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Sample records for brca2-dependent homologous recombination

  1. Recombineering Homologous Recombination Constructs in Drosophila

    OpenAIRE

    Carreira-Rosario, Arnaldo; Scoggin, Shane; Shalaby, Nevine A.; Williams, Nathan David; Hiesinger, P. Robin; Buszczak, Michael

    2013-01-01

    The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineeri...

  2. Why do bacteria engage in homologous recombination?

    NARCIS (Netherlands)

    Vos, M.

    2009-01-01

    Microbiologists have long recognized that the uptake and incorporation of homologous DNA from outside the cell is a common feature of bacteria, with important implications for their evolution. However, the exact reasons why bacteria engage in homologous recombination remain elusive. This Opinion art

  3. Cell biology of homologous recombination in yeast

    DEFF Research Database (Denmark)

    Eckert-Boulet, Nadine Valerie; Rothstein, Rodney; Lisby, Michael

    2011-01-01

    Homologous recombination is an important pathway for error-free repair of DNA lesions, such as single- and double-strand breaks, and for rescue of collapsed replication forks. Here, we describe protocols for live cell imaging of single-lesion recombination events in the yeast Saccharomyces cerevi...

  4. Regulation of Homologous Recombination by SUMOylation

    DEFF Research Database (Denmark)

    Pinela da Silva, Sonia Cristina

    factors such as the homologous recombination (HR) machinery. HR constitutes the main DSB repair pathway in Saccharomyces cerevisiae and despite being largely considered an error-free process and essential for genome stability, uncontrolled recombination can lead to loss of heterozygosity, translocations....... In this study I present new insights for the role of SUMOylation in regulating HR by dissecting the role of SUMO in the interaction between the central HR-mediator protein Rad52 and its paralogue Rad59 and the outcome of recombination. This data provides evidence for the importance of SUMO in promoting protein......-protein interactions at the sites of repair, enabling effective Rad51-mediated recombination through the concerted action of the Rad52-Rad59 complex and the helicase Srs2. In addition, I also peer into the role of Rad52 SUMOylation in the context of persistent DSBs and telomere homeostasis. Furthermore, I characterize...

  5. Regulation of homologous recombination at telomeres in budding yeast

    DEFF Research Database (Denmark)

    Eckert-Boulet, Nadine; Lisby, Michael

    2010-01-01

    Homologous recombination is suppressed at normal length telomere sequences. In contrast, telomere recombination is allowed when telomeres erode in the absence of telomerase activity or as a consequence of nucleolytic degradation or incomplete replication. Here, we review the mechanisms...... that contribute to regulating mitotic homologous recombination at telomeres and the role of these mechanisms in signalling short telomeres in the budding yeast Saccharomyces cerevisiae....

  6. Homologous recombination: from model organisms to human disease

    NARCIS (Netherlands)

    M. Modesti (Mauro); R. Kanaar (Roland)

    2001-01-01

    textabstractRecent experiments show that properly controlled recombination between homologous DNA molecules is essential for the maintenance of genome stability and for the prevention of tumorigenesis.

  7. Productive homologous and non-homologous recombination of hepatitis C virus in cell culture.

    Directory of Open Access Journals (Sweden)

    Troels K H Scheel

    2013-03-01

    Full Text Available Genetic recombination is an important mechanism for increasing diversity of RNA viruses, and constitutes a viral escape mechanism to host immune responses and to treatment with antiviral compounds. Although rare, epidemiologically important hepatitis C virus (HCV recombinants have been reported. In addition, recombination is an important regulatory mechanism of cytopathogenicity for the related pestiviruses. Here we describe recombination of HCV RNA in cell culture leading to production of infectious virus. Initially, hepatoma cells were co-transfected with a replicating JFH1ΔE1E2 genome (genotype 2a lacking functional envelope genes and strain J6 (2a, which has functional envelope genes but does not replicate in culture. After an initial decrease in the number of HCV positive cells, infection spread after 13-36 days. Sequencing of recovered viruses revealed non-homologous recombinants with J6 sequence from the 5' end to the NS2-NS3 region followed by JFH1 sequence from Core to the 3' end. These recombinants carried duplicated sequence of up to 2400 nucleotides. HCV replication was not required for recombination, as recombinants were observed in most experiments even when two replication incompetent genomes were co-transfected. Reverse genetic studies verified the viability of representative recombinants. After serial passage, subsequent recombination events reducing or eliminating the duplicated region were observed for some but not all recombinants. Furthermore, we found that inter-genotypic recombination could occur, but at a lower frequency than intra-genotypic recombination. Productive recombination of attenuated HCV genomes depended on expression of all HCV proteins and tolerated duplicated sequence. In general, no strong site specificity was observed. Non-homologous recombination was observed in most cases, while few homologous events were identified. A better understanding of HCV recombination could help identification of natural

  8. Phenylbutyrate inhibits homologous recombination induced by camptothecin and methyl methanesulfonate

    DEFF Research Database (Denmark)

    Kaiser, Gitte Schalck; Germann, Susanne Manuela; Westergaard, Tine;

    2011-01-01

    Homologous recombination is accompanied by extensive changes to chromatin organization at the site of DNA damage. Some of these changes are mediated through acetylation/deacetylation of histones. Here, we show that recombinational repair of DNA damage induced by the anti-cancer drug camptothecin...

  9. Productive homologous and non-homologous recombination of hepatitis C virus in cell culture

    DEFF Research Database (Denmark)

    Scheel, Troels K H; Galli, Andrea; Li, Yi-Ping;

    2013-01-01

    . In addition, recombination is an important regulatory mechanism of cytopathogenicity for the related pestiviruses. Here we describe recombination of HCV RNA in cell culture leading to production of infectious virus. Initially, hepatoma cells were co-transfected with a replicating JFH1ΔE1E2 genome (genotype 2a...... incompetent genomes were co-transfected. Reverse genetic studies verified the viability of representative recombinants. After serial passage, subsequent recombination events reducing or eliminating the duplicated region were observed for some but not all recombinants. Furthermore, we found that inter......-genotypic recombination could occur, but at a lower frequency than intra-genotypic recombination. Productive recombination of attenuated HCV genomes depended on expression of all HCV proteins and tolerated duplicated sequence. In general, no strong site specificity was observed. Non-homologous recombination was observed...

  10. Evidence for homologous recombination in Chikungunya Virus.

    Science.gov (United States)

    Casal, Pablo E; Chouhy, Diego; Bolatti, Elisa M; Perez, Germán R; Stella, Emma J; Giri, Adriana A

    2015-04-01

    Chikungunya Virus (CHIKV), a mosquito-transmitted alphavirus, causes acute fever and joint pain in humans. Recently, endemic CHIKV infection outbreaks have jeopardized public health in wider geographical regions. Here, we analyze the phylogenetic associations of CHIKV and explore the potential recombination events on 152 genomic isolates deposited in GenBank database. The CHIKV genotypes [West African, Asian, East/Central/South African (ECSA)], and a clear division of ECSA clade into three sub-groups (I-II-III), were defined by Bayesian analysis; similar results were obtained using E1 gene sequences. A nucleotide identity-based approach is provided to facilitate CHIKV classification within ECSA clade. Using seven methods to detect recombination, we found a statistically significant event (p-values range: 1.14×10(-7)-4.45×10(-24)) located within the nsP3 coding region. This finding was further confirmed by phylogenetic networks (PHI Test, p=0.004) and phylogenetic tree incongruence analysis. The recombinant strain, KJ679578/India/2011 (ECSA III), derives from viruses of ECSA III and ECSA I. Our study demonstrates that recombination is an additional mechanism of genetic diversity in CHIKV that might assist in the cross-species transmission process.

  11. Phylogeny and Homologous Recombination in Japanese Encephalitis Viruses

    Institute of Scientific and Technical Information of China (English)

    Li Xiao-xue; Cong Ying-ying; Wang Xin; Ren Yu-dong; Ren Xiao-feng; Lu Ai-guo; Li Guang-xing

    2015-01-01

    Japanese encephalitis virus (JEV) is a significant causative agent of arthropod-borne encephalitis and what is less clear that the factors cause the virus wide spread. The objective was to confirm whether the homologous recombination imposed on JEV. The phylogenetic and homologous recombination analyses were performed based on 163 complete JEV genomes which were recently isolated. They were still separated into five genotypes (GI-GV) and the most of recently isolated JEVs were GI rather than GIII in Asian areas including mainland China. Two recombinant events were identified in JEV and the evidence of the recombination was observed between China and Japan isolates that partitioned into two distinct subclades, but still the same genotype (GIII). Our data further suggested that most of the nucleotides in JEV genome were under negative selection; however, changes within codon 2 316 (amino acid NS4b-44) showed an evidence of the positive selection.

  12. Amifostine Metabolite WR-1065 Disrupts Homologous Recombination in Mammalian Cells

    OpenAIRE

    Dziegielewski, Jaroslaw; Goetz, Wilfried; Murley, Jeffrey S.; David J Grdina; Morgan, William F.; Janet E. Baulch

    2010-01-01

    Repair of DNA damage through homologous recombination (HR) pathways plays a crucial role in maintaining genome stability. However, overstimulation of HR pathways in response to genotoxic stress may abnormally elevate recombination frequencies, leading to increased mutation rates and delayed genomic instability. Radiation-induced genomic instability has been detected after exposure to both low- and high-linear energy transfer (LET) radiations, but the mechanisms responsible for initiating or p...

  13. Homologous recombination in DNA repair and DNA damage tolerance

    Institute of Scientific and Technical Information of China (English)

    Xuan Li; Wolf-Dietrich Heyer

    2008-01-01

    Homologous recombination (HR) comprises a series of interrelated pathways that function in the repair of DNA double-stranded breaks (DSBs) and interstrand crosslinks (ICLs). In addition, recombination provides critical sup-port for DNA replication in the recovery of stalled or broken replication forks, contributing to tolerance of DNA damage. A central core of proteins, most critically the RecA homolog Rad51, catalyzes the key reactions that typify HR: homology search and DNA strand invasion. The diverse functions of recombination are reflected in the need for context-specific factors that perform supplemental functions in conjunction with the core proteins. The inability to properly repair complex DNA damage and resolve DNA replication stress leads to genomic instability and contributes to cancer etiology. Mutations in the BRCA2 recombination gene cause predisposition to breast and ovarian cancer as well as Fanconi anemia, a cancer predisposition syndrome characterized by a defect in the repair of DNA interstrand crosslinks. The cellular functions of recombination are also germane to DNA-based treatment modaUties of cancer, which target replicating cells by the direct or indirect induction of DNA lesions that are substrates for recombination pathways. This review focuses on mechanistic aspects of HR relating to DSB and ICL repair as well as replication fork support.

  14. A PHF8 homolog in C. elegans promotes DNA repair via homologous recombination.

    Directory of Open Access Journals (Sweden)

    Changrim Lee

    Full Text Available PHF8 is a JmjC domain-containing histone demethylase, defects in which are associated with X-linked mental retardation. In this study, we examined the roles of two PHF8 homologs, JMJD-1.1 and JMJD-1.2, in the model organism C. elegans in response to DNA damage. A deletion mutation in either of the genes led to hypersensitivity to interstrand DNA crosslinks (ICLs, while only mutation of jmjd-1.1 resulted in hypersensitivity to double-strand DNA breaks (DSBs. In response to ICLs, JMJD-1.1 did not affect the focus formation of FCD-2, a homolog of FANCD2, a key protein in the Fanconi anemia pathway. However, the dynamic behavior of RPA-1 and RAD-51 was affected by the mutation: the accumulations of both proteins at ICLs appeared normal, but their subsequent disappearance was retarded, suggesting that later steps of homologous recombination were defective. Similar changes in the dynamic behavior of RPA-1 and RAD-51 were seen in response to DSBs, supporting a role of JMJD-1.1 in homologous recombination. Such a role was also supported by our finding that the hypersensitivity of jmjd-1.1 worms to ICLs was rescued by knockdown of lig-4, a homolog of Ligase 4 active in nonhomologous end-joining. The hypersensitivity of jmjd-1.1 worms to ICLs was increased by rad-54 knockdown, suggesting that JMJD-1.1 acts in parallel with RAD-54 in modulating chromatin structure. Indeed, the level of histone H3 Lys9 tri-methylation, a marker of heterochromatin, was higher in jmjd-1.1 cells than in wild-type cells. We conclude that the histone demethylase JMJD-1.1 influences homologous recombination either by relaxing heterochromatin structure or by indirectly regulating the expression of multiple genes affecting DNA repair.

  15. The many facets of homologous recombination at telomeres

    OpenAIRE

    Clémence Claussin; Michael Chang

    2015-01-01

    The ends of linear chromosomes are capped by nucleoprotein structures called telomeres. A dysfunctional telomere may resemble a DNA double-strand break (DSB), which is a severe form of DNA damage. The presence of one DSB is sufficient to drive cell cycle arrest and cell death. Therefore cells have evolved mechanisms to repair DSBs such as homologous recombination (HR). HR-mediated repair of telomeres can lead to genome instability, a hallmark of cancer cells, wh...

  16. The many facets of homologous recombination at telomeres

    Directory of Open Access Journals (Sweden)

    Clémence Claussin

    2015-07-01

    Full Text Available The ends of linear chromosomes are capped by nucleoprotein structures called telomeres. A dysfunctional telomere may resemble a DNA double-strand break (DSB, which is a severe form of DNA damage. The presence of one DSB is sufficient to drive cell cycle arrest and cell death. Therefore cells have evolved mechanisms to repair DSBs such as homologous recombination (HR. HR-mediated repair of telomeres can lead to genome instability, a hallmark of cancer cells, which is why such repair is normally inhibited. However, some HR-mediated processes are required for proper telomere function. The need for some recombination activities at telomeres but not others necessitates careful and complex regulation, defects in which can lead to catastrophic consequences. Furthermore, some cell types can maintain telomeres via telomerase-independent, recombination-mediated mechanisms. In humans, these mechanisms are called alternative lengthening of telomeres (ALT and are used in a subset of human cancer cells. In this review, we summarize the different recombination activities occurring at telomeres and discuss how they are regulated. Much of the current knowledge is derived from work using yeast models, which is the focus of this review, but relevant studies in mammals are also included.

  17. Generation of helper-dependent adenoviral vectors by homologous recombination.

    Science.gov (United States)

    Toietta, Gabriele; Pastore, Lucio; Cerullo, Vincenzo; Finegold, Milton; Beaudet, Arthur L; Lee, Brendan

    2002-02-01

    Helper-dependent adenoviral vectors (HD-Ad) represent a potentially valuable tool for safe and prolonged gene expression in vivo. The current approach for generating these vectors is based on ligation of the expression cassette into large plasmids containing the viral inverted terminal repeats flanking "stuffer" DNA to maintain a final size above the lower limit for efficient packaging into the adenovirus capsid (approximately 28 kb). The ligation to produce the viral plasmid is generally very inefficient. Similar problems in producing first-generation adenoviral (FG-Ad) vectors were circumvented with the development of a system taking advantage of efficient homologous recombination between a shuttle plasmid containing the expression cassette and a FG-Ad vector backbone in the Escherichia coli strain BJ5183. Here we describe a method for fast and efficient generation of HD-Ad vector plasmids that can accommodate expression cassettes of any size up to 35 kb. To validate the system, we generated a HD-Ad vector expressing the fusion protein between beta-galactosidase and neomycin resistance genes under the control of the SR alpha promoter, and one expressing the enhanced green fluorescent protein under the control of the cytomegalovirus promoter. The viruses were rescued and tested in vitro and for in vivo expression in mice. The data collected indicate the possibility for achieving a high level of hepatocyte transduction using HD-Ad vectors derived from plasmids obtained by homologous recombination in E. coli, with no significant alteration of liver enzymes and a less severe, transient thrombocytopenia in comparison with previous reports with similar doses of a FG-Ad vector. PMID:11829528

  18. Gimeracil sensitizes cells to radiation via inhibition of homologous recombination

    International Nuclear Information System (INIS)

    Background and purpose: 5-Chloro-2,4-dihydroxypyridine (Gimeracil) is a component of an oral fluoropyrimidine derivative S-1. Gimeracil is originally added to S-1 to yield prolonged 5-FU concentrations in tumor tissues by inhibiting dihydropyrimidine dehydrogenase, which degrades 5-FU. We found that Gimeracil by itself had the radiosensitizing effect. Methods and materials: We used various cell lines deficient in non-homologous end-joining (NHEJ) or homologous recombination (HR) as well as DLD-1 and HeLa in clonogenic assay. γ-H2AX focus formation and SCneo assay was performed to examine the effects of Gimeracil on DNA double strand break (DSB) repair mechanisms. Results: Results of γ-H2AX focus assay indicated that Gimeracil inhibited DNA DSB repair. It did not sensitize cells deficient in HR but sensitized those deficient in NHEJ. In SCneo assay, Gimeracil reduced the frequency of neo-positive clones. Additionally, it sensitized the cells in S-phase more than in G0/G1. Conclusions: Gimeracil inhibits HR. Because HR plays key roles in the repair of DSBH caused by radiotherapy, Gimeracil may enhance the efficacy of radiotherapy through the suppression of HR-mediated DNA repair pathways.

  19. [Enzymatic control of homologous recombination in Escherichia coli cells and hyper-recombination].

    Science.gov (United States)

    Bakhlanova, I V; Dudkina, A V; Baĭtin, D M

    2013-01-01

    The RecA protein is a major enzyme of homologous recombination in bacterial cell. Forming a right-handed helical filament on ssDNA, it provides a homology search between two DNA molecules and homologous strand exchange. The RecA protein not only defends the cell from exposure to ionizing radiation and UV-irradiation, but also ensures the recombination process in the course of normal cell growth. A number of wild-type or mutant RecA proteins demonstrate increased recombinogenic properties in vitro and in vivo as compared with the wild-type RecA protein from Escherichia coli, which leads to hyper-recombination. The hyper-rec activity of RecA proteins during the recombination process in many depends on the filamentation dynamics on ssDNA and DNA-transferase properties. Changes in filamentation and DNA-transferase abilities of RecA protein may be the result of not only specific amino-acid substitutions, but also the functioning of the cell enzymatic apparatus, including such proteins as RecO, RecR, RecF, RecX, DinI, SSB, PsiB. To date, the function of each of these proteins is identified at the molecular level. However, the role of some of them in the cell metabolism remains to be seen. Increase in recombination in vivo is not always useful for a cell and faces various limitations. Moreover, in the bacterial cell some mechanisms are activated, that cause genomic reorganization, directed to suppress the expression of hyper-active RecA protein. The ways of hyper-active RecA protein regulation are very interesting, and they are studied in different model systems. PMID:23808153

  20. Transcription-coupled homologous recombination after oxidative damage.

    Science.gov (United States)

    Wei, Leizhen; Levine, Arthur Samuel; Lan, Li

    2016-08-01

    Oxidative DNA damage induces genomic instability and may lead to mutagenesis and carcinogenesis. As severe blockades to RNA polymerase II (RNA POLII) during transcription, oxidative DNA damage and the associated DNA strand breaks have a profoundly deleterious impact on cell survival. To protect the integrity of coding regions, high fidelity DNA repair at a transcriptionally active site in non-dividing somatic cells, (i.e., terminally differentiated and quiescent/G0 cells) is necessary to maintain the sequence integrity of transcribed regions. Recent studies indicate that an RNA-templated, transcription-associated recombination mechanism is important to protect coding regions from DNA damage-induced genomic instability. Here, we describe the discovery that G1/G0 cells exhibit Cockayne syndrome (CS) B (CSB)-dependent assembly of homologous recombination (HR) factors at double strand break (DSB) sites within actively transcribed regions. This discovery is a challenge to the current dogma that HR occurs only in S/G2 cells where undamaged sister chromatids are available as donor templates. PMID:27233112

  1. Homologous Recombination as a Replication Fork Escort: Fork-Protection and Recovery

    Directory of Open Access Journals (Sweden)

    Audrey Costes

    2012-12-01

    Full Text Available Homologous recombination is a universal mechanism that allows DNA repair and ensures the efficiency of DNA replication. The substrate initiating the process of homologous recombination is a single-stranded DNA that promotes a strand exchange reaction resulting in a genetic exchange that promotes genetic diversity and DNA repair. The molecular mechanisms by which homologous recombination repairs a double-strand break have been extensively studied and are now well characterized. However, the mechanisms by which homologous recombination contribute to DNA replication in eukaryotes remains poorly understood. Studies in bacteria have identified multiple roles for the machinery of homologous recombination at replication forks. Here, we review our understanding of the molecular pathways involving the homologous recombination machinery to support the robustness of DNA replication. In addition to its role in fork-recovery and in rebuilding a functional replication fork apparatus, homologous recombination may also act as a fork-protection mechanism. We discuss that some of the fork-escort functions of homologous recombination might be achieved by loading of the recombination machinery at inactivated forks without a need for a strand exchange step; as well as the consequence of such a model for the stability of eukaryotic genomes.

  2. Homologous recombination-mediated cloning and manipulation of genomic DNA regions using Gateway and recombineering systems

    Directory of Open Access Journals (Sweden)

    Kagale Sateesh

    2008-11-01

    Full Text Available Abstract Background Employing genomic DNA clones to characterise gene attributes has several advantages over the use of cDNA clones, including the presence of native transcription and translation regulatory sequences as well as a representation of the complete repertoire of potential splice variants encoded by the gene. However, working with genomic DNA clones has traditionally been tedious due to their large size relative to cDNA clones and the presence, absence or position of particular restriction enzyme sites that may complicate conventional in vitro cloning procedures. Results To enable efficient cloning and manipulation of genomic DNA fragments for the purposes of gene expression and reporter-gene studies we have combined aspects of the Gateway system and a bacteriophage-based homologous recombination (i.e. recombineering system. To apply the method for characterising plant genes we developed novel Gateway and plant transformation vectors that are of small size and incorporate selectable markers which enable efficient identification of recombinant clones. We demonstrate that the genomic coding region of a gene can be directly cloned into a Gateway Entry vector by recombineering enabling its subsequent transfer to Gateway Expression vectors. We also demonstrate how the coding and regulatory regions of a gene can be directly cloned into a plant transformation vector by recombineering. This construct was then rapidly converted into a novel Gateway Expression vector incorporating cognate 5' and 3' regulatory regions by using recombineering to replace the intervening coding region with the Gateway Destination cassette. Such expression vectors can be applied to characterise gene regulatory regions through development of reporter-gene fusions, using the Gateway Entry clones of GUS and GFP described here, or for ectopic expression of a coding region cloned into a Gateway Entry vector. We exemplify the utility of this approach with the Arabidopsis

  3. Retroviral vectors for homologous recombination provide efficient cloning and expression in mammalian cells.

    Science.gov (United States)

    Kobayashi, Eiji; Kishi, Hiroyuki; Ozawa, Tatsuhiko; Horii, Masae; Hamana, Hiroshi; Nagai, Terumi; Muraguchi, Atsushi

    2014-02-14

    Homologous recombination technologies enable high-throughput cloning and the seamless insertion of any DNA fragment into expression vectors. Additionally, retroviral vectors offer a fast and efficient method for transducing and expressing genes in mammalian cells, including lymphocytes. However, homologous recombination cannot be used to insert DNA fragments into retroviral vectors; retroviral vectors contain two homologous regions, the 5'- and 3'-long terminal repeats, between which homologous recombination occurs preferentially. In this study, we have modified a retroviral vector to enable the cloning of DNA fragments through homologous recombination. To this end, we inserted a bacterial selection marker in a region adjacent to the gene insertion site. We used the modified retroviral vector and homologous recombination to clone T-cell receptors (TCRs) from single Epstein Barr virus-specific human T cells in a high-throughput and comprehensive manner and to efficiently evaluate their function by transducing the TCRs into a murine T-cell line through retroviral infection. In conclusion, the modified retroviral vectors, in combination with the homologous recombination method, are powerful tools for the high-throughput cloning of cDNAs and their efficient functional analysis. PMID:24462869

  4. Assembly and dynamics of the bacteriophage T4 homologous recombination machinery

    Directory of Open Access Journals (Sweden)

    Morrical Scott W

    2010-12-01

    Full Text Available Abstract Homologous recombination (HR, a process involving the physical exchange of strands between homologous or nearly homologous DNA molecules, is critical for maintaining the genetic diversity and genome stability of species. Bacteriophage T4 is one of the classic systems for studies of homologous recombination. T4 uses HR for high-frequency genetic exchanges, for homology-directed DNA repair (HDR processes including DNA double-strand break repair, and for the initiation of DNA replication (RDR. T4 recombination proteins are expressed at high levels during T4 infection in E. coli, and share strong sequence, structural, and/or functional conservation with their counterparts in cellular organisms. Biochemical studies of T4 recombination have provided key insights on DNA strand exchange mechanisms, on the structure and function of recombination proteins, and on the coordination of recombination and DNA synthesis activities during RDR and HDR. Recent years have seen the development of detailed biochemical models for the assembly and dynamics of presynaptic filaments in the T4 recombination system, for the atomic structure of T4 UvsX recombinase, and for the roles of DNA helicases in T4 recombination. The goal of this chapter is to review these recent advances and their implications for HR and HDR mechanisms in all organisms.

  5. Optimal Cloning of PCR Fragments by Homologous Recombination in Escherichia coli

    OpenAIRE

    Jacobus, Ana Paula; Gross, Jeferson

    2015-01-01

    PCR fragments and linear vectors containing overlapping ends are easily assembled into a propagative plasmid by homologous recombination in Escherichia coli. Although this gap-repair cloning approach is straightforward, its existence is virtually unknown to most molecular biologists. To popularize this method, we tested critical parameters influencing the efficiency of PCR fragments cloning into PCR-amplified vectors by homologous recombination in the widely used E. coli strain DH5α. We found...

  6. Gene Disruption by Homologous Recombination in the Xylella fastidiosa Citrus Variegated Chlorosis Strain

    Science.gov (United States)

    Gaurivaud, Patrice; Souza, Leonardo C. A.; Virgílio, Andrea C. D.; Mariano, Anelise G.; Palma, Renê R.; Monteiro, Patrícia B.

    2002-01-01

    Mutagenesis by homologous recombination was evaluated in Xylella fastidiosa by using the bga gene, coding for β-galactosidase, as a model. Integration of replicative plasmids by homologous recombination between the cloned truncated copy of bga and the endogenous gene was produced by one or two crossover events leading to β-galactosidase mutants. A promoterless chloramphenicol acetyltransferase gene was used to monitor the expression of the target gene and to select a cvaB mutant. PMID:12200328

  7. Transformation-associated recombination between diverged and homologous DNA repeats is induced by strand breaks

    Energy Technology Data Exchange (ETDEWEB)

    Larionov, V.; Kouprina, N. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States)]|[Institute of Cytology, St. Petersburg, (Russian Federation); Edlarov, M. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States)]|[Center of Bioengineering, Moscow, (Russian Federation); Perkins, E.; Porter, G.; Resnick, M.A. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States)

    1993-12-31

    Rearrangement and deletion within plasmid DNA is commonly observed during transformation. We have examined the mechanisms of transformation-associated recombination in the yeast Saccharomyces cerevisiae using a plasmid system which allowed the effects of physical state and/or extent of homology on recombination to be studied. The plasmid contains homologous or diverged (19%) DNA repeats separated by a genetically detectable color marker. Recombination during transformation for covalently closed circular plasmids was over 100-fold more frequent than during mitotic growth. The frequency of recombination is partly dependent on the method of transformation in that procedures involving lithium acetate or spheroplasting yield higher frequencies than electroporation. When present in the repeats, unique single-strand breaks that are ligatable, as well as double-strand breaks, lead to high levels of recombination between diverged and identical repeats. The transformation-associated recombination between repeat DNA`s is under the influence of the RADS2, RADI and the RNCI genes,

  8. Mutants of Streptomyces roseosporus that express enhanced recombination within partially homologous genes.

    Science.gov (United States)

    Hosted, T J; Baltz, R H

    1996-10-01

    Streptomyces roseosporus mutants that express enhanced recombination between partially homologous (homeologous) sequences were isolated by selection for recombination between the bacteriophage phi C31 derivative KC570 containing the Streptomyces coelicolor glucose kinase (glk) gene and the S. roseosporus chromosome. The frequencies of homeologous recombination in the ehr mutants were determined by measuring the chromosomal insertion frequencies of plasmids containing S. coelicolor glnA or whiG genes. S. roseosporus ehr mutants showed 10(2)- to 10(4)-fold increases in homeologous recombination relative to Ehr+ strains, but no increase in homologous recombination. Southern hybridization analysis revealed single unique sites for the insertion of each of the plasmids, and the crossovers occurred in frame and in proper translational register, yielding functional chimeric glnA and whiG genes.

  9. Meiotic recombination at the Lmp2 hotspot tolerates minor sequence divergence between homologous chromosomes

    Energy Technology Data Exchange (ETDEWEB)

    Yoshino, Masayasu; Sagai, Tomoko; Shiroishi, Toshihiko [National Institute of Genetics, Mishima (Japan)] [and others

    1996-06-01

    Recombination is widely considered to linearly depend on the length of the homologous sequences. An 11% mismatch decreases the rate of phage-plasmid recombination 240-fold. Two single nucleotide mismatches, which reduce the longest uninterrupted stretch of similarity from 232 base pairs (bp) to 134 bp, reduce gene conversion in mouse L cells 20-fold. The efficiency of gene targeting through homologous recombination in mouse embryonic stem cells can be increased by using an isogenic, rather than a non-isogenic, DNA construct. In this study we asked whether a high degree of sequence identity between homologous mouse chromosomes enhances meiotic recombination at a hotspot. Sites of meiotic recombination in the mouse major histocompatibility complex (MHC) class II region are not randomly distributed but are almost all clustered within short segments known as recombinational hotspots. The wm7 MHC haplotype, derived from Japanese wild mice Mus musculus molossinus, enhances meiotic recombination at a hotspot near the Lmp2 gene. Heterozygotes between the wm7 haplotype and the b or k haplotypes have yielded a high frequency of recombination (2.1%) in 1.3 kilobase kb segment of this hotspot. 20 refs., 2 figs.

  10. An Improved Homologous Recombination Method for Rapid Cloning of the Antibody Heavy Chain Gene and Its Comparison with the Homologous Recombination and Traditional Cloning Methods

    OpenAIRE

    Masoumeh Hajirezaei; Mojtaba Darbouy; Manoochehr Rasouli; Bahram Kazemi

    2015-01-01

    Background: The homologous recombination (HR) is one of the conventional cloning methods for the production of recombinant DNA. It is a quick method for in vivo DNA cloning without using the high cost restriction enzymes. A few modifications in the cloning procedure can increase the efficiency of this method.Materials and Methods: In this study, effect of heating on the rate of the IgG1 heavy chain gene cloning was investigated in the HR method and then it was compared with HR method without ...

  11. A recurrent translocation is mediated by homologous recombination between HERV-H elements

    Directory of Open Access Journals (Sweden)

    Hermetz Karen E

    2012-01-01

    Full Text Available Abstract Background Chromosome rearrangements are caused by many mutational mechanisms; of these, recurrent rearrangements can be particularly informative for teasing apart DNA sequence-specific factors. Some recurrent translocations are mediated by homologous recombination between large blocks of segmental duplications on different chromosomes. Here we describe a recurrent unbalanced translocation casued by recombination between shorter homologous regions on chromosomes 4 and 18 in two unrelated children with intellectual disability. Results Array CGH resolved the breakpoints of the 6.97-Megabase (Mb loss of 18q and the 7.30-Mb gain of 4q. Sequencing across the translocation breakpoints revealed that both translocations occurred between 92%-identical human endogenous retrovirus (HERV elements in the same orientation on chromosomes 4 and 18. In addition, we find sequence variation in the chromosome 4 HERV that makes one allele more like the chromosome 18 HERV. Conclusions Homologous recombination between HERVs on the same chromosome is known to cause chromosome deletions, but this is the first report of interchromosomal HERV-HERV recombination leading to a translocation. It is possible that normal sequence variation in substrates of non-allelic homologous recombination (NAHR affects the alignment of recombining segments and influences the propensity to chromosome rearrangement.

  12. Manipulation of Homologous and Homoeologous Chromosome Recombination in Wheat.

    Science.gov (United States)

    Lukaszewski, Adam J

    2016-01-01

    Given the sizes of the three genomes in wheat (A, B, and D) and a limited number of chiasmata formed in meiosis, recombination by crossing-over is a very rare event. It is also restricted to very similar homologues; the pairing homoeologous (Ph) system of wheat prevents differentiated chromosomes from pairing and crossing-over. This chapter presents an overview and describes several systems by which the frequency or density of crossing-over can be increased, both in homologues and homoeologues. It also presents the standard system of E.R. Sears for engineering alien chromosome transfers into wheat. PMID:27511168

  13. A universal cloning method based on yeast homologous recombination that is simple, efficient, and versatile

    OpenAIRE

    Joska, Tammy M.; Mashruwala, Ameya; Boyd, Jeffrey M.; Belden, William J.

    2014-01-01

    Cloning by homologous recombination (HR) in Saccharomyces cerevisiae is an extremely efficient and cost-effective alternative to other methods of recombinant DNA technologies. Unfortunately, it is incompatible with all the various specialized plasmids currently used in microbiology and biomedical research laboratories, and is therefore, not widely adopted. In an effort to dramatically improve the versatility of yeast gap-repair cloning and make it compatible with any DNA plasmid, we demonstra...

  14. Modification of human beta-globin locus PAC clones by homologous recombination in Escherichia coli

    NARCIS (Netherlands)

    G.P. Patrinos (George); M. de Krom (Mariken); S. Bottardi; R.J. Janssens; E. Katsantoni (Eleni); A.W. Wai; D.J. Sherratt; F.G. Grosveld (Frank); A.M.A. Imam (Ali)

    2000-01-01

    textabstractWe report here modifications of human beta-globin PAC clones by homologous recombination in Escherichia coli DH10B, utilising a plasmid temperature sensitive for replication, the recA gene and a wild-type copy of the rpsL gene which allows for an efficient selection for

  15. Ago2 facilitates Rad51 recruitment and DNA double-strand break repair by homologous recombination

    DEFF Research Database (Denmark)

    Gao, Min; Wei, Wei; Li, Ming Hua;

    2014-01-01

    (Ago) proteins and play an important role in DSB repair, though the mechanism through which they act remains unclear. Here, we report that the role of diRNAs in DSB repair is restricted to repair by homologous recombination (HR) and that it specifically relies on the effector protein Ago2 in mammalian...... to facilitate repair by HR....

  16. Ecological genomics in Xanthomonas: the nature of genetic adaptation with homologous recombination and host shifts

    KAUST Repository

    Huang, Chao-Li

    2015-03-15

    Background: Comparative genomics provides insights into the diversification of bacterial species. Bacterial speciation usually takes place with lasting homologous recombination, which not only acts as a cohering force between diverging lineages but brings advantageous alleles favored by natural selection, and results in ecologically distinct species, e.g., frequent host shift in Xanthomonas pathogenic to various plants. Results: Using whole-genome sequences, we examined the genetic divergence in Xanthomonas campestris that infected Brassicaceae, and X. citri, pathogenic to a wider host range. Genetic differentiation between two incipient races of X. citri pv. mangiferaeindicae was attributable to a DNA fragment introduced by phages. In contrast to most portions of the genome that had nearly equivalent levels of genetic divergence between subspecies as a result of the accumulation of point mutations, 10% of the core genome involving with homologous recombination contributed to the diversification in Xanthomonas, as revealed by the correlation between homologous recombination and genomic divergence. Interestingly, 179 genes were under positive selection; 98 (54.7%) of these genes were involved in homologous recombination, indicating that foreign genetic fragments may have caused the adaptive diversification, especially in lineages with nutritional transitions. Homologous recombination may have provided genetic materials for the natural selection, and host shifts likely triggered ecological adaptation in Xanthomonas. To a certain extent, we observed positive selection nevertheless contributed to ecological divergence beyond host shifting. Conclusion: Altogether, mediated with lasting gene flow, species formation in Xanthomonas was likely governed by natural selection that played a key role in helping the deviating populations to explore novel niches (hosts) or respond to environmental cues, subsequently triggering species diversification. © Huang et al.

  17. The role of Deinococcus radiodurans RecFOR proteins in homologous recombination.

    Science.gov (United States)

    Satoh, Katsuya; Kikuchi, Masahiro; Ishaque, Abu M; Ohba, Hirofumi; Yamada, Mitsugu; Tejima, Kouhei; Onodera, Takefumi; Narumi, Issay

    2012-04-01

    Deinococcus radiodurans exhibits extraordinary resistance to the lethal effect of DNA-damaging agents, a characteristic attributed to its highly proficient DNA repair capacity. Although the D. radiodurans genome is clearly devoid of recBC and addAB counterparts as RecA mediators, the genome possesses all genes associated with the RecFOR pathway. In an effort to gain insights into the role of D. radiodurans RecFOR proteins in homologous recombination, we generated recF, recO and recR disruptant strains and characterized the disruption effects. All the disruptant strains exhibited delayed growth relative to the wild-type, indicating that the RecF, RecO and RecR proteins play an important role in cell growth under normal growth conditions. A slight reduction in transformation efficiency was observed in the recF and recO disruptant strains compared to the wild-type strain. Interestingly, disruption of recR resulted in severe reduction of the transformation efficiency. On the other hand, the recF disruptant strain was the most sensitive phenotype to γ rays, UV irradiation and mitomycin C among the three disruptants. In the recF disruptant strain, the intracellular level of the LexA1 protein did not decrease following γ irradiation, suggesting that a large amount of the RecA protein remains inactive despite being induced. These results demonstrate that the RecF protein plays a crucial role in the homologous recombination repair process by facilitating RecA activation in D. radiodurans. Thus, the RecF and RecR proteins are involved in the RecA activation and the stability of incoming DNA, respectively, during RecA-mediated homologous recombination processes that initiated the ESDSA pathway in D. radiodurans. Possible mechanisms that involve the RecFOR complex in homologous intermolecular recombination and homologous recombination repair processes are also discussed. PMID:22321371

  18. Schizosaccharomyces pombe Mms1 channels repair of perturbed replication into Rhp51 independent homologous recombination

    DEFF Research Database (Denmark)

    Vejrup-Hansen, Rasmus; Mizuno, Ken'Ichi; Miyabe, Izumi;

    2011-01-01

    In both Schizosaccharomyces pombe and Saccharomyces cerevisiae, Mms22 and Mms1 form a complex with important functions in the response to DNA damage, loss of which leads to perturbations during replication. Furthermore, in S. cerevisiae, Mms1 has been suggested to function in concert with a Cullin......-like protein, Rtt101/Cul8, a potential paralog of Cullin 4. We performed epistasis analysis between ¿mms1 and mutants of pathways with known functions in genome integrity, and measured the recruitment of homologous recombination proteins to blocked replication forks and recombination frequencies. We show that...... particularly important when a single strand break is converted into a double strand break during replication. Genetic data connect Mms1 to a Mus81 and Rad22(Rad52) dependent, but Rhp51 independent, branch of homologous recombination. This is supported by results demonstrating that Mms1 is recruited to a site...

  19. Construction of the glucose isomerase deficient strain of Streptomyces M1033 by homologous recombination

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    After the establishment of the transformation conditions of Streptomyces diastaticus No.7 Strain M1033,the integration plasmid pXW for homologous recombination,which contains a 600 bp fragment of incomplete GI (G138P.G247D) gene,has been constructed in order to realize the stable overexpression of the GI (G138P.G247D) which is valuable for large-scale industrial production.The Gigene's disruption has been realized by pXW's integration into M1033 chromosomes via homologous recombination and GI deficient strain of Streptomyces M1033 has been obtained.The reliability of introduction of mutation has been proved by analysis of recombinant fragment and affirmance of existence of the mutation,as well as detection of the stability of the deficient strain.

  20. Transformation-associated recombination between diverged and homologous DNA repeats is induced by strand breaks

    Energy Technology Data Exchange (ETDEWEB)

    Larionov, V.; Kouprina, N. [National Institute of Environmental Health Sciences (NIH), Research Triangle Park, NC (United States)]|[Institute of Cytology, St. Petersburg (Russian Federation); Eldarov, M. [National Institute of Environmental Health Sciences (NIH), Research Triangle Park, NC (United States)]|[Center for Bioengineering, Moscow (Russian Federation); Perkins, E.; Porter, G.; Resnick, M.A. [National Institute of Environmental Health Sciences (NIH), Research Triangle Park, NC (United States)

    1994-10-01

    Rearrangement and deletion within plasmid DNA is commonly observed during transformation. We have examined the mechanisms of transformation-associated recombination in the yeast Saccharomyces cerevisiae using a plasmid system which allowed the effects of physical state and/or extent of homology on recombination to be studied. The plasmid contains homologous or diverged (19%) DNA repeats separated by a genetically detectable color marker. Recombination during transformation for covalently closed circular plasmids was over 100-fold more frequent than during mitotic-growth. The frequency of recombination is partly dependent on the method of transformation In that procedures involving lithium acetate or spheroplasting yield higher frequencies than electroporation. When present in the repeats, unique single-strand breaks that are ligatable, as well as double-strand breaks, lead to high levels of recombination between diverged and identical repeats. The transformation-associated recombination between repeat DNA`s is under the influence of the RAD52, RAD1 and the RNC1 genes.

  1. Cohesin Is limiting for the suppression of DNA damage-induced recombination between homologous chromosomes.

    Directory of Open Access Journals (Sweden)

    Shay Covo

    2010-07-01

    Full Text Available Double-strand break (DSB repair through homologous recombination (HR is an evolutionarily conserved process that is generally error-free. The risk to genome stability posed by nonallelic recombination or loss-of-heterozygosity could be reduced by confining HR to sister chromatids, thereby preventing recombination between homologous chromosomes. Here we show that the sister chromatid cohesion complex (cohesin is a limiting factor in the control of DSB repair and genome stability and that it suppresses DNA damage-induced interactions between homologues. We developed a gene dosage system in tetraploid yeast to address limitations on various essential components in DSB repair and HR. Unlike RAD50 and RAD51, which play a direct role in HR, a 4-fold reduction in the number of essential MCD1 sister chromatid cohesion subunit genes affected survival of gamma-irradiated G(2/M cells. The decreased survival reflected a reduction in DSB repair. Importantly, HR between homologous chromosomes was strongly increased by ionizing radiation in G(2/M cells with a single copy of MCD1 or SMC3 even at radiation doses where survival was high and DSB repair was efficient. The increased recombination also extended to nonlethal doses of UV, which did not induce DSBs. The DNA damage-induced recombinants in G(2/M cells included crossovers. Thus, the cohesin complex has a dual role in protecting chromosome integrity: it promotes DSB repair and recombination between sister chromatids, and it suppresses damage-induced recombination between homologues. The effects of limited amounts of Mcd1and Smc3 indicate that small changes in cohesin levels may increase the risk of genome instability, which may lead to genetic diseases and cancer.

  2. Change of gene structure and function by non-homologous end-joining, homologous recombination, and transposition of DNA.

    Directory of Open Access Journals (Sweden)

    Wolfgang Goettel

    2009-06-01

    Full Text Available An important objective in genome research is to relate genome structure to gene function. Sequence comparisons among orthologous and paralogous genes and their allelic variants can reveal sequences of functional significance. Here, we describe a 379-kb region on chromosome 1 of maize that enables us to reconstruct chromosome breakage, transposition, non-homologous end-joining, and homologous recombination events. Such a high-density composition of various mechanisms in a small chromosomal interval exemplifies the evolution of gene regulation and allelic diversity in general. It also illustrates the evolutionary pace of changes in plants, where many of the above mechanisms are of somatic origin. In contrast to animals, somatic alterations can easily be transmitted through meiosis because the germline in plants is contiguous to somatic tissue, permitting the recovery of such chromosomal rearrangements. The analyzed region contains the P1-wr allele, a variant of the genetically well-defined p1 gene, which encodes a Myb-like transcriptional activator in maize. The P1-wr allele consists of eleven nearly perfect P1-wr 12-kb repeats that are arranged in a tandem head-to-tail array. Although a technical challenge to sequence such a structure by shotgun sequencing, we overcame this problem by subcloning each repeat and ordering them based on nucleotide variations. These polymorphisms were also critical for recombination and expression analysis in presence and absence of the trans-acting epigenetic factor Ufo1. Interestingly, chimeras of the p1 and p2 genes, p2/p1 and p1/p2, are framing the P1-wr cluster. Reconstruction of sequence amplification steps at the p locus showed the evolution from a single Myb-homolog to the multi-gene P1-wr cluster. It also demonstrates how non-homologous end-joining can create novel gene fusions. Comparisons to orthologous regions in sorghum and rice also indicate a greater instability of the maize genome, probably due to

  3. Homologous recombination repairs secondary replication induced DNA double-strand breaks after ionizing radiation

    OpenAIRE

    Groth, Petra; Orta, Manuel Luís; Elvers, Ingegerd; Majumder, Muntasir Mamun; Lagerqvist, Anne; Helleday, Thomas

    2012-01-01

    Ionizing radiation (IR) produces direct two-ended DNA double-strand breaks (DSBs) primarily repaired by non-homologous end joining (NHEJ). It is, however, well established that homologous recombination (HR) is induced and required for repair of a subset of DSBs formed following IR. Here, we find that HR induced by IR is drastically reduced when post-DNA damage replication is inhibited in mammalian cells. Both IR-induced RAD51 foci and HR events in the hprt gene are reduced in the presence of ...

  4. Rad51 Paralogs Remodel Pre-synaptic Rad51 Filaments to Stimulate Homologous Recombination

    OpenAIRE

    Taylor, Martin R.G.; Špírek, Mário; Chaurasiya, Kathy R.; Ward, Jordan D.; Carzaniga, Raffaella; Yu, Xiong; Egelman, Edward H.; Collinson, Lucy M.; Rueda, David; Krejci, Lumir; Boulton, Simon J.

    2015-01-01

    Summary Repair of DNA double strand breaks by homologous recombination (HR) is initiated by Rad51 filament nucleation on single-stranded DNA (ssDNA), which catalyzes strand exchange with homologous duplex DNA. BRCA2 and the Rad51 paralogs are tumor suppressors and critical mediators of Rad51. To gain insight into Rad51 paralog function, we investigated a heterodimeric Rad51 paralog complex, RFS-1/RIP-1, and uncovered the molecular basis by which Rad51 paralogs promote HR. Unlike BRCA2, which ...

  5. Inhibition of homologous recombination by the PCNA-interacting protein PARI.

    Science.gov (United States)

    Moldovan, George-Lucian; Dejsuphong, Donniphat; Petalcorin, Mark I R; Hofmann, Kay; Takeda, Shunichi; Boulton, Simon J; D'Andrea, Alan D

    2012-01-13

    Inappropriate homologous recombination (HR) causes genomic instability and cancer. In yeast, the UvrD family helicase Srs2 is recruited to sites of DNA replication by SUMO-modified PCNA, where it acts to restrict HR by disassembling toxic RAD51 nucleofilaments. How human cells control recombination at replication forks is unknown. Here, we report that the protein PARI, containing a UvrD-like helicase domain, is a PCNA-interacting partner required for preservation of genome stability in human and DT40 chicken cells. Using cell-based and biochemical assays, we show that PARI restricts unscheduled recombination by interfering with the formation of RAD51-DNA HR structures. Finally, we show that PARI knockdown suppresses the genomic instability of Fanconi Anemia/BRCA pathway-deficient cells. Thus, we propose that PARI is a long sought-after factor that suppresses inappropriate recombination events at mammalian replication forks. PMID:22153967

  6. Genetic probing of homologous recombination and non-homologous end joining during meiotic prophase in irradiated mouse spermatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Ahmed, Emad A. [Department of Endocrinology and Metabolism, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht (Netherlands); Department of Zoology, Faculty of Science, Assiut University, 71516 Assiut (Egypt); Philippens, Marielle E.P.; Kal, Henk B. [Department of Radiotherapy, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht (Netherlands); Rooij, Dirk G. de, E-mail: d.g.derooij@uu.nl [Department of Endocrinology and Metabolism, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht (Netherlands); Center for Reproductive Medicine, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam (Netherlands); Boer, Peter de [Department of Obstetrics and Gynaecology, Radboud University Nijmegen Medical Centre, 6500 HB Nijmegen (Netherlands)

    2010-06-01

    This study was designed to obtain a better insight into the relative contribution of homologous recombination (HR) and non-homologous end joining (NHEJ) to the repair of radiation-induced DNA double-strand breaks (DSBs) at first meiotic prophase. Early and late pachytene and early diplotene spermatocytes that had completed crossing over were sampled. We studied the kinetics of {gamma}-H2AX chromatin foci removal after irradiation of mice deficient for HR and mice deficient for NHEJ. Analyzing {gamma}-H2AX signals in unirradiated RAD54/RAD54B deficient spermatocytes indicated incomplete meiotic recombination repair due to the pronounced increase of {gamma}-H2AX foci in late prophase primary spermatocytes. In these mice, 8 h after irradiation, early pachytene spermatocytes showed a reduction of the numbers of {gamma}-H2AX foci by 52% compared to 82% in the wild type, the difference being significant. However, after crossing over (in late pachytene and early diplotene), no effect of RAD54/RAD54B deficiency on the reduction of irradiation-induced foci was observed. In NHEJ deficient SCID mice, repair kinetics in early spermatocytes were similar to those in wild type mice. However, 1 h after irradiation in late pachytene and early diplotene spermatocytes 1.7 times more foci were found than in wild type mice. This difference might be related to the absence of a DNA-PKcs dependent fast repair component in SCID mice. As subsequent repair is normal, HR likely is taking over. Taken together, the results obtained in RAD54/RAD54B deficient mice and in SCID mice indicate that DSB repair in early pachytene spermatocytes is mainly carried out through HR. In late spermatocytes (late pachytenes and early diplotenes) NHEJ is active. However, probably there is an interplay between these repair pathways and when in late spermatocytes the NHEJ pathway is compromised HR may take over.

  7. Homologous recombination and non-homologous end-joining repair pathways in bovine embryos with different developmental competence

    International Nuclear Information System (INIS)

    This study investigated the expression of genes controlling homologous recombination (HR), and non-homologous end-joining (NHEJ) DNA-repair pathways in bovine embryos of different developmental potential. It also evaluated whether bovine embryos can respond to DNA double-strand breaks (DSBs) induced with ultraviolet irradiation by regulating expression of genes involved in HR and NHEJ repair pathways. Embryos with high, intermediate or low developmental competence were selected based on the cleavage time after in vitro insemination and were removed from in vitro culture before (36 h), during (72 h) and after (96 h) the expected period of embryonic genome activation. All studied genes were expressed before, during and after the genome activation period regardless the developmental competence of the embryos. Higher mRNA expression of 53BP1 and RAD52 was found before genome activation in embryos with low developmental competence. Expression of 53BP1, RAD51 and KU70 was downregulated at 72 h and upregulated at 168 h post-insemination in response to DSBs induced by ultraviolet irradiation. In conclusion, important genes controlling HR and NHEJ DNA-repair pathways are expressed in bovine embryos, however genes participating in these pathways are only regulated after the period of embryo genome activation in response to ultraviolet-induced DSBs.

  8. Homologous recombination and non-homologous end-joining repair pathways in bovine embryos with different developmental competence

    Energy Technology Data Exchange (ETDEWEB)

    Henrique Barreta, Marcos [Universidade Federal de Santa Catarina, Campus Universitario de Curitibanos, Curitibanos, SC (Brazil); Laboratorio de Biotecnologia e Reproducao Animal-BioRep, Universidade Federal de Santa Maria, Santa Maria, RS (Brazil); Garziera Gasperin, Bernardo; Braga Rissi, Vitor; Cesaro, Matheus Pedrotti de [Laboratorio de Biotecnologia e Reproducao Animal-BioRep, Universidade Federal de Santa Maria, Santa Maria, RS (Brazil); Ferreira, Rogerio [Centro de Educacao Superior do Oeste-Universidade do Estado de Santa Catarina, Chapeco, SC (Brazil); Oliveira, Joao Francisco de; Goncalves, Paulo Bayard Dias [Laboratorio de Biotecnologia e Reproducao Animal-BioRep, Universidade Federal de Santa Maria, Santa Maria, RS (Brazil); Bordignon, Vilceu, E-mail: vilceu.bordignon@mcgill.ca [Department of Animal Science, McGill University, Ste-Anne-De-Bellevue, QC (Canada)

    2012-10-01

    This study investigated the expression of genes controlling homologous recombination (HR), and non-homologous end-joining (NHEJ) DNA-repair pathways in bovine embryos of different developmental potential. It also evaluated whether bovine embryos can respond to DNA double-strand breaks (DSBs) induced with ultraviolet irradiation by regulating expression of genes involved in HR and NHEJ repair pathways. Embryos with high, intermediate or low developmental competence were selected based on the cleavage time after in vitro insemination and were removed from in vitro culture before (36 h), during (72 h) and after (96 h) the expected period of embryonic genome activation. All studied genes were expressed before, during and after the genome activation period regardless the developmental competence of the embryos. Higher mRNA expression of 53BP1 and RAD52 was found before genome activation in embryos with low developmental competence. Expression of 53BP1, RAD51 and KU70 was downregulated at 72 h and upregulated at 168 h post-insemination in response to DSBs induced by ultraviolet irradiation. In conclusion, important genes controlling HR and NHEJ DNA-repair pathways are expressed in bovine embryos, however genes participating in these pathways are only regulated after the period of embryo genome activation in response to ultraviolet-induced DSBs.

  9. The cell-cycle checkpoint kinase Chk1 is required for mammalian homologous recombination repair

    DEFF Research Database (Denmark)

    Sørensen, Claus Storgaard; Hansen, Lasse Tengbjerg; Dziegielewski, Jaroslaw;

    2005-01-01

    The essential checkpoint kinase Chk1 is required for cell-cycle delays after DNA damage or blocked DNA replication. However, it is unclear whether Chk1 is involved in the repair of damaged DNA. Here we establish that Chk1 is a key regulator of genome maintenance by the homologous recombination......, the essential recombination repair protein RAD51 is recruited to DNA repair foci performing a vital role in correct HRR. We demonstrate that Chk1 interacts with RAD51, and that RAD51 is phosphorylated on Thr 309 in a Chk1-dependent manner. Consistent with a functional interplay between Chk1 and RAD51...

  10. An Improved Homologous Recombination Method for Rapid Cloning of the Antibody Heavy Chain Gene and Its Comparison with the Homologous Recombination and Traditional Cloning Methods

    Directory of Open Access Journals (Sweden)

    Masoumeh Hajirezaei

    2015-10-01

    Full Text Available Background: The homologous recombination (HR is one of the conventional cloning methods for the production of recombinant DNA. It is a quick method for in vivo DNA cloning without using the high cost restriction enzymes. A few modifications in the cloning procedure can increase the efficiency of this method.Materials and Methods: In this study, effect of heating on the rate of the IgG1 heavy chain gene cloning was investigated in the HR method and then it was compared with HR method without heating and traditional cloning method. For doing this comparison, three cloning methods including HR, HR with the heat treatment, and traditional cloning were used to clone the human IgG1 heavy chain into the pcDNA3.1(+ plasmid.Results: The results showed that adding heat in the HR method converts insert and vector from the double strand DNA to the single strand DNA. This allows them to copulate with each other better and faster than the two other methods. The heat addition also helps the cell enzyme system for a faster and easier recombination and moreover it increases the cloning efficiency of the HR method in case of in vitro processing.Conclusion: The results showed that giving heat in the HR method increases cloning rate 7.5% and this increase reaches 10% in comparison with the traditional method. 

  11. High-Resolution Mapping of Homologous Recombination Events in rad3 Hyper-Recombination Mutants in Yeast.

    Directory of Open Access Journals (Sweden)

    Sabrina L Andersen

    2016-03-01

    Full Text Available The Saccharomyces cerevisae RAD3 gene is the homolog of human XPD, an essential gene encoding a DNA helicase of the TFIIH complex involved in both nucleotide excision repair (NER and transcription. Some mutant alleles of RAD3 (rad3-101 and rad3-102 have partial defects in DNA repair and a strong hyper-recombination (hyper-Rec phenotype. Previous studies showed that the hyper-Rec phenotype associated with rad3-101 and rad3-102 can be explained as a consequence of persistent single-stranded DNA gaps that are converted to recombinogenic double-strand breaks (DSBs by replication. The systems previously used to characterize the hyper-Rec phenotype of rad3 strains do not detect the reciprocal products of mitotic recombination. We have further characterized these events using a system in which the reciprocal products of mitotic recombination are recovered. Both rad3-101 and rad3-102 elevate the frequency of reciprocal crossovers about 100-fold. Mapping of these events shows that three-quarters of these crossovers reflect DSBs formed at the same positions in both sister chromatids (double sister-chromatid breaks, DSCBs. The remainder reflects DSBs formed in single chromatids (single chromatid breaks, SCBs. The ratio of DSCBs to SCBs is similar to that observed for spontaneous recombination events in wild-type cells. We mapped 216 unselected genomic alterations throughout the genome including crossovers, gene conversions, deletions, and duplications. We found a significant association between the location of these recombination events and regions with elevated gamma-H2AX. In addition, there was a hotspot for deletions and duplications at the IMA2 and HXT11 genes near the left end of chromosome XV. A comparison of these data with our previous analysis of spontaneous mitotic recombination events suggests that a sub-set of spontaneous events in wild-type cells may be initiated by incomplete NER reactions, and that DSCBs, which cannot be repaired by sister

  12. Rescue of end fragments of yeast artificial chromosomes by homologous recombination in yeast.

    OpenAIRE

    Hermanson, G G; Hoekstra, M F; McElligott, D. L.; Evans, G A

    1991-01-01

    Yeast artificial chromosomes (YACs) provide a powerful tool for the isolation and mapping of large regions of mammalian chromosomes. We developed a rapid and efficient method for the isolation of DNA fragments representing the extreme ends of YAC clones by the insertion of a rescue plasmid into the YAC vector by homologous recombination. Two rescue vectors were constructed containing a yeast LYS2 selectable gene, a bacterial origin of replication, an antibiotic resistance gene, a polylinker c...

  13. Uncoupling of sexual reproduction from homologous recombination in homozygous Oenothera species

    OpenAIRE

    Rauwolf, U; Greiner, S; Mráček, J; Rauwolf, M; Golczyk, H.; Mohler, V; Herrmann, R. G.; Meurer, J

    2011-01-01

    Salient features of the first meiotic division are independent segregation of chromosomes and homologous recombination (HR). In non-sexually reproducing, homozygous species studied to date HR is absent. In this study, we constructed the first linkage maps of homozygous, bivalent-forming Oenothera species and provide evidence that HR was exclusively confined to the chromosome ends of all linkage groups in our population. Co-segregation of complementary DNA-based markers with the major group of...

  14. Production of p53 gene knockout rats by homologous recombination in embryonic stem cells

    OpenAIRE

    Tong, Chang; Li, Ping; Wu, Nancy L; Yan, Youzhen; Ying, Qi-Long

    2010-01-01

    The use of homologous recombination to modify genes in embryonic stem (ES) cells provides a powerful means to elucidate gene function and create disease models1. Application of this technology to engineer genes in rats has previously been impossible in the absence of germline competent ES cells in this species. We have recently established authentic rat ES cells2, 3. Here we report the generation of the first gene knockout rats using the ES cell-based gene targeting technology. We designed a ...

  15. Gene Targeting Using Homologous Recombination in Embryonic Stem Cells: The Future for Behavior Genetics?

    OpenAIRE

    Gerlai, Robert

    2016-01-01

    Gene targeting with homologous recombination in embryonic stem cells created a revolution in the analysis of the function of genes in behavioral brain research. The technology allowed unprecedented precision with which one could manipulate genes and study the effect of this manipulation on the central nervous system. With gene targeting, the uncertainty inherent in psychopharmacology regarding whether a particular compound would act only through a specific target was removed. Thus, gene targe...

  16. Multi-Homologous Recombination-Based Gene Manipulation in the Rice Pathogen Fusarium fujikuroi

    OpenAIRE

    Hwang, In Sun; Ahn, Il-Pyung

    2016-01-01

    Gene disruption by homologous recombination is widely used to investigate and analyze the function of genes in Fusarium fujikuroi, a fungus that causes bakanae disease and root rot symptoms in rice. To generate gene deletion constructs, the use of conventional cloning methods, which rely on restriction enzymes and ligases, has had limited success due to a lack of unique restriction enzyme sites. Although strategies that avoid the use of restriction enzymes have been employed to overcome this ...

  17. Activities of wildtype and mutant p53 in suppression of homologous recombination as measured by a retroviral vector system

    Energy Technology Data Exchange (ETDEWEB)

    Lu Xiongbin; Lozano, Guillermina; Donehower, Lawrence A

    2003-01-28

    DNA repair of double strand breaks, interstrand DNA cross-links, and other types of DNA damage utilizes the processes of homologous recombination and non-homologous end joining to repair the damage. Aberrant homologous recombination is likely to be responsible for a significant fraction of chromosomal deletions, duplications, and translocations that are observed in cancer cells. To facilitate measurement of homologous recombination frequencies in normal cells, mutant cells, and cancer cells, we have developed a high titer retroviral vector containing tandem repeats of mutant versions of a GFP-Zeocin resistance fusion gene and an intact neomycin resistance marker. Recombination between the tandem repeats regenerates a functional GFP-Zeo{sup R} marker that can be easily scored. This retroviral vector was used to assess homologous recombination frequencies in human cancer cells and rodent fibroblasts with differing dosages of wild type or mutant p53. Absence of wild type p53 stimulated spontaneous and ionizing radiation-induced homologous recombination, confirming previous studies. Moreover, p53{sup +/-} mouse fibroblasts show elevated levels of homologous recombination compared to their p53{sup +/+} counterparts following retroviral vector infection, indicating that p53 is haploinsufficient for suppression of homologous recombination. Transfection of vector-containing p53 null Saos-2 cells with various human cancer-associated p53 mutants revealed that these altered p53 proteins retain some recombination suppression function despite being totally inactive for transcriptional transactivation. The retroviral vector utilized in these studies may be useful in performing recombination assays on a wide array of cell types, including those not readily transfected by normal vectors.

  18. A non-canonical DNA structure enables homologous recombination in various genetic systems.

    Science.gov (United States)

    Masuda, Tokiha; Ito, Yutaka; Terada, Tohru; Shibata, Takehiko; Mikawa, Tsutomu

    2009-10-30

    Homologous recombination, which is critical to genetic diversity, depends on homologous pairing (HP). HP is the switch from parental to recombinant base pairs, which requires expansion of inter-base pair spaces. This expansion unavoidably causes untwisting of the parental double-stranded DNA. RecA/Rad51-catalyzed ATP-dependent HP is extensively stimulated in vitro by negative supercoils, which compensates for untwisting. However, in vivo, double-stranded DNA is relaxed by bound proteins and thus is an unfavorable substrate for RecA/Rad51. In contrast, Mhr1, an ATP-independent HP protein required for yeast mitochondrial homologous recombination, catalyzes HP without the net untwisting of double-stranded DNA. Therefore, we questioned whether Mhr1 uses a novel strategy to promote HP. Here, we found that, like RecA, Mhr1 induced the extension of bound single-stranded DNA. In addition, this structure was induced by all evolutionarily and structurally distinct HP proteins so far tested, including bacterial RecO, viral RecT, and human Rad51. Thus, HP includes the common non-canonical DNA structure and uses a common core mechanism, independent of the species of HP proteins. We discuss the significance of multiple types of HP proteins. PMID:19729448

  19. Resolving RAD51C function in late stages of homologous recombination

    Directory of Open Access Journals (Sweden)

    Kuznetsov Sergey G

    2007-06-01

    Full Text Available Abstract DNA double strand breaks are efficiently repaired by homologous recombination. One of the last steps of this process is resolution of Holliday junctions that are formed at the sites of genetic exchange between homologous DNA. Although various resolvases with Holliday junctions processing activity have been identified in bacteriophages, bacteria and archaebacteria, eukaryotic resolvases have been elusive. Recent biochemical evidence has revealed that RAD51C and XRCC3, members of the RAD51-like protein family, are involved in Holliday junction resolution in mammalian cells. However, purified recombinant RAD51C and XRCC3 proteins have not shown any Holliday junction resolution activity. In addition, these proteins did not reveal the presence of a nuclease domain, which raises doubts about their ability to function as a resolvase. Furthermore, oocytes from infertile Rad51C mutant mice exhibit precocious separation of sister chromatids at metaphase II, a phenotype that reflects a defect in sister chromatid cohesion, not a lack of Holliday junction resolution. Here we discuss a model to explain how a Holliday junction resolution defect can lead to sister chromatid separation in mouse oocytes. We also describe other recent in vitro and in vivo evidence supporting a late role for RAD51C in homologous recombination in mammalian cells, which is likely to be resolution of the Holliday junction.

  20. Multiple regulation of Rad51-mediated homologous recombination by fission yeast Fbh1.

    Science.gov (United States)

    Tsutsui, Yasuhiro; Kurokawa, Yumiko; Ito, Kentaro; Siddique, Md Shahjahan P; Kawano, Yumiko; Yamao, Fumiaki; Iwasaki, Hiroshi

    2014-08-01

    Fbh1, an F-box helicase related to bacterial UvrD, has been proposed to modulate homologous recombination in fission yeast. We provide several lines of evidence for such modulation. Fbh1, but not the related helicases Srs2 and Rqh1, suppressed the formation of crossover recombinants from single HO-induced DNA double-strand breaks. Purified Fbh1 in complex with Skp1 (Fbh1-Skp1 complex) inhibited Rad51-driven DNA strand exchange by disrupting Rad51 nucleoprotein filaments in an ATP-dependent manner; this disruption was alleviated by the Swi5-Sfr1 complex, an auxiliary activator of Rad51. In addition, the reconstituted SCFFbh1 complex, composed of purified Fbh1-Skp1 and Pcu1-Rbx1, displayed ubiquitin-ligase E3 activity toward Rad51. Furthermore, Fbh1 reduced the protein level of Rad51 in stationary phase in an F-box-dependent, but not in a helicase domain-independent manner. These results suggest that Fbh1 negatively regulates Rad51-mediated homologous recombination via its two putative, unrelated activities, namely DNA unwinding/translocation and ubiquitin ligation. In addition to its anti-recombinase activity, we tentatively suggest that Fbh1 might also have a pro-recombination role in vivo, because the Fbh1-Skp1 complex stimulated Rad51-mediated strand exchange in vitro after strand exchange had been initiated. PMID:25165823

  1. Multiple regulation of Rad51-mediated homologous recombination by fission yeast Fbh1.

    Directory of Open Access Journals (Sweden)

    Yasuhiro Tsutsui

    2014-08-01

    Full Text Available Fbh1, an F-box helicase related to bacterial UvrD, has been proposed to modulate homologous recombination in fission yeast. We provide several lines of evidence for such modulation. Fbh1, but not the related helicases Srs2 and Rqh1, suppressed the formation of crossover recombinants from single HO-induced DNA double-strand breaks. Purified Fbh1 in complex with Skp1 (Fbh1-Skp1 complex inhibited Rad51-driven DNA strand exchange by disrupting Rad51 nucleoprotein filaments in an ATP-dependent manner; this disruption was alleviated by the Swi5-Sfr1 complex, an auxiliary activator of Rad51. In addition, the reconstituted SCFFbh1 complex, composed of purified Fbh1-Skp1 and Pcu1-Rbx1, displayed ubiquitin-ligase E3 activity toward Rad51. Furthermore, Fbh1 reduced the protein level of Rad51 in stationary phase in an F-box-dependent, but not in a helicase domain-independent manner. These results suggest that Fbh1 negatively regulates Rad51-mediated homologous recombination via its two putative, unrelated activities, namely DNA unwinding/translocation and ubiquitin ligation. In addition to its anti-recombinase activity, we tentatively suggest that Fbh1 might also have a pro-recombination role in vivo, because the Fbh1-Skp1 complex stimulated Rad51-mediated strand exchange in vitro after strand exchange had been initiated.

  2. A new thermosensitive smc-3 allele reveals involvement of cohesin in homologous recombination in C. elegans.

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    Antoine Baudrimont

    Full Text Available The cohesin complex is required for the cohesion of sister chromatids and for correct segregation during mitosis and meiosis. Crossover recombination, together with cohesion, is essential for the disjunction of homologous chromosomes during the first meiotic division. Cohesin has been implicated in facilitating recombinational repair of DNA lesions via the sister chromatid. Here, we made use of a new temperature-sensitive mutation in the Caenorhabditis elegans SMC-3 protein to study the role of cohesin in the repair of DNA double-strand breaks (DSBs and hence in meiotic crossing over. We report that attenuation of cohesin was associated with extensive SPO-11-dependent chromosome fragmentation, which is representative of unrepaired DSBs. We also found that attenuated cohesin likely increased the number of DSBs and eliminated the need of MRE-11 and RAD-50 for DSB formation in C. elegans, which suggests a role for the MRN complex in making cohesin-loaded chromatin susceptible to meiotic DSBs. Notably, in spite of largely intact sister chromatid cohesion, backup DSB repair via the sister chromatid was mostly impaired. We also found that weakened cohesins affected mitotic repair of DSBs by homologous recombination, whereas NHEJ repair was not affected. Our data suggest that recombinational DNA repair makes higher demands on cohesins than does chromosome segregation.

  3. Homologous recombination occurs in Entamoeba and is enhanced during growth stress and stage conversion.

    Science.gov (United States)

    Singh, Nishant; Bhattacharya, Alok; Bhattacharya, Sudha

    2013-01-01

    Homologous recombination (HR) has not been demonstrated in the parasitic protists Entamoeba histolytica or Entamoeba invadens, as no convenient method is available to measure it. However, HR must exist to ensure genome integrity, and possible genetic exchange, especially during stage conversion from trophozoite to cyst. Here we show the up regulation of mitotic and meiotic HR genes in Entamoeba during serum starvation, and encystation. To directly demonstrate HR we use a simple PCR-based method involving inverted repeats, which gives a reliable read out, as the recombination junctions can be determined by sequencing the amplicons. Using this read out, we demonstrate enhanced HR under growth stress in E. histolytica, and during encystation in E. invadens. We also demonstrate recombination between chromosomal inverted repeats. This is the first experimental demonstration of HR in Entamoeba and will help future investigations into this process, and to explore the possibility of meiosis in Entamoeba.

  4. Homologous recombination occurs in Entamoeba and is enhanced during growth stress and stage conversion.

    Directory of Open Access Journals (Sweden)

    Nishant Singh

    Full Text Available Homologous recombination (HR has not been demonstrated in the parasitic protists Entamoeba histolytica or Entamoeba invadens, as no convenient method is available to measure it. However, HR must exist to ensure genome integrity, and possible genetic exchange, especially during stage conversion from trophozoite to cyst. Here we show the up regulation of mitotic and meiotic HR genes in Entamoeba during serum starvation, and encystation. To directly demonstrate HR we use a simple PCR-based method involving inverted repeats, which gives a reliable read out, as the recombination junctions can be determined by sequencing the amplicons. Using this read out, we demonstrate enhanced HR under growth stress in E. histolytica, and during encystation in E. invadens. We also demonstrate recombination between chromosomal inverted repeats. This is the first experimental demonstration of HR in Entamoeba and will help future investigations into this process, and to explore the possibility of meiosis in Entamoeba.

  5. Optimal cloning of PCR fragments by homologous recombination in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Ana Paula Jacobus

    Full Text Available PCR fragments and linear vectors containing overlapping ends are easily assembled into a propagative plasmid by homologous recombination in Escherichia coli. Although this gap-repair cloning approach is straightforward, its existence is virtually unknown to most molecular biologists. To popularize this method, we tested critical parameters influencing the efficiency of PCR fragments cloning into PCR-amplified vectors by homologous recombination in the widely used E. coli strain DH5α. We found that the number of positive colonies after transformation increases with the length of overlap between the PCR fragment and linear vector. For most practical purposes, a 20 bp identity already ensures high-cloning yields. With an insert to vector ratio of 2:1, higher colony forming numbers are obtained when the amount of vector is in the range of 100 to 250 ng. An undesirable cloning background of empty vectors can be minimized during vector PCR amplification by applying a reduced amount of plasmid template or by using primers in which the 5' termini are separated by a large gap. DpnI digestion of the plasmid template after PCR is also effective to decrease the background of negative colonies. We tested these optimized cloning parameters during the assembly of five independent DNA constructs and obtained 94% positive clones out of 100 colonies probed. We further demonstrated the efficient and simultaneous cloning of two PCR fragments into a vector. These results support the idea that homologous recombination in E. coli might be one of the most effective methods for cloning one or two PCR fragments. For its simplicity and high efficiency, we believe that recombinational cloning in E. coli has a great potential to become a routine procedure in most molecular biology-oriented laboratories.

  6. Distribution of the phenotypic effects of random homologous recombination between two virus species.

    Directory of Open Access Journals (Sweden)

    Florence Vuillaume

    2011-05-01

    Full Text Available Recombination has an evident impact on virus evolution and emergence of new pathotypes, and has generated an immense literature. However, the distribution of phenotypic effects caused by genome-wide random homologous recombination has never been formally investigated. Previous data on the subject have promoted the implicit view that most viral recombinant genomes are likely to be deleterious or lethal if the nucleotide identity of parental sequences is below 90%. We decided to challenge this view by creating a bank of near-random recombinants between two viral species of the genus Begomovirus (Family Geminiviridae exhibiting 82% nucleotide identity, and by testing infectivity and in planta accumulation of recombinant clones randomly extracted from this bank. The bank was created by DNA-shuffling-a technology initially applied to the random shuffling of individual genes, and here implemented for the first time to shuffle full-length viral genomes. Together with our previously described system allowing the direct cloning of full-length infectious geminivirus genomes, it provided a unique opportunity to generate hundreds of "mosaic" virus genomes, directly testable for infectivity. A subset of 47 randomly chosen recombinants was sequenced, individually inoculated into tomato plants, and compared with the parental viruses. Surprisingly, our results showed that all recombinants were infectious and accumulated at levels comparable or intermediate to that of the parental clones. This indicates that, in our experimental system, despite the fact that the parental genomes differ by nearly 20%, lethal and/or large deleterious effects of recombination are very rare, in striking contrast to the common view that has emerged from previous studies published on other viruses.

  7. Unveiling novel RecO distant orthologues involved in homologous recombination.

    Directory of Open Access Journals (Sweden)

    Stéphanie Marsin

    Full Text Available The generation of a RecA filament on single-stranded DNA is a critical step in homologous recombination. Two main pathways leading to the formation of the nucleofilament have been identified in bacteria, based on the protein complexes mediating RecA loading: RecBCD (AddAB and RecFOR. Many bacterial species seem to lack some of the components involved in these complexes. The current annotation of the Helicobacter pylori genome suggests that this highly diverse bacterial pathogen has a reduced set of recombination mediator proteins. While it is now clear that homologous recombination plays a critical role in generating H. pylori diversity by allowing genomic DNA rearrangements and integration through transformation of exogenous DNA into the chromosome, no complete mediator complex is deduced from the sequence of its genome. Here we show by bioinformatics analysis the presence of a RecO remote orthologue that allowed the identification of a new set of RecO proteins present in all bacterial species where a RecR but not RecO was previously identified. HpRecO shares less than 15% identity with previously characterized homologues. Genetic dissection of recombination pathways shows that this novel RecO and the remote RecB homologue present in H. pylori are functional in repair and in RecA-dependent intrachromosomal recombination, defining two initiation pathways with little overlap. We found, however, that neither RecOR nor RecB contributes to transformation, suggesting the presence of a third, specialized, RecA-dependent pathway responsible for the integration of transforming DNA into the chromosome of this naturally competent bacteria. These results provide insight into the mechanisms that this successful pathogen uses to generate genetic diversity and adapt to changing environments and new hosts.

  8. Unveiling novel RecO distant orthologues involved in homologous recombination.

    Science.gov (United States)

    Marsin, Stéphanie; Mathieu, Aurélie; Kortulewski, Thierry; Guérois, Raphaël; Radicella, J Pablo

    2008-01-01

    The generation of a RecA filament on single-stranded DNA is a critical step in homologous recombination. Two main pathways leading to the formation of the nucleofilament have been identified in bacteria, based on the protein complexes mediating RecA loading: RecBCD (AddAB) and RecFOR. Many bacterial species seem to lack some of the components involved in these complexes. The current annotation of the Helicobacter pylori genome suggests that this highly diverse bacterial pathogen has a reduced set of recombination mediator proteins. While it is now clear that homologous recombination plays a critical role in generating H. pylori diversity by allowing genomic DNA rearrangements and integration through transformation of exogenous DNA into the chromosome, no complete mediator complex is deduced from the sequence of its genome. Here we show by bioinformatics analysis the presence of a RecO remote orthologue that allowed the identification of a new set of RecO proteins present in all bacterial species where a RecR but not RecO was previously identified. HpRecO shares less than 15% identity with previously characterized homologues. Genetic dissection of recombination pathways shows that this novel RecO and the remote RecB homologue present in H. pylori are functional in repair and in RecA-dependent intrachromosomal recombination, defining two initiation pathways with little overlap. We found, however, that neither RecOR nor RecB contributes to transformation, suggesting the presence of a third, specialized, RecA-dependent pathway responsible for the integration of transforming DNA into the chromosome of this naturally competent bacteria. These results provide insight into the mechanisms that this successful pathogen uses to generate genetic diversity and adapt to changing environments and new hosts. PMID:18670631

  9. Competitive repair by naturally dispersed repetitive DNA during non-allelic homologous recombination

    Energy Technology Data Exchange (ETDEWEB)

    Hoang, Margaret L.; Tan, Frederick J.; Lai, David C.; Celniker, Sue E.; Hoskins, Roger A.; Dunham, Maitreya J.; Zheng, Yixian; Koshland, Douglas

    2010-08-27

    Genome rearrangements often result from non-allelic homologous recombination (NAHR) between repetitive DNA elements dispersed throughout the genome. Here we systematically analyze NAHR between Ty retrotransposons using a genome-wide approach that exploits unique features of Saccharomyces cerevisiae purebred and Saccharomyces cerevisiae/Saccharomyces bayanus hybrid diploids. We find that DNA double-strand breaks (DSBs) induce NAHR-dependent rearrangements using Ty elements located 12 to 48 kilobases distal to the break site. This break-distal recombination (BDR) occurs frequently, even when allelic recombination can repair the break using the homolog. Robust BDR-dependent NAHR demonstrates that sequences very distal to DSBs can effectively compete with proximal sequences for repair of the break. In addition, our analysis of NAHR partner choice between Ty repeats shows that intrachromosomal Ty partners are preferred despite the abundance of potential interchromosomal Ty partners that share higher sequence identity. This competitive advantage of intrachromosomal Tys results from the relative efficiencies of different NAHR repair pathways. Finally, NAHR generates deleterious rearrangements more frequently when DSBs occur outside rather than within a Ty repeat. These findings yield insights into mechanisms of repeat-mediated genome rearrangements associated with evolution and cancer.

  10. Tankyrases Promote Homologous Recombination and Check Point Activation in Response to DSBs.

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    Zita Nagy

    2016-02-01

    Full Text Available DNA lesions are sensed by a network of proteins that trigger the DNA damage response (DDR, a signaling cascade that acts to delay cell cycle progression and initiate DNA repair. The Mediator of DNA damage Checkpoint protein 1 (MDC1 is essential for spreading of the DDR signaling on chromatin surrounding Double Strand Breaks (DSBs by acting as a scaffold for PI3K kinases and for ubiquitin ligases. MDC1 also plays a role both in Non-Homologous End Joining (NHEJ and Homologous Recombination (HR repair pathways. Here we identify two novel binding partners of MDC1, the poly (ADP-ribose Polymerases (PARPs TNKS1 and 2. We find that TNKSs are recruited to DNA lesions by MDC1 and regulate DNA end resection and BRCA1A complex stabilization at lesions leading to efficient DSB repair by HR and proper checkpoint activation.

  11. Tankyrases Promote Homologous Recombination and Check Point Activation in Response to DSBs.

    Science.gov (United States)

    Nagy, Zita; Kalousi, Alkmini; Furst, Audrey; Koch, Marc; Fischer, Benoit; Soutoglou, Evi

    2016-02-01

    DNA lesions are sensed by a network of proteins that trigger the DNA damage response (DDR), a signaling cascade that acts to delay cell cycle progression and initiate DNA repair. The Mediator of DNA damage Checkpoint protein 1 (MDC1) is essential for spreading of the DDR signaling on chromatin surrounding Double Strand Breaks (DSBs) by acting as a scaffold for PI3K kinases and for ubiquitin ligases. MDC1 also plays a role both in Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) repair pathways. Here we identify two novel binding partners of MDC1, the poly (ADP-ribose) Polymerases (PARPs) TNKS1 and 2. We find that TNKSs are recruited to DNA lesions by MDC1 and regulate DNA end resection and BRCA1A complex stabilization at lesions leading to efficient DSB repair by HR and proper checkpoint activation. PMID:26845027

  12. Tankyrases Promote Homologous Recombination and Check Point Activation in Response to DSBs

    Science.gov (United States)

    Furst, Audrey; Koch, Marc; Fischer, Benoit; Soutoglou, Evi

    2016-01-01

    DNA lesions are sensed by a network of proteins that trigger the DNA damage response (DDR), a signaling cascade that acts to delay cell cycle progression and initiate DNA repair. The Mediator of DNA damage Checkpoint protein 1 (MDC1) is essential for spreading of the DDR signaling on chromatin surrounding Double Strand Breaks (DSBs) by acting as a scaffold for PI3K kinases and for ubiquitin ligases. MDC1 also plays a role both in Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) repair pathways. Here we identify two novel binding partners of MDC1, the poly (ADP-ribose) Polymerases (PARPs) TNKS1 and 2. We find that TNKSs are recruited to DNA lesions by MDC1 and regulate DNA end resection and BRCA1A complex stabilization at lesions leading to efficient DSB repair by HR and proper checkpoint activation. PMID:26845027

  13. Srs2 and Mus81-Mms4 Prevent Accumulation of Toxic Inter-Homolog Recombination Intermediates.

    Science.gov (United States)

    Keyamura, Kenji; Arai, Kota; Hishida, Takashi

    2016-07-01

    Homologous recombination is an evolutionally conserved mechanism that promotes genome stability through the faithful repair of double-strand breaks and single-strand gaps in DNA, and the recovery of stalled or collapsed replication forks. Saccharomyces cerevisiae ATP-dependent DNA helicase Srs2 (a member of the highly conserved UvrD family of helicases) has multiple roles in regulating homologous recombination. A mutation (srs2K41A) resulting in a helicase-dead mutant of Srs2 was found to be lethal in diploid, but not in haploid, cells. In diploid cells, Srs2K41A caused the accumulation of inter-homolog joint molecule intermediates, increased the levels of spontaneous Rad52 foci, and induced gross chromosomal rearrangements. Srs2K41A lethality and accumulation of joint molecules were suppressed by inactivating Rad51 or deleting the Rad51-interaction domain of Srs2, whereas phosphorylation and sumoylation of Srs2 and its interaction with sumoylated proliferating cell nuclear antigen (PCNA) were not required for lethality. The structure-specific complex of crossover junction endonucleases Mus81 and Mms4 was also required for viability of diploid, but not haploid, SRS2 deletion mutants (srs2Δ), and diploid srs2Δ mus81Δ mutants accumulated joint molecule intermediates. Our data suggest that Srs2 and Mus81-Mms4 have critical roles in preventing the formation of (or in resolving) toxic inter-homolog joint molecules, which could otherwise interfere with chromosome segregation and lead to genetic instability. PMID:27390022

  14. Srs2 and Mus81-Mms4 Prevent Accumulation of Toxic Inter-Homolog Recombination Intermediates.

    Directory of Open Access Journals (Sweden)

    Kenji Keyamura

    2016-07-01

    Full Text Available Homologous recombination is an evolutionally conserved mechanism that promotes genome stability through the faithful repair of double-strand breaks and single-strand gaps in DNA, and the recovery of stalled or collapsed replication forks. Saccharomyces cerevisiae ATP-dependent DNA helicase Srs2 (a member of the highly conserved UvrD family of helicases has multiple roles in regulating homologous recombination. A mutation (srs2K41A resulting in a helicase-dead mutant of Srs2 was found to be lethal in diploid, but not in haploid, cells. In diploid cells, Srs2K41A caused the accumulation of inter-homolog joint molecule intermediates, increased the levels of spontaneous Rad52 foci, and induced gross chromosomal rearrangements. Srs2K41A lethality and accumulation of joint molecules were suppressed by inactivating Rad51 or deleting the Rad51-interaction domain of Srs2, whereas phosphorylation and sumoylation of Srs2 and its interaction with sumoylated proliferating cell nuclear antigen (PCNA were not required for lethality. The structure-specific complex of crossover junction endonucleases Mus81 and Mms4 was also required for viability of diploid, but not haploid, SRS2 deletion mutants (srs2Δ, and diploid srs2Δ mus81Δ mutants accumulated joint molecule intermediates. Our data suggest that Srs2 and Mus81-Mms4 have critical roles in preventing the formation of (or in resolving toxic inter-homolog joint molecules, which could otherwise interfere with chromosome segregation and lead to genetic instability.

  15. FBH1 influences DNA replication fork stability and homologous recombination through ubiquitylation of RAD51

    DEFF Research Database (Denmark)

    Chu, Wai Kit; Payne, Miranda J; Beli, Petra;

    2015-01-01

    leads to hyperrecombination, as well as several phenotypes indicative of an altered response to DNA replication stress. These effects are likely to be mediated by the enhanced nuclear matrix association of the ubiquitylation-resistant RAD51. These data are consistent with FBH1 acting as a negative......Unscheduled homologous recombination (HR) can lead to genomic instability, which greatly increases the threat of neoplastic transformation in humans. The F-box DNA helicase 1 (FBH1) is a 3'-5' DNA helicase with a putative function as a negative regulator of HR. It is the only known DNA helicase to...

  16. Targeting of human aFGF gene into silkworm, Bombyx mori L.Through homologous recombination

    Institute of Scientific and Technical Information of China (English)

    吴小锋; 曹翠平

    2004-01-01

    The long-arm and short-arm genes of fibroin light chain (L-chain) of silkworm, Bombyx Mori L., and the gene of human acidic fibroblast growth factor were cloned respectively and subsequently inserted into a transfer vector pVL1392 used as a tool to target the L-chain region of the silkworm genome. Genomic DNA from their offsprings was extracted and the expected targeting was detected using polymerase chain reaction and DNA sequencing, as well as protein analysis. The results showed that positive events occurred and that the FGF gene was integrated into the L-chain locus through homologous recombination.

  17. Early days of DNA repair: discovery of nucleotide excision repair and homology-dependent recombinational repair.

    Science.gov (United States)

    Rupp, W Dean

    2013-12-13

    The discovery of nucleotide excision repair in 1964 showed that DNA could be repaired by a mechanism that removed the damaged section of a strand and replaced it accurately by using the remaining intact strand as the template. This result showed that DNA could be actively metabolized in a process that had no precedent. In 1968, experiments describing postreplication repair, a process dependent on homologous recombination, were reported. The authors of these papers were either at Yale University or had prior Yale connections. Here we recount some of the events leading to these discoveries and consider the impact on further research at Yale and elsewhere.

  18. Construction of a novel kind of expression plasmid by homologous recombination in Saccharomyces cerevisiae

    Institute of Scientific and Technical Information of China (English)

    CHEN; Xiangling

    2005-01-01

    [1]Brunelli, J. P., Pall, M. L., A series of yeast vectors for expression of cDNAs and other DNA sequences, Yeast, 1993, 9: 1299―1308.[2]Sikorski, R. S., Hieter, P., A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae, Genetics, 1989, 122: 19―27.[3]Bonneaud, N., Ozier-Kalogerogoulos, O., Li, G. et al., A family of low and high copy replicative, integrative and single-stranded S. cerevisiae /E. coli shuttle vector, Yeast, 1991, 7: 609―615.[4]Huo, K. K., Yu, L. L., Chen, X. J., Li, Y. Y., A stable vector for high-level expression and secretion of human interferon alpha A in yeast, Science in China, Ser. B, 1993, 36(5): 557―567.[5]Zhou, Z. X., Yuan, H. Y., He, W. et al., Expression of the modified HBsAg gene SA-28 directed by a constitutive promoter, Journal of Fudan university (Natural Science), 2000, 39(3): 264―268.[6]Paques, F., Haber, J. E., Multiple pathways of recombination induces by double-strand breaks in Saccharomyces cerevisiae, Microbiology and Molecular Biology Reviews, 1999, 63(2): 349―404.[7]Martin, K., Damage-induced recombination in the yeast Saccharomyces cerevisiae, Mutation Research, 2000, 451: 91―105.[8]Alira, S., Tomoko, O., Homologous recombination and the roles of double-strand breaks, TIBS, 1995, 20: 387―391.[9]Patrick, S., Kelly, M. T., Stephen, V. K., Recombination factor of Saccharomyces cerevisiae, Mutation Research, 2000, 451: 257―275.[10]Manivasakam, P., Weber, S. C., McElver, J., Schiestl, R. H., Micro-homology mediated PCR targeting in Saccharomyces cerevisiae, Nucleic Acids Res., 1995, 23(14): 2799―2800.[11]Baudin, A., Lacroute, F., Cullin, C., A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae, Nucleic Acids Res., 1993, 21(14): 3329―3330.[12]Hua, S. B., Qiu, M., Chan, E., Zhu, L., Luo, Y., Minimum length of sequence homology required for in vivo cloning by homolo-gous recombination in yeast, Plasmid, 1997, 38

  19. Construction of recombinant industrial Saccharomyces cerevisiae strain with bglS gene insertion into PEP4 locus by homologous recombination

    Institute of Scientific and Technical Information of China (English)

    Qiang ZHANG; Qi-he CHEN; Ming-liang FU; Jin-ling WANG; Hong-bo ZHANG; Guo-qing HE

    2008-01-01

    The bglS gene encoding endo-1,3-1,4-β-glucanase from Bacillus subtilis was cloned and sequenced in this study. The bglS expression cassette, including PGK1 promoter, bglS gene fused to the signal sequence of the yeast mating pheromone α-factor (MFals), and ADH1 terminator with G418-resistance as the selected marker, was constructed. Then one of the PEP4 allele of Saccharomyces cerevisiae WZ65 strain was replaced by bglS expression cassette using chromosomal integration of polymerase chain reaction (PCR)-mediated homologous recombination, and the bglS gene was expressed simultaneously. The recombinant strain S. cerevisiae (SC-βG) was preliminarily screened by the clearing hydrolysis zone formed after the barley β-glucan was hydrolyzed in the plate and no proteinase A (PrA) activity was measured in fermenting liquor. The results of PCR analysis ofgenome DNA showed that one of the PEP4 allele had been replaced and bglS gene had been inserted into the locus of PEP4 gene in recombinant strains. Different endo-1,3-1,4-β-glucanase assay methods showed that the recombinant strain SC-βG had high endo-1,3-1,4-β-glucanase expression level with the maximum of 69.3 U/(h-ml) after 60 h of incubation. Meanwhile, the Congo Red method was suitable for the determination of endo-1,3-1,4-β-glucanase activity during the actual brewing process. The current research implies that the constructed yeast strain could be utilized to improve the industrial brewing property of beer.

  20. Gene Targeting Using Homologous Recombination in Embryonic Stem Cells: The Future for Behavior Genetics?

    Science.gov (United States)

    Gerlai, Robert

    2016-01-01

    Gene targeting with homologous recombination in embryonic stem cells created a revolution in the analysis of the function of genes in behavioral brain research. The technology allowed unprecedented precision with which one could manipulate genes and study the effect of this manipulation on the central nervous system. With gene targeting, the uncertainty inherent in psychopharmacology regarding whether a particular compound would act only through a specific target was removed. Thus, gene targeting became highly popular. However, with this popularity came the realization that like other methods, gene targeting also suffered from some technical and principal problems. For example, two decades ago, issues about compensatory changes and about genetic linkage were raised. Since then, the technology developed, and its utility has been better delineated. This review will discuss the pros and cons of the technique along with these advancements from the perspective of the neuroscientist user. It will also compare and contrast methods that may represent novel alternatives to the homologous recombination based gene targeting approach, including the TALEN and the CRISPR/Cas9 systems. The goal of the review is not to provide detailed recipes, but to attempt to present a short summary of these approaches a behavioral geneticist or neuroscientist may consider for the analysis of brain function and behavior. PMID:27148349

  1. Construction of a novel kind of expression plasmid by homologous recombination in Saccharomyces cerevisiae

    Institute of Scientific and Technical Information of China (English)

    CHEN Xiangling; YUAN Hanying; HE Wei; HU Xianghua; LU Hong; LI Yuyang

    2005-01-01

    Based on a previously used plasmid pHC11, a new plasmid pHC11R was constructed. Cutting plasmid pHC11R with proper restriction enzymes, the resulting larger DNA fragment pHC11R' was co-transformed with a PCR amplified expression cassette of human IFNα2b into yeast. By means of the homologous sequences at both ends of two DNA fragments, a novel expression plasmid pHC11R-IFNα2b was formed via homologous recombination in the yeast. Compared with pHC11-IFNα2b, the expression plasmid pHC11R-IFNα2b was smaller in size and in absence of antibiotic resistant gene. The stability and copy number of pHC11R- IFNα2b were greatly increased and the expression level of heterologous protein was improved. As the derivatives of pHC11R, a series of recombination expression vectors pHRs containing different combination of expression elements were developed. This led to a rapid and powerful method for cloning and expressing of different genes in yeast.

  2. CasHRA (Cas9-facilitated Homologous Recombination Assembly) method of constructing megabase-sized DNA.

    Science.gov (United States)

    Zhou, Jianting; Wu, Ronghai; Xue, Xiaoli; Qin, Zhongjun

    2016-08-19

    Current DNA assembly methods for preparing highly purified linear subassemblies require complex and time-consuming in vitro manipulations that hinder their ability to construct megabase-sized DNAs (e.g. synthetic genomes). We have developed a new method designated 'CasHRA (Cas9-facilitated Homologous Recombination Assembly)' that directly uses large circular DNAs in a one-step in vivo assembly process. The large circular DNAs are co-introduced into Saccharomyces cerevisiae by protoplast fusion, and they are cleaved by RNA-guided Cas9 nuclease to release the linear DNA segments for subsequent assembly by the endogenous homologous recombination system. The CasHRA method allows efficient assembly of multiple large DNA segments in vivo; thus, this approach should be useful in the last stage of genome construction. As a proof of concept, we combined CasHRA with an upstream assembly method (Gibson procedure of genome assembly) and successfully constructed a 1.03 Mb MGE-syn1.0 (Minimal Genome of Escherichia coli) that contained 449 essential genes and 267 important growth genes. We expect that CasHRA will be widely used in megabase-sized genome constructions. PMID:27220470

  3. Remodeling and Control of Homologous Recombination by DNA Helicases and Translocases that Target Recombinases and Synapsis.

    Science.gov (United States)

    Northall, Sarah J; Ivančić-Baće, Ivana; Soultanas, Panos; Bolt, Edward L

    2016-01-01

    Recombinase enzymes catalyse invasion of single-stranded DNA (ssDNA) into homologous duplex DNA forming "Displacement loops" (D-loops), a process called synapsis. This triggers homologous recombination (HR), which can follow several possible paths to underpin DNA repair and restart of blocked and collapsed DNA replication forks. Therefore, synapsis can be a checkpoint for controlling whether or not, how far, and by which pathway, HR proceeds to overcome an obstacle or break in a replication fork. Synapsis can be antagonized by limiting access of a recombinase to ssDNA and by dissociation of D-loops or heteroduplex formed by synapsis. Antagonists include DNA helicases and translocases that are identifiable in eukaryotes, bacteria and archaea, and which target synaptic and pre-synaptic DNA structures thereby controlling HR at early stages. Here we survey these events with emphasis on enabling DNA replication to be resumed from sites of blockage or collapse. We also note how knowledge of anti-recombination activities could be useful to improve efficiency of CRISPR-based genome editing. PMID:27548227

  4. Gene targeting using homologous recombination in embryonic stem cells: The future for behavior genetics?

    Directory of Open Access Journals (Sweden)

    Robert eGerlai

    2016-04-01

    Full Text Available Gene targeting with homologous recombination in embryonic stem cells created a revolution in the analysis of the function of genes in behavioral brain research. The technology allowed unprecedented precision with which one could manipulate genes and study the effect of this manipulation on the central nervous system. With gene targeting, the uncertainty inherent in psychopharmacology regarding whether a particular compound would act only through a specific target was removed. Thus, gene targeting became highly popular. However, with this popularity came the realization that like other methods, gene targeting also suffered from some technical and principal problems. For example, two decades ago, issues about compensatory changes and about genetic linkage were raised. Since then, the technology developed, and its utility has been better delineated. This review will discuss the pros and cons of the technique along with these advancements from the perspective of the neuroscientist user. It will also compare and contrast methods that may represent novel alternatives to the homologous recombination based gene targeting approach, including the TALEN and the CRISPR/Cas9 systems. The goal of the review is not to provide detailed recipes, but to attempt to present a short summary of these approaches a behavioral geneticist or neuroscientist may consider for the analysis of brain function and behavior.

  5. Gene Targeting Using Homologous Recombination in Embryonic Stem Cells: The Future for Behavior Genetics?

    Science.gov (United States)

    Gerlai, Robert

    2016-01-01

    Gene targeting with homologous recombination in embryonic stem cells created a revolution in the analysis of the function of genes in behavioral brain research. The technology allowed unprecedented precision with which one could manipulate genes and study the effect of this manipulation on the central nervous system. With gene targeting, the uncertainty inherent in psychopharmacology regarding whether a particular compound would act only through a specific target was removed. Thus, gene targeting became highly popular. However, with this popularity came the realization that like other methods, gene targeting also suffered from some technical and principal problems. For example, two decades ago, issues about compensatory changes and about genetic linkage were raised. Since then, the technology developed, and its utility has been better delineated. This review will discuss the pros and cons of the technique along with these advancements from the perspective of the neuroscientist user. It will also compare and contrast methods that may represent novel alternatives to the homologous recombination based gene targeting approach, including the TALEN and the CRISPR/Cas9 systems. The goal of the review is not to provide detailed recipes, but to attempt to present a short summary of these approaches a behavioral geneticist or neuroscientist may consider for the analysis of brain function and behavior.

  6. Emergence of pathogenic coronaviruses in cats by homologous recombination between feline and canine coronaviruses.

    Directory of Open Access Journals (Sweden)

    Yutaka Terada

    Full Text Available Type II feline coronavirus (FCoV emerged via double recombination between type I FCoV and type II canine coronavirus (CCoV. In this study, two type I FCoVs, three type II FCoVs and ten type II CCoVs were genetically compared. The results showed that three Japanese type II FCoVs, M91-267, KUK-H/L and Tokyo/cat/130627, also emerged by homologous recombination between type I FCoV and type II CCoV and their parent viruses were genetically different from one another. In addition, the 3'-terminal recombination sites of M91-267, KUK-H/L and Tokyo/cat/130627 were different from one another within the genes encoding membrane and spike proteins, and the 5'-terminal recombination sites were also located at different regions of ORF1. These results indicate that at least three Japanese type II FCoVs emerged independently. Sera from a cat experimentally infected with type I FCoV was unable to neutralize type II CCoV infection, indicating that cats persistently infected with type I FCoV may be superinfected with type II CCoV. Our previous study reported that few Japanese cats have antibody against type II FCoV. All of these observations suggest that type II FCoV emerged inside the cat body and is unable to readily spread among cats, indicating that these recombination events for emergence of pathogenic coronaviruses occur frequently.

  7. RSC facilitates Rad59-dependent homologous recombination between sister chromatids by promoting cohesin loading at DNA double-strand breaks.

    Science.gov (United States)

    Oum, Ji-Hyun; Seong, Changhyun; Kwon, Youngho; Ji, Jae-Hoon; Sid, Amy; Ramakrishnan, Sreejith; Ira, Grzegorz; Malkova, Anna; Sung, Patrick; Lee, Sang Eun; Shim, Eun Yong

    2011-10-01

    Homologous recombination repairs DNA double-strand breaks by searching for, invading, and copying information from a homologous template, typically the homologous chromosome or sister chromatid. Tight wrapping of DNA around histone octamers, however, impedes access of repair proteins to DNA damage. To facilitate DNA repair, modifications of histones and energy-dependent remodeling of chromatin are required, but the precise mechanisms by which chromatin modification and remodeling enzymes contribute to homologous DNA repair are unknown. Here we have systematically assessed the role of budding yeast RSC (remodel structure of chromatin), an abundant, ATP-dependent chromatin-remodeling complex, in the cellular response to spontaneous and induced DNA damage. RSC physically interacts with the recombination protein Rad59 and functions in homologous recombination. Multiple recombination assays revealed that RSC is uniquely required for recombination between sister chromatids by virtue of its ability to recruit cohesin at DNA breaks and thereby promoting sister chromatid cohesion. This study provides molecular insights into how chromatin remodeling contributes to DNA repair and maintenance of chromatin fidelity in the face of DNA damage.

  8. Genetic battle between Helicobacter pylori and humans. The mechanism underlying homologous recombination in bacteria, which can infect human cells.

    Science.gov (United States)

    Hanada, Katsuhiro; Yamaoka, Yoshio

    2014-10-01

    Helicobacter pylori is a gram-negative pathogenic bacterium that colonises the human stomach. The chronic infection it causes results in peptic ulcers and gastric cancers. H. pylori can easily establish a chronic infection even if the immune system attacks this pathogen with oxidative stress agents and immunoglobulins. This is attributed to bacterial defence mechanisms against these stresses. As a defence mechanism against oxidative stresses, in bacterial genomes, homologous recombination can act as a repair pathway of DNA's double-strand breaks (DSBs). Moreover, homologous recombination is also involved in the antigenic variation in H. pylori. Gene conversion alters genomic structures of babA and babB (encoding outer membrane proteins), resulting in escape from immunoglobulin attacks. Thus, homologous recombination in bacteria plays an important role in the maintenance of a chronic infection. In addition, H. pylori infection causes DSBs in human cells. Homologous recombination is also involved in the repair of DSBs in human cells. In this review, we describe the roles of homologous recombination with an emphasis on the maintenance of a chronic infection.

  9. Transient stability of DNA ends allows nonhomologous end joining to precede homologous recombination.

    Science.gov (United States)

    Frank-Vaillant, Marie; Marcand, Stéphane

    2002-11-01

    The stability of DNA ends generated by the HO endonuclease in yeast is surprisingly high with a half-life of more than an hour. This transient stability is unaffected by mutations that abolish nonhomologous end joining (NHEJ). The unprocessed ends interact with Yku70p and Yku80p, two proteins required for NHEJ, but not significantly with Rad52p, a protein involved in homologous recombination (HR). Repair of a double-strand break by NHEJ is unaffected by the possibility of HR, although the use of HR is increased in NHEJ-defective cells. Partial in vitro 5' strand processing suppresses NHEJ but not HR. These results show that NHEJ precedes HR temporally, and that the availability of substrate dictates the particular pathway used. We propose that transient stability of DNA ends is a foundation for the permanent stability of telomeres. PMID:12453425

  10. Identification of the MMS22L-TONSL complex that promotes homologous recombination

    DEFF Research Database (Denmark)

    Duro, Eris; Lundin, Cecilia; Ask, Katrine;

    2010-01-01

    Budding yeast Mms22 is required for homologous recombination (HR)-mediated repair of stalled or broken DNA replication forks. Here we identify a human Mms22-like protein (MMS22L) and an MMS22L-interacting protein, NF¿BIL2/TONSL. Depletion of MMS22L or TONSL from human cells causes a high level of...... double-strand breaks (DSBs) during DNA replication. Both proteins accumulate at stressed replication forks, and depletion of MMS22L or TONSL from cells causes hypersensitivity to agents that cause S phase-associated DSBs, such as topoisomerase (TOP) inhibitors. In this light, MMS22L and TONSL are...... stability when unscheduled DSBs occur in the vicinity of DNA replication forks....

  11. Establishing targeted carp TLR22 gene disruption via homologous recombination using CRISPR/Cas9.

    Science.gov (United States)

    Chakrapani, Vemulawada; Patra, Swagat Kumar; Panda, Rudra Prasanna; Rasal, Kiran Dashrath; Jayasankar, Pallipuram; Barman, Hirak Kumar

    2016-08-01

    Recent advances in gene editing techniques have not been exploited in farmed fishes. We established a gene targeting technique, using the CRISPR/Cas9 system in Labeo rohita, a farmed carp (known as rohu). We demonstrated that donor DNA was integrated via homologous recombination (HR) at the site of targeted double-stranded nicks created by CRISPR/Cas9 nuclease. This resulted in the successful disruption of rohu Toll-like receptor 22 (TLR22) gene, involved in innate immunity and exclusively present in teleost fishes and amphibians. The null mutant, thus, generated lacked TLR22 mRNA expression. Altogether, this is the first evidence that the CRISPR/Cas9 system is a highly efficient tool for targeted gene disruption via HR in teleosts for generating model large-bodied farmed fishes. PMID:27079451

  12. A rationally designed peptide enhances homologous recombination in vitro and resistance to DNA damaging agents in vivo

    OpenAIRE

    Chen, Li-Tzu; Wang, Andrew H.-J.

    2010-01-01

    The RecA family of proteins is essential in homologous recombination, a critical step in DNA repair. Here, we report that a rationally-designed small peptide based on the crystal structure of Escherichia coli RecA–DNA complex can promote homologous recombination through the enhancement of both RecA-mediated strand assimilation and three-strand exchange activity. Among 17 peptides tested, peptide #3 with the amino acid sequence of IRFLTARRR has the most potent activity in promoting the RecA-me...

  13. Homologous recombination mediates functional recovery of dysferlin deficiency following AAV5 gene transfer.

    Directory of Open Access Journals (Sweden)

    William E Grose

    Full Text Available The dysferlinopathies comprise a group of untreatable muscle disorders including limb girdle muscular dystrophy type 2B, Miyoshi myopathy, distal anterior compartment syndrome, and rigid spine syndrome. As with other forms of muscular dystrophy, adeno-associated virus (AAV gene transfer is a particularly auspicious treatment strategy, however the size of the DYSF cDNA (6.5 kb negates packaging into traditional AAV serotypes known to express well in muscle (i.e. rAAV1, 2, 6, 8, 9. Potential advantages of a full cDNA versus a mini-gene include: maintaining structural-functional protein domains, evading protein misfolding, and avoiding novel epitopes that could be immunogenic. AAV5 has demonstrated unique plasticity with regards to packaging capacity and recombination of virions containing homologous regions of cDNA inserts has been implicated in the generation of full-length transcripts. Herein we show for the first time in vivo that homologous recombination following AAV5.DYSF gene transfer leads to the production of full length transcript and protein. Moreover, gene transfer of full-length dysferlin protein in dysferlin deficient mice resulted in expression levels sufficient to correct functional deficits in the diaphragm and importantly in skeletal muscle membrane repair. Intravascular regional gene transfer through the femoral artery produced high levels of transduction and enabled targeting of specific muscle groups affected by the dysferlinopathies setting the stage for potential translation to clinical trials. We provide proof of principle that AAV5 mediated delivery of dysferlin is a highly promising strategy for treatment of dysferlinopathies and has far-reaching implications for the therapeutic delivery of other large genes.

  14. p53 gene targeting by homologous recombination in fish ES cells.

    Directory of Open Access Journals (Sweden)

    Yan Yan

    Full Text Available BACKGROUND: Gene targeting (GT provides a powerful tool for the generation of precise genetic alterations in embryonic stem (ES cells to elucidate gene function and create animal models for human diseases. This technology has, however, been limited to mouse and rat. We have previously established ES cell lines and procedures for gene transfer and selection for homologous recombination (HR events in the fish medaka (Oryzias latipes. METHODOLOGY AND PRINCIPAL FINDINGS: Here we report HR-mediated GT in this organism. We designed a GT vector to disrupt the tumor suppressor gene p53 (also known as tp53. We show that all the three medaka ES cell lines, MES1∼MES3, are highly proficient for HR, as they produced detectable HR without drug selection. Furthermore, the positive-negative selection (PNS procedure enhanced HR by ∼12 folds. Out of 39 PNS-resistant colonies analyzed, 19 (48.7% were positive for GT by PCR genotyping. When 11 of the PCR-positive colonies were further analyzed, 6 (54.5% were found to be bona fide homologous recombinants by Southern blot analysis, sequencing and fluorescent in situ hybridization. This produces a high efficiency of up to 26.6% for p53 GT under PNS conditions. We show that p53 disruption and long-term propagation under drug selection conditions do not compromise the pluripotency, as p53-targeted ES cells retained stable growth, undifferentiated phenotype, pluripotency gene expression profile and differentiation potential in vitro and in vivo. CONCLUSIONS: Our results demonstrate that medaka ES cells are proficient for HR-mediated GT, offering a first model organism of lower vertebrates towards the development of full ES cell-based GT technology.

  15. Biochemical analysis of the human ENA/VASP-family proteins, MENA, VASP and EVL, in homologous recombination.

    Science.gov (United States)

    Takaku, Motoki; Ueno, Hiroyuki; Kurumizaka, Hitoshi

    2011-06-01

    MENA, VASP and EVL are members of the ENA/VASP family of proteins and are involved in cytoplasmic actin remodeling. Previously, we found that EVL directly interacts with RAD51, an essential protein in the homologous recombinational repair of double-strand breaks (DSBs) and stimulates the RAD51-mediated recombination reactions in vitro. The EVL-knockdown MCF7 cells exhibited a clear reduction in RAD51-foci formation, suggesting that EVL may function in the DSB repair pathway through RAD51-mediated homologous recombination. However, the DSB repair defects were less significant in the EVL-knockdown cells, implying that two EVL paralogues, MENA and VASP, may complement the EVL function in human cells. Therefore, in the present study, we purified human MENA, VASP and EVL as recombinant proteins, and compared their biochemical activities in vitro. We found that all three proteins commonly exhibited the RAD51 binding, DNA binding and DNA-annealing activities. Stimulation of the RAD51-mediated homologous pairing was also observed with all three proteins. In addition, surface plasmon resonance analyses revealed that MENA, VASP and EVL mutually interacted. These results support the ideas that the ENA/VASP-family proteins are functionally redundant in homologous recombination, and that all three may be involved in the DSB repair pathway in humans.

  16. Promotion of Homologous Recombination by SWS-1 in Complex with RAD-51 Paralogs in Caenorhabditis elegans.

    Science.gov (United States)

    McClendon, T Brooke; Sullivan, Meghan R; Bernstein, Kara A; Yanowitz, Judith L

    2016-05-01

    Homologous recombination (HR) repairs cytotoxic DNA double-strand breaks (DSBs) with high fidelity. Deficiencies in HR result in genome instability. A key early step in HR is the search for and invasion of a homologous DNA template by a single-stranded RAD-51 nucleoprotein filament. The Shu complex, composed of a SWIM domain-containing protein and its interacting RAD51 paralogs, promotes HR by regulating RAD51 filament dynamics. Despite Shu complex orthologs throughout eukaryotes, our understanding of its function has been most extensively characterized in budding yeast. Evolutionary analysis of the SWIM domain identified Caenorhabditis elegans sws-1 as a putative homolog of the yeast Shu complex member Shu2. Using a CRISPR-induced nonsense allele of sws-1, we show that sws-1 promotes HR in mitotic and meiotic nuclei. sws-1 mutants exhibit sensitivity to DSB-inducing agents and fail to form mitotic RAD-51 foci following treatment with camptothecin. Phenotypic similarities between sws-1 and the two RAD-51 paralogs rfs-1 and rip-1 suggest that they function together. Indeed, we detect direct interaction between SWS-1 and RIP-1 by yeast two-hybrid assay that is mediated by the SWIM domain in SWS-1 and the Walker B motif in RIP-1 Furthermore, RIP-1 bridges an interaction between SWS-1 and RFS-1, suggesting that RIP-1 facilitates complex formation with SWS-1 and RFS-1 We propose that SWS-1, RIP-1, and RFS-1 compose a C. elegans Shu complex. Our work provides a new model for studying Shu complex disruption in the context of a multicellular organism that has important implications as to why mutations in the human RAD51 paralogs are associated with genome instability. PMID:26936927

  17. Target-selective homologous recombination cloning for high-throughput generation of monoclonal antibodies from single plasma cells

    OpenAIRE

    Isobe Masaharu; Yoshioka Megumi; Kurosawa Nobuyuki

    2011-01-01

    Abstract Background Molecular cloning of functional immunoglobulin genes from single plasma cells is one of the most promising technologies for the rapid development of monoclonal antibody drugs. However, the proper insertion of PCR-amplified immunoglobulin genes into expression vectors remains an obstacle to the high-throughput production of recombinant monoclonal antibodies. Results We developed a single-step cloning method, target-selective homologous recombination (TS-HR), in which PCR-am...

  18. A versatile bacterial expression vector designed for single-step cloning of multiple DNA fragments using homologous recombination

    OpenAIRE

    Holmberg, Mats A.; Gowda, Naveen Kumar Chandappa; Andréasson, Claes

    2014-01-01

    Production of recombinant proteins is the starting point for biochemical and biophysical analyses and requires methodology to efficiently proceed from gene sequence to purified protein. While optimized strategies for the efficient cloning of single-gene fragments for bacterial expression is available, efficient multiple DNA fragment cloning still presents a challenge. To facilitate this step, we have developed an efficient cloning strategy based on yeast homologous recombination cloning (YHRC...

  19. A pan-cancer analysis of inferred homologous recombination deficiency identifies potential platinum benefit in novel subtypes

    DEFF Research Database (Denmark)

    Marquard, Andrea Marion; Eklund, Aron Charles; Wang, Zhigang C.;

    2014-01-01

    Personalized medicine in cancer aims to improve treatment outcome, by exploiting the molecular alterations of the individual tumor to inform therapeutic decisions. Ovarian and triple-negative breast cancers with defects in homologous recombination (HR) DNA repair are highly sensitive to treatment...

  20. Target-selective homologous recombination cloning for high-throughput generation of monoclonal antibodies from single plasma cells

    Directory of Open Access Journals (Sweden)

    Isobe Masaharu

    2011-04-01

    Full Text Available Abstract Background Molecular cloning of functional immunoglobulin genes from single plasma cells is one of the most promising technologies for the rapid development of monoclonal antibody drugs. However, the proper insertion of PCR-amplified immunoglobulin genes into expression vectors remains an obstacle to the high-throughput production of recombinant monoclonal antibodies. Results We developed a single-step cloning method, target-selective homologous recombination (TS-HR, in which PCR-amplified immunoglobulin variable genes were selectively inserted into vectors, even in the presence of nonspecifically amplified DNA. TS-HR utilizes Red/ET-mediated homologous recombination with a target-selective vector (TS-vector with unique homology arms on its termini. Using TS-HR, immunoglobulin variable genes were cloned directly into expression vectors by co-transforming unpurified PCR products and the TS-vector into E. coli. Furthermore, the high cloning specificity of TS-HR allowed plasmids to be extracted from pools of transformed bacteria without screening single colonies for correct clones. We present a one-week protocol for the production of recombinant mouse monoclonal antibodies from large numbers of single plasma cells. Conclusion The time requirements and limitations of traditional cloning procedures for the production of recombinant immunoglobulins have been significantly reduced with the development of the TS-HR cloning technique.

  1. Prolonged Particulate Hexavalent Chromium Exposure Suppresses Homologous Recombination Repair in Human Lung Cells.

    Science.gov (United States)

    Browning, Cynthia L; Qin, Qin; Kelly, Deborah F; Prakash, Rohit; Vanoli, Fabio; Jasin, Maria; Wise, John Pierce

    2016-09-01

    Genomic instability is one of the primary models of carcinogenesis and a feature of almost all cancers. Homologous recombination (HR) repair protects against genomic instability by maintaining high genomic fidelity during the repair of DNA double strand breaks. The defining step of HR repair is the formation of the Rad51 nucleofilament, which facilitates the search for a homologous sequence and invasion of the template DNA strand. Particulate hexavalent chromium (Cr(VI)), a human lung carcinogen, induces DNA double strand breaks and chromosome instability. Since the loss of HR repair increases Cr(VI)-induced chromosome instability, we investigated the effect of extended Cr(VI) exposure on HR repair. We show acute (24 h) Cr(VI) exposure induces a normal HR repair response. In contrast, prolonged (120 h) exposure to particulate Cr(VI) inhibited HR repair and Rad51 nucleofilament formation. Prolonged Cr(VI) exposure had a profound effect on Rad51, evidenced by reduced protein levels and Rad51 mislocalization to the cytoplasm. The response of proteins involved in Rad51 nuclear import and nucleofilament formation displayed varying responses to prolonged Cr(VI) exposure. BRCA2 formed nuclear foci after prolonged Cr(VI) exposure, while Rad51C foci formation was suppressed. These results suggest that particulate Cr(VI), a major chemical carcinogen, inhibits HR repair by targeting Rad51, causing DNA double strand breaks to be repaired by a low fidelity, Rad51-independent repair pathway. These results further enhance our understanding of the underlying mechanism of Cr(VI)-induced chromosome instability and thus, carcinogenesis. PMID:27449664

  2. Mitochondrial genome rearrangements in glomus species triggered by homologous recombination between distinct mtDNA haplotypes.

    Science.gov (United States)

    Beaudet, Denis; Terrat, Yves; Halary, Sébastien; de la Providencia, Ivan Enrique; Hijri, Mohamed

    2013-01-01

    Comparative mitochondrial genomics of arbuscular mycorrhizal fungi (AMF) provide new avenues to overcome long-lasting obstacles that have hampered studies aimed at understanding the community structure, diversity, and evolution of these multinucleated and genetically polymorphic organisms.AMF mitochondrial (mt) genomes are homogeneous within isolates, and their intergenic regions harbor numerous mobile elements that have rapidly diverged, including homing endonuclease genes, small inverted repeats, and plasmid-related DNA polymerase genes (dpo), making them suitable targets for the development of reliable strain-specific markers. However, these elements may also lead to genome rearrangements through homologous recombination, although this has never previously been reported in this group of obligate symbiotic fungi. To investigate whether such rearrangements are present and caused by mobile elements in AMF, the mitochondrial genomes from two Glomeraceae members (i.e., Glomus cerebriforme and Glomus sp.) with substantial mtDNA synteny divergence,were sequenced and compared with available glomeromycotan mitochondrial genomes. We used an extensive nucleotide/protein similarity network-based approach to investigated podiversity in AMF as well as in other organisms for which sequences are publicly available. We provide strong evidence of dpo-induced inter-haplotype recombination, leading to a reshuffled mitochondrial genome in Glomus sp. These findings raise questions as to whether AMF single spore cultivations artificially underestimate mtDNA genetic diversity.We assessed potential dpo dispersal mechanisms in AMF and inferred a robust phylogenetic relationship with plant mitochondrial plasmids. Along with other indirect evidence, our analyses indicate that members of the Glomeromycota phylum are potential donors of mitochondrial plasmids to plants.

  3. Homologous recombination in mammalian cells: effect of p53 and Bcl-2 proteins, replication inhibition and ionizing radiations

    International Nuclear Information System (INIS)

    The control of cell cycle, associated with the mechanisms of replication, DNA repair/recombination allows the cells to maintain their genetic integrity. The p53 protein ensures the control of G1/S transition. Its inactivation would allow to initial replication on damaged matrix and lead to the block of replication forks followed by DNA strand breaks, good substrates for recombination. This work shows that the expression of mutant p53 protein stimulates both spontaneous and radio-induced homologous recombination, independently of the control of cell cycle. Moreover, the use of a set of replication inhibitors show that inhibition of the replication elongation stimulates recombination more strongly than the initiation inhibition. Replication arrest by these inhibitors also significantly increases the number of DNA strand breaks. These results highlighted a point of action of p53 protein on the ultimate stages of the homologous recombination mechanism. Lastly, the expression of Bcl-2 protein inhibits apoptosis and increases survival, but specifically inhibits conservative recombination, after radiation as well as in absence of apoptotic stress. The extinction of this mechanism of DNA repair is associated with an increase of mutagenesis. Taken together, these results allow ta consider the maintenance of the genetic stability as a cellular network involving different pathways. A multiple stages model for tumoral progression can be deduced. (author)

  4. DNA homologous recombination factor SFR1 physically and functionally interacts with estrogen receptor alpha.

    Directory of Open Access Journals (Sweden)

    Yuxin Feng

    Full Text Available Estrogen receptor alpha (ERα, a ligand-dependent transcription factor, mediates the expression of its target genes by interacting with corepressors and coactivators. Since the first cloning of SRC1, more than 280 nuclear receptor cofactors have been identified, which orchestrate target gene transcription. Aberrant activity of ER or its accessory proteins results in a number of diseases including breast cancer. Here we identified SFR1, a protein involved in DNA homologous recombination, as a novel binding partner of ERα. Initially isolated in a yeast two-hybrid screen, the interaction of SFR1 and ERα was confirmed in vivo by immunoprecipitation and mammalian one-hybrid assays. SFR1 co-localized with ERα in the nucleus, potentiated ER's ligand-dependent and ligand-independent transcriptional activity, and occupied the ER binding sites of its target gene promoters. Knockdown of SFR1 diminished ER's transcriptional activity. Manipulating SFR1 expression by knockdown and overexpression revealed a role for SFR1 in ER-dependent and -independent cancer cell proliferation. SFR1 differs from SRC1 by the lack of an intrinsic activation function. Taken together, we propose that SFR1 is a novel transcriptional modulator for ERα and a potential target in breast cancer therapy.

  5. Gene targeting and transgene stacking using intra genomic homologous recombination in plants.

    Science.gov (United States)

    Kumar, Sandeep; Barone, Pierluigi; Smith, Michelle

    2016-01-01

    Modern agriculture has created a demand for plant biotechnology products that provide durable resistance to insect pests, tolerance of herbicide applications for weed control, and agronomic traits tailored for specific geographies. These transgenic trait products require a modular and sequential multigene stacking platform that is supported by precise genome engineering technology. Designed nucleases have emerged as potent tools for creating targeted DNA double strand breaks (DSBs). Exogenously supplied donor DNA can repair the targeted DSB by a process known as gene targeting (GT), resulting in a desired modification of the target genome. The potential of GT technology has not been fully realized for trait deployment in agriculture, mainly because of inefficient transformation and plant regeneration systems in a majority of crop plants and genotypes. This challenge of transgene stacking in plants could be overcome by Intra-Genomic Homologous Recombination (IGHR) that converts independently segregating unlinked donor and target transgenic loci into a genetically linked molecular stack. The method requires stable integration of the donor DNA into the plant genome followed by intra-genomic mobilization. IGHR complements conventional breeding with genetic transformation and designed nucleases to provide a flexible transgene stacking and trait deployment platform.

  6. Nanog reporter system in mouse embryonic stem cells based on highly efficient BAC homologous recombination

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Nanog is a novel transcription factor specifically expressed in mouse embryonic stem cells (mES cells). It has been reported that Nanog plays an essential role in maintaining multi-potency of ES cells. The expression of Nanog is very sensitive to ES cells differentiation, making Nanog one of the best markers to indicate the status of ES cells. In this study, we developed an efficient method to construct Nanog promoter driven EGFP reporter system based on the BAC homologous recombination. We further generated a Nanog-EGFP reporter mES cell line. This reporter mES cell line exhibited features similar to those of normal mES cells, and the EGFP reporter efficiently reflected the expression of Nanog, indicating the differentiation status of mES cells. We achieved a reliable experimental reporter system to research self-renewal and differentiation of mES cells. The system could facilitate research on culture system of mES cells and researches on the expression and regulation of Nanog and other related factors in mES cells.

  7. Suppression of mutagenesis by Rad51D-mediated homologous recombination

    Energy Technology Data Exchange (ETDEWEB)

    Hinz, J M; Tebbs, R S; Wilson, P F; Nham, P B; Salazar, E P; Nagasawa, H; Urbin, S S; Thompson, L H

    2005-11-15

    Homologous recombinational repair (HRR) restores chromatid breaks arising during DNA replication and prevents chromosomal rearrangements that can occur from the misrepair of such breaks. In vertebrates, five Rad51 paralogs are identified that contribute in a nonessential but critical manner to HRR efficiency. We constructed and characterized a Rad51D knockout cell line in widely studied CHO cells. The rad51d mutant (51D1) displays sensitivity to a wide spectrum of induced DNA damage, indicating the broad relevance of HRR to genotoxicity. Untreated 51D1 cells exhibit {approx}5-fold elevated chromosomal breaks, a 12-fold increased rate of hprt mutation, and 4- to 10-fold increased rates of gene amplification at the dhfr and CAD loci, respectively. These results explicitly show the quantitative importance of HHR in preventing these types genetic alterations, which are associated with carcinogenesis. Thus, HRR copes in an error-free manner with spontaneous DNA damage encountered during DNA replication, and Rad51D is essential for this fidelity.

  8. Perturbing A-to-I RNA editing using genetics and homologous recombination.

    Science.gov (United States)

    Staber, Cynthia J; Gell, Selena; Jepson, James E C; Reenan, Robert A

    2011-01-01

    Evidence for the chemical conversion of adenosine-to-inosine (A-to-I) in messenger RNA (mRNA) has been detected in numerous metazoans, especially those "most successful" phyla: Arthropoda, Mollusca, and Chordata. The requisite enzymes for A-to-I editing, ADARs (adenosine deaminases acting on RNA) are highly conserved and are present in every higher metazoan genome sequenced to date. The fruit fly, Drosophila melanogaster, represents an ideal model organism for studying A-to-I editing, both in terms of fundamental biochemistry and in relation to determining adaptive downstream effects on physiology and behavior. The Drosophila genome contains a single structural gene for ADAR (dAdar), yet the fruit fly transcriptome has the widest range of conserved and validated ADAR targets in coding mRNAs of any known organism. In addition, many of the genes targeted by dADAR have been genetically identified as playing a role in nervous system function, providing a rich source of material to investigate the biological relevance of this intriguing process. Here, we discuss how recent advances in the use of ends-out homologous recombination (HR) in Drosophila make possible both the precise control of the editing status for defined adenosine residues and the engineering of flies with globally altered RNA editing of the fly transcriptome. These new approaches promise to significantly improve our understanding of how mRNA modification contributes to insect physiology and ethology.

  9. Multi-Homologous Recombination-Based Gene Manipulation in the Rice Pathogen Fusarium fujikuroi

    Directory of Open Access Journals (Sweden)

    In Sun Hwang

    2016-06-01

    Full Text Available Gene disruption by homologous recombination is widely used to investigate and analyze the function of genes in Fusarium fujikuroi, a fungus that causes bakanae disease and root rot symptoms in rice. To generate gene deletion constructs, the use of conventional cloning methods, which rely on restriction enzymes and ligases, has had limited success due to a lack of unique restriction enzyme sites. Although strategies that avoid the use of restriction enzymes have been employed to overcome this issue, these methods require complicated PCR steps or are frequently inefficient. Here, we introduce a cloning system that utilizes multi-fragment assembly by In-Fusion to generate a gene disruption construct. This method utilizes DNA fragment fusion and requires only one PCR step and one reaction for construction. Using this strategy, a gene disruption construct for Fusarium cyclin C1 (FCC1 , which is associated with fumonisin B1 biosynthesis, was successfully created and used for fungal transformation. In vivo and in vitro experiments using confirmed fcc1 mutants suggest that fumonisin production is closely related to disease symptoms exhibited by F. fujikuroi strain B14. Taken together, this multi-fragment assembly method represents a simpler and a more convenient process for targeted gene disruption in fungi.

  10. ATM and Artemis promote homologous recombination of radiation-induced DNA double-strand breaks in G2

    OpenAIRE

    Beucher, Andrea; Birraux, Julie; Tchouandong, Leopoldine; Barton, Olivia; Shibata, Atsushi; Conrad, Sandro; Goodarzi, Aaron A.; KREMPLER, ANDREA; Jeggo, Penny; Lo¨brich, Markus

    2009-01-01

    Homologous recombination (HR) and non-homologous end joining (NHEJ) represent distinct pathways for repairing DNA double-strand breaks (DSBs). Previous work implicated Artemis and ATM in an NHEJ-dependent process, which repairs a defined subset of radiation-induced DSBs in G1-phase. Here, we show that in G2, as in G1, NHEJ represents the major DSB-repair pathway whereas HR is only essential for repair of ∼15% of X- or γ-ray-induced DSBs. In addition to requiring the known HR proteins, Brca2, ...

  11. High-throughput plasmid construction using homologous recombination in yeast: its mechanisms and application to protein production for X-ray crystallography.

    Science.gov (United States)

    Mizutani, Kimihiko

    2015-01-01

    Homologous recombination is a system for repairing the broken genomes of living organisms by connecting two DNA strands at their homologous sequences. Today, homologous recombination in yeast is used for plasmid construction as a substitute for traditional methods using restriction enzymes and ligases. This method has various advantages over the traditional method, including flexibility in the position of DNA insertion and ease of manipulation. Recently, the author of this review reported the construction of plasmids by homologous recombination in the methanol-utilizing yeast Pichia pastoris, which is known to be an excellent expression host for secretory proteins and membrane proteins. The method enabled high-throughput construction of expression systems of proteins using P. pastoris; the constructed expression systems were used to investigate the expression conditions of membrane proteins and to perform X-ray crystallography of secretory proteins. This review discusses the mechanisms and applications of homologous recombination, including the production of proteins for X-ray crystallography.

  12. A New Method for Rapidly Generating Gene-Targeting Vectors by Engineering BACs Through Homologous Recombination in Bacteria

    OpenAIRE

    Cotta-de-Almeida, Vinícius; Schonhoff, Susan; Shibata, Tomoyuki; Leiter, Andrew; Snapper, Scott B.

    2003-01-01

    Generating knockout mice is still an expensive and highly time-consuming process. Target construct generation, the first labor-intensive step in this process, requires the manipulation of large fragments of DNA and numerous, and often cumbersome, cloning steps. Here we show the development of a rapid approach for generating targeting constructs that capitalizes on efficient homologous recombination between linear DNA fragments and circular plasmids in Escherichia coli (“recombineering”)...

  13. Generating in vivo cloning vectors for parallel cloning of large gene clusters by homologous recombination.

    Directory of Open Access Journals (Sweden)

    Jeongmin Lee

    Full Text Available A robust method for the in vivo cloning of large gene clusters was developed based on homologous recombination (HR, requiring only the transformation of PCR products into Escherichia coli cells harboring a receiver plasmid. Positive clones were selected by an acquired antibiotic resistance, which was activated by the recruitment of a short ribosome-binding site plus start codon sequence from the PCR products to the upstream position of a silent antibiotic resistance gene in receiver plasmids. This selection was highly stringent and thus the cloning efficiency of the GFPuv gene (size: 0.7 kb was comparable to that of the conventional restriction-ligation method, reaching up to 4.3 × 10(4 positive clones per μg of DNA. When we attempted parallel cloning of GFPuv fusion genes (size: 2.0 kb and carotenoid biosynthesis pathway clusters (sizes: 4 kb, 6 kb, and 10 kb, the cloning efficiency was similarly high regardless of the DNA size, demonstrating that this would be useful for the cloning of large DNA sequences carrying multiple open reading frames. However, restriction analyses of the obtained plasmids showed that the selected cells may contain significant amounts of receiver plasmids without the inserts. To minimize the amount of empty plasmid in the positive selections, the sacB gene encoding a levansucrase was introduced as a counter selection marker in receiver plasmid as it converts sucrose to a toxic levan in the E. coli cells. Consequently, this method yielded completely homogeneous plasmids containing the inserts via the direct transformation of PCR products into E. coli cells.

  14. Gimeracil, an inhibitor of dihydropyrimidine dehydrogenase, inhibits the early step in homologous recombination

    International Nuclear Information System (INIS)

    Gimeracil (5-chloro-2, 4-dihydroxypyridine) is an inhibitor of dihydropyrimidine dehydrogenase (DPYD), which degrades pyrimidine including 5-fluorouracil in the blood. Gimeracil was originally added to an oral fluoropyrimidine derivative S-1 to yield prolonged 5-fluorouracil concentrations in serum and tumor tissues. We have already reported that gimeracil had radiosensitizing effects by partially inhibiting homologous recombination (HR) in the repair of DNA double strand breaks. We investigated the mechanisms of gimeracil radiosensitization. Comet assay and radiation-induced focus formation of various kinds of proteins involved in HR was carried out. Small interfering RNA (siRNA) for DPYD were transfected to HeLa cells to investigate the target protein for radiosensitization with gimeracil. SCneo assay was carried out to examine whether DPYD depletion by siRNA inhibited HR repair of DNA double strand breaks. Tail moments in neutral comet assay increased in gimeracil-treated cells. Gimeracil restrained the formation of foci of Rad51 and replication protein A (RPA), whereas it increased the number of foci of Nbs1, Mre11, Rad50, and FancD2. When HeLa cells were transfected with the DPYD siRNA before irradiation, the cells became more radiosensitive. The degree of radiosensitization by transfection of DPYD siRNA was similar to that of gimeracil. Gimeracil did not sensitize DPYD-depleted cells. Depletion of DPYD by siRNA significantly reduced the frequency of neopositive clones in SCneo assay. Gimeracil partially inhibits the early step in HR. It was found that DPYD is the target protein for radiosensitization by gimeracil. The inhibitors of DPYD, such as gimeracil, could enhance the efficacy of radiotherapy through partial suppression of HR-mediated DNA repair. (author)

  15. Homologous recombination in Arabidopsis seeds along the track of energetic carbon ions

    Energy Technology Data Exchange (ETDEWEB)

    Wang Ting [University of Science and Technology of China, 96 Jinzhai Road, Hefei 230026 (China); Key Laboratory of Ion Beam Bio-engineering, Institute of Technical Biology and Agricultural Engineering, Chinese Academy of Sciences, 350 Shushanhu Road, Hefei 230031 (China); Li Fanghua [Key Laboratory of Ion Beam Bio-engineering, Institute of Technical Biology and Agricultural Engineering, Chinese Academy of Sciences, 350 Shushanhu Road, Hefei 230031 (China); Liu Qingfang [Radiobiology Laboratory, Institute of Modern Physics, Chinese Academy of Sciences, 509 Nanchang Road, Lanzhou 730000 (China); Bian Po, E-mail: bianpo@ipp.ac.cn [Key Laboratory of Ion Beam Bio-engineering, Institute of Technical Biology and Agricultural Engineering, Chinese Academy of Sciences, 350 Shushanhu Road, Hefei 230031 (China); Wang Jufang [Radiobiology Laboratory, Institute of Modern Physics, Chinese Academy of Sciences, 509 Nanchang Road, Lanzhou 730000 (China); Wu Yuejin; Wu Lijun [Key Laboratory of Ion Beam Bio-engineering, Institute of Technical Biology and Agricultural Engineering, Chinese Academy of Sciences, 350 Shushanhu Road, Hefei 230031 (China); Li Wenjian [Radiobiology Laboratory, Institute of Modern Physics, Chinese Academy of Sciences, 509 Nanchang Road, Lanzhou 730000 (China)

    2012-09-01

    Heavy ion irradiation has been used as radiotherapy of deep-seated tumors, and is also an inevitable health concern for astronauts in space mission. Unlike photons such as X-rays and {gamma}-rays, a high linear energy transfer (LET) heavy ion has a varying energy distribution along its track. Therefore, it is important to determine the correlation of biological effects with the Bragg curve energy distribution of heavy ions. In this study, a continuous biological tissue equivalent was constructed using a layered cylinder of Arabidopsis seeds, which was irradiated with carbon ions of 87.5 MeV/nucleon. The position of energy loss peak in the seed pool was determined with CR-39 track detectors. The mutagenic effect in vivo along the path of carbon ions was investigated with the seeds in each layer as an assay unit, which corresponded to a given position in physical Bragg curve. Homologous recombination frequency (HRF), expression level of AtRAD54 gene, germination rate of seeds, and survival rate of young seedlings were used as checking endpoints, respectively. Our results showed that Arabidopsis S0 and S1 plants exhibited significant increases in HRF compared to their controls, and the expression level of AtRAD54 gene in S0 plants was significantly up-regulated. The depth-biological effect curves for HRF and the expression of AtRAD54 gene were not consistent with the physical Bragg curve. Differently, the depth-biological effect curves for the developmental endpoints matched generally with the physical Bragg curve. The results suggested a different response pattern of various types of biological events to heavy ion irradiation. It is also interesting that except for HRF in S0 plants, the depth-biological effect curves for each biological endpoint were similar for 5 Gy and 30 Gy of carbon irradiation.

  16. Efficient assembly of full-length infectious clone of Brazilian IBDV isolate by homologous recombination in yeast

    Directory of Open Access Journals (Sweden)

    J.V.J. Silva Jr.

    2014-12-01

    Full Text Available The Infectious Bursal Disease Virus (IBDV causes immunosuppression in young chickens. Advances in molecular virology and vaccines for IBDV have been achieved by viral reverse genetics (VRG. VRG for IBDV has undergone changes over time, however all strategies used to generate particles of IBDV involves multiple rounds of amplification and need of in vitro ligation and restriction sites. The aim of this research was to build the world's first VRG for IBDV by yeast-based homologous recombination; a more efficient, robust and simple process than cloning by in vitro ligation. The wild type IBDV (Wt-IBDV-Br was isolated in Brazil and had its genome cloned in pJG-CMV-HDR vector by yeast-based homologous recombination. The clones were transfected into chicken embryo fibroblasts and the recovered virus (IC-IBDV-Br showed genetic stability and similar phenotype to Wt-IBDV-Br, which were observed by nucleotide sequence, focus size/morphology and replication kinetics, respectively. Thus, IBDV reverse genetics by yeast-based homologous recombination provides tools to IBDV understanding and vaccines/viral vectors development.

  17. Restriction-ligation-free (RLF) cloning: a high-throughput cloning method by in vivo homologous recombination of PCR products.

    Science.gov (United States)

    Wang, Y; Liu, Y; Chen, J; Tang, M J; Zhang, S L; Wei, L N; Li, C H; Wei, D B

    2015-01-01

    In this study, we optimized a restriction-ligation-free (RLF) method to save time and cost of constructing multiple plasmids with the same gene insert, and examined the efficacy of RLF on high-throughput multi-plasmid cloning. This method utilizes the precise DNA repair and recombination systems within Escherichia coli, which allows to bypass the in vitro restriction and ligation enzyme reactions commonly included in routine cloning procedures. A homologous arm is linked to the 5'-end of the forward primer used to amplify both the target gene and vector. A different homologous arm is linked to the 5'-end of the reverse primer. Therefore, genes can be cloned into the vectors by homologous recombination after co-transformation of the amplified target gene and the linearized vector, which bear the same homologous arm on either end. More than twenty-four different plasmids were generated by this method, which uses two simple polymerase chain reaction steps. This method is highly efficient in cloning any gene of interest into any vector at any site without sequence constraints, as no restriction and ligation reactions are required. PMID:26505379

  18. Gastroesophageal junction adenocarcinoma displays abnormalities in homologous recombination and nucleotide excision repair

    Directory of Open Access Journals (Sweden)

    Dewalt RI

    2014-02-01

    Full Text Available Robin I Dewalt,1 Kenneth A Kesler,2 Zane T Hammoud,3 LeeAnn Baldridge,4 Eyas M Hattab,4 Shadia I Jalal1,5 1Division of Hematology/Oncology, Department of Medicine, 2Cardiothoracic Division, Department of Surgery, Indiana University School of Medicine, Indianapolis, IN, USA; 3Henry Ford Hospital, Detroit, MI, USA; 4Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN, USA; 5Indiana University Melvin and Bren Simon Cancer Center, Indianapolis, IN, USA Objective: Esophageal adenocarcinoma (EAC continues to be a disease associated with high mortality. Among the factors leading to poor outcomes are innate resistance to currently available therapies, advanced stage at diagnosis, and complex biology. Platinum and ionizing radiation form the backbone of treatment for the majority of patients with EAC. Of the multiple processes involved in response to platinum chemotherapy or ionizing radiation, deoxyribonucleic acid (DNA repair has been a major player in cancer sensitivity to these agents. DNA repair defects have been described in various malignancies. The purpose of this study was to determine whether alterations in DNA repair are present in EAC compared with normal gastroesophageal tissues. Methods: We analyzed the expression of genes involved in homologous recombination (HR, nonhomologous end-joining, and nucleotide excision repair (NER pathways in 12 EAC tumor samples with their matched normal counterparts. These pathways were chosen because they are the main pathways involved in the repair of platinum- or ionizing-radiation-induced damage. In addition, abnormalities in these pathways have not been well characterized in EAC. Results: We identified increased expression of at least one HR gene in eight of the EAC tumor samples. Alterations in the expression of EME1, a structure-specific endonuclease involved in HR, were the most prevalent, with messenger (mRNA overexpression in six of the EAC samples

  19. Mutation of the BRCA1 SQ-cluster results in aberrant mitosis, reduced homologous recombination, and a compensatory increase in non-homologous end joining.

    Science.gov (United States)

    Beckta, Jason M; Dever, Seth M; Gnawali, Nisha; Khalil, Ashraf; Sule, Amrita; Golding, Sarah E; Rosenberg, Elizabeth; Narayanan, Aarthi; Kehn-Hall, Kylene; Xu, Bo; Povirk, Lawrence F; Valerie, Kristoffer

    2015-09-29

    Mutations in the breast cancer susceptibility 1 (BRCA1) gene are catalysts for breast and ovarian cancers. Most mutations are associated with the BRCA1 N- and C-terminal domains linked to DNA double-strand break (DSB) repair. However, little is known about the role of the intervening serine-glutamine (SQ) - cluster in the DNA damage response beyond its importance in regulating cell cycle checkpoints. We show that serine-to-alanine alterations at critical residues within the SQ-cluster known to be phosphorylated by ATM and ATR result in reduced homologous recombination repair (HRR) and aberrant mitosis. While a S1387A BRCA1 mutant - previously shown to abrogate S-phase arrest in response to radiation - resulted in only a modest decrease in HRR, S1387A together with an additional alteration, S1423A (BRCA12P), reduced HRR to vector control levels and similar to a quadruple mutant also including S1457A and S1524A (BRCA14P). These effects appeared to be independent of PALB2. Furthermore, we found that BRCA14P promoted a prolonged and struggling HRR late in the cell cycle and shifted DSB repair from HRR to non-homologous end joining which, in the face of irreparable chromosomal damage, resulted in mitotic catastrophe. Altogether, SQ-cluster phosphorylation is critical for allowing adequate time for completing normal HRR prior to mitosis and preventing cells from entering G1 prematurely resulting in gross chromosomal aberrations.

  20. Either non-homologous ends joining or homologous recombination is required to repair double-strand breaks in the genome of macrophage-internalized Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Anna Brzostek

    Full Text Available The intracellular pathogen Mycobacterium tuberculosis (Mtb is constantly exposed to a multitude of hostile conditions and is confronted by a variety of potentially DNA-damaging assaults in vivo, primarily from host-generated antimicrobial toxic radicals. Exposure to reactive nitrogen species and/or reactive oxygen species causes different types of DNA damage, including oxidation, depurination, methylation and deamination, that can result in single- or double-strand breaks (DSBs. These breaks affect the integrity of the whole genome and, when left unrepaired, can lead to cell death. Here, we investigated the role of the DSB repair pathways, homologous recombination (HR and non-homologous ends joining (NHEJ, in the survival of Mtb inside macrophages. To this end, we constructed Mtb strains defective for HR (ΔrecA, NHEJ [Δ(ku,ligD], or both DSB repair systems [Δ(ku,ligD,recA]. Experiments using these strains revealed that either HR or NHEJ is sufficient for the survival and propagation of tubercle bacilli inside macrophages. Inhibition of nitric oxide or superoxide anion production with L-NIL or apocynin, respectively, enabled the Δ(ku,ligD,recA mutant strain lacking both systems to survive intracellularly. Complementation of the Δ(ku,ligD,recA mutant with an intact recA or ku-ligD rescued the ability of Mtb to propagate inside macrophages.

  1. Loss of Ubr2, an E3 ubiquitin ligase, leads to chromosome fragility and impaired homologous recombinational repair

    Energy Technology Data Exchange (ETDEWEB)

    Ouyang, Yan [Department of Human Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 (United States); Kwon, Yong Tae [Center for Pharmacogenetics and Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh, PA 15261 (United States); An, Jee Young [Center for Pharmacogenetics and Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Eller, Danny [Department of Human Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 (United States); Tsai, S.-C. [Department of Human Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 (United States); Diaz-Perez, Silvia [Department of Human Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 (United States); Troke, Joshua J. [Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 (United States); Teitell, Michael A. [Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 (United States); Marahrens, York [Department of Human Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 (United States)]. E-mail: ymarahrens@mednet.ucla.edu

    2006-04-11

    The N-end rule pathway of protein degradation targets proteins with destabilizing N-terminal residues. Ubr2 is one of the E3 ubiquitin ligases of the mouse N-end rule pathway. We have previously shown that Ubr2{sup -/-} male mice are infertile, owing to the arrest of spermatocytes between the leptotene/zygotene and pachytene of meiosis I, the failure of chromosome pairing, and subsequent apoptosis. Here, we report that mouse fibroblast cells derived from Ubr2{sup -/-} embryos display genome instability. The frequency of chromosomal bridges and micronuclei were much higher in Ubr2{sup -/-} fibroblasts than in +/+ controls. Metaphase chromosome spreads from Ubr2{sup -/-} cells revealed a high incidence of spontaneous chromosomal gaps, indicating chromosomal fragility. These fragile sites were generally replicated late in S phase. Ubr2{sup -/-} cells were hypersensitive to mitomycin C, a DNA cross-linking agent, but displayed normal sensitivity to gamma-irradiation. A reporter assay showed that Ubr2{sup -/-} cells are significantly impaired in the homologous recombination repair of a double strand break. In contrast, Ubr2{sup -/-} cells appeared normal in an assay for non-homologous end joining. Our results therefore unveil the role of the ubiquitin ligase Ubr2 in maintaining genome integrity and in homologous recombination repair.

  2. Various applications of TALEN- and CRISPR/Cas9-mediated homologous recombination to modify the Drosophila genome

    Directory of Open Access Journals (Sweden)

    Zhongsheng Yu

    2014-03-01

    Full Text Available Modifying the genomes of many organisms is becoming as easy as manipulating DNA in test tubes, which is made possible by two recently developed techniques based on either the customizable DNA binding protein, TALEN, or the CRISPR/Cas9 system. Here, we describe a series of efficient applications derived from these two technologies, in combination with various homologous donor DNA plasmids, to manipulate the Drosophila genome: (1 to precisely generate genomic deletions; (2 to make genomic replacement of a DNA fragment at single nucleotide resolution; and (3 to generate precise insertions to tag target proteins for tracing their endogenous expressions. For more convenient genomic manipulations, we established an easy-to-screen platform by knocking in a white marker through homologous recombination. Further, we provided a strategy to remove the unwanted duplications generated during the “ends-in” recombination process. Our results also indicate that TALEN and CRISPR/Cas9 had comparable efficiency in mediating genomic modifications through HDR (homology-directed repair; either TALEN or the CRISPR/Cas9 system could efficiently mediate in vivo replacement of DNA fragments of up to 5 kb in Drosophila, providing an ideal genetic tool for functional annotations of the Drosophila genome.

  3. RSC Facilitates Rad59-Dependent Homologous Recombination between Sister Chromatids by Promoting Cohesin Loading at DNA Double-Strand Breaks ▿

    OpenAIRE

    Oum, Ji-Hyun; Seong, Changhyun; Kwon, YoungHo; Ji, Jae-Hoon; Sid, Amy; Ramakrishnan, Sreejith; Ira, Grzegorz; Malkova, Anna; Sung, Patrick; Lee, Sang Eun; Shim, Eun Yong

    2011-01-01

    Homologous recombination repairs DNA double-strand breaks by searching for, invading, and copying information from a homologous template, typically the homologous chromosome or sister chromatid. Tight wrapping of DNA around histone octamers, however, impedes access of repair proteins to DNA damage. To facilitate DNA repair, modifications of histones and energy-dependent remodeling of chromatin are required, but the precise mechanisms by which chromatin modification and remodeling enzymes cont...

  4. Involvement of a periplasmic protein kinase in DNA strand break repair and homologous recombination in Escherichia coli.

    Science.gov (United States)

    Khairnar, Nivedita P; Kamble, Vidya A; Mangoli, Suhas H; Apte, Shree K; Misra, Hari S

    2007-07-01

    The involvement of signal transduction in the repair of radiation-induced damage to DNA has been known in eukaryotes but remains understudied in bacteria. This article for the first time demonstrates a role for the periplasmic lipoprotein (YfgL) with protein kinase activity transducing a signal for DNA strand break repair in Escherichia coli. Purified YfgL protein showed physical as well as functional interaction with pyrroloquinoline-quinone in solution and the protein kinase activity of YfgL was strongly stimulated in the presence of pyrroloquinoline-quinone. Transgenic E. coli cells producing Deinococcus radiodurans pyrroloquinoline-quinone synthase showed nearly four log cycle improvement in UVC dark survival and 10-fold increases in gamma radiation resistance as compared with untransformed cells. Pyrroloquinoline-quinone enhanced the UV resistance of E. coli through the YfgL protein and required the active recombination repair proteins. The yfgL mutant showed higher sensitivity to UVC, mitomycin C and gamma radiation as compared with wild-type cells and showed a strong impairment in homologous DNA recombination. The mutant expressing an active YfgL in trans recovered the lost phenotypes to nearly wild-type levels. The results strongly suggest that the periplasmic phosphoquinolipoprotein kinase YfgL plays an important role in radiation-induced DNA strand break repair and homologous recombination in E. coli. PMID:17630970

  5. Structural organization of glycophorin A and B genes: Glycophorin B gene evolved by homologous recombination at Alu repeat sequences

    International Nuclear Information System (INIS)

    Glycophorins A (GPA) and B (GPB) are two major sialoglycoproteins of the human erythrocyte membrane. Here the authors present a comparison of the genomic structures of GPA and GPB developed by analyzing DNA clones isolated from a K562 genomic library. Nucleotide sequences of exon-intron junctions and 5' and 3' flanking sequences revealed that the GPA and GPB genes consist of 7 and 5 exons, respectively, and both genes have >95% identical sequence from the 5' flanking region to the region ∼ 1 kilobase downstream from the exon encoding the transmembrane regions. In this homologous part of the genes, GPB lacks one exon due to a point mutation at the 5' splicing site of the third intron, which inactivates the 5' cleavage event of splicing and leads to ligation of the second to the fourth exon. Following these very homologous sequences, the genomic sequences for GPA and GPB diverge significantly and no homology can be detected in their 3' end sequences. The analysis of the Alu sequences and their flanking direct repeat sequences suggest that an ancestral genomic structure has been maintained in the GPA gene, whereas the GPB gene has arisen from the acquisition of 3' sequences different from those of the GPA gene by homologous recombination at the Alu repeats during or after gene duplication

  6. Arabidopsis RecQl4A suppresses homologous recombination and modulates DNA damage responses

    NARCIS (Netherlands)

    Bagherieh-Najjar, MB; de Vries, OMH; Hille, J; Dijkwel, PP; Bagherieh-Najjar, Mohammad B.

    2005-01-01

    The DNA damage response and DNA recombination are two interrelated mechanisms involved in maintaining the integrity of the genome, but in plants they are poorly understood. RecO is a family of genes with conserved roles in the regulation of DNA recombination in eukaryotes; there are seven members in

  7. Srs2 and Mus81–Mms4 Prevent Accumulation of Toxic Inter-Homolog Recombination Intermediates

    Science.gov (United States)

    Keyamura, Kenji; Arai, Kota

    2016-01-01

    Homologous recombination is an evolutionally conserved mechanism that promotes genome stability through the faithful repair of double-strand breaks and single-strand gaps in DNA, and the recovery of stalled or collapsed replication forks. Saccharomyces cerevisiae ATP-dependent DNA helicase Srs2 (a member of the highly conserved UvrD family of helicases) has multiple roles in regulating homologous recombination. A mutation (srs2K41A) resulting in a helicase-dead mutant of Srs2 was found to be lethal in diploid, but not in haploid, cells. In diploid cells, Srs2K41A caused the accumulation of inter-homolog joint molecule intermediates, increased the levels of spontaneous Rad52 foci, and induced gross chromosomal rearrangements. Srs2K41A lethality and accumulation of joint molecules were suppressed by inactivating Rad51 or deleting the Rad51-interaction domain of Srs2, whereas phosphorylation and sumoylation of Srs2 and its interaction with sumoylated proliferating cell nuclear antigen (PCNA) were not required for lethality. The structure-specific complex of crossover junction endonucleases Mus81 and Mms4 was also required for viability of diploid, but not haploid, SRS2 deletion mutants (srs2Δ), and diploid srs2Δ mus81Δ mutants accumulated joint molecule intermediates. Our data suggest that Srs2 and Mus81–Mms4 have critical roles in preventing the formation of (or in resolving) toxic inter-homolog joint molecules, which could otherwise interfere with chromosome segregation and lead to genetic instability. PMID:27390022

  8. The PCNA-associated protein PARI negatively regulates homologous recombination via the inhibition of DNA repair synthesis.

    Science.gov (United States)

    Burkovics, Peter; Dome, Lili; Juhasz, Szilvia; Altmannova, Veronika; Sebesta, Marek; Pacesa, Martin; Fugger, Kasper; Sorensen, Claus Storgaard; Lee, Marietta Y W T; Haracska, Lajos; Krejci, Lumir

    2016-04-20

    Successful and accurate completion of the replication of damage-containing DNA requires mainly recombination and RAD18-dependent DNA damage tolerance pathways. RAD18 governs at least two distinct mechanisms: translesion synthesis (TLS) and template switching (TS)-dependent pathways. Whereas TS is mainly error-free, TLS can work in an error-prone manner and, as such, the regulation of these pathways requires tight control to prevent DNA errors and potentially oncogenic transformation and tumorigenesis. In humans, the PCNA-associated recombination inhibitor (PARI) protein has recently been shown to inhibit homologous recombination (HR) events. Here, we describe a biochemical mechanism in which PARI functions as an HR regulator after replication fork stalling and during double-strand break repair. In our reconstituted biochemical system, we show that PARI inhibits DNA repair synthesis during recombination events in a PCNA interaction-dependent way but independently of its UvrD-like helicase domain. In accordance, we demonstrate that PARI inhibits HRin vivo, and its knockdown suppresses the UV sensitivity of RAD18-depleted cells. Our data reveal a novel human regulatory mechanism that limits the extent of HR and represents a new potential target for anticancer therapy. PMID:26792895

  9. Mutagenesis and homologous recombination in Drosophila cell lines using CRISPR/Cas9

    Directory of Open Access Journals (Sweden)

    Andrew R. Bassett

    2013-12-01

    We have applied the CRISPR/Cas9 system to Drosophila S2 cells to generate targeted genetic mutations in more than 85% of alleles. By targeting a constitutive exon of the AGO1 gene, we demonstrate homozygous mutation in up to 82% of cells, thereby allowing the study of genetic knockouts in a Drosophila cell line for the first time. We have shown that homologous gene targeting is possible at 1–4% efficiency using this system, allowing for the construction of defined insertions and deletions. We demonstrate that a 1 kb homology arm length is optimal for integration by homologous gene targeting, and demonstrate its efficacy by tagging the endogenous AGO1 protein. This technology enables controlled genetic manipulation in Drosophila cell lines, and its simplicity offers the opportunity to study cellular phenotypes genome-wide.

  10. Germline Chromothripsis Driven by L1-Mediated Retrotransposition and Alu/Alu Homologous Recombination

    DEFF Research Database (Denmark)

    Nazaryan-Petersen, Lusine; Bertelsen, Birgitte; Bak, Mads;

    2016-01-01

    L1-endonuclease potential target sites in other breakpoints. In addition, we found four Alu elements flanking the 110-kb deletion and associated with an inversion. We suggest that chromatin looping mediated by homologous Alu elements may have brought distal DNA regions into close proximity...

  11. FBH1 helicase disrupts RAD51 filaments in vitro and modulates homologous recombination in mammalian cells

    DEFF Research Database (Denmark)

    Simandlova, Jitka; Zagelbaum, Jennifer; Payne, Miranda J;

    2013-01-01

    filaments on DNA through its ssDNA translocase function. Consistent with this, a mutant mouse embryonic stem cell line with a deletion in the FBH1 helicase domain fails to limit RAD51 chromatin association and shows hyper-recombination. Our data are consistent with FBH1 restraining RAD51 DNA binding under...

  12. Abiotic stress leads to somatic and heritable changes in homologous recombination frequency, point mutation frequency and microsatellite stability in Arabidopsis plants

    International Nuclear Information System (INIS)

    In earlier studies, we showed that abiotic stresses, such as ionizing radiation, heavy metals, temperature and water, trigger an increase in homologous recombination frequency (HRF). We also demonstrated that many of these stresses led to inheritance of high-frequency homologous recombination, HRF. Although an increase in recombination frequency is an important indicator of genome rearrangements, it only represents a minor portion of possible stress-induced mutations. Here, we analyzed the influence of heat, cold, drought, flood and UVC abiotic stresses on two major types of mutations in the genome, point mutations and small deletions/insertions. We used two transgenic lines of Arabidopsis thaliana, one allowing an analysis of reversions in a stop codon-containing inactivated β-glucuronidase transgene and another one allowing an analysis of repeat stability in a microsatellite-interrupted β-glucuronidase transgene. The transgenic Arabidopsis line carrying the β-glucuronidase-based homologous recombination substrate was used as a positive control. We showed that the majority of stresses increased the frequency of point mutations, homologous recombination and microsatellite instability in somatic cells, with the frequency of homologous recombination being affected the most. The analysis of transgenerational changes showed an increase in HRF to be the most prominent effect observed in progeny. Significant changes in recombination frequency were observed upon exposure to all types of stress except drought, whereas changes in microsatellite instability were observed upon exposure to UVC, heat and cold. The frequency of point mutations in the progeny of stress-exposed plants was the least affected; an increase in mutation frequency was observed only in the progeny of plants exposed to UVC. We thus conclude that transgenerational changes in genome stability in response to stress primarily involve an increase in recombination frequency.

  13. Abiotic stress leads to somatic and heritable changes in homologous recombination frequency, point mutation frequency and microsatellite stability in Arabidopsis plants

    Energy Technology Data Exchange (ETDEWEB)

    Yao Youli, E-mail: youli.yao@uleth.ca [Department of Biological Sciences, University of Lethbridge, Lethbridge, T1K 3M4 Alberta (Canada); Kovalchuk, Igor, E-mail: igor.kovalchuk@uleth.ca [Department of Biological Sciences, University of Lethbridge, Lethbridge, T1K 3M4 Alberta (Canada)

    2011-02-10

    In earlier studies, we showed that abiotic stresses, such as ionizing radiation, heavy metals, temperature and water, trigger an increase in homologous recombination frequency (HRF). We also demonstrated that many of these stresses led to inheritance of high-frequency homologous recombination, HRF. Although an increase in recombination frequency is an important indicator of genome rearrangements, it only represents a minor portion of possible stress-induced mutations. Here, we analyzed the influence of heat, cold, drought, flood and UVC abiotic stresses on two major types of mutations in the genome, point mutations and small deletions/insertions. We used two transgenic lines of Arabidopsis thaliana, one allowing an analysis of reversions in a stop codon-containing inactivated {beta}-glucuronidase transgene and another one allowing an analysis of repeat stability in a microsatellite-interrupted {beta}-glucuronidase transgene. The transgenic Arabidopsis line carrying the {beta}-glucuronidase-based homologous recombination substrate was used as a positive control. We showed that the majority of stresses increased the frequency of point mutations, homologous recombination and microsatellite instability in somatic cells, with the frequency of homologous recombination being affected the most. The analysis of transgenerational changes showed an increase in HRF to be the most prominent effect observed in progeny. Significant changes in recombination frequency were observed upon exposure to all types of stress except drought, whereas changes in microsatellite instability were observed upon exposure to UVC, heat and cold. The frequency of point mutations in the progeny of stress-exposed plants was the least affected; an increase in mutation frequency was observed only in the progeny of plants exposed to UVC. We thus conclude that transgenerational changes in genome stability in response to stress primarily involve an increase in recombination frequency.

  14. DNA annealing by Redβ is insufficient for homologous recombination and the additional requirements involve intra- and inter-molecular interactions

    Science.gov (United States)

    Subramaniam, Sivaraman; Erler, Axel; Fu, Jun; Kranz, Andrea; Tang, Jing; Gopalswamy, Mohanraj; Ramakrishnan, Saminathan; Keller, Adrian; Grundmeier, Guido; Müller, Daniel; Sattler, Michael; Stewart, A. Francis

    2016-01-01

    Single strand annealing proteins (SSAPs) like Redβ initiate homologous recombination by annealing complementary DNA strands. We show that C-terminally truncated Redβ, whilst still able to promote annealing and nucleoprotein filament formation, is unable to mediate homologous recombination. Mutations of the C-terminal domain were evaluated using both single- and double stranded (ss and ds) substrates in recombination assays. Mutations of critical amino acids affected either dsDNA recombination or both ssDNA and dsDNA recombination indicating two separable functions, one of which is critical for dsDNA recombination and the second for recombination per se. As evaluated by co-immunoprecipitation experiments, the dsDNA recombination function relates to the Redα-Redβ protein-protein interaction, which requires not only contacts in the C-terminal domain but also a region near the N-terminus. Because the nucleoprotein filament formed with C-terminally truncated Redβ has altered properties, the second C-terminal function could be due to an interaction required for functional filaments. Alternatively the second C-terminal function could indicate a requirement for a Redβ-host factor interaction. These data further advance the model for Red recombination and the proposition that Redβ and RAD52 SSAPs share ancestral and mechanistic roots. PMID:27708411

  15. Processing of homologous recombination repair intermediates by the Sgs1-Top3-Rmi1 and Mus81-Mms4 complexes

    DEFF Research Database (Denmark)

    Hickson, Ian D; Mankouri, Hocine W

    2011-01-01

    Homologous recombination repair (HRR) is an evolutionarily conserved cellular process that is important for the maintenance of genome stability during S phase. Inactivation of the Saccharomyces cerevisiae Sgs1-Top3-Rmi1 complex leads to the accumulation of unprocessed, X-shaped HRR intermediates (X...... structures) following replicative stress. Further characterization of these X structures may reveal why loss of BLM (the human Sgs1 ortholog) leads to the human cancer predisposition disorder, Bloom syndrome. In two recent complementary studies, we examined the nature of the X structures arising in yeast strains...... lacking Sgs1, Top3 or Rmi1 by identifying which proteins could process these structures in vivo. We revealed that the unprocessed X structures that accumulate in these strains could be resolved by the ectopic overexpression of two different Holliday junction (HJ) resolvases, and that the endogenous Mus81...

  16. Shu proteins promote the formation of homologous recombination intermediates that are processed by Sgs1-Rmi1-Top3

    DEFF Research Database (Denmark)

    Mankouri, Hocine W; Ngo, Hien-Ping; Hickson, Ian D

    2007-01-01

    CSM2, PSY3, SHU1, and SHU2 (collectively referred to as the SHU genes) were identified in Saccharomyces cerevisiae as four genes in the same epistasis group that suppress various sgs1 and top3 mutant phenotypes when mutated. Although the SHU genes have been implicated in homologous recombination...... formation of MMS-induced HRR intermediates (X-molecules) arising during replication in sgs1 cells, mutation of SHU genes attenuated the level of these structures. Similar findings were also observed in shu1 cells in which Rmi1 or Top3 function was impaired. We propose a model in which the Shu proteins act...... in HRR to promote the formation of HRR intermediates that are processed by the Sgs1-Rmi1-Top3 complex....

  17. Hybrid pseudomonads engineered by two-step homologous recombination acquire novel degradation abilities toward aromatics and polychlorinated biphenyls

    Energy Technology Data Exchange (ETDEWEB)

    Suenaga, Hikaru [National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba (Japan). Bioproduction Research Inst.; Nonaka, Kazuhiko; Goto, Masatoshi [Kyushu Univ., Fukuoka (Japan). Dept. of Bioscience and Biotechnology; Fujihara, Hidehiko; Furukawa, Kensuke [Beppu Univ. (Japan). Dept. of Fermentation and Food Science

    2010-10-15

    Pseudomonas pseudoalcaligenes KF707 possesses a chromosomally encoded bph gene cluster responsible for the catabolism of biphenyl and polychlorinated biphenyls. Previously, we constructed chimeric versions of the bphA1 gene, which encodes a large subunit of biphenyl dioxygenase, by using DNA shuffling between bphA1 genes from P. pseudoalcaligenes KF707 and Burkholderia xenovorans LB400. In this study, we demonstrate replacement of the bphA1 gene with chimeric bphA1 sequence within the chromosomal bph gene cluster by two-step homologous recombination. Notably, some of the hybrid strains acquired enhanced and/or expanded degradation capabilities for specific aromatic compounds, including single aromatic hydrocarbons and polychlorinated biphenyls. (orig.)

  18. The response of mammalian cells to UV-light reveals Rad54-dependent and independent pathways of homologous recombination

    DEFF Research Database (Denmark)

    Eppink, Berina; Tafel, Agnieszka A; Hanada, Katsuhiro;

    2011-01-01

    Ultraviolet (UV) radiation-induced DNA lesions can be efficiently repaired by nucleotide excision repair (NER). However, NER is less effective during replication of UV-damaged chromosomes. In contrast, translesion DNA synthesis (TLS) and homologous recombination (HR) are capable of dealing...... with lesions in replicating DNA. The core HR protein in mammalian cells is the strand exchange protein RAD51, which is aided by numerous proteins, including RAD54. We used RAD54 as a cellular marker for HR to study the response of mammalian embryonic stem (ES) cells to UV irradiation. In contrast to yeast, ES...... cells lacking RAD54 are not UV sensitive. Here we show that the requirement for mammalian RAD54 is masked by active NER. By genetically inactivating NER and HR through disruption of the Xpa and Rad54 genes, respectively, we demonstrate the contribution of HR to chromosomal integrity upon UV irradiation...

  19. Efficient generation of double heterologous promoter controlled oncolytic adenovirus vectors by a single homologous recombination step in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Wildner Oliver

    2006-08-01

    Full Text Available Abstract Background Oncolytic adenoviruses are promising agents for the multimodal treatment of cancer. However, tumor-selectivity is crucial for their applicability in patients. Recent studies by several groups demonstrated that oncolytic adenoviruses with tumor-/tissue-specific expression of the E1 and E4 genes, which are pivotal for adenoviral replication, have a specificity profile that is superior to viruses that solely target the expression of E1 or E4 genes. Presently the E1 and E4 regions are modified in a time consuming sequential fashion. Results Based on the widely used adenoviral cloning system AdEasy we generated a novel transfer vector that allows efficient and rapid generation of conditionally replication-competent adenovirus type 5 based vectors with the viral E1 and E4 genes under the transcriptional control of heterologous promoters. For insertion of the promoters of interest our transfer vector has two unique multiple cloning sites. Additionally, our shuttle plasmid allows encoding of a transgene within the E1A transcription unit. The modifications, including E1 mutations, are introduced into the adenoviral genome by a single homologous recombination step in Escherichia coli. Subsequently infectious viruses are rescued from plasmids. As a proof-of-concept we generated two conditionally replication-competent adenoviruses Ad.Ki•COX and Ad.COX•Ki with the promoters of the Ki-67 protein and the cyclooxygenase-2 (COX-2 driving E1 and E4 and vice versa. Conclusion We demonstrated with our cloning system efficient generation of double heterologous promoter controlled oncolytic adenoviral vectors by a single homologous recombination step in bacteria. The generated viruses showed preferential replication in tumor cells and in a subcutaneous HT-29 colon cancer xenograft model the viruses demonstrated significant oncolytic activity comparable with dl327.

  20. Autoexcision of Bacterial Artificial Chromosome Facilitated by Terminal Repeat-Mediated Homologous Recombination: a Novel Approach for Generating Traceless Genetic Mutants of Herpesviruses ▿

    OpenAIRE

    Zhou, Fuchun; Li, Qiuhua; Wong, Scott W.; Gao, Shou-jiang

    2010-01-01

    Infectious bacterial artificial chromosomes (BACs) of herpesviruses are powerful tools for genetic manipulation. However, the presence of BAC vector sequence in the viral genomes often causes genetic and phenotypic alterations. While the excision of the BAC vector cassette can be achieved by homologous recombination between extra duplicate viral sequences or loxP site-mediated recombination, these methods either are inefficient or leave a loxP site mark in the viral genome. Here we describe t...

  1. Regulation of Rad51-Mediated Homologous Recombination by BRCA2, DSS1 and RAD52

    DEFF Research Database (Denmark)

    Rants, Louise Olthaver Juhl

    in governing the activity of Rad51 and to learn how other recombination-associated proteins such as DSS1 and RAD52 contribute to its regulation. We use the yeast-like fungus Ustilago maydis and the avian DT40 cell line as experimental systems since both have a well-conserved BRCA2-based recombinational repair...... system that resembles the one seen in human. In U. maydis, we show that Brh2, the BRCA2 homologue, and Dss1 colocalize at DNA damage-induced foci, with Dss1 exhibiting a dynamic association with Brh2 foci. Dss1 focus formation is dependent on interaction with full-length Brh2, and the Dss1-Brh2...... interaction is required for resistance to DNA damage. In avian DT40 cells, we show that endogenously tagged DSS1 redistributes into subnuclear foci in response to DNA damaging agents. However, DSS1 rarely colocalizes with BRCA2. Our data also indicate that both U. maydis Dss1 and avian DSS1 are involved...

  2. A versatile bacterial expression vector designed for single-step cloning of multiple DNA fragments using homologous recombination.

    Science.gov (United States)

    Holmberg, Mats A; Gowda, Naveen Kumar Chandappa; Andréasson, Claes

    2014-06-01

    Production of recombinant proteins is the starting point for biochemical and biophysical analyses and requires methodology to efficiently proceed from gene sequence to purified protein. While optimized strategies for the efficient cloning of single-gene fragments for bacterial expression is available, efficient multiple DNA fragment cloning still presents a challenge. To facilitate this step, we have developed an efficient cloning strategy based on yeast homologous recombination cloning (YHRC) into the new pET-based bacterial expression vector pSUMO-YHRC. The vector supports cloning for untagged expression as well as fusions to His6-SUMO or His6 tags. We demonstrate that YHRC from single PCR products of 6 independent genes into the vector results in virtually no background. Importantly, in a quantitative assay for functional expression we find that single-step YHRC of 7 DNA fragments can be performed with very high cloning efficiencies. The method and reagents described in this paper significantly simplifies the construction of expression plasmids from multiple DNA fragments, including complex gene fusions, chimeric genes and polycistronic constructs. PMID:24631626

  3. Tetracycline Selective Pressure and Homologous Recombination Shape the Evolution of Chlamydia suis: A Recently Identified Zoonotic Pathogen.

    Science.gov (United States)

    Joseph, Sandeep J; Marti, Hanna; Didelot, Xavier; Read, Timothy D; Dean, Deborah

    2016-01-01

    Species closely related to the human pathogen Chlamydia trachomatis (Ct) have recently been found to cause zoonotic infections, posing a public health threat especially in the case of tetracycline resistant Chlamydia suis (Cs) strains. These strains acquired a tet(C)-containing cassette via horizontal gene transfer (HGT). Genomes of 11 Cs strains from various tissues were sequenced to reconstruct evolutionary pathway(s) for tet(C) HGT. Cs had the highest recombination rate of Chlamydia species studied to date. Admixture occurred among Cs strains and with Chlamydia muridarum but not with Ct Although in vitro tet(C) cassette exchange with Ct has been documented, in vivo evidence may require examining human samples from Ct and Cs co-infected sites. Molecular-clock dating indicated that ancestral clades of resistant Cs strains predated the 1947 discovery of tetracycline, which was subsequently used in animal feed. The cassette likely spread throughout Cs strains by homologous recombination after acquisition from an external source, and our analysis suggests Betaproteobacteria as the origin. Selective pressure from tetracycline may be responsible for recent bottlenecks in Cs populations. Since tetracycline is an important antibiotic for treating Ct, zoonotic infections at mutual sites of infection indicate the possibility for cassette transfer and major public health repercussions. PMID:27576537

  4. Promotion of Homologous Recombination and Genomic Stability byRAD51AP1 via RAD51 Recombinase Enhancement

    Energy Technology Data Exchange (ETDEWEB)

    Wiese, Claudia; Dray, Eloise; Groesser, Torsten; San Filippo,Joseph; Shi, Idina; Collins, David W.; Tsai, Miaw-Sheue; Williams,Gareth; Rydberg, Bjorn; Sung, Patrick; Schild, David

    2007-04-11

    Homologous recombination (HR) repairs chromosome damage and is indispensable for tumor suppression in humans. RAD51 mediates the DNA strand pairing step in HR. RAD51AP1 (RAD51 Associated Protein 1) is a RAD51-interacting protein whose function has remained elusive. Knockdown of RAD51AP1 in human cells by RNA interference engenders sensitivity to different types of genotoxic stress. Moreover, RAD51AP1-depleted cells are impaired for the recombinational repair of a DNA double-strand break and exhibit chromatid breaks both spontaneously and upon DNA damaging treatment. Purified RAD51AP1 binds dsDNA and RAD51, and it greatly stimulates the RAD51-mediated D-loop reaction. Biochemical and cytological results show that RAD51AP1 functions at a step subsequent to the assembly of the RAD51-ssDNA nucleoprotein filament. Our findings provide the first evidence that RAD51AP1 helps maintain genomic integrity via RAD51 recombinase enhancement.

  5. New Coffee Plant-Infecting Xylella fastidiosa Variants Derived via Homologous Recombination.

    Science.gov (United States)

    Jacques, Marie-Agnès; Denancé, Nicolas; Legendre, Bruno; Morel, Emmanuelle; Briand, Martial; Mississipi, Stelly; Durand, Karine; Olivier, Valérie; Portier, Perrine; Poliakoff, Françoise; Crouzillat, Dominique

    2016-03-01

    Xylella fastidiosa is a xylem-limited phytopathogenic bacterium endemic to the Americas that has recently emerged in Asia and Europe. Although this bacterium is classified as a quarantine organism in the European Union, importation of plant material from contaminated areas and latent infection in asymptomatic plants have engendered its inevitable introduction. In 2012, four coffee plants (Coffea arabica and Coffea canephora) with leaf scorch symptoms growing in a confined greenhouse were detected and intercepted in France. After identification of the causal agent, this outbreak was eradicated. Three X. fastidiosa strains were isolated from these plants, confirming a preliminary identification based on immunology. The strains were characterized by multiplex PCR and by multilocus sequence analysis/typing (MLSA-MLST) based on seven housekeeping genes. One strain, CFBP 8073, isolated from C. canephora imported from Mexico, was assigned to X. fastidiosa subsp. fastidiosa/X. fastidiosa subsp. sandyi. This strain harbors a novel sequence type (ST) with novel alleles at two loci. The two other strains, CFBP 8072 and CFBP 8074, isolated from Coffea arabica imported from Ecuador, were allocated to X. fastidiosa subsp. pauca. These two strains shared a novel ST with novel alleles at two loci. These MLST profiles showed evidence of recombination events. We provide genome sequences for CFBP 8072 and CFBP 8073 strains. Comparative genomic analyses of these two genome sequences with publicly available X. fastidiosa genomes, including the Italian strain CoDiRO, confirmed these phylogenetic positions and provided candidate alleles for coffee plant adaptation. This study demonstrates the global diversity of X. fastidiosa and highlights the diversity of strains isolated from coffee plants. PMID:26712553

  6. Tousled kinase activator, gallic acid, promotes homologous recombinational repair and suppresses radiation cytotoxicity in salivary gland cells.

    Science.gov (United States)

    Timiri Shanmugam, Prakash Srinivasan; Nair, Renjith Parameshwaran; De Benedetti, Arrigo; Caldito, Gloria; Abreo, Fleurette; Sunavala-Dossabhoy, Gulshan

    2016-04-01

    Accidental or medical radiation exposure of the salivary glands can gravely impact oral health. Previous studies have shown the importance of Tousled-like kinase 1 (TLK1) and its alternate start variant TLK1B in cell survival against genotoxic stresses. Through a high-throughput library screening of natural compounds, the phenolic phytochemical, gallic acid (GA), was identified as a modulator of TLK1/1B. This small molecule possesses anti-oxidant and free radical scavenging properties, but in this study, we report that in vitro it promotes survival of human salivary acinar cells, NS-SV-AC, through repair of ionizing radiation damage. Irradiated cells treated with GA show improved clonogenic survival compared to untreated controls. And, analyses of DNA repair kinetics by alkaline single-cell gel electrophoresis and γ-H2AX foci immunofluorescence indicate rapid resolution of DNA breaks in drug-treated cells. Study of DR-GFP transgene repair indicates GA facilitates homologous recombinational repair to establish a functional GFP gene. In contrast, inactivation of TLK1 or its shRNA knockdown suppressed resolution of radiation-induced DNA tails in NS-SV-AC, and homology directed repair in DR-GFP cells. Consistent with our results in culture, animals treated with GA after exposure to fractionated radiation showed better preservation of salivary function compared to saline-treated animals. Our results suggest that GA-mediated transient modulation of TLK1 activity promotes DNA repair and suppresses radiation cytoxicity in salivary gland cells.

  7. Roles of Rad51 protein in homologous recombination in mammalian cells: relation with repair, replication and cell cycle

    International Nuclear Information System (INIS)

    Homologous recombination (HR) is a fundamental process, allowing a faithful repair. In mammalian, MmRAD51, which is the homologue of Saccharomyces cerevisiae ScRAD51 key protein for HR, is an essential gene. This work is based on the characterisation of viable hyper and hypo-recombinant cell lines specifically affected in the Rad51 pathway. By expressing wild type and dominant negative forms of MmRad51, we demonstrated that Rad51 pathway participates to the repair by HR to induced DNA damages. However, inhibition of the Rad 51 pathway does not affect cell viability, spontaneously or after irradiation, whereas, radiation induced HR is inhibited. In the presence of DNA damages during late S and G2/M phase, inhibition of Rad51 pathway induced chromosomal aberrations, leading to a transient arrest in mitosis. This arrest is associated with an increased of cell death. However, a fraction of cells can escape from this transient arrest by forming tetraploid cells, associated with an absence of chromalid separation. Thus, in response to impaired Rad51 pathway, mitotic checkpoints seems to play an essential role. In line with this, we showed that the essential function of Rad51 is p53-dependent, which is in agreement with the role of p53 in tetraploidy inhibition. Our results suggest that the Rad51 protein could participate to the control of mitotic checkpoints and thus to the maintenance of genetic stability. This function could involve other Rad51 partners such as the tumour suppressors BRCA1, BRCA2 and p53. (author)

  8. I-SceI-mediated double-strand break does not increase the frequency of homologous recombination at the Dct locus in mouse embryonic stem cells.

    Science.gov (United States)

    Fenina, Myriam; Simon-Chazottes, Dominique; Vandormael-Pournin, Sandrine; Soueid, Jihane; Langa, Francina; Cohen-Tannoudji, Michel; Bernard, Bruno A; Panthier, Jean-Jacques

    2012-01-01

    Targeted induction of double-strand breaks (DSBs) at natural endogenous loci was shown to increase the rate of gene replacement by homologous recombination in mouse embryonic stem cells. The gene encoding dopachrome tautomerase (Dct) is specifically expressed in melanocytes and their precursors. To construct a genetic tool allowing the replacement of Dct gene by any gene of interest, we generated an embryonic stem cell line carrying the recognition site for the yeast I-SceI meganuclease embedded in the Dct genomic segment. The embryonic stem cell line was electroporated with an I-SceI expression plasmid, and a template for the DSB-repair process that carried sequence homologies to the Dct target. The I-SceI meganuclease was indeed able to introduce a DSB at the Dct locus in live embryonic stem cells. However, the level of gene targeting was not improved by the DSB induction, indicating a limited capacity of I-SceI to mediate homologous recombination at the Dct locus. These data suggest that homologous recombination by meganuclease-induced DSB may be locus dependent in mammalian cells.

  9. Non-catalytic Roles for XPG with BRCA1 and BRCA2 in Homologous Recombination and Genome Stability.

    Science.gov (United States)

    Trego, Kelly S; Groesser, Torsten; Davalos, Albert R; Parplys, Ann C; Zhao, Weixing; Nelson, Michael R; Hlaing, Ayesu; Shih, Brian; Rydberg, Björn; Pluth, Janice M; Tsai, Miaw-Sheue; Hoeijmakers, Jan H J; Sung, Patrick; Wiese, Claudia; Campisi, Judith; Cooper, Priscilla K

    2016-02-18

    XPG is a structure-specific endonuclease required for nucleotide excision repair, and incision-defective XPG mutations cause the skin cancer-prone syndrome xeroderma pigmentosum. Truncating mutations instead cause the neurodevelopmental progeroid disorder Cockayne syndrome, but little is known about how XPG loss results in this devastating disease. We identify XPG as a partner of BRCA1 and BRCA2 in maintaining genomic stability through homologous recombination (HRR). XPG depletion causes DNA double-strand breaks, chromosomal abnormalities, cell-cycle delays, defective HRR, inability to overcome replication fork stalling, and replication stress. XPG directly interacts with BRCA2, RAD51, and PALB2, and XPG depletion reduces their chromatin binding and subsequent RAD51 foci formation. Upstream in HRR, XPG interacts directly with BRCA1. Its depletion causes BRCA1 hyper-phosphorylation and persistent chromatin binding. These unexpected findings establish XPG as an HRR protein with important roles in genome stability and suggest how XPG defects produce severe clinical consequences including cancer and accelerated aging. PMID:26833090

  10. Excess Polθ functions in response to replicative stress in homologous recombination-proficient cancer cells

    Science.gov (United States)

    de Rugy, T. Goullet; Bashkurov, M.; Datti, A.; Betous, R.; Guitton-Sert, L.; Cazaux, C.; Durocher, D.

    2016-01-01

    ABSTRACT DNA polymerase theta (Polθ) is a specialized A-family DNA polymerase that functions in processes such as translesion synthesis (TLS), DNA double-strand break repair and DNA replication timing. Overexpression of POLQ, the gene encoding Polθ, is a prognostic marker for an adverse outcome in a wide range of human cancers. While increased Polθ dosage was recently suggested to promote survival of homologous recombination (HR)-deficient cancer cells, it remains unclear whether POLQ overexpression could be also beneficial to HR-proficient cancer cells. By performing a short interfering (si)RNA screen in which genes encoding druggable proteins were knocked down in Polθ-overexpressing cells as a means to uncover genetic vulnerabilities associated with POLQ overexpression, we could not identify genes that were essential for viability in Polθ-overexpressing cells in normal growth conditions. We also showed that, upon external DNA replication stress, Polθ expression promotes cell survival and limits genetic instability. Finally, we report that POLQ expression correlates with the expression of a set of HR genes in breast, lung and colorectal cancers. Collectively, our data suggest that Polθ upregulation, besides its importance for survival of HR-deficient cancer cells, may be crucial also for HR-proficient cells to better tolerate DNA replication stress, as part of a global gene deregulation response, including HR genes. PMID:27612511

  11. Homologous recombination contributes to the repair of DNA double-strand breaks induced by high-energy iron ions

    Energy Technology Data Exchange (ETDEWEB)

    Zafar, Faria; Seidler, Sara B.; Kronenberg, Amy; Schild, David; Wiese, Claudia

    2010-06-29

    To test the contribution of homologous recombinational repair (HRR) in repairing DNA damaged sites induced by high-energy iron ions, we used: (1) HRR-deficient rodent cells carrying a deletion in the RAD51D gene and (2) syngeneic human cells impaired for HRR by RAD51D or RAD51 knockdown using RNA interference. We show that in response to iron ions, HRR contributes to cell survival in rodent cells, and that HRR-deficiency abrogates RAD51 foci formation. Complementation of the HRR defect by human RAD51D rescues both enhanced cytotoxicity and RAD51 foci formation. For human cells irradiated with iron ions, cell survival is decreased, and, in p53 mutant cells, the levels of mutagenesis are increased when HRR is impaired. Human cells synchronized in S phase exhibit more pronounced resistance to iron ions as compared with cells in G1 phase, and this increase in radioresistance is diminished by RAD51 knockdown. These results implicate a role for RAD51-mediated DNA repair (i.e. HRR) in removing a fraction of clustered lesions induced by charged particle irradiation. Our results are the first to directly show the requirement for an intact HRR pathway in human cells in ensuring DNA repair and cell survival in response to high-energy high LET radiation.

  12. Homologous recombination contributes to the repair of DNA double-strand breaks induced by high-energy iron ions

    International Nuclear Information System (INIS)

    To test the contribution of homologous recombinational repair (HRR) in repairing DNA damaged sites induced by high-energy iron ions, we used: (1) HRR-deficient rodent cells carrying a deletion in the RAD51D gene and (2) syngeneic human cells impaired for HRR by RAD51D or RAD51 knockdown using RNA interference. We show that in response to iron ions, HRR contributes to cell survival in rodent cells, and that HRR-deficiency abrogates RAD51 foci formation. Complementation of the HRR defect by human RAD51D rescues both enhanced cytotoxicity and RAD51 foci formation. For human cells irradiated with iron ions, cell survival is decreased, and, in p53 mutant cells, the levels of mutagenesis are increased when HRR is impaired. Human cells synchronized in S phase exhibit more pronounced resistance to iron ions as compared with cells in G1 phase, and this increase in radioresistance is diminished by RAD51 knockdown. These results implicate a role for RAD51-mediated DNA repair (i.e. HRR) in removing a fraction of clustered lesions induced by charged particle irradiation. Our results are the first to directly show the requirement for an intact HRR pathway in human cells in ensuring DNA repair and cell survival in response to high-energy high LET radiation.

  13. Targeting Human α-Lactalbumin Gene Insertion into the Goat β-Lactoglobulin Locus by TALEN-Mediated Homologous Recombination.

    Directory of Open Access Journals (Sweden)

    Hongmei Zhu

    Full Text Available Special value of goat milk in human nutrition and well being is associated with medical problems of food allergies which are caused by milk proteins such as β-lactoglobulin (BLG. Here, we employed transcription activator-like effector nuclease (TALEN-assisted homologous recombination in goat fibroblasts to introduce human α-lactalbumin (hLA genes into goat BLG locus. TALEN-mediated targeting enabled isolation of colonies with mono- and bi-allelic transgene integration in up to 10.1% and 1.1%, respectively, after selection. Specifically, BLG mRNA levels were gradually decreasing in both mo- and bi-allelic goat mammary epithelial cells (GMECs while hLA demonstrated expression in GMECs in vitro. Gene-targeted fibroblast cells were efficiently used in somatic cell nuclear transfer, resulting in production of hLA knock-in goats directing down-regulated BLG expression and abundant hLA secretion in animal milk. Our findings provide valuable background for animal milk optimization and expedited development for agriculture and biomedicine.

  14. Inhibition of homologous recombination repair in irradiated tumor cells pretreated with Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin

    International Nuclear Information System (INIS)

    In order to investigate the mechanism of radio-sensitization by an Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG), we studied repair of DNA double strand breaks (DSBs) in irradiated human cells pre-treated with 17-AAG. DSBs are thought to be the critical target for radiation-induced cell death. Two human tumor cell lines DU145 and SQ-5 which showed clear radio-sensitization by 17-AAG revealed a significant inhibition of DSB repair, while normal human cells which did not show radio-sensitization by the drug indicated no change in the DSB repair kinetics with 17-AAG. We further demonstrated that BRCA2 was a novel client protein for Hsp90, and 17-AAG caused the degradation of BRCA2 and in turn altered the behavior of Rad51, a critical protein for homologous recombination (HR) pathway of DSB repair. Our data demonstrate for the first time that 17-AAG inhibits the HR repair process and could provide a new therapeutic strategy to selectively result in higher tumor cell killing

  15. Yeast homologous recombination-based promoter engineering for the activation of silent natural product biosynthetic gene clusters.

    Science.gov (United States)

    Montiel, Daniel; Kang, Hahk-Soo; Chang, Fang-Yuan; Charlop-Powers, Zachary; Brady, Sean F

    2015-07-21

    Large-scale sequencing of prokaryotic (meta)genomic DNA suggests that most bacterial natural product gene clusters are not expressed under common laboratory culture conditions. Silent gene clusters represent a promising resource for natural product discovery and the development of a new generation of therapeutics. Unfortunately, the characterization of molecules encoded by these clusters is hampered owing to our inability to express these gene clusters in the laboratory. To address this bottleneck, we have developed a promoter-engineering platform to transcriptionally activate silent gene clusters in a model heterologous host. Our approach uses yeast homologous recombination, an auxotrophy complementation-based yeast selection system and sequence orthogonal promoter cassettes to exchange all native promoters in silent gene clusters with constitutively active promoters. As part of this platform, we constructed and validated a set of bidirectional promoter cassettes consisting of orthogonal promoter sequences, Streptomyces ribosome binding sites, and yeast selectable marker genes. Using these tools we demonstrate the ability to simultaneously insert multiple promoter cassettes into a gene cluster, thereby expediting the reengineering process. We apply this method to model active and silent gene clusters (rebeccamycin and tetarimycin) and to the silent, cryptic pseudogene-containing, environmental DNA-derived Lzr gene cluster. Complete promoter refactoring and targeted gene exchange in this "dead" cluster led to the discovery of potent indolotryptoline antiproliferative agents, lazarimides A and B. This potentially scalable and cost-effective promoter reengineering platform should streamline the discovery of natural products from silent natural product biosynthetic gene clusters.

  16. Disparate requirements for the Walker A and B ATPase motifs ofhuman RAD51D in homologous recombination

    Energy Technology Data Exchange (ETDEWEB)

    Wiese, Claudia; Hinz, John M.; Tebbs, Robert S.; Nham, Peter B.; Urbin, Salustra S.; Collins, David W.; Thompson, Larry H.; Schild, David

    2006-04-21

    In vertebrates, homologous recombinational repair (HRR) requires RAD51 and five RAD51 paralogs (XRCC2, XRCC3, RAD51B, RAD51C, and RAD51D) that all contain conserved Walker A and B ATPase motifs. In human RAD51D we examined the requirement for these motifs in interactions with XRCC2 and RAD51C, and for survival of cells in response to DNA interstrand crosslinks. Ectopic expression of wild type human RAD51D or mutants having a non-functional A or B motif was used to test for complementation of a rad51d knockout hamster CHO cell line. Although A-motif mutants complement very efficiently, B-motif mutants do not. Consistent with these results, experiments using the yeast two- and three-hybrid systems show that the interactions between RAD51D and its XRCC2 and RAD51C partners also require a functional RAD51D B motif, but not motif A. Similarly, hamster Xrcc2 is unable to bind to the non-complementing human RAD51D B-motif mutants in co-immunoprecipitation assays. We conclude that a functional Walker B motif, but not A motif, is necessary for RAD51D's interactions with other paralogs and for efficient HRR. We present a model in which ATPase sites are formed in a bipartite manner between RAD51D and other RAD51 paralogs.

  17. DNA Ligase IV and Artemis Act Cooperatively to Suppress Homologous Recombination in Human Cells: Implications for DNA Double-Strand Break Repair

    OpenAIRE

    Aya Kurosawa; Shinta Saito; Sairei So; Mitsumasa Hashimoto; Kuniyoshi Iwabuchi; Haruka Watabe; Noritaka Adachi

    2013-01-01

    Nonhomologous end-joining (NHEJ) and homologous recombination (HR) are two major pathways for repairing DNA double-strand breaks (DSBs); however, their respective roles in human somatic cells remain to be elucidated. Here we show using a series of human gene-knockout cell lines that NHEJ repairs nearly all of the topoisomerase II- and low-dose radiation-induced DNA damage, while it negatively affects survival of cells harbouring replication-associated DSBs. Intriguingly, we find that loss of ...

  18. 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.

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    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.

  19. Progress in the treatment of ovarian cancer-lessons from homologous recombination deficiency-the first 10 years.

    Science.gov (United States)

    Kaye, S B

    2016-04-01

    For several years, a major obstacle in the systemic treatment of ovarian cancer has been the lack of a therapeutic strategy tailored to specific biomarkers present in the individual patient's tumour. However, considerable progress has been made recently through the development of drugs targeting cells deficient in the key mechanism of double-strand DNA repair, known as homologous recombination (HRD). These drugs, inhibitors of the enzyme poly (ADP) ribose polymerase (PARP), selectively kill HRD cells through a process known as tumour-selective synthetic lethality. Olaparib is the first such agent, now approved for the treatment of ovarian cancer associated with mutations in the BRCA 1/2 genes, since these are characterised by cells with HRD. Importantly, another group of patients with tumours bearing a similar repair deficiency but without BRCA mutations may also be susceptible to PARP inhibition and efforts to develop an HRD assay are therefore a priority so that these patients can be identified as PARPi candidates. In addition, combination strategies are an area of intense research; these include combinations with antiangiogenic agents and with inhibitors of the P13K/AKT pathway and others are likely to merit assessment since resistance to PARP inhibitors will certainly emerge as the next challenge. While olaparib is the first PARP inhibitor to receive approval for ovarian cancer treatment, others including rucaparib and niraparib are clearly effective in this disease and, within the next year or two, the results of ongoing randomised trials will clarify their respective roles. PARP inhibitors are generally well tolerated; regulatory approval at present supports their use as a maintenance therapy (in Europe) and as treatment for advanced recurrent disease (in the United States), but it is likely that these indications will extend as the results of ongoing trials become available. Ten years have elapsed between the first pre-clinical publications and the

  20. Sensitization of Pancreatic Cancers to Gemcitabine Chemoradiation by WEE1 Kinase Inhibition Depends on Homologous Recombination Repair

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    Tasneem Kausar

    2015-10-01

    Full Text Available To improve the efficacy of chemoradiation therapy for locally advanced pancreatic cancer and begin to establish patient selection criteria, we investigated the combination of the WEE1 inhibitor AZD1775 with gemcitabine-radiation in homologous recombination (HR repair proficient and deficient pancreatic cancers. Sensitization to gemcitabine-radiation by AZD1775 was assessed in pancreatic cancer cells by clonogenic survival and in patient-derived xenografts by tumor growth. The contributions of HR repair inhibition and G2 checkpoint abrogation to sensitization were assessed by γH2AX, BRCA2 manipulation, and RAD51 focus formation and pHistone H3 flow cytometry, respectively. We found that AZD1775 sensitized to gemcitabine-radiation in BRCA2 wild-type but not BRCA2 mutant pancreatic cancer cells. In all cells, AZD1775 caused inhibition of CDK1 phosphorylation and G2 checkpoint abrogation. However, sensitization by AZD1775 was associated with persistent γH2AX and inhibition of RAD51 focus formation. In HR-proficient (BRCA2 wild-type or -deficient (BRAC2 null isogenic cells, AZD1775 sensitized to gemcitabine-radiation in BRCA2 wild-type, but not in BRCA2 null cells, despite significant G2 checkpoint abrogation. In patient-derived pancreatic tumor xenografts, AZD1775 significantly inhibited tumor growth and impaired RAD51 focus formation in response to gemcitabine-radiation. In conclusion, WEE1 inhibition by AZD1775 is an effective strategy for sensitizing pancreatic cancers to gemcitabine chemoradiation. Although this sensitization is accompanied by inhibition of CDK1 phosphorylation and G2 checkpoint abrogation, this mechanism is not sufficient for sensitization. Our findings demonstrate that sensitization to chemoradiation by WEE1 inhibition results from inhibition of HR repair and suggest that patient tumors without underlying HR defects would benefit most from this therapy.

  1. The rate of nonallelic homologous recombination in males is highly variable, correlated between monozygotic twins and independent of age.

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    Jacqueline A L MacArthur

    2014-03-01

    Full Text Available Nonallelic homologous recombination (NAHR between highly similar duplicated sequences generates chromosomal deletions, duplications and inversions, which can cause diverse genetic disorders. Little is known about interindividual variation in NAHR rates and the factors that influence this. We estimated the rate of deletion at the CMT1A-REP NAHR hotspot in sperm DNA from 34 male donors, including 16 monozygotic (MZ co-twins (8 twin pairs aged 24 to 67 years old. The average NAHR rate was 3.5 × 10(-5 with a seven-fold variation across individuals. Despite good statistical power to detect even a subtle correlation, we observed no relationship between age of unrelated individuals and the rate of NAHR in their sperm, likely reflecting the meiotic-specific origin of these events. We then estimated the heritability of deletion rate by calculating the intraclass correlation (ICC within MZ co-twins, revealing a significant correlation between MZ co-twins (ICC = 0.784, p = 0.0039, with MZ co-twins being significantly more correlated than unrelated pairs. We showed that this heritability cannot be explained by variation in PRDM9, a known regulator of NAHR, or variation within the NAHR hotspot itself. We also did not detect any correlation between Body Mass Index (BMI, smoking status or alcohol intake and rate of NAHR. Our results suggest that other, as yet unidentified, genetic or environmental factors play a significant role in the regulation of NAHR and are responsible for the extensive variation in the population for the probability of fathering a child with a genomic disorder resulting from a pathogenic deletion.

  2. Genetic polymorphisms in homologous recombination repair genes in healthy Slovenian population and their influence on DNA damage

    International Nuclear Information System (INIS)

    Homologous recombination (HR) repair is an important mechanism involved in repairing double-strand breaks in DNA and for maintaining genomic stability. Polymorphisms in genes coding for enzymes involved in this pathway may influence the capacity for DNA repair. The aim of this study was to select tag single nucleotide polymorphisms (SNPs) in specific genes involved in HR repair, to determine their allele frequencies in a healthy Slovenian population and their influence on DNA damage detected with comet assay. In total 373 individuals were genotyped for nine tag SNPs in three genes: XRCC3 722C>T, XRCC3 -316A>G, RAD51 -98G>C, RAD51 -61G>T, RAD51 1522T>G, NBS1 553G>C, NBS1 1197A>G, NBS1 37117C>T and NBS1 3474A>C using competitive allele-specific amplification (KASPar assay). Comet assay was performed in a subgroup of 26 individuals to determine the influence of selected SNPs on DNA damage. We observed that age significantly affected genotype frequencies distribution of XRCC3 -316A>G (P = 0.039) in healthy male blood donors. XRCC3 722C>T (P = 0.005), RAD51 -61G>T (P = 0.023) and NBS1 553G>C (P = 0.008) had a statistically significant influence on DNA damage. XRCC3 722C>T, RAD51 -61G>T and NBS1 553G>C polymorphisms significantly affect the repair of damaged DNA and may be of clinical importance as they are common in Slovenian population

  3. Plasticity of BRCA2 function in homologous recombination: genetic interactions of the PALB2 and DNA binding domains.

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    Nicolas Siaud

    2011-12-01

    Full Text Available The breast cancer suppressor BRCA2 is essential for the maintenance of genomic integrity in mammalian cells through its role in DNA repair by homologous recombination (HR. Human BRCA2 is 3,418 amino acids and is comprised of multiple domains that interact with the RAD51 recombinase and other proteins as well as with DNA. To gain insight into the cellular function of BRCA2 in HR, we created fusions consisting of various BRCA2 domains and also introduced mutations into these domains to disrupt specific protein and DNA interactions. We find that a BRCA2 fusion peptide deleted for the DNA binding domain and active in HR is completely dependent on interaction with the PALB2 tumor suppressor for activity. Conversely, a BRCA2 fusion peptide deleted for the PALB2 binding domain is dependent on an intact DNA binding domain, providing a role for this conserved domain in vivo; mutagenesis suggests that both single-stranded and double-stranded DNA binding activities in the DNA binding domain are required for its activity. Given that PALB2 itself binds DNA, these results suggest alternative mechanisms to deliver RAD51 to DNA. In addition, the BRCA2 C terminus contains both RAD51-dependent and -independent activities which are essential to HR in some contexts. Finally, binding the small peptide DSS1 is essential for activity when its binding domain is present, but not when it is absent. Our results reveal functional redundancy within the BRCA2 protein and emphasize the plasticity of this large protein built for optimal HR function in mammalian cells. The occurrence of disease-causing mutations throughout BRCA2 suggests sub-optimal HR from a variety of domain modulations.

  4. Sensitization of Pancreatic Cancers to Gemcitabine Chemoradiation by WEE1 Kinase Inhibition Depends on Homologous Recombination Repair.

    Science.gov (United States)

    Kausar, Tasneem; Schreiber, Jason S; Karnak, David; Parsels, Leslie A; Parsels, Joshua D; Davis, Mary A; Zhao, Lili; Maybaum, Jonathan; Lawrence, Theodore S; Morgan, Meredith A

    2015-10-01

    To improve the efficacy of chemoradiation therapy for locally advanced pancreatic cancer and begin to establish patient selection criteria, we investigated the combination of the WEE1 inhibitor AZD1775 with gemcitabine-radiation in homologous recombination (HR) repair proficient and deficient pancreatic cancers. Sensitization to gemcitabine-radiation by AZD1775 was assessed in pancreatic cancer cells by clonogenic survival and in patient-derived xenografts by tumor growth. The contributions of HR repair inhibition and G2 checkpoint abrogation to sensitization were assessed by γH2AX, BRCA2 manipulation, and RAD51 focus formation and pHistone H3 flow cytometry, respectively. We found that AZD1775 sensitized to gemcitabine-radiation in BRCA2 wild-type but not BRCA2 mutant pancreatic cancer cells. In all cells, AZD1775 caused inhibition of CDK1 phosphorylation and G2 checkpoint abrogation. However, sensitization by AZD1775 was associated with persistent γH2AX and inhibition of RAD51 focus formation. In HR-proficient (BRCA2 wild-type) or -deficient (BRAC2 null) isogenic cells, AZD1775 sensitized to gemcitabine-radiation in BRCA2 wild-type, but not in BRCA2 null cells, despite significant G2 checkpoint abrogation. In patient-derived pancreatic tumor xenografts, AZD1775 significantly inhibited tumor growth and impaired RAD51 focus formation in response to gemcitabine-radiation. In conclusion, WEE1 inhibition by AZD1775 is an effective strategy for sensitizing pancreatic cancers to gemcitabine chemoradiation. Although this sensitization is accompanied by inhibition of CDK1 phosphorylation and G2 checkpoint abrogation, this mechanism is not sufficient for sensitization. Our findings demonstrate that sensitization to chemoradiation by WEE1 inhibition results from inhibition of HR repair and suggest that patient tumors without underlying HR defects would benefit most from this therapy. PMID:26585231

  5. Low doses of alpha particles do not induce sister chromatid exchanges in bystander Chinese hamster cells defective in homologous recombination

    Energy Technology Data Exchange (ETDEWEB)

    Nagasawa, H; Wilson, P F; Chen, D J; Thompson, L H; Bedford, J S; Little, J B

    2007-10-26

    We reported previously that the homologous recombinational repair (HRR)-deficient Chinese hamster mutant cell line irs3 (deficient in the Rad51 paralog Rad51C) showed only a 50% spontaneous frequency of sister chromatid exchange (SCE) as compared to parental wild-type V79 cells. Furthermore, when irradiated with very low doses of alpha particles, SCEs were not induced in irs3 cells, as compared to a prominent bystander effect observed in V79 cells (Nagasawa et al., Radiat. Res. 164, 141-147, 2005). In the present study, we examined additional Chinese hamster cell lines deficient in the Rad51 paralogs Rad51C, Rad51D, Xrcc2, and Xrcc3 as well as another essential HRR protein, Brca2. Spontaneous SCE frequencies in non-irradiated wild-type cell lines CHO, AA8 and V79 were 0.33 SCE/chromosome, whereas two Rad51C-deficient cell lines showed only 0.16 SCE/chromosome. Spontaneous SCE frequencies in cell lines defective in Rad51D, Xrcc2, Xrcc3, and Brca2 ranged from 0.23-0.33 SCE/chromosome, 0-30% lower than wild-type cells. SCEs were induced significantly 20-50% above spontaneous levels in wild-type cells exposed to a mean dose of 1.3 mGy of alpha particles (<1% of nuclei traversed by an alpha particle). However, induction of SCEs above spontaneous levels was minimal or absent after {alpha}-particle irradiation in all of the HRR-deficient cell lines. These data suggest that Brca2 and the Rad51 paralogs contribute to DNA damage repair processes induced in bystander cells (presumably oxidative damage repair in S-phase cells) following irradiation with very low doses of alpha particles.

  6. Differential contributory roles of nucleotide excision and homologous recombination repair for enhancing cisplatin sensitivity in human ovarian cancer cells

    Directory of Open Access Journals (Sweden)

    Wani Gulzar

    2011-03-01

    Full Text Available Abstract Background While platinum-based chemotherapeutic agents are widely used to treat various solid tumors, the acquired platinum resistance is a major impediment in their successful treatment. Since enhanced DNA repair capacity is a major factor in conferring cisplatin resistance, targeting of DNA repair pathways is an effective stratagem for overcoming cisplatin resistance. This study was designed to delineate the role of nucleotide excision repair (NER, the principal mechanism for the removal of cisplatin-induced DNA intrastrand crosslinks, in cisplatin resistance and reveal the impact of DNA repair interference on cisplatin sensitivity in human ovarian cancer cells. Results We assessed the inherent NER efficiency of multiple matched pairs of cisplatin-sensitive and -resistant ovarian cancer cell lines and their expression of NER-related factors at mRNA and protein levels. Our results showed that only the cisplatin-resistant ovarian cancer cell line PEO4 possessed an increased NER capacity compared to its inherently NER-inefficient parental line PEO1. Several other cisplatin-resistant cell lines, including CP70, CDDP and 2008C13, exhibited a normal and parental cell-comparable NER capacity for removing cisplatin-induced DNA intrastrand cross-links (Pt-GG. Concomitant gene expression analysis revealed discordance in mRNA and protein levels of NER factors in various ovarian cancer cell lines and NER proteins level were unrelated to the cisplatin sensitivity of these cell lines. Although knockdown of NER factors was able to compromise the NER efficiency, it only caused a minimal effect on cisplatin sensitivity. On the contrary, downregulation of BRCA2, a critical protein for homologous recombination repair (HRR, significantly enhanced the efficacy of cisplatin in killing ovarian cancer cell line PEO4. Conclusion Our studies indicate that the level of NER factors in ovarian cancer cell lines is neither a determinant of their NER capacity nor

  7. A small molecule inhibitor of RAD51 reduces homologous recombination and sensitizes multiple myeloma cells to doxorubicin

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    David A. Alagpulinsa

    2014-10-01

    Full Text Available We previously reported high expression of RAD51 and increased homologous recombination (HR rates in multiple myeloma (MM cells, and showed that genomic instability and disease progression are commensurate with HR levels. Moreover, high RAD51 expression in vivo is associated with chemoresistance and poor patient survival. Doxorubicin (DOX is one of the most widely used drug treatments in MM chemotherapy. DOX is cytotoxic because it induces DNA double-strand breaks, which can be repaired by RAD51-mediated HR; activation of this pathway thus contributes to resistance. To investigate the role of RAD51 in MM drug resistance, we assessed the ability of B02, a small-molecule inhibitor of RAD51, to enhance DOX sensitivity of MM cells. Combining low-toxicity doses of DOX and B02 resulted in significant synthetic lethality, observed as increased apoptosis and reduced viability compared to either agent alone, or to the product of their individual effects. In contrast, the combination did not produce significant synergy against normal human CD19+ B cells from peripheral blood. DOX induced RAD51 at both mRNA and protein levels, while arresting cells in S and G2. DOX treatment also increased the number of RAD51 foci, a marker of HR repair, so that the fraction of cells with ≥5 foci rose 4-fold, whereas γH2AX foci rose far less, implying that most new breaks are repaired. When B02 treatment preceded DOX exposure, the induction of RAD51 foci was severely blunted, whereas γH2AX foci rose significantly relative to basal levels or either agent alone. In MM cells carrying a chromosomally integrated reporter of HR repair, DOX increased HR events while B02 inhibition of RAD51 blocked the HR response. These studies demonstrate the crucial role of RAD51 in protecting MM cells from genotoxic agents such as DOX, and suggest that specific inhibition of RAD51 may be an effective means to block DNA repair in MM cells and thus to enhance the efficacy of chemotherapy.

  8. RecF and RecR Play Critical Roles in the Homologous Recombination and Single-Strand Annealing Pathways of Mycobacteria.

    Science.gov (United States)

    Gupta, Richa; Shuman, Stewart; Glickman, Michael S

    2015-10-01

    Mycobacteria encode three DNA double-strand break repair pathways: (i) RecA-dependent homologous recombination (HR), (ii) Ku-dependent nonhomologous end joining (NHEJ), and (iii) RecBCD-dependent single-strand annealing (SSA). Mycobacterial HR has two presynaptic pathway options that rely on the helicase-nuclease AdnAB and the strand annealing protein RecO, respectively. Ablation of adnAB or recO individually causes partial impairment of HR, but loss of adnAB and recO in combination abolishes HR. RecO, which can accelerate annealing of single-stranded DNA in vitro, also participates in the SSA pathway. The functions of RecF and RecR, which, in other model bacteria, function in concert with RecO as mediators of RecA loading, have not been examined in mycobacteria. Here, we present a genetic analysis of recF and recR in mycobacterial recombination. We find that RecF, like RecO, participates in the AdnAB-independent arm of the HR pathway and in SSA. In contrast, RecR is required for all HR in mycobacteria and for SSA. The essentiality of RecR as an agent of HR is yet another distinctive feature of mycobacterial DNA repair.IMPORTANCE This study clarifies the molecular requirements for homologous recombination in mycobacteria. Specifically, we demonstrate that RecF and RecR play important roles in both the RecA-dependent homologous recombination and RecA-independent single-strand annealing pathways. Coupled with our previous findings (R. Gupta, M. Ryzhikov, O. Koroleva, M. Unciuleac, S. Shuman, S. Korolev, and M. S. Glickman, Nucleic Acids Res 41:2284-2295, 2013, http://dx.doi.org/10.1093/nar/gks1298), these results revise our view of mycobacterial recombination and place the RecFOR system in a central position in homology-dependent DNA repair. PMID:26195593

  9. A role for the malignant brain tumour (MBT domain protein LIN-61 in DNA double-strand break repair by homologous recombination.

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    Nicholas M Johnson

    Full Text Available Malignant brain tumour (MBT domain proteins are transcriptional repressors that function within Polycomb complexes. Some MBT genes are tumour suppressors, but how they prevent tumourigenesis is unknown. The Caenorhabditis elegans MBT protein LIN-61 is a member of the synMuvB chromatin-remodelling proteins that control vulval development. Here we report a new role for LIN-61: it protects the genome by promoting homologous recombination (HR for the repair of DNA double-strand breaks (DSBs. lin-61 mutants manifest numerous problems associated with defective HR in germ and somatic cells but remain proficient in meiotic recombination. They are hypersensitive to ionizing radiation and interstrand crosslinks but not UV light. Using a novel reporter system that monitors repair of a defined DSB in C. elegans somatic cells, we show that LIN-61 contributes to HR. The involvement of this MBT protein in HR raises the possibility that MBT-deficient tumours may also have defective DSB repair.

  10. A dual role for mycobacterial RecO in RecA-dependent homologous recombination and RecA-independent single-strand annealing.

    Science.gov (United States)

    Gupta, Richa; Ryzhikov, Mikhail; Koroleva, Olga; Unciuleac, Mihaela; Shuman, Stewart; Korolev, Sergey; Glickman, Michael S

    2013-02-01

    Mycobacteria have two genetically distinct pathways for the homology-directed repair of DNA double-strand breaks: homologous recombination (HR) and single-strand annealing (SSA). HR is abolished by deletion of RecA and reduced in the absence of the AdnAB helicase/nuclease. By contrast, SSA is RecA-independent and requires RecBCD. Here we examine the function of RecO in mycobacterial DNA recombination and repair. Loss of RecO elicits hypersensitivity to DNA damaging agents similar to that caused by deletion of RecA. We show that RecO participates in RecA-dependent HR in a pathway parallel to the AdnAB pathway. We also find that RecO plays a role in the RecA-independent SSA pathway. The mycobacterial RecO protein displays a zinc-dependent DNA binding activity in vitro and accelerates the annealing of SSB-coated single-stranded DNA. These findings establish a role for RecO in two pathways of mycobacterial DNA double-strand break repair and suggest an in vivo function for the DNA annealing activity of RecO proteins, thereby underscoring their similarity to eukaryal Rad52. PMID:23295671

  11. Expression of human poly (ADP-ribose) polymerase 1 in Saccharomyces cerevisiae: Effect on survival, homologous recombination and identification of genes involved in intracellular localization

    Energy Technology Data Exchange (ETDEWEB)

    La Ferla, Marco; Mercatanti, Alberto; Rocchi, Giulia; Lodovichi, Samuele; Cervelli, Tiziana; Pignata, Luca [Yeast Genetics and Genomics, Institute of Clinical Physiology, National Council of Research (CNR), via Moruzzi 1, 56122 Pisa (Italy); Caligo, Maria Adelaide [Section of Genetic Oncology, University Hospital and University of Pisa, via Roma 57, 56125 Pisa (Italy); Galli, Alvaro, E-mail: alvaro.galli@ifc.cnr.it [Yeast Genetics and Genomics, Institute of Clinical Physiology, National Council of Research (CNR), via Moruzzi 1, 56122 Pisa (Italy)

    2015-04-15

    Highlights: • The human poly (ADP-ribose) polymerase 1 (PARP-1) gene affects growth and UV-induced homologous recombination in yeast. • PARP-1 chemical inhibition impacts yeast growth and UV-induced recombination. • A genome-wide screen identifies 99 yeast genes that suppress the growth defect inferred by PARP-1. • Bioinformatics analysis identifies 41 human orthologues that may have a role in PARP-1 intracellular localization. • The findings suggest that PARP-1 nuclear localization may affect the response to PARP inhibitors in cancer therapy. - Abstract: The poly (ADP-ribose) polymerase 1 (PARP-1) actively participates in a series of functions within the cell that include: mitosis, intracellular signaling, cell cycle regulation, transcription and DNA damage repair. Therefore, inhibition of PARP1 has a great potential for use in cancer therapy. As resistance to PARP inhibitors is starting to be observed in patients, thus the function of PARP-1 needs to be studied in depth in order to find new therapeutic targets. To gain more information on the PARP-1 activity, we expressed PARP-1 in yeast and investigated its effect on cell growth and UV induced homologous recombination. To identify candidate genes affecting PARP-1 activity and cellular localization, we also developed a yeast genome wide genetic screen. We found that PARP-1 strongly inhibited yeast growth, but when yeast was exposed to the PARP-1 inhibitor 6(5-H) phenantridinone (PHE), it recovered from the growth suppression. Moreover, we showed that PARP-1 produced PAR products in yeast and we demonstrated that PARP-1 reduced UV-induced homologous recombination. By genome wide screening, we identified 99 mutants that suppressed PARP-1 growth inhibition. Orthologues of human genes were found for 41 of these yeast genes. We determined whether the PARP-1 protein level was altered in strains which are deleted for the transcription regulator GAL3, the histone H1 gene HHO1, the HUL4 gene, the

  12. Expression of human poly (ADP-ribose) polymerase 1 in Saccharomyces cerevisiae: Effect on survival, homologous recombination and identification of genes involved in intracellular localization

    International Nuclear Information System (INIS)

    Highlights: • The human poly (ADP-ribose) polymerase 1 (PARP-1) gene affects growth and UV-induced homologous recombination in yeast. • PARP-1 chemical inhibition impacts yeast growth and UV-induced recombination. • A genome-wide screen identifies 99 yeast genes that suppress the growth defect inferred by PARP-1. • Bioinformatics analysis identifies 41 human orthologues that may have a role in PARP-1 intracellular localization. • The findings suggest that PARP-1 nuclear localization may affect the response to PARP inhibitors in cancer therapy. - Abstract: The poly (ADP-ribose) polymerase 1 (PARP-1) actively participates in a series of functions within the cell that include: mitosis, intracellular signaling, cell cycle regulation, transcription and DNA damage repair. Therefore, inhibition of PARP1 has a great potential for use in cancer therapy. As resistance to PARP inhibitors is starting to be observed in patients, thus the function of PARP-1 needs to be studied in depth in order to find new therapeutic targets. To gain more information on the PARP-1 activity, we expressed PARP-1 in yeast and investigated its effect on cell growth and UV induced homologous recombination. To identify candidate genes affecting PARP-1 activity and cellular localization, we also developed a yeast genome wide genetic screen. We found that PARP-1 strongly inhibited yeast growth, but when yeast was exposed to the PARP-1 inhibitor 6(5-H) phenantridinone (PHE), it recovered from the growth suppression. Moreover, we showed that PARP-1 produced PAR products in yeast and we demonstrated that PARP-1 reduced UV-induced homologous recombination. By genome wide screening, we identified 99 mutants that suppressed PARP-1 growth inhibition. Orthologues of human genes were found for 41 of these yeast genes. We determined whether the PARP-1 protein level was altered in strains which are deleted for the transcription regulator GAL3, the histone H1 gene HHO1, the HUL4 gene, the

  13. Rosa26-GFP direct repeat (RaDR-GFP mice reveal tissue- and age-dependence of homologous recombination in mammals in vivo.

    Directory of Open Access Journals (Sweden)

    Michelle R Sukup-Jackson

    2014-06-01

    Full Text Available Homologous recombination (HR is critical for the repair of double strand breaks and broken replication forks. Although HR is mostly error free, inherent or environmental conditions that either suppress or induce HR cause genomic instability. Despite its importance in carcinogenesis, due to limitations in our ability to detect HR in vivo, little is known about HR in mammalian tissues. Here, we describe a mouse model in which a direct repeat HR substrate is targeted to the ubiquitously expressed Rosa26 locus. In the Rosa26 Direct Repeat-GFP (RaDR-GFP mice, HR between two truncated EGFP expression cassettes can yield a fluorescent signal. In-house image analysis software provides a rapid method for quantifying recombination events within intact tissues, and the frequency of recombinant cells can be evaluated by flow cytometry. A comparison among 11 tissues shows that the frequency of recombinant cells varies by more than two orders of magnitude among tissues, wherein HR in the brain is the lowest. Additionally, de novo recombination events accumulate with age in the colon, showing that this mouse model can be used to study the impact of chronic exposures on genomic stability. Exposure to N-methyl-N-nitrosourea, an alkylating agent similar to the cancer chemotherapeutic temozolomide, shows that the colon, liver and pancreas are susceptible to DNA damage-induced HR. Finally, histological analysis of the underlying cell types reveals that pancreatic acinar cells and liver hepatocytes undergo HR and also that HR can be specifically detected in colonic somatic stem cells. Taken together, the RaDR-GFP mouse model provides new understanding of how tissue and age impact susceptibility to HR, and enables future studies of genetic, environmental and physiological factors that modulate HR in mammals.

  14. Efficient assembly of full-length infectious clone of Brazilian IBDV isolate by homologous recombination in yeast

    OpenAIRE

    J.V.J. Silva Jr.; Arenhart, S.; Santos, H.F.; S.R. Almeida-Queiroz; Silva, A.N.M.R.; Trevisol, I.M.; Bertani, G.R.; L.H.V.G. Gil

    2015-01-01

    The Infectious Bursal Disease Virus (IBDV) causes immunosuppression in young chickens. Advances in molecular virology and vaccines for IBDV have been achieved by viral reverse genetics (VRG). VRG for IBDV has undergone changes over time, however all strategies used to generate particles of IBDV involves multiple rounds of amplification and need of in vitro ligation and restriction sites. The aim of this research was to build the world's first VRG for IBDV by yeast-based homologous recombinati...

  15. Independent intrachromosomal recombination events underlie the pericentric inversions of chimpanzee and gorilla chromosomes homologous to human chromosome 16

    OpenAIRE

    Goidts, Violaine; Szamalek, Justyna M.; de Jong, Pieter J; Cooper, David N.; Chuzhanova, Nadia; Hameister, Horst; Kehrer-Sawatzki, Hildegard

    2005-01-01

    Analyses of chromosomal rearrangements that have occurred during the evolution of the hominoids can reveal much about the mutational mechanisms underlying primate chromosome evolution. We characterized the breakpoints of the pericentric inversion of chimpanzee chromosome 18 (PTR XVI), which is homologous to human chromosome 16 (HSA 16). A conserved 23-kb inverted repeat composed of satellites, LINE and Alu elements was identified near the breakpoints and could have mediated the inversion by b...

  16. Rapid construction of phosphatase and tensin homolog-deleted on chromosome ten gene recombinant adenovirus using the AdEasy system

    Institute of Scientific and Technical Information of China (English)

    Yongqiong Wei; Lixue Chen; Zhaofang Zeng; Chongbiao Shen

    2010-01-01

    Recent studies have shown that phosphatase and tensin homolog-deleted on chromosome ten(PTEN)gene plays an important role in ischemic brain damage and synaptic plasticity.The AdEasy system,which has been widely used,greatly simplifies preparation of recombinant adenovirus.Therefore,recombinant defective adenovirus vector carrying human PTEN tumor suppressor gene(Ad-PTEN)was constructed using the AdEasy-1 system and was transfected into HEK293 cells for packaging and amplification.Infection efficiency and expression intensity were observed in primary cultured rat hippocampal neurons infected with Ad-PTEN in vitro.Results revealed a cytopathic effect in green fluorescent protein expression,which increased with prolonged time.After three cycles of amplification,the adenovirus titer was increased to an adequate titer for infecting hippocampal neurons.The entire process typically requires 4-5 weeks for completion.Results suggested that recombinant defective adenovirus vector carrying the PTEN gene was successfully and rapidly constructed using the AdEasy system.

  17. Increasing the efficiency of homologous recombination vector-mediated end joining repair by inhibition of Lig4 gene using siRNA in sheep embryo fibroblasts.

    Science.gov (United States)

    Wei, Wang; Yushuang, Wang; Lanlan, Huang; Zijian, Jian; Xinhua, Wang; Shouren, Liu; Wenhui, Pi

    2016-09-01

    In animal cells, inhibition of non-homologous end joining (NHEJ) pathway improves the efficiency of homologous recombination (HR)-mediated double-strand brakes (DSBs) repair. To improve the efficiency of HR in sheep embryo fibroblasts, the NHEJ key molecule DNA ligase 4 (Lig4) was suppressed by siRNA interference. Four pairs of siRNA targeting Lig4 were designed and chemically synthesized. These siRNA were electro-transferred into sheep embryo fibroblasts respectively. Compared with the control groups, two pairs of siRNA were identified to effectively inhibit the expression of sheep Lig4 gene by qRT-PCR and Western blotting. The plasmid rejoining assay was adopted for examining the efficiency of HR-mediated DSB repair. I-SceⅠ endonuclease linearized vector and siRNA were co-transfected into sheep embryo fibroblasts. Flow cytometry analysis of cells after transfection for 72 h showed that suppression of Lig4 using siRNAs increased the rejoining efficiency of HR vector by 3-4 times compared with the control groups. Therefore, enhanced HR vector rejoining frequency by instant inhabition of Lig4 gene provides theoretical basis for improving gene targeting efficiency of sheep embryo fibroblasts. PMID:27644744

  18. Constitutive expression of human coagulating factor IX in HeLa cells by homologous recombination of the promoter

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Constitutive expression of hFIX protein in nonhepatocytes wasstudied. The gene targeting vector was constructed and transferred into HeLa cells. With the detection system of PCR, we demonstrated that the endogenous hFIX promoter was replaced with an hCMV promoter when targeted insertion of the constructor was directed by the sequence homology. The expression of hFIX in the modified HeLa cells, 11.2 ng/106 cell/24 h, strongly suggested that hFIX gene could be activated by a powerful promoter in nonhepatocytes. The results would make it possible to examine the feasibility of re-regulate gene expression by promoter replacement.

  19. Construction and Identification of Lactobacillus Case Homologous Recombination Vector%干酪乳杆菌同源整合载体的构建与鉴定

    Institute of Scientific and Technical Information of China (English)

    郭建军; 龚小华; 袁林; 曾静; 付锦楠; 何顺华

    2015-01-01

    The purpose of this study was to construct homologous integration vector of Lactobacillus Case and to implement the ectogenic genome integration into Lactobacillus Case genome successfully. The lacZ gene with its promoter and catabolite activator protein ( CAP ) binding site was mplified from the pUC19 vector,and the Lactobacillus Casei 393 upp gene with its putative promoter region was PCR amplified,Both purified PCR amplicons were joined by SOE-PCR,The resulting lacZ-P-upp fusion products were ligated into the blunt-ended pORI28 backbone. Using chromosome DNA of Lactobacillus Casei 393 as template,the left and right arm of lai were amplified by PCR and purified by purification kit. The PCR product was cloned into pORZP vector,then the homologous recombina-tion pORZP-lai vector was obtained. The results showed that the target genes were amplified success-fully,double enzyme digestion and DNA sequencing confirmed that homologous recombination vector pORZP-lai was successfully constructed.%构建乳酸菌同源重组载体,以实现外源基因在乳酸菌中的整合型表达。 PCR 扩增 lacZ 基因表达盒lacZ-cassette,和upp基因表达盒upp-cassette,SOE-PCR扩增lacZ-P-upp,产物回收后连接到pORI28载体中,构建载体pORZP;根据干酪乳杆菌Lactobacillus casei 393全基因组中lai基因序列设计引物,PCR扩增lai基因两端同源重组臂lai-up和lai-down基因序列,产物经回收后,连接至pORZP载体中,得同源重组载体pORZP-lai。双酶切及DNA测序结果证实,成功构建了同源重组载体pORZP-lai,为实现外源基因在乳酸菌中的非抗性、整合型表达奠定了基础。

  20. Nrf2 facilitates repair of radiation induced DNA damage through homologous recombination repair pathway in a ROS independent manner in cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Jayakumar, Sundarraj; Pal, Debojyoti; Sandur, Santosh K., E-mail: sskumar@barc.gov.in

    2015-09-15

    Highlights: • Nrf2 inhibition in A549 cells led to attenuated DNA repair and radiosensitization. • Influence of Nrf2 on DNA repair is not linked to its antioxidant function. • Nrf2 influences DNA repair through homologous recombination (HR) repair pathway. • Many genes involved in HR pathway show ARE sequences in their upstream region. - Abstract: Nrf2 is a redox sensitive transcription factor that is involved in the co-ordinated transcription of genes involved in redox homeostasis. But the role of Nrf2 in DNA repair is not investigated in detail. We have employed A549 and MCF7 cells to study the role of Nrf2 on DNA repair by inhibiting Nrf2 using all-trans retinoic acid (ATRA) or by knock down approach prior to radiation exposure (4 Gy). DNA damage and repair analysis was studied by γH2AX foci formation and comet assay. Results suggested that the inhibition of Nrf2 in A549 or MCF7 cells led to significant slowdown in DNA repair as compared to respective radiation controls. The persistence of residual DNA damage even in the presence of free radical scavenger N-acetyl cysteine, suggested that the influence of Nrf2 on DNA repair was not linked to its antioxidant functions. Further, its influence on non-homologous end joining repair pathway was studied by inhibiting both Nrf2 and DNA-PK together. This led to synergistic reduction of survival fraction, indicating that Nrf2 may not be influencing the NHEJ pathway. To investigate the role of homologous recombination repair (HR) pathway, RAD51 foci formation was monitored. There was a significant reduction in the foci formation in cells treated with ATRA or shRNA against Nrf2 as compared to their respective radiation controls. Further, Nrf2 inhibition led to significant reduction in mRNA levels of RAD51. BLAST analysis was also performed on upstream regions of DNA repair genes to identify antioxidant response element and found that many repair genes that are involved in HR pathway may be regulated by Nrf2

  1. Immunization of Mastomys coucha with Brugia malayi recombinant trehalose-6-phosphate phosphatase results in significant protection against homologous challenge infection.

    Directory of Open Access Journals (Sweden)

    Susheela Kushwaha

    Full Text Available Development of a vaccine to prevent or reduce parasite development in lymphatic filariasis would be a complementary approach to existing chemotherapeutic tools. Trehalose-6-phosphate phosphatase of Brugia malayi (Bm-TPP represents an attractive vaccine target due to its absence in mammals, prevalence in the major life stages of the parasite and immunoreactivity with human bancroftian antibodies, especially from endemic normal subjects. We have recently reported on the cloning, expression, purification and biochemical characterization of this vital enzyme of B. malayi. In the present study, immunoprophylactic evaluation of Bm-TPP was carried out against B. malayi larval challenge in a susceptible host Mastomys coucha and the protective ability of the recombinant protein was evaluated by observing the adverse effects on microfilarial density and adult worm establishment. Immunization caused 78.4% decrease in microfilaremia and 71.04% reduction in the adult worm establishment along with sterilization of 70.06% of the recovered live females. The recombinant protein elicited a mixed Th1/Th2 type of protective immune response as evidenced by the generation of both pro- and anti-inflammatory cytokines IL-2, IFN-γ, TNF-α, IL-4 and an increased production of antibody isotypes IgG1, IgG2a, IgG2b and IgA. Thus immunization with Bm-TPP conferred considerable protection against B. malayi establishment by engendering a long-lasting effective immune response and therefore emerges as a potential vaccine candidate against lymphatic filariasis (LF.

  2. A novel partial deletion of the Y chromosome azoospermia factor c region is caused by non-homologous recombination between palindromes and may be associated with increased sperm counts

    NARCIS (Netherlands)

    M.J. Noordam; S.K.M. van Daalen; S.E. Hovingh; C.M. Korver; F. van der Veen; S. Repping

    2011-01-01

    BACKGROUND: The male-specific region of the human Y chromosome (MSY) contains multiple testis-specific genes. Most deletions in the MSY lead to inadequate or absent sperm production. Nearly all deletions occur via homologous recombination between amplicons. Previously, we identified two P5/distal-P1

  3. Efficacy of soluble recombinant FliC protein from Salmonella enterica serovar enteritidis as a potential vaccine candidate against homologous challenge in chickens.

    Science.gov (United States)

    Okamura, Masashi; Matsumoto, Wakako; Seike, Fumio; Tanaka, Yuuya; Teratani, Chie; Tozuka, Maki; Kashimoto, Takashige; Takehara, Kazuaki; Nakamura, Masayuki; Yoshikawa, Yasuhiro

    2012-06-01

    FliC, the flagellin antigen of Salmonella Enteritidis, was tested as a vaccine candidate for protective effect against a homologous challenge in chickens. After immunization with recombinant FliC (rFliC) or administration of phosphate-buffered saline (PBS) at 56 days old, the chickens were challenged with 10(9) colony-forming units of Salmonella Enteritidis at 76 days old. The vaccinated birds showed significantly decreased bacterial counts in the liver and cecal contents compared to those administered PBS at 7 days postchallenge, but the protection was partial. The replication experiment also showed a similar result. In both experiments, vaccination induced an increased level of serum anti-rFliC IgG, which was also reactive to the native flagella. The intestinal IgA level was slightly higher in the vaccinated birds than in the control. However, neither the proliferative response nor interferon-gamma secretion of splenic cells upon stimulation with rFliC was induced. Therefore, the effect of rFliC as a vaccine is limited, and further improvement is needed.

  4. Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids.

    Science.gov (United States)

    Sakuma, Tetsushi; Takenaga, Mitsumasa; Kawabe, Yoshinori; Nakamura, Takahiro; Kamihira, Masamichi; Yamamoto, Takashi

    2015-01-01

    Gene knock-in techniques have rapidly evolved in recent years, along with the development and maturation of genome editing technology using programmable nucleases. We recently reported a novel strategy for microhomology-mediated end-joining-dependent integration of donor DNA by using TALEN or CRISPR/Cas9 and optimized targeting vectors, named PITCh (Precise Integration into Target Chromosome) vectors. Here we describe TALEN and PITCh vector-mediated integration of long gene cassettes, including a single-chain Fv-Fc (scFv-Fc) gene, in Chinese hamster ovary (CHO) cells, with comparison of targeting and cloning efficiency among several donor design and culture conditions. We achieved 9.6-kb whole plasmid integration and 7.6-kb backbone-free integration into a defined genomic locus in CHO cells. Furthermore, we confirmed the reasonable productivity of recombinant scFv-Fc protein of the knock-in cells. Using our protocol, the knock-in cell clones could be obtained by a single transfection and a single limiting dilution using a 96-well plate, without constructing targeting vectors containing long homology arms. Thus, the study described herein provides a highly practical strategy for gene knock-in of large DNA in CHO cells, which accelerates high-throughput generation of cell lines stably producing any desired biopharmaceuticals, including huge antibody proteins. PMID:26473830

  5. DNA ligase IV and artemis act cooperatively to suppress homologous recombination in human cells: implications for DNA double-strand break repair.

    Directory of Open Access Journals (Sweden)

    Aya Kurosawa

    Full Text Available Nonhomologous end-joining (NHEJ and homologous recombination (HR are two major pathways for repairing DNA double-strand breaks (DSBs; however, their respective roles in human somatic cells remain to be elucidated. Here we show using a series of human gene-knockout cell lines that NHEJ repairs nearly all of the topoisomerase II- and low-dose radiation-induced DNA damage, while it negatively affects survival of cells harbouring replication-associated DSBs. Intriguingly, we find that loss of DNA ligase IV, a critical NHEJ ligase, and Artemis, an NHEJ factor with endonuclease activity, independently contribute to increased resistance to replication-associated DSBs. We also show that loss of Artemis alleviates hypersensitivity of DNA ligase IV-null cells to low-dose radiation- and topoisomerase II-induced DSBs. Finally, we demonstrate that Artemis-null human cells display increased gene-targeting efficiencies, particularly in the absence of DNA ligase IV. Collectively, these data suggest that DNA ligase IV and Artemis act cooperatively to promote NHEJ, thereby suppressing HR. Our results point to the possibility that HR can only operate on accidental DSBs when NHEJ is missing or abortive, and Artemis may be involved in pathway switching from incomplete NHEJ to HR.

  6. The receptor tyrosine kinase inhibitor amuvatinib (MP470) sensitizes tumor cells to radio- and chemo-therapies in part by inhibiting homologous recombination

    International Nuclear Information System (INIS)

    Background and purpose: RAD51 is a key protein involved in homologous recombination (HR) and a potential target for radiation- and chemotherapies. Amuvatinib (formerly known as MP470) is a novel receptor tyrosine kinase inhibitor that targets c-KIT and PDGFRα and can sensitize tumor cells to ionizing radiation (IR). Here, we studied amuvatinib mechanism on RAD51 and functional HR. Materials and methods: Protein and RNA analyses, direct repeat green fluorescent protein (DR-GFP) assay and polysomal fractioning were used to measure HR efficiency and global translation in amuvatinib-treated H1299 lung carcinoma cells. Synergy of amuvatinib with IR or mitomycin c (MMC) was assessed by clonogenic survival assay. Results: Amuvaninib inhibited RAD51 protein expression and HR. This was associated with reduced ribosomal protein S6 phosphorylation and inhibition of global translation. Amuvatinib sensitized cells to IR and MMC, agents that are selectively toxic to HR-deficient cells. Conclusions: Amuvatinib is a promising agent that may be used to decrease tumor cell resistance. Our work suggests that this is associated with decreased RAD51 expression and function and supports the further study of amuvatinib in combination with chemotherapy and radiotherapy.

  7. The impact of homologous recombination repair deficiency on depleted uranium clastogenicity in Chinese hamster ovary cells: XRCC3 protects cells from chromosome aberrations, but increases chromosome fragmentation.

    Science.gov (United States)

    Holmes, Amie L; Joyce, Kellie; Xie, Hong; Falank, Carolyne; Hinz, John M; Wise, John Pierce

    2014-04-01

    Depleted uranium (DU) is extensively used in both industry and military applications. The potential for civilian and military personnel exposure to DU is rising, but there are limited data on the potential health hazards of DU exposure. Previous laboratory research indicates DU is a potential carcinogen, but epidemiological studies remain inconclusive. DU is genotoxic, inducing DNA double strand breaks, chromosome damage and mutations, but the mechanisms of genotoxicity or repair pathways involved in protecting cells against DU-induced damage remain unknown. The purpose of this study was to investigate the effects of homologous recombination repair deficiency on DU-induced genotoxicity using RAD51D and XRCC3-deficient Chinese hamster ovary (CHO) cell lines. Cells deficient in XRCC3 (irs1SF) exhibited similar cytotoxicity after DU exposure compared to wild-type (AA8) and XRCC3-complemented (1SFwt8) cells, but DU induced more break-type and fusion-type lesions in XRCC3-deficient cells compared to wild-type and XRCC3-complemented cells. Surprisingly, loss of RAD51D did not affect DU-induced cytotoxicity or genotoxicity. DU induced selective X-chromosome fragmentation irrespective of RAD51D status, but loss of XRCC3 nearly eliminated fragmentation observed after DU exposure in wild-type and XRCC3-complemented cells. Thus, XRCC3, but not RAD51D, protects cells from DU-induced breaks and fusions and also plays a role in DU-induced chromosome fragmentation. PMID:24561002

  8. Site-specific DNA double-strand break generated by I-SceI endonuclease enhances ectopic homologous recombination in Pyricularia oryzae.

    Science.gov (United States)

    Arazoe, Takayuki; Younomaru, Tetsuya; Ohsato, Shuichi; Kimura, Makoto; Arie, Tsutomu; Kuwata, Shigeru

    2014-03-01

    To evaluate the contribution of DNA double-strand breaks (DSBs) to somatic homologous recombination (HR) in Pyricularia oryzae, we established a novel detection/selection system of DSBs-mediated ectopic HR. This system consists of donor and recipient nonfunctional yellow fluorescent protein (YFP)/blasticidin S deaminase (BSD) fusion genes and the yeast endonuclease I-SceI gene as a recipient-specific DSB inducer. The system enables to detect and select ectopic HR events by the restoration of YFP fluorescence and blasticidin S resistance. The transformed lines with donor and recipient showed low frequencies of endogenous ectopic HR (> 2.1%). Compared with spontaneous HR, c. 20-fold increases in HR and absolute frequency of HR as high as 40% were obtained by integration of I-SceI gene, indicating that I-SceI-mediated DSB was efficiently repaired via ectopic HR. Furthermore, to validate the impact of DSB on targeted gene replacement (TGR), the transformed lines with a recipient gene were transfected with an exogenous donor plasmid in combination with the DSB inducer. TGR events were not observed without the DSB inducer, whereas hundreds of colonies resulting from TGR events were obtained with the DSB inducer. These results clearly demonstrated that the introduction of site-specific DSB promotes ectopic HR repair in P. oryzae. PMID:24517488

  9. The DNA damage checkpoint pathway promotes extensive resection and nucleotide synthesis to facilitate homologous recombination repair and genome stability in fission yeast.

    Science.gov (United States)

    Blaikley, Elizabeth J; Tinline-Purvis, Helen; Kasparek, Torben R; Marguerat, Samuel; Sarkar, Sovan; Hulme, Lydia; Hussey, Sharon; Wee, Boon-Yu; Deegan, Rachel S; Walker, Carol A; Pai, Chen-Chun; Bähler, Jürg; Nakagawa, Takuro; Humphrey, Timothy C

    2014-05-01

    DNA double-strand breaks (DSBs) can cause chromosomal rearrangements and extensive loss of heterozygosity (LOH), hallmarks of cancer cells. Yet, how such events are normally suppressed is unclear. Here we identify roles for the DNA damage checkpoint pathway in facilitating homologous recombination (HR) repair and suppressing extensive LOH and chromosomal rearrangements in response to a DSB. Accordingly, deletion of Rad3(ATR), Rad26ATRIP, Crb2(53BP1) or Cdc25 overexpression leads to reduced HR and increased break-induced chromosome loss and rearrangements. We find the DNA damage checkpoint pathway facilitates HR, in part, by promoting break-induced Cdt2-dependent nucleotide synthesis. We also identify additional roles for Rad17, the 9-1-1 complex and Chk1 activation in facilitating break-induced extensive resection and chromosome loss, thereby suppressing extensive LOH. Loss of Rad17 or the 9-1-1 complex results in a striking increase in break-induced isochromosome formation and very low levels of chromosome loss, suggesting the 9-1-1 complex acts as a nuclease processivity factor to facilitate extensive resection. Further, our data suggest redundant roles for Rad3ATR and Exo1 in facilitating extensive resection. We propose that the DNA damage checkpoint pathway coordinates resection and nucleotide synthesis, thereby promoting efficient HR repair and genome stability. PMID:24623809

  10. The Over-expression of the β2 Catalytic Subunit of the Proteasome Decreases Homologous Recombination and Impairs DNA Double-Strand Break Repair in Human Cells

    Directory of Open Access Journals (Sweden)

    Anita Collavoli

    2011-01-01

    Full Text Available By a human cDNA library screening, we have previously identified two sequences coding two different catalytic subunits of the proteasome which increase homologous recombination (HR when overexpressed in the yeast Saccharomyces cerevisiae. Here, we investigated the effect of proteasome on spontaneous HR and DNA repair in human cells. To determine if the proteasome has a role in the occurrence of spontaneous HR in human cells, we overexpressed the β2 subunit of the proteasome in HeLa cells and determined the effect on intrachromosomal HR. Results showed that the overexpression of β2 subunit decreased HR in human cells without altering the cell proteasome activity and the Rad51p level. Moreover, exposure to MG132 that inhibits the proteasome activity reduced HR in human cells. We also found that the expression of the β2 subunit increases the sensitivity to the camptothecin that induces DNA double-strand break (DSB. This suggests that the β2 subunit has an active role in HR and DSB repair but does not alter the intracellular level of the Rad51p.

  11. Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids

    Directory of Open Access Journals (Sweden)

    Tetsushi Sakuma

    2015-10-01

    Full Text Available Gene knock-in techniques have rapidly evolved in recent years, along with the development and maturation of genome editing technology using programmable nucleases. We recently reported a novel strategy for microhomology-mediated end-joining-dependent integration of donor DNA by using TALEN or CRISPR/Cas9 and optimized targeting vectors, named PITCh (Precise Integration into Target Chromosome vectors. Here we describe TALEN and PITCh vector-mediated integration of long gene cassettes, including a single-chain Fv-Fc (scFv-Fc gene, in Chinese hamster ovary (CHO cells, with comparison of targeting and cloning efficiency among several donor design and culture conditions. We achieved 9.6-kb whole plasmid integration and 7.6-kb backbone-free integration into a defined genomic locus in CHO cells. Furthermore, we confirmed the reasonable productivity of recombinant scFv-Fc protein of the knock-in cells. Using our protocol, the knock-in cell clones could be obtained by a single transfection and a single limiting dilution using a 96-well plate, without constructing targeting vectors containing long homology arms. Thus, the study described herein provides a highly practical strategy for gene knock-in of large DNA in CHO cells, which accelerates high-throughput generation of cell lines stably producing any desired biopharmaceuticals, including huge antibody proteins.

  12. Genomic rearrangements at the FRA2H common fragile site frequently involve non-homologous recombination events across LTR and L1(LINE) repeats.

    Science.gov (United States)

    Brueckner, Lena M; Sagulenko, Evgeny; Hess, Elisa M; Zheglo, Diana; Blumrich, Anne; Schwab, Manfred; Savelyeva, Larissa

    2012-08-01

    Common fragile sites (cFSs) are non-random chromosomal regions that are prone to breakage under conditions of replication stress. DNA damage and chromosomal alterations at cFSs appear to be critical events in the development of various human diseases, especially carcinogenesis. Despite the growing interest in understanding the nature of cFS instability, only a few cFSs have been molecularly characterised. In this study, we fine-mapped the location of FRA2H using six-colour fluorescence in situ hybridisation and showed that it is one of the most active cFSs in the human genome. FRA2H encompasses approximately 530 kb of a gene-poor region containing a novel large intergenic non-coding RNA gene (AC097500.2). Using custom-designed array comparative genomic hybridisation, we detected gross and submicroscopic chromosomal rearrangements involving FRA2H in a panel of 54 neuroblastoma, colon and breast cancer cell lines. The genomic alterations frequently involved different classes of long terminal repeats and long interspersed nuclear elements. An analysis of breakpoint junction sequence motifs predominantly revealed signatures of microhomology-mediated non-homologous recombination events. Our data provide insight into the molecular structure of cFSs and sequence motifs affected by their activation in cancer. Identifying cFS sequences will accelerate the search for DNA biomarkers and targets for individualised therapies.

  13. Contribution of RecFOR machinery of homologous recombination to cell survival after loss of a restriction-modification gene complex.

    Science.gov (United States)

    Handa, Naofumi; Ichige, Asao; Kobayashi, Ichizo

    2009-07-01

    Loss of a type II restriction-modification (RM) gene complex, such as EcoRI, from a bacterial cell leads to death of its descendent cells through attack by residual restriction enzymes on undermethylated target sites of newly synthesized chromosomes. Through such post-segregational host killing, these gene complexes impose their maintenance on their host cells. This finding led to the rediscovery of type II RM systems as selfish mobile elements. The host prokaryote cells were found to cope with such attacks through a variety of means. The RecBCD pathway of homologous recombination in Escherichia coli repairs the lethal lesions on the chromosome, whilst it destroys restricted non-self DNA. recBCD homologues, however, appear very limited in distribution among bacterial genomes, whereas homologues of the RecFOR proteins, responsible for another pathway, are widespread in eubacteria, just like the RM systems. In the present work, therefore, we examined the possible contribution of the RecFOR pathway to cell survival after loss of an RM gene complex. A recF mutation reduced survival in an otherwise rec-positive background and, more severely, in a recBC sbcBC background. We also found that its effect is prominent in the presence of specific non-null mutant forms of the RecBCD enzyme: the resistance to killing seen with recC1002, recC1004, recC2145 and recB2154 is severely reduced to the level of a null recBC allele when combined with a recF, recO or recR mutant allele. Such resistance was also dependent on RecJ and RecQ functions. UV resistance of these non-null recBCD mutants is also reduced by recF, recJ or recQ mutation. These results demonstrate that the RecFOR pathway of recombination can contribute greatly to resistance to RM-mediated host killing, depending on the genetic background. PMID:19389761

  14. Hyperthermia adds to trabectedin effectiveness and thermal enhancement is associated with BRCA2 degradation and impairment of DNA homologous recombination repair.

    Science.gov (United States)

    Harnicek, Dominique; Kampmann, Eric; Lauber, Kirsten; Hennel, Roman; Cardoso Martins, Ana Sofia; Guo, Yang; Belka, Claus; Mörtl, Simone; Gallmeier, Eike; Kanaar, Roland; Mansmann, Ulrich; Hucl, Tomas; Lindner, Lars H; Hiddemann, Wolfgang; Issels, Rolf D

    2016-07-15

    The tetrahydroisoquinoline trabectedin is a marine compound with approved activity against human soft-tissue sarcoma. It exerts antiproliferative activity mainly by specific binding to the DNA and inducing DNA double-strand breaks (DSB). As homologous recombination repair (HRR)-deficient tumors are more susceptible to trabectedin, hyperthermia-mediated on-demand induction of HRR deficiency represents a novel and promising strategy to boost trabectedin treatment. For the first time, we demonstrate enhancement of trabectedin effectiveness in human sarcoma cell lines by heat and characterize cellular events and molecular mechanisms related to heat-induced effects. Hyperthermic temperatures (41.8 or 43°C) enhanced significantly trabectedin-related clonogenic cell death and G2/M cell cycle arrest followed by cell type-dependent induction of apoptosis or senescence. Heat combination increased accumulation of γH2AX foci as key marker of DSBs. Expression of BRCA2 protein, an integral protein of the HRR machinery, was significantly decreased by heat. Consequently, recruitment of downstream RAD51 to γH2AX-positive repair foci was almost abolished indicating relevant impairment of HRR by heat. Accordingly, enhancement of trabectedin effectiveness was significantly augmented in BRCA2-proficient cells by hyperthermia and alleviated in BRCA2 knockout or siRNA-transfected BRCA2 knockdown cells. In peripheral blood mononuclear cells isolated from sarcoma patients, increased numbers of nuclear γH2AX foci were detected after systemic treatment with trabectedin and hyperthermia of the tumor region. The findings establish BRCA2 degradation by heat as a key factor for a novel treatment strategy that allows targeted chemosensitization to trabectedin and other DNA damaging antitumor drugs by on-demand induction of HRR deficiency. PMID:26933761

  15. ATR-p53 restricts homologous recombination in response to replicative stress but does not limit DNA interstrand crosslink repair in lung cancer cells.

    Directory of Open Access Journals (Sweden)

    Bianca M Sirbu

    Full Text Available Homologous recombination (HR is required for the restart of collapsed DNA replication forks and error-free repair of DNA double-strand breaks (DSB. However, unscheduled or hyperactive HR may lead to genomic instability and promote cancer development. The cellular factors that restrict HR processes in mammalian cells are only beginning to be elucidated. The tumor suppressor p53 has been implicated in the suppression of HR though it has remained unclear why p53, as the guardian of the genome, would impair an error-free repair process. Here, we show for the first time that p53 downregulates foci formation of the RAD51 recombinase in response to replicative stress in H1299 lung cancer cells in a manner that is independent of its role as a transcription factor. We find that this downregulation of HR is not only completely dependent on the binding site of p53 with replication protein A but also the ATR/ATM serine 15 phosphorylation site. Genetic analysis suggests that ATR but not ATM kinase modulates p53's function in HR. The suppression of HR by p53 can be bypassed under experimental conditions that cause DSB either directly or indirectly, in line with p53's role as a guardian of the genome. As a result, transactivation-inactive p53 does not compromise the resistance of H1299 cells to the interstrand crosslinking agent mitomycin C. Altogether, our data support a model in which p53 plays an anti-recombinogenic role in the ATR-dependent mammalian replication checkpoint but does not impair a cell's ability to use HR for the removal of DSB induced by cytotoxic agents.

  16. The HSP90 inhibitor NVP-AUY922 radiosensitizes by abrogation of homologous recombination resulting in mitotic entry with unresolved DNA damage.

    Directory of Open Access Journals (Sweden)

    Shane Zaidi

    Full Text Available Heat shock protein 90 (HSP90 is a molecular chaperone responsible for the conformational maintenance of a number of client proteins that play key roles in cell cycle arrest, DNA damage repair and apoptosis following radiation. HSP90 inhibitors exhibit antitumor activity by modulating the stabilisation and activation of HSP90 client proteins. We sought to evaluate NVP-AUY922, the most potent HSP90 inhibitor yet reported, in preclinical radiosensitization studies.NVP-AUY922 potently radiosensitized cells in vitro at low nanomolar concentrations with a concurrent depletion of radioresistance-linked client proteins. Radiosensitization by NVP-AUY922 was verified for the first time in vivo in a human head and neck squamous cell carcinoma xenograft model in athymic mice, as measured by delayed tumor growth and increased surrogate end-point survival (p = <0.0001. NVP-AUY922 was shown to ubiquitously inhibit resolution of dsDNA damage repair correlating to delayed Rad51 foci formation in all cell lines tested. Additionally, NVP-AUY922 induced a stalled mitotic phenotype, in a cell line-dependent manner, in HeLa and HN5 cell lines irrespective of radiation exposure. Cell cycle analysis indicated that NVP-AUY922 induced aberrant mitotic entry in all cell lines tested in the presence of radiation-induced DNA damage due to ubiquitous CHK1 depletion, but resultant downstream cell cycle effects were cell line dependent.These results identify NVP-AUY922 as the most potent HSP90-mediated radiosensitizer yet reported in vitro, and for the first time validate it in a clinically relevant in vivo model. Mechanistic analysis at clinically achievable concentrations demonstrated that radiosensitization is mediated by the combinatorial inhibition of cell growth and survival pathways, ubiquitous delay in Rad51-mediated homologous recombination and CHK1-mediated G(2/M arrest, but that the contribution of cell cycle perturbation to radiosensitization may be cell line

  17. Rad51c- and Trp53-double-mutant mouse model reveals common features of homologous recombination-deficient breast cancers.

    Science.gov (United States)

    Tumiati, M; Munne, P M; Edgren, H; Eldfors, S; Hemmes, A; Kuznetsov, S G

    2016-09-01

    Almost half of all hereditary breast cancers (BCs) are associated with germ-line mutations in homologous recombination (HR) genes. However, the tumor phenotypes associated with different HR genes vary, making it difficult to define the role of HR in BC predisposition. To distinguish between HR-dependent and -independent features of BCs, we generated a mouse model in which an essential HR gene, Rad51c, is knocked-out specifically in epidermal tissues. Rad51c is one of the key mediators of HR and a well-known BC predisposition gene. Here, we demonstrate that deletion of Rad51c invariably requires inactivation of the Trp53 tumor suppressor (TP53 in humans) to produce mammary carcinomas in 63% of female mice. Nonetheless, loss of Rad51c shortens the latency of Trp53-deficient mouse tumors from 11 to 6 months. Remarkably, the histopathological features of Rad51c-deficient mammary carcinomas, such as expression of hormone receptors and luminal epithelial markers, faithfully recapitulate the histopathology of human RAD51C-mutated BCs. Similar to other BC models, Rad51c/p53 double-mutant mouse mammary tumors also reveal a propensity for genomic instability, but lack the focal amplification of the Met locus or distinct mutational signatures reported for other HR genes. Using the human mammary epithelial cell line MCF10A, we show that deletion of TP53 can rescue RAD51C-deficient cells from radiation-induced cellular senescence, whereas it exacerbates their centrosome amplification and nuclear abnormalities. Altogether, our data indicate that a trend for genomic instability and inactivation of Trp53 are common features of HR-mediated BCs, whereas histopathology and somatic mutation patterns are specific for different HR genes. PMID:26820992

  18. Combining Heavy Ion Radiation and Artificial MicroRNAs to Target the Homologous Recombination Repair Gene Efficiently Kills Human Tumor Cells

    International Nuclear Information System (INIS)

    Purpose: Previously, we demonstrated that heavy ions kill more cells at the same dose than X-rays because DNA-clustered lesions produced by heavy ions affect nonhomologous end-joining (NHEJ) repair but not homologous recombination repair (HRR). We have also shown that our designed artificial microRNAs (amiRs) could efficiently target XRCC4 (an essential factor for NHEJ) or XRCC2 (an essential factor for HRR) and sensitize human tumor cells to X-rays. Based on these data, we were interested in testing the hypothesis that combining heavy ions and amiRs to target HRR but not NHEJ should more efficiently kill human tumor cells. Methods and Materials: Human tumor cell lines (U87MG, a brain tumor cell line, and A549, a lung cancer cell line) and their counterparts, overexpressed with amiR to target XRCC2, XRCC4 or both, were used in this study. Survival sensitivities were examined using a clonogenic assay after these cells were exposed to X-rays or heavy ions. In addition, these cell lines were subcutaneously injected into nude mice to form xenografts and the tumor size was compared after the tumor areas were exposed to X-rays or heavy ions. Results: Although targeting either XRCC4 (NHEJ factor) or XRCC2 (HRR factor) sensitized the human tumor cells to X-rays, in vitro and the xenograft animal model, targeting only XRCC2 but not XRCC4 sensitized the human tumor cells to heavy ions in vitro and in the xenograft animal model. Conclusions: Combining heavy ions with targeting the HRR pathway, but not the NHEJ pathway, could significantly improve the efficiency of tumor cell death.

  19. Combining Heavy Ion Radiation and Artificial MicroRNAs to Target the Homologous Recombination Repair Gene Efficiently Kills Human Tumor Cells

    Energy Technology Data Exchange (ETDEWEB)

    Zheng Zhiming [Department of Neurosurgery, Provincial Hospital Affiliated to Shandong University, Shandong University, Jinan (China); Department of Radiation Oncology, School of Medicine, Winship Cancer Institute, Emory University, Atlanta, Georgia (United States); Wang Ping; Wang Hongyan; Zhang Xiangming [Department of Radiation Oncology, School of Medicine, Winship Cancer Institute, Emory University, Atlanta, Georgia (United States); Wang Minli [Division of Life Sciences, Universities Space Research Association, Houston, Texas (United States); Cucinotta, Francis A. [National Aeronautics and Space Administration, Lyndon B. Johnson Space Center, Houston, Texas (United States); Wang Ya, E-mail: ywang94@emory.edu [Department of Radiation Oncology, School of Medicine, Winship Cancer Institute, Emory University, Atlanta, Georgia (United States)

    2013-02-01

    Purpose: Previously, we demonstrated that heavy ions kill more cells at the same dose than X-rays because DNA-clustered lesions produced by heavy ions affect nonhomologous end-joining (NHEJ) repair but not homologous recombination repair (HRR). We have also shown that our designed artificial microRNAs (amiRs) could efficiently target XRCC4 (an essential factor for NHEJ) or XRCC2 (an essential factor for HRR) and sensitize human tumor cells to X-rays. Based on these data, we were interested in testing the hypothesis that combining heavy ions and amiRs to target HRR but not NHEJ should more efficiently kill human tumor cells. Methods and Materials: Human tumor cell lines (U87MG, a brain tumor cell line, and A549, a lung cancer cell line) and their counterparts, overexpressed with amiR to target XRCC2, XRCC4 or both, were used in this study. Survival sensitivities were examined using a clonogenic assay after these cells were exposed to X-rays or heavy ions. In addition, these cell lines were subcutaneously injected into nude mice to form xenografts and the tumor size was compared after the tumor areas were exposed to X-rays or heavy ions. Results: Although targeting either XRCC4 (NHEJ factor) or XRCC2 (HRR factor) sensitized the human tumor cells to X-rays, in vitro and the xenograft animal model, targeting only XRCC2 but not XRCC4 sensitized the human tumor cells to heavy ions in vitro and in the xenograft animal model. Conclusions: Combining heavy ions with targeting the HRR pathway, but not the NHEJ pathway, could significantly improve the efficiency of tumor cell death.

  20. Association between single nucleotide polymorphisms (SNPs) of XRCC2 and XRCC3 homologous recombination repair genes and ovarian cancer in Polish women.

    Science.gov (United States)

    Michalska, Magdalena M; Samulak, Dariusz; Romanowicz, Hanna; Jabłoński, Filip; Smolarz, Beata

    2016-04-01

    The variability, perceived in DNA repair genes, may be of clinical importance for evaluation of the risk of occurrence of a given type of cancer, its prophylactics and therapy. The aim of the present work was to evaluate associations between the risk of ovarian cancer and polymorphisms in the genes, encoding for two key proteins of homologous recombination: XRCC2 Arg188His (c. 563 G>A; rs3218536) and XRCC3 Thr241Met (c. 722 C>T; rs861539). The study consisted of 700 patients with ovarian cancer and 700 healthy subjects. Analysis of the gene polymorphisms was performed using PCR-RFLP (restriction length fragment polymorphism). We found a statistically significant increase of the 188His allele frequency (OR=4.01; 95% CI=3.40-4.72; pcancer compared to healthy controls. There were no differences in the genotype and allele distributions and odds ratios of the XRCC3 Thr241Met polymorphism between patient and control groups. Association of these genetic polymorphisms with histological grading showed increased XRCC2 188Arg/His (OR=33.0; 95% CI=14.51-75.05; p<.0001) and 188His/His genotypes (OR=9.37; 95% CI=4.79-18.32; p<.0001) and XRCC3 241Thr/Met (OR=24.28; 95% CI=12.38-47.61; p<.0001) and 241Met/Met genotype frequencies (OR=17.00; 95% CI=8.42-34.28; p<.0001) in grading 1 (G1) as well as 188His (OR=2.78; 95% CI=2.11-3.69; p<.0001) and 241Met allele overrepresentation (OR=2.59; 95% CI=2.08-3.22; p<.0001) in G1 ovarian patients. Finally, with clinical FIGO staging under evaluation, an increase in XRCC2 188His/His homozygote and 188Arg/His heterozygote frequencies in staging I (SI) and XRCC3 Thr/Met heterozygote frequencies in SI was observed. The obtained results indicate that XRCC2 Arg188His and XRCC3 Thr241Met polymorphisms may be positively associated with the incidence of ovarian carcinoma in the population of Polish women.

  1. DNA-PK inhibition causes a low level of H2AX phosphorylation and homologous recombination repair in Medaka (Oryzias latipes) cells

    International Nuclear Information System (INIS)

    Highlights: ► We investigated the effect of DNA-PK inhibition on DSB repair using fish cells. ► A radiation sensitive mutant RIC1 strain showed a low level of DNA-PK activity. ► DNA-PK dysfunction leads defects in HR repair and DNA-PKcs autophosphorylation. ► DNA-PK dysfunction leads a slight increase in the number of 53BP1 foci after DSBs. ► DNA-PK dysfunction leads an alternative NHEJ that depends on 53BP1. -- Abstract: Nonhomologous end joining (NHEJ) and homologous recombination (HR) are known as DNA double-strand break (DSB) repair pathways. It has been reported that DNA-PK, a member of PI3 kinase family, promotes NHEJ and aberrant DNA-PK causes NHEJ deficiency. However, in this study, we demonstrate that a wild-type cell line treated with DNA-PK inhibitor and a mutant cell line with dysfunctional DNA-PK showed decreased HR efficiency in fish cells (Medaka, Oryzias latipes). Previously, we reported that the radiation-sensitive mutant RIC1 strain has a defect in the Histone H2AX phosphorylation after γ-irradiation. Here, we showed that a DNA-PK inhibitor, NU7026, treatment resulted in significant reduction in the number of γH2AX foci after γ-irradiation in wild-type cells, but had no significant effect in RIC1 cells. In addition, RIC1 cells showed significantly lower levels of DNA-PK kinase activity compared with wild-type cells. We investigated NHEJ and HR efficiency after induction of DSBs. Wild-type cells treated with NU7026 and RIC1 cells showed decreased HR efficiency. These results indicated that aberrant DNA-PK causes the reduction in the number of γH2AX foci and HR efficiency in RIC1 cells. We performed phosphorylated DNA-PKcs (Thr2609) and 53BP1 focus assay after γ-irradiation. RIC1 cells showed significant reduction in the number of phosphorylated DNA-PKcs foci and no deference in the number of 53BP1 foci compared with wild-type cells. These results suggest that low level of DNA-PK activity causes aberrant DNA-PKcs autophosphorylation

  2. DNA-PK inhibition causes a low level of H2AX phosphorylation and homologous recombination repair in Medaka (Oryzias latipes) cells

    Energy Technology Data Exchange (ETDEWEB)

    Urushihara, Yusuke [Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba 277-8562 (Japan); Kobayashi, Junya [Department of Genome Repair Dynamics, Radiation Biology Center, Kyoto University, Kyoto 606-8501 (Japan); Matsumoto, Yoshihisa [Research Laboratory for Nuclear Reactors, Tokyo Institute of Technology, Tokyo 152-8550 (Japan); Komatsu, Kenshi [Department of Genome Repair Dynamics, Radiation Biology Center, Kyoto University, Kyoto 606-8501 (Japan); Oda, Shoji [Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba 277-8562 (Japan); Mitani, Hiroshi, E-mail: mitani@k.u-tokyo.ac.jp [Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba 277-8562 (Japan)

    2012-12-14

    Highlights: Black-Right-Pointing-Pointer We investigated the effect of DNA-PK inhibition on DSB repair using fish cells. Black-Right-Pointing-Pointer A radiation sensitive mutant RIC1 strain showed a low level of DNA-PK activity. Black-Right-Pointing-Pointer DNA-PK dysfunction leads defects in HR repair and DNA-PKcs autophosphorylation. Black-Right-Pointing-Pointer DNA-PK dysfunction leads a slight increase in the number of 53BP1 foci after DSBs. Black-Right-Pointing-Pointer DNA-PK dysfunction leads an alternative NHEJ that depends on 53BP1. -- Abstract: Nonhomologous end joining (NHEJ) and homologous recombination (HR) are known as DNA double-strand break (DSB) repair pathways. It has been reported that DNA-PK, a member of PI3 kinase family, promotes NHEJ and aberrant DNA-PK causes NHEJ deficiency. However, in this study, we demonstrate that a wild-type cell line treated with DNA-PK inhibitor and a mutant cell line with dysfunctional DNA-PK showed decreased HR efficiency in fish cells (Medaka, Oryzias latipes). Previously, we reported that the radiation-sensitive mutant RIC1 strain has a defect in the Histone H2AX phosphorylation after {gamma}-irradiation. Here, we showed that a DNA-PK inhibitor, NU7026, treatment resulted in significant reduction in the number of {gamma}H2AX foci after {gamma}-irradiation in wild-type cells, but had no significant effect in RIC1 cells. In addition, RIC1 cells showed significantly lower levels of DNA-PK kinase activity compared with wild-type cells. We investigated NHEJ and HR efficiency after induction of DSBs. Wild-type cells treated with NU7026 and RIC1 cells showed decreased HR efficiency. These results indicated that aberrant DNA-PK causes the reduction in the number of {gamma}H2AX foci and HR efficiency in RIC1 cells. We performed phosphorylated DNA-PKcs (Thr2609) and 53BP1 focus assay after {gamma}-irradiation. RIC1 cells showed significant reduction in the number of phosphorylated DNA-PKcs foci and no deference in the

  3. A novel third type of recurrent NF1 microdeletion mediated by nonallelic homologous recombination between LRRC37B-containing low-copy repeats in 17q11.2.

    Science.gov (United States)

    Bengesser, Kathrin; Cooper, David N; Steinmann, Katharina; Kluwe, Lan; Chuzhanova, Nadia A; Wimmer, Katharina; Tatagiba, Marcos; Tinschert, Sigrid; Mautner, Victor-Felix; Kehrer-Sawatzki, Hildegard

    2010-06-01

    Large microdeletions encompassing the neurofibromatosis type-1 (NF1) gene and its flanking regions at 17q11.2 belong to the group of genomic disorders caused by aberrant recombination between segmental duplications. The most common NF1 microdeletions (type-1) span 1.4-Mb and have breakpoints located within NF1-REPs A and C, low-copy repeats (LCRs) containing LRRC37-core duplicons. We have identified a novel type of recurrent NF1 deletion mediated by nonallelic homologous recombination (NAHR) between the highly homologous NF1-REPs B and C. The breakpoints of these approximately 1.0-Mb ("type-3") NF1 deletions were characterized at the DNA sequence level in three unrelated patients. Recombination regions, spanning 275, 180, and 109-bp, respectively, were identified within the LRRC37B-P paralogues of NF1-REPs B and C, and were found to contain sequences capable of non-B DNA formation. Both LCRs contain LRRC37-core duplicons, abundant and highly dynamic sequences in the human genome. NAHR between LRRC37-containing LCRs at 17q21.31 is known to have mediated the 970-kb polymorphic inversions of the MAPT-locus that occurred independently in different primate species, but also underlies the syndromes associated with recurrent 17q21.31 microdeletions and reciprocal microduplications. The novel NF1 microdeletions reported here provide further evidence for the unusually high recombinogenic potential of LRRC37-containing LCRs in the human genome. PMID:20506354

  4. Spiroplasma citri virus SpV1-derived cloning vector: deletion formation by illegitimate and homologous recombination in a spiroplasmal host strain which probably lacks a functional recA gene.

    OpenAIRE

    Marais, A.; Bové, J M; Renaudin, J

    1996-01-01

    We have previously described the use of the replicative form (RF) of Spiroplasma citri virus SpV1 as a vector for expressing an epitope of the P1 adhesin protein from Mycoplasma pneumoniae in S. citri (A. Marais, J. M. Bové, S.F. Dallo, J. B. Baseman, and J. Renaudin, J. Bacteriol. 175:2783-2787, 1993). We have now studied the structural instability of the recombinant RF leading to loss of the DNA insert. Analyses of viral clones with deletions have shown that both illegitimate and homologous...

  5. A novel third type of recurrent NF1 microdeletion mediated by non-allelic homologous recombination between LRRC37B-containing low-copy repeats in 17q11.2

    OpenAIRE

    Bengesser, Kathrin; Cooper, David N.; Steinmann, Katharina; Kluwe, Lan; Chuzhanova, Nadia; Wimmer, Katharina; Tatagiba, Marcos; Tinschert, Sigrid; Mautner, Victor; Kehrer-Sawatzki, Hildegard

    2010-01-01

    Abstract Large microdeletions encompassing the neurofibromatosis type-1 (NF1) gene and its flanking regions at 17q11.2 belong to the group of genomic disorders caused by aberrant recombination between segmental duplications. The most common NF1 microdeletions (type-1) span 1.4-Mb and have breakpoints located within NF1-REPs A and C, low-copy repeats (LCRs) containing LRRC37-core duplicons. We have identified a novel type of recurrent NF1 deletion mediated by non-allelic homologous ...

  6. DNA apoptosis and stability in B-cell chronic lymphoid leukaemia: implication of the DNA double-strand breaks repair system by non homologous recombination

    International Nuclear Information System (INIS)

    After an introduction presenting the diagnosis and treatment of chronic lymphoid leukaemia, its molecular and genetic characteristics, and its cellular origin and clonal evolution, this research thesis describes the apoptosis (definition and characteristics, cancer and chemotherapy, apoptotic ways induced by gamma irradiation), the genotoxic stresses, the different repair mechanisms for different damages, and the DNA repair processes. It reports how human chronic lymphocytic leukaemia B cells can escape DNA damage-induced apoptosis through the non-homologous end-joining DNA repair pathway, and presents non-homologous end-joining DNA repair as a potent mutagenic process in human chronic lymphocytic leukaemia B cells

  7. Directed homology

    DEFF Research Database (Denmark)

    Fahrenberg, Uli

    2004-01-01

    We introduce a new notion of directed homology for semicubical sets. We show that it respects directed homotopy and is functorial, and that it appears to enjoy some good algebraic properties. Our work has applications to higher-dimensional automata.......We introduce a new notion of directed homology for semicubical sets. We show that it respects directed homotopy and is functorial, and that it appears to enjoy some good algebraic properties. Our work has applications to higher-dimensional automata....

  8. Detection of rbf via homologous recombination in a clinical isolate of Staphylococcus aureus%同源重组构建临床分离金黄色葡萄球菌rbf基因阴性突变株

    Institute of Scientific and Technical Information of China (English)

    薛跃华; 林亦海; 李招云; 徐正保; 张黎明; 张春玲

    2013-01-01

    目的 利用同源重组技术敲除金黄色葡萄球菌rbf基因,构建金黄色葡萄球菌临床分离株金黄色葡萄球菌20(SAU20)rbf基因的阴性突变株.方法 构建pBT2-Δrbf质粒后将之电转入金黄色葡萄球菌RN 4220中,再次将pBT2-Δrbf质粒转入金黄色葡萄球菌临床分离株SAU20;利用pBT2载体对温度敏感的特点,使含重组质粒的SAU20在40℃多次传代,最终筛选出金黄色葡萄球菌rbf基因的阴性突变株.结果 成功构建pBT2-Δrbf质粒,pBT2-Δrbf质粒被转入SAU20后,筛选到可疑突变株,经PCR及DNA测序证实SAU20 rbf基因被ermB基因置换.结论 利用同源重组技术敲除了金黄色葡萄球菌rbf基因,完成了金黄色葡萄球菌临床分离株SAU20 rbf基因的阴性突变株的构建,为今后研究金黄色葡萄球菌生物膜形成过程中rbf基因的作用及其机制提供了实验载体.%OBJECTIVE To delete rbf in a clinical isolate of Staphylococcus aureus homologous recombination and to get a S. aureus strain 20(SAU20)of the rbf negative mutant. METHODS A plasmid pBT2-△rbf was constructed with inserting erythromycin resistance gene and two PCR-amplified rbf/-flanking regions for homologous recombination of S. aureus. The recombination vector was transformed to S. aureus RN4220 by electroporation firstly and then transformed to S. aureus 20. SAU20 with recombination vector was incubated at 40~*C until the rbf delectation strain was selected. RESULTS _pBT2-Arbf has been successfully constructed, the suspected mutant strains were screened out after the _pBT2-△rbf was transferred to the SAU20, the restriction endonucleases results indicated that the homologous recombination vector was correct and SAU20 accepted the recombination vector. The rbf of SAU20 was replaced by ermB, which was proved by PCR and DNA sequencing. CONCLUSION S. aureus rbf deletiou mutation has been constructed successfully. The sequence of agr gene is replaced by ermB gene in SAU20, which

  9. NRAGE is involved in homologous recombination repair to resist the DNA-damaging chemotherapy and composes a ternary complex with RNF8-BARD1 to promote cell survival in squamous esophageal tumorigenesis.

    Science.gov (United States)

    Yang, Q; Pan, Q; Li, C; Xu, Y; Wen, C; Sun, F

    2016-08-01

    NRAGE, a neurotrophin receptor-interacting melanoma antigen-encoding gene homolog, is significantly increased in the nucleus of radioresistant esophageal tumor cell lines and is highly upregulated to promote cell proliferation in esophageal carcinomas (ECs). However, whether the overexpressed NRAGE promotes cell growth by participating in DNA-damage response (DDR) is still unclear. Here we show that NRAGE is required for efficient double-strand breaks (DSBs) repair via homologous recombination repair (HRR) and downregulation of NRAGE greatly sensitizes EC cells to DNA-damaging agents both in vitro and in vivo. Moreover, NRAGE not only regulates the stability of DDR factors, RNF8 and BARD1, in a ubiquitin-proteolytic pathway, but also chaperons the interaction between BARD1 and RNF8 via their RING domains to form a novel ternary complex. Additionally, the expression of NRAGE is closely correlated with RNF8 and BARD1 in esophageal tumor tissues. In summary, our findings reveal a novel function of NRAGE that will help to guide personalized esophageal cancer treatments by targeting NRAGE to increase cell sensitivity to DNA-damaging therapeutics in the long run. PMID:27035619

  10. Homologous elements hs3a and hs3b in the 3' regulatory region of the murine immunoglobulin heavy chain (Igh) locus are both dispensable for class-switch recombination.

    Science.gov (United States)

    Yan, Yi; Pieretti, Joyce; Ju, Zhongliang; Wei, Shiniu; Christin, John R; Bah, Fatmata; Birshtein, Barbara K; Eckhardt, Laurel A

    2011-08-01

    Immunoglobulin heavy chain (IgH) genes are formed, tested, and modified to yield diverse, specific, and high affinity antibody responses to antigen. The processes involved must be regulated, however, to avoid unintended damage to chromosomes. The 3' regulatory region of the Igh locus plays a major role in regulating class-switch recombination (CSR), the process by which antibody effector functions are modified during an immune response. Loss of all known enhancer-like elements in this region dramatically impairs CSR, but individual element deletions have no effect on this process. In the present study, we explored the hypothesis that an underlying functional redundancy in the homologous elements hs3a and hs3b was masking the importance of either element to CSR. Several transgenic mouse lines were generated, each carrying a bacterial artificial chromosome transgene that mimicked Igh locus structure but in which hs3a was missing and hs3b was flanked by loxP sites. Matings to Cyclization Recombination Enzyme-expressing mice established "pairs" of lines that differed only in the presence or absence of hs3b. Remarkably, CSR remained robust in the absence of both hs3a and hs3b, suggesting that the remaining two elements of the 3' regulatory region, hs1.2 and hs4, although individually dispensable for CSR, are, together, sufficient to support CSR.

  11. Homologous elements hs3a and hs3b in the 3' regulatory region of the murine immunoglobulin heavy chain (Igh) locus are both dispensable for class-switch recombination.

    Science.gov (United States)

    Yan, Yi; Pieretti, Joyce; Ju, Zhongliang; Wei, Shiniu; Christin, John R; Bah, Fatmata; Birshtein, Barbara K; Eckhardt, Laurel A

    2011-08-01

    Immunoglobulin heavy chain (IgH) genes are formed, tested, and modified to yield diverse, specific, and high affinity antibody responses to antigen. The processes involved must be regulated, however, to avoid unintended damage to chromosomes. The 3' regulatory region of the Igh locus plays a major role in regulating class-switch recombination (CSR), the process by which antibody effector functions are modified during an immune response. Loss of all known enhancer-like elements in this region dramatically impairs CSR, but individual element deletions have no effect on this process. In the present study, we explored the hypothesis that an underlying functional redundancy in the homologous elements hs3a and hs3b was masking the importance of either element to CSR. Several transgenic mouse lines were generated, each carrying a bacterial artificial chromosome transgene that mimicked Igh locus structure but in which hs3a was missing and hs3b was flanked by loxP sites. Matings to Cyclization Recombination Enzyme-expressing mice established "pairs" of lines that differed only in the presence or absence of hs3b. Remarkably, CSR remained robust in the absence of both hs3a and hs3b, suggesting that the remaining two elements of the 3' regulatory region, hs1.2 and hs4, although individually dispensable for CSR, are, together, sufficient to support CSR. PMID:21673112

  12. Members of the RAD52 Epistasis Group Contribute to Mitochondrial Homologous Recombination and Double-Strand Break Repair in Saccharomyces cerevisiae.

    Science.gov (United States)

    Stein, Alexis; Kalifa, Lidza; Sia, Elaine A

    2015-11-01

    Mitochondria contain an independently maintained genome that encodes several proteins required for cellular respiration. Deletions in the mitochondrial genome have been identified that cause several maternally inherited diseases and are associated with certain cancers and neurological disorders. The majority of these deletions in human cells are flanked by short, repetitive sequences, suggesting that these deletions may result from recombination events. Our current understanding of the maintenance and repair of mtDNA is quite limited compared to our understanding of similar events in the nucleus. Many nuclear DNA repair proteins are now known to also localize to mitochondria, but their function and the mechanism of their action remain largely unknown. This study investigated the contribution of the nuclear double-strand break repair (DSBR) proteins Rad51p, Rad52p and Rad59p in mtDNA repair. We have determined that both Rad51p and Rad59p are localized to the matrix of the mitochondria and that Rad51p binds directly to mitochondrial DNA. In addition, a mitochondrially-targeted restriction endonuclease (mtLS-KpnI) was used to produce a unique double-strand break (DSB) in the mitochondrial genome, which allowed direct analysis of DSB repair in vivo in Saccharomyces cerevisiae. We find that loss of these three proteins significantly decreases the rate of spontaneous deletion events and the loss of Rad51p and Rad59p impairs the repair of induced mtDNA DSBs.

  13. The effective expression of xylanase gene in Candida utilis by 18S rDNA targeted homologous recombination in pGLR9K.

    Science.gov (United States)

    Wei, Wang; Hong-Lan, Yang; HuiFang, Bao; Daoyuan, Zhang; Qi-mu-ge, Shan; Woof, Andrew J

    2010-07-01

    In order to test whether 18S rDNA can influence positively xylanase gene effective expression in the yeast of Candida utilis, a targeting vector pGLR9K-XA was constructed by adding an interested gene xynA from Streptomyces olivaceoviridis into the vector pGLR9K which is constructed by ourselves. pGLR9K contains the 18S rDNA, GAP promoter and CYH resistance gene sequence, all of which is from C. utilis. Then the vector pGLR9K-XA was transformed into C. utilis. To test the vector and transformed system, PCR, Southern blot and DNS methods were used. The results showed that xylanase gene can be detected in the chromosome DNA of recombinant C. utilis and the enzyme activity of xylanase is up to 60 IU ml(-1) in the study. It is suggested that this system can be used to express exogenous genes in C. utilis as a bioreactors. This is the first report that xylanase gene was expressed in C. utilis. PMID:19731075

  14. Single Nucleotide Polymorphisms (SNPs) of RAD51-G172T and XRCC2-41657C/T Homologous Recombination Repair Genes and the Risk of Triple- Negative Breast Cancer in Polish Women.

    Science.gov (United States)

    Michalska, Magdalena M; Samulak, Dariusz; Romanowicz, Hanna; Smolarz, Beata

    2015-09-01

    Double strand DNA breaks are the most dangerous DNA damage which, if non-repaired or misrepaired, may result in genomic instability, cancer transformation or cell death. RAD51 and XRCC2 encode proteins that are important for the repair of double-strand DNA breaks by homologous recombination. Therefore, genetic variability in these genes may contribute to the occurrence and progression of triple-negative breast cancer. The polymorphisms of the XRCC2 gene -41657C/T (rs718282) and of the RAD51 gene, -172G/T (rs1801321), were investigated by PCR-RFLP in 70 patients with triple-negative breast cancer and 70 age- and sex matched non-cancer controls. The obtained results demonstrated a significant positive association between the RAD51 T/T genotype and TNBC, with an adjusted odds ratio (OR) of 4.94 (p = 0.001). The homozygous T/T genotype was found in 60 % of TNBC cases and in 14 % of the used controls. Variant 172 T allele of RAD51 increased cancer risk (OR = 2.81 (1.72-4.58), p < .0001). No significant associations were observed between -41657C/T genotype of XRCC2 and the incidence of TNBC. There were no significant differences between the distribution of XRCC2 -41657C/T genotypes in the subgroups assigned to histological grades. The obtained results indicate that the polymorphism of RAD51, but not of XRCC2 gene, may be positively associated with the incidence of triple-negative breast carcinoma in the population of Polish women.

  15. DNA双链断裂与同源重组修复的研究进展%Advance in Research of Homologous Recombination Repair in DNA Double Strands Breakage

    Institute of Scientific and Technical Information of China (English)

    董隽; 张天; 碧秀

    2015-01-01

    DNA双链断裂(DSB)是细胞受到电离辐射后最严重的DNA损伤,导致细胞凋亡、细胞周期阻滞以及DNA损伤修复。DNA损伤发生后,激活细胞内DNA损伤应答,启动DSB修复通路同源重组(HR)和非同源重组末端连接(NHEJ)。HR修复分为联会前期、联会期和联会后期,以姐妹染色单体为模板,进行无错误修复,是保护基因组完整性的主要机制。对IR导致的DSB HR和NHEJ具有互补关系,G2和S期HR是主要修复方式。HR是肿瘤发病风险、预后指标和治疗靶点,合成致死是HR用于肿瘤靶向治疗的重要机制。本文主要对DSB修复过程中所涉及HR修复通路中的分子机制、合成致死概念及其与NHEJ修复的关系作一综述,并探讨其成为转化医学研究和潜在临床应用的可能性。%DNA double strand breakage (DSB) is the most significantly biological effect when cells are exposed to ionizing radiation (IR) which may result in apoptosis, checkpoint arrest, cellular senescence and DSB repair. DNA damage response (DDR) is activated with induction of DNA damage. The mechanisms involved in DSB repair include homologous recombination (HR) and non-homologous end-joining (NHEJ). HR, a template-dependent and mostly error-free pathway, plays a crucial role in protecting genome fidelity from DSB. It can be divided into three phases including presynaptic, synaptic and postsynaptic phases. For the repair of DSBs caused by IR, HR is mainly restricted in G2 and S phases while NHEJ and HR function complementarily. HR is related to the risk of tumorigenesis, predicts the survival of several kinds of carcinoma and is a novel target of cancer therapy. This article has comprehensively reviewed the progress in understanding of the mechanism of HR repair, its associated factors affecting the fidelity in DSB repair, the concept of synthetic lethality and its association with NHEJ repair. The potential of its clinical application by

  16. Cell biology of mitotic recombination

    DEFF Research Database (Denmark)

    Lisby, Michael; Rothstein, Rodney

    2015-01-01

    Homologous recombination provides high-fidelity DNA repair throughout all domains of life. Live cell fluorescence microscopy offers the opportunity to image individual recombination events in real time providing insight into the in vivo biochemistry of the involved proteins and DNA molecules as w...

  17. Recombinant methods and materials

    Energy Technology Data Exchange (ETDEWEB)

    Roizman, B.; Post, L.E.

    1988-09-06

    This patent describes a method for stably effecting the insertion or deletion of a selected DNA sequence at a specific site in a viral genome. The method consists of: (1) isolating from the genome a linear DNA fragment comprising both (a) the specific site determined for insertion or deletion of selected DNA sequence and (b) flanking DNA sequences normally preceding and following the site; (2) preparing first and second altered genome fragments from the fragment isolated in step (1). (a) the first altered fragment comprising the fragment comprising a thymidine kinase gene in a position intermediate the ends of the fragment, and (b) the second altered fragment comprising the fragment having the selected DNA sequence inserted therein or deleted therefrom; (3) contacting the genome with the first altered fragment under conditions permitting recombination at sites of DNA sequence homology, selecting for a recombinant genome comprising the thymidine kinase gene, and isolating the recombinant genome; and (4) contacting the recombinant genome isolated in step (3) with the second altered fragment under conditions permitting recombination at sites of DNA sequence homology, selecting for a recombinant genome lacking the thymidine kinase gene, and isolating the recombinant genome product.

  18. Intersection homology Betti numbers

    CERN Document Server

    Durfee, A H

    1993-01-01

    A generalization of the formula of Fine and Rao for the ranks of the intersection homology groups of a complex algebraic variety is given. The proof uses geometric properties of intersection homology and mixed Hodge theory.

  19. Combinatorial Floer Homology

    CERN Document Server

    de Silva, Vin; Salamon, Dietmar

    2012-01-01

    We define combinatorial Floer homology of a transverse pair of noncontractibe nonisotopic embedded loops in an oriented 2-manifold without boundary, prove that it is invariant under isotopy, and prove that it is isomorphic to the original Lagrangian Floer homology.

  20. Meiotic recombination in normal and cloned bulls and their offspring

    Science.gov (United States)

    Homologous chromosome pairing and recombination are essential components of meiosis and sexual reproduction. The reshuffling of genetic material through breakage and reunion of chromatids ensure proper segregation of homologous chromosomes in reduction division and genetic diversity in the progeny....

  1. 应用体外DNA同源重组技术构建pcDNA3.1-NGF和pcDNA3.1-TrkA%Application of homologous recombination in vitro to construct pcDNA3.1-NGF and pcDNA3.1-TrkA

    Institute of Scientific and Technical Information of China (English)

    张严; 龚爱华; 金洁; 邵根宝; 彭琬昕

    2011-01-01

    目的:利用体外DNA 同源重组的方法分别构建含神经生长因子(NGF)和神经生长因子受体(TrkA)基因的真核表达载体.方法:在引物5′加上一段与载体克隆位点两端碱基序列相同的序列,PCR扩增目的基因NGF和TrkA,线性载体片段和目的基因片段经T4 DNA 聚合酶的外切产生互补的单链DNA,然后37℃退火实现体外同源重组,转化并鉴定;将重组质粒转染293A细胞,免疫印迹鉴定目的基因的表达情况.结果:成功构建真核载体pcDNA3.1-NGF和pcDNA3.1-TrkA,转染细胞后的表达产物相对分子质量分别是31×103和140×103.结论:与常规重组技术相比,体外DNA 同源重组技术是一种高效的DNA 重组方法,且不需考虑目的片段的限制性酶切位点.%Objective: In this study, we use the DNA homologous recombination in vitro to construct NGF and tyrosine kinase A (TrkA) eukaryotic expression vector. Methods: PCR primers for inserts were designed to contain appropriate 5' extension sequences. For the cloning reaction we generated the vector by cleavage with double restriction enzyme and generated the inserts TrkA and NGF by PCR. We treated both the vector and the inserts with T4 DNA polymerase to generate overhangs, then incubated vector and insert to promote recombination, and transformed the products into E. Coli and identified. Transfect the recombination DNA into 293A and identify the gene expression. Results: The expression vector pcDNA3. 1-NGF and pcDNA3.1-TrkA were constructed successfully by homologous recombination. Western blot showed TrkA protein expression in infected 293A cells was 140 x 103 and NGF was 31 x 103. Conclusion: Compared with conventional recombination technology, homologous recombination in vitro was a kind of high efficient DNA recombination method, and with no need for considering the restriction enzymes' site.

  2. Lectures on functor homology

    CERN Document Server

    Touzé, Antoine

    2015-01-01

    This book features a series of lectures that explores three different fields in which functor homology (short for homological algebra in functor categories) has recently played a significant role. For each of these applications, the functor viewpoint provides both essential insights and new methods for tackling difficult mathematical problems. In the lectures by Aurélien Djament, polynomial functors appear as coefficients in the homology of infinite families of classical groups, e.g. general linear groups or symplectic groups, and their stabilization. Djament’s theorem states that this stable homology can be computed using only the homology with trivial coefficients and the manageable functor homology. The series includes an intriguing development of Scorichenko’s unpublished results. The lectures by Wilberd van der Kallen lead to the solution of the general cohomological finite generation problem, extending Hilbert’s fourteenth problem and its solution to the context of cohomology. The focus here is o...

  3. Sutures and contact homology I

    OpenAIRE

    Colin, Vincent; Ghiggini, Paolo; Honda, Ko; Hutchings, Michael

    2010-01-01

    We define a relative version of contact homology for contact manifolds with convex boundary, and prove basic properties of this relative contact homology. Similar considerations also hold for embedded contact homology.

  4. Enhanced levels of λ Red-mediated recombinants in mismatch repair mutants

    OpenAIRE

    Costantino, Nina; Court, Donald L.

    2003-01-01

    Homologous recombination can be used to generate recombinants on episomes or directly on the Escherichia coli chromosome with PCR products or synthetic single-stranded DNA (ssDNA) oligonucleotides (oligos). Such recombination is possible because bacteriophage λ-encoded functions, called Red, efficiently recombine linear DNA with homologies as short as 20–70 bases. This technology, termed recombineering, provides ways to modify genes and segments of the chromosome as well as to study homologou...

  5. Regulation of Meiotic Recombination

    Energy Technology Data Exchange (ETDEWEB)

    Gregory p. Copenhaver

    2011-11-09

    Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diverse as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a facile system

  6. Braid Floer homology

    Science.gov (United States)

    van den Berg, J. B.; Ghrist, R.; Vandervorst, R. C.; Wójcik, W.

    2015-09-01

    Area-preserving diffeomorphisms of a 2-disc can be regarded as time-1 maps of (non-autonomous) Hamiltonian flows on R / Z ×D2. The periodic flow-lines define braid (conjugacy) classes, up to full twists. We examine the dynamics relative to such braid classes and define a new invariant for such classes, the BRAID FLOER HOMOLOGY. This refinement of Floer homology, originally used for the Arnol'd Conjecture, yields a Morse-type forcing theory for periodic points of area-preserving diffeomorphisms of the 2-disc based on braiding. Contributions of this paper include (1) a monotonicity lemma for the behavior of the nonlinear Cauchy-Riemann equations with respect to algebraic lengths of braids; (2) establishment of the topological invariance of the resulting braid Floer homology; (3) a shift theorem describing the effect of twisting braids in terms of shifting the braid Floer homology; (4) computation of examples; and (5) a forcing theorem for the dynamics of Hamiltonian disc maps based on braid Floer homology.

  7. Role of ubiquitination in meiotic recombination repair

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Programmed and unprogrammed double-strand breaks (DSBs) often arise from such physiological requirements as meiotic recombination, and exogenous insults, such as ionizing radiation (IR). Due to deleterious impacts on genome stability, DSBs must be appropriately processed and repaired in a regulatory manner. Recent investigations have indicated that ubiquitination is a critical factor in DNA damage response and meiotic recombination repair. This review summarizes the effects of proteins and complexes associated with ubiquitination with regard to homologous recombination (HR)-dependent DSB repair.

  8. Meiotic Recombination in Somatic Cell Nuclear Transfer Bulls and Their Offspring

    Science.gov (United States)

    In mammals, homologous chromosome pairing and recombination are essential events for meiosis. The generation of reciprocal exchanges of genetic material ensure both genetic diversity and the proper segregation of homologous chromosomes. With the advent of reproductive biotechnologies such as somat...

  9. The unconventional xer recombination machinery of Streptococci/Lactococci

    NARCIS (Netherlands)

    Le Bourgeois, Pascal; Bugarel, Marie; Campo, Nathalie; Daveran-Mingot, Marie-Line; Labonte, Jessica; Lanfranchi, Daniel; Lautier, Thomas; Pages, Carine; Ritzenthaler, Paul

    2007-01-01

    Homologous recombination between circular sister chromosomes during DNA replication in bacteria can generate chromosome dimers that must be resolved into monomers prior to cell division. In Escherichia coli, dimer resolution is achieved by site-specific recombination, Xer recombination, involving tw

  10. Pseudocycles and Integral Homology

    OpenAIRE

    Zinger, Aleksey

    2006-01-01

    We describe a natural isomorphism between the set of equivalence classes of pseudocycles and the integral homology groups of a smooth manifold. Our arguments generalize to settings well-suited for applications in enumerative algebraic geometry and for construction of the virtual fundamental class in the Gromov-Witten theory.

  11. Gorenstein homological dimensions

    DEFF Research Database (Denmark)

    Holm, Henrik Granau

    2004-01-01

    In basic homological algebra, the projective, injective and 2at dimensions of modules play an important and fundamental role. In this paper, the closely related Gorenstein projective, Gorenstein injective and Gorenstein 2at dimensions are studied. There is a variety of nice results about Gorenste...

  12. 小鼠LRP16基因打靶载体的构建和同源重组型胚胎干细胞筛选%Construction of a LRP16 gene targeting vector and screening of homologously recombinant clone of embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    伍志强; 韩为东; 赵亚力; 司艺玲; 母义明; 孟元光; Masatoshi Nomura

    2008-01-01

    BACKGROUND: Previous studies have demonstrated that LRP16 is an estrogen-responsive gene. Its expression level is strongly associated with the proliferation and invasive growth of human breast cancer cells.OBJECTIVE: To construct a LRP16 targeting vector and screen mouse embryonic stem cell clones with homolougous recombination of an inactive LRP16 gene.DESIGN: Constructing an inserting inactivation target by inserting SA-RIES-β geo expression cassette.SETTING: Bioregulatory Laboratory of the Third Medical Department of Kyushu University in Japan and Department of Molecular Biology, General Hospital of Chinese PLA.MATERIALS: The materials used here were mainly provided by the Bioregulatory Laboratory, the Third Medical Department of Kyushu University in Japan. The mouse genomic library in pBeloBAC11 Vector was purchased from lnvitrogen Corp. The competent TopF10 was purchased from Beijing Tiangen Biotech Corp. pcDNA3.1(+) vector was kept in our laboratory. Mouse ES cells were provided by Kyushu University.METHODS: The experiment was performed in Kyushu University and Department of Molecular Biology of PLA General Hospital from November 2004 to May 2005. Targeting sequence of LRP16 gene was obtained from 129 mouse genomic Bacterial Artificial Chromosomes library based on polymerase chain reaction (PCR) screening. The SA-RIES-β geo fragment was inserted within LRP16 fifth exon to inactivate LRP16. ES cells were screened with G418 and the homologously recombinant clone was identified by Southern blot analysis.MAIN OUTCOME MEASURES: Clones with homologous recombination.RESULTS: The LRP16 fragment including exon 5 to 11 was subcloned into the pBluescript SK Ⅱvector. Restriction map demonstrated that the SA-IRES-β geo fragment was correctly inserted into the LRP16 fifth exon. Southern blot results showed that there was an ES clone with targeting sequence homologously inserted.CONCLUSION: A LRP16 gene targeting vector is constructed and a homologous recombinant is

  13. Algebra V homological algebra

    CERN Document Server

    Shafarevich, I

    1994-01-01

    This book, the first printing of which was published as volume 38 of the Encyclopaedia of Mathematical Sciences, presents a modern approach to homological algebra, based on the systematic use of the terminology and ideas of derived categories and derived functors. The book contains applications of homological algebra to the theory of sheaves on topological spaces, to Hodge theory, and to the theory of modules over rings of algebraic differential operators (algebraic D-modules). The authors Gelfand and Manin explain all the main ideas of the theory of derived categories. Both authors are well-known researchers and the second, Manin, is famous for his work in algebraic geometry and mathematical physics. The book is an excellent reference for graduate students and researchers in mathematics and also for physicists who use methods from algebraic geometry and algebraic topology.

  14. Bacteriophage recombination systems and biotechnical applications.

    Science.gov (United States)

    Nafissi, Nafiseh; Slavcev, Roderick

    2014-04-01

    Bacteriophage recombination systems have been widely used in biotechnology for modifying prokaryotic species, for creating transgenic animals and plants, and more recently, for human cell gene manipulation. In contrast to homologous recombination, which benefits from the endogenous recombination machinery of the cell, site-specific recombination requires an exogenous source of recombinase in mammalian cells. The mechanism of bacteriophage evolution and their coexistence with bacterial cells has become a point of interest ever since bacterial viruses' life cycles were first explored. Phage recombinases have already been exploited as valuable genetic tools and new phage enzymes, and their potential application to genetic engineering and genome manipulation, vectorology, and generation of new transgene delivery vectors, and cell therapy are attractive areas of research that continue to be investigated. The significance and role of phage recombination systems in biotechnology is reviewed in this paper, with specific focus on homologous and site-specific recombination conferred by the coli phages, λ, and N15, the integrase from the Streptomyces phage, ΦC31, the recombination system of phage P1, and the recently characterized recombination functions of Yersinia phage, PY54. Key steps of the molecular mechanisms involving phage recombination functions and their application to molecular engineering, our novel exploitations of the PY54-derived recombination system, and its application to the development of new DNA vectors are discussed.

  15. Bacteriophage recombination systems and biotechnical applications.

    Science.gov (United States)

    Nafissi, Nafiseh; Slavcev, Roderick

    2014-04-01

    Bacteriophage recombination systems have been widely used in biotechnology for modifying prokaryotic species, for creating transgenic animals and plants, and more recently, for human cell gene manipulation. In contrast to homologous recombination, which benefits from the endogenous recombination machinery of the cell, site-specific recombination requires an exogenous source of recombinase in mammalian cells. The mechanism of bacteriophage evolution and their coexistence with bacterial cells has become a point of interest ever since bacterial viruses' life cycles were first explored. Phage recombinases have already been exploited as valuable genetic tools and new phage enzymes, and their potential application to genetic engineering and genome manipulation, vectorology, and generation of new transgene delivery vectors, and cell therapy are attractive areas of research that continue to be investigated. The significance and role of phage recombination systems in biotechnology is reviewed in this paper, with specific focus on homologous and site-specific recombination conferred by the coli phages, λ, and N15, the integrase from the Streptomyces phage, ΦC31, the recombination system of phage P1, and the recently characterized recombination functions of Yersinia phage, PY54. Key steps of the molecular mechanisms involving phage recombination functions and their application to molecular engineering, our novel exploitations of the PY54-derived recombination system, and its application to the development of new DNA vectors are discussed. PMID:24442504

  16. Which way up? Recognition of homologous DNA segments in parallel and antiparallel alignment

    CERN Document Server

    Lee, Dominic J; Albrecht, Tim; Kornyshev, Alexei A

    2014-01-01

    Homologous gene shuffling between DNA promotes genetic diversity and is an important pathway for DNA repair. For this to occur, homologous genes need to find and recognize each other. However, despite its central role in homologous recombination, the mechanism of homology recognition is still an unsolved puzzle. While specific proteins are known to play a role at later stages of recombination, an initial coarse grained recognition step has been proposed. This relies on the sequence dependence of the DNA structural parameters, such as twist and rise, mediated by intermolecular interactions, in particular electrostatic ones. In this proposed mechanism, sequences having the same base pair text, or are homologous, have lower interaction energy than those sequences with uncorrelated base pair texts; the difference termed the recognition energy. Here, we probe how the recognition energy changes when one DNA fragment slides past another, and consider, for the first time, homologous sequences in antiparallel alignmen...

  17. Stimulation of homology-directed gene targeting at an endogenous human locus by a nicking endonuclease

    NARCIS (Netherlands)

    G.P. van Nierop (Gijsbert); A.A.F. de Vries (Antoine); M. Holkers (Maarten); K.R. Vrijsen (Krijn); M.A.F.V. Gonçalves (Manuel)

    2009-01-01

    textabstractHomologous recombination (HR) is a highly accurate mechanism of DNA repair that can be exploited for homology-directed gene targeting. Since in most cell types HR occurs very infrequently (̃10.-6to 10.-8), its practical application has been largely restricted to specific experimental sys

  18. Plasmid recombination in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, D.

    1982-01-01

    DNA recombination in exponential phase and competent Haemophilus influenzae was measured by an electron microscopic assay that relies on the conversion of plasmid RSF0885 monomers into multimeric forms. Dimer circles were present at a frequency of 2% in plasmid preparations from competent Rd (wild-type) cells; multimers were present at a frequency of 0.2% in preparations from exponential phase cells. Thus, plasmid recombination was stimulated in competent cells. Multimer formation occurred efficiently in cells of the transformation defective mutant rec2, implying that the rec2 gene product is not required for plasmid recombination. However, the absence of multimer plasmids in preparations from competent cells of the transformation defective mutant rec1 suggests that the rec1 gene product is required. Digestion of purified plasmids with restriction endonuclease PvuII, which makes a single cut in the monomer, revealed the presence of recombination intermediates composed of two linear plasmids joined to form two pairs of arms resembling the Greek letter chi. Length measurements of these arms taken from a population of recombination intermediates gave evidence that the plasmids were joined at sites of homology. The distributions of individual DNA strands, at the intersections of the four arms, could be resolved in some recombination intermediates and were of two types. The first type of junction appeared as a single-stranded arm appended to each corner. The second type of junction consisted of a single strand of DNA linking the two linear plasmids at a site of homology. The single-stranded linker was frequently situated at the edge of a short gap on one of the plasmids in the pair. The fine structures of the recombinational joints have been interpreted in terms of previously proposed models of recombination.

  19. Initiation of meiotic recombination in Ustilago maydis.

    Science.gov (United States)

    Kojic, Milorad; Sutherland, Jeanette H; Pérez-Martín, José; Holloman, William K

    2013-12-01

    A central feature of meiosis is the pairing and recombination of homologous chromosomes. Ustilago maydis, a biotrophic fungus that parasitizes maize, has long been utilized as an experimental system for studying recombination, but it has not been clear when in the life cycle meiotic recombination initiates. U. maydis forms dormant diploid teliospores as the end product of the infection process. Upon germination, teliospores complete meiosis to produce four haploid basidiospores. Here we asked whether the meiotic process begins when teliospores germinate or at an earlier stage in development. When teliospores homozygous for a cdc45 mutation temperature sensitive for DNA synthesis were germinated at the restrictive temperature, four nuclei became visible. This implies that teliospores have already undergone premeiotic DNA synthesis and suggests that meiotic recombination initiates at a stage of infection before teliospores mature. Determination of homologous recombination in plant tissue infected with U. maydis strains heteroallelic for the nar1 gene revealed that Nar(+) recombinants were produced at a stage before teliospore maturation. Teliospores obtained from a spo11Δ cross were still able to germinate but the process was highly disturbed and the meiotic products were imbalanced in chromosomal complement. These results show that in U. maydis, homologous recombination initiates during the infection process and that meiosis can proceed even in the absence of Spo11, but with loss of genomic integrity.

  20. Recombination instability

    DEFF Research Database (Denmark)

    D'Angelo, N.

    1967-01-01

    A recombination instability is considered which may arise in a plasma if the temperature dependence of the volume recombination coefficient, alpha, is sufficiently strong. Two cases are analyzed: (a) a steady-state plasma produced in a neutral gas by X-rays or high energy electrons; and (b...

  1. An RNA secondary structure bias for non-homologous reverse transcriptase-mediated deletions in vivo

    DEFF Research Database (Denmark)

    Duch, Mogens; Carrasco, Maria L; Jespersen, Thomas;

    2004-01-01

    result from template switching during first-strand cDNA synthesis and that the choice of acceptor sites for non-homologous recombination are guided by non-paired regions. Our results may have implications for recombination events taking place within structured regions of retroviral RNA genomes......, especially in the absence of longer stretches of sequence similarity....

  2. The many facets of homologous recombination at telomeres

    NARCIS (Netherlands)

    Claussin, Clémence; Chang, Michael

    2015-01-01

    The ends of linear chromosomes are capped by nucleoprotein structures called telomeres. A dysfunctional telomere may resemble a DNA double-strand break (DSB), which is a severe form of DNA damage. The presence of one DSB is sufficient to drive cell cycle arrest and cell death. Therefore cells have e

  3. Defining the minimal length of sequence homology required for selective gene isolation by TAR cloning

    OpenAIRE

    Noskov, V. N.; Koriabine, M.; Solomon, G.; Randolph, M; Barrett, J C; Leem, S.-H.; Stubbs, L; Kouprina, N; Larionov, V.

    2001-01-01

    The transformation-associated recombination (TAR) cloning technique allows selective and accurate isolation of chromosomal regions and genes from complex genomes. The technique is based on in vivo recombination between genomic DNA and a linearized vector containing homologous sequences, or hooks, to the gene of interest. The recombination occurs during transformation of yeast spheroplasts that results in the generation of a yeast artificial chromosome (YAC) contain...

  4. Single-Molecule Imaging of DNA Pairing by RecA Reveals a 3-Dimensional Homology Search

    OpenAIRE

    Forget, Anthony L.; Kowalczykowski, Stephen C.

    2012-01-01

    DNA breaks can be repaired with high-fidelity by homologous recombination. A ubiquitous protein that is essential for this DNA template-directed repair is RecA 1 . After resection of broken DNA to produce single-stranded DNA (ssDNA), RecA assembles on this ssDNA into a filament with the unique capacity to search and find DNA sequences in double-stranded DNA (dsDNA) that are homologous to the ssDNA. This homology search is vital to recombinational DNA repair, and results in homologous pairing ...

  5. Homologous and non-homologous recombination differentially affect DNA damage repair in mice.

    NARCIS (Netherlands)

    J. Essers (Jeroen); H. van Steeg (Harry); J. de Wit (Jan); M. Vermeij (Marcel); J.H.J. Hoeijmakers (Jan); R. Kanaar (Roland); S.M.A. Swagemakers (Sigrid)

    2000-01-01

    textabstractIonizing radiation and interstrand DNA crosslinking compounds provide important treatments against cancer due to their extreme genotoxicity for proliferating cells. Both the efficacies of such treatments and the mutagenic potential of these agents are modulated by the a

  6. 2-categories and cyclic homology

    OpenAIRE

    Slevin, Paul

    2016-01-01

    The topic of this thesis is the application of distributive laws between comonads to the theory of cyclic homology. Explicitly, our main aims are: 1) To study how the cyclic homology of associative algebras and of Hopf algebras in the original sense of Connes and Moscovici arises from a distributive law, and to clarify the role of different notions of bimonad in this generalisation. 2) To extend the procedure of twisting the cyclic homology of a unital associative algebra to any duplicial obj...

  7. The Geometry of Homological Triangles

    CERN Document Server

    Smarandache, Florentin

    2012-01-01

    This book is addressed to students, professors and researchers of geometry, who will find herein many interesting and original results. The originality of the book The Geometry of Homological Triangles consists in using the homology of triangles as a "filter" through which remarkable notions and theorems from the geometry of the triangle are unitarily passed. Our research is structured in seven chapters, the first four are dedicated to the homology of the triangles, while the last ones to their applications.

  8. Matrix Factorizations and Kauffman Homology

    CERN Document Server

    Gukov, S; Gukov, Sergei; Walcher, Johannes

    2005-01-01

    The topological string interpretation of homological knot invariants has led to several insights into the structure of the theory in the case of sl(N). We study possible extensions of the matrix factorization approach to knot homology for other Lie groups and representations. In particular, we introduce a new triply graded theory categorifying the Kauffman polynomial, test it, and predict the Kauffman homology for several simple knots.

  9. Homology of L_{\\infty}-Algebras and Cyclic Homology

    OpenAIRE

    Khalkhali, Masoud

    1998-01-01

    A classical result of Loday-Quillen and Tsygan states that the Lie algebra homology of the algebra of stable matrices over an associative algebra is isomorphic, as a Hopf algebra, to the exterior algebra of the cyclic homology of the algebra. In this paper we develop the necessary tools needed to extend extend this result to the category of L_{\\infty} algebras.

  10. The endless tale of non-homologous end-joining

    Institute of Scientific and Technical Information of China (English)

    Eric Weterings; David J Chen

    2008-01-01

    DNA double-strand breaks (DSBs) are introduced in cells by ionizing radiation and reactive oxygen species. In addi-tion, they are commonly generated during V(D)J recombination, an essential aspect of the developing immune system. Failure to effectively repair these DSBs can result in chromosome breakage, cell death, onset of cancer, and defects in the immune system of higher vertebrates. Fortunately, all mammalian cells possess two enzymatic pathways that mediate the repair of DSBs: homologous recombination and non-homologous end-joining (NHEJ). The NHEJ process utilizes enzymes that capture both ends of the broken DNA molecule, bring them together in a synaptic DNA-protein complex, and finally repair the DNA break. In this review, all the known enzymes that play a role in the NHEJ process are discussed and a working model for the co-operation of these enzymes during DSB repair is presented.

  11. K-Kolmogorov homology groups

    International Nuclear Information System (INIS)

    In the present work we use the idea of the K-groups to define the K-Kolmogorov homology groups, and their induced homomorphisms and boundary operators for the case of a pair of discrete coefficient groups, where K denotes a locally-finite simplicial complex. Moreover, we prove that our homology construction is exact. (author)

  12. Mitochondrial recombination increases with age in Podospora anserina

    NARCIS (Netherlands)

    van Diepeningen, Anne D; Goedbloed, Daniël J; Slakhorst, S Marijke; Koopmanschap, A Bertha; Maas, Marc F P M; Hoekstra, Rolf F; Debets, Alfons J M

    2010-01-01

    With uniparental inheritance of mitochondria, there seems little reason for homologous recombination in mitochondria, but the machinery for mitochondrial recombination is quite well-conserved in many eukaryote species. In fungi and yeasts heteroplasmons may be formed when strains fuse and transfer o

  13. An efficient method of constructing homologous recom binant baculovirus with PCR-amplified fragments

    Institute of Scientific and Technical Information of China (English)

    HOU; Songwang; (侯松旺); CHEN; Xinwen; (陈新文); WANG; Hanzhong; (王汉中); HU; Zhihong; (胡志红)

    2003-01-01

    This paper describes a rapid method of constructing homologous recombinant baculovirus in E. coli with PCR-amplified fragments. By using this method, the traditional steps of constructing transfer vector are omitted. The method is based on phage λ red system which can promote the recombination between the homologous fragments with the length above 36 bp. Taking HaSNPV as an example, this paper describes the rapid recombination process by using chloramphenicol resistance gene (CmR) to replace orf135 in HaSNPV genome. A pair of primers with length of 60 bp was synthesized, in which 40 bp was homologous to the each end sequence of orf135, and the rest 20 bp was homologous to the each end sequence of CmR. By using these primers, a linear fragment containing the complete CmR gene between 40 bp of homologous arms of orf135 was generated by PCR with the plasmid pKD3 which contains CmR as the template. By transforming the linear fragment into the E. coli containing the bacterial artificial chromosome of HaSNPV and with the help of a plasmid expressing λ recombinase, the recombinants on which the homologue replacement had taken place were selected by chloramphenicol resistance. This method greatly shortens the process of constructing recombinant baculovirus since the process was performed in E. coli and does not need to construct transfer vectors. It can be further used for gene replacement and gene deletion of other large viral genomes.

  14. Linker-Mediated Recombinational Subcloning of Large DNA Fragments Using Yeast

    OpenAIRE

    Raymond, Christopher K.; Sims, Elizabeth H.; Olson, Maynard V.

    2002-01-01

    The homologous recombination pathway in yeast is an ideal tool for the sequence-specific assembly of plasmids. Complementary 80-nucleotide oligonucleotides that overlap a vector and a target fragment were found to serve as efficient recombination linkers for fragment subcloning. Using electroporation, single-stranded 80-mers were adequate for routine plasmid construction. A cycloheximide-based counterselection was introduced to increase the specificity of cloning by homologous recombination r...

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

    Science.gov (United States)

    Kowalczykowski, Stephen C

    2015-11-01

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

  16. Mod two homology and cohomology

    CERN Document Server

    Hausmann, Jean-Claude

    2014-01-01

    Cohomology and homology modulo 2 helps the reader grasp more readily the basics of a major tool in algebraic topology. Compared to a more general approach to (co)homology this refreshing approach has many pedagogical advantages: It leads more quickly to the essentials of the subject, An absence of signs and orientation considerations simplifies the theory, Computations and advanced applications can be presented at an earlier stage, Simple geometrical interpretations of (co)chains. Mod 2 (co)homology was developed in the first quarter of the twentieth century as an alternative to integral homology, before both became particular cases of (co)homology with arbitrary coefficients. The first chapters of this book may serve as a basis for a graduate-level introductory course to (co)homology. Simplicial and singular mod 2 (co)homology are introduced, with their products and Steenrod squares, as well as equivariant cohomology. Classical applications include Brouwer's fixed point theorem, Poincaré duality, Borsuk-Ula...

  17. Compositional Homology and Creative Thinking

    Directory of Open Access Journals (Sweden)

    Salvatore Tedesco

    2015-05-01

    Full Text Available The concept of homology is the most solid theoretical basis elaborated by the morphological thinking during its history. The enucleation of some general criteria for the interpretation of homology is today a fundamental tool for life sciences, and for restoring their own opening to the question of qualitative innovation that arose so powerfully in the original Darwinian project. The aim of this paper is to verify the possible uses of the concept of compositional homology in order to provide of an adequate understanding of the dynamics of creative thinking.

  18. Fivebranes and 3-manifold homology

    CERN Document Server

    Gukov, Sergei; Vafa, Cumrun

    2016-01-01

    Motivated by physical constructions of homological knot invariants, we study their analogs for closed 3-manifolds. We show that fivebrane compactifications provide a universal description of various old and new homological invariants of 3-manifolds. In terms of 3d/3d correspondence, such invariants are given by the Q-cohomology of the Hilbert space of partially topologically twisted 3d N=2 theory T[M_3] on a Riemann surface with defects. We demonstrate this by concrete and explicit calculations in the case of monopole/Heegaard Floer homology and a 3-manifold analog of Khovanov-Rozansky link homology. The latter gives a categorification of Chern-Simons partition function. Some of the new key elements include the explicit form of the S-transform and a novel connection between categorification and a previously mysterious role of Eichler integrals in Chern-Simons theory.

  19. RecA bundles mediate homology pairing between distant sisters during DNA break repair

    Science.gov (United States)

    Lesterlin, Christian; Ball, Graeme; Schermelleh, Lothar; Sherratt, David J.

    2014-02-01

    DNA double-strand break (DSB) repair by homologous recombination has evolved to maintain genetic integrity in all organisms. Although many reactions that occur during homologous recombination are known, it is unclear where, when and how they occur in cells. Here, by using conventional and super-resolution microscopy, we describe the progression of DSB repair in live Escherichia coli. Specifically, we investigate whether homologous recombination can occur efficiently between distant sister loci that have segregated to opposite halves of an E. coli cell. We show that a site-specific DSB in one sister can be repaired efficiently using distant sister homology. After RecBCD processing of the DSB, RecA is recruited to the cut locus, where it nucleates into a bundle that contains many more RecA molecules than can associate with the two single-stranded DNA regions that form at the DSB. Mature bundles extend along the long axis of the cell, in the space between the bulk nucleoid and the inner membrane. Bundle formation is followed by pairing, in which the two ends of the cut locus relocate at the periphery of the nucleoid and together move rapidly towards the homology of the uncut sister. After sister locus pairing, RecA bundles disassemble and proteins that act late in homologous recombination are recruited to give viable recombinants 1-2-generation-time equivalents after formation of the initial DSB. Mutated RecA proteins that do not form bundles are defective in sister pairing and in DSB-induced repair. This work reveals an unanticipated role of RecA bundles in channelling the movement of the DNA DSB ends, thereby facilitating the long-range homology search that occurs before the strand invasion and transfer reactions.

  20. Optimizing the design of oligonucleotides for homology directed gene targeting.

    Directory of Open Access Journals (Sweden)

    Judith Miné-Hattab

    Full Text Available BACKGROUND: Gene targeting depends on the ability of cells to use homologous recombination to integrate exogenous DNA into their own genome. A robust mechanistic model of homologous recombination is necessary to fully exploit gene targeting for therapeutic benefit. METHODOLOGY/PRINCIPAL FINDINGS: In this work, our recently developed numerical simulation model for homology search is employed to develop rules for the design of oligonucleotides used in gene targeting. A Metropolis Monte-Carlo algorithm is used to predict the pairing dynamics of an oligonucleotide with the target double-stranded DNA. The model calculates the base-alignment between a long, target double-stranded DNA and a probe nucleoprotein filament comprised of homologous recombination proteins (Rad51 or RecA polymerized on a single strand DNA. In this study, we considered different sizes of oligonucleotides containing 1 or 3 base heterologies with the target; different positions on the probe were tested to investigate the effect of the mismatch position on the pairing dynamics and stability. We show that the optimal design is a compromise between the mean time to reach a perfect alignment between the two molecules and the stability of the complex. CONCLUSION AND SIGNIFICANCE: A single heterology can be placed anywhere without significantly affecting the stability of the triplex. In the case of three consecutive heterologies, our modeling recommends using long oligonucleotides (at least 35 bases in which the heterologous sequences are positioned at an intermediate position. Oligonucleotides should not contain more than 10% consecutive heterologies to guarantee a stable pairing with the target dsDNA. Theoretical modeling cannot replace experiments, but we believe that our model can considerably accelerate optimization of oligonucleotides for gene therapy by predicting their pairing dynamics with the target dsDNA.

  1. Object-oriented persistent homology

    Science.gov (United States)

    Wang, Bao; Wei, Guo-Wei

    2016-01-01

    Persistent homology provides a new approach for the topological simplification of big data via measuring the life time of intrinsic topological features in a filtration process and has found its success in scientific and engineering applications. However, such a success is essentially limited to qualitative data classification and analysis. Indeed, persistent homology has rarely been employed for quantitative modeling and prediction. Additionally, the present persistent homology is a passive tool, rather than a proactive technique, for classification and analysis. In this work, we outline a general protocol to construct object-oriented persistent homology methods. By means of differential geometry theory of surfaces, we construct an objective functional, namely, a surface free energy defined on the data of interest. The minimization of the objective functional leads to a Laplace-Beltrami operator which generates a multiscale representation of the initial data and offers an objective oriented filtration process. The resulting differential geometry based object-oriented persistent homology is able to preserve desirable geometric features in the evolutionary filtration and enhances the corresponding topological persistence. The cubical complex based homology algorithm is employed in the present work to be compatible with the Cartesian representation of the Laplace-Beltrami flow. The proposed Laplace-Beltrami flow based persistent homology method is extensively validated. The consistence between Laplace-Beltrami flow based filtration and Euclidean distance based filtration is confirmed on the Vietoris-Rips complex for a large amount of numerical tests. The convergence and reliability of the present Laplace-Beltrami flow based cubical complex filtration approach are analyzed over various spatial and temporal mesh sizes. The Laplace-Beltrami flow based persistent homology approach is utilized to study the intrinsic topology of proteins and fullerene molecules. Based on a

  2. Homologous recombination-mediated cloning and manipulation of genomic DNA regions using Gateway and recombineering systems

    OpenAIRE

    Kagale Sateesh; Yang Wen; Rozwadowski Kevin

    2008-01-01

    Abstract Background Employing genomic DNA clones to characterise gene attributes has several advantages over the use of cDNA clones, including the presence of native transcription and translation regulatory sequences as well as a representation of the complete repertoire of potential splice variants encoded by the gene. However, working with genomic DNA clones has traditionally been tedious due to their large size relative to cDNA clones and the presence, absence or position of particular res...

  3. Role of enzymes of homologous recombination in illegitimate plasmid recombination in Bacillus subtilis

    NARCIS (Netherlands)

    Meima, R; Haijema, BJ; Haan, GJ; Venema, G; Bron, S

    1997-01-01

    The structural stability of plasmid pGP1, which encodes a fusion between the penicillinase gene (penP) of Bacillus licheniformis and the Escherichia coli lacZ gene, was investigated in Bacillus subtilis strains expressing mutated subunits of the ATP-dependent nuclease, AddAB, and strains lacking the

  4. Localization theorems in topological Hochschild homology and topological cyclic homology

    CERN Document Server

    Blumberg, Andrew J

    2008-01-01

    We construct localization cofiber sequences for the topological Hochschild homology (THH) and topological cyclic homology (TC) of spectral categories. Using a ``global'' construction of the THH and TC of a scheme in terms of the perfect complexes in a spectrally enriched version of the category of unbounded complexes, the sequences specialize to localization cofiber sequences associated to the inclusion of an open subscheme. These are the targets of the cyclotomic trace from the localization sequence of Thomason-Trobaugh in K-theory. We also deduce a version of Thomason's blow-up formula for THH and TC.

  5. Recombination analysis and homology alignment of full-length genome sequences of die novel A/H1N1 influenza virus in 2009%2009年新型甲型H1N1流感病毒全基因组序列重组分析及同源性比对

    Institute of Scientific and Technical Information of China (English)

    鹿文英; 殷建华; 李淑华; 韩磊; 韩一芳; 苏彤; 曹广文

    2009-01-01

    Objective To analyze the genetic variation and recombination of the novel A/H1N1 influenza pandemic virus in 2009. Methods Full-length sequence of typical novel A/H1N1 influenza virus was downloaded from NCBI database. MEGA4.0 software was used to connect and align the eight fragments of the virus. Then the fragments of different subtypes such as H1N1, H5N1 and H3N2 of the historical strains from different hosts, including human, poultry and pigs, were connected and aligned in the same way. A phylogenetic tree was constructed by NJ method. The recombination analysis of 2009 pandemic virus was made with Simplot 3. 5.1 software. Results There was no clear variation (identity was 99.69% - 99. 93%) in the novel A/H1N1 influenza virus from April to September, 2009. Simplot and MEGA analysis indicated that the PB2, PB1, PA, HA, NP and NS of the novel A/H1N1 virus might originally evolve from the swine and human H1N1 virus isolated in North America (identity was 95. 25%, 95.08%, 95.21%, 93.52%, 95.23% and 94.78%, respectively). NA and MP showed high homology with the European swine H1N1 virus, the identity was 90.21% and 94.43%, respectively. Full-length sequence of the novel A/H1N1 influenza virus had a highest similarity with swine H1N1 virus isolated from North America (identity was 92.22%). Conclusions The novel A/H1N1 influenza pandemic virus in 2009 was originated from the reassortment and evolution of swine H1N1 2005 pandemic virus in North America, and the NA and MP fragments of European swine H1N1. There is no clear variation in novel influenza virus up to now. The novel A/H1N1 influenza vaccine possesses protective effect.%目的 分析2009年新型甲型H1N1流感爆发以来流感病毒的全基因组进化变异及重组情况.方法 从NCBI基因数据库下载2009年新型甲型H1N1流感病毒(A/H1N1)代表性全基因组序列,先用MEGA4.0软件对8个基因序列片段进行比对和拼接;然后将历史上流行的H1N1、H5N1、H3N2等不同宿

  6. A cohesin-based structural platform supporting homologous chromosome pairing in meiosis.

    Science.gov (United States)

    Ding, Da-Qiao; Haraguchi, Tokuko; Hiraoka, Yasushi

    2016-08-01

    The pairing and recombination of homologous chromosomes during the meiotic prophase is necessary for the accurate segregation of chromosomes in meiosis. However, the mechanism by which homologous chromosomes achieve this pairing has remained an open question. Meiotic cohesins have been shown to affect chromatin compaction; however, the impact of meiotic cohesins on homologous pairing and the fine structures of cohesion-based chromatin remain to be determined. A recent report using live-cell imaging and super-resolution microscopy demonstrated that the lack of meiotic cohesins alters the chromosome axis structures and impairs the pairing of homologous chromosomes. These results suggest that meiotic cohesin-based chromosome axis structures are crucial for the pairing of homologous chromosomes. PMID:26856595

  7. Genetic Analyses of Meiotic Recombination in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Meiosis is essential for sexual reproduction and recombination is a critical step required for normal meiosis. Understanding the underlying molecular mechanisms that regulate recombination ie important for medical, agricultural and ecological reasons. Readily available molecular and cytological tools make Arabidopsis an excellent system to study meiosis. Here we review recent developments in molecular genetic analyses on meiotic recombination. These Include studies on plant homologs of yeast and animal genes, as well as novel genes that were first identified in plants. The characterizations of these genes have demonstrated essential functions from the initiation of recombination by double-strand breaks to repair of such breaks, from the formation of double-Holliday junctions to possible resolution of these junctions, both of which are critical for crossover formation. The recent advances have ushered a new era in plant meiosis, in which the combination of genetics, genomics, and molecular cytology can uncover important gene functions.

  8. Homology group on manifolds and their foldings

    Directory of Open Access Journals (Sweden)

    M. Abu-Saleem

    2010-03-01

    Full Text Available In this paper, we introduce the definition of the induced unfolding on the homology group. Some types of conditional foldings restricted on the elements of the homology groups are deduced. The effect of retraction on the homology group of a manifold is dicussed. The unfolding of variation curvature of manifolds on their homology group are represented. The relations between homology group of the manifold and its folding are deduced.

  9. Grid diagrams and Khovanov homology

    DEFF Research Database (Denmark)

    Droz, Jean-Marie; Wagner, Emmanuel

    2009-01-01

    We explain how to compute the Jones polynomial of a link from one of its grid diagrams and we observe a connection between Bigelow’s homological definition of the Jones polynomial and Kauffman’s definition of the Jones polynomial. Consequently, we prove that the Maslov grading on the Seidel–Smith...

  10. Molecular evolution of a Drosophila homolog of human BRCA2.

    Science.gov (United States)

    Bennett, Sarah M; Noor, Mohamed A F

    2009-11-01

    The human cancer susceptibility gene, BRCA2, functions in double-strand break repair by homologous recombination, and it appears to function via interaction of a repetitive region ("BRC repeats") with RAD-51. A putatively simpler homolog, dmbrca2, was identified in Drosophila melanogaster recently and also affects mitotic and meiotic double-strand break repair. In this study, we examined patterns of repeat variation both within Drosophila pseudoobscura and among available Drosophila genome sequences. We identified extensive variation within and among closely related Drosophila species in BRC repeat number, to the extent that variation within this genus recapitulates the extent of variation found across the entire animal kingdom. We describe patterns of evolution across species by documenting recent repeat expansions (sometimes in tandem arrays) and homogenizations within available genome sequences. Overall, we have documented patterns and modes of evolution in a new model system of a gene which is important to human health.

  11. Recombination-assisted megaprimer (RAM) cloning

    OpenAIRE

    Jacques Mathieu; Emilia Alvarez; Alvarez, Pedro J. J.

    2014-01-01

    No molecular cloning technique is considered universally reliable, and many suffer from being too laborious, complex, or expensive. Restriction-free cloning is among the simplest, most rapid, and cost-effective methods, but does not always provide successful results. We modified this method to enhance its success rate through the use of exponential amplification coupled with homologous end-joining. This new method, recombination-assisted megaprimer (RAM) cloning, significantly extends the app...

  12. Sutured Floer homology and hypergraphs

    CERN Document Server

    Juhász, András; Rasmussen, Jacob

    2011-01-01

    By applying Seifert's algorithm to a special alternating diagram of a link L, one obtains a Seifert surface F of L. We show that the support of the sutured Floer homology of the sutured manifold complementary to F is affine isomorphic to the set of lattice points given as hypertrees in a certain hypergraph that is naturally associated to the diagram. This implies that the Floer groups in question are supported in a set of Spin^c structures that are the integer lattice points of a convex polytope. This property has an immediate extension to Seifert surfaces arising from homogeneous link diagrams (including all alternating and positive diagrams). In another direction, together with work in progress of the second author and others, our correspondence suggests a method for computing the "top" coefficients of the HOMFLY polynomial of a special alternating link from the sutured Floer homology of a Seifert surface complement for a certain dual link.

  13. Representation theory and homological stability

    CERN Document Server

    Church, Thomas

    2010-01-01

    We introduce the idea of *representation stability* (and several variations) for a sequence of representations V_n of groups G_n. One main goal is to expand the important and well-studied concept of homological stability so that it applies to a much broader variety of examples. Representation stability also provides a framework in which to find and to predict patterns, from classical representation theory (Littlewood--Richardson and Murnaghan rules, stability of Schur functors), to cohomology of groups (pure braid, Torelli and congruence groups), to Lie algebras and their homology, to the (equivariant) cohomology of flag and Schubert varieties, to combinatorics (the (n+1)^(n-1) conjecture). The majority of this paper is devoted to exposing this phenomenon through examples. In doing this we obtain applications, theorems, and conjectures. Beyond the discovery of new phenomena, the viewpoint of representation stability can be useful in solving problems outside the theory. In addition to the applications given in...

  14. Persistent Homology of Collaboration Networks

    Directory of Open Access Journals (Sweden)

    C. J. Carstens

    2013-01-01

    Full Text Available Over the past few decades, network science has introduced several statistical measures to determine the topological structure of large networks. Initially, the focus was on binary networks, where edges are either present or not. Thus, many of the earlier measures can only be applied to binary networks and not to weighted networks. More recently, it has been shown that weighted networks have a rich structure, and several generalized measures have been introduced. We use persistent homology, a recent technique from computational topology, to analyse four weighted collaboration networks. We include the first and second Betti numbers for the first time for this type of analysis. We show that persistent homology corresponds to tangible features of the networks. Furthermore, we use it to distinguish the collaboration networks from similar random networks.

  15. Inter-telomeric recombination is present in telomerase-positive human cells

    OpenAIRE

    Dlaska, Margit; Schöffski, Patrick; Bechter, Oliver E.

    2013-01-01

    Immortal cells require a mechanism of telomere length control in order to divide infinitely. One mechanism is telomerase, an enzyme that compensates the loss of telomeric DNA. The second mechanism is the alternative lengthening of telomeres (ALT) pathway. In ALT pathway cells, homologous recombination between telomeric DNA is the mechanism by which telomere homeostasis is achieved. We developed a novel homologous recombination reporter system that is able to measure inter-telomeric recombinat...

  16. FASEB Summer Research Conference. Genetic Recombination and Chromosome Rearrangements

    Energy Technology Data Exchange (ETDEWEB)

    Jinks-Robertson, Sue

    2002-02-01

    The 2001 meeting entitled ''Genetic Recombination and Genome Rearrangements'' was held July 21-26 in Snowmass, Colorado. The goal of the meeting was to bring together scientists using diverse approaches to study all aspects of genetic recombination. This goal was achieved by integrating talks covering the genetics, biochemistry and structural biology of homologous recombination, site-specific recombination, and nonhomologous recombination. The format of the meeting consisted of a keynote address on the opening evening, two formal plenary sessions on each of the four full meeting days, a single afternoon workshop consisting of short talks chosen from among submitted abstracts, and afternoon poster sessions on each of the four full meeting days. The eight plenary session were entitled: (1) Recombination Mechanisms, (2) Prokaryotic Recombination, (3) Repair and Recombination, (4) Site-specific Recombination and Transposition, (5) Eukaryotic Recombination I, (6) Genome Rearrangements, (7) Meiosis, and (8) Eukaryotic Recombination II. Each session included a mix of genetic, biochemical and structural talks; talks were limited to 20 minutes, followed by 10 minutes of very lively, general discussion. Much of the data presented in the plenary sessions was unpublished, thus providing attendees with the most up-to-date knowledge of this rapidly-moving field.

  17. Rad52 forms DMA repair and recombination centers during S phase

    DEFF Research Database (Denmark)

    Lisby, M.; Rothstein, R.; Mortensen, Uffe Hasbro

    2001-01-01

    cerevisiae, homologous recombination is the major pathway for repairing DNA double-strand breaks. The key role played by Rad52 in this pathway has been attributed to its ability to seek out and mediate annealing of homologous DNA strands. In this study, we find that S. cerevisiae Rad52 fused to green...

  18. The dynamics of homologous pairing during mating type interconversion in budding yeast.

    Directory of Open Access Journals (Sweden)

    Peter L Houston

    2006-06-01

    Full Text Available Cells repair most double-strand breaks (DSBs that arise during replication or by environmental insults through homologous recombination, a high-fidelity process critical for maintenance of genomic integrity. However, neither the detailed mechanism of homologous recombination nor the specific roles of critical components of the recombination machinery-such as Bloom and Werner syndrome proteins-have been resolved. We have taken a novel approach to examining the mechanism of homologous recombination by tracking both a DSB and the template from which it is repaired during the repair process in individual yeast cells. The two loci were labeled with arrays of DNA binding sites and visualized in live cells expressing green fluorescent protein-DNA binding protein chimeras. Following induction of an endonuclease that introduces a DSB next to one of the marked loci, live cells were imaged repeatedly to determine the relative positions of the DSB and the template locus. We found a significant increase in persistent associations between donor and recipient loci following formation of the DSB, demonstrating DSB-induced pairing between donor and template. However, such associations were transient and occurred repeatedly in every cell, a result not predicted from previous studies on populations of cells. Moreover, these associations were absent in sgs1 or srs2 mutants, yeast homologs of the Bloom and Werner syndrome genes, but were enhanced in a rad54 mutant, whose protein product promotes efficient strand exchange in vitro. Our results indicate that a DSB makes multiple and reversible contacts with a template during the repair process, suggesting that repair could involve interactions with multiple templates, potentially creating novel combinations of sequences at the repair site. Our results further suggest that both Sgs1 and Srs2 are required for efficient completion of recombination and that Rad54 may serve to dissociate such interactions. Finally, these

  19. DNA helicases in recombination and repair: construction of a delta uvrD delta helD delta recQ mutant deficient in recombination and repair.

    OpenAIRE

    Mendonca, V M; Klepin, H D; Matson, S W

    1995-01-01

    DNA helicases play pivotal roles in homologous recombination and recombinational DNA repair. They are involved in both the generation of recombinogenic single-stranded DNA ends and branch migration of synapsed Holliday junctions. Escherichia coli helicases II (uvrD), IV (helD), and RecQ (recQ) have all been implicated in the presynaptic stage of recombination in the RecF pathway. To probe for functional redundancy among these helicases, mutant strains containing single, double, and triple del...

  20. Recombinant Technology and Probiotics

    Directory of Open Access Journals (Sweden)

    Icy D’Silva

    2011-09-01

    Full Text Available Recombinant technology has led the way to monumental advances in the development of useful molecules, including the development of safe probiotics. The development of novel approaches using recombinant technology and probiotics that allow accurate targeting of therapeutics to the mucosa is an interesting area of research. The creation and use of recombinant probiotics expressing recombinantovalbumin, recombinant ovalbumin mutants and yet-to-be-designed recombinant hypo/non-allergenic molecules offer the opportunity to further investigate their effects for food, nutrition, environment andhealth. This review highlights advances in native probiotics and recombinant probiotics expressing native and recombinant molecules for food, nutrition, environment and health.

  1. SUMOylation of Rad52-Rad59 synergistically change the outcome of mitotic recombination

    DEFF Research Database (Denmark)

    Silva, Sonia; Altmannova, Veronika; Eckert-Boulet, Nadine;

    2016-01-01

    Homologous recombination (HR) is essential for maintenance of genome stability through double-strand break (DSB) repair, but at the same time HR can lead to loss of heterozygosity and uncontrolled recombination can be genotoxic. The post-translational modification by SUMO (small ubiquitin-like mo...

  2. Weak homology of elliptical galaxies

    CERN Document Server

    Bertin, G; Principe, M D

    2002-01-01

    We start by studying a small set of objects characterized by photometric profiles that have been pointed out to deviate significantly from the standard R^{1/4} law. For these objects we confirm that a generic R^{1/n} law, with n a free parameter, can provide superior fits (the best-fit value of n can be lower than 2.5 or higher than 10), better than those that can be obtained by a pure R^{1/4} law, by an R^{1/4}+exponential model, and by other dynamically justified self--consistent models. Therefore, strictly speaking, elliptical galaxies should not be considered homologous dynamical systems. Still, a case for "weak homology", useful for the interpretation of the Fundamental Plane of elliptical galaxies, could be made if the best-fit parameter n, as often reported, correlates with galaxy luminosity L, provided the underlying dynamical structure also follows a systematic trend with luminosity. We demonstrate that this statement may be true even in the presence of significant scatter in the correlation n(L). Pr...

  3. Syrinx 2A: an improved lambda phage vector designed for screening DNA libraries by recombination in vivo.

    OpenAIRE

    Lutz, C T; Hollifield, W C; Seed, B.; Davie, J M; Huang, H V

    1987-01-01

    The Syrinx 2A phage and pi AN13 plasmid were designed for screening of DNA libraries by homologous recombination in vivo. Syrinx 2A carries multiple cloning sites and a recently identified lambda gene, rap (recombination adept with plasmid), required for efficient phage-plasmid recombination. We describe a rapid, reliable, and technically easy method to screen Syrinx 2A libraries, expand the resulting phage-plasmid cointegrates, and subclone plasmid in as little as 2 days. Recombination scree...

  4. Links between replication, recombination and genome instability in eukaryotes

    OpenAIRE

    Flores-Rozas, Hernan; Kolodner, Richard D.

    2000-01-01

    Double-strand breaks in DNA can be repaired by homologous recombination including break-induced replication. In this reaction, the end of a broken DNA invades an intact chromosome and primes DNA replication resulting in the synthesis of an intact chromosome. Break-induced replication has also been suggested to cause different types of genome rearrangements.

  5. Meiotic sister chromatid cohesion and recombination in two filamentous fungi

    NARCIS (Netherlands)

    Heemst, van D.

    2000-01-01

    Homologous recombination and sister chromatid cohesion play important roles in the maintenance of genome integrity and the fidelity of chromosome segregation in mitosis and meiosis. Within the living cell, the integrity of the DNA is threatened by various factors that cause DNA-lesions, of which DNA

  6. Construction of Recombinant Plasmid Containing S. Mutans F-ATPase β Subunit Gene

    Institute of Scientific and Technical Information of China (English)

    YU Dan-ni; JIANG Li

    2005-01-01

    objective: construct a homologous recombinant plasmid which was expected to be transformed into S. mutans Methods: a region at the 5' terminus of the S. mutans F-ATPase β subunit gene was amplified by PCR, the PCR product was inserted into vector pVA891, yielding recombinant plasmid. Results: the DNA sequence of the recombinant plasmid was identified correct in whole by restriction endonuclease and DNA sequence techniques. Conclusion: the recombinant plasmid of S. mutans DNA was cloned in effect ,it may assist in construction of homologues recombinant mutant.

  7. A genetic system for direct selection of gene-positive clones during recombinational cloning in yeast

    OpenAIRE

    Noskov, Vladimir; Kouprina, Natalay; Leem, Sun-Hee; Koriabine, Maxim; Barrett, J. Carl; Larionov, Vladimir

    2002-01-01

    Transformation-associated recombination (TAR) is a cloning technique that allows specific chromosomal regions or genes to be isolated directly from genomic DNA without prior construction of a genomic library. This technique involves homologous recombination during spheroplast transformation between genomic DNA and a TAR vector that has 5′ and 3′ gene targeting sequences (hooks). Typically, TAR cloning produces positive YAC recombinants at a frequency of ∼0.5%; the positive clones are identifi...

  8. Critical Role of RecN in Recombinational DNA Repair and Survival of Helicobacter pylori▿

    OpenAIRE

    Wang, Ge; Maier, Robert J.

    2007-01-01

    Homologous recombination is one of the key mechanisms responsible for the repair of DNA double-strand breaks. Recombinational repair normally requires a battery of proteins, each with specific DNA recognition, strand transfer, resolution, or other functions. Helicobacter pylori lacks many of the proteins normally involved in the early stage (presynapsis) of recombinational repair, but it has a RecN homologue with an unclear function. A recN mutant strain of H. pylori was shown to be much more...

  9. Homologous recombinatorial cloning without the creation of single-stranded ends: exonuclease and ligation-independent cloning (ELIC).

    Science.gov (United States)

    Koskela, Essi V; Frey, Alexander D

    2015-03-01

    We describe a new type of molecular cloning that complements the available strategies for homologous recombinatorial cloning. Purified, linear double-stranded DNA molecules with homologous ends are simply mixed in water and they transform readily into E. coli. Insert and linear vector need as few as ten base pairs of homologous sequence at their ends and essentially no incubation or enzyme treatments are needed for creating recombinants from linear fragments. Our method outcompetes most existing cloning methods in simplicity and affordability and is well-suited for high-throughput applications. PMID:25370826

  10. Cas9-assisted recombineering in C. elegans: genome editing using in vivo assembly of linear DNAs

    Science.gov (United States)

    Paix, Alexandre; Schmidt, Helen; Seydoux, Geraldine

    2016-01-01

    Recombineering, the use of endogenous homologous recombination systems to recombine DNA in vivo, is a commonly used technique for genome editing in microbes. Recombineering has not yet been developed for animals, where non-homology-based mechanisms have been thought to dominate DNA repair. Here, we demonstrate, using Caenorhabditis elegans, that linear DNAs with short homologies (∼35 bases) engage in a highly efficient gene conversion mechanism. Linear DNA repair templates with homology to only one side of a double-strand break (DSB) initiate repair efficiently, and short overlaps between templates support template switching. We demonstrate the use of single-stranded, bridging oligonucleotides (ssODNs) to target PCR fragments for repair of DSBs induced by CRISPR/Cas9 on chromosomes. Based on these findings, we develop recombineering strategies for precise genome editing that expand the utility of ssODNs and eliminate in vitro cloning steps for template construction. We apply these methods to the generation of GFP knock-in alleles and gene replacements without co-integrated markers. We conclude that, like microbes, metazoans possess robust homology-dependent repair mechanisms that can be harnessed for recombineering and genome editing. PMID:27257074

  11. Link homology and equivariant gauge theory

    OpenAIRE

    Poudel, Prayat; Saveliev, Nikolai

    2015-01-01

    The singular instanton Floer homology was defined by Kronheimer and Mrowka in connection with their proof that the Khovanov homology is an unknot detector. We study this theory for knots and two-component links using equivariant gauge theory on their double branched covers. We show that the special generator in the singular instanton Floer homology of a knot is graded by the knot signature mod 4, thereby providing a purely topological way of fixing the absolute grading in the theory. Our appr...

  12. Homology in Electromagnetic Boundary Value Problems

    Directory of Open Access Journals (Sweden)

    Matti Pellikka

    2010-01-01

    Full Text Available We discuss how homology computation can be exploited in computational electromagnetism. We represent various cellular mesh reduction techniques, which enable the computation of generators of homology spaces in an acceptable time. Furthermore, we show how the generators can be used for setting up and analysis of an electromagnetic boundary value problem. The aim is to provide a rationale for homology computation in electromagnetic modeling software.

  13. Including Biological Literature Improves Homology Search

    OpenAIRE

    Chang, Jeffrey T.; Raychaudhuri, Soumya; Altman, Russ B

    2001-01-01

    Annotating the tremendous amount of sequence information being generated requires accurate automated methods for recognizing homology. Although sequence similarity is only one of many indicators of evolutionary homology, it is often the only one used. Here we find that supplementing sequence similarity with information from biomedical literature is successful in increasing the accuracy of homology search results. We modified the PSI-BLAST algorithm to use literature similarity in each iterati...

  14. Recombinant DNA in Medicine

    OpenAIRE

    Cederbaum, Stephen D.; Fareed, George C.; Lovett, Michael A.; Shapiro, Larry J.

    1984-01-01

    Studies in bacteria and bacterial viruses have led to methods to manipulate and recombine DNA in unique and reproducible ways and to amplify these recombined molecules millions of times. Once properly identified, the recombinant DNA molecules can be used in various ways useful in medicine and human biology. There are many applications for recombinant DNA technology. Cloned complementary DNA has been used to produce various human proteins in microorganisms. Insulin and growth hormone have been...

  15. Improving baculovirus recombination

    OpenAIRE

    Zhao, Yuguang; Chapman, David A. G.; Jones, Ian M.

    2003-01-01

    Recombinant baculoviruses have established themselves as a favoured technology for the high-level expression of recombinant proteins. The construction of recombinant viruses, however, is a time consuming step that restricts consideration of the technology for high throughput developments. Here we use a targeted gene knockout technology to inactivate an essential viral gene that lies adjacent to the locus used for recombination. Viral DNA prepared from the knockout fails to initiate an infecti...

  16. Sex, not genotype, determines recombination levels in mice.

    Science.gov (United States)

    Lynn, Audrey; Schrump, Stefanie; Cherry, Jonathan; Hassold, Terry; Hunt, Patricia

    2005-10-01

    Recombination, the precise physical breakage and rejoining of DNA between homologous chromosomes, plays a central role in mediating the orderly segregation of meiotic chromosomes in most eukaryotes. Despite its importance, the factors that control the number and placement of recombination events within a cell remain poorly defined. The rate of recombination exhibits remarkable species specificity, and, within a species, recombination is affected by the physical size of the chromosome, chromosomal location, proximity to other recombination events (i.e., chiasma interference), and, intriguingly, the sex of the transmitting parent. To distinguish between simple genetic and nongenetic explanations of sex-specific recombination differences in mammals, we compared recombination in meiocytes from XY sex-reversed and XO females with that in meiocytes from XX female and XY male mice. The rate and pattern of recombination in XY and XO oocytes were virtually identical to those in normal XX females, indicating that sex, not genotype, is the primary determinant of meiotic recombination patterns in mammals.

  17. Buoyancy instability of homologous implosions

    Science.gov (United States)

    Johnson, Bryan

    2015-11-01

    Hot spot turbulence is a potential contributor to yield degradation in inertial confinement fusion (ICF) capsules, although its origin, if present, remains unclear. In this work, a perturbation analysis is performed of an analytical homologous solution that mimics the hot spot and surrounding cold fuel during the late stages of an ICF implosion. It is shown that the flow is governed by the Schwarzschild criterion for buoyant stability, and that during stagnation, short wavelength entropy and vorticity fluctuations amplify by a factor exp (π |N0 | ts) , where N0 is the buoyancy frequency at stagnation and ts is the stagnation time scale. This amplification factor is exponentially sensitive to mean flow gradients and varies from 103-107 for realistic gradients. Comparisons are made with a Lagrangian hydrodynamics code, and it is found that a numerical resolution of ~ 30 zones per wavelength is required to capture the evolution of vorticity accurately. This translates to an angular resolution of ~(12 / l) ∘ , or ~ 0 .1° to resolve the fastest growing modes (Legendre mode l > 100).

  18. Stability of p53 homologs.

    Directory of Open Access Journals (Sweden)

    Tobias Brandt

    Full Text Available Most proteins have not evolved for maximal thermal stability. Some are only marginally stable, as for example, the DNA-binding domains of p53 and its homologs, whose kinetic and thermodynamic stabilities are strongly correlated. Here, we applied high-throughput methods using a real-time PCR thermocycler to study the stability of several full-length orthologs and paralogs of the p53 family of transcription factors, which have diverse functions, ranging from tumour suppression to control of developmental processes. From isothermal denaturation fluorimetry and differential scanning fluorimetry, we found that full-length proteins showed the same correlation between kinetic and thermodynamic stability as their isolated DNA-binding domains. The stabilities of the full-length p53 orthologs were marginal and correlated with the temperature of their organism, paralleling the stability of the isolated DNA-binding domains. Additionally, the paralogs p63 and p73 were significantly more stable and long-lived than p53. The short half-life of p53 orthologs and the greater persistence of the paralogs may be biologically relevant.

  19. Use of Recombination-Mediated Genetic Engineering for Construction of Rescue Human Cytomegalovirus Bacterial Artificial Chromosome Clones

    Directory of Open Access Journals (Sweden)

    Kalpana Dulal

    2012-01-01

    Full Text Available Bacterial artificial chromosome (BAC technology has contributed immensely to manipulation of larger genomes in many organisms including large DNA viruses like human cytomegalovirus (HCMV. The HCMV BAC clone propagated and maintained inside E. coli allows for accurate recombinant virus generation. Using this system, we have generated a panel of HCMV deletion mutants and their rescue clones. In this paper, we describe the construction of HCMV BAC mutants using a homologous recombination system. A gene capture method, or gap repair cloning, to seize large fragments of DNA from the virus BAC in order to generate rescue viruses, is described in detail. Construction of rescue clones using gap repair cloning is highly efficient and provides a novel use of the homologous recombination-based method in E. coli for molecular cloning, known colloquially as recombineering, when rescuing large BAC deletions. This method of excising large fragments of DNA provides important prospects for in vitro homologous recombination for genetic cloning.

  20. Homotopic Chain Maps Have Equal s-Homology and d-Homology

    Directory of Open Access Journals (Sweden)

    M. Z. Kazemi-Baneh

    2016-01-01

    Full Text Available The homotopy of chain maps on preabelian categories is investigated and the equality of standard homologies and d-homologies of homotopic chain maps is established. As a special case, if X and Y are the same homotopy type, then their nth d-homology R-modules are isomorphic, and if X is a contractible space, then its nth d-homology R-modules for n≠0 are trivial.

  1. Photoionization and Recombination

    Science.gov (United States)

    Nahar, Sultana N.

    2000-01-01

    Theoretically self-consistent calculations for photoionization and (e + ion) recombination are described. The same eigenfunction expansion for the ion is employed in coupled channel calculations for both processes, thus ensuring consistency between cross sections and rates. The theoretical treatment of (e + ion) recombination subsumes both the non-resonant recombination ("radiative recombination"), and the resonant recombination ("di-electronic recombination") processes in a unified scheme. In addition to the total, unified recombination rates, level-specific recombination rates and photoionization cross sections are obtained for a large number of atomic levels. Both relativistic Breit-Pauli, and non-relativistic LS coupling, calculations are carried out in the close coupling approximation using the R-matrix method. Although the calculations are computationally intensive, they yield nearly all photoionization and recombination parameters needed for astrophysical photoionization models with higher precision than hitherto possible, estimated at about 10-20% from comparison with experimentally available data (including experimentally derived DR rates). Results are electronically available for over 40 atoms and ions. Photoionization and recombination of He-, and Li-like C and Fe are described for X-ray modeling. The unified method yields total and complete (e+ion) recombination rate coefficients, that can not otherwise be obtained theoretically or experimentally.

  2. Detecting atmospheric rivers using persistent homology

    OpenAIRE

    Alfsvåg, Kristian Stusdal

    2015-01-01

    This master's thesis is a first investigation on the problem of seeing whether it is possible to detect atmospheric rivers using persistent homology. Two different computations are done and a basic analysis is made. In addition an implementation of persistent homology made during the thesis is described.

  3. Relative K-homology and normal operators

    DEFF Research Database (Denmark)

    Manuilov, Vladimir; Thomsen, Klaus

    2009-01-01

    -term exact sequence which generalizes the excision six-term exact sequence in the first variable of KK-theory. Subsequently we investigate the relative K-homology which arises from the group of relative extensions by specializing to abelian $C^*$-algebras. It turns out that this relative K-homology carries...

  4. RecA bundles mediate homology pairing between distant sisters during DNA break repair

    OpenAIRE

    Lesterlin, Christian; Ball, Graeme; Schermelleh, Lothar; Sherratt, David J.

    2013-01-01

    DNA double-strand break (DSB) repair by homologous recombination (HR) has evolved to maintain genetic integrity in all organisms 1 . Although many reactions that occur during HR are known 1-3 , it is unclear where, when and how they occur in cells is lacking. Here, by using conventional and super-resolution microscopy we describe the progression of DSB repair in live Escherichia coli. Specifically, we investigate whether HR can occur efficiently between distant sister loci that have segregate...

  5. Escherichia coli F41 adhesin: genetic organization, nucleotide sequence, and homology with the K88 determinant.

    OpenAIRE

    1988-01-01

    The genetic organization of the polypeptides required for the biosynthesis of the F41 adhesin of enterotoxigenic Escherichia coli strains was investigated. Maxicell analysis demonstrated that a recombinant plasmid which mediated mannose-resistant hemagglutination and F41 antigen production encoded four polypeptides of 29, 30, 32, and 86 kilodaltons. The 29-kilodalton protein was identified as the F41 antigen, and the nucleotide sequence of the gene was determined. Extensive homology was obser...

  6. A universal BMV-based RNA recombination system--how to search for general rules in RNA recombination.

    Science.gov (United States)

    Alejska, Magdalena; Figlerowicz, Magdalena; Malinowska, Nelli; Urbanowicz, Anna; Figlerowicz, Marek

    2005-07-07

    At present, there is no doubt that RNA recombination is one of the major factors responsible for the generation of new RNA viruses and retroviruses. Numerous experimental systems have been created to investigate this complex phenomenon. Consequently, specific RNA structural motifs mediating recombination have been identified in several viruses. Unfortunately, up till now a unified model of genetic RNA recombination has not been formulated, mainly due to difficulties with the direct comparison of data obtained for different RNA-based viruses. To solve this problem, we have attempted to construct a universal system in which the recombination activity of various RNA sequences could be tested. To this end, we have used brome mosaic virus, a model (+)RNA virus of plants, for which the structural requirements of RNA recombination are well defined. The effectiveness of the new homomolecular system has been proven in an experiment involving two RNA sequences derived from the hepatitis C virus genome. In addition, comparison of the data obtained with the homomolecular system with those generated earlier using the heteromolecular one has provided new evidence that the mechanisms of homologous and non-homologous recombination are different and depend on the virus' mode of replication.

  7. Meiotic crossover control by concerted action of Rad51-Dmc1 in homolog template bias and robust homeostatic regulation.

    Directory of Open Access Journals (Sweden)

    Jessica P Lao

    Full Text Available During meiosis, repair of programmed DNA double-strand breaks (DSBs by recombination promotes pairing of homologous chromosomes and their connection by crossovers. Two DNA strand-exchange proteins, Rad51 and Dmc1, are required for meiotic recombination in many organisms. Studies in budding yeast imply that Rad51 acts to regulate Dmc1's strand exchange activity, while its own exchange activity is inhibited. However, in a dmc1 mutant, elimination of inhibitory factor, Hed1, activates Rad51's strand exchange activity and results in high levels of recombination without participation of Dmc1. Here we show that Rad51-mediated meiotic recombination is not subject to regulatory processes associated with high-fidelity chromosome segregation. These include homolog bias, a process that directs strand exchange between homologs rather than sister chromatids. Furthermore, activation of Rad51 does not effectively substitute for Dmc1's chromosome pairing activity, nor does it ensure formation of the obligate crossovers required for accurate homolog segregation. We further show that Dmc1's dominance in promoting strand exchange between homologs involves repression of Rad51's strand-exchange activity. This function of Dmc1 is independent of Hed1, but requires the meiotic kinase, Mek1. Hed1 makes a relatively minor contribution to homolog bias, but nonetheless this is important for normal morphogenesis of synaptonemal complexes and efficient crossing-over especially when DSB numbers are decreased. Super-resolution microscopy shows that Dmc1 also acts to organize discrete complexes of a Mek1 partner protein, Red1, into clusters along lateral elements of synaptonemal complexes; this activity may also contribute to homolog bias. Finally, we show that when interhomolog bias is defective, recombination is buffered by two feedback processes, one that increases the fraction of events that yields crossovers, and a second that we propose involves additional DSB formation

  8. Bacterial Artificial Chromosome Mutagenesis Using Recombineering

    Directory of Open Access Journals (Sweden)

    Kumaran Narayanan

    2011-01-01

    Full Text Available Gene expression from bacterial artificial chromosome (BAC clones has been demonstrated to facilitate physiologically relevant levels compared to viral and nonviral cDNA vectors. BACs are large enough to transfer intact genes in their native chromosomal setting together with flanking regulatory elements to provide all the signals for correct spatiotemporal gene expression. Until recently, the use of BACs for functional studies has been limited because their large size has inherently presented a major obstacle for introducing modifications using conventional genetic engineering strategies. The development of in vivo homologous recombination strategies based on recombineering in E. coli has helped resolve this problem by enabling facile engineering of high molecular weight BAC DNA without dependence on suitably placed restriction enzymes or cloning steps. These techniques have considerably expanded the possibilities for studying functional genetics using BACs in vitro and in vivo.

  9. Assisted large fragment insertion by Red/ET-recombination (ALFIRE)—an alternative and enhanced method for large fragment recombineering

    OpenAIRE

    Rivero-Müller, Adolfo; Lajić, Svetlana; Huhtaniemi, Ilpo

    2007-01-01

    Functional genomics require manipulation and modification of large fragments of the genome. Such manipulation has only recently become more efficient due to the discovery of different techniques based on homologous recombination. However, certain limitations of these strategies still exist since insertion of homology arms (HAs) is often based on amplification of DNA sequences with PCR. Large quantities of PCR products longer than 4–5 kb can be difficult to obtain and the risk of mutations or ...

  10. Non-homologous end joining: advances and frontiers.

    Science.gov (United States)

    Yang, Kai; Guo, Rong; Xu, Dongyi

    2016-07-01

    DNA double-strand breaks (DSBs) are the most serious form of DNA damage. In human cells, non-homologous end joining (NHEJ) is the major pathway for the repair of DSBs. Different types of DSBs result in different subsets of NHEJ repair strategies. These variations in NHEJ repair strategies depend on numerous elements, such as the flexible recruitment of NHEJ-related proteins, the complexity of the DSB ends, and the spatial- and temporal-ordered formation of the multi-protein complex. On the one hand, current studies of DNA DSBs repair focus on the repair pathway choices between homologous recombination and classic or alternative NHEJ. On the other hand, increasing researches have also deepened the significance and dug into the cross-links between the NHEJ pathway and the area of genome organization and aging. Although remarkable progress has been made in elucidating the underlying principles during the past decades, the detailed mechanism of action in response to different types of DSBs remains largely unknown and needs further evaluation in the future study. PMID:27217473

  11. The PIKE homolog Centaurin gamma regulates developmental timing in Drosophila.

    Directory of Open Access Journals (Sweden)

    Anna Lisa Gündner

    Full Text Available Phosphoinositide-3-kinase enhancer (PIKE proteins encoded by the PIKE/CENTG1 gene are members of the gamma subgroup of the Centaurin superfamily of small GTPases. They are characterized by their chimeric protein domain architecture consisting of a pleckstrin homology (PH domain, a GTPase-activating (GAP domain, Ankyrin repeats as well as an intrinsic GTPase domain. In mammals, three PIKE isoforms with variations in protein structure and subcellular localization are encoded by the PIKE locus. PIKE inactivation in mice results in a broad range of defects, including neuronal cell death during brain development and misregulation of mammary gland development. PIKE -/- mutant mice are smaller, contain less white adipose tissue, and show insulin resistance due to misregulation of AMP-activated protein kinase (AMPK and insulin receptor/Akt signaling. here, we have studied the role of PIKE proteins in metabolic regulation in the fly. We show that the Drosophila PIKE homolog, ceng1A, encodes functional GTPases whose internal GAP domains catalyze their GTPase activity. To elucidate the biological function of ceng1A in flies, we introduced a deletion in the ceng1A gene by homologous recombination that removes all predicted functional PIKE domains. We found that homozygous ceng1A mutant animals survive to adulthood. In contrast to PIKE -/- mouse mutants, genetic ablation of Drosophila ceng1A does not result in growth defects or weight reduction. Although metabolic pathways such as insulin signaling, sensitivity towards starvation and mobilization of lipids under high fed conditions are not perturbed in ceng1A mutants, homozygous ceng1A mutants show a prolonged development in second instar larval stage, leading to a late onset of pupariation. In line with these results we found that expression of ecdysone inducible genes is reduced in ceng1A mutants. Together, we propose a novel role for Drosophila Ceng1A in regulating ecdysone signaling-dependent second to

  12. A Heritable Recombination system for synthetic Darwinian evolution in yeast.

    Science.gov (United States)

    Romanini, Dante W; Peralta-Yahya, Pamela; Mondol, Vanessa; Cornish, Virginia W

    2012-12-21

    Genetic recombination is central to the generation of molecular diversity and enhancement of evolutionary fitness in living systems. Methods such as DNA shuffling that recapitulate this diversity mechanism in vitro are powerful tools for engineering biomolecules with useful new functions by directed evolution. Synthetic biology now brings demand for analogous technologies that enable the controlled recombination of beneficial mutations in living cells. Thus, here we create a Heritable Recombination system centered around a library cassette plasmid that enables inducible mutagenesis via homologous recombination and subsequent combination of beneficial mutations through sexual reproduction in Saccharomyces cerevisiae. Using repair of nonsense codons in auxotrophic markers as a model, Heritable Recombination was optimized to give mutagenesis efficiencies of up to 6% and to allow successive repair of different markers through two cycles of sexual reproduction and recombination. Finally, Heritable Recombination was employed to change the substrate specificity of a biosynthetic enzyme, with beneficial mutations in three different active site loops crossed over three continuous rounds of mutation and selection to cover a total sequence diversity of 10(13). Heritable Recombination, while at an early stage of development, breaks the transformation barrier to library size and can be immediately applied to combinatorial crossing of beneficial mutations for cell engineering, adding important features to the growing arsenal of next generation molecular biology tools for synthetic biology. PMID:23412545

  13. Threading homology through algebra selected patterns

    CERN Document Server

    Boffi, Giandomenico

    2006-01-01

    Aimed at graduate students and researchers in mathematics, this book takes homological themes, such as Koszul complexes and their generalizations, and shows how these can be used to clarify certain problems in selected parts of algebra, as well as their success in solving a number of them. - ;Threading Homology through Algebra takes homological themes (Koszul complexes and their variations, resolutions in general) and shows how these affect the perception of certain problems in selected parts of algebra, as well as their success in solving a number of them. The text deals with regular local ri

  14. Exceptional cosmetic surgeries on homology spheres

    OpenAIRE

    Ravelomanana, Huygens C.

    2016-01-01

    We investigate the cosmetic surgery conjecture for hyperbolic knots in integer homology spheres, focusing on exceptional surgeries. We give some restrictions on the slopes of exceptional truly cosmetic surgeries according to the type of surgery.

  15. Superconformal field theories and cyclic homology

    CERN Document Server

    Eager, Richard

    2015-01-01

    One of the predictions of the AdS/CFT correspondence is the matching of protected operators between a superconformal field theory and its holographic dual. We review the spectrum of protected operators in quiver gauge theories that flow to superconformal field theories at low energies. The spectrum is determined by the cyclic homology of an algebra associated to the quiver gauge theory. Identifying the spectrum of operators with cyclic homology allows us to apply the Hochschild-Kostant-Rosenberg theorem to relate the cyclic homology groups to deRham cohomology groups. The map from cyclic homology to deRham cohomology can be viewed as a mathematical avatar of the passage from open to closed strings under the AdS/CFT correspondence.

  16. Cylindrical contact homology and topological entropy

    OpenAIRE

    Alves, Marcelo R. R.

    2014-01-01

    We establish a relation between the growth of the cylindrical contact homology of a contact manifold and the topological entropy of Reeb flows on this manifold. We show that if a contact manifold $(M,\\xi)$ admits a hypertight contact form $\\lambda_0$ for which the cylindrical contact homology has exponential homotopical growth rate, then the Reeb flow of every contact form on $(M,\\xi)$ has positive topological entropy. Using this result, we provide numerous new examples of contact 3-manifolds...

  17. Dualities in Persistent (Co)Homology

    Energy Technology Data Exchange (ETDEWEB)

    de Silva, Vin; Morozov, Dmitriy; Vejdemo-Johansson, Mikael

    2011-09-16

    We consider sequences of absolute and relative homology and cohomology groups that arise naturally for a filtered cell complex. We establishalgebraic relationships between their persistence modules, and show that they contain equivalent information. We explain how one can use the existingalgorithm for persistent homology to process any of the four modules, and relate it to a recently introduced persistent cohomology algorithm. Wepresent experimental evidence for the practical efficiency of the latter algorithm.

  18. Preserved irradiated homologous cartilage for orbital reconstruction

    Energy Technology Data Exchange (ETDEWEB)

    Linberg, J.V.; Anderson, R.L.; Edwards, J.J.; Panje, W.R.; Bardach, J.

    1980-07-01

    Human costal cartilage is an excellent implant material for orbital and periorbital reconstruction because of its light weight, strength, homogeneous consistency and the ease with which it can be carved. Its use has been limited by the necessity of a separate surgical procedure to obtain the material. Preserved irradiated homologous cartilage has been shown to have almost all the autogenous cartilage and is convenient to use. Preserved irradiated homologous cartilage transplants do not elicit rejection reactions, resist infection and rarely undergo absorption.

  19. On the hodological criterion for homology

    Science.gov (United States)

    Faunes, Macarena; Francisco Botelho, João; Ahumada Galleguillos, Patricio; Mpodozis, Jorge

    2015-01-01

    Owen's pre-evolutionary definition of a homolog as “the same organ in different animals under every variety of form and function” and its redefinition after Darwin as “the same trait in different lineages due to common ancestry” entail the same heuristic problem: how to establish “sameness.”Although different criteria for homology often conflict, there is currently a generalized acceptance of gene expression as the best criterion. This gene-centered view of homology results from a reductionist and preformationist concept of living beings. Here, we adopt an alternative organismic-epigenetic viewpoint, and conceive living beings as systems whose identity is given by the dynamic interactions between their components at their multiple levels of composition. We posit that there cannot be an absolute homology criterion, and instead, homology should be inferred from comparisons at the levels and developmental stages where the delimitation of the compared trait lies. In this line, we argue that neural connectivity, i.e., the hodological criterion, should prevail in the determination of homologies between brain supra-cellular structures, such as the vertebrate pallium. PMID:26157357

  20. A novel allele of Saccharomyces cerevisiae RFA1 that is deficient in recombination and repair and suppressible by RAD52.

    OpenAIRE

    Firmenich, A A; Elias-Arnanz, M; Berg, P

    1995-01-01

    To understand the mechanisms involved in homologous recombination, we have performed a search for Saccharomyces cerevisiae mutants unable to carry out plasmid-to-chromosome gene conversion. For this purpose, we have developed a colony color assay in which recombination is induced by the controlled delivery of double-strand breaks (DSBs). Recombination occurs between a chromosomal mutant ade2 allele and a second plasmid-borne ade2 allele where DSBs are introduced via the site-specific HO endon...

  1. Heterologous Overexpression and Mutagenesis of the Human Bile Salt Export Pump (ABCB11) Using DREAM (Directed REcombination-Assisted Mutagenesis)

    OpenAIRE

    Jan Stindt; Philipp Ellinger; Claudia Stross; Verena Keitel; Dieter Häussinger; Smits, Sander H. J.; Ralf Kubitz; Lutz Schmitt

    2011-01-01

    Homologous recombination in Saccharomyces cerevisiae is a well-studied process. Here, we describe a yeast-recombination-based approach to construct and mutate plasmids containing the cDNA of the human bile salt export pump (BSEP) that has been shown to be unstable in E. coli. Using this approach, we constructed the necessary plasmids for a heterologous overexpression of BSEP in the yeast Pichia pastoris. We then applied a new site-directed mutagenesis method, DREAM (Directed REcombination-Ass...

  2. Plasmid-Chromosome Recombination of Irradiated Shuttle Vector DNA in African Green Monkey Kidney Cells.

    Science.gov (United States)

    Mudgett, John Stuart

    1987-09-01

    An autonomously replicating shuttle vector was used to investigate the enhancement of plasmid-chromosome recombination in mammalian host cells by ultraviolet light and gamma radiation. Sequences homologous to the shuttle vector were stably inserted into the genome of African Green Monkey kidney cells to act as the target substrate for these recombination events. The SV40- and pBR322-derived plasmid DNA was irradiated with various doses of radiation before transfection into the transformed mammalian host cells. The successful homologous transfer of the bacterial ampicillin resistance (amp^{rm r}) gene from the inserted sequences to replace a mutant amp^->=ne on the shuttle vector was identified by plasmid extraction and transformation into E. coli host cells. Ultraviolet light (UV) was found not to induce homologous plasmid-chromosome recombination, while gamma radiation increased the frequency of recombinant plasmids detected. The introduction of specific double -strand breaks in the plasmid or prolonging the time of plasmid residence in the mammalian host cells also enhanced plasmid-chromosome recombination. In contrast, plasmid mutagenesis was found to be increased by plasmid UV irradiation, but not to change with time. Plasmid survival, recombination, and mutagenesis were not affected by treating the mammalian host cells with UV light prior to plasmid transfection. The amp^{rm r} recombinant plasmid molecules analyzed were found to be mostly the result of nonconservative exchanges which appeared to involve both homologous and possibly nonhomologous interactions with the host chromosome. The observation that these recombinant structures were obtained from all of the plasmid alterations investigated suggests a common mechanistic origin for plasmid -chromosome recombination in these mammalian cells.

  3. A Khovanov Type Link Homology with Geometric Interpretation

    Institute of Scientific and Technical Information of China (English)

    Mei Li ZHANG; Feng Chun LEI

    2016-01-01

    We study a Khovanov type homology close to the original Khovanov homology theory from Frobenius system. The homology is an invariant for oriented links up to isotopy by applying a tautological functor on the geometric complex. The homology has also geometric descriptions by introducing the genus generating operations. We prove that Jones Polynomial is equal to a suitable Euler characteristic of the homology groups. As an application, we compute the homology groups of (2, k)-torus knots for every k∈N.

  4. Expression of Recombinant Baculovirus Carrying Schistosoma japonicum 26 ku GST in Mammalian Cells

    Institute of Scientific and Technical Information of China (English)

    YU Guangqing; SONG Jianhua; LIU Wenqi; LONG Xiaochun; MO Hongmei; LI Yonglong; CHEN Xinwen

    2006-01-01

    In order to construct recombinant baculovirus carrying Schistosoma japonicum 26 ku glutathione S-transferase gene (Sj26), and observe the expression of Sj26 in mammalian cells, the Sj26 gene was amplified with plasmid pGEX-3X as template by PCR, and then recombined into Tvector for sequencing. Sj26 gene was inserted into the downstream of CMV promoter of donor plasmid pFBDGC, and the recombinant donor plasmid pFBDGC-Sj26 transformed into DH10Bac,then the recombinant bacmid AcCMVSj26 was isolated and transfected into Sf9 cells. The recombinant baculovirus was harvested and final titer of vAcCMVSj26 was measured. BHK cells were transducted with recombinant baculovirus in vitro. By using Western blot, the expression of 26 ku glutathione S-transferase (GST) was detected. The results showed that after enzyme digestion and sequencing, the donor plasmid was successfully constructed. PCR confirmed that pFBDGC-Sj26 and Bacmid homologous recombination occurred in E. coli. After transfection of Sf9 cells with recombinant Bacmid, recombinant baculovirus was replicated in Sf9 cells and expressed green fluorescent protein. PCR further revealed recombinant baculovirus contained Sj26. The titer of the harvested baculovirus was 1.24 × 108. Western blot demonstrated that recombinant baculovirus could express 26 ku GST in BHK cells. It was concluded that Sj26 recombinant baculovirus was successfully constructed, and the 26 ku GST was expressed in mammalian cells.

  5. Investigating homology between proteins using energetic profiles.

    Directory of Open Access Journals (Sweden)

    James O Wrabl

    2010-03-01

    Full Text Available Accumulated experimental observations demonstrate that protein stability is often preserved upon conservative point mutation. In contrast, less is known about the effects of large sequence or structure changes on the stability of a particular fold. Almost completely unknown is the degree to which stability of different regions of a protein is generally preserved throughout evolution. In this work, these questions are addressed through thermodynamic analysis of a large representative sample of protein fold space based on remote, yet accepted, homology. More than 3,000 proteins were computationally analyzed using the structural-thermodynamic algorithm COREX/BEST. Estimated position-specific stability (i.e., local Gibbs free energy of folding and its component enthalpy and entropy were quantitatively compared between all proteins in the sample according to all-vs.-all pairwise structural alignment. It was discovered that the local stabilities of homologous pairs were significantly more correlated than those of non-homologous pairs, indicating that local stability was indeed generally conserved throughout evolution. However, the position-specific enthalpy and entropy underlying stability were less correlated, suggesting that the overall regional stability of a protein was more important than the thermodynamic mechanism utilized to achieve that stability. Finally, two different types of statistically exceptional evolutionary structure-thermodynamic relationships were noted. First, many homologous proteins contained regions of similar thermodynamics despite localized structure change, suggesting a thermodynamic mechanism enabling evolutionary fold change. Second, some homologous proteins with extremely similar structures nonetheless exhibited different local stabilities, a phenomenon previously observed experimentally in this laboratory. These two observations, in conjunction with the principal conclusion that homologous proteins generally conserved

  6. Improved and simplified recombineering approach for influenza virus reverse genetics

    OpenAIRE

    Liu, Qinfang; Wang, Shuai; Ma, Guangpeng; Pu, Juan; Forbes, Nicole E.; Brown, Earl G.; Liu, Jin-Hua

    2009-01-01

    Typical reverse genetics systems for generating influenza viruses require the insertion of each genome segments by DNA ligation into vectors for genome synthesis and expression. Herein is described the construction and use of a novel pair of plasmid vectors for cloning all eight genome segments of influenza A virus by homologous recombination for influenza virus reverse genetics. Plasmids, pLLBA and pLLBG, were constructed to possess opposing RNA polymerase I and RNA polymerase II transcripti...

  7. Meiotic sister chromatid cohesion and recombination in two filamentous fungi

    OpenAIRE

    Heemst, van, D.

    2000-01-01

    Homologous recombination and sister chromatid cohesion play important roles in the maintenance of genome integrity and the fidelity of chromosome segregation in mitosis and meiosis. Within the living cell, the integrity of the DNA is threatened by various factors that cause DNA-lesions, of which DNA double-strand breaks (DSBs) are considered particularly deleterious. The causative agents can be of endogenous origin, such as metabolically produced free radicals, and of exogenous origin, such a...

  8. Choreography of recombination proteins during the DNA damage response

    DEFF Research Database (Denmark)

    Lisby, Michael; Rothstein, Rodney

    2009-01-01

    Genome integrity is frequently challenged by DNA lesions from both endogenous and exogenous sources. A single DNA double-strand break (DSB) is lethal if unrepaired and may lead to loss of heterozygosity, mutations, deletions, genomic rearrangements and chromosome loss if repaired improperly. Such...... research. Here we review the cell biological response to DSBs in mitotically growing cells with an emphasis on homologous recombination pathways in yeast Saccharomyces cerevisiae and in mammalian cells....

  9. Dynamics of rye chromosome 1R regions with high or low crossover frequency in homology search and synapsis development.

    Directory of Open Access Journals (Sweden)

    Nohelia T Valenzuela

    Full Text Available In many organisms, homologous pairing and synapsis depend on the meiotic recombination machinery that repairs double-strand DNA breaks (DSBs produced at the onset of meiosis. The culmination of recombination via crossover gives rise to chiasmata, which locate distally in many plant species such as rye, Secale cereale. Although, synapsis initiates close to the chromosome ends, a direct effect of regions with high crossover frequency on partner identification and synapsis initiation has not been demonstrated. Here, we analyze the dynamics of distal and proximal regions of a rye chromosome introgressed into wheat to define their role on meiotic homology search and synapsis. We have used lines with a pair of two-armed chromosome 1R of rye, or a pair of telocentrics of its long arm (1RL, which were homozygous for the standard 1RL structure, homozygous for an inversion of 1RL that changes chiasma location from distal to proximal, or heterozygous for the inversion. Physical mapping of recombination produced in the ditelocentric heterozygote (1RL/1RL(inv showed that 70% of crossovers in the arm were confined to a terminal segment representing 10% of the 1RL length. The dynamics of the arms 1RL and 1RL(inv during zygotene demonstrates that crossover-rich regions are more active in recognizing the homologous partner and developing synapsis than crossover-poor regions. When the crossover-rich regions are positioned in the vicinity of chromosome ends, their association is facilitated by telomere clustering; when they are positioned centrally in one of the two-armed chromosomes and distally in the homolog, their association is probably derived from chromosome elongation. On the other hand, chromosome movements that disassemble the bouquet may facilitate chromosome pairing correction by dissolution of improper chromosome associations. Taken together, these data support that repair of DSBs via crossover is essential in both the search of the homologous partner

  10. On the hodological criterion for homology

    Directory of Open Access Journals (Sweden)

    Macarena eFaunes

    2015-06-01

    Full Text Available Owen’s pre-evolutionary definition of a homologue as the same organ in different animals under every variety of form and function and its redefinition after Darwin as the same trait in different lineages due to common ancestry entail the same heuristic problem: how to establish sameness. Although different criteria for homology often conflict, there is currently a generalized acceptance of gene expression as the best criterion. This gene-centered view of homology results from a reductionist and preformationist concept of living beings. Here, we adopt an alternative organismic-epigenetic viewpoint, and conceive living beings as systems whose identity is given by the dynamic interactions between their components at their multiple levels of composition. We posit that there cannot be an absolute homology criterion, and instead, homology should be inferred from comparisons at the levels and developmental stages where the delimitation of the compared trait lies. In this line, we argue that neural connectivity, i.e., the hodological criterion, should prevail in the determination of homologies between brain supra-cellular structures, such as the vertebrate pallium.

  11. The Arabidopsis MutS homolog AtMSH5 is required for normal meiosis

    Institute of Scientific and Technical Information of China (English)

    Xiaoduo Lu; Xiaolin Liu; Lizhe An; Wei Zhang; Jian Sun; Huijuan Pei; Hongyan Meng; Yunliu Fan; Chunyi Zhang

    2008-01-01

    MSH5,a member of the MutS homolog DNA mismatch repair protein family,has been shown to be required for proper homologous chromosome recombination in diverse organisms such as mouse,budding yeast and Caenorhabditis elegans.In this paper,we show that a mutant Arabidopsis plant carrying the putative disrupted AtMSH5 gene exhibits defects during meiotic division,producing a proportion of nonviable pollen grains and abnormal embryo sacs,and thereby leading to a decrease in fertility.AtMSH5 expression is confined to meiotic floral buds,which is consistent with a possible role during meiosis.Cytological analysis of male meiosis revealed the presence of numerous univalents from diplotene to metaphase I,which were associated with a great reduction in chiasma frequencies.The average number of residual chiasmata in the mutant is reduced to 2.54 per meiocyte,which accounts for~25% of the amount in the wild type.Here,quantitative cytogenetical analysis reveals that the residual chiasmata in Atmsh5 mutants are randomly distributed among meiocytes,suggesting that AtMSH5 has an essential role during interferencesensitive chiasma formation.Taken together,the evidence indicates that AtMSH5 promotes homologous recombination through facilitating chiasma formation during prophase I in Arabidopsis.

  12. The function of classical and alternative non-homologous end-joining pathways in the fusion of dysfunctional telomeres

    OpenAIRE

    Rai, Rekha; Zheng, Hong; He, Hua; Luo, Ying; Multani, Asha; Carpenter, Phillip B.; Chang, Sandy

    2010-01-01

    Repair of DNA double-stranded breaks (DSBs) is crucial for the maintenance of genome stability. DSBs are repaired by either error prone non-homologous end-joining (NHEJ) or error-free homologous recombination. NHEJ precedes either by a classic, Lig4-dependent process (C-NHEJ) or an alternative, Lig4-independent one (A-NHEJ). Dysfunctional telomeres arising either through natural attrition due to telomerase deficiency or by removal of telomere-binding proteins are recognized as DSBs. In this r...

  13. Optimum conditions for selective isolation of genes from complex genomes by transformation-associated recombination cloning

    OpenAIRE

    Leem, Sun-Hee; Noskov, Vladimir N.; Park, Jung-Eun; Kim, Seung Il; Larionov, Vladimir; Kouprina, Natalay

    2003-01-01

    Transformation-associated recombination (TAR) cloning in yeast is used to isolate a desired chromosomal region or gene from a complex genome without construction of a genomic library. The technique involves homologous recombination during yeast spheroplast transformation between genomic DNA and a TAR vector containing short 5′ and 3′ gene-specific targeting hooks. Efficient gene capture requires a high yield of transformants, and we demonstrate here that the transformant yield increases ∼10-f...

  14. UvrD Helicase Suppresses Recombination and DNA Damage-Induced Deletions†

    OpenAIRE

    Kang, Josephine; Martin J Blaser

    2006-01-01

    UvrD, a highly conserved helicase involved in mismatch repair, nucleotide excision repair (NER), and recombinational repair, plays a critical role in maintaining genomic stability and facilitating DNA lesion repair in many prokaryotic species. In this report, we focus on the UvrD homolog in Helicobacter pylori, a genetically diverse organism that lacks many known DNA repair proteins, including those involved in mismatch repair and recombinational repair, and that is noted for high levels of i...

  15. Transmission distortion affecting human noncrossover but not crossover recombination: a hidden source of meiotic drive.

    OpenAIRE

    Linda Odenthal-Hesse; Ingrid L Berg; Amelia Veselis; Jeffreys, Alec J.; May, Celia A

    2014-01-01

    Meiotic recombination ensures the correct segregation of homologous chromosomes during gamete formation and contributes to DNA diversity through both large-scale reciprocal crossovers and very localised gene conversion events, also known as noncrossovers. Considerable progress has been made in understanding factors such as PRDM9 and SNP variants that influence the initiation of recombination at human hotspots but very little is known about factors acting downstream. To address this, we simult...

  16. Processing of triplex-directed psoralen DNA interstrand crosslinks by recombination mechanisms

    OpenAIRE

    Liu, Yaobin; Nairn, Rodney S.; Vasquez, Karen M.

    2008-01-01

    Gene targeting via homologous recombination (HR) is an important application in biotechnology and medicine. However, in mammalian cells HR is much less efficient than random integration. Triplex-forming oligonucleotides (TFOs) linked to DNA damaging agents (e.g. psoralen) can stimulate HR, providing the potential to improve gene therapy applications. To elucidate factors affecting TFO-directed psoralen interstrand crosslink (ICL)-induced recombination, we constructed a series of plasmids with...

  17. Homological stability for oriented configuration spaces

    CERN Document Server

    Palmer, Martin

    2011-01-01

    We prove homological stability for sequences of "oriented configuration spaces" as the number of points in the configuration goes to infinity. These are spaces of configurations of n points in a connected manifold M of dimension at least 2 which 'admits a boundary', with labels in a path-connected space X, and with an orientation: an ordering of the points up to even permutations. They are double covers of the corresponding unordered configuration spaces, where the points do not have this orientation. To prove our result we adapt methods from a paper of Randal-Williams, which proves homological stability in the unordered case. Interestingly the oriented configuration spaces stabilise more slowly than the unordered ones: the stability slope we obtain is one-third, compared to one-half in the unordered case (these are the best possible slopes in their respective cases). This result can also be interpreted as homological stability for unordered configuration spaces with certain twisted coefficients.

  18. Crystal structure of an archaeal actin homolog.

    Science.gov (United States)

    Roeben, Annette; Kofler, Christine; Nagy, István; Nickell, Stephan; Hartl, F Ulrich; Bracher, Andreas

    2006-04-21

    Prokaryotic homologs of the eukaryotic structural protein actin, such as MreB and ParM, have been implicated in determination of bacterial cell shape, and in the segregation of genomic and plasmid DNA. In contrast to these bacterial actin homologs, little is known about the archaeal counterparts. As a first step, we expressed a predicted actin homolog of the thermophilic archaeon Thermoplasma acidophilum, Ta0583, and determined its crystal structure at 2.1A resolution. Ta0583 is expressed as a soluble protein in T.acidophilum and is an active ATPase at physiological temperature. In vitro, Ta0583 forms sheets with spacings resembling the crystal lattice, indicating an inherent propensity to form filamentous structures. The fold of Ta0583 contains the core structure of actin and clearly belongs to the actin/Hsp70 superfamily of ATPases. Ta0583 is approximately equidistant from actin and MreB on the structural level, and combines features from both eubacterial actin homologs, MreB and ParM. The structure of Ta0583 co-crystallized with ADP indicates that the nucleotide binds at the interface between the subdomains of Ta0583 in a manner similar to that of actin. However, the conformation of the nucleotide observed in complex with Ta0583 clearly differs from that in complex with actin, but closely resembles the conformation of ParM-bound nucleotide. On the basis of sequence and structural homology, we suggest that Ta0583 derives from a ParM-like actin homolog that was once encoded by a plasmid and was transferred into a common ancestor of Thermoplasma and Ferroplasma. Intriguingly, both genera are characterized by the lack of a cell wall, and therefore Ta0583 could have a function in cellular organization.

  19. Flare build-up study: Homologous flares group - Interim report

    Science.gov (United States)

    Woodgate, B. E.

    1982-01-01

    When homologous flares are broadly defined as having footpoint structures in common, it is found that a majority of flares fall into homologous sets. Filament eruptions and mass ejection in members of an homologous flare set show that maintainance of the magnetic structure is not a necessary condition for homology.

  20. Homological and homotopical Dehn functions are different

    CERN Document Server

    Abrams, Aaron; Dani, Pallavi; Young, Robert

    2012-01-01

    The homological and homotopical Dehn functions are different ways of measuring the difficulty of filling a closed curve inside a group or a space. The homological Dehn function measures fillings of cycles by chains, while the homotopical Dehn function measures fillings of curves by disks. Since the two definitions involve different sorts of boundaries and fillings, there is no a priori relationship between the two functions, but prior to this work there were no known examples of finitely-presented groups for which the two functions differ. This paper gives the first such examples, constructed by amalgamating a free-by-cyclic group with several Bestvina-Brady groups.

  1. Sheaves on Graphs and Their Homological Invariants

    OpenAIRE

    Friedman, Joel

    2011-01-01

    We introduce a notion of a sheaf of vector spaces on a graph, and develop the foundations of homology theories for such sheaves. One sheaf invariant, its "maximum excess," has a number of remarkable properties. It has a simple definition, with no reference to homology theory, that resembles graph expansion. Yet it is a "limit" of Betti numbers, and hence has a short/long exact sequence theory and resembles the $L^2$ Betti numbers of Atiyah. Also, the maximum excess is defined via a supermodul...

  2. New mesogenic homologous series of -methylcinnamates

    Indian Academy of Sciences (India)

    R A Vora; A K Prajapati

    2001-04-01

    Compounds of a new smectogenic homologous series of -methylcinnamates were prepared by condensing different 4--alkoxybenzoyl chloride with methoxyethyl trans-4-hydroxy- -methylcinnamate. In this series, the first six members are non-mesogenic. -Heptyloxy derivative exhibits monotropic smectic A phase whereas rest of the members exhibit enantiotropic smectic A mesophase. The compounds are characterized by combination of elemental analysis and spectroscopic techniques. Enthalpies of few homologues are measured by DSC techniques. Fluorescent properties are also observed. The thermal stabilities of the present series are compared with those of other structurally related mesogenic homologous series.

  3. Relative Derived Equivalences and Relative Homological Dimensions

    Institute of Scientific and Technical Information of China (English)

    Sheng Yong PAN

    2016-01-01

    Let A be a small abelian category. For a closed subbifunctor F of Ext1A (−,−), Buan has generalized the construction of Verdier’s quotient category to get a relative derived category, where he localized with respect to F-acyclic complexes. In this paper, the homological properties of relative derived categories are discussed, and the relation with derived categories is given. For Artin algebras, using relative derived categories, we give a relative version on derived equivalences induced by F-tilting complexes. We discuss the relationships between relative homological dimensions and relative derived equivalences.

  4. Sutured Floer homology distinguishes between Seifert surfaces

    CERN Document Server

    Altman, Irida

    2010-01-01

    In this note we exhibit the first example of a knot in the three-sphere with a pair of minimal genus Seifert surfaces that can be distinguished using the sutured Floer homology of their complementary manifolds together with the Spin^c-grading. This answers a question of Juh\\'asz. More precisely, we show that the Euler characteristic of the sutured Floer homology of the complementary manifolds distinguishes between the two surfaces, and we exhibit an infinite family of knots with pairs of Seifert surfaces that can be distinguished in such a way.

  5. Expression of Recombinant Antibodies

    OpenAIRE

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transg...

  6. Dpb11/TopBP1 contributes to genomicstability via homologous recombinationand checkpoint signaling

    DEFF Research Database (Denmark)

    Germann, Susanne Manuela

    Homologous recombination (HR) is essential for maintaining genome integrity and is a major pathway for repairing (DSBs). DPB11 is an essential gene conserved from yeast to human (TopBP1), which is involved in initiation of DNA replication and DNA checkpoint signaling. We found that Dpb11 forms foci...... signaling. Importantly, Dpb11 foci are independent of checkpoint kinases Mec1 and Tel1, as well as Rad9, further strengthening the upstream position of Dpb11 in the DNA damage checkpoint response. Moreover, dpb11-PF has a defect in S-phase checkpoint function, albeit to a lesser extent than dpb11-1. Altered...... rates of heteroallelic and direct repeat recombination implicate a role for Dpb11 in homologous recombination. Physical monitoring of mating-type switching as a model for DSB repair revealed that the repair kinetics of dpb11-PF are delayed. Finally, we found Dpb11 in budding yeast as well as TopBP1...

  7. The Contribution of Genetic Recombination to CRISPR Array Evolution.

    Science.gov (United States)

    Kupczok, Anne; Landan, Giddy; Dagan, Tal

    2015-06-16

    CRISPR (clustered regularly interspaced short palindromic repeats) is a microbial immune system against foreign DNA. Recognition sequences (spacers) encoded within the CRISPR array mediate the immune reaction in a sequence-specific manner. The known mechanisms for the evolution of CRISPR arrays include spacer acquisition from foreign DNA elements at the time of invasion and array erosion through spacer deletion. Here, we consider the contribution of genetic recombination between homologous CRISPR arrays to the evolution of spacer repertoire. Acquisition of spacers from exogenic arrays via recombination may confer the recipient with immunity against unencountered antagonists. For this purpose, we develop a novel method for the detection of recombination in CRISPR arrays by modeling the spacer order in arrays from multiple strains from the same species. Because the evolutionary signal of spacer recombination may be similar to that of pervasive spacer deletions or independent spacer acquisition, our method entails a robustness analysis of the recombination inference by a statistical comparison to resampled and perturbed data sets. We analyze CRISPR data sets from four bacterial species: two Gammaproteobacteria species harboring CRISPR type I and two Streptococcus species harboring CRISPR type II loci. We find that CRISPR array evolution in Escherichia coli and Streptococcus agalactiae can be explained solely by vertical inheritance and differential spacer deletion. In Pseudomonas aeruginosa, we find an excess of single spacers potentially incorporated into the CRISPR locus during independent acquisition events. In Streptococcus thermophilus, evidence for spacer acquisition by recombination is present in 5 out of 70 strains. Genetic recombination has been proposed to accelerate adaptation by combining beneficial mutations that arose in independent lineages. However, for most species under study, we find that CRISPR evolution is shaped mainly by spacer acquisition and

  8. Betti numbers and stability for configuration spaces via factorization homology

    OpenAIRE

    Knudsen, Ben

    2014-01-01

    Using factorization homology, we realize the rational homology of the unordered configuration spaces of an arbitrary manifold $M$, possibly with boundary, as the homology of a Lie algebra constructed from the compactly supported cohomology of $M$. By locating the homology of each configuration space within the Chevalley-Eilenberg complex of this Lie algebra, we extend theorems of B\\"{o}digheimer-Cohen-Taylor and F\\'{e}lix-Thomas and give a new, combinatorial proof of the homological stability...

  9. Einstein Metrics on Rational Homology Spheres

    OpenAIRE

    Boyer, Charles P.; Galicki, Krzysztof

    2003-01-01

    We prove the existence of Sasakian-Einstein metrics on infinitely many rational homology spheres in all odd dimensions greater than 3. In dimension 5 we obain somewhat sharper results. There are examples where the number of effective parameters in the Einstein metric grows exponentially with dimension.

  10. Homological Perturbation Theory and Mirror Symmetry

    Institute of Scientific and Technical Information of China (English)

    Jian ZHOU

    2003-01-01

    We explain how deformation theories of geometric objects such as complex structures,Poisson structures and holomorphic bundle structures lead to differential Gerstenhaber or Poisson al-gebras. We use homological perturbation theory to construct A∞ algebra structures on the cohomology,and their canonically defined deformations. Such constructions are used to formulate a version of A∞algebraic mirror symmetry.

  11. Gorenstein Homological Dimensions and Change of Rings

    Institute of Scientific and Technical Information of China (English)

    Xiaoyan YANG

    2012-01-01

    In this paper,we shall be concerned with what happens of Gorenstein homological dimensions when certain modifications are made to a ring. The five structural operations addressed later are the formation of excellent extensions,localizations,Morita equivalences,polynomial extensions and power series extensions.

  12. Homological stability for unordered configuration spaces

    CERN Document Server

    Randal-Williams, Oscar

    2011-01-01

    This paper consists of two related parts. In the first part we give a self-contained proof of homological stability for the spaces C_n(M;X) of configurations of n unordered points in a connected open manifold M with labels in a path-connected space X, with the best possible integral stability range of 2* \\leq n. Along the way we give a new proof of the high connectivity of the complex of injective words. If the manifold has dimension at least three, we show that in rational homology the stability range may be improved to * \\leq n. In the second part we study to what extent the homology of the spaces C_n(M) can be considered stable when M is a closed manifold. In this case there are no stabilisation maps, but one may still ask if the dimensions of the homology groups over some field stabilise with n. We prove that this is true when M is odd-dimensional, or when the field is F_2 or Q. It is known to be false in the remaining cases.

  13. Homological stability for configuration spaces of manifolds

    CERN Document Server

    Church, Thomas

    2011-01-01

    Let C_n(M) be the configuration space of n distinct ordered points in M. We prove that if M is any connected orientable manifold (closed or open), the homology groups H_i(C_n(M); Q) are representation stable in the sense of [Church-Farb]. Applying this to the trivial representation, we obtain as a corollary that the unordered configuration space B_n(M) satisfies classical homological stability: for each i, H_i(B_n(M); Q) is isomorphic to H_i(B_{n+1}(M); Q) for n > i. This improves on results of McDuff, Segal, and others for open manifolds. Applied to closed manifolds, this provides natural examples where rational homological stability holds even though integral homological stability fails. To prove the main theorem, we introduce the notion of monotonicity for a sequence of S_n--representations, which is of independent interest. Sequences that are both monotone and uniformly representation stable form an abelian category. Monotonicity provides a new mechanism for proving representation stability using spectral...

  14. Planar open books and Floer homology

    OpenAIRE

    Ozsvath, Peter; Stipsicz, Andras I.; Szabo, Zoltan

    2005-01-01

    Giroux has described a correspondence between open book decompositions on a 3--manifold and contact structures. In this paper we use Heegaard Floer homology to give restrictions on contact structures which correspond to open book decompositions with planar pages, generalizing a recent result of Etnyre.

  15. Khovanov-Rozansky homology and Directed Cycles

    OpenAIRE

    Wu, Hao

    2015-01-01

    We determine the cycle packing number of a directed graph using elementary projective algebraic geometry. Our idea is rooted in the Khovanov-Rozansky theory. In fact, using the Khovanov-Rozansky homology of a graph, we also obtain algebraic methods of detecting directed and undirected cycles containing a particular vertex or edge.

  16. Homological aperiodic tilings of 3-dimensional geometries

    CERN Document Server

    Nowak, Piotr W

    2012-01-01

    We construct the first aperiodic tiles for two amenable 3-dimensional Lie groups: Sol and the Heisenberg group. Our construction relies on the use of higher-dimensional uniformly finite homology. In particular, we settle completely the existence of aperiodic tiles for all of the non-compact geometries of 3-manifolds appearing in the geometrization conjecture.

  17. Chromosome sites play dual roles to establish homologous synapsisduring meiosis in C. elegans

    Energy Technology Data Exchange (ETDEWEB)

    MacQueen, Amy J.; Phillips, Carolyn M.; Bhalla, Needhi; Weiser,Pinky; Villeneuve, Anne M.; Dernburg, Abby F.

    2005-06-05

    required for accurate segregation of homologous chromosomesduring meiosisin C. elegans. We find that these sites play two distinctroles that contribute to proper segregation. Chromosomes lacking PCsusually fail to synapse and also lack a synapsis-independentstabilization activity. The presence of a PC on justone copy of achromosome pair promotes synapsis but does not supportsynapsis-independent pairing stabilization, indicating that thesefunctions are separable. Once initiated, synapsis is highly processive,even between non homologous chromosomes of disparate lengths, elucidatinghow translocations suppress meiotic recombination in C. elegans. Thesefindings suggest a multistep pathway for chromosome synapsis in which PCsimpart selectivity and efficiency through a kinetic proofreadingmechanism. We speculate that concentration of these activities at oneregion per chromosome may have co-evolved with the loss of a pointcentromere to safeguard karyotype stability.

  18. The role of RecQ helicases in non-homologous end-joining

    DEFF Research Database (Denmark)

    Keijzers, Guido; Maynard, Scott; Shamanna, Raghavendra A;

    2014-01-01

    Abstract DNA double-strand breaks are highly toxic DNA lesions that cause genomic instability, if not efficiently repaired. RecQ helicases are a family of highly conserved proteins that maintain genomic stability through their important roles in several DNA repair pathways, including DNA double......-strand break repair. Double-strand breaks can be repaired by homologous recombination (HR) using sister chromatids as templates to facilitate precise DNA repair, or by an HR-independent mechanism known as non-homologous end-joining (NHEJ) (error-prone). NHEJ is a non-templated DNA repair process, in which DNA...... termini are directly ligated. Canonical NHEJ requires DNA-PKcs and Ku70/80, while alternative NHEJ pathways are DNA-PKcs and Ku70/80 independent. This review discusses the role of RecQ helicases in NHEJ, alternative (or back-up) NHEJ (B-NHEJ) and microhomology-mediated end-joining (MMEJ) in V...

  19. Making ends meet: repairing breaks in bacterial DNA by non-homologous end-joining.

    Directory of Open Access Journals (Sweden)

    Richard Bowater

    2006-02-01

    Full Text Available DNA double-strand breaks (DSBs are one of the most dangerous forms of DNA lesion that can result in genomic instability and cell death. Therefore cells have developed elaborate DSB-repair pathways to maintain the integrity of genomic DNA. There are two major pathways for the repair of DSBs in eukaryotes: homologous recombination and non-homologous end-joining (NHEJ. Until very recently, the NHEJ pathway had been thought to be restricted to the eukarya. However, an evolutionarily related NHEJ apparatus has now been identified and characterized in the prokarya. Here we review the recent discoveries concerning bacterial NHEJ and discuss the possible origins of this repair system. We also examine the insights gained from the recent cellular and biochemical studies of this DSB-repair process and discuss the possible cellular roles of an NHEJ pathway in the life-cycle of prokaryotes and phages.

  20. Parametric representation of centrifugal pump homologous curves

    Energy Technology Data Exchange (ETDEWEB)

    Veloso, Marcelo A.; Mattos, Joao R.L. de, E-mail: velosom@cdtn.br, E-mail: jrmattos@cdtn.br [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil)

    2015-07-01

    Essential for any mathematical model designed to simulate flow transient events caused by pump operations is the pump performance data. The performance of a centrifugal pump is characterized by four basic quantities: the rotational speed, the volumetric flow rate, the dynamic head, and the hydraulic torque. The curves showing the relationships between these four variables are called the pump characteristic curves. The characteristic curves are empirically developed by the pump manufacturer and uniquely describe head and torque as functions of volumetric flow rate and rotation speed. Because of comprising a large amount of points, this configuration is not suitable for computational purposes. However, it can be converted to a simpler form by the development of the homologous curves, in which dynamic head and hydraulic torque ratios are expressed as functions of volumetric flow and rotation speed ratios. The numerical use of the complete set of homologous curves requires specification of sixteen partial curves, being eight for the dynamic head and eight for the hydraulic torque. As a consequence, the handling of homologous curves is still somewhat complicated. In solving flow transient problems that require the pump characteristic data for all the operation zones, the parametric form appears as the simplest way to deal with the homologous curves. In this approach, the complete characteristics of a pump can be described by only two closed curves, one for the dynamic head and other for the hydraulic torque, both in function of a single angular coordinate defined adequately in terms of the quotient between volumetric flow ratio and rotation speed ratio. The usefulness and advantages of this alternative method are demonstrated through a practical example in which the homologous curves for a pump of the type used in the main coolant loops of a pressurized water reactor (PWR) are transformed to the parametric form. (author)

  1. Pro-recombination role of Srs2 protein requires SUMO (small ubiquitin-like modifier) but is independent of PCNA (proliferating cell nuclear antigen) interaction

    DEFF Research Database (Denmark)

    Kolesar, Peter; Altmannova, Veronika; Pinela da Silva, Sonia Cristina;

    2016-01-01

    -interacting motif (SIM) of Srs2 is important for the interaction with several recombination factors. Lack of SIM, but not proliferating cell nuclear antigen (PCNA)-interacting motif (PIM), leads to increased cell death under circumstances requiring homologous recombination for DNA repair. Simultaneous mutation...

  2. Recombination induced by triple-helix-targeted DNA damage in mammalian cells.

    OpenAIRE

    Faruqi, A F; Seidman, M M; Segal, D J; Carroll, D; Glazer, P M

    1996-01-01

    Gene therapy has been hindered by the low frequency of homologous recombination in mammalian cells. To stimulate recombination, we investigated the use of triple-helix-forming oligonucleotides (TFOs) to target DNA damage to a selected site within cells. By treating cells with TFOs linked to psoralen, recombination was induced within a simian virus 40 vector carrying two mutant copies of the supF tRNA reporter gene. Gene conversion events, as well as mutations at the target site, were also obs...

  3. Analysis of the role of the gene bipA, encoding the major endoplasmic reticulum chaperone protein in the secretion of homologous and heterologous proteins in black Aspergilli

    NARCIS (Netherlands)

    Punt, P.J.; Gemeren, I.A. van; Drint-Kuijvenhoven, J.; Hessing, J.G.M.; Muijlwijk van - Harteveld, G.M.; Beijersbergen, A.; Verrips, C.T.; Hondel, C.A.M.J.J. van den

    1998-01-01

    The function of the endoplasmic-reticulum-localized chaperone binding protein (BiP) in relation to protein secretion in filamentous fungi was studied. It was shown that the overproduction of several homologous and heterologous recombinant proteins by Aspergillus strains induces the expression of bip

  4. Molecular cloning, DNA sequence analysis, and characterization of the Corynebacterium diphtheriae dtxR homolog from Brevibacterium lactofermentum.

    OpenAIRE

    Oguiza, J A; Tao, X; Marcos, A T; Martín, J F; Murphy, J R

    1995-01-01

    A homolog of the Corynebacterium diphtheriae dtxR gene was isolated from Brevibacterium lactofermentum. The product of the B. lactofermentum dtxR gene was immunoreactive with polyclonal anti-DtxR antibodies and functioned as an iron-activated repressor capable of regulating the expression of beta-galactosidase from a diphtheria tox promoter/operator transcriptional fusion in recombinant Escherichia coli. The extents of induction by increasing concentrations of the chelator 2,2'-dipyridyl were...

  5. The spatial organization of non-homologous end joining: From bridging to end joining

    OpenAIRE

    Ochi, Takashi; Wu, Qian; Blundell, Tom L

    2014-01-01

    Non-homologous end joining (NHEJ) repairs DNA double-strand breaks generated by DNA damage and also those occurring in V(D)J recombination in immunoglobulin and T cell receptor production in the immune system. In NHEJ DNA-PKcs assembles with Ku heterodimer on the DNA ends at double-strand breaks, in order to bring the broken ends together and to assemble other proteins, including DNA ligase IV (LigIV), required for DNA repair. Here we focus on structural aspects of the interactions of LigIV w...

  6. Failure of homologous synapsis and sex-specific reproduction problems

    Directory of Open Access Journals (Sweden)

    Hiroki eKurahashi

    2012-06-01

    Full Text Available The prophase of meiosis I ensures the correct segregation of chromosomes to each daughter cell. This includes the pairing, synapsis and recombination of homologous chromosomes. A subset of chromosomal abnormalities, including translocation and inversion, disturbs these processes, resulting in the failure to complete synapsis. This activates the meiotic pachytene checkpoint, and the gametes are fated to undergo cell cycle arrest and subsequent apoptosis. Spermatogenic cells appear to be more vulnerable to the pachytene checkpoint, and male carriers of chromosomal abnormalities are more susceptible to infertility. In contrast, oocytes tend to bypass the checkpoint and instead generate other problems, such as chromosome imbalance that often leads to recurrent pregnancy loss in female carriers. Recent advances in genetic manipulation technologies have increased our knowledge about the pachytene checkpoint and surveillance systems that detect chromosomal synapsis. This review focuses on the consequences of synapsis failure in humans and provides an overview of the mechanisms involved. We also discuss the sexual dimorphism of the involved pathways that leads to the differences in reproductive outcomes between males and females.

  7. Problem-Solving Test: Conditional Gene Targeting Using the Cre/loxP Recombination System

    Science.gov (United States)

    Szeberényi, József

    2013-01-01

    Terms to be familiar with before you start to solve the test: gene targeting, knock-out mutation, bacteriophage, complementary base-pairing, homologous recombination, deletion, transgenic organisms, promoter, polyadenylation element, transgene, DNA replication, RNA polymerase, Shine-Dalgarno sequence, restriction endonuclease, polymerase chain…

  8. Brh2-Dss1 interplay enables properly controlled recombination in Ustilago maydis

    DEFF Research Database (Denmark)

    Kojic, Milorad; Zhou, Qingwen; Lisby, Michael;

    2005-01-01

    Brh2, the BRCA2 homolog in Ustilago maydis, functions in recombinational repair of DNA damage by regulating Rad51 and is, in turn, regulated by Dss1. Dss1 is not required for Brh2 stability in vivo, nor for Brh2 to associate with Rad51, but is required for formation of green fluorescent protein...

  9. Differential contributions of mammalian Rad54 paralogs to recombination, DNA damage repair, and meiosis.

    NARCIS (Netherlands)

    J. Wesoly (Joanna); S. Agarwal (Sheba); S. Sigurdsson (Stefan); W. Bussen (Wendy); S. Komen (Stephen); J. Qin (Jian); H. van Steeg (Harry); J. van Benthem (Jan); E. Wassenaar (Evelyne); W.M. Baarends (Willy); M. Ghazvini (Mehrnaz); A. Tafel (Agnieszka); H. Heath (Helen); N.J. Galjart (Niels); J. Essers (Jeroen); J.A. Grootegoed (Anton); N. Arnheim (Norman); O.Y. Bezzubova (Olga); J-M. Buerstedde; P. Sung (Patrick); R. Kanaar (Roland)

    2006-01-01

    textabstractHomologous recombination is a versatile DNA damage repair pathway requiring Rad51 and Rad54. Here we show that a mammalian Rad54 paralog, Rad54B, displays physical and functional interactions with Rad51 and DNA that are similar to those of Rad54. While ablation of Rad54 in mouse embryoni

  10. Chromosomal transformation in Bacillus subtilis is a non-polar recombination reaction

    NARCIS (Netherlands)

    B. Carrasco (Begoña); E. Serrano (Ester); H. Sanchez (Humberto); C. Wyman (Claire); J.C. Alonso (Juan)

    2016-01-01

    textabstractNatural chromosomal transformation is one of the primary driving forces of bacterial evolution. This reaction involves the recombination of the internalized linear single-stranded (ss) DNA with the homologous resident duplex via RecA-mediated integration in concert with SsbA and DprA or

  11. The role of homologous recombination in mitotic and meiotic double-strand break repair

    NARCIS (Netherlands)

    Vries, Femke Adriana Theodora de

    2007-01-01

    All organisms are composed of cells and the cell’s nucleus contains DNA. The induction of DNA damage is a threat to organisms. Signalling of DNA damage and subsequent repair is of substantial importance. Double-strand breaks (DSBs) in DNA can be induced by ionising radiation and DNA damaging agents

  12. The role of homologous recombination in mitotic and meiotic double-strand break repair

    OpenAIRE

    Vries, Femke Adriana Theodora de

    2007-01-01

    All organisms are composed of cells and the cell’s nucleus contains DNA. The induction of DNA damage is a threat to organisms. Signalling of DNA damage and subsequent repair is of substantial importance. Double-strand breaks (DSBs) in DNA can be induced by ionising radiation and DNA damaging agents but also arise as intermediates in several cellular processes (e.g. meiosis). DSBs are among the most genotoxic DNA lesions and their accurate repair is crucial. Genetic instability resulting from ...

  13. Homologous recombination control by the anti-apoptotic onco-protein Bcl-2

    International Nuclear Information System (INIS)

    This research thesis deals with the different biological mechanisms, notably the repair and apoptosis mechanisms induced by irradiation in cells. After a presentation of the genotoxic stress and DNA repair mechanisms, the author discusses the cellular response to a DNA double-strand break, and the regulation of these response mechanisms (how a cellular response emerges: life or death). The next part deals with the apoptosis (cell death by necrosis or apoptosis), and presents the BCL-2 protein family. Results are then reported on laboratory studies of the effect of this protein family

  14. BLM has early and late functions in homologous recombination repair in mouse embryonic stem cells

    DEFF Research Database (Denmark)

    Chu, W K; Hanada, K; Kanaar, R;

    2010-01-01

    ' roles, such as dissolution of double Holliday junctions. However, most of the evidence for these putative roles comes from in vitro biochemical data. In this study, we report the characterization of mouse embryonic stem cells with disruption of Blm and/or Rad54 genes. We show that Blm has roles both...... in Rad54(-/-) cells rescued their mitomycin C (MMC) sensitivity, and decreased both the level of DNA damage and cell cycle perturbation induced by MMC, suggesting an early role for Blm. Our data are consistent with Blm having at least two roles in HR repair in mammalian cells....

  15. A murine ESC-like state facilitates transgenesis and homologous recombination in human pluripotent stem cells

    NARCIS (Netherlands)

    C. Buecker (Christa); H.H. Chen; J.M. Polo; L. Daheron (Laurence); L. Bu (Lei); T.S. Barakat (Tahsin Stefan); P. Okwieka (Patricia); A. Porter (Andrew); J.H. Gribnau (Joost); K. Hochedlinger (Konrad); N. Geijsen (Niels)

    2010-01-01

    textabstractMurine pluripotent stem cells can exist in two functionally distinct states, LIF-dependent embryonic stem cells (ESCs) and bFGF-dependent epiblast stem cells (EpiSCs). However, human pluripotent cells so far seemed to assume only an epiblast-like state. Here we demonstrate that human iPS

  16. A Murine ESC-like State Facilitates Transgenesis and Homologous Recombination in Human Pluripotent Stem Cells

    NARCIS (Netherlands)

    Buecker, Christa; Chen, Hsu-Hsin; Polo, Jose Maria; Daheron, Laurence; Bu, Lei; Barakat, Tahsin Stefan; Okwieka, Patricia; Porter, Andrew; Gribnau, Joost; Hochedlinger, Konrad; Geijsen, Niels

    2010-01-01

    Murine pluripotent stem cells can exist in two functionally distinct states, LIF-dependent embryonic stem cells (ESCs) and bFGF-dependent epiblast stem cells (EpiSCs). However, human pluripotent cells so far seemed to assume only an epiblast-like state. Here we demonstrate that human iPSC reprogramm

  17. Analysis of the hormone-binding domain of steroid receptors using chimeras generated by homologous recombination

    International Nuclear Information System (INIS)

    The glucocorticoid receptor and the mineralocorticoid receptor are members of the steroid receptor family that exhibit ligand cross-reactivity. Specificity of steroid receptor action is investigated in the present work by the construction and characterization of chimeras between the glucocorticoid receptor and the mineralocorticoid receptor. We used an innovative approach to make novel steroid receptor proteins in vivo that in general, contrary to our expectations, show increased ligand specificity compared to the parental receptors. We describe a receptor that is specific for the potent synthetic glucocorticoid triamcinolone acetonide and does not bind aldosterone. A further set of chimeras has an increased ability to discriminate between ligands, responding potently to mineralocorticoids and only very weakly to synthetic glucocorticoids. A chimera with the fusion site in the hinge highlights the importance of the region between the DNA-binding and the hormone-binding domains since, unlike both the glucocorticoid and mineralocorticoid receptors, it only responds to mineralocorticoids. One chimera has reduced specificity in that it acts as a general corticoid receptor, responding to glucocorticoids and mineralocorticoids with similar potency and efficacy. Our data suggest that regions of the glucocorticoid and mineralocorticoid receptor hormone-binding domains are functionally non-reciprocal. We present transcriptional, hormone-binding, and structure-modeling evidence that suggests that receptor-specific interactions within and across domains mediate aspects of specificity in transcriptional responses to steroids

  18. Regulated expression of genes inserted at the human chromosomal β-globin locus by homologous recombination

    International Nuclear Information System (INIS)

    The authors have examined the effect of the site of integration on the expression of cloned genes introduced into cultured erythroid cells. Smithies et al. reported the targeted integration of DNA into the human β-globin locus on chromosome 11 in a mouse erythroleukemia-human cell hybrid. These hybrid cells can undergo erythroid differentiation leading to greatly increased mouse and human β-globin synthesis. By transfection of these hybrid cells with a plasmid carrying a modified human β-globin gene and a foreign gene composed of the coding sequence of the bacterial neomycin-resistance gene linked to simian virus 40 transcription signals (SVneo), cells were obtained in which the two genes are integrated at the β-globin locus on human chromosome 11 or at random sites. When they examined the response of the integrated genes to cell differentation, they found that the genes inserted at the β-globin locus were induced during differentiation, whereas randomly positioned copies were not induced. Even the foreign SVneo gene was inducible when it had been integrated at the β-globin locus. The results show that genes introduced at the β-globin locus acquire some of the regulatory properties of globin genes during erythroid differentiation

  19. The role of homologous recombination in radiation-induced double-strand break repair

    International Nuclear Information System (INIS)

    DNA double-strand breaks (DSBs) represent the most biologically significant lesions induced by ionizing radiation (IR). HR is the predominant pathway for repairing one-ended DSBs arising in S-phase when the replication fork encounters single-stranded breaks or base damages. Here, we discuss recent findings that two-ended DSBs directly induced by X- or γ-rays in late S- or G2-phase are repaired predominantly by NHEJ, with HR only repairing a sub-fraction of such DSBs. This sub-fraction represents DSBs which localize to heterochromatic DNA regions and, which in control cells, are repaired with slow kinetics over many hours post irradiation. The observation that defined DSB populations are repaired by either NHEJ or HR suggests an assignment of specific tasks for each of the two processes. Furthermore, heavy ion induced complex DSBs, which are in general more slowly repaired than X- or γ-ray induced breaks, are nearly always repaired by HR independent of chromatin localization suggesting that the speed of repair is an important factor determining the DSB repair pathway usage. Finally, NHEJ and HR can, under certain conditions, also compensate for each other such that DSBs normally repaired by one pathway can undergo repair by the other if genetic failures necessitate the pathway switch.

  20. The role of DNA double-strand breaks in spontaneous homologous recombination in S. cerevisiae

    DEFF Research Database (Denmark)

    Lettier, Gaëlle; Feng, Q.; Mayolo, A.A. de;

    2006-01-01

    of meiosis and result from the induction of a large number of DNA double-strand breaks (DSBs). By analogy, it is generally believed that the rare spontaneous mitotic HR events are due to repair of DNA DSBs that accidentally occur during mitotic growth. Here we provide the first direct evidence that most...

  1. DNA synthesis on discontinuous templates by human DNA polymerases: implications for non-homologous DNA recombination.

    OpenAIRE

    Islas, L; Fairley, C F; Morgan, W. F.

    1998-01-01

    DNA polymerases catalyze the synthesis of DNA using a continuous uninterrupted template strand. However, it has been shown that a 3'-->5' exonuclease-deficient form of the Klenow fragment of Escherichia coli DNA polymerase I as well as DNA polymerase of Thermus aquaticus can synthesize DNA across two unlinked DNA templates. In this study, we used an oligonucleotide-based assay to show that discontinuous DNA synthesis was present in HeLa cell extracts. DNA synthesis inhibitor studies as well a...

  2. Forced activation of Cdk1 via wee1 inhibition impairs homologous recombination

    NARCIS (Netherlands)

    Krajewska, M.; Heijink, A. M.; Bisselink, Y. J. W. M.; Seinstra, R. I.; Sillje, H. H. W.; de Vries, E. G. E.; van Vugt, M. A. T. M.

    2013-01-01

    In response to DNA breaks, the 'DNA damage response' provokes a cell cycle arrest to facilitate DNA repair. Recent findings have indicated that cells can respond to DNA damage throughout the cell cycle, except during mitosis. Specifically, various mitotic kinases, including Cdk1, Aurora A and Plk1,

  3. Homologous recombination, sister chromatid cohesion, and chromosome condensation in mammalian meiosis

    NARCIS (Netherlands)

    Eijpe, M.

    2002-01-01

    In the life cycle of sexually reproducing eukaryotes, haploid and diploid generations of cells alternate. Two types of cell division occur in such a life cycle: mitosis and meiosis. They are compared in chapter 1 . Haploid and diploid cells can multiply by mitoses.

  4. Efficient genome editing in Caenorhabditis elegans by CRISPR-targeted homologous recombination

    OpenAIRE

    Chen, C.; Fenk, L. A.; Bono, M.

    2013-01-01

    Cas9 is an RNA-guided double-stranded DNA nuclease that participates in clustered regularly interspaced short palindromic repeats (CRISPR)-mediated adaptive immunity in prokaryotes. CRISPR–Cas9 has recently been used to generate insertion and deletion mutations in Caenorhabditis elegans, but not to create tailored changes (knock-ins). We show that the CRISPR–CRISPR-associated (Cas) system can be adapted for efficient and precise editing of the C. elegans genome. The targeted double-strand bre...

  5. Initiation of Meiotic Recombination in Mammals

    Directory of Open Access Journals (Sweden)

    Rajeev Kumar

    2010-12-01

    Full Text Available Meiotic recombination is initiated by the induction of programmed DNA double strand breaks (DSBs. DSB repair promotes homologous interactions and pairing and leads to the formation of crossovers (COs, which are required for the proper reductional segregation at the first meiotic division. In mammals, several hundred DSBs are generated at the beginning of meiotic prophase by the catalytic activity of SPO11. Currently it is not well understood how the frequency and timing of DSB formation and their localization are regulated. Several approaches in humans and mice have provided an extensive description of the localization of initiation events based on CO mapping, leading to the identification and characterization of preferred sites (hotspots of initiation. This review presents the current knowledge about the proteins known to be involved in this process, the sites where initiation takes place, and the factors that control hotspot localization.

  6. Recombinant Helicobacter pylori catalase

    Institute of Scientific and Technical Information of China (English)

    Yang Bai; Ya-Li Zhang; Jian-Feng Jin; Ji-De Wang; Zhao-Shan Zhang

    2003-01-01

    AIM: To construct a recombinant strain which highly expresses catalase of Helicobacter pylori(H.pylori) and assay the activity of H. pylori catalase.METHODS: The catalase DNA was amplified from H. pylori chromosomal DNA with PCR techniques and inserted into the prokaryotie expression vector pET-22b (+), and then was transformed into the BL21 (DE3) E. coli strain which expressed catalase recombinant protein. The activity of H.pylori catalase was assayed by the Beers & Sizers.RESULTS: DNA sequence analysis showed that the sequence of catalase DNA was the same as GenBank's research. The catalase recombinant protein amounted to 24.4 % of the total bacterial protein after induced with IPTG for 3 hours at 37 ℃ and the activity of H. pylori catalase was high in the BL21 (DE3) E. coli strain.CONCLUSION: A clone expressing high activity H. pylori catalase is obtained, laying a good foundation for further studies.

  7. Exploitation of sulfonylurea resistance marker and non-homologous end joining mutants for functional analysis in Zymoseptoria tritici.

    Science.gov (United States)

    Sidhu, Y S; Cairns, T C; Chaudhari, Y K; Usher, J; Talbot, N J; Studholme, D J; Csukai, M; Haynes, K

    2015-06-01

    The lack of techniques for rapid assembly of gene deletion vectors, paucity of selectable marker genes available for genetic manipulation and low frequency of homologous recombination are major constraints in construction of gene deletion mutants in Zymoseptoria tritici. To address these issues, we have constructed ternary vectors for Agrobacterium tumefaciens mediated transformation of Z. tritici, which enable the single step assembly of multiple fragments via yeast recombinational cloning. The sulfonylurea resistance gene, which is a mutated allele of the Magnaporthe oryzae ILV2 gene, was established as a new dominant selectable marker for Z. tritici. To increase the frequency of homologous recombination, we have constructed Z. tritici strains deficient in the non-homologous end joining pathway of DNA double stranded break repair by inactivating the KU70 and KU80 genes. Targeted gene deletion frequency increased to more than 85% in both Z. tritici ku70 and ku80 null strains, compared to ⩽10% seen in the wild type parental strain IPO323. The in vitro growth and in planta pathogenicity of the Z. tritici ku70 and ku80 null strains were comparable to strain IPO323. Together these molecular tools add significantly to the platform available for genomic analysis through targeted gene deletion or promoter replacements and will facilitate large-scale functional characterization projects in Z. tritici. PMID:26092796

  8. Recombination in ionized gases

    International Nuclear Information System (INIS)

    In this paper it is shown how capture-stabilized methodology (both macroscopic and microscopic) can provide a generic basis for a unified treatment of all of the above recombination mechanisms. A new semiclassical theory of dissociative recombination is also presented in an effort to gain further insight into the physics not included in the first-order treatment and difficult to extract from numerical quantal treatments based on configuration mixing and on multichannel quantum defect theory. A simple analytical expression more accurate than the standard first-order result is obtained for the cross section σ and rate coefficient α. (author)

  9. Homological stability for unordered configuration spaces

    DEFF Research Database (Denmark)

    Randal-Williams, Oscar

    2013-01-01

    This paper consists of two related parts. In the first part we give a self-contained proof of homological stability for the spaces C_n(M;X) of configurations of n unordered points in a connected open manifold M with labels in a path-connected space X, with the best possible integral stability range...... of the spaces C_n(M) can be considered stable when M is a closed manifold. In this case there are no stabilisation maps, but one may still ask if the dimensions of the homology groups over some field stabilise with n. We prove that this is true when M is odd-dimensional, or when the field is F_2 or Q...

  10. Homological mirror symmetry and tropical geometry

    CERN Document Server

    Catanese, Fabrizio; Kontsevich, Maxim; Pantev, Tony; Soibelman, Yan; Zharkov, Ilia

    2014-01-01

    The relationship between Tropical Geometry and Mirror Symmetry goes back to the work of Kontsevich and Y. Soibelman (2000), who applied methods of non-archimedean geometry (in particular, tropical curves) to Homological Mirror Symmetry. In combination with the subsequent work of Mikhalkin on the “tropical” approach to Gromov-Witten theory, and the work of Gross and Siebert, Tropical Geometry has now become a powerful tool. Homological Mirror Symmetry is the area of mathematics concentrated around several categorical equivalences connecting symplectic and holomorphic (or algebraic) geometry. The central ideas first appeared in the work of Maxim Kontsevich (1993). Roughly speaking, the subject can be approached in two ways: either one uses Lagrangian torus fibrations of Calabi-Yau manifolds (the so-called Strominger-Yau-Zaslow picture, further developed by Kontsevich and Soibelman) or one uses Lefschetz fibrations of symplectic manifolds (suggested by Kontsevich and further developed by Seidel). Tropical Ge...

  11. Homological Pisot Substitutions and Exact Regularity

    CERN Document Server

    Barge, Marcy; Jones, Leslie; Sadun, Lorenzo

    2010-01-01

    We consider one-dimensional substitution tiling spaces where the dilatation (stretching factor) is a degree d Pisot number, and where the first rational Cech cohomology is d-dimensional. We construct examples of such "homological Pisot" substitutions that do not have pure discrete spectra. These examples are not unimodular, and we conjecture that the coincidence rank must always divide a power of the norm of the dilatation. To support this conjecture, we show that homological Pisot substitutions exhibit an Exact Regularity Property (ERP), in which the number of occurrences of a patch for a return length is governed strictly by the length. The ERP puts strong constraints on the measure of any cylinder set in the corresponding tiling space.

  12. Hochschild homology, lax codescent, and duplicial structure

    OpenAIRE

    Garner, Richard; Lack, Stephen; Slevin, Paul

    2015-01-01

    We study the duplicial objects of Dwyer and Kan, which generalize the cyclic objects of Connes. We describe duplicial objects in terms of the decalage comonads, and we give a conceptual account of the construction of duplicial objects due to Bohm and Stefan. This is done in terms of a 2-categorical generalization of Hochschild homology. We also study duplicial structure on nerves of categories, bicategories, and monoidal categories.

  13. On the definition of homological critical value

    OpenAIRE

    Govc, Dejan

    2013-01-01

    We point out that there is a problem with the definition of homological critical value (as defined in the widely cited paper \\cite{stability} by Cohen-Steiner, Edelsbrunner and Harer). Under that definition, the critical value lemma of \\cite{stability} in fact fails. We provide several counterexamples and a definition (due to Bubenik and Scott \\cite{categorification}) we feel should be preferred and under which the critical value lemma does indeed hold. One of the counterexamples we have foun...

  14. Nash equilibria via duality and homological selection

    Indian Academy of Sciences (India)

    Arnab Basu; Samik Basu; Mahan MJ

    2014-11-01

    Given a multifunction from to the -fold symmetric product Sym$_{k}(X)$, we use the Dold–Thom theorem to establish a homological selection theorem. This is used to establish existence of Nash equilibria. Cost functions in problems concerning the existence of Nash equilibria are traditionally multilinear in the mixed strategies. The main aim of this paper is to relax the hypothesis of multilinearity. We use basic intersection theory, Poincaré duality in addition to the Dold–Thom theorem.

  15. Persistent Homology and Partial Similarity of Shapes

    OpenAIRE

    Di Fabio, Barbara; Landi, Claudia

    2011-01-01

    The ability to perform shape retrieval based not only on full similarity, but also partial similarity is a key property for any content-based search engine. We prove that persistence diagrams can reveal a partial similarity between two shapes by showing a common subset of points. This can be explained using the Mayer-Vietoris formulas that we develop for ordinary, relative and extended persistent homology. An experiment outlines the potential of persistence diagrams as shape descriptors in re...

  16. Indirect stimulation of recombination in Escherichia coli K-12: dependence on recJ, uvrA, and uvrD.

    OpenAIRE

    Schellhorn, H E; Low, K B

    1991-01-01

    Direct and indirect UV-stimulated homologous genetic recombination was investigated in Escherichia coli strains blocked in several host-encoded functions. Genetic recombination was assayed by measuring beta-galactosidase produced after recombination between two noncomplementing lacZ ochre alleles. Both types of stimulation (direct and indirect) were found to be primarily RecF pathway-mediated. In a rec+ background, both direct and indirect stimulation were found to be dependent on uvrD (codin...

  17. The fate of linear DNA in Saccharomyces cerevisiae and Candida glabrata: the role of homologous and non-homologous end joining.

    Directory of Open Access Journals (Sweden)

    Mary W Corrigan

    Full Text Available In vivo assembly of plasmids has become an increasingly used process, as high throughput studies in molecular biology seek to examine gene function. In this study, we investigated the plasmid construction technique called gap repair cloning (GRC in two closely related species of yeast - Saccharomyces cerevisiae and Candida glabrata. GRC utilizes homologous recombination (HR activity to join a linear vector and a linear piece of DNA that contains base pair homology. We demonstrate that a minimum of 20 bp of homology on each side of the linear DNA is required for GRC to occur with at least 10% efficiency. Between the two species, we determine that S. cerevisiae is slightly more efficient at performing GRC. GRC is less efficient in rad52 deletion mutants, which are defective in HR in both species. In dnl4 deletion mutants, which perform less non-homologous end joining (NHEJ, the frequency of GRC increases in C. glabrata, whereas GRC frequency only minimally increases in S. cerevisiae, suggesting that NHEJ is more prevalent in C. glabrata. Our studies allow for a model of the fate of linear DNA when transformed into yeast cells. This model is not the same for both species. Most significantly, during GRC, C. glabrata performs NHEJ activity at a detectable rate (>5%, while S. cerevisiae does not. Our model suggests that S. cerevisiae is more efficient at HR because NHEJ is less prevalent than in C. glabrata. This work demonstrates the determinants for GRC and that while C. glabrata has a lower efficiency of GRC, this species still provides a viable option for GRC.

  18. Dental homologies in lamniform sharks (Chondrichthyes: Elasmobranchii).

    Science.gov (United States)

    Shimada, Kenshu

    2002-01-01

    The dentitions of lamniform sharks are said to exhibit a unique heterodonty called the "lamnoid tooth pattern." The presence of an inflated hollow "dental bulla" on each jaw cartilage allows the recognition of homologous teeth across most modern macrophagous lamniforms based on topographic correspondence through the "similarity test." In most macrophagous lamniforms, three tooth rows are supported by the upper dental bulla: two rows of large anterior teeth followed by a row of small intermediate teeth. The lower tooth row occluding between the two rows of upper anterior teeth is the first lower anterior tooth row. Like the first and second lower anterior tooth rows, the third lower tooth row is supported by the dental bulla and may be called the first lower intermediate tooth row. The lower intermediate tooth row occludes between the first and second upper lateral tooth rows situated distal to the upper dental bulla, and the rest of the upper and lower tooth rows, all called lateral tooth rows, occlude alternately. Tooth symmetry cannot be used to identify their dental homology. The presence of dental bullae can be regarded as a synapomorphy of Lamniformes and this character is more definable than the "lamnoid tooth pattern." The formation of the tooth pattern appears to be related to the evolution of dental bullae. This study constitutes the first demonstration of supraspecific tooth-to-tooth dental homologies in nonmammalian vertebrates.

  19. Irradiated homologous costal cartilage for augmentation rhinoplasty

    Energy Technology Data Exchange (ETDEWEB)

    Lefkovits, G. (Lenox Hill Hospital, New York, NY (USA))

    1990-10-01

    Although the ideal reconstructive material for augmentation rhinoplasty continues to challenge plastic surgeons, there exists no report in the literature that confines the use of irradiated homologous costal cartilage, first reported by Dingman and Grabb in 1961, to dorsal nasal augmentation. The purpose of this paper is to present a retrospective analysis of the author's experience using irradiated homologous costal cartilage in augmentation rhinoplasty. Twenty-seven dorsal nasal augmentations were performed in 24 patients between 16 and 49 years of age with a follow-up ranging from 1 to 27 months. Good-to-excellent results were achieved in 83.3% (20 of 24). Poor results requiring revision were found in 16.7% (4 of 24). Complication rates included 7.4% infection (2 of 27) and 14.8% warping (4 of 27). The resorption rate was zero. These results compare favorably with other forms of nasal augmentation. Advantages and disadvantages of irradiated homologous costal cartilage are discussed.

  20. DNA intermediates of meiotic recombination in synchronous S. pombe at optimal temperature.

    Science.gov (United States)

    Hyppa, Randy W; Fowler, Kyle R; Cipak, Lubos; Gregan, Juraj; Smith, Gerald R

    2014-01-01

    Crossovers formed by recombination between homologous chromosomes are important for proper homolog segregation during meiosis and for generation of genetic diversity. Optimal molecular analysis of DNA intermediates of recombination requires synchronous cultures. We previously described a mutant, pat1-as2, of the fission yeast Schizosaccharomyces pombe that undergoes synchronous meiosis at 25°C when an ATP analog is added to the culture. Here, we compare recombination intermediates in pat1-as2 at 25°C with those in the widely used pat1-114 temperature-sensitive mutant at 34°C, a temperature higher than optimal. DNA double-strand breaks at most hotspots are similarly abundant in the two conditions but, remarkably, a few hotspots are distinctly deficient at 25°C. In both conditions, Holliday junctions at DNA break hotspots form more frequently between sister chromatids than between homologs, but a novel species, perhaps arising from invasion by only one end of broken DNA, is more readily observed at 25°C. Our results confirm the validity of previous assays of recombination intermediates in S. pombe and provide new information on the mechanism of meiotic recombination. PMID:24089141

  1. Recombinant DNA for Teachers.

    Science.gov (United States)

    Duvall, James G., III

    1992-01-01

    A science teacher describes his experience at a workshop to learn to teach the Cold Spring Harbor DNA Science Laboratory Protocols. These protocols lead students through processes for taking E. coli cells and transforming them into a new antibiotic resistant strain. The workshop featured discussions of the role of DNA recombinant technology in…

  2. Recombineering Pseudomonas syringae

    Science.gov (United States)

    Here we report the identification of functions that promote genomic recombination of linear DNA introduced into Pseudomonas cells by electroporation. The genes encoding these functions were identified in Pseudomonas syringae pv. syringae B728a based on similarity to the lambda Red Exo/Beta and RecE...

  3. Multiple host-cell recombination pathways act in Agrobacterium-mediated transformation of plant cells.

    Science.gov (United States)

    Mestiri, Imen; Norre, Frédéric; Gallego, Maria E; White, Charles I

    2014-02-01

    Using floral-dip, tumorigenesis and root callus transformation assays of both germline and somatic cells, we present here results implicating the four major non-homologous and homologous recombination pathways in Agrobacterium-mediated transformation of Arabidopsis thaliana. All four single mutant lines showed similar mild reductions in transformability, but knocking out three of four pathways severely compromised Agrobacterium-mediated transformation. Although integration of T-DNA into the plant genome is severely compromised in the absence of known DNA double-strand break repair pathways, it does still occur, suggesting the existence of other pathways involved in T-DNA integration. Our results highlight the functional redundancy of the four major plant recombination pathways in transformation, and provide an explanation for the lack of strong effects observed in previous studies on the roles of plant recombination functions in transformation.

  4. Tocopherol and tocotrienol homologs in parenteral lipid emulsions

    OpenAIRE

    Xu, Zhidong; Harvey, Kevin A.; Pavlina, Thomas M; Zaloga, Gary P.; Siddiqui, Rafat A.

    2014-01-01

    Parenteral lipid emulsions, which are made of oils from plant and fish sources, contain different types of tocopherols and tocotrienols (vitamin E homologs). The amount and types of vitamin E homologs in various lipid emulsions vary considerably and are not completely known. The objective of this analysis was to develop a quantitative method to determine levels of all vitamin E homologs in various lipid emulsions. An HPLC system was used to measure vitamin E homologs using a Pinnacle DB Silic...

  5. Computing Small 1-Homological Models for Commutative Differential Graded Algebras

    OpenAIRE

    Alvarez, Victor; Armario, Jose Andres; Frau, Maria Dolores; Gonzalez-Diaz, Rocio; Jimenez, Maria Jose; Real, Pedro; Silva, Beatriz

    2001-01-01

    We use homological perturbation machinery specific for the algebra category [P. Real. Homological Perturbation Theory and Associativity. Homology, Homotopy and Applications vol. 2, n. 5 (2000) 51-88] to give an algorithm for computing the differential structure of a small 1--homological model for commutative differential graded algebras (briefly, CDGAs). The complexity of the procedure is studied and a computer package in Mathematica is described for determining such models.

  6. Equivariant geometric K-homology for compact Lie group actions

    CERN Document Server

    Baum, Paul; Schick, Thomas

    2009-01-01

    Let G be a compact Lie-group, X a compact G-CW-complex. We define equivariant geometric K-homology groups K^G_*(X), using an obvious equivariant version of the (M,E,f)-picture of Baum-Douglas for K-homology. We define explicit natural transformations to and from equivariant K-homology defined via KK-theory (the "official" equivariant K-homology groups) and show that these are isomorphism.

  7. [Telomere Recombination in Normal Mammalian Cells].

    Science.gov (United States)

    Zhdanova, N S; Rubtsov, N B

    2016-01-01

    Two mechanisms of telomere length maintenance are known to date. The first includes the use of a special enzymatic telomerase complex to solve the problems that arise during the replication of linear DNA in a normal diploid and part of tumor cells. Alternative lengthening of telomeres (ALT), which is based on the homologous recombination of telomere DNA, represents the second mechanism. Until recently, ALT was assumed to be expressed only in 15-20% of tumors lacking active telomerase and, together with telomerase reactivation represented one of two possibilities to overcome the replicative senescence observed in somatic mammalian cells due to aging or during cell culturing in vitro. Previously described sporadic cases of combinations of the two mechanisms of telomere length maintenance in several cell lines in vitro were attributed to the experimental design rather than to a real biological phenomenon, since active cellular division without active telomerase was considered to be the "gold standard" of ALT. The present review describes the morphological and functional reorganizations of mammalian telomeres observed with ALT activation, as well as recently observed,and well-documented cases of combinations between ALT-like and telomerase-dependent mechanisms in mammalian cells. The possible role of telomere recombination in telomerase-dependent cells is discussed. PMID:27183789

  8. Extensive Recombination of a Yeast Diploid Hybrid through Meiotic Reversion.

    Directory of Open Access Journals (Sweden)

    Raphaëlle Laureau

    2016-02-01

    Full Text Available In somatic cells, recombination between the homologous chromosomes followed by equational segregation leads to loss of heterozygosity events (LOH, allowing the expression of recessive alleles and the production of novel allele combinations that are potentially beneficial upon Darwinian selection. However, inter-homolog recombination in somatic cells is rare, thus reducing potential genetic variation. Here, we explored the property of S. cerevisiae to enter the meiotic developmental program, induce meiotic Spo11-dependent double-strand breaks genome-wide and return to mitotic growth, a process known as Return To Growth (RTG. Whole genome sequencing of 36 RTG strains derived from the hybrid S288c/SK1 diploid strain demonstrates that the RTGs are bona fide diploids with mosaic recombined genome, derived from either parental origin. Individual RTG genome-wide genotypes are comprised of 5 to 87 homozygous regions due to the loss of heterozygous (LOH events of various lengths, varying between a few nucleotides up to several hundred kilobases. Furthermore, we show that reiteration of the RTG process shows incremental increases of homozygosity. Phenotype/genotype analysis of the RTG strains for the auxotrophic and arsenate resistance traits validates the potential of this procedure of genome diversification to rapidly map complex traits loci (QTLs in diploid strains without undergoing sexual reproduction.

  9. Duality and products in algebraic (co)homology theories

    OpenAIRE

    Kowalzig, N.; Kraehmer, U.

    2008-01-01

    The origin and interplay of products and dualities in algebraic (co)homology theories is ascribed to a ×A-Hopf algebra structure on the relevant universal enveloping algebra. This provides a unified treatment for example of results by Van den Bergh about Hochschild (co)homology and by Huebschmann about Lie–Rinehart (co)homology.

  10. Characterization of cDNA encoding human placental anticoagulant protein (PP4): Homology with the lipocortin family

    Energy Technology Data Exchange (ETDEWEB)

    Grundmann, U.; Abel, K.J.; Bohn, H.; Loebermann, H.; Lottspeich, F.; Kuepper, H. (Research Institutes, Postfach (West Germany))

    1988-06-01

    A cDNA library prepared from human placenta was screened for sequences encoding the placental protein 4 (PP4). PP4 is an anticoagulant protein that acts as an indirect inhibitor of the thromboplastin-specific complex, which is involved in the blood coagulation cascade. Partial amino acid sequence information from PP4-derived cyanogen bromide fragments was used to design three oligonucleotide probes for screening the library. From 10{sup 6} independent recombinants, 18 clones were identified that hybridized to all three probes. These 18 recombinants contained cDNA inserts encoding a protein of 320 amino acid residues. In addition to the PP4 cDNA the authors identified 9 other recombinants encoding a protein with considerable similarity (74%) to PP4, which was termed PP4-X. PP4 and PP4-X belong to the lipocortin family, as judged by their homology to lipocortin I and calpactin I.

  11. Characterization of cDNA encoding human placental anticoagulant protein (PP4): Homology with the lipocortin family

    International Nuclear Information System (INIS)

    A cDNA library prepared from human placenta was screened for sequences encoding the placental protein 4 (PP4). PP4 is an anticoagulant protein that acts as an indirect inhibitor of the thromboplastin-specific complex, which is involved in the blood coagulation cascade. Partial amino acid sequence information from PP4-derived cyanogen bromide fragments was used to design three oligonucleotide probes for screening the library. From 106 independent recombinants, 18 clones were identified that hybridized to all three probes. These 18 recombinants contained cDNA inserts encoding a protein of 320 amino acid residues. In addition to the PP4 cDNA the authors identified 9 other recombinants encoding a protein with considerable similarity (74%) to PP4, which was termed PP4-X. PP4 and PP4-X belong to the lipocortin family, as judged by their homology to lipocortin I and calpactin I

  12. RecO-mediated DNA homology search and annealing is facilitated by SsbA.

    Science.gov (United States)

    Manfredi, Candela; Suzuki, Yuki; Yadav, Tribhuwan; Takeyasu, Kunio; Alonso, Juan C

    2010-11-01

    Bacillus subtilis RecO plays a central role in recombinational repair and genetic recombination by (i) stimulating RecA filamentation onto SsbA-coated single-stranded (ss) DNA, (ii) modulating the extent of RecA-mediated DNA strand exchange and (iii) promoting annealing of complementary DNA strands. Here, we report that RecO-mediated strand annealing is facilitated by cognate SsbA, but not by a heterologous one. Analysis of non-productive intermediates reveals that RecO interacts with SsbA-coated ssDNA, resulting in transient ternary complexes. The self-interaction of ternary complexes via RecO led to the formation of large nucleoprotein complexes. In the presence of homology, SsbA, at the nucleoprotein, removes DNA secondary structures, inhibits spontaneous strand annealing and facilitates RecO loading onto SsbA-ssDNA complex. RecO relieves SsbA inhibition of strand annealing and facilitates transient and random interactions between homologous naked ssDNA molecules. Finally, both proteins lose affinity for duplex DNA. Our results provide a mechanistic framework for rationalizing protein release and dsDNA zippering as coordinated events that are crucial for RecA-independent plasmid transformation. PMID:20581116

  13. Creating Porcine Biomedical Models Through Recombineering

    Directory of Open Access Journals (Sweden)

    Lawrence B. Schook

    2006-03-01

    Full Text Available Recent advances in genomics provide genetic information from humans and other mammals (mouse, rat, dog and primates traditionally used as models as well as new candidates (pigs and cattle. In addition, linked enabling technologies, such as transgenesis and animal cloning, provide innovative ways to design and perform experiments to dissect complex biological systems. Exploitation of genomic information overcomes the traditional need to choose naturally occurring models. Thus, investigators can utilize emerging genomic knowledge and tools to create relevant animal models. This approach is referred to as reverse genetics. In contrast to ‘forward genetics’, in which gene(s responsible for a particular phenotype are identified by positional cloning (phenotype to genotype, the ‘reverse genetics’ approach determines the function of a gene and predicts the phenotype of a cell, tissue, or organism (genotype to phenotype. The convergence of classical and reverse genetics, along with genomics, provides a working definition of a ‘genetic model’ organism (3. The recent construction of phenotypic maps defining quantitative trait loci (QTL in various domesticated species provides insights into how allelic variations contribute to phenotypic diversity. Targeted chromosomal regions are characterized by the construction of bacterial artificial chromosome (BAC contigs to isolate and characterize genes contributing towards phenotypic variation. Recombineering provides a powerful methodology to harvest genetic information responsible for phenotype. Linking recombineering with gene-targeted homologous recombination, coupled with nuclear transfer (NT technology can provide ‘clones’ of genetically modified animals.

  14. L^2-homology for compact quantum groups

    OpenAIRE

    Kyed, David

    2006-01-01

    A notion of L^2-homology for compact quantum groups is introduced, generalizing the classical notion for countable, discrete groups. If the compact quantum group in question has tracial Haar state, it is possible to define its L^2-Betti numbers and Novikov-Shubin invariants/capacities. It is proved that these L^2-Betti numbers vanish for the Gelfand dual of a compact Lie group and that the zeroth Novikov-Shubin invariant equals the dimension of the underlying Lie group. Finally, we relate our...

  15. Homology of lipoprotein lipase to pancreatic lipase.

    OpenAIRE

    Ben-Avram, C M; Ben-Zeev, O; Lee, T.D. (Taunia D.); Haaga, K; Shively, J. E.; Goers, J; Pedersen, M.E; Reeve, J R; Schotz, M C

    1986-01-01

    Bovine milk lipoprotein lipase was subjected to amino acid sequence analysis. The first 19 amino-terminal residues were Asp-Arg-Ile-Thr-Gly-Gly-Lys-Asp-Phe-Arg-Asp-Ile-Glu-Ser-Lys-Phe-Ala-Leu- Arg. In addition, reversed-phase high-performance liquid chromatography of a tryptic digest of reduced and alkylated lipase resolved a number of peptides, five of which contained cysteine. Sequence analysis of the tryptic peptides revealed in most instances a close homology to porcine pancreatic lipase....

  16. Fukaya categories as categorical Morse homology

    CERN Document Server

    Nadler, David

    2011-01-01

    The Fukaya category of a Weinstein manifold is an intricate symplectic invariant of high interest in mirror symmetry and geometric representation theory. We show in analogy with Morse homology that the Fukaya category can be obtained by gluing together Fukaya categories of Weinstein cells. Our main technical result is a d\\'evissage pattern for Lagrangian branes parallel to that for constructible sheaves. As an application, we exhibit the Fukaya category as the global sections of a sheaf on the conic topology of the Weinstein manifold. This can be viewed as a symplectic analogue of the well-known algebraic and topological theories of (micro)localization.

  17. Railway vehicle performance optimisation using virtual homologation

    Science.gov (United States)

    Magalhães, H.; Madeira, J. F. A.; Ambrósio, J.; Pombo, J.

    2016-09-01

    Unlike regular automotive vehicles, which are designed to travel in different types of roads, railway vehicles travel mostly in the same route during their life cycle. To accept the operation of a railway vehicle in a particular network, a homologation process is required according to local standard regulations. In Europe, the standards EN 14363 and UIC 518, which are used for railway vehicle acceptance, require on-track tests and/or numerical simulations. An important advantage of using virtual homologation is the reduction of the high costs associated with on-track tests by studying the railway vehicle performance in different operation conditions. This work proposes a methodology for the improvement of railway vehicle design with the objective of its operation in selected railway tracks by using optimisation. The analyses required for the vehicle improvement are performed under control of the optimisation method global and local optimisation using direct search. To quantify the performance of the vehicle, a new objective function is proposed, which includes: a Dynamic Performance Index, defined as a weighted sum of the indices obtained from the virtual homologation process; the non-compensated acceleration, which is related to the operational velocity; and a penalty associated with cases where the vehicle presents an unacceptable dynamic behaviour according to the standards. Thus, the optimisation process intends not only to improve the quality of the vehicle in terms of running safety and ride quality, but also to increase the vehicle availability via the reduction of the time for a journey while ensuring its operational acceptance under the standards. The design variables include the suspension characteristics and the operational velocity of the vehicle, which are allowed to vary in an acceptable range of variation. The results of the optimisation lead to a global minimum of the objective function in which the suspensions characteristics of the vehicle are

  18. Periodic cyclic homology of affine Hecke algebras

    CERN Document Server

    Solleveld, Maarten

    2009-01-01

    This is the author's PhD-thesis, which was written in 2006. The version posted here is identical to the printed one. Instead of an abstract, the short list of contents: Preface 5 1 Introduction 9 2 K-theory and cyclic type homology theories 13 3 Affine Hecke algebras 61 4 Reductive p-adic groups 103 5 Parameter deformations in affine Hecke algebras 129 6 Examples and calculations 169 A Crossed products 223 Bibliography 227 Index 237 Samenvatting 245 Curriculum vitae 253

  19. AECL passive autocatalytic recombiners

    Energy Technology Data Exchange (ETDEWEB)

    Gardner, L.B.; Marcinkowska, K. [Atomic Energy of Canada Limited, Chalk River, Ontario (Canada)

    2012-03-15

    Atomic Energy of Canada Limited's (AECL) Passive Autocatalytic Recombiner (PAR) is a passive device used for hydrogen mitigation under post-accident conditions in nuclear reactor containment. The PAR employs a proprietary AECL catalyst which promotes the exothermal reaction between hydrogen and oxygen to form water vapour. The heat of reaction combined with the PAR geometry establishes a convective flow through the recombiner, where ambient hydrogen-rich gas enters the PAR inlet and hot, humid, hydrogen-depleted gas exits the outlet. AECL's PAR has been extensively qualified for CANDU and light water reactors (LWRs), and has been supplied to France, Finland, Ukraine, South Korea and is currently being deployed in Canadian nuclear power plants. (author)

  20. AECL passive autocatalytic recombiners

    Energy Technology Data Exchange (ETDEWEB)

    Gardner, L.B.; Marcinkowska, K. [Atomic Energy of Canada Limited, Chalk River, Ontario (Canada)

    2011-07-01

    Atomic Energy of Canada Limited's (AECL) Passive Autocatalytic Recombiner (PAR) is a passive device used for hydrogen mitigation under post-accident conditions in nuclear reactor containment. The PAR employs a proprietary AECL catalyst which promotes the exothermal reaction between hydrogen and oxygen to form water vapour. The heat of reaction combined with the PAR geometry establishes a convective flow through the recombiner, where ambient hydrogen-rich gas enters the PAR inlet and hot, humid, hydrogen-depleted gas exits the outlet. AECL's PAR has been extensively qualified for CANDU and light water reactors (LWRs), and has been supplied to France, Finland, Ukraine, South Korea and is currently being deployed in Canadian nuclear power plants. (author)

  1. Exponential growth of colored HOMFLY-PT homology

    CERN Document Server

    Wedrich, Paul

    2016-01-01

    We define reduced colored sl(N) link homologies and use deformation spectral sequences to characterize their dependence on color and rank. We then define reduced colored HOMFLY-PT homologies and prove that they arise as large N limits of sl(N) homologies. Together, these results allow proofs of many aspects of the physically conjectured structure of the family of type A link homologies. In particular, we verify a conjecture of Gorsky, Gukov and Sto\\v{s}i\\'c about the growth of colored HOMFLY-PT homologies.

  2. Excluded volume effect enhances the homology pairing of model chromosomes

    CERN Document Server

    Takamiya, Kazunori; Isami, Shuhei; Nishimori, Hiraku; Awazu, Akinori

    2015-01-01

    To investigate the structural dynamics of the homology pairing of polymers, we mod- eled the scenario of homologous chromosome pairings during meiosis in Schizosaccharomyces pombe, one of the simplest model organisms of eukaryotes. We consider a simple model consist- ing of pairs of homologous polymers with the same structures that are confined in a cylindrical container, which represents the local parts of chromosomes contained in an elongated nucleus of S. pombe. Brownian dynamics simulations of this model showed that the excluded volume effects among non-homological chromosomes and the transitional dynamics of nuclear shape serve to enhance the pairing of homologous chromosomes.

  3. Use of Recombination-Mediated Genetic Engineering for Construction of Rescue Human Cytomegalovirus Bacterial Artificial Chromosome Clones

    OpenAIRE

    Kalpana Dulal; Benjamin Silver; Hua Zhu

    2012-01-01

    Bacterial artificial chromosome (BAC) technology has contributed immensely to manipulation of larger genomes in many organisms including large DNA viruses like human cytomegalovirus (HCMV). The HCMV BAC clone propagated and maintained inside E. coli allows for accurate recombinant virus generation. Using this system, we have generated a panel of HCMV deletion mutants and their rescue clones. In this paper, we describe the construction of HCMV BAC mutants using a homologous recombination syste...

  4. RECOMBINANT INFLUENZA VACCINES

    OpenAIRE

    Sedova, E.; Shcherbinin, D.; Migunov, A.; Smirnov, Iu; Logunov, D.; Shmarov, M.; Tsybalova, L.; Naroditskiĭ, B.; O. Kiselev; Gintsburg, A.

    2012-01-01

    This review covers the problems encountered in the construction and production of new recombinant influenza vaccines. New approaches to the development of influenza vaccines are investigated; they include reverse genetics methods, production of virus-like particles, and DNA- and viral vector-based vaccines. Such approaches as the delivery of foreign genes by DNA- and viral vector-based vaccines can preserve the native structure of antigens. Adenoviral vectors are a promising gene-delivery pla...

  5. Soluble recombinant influenza vaccines.

    OpenAIRE

    Fiers, W; Neirynck, S; Deroo, T; Saelens, X; Jou, W M

    2001-01-01

    Soluble, recombinant forms of influenza A virus haemagglutinin and neuraminidase have been produced in cells of lower eukaryotes, and shown in a mouse model to induce complete protective immunity against a lethal virus challenge. Soluble neuraminidase, produced in a baculovirus system, consisted of tetramers, dimers and monomers. Only the tetramers were enzymatically active. The immunogenicity decreased very considerably in the order tetra > di > mono. Therefore, we fused the head part of the...

  6. Germline progenitors escape the widespread phenomenon of homolog pairing during Drosophila development.

    Directory of Open Access Journals (Sweden)

    Eric F Joyce

    Full Text Available Homolog pairing, which plays a critical role in meiosis, poses a potential risk if it occurs in inappropriate tissues or between nonallelic sites, as it can lead to changes in gene expression, chromosome entanglements, and loss-of-heterozygosity due to mitotic recombination. This is particularly true in Drosophila, which supports organismal-wide pairing throughout development. Discovered over a century ago, such extensive pairing has led to the perception that germline pairing in the adult gonad is an extension of the pairing established during embryogenesis and, therefore, differs from the mechanism utilized in most species to initiate pairing specifically in the germline. Here, we show that, contrary to long-standing assumptions, Drosophila meiotic pairing in the gonad is not an extension of pairing established during embryogenesis. Instead, we find that homologous chromosomes are unpaired in primordial germ cells from the moment the germline can be distinguished from the soma in the embryo and remain unpaired even in the germline stem cells of the adult gonad. We further establish that pairing originates immediately after the stem cell stage. This pairing occurs well before the initiation of meiosis and, strikingly, continues through the several mitotic divisions preceding meiosis. These discoveries indicate that the spatial organization of the Drosophila genome differs between the germline and the soma from the earliest moments of development and thus argue that homolog pairing in the germline is an active process as versus a passive continuation of pairing established during embryogenesis.

  7. Exploring optimization parameters to increase ssDNA recombineering in Lactococcus lactis and Lactobacillus reuteri.

    Science.gov (United States)

    Van Pijkeren, Jan-Peter; Neoh, Kar Mun; Sirias, Denise; Findley, Anthony S; Britton, Robert A

    2012-01-01

    Single-stranded DNA (ssDNA) recombineering is a technology which is used to make subtle changes in the chromosome of several bacterial genera. Cells which express a single-stranded DNA binding protein (RecT or Bet) are transformed with an oligonucleotide which is incorporated via an annealing and replication-dependent mechanism. By in silico analysis we identified ssDNA binding protein homologs in the genus Lactobacillus and Lactococcus lactis. To assess whether we could further improve the recombineering efficiency in Lactobacillus reuteri ATCC PTA 6475 we expressed several RecT homologs in this strain. RecT derived from Enterococcus faecalis CRMEN 19 yielded comparable efficiencies compared with a native RecT protein, but none of the other proteins further increased the recombineering efficiency. We successfully improved recombineering efficiency 10-fold in L. lactis by increasing oligonucleotide concentration combined with the use of oligonucleotides containing phosphorothioate-linkages (PTOs). Surprisingly, neither increased oligonucleotide concentration nor PTO linkages enhanced recombineering in L. reuteri 6475. To emphasize the utility of this technology in improving probiotic features we modified six bases in a transcriptional regulatory element region of the pdu-operon of L. reuteri 6475, yielding a 3-fold increase in the production of the antimicrobial compound reuterin. Directed genetic modification of lactic acid bacteria through ssDNA recombineering will simplify strain improvement in a way that, when mutating a single base, is genetically indistinguishable from strains obtained through directed evolution. PMID:22750793

  8. Chemical Inhibitors of Non-Homologous End Joining Increase Targeted Construct Integration in Cryptococcus neoformans.

    Science.gov (United States)

    Arras, Samantha D M; Fraser, James A

    2016-01-01

    The development of a biolistic transformation protocol for Cryptococcus neoformans over 25 years ago ushered in a new era of molecular characterization of virulence in this previously intractable fungal pathogen. However, due to the low rate of homologous recombination in this species, the process of creating targeted gene deletions using biolistic transformation remains inefficient. To overcome the corresponding difficulty achieving molecular genetic modifications, members of the Cryptococcus community have investigated the use of specific genetic backgrounds or construct design strategies aimed at reducing ectopic construct integration via non-homologous end joining (NHEJ). One such approach involves deletion of components of the NHEJ-associated Ku heterodimer. While this strategy increases homologous recombination to nearly 100%, it also restricts strain generation to a ku80Δ genetic background and requires subsequent complex mating procedures to reestablish wild-type DNA repair. In this study, we have investigated the ability of known inhibitors of mammalian NHEJ to transiently phenocopy the C. neoformans Ku deletion strains. Testing of eight candidate inhibitors revealed a range of efficacies in C. neoformans, with the most promising compound (W7) routinely increasing the rate of gene deletion to over 50%. We have successfully employed multiple inhibitors to reproducibly enhance the deletion rate at multiple loci, demonstrating a new, easily applied methodology to expedite acquisition of precise genetic alterations in C. neoformans. Based on this success, we anticipate that the use of these inhibitors will not only become widespread in the Cryptococcus community, but may also find use in other fungal species as well. PMID:27643854

  9. Chemical Inhibitors of Non-Homologous End Joining Increase Targeted Construct Integration in Cryptococcus neoformans.

    Science.gov (United States)

    Arras, Samantha D M; Fraser, James A

    2016-01-01

    The development of a biolistic transformation protocol for Cryptococcus neoformans over 25 years ago ushered in a new era of molecular characterization of virulence in this previously intractable fungal pathogen. However, due to the low rate of homologous recombination in this species, the process of creating targeted gene deletions using biolistic transformation remains inefficient. To overcome the corresponding difficulty achieving molecular genetic modifications, members of the Cryptococcus community have investigated the use of specific genetic backgrounds or construct design strategies aimed at reducing ectopic construct integration via non-homologous end joining (NHEJ). One such approach involves deletion of components of the NHEJ-associated Ku heterodimer. While this strategy increases homologous recombination to nearly 100%, it also restricts strain generation to a ku80Δ genetic background and requires subsequent complex mating procedures to reestablish wild-type DNA repair. In this study, we have investigated the ability of known inhibitors of mammalian NHEJ to transiently phenocopy the C. neoformans Ku deletion strains. Testing of eight candidate inhibitors revealed a range of efficacies in C. neoformans, with the most promising compound (W7) routinely increasing the rate of gene deletion to over 50%. We have successfully employed multiple inhibitors to reproducibly enhance the deletion rate at multiple loci, demonstrating a new, easily applied methodology to expedite acquisition of precise genetic alterations in C. neoformans. Based on this success, we anticipate that the use of these inhibitors will not only become widespread in the Cryptococcus community, but may also find use in other fungal species as well.

  10. Multiple evolutionary events involved in maintaining homologs of Resistance to Powdery Mildew 8 in Brassica napus

    Directory of Open Access Journals (Sweden)

    Qin Li

    2016-07-01

    Full Text Available The Resistance to Powdery Mildew 8 (RPW8 locus confers broad-spectrum resistance to powdery mildew in Arabidopsis thaliana. There are four Homologous to RPW8s (BrHRs in Brassica rapa and three in B. oleracea (BoHRs. B. napus (Bn is derived from diploidization of a hybrid between B. rapa and B. oleracea, thus should have seven homologs of RPW8 (BnHRs. It is unclear whether these genes are still maintained or lost in B. napus after diploidization and how they might have been evolved. Here we reported the identification and sequence polymorphisms of BnHRs from a set of B. napus accessions. Our data indicated that while the BoHR copy from B. oleracea is highly conserved, the BrHR copy from B. rapa is relatively variable in the B. napus genome owing to multiple evolutionary events, such as gene loss, point mutation, insertion, deletion and intragenic recombination. Given the overall high sequence homology of BnHR genes, it is not surprising that both intragenic recombination between two orthologs and two paralogs were detected in B. napus, which may explain the loss of BoHR genes in some B. napus accessions. When ectopically expressed in Arabidopsis, a C-terminally truncated version of BnHRa and BnHRb, as well as the full length BnHRd fused with YFP at their C-termini could trigger cell death in the absence of pathogens and enhanced resistance to powdery mildew disease. Moreover, subcellular localization analysis showed that both BnHRa-YFP and BnHRb-YFP were mainly localized to the extra-haustorial membrane (EHM encasing the haustorium of powdery mildew. Taken together, our data suggest that the duplicated BnHR genes might have been subjected to differential selection and at least some may play a role in defense and could serve as resistance resource in engineering disease-resistant plants.

  11. Multiple Evolutionary Events Involved in Maintaining Homologs of Resistance to Powdery Mildew 8 in Brassica napus

    Science.gov (United States)

    Li, Qin; Li, Jing; Sun, Jin-Long; Ma, Xian-Feng; Wang, Ting-Ting; Berkey, Robert; Yang, Hui; Niu, Ying-Ze; Fan, Jing; Li, Yan; Xiao, Shunyuan; Wang, Wen-Ming

    2016-01-01

    The Resistance to Powdery Mildew 8 (RPW8) locus confers broad-spectrum resistance to powdery mildew in Arabidopsis thaliana. There are four Homologous to RPW8s (BrHRs) in Brassica rapa and three in Brassica oleracea (BoHRs). Brassica napus (Bn) is derived from diploidization of a hybrid between B. rapa and B. oleracea, thus should have seven homologs of RPW8 (BnHRs). It is unclear whether these genes are still maintained or lost in B. napus after diploidization and how they might have been evolved. Here, we reported the identification and sequence polymorphisms of BnHRs from a set of B. napus accessions. Our data indicated that while the BoHR copy from B. oleracea is highly conserved, the BrHR copy from B. rapa is relatively variable in the B. napus genome owing to multiple evolutionary events, such as gene loss, point mutation, insertion, deletion, and intragenic recombination. Given the overall high sequence homology of BnHR genes, it is not surprising that both intragenic recombination between two orthologs and two paralogs were detected in B. napus, which may explain the loss of BoHR genes in some B. napus accessions. When ectopically expressed in Arabidopsis, a C-terminally truncated version of BnHRa and BnHRb, as well as the full length BnHRd fused with YFP at their C-termini could trigger cell death in the absence of pathogens and enhanced resistance to powdery mildew disease. Moreover, subcellular localization analysis showed that both BnHRa-YFP and BnHRb-YFP were mainly localized to the extra-haustorial membrane encasing the haustorium of powdery mildew. Taken together, our data suggest that the duplicated BnHR genes might have been subjected to differential selection and at least some may play a role in defense and could serve as resistance resource in engineering disease-resistant plants. PMID:27493652

  12. Multiple Evolutionary Events Involved in Maintaining Homologs of Resistance to Powdery Mildew 8 in Brassica napus.

    Science.gov (United States)

    Li, Qin; Li, Jing; Sun, Jin-Long; Ma, Xian-Feng; Wang, Ting-Ting; Berkey, Robert; Yang, Hui; Niu, Ying-Ze; Fan, Jing; Li, Yan; Xiao, Shunyuan; Wang, Wen-Ming

    2016-01-01

    The Resistance to Powdery Mildew 8 (RPW8) locus confers broad-spectrum resistance to powdery mildew in Arabidopsis thaliana. There are four Homologous to RPW8s (BrHRs) in Brassica rapa and three in Brassica oleracea (BoHRs). Brassica napus (Bn) is derived from diploidization of a hybrid between B. rapa and B. oleracea, thus should have seven homologs of RPW8 (BnHRs). It is unclear whether these genes are still maintained or lost in B. napus after diploidization and how they might have been evolved. Here, we reported the identification and sequence polymorphisms of BnHRs from a set of B. napus accessions. Our data indicated that while the BoHR copy from B. oleracea is highly conserved, the BrHR copy from B. rapa is relatively variable in the B. napus genome owing to multiple evolutionary events, such as gene loss, point mutation, insertion, deletion, and intragenic recombination. Given the overall high sequence homology of BnHR genes, it is not surprising that both intragenic recombination between two orthologs and two paralogs were detected in B. napus, which may explain the loss of BoHR genes in some B. napus accessions. When ectopically expressed in Arabidopsis, a C-terminally truncated version of BnHRa and BnHRb, as well as the full length BnHRd fused with YFP at their C-termini could trigger cell death in the absence of pathogens and enhanced resistance to powdery mildew disease. Moreover, subcellular localization analysis showed that both BnHRa-YFP and BnHRb-YFP were mainly localized to the extra-haustorial membrane encasing the haustorium of powdery mildew. Taken together, our data suggest that the duplicated BnHR genes might have been subjected to differential selection and at least some may play a role in defense and could serve as resistance resource in engineering disease-resistant plants. PMID:27493652

  13. The Homology Groups of a Partial Trace Monoid Action

    CERN Document Server

    Husainov, Ahmet A

    2011-01-01

    The aim of this paper is to investigate the homology groups of mathematical models of concurrency. We study the Baues-Wirsching homology groups of a small category associated with a partial monoid action on a set. We prove that these groups can be reduced to the Leech homology groups of the monoid. For a trace monoid with an action on a set, we will build a cubical complex of free Abelian groups with homology groups isomorphic to the integral homology groups of the action category. It allows us to solve the problem posed by the author in 2004 of the constructing an algorithm for computing homology groups of the CE nets. We describe the algorithm and give examples of calculating the homology groups.

  14. Detailed assessment of homology detection using different substitution matrices

    Institute of Scientific and Technical Information of China (English)

    LI Jing; WANG Wei

    2006-01-01

    Homology detection plays a key role in bioinformatics, whereas substitution matrix is one of the most important components in homology detection. Thus, besides the improvement of alignment algorithms, another effective way to enhance the accuracy of homology detection is to use proper substitution matrices or even construct new matrices.A study on the features of various matrices and on the comparison of the performances between different matrices in homology detection enable us to choose the most proper or optimal matrix for some specific applications. In this paper, by taking BLOSUM matrices as an example, some detailed features of matrices in homology detection are studied by calculating the distributions of numbers of recognized proteins over different sequence identities and sequence lengths. Our results clearly showed that different matrices have different preferences and abilities to the recognition of remote homologous proteins. Furthermore, detailed features of the various matrices can be used to improve the accuracy of homology detection.

  15. Sheaves on Graphs and Their Homological Invariants

    CERN Document Server

    Friedman, Joel

    2011-01-01

    We introduce a notion of a sheaf of vector spaces on a graph, and develop the foundations of homology theories for such sheaves. One sheaf invariant, its "maximum excess," has a number of remarkable properties. It has a simple definition, with no reference to homology theory, that resembles graph expansion. Yet it is a "limit" of Betti numbers, and hence has a short/long exact sequence theory and resembles the $L^2$ Betti numbers of Atiyah. Also, the maximum excess is defined via a supermodular function, which gives the maximum excess much stronger properties than one has of a typical Betti number. The maximum excess gives a simple interpretation of an important graph invariant, which will be used to study the Hanna Neumann Conjecture in a future paper. Our sheaf theory can be viewed as a vast generalization of algebraic graph theory: each sheaf has invariants associated to it---such as Betti numbers and Laplacian matrices---that generalize those in classical graph theory.

  16. Recombinant protein production from stable mammalian cell lines and pools.

    Science.gov (United States)

    Hacker, David L; Balasubramanian, Sowmya

    2016-06-01

    We highlight recent developments for the production of recombinant proteins from suspension-adapted mammalian cell lines. We discuss the generation of stable cell lines using transposons and lentivirus vectors (non-targeted transgene integration) and site-specific recombinases (targeted transgene integration). Each of these methods results in the generation of cell lines with protein yields that are generally superior to those achievable through classical plasmid transfection that depends on the integration of the transfected DNA by non-homologous DNA end-joining. This is the main reason why these techniques can also be used for the generation of stable cell pools, heterogenous populations of recombinant cells generated by gene delivery and genetic selection without resorting to single cell cloning. This allows the time line from gene transfer to protein production to be reduced.

  17. Transmission distortion affecting human noncrossover but not crossover recombination: a hidden source of meiotic drive.

    Directory of Open Access Journals (Sweden)

    Linda Odenthal-Hesse

    2014-02-01

    Full Text Available Meiotic recombination ensures the correct segregation of homologous chromosomes during gamete formation and contributes to DNA diversity through both large-scale reciprocal crossovers and very localised gene conversion events, also known as noncrossovers. Considerable progress has been made in understanding factors such as PRDM9 and SNP variants that influence the initiation of recombination at human hotspots but very little is known about factors acting downstream. To address this, we simultaneously analysed both types of recombinant molecule in sperm DNA at six highly active hotspots, and looked for disparity in the transmission of allelic variants indicative of any cis-acting influences. At two of the hotspots we identified a novel form of biased transmission that was exclusive to the noncrossover class of recombinant, and which presumably arises through differences between crossovers and noncrossovers in heteroduplex formation and biased mismatch repair. This form of biased gene conversion is not predicted to influence hotspot activity as previously noted for SNPs that affect recombination initiation, but does constitute a powerful and previously undetected source of recombination-driven meiotic drive that by extrapolation may affect thousands of recombination hotspots throughout the human genome. Intriguingly, at both of the hotspots described here, this drive favours strong (G/C over weak (A/T base pairs as might be predicted from the well-established correlations between high GC content and recombination activity in mammalian genomes.

  18. Transmission distortion affecting human noncrossover but not crossover recombination: a hidden source of meiotic drive.

    Science.gov (United States)

    Odenthal-Hesse, Linda; Berg, Ingrid L; Veselis, Amelia; Jeffreys, Alec J; May, Celia A

    2014-02-01

    Meiotic recombination ensures the correct segregation of homologous chromosomes during gamete formation and contributes to DNA diversity through both large-scale reciprocal crossovers and very localised gene conversion events, also known as noncrossovers. Considerable progress has been made in understanding factors such as PRDM9 and SNP variants that influence the initiation of recombination at human hotspots but very little is known about factors acting downstream. To address this, we simultaneously analysed both types of recombinant molecule in sperm DNA at six highly active hotspots, and looked for disparity in the transmission of allelic variants indicative of any cis-acting influences. At two of the hotspots we identified a novel form of biased transmission that was exclusive to the noncrossover class of recombinant, and which presumably arises through differences between crossovers and noncrossovers in heteroduplex formation and biased mismatch repair. This form of biased gene conversion is not predicted to influence hotspot activity as previously noted for SNPs that affect recombination initiation, but does constitute a powerful and previously undetected source of recombination-driven meiotic drive that by extrapolation may affect thousands of recombination hotspots throughout the human genome. Intriguingly, at both of the hotspots described here, this drive favours strong (G/C) over weak (A/T) base pairs as might be predicted from the well-established correlations between high GC content and recombination activity in mammalian genomes. PMID:24516398

  19. Recombinant Collagenlike Proteins

    Science.gov (United States)

    Fertala, Andzej

    2007-01-01

    A group of collagenlike recombinant proteins containing high densities of biologically active sites has been invented. The method used to express these proteins is similar to a method of expressing recombinant procollagens and collagens described in U. S. Patent 5,593,859, "Synthesis of human procollagens and collagens in recombinant DNA systems." Customized collagenous proteins are needed for biomedical applications. In particular, fibrillar collagens are attractive for production of matrices needed for tissue engineering and drug delivery. Prior to this invention, there was no way of producing customized collagenous proteins for these and other applications. Heretofore, collagenous proteins have been produced by use of such biological systems as yeasts, bacteria, and transgenic animals and plants. These products are normal collagens that can also be extracted from such sources as tendons, bones, and hides. These products cannot be made to consist only of biologically active, specific amino acid sequences that may be needed for specific applications. Prior to this invention, it had been established that fibrillar collagens consist of domains that are responsible for such processes as interaction with cells, binding of growth factors, and interaction with a number of structural proteins present in the extracellular matrix. A normal collagen consists of a sequence of domains that can be represented by a corresponding sequence of labels, e.g., D1D2D3D4. A collagenlike protein of the present invention contains regions of collagen II that contain multiples of a single domain (e.g., D1D1D1D1 or D4D4D4D4) chosen for its specific biological activity. By virtue of the multiplicity of the chosen domain, the density of sites having that specific biological activity is greater than it is in a normal collagen. A collagenlike protein according to this invention can thus be made to have properties that are necessary for tissue engineering.

  20. Primordial magnetogenesis before recombination

    CERN Document Server

    Fabre, Ophélia

    2015-01-01

    The origin of large magnetic fields in the Universe remains currently unknown. We investigate here a mechanism before recombination based on known physics. The source of the vorticity is due to the changes in the photon distribution function caused by the fluctuations in the background photons. We show that the magnetic field generated in the MHD limit, due to the Coulomb scattering, is of the order $10^{-49}$ G. We explicitly show that the magnetic fields generated from this process are sustainable and are not erased by resistive diffusion. We compare the results with current observations and discuss the implications.

  1. Role of discs large homolog 5

    Institute of Scientific and Technical Information of China (English)

    Frauke Friedrichs; Monika Stoll

    2006-01-01

    In 2004, an association of genetic variation in the discs large homolog 5 (DLG5) gene with inflammatory bowel disease (IBD) was described in two large European study samples[1]. The initial report of DLG5 as a novel IBD susceptibility gene sparked a multitude of studies investigating its effect on CD and IBD, respectively,leading to controversial findings and ongoing discussions concerning the validity of the initial association finding and its role in the aetiology of Crohn disease. This review aims to summarize the current state of knowledge and to place the reported findings in the context of current concepts of complex diseases. This includes aspects of statistical power, phenotype differences and genetic heterogeneity between different populations as well as gene-gene and gene-environment interactions.

  2. REV7/MAD2L2: the multitasking maestro emerges as a barrier to recombination

    OpenAIRE

    Julian E Sale

    2015-01-01

    REV7/MAD2L2 plays important roles in translesion DNA synthesis and mitotic control. Two new papers extend its gamut by revealing its unexpected participation in pathway choice during DNA double-strand break repair. By inhibiting 5′ DNA end resection downstream of 53BP1 and RIF1, REV7/MAD2L2 promotes non-homologous end joining at the expense of homologous recombination. Importantly, loss of REV7/MAD2L2 renders PARP inhibitors ineffective in BRCA1-deficient tumours, suggesting another possible ...

  3. Loss of ATRX Suppresses Resolution of Telomere Cohesion to Control Recombination in ALT Cancer Cells.

    Science.gov (United States)

    Ramamoorthy, Mahesh; Smith, Susan

    2015-09-14

    The chromatin-remodeler ATRX is frequently lost in cancer cells that use ALT (alternative lengthening of telomeres) for telomere maintenance, but its function in telomere recombination is unknown. Here we show that loss of ATRX suppresses the timely resolution of sister telomere cohesion that normally occurs prior to mitosis. In the absence of ATRX, the histone variant macroH2A1.1 binds to the poly(ADP-ribose) polymerase tankyrase 1, preventing it from localizing to telomeres and resolving cohesion. The resulting persistent telomere cohesion promotes recombination between sister telomeres, while it suppresses inappropriate recombination between non-sisters. Forced resolution of sister telomere cohesion induces excessive recombination between non-homologs, genomic instability, and impaired cell growth, indicating the ATRX-macroH2A1.1-tankyrase axis as a potential therapeutic target in ALT tumors. PMID:26373281

  4. A roadmap for the computation of persistent homology

    CERN Document Server

    Otter, Nina; Tillmann, Ulrike; Grindrod, Peter; Harrington, Heather A

    2015-01-01

    Persistent homology is a method used in topological data analysis to study qualitative features of data, which is robust to perturbations, dimension independent and provides statistical summaries of the outputs. Despite recent progress, the computation of persistent homology for large data sets remains an open problem. We investigate the challenges of computing persistent homology and navigate through the different algorithms and data structures. Specifically, we evaluate the (currently available) open source implementations of persistent homology computations on a wide range of synthetic and real-world data sets, and indicate which algorithms and implementations are best suited to these data. We provide guidelines for the computation of persistent homology, make our own implementations used in this study available, and put forward measures to quantify the challenges of the computation of persistent homology.

  5. Variants of equivariant Seiberg-Witten Floer homology

    OpenAIRE

    Marcolli, M.; Wang, B-L

    2005-01-01

    For a rational homology 3-sphere Y with a Spin c structure s, we show that simple algebraic manipulations of our construction of equivariant Seiberg-Witten Floer homology in lead to a collection of variants, which are all topological invariants. We establish a long exact sequence relating them and we show that they satisfy a duality under orientation reversal. We explain their relation to the equivariant Seiberg-Witten Floer (co)homologies introduced in [loc. cit.]. We conjecture the equivale...

  6. Rad51C deficiency destabilizes XRCC3, impairs recombination and radiosensitizes S/G2-phase cells

    Energy Technology Data Exchange (ETDEWEB)

    Lio, Yi-Ching; Schild, David; Brenneman, Mark A.; Redpath, J. Leslie; Chen, David J.

    2004-05-01

    The highly conserved Rad51 protein plays an essential role in repairing DNA damage through homologous recombination. In vertebrates, five Rad51 paralogs (Rad51B, Rad51C, Rad51D, XRCC2, XRCC3) are expressed in mitotically growing cells, and are thought to play mediating roles in homologous recombination, though their precise functions remain unclear. Here we report the use of RNA interference to deplete expression of Rad51C protein in human HT1080 and HeLa cells. In HT1080 cells, depletion of Rad51C by small interfering RNA caused a significant reduction of frequency in homologous recombination. The level of XRCC3 protein was also sharply reduced in Rad51C-depleted HeLa cells, suggesting that XRCC3 is dependent for its stability upon heterodimerization with Rad51C. In addition, Rad51C-depleted HeLa cells showed hypersensitivity to the DNA cross-linking agent mitomycin C, and moderately increased sensitivity to ionizing radiation. Importantly, the radiosensitivity of Rad51C-deficient HeLa cells was evident in S and G{sub 2}/M phases of the cell cycle but not in G{sub 1} phase. Together, these results provide direct cellular evidence for the importance of human Rad51C in homologous recombinational repair.

  7. Long G2 accumulates recombination intermediates and disturbs chromosome segregation at dysfunction telomere in Schizosaccharomyces pombe

    Energy Technology Data Exchange (ETDEWEB)

    Habib, Ahmed G.K.; Masuda, Kenta; Yukawa, Masashi; Tsuchiya, Eiko [Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8530 (Japan); Ueno, Masaru, E-mail: scmueno@hiroshima-u.ac.jp [Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8530 (Japan); Research Center for the Mathematics on Chromatin Live Dynamics, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8530 (Japan)

    2015-08-14

    Protection of telomere (Pot1) is a single-stranded telomere binding protein which is essential for chromosome ends protection. Fission yeast Rqh1 is a member of RecQ helicases family which has essential roles in the maintenance of genomic stability and regulation of homologous recombination. Double mutant between fission yeast pot1Δ and rqh1 helicase dead (rqh1-hd) maintains telomere by homologous recombination. In pot1Δ rqh1-hd double mutant, recombination intermediates accumulate near telomere which disturb chromosome segregation and make cells sensitive to microtubule inhibitors thiabendazole (TBZ). Deletion of chk1{sup +} or mutation of its kinase domain shortens the G2 of pot1Δ rqh1-hd double mutant and suppresses both the accumulation of recombination intermediates and the TBZ sensitivity of that double mutant. In this study, we asked whether the long G2 is the reason for the TBZ sensitivity of pot1Δ rqh1-hd double mutant. We found that shortening the G2 of pot1Δ rqh1-hd double mutant by additional mutations of wee1 and mik1 or gain of function mutation of Cdc2 suppresses both the accumulation of recombination intermediates and the TBZ sensitivity of pot1Δ rqh1-hd double mutant. Our results suggest that long G2 of pot1Δ rqh1-hd double mutant may allow time for the accumulation of recombination intermediates which disturb chromosome segregation and make cells sensitive to TBZ. - Ηighlights: • We show link between long G2 and accumulation of toxic recombination intermediates. • Accumulation of recombination intermediates at telomere results in TBZ sensitivity. • Activation of DNA damage checkpoint worsens cells' viability in presence of TBZ.

  8. Gene prediction by pattern recognition and homology search

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Y.; Uberbacher, E.C.

    1996-05-01

    This paper presents an algorithm for combining pattern recognition-based exon prediction and database homology search in gene model construction. The goal is to use homologous genes or partial genes existing in the database as reference models while constructing (multiple) gene models from exon candidates predicted by pattern recognition methods. A unified framework for gene modeling is used for genes ranging from situations with strong homology to no homology in the database. To maximally use the homology information available, the algorithm applies homology on three levels: (1) exon candidate evaluation, (2) gene-segment construction with a reference model, and (3) (complete) gene modeling. Preliminary testing has been done on the algorithm. Test results show that (a) perfect gene modeling can be expected when the initial exon predictions are reasonably good and a strong homology exists in the database; (b) homology (not necessarily strong) in general helps improve the accuracy of gene modeling; (c) multiple gene modeling becomes feasible when homology exists in the database for the involved genes.

  9. On the geography and botany of knot Floer homology

    OpenAIRE

    Hedden, Matthew; Watson, Liam

    2014-01-01

    This note explores two questions: (1) Which bigraded groups arise as the knot Floer homology of a knot in the three-sphere? (2) Given a knot, how many distinct knots share its Floer homology? Regarding the first, we show there exist bigraded groups satisfying all previously known constraints of knot Floer homology which do not arise as the invariant of a knot. This leads to a new constraint for knots admitting lens space surgeries, as well as a proof that the rank of knot Floer homology detec...

  10. A novel computational method identifies intra- and inter-species recombination events in Staphylococcus aureus and Streptococcus pneumoniae.

    Science.gov (United States)

    Sanguinetti, Lisa; Toti, Simona; Reguzzi, Valerio; Bagnoli, Fabio; Donati, Claudio

    2012-01-01

    Advances in high-throughput DNA sequencing technologies have determined an explosion in the number of sequenced bacterial genomes. Comparative sequence analysis frequently reveals evidences of homologous recombination occurring with different mechanisms and rates in different species, but the large-scale use of computational methods to identify recombination events is hampered by their high computational costs. Here, we propose a new method to identify recombination events in large datasets of whole genome sequences. Using a filtering procedure of the gene conservation profiles of a test genome against a panel of strains, this algorithm identifies sets of contiguous genes acquired by homologous recombination. The locations of the recombination breakpoints are determined using a statistical test that is able to account for the differences in the natural rate of evolution between different genes. The algorithm was tested on a dataset of 75 genomes of Staphylococcus aureus and 50 genomes comprising different streptococcal species, and was able to detect intra-species recombination events in S. aureus and in Streptococcus pneumoniae. Furthermore, we found evidences of an inter-species exchange of genetic material between S. pneumoniae and Streptococcus mitis, a closely related commensal species that colonizes the same ecological niche. The method has been implemented in an R package, Reco, which is freely available from supplementary material, and provides a rapid screening tool to investigate recombination on a genome-wide scale from sequence data. PMID:22969418

  11. A novel computational method identifies intra- and inter-species recombination events in Staphylococcus aureus and Streptococcus pneumoniae.

    Directory of Open Access Journals (Sweden)

    Lisa Sanguinetti

    Full Text Available Advances in high-throughput DNA sequencing technologies have determined an explosion in the number of sequenced bacterial genomes. Comparative sequence analysis frequently reveals evidences of homologous recombination occurring with different mechanisms and rates in different species, but the large-scale use of computational methods to identify recombination events is hampered by their high computational costs. Here, we propose a new method to identify recombination events in large datasets of whole genome sequences. Using a filtering procedure of the gene conservation profiles of a test genome against a panel of strains, this algorithm identifies sets of contiguous genes acquired by homologous recombination. The locations of the recombination breakpoints are determined using a statistical test that is able to account for the differences in the natural rate of evolution between different genes. The algorithm was tested on a dataset of 75 genomes of Staphylococcus aureus and 50 genomes comprising different streptococcal species, and was able to detect intra-species recombination events in S. aureus and in Streptococcus pneumoniae. Furthermore, we found evidences of an inter-species exchange of genetic material between S. pneumoniae and Streptococcus mitis, a closely related commensal species that colonizes the same ecological niche. The method has been implemented in an R package, Reco, which is freely available from supplementary material, and provides a rapid screening tool to investigate recombination on a genome-wide scale from sequence data.

  12. Primordial magnetogenesis before recombination

    Science.gov (United States)

    Fabre, Ophélia; Shankaranarayanan, S.

    2016-04-01

    The origin of large magnetic fields in the Universe remains currently unknown. We investigate here a mechanism before recombination based on known physics. The source of the vorticity is due to the changes in the photon distribution function caused by the fluctuations in the background photons. We show that the magnetic field generated in the MHD limit, due to the Coulomb scattering, is of the order 10-49 G on a coherence scale of 10 kpc. We explicitly show that the magnetic fields generated from this process are sustainable and are not erased by resistive diffusion. We compare the results with current observations and discuss the implications. Our seed magnetic fields are generated on small scales whereas the main mechanisms studied in the literature are on scale bigger than 1 Mpc. However, compared to more exotic theories generating seed magnetic fields on similar scales, the strength of our fields are generally smaller.

  13. Overlapping contributions of Msh1p and putative recombination proteins Cce1p, Din7p, and Mhr1p in large-scale recombination and genome sorting events in the mitochondrial genome of Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Mookerjee, Shona A. [Department of Biology, University of Rochester, Rochester, NY 14627-0211 (United States); Sia, Elaine A. [Department of Biology, University of Rochester, Rochester, NY 14627-0211 (United States)]. E-mail: esia@mail.rochester.edu

    2006-03-20

    The mechanisms that govern mutation avoidance in the mitochondrial genome, though believed to be numerous, are poorly understood. The identification of individual genes has implicated mismatch repair and several recombination pathways in maintaining the fidelity and structural stability of mitochondrial DNA. However, the majority of genes in these pathways have not been identified and the interactions between different pathways have not been extensively studied. Additionally, the multicopy presence of the mitochondrial genome affects the occurrence and persistence of mutant phenotypes, making mitochondrial DNA transmission and sorting important factors affecting mutation accumulation. We present new evidence that the putative recombination genes CCE1, DIN7, and MHR1 have overlapping function with the mismatch repair homolog MSH1 in point mutation avoidance and suppression of aberrant recombination events. In addition, we demonstrate a novel role for Msh1p in mtDNA transmission, a role not predicted by studies of its nuclear homologs.

  14. Cdk1 targets Srs2 to complete synthesis-dependent strand annealing and to promote recombinational repair.

    Directory of Open Access Journals (Sweden)

    Marco Saponaro

    2010-02-01

    Full Text Available Cdk1 kinase phosphorylates budding yeast Srs2, a member of UvrD protein family, displays both DNA translocation and DNA unwinding activities in vitro. Srs2 prevents homologous recombination by dismantling Rad51 filaments and is also required for double-strand break (DSB repair. Here we examine the biological significance of Cdk1-dependent phosphorylation of Srs2, using mutants that constitutively express the phosphorylated or unphosphorylated protein isoforms. We found that Cdk1 targets Srs2 to repair DSB and, in particular, to complete synthesis-dependent strand annealing, likely controlling the disassembly of a D-loop intermediate. Cdk1-dependent phosphorylation controls turnover of Srs2 at the invading strand; and, in absence of this modification, the turnover of Rad51 is not affected. Further analysis of the recombination phenotypes of the srs2 phospho-mutants showed that Srs2 phosphorylation is not required for the removal of toxic Rad51 nucleofilaments, although it is essential for cell survival, when DNA breaks are channeled into homologous recombinational repair. Cdk1-targeted Srs2 displays a PCNA-independent role and appears to have an attenuated ability to inhibit recombination. Finally, the recombination defects of unphosphorylatable Srs2 are primarily due to unscheduled accumulation of the Srs2 protein in a sumoylated form. Thus, the Srs2 anti-recombination function in removing toxic Rad51 filaments is genetically separable from its role in promoting recombinational repair, which depends exclusively on Cdk1-dependent phosphorylation. We suggest that Cdk1 kinase counteracts unscheduled sumoylation of Srs2 and targets Srs2 to dismantle specific DNA structures, such as the D-loops, in a helicase-dependent manner during homologous recombinational repair.

  15. Cdk1 targets Srs2 to complete synthesis-dependent strand annealing and to promote recombinational repair.

    Science.gov (United States)

    Saponaro, Marco; Callahan, Devon; Zheng, Xiuzhong; Krejci, Lumir; Haber, James E; Klein, Hannah L; Liberi, Giordano

    2010-02-01

    Cdk1 kinase phosphorylates budding yeast Srs2, a member of UvrD protein family, displays both DNA translocation and DNA unwinding activities in vitro. Srs2 prevents homologous recombination by dismantling Rad51 filaments and is also required for double-strand break (DSB) repair. Here we examine the biological significance of Cdk1-dependent phosphorylation of Srs2, using mutants that constitutively express the phosphorylated or unphosphorylated protein isoforms. We found that Cdk1 targets Srs2 to repair DSB and, in particular, to complete synthesis-dependent strand annealing, likely controlling the disassembly of a D-loop intermediate. Cdk1-dependent phosphorylation controls turnover of Srs2 at the invading strand; and, in absence of this modification, the turnover of Rad51 is not affected. Further analysis of the recombination phenotypes of the srs2 phospho-mutants showed that Srs2 phosphorylation is not required for the removal of toxic Rad51 nucleofilaments, although it is essential for cell survival, when DNA breaks are channeled into homologous recombinational repair. Cdk1-targeted Srs2 displays a PCNA-independent role and appears to have an attenuated ability to inhibit recombination. Finally, the recombination defects of unphosphorylatable Srs2 are primarily due to unscheduled accumulation of the Srs2 protein in a sumoylated form. Thus, the Srs2 anti-recombination function in removing toxic Rad51 filaments is genetically separable from its role in promoting recombinational repair, which depends exclusively on Cdk1-dependent phosphorylation. We suggest that Cdk1 kinase counteracts unscheduled sumoylation of Srs2 and targets Srs2 to dismantle specific DNA structures, such as the D-loops, in a helicase-dependent manner during homologous recombinational repair. PMID:20195513

  16. A cytohesin homolog in Dictyostelium amoebae.

    Directory of Open Access Journals (Sweden)

    Maria Christina Shina

    Full Text Available BACKGROUND: Dictyostelium, an amoeboid motile cell, harbors several paralogous Sec7 genes that encode members of three distinct subfamilies of the Sec7 superfamily of Guanine nucleotide exchange factors. Among them are proteins of the GBF/BIG family present in all eukaryotes. The third subfamily represented with three members in D. discoideum is the cytohesin family that has been thought to be metazoan specific. Cytohesins are characterized by a Sec7 PH tandem domain and have roles in cell adhesion and migration. PRINCIPAL FINDINGS: Dictyostelium SecG exhibits highest homologies to the cytohesins. It harbors at its amino terminus several ankyrin repeats that are followed by the Sec7 PH tandem domain. Mutants lacking SecG show reduced cell-substratum adhesion whereas cell-cell adhesion that is important for development is not affected. Accordingly, multicellular development proceeds normally in the mutant. During chemotaxis secG(- cells elongate and migrate in a directed fashion towards cAMP, however speed is moderately reduced. SIGNIFICANCE: The data indicate that SecG is a relevant factor for cell-substrate adhesion and reveal the basic function of a cytohesin in a lower eukaryote.

  17. An adelic resolution for homology sheaves

    Energy Technology Data Exchange (ETDEWEB)

    Gorchinskii, S O [Steklov Mathematical Institute, Russian Academy of Sciences, Moscow (Russian Federation)

    2008-12-31

    We propose a generalization of the ordinary idele group by constructing certain adelic complexes for sheaves of K-groups on schemes. Such complexes are defined for any abelian sheaf on a scheme. We focus on the case when the sheaf is associated with the presheaf of a homology theory with certain natural axioms satisfied, in particular, by K-theory. In this case it is proved that the adelic complex provides a flabby resolution for this sheaf on smooth varieties over an infinite perfect field and that the natural morphism to the Gersten complex is a quasi-isomorphism. The main advantage of the new adelic resolution is that it is contravariant and multiplicative. In particular, this enables us to reprove that the intersection in Chow groups coincides (up to a sign) with the natural product in the corresponding K-cohomology groups. Also, we show that the Weil pairing can be expressed as a Massey triple product in K-cohomology groups with certain indices.

  18. Circular Ribbon Flares and Homologous Jets

    CERN Document Server

    Wang, Haimin

    2012-01-01

    Solar flare emissions in the chromosphere often appear as elongated ribbons on both sides of the magnetic polarity inversion line (PIL), and this has been regarded as evidence of a typical configuration of magnetic reconnection. However, flares having a closed circular ribbon have rarely been reported, although it is expected in the fan--spine magnetic topology involving reconnection at a three-dimensional (3D) coronal null point. We present five circular ribbon flares with associated surges, using high-resolution and high-cadence \\ha blue wing observations obtained from the recently digitized films of Big Bear Solar Observatory (BBSO). In all the events, a central parasitic magnetic field is encompassed by the opposite magnetic polarity, forming a circular PIL that is also traced by filament material. Consequently, a flare kernel at the center is surrounded by a circular flare ribbon. The four homologous jet-related flares on 1991 March 17 and 18 are of particular interest, as (1) the circular ribbons bright...

  19. Following tetraploidy in maize, a short deletion mechanism removed genes preferentially from one of the two homologs.

    Directory of Open Access Journals (Sweden)

    Margaret R Woodhouse

    Full Text Available Previous work in Arabidopsis showed that after an ancient tetraploidy event, genes were preferentially removed from one of the two homologs, a process known as fractionation. The mechanism of fractionation is unknown. We sought to determine whether such preferential, or biased, fractionation exists in maize and, if so, whether a specific mechanism could be implicated in this process. We studied the process of fractionation using two recently sequenced grass species: sorghum and maize. The maize lineage has experienced a tetraploidy since its divergence from sorghum approximately 12 million years ago, and fragments of many knocked-out genes retain enough sequence similarity to be easily identifiable. Using sorghum exons as the query sequence, we studied the fate of both orthologous genes in maize following the maize tetraploidy. We show that genes are predominantly lost, not relocated, and that single-gene loss by deletion is the rule. Based on comparisons with orthologous sorghum and rice genes, we also infer that the sequences present before the deletion events were flanked by short direct repeats, a signature of intra-chromosomal recombination. Evidence of this deletion mechanism is found 2.3 times more frequently on one of the maize homologs, consistent with earlier observations of biased fractionation. The over-fractionated homolog is also a greater than 3-fold better target for transposon removal, but does not have an observably higher synonymous base substitution rate, nor could we find differentially placed methylation domains. We conclude that fractionation is indeed biased in maize and that intra-chromosomal or possibly a similar illegitimate recombination is the primary mechanism by which fractionation occurs. The mechanism of intra-chromosomal recombination explains the observed bias in both gene and transposon loss in the maize lineage. The existence of fractionation bias demonstrates that the frequency of deletion is modulated

  20. Bimolecular recombination in organic photovoltaics.

    Science.gov (United States)

    Lakhwani, Girish; Rao, Akshay; Friend, Richard H

    2014-01-01

    The recombination of electrons and holes is a major loss mechanism in photovoltaic devices that controls their performance. We review scientific literature on bimolecular recombination (BR) in bulk heterojunction organic photovoltaic devices to bring forward existing ideas on the origin and nature of BR and highlight both experimental and theoretical work done to quantify its extent. For these systems, Langevin theory fails to explain BR, and recombination dynamics turns out to be dependent on mobility, temperature, electric field, charge carrier concentration, and trapped charges. Relationships among the photocurrent, open-circuit voltage, fill factor, and morphology are discussed. Finally, we highlight the recent emergence of a molecular-level picture of recombination, taking into account the spin and delocalization of charges. Together with the macroscopic picture of recombination, these new insights allow for a comprehensive understanding of BR and provide design principles for future materials and devices.

  1. RPA mediates recombination repair during replication stress and is displaced from DNA by checkpoint signalling in human cells

    DEFF Research Database (Denmark)

    Sleeth, Kate M; Sørensen, Claus Storgaard; Issaeva, Natalia;

    2007-01-01

    The replication protein A (RPA) is involved in most, if not all, nuclear metabolism involving single-stranded DNA. Here, we show that RPA is involved in genome maintenance at stalled replication forks by the homologous recombination repair system in humans. Depletion of the RPA protein inhibited...... the formation of RAD51 nuclear foci after hydroxyurea-induced replication stalling leading to persistent unrepaired DNA double-strand breaks (DSBs). We demonstrate a direct role of RPA in homology directed recombination repair. We find that RPA is dispensable for checkpoint kinase 1 (Chk1) activation...... and that RPA directly binds RAD52 upon replication stress, suggesting a direct role in recombination repair. In addition we show that inhibition of Chk1 with UCN-01 decreases dissociation of RPA from the chromatin and inhibits association of RAD51 and RAD52 with DNA. Altogether, our data suggest a...

  2. Cylindrical contact homology of subcritical Stein-fillable contact manifolds

    OpenAIRE

    Yau, Mei-Lin

    2004-01-01

    We use contact handle decompositions and a stabilization process to compute the cylindrical contact homology of a subcritical Stein-fillable contact manifold with vanishing first Chern class, and show that it is completely determined by the homology of a subcritical Stein-filling of the contact manifold.

  3. CBH1 homologs and varian CBH1 cellulase

    Energy Technology Data Exchange (ETDEWEB)

    Goedegebuur, Frits; Gualfetti, Peter; Mitchinson, Colin; Neefe, Paulien

    2014-07-01

    Disclosed are a number of homologs and variants of Hypocrea jecorina Cel7A (formerly Trichoderma reesei cellobiohydrolase I or CBH1), nucleic acids encoding the same and methods for producing the same. The homologs and variant cellulases have the amino acid sequence of a glycosyl hydrolase of family 7A wherein one or more amino acid residues are substituted and/or deleted.

  4. Cycles in the chamber homology of GL(3)

    OpenAIRE

    Aubert, Anne-Marie; Hasan, Samir; Plymen, Roger

    2004-01-01

    Let F be a nonarchimedean local field and let GL(N) = GL(N,F). We prove the existence of parahoric types for GL(N). We construct representative cycles in all the homology classes of the chamber homology of GL(3).

  5. A configuration space for equivariant connective K-homology

    CERN Document Server

    Velasquez, Mario

    2012-01-01

    Following ideas of Graeme Segal we construct a configuration space that represents equivariant connective K-homology for group actions of finite groups and furthermore we describe explicitly the complex homology of this configuration space as a Hopf algebra. As a consequence of this work we obtain models of representing spaces for equivariant K-theory.

  6. Assignment of the locus for Waardenburg syndrome type I to human chromosome 2q37 and possible homology to the Splotch mouse.

    OpenAIRE

    Foy, C; Newton, V; Wellesley, D.; Harris, R.; Read, A P

    1990-01-01

    We have demonstrated close linkage between the locus for the autosomal dominant Waardenburg syndrome type I and the placental alkaline phosphatase locus on chromosome 2q37. In five families the peak lod score was 4.76 at a recombination fraction of .023. In the mouse the Splotch locus maps to near the homologous position. Splotch mice have white spotting and hearing defects, suggesting that Splotch may be the murine homologue of Waardenburg syndrome type I.

  7. Interaction of TAPBPR, a tapasin homolog, with MHC-I molecules promotes peptide editing

    Science.gov (United States)

    Morozov, Giora I.; Zhao, Huaying; Mage, Michael G.; Boyd, Lisa F.; Jiang, Jiansheng; Dolan, Michael A.; Venna, Ramesh; Norcross, Michael A.; McMurtrey, Curtis P.; Hildebrand, William; Schuck, Peter; Natarajan, Kannan; Margulies, David H.

    2016-01-01

    Peptide loading of major histocompatibility complex class I (MHC-I) molecules is central to antigen presentation, self-tolerance, and CD8+ T-cell activation. TAP binding protein, related (TAPBPR), a widely expressed tapasin homolog, is not part of the classical MHC-I peptide-loading complex (PLC). Using recombinant MHC-I molecules, we show that TAPBPR binds HLA-A*02:01 and several other MHC-I molecules that are either peptide-free or loaded with low-affinity peptides. Fluorescence polarization experiments establish that TAPBPR augments peptide binding by MHC-I. The TAPBPR/MHC-I interaction is reversed by specific peptides, related to their affinity. Mutational and small-angle X-ray scattering (SAXS) studies confirm the structural similarities of TAPBPR with tapasin. These results support a role of TAPBPR in stabilizing peptide-receptive conformation(s) of MHC-I, permitting peptide editing. PMID:26869717

  8. Flare build-up study - Homologous flares group. I

    Science.gov (United States)

    Martres, M.-J.; Mein, N.; Mouradian, Z.; Rayrole, J.; Schmieder, B.; Simon, G.; Soru-Escaut, I.; Woodgate, B. E.

    1984-01-01

    Solar Maximum Mission observations have been used to study the origin and amount of energy, mechanism of storage and release, and conditions for the occurrence of solar flares, and some results of these studies as they pertain to homologous flares are briefly discussed. It was found that every set of flares produced 'rafales' of homologous flares, i.e., two, three, four, or more flares separated in time by an hour or less. No great changes in macroscopic photospheric patterns were observed during these flaring periods. A quantitative brightness parameter of the relation between homologous flares is defined. Scale changes detected in the dynamic spectrum of flare sites are in good agreement with a theoretical suggestion by Sturrock. Statistical results for different homologous flare active regions show the existence in homologous flaring areas of a 'pivot' of previous filaments interpreted as a signature of an anomaly in the solar rotation.

  9. Genomic lineages of Rhizobium etli revealed by the extent of nucleotide polymorphisms and low recombination

    Directory of Open Access Journals (Sweden)

    González Víctor

    2011-10-01

    Full Text Available Abstract Background Most of the DNA variations found in bacterial species are in the form of single nucleotide polymorphisms (SNPs, but there is some debate regarding how much of this variation comes from mutation versus recombination. The nitrogen-fixing symbiotic bacteria Rhizobium etli is highly variable in both genomic structure and gene content. However, no previous report has provided a detailed genomic analysis of this variation at nucleotide level or the role of recombination in generating diversity in this bacterium. Here, we compared draft genomic sequences versus complete genomic sequences to obtain reliable measures of genetic diversity and then estimated the role of recombination in the generation of genomic diversity among Rhizobium etli. Results We identified high levels of DNA polymorphism in R. etli, and found that there was an average divergence of 4% to 6% among the tested strain pairs. DNA recombination events were estimated to affect 3% to 10% of the genomic sample analyzed. In most instances, the nucleotide diversity (π was greater in DNA segments with recombinant events than in non-recombinant segments. However, this degree of recombination was not sufficiently large to disrupt the congruence of the phylogenetic trees, and further evaluation of recombination in strains quartets indicated that the recombination levels in this species are proportionally low. Conclusion Our data suggest that R. etli is a species composed of separated lineages with low homologous recombination among the strains. Horizontal gene transfer, particularly via the symbiotic plasmid characteristic of this species, seems to play an important role in diversity but the lineages maintain their evolutionary cohesiveness.

  10. Insertion DNA Accelerates Meiotic Interchromosomal Recombination in Arabidopsis thaliana.

    Science.gov (United States)

    Sun, Xiao-Qin; Li, Ding-Hong; Xue, Jia-Yu; Yang, Si-Hai; Zhang, Yan-Mei; Li, Mi-Mi; Hang, Yue-Yu

    2016-08-01

    Nucleotide insertions/deletions are ubiquitous in eukaryotic genomes, and the resulting hemizygous (unpaired) DNA has significant, heritable effects on adjacent DNA. However, little is known about the genetic behavior of insertion DNA. Here, we describe a binary transgenic system to study the behavior of insertion DNA during meiosis. Transgenic Arabidopsis lines were generated to carry two different defective reporter genes on nonhomologous chromosomes, designated as "recipient" and "donor" lines. Double hemizygous plants (harboring unpaired DNA) were produced by crossing between the recipient and the donor, and double homozygous lines (harboring paired DNA) via self-pollination. The transfer of the donor's unmutated sequence to the recipient generated a functional β-glucuronidase gene, which could be visualized by histochemical staining and corroborated by polymerase chain reaction amplification and sequencing. More than 673 million seedlings were screened, and the results showed that meiotic ectopic recombination in the hemizygous lines occurred at a frequency  >6.49-fold higher than that in the homozygous lines. Gene conversion might have been exclusively or predominantly responsible for the gene correction events. The direct measurement of ectopic recombination events provided evidence that an insertion, in the absence of an allelic counterpart, could scan the entire genome for homologous counterparts with which to pair. Furthermore, the unpaired (hemizygous) architectures could accelerate ectopic recombination between itself and interchromosomal counterparts. We suggest that the ectopic recombination accelerated by hemizygous architectures may be a general mechanism for interchromosomal recombination through ubiquitously dispersed repeat sequences in plants, ultimately contributing to genetic renovation and eukaryotic evolution. PMID:27189569

  11. Recombination in the human Pseudoautosomal region PAR1.

    Science.gov (United States)

    Hinch, Anjali G; Altemose, Nicolas; Noor, Nudrat; Donnelly, Peter; Myers, Simon R

    2014-07-01

    The pseudoautosomal region (PAR) is a short region of homology between the mammalian X and Y chromosomes, which has undergone rapid evolution. A crossover in the PAR is essential for the proper disjunction of X and Y chromosomes in male meiosis, and PAR deletion results in male sterility. This leads the human PAR with the obligatory crossover, PAR1, to having an exceptionally high male crossover rate, which is 17-fold higher than the genome-wide average. However, the mechanism by which this obligatory crossover occurs remains unknown, as does the fine-scale positioning of crossovers across this region. Recent research in mice has suggested that crossovers in PAR may be mediated independently of the protein PRDM9, which localises virtually all crossovers in the autosomes. To investigate recombination in this region, we construct the most fine-scale genetic map containing directly observed crossovers to date using African-American pedigrees. We leverage recombination rates inferred from the breakdown of linkage disequilibrium in human populations and investigate the signatures of DNA evolution due to recombination. Further, we identify direct PRDM9 binding sites using ChIP-seq in human cells. Using these independent lines of evidence, we show that, in contrast with mouse, PRDM9 does localise peaks of recombination in the human PAR1. We find that recombination is a far more rapid and intense driver of sequence evolution in PAR1 than it is on the autosomes. We also show that PAR1 hotspot activities differ significantly among human populations. Finally, we find evidence that PAR1 hotspot positions have changed between human and chimpanzee, with no evidence of sharing among the hottest hotspots. We anticipate that the genetic maps built and validated in this work will aid research on this vital and fascinating region of the genome. PMID:25033397

  12. Recombination in the human Pseudoautosomal region PAR1.

    Directory of Open Access Journals (Sweden)

    Anjali G Hinch

    2014-07-01

    Full Text Available The pseudoautosomal region (PAR is a short region of homology between the mammalian X and Y chromosomes, which has undergone rapid evolution. A crossover in the PAR is essential for the proper disjunction of X and Y chromosomes in male meiosis, and PAR deletion results in male sterility. This leads the human PAR with the obligatory crossover, PAR1, to having an exceptionally high male crossover rate, which is 17-fold higher than the genome-wide average. However, the mechanism by which this obligatory crossover occurs remains unknown, as does the fine-scale positioning of crossovers across this region. Recent research in mice has suggested that crossovers in PAR may be mediated independently of the protein PRDM9, which localises virtually all crossovers in the autosomes. To investigate recombination in this region, we construct the most fine-scale genetic map containing directly observed crossovers to date using African-American pedigrees. We leverage recombination rates inferred from the breakdown of linkage disequilibrium in human populations and investigate the signatures of DNA evolution due to recombination. Further, we identify direct PRDM9 binding sites using ChIP-seq in human cells. Using these independent lines of evidence, we show that, in contrast with mouse, PRDM9 does localise peaks of recombination in the human PAR1. We find that recombination is a far more rapid and intense driver of sequence evolution in PAR1 than it is on the autosomes. We also show that PAR1 hotspot activities differ significantly among human populations. Finally, we find evidence that PAR1 hotspot positions have changed between human and chimpanzee, with no evidence of sharing among the hottest hotspots. We anticipate that the genetic maps built and validated in this work will aid research on this vital and fascinating region of the genome.

  13. Detecting Genetic Introgression: High Levels of Intersubspecific Recombination Found in Xylella fastidiosa in Brazil

    OpenAIRE

    Nunney, Leonard; Yuan, Xiaoli; Bromley, Robin E.; Stouthamer, Richard

    2012-01-01

    Documenting the role of novel mutation versus homologous recombination in bacterial evolution, and especially in the invasion of new hosts, is central to understanding the long-term dynamics of pathogenic bacteria. We used multilocus sequence typing (MLST) to study this issue in Xylella fastidiosa subsp. pauca from Brazil, a bacterium causing citrus variegated chlorosis (CVC) and coffee leaf scorch (CLS). All 55 citrus isolates typed (plus one coffee isolate) defined three similar sequence ty...

  14. Conformational variability of recombination R-triplex formed by the mammalian telomeric sequence

    OpenAIRE

    Shchyolkina, A.; Kaluzhny, D.; O. Borisova; Arndt-Jovin, D; Jovin, T.; Zhurkin, V.

    2015-01-01

    Alignment of three nucleic acids strands, in which the third strand is identical to one of the DNA duplex strands, occurs in various cellular systems. In the case of telomeric t-loops, recognition between the DNA duplex and the homologous single strand is likely to be mediated by proteins through formation of the transient recombination-type R-triplex. Earlier, using 2-aminopurine as a fluorescent reporting base, we evaluated the thermodynamic characteristics of intramolecular R-triplex forme...

  15. Peridinialean dinoflagellate plate patterns, labels and homologies

    Science.gov (United States)

    Edwards, L.E.

    1990-01-01

    Tabulation patterns for peridinialean dinoflagellate thecae and cysts have been traditionally expressed using a plate labelling system described by C.A. Kofoid in the early 1900's. This system can obscure dinoflagellate plate homologies and has not always been strictly applied. The plate-labelling system presented here introduces new series labels but incorporates key features and ideas from the more recently proposed systems of G.L. Eaton and F.J.R. Taylor, as modified by W.R. Evitt. Plate-series recognition begins with the cingulum (C-series) and proceeds from the cingulum toward the apex for the three series of the epitheca/epicyst and proceeds from the cingulum toward the antapex for the two series of the hypotheca/hypocyst. The epithecal/epicystal model consists of eight plates that touch the anterior margin of the cingulum (E-series: plates E1-E7, ES), seven plates toward the apex that touch the E-series plates (M-series: R, M1-M6), and up to seven plates near the apex that do not touch E-series plates (D-series: Dp-Dv). The hypothecal/hypocystal model consists of eight plates that touch the posterior margin of the cingulum (H-series: H1-H6,HR,HS) and three plates toward the antapex (T1-T3). Epithecal/epicystal tabulation patterns come in both 8- and 7- models, corresponding to eight and seven plates, respectively, in the E-series. Hypothecal/hypocystal tabulation patterns also come in both 8- and 7-models, corresponding to eight and seven plates, respectively, in the H-series. By convention, the 7-model epitheca/epicyst has no plates E1 and M1; the 7-model hypotheca/hypocyst has no plate H6. Within an 8-model or 7-model, the system emphasizes plates that are presumed to be homologous by giving them identical labels. I introduce the adjectives "monothigmate", "dithigmate," and "trithigmate" to designate plates touching one, two, and three plates, respectively, of the adjacent series. The term "thigmation" applies to the analysis of plate contacts between

  16. HIV-specific humoral and cellular immunity in rabbits vaccinated with recombinant human immunodeficiency virus-like gag-env particles

    International Nuclear Information System (INIS)

    Recombinant human immunodeficiency virus type-1 (HIV-1)-like gag-env particles produced in mammalian cells were inoculated into two New Zealand white rabbits. In parallel, two control rabbits were inoculated with the homologous HIV-1 virions inactivated by ultraviolet light (uv) and psoralen treatments. The humoral and cellular immune responses to HIV-1 were evaluated for both groups of animals. Recombinant particles elicited humoral immunity that was specific for all the viral structural proteins. The antibodies recognized both denatured and nondenatured proteins. Moreover, the sera neutralized the in vitro infectivity of the homologous virus in CEM cells. Importantly, the recombinant particles also generated a T helper response by priming with the HIV proteins. Similar results were observed with inactivated virus immunization. Therefore, the authors results suggest that the recombinant HIV-like particles elicit functional humoral immunity as well as cellular immunity and represent a novel vaccine candidate for AIDS

  17. Analysis of interchromosomal mitotic recombination.

    Science.gov (United States)

    McGill, C B; Shafer, B K; Higgins, D R; Strathern, J N

    1990-07-01

    A novel synthetic locus is described that provides a simple assay system for characterizing mitotic recombinants. The locus consists of the TRP1 and HIS3 genes inserted into chromosome III of S. cerevisiae between the CRY1 and MAT loci. Defined trp1 and his3 alleles have been generated that allow the selection of interchromosomal recombinants in this interval. Trp+ or His+ recombinants can be divided into several classes based on coupling of the other alleles in the interval. The tight linkage of the CRY1 and MAT loci, combined with the drug resistance and cell type phenotypes that they respectively control, facilitates the classification of the recombinants without resorting to tetrad dissection. We present the distribution of spontaneous recombinants among the classes defined by this analysis. The data suggest that the recombination intermediate can have regions of symmetric strand exchange and that co-conversion tracts can extend over 1-3 kb. Continuous conversion tracts are favored over discontinuous tracts. The distribution among the classes defined by this analysis is altered in recombinants induced by UV irradiation.

  18. Rapid pairing and resegregation of distant homologous loci enables double-strand break repair in bacteria.

    Science.gov (United States)

    Badrinarayanan, Anjana; Le, Tung B K; Laub, Michael T

    2015-08-01

    Double-strand breaks (DSBs) can lead to the loss of genetic information and cell death. Although DSB repair via homologous recombination has been well characterized, the spatial organization of this process inside cells remains poorly understood, and the mechanisms used for chromosome resegregation after repair are unclear. In this paper, we introduced site-specific DSBs in Caulobacter crescentus and then used time-lapse microscopy to visualize the ensuing chromosome dynamics. Damaged loci rapidly mobilized after a DSB, pairing with their homologous partner to enable repair, before being resegregated to their original cellular locations, independent of DNA replication. Origin-proximal regions were resegregated by the ParABS system with the ParA structure needed for resegregation assembling dynamically in response to the DSB-induced movement of an origin-associated ParB away from one cell pole. Origin-distal regions were resegregated in a ParABS-independent manner and instead likely rely on a physical, spring-like force to segregate repaired loci. Collectively, our results provide a mechanistic basis for the resegregation of chromosomes after a DSB. PMID:26240183

  19. Aspergillus: sex and recombination.

    Science.gov (United States)

    Varga, János; Szigeti, Gyöngyi; Baranyi, Nikolett; Kocsubé, Sándor; O'Gorman, Céline M; Dyer, Paul S

    2014-12-01

    The genus Aspergillus is one of the most widespread groups of fungi on Earth, comprised of about 300-350 species with very diverse lifestyles. Most species produce asexual propagula (conidia) on conidial heads. Despite their ubiquity, a sexual cycle has not yet been identified for most of the aspergilli. Where sexual reproduction is present, species exhibit either homothallic (self fertile) or heterothallic (obligate outcrossing) breeding systems. A parasexual cycle has also been described in some Aspergillus species. As in other fungi, sexual reproduction is governed by mating-type (MAT) genes, which determine sexual identity and are involved in regulating later stages of sexual development. Previous population genetic studies have indicated that some supposedly asexual aspergilli exhibit evidence of a recombining population structure, suggesting the presence of a cryptic sexual cycle. In addition, genome analyses have revealed networks of genes necessary for sexual reproduction in several Aspergillus species, again consistent with latent sexuality in these fungi. Knowledge of MAT gene presence has then successfully been applied to induce sexual reproduction between MAT1-1 and MAT1-2 isolates of certain supposedly asexual aspergilli. Recent progress in understanding the extent and significance of sexual reproduction is described here, with special emphasis on findings that are relevant to clinically important aspergilli. PMID:25118872

  20. Scientific and forensic standards for homologous blood transfusion anti-doping analyses.

    Science.gov (United States)

    Giraud, Sylvain; Robinson, Neil; Mangin, Patrice; Saugy, Martial

    2008-07-18

    Since the introduction in 2001 of a urine-based detection method for recombinant erythropoietin (rHuEPO), transfusion-doping practices have regained interest. To address this problem, an efficient antidoping test designed to obtain direct proof of allogeneic blood transfusion was developed and validated. This test, based on flow cytometry analysis of red blood cell (RBCs) phenotypes, was used to determine the absence or the presence of numerous RBCs populations in a blood sample. A such, it may constitute a direct proof of an abnormal blood population resulting from homologous transfusion. Single-blind and single-site studies were carried out to validate this method as a forensic quality standard analysis and to allow objective interpretation of real cases. The analysis of 140 blood samples containing different percentages (0-5%) of a minor RBCs population were carried on by four independent analysts. Robustness, sensitivity, specificity, precision and stability were assessed. ISO-accredited controls samples were used to demonstrate that the method was robust, stable and precise. No false positive results were observed, resulting in a 100% specificity of the method. Most samples containing a 1.5% minor RBCs population were unambiguously detected, yielding a 78.1% sensitivity. These samples mimicked blood collected from an athlete 3 months after a homologous blood transfusion event where 10% of the total RBCs present in the recipient originated in the donor. The observed false negative results could be explained by differences in antigen expression between the donor and the recipient. False negatives were more numerous with smaller minor RBCs populations. The method described here fulfils the ISO-17025 accreditation and validation requirements. The controls and the methodology are solid enough to determine with certainty whether a sample contains one or more RBCs populations. This variable is currently the best indicator for homologous blood transfusion doping.

  1. PXA1, a putative S. cerevisiae homolog of the human adrenoleukyodystrophy gene

    Energy Technology Data Exchange (ETDEWEB)

    Shani, N.; Watkins, P.A.; Valle, D. [Johns Hopkins Univ. School of Medicine, Baltimore, MD (United States)

    1994-09-01

    The adrenoleukodystrophy protein (ALDP) and the 70 kD peroxisomal membrane protein (PMP70) are ATP-binding cassette transporters in the peroxisome membrane. The former is defective in X-linked adrenoleukodystrophy (ALD), a neurodegenerative disorder with defective peroxisome oxidation of very long chain fatty acids; the latter is implicated in Zellweger syndrome, a defect in peroxisome biogenesis. The functions and interactions of ALDP and PMP70 in the peroxisomal membrane are not known. To develop a system in which these questions could be addressed, we sought to clone their yeast homologs. Using RT/PCR with degenerate primers and oleic acid (C18:1) induced yeast RNA as template, we amplified a cDNA segment corresponding to a conserved region of ALDP and PMP70. By sequencing amplified products, we found one with homology to both proteins and used it to clone the corresponding full length yeast gene (PXA1). PXA1 encodes a 758 amino acid protein with 28% and 21% overall identity to ALDP and PMP70, respectively which increases to 47% and 39% in a C terminal region of 178 amino acids. The PXA1 protein precipitates with peroxisomes as shown by immunoblot analysis of cell fractionation gradients. Disruption of PXA1 by homologous recombination results in impaired growth on oleic acid and reduced ability to oxidize oleate. The growth phenotype can be corrected by expression of the wild type PXA1 in the mutant strain. Peroxisomes in the PXA1 mutant yeast strain are intact as judged by catalase distribution and electron microscopy. Given the amino acid similarity, fatty acid oxidation defect and lack of an effect on peroxisomal integrity, we hypothesize that PXA1 may be the yeast ortholog of ALDP. Complementation studies to examine this hypothesis are in progress.

  2. [Construction of recombinant yellow fever virus 17D containing 2A fragment as a vaccine vector].

    Science.gov (United States)

    Xiaowu, Pang; Fu, Wen-Chuan; Guo, Yin-Han; Zhang, Li-Shu; Xie, Tian-Pei; Xinbin, Gu

    2006-05-01

    The Yellow Fever (YF) vaccine, an attenuated yellow fever 17D (YF-17D) live vaccine, is one of the most effective and safest vaccines in the world and is regarded as one of the best candidates for viral expression vector. We here first reported in China the construction and characterization of the recombinant expression vector of yellow fever 17D which contained the proteinase 2A fragment of foot-and-mouth disease virus (FMDV). Three cDNA fragments representing the full-length YF-17D genome, named 5'-end cDNA (A), 3'-end cDNA (B) and middle cDNA (C), were obtained by reverse transcription polymerase chain reaction (RT-PCR), together with the introduction of SP6 enhancer, necessary restriction sites and overlaps for homologous recombination in yeast. Fragment A and B were then introduced into pRS424 in turn by DNA recombination, followed by transfection of fragment C and the recombinant pRS424 containing A and B (pRS-A-B) into yeast. A recombinant vector containing full length cDNA of YF-17D (pRS-YF) was obtained by screening on medium lack of tryptophan and uracil. A recombinant YF-17D expression vector containing FMDV-2A gene fragment (pRS-YF-2A1) was then constructed by methods of DNA recombination and homologous recombination in yeast described above. In vitro transcription of the recombinant vector pRS-YF-2A1 was then carried out and introduced into BHK-21 cells by electroporation. Results of indirect immunofluorescence assay (IFA) and titer determination showed a stable infectious recombinant virus was gotten, whose features such as growth curve were similar to those of the parental YF-17D. The results suggest that the recombinant vector pRS-YF-2A1, by introduction of heterogenous genes via 2A region, is potential to be an effective live vaccine expression vector. PMID:16755933

  3. Gentle masking of low-complexity sequences improves homology search.

    Directory of Open Access Journals (Sweden)

    Martin C Frith

    Full Text Available Detection of sequences that are homologous, i.e. descended from a common ancestor, is a fundamental task in computational biology. This task is confounded by low-complexity tracts (such as atatatatatat, which arise frequently and independently, causing strong similarities that are not homologies. There has been much research on identifying low-complexity tracts, but little research on how to treat them during homology search. We propose to find homologies by aligning sequences with "gentle" masking of low-complexity tracts. Gentle masking means that the match score involving a masked letter is min(0,S, where S is the unmasked score. Gentle masking slightly but noticeably improves the sensitivity of homology search (compared to "harsh" masking, without harming specificity. We show examples in three useful homology search problems: detection of NUMTs (nuclear copies of mitochondrial DNA, recruitment of metagenomic DNA reads to reference genomes, and pseudogene detection. Gentle masking is currently the best way to treat low-complexity tracts during homology search.

  4. Purification and Characterization of Recombinant Darbepoetin Alfa from Leishmania tarentolae.

    Science.gov (United States)

    Kianmehr, Anvarsadat; Mahrooz, Abdolkarim; Oladnabi, Morteza; Safdari, Yaghoub; Ansari, Javad; Veisi, Kamal; Evazalipour, Mehdi; Shahbazmohammadi, Hamid; Omidinia, Eskandar

    2016-09-01

    Darbepoetin alfa is a biopharmaceutical glycoprotein that stimulates erythropoiesis and is used to treat anemia, which associated with renal failure and cancer chemotherapy. We herein describe the structural characterization of recombinant darbepoetin alfa produced by Leishmania tarentolae T7-TR host. The DNA expression cassette was integrated into the L. tarentolae genome through homologous recombination. Transformed clones were selected by antibiotic resistance, diagnostic PCRs, and protein expression analysis. The structure of recombinant darbepoetin alfa was analyzed by isoelectric focusing, ultraviolet-visible spectrum, and circular dichroism (CD) spectroscopy. Expression analysis showed the presence of a protein band at 40 kDa, and its expression level was 51.2 mg/ml of culture medium. Darbepoetin alfa have 5 isoforms with varying degree of sialylation. The UV absorption and CD spectra were analogous to original drug (Aranesp), which confirmed that the produced protein was darbepoetin alfa. Potency test results revealed that the purified protein was biologically active. In brief, the structural and biological characteristics of expressed darbepoetin alfa were very similar to Aranesp which has been normally expressed in CHO. Our data also suggest that produced protein has potential to be developed for clinical use. PMID:27282622

  5. Telomerase-null survivor screening identifies novel telomere recombination regulators.

    Directory of Open Access Journals (Sweden)

    Yan Hu

    Full Text Available Telomeres are protein-DNA structures found at the ends of linear chromosomes and are crucial for genome integrity. Telomeric DNA length is primarily maintained by the enzyme telomerase. Cells lacking telomerase will undergo senescence when telomeres become critically short. In Saccharomyces cerevisiae, a very small percentage of cells lacking telomerase can remain viable by lengthening telomeres via two distinct homologous recombination pathways. These "survivor" cells are classified as either Type I or Type II, with each class of survivor possessing distinct telomeric DNA structures and genetic requirements. To elucidate the regulatory pathways contributing to survivor generation, we knocked out the telomerase RNA gene TLC1 in 280 telomere-length-maintenance (TLM gene mutants and examined telomere structures in post-senescent survivors. We uncovered new functional roles for 10 genes that affect the emerging ratio of Type I versus Type II survivors and 22 genes that are required for Type II survivor generation. We further verified that Pif1 helicase was required for Type I recombination and that the INO80 chromatin remodeling complex greatly affected the emerging frequency of Type I survivors. Finally, we found the Rad6-mediated ubiquitination pathway and the KEOPS complex were required for Type II recombination. Our data provide an independent line of evidence supporting the idea that these genes play important roles in telomere dynamics.

  6. Importing the homology concept from biology into developmental psychology.

    Science.gov (United States)

    Moore, David S

    2013-01-01

    To help introduce the idea of homology into developmental psychology, this article presents some of the concepts, distinctions, and guidelines biologists and philosophers of biology have devised to study homology. Some unresolved issues related to this idea are considered as well. Because homology reflects continuity across time, developmental scientists should find this concept to be useful in the study of psychological/behavioral development, just as biologists have found it essential in the study of the evolution and development of morphological and other characteristics.

  7. Controlled release from recombinant polymers.

    Science.gov (United States)

    Price, Robert; Poursaid, Azadeh; Ghandehari, Hamidreza

    2014-09-28

    Recombinant polymers provide a high degree of molecular definition for correlating structure with function in controlled release. The wide array of amino acids available as building blocks for these materials lend many advantages including biorecognition, biodegradability, potential biocompatibility, and control over mechanical properties among other attributes. Genetic engineering and DNA manipulation techniques enable the optimization of structure for precise control over spatial and temporal release. Unlike the majority of chemical synthetic strategies used, recombinant DNA technology has allowed for the production of monodisperse polymers with specifically defined sequences. Several classes of recombinant polymers have been used for controlled drug delivery. These include, but are not limited to, elastin-like, silk-like, and silk-elastinlike proteins, as well as emerging cationic polymers for gene delivery. In this article, progress and prospects of recombinant polymers used in controlled release will be reviewed.

  8. Cell encoding recombinant human erythropoietin

    Energy Technology Data Exchange (ETDEWEB)

    Beck, A.K.; Withy, R.M.; Zabrecky, J.R.; Masiello, N.C.

    1990-09-04

    This patent describes a C127 cell transformed with a recombinant DNA vector. It comprises: a DNA sequence encoding human erythropoietin, the transformed cell being capable of producing N-linked and O-linked glycosylated human erythropoietin.

  9. Progenitors of Recombining Supernova Remnants

    OpenAIRE

    Moriya, Takashi J.

    2012-01-01

    Usual supernova remnants have either ionizing plasma or plasma in collisional ionization equilibrium, i.e., the ionization temperature is lower than or equal to the electron temperature. However, the existence of recombining supernova remnants, i.e., supernova remnants with the ionization temperature higher than the electron temperature, is recently confirmed. One suggested way to have recombining plasma in a supernova remnant is to have a dense circumstellar medium at the time of the superno...

  10. Recombinant snake venom prothrombin activators

    OpenAIRE

    Lövgren, Ann

    2012-01-01

    Three prothrombin activators; ecarin, which was originally isolated from the venom of the saw-scaled viper Echis carinatus, trocarin from the rough-scaled snake Tropidechis carinatus, and oscutarin from the Taipan snake Oxyuranus scutellatus, were expressed in mammalian cells with the purpose to obtain recombinant prothrombin activators that could be used to convert prothrombin to thrombin. We have previously reported that recombinant ecarin can efficiently generate thrombin without the need ...

  11. Molecular and population analyses of a recombination event in the catabolic plasmid pJP4.

    Science.gov (United States)

    Larraín-Linton, Juanita; De la Iglesia, Rodrigo; Melo, Francisco; González, Bernardo

    2006-10-01

    Cupriavidus necator JMP134(pJP4) harbors a catabolic plasmid, pJP4, which confers the ability to grow on chloroaromatic compounds. Repeated growth on 3-chlorobenzoate (3-CB) results in selection of a recombinant strain, which degrades 3-CB better but no longer grows on 2,4-dichlorophenoxyacetate (2,4-D). We have previously proposed that this phenotype is due to a double homologous recombination event between inverted repeats of the multicopies of this plasmid within the cell. One recombinant form of this plasmid (pJP4-F3) explains this phenotype, since it harbors two copies of the chlorocatechol degradation tfd gene clusters, which are essential to grow on 3-CB, but has lost the tfdA gene, encoding the first step in degradation of 2,4-D. The other recombinant plasmid (pJP4-FM) should harbor two copies of the tfdA gene but no copies of the tfd gene clusters. A molecular analysis using a multiplex PCR approach to distinguish the wild-type plasmid pJP4 from its two recombinant forms, was carried out. Expected PCR products confirming this recombination model were found and sequenced. Few recombinant plasmid forms in cultures grown in several carbon sources were detected. Kinetic studies indicated that cells containing the recombinant plasmid pJP4-FM were not selectable by sole carbon source growth pressure, whereas those cells harboring recombinant plasmid pJP4-F3 were selected upon growth on 3-CB. After 12 days of repeated growth on 3-CB, the complete plasmid population in C. necator JMP134 apparently corresponds to this form. However, wild-type plasmid forms could be recovered after growing this culture on 2,4-D, indicating that different plasmid forms can be found in C. necator JMP134 at the population level. PMID:16980481

  12. Hybrid Sterility Locus on Chromosome X Controls Meiotic Recombination Rate in Mouse.

    Directory of Open Access Journals (Sweden)

    Maria Balcova

    2016-04-01

    Full Text Available Meiotic recombination safeguards proper segregation of homologous chromosomes into gametes, affects genetic variation within species, and contributes to meiotic chromosome recognition, pairing and synapsis. The Prdm9 gene has a dual role, it controls meiotic recombination by determining the genomic position of crossover hotspots and, in infertile hybrids of house mouse subspecies Mus m. musculus (Mmm and Mus m. domesticus (Mmd, it further functions as the major hybrid sterility gene. In the latter role Prdm9 interacts with the hybrid sterility X 2 (Hstx2 genomic locus on Chromosome X (Chr X by a still unknown mechanism. Here we investigated the meiotic recombination rate at the genome-wide level and its possible relation to hybrid sterility. Using immunofluorescence microscopy we quantified the foci of MLH1 DNA mismatch repair protein, the cytological counterparts of reciprocal crossovers, in a panel of inter-subspecific chromosome substitution strains. Two autosomes, Chr 7 and Chr 11, significantly modified the meiotic recombination rate, yet the strongest modifier, designated meiotic recombination 1, Meir1, emerged in the 4.7 Mb Hstx2 genomic locus on Chr X. The male-limited transgressive effect of Meir1 on recombination rate parallels the male-limited transgressive role of Hstx2 in hybrid male sterility. Thus, both genetic factors, the Prdm9 gene and the Hstx2/Meir1 genomic locus, indicate a link between meiotic recombination and hybrid sterility. A strong female-specific modifier of meiotic recombination rate with the effect opposite to Meir1 was localized on Chr X, distally to Meir1. Mapping Meir1 to a narrow candidate interval on Chr X is an important first step towards positional cloning of the respective gene(s responsible for variation in the global recombination rate between closely related mouse subspecies.

  13. Hybrid Sterility Locus on Chromosome X Controls Meiotic Recombination Rate in Mouse.

    Science.gov (United States)

    Balcova, Maria; Faltusova, Barbora; Gergelits, Vaclav; Bhattacharyya, Tanmoy; Mihola, Ondrej; Trachtulec, Zdenek; Knopf, Corinna; Fotopulosova, Vladana; Chvatalova, Irena; Gregorova, Sona; Forejt, Jiri

    2016-04-01

    Meiotic recombination safeguards proper segregation of homologous chromosomes into gametes, affects genetic variation within species, and contributes to meiotic chromosome recognition, pairing and synapsis. The Prdm9 gene has a dual role, it controls meiotic recombination by determining the genomic position of crossover hotspots and, in infertile hybrids of house mouse subspecies Mus m. musculus (Mmm) and Mus m. domesticus (Mmd), it further functions as the major hybrid sterility gene. In the latter role Prdm9 interacts with the hybrid sterility X 2 (Hstx2) genomic locus on Chromosome X (Chr X) by a still unknown mechanism. Here we investigated the meiotic recombination rate at the genome-wide level and its possible relation to hybrid sterility. Using immunofluorescence microscopy we quantified the foci of MLH1 DNA mismatch repair protein, the cytological counterparts of reciprocal crossovers, in a panel of inter-subspecific chromosome substitution strains. Two autosomes, Chr 7 and Chr 11, significantly modified the meiotic recombination rate, yet the strongest modifier, designated meiotic recombination 1, Meir1, emerged in the 4.7 Mb Hstx2 genomic locus on Chr X. The male-limited transgressive effect of Meir1 on recombination rate parallels the male-limited transgressive role of Hstx2 in hybrid male sterility. Thus, both genetic factors, the Prdm9 gene and the Hstx2/Meir1 genomic locus, indicate a link between meiotic recombination and hybrid sterility. A strong female-specific modifier of meiotic recombination rate with the effect opposite to Meir1 was localized on Chr X, distally to Meir1. Mapping Meir1 to a narrow candidate interval on Chr X is an important first step towards positional cloning of the respective gene(s) responsible for variation in the global recombination rate between closely related mouse subspecies.

  14. Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains

    Directory of Open Access Journals (Sweden)

    Penn Charles W

    2009-12-01

    Full Text Available Abstract Background Homologous recombination mediated by the λ-Red genes is a common method for making chromosomal modifications in Escherichia coli. Several protocols have been developed that differ in the mechanisms by which DNA, carrying regions homologous to the chromosome, are delivered into the cell. A common technique is to electroporate linear DNA fragments into cells. Alternatively, DNA fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. In both cases the λ-Red gene products recombine homologous regions carried on the linear DNA fragments with the chromosome. We have successfully used both techniques to generate chromosomal mutations in E. coli K-12 strains. However, we have had limited success with these λ-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains. Results Our goal was to develop a high-throughput recombineering system, primarily for the coupling of genes to epitope tags, which could also be used for deletion of genes in both pathogenic and K-12 E. coli strains. To that end we have designed a series of donor plasmids for use with the λ-Red recombination system, which when cleaved in vivo by the I-SceI meganuclease generate a discrete linear DNA fragment, allowing for C-terminal tagging of chromosomal genes with a 6 × His, 3 × FLAG, 4 × ProteinA or GFP tag or for the deletion of chromosomal regions. We have enhanced existing protocols and technologies by inclusion of a cassette conferring kanamycin resistance and, crucially, by including the sacB gene on the donor plasmid, so that all but true recombinants are counter-selected on kanamycin and sucrose containing media, thus eliminating the need for extensive screening. This method has the added advantage of limiting the exposure of cells to the potential damaging effects of the λ-Red system, which can lead

  15. What is "homology thinking" and what is it for?

    Science.gov (United States)

    Wagner, Günter P

    2016-01-01

    In this paper I examine the thesis by Marc Ereshefsky that, in evolutionary biology, there is a third style of thinking, besides the well-known "population thinking" and "tree thinking." Ereshefsky proposes "homology thinking" as a third approach, focused on the transformation of organismal phenotypes. In this short commentary, I aim at identifying the underlying biological assumptions for homology thinking and how they can be put to work in a research program within evolutionary biology. I propose that homology thinking is based on three insights: 1) multicellular organisms consist of developmentally individualized parts (sub-systems); 2) that developmental individuation entails evolutionary individuation, that is, variational quasi-independence; and 3) these individuated body parts are inherited, though indirectly, and form lineages that are recognized as homologies. These facts support a research program focused on the modification and origination of individuated body parts that supplements and puts into perspective the population genetic and phylogenetic approaches to the study of evolution. PMID:26486321

  16. Homologous prominence non-radial eruptions: A case study

    CERN Document Server

    Duchlev, P; Madjarska, M S; Dechev, M

    2016-01-01

    The present study provides important details on homologous eruptions of a solar prominence that occurred in active region NOAA 10904 on 2006 August 22. We report on the preeruptive phase of the homologous feature as well as the kinematics and the morphology of a forth from a series of prominence eruptions that is critical in defining the nature of the previous consecutive eruptions. The evolution of the overlying coronal field during homologous eruptions is discussed and a new observational criterion for homologous eruptions is provided. We find a distinctive sequence of three activation periods each of them containing preeruptive precursors such as a brightening and enlarging of the prominence body followed by small surge- like ejections from its southern end observed in the radio 17 GHz. We analyse a fourth eruption that clearly indicates a full reformation of the prominence after the third eruption. The fourth eruption although occurring 11 hrs later has an identical morphology, the same angle of propagati...

  17. Homological Dimensions of the Extension Algebras of Monomial Algebras

    Institute of Scientific and Technical Information of China (English)

    Hong Bo SHI

    2015-01-01

    The main objective of this paper is to study the dimension trees and further the homo-logical dimensions of the extension algebras — dual and trivially twisted extensions — with a unified combinatorial approach using the two combinatorial algorithms — Topdown and Bottomup. We first present a more complete and clearer picture of a dimension tree, with which we are then able, on the one hand, to sharpen some results obtained before and furthermore reveal a few more hidden sub-tle homological phenomenons of or connections between the involved algebras; on the other hand, to provide two more effi cient combinatorial algorithms for computing dimension trees, and consequently the homological dimensions as an application. We believe that the more refined complete structural information on dimension trees will be useful to study other homological properties of this class of extension algebras.

  18. Overexpressed of RAD51 suppresses recombination defects: a possible mechanism to reverse genomic instability

    Energy Technology Data Exchange (ETDEWEB)

    Schild, David; Wiese, Claudia

    2009-10-15

    RAD51, a key protein in the homologous recombinational DNA repair (HRR) pathway, is the major strand-transferase required for mitotic recombination. An important early step in HRR is the formation of single-stranded DNA (ss-DNA) coated by RPA (a ss-DNA binding protein). Displacement of RPA by RAD51 is highly regulated and facilitated by a number of different proteins known as the 'recombination mediators'. To assist these recombination mediators, a second group of proteins also is required and we are defining these proteins here as 'recombination co-mediators'. Defects in either recombination mediators or comediators, including BRCA1 and BRCA2, lead to impaired HRR that can genetically be complemented for (i.e. suppressed) by overexpression of RAD51. Defects in HRR have long been known to contribute to genomic instability leading to tumor development. Since genomic instability also slows cell growth, precancerous cells presumably require genomic restabilization to gain a growth advantage. RAD51 is overexpressed in many tumors, and therefore, we hypothesize that the complementing ability of elevated levels of RAD51 in tumors with initial HRR defects limits genomic instability during carcinogenic progression. Of particular interest, this model may also help explain the high frequency of TP53 mutations in human cancers, since wild-type p53 represses RAD51.

  19. Inhibition of telomere recombination by inactivation of KEOPS subunit Cgi121 promotes cell longevity.

    Directory of Open Access Journals (Sweden)

    Jing Peng

    2015-03-01

    Full Text Available DNA double strand break (DSB is one of the major damages that cause genome instability and cellular aging. The homologous recombination (HR-mediated repair of DSBs plays an essential role in assurance of genome stability and cell longevity. Telomeres resemble DSBs and are competent for HR. Here we show that in budding yeast Saccharomyces cerevisiae telomere recombination elicits genome instability and accelerates cellular aging. Inactivation of KEOPS subunit Cgi121 specifically inhibits telomere recombination, and significantly extends cell longevity in both telomerase-positive and pre-senescing telomerase-negative cells. Deletion of CGI121 in the short-lived yku80(tel mutant restores lifespan to cgi121Δ level, supporting the function of Cgi121 in telomeric single-stranded DNA generation and thus in promotion of telomere recombination. Strikingly, inhibition of telomere recombination is able to further slow down the aging process in long-lived fob1Δ cells, in which rDNA recombination is restrained. Our study indicates that HR activity at telomeres interferes with telomerase to pose a negative impact on cellular longevity.

  20. Constructing recombinant replication-defective adenoviral vectors that express glucose transporter-1 through in vitro ligation

    Institute of Scientific and Technical Information of China (English)

    Fangcheng Li; Junliang Li; Ranyi Liu; Xinke Xu; Kaichang Yuan; Zhonghua Wu

    2008-01-01

    BACKGROUND: We constructed a homologous recombination bacterial method based on the pAdEasy system, a widely used system, for generating recombinant adenoviral vectors that express glucose transporter-1 (GLUT1) in rats.OBJECTIVE: This study was designed to investigate the feasibility of generating recombinant replication-defective adenoviral vectors that express GLUT1 in rats by in vitro ligation based on the Adeno-XTM system. DESIGN: An in vitro cell-based experiment. SETTING: This study was performed at the Linbaixin Medical Research Center of the Second Hospital Affiliated to Sun Yat-sen University and Central Laboratory for Prevention and Treatment of Tumor, Sun Yat-sen University between January and August 2004. MATERIALS: Male, adult, Sprague Dawley rats were used to extract total RNA from brain tissue. E. coli DH5?and human embryonic kidney 293 cells (HEK293 cells) used in the present study were cryo-preserved by the Second Hospital Affiliated to Sun Yat-sen University. Rabbit anti-rat GLUT1 polyclonal antibody (Chemicon, U.S.A.) and primers (Shanghai Boya Bioengineering Co., Ltd) were also used. METHODS: E1/E3-deleted replication-defective adenoviral vectors were used. Using in vitro ligation, the target gene was first sub-cloned into a shuttle vector plasmid to obtain the fragment containing target gene expression cassettes by enzyme digestion. Subsequently, the fragment was co-transformed with linearized adenoviral backbone vector into the E. coli strain. The recombinant adenoviral plasmid was transfected into HEK293 cells to assembly recombinant adenoviral vectors with replication capabilities. The procedure was repeated several times for recombinant adenoviral vectors amplification. MAIN OUTCOME MEASURES: Efficiency of recombinant adenoviral vectors to express the target gene was measured by gene and protein expression through polymerase chain reaction and Western Blot assays, respectively.RESULTS: Results demonstrated that recombinant adenoviral