression was demonstrated for the lymphocyte imprinted gene ZNF331 in human placenta. Two potential differentially methylated regions (DMRs were found in the vicinity of ZNF331. None of the bioinformatically predicted candidates tested showed imprinting except for a skewed allelic expression in a parent-specific manner observed for PHACTR2, a neighbour of the imprinted PLAGL1 gene. ASE was detected for two or more individuals in 39 candidate genes (18%. Conclusions Both Sequenom and Illumina assays were sensitive enough to study imprinting and strong allelic bias. Previous bioinformatics approaches were not predictive of new imprinted genes

  6. In vivo effects of UV radiation on multiple endpoints and expression profiles of DNA repair and heat shock protein (Hsp) genes in the cycloid copepod Paracyclopina nana

    Energy Technology Data Exchange (ETDEWEB)

    Won, Eun-Ji; Han, Jeonghoon [Department of Biological Science, College of Science, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Lee, Yeonjung; Kumar, K. Suresh; Shin, Kyung-Hoon [Department of Marine Sciences and Convergent Technology, College of Science and Technology, Hanyang University, Ansan 426-791 (Korea, Republic of); Lee, Su-Jae [Department of Life Sciences, College of Natural Sciences, Hanyang University, Seoul 133-791 (Korea, Republic of); Park, Heum Gi, E-mail: hgpark@gwnu.ac.kr [Department of Marine Resource Development, College of Life Sciences, Gangneung-Wonju National University, Gangneung 210-702 (Korea, Republic of); Lee, Jae-Seong, E-mail: jslee2@skku.edu [Department of Biological Science, College of Science, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of)

    2015-08-15

    Highlights: • UV-B radiation induced a significant reduction of the re-brooding rate of ovigerous females. • A dose-dependent decrease in food ingestion and the rate of assimilation to the body upon UV radiation. • Expression of base excision repair-associated and hsp chaperoning genes was significantly increased upon UV radiation in P. nana. - Abstract: To evaluate the effects of ultraviolet (UV) radiation on energy acquisition and consumption, the copepod Paracyclopina nana was irradiated with several doses (0–3 kJ/m{sup 2}) of UV. After UV radiation, we measured the re-brooding success, growth pattern of newly hatched nauplii, ingestion rate, and assimilation of diet. In addition, we checked the modulated patterns of DNA repair and heat shock protein (hsp) chaperoning genes of P. nana. UV-B radiation induced a significant reduction (7–87%) of the re-brooding rate of ovigerous females, indicating that UV-induced egg sac damage is closely correlated with a reduction in the hatching rate of UV-irradiated ovigerous female offspring. Using chlorophyll a and stable carbon isotope incubation experiments, we found a dose-dependent decrease (P < 0.05) in food ingestion and the rate of assimilation to the body in response to UV radiation, implying that P. nana has an underlying ability to shift its balanced-energy status from growth and reproduction to DNA repair and adaptation. Also, expression of P. nana base excision repair (BER)-associated genes and hsp chaperoning genes was significantly increased in response to UV radiation in P. nana. These findings indicate that even 1 kJ/m{sup 2} of UV radiation induces a reduction in reproduction and growth patterns, alters the physiological balance and inhibits the ability to cope with UV-induced damage in P. nana.

  7. Association between Genetic Polymorphisms of DNA Repair Genes and Chromosomal Damage for 1,3-Butadiene-Exposed Workers in a Matched Study in China

    OpenAIRE

    2015-01-01

    The aim of the study was to examine the association between polymorphisms of DNA repair genes and chromosomal damage of 1,3-butadiene- (BD-) exposed workers. The study was conducted in 45 pairs of occupationally exposed workers in a BD product workshop and matched control workers in an administrative office and a circulatory water workshop in China. Newly developed biomarkers (micronuclei, MNi; nucleoplasmic bridges, NPBs; nuclear buds, NBUDs) in the cytokinesis-blocked micronucleus (CBMN) cy...

  8. Germ line mutations of mismatch repair genes in hereditary nonpolyposis colorectal cancer patients with small bowel cancer: International Society for Gastrointestinal Hereditary Tumours Collaborative Study

    DEFF Research Database (Denmark)

    Park, Jae-Gahb; Kim, Duck-Woo; Hong, Chang Won;

    2006-01-01

    PURPOSE: The aim of study was to determine the clinical characteristics and mutational profiles of the mismatch repair genes in hereditary nonpolyposis colorectal cancer (HNPCC) patients with small bowel cancer (SBC). EXPERIMENTAL DESIGN: A questionnaire was mailed to 55 members of the Internatio.......8%, P teens. The distribution of MSH2 mutations found in patients with HNPCC-associated SBCs significantly differed from that found in the control group (P

  9. Specific genetic modifications of domestic animals by gene targeting and animal cloning.

    Science.gov (United States)

    Wang, Bin; Zhou, Jiangfeng

    2003-11-13

    The technology of gene targeting through homologous recombination has been extremely useful for elucidating gene functions in mice. The application of this technology was thought impossible in the large livestock species until the successful creation of the first mammalian clone "Dolly" the sheep. The combination of the technologies for gene targeting of somatic cells with those of animal cloning made it possible to introduce specific genetic mutations into domestic animals. In this review, the principles of gene targeting in somatic cells and the challenges of nuclear transfer using gene-targeted cells are discussed. The relevance of gene targeting in domestic animals for applications in bio-medicine and agriculture are also examined.

  10. Defining diversity, specialization, and gene specificity in transcriptomes through information theory

    Science.gov (United States)

    Martínez, Octavio; Reyes-Valdés, M. Humberto

    2008-01-01

    The transcriptome is a set of genes transcribed in a given tissue under specific conditions and can be characterized by a list of genes with their corresponding frequencies of transcription. Transcriptome changes can be measured by counting gene tags from mRNA libraries or by measuring light signals in DNA microarrays. In any case, it is difficult to completely comprehend the global changes that occur in the transcriptome, given that thousands of gene expression measurements are involved. We propose an approach to define and estimate the diversity and specialization of transcriptomes and gene specificity. We define transcriptome diversity as the Shannon entropy of its frequency distribution. Gene specificity is defined as the mutual information between the tissues and the corresponding transcript, allowing detection of either housekeeping or highly specific genes and clarifying the meaning of these concepts in the literature. Tissue specialization is measured by average gene specificity. We introduce the formulae using a simple example and show their application in two datasets of gene expression in human tissues. Visualization of the positions of transcriptomes in a system of diversity and specialization coordinates makes it possible to understand at a glance their interrelations, summarizing in a powerful way which transcriptomes are richer in diversity of expressed genes, or which are relatively more specialized. The framework presented enlightens the relation among transcriptomes, allowing a better understanding of their changes through the development of the organism or in response to environmental stimuli. PMID:18606989

  11. Allele specific expression in worker reproduction genes in the bumblebee Bombus terrestris

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    Harindra E. Amarasinghe

    2015-07-01

    Full Text Available Methylation has previously been associated with allele specific expression in ants. Recently, we found methylation is important in worker reproduction in the bumblebee Bombus terrestris. Here we searched for allele specific expression in twelve genes associated with worker reproduction in bees. We found allele specific expression in Ecdysone 20 monooxygenase and IMP-L2-like. Although we were unable to confirm a genetic or epigenetic cause for this allele specific expression, the expression patterns of the two genes match those predicted for imprinted genes.

  12. Association between Genetic Polymorphisms of DNA Repair Genes and Chromosomal Damage for 1,3-Butadiene-Exposed Workers in a Matched Study in China.

    Science.gov (United States)

    Xiang, Menglong; Sun, Lei; Dong, Xiaomei; Yang, Huan; Liu, Wen-bin; Zhou, Niya; Han, Xue; Zhou, Ziyuan; Cui, Zhihong; Liu, Jing-yi; Cao, Jia; Ao, Lin

    2015-01-01

    The aim of the study was to examine the association between polymorphisms of DNA repair genes and chromosomal damage of 1,3-butadiene- (BD-) exposed workers. The study was conducted in 45 pairs of occupationally exposed workers in a BD product workshop and matched control workers in an administrative office and a circulatory water workshop in China. Newly developed biomarkers (micronuclei, MNi; nucleoplasmic bridges, NPBs; nuclear buds, NBUDs) in the cytokinesis-blocked micronucleus (CBMN) cytome assay were adopted to detect chromosomal damage. PCR and PCR-restriction fragment length polymorphism (RFLP) are adopted to analyze polymorphisms of DNA repair genes, such as X-ray repair cross-complementing Group 1 (XRCC1), O6-methylguanine-DNA methyltransferase (MGMT), poly (adenosine diphosphate-ribose) polymerases (ADPRT), and apurinic/apyrimidinic endonucleases (APE1). The BD-exposed workers exhibited increased frequencies of MNi and NPBs when compared to subjects in the control group. The results also show that the BD-exposed workers carrying XRCC1 diplotypes TCGA-CCGG (4.25 ± 2.06 ‰) (FR = 2.10, 95% CI: 1.03-4.28) and TCGG-TCGA (5.80 ± 3.56 ‰) (FR = 2.75, 95% CI: 0.76-2.65) had statistically higher NBUD frequencies than those who carried diplotype TCGG-TCGG (1.89 ± 1.27 ‰). Our study suggests that polymorphisms of XRCC1 gene may influence chromosomal damage in BD-exposed workers.

  13. In vivo effects of UV radiation on multiple endpoints and expression profiles of DNA repair and heat shock protein (Hsp) genes in the cycloid copepod Paracyclopina nana.

    Science.gov (United States)

    Won, Eun-Ji; Han, Jeonghoon; Lee, Yeonjung; Kumar, K Suresh; Shin, Kyung-Hoon; Lee, Su-Jae; Park, Heum Gi; Lee, Jae-Seong

    2015-08-01

    To evaluate the effects of ultraviolet (UV) radiation on energy acquisition and consumption, the copepod Paracyclopina nana was irradiated with several doses (0-3kJ/m(2)) of UV. After UV radiation, we measured the re-brooding success, growth pattern of newly hatched nauplii, ingestion rate, and assimilation of diet. In addition, we checked the modulated patterns of DNA repair and heat shock protein (hsp) chaperoning genes of P. nana. UV-B radiation induced a significant reduction (7-87%) of the re-brooding rate of ovigerous females, indicating that UV-induced egg sac damage is closely correlated with a reduction in the hatching rate of UV-irradiated ovigerous female offspring. Using chlorophyll a and stable carbon isotope incubation experiments, we found a dose-dependent decrease (Pnana has an underlying ability to shift its balanced-energy status from growth and reproduction to DNA repair and adaptation. Also, expression of P. nana base excision repair (BER)-associated genes and hsp chaperoning genes was significantly increased in response to UV radiation in P. nana. These findings indicate that even 1kJ/m(2) of UV radiation induces a reduction in reproduction and growth patterns, alters the physiological balance and inhibits the ability to cope with UV-induced damage in P. nana.

  14. Normal repair of ultraviolet radiation-induced DNA damage in familial melanoma without CDKN2A or CDK4 gene mutation.

    Science.gov (United States)

    Shannon, J A; Matias, C; Luxford, C; Kefford, R F; Mann, G J

    1999-04-01

    Excessive sun exposure and family history are strong risk factors for the development of cutaneous melanoma. Inherited susceptibility to this type of skin cancer could therefore result from constitutively impaired capacity to repair ultraviolet (UV)-induced DNA lesions. While a proportion of familial melanoma kindreds exhibit germline mutations in the cell cycle regulatory gene CDKN2A (p16INK4a) or its protein target, cyclin-dependent kinase 4 (CDK4), the biochemical basis of most familial melanoma is unknown. We have examined lymphoblastoid cell lines from melanoma-affected and unaffected individuals from large hereditary melanoma kindreds which are not attributable to CDKN2A or CDK4 gene mutation. These lines were tested for sensitivity of clonogenic growth to UV radiation and for their ability to repair transfected UV-damaged plasmid templates (host cell reactivation). Two of seven affected-unaffected pairs differed in colony survival after exposure to UVB radiation; however, no significant differences were observed in the host-cell reactivation assays. These results indicate that melanoma susceptibility genes other than CDKN2A and CDK4 do not impair net capacity to repair UV-induced DNA damage.

  15. Polymorphisms in DNA Repair Gene XRCC3 and Susceptibility to Breast Cancer in Saudi Females

    Directory of Open Access Journals (Sweden)

    Alaa Mohammed Ali

    2016-01-01

    Full Text Available We investigated three common polymorphisms (SNPs in the XRCC3 gene (rs861539, rs1799794, and rs1799796 in 143 Saudi females suffering from breast cancer (median age = 51.4 years and 145 age matched normal healthy controls. DNA was extracted from whole blood and genotyping was conducted using PCR-RFLP. rs1799794 showed significant association, where AA and AA+AG occurred at a significantly higher frequency in the cancer patients compared to the control group (OR: 28.1; 95% CI: 3.76–21.12; χ2: 22.82; pT and rs1799796 A>G did not show a significant difference when the results in the patients and controls were compared. However, the frequency of rs1799796 differed significantly in patients with different age of diagnosis, tumor grade, and ER and HER2 status. The wild type A allele occurred at a higher frequency in the ER− and HER2− group. Our results among Saudis suggest that some variations in XRCC3 may contribute to breast cancer susceptibility. In conclusion, the results obtained during this study suggest that rs1799794 in XRCC3 shows strong association with breast cancer development in Saudi females.

  16. Lineage-Specific and Non-specific Cytokine-Sensing Genes Respond Differentially to the Master Regulator STAT5.

    Science.gov (United States)

    Zeng, Xianke; Willi, Michaela; Shin, Ha Youn; Hennighausen, Lothar; Wang, Chaochen

    2016-12-20

    STAT5, a member of the family of signal transducers and activators of transcription, senses cytokines and controls the biology of cell lineages, including mammary, liver, and T cells. Here, we show that STAT5 activates lineage-specific and widely expressed genes through different mechanisms. STAT5 preferentially binds to promoter sequences of cytokine-responsive genes expressed across cell types and to putative enhancers of lineage-specific genes. While chromatin accessibility of STAT5-based enhancers was dependent on cytokine exposure, STAT5-responsive promoters of widely expressed target genes were generally constitutively accessible. While the contribution of STAT5 to enhancers is well established, its role on promoters is poorly understood. To address this, we focused on Socs2, a widely expressed cytokine-sensing gene. Upon deletion of the STAT5 response elements from the Socs2 promoter in mice, cytokine induction was abrogated, while basal activity remained intact. Our data suggest that promoter-bound STAT5 modulates cytokine responses and enhancer-bound STAT5 is mandatory for gene activation.

  17. Endometrial tumour BRAF mutations and MLH1 promoter methylation as predictors of germline mismatch repair gene mutation status: a literature review.

    Science.gov (United States)

    Metcalf, Alexander M; Spurdle, Amanda B

    2014-03-01

    Colorectal cancer (CRC) that displays high microsatellite instability (MSI-H) can be caused by either germline mutations in mismatch repair (MMR) genes, or non-inherited transcriptional silencing of the MLH1 promoter. A correlation between MLH1 promoter methylation, specifically the 'C' region, and BRAF V600E status has been reported in CRC studies. Germline MMR mutations also greatly increase risk of endometrial cancer (EC), but no systematic review has been undertaken to determine if these tumour markers may be useful predictors of MMR mutation status in EC patients. Endometrial cancer cohorts meeting review inclusion criteria encompassed 2675 tumours from 20 studies for BRAF V600E, and 447 tumours from 11 studies for MLH1 methylation testing. BRAF V600E mutations were reported in 4/2675 (0.1%) endometrial tumours of unknown MMR mutation status, and there were 7/823 (0.9%) total sequence variants in exon 11 and 27/1012 (2.7%) in exon 15. Promoter MLH1 methylation was not observed in tumours from 32 MLH1 mutation carriers, or for 13 MSH2 or MSH6 mutation carriers. MMR mutation-negative individuals with tumour MLH1 and PMS2 IHC loss displayed MLH1 methylation in 48/51 (94%) of tumours. We have also detailed specific examples that show the importance of MLH1 promoter region, assay design, and quantification of methylation. This review shows that BRAF mutations occurs so infrequently in endometrial tumours they can be discounted as a useful marker for predicting MMR-negative mutation status, and further studies of endometrial cohorts with known MMR mutation status are necessary to quantify the utility of tumour MLH1 promoter methylation as a marker of negative germline MMR mutation status in EC patients.

  18. Functional Enhancers As Master Regulators of Tissue-Specific Gene Regulation and Cancer Development

    Science.gov (United States)

    Ko, Je Yeong; Oh, Sumin; Yoo, Kyung Hyun

    2017-01-01

    Tissue-specific transcription is critical for normal development, and abnormalities causing undesirable gene expression may lead to diseases such as cancer. Such highly organized transcription is controlled by enhancers with specific DNA sequences recognized by transcription factors. Enhancers are associated with chromatin modifications that are distinct epigenetic features in a tissue-specific manner. Recently, super-enhancers comprising enhancer clusters co-occupied by lineage-specific factors have been identified in diverse cell types such as adipocytes, hair follicle stem cells, and mammary epithelial cells. In addition, noncoding RNAs, named eRNAs, are synthesized at super-enhancer regions before their target genes are transcribed. Many functional studies revealed that super-enhancers and eRNAs are essential for the regulation of tissue-specific gene expression. In this review, we summarize recent findings concerning enhancer function in tissue-specific gene regulation and cancer development. PMID:28359147

  19. Development of a screening method for R gene specificity in wheat

    DEFF Research Database (Denmark)

    Kronbak, Remy; Yin, Chang; Hovmøller, Mogens Støvring;

    2011-01-01

    Crops and their wild relatives contain several hundred genes that confer disease resistance (R genes). This pool of genes represents a vast resource for breeding resistant crops, but the identification of R genes is lengthy and laborious by classical genetic approaches. Thus, the lack of knowledge...... about R genes, and the pests individual R genes may provide resistance against, is a major bottleneck in plant breeding. Recently our cooperators from KU found that R genes mutated in specific domains act as dominant negatives conferring susceptibility, when introduced into plants resistant....... The yellow rust R gene Yr10 will be transformed into Bobwhite, and transgenic plants tested for resistance against an appropriate avirulent P. striiformis isolate harboring the AvrYr10 gene. Regenerated transgenic plants will be re-transformed with a mutated form of Yr10. The host-pathogen phenotype...

  20. Cortical gene expression in spinal cord injury and repair: insight into the functional complexity of the neural regeneration program

    Directory of Open Access Journals (Sweden)

    Fabian eKruse

    2011-09-01

    Full Text Available Traumatic spinal cord injury (SCI results in the formation of a fibrous scar acting as a growth barrier for regenerating axons at the lesion site. We have previously shown (Klapka et al., 2005 that transient suppression of the inhibitory lesion scar in rat spinal cord leads to long distance axon regeneration, retrograde rescue of axotomized cortical motoneurons and improvement of locomotor function. Here we applied a systemic approach to investigate for the first time specific and dynamic alterations in the cortical gene expression profile following both thoracic SCI and regeneration-promoting anti-scarring treatment (AST. In order to monitor cortical gene expression we carried out microarray analyses using total RNA isolated from layer V/VI of rat sensorimotor cortex at 1-60 days post-operation (dpo. We demonstrate that cortical neurons respond to injury by massive changes in gene expression, starting as early as 1 dpo. AST, in turn, results in profound modifications of the lesion-induced expression profile. The treatment attenuates SCI-triggered transcriptional changes of genes related to inhibition of axon growth and impairment of cell survival, while upregulating the expression of genes associated with axon outgrowth, cell protection and neural development. Thus, AST not only modifies the local environment impeding spinal cord regeneration by reduction of fibrous scarring in the injured spinal cord, but, in addition, strikingly changes the intrinsic capacity of cortical pyramidal neurons towards enhanced cell maintenance and axonal regeneration.

  1. Modulation of DNA binding by gene-specific transcription factors.

    Science.gov (United States)

    Schleif, Robert F

    2013-10-01

    The transcription of many genes, particularly in prokaryotes, is controlled by transcription factors whose activity can be modulated by controlling their DNA binding affinity. Understanding the molecular mechanisms by which DNA binding affinity is regulated is important, but because forming definitive conclusions usually requires detailed structural information in combination with data from extensive biophysical, biochemical, and sometimes genetic experiments, little is truly understood about this topic. This review describes the biological requirements placed upon DNA binding transcription factors and their consequent properties, particularly the ways that DNA binding affinity can be modulated and methods for its study. What is known and not known about the mechanisms modulating the DNA binding affinity of a number of prokaryotic transcription factors, including CAP and lac repressor, is provided.

  2. Knock down of Whitefly Gut Gene Expression and Mortality by Orally Delivered Gut Gene-Specific dsRNAs.

    Science.gov (United States)

    Vyas, Meenal; Raza, Amir; Ali, Muhammad Yousaf; Ashraf, Muhammad Aleem; Mansoor, Shahid; Shahid, Ahmad Ali; Brown, Judith K

    2017-01-01

    Control of the whitefly Bemisia tabaci (Genn.) agricultural pest and plant virus vector relies on the use of chemical insecticides. RNA-interference (RNAi) is a homology-dependent innate immune response in eukaryotes, including insects, which results in degradation of the corresponding transcript following its recognition by a double-stranded RNA (dsRNA) that shares 100% sequence homology. In this study, six whitefly 'gut' genes were selected from an in silico-annotated transcriptome library constructed from the whitefly alimentary canal or 'gut' of the B biotype of B. tabaci, and tested for knock down efficacy, post-ingestion of dsRNAs that share 100% sequence homology to each respective gene target. Candidate genes were: Acetylcholine receptor subunit α, Alpha glucosidase 1, Aquaporin 1, Heat shock protein 70, Trehalase1, and Trehalose transporter1. The efficacy of RNAi knock down was further tested in a gene-specific functional bioassay, and mortality was recorded in 24 hr intervals, six days, post-treatment. Based on qPCR analysis, all six genes tested showed significantly reduced gene expression. Moderate-to-high whitefly mortality was associated with the down-regulation of osmoregulation, sugar metabolism and sugar transport-associated genes, demonstrating that whitefly survivability was linked with RNAi results. Silenced Acetylcholine receptor subunit α and Heat shock protein 70 genes showed an initial low whitefly mortality, however, following insecticide or high temperature treatments, respectively, significantly increased knockdown efficacy and death was observed, indicating enhanced post-knockdown sensitivity perhaps related to systemic silencing. The oral delivery of gut-specific dsRNAs, when combined with qPCR analysis of gene expression and a corresponding gene-specific bioassay that relates knockdown and mortality, offers a viable approach for functional genomics analysis and the discovery of prospective dsRNA biopesticide targets. The approach can

  3. Positional bias of general and tissue-specific regulatory motifs in mouse gene promoters

    Directory of Open Access Journals (Sweden)

    Farré Domènec

    2007-12-01

    Full Text Available Abstract Background The arrangement of regulatory motifs in gene promoters, or promoter architecture, is the result of mutation and selection processes that have operated over many millions of years. In mammals, tissue-specific transcriptional regulation is related to the presence of specific protein-interacting DNA motifs in gene promoters. However, little is known about the relative location and spacing of these motifs. To fill this gap, we have performed a systematic search for motifs that show significant bias at specific promoter locations in a large collection of housekeeping and tissue-specific genes. Results We observe that promoters driving housekeeping gene expression are enriched in particular motifs with strong positional bias, such as YY1, which are of little relevance in promoters driving tissue-specific expression. We also identify a large number of motifs that show positional bias in genes expressed in a highly tissue-specific manner. They include well-known tissue-specific motifs, such as HNF1 and HNF4 motifs in liver, kidney and small intestine, or RFX motifs in testis, as well as many potentially novel regulatory motifs. Based on this analysis, we provide predictions for 559 tissue-specific motifs in mouse gene promoters. Conclusion The study shows that motif positional bias is an important feature of mammalian proximal promoters and that it affects both general and tissue-specific motifs. Motif positional constraints define very distinct promoter architectures depending on breadth of expression and type of tissue.

  4. Data in support of genome-wide identification of lineage-specific genes within Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Kun Zhou

    2015-09-01

    Full Text Available Two sets of LSGs were identified using BLAST: Caenorhabditis elegans species-specific genes (SSGs, 1423, and Caenorhabditis genus-specific genes (GSGs, 4539. The data contained in this article show SSGs and GSGs have significant differences in evolution and that most of them were formed by gene duplication and integration of transposable elements (TEs. Subsequent observation of temporal expression and protein function presents that many SSGs and GSGs are expressed and that genes involved with sex determination, specific stress, immune response, and morphogenesis are most represented. The data are related to research article “Genome-wide identification of lineage-specific genes within Caenorhabditis elegans” in Journal of Genomics [1].

  5. A comparison of reptilian and avian olfactory receptor gene repertoires: Species-specific expansion of group γ genes in birds

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    Kempenaers Bart

    2009-09-01

    Full Text Available Abstract Background The detection of odorants is mediated by olfactory receptors (ORs. ORs are G-protein coupled receptors that form a remarkably large protein superfamily in vertebrate genomes. We used data that became available through recent sequencing efforts of reptilian and avian genomes to identify the complete OR gene repertoires in a lizard, the green anole (Anolis carolinensis, and in two birds, the chicken (Gallus gallus and the zebra finch (Taeniopygia guttata. Results We identified 156 green anole OR genes, including 42 pseudogenes. The OR gene repertoire of the two bird species was substantially larger with 479 and 553 OR gene homologs in the chicken and zebra finch, respectively (including 111 and 221 pseudogenes, respectively. We show that the green anole has a higher fraction of intact OR genes (~72% compared with the chicken (~66% and the zebra finch (~38%. We identified a larger number and a substantially higher proportion of intact OR gene homologs in the chicken genome than previously reported (214 versus 82 genes and 66% versus 15%, respectively. Phylogenetic analysis showed that lizard and bird OR gene repertoires consist of group α, θ and γ genes. Interestingly, the vast majority of the avian OR genes are confined to a large expansion of a single branch (the so called γ-c clade. An analysis of the selective pressure on the paralogous genes of each γ-c clade revealed that they have been subjected to adaptive evolution. This expansion appears to be bird-specific and not sauropsid-specific, as it is lacking from the lizard genome. The γ-c expansions of the two birds do not intermix, i.e., they are lineage-specific. Almost all (group γ-c OR genes mapped to the unknown chromosome. The remaining OR genes mapped to six homologous chromosomes plus three to four additional chromosomes in the zebra finch and chicken. Conclusion We identified a surprisingly large number of potentially functional avian OR genes. Our data

  6. Global genome nucleotide excision repair is organized into domains that promote efficient DNA repair in chromatin

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    Yu, Shirong; Evans, Katie; Bennett, Mark; Webster, Richard M.; Leadbitter, Matthew; Teng, Yumin; Waters, Raymond

    2016-01-01

    The rates at which lesions are removed by DNA repair can vary widely throughout the genome, with important implications for genomic stability. To study this, we measured the distribution of nucleotide excision repair (NER) rates for UV-induced lesions throughout the budding yeast genome. By plotting these repair rates in relation to genes and their associated flanking sequences, we reveal that, in normal cells, genomic repair rates display a distinctive pattern, suggesting that DNA repair is highly organized within the genome. Furthermore, by comparing genome-wide DNA repair rates in wild-type cells and cells defective in the global genome–NER (GG-NER) subpathway, we establish how this alters the distribution of NER rates throughout the genome. We also examined the genomic locations of GG-NER factor binding to chromatin before and after UV irradiation, revealing that GG-NER is organized and initiated from specific genomic locations. At these sites, chromatin occupancy of the histone acetyl-transferase Gcn5 is controlled by the GG-NER complex, which regulates histone H3 acetylation and chromatin structure, thereby promoting efficient DNA repair of UV-induced lesions. Chromatin remodeling during the GG-NER process is therefore organized into these genomic domains. Importantly, loss of Gcn5 significantly alters the genomic distribution of NER rates; this has implications for the effects of chromatin modifiers on the distribution of mutations that arise throughout the genome. PMID:27470111

  7. Gene expression profiling in porcine mammary gland during lactation and identification of breed- and developmental-stage-specific genes

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    SU; Zhixi; DONG; Xinjiao; ZHANG; Bing; ZENG; Yanwu; FU; Yan; YU; Jun; HU; Songnian

    2006-01-01

    A total of 28941 ESTs were sequenced from five 5(-directed non-normalized cDNA libraries, which were assembled into 2212 contigs and 5642 singlets using CAP3. These sequences were annotated and clustered into 6857 unique genes, 2072 of which having no functional annotations were considered as novel genes. These genes were further classified into Gene Ontology categories. By comparing the expression profiles, we identified some breed- and developmental-stage-specific gene groups. These genes may be relative to reproductive performance or play important roles in milk synthesis, secretion and mammary involution. The unknown EST sequences and expression profiles at different developmental stages and breeds are very important resources for further research.

  8. Influence of DNA repair gene polymorphisms of hOGG1, XRCC1, XRCC3, ERCC2 and the folate metabolism gene MTHFR on chromosomal aberration frequencies.

    Science.gov (United States)

    Skjelbred, Camilla Furu; Svendsen, Marit; Haugan, Vera; Eek, Anette Kildal; Clausen, Kjell Oskar; Svendsen, Martin Veel; Hansteen, Inger-Lise

    2006-12-01

    We have studied the effect of genetic polymorphisms in the DNA repair genes hOGG1, XRCC1, XRCC3, ERCC2 and the MTHFR gene in the folate metabolism on the frequencies of cells with chromosomal aberrations (CA), chromosome-type aberrations (CSA), chromatid-type aberrations (CTA), chromatid breaks (CTB) and chromatid gaps (CTG) scored in peripheral blood lymphocytes from 651 Norwegian subjects of Caucasian descendant. DNA was extracted from fixed cell suspensions. The log-linear Poisson regression model was used for the combined data which included age, smoking, occupational exposure and genotype for 449 subjects. Our results suggest that individuals carrying the hOGG1 326Cys or the XRCC1 399Gln allele have an increased risk of chromosomal damage, while individuals carrying the XRCC1 194Trp or the ERCC2 751Gln allele have a reduced risk regardless of smoking habits and age. Individuals carrying the XRCC1 280His allele had an increased risk of CSA which was only apparent in non-smokers. This was independent of age. A protective effect of the XRCC3 241Met allele was only found in the older age group in non-smokers for CA, CSA and CTA, and in smokers for CSA. In the youngest age group, the opposite effect was found, with an increased risk for CA, CTA and CT