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Sample records for chromosomal integration sites

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

    PubMed

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

    2016-06-01

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

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

    PubMed

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

    2013-06-01

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

  3. HIV Provirus Stably Reproduces Parental Latent and Induced Transcription Phenotypes Regardless of the Chromosomal Integration Site

    PubMed Central

    Hashemi, Farhad B.; Barreto, Kris; Bernhard, Wendy; Hashemi, Pargol; Lomness, Adam

    2016-01-01

    ABSTRACT Understanding the mechanisms of HIV proviral latency is essential for development of a means to eradicate infection and achieve a cure. We have previously described an in vitro latency model that reliably identifies HIV expression phenotypes of infected cells using a dual-fluorescence reporter virus. Our results have demonstrated that ∼50% of infected cells establish latency immediately upon integration of provirus, a phenomenon termed early latency, which appears to occur by mechanisms that are distinct from epigenetic silencing observed with HIV provirus that establishes productive infections. In this study, we have used a mini-dual HIV reporter virus (mdHIV) to compare the long-term stability of provirus produced as early latent or productive infections using Jurkat-Tat T cell clones. Cloned lines bearing mdHIV provirus integrated at different chromosomal locations display unique differences in responsiveness to signaling agonists and chromatin-modifying compounds, and they also produce characteristic expression patterns from the 5′ long terminal repeat (LTR) dsRed and internal EIF1α-enhanced green fluorescent protein (EIF1α-eGFP) reporters. Furthermore, reporter expression profiles of single cell sorted subcultures faithfully reproduce expression profiles identical to that of their original parental population, following prolonged growth in culture, without shifting toward expression patterns resembling that of cell subclones at the time of sorting. Comparison of population dispersion coefficient (CV) and mean fluorescence intensity (MFI) of the subcloned lines showed that both untreated and phorbol myristate acetate (PMA)-ionomycin-stimulated cultures produce expression patterns identical to those of their parental lines. These results indicate that HIV provirus expression characteristics are strongly influenced by the epigenetic landscape at the site of chromosomal integration. IMPORTANCE There is currently considerable interest in development

  4. Multicopy integration of mini-Tn7 transposons into selected chromosomal sites of a Salmonella vaccine strain

    PubMed Central

    Roos, Karen; Werner, Esther; Loessner, Holger

    2015-01-01

    Chromosomal integration of expression modules for transgenes is an important aspect for the development of novel Salmonella vectors. Mini-Tn7 transposons have been used for the insertion of one such module into the chromosomal site attTn7, present only once in most Gram-negative bacteria. However, integration of multiple mini-Tn7 copies might be suitable for expression of appropriate amounts of antigen or combination of different modules. Here we demonstrate that integration of a 9.6 kb mini-Tn7 harbouring the luciferase luxCDABE (lux) occurs at the natural attTn7 site and simultaneously other locations of the Salmonella chromosome, which were engineered using λ-Red recombinase to contain one or two additional artificial attTn7 sites (a-attTn7). Multicopy integration even at closely spaced attTn7 sites was unexpected in light of the previously reported distance-dependent Tn7 target immunity. Integration of multiple copies of a mini-Tn7 containing a gfp cassette resulted in increasing green fluorescence of bacteria. Stable consecutive integration of two mini-Tn7 encoding lacZ and lux was achieved by initial transposition of lacZ-mini-Tn7, subsequent chromosomal insertion of a-attTn7 and a second round of transposition with lux-mini-Tn7. Mini-Tn7 thus constitutes a versatile method for multicopy integration of expression cassettes into the chromosome of Salmonella and possibly other bacteria. PMID:25488129

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

    PubMed

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

    2014-05-20

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

  6. Transgene expression after rep-mediated site-specific integration into chromosome 19.

    PubMed

    Philpott, Nicola J; Gomos, Janette; Falck-Pedersen, Erik

    2004-01-01

    We have used a plasmid-based transfection model of the adeno-associated virus (AAV) Rep-mediated site-specific integration (RMSSI) pathway to characterize the stability and expression of a site-specifically integrated transgene (either green fluorescent protein [GFP] or chloramphenicol acetyltransferase [CAT]). Three plasmids containing the AAV p5 integration efficiency element (p5IEE) have been used to study integration and transgene expression in HeLa cells: (1) pRepGFP(itr+) contains both AAV ITRs, rep, and p5IEE and can be used as either a plasmid or rAAV vehicle for integration; (2) pRepGFP(itr-) contains the AAV rep gene and the p5IEE; (3) pAd-p5CAT contains only the 138-bp p5IEE of AAV. The data presented demonstrate that in the absence of drug selection, all three constructs undergo site-specific integration (efficiencies of between 10 and 40% of transduced cell lines). At 6 weeks posttransfection most cell lines that underwent RMSSI also expressed the appropriate transgene product. By 18 weeks posttransfection cell lines that were established with rep in cis to the transgene showed a decline in transgene expression as well as a loss of transgene DNA. In many cell lines, there appears to be transgene-containing DNA that does not contribute to gene expression. Data support a model of gene expression and transgene instability through a Rep-mediated pathway. In contrast to rep-containing cell lines, clonal cell lines containing p5IEECAT (with Rep provided in trans) maintained both the integrated transgene and transgene expression throughout the entire experimental time course (18 weeks). PMID:14965377

  7. Interstitial telomeric sequences in human chromosomes cluster with common fragile sites, mutagen sensitive sites, viral integration sites, cancer breakpoints, proto-oncogenes and breakpoints involved in primate evolution

    SciTech Connect

    Adekunle, S.S.A.; Wyandt, H.; Mark, H.F.L.

    1994-09-01

    Recently we mapped the telomeric repeat sequences to 111 interstitial sites in the human genome and to sites of gaps and breaks induced by aphidicolin and sister chromatid exchange sites detected by BrdU. Many of these sites correspond to conserved fragile sites in man, gorilla and chimpazee, to sites of conserved sister chromatid exchange in the mammalian X chromosome, to mutagenic sensitive sites, mapped locations of proto-oncogenes, breakpoints implicated in primate evolution and to breakpoints indicated as the sole anomaly in neoplasia. This observation prompted us to investigate if the interstitial telomeric sites cluster with these sites. An extensive literature search was carried out to find all the available published sites mentioned above. For comparison, we also carried out a statistical analysis of the clustering of the sites of the telomeric repeats with the gene locations where only nucleotide mutations have been observed as the only chromosomal abnormality. Our results indicate that the telomeric repeats cluster most with fragile sites, mutagenic sensitive sites and breakpoints implicated in primate evolution and least with cancer breakpoints, mapped locations of proto-oncogenes and other genes with nucleotide mutations.

  8. Site-specific integration and constitutive expression of key genes into Escherichia coli chromosome increases shikimic acid yields.

    PubMed

    Liu, Xianglei; Lin, Jun; Hu, Haifeng; Zhou, Bin; Zhu, Baoquan

    2016-01-01

    As the key starting material for the chemical synthesis of Oseltamivir, shikimic acid (SA) has captured worldwide attention. Many researchers have tried to improve SA production by metabolic engineering, yet expression plasmids were used generally. In recent years, site-specific integration of key genes into chromosome to increase the yield of metabolites showed considerable advantages. The genes could maintain stably and express constitutively without induction. Herein, crucial genes aroG, aroB, tktA, aroE (encoding 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase, dehydroquinate synthase, transketolase and shikimate dehydrogenase, respectively) of SA pathway and glk, galP (encoding glucokinase and galactose permease) were integrated into the locus of ptsHIcrr (phosphoenolpyruvate: carbohydrate phosphotransferase system operon) in a shikimate kinase genetic defect strain Escherichia coli BW25113 (ΔaroL/aroK, DE3). Furthermore, another key gene ppsA (encoding phosphoenolpyruvate synthase) was integrated into tyrR (encoding Tyr regulator protein). As a result, SA production of the recombinant (SA5/pGBAE) reached to 4.14 g/L in shake flask and 27.41 g/L in a 5-L bioreactor. These data suggested that integration of key genes increased SA yields effectively. This strategy is environmentally friendly for no antibiotic is added, simple to handle without induction, and suitable for industrial production. PMID:26672454

  9. HIV gene expression from intact proviruses positioned in bacterial artificial chromosomes at integration sites previously identified in latently infected T cells

    SciTech Connect

    Eipers, Peter G.; Salazar-Gonzalez, Jesus F.; Morrow, Casey D.

    2011-02-05

    HIV integration predominantly occurs in introns of transcriptionally active genes. To study the impact of the integration site on HIV gene expression, a complete HIV-1 provirus (with GFP as a fusion with Nef) was inserted into bacterial artificial chromosomes (BACs) at three sites previously identified in latent T cells of patients: topoisomerase II (Top2A), DNA methyltransferase 1 (DNMT1), or basic leucine transcription factor 2 (BACH2). Transfection of BAC-HIV into 293 T cells resulted in a fourfold difference in production of infectious HIV-1. Cell lines were established that contained BAC-Top2A, BAC-DNMT1, or BAC-BACH2, but only BAC-DNMT1 spontaneously produced virus, albeit at a low level. Stimulation with TNF-{alpha} resulted in virus production from four of five BAC-Top2A and all BAC-DNMT1 cell lines, but not from the BAC-BACH2 lines. The results of these studies highlight differences between integration sites identified in latent T cells to support virus production and reactivation from latency.

  10. Complete Genome Sequence of Germline Chromosomally Integrated Human Herpesvirus 6A and Analyses Integration Sites Define a New Human Endogenous Virus with Potential to Reactivate as an Emerging Infection.

    PubMed

    Tweedy, Joshua; Spyrou, Maria Alexandra; Pearson, Max; Lassner, Dirk; Kuhl, Uwe; Gompels, Ursula A

    2016-01-01

    Human herpesvirus-6A and B (HHV-6A, HHV-6B) have recently defined endogenous genomes, resulting from integration into the germline: chromosomally-integrated "CiHHV-6A/B". These affect approximately 1.0% of human populations, giving potential for virus gene expression in every cell. We previously showed that CiHHV-6A was more divergent than CiHHV-6B by examining four genes in 44 European CiHHV-6A/B cardiac/haematology patients. There was evidence for gene expression/reactivation, implying functional non-defective genomes. To further define the relationship between HHV-6A and CiHHV-6A we used next-generation sequencing to characterize genomes from three CiHHV-6A cardiac patients. Comparisons to known exogenous HHV-6A showed CiHHV-6A genomes formed a separate clade; including all 85 non-interrupted genes and necessary cis-acting signals for reactivation as infectious virus. Greater single nucleotide polymorphism (SNP) density was defined in 16 genes and the direct repeats (DR) terminal regions. Using these SNPs, deep sequencing analyses demonstrated superinfection with exogenous HHV-6A in two of the CiHHV-6A patients with recurrent cardiac disease. Characterisation of the integration sites in twelve patients identified the human chromosome 17p subtelomere as a prevalent site, which had specific repeat structures and phylogenetically related CiHHV-6A coding sequences indicating common ancestral origins. Overall CiHHV-6A genomes were similar, but distinct from known exogenous HHV-6A virus, and have the capacity to reactivate as emerging virus infections. PMID:26784220

  11. Complete Genome Sequence of Germline Chromosomally Integrated Human Herpesvirus 6A and Analyses Integration Sites Define a New Human Endogenous Virus with Potential to Reactivate as an Emerging Infection

    PubMed Central

    Tweedy, Joshua; Spyrou, Maria Alexandra; Pearson, Max; Lassner, Dirk; Kuhl, Uwe; Gompels, Ursula A.

    2016-01-01

    Human herpesvirus-6A and B (HHV-6A, HHV-6B) have recently defined endogenous genomes, resulting from integration into the germline: chromosomally-integrated “CiHHV-6A/B”. These affect approximately 1.0% of human populations, giving potential for virus gene expression in every cell. We previously showed that CiHHV-6A was more divergent than CiHHV-6B by examining four genes in 44 European CiHHV-6A/B cardiac/haematology patients. There was evidence for gene expression/reactivation, implying functional non-defective genomes. To further define the relationship between HHV-6A and CiHHV-6A we used next-generation sequencing to characterize genomes from three CiHHV-6A cardiac patients. Comparisons to known exogenous HHV-6A showed CiHHV-6A genomes formed a separate clade; including all 85 non-interrupted genes and necessary cis-acting signals for reactivation as infectious virus. Greater single nucleotide polymorphism (SNP) density was defined in 16 genes and the direct repeats (DR) terminal regions. Using these SNPs, deep sequencing analyses demonstrated superinfection with exogenous HHV-6A in two of the CiHHV-6A patients with recurrent cardiac disease. Characterisation of the integration sites in twelve patients identified the human chromosome 17p subtelomere as a prevalent site, which had specific repeat structures and phylogenetically related CiHHV-6A coding sequences indicating common ancestral origins. Overall CiHHV-6A genomes were similar, but distinct from known exogenous HHV-6A virus, and have the capacity to reactivate as emerging virus infections. PMID:26784220

  12. Clinical and laboratory features of human herpesvirus 6 chromosomal integration.

    PubMed

    Clark, D A

    2016-04-01

    Human herpesvirus 6 (HHV-6) comprises two separate viruses, HHV-6A and HHV-6B, although this distinction is not commonly made. HHV-6B is ubiquitous in the population with primary infection usually occurring in early childhood, and often resulting in febrile illness. HHV-6B is also recognized as a pathogen in the immunocompromised host, particularly in transplant recipients. HHV-6A is less well characterized and may have a more restricted prevalence. Both viruses are unique among the human herpesviruses in that the entire viral genome can be found integrated into the telomeric regions of host cell chromosomes. Approximately 1% of persons have inherited integrated viral sequences through the germline, and these individuals characteristically have very high viral loads in blood and other sample types. Emerging evidence suggests that HHV-6A and HHV-6B chromosomal integration may not just be an uncommon biological observation, but more likely a characteristic of the replication properties of these viruses. The integrated viral genome appears capable of excision from the chromosomal site and potentially allows viral replication. The clinical consequences of inherited chromosomally integrated HHV-6 have yet to be fully appreciated. PMID:26802216

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

    PubMed

    Juhas, Mario; Ajioka, James W

    2016-06-01

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

  14. [Integration of plasmids into E. coli K12 chromosomes, caused by a Bordetella transposon].

    PubMed

    Sivov, I G; Beliavskiĭ, O A; karataev, G I

    2000-01-01

    Transposition of Bordetella pertussis transposon in E. coli chromosome has been studied on a model of exclusion of donor multicopy pKK3 plasmid with coumermicin. TnBP3 induced the formation of co-integrates between the plasmid and chromosome. The structure of co-integrate was determined. Facts of exclusion of integrated structure and transposon transposition within integrated plasmid into new sites on a recipient chromosome were detected. Relationship between these processes and activity of bacterial cell recombination system has been determined. PMID:10876766

  15. FLP-mediated DNA mobilization to specific target sites in Drosophila chromosomes.

    PubMed Central

    Golic, M M; Rong, Y S; Petersen, R B; Lindquist, S L; Golic, K G

    1997-01-01

    The ability to place a series of gene constructs at a specific site in the genome opens new possibilities for the experimental examination of gene expression and chromosomal position effects. We report that the FLP- FRT site-specific recombination system of the yeast 2mu plasmid can be used to integrate DNA at a chromosomal FRT target site in Drosophila. The technique we used was to first integrate an FRT- flanked gene by standard P element-mediated transformation. FLP was then used to excise the FRT- flanked donor DNA and screen for FLP-mediated re-integration at an FRT target at a different chromosome location. Such events were recovered from up to 5% of the crosses used to screen for mobilization and are easily detectable by altered linkage of a white reporter gene or by the generation of a white + gene upon integration. PMID:9278488

  16. Chromosomal fragile site expression in dogs: I. Breed specific differences.

    PubMed

    Stone, D M; Jacky, P B; Hancock, D D; Prieur, D J

    1991-08-01

    Peripheral blood lymphocytes from clinically normal Doberman pinscher and boxer dogs were cultured for folate-sensitive and, in preliminary studies, aphidicolin-inducible fragile site expression. Both autosomal and X chromosomal fragile sites were observed in canine cells cultured under folate/thymidine depletion and in cells cultured in medium containing aphidicolin. Results from the three dogs evaluated for both folate-sensitive and aphidicolin-inducible fragile site expression showed that the frequency of fragile site expression was significantly (P less than 0.05) greater in cells cultured in medium containing aphidicolin than in cells cultured in folate/thymidine-depleted medium. Cells from the boxer dog expressed a high percentage (66.67%) of aphidicolin-inducible fragile sites in contrast to the Doberman pinscher dog in which only 21.10% of the lymphocytes expressed aphidicolin-inducible fragile sites. The frequencies of spontaneous and folate-sensitive fragile site expression did not vary significantly by breed of dog. Age of dog was significantly and positively correlated with frequency of folate-sensitive fragile site expression in dogs of the boxer breed, but not in dogs of the Doberman pinscher breed. The dog X chromosome expressed three folate-sensitive and aphidicolin-inducible fragile sites. The G-band location of these three fragile sites showed homology with three recognized constitutive common fragile sites on the human X chromosome: Xp22, Xq21, and Xq27.2. Two specific autosomal fragile sites were identified, one on the distal end of the long arm of chromosome 1 and one on the distal end of the long arm of chromosome 8. Other autosomal fragile sites were also apparent but could not be assigned reliably to specific chromosomes. PMID:1910263

  17. Site-specific genetic engineering of the Anopheles gambiae Y chromosome.

    PubMed

    Bernardini, Federica; Galizi, Roberto; Menichelli, Miriam; Papathanos, Philippos-Aris; Dritsou, Vicky; Marois, Eric; Crisanti, Andrea; Windbichler, Nikolai

    2014-05-27

    Despite its function in sex determination and its role in driving genome evolution, the Y chromosome remains poorly understood in most species. Y chromosomes are gene-poor, repeat-rich and largely heterochromatic and therefore represent a difficult target for genetic engineering. The Y chromosome of the human malaria vector Anopheles gambiae appears to be involved in sex determination although very little is known about both its structure and function. Here, we characterize a transgenic strain of this mosquito species, obtained by transposon-mediated integration of a transgene construct onto the Y chromosome. Using meganuclease-induced homologous repair we introduce a site-specific recombination signal onto the Y chromosome and show that the resulting docking line can be used for secondary integration. To demonstrate its utility, we study the activity of a germ-line-specific promoter when located on the Y chromosome. We also show that Y-linked fluorescent transgenes allow automated sex separation of this important vector species, providing the means to generate large single-sex populations. Our findings will aid studies of sex chromosome function and enable the development of male-exclusive genetic traits for vector control. PMID:24821795

  18. A sequence-tagged site map of human chromosome 11.

    PubMed

    Smith, M W; Clark, S P; Hutchinson, J S; Wei, Y H; Churukian, A C; Daniels, L B; Diggle, K L; Gen, M W; Romo, A J; Lin, Y

    1993-09-01

    We report the construction of 370 sequence-tagged sites (STSs) that are detectable by PCR amplification under sets of standardized conditions and that have been regionally mapped to human chromosome 11. DNA sequences were determined by sequencing directly from cosmid templates using primers complementary to T3 and T7 promoters present in the cloning vector. Oligonucleotide PCR primers were predicted by computer and tested using a battery of genomic DNAs. Cosmids were regionally localized on chromosome 11 by using fluorescence in situ hybridization or by analyzing a somatic cell hybrid panel. Additional STSs corresponding to known genes and markers on chromosome 11 were also produced under the same series of standardized conditions. The resulting STSs provide uniform coverage of chromosome 11 with an average spacing of 340 kb. The DNA sequence determined for use in STS production corresponds to about 0.1% (116 kb) of chromosome 11 and has been analyzed for the presence of repetitive sequences, similarities to known genes and motifs, and possible exons. Computer analysis of this sequence has identified and therefore mapped at least eight new genes on chromosome 11. PMID:8244387

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

    PubMed

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

    2013-09-20

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

  20. DNA Instability at Chromosomal Fragile Sites in Cancer

    PubMed Central

    Dillon, Laura W; Burrow, Allison A; Wang, Yuh-Hwa

    2010-01-01

    Human chromosomal fragile sites are specific genomic regions which exhibit gaps or breaks on metaphase chromosomes following conditions of partial replication stress. Fragile sites often coincide with genes that are frequently rearranged or deleted in human cancers, with over half of cancer-specific translocations containing breakpoints within fragile sites. But until recently, little direct evidence existed linking fragile site breakage to the formation of cancer-causing chromosomal aberrations. Studies have revealed that DNA breakage at fragile sites can induce formation of RET/PTC rearrangements, and deletions within the FHIT gene, resembling those observed in human tumors. These findings demonstrate the important role of fragile sites in cancer development, suggesting that a better understanding of the molecular basis of fragile site instability is crucial to insights in carcinogenesis. It is hypothesized that under conditions of replication stress, stable secondary structures form at fragile sites and stall replication fork progress, ultimately resulting in DNA breaks. A recent study examining an FRA16B fragment confirmed the formation of secondary structure and DNA polymerase stalling within this sequence in vitro, as well as reduced replication efficiency and increased instability in human cells. Polymerase stalling during synthesis of FRA16D has also been demonstrated. The ATR DNA damage checkpoint pathway plays a critical role in maintaining stability at fragile sites. Recent findings have confirmed binding of the ATR protein to three regions of FRA3B under conditions of mild replication stress. This review will discuss recent advances made in understanding the role and mechanism of fragile sites in cancer development. PMID:21286310

  1. Comprehensive prediction of chromosome dimer resolution sites in bacterial genomes

    PubMed Central

    2011-01-01

    Background During the replication process of bacteria with circular chromosomes, an odd number of homologous recombination events results in concatenated dimer chromosomes that cannot be partitioned into daughter cells. However, many bacteria harbor a conserved dimer resolution machinery consisting of one or two tyrosine recombinases, XerC and XerD, and their 28-bp target site, dif. Results To study the evolution of the dif/XerCD system and its relationship with replication termination, we report the comprehensive prediction of dif sequences in silico using a phylogenetic prediction approach based on iterated hidden Markov modeling. Using this method, dif sites were identified in 641 organisms among 16 phyla, with a 97.64% identification rate for single-chromosome strains. The dif sequence positions were shown to be strongly correlated with the GC skew shift-point that is induced by replicational mutation/selection pressures, but the difference in the positions of the predicted dif sites and the GC skew shift-points did not correlate with the degree of replicational mutation/selection pressures. Conclusions The sequence of dif sites is widely conserved among many bacterial phyla, and they can be computationally identified using our method. The lack of correlation between dif position and the degree of GC skew suggests that replication termination does not occur strictly at dif sites. PMID:21223577

  2. Analysis of chromosomal integration and deletions of yeast plasmids.

    PubMed Central

    Cameron, J R; Philippsen, P; Davis, R W

    1977-01-01

    Plasmid DNAs from six strains of Saccharomyces cerevisiae were compared. Three different plasmids were found, designated Scp 1, Scp 2 and Scp 3, with monomer lengths of 6.19, 6.06 and 5.97 kilobases as referenced to sequenced phiX174 DNA. DNA from each of the plasmids was inserted into a lambda vector DNA. Hybrid phage containing inserted DNA of the desired size were enriched by genetic selection and their DNAs analysed by rapid techniques. All three plasmids share the same organization, two unique sequences separated by two inverted repeats, and share basically the same DNA sequences. Scp 2 and Scp 3 differ from Scp 1 by missing a unique HpaI site and by having small overlapping deletions in the same region. The HpaI site in Scp 1 is, therefore, in a nonessential region and suitable for insertion of foreign DNA in the potential use of the yeast plasmid as a vector. Hybridization of labelled cloned plasmid DNA to restriction fragments of linear yeast DNA separated on agarose gels showed that the plasmid DNA was not stably integrated into the yeast chromosomal DNA. Images PMID:331256

  3. Increased recombinant protein production owing to expanded opportunities for vector integration in high chromosome number Chinese hamster ovary cells.

    PubMed

    Yamano, Noriko; Takahashi, Mai; Ali Haghparast, Seyed Mohammad; Onitsuka, Masayoshi; Kumamoto, Toshitaka; Frank, Jana; Omasa, Takeshi

    2016-08-01

    Chromosomal instability is a characteristic of Chinese hamster ovary (CHO) cells. Cultures of these cells gradually develop heterogeneity even if established from a single cell clone. We isolated cells containing different numbers of chromosomes from a CHO-DG44-based human granulocyte-macrophage colony stimulating factor (hGM-CSF)-producing cell line and found that high chromosome number cells showed higher hGM-CSF productivity. Therefore, we focused on the relationship between chromosome aneuploidy of CHO cells and high recombinant protein-producing cell lines. Distribution and stability of chromosomes were examined in CHO-DG44 cells, and two cell lines expressing different numbers of chromosomes were isolated from the original CHO-DG44 cell line to investigate the effect of aneuploid cells on recombinant protein production. Both cell lines were stably transfected with a vector that expresses immunoglobulin G3 (IgG3), and specific antibody production rates were compared. Cells containing more than 30 chromosomes had higher specific antibody production rates than those with normal chromosome number. Single cell analysis of enhanced green fluorescent protein (Egfp)-gene transfected cells revealed that increased GFP expression was relative to the number of gene integration sites rather than the difference in chromosome numbers or vector locations. Our results suggest that CHO cells with high numbers of chromosomes contain more sites for vector integration, a characteristic that could be advantageous in biopharmaceutical production. PMID:26850366

  4. Genomic structure and evolution of the ancestral chromosome fusion site in 2q13-2q14.1 and paralogous regions on other human chromosomes.

    PubMed

    Fan, Yuxin; Linardopoulou, Elena; Friedman, Cynthia; Williams, Eleanor; Trask, Barbara J

    2002-11-01

    Human chromosome 2 was formed by the head-to-head fusion of two ancestral chromosomes that remained separate in other primates. Sequences that once resided near the ends of the ancestral chromosomes are now interstitially located in 2q13-2q14.1. Portions of these sequences had duplicated to other locations prior to the fusion. Here we present analyses of the genomic structure and evolutionary history of >600 kb surrounding the fusion site and closely related sequences on other human chromosomes. Sequence blocks that closely flank the inverted arrays of degenerate telomere repeats marking the fusion site are duplicated at many, primarily subtelomeric, locations. In addition, large portions of a 168-kb centromere-proximal block are duplicated at 9pter, 9p11.2, and 9q13, with 98%-99% average sequence identity. A 67-kb block on the distal side of the fusion site is highly homologous to sequences at 22qter. A third ~100-kb segment is 96% identical to a region in 2q11.2. By integrating data on the extent and similarity of these paralogous blocks, including the presence of phylogenetically informative repetitive elements, with observations of their chromosomal distribution in nonhuman primates, we infer the order of the duplications that led to their current arrangement. Several of these duplicated blocks may be associated with breakpoints of inversions that occurred during primate evolution and of recurrent chromosome rearrangements in humans. PMID:12421751

  5. Chromosome

    MedlinePlus

    ... if you are born a boy or a girl (your gender). They are called sex chromosomes: Females have 2 X chromosomes. Males have 1 X and 1 Y chromosome. The mother gives an X chromosome to the ... baby is a girl or a boy. The remaining chromosomes are called ...

  6. Viral induction of site-specific chromosome damage.

    PubMed

    Fortunato, Elizabeth A; Spector, Deborah H

    2003-01-01

    The advent of advanced cell culture and cytogenetics techniques in the 1950s opened a new avenue for research on the pathogenic interactions between animal viruses and their hosts. Studies of many viruses revealed their ability to nonspecifically induce cytogenetic damage to their host cell's chromosomes. However, only three viruses, the oncogenic adenoviruses, herpes simplex virus (HSV) and human cytomegalovirus (HCMV), have been found to cause non-random, site-specific chromosomal damage. Adenovirus (Ad) type 12 induces fragility at four distinct loci (RNU1, RNU2, RN5S and PSU1) in many different types of human cells. A common feature of these loci is that they contain a repeated array of transcriptionally active genes encoding small structural RNAs. Site-specific induction of breaks also requires the virally encoded E1B protein of M(r) 55000 and the C-terminus of the cellular p53 protein. Analysis of the induction of damage by HSV and HCMV necessitates consideration of several factors, including the strain of virus used, the timing of infection, the type of cell used, and the multiplicity of infection. Both HSV strains 1 and 2 are cytotoxic, although the former seems to be more proficient at inducing damage. At early times post infection, HSV induces breaks and specific uncoiling of the centromeres of chromosomes 1, 9 and 16. This is followed at later times by a more complete severing of all of the chromosomes, termed pulverisation. Damage by HSV requires viral entry and de novo viral protein synthesis, with immediate early viral proteins responsible for the induction of breaks and uncoiling and early gene products (most likely nucleases) involved in the extensive pulverisation seen later. HCMV has been studied primarily in permissive human fibroblasts. Its ability to induce specific damage in chromosome 1 at two loci, 1q21 and 1q42, was only recently revealed as the cells must be in S-phase when they are infected for the breaks to be observed. In contrast to

  7. Chromosome

    MedlinePlus

    ... genes . It is the building block of the human body. Chromosomes also contain proteins that help DNA exist ... come in pairs. Normally, each cell in the human body has 23 pairs of chromosomes (46 total chromosomes). ...

  8. An integrated YAC contig map for human chromosome 3

    SciTech Connect

    Gemmill, R.M.; Scott, P.; Cypser, J.

    1994-09-01

    An integrated physical map of human chromosome 3, containing 590 primary markers and 694 corresponding megabase-sized YACs, has been developed from the fusion of two large datasets. YACs identified with 462 physically localized hybridization-based probes and 120 genetically linked polymorphic STSs form the basis of the contigs. Many additional chromosome 3-specific YACs were identified by Alu-PCR hybridization and fingerprint analyses. Contigs defined entirely by primary marker content (level 1) provide >70% coverage. Since the markers used are chromosome 3-specific and have been previously localized by genetic or regional positioning, the reliability of the data at this level is extremely high. Alu-PCR and fingerprint analyses identified overlapping YACs which have permitted fusion and extension of these primary contigs. Selected incorporation of these data has resulted in an integrated coverage of over 80% at level 3. This level corresponds to the use of a single YAC to bridge gaps between YACs or contigs identified by primary markers. Limiting level 3 connections such that the STS or hybridization probe-containing YACs are derived from a common chromosomal segment has helped to ensure their authenticity. The chromosome is now represented by a number of very large YAC contigs whose order is known. Some of the gaps which separate these contigs can be bridged using Alu-PCR and fingerprint data at level 4, although these connections require STS confirmation. The map generated by fusion of the hybridization and STS based datasets is a major advance over maps possible from either dataset alone, both in terms of overall coverage and reliability, and should provide the foundation for development of a transcriptional map and a higher resolution physical map.

  9. Integrating risks at contaminated sites

    SciTech Connect

    MacDonell, M.; Habegger, L.; Nieves, L.; Schreiber, Z.; Travis, C.

    2000-02-17

    The U.S. Department of Energy (DOE) is responsible for a number of large sites across the country that were radioactively and chemically contaminated by past nuclear research, development, and production activities. Multiple risk assessments are being conducted for these sites to evaluate current conditions and determine what measures are needed to protect human health and the environment from today through the long term. Integrating the risks associated with multiple contaminants in different environmental media across extensive areas, over time periods that extend beyond 1,000 years, and for a number of different impact categories--from human health and ecological to social and economic--represents a considerable challenge. A central element of these integrated analyses is the ability to reflect key interrelationships among environmental resources and human communities that may be adversely affected by the actions or inactions being considered for a given site. Complicating the already difficult task of integrating many kinds of risk is the importance of reflecting the diverse values and preferences brought to bear by the multiple parties interested in the risk analysis process and outcome. An initial conceptual framework has been developed to provide an organized structure to this risk integration, with the aim of supporting effective environmental management decisions. This paper highlights key issues associated with comprehensive risk integration and offers suggestions developed from preliminary work at a complex DOE site.

  10. Integrating multi-omic features exploiting Chromosome Conformation Capture data

    PubMed Central

    Merelli, Ivan; Tordini, Fabio; Drocco, Maurizio; Aldinucci, Marco; Liò, Pietro; Milanesi, Luciano

    2015-01-01

    The representation, integration, and interpretation of omic data is a complex task, in particular considering the huge amount of information that is daily produced in molecular biology laboratories all around the world. The reason is that sequencing data regarding expression profiles, methylation patterns, and chromatin domains is difficult to harmonize in a systems biology view, since genome browsers only allow coordinate-based representations, discarding functional clusters created by the spatial conformation of the DNA in the nucleus. In this context, recent progresses in high throughput molecular biology techniques and bioinformatics have provided insights into chromatin interactions on a larger scale and offer a formidable support for the interpretation of multi-omic data. In particular, a novel sequencing technique called Chromosome Conformation Capture allows the analysis of the chromosome organization in the cell’s natural state. While performed genome wide, this technique is usually called Hi–C. Inspired by service applications such as Google Maps, we developed NuChart, an R package that integrates Hi–C data to describe the chromosomal neighborhood starting from the information about gene positions, with the possibility of mapping on the achieved graphs genomic features such as methylation patterns and histone modifications, along with expression profiles. In this paper we show the importance of the NuChart application for the integration of multi-omic data in a systems biology fashion, with particular interest in cytogenetic applications of these techniques. Moreover, we demonstrate how the integration of multi-omic data can provide useful information in understanding why genes are in certain specific positions inside the nucleus and how epigenetic patterns correlate with their expression. PMID:25717338

  11. A simple cytogenetic method to detect chromosomally integrated human herpesvirus-6.

    PubMed

    Ohye, Tamae; Kawamura, Yoshiki; Inagaki, Hidehito; Yoshikawa, Akiko; Ihira, Masaru; Yoshikawa, Tetsushi; Kurahashi, Hiroki

    2016-02-01

    Some healthy individuals carry human herpesvirus-6 (HHV-6) within a host chromosome, which is called inherited chromosomally integrated human herpesvirus-6 (iciHHV-6). Because iciHHV-6 is generally considered a non-pathogenic condition, it is important to distinguish iciHHV-6 from HHV-6 reactivation in immunocompromised hosts because both conditions manifest high copy numbers of the HHV-6 in peripheral blood mononuclear cells. Although fluorescent in situ hybridization (FISH) is a reliable method for the diagnosis of iciHHV-6, HHV-6-specific FISH probes are not commercially available. In our present study, we established a simple PCR-based method for producing FISH probes that can detect the chromosomal integration site of iciHHV-6 at high sensitivity. Using these probes, we confirmed that HHV-6 signals were consistently located at the telomeric region in all of the 13 iciHHV-6 individuals examined. Interestingly, in all seven Japanese iciHHV-6A patients, signals were detected exclusively on chromosome 22q. This method provides a simple and fast approach for iciHHV-6 diagnosis in the clinical laboratory. PMID:26549829

  12. Tagged Chromosomal Insertion Site System: A Method to Study Lamina-Associated Chromatin.

    PubMed

    Harr, Jennifer C; Reddy, Karen L

    2016-01-01

    The three-dimensional (3D) organization of the genome is important for chromatin regulation. This organization is nonrandom and appears to be tightly correlated with or regulated by chromatin state and scaffolding proteins. To understand how specific DNA and chromatin elements contribute to the functional organization of the genome, we developed a new tool-the tagged chromosomal insertion site (TCIS) system-to identify and study minimal DNA sequences that drive nuclear compartmentalization and applied this system to specifically study the role of cis elements in targeting DNA to the nuclear lamina. The TCIS system allows Cre-recombinase-mediated site-directed integration of any DNA fragment into a locus tagged with lacO arrays, thus enabling both functional molecular studies and positional analysis of the altered locus. This system can be used to study the minimal DNA sequences that target the nuclear periphery (or other nuclear compartments), allowing researchers to understand how genome-wide results obtained, for example, by DNA adenine methyltransferase identification, chromosome conformation capture (HiC), or related methods, connect to the actual organization of DNA and chromosomes at the single-cell level. Finally, TCIS allows one to test roles for specific proteins in chromatin reorganization and to determine how changes in nuclear environment affect chromatin state and gene regulation at a single locus. PMID:26778570

  13. Recombinant cells that highly express chromosomally-integrated heterologous genes

    DOEpatents

    Ingram, L.O.; Ohta, Kazuyoshi; Wood, B.E.

    1998-10-13

    Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol. 13 figs.

  14. Recombinant cells that highly express chromosomally-integrated heterologous gene

    DOEpatents

    Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

    2007-03-20

    Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

  15. Recombinant cells that highly express chromosomally-integrated heterologous genes

    DOEpatents

    Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

    1998-01-01

    Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

  16. Recombinant cells that highly express chromosomally-integrated heterologous genes

    DOEpatents

    Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

    2000-08-22

    Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

  17. Selective pressures to maintain attachment site specificity of integrative and conjugative elements.

    PubMed

    Menard, Kayla L; Grossman, Alan D

    2013-01-01

    Integrative and conjugative elements (ICEs) are widespread mobile genetic elements that are usually found integrated in bacterial chromosomes. They are important agents of evolution and contribute to the acquisition of new traits, including antibiotic resistances. ICEs can excise from the chromosome and transfer to recipients by conjugation. Many ICEs are site-specific in that they integrate preferentially into a primary attachment site in the bacterial genome. Site-specific ICEs can also integrate into secondary locations, particularly if the primary site is absent. However, little is known about the consequences of integration of ICEs into alternative attachment sites or what drives the apparent maintenance and prevalence of the many ICEs that use a single attachment site. Using ICEBs1, a site-specific ICE from Bacillus subtilis that integrates into a tRNA gene, we found that integration into secondary sites was detrimental to both ICEBs1 and the host cell. Excision of ICEBs1 from secondary sites was impaired either partially or completely, limiting the spread of ICEBs1. Furthermore, induction of ICEBs1 gene expression caused a substantial drop in proliferation and cell viability within three hours. This drop was dependent on rolling circle replication of ICEBs1 that was unable to excise from the chromosome. Together, these detrimental effects provide selective pressure against the survival and dissemination of ICEs that have integrated into alternative sites and may explain the maintenance of site-specific integration for many ICEs. PMID:23874222

  18. Induction of site-specific chromosomal translocations in embryonic stem cells by CRISPR/Cas9

    PubMed Central

    Jiang, Junfeng; Zhang, Li; Zhou, Xingliang; Chen, Xi; Huang, Guanyi; Li, Fengsheng; Wang, Ruizhe; Wu, Nancy; Yan, Youzhen; Tong, Chang; Srivastava, Sankalp; Wang, Yue; Liu, Houqi; Ying, Qi-Long

    2016-01-01

    Chromosomal translocation is the most common form of chromosomal abnormality and is often associated with congenital genetic disorders, infertility, and cancers. The lack of cellular and animal models for chromosomal translocations, however, has hampered our ability to understand the underlying disease mechanisms and to develop new therapies. Here, we show that site-specific chromosomal translocations can be generated in mouse embryonic stem cells (mESCs) via CRISPR/Cas9. Mouse ESCs carrying translocated chromosomes can be isolated and expanded to establish stable cell lines. Furthermore, chimeric mice can be generated by injecting these mESCs into host blastocysts. The establishment of ESC-based cellular and animal models of chromosomal translocation by CRISPR/Cas9 provides a powerful platform for understanding the effect of chromosomal translocation and for the development of new therapeutic strategies. PMID:26898344

  19. Chromosome Sites Play Dual Roles to Establish Homologous Synapsis during Meiosis in C. elegans

    PubMed Central

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

    2015-01-01

    SUMMARY We have investigated the role of pairing centers (PCs), cis-acting sites required for accurate segregation of homologous chromosomes during meiosis in C. elegans. We find that these sites play two distinct roles that contribute to proper segregation. Chromosomes lacking PCs usually fail to synapse and also lack a synapsis-independent stabilization activity. The presence of a PC on just one copy of a chromosome pair promotes synapsis but does not support synapsis-independent pairing stabilization, indicating that these functions are separable. Once initiated, synapsis is highly processive, even between nonhomologous chromosomes of disparate lengths, elucidating how translocations suppress meiotic recombination in C. elegans. These findings suggest a multistep pathway for chromosome synapsis in which PCs impart selectivity and efficiency through a “kinetic proofreading” mechanism. We speculate that concentration of these activities at one region per chromosome may have coevolved with the loss of a point centromere to safeguard karyotype stability. PMID:16360034

  20. Chromosome site for Epstein-Barr virus DNA in a Burkitt tumor cell line and in lymphocytes growth-transformed in vitro.

    PubMed Central

    Henderson, A; Ripley, S; Heller, M; Kieff, E

    1983-01-01

    The Epstein-Barr virus (EBV) genome stably persists in latently infected Burkitt tumor cells and growth-transformed B lymphocytes. These cells usually contain multiple copies of episomal viral DNA. Cytological hybridization of recombinant viral DNA fragments to metaphase chromosomes of two latently infected cell lines demonstrates that viral DNA localizes to both chromatids of one homologue of chromosome 1 in Namalwa, a Burkitt tumor cell line, and to both chromatids of one homologue of chromosome 4 in IB4, a cell line with transformed growth properties in vitro. The site of chromosome association remains stable in a clone of IB4 cells. Probes from five separate regions of the EBV genome hybridize to the same chromosome regions. A previously undescribed achromatic site is identified within the region of EBV chromosome cytological hybridization. These observations suggest that most or all of the EBV genome is integrated into the chromosomal DNA of Namalwa and IB4 cells. Although the chromosomal sites of EBV DNA association are among those regions with homology to the EBV IR3 repeated DNA sequence, EBV IR3 did not mediate recombination between EBV and chromosomal DNA. Images PMID:6300885

  1. Effect of LexA on Chromosomal Integration of CTXϕ in Vibrio cholerae

    PubMed Central

    Pant, Archana; Anbumani, D; Bag, Satyabrata; Mehta, Ojasvi; Kumar, Pawan; Saxena, Shruti; Nair, G. Balakrish

    2015-01-01

    ABSTRACT The genesis of toxigenic Vibrio cholerae involves acquisition of CTXϕ, a single-stranded DNA (ssDNA) filamentous phage that encodes cholera toxin (CT). The phage exploits host-encoded tyrosine recombinases (XerC and XerD) for chromosomal integration and lysogenic conversion. The replicative genome of CTXϕ produces ssDNA by rolling-circle replication, which may be used either for virion production or for integration into host chromosome. Fine-tuning of different ssDNA binding protein (Ssb) levels in the host cell is crucial for cellular functioning and important for CTXϕ integration. In this study, we mutated the master regulator gene of SOS induction, lexA, of V. cholerae because of its known role in controlling levels of Ssb proteins in other bacteria. CTXϕ integration decreased in cells with a ΔlexA mutation and increased in cells with an SOS-noninducing mutation, lexA (Ind−). We also observed that overexpression of host-encoded Ssb (VC0397) decreased integration of CTXϕ. We propose that LexA helps CTXϕ integration, possibly by fine-tuning levels of host- and phage-encoded Ssbs. IMPORTANCE Cholera toxin is the principal virulence factor responsible for the acute diarrheal disease cholera. CT is encoded in the genome of a lysogenic filamentous phage, CTXϕ. Vibrio cholerae has a bipartite genome and harbors single or multiple copies of CTXϕ prophage in one or both chromosomes. Two host-encoded tyrosine recombinases (XerC and XerD) recognize the folded ssDNA genome of CTXϕ and catalyze its integration at the dimer resolution site of either one or both chromosomes. Fine-tuning of ssDNA binding proteins in host cells is crucial for CTXϕ integration. We engineered the V. cholerae genome and created several reporter strains carrying ΔlexA or lexA (Ind−) alleles. Using the reporter strains, the importance of LexA control of Ssb expression in the integration efficiency of CTXϕ was demonstrated. PMID:26503849

  2. Beyond the chromosome: the prevalence of unique extra-chromosomal bacteriophages with integrated virulence genes in pathogenic Staphylococcus aureus.

    PubMed

    Utter, Bryan; Deutsch, Douglas R; Schuch, Raymond; Winer, Benjamin Y; Verratti, Kathleen; Bishop-Lilly, Kim; Sozhamannan, Shanmuga; Fischetti, Vincent A

    2014-01-01

    In Staphylococcus aureus, the disease impact of chromosomally integrated prophages on virulence is well described. However, the existence of extra-chromosomal prophages, both plasmidial and episomal, remains obscure. Despite the recent explosion in bacterial and bacteriophage genomic sequencing, studies have failed to specifically focus on extra-chromosomal elements. We selectively enriched and sequenced extra-chromosomal DNA from S. aureus isolates using Roche-454 technology and uncovered evidence for the widespread distribution of multiple extra-chromosomal prophages (ExPΦs) throughout both antibiotic-sensitive and -resistant strains. We completely sequenced one such element comprised of a 43.8 kbp, circular ExPΦ (designated ФBU01) from a vancomycin-intermediate S. aureus (VISA) strain. Assembly and annotation of ФBU01 revealed a number of putative virulence determinants encoded within a bacteriophage immune evasion cluster (IEC). Our identification of several potential ExPΦs and mobile genetic elements (MGEs) also revealed numerous putative virulence factors and antibiotic resistance genes. We describe here a previously unidentified level of genetic diversity of stealth extra-chromosomal elements in S. aureus, including phages with a larger presence outside the chromosome that likely play a prominent role in pathogenesis and strain diversity driven by horizontal gene transfer (HGT). PMID:24963913

  3. Methods for integration site distribution analyses in animal cell genomes

    PubMed Central

    Ciuffi, Angela; Ronen, Keshet; Brady, Troy; Malani, Nirav; Wang, Gary; Berry, Charles C.; Bushman, Frederic D.

    2014-01-01

    The question of where retroviral DNA becomes integrated in chromosomes is important for understanding (i) the mechanisms of viral growth, (ii) devising new anti-retroviral therapy, (iii) understanding how genomes evolve, and (iv) developing safer methods for gene therapy. With the completion of genome sequences for many organisms, it has become possible to study integration targeting by cloning and sequencing large numbers of host–virus DNA junctions, then mapping the host DNA segments back onto the genomic sequence. This allows statistical analysis of the distribution of integration sites relative to the myriad types of genomic features that are also being mapped onto the sequence scaffold. Here we present methods for recovering and analyzing integration site sequences. PMID:19038346

  4. Chromatin Folding, Fragile Sites, and Chromosome Aberrations Induced by Low- and High- LET Radiation

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Cox, Bradley; Asaithamby, Aroumougame; Chen, David J.; Wu, Honglu

    2013-01-01

    We previously demonstrated non-random distributions of breaks involved in chromosome aberrations induced by low- and high-LET radiation. To investigate the factors contributing to the break point distribution in radiation-induced chromosome aberrations, human epithelial cells were fixed in G1 phase. Interphase chromosomes were hybridized with a multicolor banding in situ hybridization (mBAND) probe for chromosome 3 which distinguishes six regions of the chromosome in separate colors. After the images were captured with a laser scanning confocal microscope, the 3-dimensional structure of interphase chromosome 3 was reconstructed at multimega base pair scale. Specific locations of the chromosome, in interphase, were also analyzed with bacterial artificial chromosome (BAC) probes. Both mBAND and BAC studies revealed non-random folding of chromatin in interphase, and suggested association of interphase chromatin folding to the radiation-induced chromosome aberration hotspots. We further investigated the distribution of genes, as well as the distribution of breaks found in tumor cells. Comparisons of these distributions to the radiation hotspots showed that some of the radiation hotspots coincide with the frequent breaks found in solid tumors and with the fragile sites for other environmental toxins. Our results suggest that multiple factors, including the chromatin structure and the gene distribution, can contribute to radiation-induced chromosome aberrations.

  5. Isolation of the human chromosomal band Xq28 within somatic cell hybrids by fragile X site breakage.

    PubMed Central

    Warren, S T; Knight, S J; Peters, J F; Stayton, C L; Consalez, G G; Zhang, F P

    1990-01-01

    The chromosomal fragile-site mapping to Xq27.3 is associated with a frequent form of mental retardation and is prone to breakage after induced deoxyribonucleotide pool perturbation. The human hypoxanthine phosphoribosyltransferase (HPRT) and glucose-6-phosphate dehydrogenase (G6PD) genes flank the fragile X chromosome site and can be used to monitor integrity of the site in human-hamster somatic cell hybrids deficient in the rodent forms of these activities. After induction of the fragile X site, negative selection for HPRT and positive enrichment for G6PD resulted in 31 independent colonies of HPRT-,G6PD+ phenotype. Southern blot analysis demonstrated the loss of all tested markers proximal to the fragile X site with retention of all tested human Xq28 loci in a majority of the hybrids. In situ hybridization with a human-specific probe demonstrated the translocation of a small amount of human DNA to rodent chromosomes in these hybrids, suggesting chromosome breakage at the fragile X site and the subsequent translocation of Xq28. Southern blot hybridization of hybrid-cell DNA, resolved by pulsed-field gel electrophoresis, for human-specific repetitive sequences revealed abundant CpG-islands within Xq28, consistent with its known gene density. The electrophoretic banding patterns of human DNA among the hybrids were remarkably consistent, suggesting that fragile X site breakage is limited to a relatively small region in Xq27-28. These somatic cell hybrids, containing Xq27.3-qter as the sole human DNA, will aid the search for DNA associated with the fragile X site and will augment the high resolution genomic analysis of Xq28, including the identification of candidate genes for genetic-disease loci mapping to this region. Images PMID:2339126

  6. Flagellar region 3b supports strong expression of integrated DNA and the highest chromosomal integration efficiency of the Escherichia coli flagellar regions

    PubMed Central

    Juhas, Mario; Ajioka, James W

    2015-01-01

    The Gram-negative bacterium Escherichia coli is routinely used as the chassis for a variety of biotechnology and synthetic biology applications. Identification and analysis of reliable chromosomal integration and expression target loci is crucial for E. coli engineering. Chromosomal loci differ significantly in their ability to support integration and expression of the integrated genetic circuits. In this study, we investigate E. coli K12 MG1655 flagellar regions 2 and 3b. Integration of the genetic circuit into seven and nine highly conserved genes of the flagellar regions 2 (motA, motB, flhD, flhE, cheW, cheY and cheZ) and 3b (fliE, F, G, J, K, L, M, P, R), respectively, showed significant variation in their ability to support chromosomal integration and expression of the integrated genetic circuit. While not reducing the growth of the engineered strains, the integrations into all 16 target sites led to the loss of motility. In addition to high expression, the flagellar region 3b supports the highest efficiency of integration of all E. coli K12 MG1655 flagellar regions and is therefore potentially the most suitable for the integration of synthetic genetic circuits. PMID:26074421

  7. Distribution of 45S rDNA sites in chromosomes of plants: Structural and evolutionary implications

    PubMed Central

    2012-01-01

    Background 45S rDNA sites are the most widely documented chromosomal regions in eukaryotes. The analysis of the distribution of these sites along the chromosome in several genera has suggested some bias in their distribution. In order to evaluate if these loci are in fact non-randomly distributed and what is the influence of some chromosomal and karyotypic features on the distribution of these sites, a database was built with the position and number of 45S rDNA sites obtained by FISH together with other karyotypic data from 846 plant species. Results In angiosperms the most frequent numbers of sites per diploid karyotype were two and four, suggesting that in spite of the wide dispersion capacity of these sequences the number of rDNA sites tends to be restricted. The sites showed a preferential distribution on the short arms, mainly in the terminal regions. Curiously, these sites were frequently found on the short arms of acrocentric chromosomes where they usually occupy the whole arm. The trend to occupy the terminal region is especially evident in holokinetic chromosomes, where all of them were terminally located. In polyploids there is a trend towards reduction in the number of sites per monoploid complement. In gymnosperms, however, the distribution of rDNA sites varied strongly among the sampled families. Conclusions The location of 45S rDNA sites do not vary randomly, occurring preferentially on the short arm and in the terminal region of chromosomes in angiosperms. The meaning of this preferential location is not known, but some hypotheses are considered and the observed trends are discussed. PMID:23181612

  8. Chromosomal integration of recombinant alpha-amylase and glucoamylase genes in saccharomyces cerevisiae for starch conversion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recombinant constructs of barley '-amylase and Lentinula edodes glucoamylase genes were integrated into the chromosomes of Saccharomyces cerevisiae. The insertion was confirmed by PCR amplification of the gene sequence in the chromosomes. The expression was analyzed by SDS-PAGE of the enzymes puri...

  9. An improved Tn7-lux reporter for broad host range, chromosomally-integrated promoter fusions in Gram-negative bacteria.

    PubMed

    Glassing, Angela; Lewis, Thomas A

    2015-11-01

    An improved vector for chromosomally-integrated promoter-lux fusions is described. The modified vector was tested in parallel with the unmodified vector using the well-characterized Escherichia coli araBAD promoter in the Pseudomonas aeruginosa attTn7 site. The modified mini-Tn7 showed reduced background luminescence, and increased luminescence upon induction, giving >16-fold higher induction ratio. PMID:26341612

  10. Integrated Genomic and Transcriptional Profiling Identifies Chromosomal Loci with Altered Gene Expression in Cervical Cancer

    PubMed Central

    Wilting, Saskia M.; de Wilde, Jillian; Meijer, Chris J. L. M.; Berkhof, Johannes; Yi, Yajun; van Wieringen, Wessel N.; Braakhuis, Boudewijn J. M.; Meijer, Gerrit A.; Ylstra, Bauke; Snijders, Peter J. F.; Steenbergen, Renske D. M.

    2009-01-01

    For a better understanding of the consequences of recurrent chromosomal alterations in cervical carcinomas, we integrated genome-wide chromosomal and transcriptional profiles of 10 squamous cell carcinomas (SCCs), 5 adenocarcinomas (AdCAs) and 6 normal controls. Previous genomic profiling showed that gains at chromosome arms 1q, 3q, and 20q as well as losses at 8q, 10q, 11q, and 13q were common in cervical carcinomas. Altered regions spanned multiple megabases, and the extent to which expression of genes located there is affected remains unclear. Expression analysis of these previously chromosomally profiled carcinomas yielded 83 genes with significantly differential expression between carcinomas and normal epithelium. Application of differential gene locus mapping (DIGMAP) analysis and the array CGH expression integration tool (ACE-it) identified hotspots within large chromosomal alterations in which gene expression was altered as well. Chromosomal gains of the long arms of chromosome 1, 3, and 20 resulted in increased expression of genes located at 1q32.1-32.2, 3q13.32-23, 3q26.32-27.3, and 20q11.21-13.33, whereas a chromosomal loss of 11q22.3-25 was related to decreased expression of genes located in this region. Overexpression of DTX3L, PIK3R4, ATP2C1, and SLC25A36, all located at 3q21.1-23 and identified by DIGMAP, ACE-it or both, was confirmed in an independent validation sample set consisting of 12 SCCs and 13 normal ectocervical samples. In conclusion, integrated chromosomal and transcriptional profiling identified chromosomal hotspots at 1q, 3q, 11q, and 20q with altered gene expression within large commonly altered chromosomal regions in cervical cancer. PMID:18618715

  11. Transfer of plasmid RP4 to Myxococcus xanthus and evidence for its integration into the chromosome.

    PubMed Central

    Breton, A M; Jaoua, S; Guespin-Michel, J

    1985-01-01

    The broad-host-range plasmid RP4 and its derivative R68.45 were transferred to Myxococcus xanthus DK101 and DZ1; RP4 was maintained integrated in the chromosome. Loss of plasmid markers occurred during the growth of the transconjugants, which could be prevented by selective pressure with oxytetracycline. The integrated plasmid was transferred back to Escherichia coli often as RP4-prime plasmids carrying various segments of the M. xanthus chromosome. It also mediated chromosomal transfer between M. xanthus strains. Images PMID:3918015

  12. Hanford site integrated pest management plan

    SciTech Connect

    Giddings, R.F.

    1996-04-09

    The Hanford Site Integrated Pest Management Plan (HSIPMP) defines the Integrated Pest Management (IPM) decision process and subsequent strategies by which pest problems are to be solved at all Hanford Site properties per DOE-RL Site Infrastructure Division memo (WHC 9505090). The HSIPMP defines the roles that contractor organizations play in supporting the IPM process. In short the IPM process anticipates and prevents pest activity and infestation by combining several strategies to achieve long-term pest control solutions.

  13. Fragile Sites of 'Valencia' Sweet Orange (Citrus sinensis) Chromosomes Are Related with Active 45s rDNA.

    PubMed

    Lan, Hong; Chen, Chun-Li; Miao, Yin; Yu, Chang-Xiu; Guo, Wen-Wu; Xu, Qiang; Deng, Xiu-Xin

    2016-01-01

    Citrus sinensis chromosomes present a morphological differentiation of bands after staining by the fluorochromes CMA and DAPI, but there is still little information on its chromosomal characteristics. In this study, the chromosomes in 'Valencia' C. sinensis were analyzed by fluorescence in situ hybridization (FISH) using telomere DNA and the 45S rDNA gene as probes combining CMA/DAPI staining, which showed that there were two fragile sites in sweet orange chromosomes co-localizing at distended 45S rDNA regions, one proximally locating on B-type chromosome and the other subterminally locating on D-type chromosome. While the chromosomal CMA banding and 45S rDNA FISH mapping in the doubled haploid line of 'Valencia' C. sinensis indicated six 45S rDNA regions, four were identified as fragile sites as doubled comparing its parental line, which confirmed the cytological heterozygosity and chromosomal heteromorphisms in sweet orange. Furthermore, Ag-NOR identified two distended 45S rDNA regions to be active nucleolar organizing regions (NORs) in diploid 'Valencia' C. sinensis. The occurrence of quadrivalent in meiosis of pollen mother cells (PMCs) in 'Valencia' sweet orange further confirmed it was a chromosomal reciprocal translocation line. We speculated this chromosome translocation was probably related to fragile sites. Our data provide insights into the chromosomal characteristics of the fragile sites in 'Valencia' sweet orange and are expected to facilitate the further investigation of the possible functions of fragile sites. PMID:26977938

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

    SciTech Connect

    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.

  15. Progesterone receptor gene maps to human chromosome band 11q13, the site of the mammary oncogene int-2

    SciTech Connect

    Law, M.L.; Kao, F.T.; Wei, Q.; Hartz, J.A.; Greene, G.L.; Zarucki-Schulz, T.; Conneely, O.M.; Jones, C.; Puck, T.T.; O'Malley, B.W.; Horwitz, K.B.

    1987-05-01

    Progesterone is involved in the development and progression of breast cancers, and progesterone receptors (PR) are important markers of hormone dependence and disease prognosis. The authors have used a human PR cDNA probe, genomic DNA blotting of a series of Chinese hamster-human cell hybrids, and in situ hybridization to map the human PR gene to chromosome 11, band q13. This band also contains the human homolog of the mouse mammary tumor virus integration site, int-2, which surrounds a protooncogene thought to be involved in the development of murine mammary cancers. That these two genes share the same chromosomal location raises important questions about their possible linkage and about the relationship between the mammary-specific oncogene and the steroid hormone in the development, growth, and hormone dependence of human breast cancers.

  16. An Integrated Linkage, Chromosome, and Genome Map for the Yellow Fever Mosquito Aedes aegypti

    PubMed Central

    Timoshevskiy, Vladimir A.; Severson, David W.; deBruyn, Becky S.; Black, William C.; Sharakhov, Igor V.; Sharakhova, Maria V.

    2013-01-01

    Background Aedes aegypti, the yellow fever mosquito, is an efficient vector of arboviruses and a convenient model system for laboratory research. Extensive linkage mapping of morphological and molecular markers localized a number of quantitative trait loci (QTLs) related to the mosquito's ability to transmit various pathogens. However, linking the QTLs to Ae. aegypti chromosomes and genomic sequences has been challenging because of the poor quality of polytene chromosomes and the highly fragmented genome assembly for this species. Methodology/Principal Findings Based on the approach developed in our previous study, we constructed idiograms for mitotic chromosomes of Ae. aegypti based on their banding patterns at early metaphase. These idiograms represent the first cytogenetic map developed for mitotic chromosomes of Ae. aegypti. One hundred bacterial artificial chromosome clones carrying major genetic markers were hybridized to the chromosomes using fluorescent in situ hybridization. As a result, QTLs related to the transmission of the filarioid nematode Brugia malayi, the avian malaria parasite Plasmodium gallinaceum, and the dengue virus, as well as sex determination locus and 183 Mbp of genomic sequences were anchored to the exact positions on Ae. aegypti chromosomes. A linear regression analysis demonstrated a good correlation between positions of the markers on the physical and linkage maps. As a result of the recombination rate variation along the chromosomes, 12 QTLs on the linkage map were combined into five major clusters of QTLs on the chromosome map. Conclusion This study developed an integrated linkage, chromosome, and genome map—iMap—for the yellow fever mosquito. Our discovery of the localization of multiple QTLs in a few major chromosome clusters suggests a possibility that the transmission of various pathogens is controlled by the same genomic loci. Thus, the iMap will facilitate the identification of genomic determinants of traits responsible

  17. A database system for constructing, integrating, and displaying physical maps of chromosome 19

    SciTech Connect

    Slezak, T.; Wagner, M.; Yeh, Mimi; Ashworth, L.; Nelson, D.; Ow, D.; Branscomb, E.; Carrano, A.

    1994-06-01

    Efforts are underway at numerous sites around the world to construct physical maps of all human chromosomes. These maps will enable researchers to locate, characterize, and eventually understand the genes that control human structure and function. Accomplishing this goal will require a staggering amount of innovation and advancement of biological technology. The volume and complexity of the data already generated requires a sophisticated array of computational support to collect, store, analyze, integrate, and display it in biologically meaningful ways. The Human Genome Center at Livermore has spent the last 6 years constructing a database system to support its physical mapping efforts on human chromosome 19. Our computational support team is composed of experienced computer professionals who share a common pragmatic primary goal of rapidly supplying tools that meet the ever-changing needs of the biologists. Most papers describing computational support of genome research concentrate on mathematical details of key algorithms. However, in this paper we would like to concentrate on the design issues, tradeoffs, and consequences from the point of view of building a complex database system to support leading-edge genomic research. We introduce the topic of physical mapping, discuss the key design issues involved in our databases, and discuss the use of this data by our major tools (DNA fingerprint analysis and overlap computation, contig assembly, map integration, and database browsing.) Given the advantage of hindsight, we discuss what worked, what didn`t, and how we will evolve from here. As early pioneers in this field we hope that our experience may prove useful to others who are now beginning to design and construct similar systems.

  18. Host factors in retroviral integration and the selection of integration target sites

    PubMed Central

    Craigie, Robert; Bushman, Frederic D.

    2015-01-01

    In order to replicate, a retrovirus must integrate a DNA copy of the viral RNA genome into a chromosome of the host cell. The study of retroviral integration has advanced considerably in the last few years. Here we focus on host factor interactions and the linked area of integration targeting. Genome-wide screens for cellular factors affecting HIV replication have identified a series of host cell proteins that may mediate subcellular trafficking of integration complexes, nuclear import, and integration target site selection. The cell transcriptional co-activator protein LEDGF/p75 has been identified as a tethering factor important for HIV integration, and recently, BET proteins (Brd2, 4, and 4) have been identified as tethering factors for the gammaretroviruses. A new class of HIV inhibitors has been developed targeting the HIV-1 IN-LEDGF binding site, though surprisingly these inhibitors appear to block assembly late during replication and do not act at the integration step. Going forward, genome-wide studies of HIV-host interactions offer many new starting points to investigate HIV replication and identify potential new inhibitor targets. PMID:26104434

  19. Dgcr8 and Dicer are essential for sex chromosome integrity during meiosis in males

    PubMed Central

    Modzelewski, Andrew J.; Hilz, Stephanie; Crate, Elizabeth A.; Schweidenback, Caterina T. H.; Fogarty, Elizabeth A.; Grenier, Jennifer K.; Freire, Raimundo; Cohen, Paula E.; Grimson, Andrew

    2015-01-01

    ABSTRACT Small RNAs play crucial roles in regulating gene expression during mammalian meiosis. To investigate the function of microRNAs (miRNAs) and small interfering RNAs (siRNAs) during meiosis in males, we generated germ-cell-specific conditional deletions of Dgcr8 and Dicer in mice. Analysis of spermatocytes from both conditional knockout lines revealed that there were frequent chromosomal fusions during meiosis, always involving one or both sex chromosomes. RNA sequencing indicates upregulation of Atm in spermatocytes from miRNA-deficient mice, and immunofluorescence imaging demonstrates an increased abundance of activated ATM kinase and mislocalization of phosphorylated MDC1, an ATM phosphorylation substrate. The Atm 3′UTR contains many potential microRNA target sites, and, notably, target sites for several miRNAs depleted in both conditional knockout mice were highly effective at promoting repression. RNF8, a telomere-associated protein whose localization is controlled by the MDC1–ATM kinase cascade, normally associates with the sex chromosomes during pachytene, but in both conditional knockouts redistributed to the autosomes. Taken together, these results suggest that Atm dysregulation in microRNA-deficient germ lines contributes to the redistribution of proteins involved in chromosomal stability from the sex chromosomes to the autosomes, resulting in sex chromosome fusions during meiotic prophase I. PMID:25934699

  20. [Integration sites and their characteristic analysis of piggyBac transposon in cattle genome].

    PubMed

    Du, Xin-Hua; Gao, Xue; Zhang, Lu-Pei; Gao, Hui-Jiang; Li, Jun-Ya; Xu, Shang-Zhong

    2013-06-01

    As a useful tool for genetic engineering, piggyBac (PB) transposons have been widely used in more than one species of transgenosis or generating mutation studies. At present, the studies about PB transposons in cattle were few. In order to get the PB transposon integration sites and summarize its characteristics in bovine genome, donor plasmid of PB[CMV-EGFP] and helper-dependent plasmid of pcDNA-PBase were constructed and transferred into bovine fibroblasts by Amaxa basic nucleofector kit for primary mammalian fibroblasts. Cell clones stably transfected were obtained after screening by G-418. Genomic DNA of transgenic cells was extracted and the integration sites of PB transposon were detected by genome walking technology. Eight integration sites were obtained in bovine genome, although only 5 sites were mapped on chromosomes 1, 2, 11, and X chromosome. We found that PB transposon was inserted into the "TTAA" location and integrated into the intergenic non-regulatory sites between two genes. Analysis of the composition of the five bases, which was close to the side of the PB integration sites "TTAA", showed that PB 5' tended to be inserted into region rich in GC (62.5%). From the study, we got that transposition occurred in cattle genome by PB transposons and the integration site information acquired from the research will provide theoretical references for bovine study by PB transposon. PMID:23774022

  1. Influence of chromosomal integration on glucocorticoid-regulated transcription of growth-stimulating papillomavirus genes E6 and E7 in cervical carcinoma cells

    SciTech Connect

    Von Knebel Doeberitz, M.; Bauknecht, T.; Bartsch, D.; Zur Hausen, H. )

    1991-02-15

    In most cervical carcinoma cells the E6 and E7 genes of specific human papillomaviruses are transcribed from viral sequences integrated into host cell chromosomes. Glucocorticoids activate the promoter elements of various human papillomaviruses in transient-expression assays. The authors have analyzed the effect of dexamethasone on the transcription rate of human papillomaviruses 18 E6 and E7 genes integrated at different chromosomal sites in four cervical cancer cell lines. Dexamethasone led to an increase in the transcription rate of the integrated E6-E7 sequences in C4-1 and C4-2 cells but led to a decrease in SW 756 cells and did not affect the transcription rate in HeLa cells. It thus appears that dominant regulatory mechanisms presumably depending on the chromosomal integration site are able to override the response of the viral promoter to steroid hormones. The growth rate of all dexamethasone-treated cell lines correlated consistently with the expression of the papillomavirus E6 and E7 genes, supporting their role in the maintenance of the proliferative phenotype of cervical carcinoma cells. Since human papillomaviruses are integrated into the host cell genome at variable, presumably randomly selected chromosomal loci, regulatory mechanisms that influence viral gene expression, and hence cell growth, may differ among cancers of independent clonal origin.

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

    PubMed

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

    2016-06-01

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

  3. Sequential cloning of chromosomes

    DOEpatents

    Lacks, Sanford A.

    1995-07-18

    A method for sequential cloning of chromosomal DNA of a target organism is disclosed. A first DNA segment homologous to the chromosomal DNA to be sequentially cloned is isolated. The first segment has a first restriction enzyme site on either side. A first vector product is formed by ligating the homologous segment into a suitably designed vector. The first vector product is circularly integrated into the target organism's chromosomal DNA. The resulting integrated chromosomal DNA segment includes the homologous DNA segment at either end of the integrated vector segment. The integrated chromosomal DNA is cleaved with a second restriction enzyme and ligated to form a vector-containing plasmid, which is replicated in a host organism. The replicated plasmid is then cleaved with the first restriction enzyme. Next, a DNA segment containing the vector and a segment of DNA homologous to a distal portion of the previously isolated DNA segment is isolated. This segment is then ligated to form a plasmid which is replicated within a suitable host. This plasmid is then circularly integrated into the target chromosomal DNA. The chromosomal DNA containing the circularly integrated vector is treated with a third, retrorestriction (class IIS) enzyme. The cleaved DNA is ligated to give a plasmid that is used to transform a host permissive for replication of its vector. The sequential cloning process continues by repeated cycles of circular integration and excision. The excision is carried out alternately with the second and third enzymes.

  4. Sequential cloning of chromosomes

    DOEpatents

    Lacks, S.A.

    1995-07-18

    A method for sequential cloning of chromosomal DNA of a target organism is disclosed. A first DNA segment homologous to the chromosomal DNA to be sequentially cloned is isolated. The first segment has a first restriction enzyme site on either side. A first vector product is formed by ligating the homologous segment into a suitably designed vector. The first vector product is circularly integrated into the target organism`s chromosomal DNA. The resulting integrated chromosomal DNA segment includes the homologous DNA segment at either end of the integrated vector segment. The integrated chromosomal DNA is cleaved with a second restriction enzyme and ligated to form a vector-containing plasmid, which is replicated in a host organism. The replicated plasmid is then cleaved with the first restriction enzyme. Next, a DNA segment containing the vector and a segment of DNA homologous to a distal portion of the previously isolated DNA segment is isolated. This segment is then ligated to form a plasmid which is replicated within a suitable host. This plasmid is then circularly integrated into the target chromosomal DNA. The chromosomal DNA containing the circularly integrated vector is treated with a third, retrorestriction (class IIS) enzyme. The cleaved DNA is ligated to give a plasmid that is used to transform a host permissive for replication of its vector. The sequential cloning process continues by repeated cycles of circular integration and excision. The excision is carried out alternately with the second and third enzymes. 9 figs.

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

    SciTech Connect

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

    1986-09-01

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

  6. Launch site integration for mixed fleet operations

    NASA Technical Reports Server (NTRS)

    Scott, L. P.

    1990-01-01

    Launch site impacts and integration planning issues are presented to support launch operations for a mixed vehicle fleet (manned and cargo). Proposed ground systems and launch site configurations are described. Prelaunch processing scenarios and schedules are developed for candidate launch vehicles. Earth-to-orbit (ETO) vehicle architectures are presented to meet future launch requirements, including the Space Exploration Initiative (SEI). Flight vehicle design recommendations to enhance launch processing are discussed. The significance of operational designs for future launch vehicles is shown to be a critical factor in planning for mixed fleet launch site operations.

  7. Fragile Sites of ‘Valencia’ Sweet Orange (Citrus sinensis) Chromosomes Are Related with Active 45s rDNA

    PubMed Central

    Lan, Hong; Chen, Chun-Li; Miao, Yin; Yu, Chang-Xiu; Guo, Wen-Wu; Xu, Qiang; Deng, Xiu-Xin

    2016-01-01

    Citrus sinensis chromosomes present a morphological differentiation of bands after staining by the fluorochromes CMA and DAPI, but there is still little information on its chromosomal characteristics. In this study, the chromosomes in ‘Valencia’ C. sinensis were analyzed by fluorescence in situ hybridization (FISH) using telomere DNA and the 45S rDNA gene as probes combining CMA/DAPI staining, which showed that there were two fragile sites in sweet orange chromosomes co-localizing at distended 45S rDNA regions, one proximally locating on B-type chromosome and the other subterminally locating on D-type chromosome. While the chromosomal CMA banding and 45S rDNA FISH mapping in the doubled haploid line of ‘Valencia’ C. sinensis indicated six 45S rDNA regions, four were identified as fragile sites as doubled comparing its parental line, which confirmed the cytological heterozygosity and chromosomal heteromorphisms in sweet orange. Furthermore, Ag-NOR identified two distended 45S rDNA regions to be active nucleolar organizing regions (NORs) in diploid ‘Valencia’ C. sinensis. The occurrence of quadrivalent in meiosis of pollen mother cells (PMCs) in ‘Valencia’ sweet orange further confirmed it was a chromosomal reciprocal translocation line. We speculated this chromosome translocation was probably related to fragile sites. Our data provide insights into the chromosomal characteristics of the fragile sites in ‘Valencia’ sweet orange and are expected to facilitate the further investigation of the possible functions of fragile sites. PMID:26977938

  8. Chromosomal instability in Afrotheria: fragile sites, evolutionary breakpoints and phylogenetic inference from genome sequence assemblies

    PubMed Central

    Ruiz-Herrera, Aurora; Robinson, Terence J

    2007-01-01

    Background Extant placental mammals are divided into four major clades (Laurasiatheria, Supraprimates, Xenarthra and Afrotheria). Given that Afrotheria is generally thought to root the eutherian tree in phylogenetic analysis of large nuclear gene data sets, the study of the organization of the genomes of afrotherian species provides new insights into the dynamics of mammalian chromosomal evolution. Here we test if there are chromosomal bands with a high tendency to break and reorganize in Afrotheria, and by analyzing the expression of aphidicolin-induced common fragile sites in three afrotherian species, whether these are coincidental with recognized evolutionary breakpoints. Results We described 29 fragile sites in the aardvark (OAF) genome, 27 in the golden mole (CAS), and 35 in the elephant-shrew (EED) genome. We show that fragile sites are conserved among afrotherian species and these are correlated with evolutionary breakpoints when compared to the human (HSA) genome. Inddition, by computationally scanning the newly released opossum (Monodelphis domestica) and chicken sequence assemblies for use as outgroups to Placentalia, we validate the HSA 3/21/5 chromosomal synteny as a rare genomic change that defines the monophyly of this ancient African clade of mammals. On the other hand, support for HSA 1/19p, which is also thought to underpin Afrotheria, is currently ambiguous. Conclusion We provide evidence that (i) the evolutionary breakpoints that characterise human syntenies detected in the basal Afrotheria correspond at the chromosomal band level with fragile sites, (ii) that HSA 3p/21 was in the amniote ancestor (i.e., common to turtles, lepidosaurs, crocodilians, birds and mammals) and was subsequently disrupted in the lineage leading to marsupials. Its expansion to include HSA 5 in Afrotheria is unique and (iii) that its fragmentation to HSA 3p/21 + HSA 5/21 in elephant and manatee was due to a fission within HSA 21 that is probably shared by all

  9. Integration of 28 STSs into the physical map of human chromosome 18

    SciTech Connect

    Gerken, S.; White, R.; Bradley, P.

    1994-12-01

    Genes on human chromosome 18 are associated with familial glucocorticoid deficiency (MC2R), pemphigus vulgaris (DSG3) and foliaceus (DSG1), familial amyloidosis (TTR), colorectal carcinoma (DCC), erythropoietic protoporphyria (FECH), follicular lymphoma (BCL2, FVT1), and congenital methemoglobinemia (CYB5). As the resolution of human genetic maps improves, linkage between other diseases and specific regions of chromosome 18 will occur. A physical map of human chromosome 18 will prove useful in identifying candidate genes that are associated with these disorders. Using various physical and genetic mapping techniques, over 35 genes and 19 expressed sequence tags (ESTs) are assigned to human chromosome 18. Most of these genes and several of the ESTs were sublocalized using a well-defined panel of somatic cell hybrids that contain different segments of human chromosome 18. Despite recent efforts, progress in mapping human chromosome 18 has lagged behind that achieved for other chromosomes. Thus, the purpose of this study was to integrate 9 new transcriptional tags [8 brain ESTs (8) and the melanocortin 4 receptor (MC4R) (3)] and 19 simple sequence repeats (SSRs) into the physical map of human chromosome 18. The SSRs were isolated by screening genomic DNA libraries constructed in M13mp18 vectors with oligonucleotide probes that detected dinucleotide d(CA)- and tetranucleotide-repeat motifs. DNA sequences of clones that contained microsatellite repeats were obtained by thermal-cycle sequencing, and STSs were developed from clones that contained numerous repeats. STSs that identified highly polymorphic loci in eight unrelated CEPH parents were used for genotyping. Results of linkage analyses and estimates of heterozygosity for these markers will be reported. 9 refs., 1 fig., 1 tab.

  10. Comprehensive, integrated, remote sensing at DOE sites

    SciTech Connect

    Lackey, J.G.; Burson, Z.G.

    1984-01-01

    The Department of Energy has established a program called Comprehensive, Integrated Remote Sensing (CIRS). The overall objective is to provide a state-of-the-art data base of remotely sensed data for all users of such information at large DOE sites. The primary types of remote sensing provided consist of the following: (1) large format aerial photography; (2) video from aerial platforms; (3) multispectral scanning; and (4) airborne nuclear radiometric surveys. Implementation of the CIRS Program began with field operations at the Savannah River Plant in 1982 and is continuing at that DOE site at a level of effort of about $1.5 m per year. Integrated remote sensing studies were subsequently extended to the West Valley Demonstration Project in the summer and fall of 1984. It is expected that the Program will eventually be extended to cover all large DOE sites on a continuing basis. 2 figures.

  11. Development of an integrated computerized scheme for metaphase chromosome image analysis: a robustness experiment

    NASA Astrophysics Data System (ADS)

    Wang, Xingwei; Zheng, Bin; Li, Shibo; Mulvihill, John J.; Wood, Marc C.; Yuan, Chaowei; Chen, Wei; Liu, Hong

    2008-02-01

    Our integrated computer-aided detection (CAD) scheme includes three basic modules. The first module detects whether a microscopic digital image depicts a metaphase chromosome cell. If a cell is detected, the scheme will justify whether it is analyzable with a decision tree. Once an analyzable cell is detected, the second module is applied to segment individual chromosomes and to compute two important features. Specifically, the scheme utilizes a modified thinning algorithm to identify the medial axis of a chromosome. By tracking perpendicular lines along the medial axis, the scheme computes four feature profiles, identifies centromeres, and assigns polarities of chromosomes based on a set of pre-optimized rules. The third module is followed to classify chromosomes into 24 types. In this module, each chromosome is initially represented by a vector of 31 features. A two-layer classifier with 8 artificial neural networks (ANN) is optimized by a genetic algorithm. A testing chromosome is first classified into one of the seven groups by the ANN in the first layer. Another ANN is then automatically selected from the seven ANNs in the second layer (one for each group) to further classify this chromosome into one of 24 types. To test the performance and robustness of this CAD scheme, we randomly selected and assembled an independent testing dataset. The dataset contains 100 microscopic digital images including 50 analyzable and 50 un-analyzable metphase cells identified by the experts. The centromere location, the corresponding polarity, and karyotype for each individual chromosome were recorded in the "truth" file. The performance of the CAD scheme applied to this image dataset is analyzed and compared with the results in the true file. The assessment accuracies are 93% for the first module, 90.8% for centromere identification and 93.2% for polarity assignment in the second module, over 96% for six chromosome groups and 81.8% for one group in the third module

  12. A physical map of the X chromosome of Drosophila melanogaster: Cosmid contigs and sequence tagged sites

    SciTech Connect

    Madueno, E.; Modolell, J.; Papagiannakis, G.

    1995-04-01

    A physical map of the euchromatic X chromosome of Drosophila melanogaster has been constructed by assembling contiguous arrays of cosmids that were selected by screening a library with DNA isolated from microamplified chromosomal divisions. This map, consisting of 893 cosmids, covers {approximately}64% of the euchromatic part of the chromosome. In addition, 568 sequence tagged sites (STS), in aggregate representing 120 kb of sequenced DNA, were derived from selected cosmids. Most of these STSs, spaced at an average distance of {approximately} 35 kb along the euchromatic region of the chromosome, represent DNA tags that can be used as entry points to the fruitfly genome. Furthermore, 42 genes have been placed on the physical map, either through the hybridization of specific probes to the cosmids or through the fact that they were represented among the STSs. These provide a link between the physical and the genetic maps of D. melanogaster. Nine novel genes have been tentatively identified in Drosophila on the basis of matches between STS sequences and sequences from other species. 32 refs., 3 figs., 4 tabs.

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

    PubMed

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

    2016-02-01

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

  14. Site-specific integration of mycobacteriophage L5: integration-proficient vectors for Mycobacterium smegmatis, Mycobacterium tuberculosis, and bacille Calmette-Guérin.

    PubMed Central

    Lee, M H; Pascopella, L; Jacobs, W R; Hatfull, G F

    1991-01-01

    Mycobacteriophage L5, a temperate phage of mycobacteria, integrates site-specifically into the Mycobacterium smegmatis chromosome. We have identified the int gene and attP site of L5, characterized the chromosomal attachment site (attB), and constructed plasmid vectors that efficiently transform M. smegmatis through stable site-specific integration of the plasmid into the bacterial genome. These integration-proficient plasmids also efficiently transform slow-growing mycobacteria such as the pathogen Mycobacterium tuberculosis and the vaccine strain bacille Calmette-Guérin (BCG). The ability to easily generate stable recombinants in these slow-growing mycobacteria without the requirement for continual selection is of particular importance for the construction of recombinant BCG vaccines and for the isolation and characterization of mycobacterial pathogenic determinants in animal model systems. Integration vectors of this type should be of general use in a number of additional bacterial systems where temperate phages have been identified. Images PMID:1901654

  15. Chromosome-Specific Single-Locus FISH Probes Allow Anchorage of an 1800-Marker Integrated Radiation-Hybrid/Linkage Map of the Domestic Dog Genome to All Chromosomes

    PubMed Central

    Breen, Matthew; Jouquand, Sophie; Renier, Corinne; Mellersh, Cathryn S.; Hitte, Christophe; Holmes, Nigel G.; Chéron, Angélique; Suter, Nicola; Vignaux, Françoise; Bristow, Anna E.; Priat, Catherine; McCann, E.; André, Catherine; Boundy, Sam; Gitsham, Paul; Thomas, Rachael; Bridge, Wendy L.; Spriggs, Helen F.; Ryder, Ed J.; Curson, Alistair; Sampson, Jeff; Ostrander, Elaine A.; Binns, Matthew M.; Galibert, Francis

    2001-01-01

    We present here the first fully integrated, comprehensive map of the canine genome, incorporating detailed cytogenetic, radiation hybrid (RH), and meiotic information. We have mapped a collection of 266 chromosome-specific cosmid clones, each containing a microsatellite marker, to all 38 canine autosomes by fluorescence in situ hybridization (FISH). A 1500-marker RH map, comprising 1078 microsatellites, 320 dog gene markers, and 102 chromosome-specific markers, has been constructed using the RHDF5000-2 whole-genome radiation hybrid panel. Meiotic linkage analysis was performed, with at least one microsatellite marker from each dog autosome on a panel of reference families, allowing one meiotic linkage group to be anchored to all 38 dog autosomes. We present a karyotype in which each chromosome is identified by one meiotic linkage group and one or more RH groups. This updated integrated map, containing a total of 1800 markers, covers >90% of the dog genome. Positional selection of anchor clones enabled us, for the first time, to orientate nearly all of the integrated groups on each chromosome and to evaluate the extent of individual chromosome coverage in the integrated genome map. Finally, the inclusion of 320 dog genes into this integrated map enhances existing comparative mapping data between human and dog, and the 1000 mapped microsatellite markers constitute an invaluable tool with which to perform genome scanning studies on pedigrees of interest. PMID:11591656

  16. Chromosomal integration of hyaluronic acid synthesis (has) genes enhances the molecular weight of hyaluronan produced in Lactococcus lactis.

    PubMed

    Hmar, Rothangmawi Victoria; Prasad, Shashi Bala; Jayaraman, Guhan; Ramachandran, Kadathur B

    2014-12-01

    Microbial production of hyaluronic acid (HA) is an attractive substitute for extraction of this biopolymer from animal tissues. Natural producers such as Streptococcus zooepidemicus are potential pathogens; therefore, production of HA by recombinant bacteria that are generally recognized as safe (GRAS) organisms is a viable alternative that is being extensively explored. However, plasmid-based expression systems for HA production by recombinant bacteria have the inherent disadvantage of reduced productivity because of plasmid instability. To overcome this problem, the HA synthesis genes (hasA-hasB and hasA-hasB-hasC) from has-operon of S. zooepidemicus were integrated into the chromosome of Lactococcus lactis by site-directed, double-homologous recombination developing strains VRJ2AB and VRJ3ABC. The chromosomal integration stabilized the genes and obviated the instability observed in plasmid-expressed recombinant strains. The genome-integrated strains produced higher molecular weight (3.5-4 million Dalton [MDa]) HA compared to the plasmid-expressed strains (2 MDa). High molecular weight HA was produced when the intracellular concentration of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) and uridine diphosphate-glucuronic acid (UDP-GlcUA) was almost equal and hasA to hasB ratio was low. This work suggests an optimal approach to obtain high molecular weight HA in recombinant strains. PMID:25044639

  17. Phosphorylation of the Scc2 cohesin deposition complex subunit regulates chromosome condensation through cohesin integrity

    PubMed Central

    Woodman, Julie; Hoffman, Matthew; Dzieciatkowska, Monika; Hansen, Kirk C.; Megee, Paul C.

    2015-01-01

    The cohesion of replicated sister chromatids promotes chromosome biorientation, gene regulation, DNA repair, and chromosome condensation. Cohesion is mediated by cohesin, which is deposited on chromosomes by a separate conserved loading complex composed of Scc2 and Scc4 in Saccharomyces cerevisiae. Although it is known to be required, the role of Scc2/Scc4 in cohesin deposition remains enigmatic. Scc2 is a phosphoprotein, although the functions of phosphorylation in deposition are unknown. We identified 11 phosphorylated residues in Scc2 by mass spectrometry. Mutants of SCC2 with substitutions that mimic constitutive phosphorylation retain normal Scc2–Scc4 interactions and chromatin association but exhibit decreased viability, sensitivity to genotoxic agents, and decreased stability of the Mcd1 cohesin subunit in mitotic cells. Cohesin association on chromosome arms, but not pericentromeric regions, is reduced in the phosphomimetic mutants but remains above a key threshold, as cohesion is only modestly perturbed. However, these scc2 phosphomimetic mutants exhibit dramatic chromosome condensation defects that are likely responsible for their high inviability. From these data, we conclude that normal Scc2 function requires modulation of its phosphorylation state and suggest that scc2 phosphomimetic mutants cause an increased incidence of abortive cohesin deposition events that result in compromised cohesin complex integrity and Mcd1 turnover. PMID:26354421

  18. Impact of Nucleoporin-Mediated Chromatin Localization and Nuclear Architecture on HIV Integration Site Selection.

    PubMed

    Wong, Richard W; Mamede, João I; Hope, Thomas J

    2015-10-01

    It has been known for a number of years that integration sites of human immunodeficiency virus type 1 (HIV-1) DNA show a preference for actively expressed chromosomal locations. A number of viral and cellular proteins are implicated in this process, but the underlying mechanism is not clear. Two recent breakthrough publications advance our understanding of HIV integration site selection by focusing on the localization of the preferred target genes of integration. These studies reveal that knockdown of certain nucleoporins and components of nucleocytoplasmic trafficking alter integration site preference, not by altering the trafficking of the viral genome but by altering the chromatin subtype localization relative to the structure of the nucleus. Here, we describe the link between the nuclear basket nucleoporins (Tpr and Nup153) and chromatin organization and how altering the host environment by manipulating nuclear structure may have important implications for the preferential integration of HIV into actively transcribed genes, facilitating efficient viral replication. PMID:26136574

  19. Methods of Reprogramming to Induced Pluripotent Stem Cell Associated with Chromosomal Integrity and Delineation of a Chromosome 5q Candidate Region for Growth Advantage.

    PubMed

    Sobol, Maria; Raykova, Doroteya; Cavelier, Lucia; Khalfallah, Ayda; Schuster, Jens; Dahl, Niklas

    2015-09-01

    Induced pluripotent stem cells (iPSCs) have brought great promises for disease modeling and cell-based therapies. One concern related to the use of reprogrammed somatic cells is the loss of genomic integrity and chromosome stability, a hallmark for cancer and many other human disorders. We investigated 16 human iPSC lines reprogrammed by nonintegrative Sendai virus (SeV) and another 16 iPSC lines generated by integrative lentivirus for genetic changes. At early passages we detected cytogenetic rearrangements in 44% (7/16) of iPSC lines generated by lentiviral integration whereas the corresponding figure was 6% (1/16) using SeV-based delivery. The rearrangements were numerical and/or structural with chromosomes 5 and 12 as the most frequently involved chromosomes. Three iPSC lines with chromosome 5 aberrations were derived from one and the same donor. We present in this study the aberrant karyotypes including a duplication of chromosome 5q13q33 that restricts a candidate region for growth advantage. Our results suggest that the use of integrative lentivirus confers a higher risk for cytogenetic abnormalities at early passages when compared to SeV-based reprogramming. In combination, our findings expand the knowledge on acquired cytogenetic aberrations in iPSC after reprogramming and during culture. PMID:25867454

  20. Sequential cloning of chromosomes

    SciTech Connect

    Lacks, S.A.

    1991-12-31

    A method for sequential cloning of chromosomal DNA and chromosomal DNA cloned by this method are disclosed. The method includes the selection of a target organism having a segment of chromosomal DNA to be sequentially cloned. A first DNA segment, having a first restriction enzyme site on either side. homologous to the chromosomal DNA to be sequentially cloned is isolated. A first vector product is formed by ligating the homologous segment into a suitably designed vector. The first vector product is circularly integrated into the target organism`s chromosomal DNA. The resulting integrated chromosomal DNA segment includes the homologous DNA segment at either end of the integrated vector segment. The integrated chromosomal DNA is cleaved with a second restriction enzyme and ligated to form a vector-containing plasmid, which is replicated in a host organism. The replicated plasmid is then cleaved with the first restriction enzyme. Next, a DNA segment containing the vector and a segment of DNA homologous to a distal portion of the previously isolated DNA segment is isolated. This segment is then ligated to form a plasmid which is replicated within a suitable host. This plasmid is then circularly integrated into the target chromosomal DNA. The chromosomal DNA containing the circularly integrated vector is treated with a third, retrorestriction enzyme. The cleaved DNA is ligated to give a plasmid that is used to transform a host permissive for replication of its vector. The sequential cloning process continues by repeated cycles of circular integration and excision. The excision is carried out alternately with the second and third enzymes.

  1. Structural Maintenance of Chromosome (SMC) Proteins Link Microtubule Stability to Genome Integrity*

    PubMed Central

    Laflamme, Guillaume; Tremblay-Boudreault, Thierry; Roy, Marc-André; Andersen, Parker; Bonneil, Éric; Atchia, Kaleem; Thibault, Pierre; D'Amours, Damien; Kwok, Benjamin H.

    2014-01-01

    Structural maintenance of chromosome (SMC) proteins are key organizers of chromosome architecture and are essential for genome integrity. They act by binding to chromatin and connecting distinct parts of chromosomes together. Interestingly, their potential role in providing connections between chromatin and the mitotic spindle has not been explored. Here, we show that yeast SMC proteins bind directly to microtubules and can provide a functional link between microtubules and DNA. We mapped the microtubule-binding region of Smc5 and generated a mutant with impaired microtubule binding activity. This mutant is viable in yeast but exhibited a cold-specific conditional lethality associated with mitotic arrest, aberrant spindle structures, and chromosome segregation defects. In an in vitro reconstitution assay, this Smc5 mutant also showed a compromised ability to protect microtubules from cold-induced depolymerization. Collectively, these findings demonstrate that SMC proteins can bind to and stabilize microtubules and that SMC-microtubule interactions are essential to establish a robust system to maintain genome integrity. PMID:25135640

  2. Comparative Genomic Hybridization of Human Malignant Gliomas Reveals Multiple Amplification Sites and Nonrandom Chromosomal Gains and Losses

    PubMed Central

    Schròck, Evelin; Thiel, Gundula; Lozanova, Tanka; du Manoir, Stanislas; Meffert, Marie-Christine; Jauch, Anna; Speicher, Michael R.; Nürnberg, Peter; Vogel, Siegfried; Janisch, Werner; Donis-Keller, Helen; Ried, Thomas; Witkowski, Regine; Cremer, Thomas

    1994-01-01

    Nine human malignant gliomas (2 astrocytomas grade III and 7 glioblastomas) were analyzed using comparative genomic hybridization (CGH). In addition to the amplification of the EGFR gene at 7p12 in 4 of 9 cases, six new amplification sites were mapped to 1q32, 4q12, 7q21.1, 7q21.2-3, 12p, and 22q12. Nonrandom chromosomal gains and losses were identified with overrepresentation of chromosome 7 and underrepresentation of chromosome 10 as the most frequent events (1 of 2 astrocytomas, 7 of 7 glioblastomas). Gain of a part or the whole chromosome 19 and losses of chromosome bands 9pter-23 and 22q13 were detected each in five cases. Loss of chromosome band 17p13 and gain of chromosome 20 were revealed each in three cases. The validity of the CGH data was confirmed using interphase cytogenetics with YAC clones, chromosome painting in tumor metaphase spreads, and DNA fingerprinting. A comparison of CGH data with the results of chromosome banding analyses indicates that metaphase spreads accessible in primary tumor cell cultures may not represent the clones predominant in the tumor tissue ImagesFigure 1Figure 4Figure 6 PMID:8203461

  3. Module-based construction of plasmids for chromosomal integration of the fission yeast Schizosaccharomyces pombe.

    PubMed

    Kakui, Yasutaka; Sunaga, Tomonari; Arai, Kunio; Dodgson, James; Ji, Liang; Csikász-Nagy, Attila; Carazo-Salas, Rafael; Sato, Masamitsu

    2015-06-01

    Integration of an external gene into a fission yeast chromosome is useful to investigate the effect of the gene product. An easy way to knock-in a gene construct is use of an integration plasmid, which can be targeted and inserted to a chromosome through homologous recombination. Despite the advantage of integration, construction of integration plasmids is energy- and time-consuming, because there is no systematic library of integration plasmids with various promoters, fluorescent protein tags, terminators and selection markers; therefore, researchers are often forced to make appropriate ones through multiple rounds of cloning procedures. Here, we establish materials and methods to easily construct integration plasmids. We introduce a convenient cloning system based on Golden Gate DNA shuffling, which enables the connection of multiple DNA fragments at once: any kind of promoters and terminators, the gene of interest, in combination with any fluorescent protein tag genes and any selection markers. Each of those DNA fragments, called a 'module', can be tandemly ligated in the order we desire in a single reaction, which yields a circular plasmid in a one-step manner. The resulting plasmids can be integrated through standard methods for transformation. Thus, these materials and methods help easy construction of knock-in strains, and this will further increase the value of fission yeast as a model organism. PMID:26108218

  4. Module-based construction of plasmids for chromosomal integration of the fission yeast Schizosaccharomyces pombe

    PubMed Central

    Kakui, Yasutaka; Sunaga, Tomonari; Arai, Kunio; Dodgson, James; Ji, Liang; Csikász-Nagy, Attila; Carazo-Salas, Rafael; Sato, Masamitsu

    2015-01-01

    Integration of an external gene into a fission yeast chromosome is useful to investigate the effect of the gene product. An easy way to knock-in a gene construct is use of an integration plasmid, which can be targeted and inserted to a chromosome through homologous recombination. Despite the advantage of integration, construction of integration plasmids is energy- and time-consuming, because there is no systematic library of integration plasmids with various promoters, fluorescent protein tags, terminators and selection markers; therefore, researchers are often forced to make appropriate ones through multiple rounds of cloning procedures. Here, we establish materials and methods to easily construct integration plasmids. We introduce a convenient cloning system based on Golden Gate DNA shuffling, which enables the connection of multiple DNA fragments at once: any kind of promoters and terminators, the gene of interest, in combination with any fluorescent protein tag genes and any selection markers. Each of those DNA fragments, called a ‘module’, can be tandemly ligated in the order we desire in a single reaction, which yields a circular plasmid in a one-step manner. The resulting plasmids can be integrated through standard methods for transformation. Thus, these materials and methods help easy construction of knock-in strains, and this will further increase the value of fission yeast as a model organism. PMID:26108218

  5. Failure of origin activation in response to fork stalling leads to chromosomal instability at fragile sites.

    PubMed

    Ozeri-Galai, Efrat; Lebofsky, Ronald; Rahat, Ayelet; Bester, Assaf C; Bensimon, Aaron; Kerem, Batsheva

    2011-07-01

    Perturbed DNA replication in early stages of cancer development induces chromosomal instability preferentially at fragile sites. However, the molecular basis for this instability is unknown. Here, we show that even under normal growth conditions, replication fork progression along the fragile site, FRA16C, is slow and forks frequently stall at AT-rich sequences, leading to activation of additional origins to enable replication completion. Under mild replication stress, the frequency of stalling at AT-rich sequences is further increased. Strikingly, unlike in the entire genome, in the FRA16C region additional origins are not activated, suggesting that all potential origins are already activated under normal conditions. Thus, the basis for FRA16C fragility is replication fork stalling at AT-rich sequences and inability to activate additional origins under replication stress. Our results provide a mechanism explaining the replication stress sensitivity of fragile sites and thus, the basis for genomic instability during early stages of cancer development. PMID:21726815

  6. Type I bipolar disorder associated with a fragile site on chromosome 1

    SciTech Connect

    Turecki, G.; Mari, J.J.; M. de Smith, A.C.

    1995-06-19

    The objective of this paper is to study the association between chromosomal fragile sites and type I bipolar disorder. This case-control study compares bipolar patients with normal controls. Ten cases of type I bipolar disorder diagnosed according to DSM-III-R criteria and the Composite International Diagnostic Interview (CIDI) were selected from the Escola Paulista affective disorders outpatient clinic and 10 healthy controls (CIDI negative for psychiatric diagnoses) matched for sex and age were drawn from the otorhinolaryngologic outpatient clinic of the same hospital. The cytogenetic analysis was carried out with blood lymphocytes, which were cultured in a folic acid-free medium. A total of 100 mitoses per subject were blindly analyzed to the psychiatric diagnostic assignment, and fragile sites were identified according to a minimum expected frequency of events per band in conformity with a Poisson distribution. A higher frequency of chromosomal lesions for cases than controls was found for the following bands: 1q32, 5q31, and 11q23, the 1q32 being considered a fragile site. Although no evident neuropsychiatric etiological component has been mapped to the 1q32 region so far, this finding may lead to further investigation of a possible linkage between genetic markers of this region and bipolar disorder. 40 refs., 2 tabs.

  7. New methods for tightly regulated gene expression and highly efficient chromosomal integration of cloned genes for Methanosarcina species

    DOE PAGESBeta

    Guss, Adam M.; Rother, Michael; Zhang, Jun Kai; Kulkkarni, Gargi; Metcalf, William W.

    2008-01-01

    A highly efficient method for chromosomal integration of cloned DNA into Methanosarcina spp. was developed utilizing the site-specific recombination system from the Streptomyces phage φC31. Host strains expressing the φC31 integrase gene and carrying an appropriate recombination site can be transformed with non-replicating plasmids carrying the complementary recombination site at efficiencies similar to those obtained with self-replicating vectors. We have also constructed a series of hybrid promoters that combine the highly expressed M. barkeri P mcrB promoter with binding sites for the tetracycline-responsive, bacterial TetR protein. These promoters are tightly regulated by the presence or absence of tetracycline inmore » strains that express the tetR gene. The hybrid promoters can be used in genetic experiments to test gene essentiality by placing a gene of interest under their control. Thus, growth of strains with tetR -regulated essential genes becomes tetracycline-dependent. A series of plasmid vectors that utilize the site-specific recombination system for construction of reporter gene fusions and for tetracycline regulated expression of cloned genes are reported. These vectors were used to test the efficiency of translation at a variety of start codons. Fusions using an ATG start site were the most active, whereas those using GTG and TTG were approximately one half or one fourth as active, respectively. The CTG fusion was 95% less active than the ATG fusion.« less

  8. Individual chromosome identification, chromosomal collinearity and genetic-physical integrated map in Gossypium darwinii and four D genome cotton species revealed by BAC-FISH.

    PubMed

    Gan, Yimei; Liu, Fang; Peng, Renhai; Wang, Chunying; Li, Shaohui; Zhang, Xiangdi; Wang, Yuhong; Wang, Kunbo

    2012-01-01

    The study was conducted on the individual chromosome identification in Gossypium darwinii (A(d)D(d)), G. klotzschianum (D(3k)), G. davidsonii (D(3d)), G. armourianum (D(2-1)) and G. aridum (D(4)) using a multi-probe fluorescence of in situ hybridization (FISH) system. Comparative analysis on their genetic maps with that of physical maps was made as well. The FISH probes used contained two sets of bacterial artificial chromosome (BAC) clones [one is specific to 26 individual chromosomes from A and D subgenomes of G. hirsutum (A(h) and D(h)) while the other is a D genome centromere-specific BAC clone 150D24], 45S and 5S rDNA clones. The results showed that all A(d) chromosomes were marked with the 13 A(h) chromosome-specific BAC clones, whilst all D(d), D(3k), D(3d), D(2-1) and D(4) chromosomes and chromosomal arms were identified with the 13 D(h) chromosome-specific BAC clones and the D genome centromere-specific BAC. According to the homology within D subgenomes which are between A (D) genome and A (D) subgenome, the systematic nomenclature for individual chromosome in the five species was established. The chromosomes of A (D) subgenomes in G. darwinii were named as A(d)01-A(d)13 (D(d)01-D(d)13). The chromosomes in D(3k), D(3d), D(2-1) and D(4) were named as D(3k)01-D(3k)13, D(3d)01-D(3d)13, D(2-1)01-D(2-1)13 and D(4)01-D(4)13, respectively. Based on the successful identification for individual chromosomes, 45S and 5S rDNA were located to the special chromosomes and chromosomal arms for all five species. And there appeared chromosomal collinearity from the BAC clones among different species by comparing BACs positions, which suggested that the majority of chromosome segment homology may exist between D genomes and D subgenome. Moreover, as the genetic map and physical map were integrated, the orientations of genetic maps for D(d) and D genomes of diploid cotton were established. The orientations of some of chromosomes in genetic maps (D(d)03, D(d)04, D(d)06, D(d)09

  9. Integration Site of Noninducible Coliphage 186

    PubMed Central

    Woods, Walter H.; Egan, J. Barry

    1972-01-01

    From conjugational data, the attachment site for noninducible coliphage 186 (att186) was located between the origins of Hfr strains KL16 and KL98, and close to the pheA gene in Escherichia coli K-12. P1 transductions indicated that att186 lies at 51 min on the standard genetic map of E. coli, with the order cysC-nalB-att186-pheA. The presence of prophage 186 in the donor destroyed linkage between nalB and pheA, which is taken as evidence for the integration of the 186 prophage between these genes. PMID:4559723

  10. Influence of chromosomal integration on glucocorticoid-regulated transcription of growth-stimulating papillomavirus genes E6 and E7 in cervical carcinoma cells.

    PubMed Central

    von Knebel Doeberitz, M; Bauknecht, T; Bartsch, D; zur Hausen, H

    1991-01-01

    In most cervical carcinoma cells the E6 and E7 genes of specific human papillomaviruses are transcribed from viral sequences integrated into host cell chromosomes. Glucocorticoids activate the promoter elements of various human papillomaviruses in transient-expression assays. We have analyzed the effect of dexamethasone on the transcription rate of human papillomavirus 18 E6 and E7 genes integrated at different chromosomal sites in four cervical cancer cell lines. Dexamethasone led to an increase in the transcription rate of the integrated E6-E7 sequences in C4-1 and C4-2 cells but led to a decrease in SW 756 cells and did not affect the transcription rate in HeLa cells. However, when the viral promoter elements derived from HeLa or SW 756 cells, in which dexamethasone does not activate transcription of the integrated E6-E7 sequences, were tested in transient-expression assays within the same cell lines, dexamethasone consistently activated the viral promoter. It thus appears that dominant regulatory mechanisms presumably depending on the chromosomal integration site are able to override the response of the viral promoter to steroid hormones. The growth rate of all dexamethasone-treated cell lines correlated consistently with the expression of the papillomavirus E6 and E7 genes, supporting their role in the maintenance of the proliferative phenotype of cervical carcinoma cells. Since human papillomaviruses are integrated into the host cell genome at variable, presumably randomly selected chromosomal loci, regulatory mechanisms that influence viral gene expression, and hence cell growth, may differ among cancers of independent clonal origin. Images PMID:1847520

  11. Escherichia coli flagellar genes as target sites for integration and expression of genetic circuits.

    PubMed

    Juhas, Mario; Evans, Lewis D B; Frost, Joe; Davenport, Peter W; Yarkoni, Orr; Fraser, Gillian M; Ajioka, James W

    2014-01-01

    E. coli is a model platform for engineering microbes, so genetic circuit design and analysis will be greatly facilitated by simple and effective approaches to introduce genetic constructs into the E. coli chromosome at well-characterised loci. We combined the Red recombinase system of bacteriophage λ and Isothermal Gibson Assembly for rapid integration of novel DNA constructs into the E. coli chromosome. We identified the flagellar region as a promising region for integration and expression of genetic circuits. We characterised integration and expression at four candidate loci, fliD, fliS, fliT, and fliY, of the E. coli flagellar region 3a. The integration efficiency and expression from the four integrations varied considerably. Integration into fliD and fliS significantly decreased motility, while integration into fliT and fliY had only a minor effect on the motility. None of the integrations had negative effects on the growth of the bacteria. Overall, we found that fliT was the most suitable integration site. PMID:25350000

  12. Escherichia coli Flagellar Genes as Target Sites for Integration and Expression of Genetic Circuits

    PubMed Central

    Juhas, Mario; Evans, Lewis D. B.; Frost, Joe; Davenport, Peter W.; Yarkoni, Orr; Fraser, Gillian M.; Ajioka, James W.

    2014-01-01

    E. coli is a model platform for engineering microbes, so genetic circuit design and analysis will be greatly facilitated by simple and effective approaches to introduce genetic constructs into the E. coli chromosome at well-characterised loci. We combined the Red recombinase system of bacteriophage λ and Isothermal Gibson Assembly for rapid integration of novel DNA constructs into the E. coli chromosome. We identified the flagellar region as a promising region for integration and expression of genetic circuits. We characterised integration and expression at four candidate loci, fliD, fliS, fliT, and fliY, of the E. coli flagellar region 3a. The integration efficiency and expression from the four integrations varied considerably. Integration into fliD and fliS significantly decreased motility, while integration into fliT and fliY had only a minor effect on the motility. None of the integrations had negative effects on the growth of the bacteria. Overall, we found that fliT was the most suitable integration site. PMID:25350000

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

    SciTech Connect

    Pozzi, G.; Guild, W.R.

    1985-03-01

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

  14. Heritable fragile sites on human chromosomes. IX. Population cytogenetics and segregation analysis of the BrdU-requiring fragile site at 10q25

    PubMed Central

    Sutherland, Grant R.

    1982-01-01

    The frequencies of the bromodeoxyuridine (BrdU)-requiring fragile site at 10q25 in 1,026 unselected neonates, 901 patients referred for chromosome studies, and 87 institutionalized retardates were not significantly different from each other. The gene frequency was .013, and the population was in Hardy-Weinberg equilibrium. Segregation analysis confirmed that the fragile site followed codominant inheritance. This fragile site and its nonfragile allelomorph can be considered to constitute the first true chromosomal polymorphism to be described in man. PMID:7124730

  15. A Novel Ty1-Mediated Fragmentation Method for Native and Artificial Yeast Chromosomes Reveals That the Mouse Steel Gene Is a Hotspot for Ty1 Integration

    PubMed Central

    Dalgaard, J. Z.; Banerjee, M.; Curcio, M. J.

    1996-01-01

    We have developed a powerful new tool for the physical analysis of genomes called Ty1-mediated chromosomal fragmentation and have used the method to map 24 retrotransposon insertions into two different mouse-derived yeast artificial chromosomes (YACs). Expression of a plasmid-encoded GAL1:Ty1 fusion element marked with the retrotransposition indicator gene, ade2AI, resulted in a high fraction of cells that sustained a single Ty1 insertion marked with ADE2. Strains in which Ty1ADE2 inserted into a YAC were identified by cosegregation of the ADE2 gene with the URA3-marked YAC. Ty1ADE2 elements also carried a site for the endonuclease I-DmoI, which we demonstrate is not present anywhere in the yeast genome. Consequently, I-DmoI cleaved a single chromosome or YAC at the unique site of Ty1ADE2 insertion, allowing rapid mapping of integration events. Our analyses showed that the frequency of Ty1ADE2 integration into YACs is equivalent to or higher than that expected based on random insertion. Remarkably, the 50-kb transcription unit of the mouse Steel locus was shown to be a highly significant hotspot for Ty1 integration. The accessibility of mammalian transcription units to Ty1 insertion stands in contrast to that of yeast transcription units. PMID:8725218

  16. Chromosome breakage at a major fragile site associated with P-glycoprotein gene amplification in multidrug-resistant CHO cells.

    PubMed Central

    Kuo, M T; Vyas, R C; Jiang, L X; Hittelman, W N

    1994-01-01

    Recent studies of several drug-resistant Chinese hamster cell lines suggested that a breakage-fusion-bridge mechanism is frequently involved in the amplification of drug resistance genes. These observations underscore the importance of chromosome breakage in the initiation of DNA amplification in mammalian cells. However, the mechanism of this breakage is unknown. Here, we propose that the site of chromosome breakage consistent with the initial event of P-glycoprotein (P-gp) gene amplification via the breakage-fusion-bridge cycle in three independently established multidrug-resistant CHO cells was located at 1q31. This site is a major chromosome fragile site that can be induced by methotrexate and aphidicolin treatments. Pretreatments of CHO cells with methotrexate or aphidicolin enhanced the frequencies of resistance to vinca alkaloid and amplification of the P-gp gene. These observations suggest that chromosome fragile sites play a pivotal role in DNA amplification in mammalian cells. Our data are also consistent with the hypothesis that gene amplification can be initiated by stress-induced chromosome breakage that is independent of modes of action of cytotoxic agents. Drug-resistant variants may arise by their growth advantage due to overproduction of cellular target molecules via gene amplification. Images PMID:7913517

  17. Chromosome breakage at a major fragile site associated with P-glycoprotein gene amplification in multidrug-resistant CHO cells.

    PubMed

    Kuo, M T; Vyas, R C; Jiang, L X; Hittelman, W N

    1994-08-01

    Recent studies of several drug-resistant Chinese hamster cell lines suggested that a breakage-fusion-bridge mechanism is frequently involved in the amplification of drug resistance genes. These observations underscore the importance of chromosome breakage in the initiation of DNA amplification in mammalian cells. However, the mechanism of this breakage is unknown. Here, we propose that the site of chromosome breakage consistent with the initial event of P-glycoprotein (P-gp) gene amplification via the breakage-fusion-bridge cycle in three independently established multidrug-resistant CHO cells was located at 1q31. This site is a major chromosome fragile site that can be induced by methotrexate and aphidicolin treatments. Pretreatments of CHO cells with methotrexate or aphidicolin enhanced the frequencies of resistance to vinca alkaloid and amplification of the P-gp gene. These observations suggest that chromosome fragile sites play a pivotal role in DNA amplification in mammalian cells. Our data are also consistent with the hypothesis that gene amplification can be initiated by stress-induced chromosome breakage that is independent of modes of action of cytotoxic agents. Drug-resistant variants may arise by their growth advantage due to overproduction of cellular target molecules via gene amplification. PMID:7913517

  18. Construction of reference chromosome-scale pseudomolecules for potato: integrating the potato genome with genetic and physical maps.

    PubMed

    Sharma, Sanjeev Kumar; Bolser, Daniel; de Boer, Jan; Sønderkær, Mads; Amoros, Walter; Carboni, Martin Federico; D'Ambrosio, Juan Martín; de la Cruz, German; Di Genova, Alex; Douches, David S; Eguiluz, Maria; Guo, Xiao; Guzman, Frank; Hackett, Christine A; Hamilton, John P; Li, Guangcun; Li, Ying; Lozano, Roberto; Maass, Alejandro; Marshall, David; Martinez, Diana; McLean, Karen; Mejía, Nilo; Milne, Linda; Munive, Susan; Nagy, Istvan; Ponce, Olga; Ramirez, Manuel; Simon, Reinhard; Thomson, Susan J; Torres, Yerisf; Waugh, Robbie; Zhang, Zhonghua; Huang, Sanwen; Visser, Richard G F; Bachem, Christian W B; Sagredo, Boris; Feingold, Sergio E; Orjeda, Gisella; Veilleux, Richard E; Bonierbale, Merideth; Jacobs, Jeanne M E; Milbourne, Dan; Martin, David Michael Alan; Bryan, Glenn J

    2013-11-01

    The genome of potato, a major global food crop, was recently sequenced. The work presented here details the integration of the potato reference genome (DM) with a new sequence-tagged site marker-based linkage map and other physical and genetic maps of potato and the closely related species tomato. Primary anchoring of the DM genome assembly was accomplished by the use of a diploid segregating population, which was genotyped with several types of molecular genetic markers to construct a new ~936 cM linkage map comprising 2469 marker loci. In silico anchoring approaches used genetic and physical maps from the diploid potato genotype RH89-039-16 (RH) and tomato. This combined approach has allowed 951 superscaffolds to be ordered into pseudomolecules corresponding to the 12 potato chromosomes. These pseudomolecules represent 674 Mb (~93%) of the 723 Mb genome assembly and 37,482 (~96%) of the 39,031 predicted genes. The superscaffold order and orientation within the pseudomolecules are closely collinear with independently constructed high density linkage maps. Comparisons between marker distribution and physical location reveal regions of greater and lesser recombination, as well as regions exhibiting significant segregation distortion. The work presented here has led to a greatly improved ordering of the potato reference genome superscaffolds into chromosomal "pseudomolecules". PMID:24062527

  19. Construction of Reference Chromosome-Scale Pseudomolecules for Potato: Integrating the Potato Genome with Genetic and Physical Maps

    PubMed Central

    Sharma, Sanjeev Kumar; Bolser, Daniel; de Boer, Jan; Sønderkær, Mads; Amoros, Walter; Carboni, Martin Federico; D’Ambrosio, Juan Martín; de la Cruz, German; Di Genova, Alex; Douches, David S.; Eguiluz, Maria; Guo, Xiao; Guzman, Frank; Hackett, Christine A.; Hamilton, John P.; Li, Guangcun; Li, Ying; Lozano, Roberto; Maass, Alejandro; Marshall, David; Martinez, Diana; McLean, Karen; Mejía, Nilo; Milne, Linda; Munive, Susan; Nagy, Istvan; Ponce, Olga; Ramirez, Manuel; Simon, Reinhard; Thomson, Susan J.; Torres, Yerisf; Waugh, Robbie; Zhang, Zhonghua; Huang, Sanwen; Visser, Richard G. F.; Bachem, Christian W. B.; Sagredo, Boris; Feingold, Sergio E.; Orjeda, Gisella; Veilleux, Richard E.; Bonierbale, Merideth; Jacobs, Jeanne M. E.; Milbourne, Dan; Martin, David Michael Alan; Bryan, Glenn J.

    2013-01-01

    The genome of potato, a major global food crop, was recently sequenced. The work presented here details the integration of the potato reference genome (DM) with a new sequence-tagged site marker−based linkage map and other physical and genetic maps of potato and the closely related species tomato. Primary anchoring of the DM genome assembly was accomplished by the use of a diploid segregating population, which was genotyped with several types of molecular genetic markers to construct a new ~936 cM linkage map comprising 2469 marker loci. In silico anchoring approaches used genetic and physical maps from the diploid potato genotype RH89-039-16 (RH) and tomato. This combined approach has allowed 951 superscaffolds to be ordered into pseudomolecules corresponding to the 12 potato chromosomes. These pseudomolecules represent 674 Mb (~93%) of the 723 Mb genome assembly and 37,482 (~96%) of the 39,031 predicted genes. The superscaffold order and orientation within the pseudomolecules are closely collinear with independently constructed high density linkage maps. Comparisons between marker distribution and physical location reveal regions of greater and lesser recombination, as well as regions exhibiting significant segregation distortion. The work presented here has led to a greatly improved ordering of the potato reference genome superscaffolds into chromosomal “pseudomolecules”. PMID:24062527

  20. Germ-Line Transmitted, Chromosomally Integrated HHV-6 and Classical Hodgkin Lymphoma

    PubMed Central

    Bell, Adam J.; Gallagher, Alice; Mottram, Timothy; Lake, Annette; Kane, Eleanor V.; Lightfoot, Tracy; Roman, Eve; Jarrett, Ruth F.

    2014-01-01

    A unique feature of both human herpesvirus 6A and B (HHV-6A and B) among human herpesviruses is their ability to integrate into chromosomal telomeres. In some individuals integrated viral genomes are present in the germ-line and result in the vertical transmission of HHV-6; however, little is known about the disease associations of germ-line transmitted, chromosomally integrated HHV-6 (ciHHV-6). Recent publications suggest that HHV-6 is associated with classical Hodgkin lymphoma (cHL). Here we examine the prevalence of ciHHV-6 in 936 cases of cHL and 563 controls by screening with a duplex TaqMan assay and confirming with droplet digital PCR. ciHHV-6 was detected in 10/563 (1.8%) controls and in all but one individual the virus was HHV-6B. Amongst cases 16/936 (1.7%) harboured ciHHV-6, thus demonstrating no association between ciHHV-6 and risk of cHL. PMID:25384040

  1. Learning with two sites of synaptic integration.

    PubMed

    Körding, K P; König, P

    2000-02-01

    Since the classical work of D O Hebb 1949 The Organization of Behaviour (New York: Wiley) it is assumed that synaptic plasticity solely depends on the activity of the pre- and the postsynaptic cells. Synapses influence the plasticity of other synapses exclusively via the post-synaptic activity. This confounds effects on synaptic plasticity and neuronal activation and, thus, makes it difficult to implement networks which optimize global measures of performance. Exploring solutions to this problem, inspired by recent research on the properties of apical dendrites, we examine a network of neurons with two sites of synaptic integration. These communicate in such a way that one set of synapses mainly influences the neurons' activity; the other set gates synaptic plasticity. Analysing the system with a constant set of parameters reveals: (1) the afferents that gate plasticity act as supervisors, individual to every cell. (2) While the neurons acquire specific receptive fields the net activity remains constant for different stimuli. This ensures that all stimuli are represented and, thus, contributes to information maximization. (3) Mechanisms for maximization of coherent information can easily be implemented. Neurons with non-overlapping receptive fields learn to fire correlated and preferentially transmit information that is correlated over space. (4) We demonstrate how a new measure of performance can be implemented: cells learn to represent only the part of the input that is relevant to the processing at higher stages. This criterion is termed 'relevant infomax'. PMID:10735527

  2. Retrotransposons. An RNA polymerase III subunit determines sites of retrotransposon integration.

    PubMed

    Bridier-Nahmias, Antoine; Tchalikian-Cosson, Aurélie; Baller, Joshua A; Menouni, Rachid; Fayol, Hélène; Flores, Amando; Saïb, Ali; Werner, Michel; Voytas, Daniel F; Lesage, Pascale

    2015-05-01

    Mobile genetic elements are ubiquitous. Their integration site influences genome stability and gene expression. The Ty1 retrotransposon of the yeast Saccharomyces cerevisiae integrates upstream of RNA polymerase III (Pol III)-transcribed genes, yet the primary determinant of target specificity has remained elusive. Here we describe an interaction between Ty1 integrase and the AC40 subunit of Pol III and demonstrate that AC40 is the predominant determinant targeting Ty1 integration upstream of Pol III-transcribed genes. Lack of an integrase-AC40 interaction dramatically alters target site choice, leading to a redistribution of Ty1 insertions in the genome, mainly to chromosome ends. The mechanism of target specificity allows Ty1 to proliferate and yet minimizes genetic damage to its host. PMID:25931562

  3. Chromosomal location of 18S and 5S rDNA sites in Triportheus fish species (Characiformes, Characidae)

    PubMed Central

    2009-01-01

    The location of 18S and 5S rDNA sites was determined in eight species and populations of the fish genus Triportheus by using fluorescent in situ hybridization (FISH). The males and females of all species had 2n = 52 chromosomes and a ZZ/ZW sex chromosome system. A single 18S rDNA site that was roughly equivalent to an Ag-NOR was detected on the short arms of a submetacentric pair in nearly all species, and up to two additional sites were also observed in some species. In addition, another 18S rDNA cluster was identified in a distal region on the long arms of the W chromosome; this finding corroborated previous evidence that this cluster would be a shared feature amongst Triportheus species. In T. angulatus, a heterozygotic paracentric inversion involving the short arms of one homolog of a metacentric pair was associated with NORs. The 5S rDNA sites were located on the short arms of a single submetacentric chromosomal pair, close to the centromeres, except in T. auritus, which had up to ten 5S rDNA sites. The 18S and 5S rDNA sites were co-localized and adjacent on the short arms of a chromosomal pair in two populations of T. nematurus. Although all Triportheus species have a similar karyotypic macrostructure, the results of this work show that in some species ribosomal genes may serve as species-specific markers when used in conjunction with other putatively synapomorphic features. PMID:21637644

  4. Chromosomal Distribution of Transposable Elements in Drosophila Melanogaster: Test of the Ectopic Recombination Model for Maintenance of Insertion Site Number

    PubMed Central

    Hoogland, C.; Biemont, C.

    1996-01-01

    Data of insertion site localization and site occupancy frequency of P, hobo, I, copia, mdg1, mdg3, 412, 297, and roo transposable elements (TEs) on the polytene chromosomes of Drosophila melanogaster were extracted from the literature. We show that TE insertion site number per chromosomal division was significantly correlated with the amount of DNA. The insertion site number weighted by DNA content was not correlated with recombination rate for all TEs except hobo, for which a positive correlation was detected. No global tendency emerged in the relationship between TE site occupancy frequency, weighted by DNA content, and recombination rate; a strong negative correlation was, however, found for the 3L arm. A possible dominant deleterious effect of chromosomal rearrangements due to recombination between TE insertions is thus not the main factor explaining the dynamics of TEs, since this hypothesis implies a negative relationship between recombination rate and both TE insertion site number and site occupancy frequency. The alternative hypothesis of selection against deleterious effects of insertional mutations is discussed. PMID:8878685

  5. Conformational Variants of Duplex DNA Correlated with Cytosine-rich Chromosomal Fragile Sites*S⃞

    PubMed Central

    Tsai, Albert G.; Engelhart, Aaron E.; Hatmal, Ma'mon M.; Houston, Sabrina I.; Hud, Nicholas V.; Haworth, Ian S.; Lieber, Michael R.

    2009-01-01

    We found that several major chromosomal fragile sites in human lymphomas, including the bcl-2 major breakpoint region, bcl-1 major translocation cluster, and c-Myc exon 1-intron 1 boundary, contain distinctive sequences of consecutive cytosines exhibiting a high degree of reactivity with the structure-specific chemical probe bisulfite. To assess the inherent structural variability of duplex DNA in these regions and to determine the range of structures reactive to bisulfite, we have performed bisulfite probing on genomic DNA in vitro and in situ; on duplex DNA in supercoiled and linearized plasmids; and on oligonucleotide DNA/DNA and DNA/2′-O-methyl RNA duplexes. Bisulfite is significantly more reactive at the frayed ends of DNA duplexes, which is expected given that bisulfite is an established probe of single-stranded DNA. We observed that bisulfite also distinguishes between more subtle sequence/structural differences in duplex DNA. Supercoiled plasmids are more reactive than linear DNA; and sequences containing consecutive cytosines, namely GGGCCC, are more reactive than those with alternating guanine and cytosine, namely GCGCGC. Circular dichroism and x-ray crystallography show that the GGGCCC sequence forms an intermediate B/A structure. Molecular dynamics simulations also predict an intermediate B/A structure for this sequence, and probe calculations suggest greater bisulfite accessibility of cytosine bases in the intermediate B/A structure over canonical B- or A-form DNA. Electrostatic calculations reveal that consecutive cytosine bases create electropositive patches in the major groove, predicting enhanced localization of the bisulfite anion at homo-C tracts over alternating G/C sequences. These characteristics of homo-C tracts in duplex DNA may be associated with DNA-protein interactions in vivo that predispose certain genomic regions to chromosomal fragility. PMID:19106104

  6. The use of HIV-1 integration site analysis information in clinical studies aiming at HIV cure.

    PubMed

    Kiselinova, Maja; De Spiegelaere, Ward; Vandekerckhove, Linos

    2016-01-01

    The mechanisms for the establishment and the persistence of the latent HIV-1 reservoir remain to be completely defined. HIV-1 infection is characterised by the integration of the reverse transcribed proviral DNA into the host's genome. This integrated proviral DNA can remain replication silent, but a small part of it is fully competent to restart viral replication when treatment is interrupted. Hence, this replication-competent provirus is the cause of viral rebound and is called the viral reservoir. The exact site of proviral integration within the host's cellular chromosome may affect the transcriptional activity of HIV. Thanks to recent technological advances, HIV-1 integration site analysis has been used to assess HIV-1 reservoirs in HIV-infected individuals. Analysis of HIV-1 integration sites in infected individuals undergoing suppressive ART led to identification of expanded clonal cell populations, indicating that clonal proliferation of the proviral reservoir may contribute to the long-term persistence of viral reservoirs. Here we describe the findings of several clinical studies, where a comprehensive HIV-1 integration site analysis was performed. PMID:27482458

  7. The ripX Locus of Bacillus subtilis Encodes a Site-Specific Recombinase Involved in Proper Chromosome Partitioning

    PubMed Central

    Sciochetti, Stephen A.; Piggot, Patrick J.; Sherratt, David J.; Blakely, Garry

    1999-01-01

    The Bacillus subtilis ripX gene encodes a protein that has 37 and 44% identity with the XerC and XerD site-specific recombinases of Escherichia coli. XerC and XerD are hypothesized to act in concert at the dif site to resolve dimeric chromosomes formed by recombination during replication. Cultures of ripX mutants contained a subpopulation of unequal-size cells held together in long chains. The chains included anucleate cells and cells with aberrantly dense or diffuse nucleoids, indicating a chromosome partitioning failure. This result is consistent with RipX having a role in the resolution of chromosome dimers in B. subtilis. Spores contain a single uninitiated chromosome, and analysis of germinated, outgrowing spores showed that the placement of FtsZ rings and septa is affected in ripX strains by the first division after the initiation of germination. The introduction of a recA mutation into ripX strains resulted in only slight modifications of the ripX phenotype, suggesting that chromosome dimers can form in a RecA-independent manner in B. subtilis. In addition to RipX, the CodV protein of B. subtilis shows extensive similarity to XerC and XerD. The RipX and CodV proteins were shown to bind in vitro to DNA containing the E. coli dif site. Together they functioned efficiently in vitro to catalyze site-specific cleavage of an artificial Holliday junction containing a dif site. Inactivation of codV alone did not cause a discernible change in phenotype, and it is speculated that RipX can substitute for CodV in vivo. PMID:10498718

  8. Repetitive genome elements in a European corn borer, Ostrinia nubilalis, bacterial artificial chromosome library were indicated by bacterial artificial chromosome end sequencing and development of sequence tag site markers: implications for lepidopteran genomic research.

    PubMed

    Coates, Brad S; Sumerford, Douglas V; Hellmich, Richard L; Lewis, Leslie C

    2009-01-01

    The European corn borer, Ostrinia nubilalis, is a serious pest of food, fiber, and biofuel crops in Europe, North America, and Asia and a model system for insect olfaction and speciation. A bacterial artificial chromosome library constructed for O. nubilalis contains 36 864 clones with an estimated average insert size of >or=120 kb and genome coverage of 8.8-fold. Screening OnB1 clones comprising approximately 2.76 genome equivalents determined the physical position of 24 sequence tag site markers, including markers linked to ecologically important and Bacillus thuringiensis toxin resistance traits. OnB1 bacterial artificial chromosome end sequence reads (GenBank dbGSS accessions ET217010 to ET217273) showed homology to annotated genes or expressed sequence tags and identified repetitive genome elements, O. nubilalis miniature subterminal inverted repeat transposable elements (OnMITE01 and OnMITE02), and ezi-like long interspersed nuclear elements. Mobility of OnMITE01 was demonstrated by the presence or absence in O. nubilalis of introns at two different loci. A (GTCT)n tetranucleotide repeat at the 5' ends of OnMITE01 and OnMITE02 are evidence for transposon-mediated movement of lepidopteran microsatellite loci. The number of repetitive elements in lepidopteran genomes will affect genome assembly and marker development. Single-locus sequence tag site markers described here have downstream application for integration within linkage maps and comparative genomic studies. PMID:19132072

  9. Efficient identification of inherited chromosomally integrated human herpesvirus 6 using specimen pooling☆

    PubMed Central

    Hill, Joshua A.; HallSedlak, Ruth; Magaret, Amalia; Huang, Meei-Li; Zerr, Danielle M.; Jerome, Keith R.; Boeckh, Michael

    2016-01-01

    Background Human herpesvirus 6 (HHV-6) has a unique ability to integrate into chromosomal telomeres. Vertical transmission via germ cell integration results in offspring with inherited chromosomally integrated (ci)HHV-6 in all nucleated cells, affecting ~1% of the population. Objectives Inherited ciHHV-6 may be a direct or indirect mediator of human disease, but efficient identification of affected individuals is a fundamental roadblock to larger studies exploring the clinical importance of this condition. Study design A group testing strategy was designed to efficiently identify individuals with inherited ciHHV-6. DNA was extracted from 2496 cellular samples from hematopoietic cell transplant (HCT) donor–recipient pairs. Pools of 12 samples were screened for HHV-6 DNA with quantitative (q)PCR. Individual samples from high positive pools were tested with qPCR, and high positive individual samples were tested for inherited ciHHV-6 using droplet digital (dd)PCR to determine HHV-6 DNA copies/cellular genome. Results Thirty-one pools had high positive HHV-6 DNA detection with >103 HHV-6 DNA copies/μg. Each pool had one sample with >104 copies/μg HHV-6 DNA. Inherited ciHHV-6 was confirmed by ddPCR in every high positive sample (>103 HHV-6 DNA copies/μg), yielding a prevalence of 1.5% in HCT recipients and 0.96% in donors. We performed 580 qPCR tests to screen 2496 samples for inherited ciHHV-6, a 77% reduction in testing. Conclusions Inherited ciHHV-6 can be efficiently identified by specimen pooling coupled with modern molecular techniques. This algorithm can be used to facilitate cost-effective identification of patients with inherited ciHHV-6, thereby removing a major hurdle for large-scale study of its clinical impact. PMID:26921738

  10. Exclusion of the retinoblastoma gene and chromosome 13q as the site of a primary lesion for human breast cancer.

    PubMed Central

    Bowcock, A M; Hall, J M; Hebert, J M; King, M C

    1990-01-01

    Chromosome 13q has been suggested as the site of a gene predisposing to human breast cancer, because loss of heterozygosity of alleles on this chromosome has been observed in some ductal breast tumors and because two breast cancer lines are altered at the retinoblastoma gene (RB1) at 13q14. To test this possibility, linkage of breast cancer susceptibility to 14 loci on chromosome 13q loci was assessed in extended families in which breast cancer is apparently inherited as an autosomal dominant trait. RB1 was excluded as the site of a breast cancer gene by a lod score of Z = -7.60 at close linkage for 13 families. Multipoint analysis yielded negative lod scores throughout the region between 13q12 and 13q34; over most of this distance, Z less than -2.0. Therefore, chromosome 13q appears to be excluded as the site of primary lesion for breast cancer in these families. In addition, comparison of tumor versus normal tissues of nonfamilial breast cancer patients revealed an alteration at the 5' end of RB1 in a mucoid carcinoma but no alterations of RB1 in five informative ductal adenocarcinomas. Linkage data and comparisons of tumor and normal tissues suggest that changes in the RBI locus either are secondary alterations associated with progression of some tumors or occur by chance. Images Figure 2 PMID:2294744

  11. Knock-in/Knock-out (KIKO) vectors for rapid integration of large DNA sequences, including whole metabolic pathways, onto the Escherichia coli chromosome at well-characterised loci

    PubMed Central

    2013-01-01

    Background Metabolic engineering projects often require integration of multiple genes in order to control the desired phenotype. However, this often requires iterative rounds of engineering because many current insertion approaches are limited by the size of the DNA that can be transferred onto the chromosome. Consequently, construction of highly engineered strains is very time-consuming. A lack of well-characterised insertion loci is also problematic. Results A series of knock-in/knock-out (KIKO) vectors was constructed for integration of large DNA sequences onto the E. coli chromosome at well-defined loci. The KIKO plasmids target three nonessential genes/operons as insertion sites: arsB (an arsenite transporter); lacZ (β-galactosidase); and rbsA-rbsR (a ribose metabolism operon). Two homologous ‘arms’ target each insertion locus; insertion is mediated by λ Red recombinase through these arms. Between the arms is a multiple cloning site for the introduction of exogenous sequences and an antibiotic resistance marker (either chloramphenicol or kanamycin) for selection of positive recombinants. The resistance marker can subsequently be removed by flippase-mediated recombination. The insertion cassette is flanked by hairpin loops to isolate it from the effects of external transcription at the integration locus. To characterize each target locus, a xylanase reporter gene (xynA) was integrated onto the chromosomes of E. coli strains W and K-12 using the KIKO vectors. Expression levels varied between loci, with the arsB locus consistently showing the highest level of expression. To demonstrate the simultaneous use of all three loci in one strain, xynA, green fluorescent protein (gfp) and a sucrose catabolic operon (cscAKB) were introduced into lacZ, arsB and rbsAR respectively, and shown to be functional. Conclusions The KIKO plasmids are a useful tool for efficient integration of large DNA fragments (including multiple genes and pathways) into E. coli. Chromosomal

  12. Biosynthetic burden and plasmid burden limit expression of chromosomally integrated heterologous genes (pdc, adhB) in Escherichia coli

    SciTech Connect

    Martinez, A.; York, S.W.; Yomano, L.P.; Pineda, V.L.; Davis, F.C.; Shelton, J.C.; Ingram, L.O.

    1999-10-01

    Previous studies have shown an unexpectedly high nutrient requirement for efficient ethanol production by ethanologenic recombinants of Escherichia coli B such as LY01 which contain chromosomally integrated Zymomonas mobilis genes (pdc, adhB) encoding the ethanol pathway. The basis for this requirement has been identified as a media-dependent effect on the expression of the Z. mobilis genes rather than a nutritional limitation. Ethanol production was substantially increased without additional nutrients simply by increasing the level of pyruvate decarboxylase activity. This was accomplished by adding a multicopy plasmid containing pdc alone (but not adhB alone) to strain LY01, and by adding multicopy plasmids which express pdc and adhB from strong promoters. New strong promoters were isolated from random fragments of Z. mobilis DNA and characterized but were not used to construct integrated biocatalysts. These promoters contained regions resembling recognition sites for 3 different E. coli sigma factors: {sigma}{sup 70}, {sigma}{sup 38}, and {sigma}{sup 28}. The most effective plasmid-based promoters for fermentation were recognized by multiple sigma factors, expressed both pdc and adhB at high levels, and produced ethanol efficiently while allowing up to 80% reduction in complex nutrients as compared to LY01. The ability to utilize multiple sigma factors may be advantageous to maintain the high levels of PDC and ADH needed for efficient ethanol production throughout batch fermentation.

  13. Chromosome Mapping of 18S Ribosomal RNA Genes in Eleven Hypostomus Species (Siluriformes, Loricariidae): Diversity Analysis of the Sites.

    PubMed

    Rubert, Marceléia; da Rosa, Renata; Zawadzki, Claudio H; Mariotto, Sandra; Moreira-Filho, Orlando; Giuliano-Caetano, Lucia

    2016-08-01

    We investigated the chromosomal distribution of 18S ribosomal DNA (rDNA) in different populations of 11 species of Hypostomus collected in important Brazilian basins, namely South Atlantic, Upper Paraná, and Paraguay applying the fluorescence in situ hybridization (FISH). Hypostomus cochliodon, Hypostomus commersoni, Hypostomus hermanni, Hypostomus regani, Hypostomus albopunctatus, Hypostomus paulinus, Hypostomus aff. paulinus, Hypostomus iheringii, and Hypostomus mutucae presented multiple 18S rDNA sites while Hypostomus strigaticeps and Hypostomus nigromaculatus exhibited a single pair of chromosomes with 18S rDNA sites. The studied species presented variations in the number and position of these sites. The results accomplished were similar to those obtained by the analysis of AgNORs, revealing the same interspecific variability. Each species exhibited distinctive patterns of AgNOR and 18S rDNA distribution, which can be considered cytogenetic markers in each species of the genus and help improve the discussions on the phylogeny of the group. PMID:27192329

  14. PBO Integrated Real-Time Observing Sites at Volcanic Sites

    NASA Astrophysics Data System (ADS)

    Mencin, D.; Jackson, M.; Borsa, A.; Feaux, K.; Smith, S.

    2009-05-01

    The Plate Boundary Observatory, an element of NSF's EarthScope program, has six integrated observatories in Yellowstone and four on Mt St Helens. These observatories consist of some combination of borehole strainmeters, borehole seismometers, GPS, tiltmeters, pore pressure, thermal measurements and meteorological data. Data from all these instruments have highly variable data rates and formats, all synchronized to GPS time which can cause significant congestion of precious communication resources. PBO has been experimenting with integrating these data streams to both maximize efficiency and minimize latency through the use of software that combines the streams, like Antelope, and VPN technologies.

  15. Integration of erm(B)-containing elements through large chromosome fragment exchange in Clostridium difficile

    PubMed Central

    Wasels, François; Spigaglia, Patrizia; Barbanti, Fabrizio; Monot, Marc; Villa, Laura; Dupuy, Bruno; Carattoli, Alessandra; Mastrantonio, Paola

    2015-01-01

    In Clostridium difficile, erm(B) genes are located on mobile elements like Tn5398 and Tn6215. In previous studies, some of these elements were transferred by conjugation-like mechanisms, mobilized in trans by helper conjugative systems. In this study, we analyzed the genomes of several recipient strains that acquired either Tn5398 or Tn6215-like elements. We demonstrated that the integration of the transposons in the genome of the recipient cell was always due to homologous recombination events, involving exchange of large chromosomal segments. We did not observed transposon transfer to a C. difficile strain in presence of DNAse, suggesting that a possible transformation-like mechanism occurred in this recipient.

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

    PubMed Central

    Mogen, Bradley D.; Oleson, Arland E.

    1987-01-01

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

  17. Single inverted terminal repeats of the Junonia coenia Densovirus promotes somatic chromosomal integration of vector plasmids in insect cells and supports high efficiency expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plasmids that contain a disrupted genome of the Junonia coenia densovirus (JcDNV) integrate into the chromosomes of the somatic cells of insects. When subcloned individually, both the P9 inverted terminal repeat (P9-ITR) and the P93-ITR promote the chromosomal integration of vector plasmids in insec...

  18. RELATIVE EXPRESSION AND STABILITY OF A CHROMOSOMALLY INTEGRATED AND PLASMID-BORNE MARKER GENE FUSION IN ENVIRONMENTALLY COMPETENT BACTERIA

    EPA Science Inventory

    A xyIE-iceC transcriptional fusion was created by ligating a DNA fragment harboring the cloned xyIE structural gene from the TOL plasmid of Pseudomonas putida mt-2 into the cloned iceC gene of Pseudomonas syringae Cit7. This fusion construct was integrated into chromosome of Pseu...

  19. Replicon-free and markerless methods for genomic insertion of DNAs in phage attachment sites and controlled expression of chromosomal genes in Escherichia coli.

    PubMed

    Chiang, Chung-Jen; Chen, Po Ting; Chao, Yun-Peng

    2008-12-01

    Genetic manipulation of cells for desired traits is the most appreciable strategy implemented in the field of bioengineering. However, this approach closely relies on the use of plasmids and is commonly afflicted by the potential problem of plasmid instability and safety caution. Meanwhile, it may also lead to the spread of antibiotic-resistant markers with replicons of plasmids to the environment. However, this issue has long been neglected. In this study, we have addressed these subjects by developing replicon-free and markerless methods for chromosomal insertion of genes and controlled expression of genomic genes in Escherichia coli. For the former application, the integration vectors of conditional replication were incorporated with the prophage attachment site and duplicated FRT sites. Their utility was illustrated by site-specific insertion of target genes, the endogenous dxs gene and three heterologous genes consisting of gps, crtI, and crtB, fused to T7 promoter into E. coli genome. For the latter application, the template vectors for promoter replacement were constructed to carry a DNA cassette containing the T7 promoter linked to a selective marker flanked with the FRT site. Subsequently, it was illustrated by replacement of the native promoter of chromosomal pckA by the T7 promoter. Finally, with the aid of FLP recombinase supplied from a helper plasmid, the regions containing replicon and/or selective markers in inserted DNAs were eliminated from integrants for both approaches. As a consequence, the expression of these five genes was subject to control by one response regulator, T7 RNA polymerase, in a regulon way, resulting in a high and stable production of lycopene in the cell. This result indicates the promise of developed methods for genome engineering in E. coli. PMID:18553504

  20. Mapping strategies: Chromosome 16 workshop

    SciTech Connect

    Not Available

    1989-01-01

    The following topics from a workshop on chromosome 16 are briefly discussed: genetic map of chromosome 16; chromosome breakpoint map of chromosome 16; integrated physical/genetic map of chromosome 16; pulsed field map of the 16p13.2--p13.3 region (3 sheets); and a report of the HGM10 chromosome 16 committee.

  1. Characterization of the genetic elements required for site-specific integration of plasmid pSE211 in Saccharopolyspora erythraea.

    PubMed

    Brown, D P; Idler, K B; Katz, L

    1990-04-01

    The 18.1-kilobase plasmid pSE211 integrates into the chromosome of Saccharopolyspora erythraea at a specific attB site. Restriction analysis of the integrated plasmid, pSE211int, and adjacent chromosomal sequences allowed identification of attP, the plasmid attachment site. Nucleotide sequencing of attP, attB, attL, and attR revealed a 57-base-pair sequence common to all sites with no duplications of adjacent plasmid or chromosomal sequences in the integrated state, indicating that integration takes place through conservative, reciprocal strand exchange. An analysis of the sequences indicated the presence of a putative gene for Phe-tRNA at attB which is preserved at attL after integration has occurred. A comparison of the attB site for a number of actinomycete plasmids is presented. Integration at attB was also observed when a 2.4-kilobase segment of pSE211 containing attP and the adjacent plasmid sequence was used to transform a pSE211- host. Nucleotide sequencing of this segment revealed the presence of two complete open reading frames (ORFs) and a segment of a third ORF. The ORF adjacent to attP encodes a putative polypeptide 437 amino acids in length that shows similarity, at its C-terminal domain, to sequences of site-specific recombinases of the integrase family. The adjacent ORF encodes a putative 98-amino-acid basic polypeptide that contains a helix-turn-helix motif at its N terminus which corresponds to domains in the Xis proteins of a number of bacteriophages. A proposal for the function of this polypeptide is presented. The deduced amino acid sequence of the third ORF did not reveal similarities to polypeptide sequences in the current data banks. PMID:2180909

  2. Blood transport method for chromosome analysis of residents living near Semipalatinsk nuclear test site.

    PubMed

    Rodzi, Mohd; Ihda, Shozo; Yokozeki, Masako; Takeichi, Nobuo; Tanaka, Kimio; Hoshi, Masaharu

    2009-12-01

    A study was conducted to compare the storage conditions and transportation period for blood samples collected from residents living in areas near the Semipalatinsk nuclear test site (SNTS). Experiments were performed to simulate storage and shipping environments. Phytohaemagglutinin (PHA)-stimulated blood was stored in 15-ml tubes (condition A: current transport method) in the absence or in 50-ml flasks (condition B: previous transport method) in the presence of RPMI-1640 and 20% fetal bovine serum (FBS). Samples were kept refrigerated at 4 degrees C and cell viability was assessed after 3, 8, 12 and 14 days of storage. RPMI-1640, 20% FBS and further PHA were added to blood samples under condition A in 50-ml flasks for culture. Whole-blood samples under condition B were directly incubated without further sub-culturing process, neither media nor PHA were added, to adopt a similar protocol to that employed in the previous transport method. Samples in condition A and condition B were incubated for 48 hr at 37 degrees C and their mitotic index was determined. The results showed that viable lymphocytes were consistent in both storage conditions but the mitotic index was higher in condition A than in condition B. Although further confirmation studies have to be carried out, previous chromosomal studies and the present experiment have shown that PHA-stimulated blood could be stored without culture medium for up to 8 days under condition A. The present results will be useful for cytogenetic analysis of blood samples that have been transported long distances wherever a radiation accident has occurred. PMID:20349749

  3. Integrated Propulsion Data System Public Web Site

    NASA Technical Reports Server (NTRS)

    Hamilton, Kimberly

    2001-01-01

    The Integrated Propulsion Data System's (IPDS) focus is to provide technologically-advanced philosophies of doing business at SSC that will enhance the existing operations, engineering and management strategies and provide insight and metrics to assess their daily impacts, especially as related to the Propulsion Test Directorate testing scenarios for the 21st Century.

  4. Integrated map of new CA-repeats of human chromosome 21

    SciTech Connect

    Bosch, A.; Guimera, J.; Patterson, D.

    1994-09-01

    To contribute to the map of human chromosome 21 (HC21), 35 new CA-repeats isolated from an HC21 phage library (LA21NS01) have been characterized and localized on HC21 using somatic cell hybrids and the Genethon YAC panel. Their heterozygosities range between 0.10 and 0.90 with an average of 0.70. Twenty of them, with heterozygosities between 0.47 and 0.83, have been analyzed in the CEPH reference families. A linkage map was constructed using the CRI-MAP program with 46 markers. This linkage map contains 22 uniquely placed loci, ordered with odds of at least 1000:1, with an average size interval of 3.5 cM. 10 markers were approximately positioned on the map. The sex average length of the map is 73.4 cM, with male and female lengths being 64.2 and 83.5 cM, respectively. D21S1262 was placed in the same megalocus as the IFNAR and GART genes. D21S369 seems to be the most pericentromeric microsatellite marker on HC21 based on linkage analysis, with a heterozygosity of 0.60, and may be very useful for nondisjunction studies. Integration of the somatic cell hybrid, TAC and/or linkage maps have provided a more defined location for these 35 new CA-repeat markers which will contribute greatly to the integrated map of HC21.

  5. Repair of chromosomal RAG-mediated DNA breaks by mutant RAG proteins lacking phosphatidylinositol 3-like kinase consensus phosphorylation sites.

    PubMed

    Gapud, Eric J; Lee, Baeck-Seung; Mahowald, Grace K; Bassing, Craig H; Sleckman, Barry P

    2011-08-15

    Ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase catalytic subunits (DNA-PKcs) are members of the phosphatidylinositol 3-like family of serine/threonine kinases that phosphorylate serines or threonines when positioned adjacent to a glutamine residue (SQ/TQ). Both kinases are activated rapidly by DNA double-strand breaks (DSBs) and regulate the function of proteins involved in DNA damage responses. In developing lymphocytes, DSBs are generated during V(D)J recombination, which is required to assemble the second exon of all Ag receptor genes. This reaction is initiated through a DNA cleavage step by the RAG1 and RAG2 proteins, which together comprise an endonuclease that generates DSBs at the border of two recombining gene segments and their flanking recombination signals. This DNA cleavage step is followed by a joining step, during which pairs of DNA coding and signal ends are ligated to form a coding joint and a signal joint, respectively. ATM and DNA-PKcs are integrally involved in the repair of both signal and coding ends, but the targets of these kinases involved in the repair process have not been fully elucidated. In this regard, the RAG1 and RAG2 proteins, which each have several SQ/TQ motifs, have been implicated in the repair of RAG-mediated DSBs. In this study, we use a previously developed approach for studying chromosomal V(D)J recombination that has been modified to allow for the analysis of RAG1 and RAG2 function. We show that phosphorylation of RAG1 or RAG2 by ATM or DNA-PKcs at SQ/TQ consensus sites is dispensable for the joining step of V(D)J recombination. PMID:21742970

  6. Genome-wide analysis of host-chromosome binding sites for Epstein-Barr Virus Nuclear Antigen 1 (EBNA1)

    PubMed Central

    2010-01-01

    The Epstein-Barr Virus (EBV) Nuclear Antigen 1 (EBNA1) protein is required for the establishment of EBV latent infection in proliferating B-lymphocytes. EBNA1 is a multifunctional DNA-binding protein that stimulates DNA replication at the viral origin of plasmid replication (OriP), regulates transcription of viral and cellular genes, and tethers the viral episome to the cellular chromosome. EBNA1 also provides a survival function to B-lymphocytes, potentially through its ability to alter cellular gene expression. To better understand these various functions of EBNA1, we performed a genome-wide analysis of the viral and cellular DNA sites associated with EBNA1 protein in a latently infected Burkitt lymphoma B-cell line. Chromatin-immunoprecipitation (ChIP) combined with massively parallel deep-sequencing (ChIP-Seq) was used to identify cellular sites bound by EBNA1. Sites identified by ChIP-Seq were validated by conventional real-time PCR, and ChIP-Seq provided quantitative, high-resolution detection of the known EBNA1 binding sites on the EBV genome at OriP and Qp. We identified at least one cluster of unusually high-affinity EBNA1 binding sites on chromosome 11, between the divergent FAM55 D and FAM55B genes. A consensus for all cellular EBNA1 binding sites is distinct from those derived from the known viral binding sites, suggesting that some of these sites are indirectly bound by EBNA1. EBNA1 also bound close to the transcriptional start sites of a large number of cellular genes, including HDAC3, CDC7, and MAP3K1, which we show are positively regulated by EBNA1. EBNA1 binding sites were enriched in some repetitive elements, especially LINE 1 retrotransposons, and had weak correlations with histone modifications and ORC binding. We conclude that EBNA1 can interact with a large number of cellular genes and chromosomal loci in latently infected cells, but that these sites are likely to represent a complex ensemble of direct and indirect EBNA1 binding sites. PMID

  7. A Bayesian analysis of the chromosome architecture of human disorders by integrating reductionist data.

    PubMed

    Emmert-Streib, Frank; de Matos Simoes, Ricardo; Tripathi, Shailesh; Glazko, Galina V; Dehmer, Matthias

    2012-01-01

    In this paper, we present a Bayesian approach to estimate a chromosome and a disorder network from the Online Mendelian Inheritance in Man (OMIM) database. In contrast to other approaches, we obtain statistic rather than deterministic networks enabling a parametric control in the uncertainty of the underlying disorder-disease gene associations contained in the OMIM, on which the networks are based. From a structural investigation of the chromosome network, we identify three chromosome subgroups that reflect architectural differences in chromosome-disorder associations that are predictively exploitable for a functional analysis of diseases. PMID:22822426

  8. Preferential integration of a transfected marker gene into spontaneously expressed fragile sites of a breast cancer cell line.

    PubMed

    Matzner, Isabel; Savelyeva, Larissa; Schwab, Manfred

    2003-01-28

    Common fragile sites are non-randomly distributed unstable chromosomal regions thought to be hot spots for recombination. They appear as gaps, breaks and triradial figures when cells are cultured under conditions that inhibit replication or repair of DNA. The removal of replication-inhibitory challenges is followed by repair activation to restore the DNA damage at the fragile site. The breast cancer cell line MDA-MB-436 has a spontaneous and non-random expression pattern of fragile sites that appear to be related to the complex pattern of chromosomal rearrangements. The high frequency of which fragile sites are spontaneously activated should make MDA-MB-436 cells a powerful tool to study in greater detail the DNA sequences of a multiplicity of fragile sites. Here, we have explored if the DNA at spontaneously activated fragile sites in MDA-MB-436 cells can be genetically tagged by the repair-mediated insertion of an exogenously supplied drug resistance gene. The cells were transfected with pSV2Neo, stably transfected clones were selected with neomycin, and the sites of pSV2Neo integration were determined by fluorescent in situ hybridization. Eighty-eight of 100 isolated clones had a non-random distribution of a total of 112 pSV2Neo integrations. Of these, 95 integrations (85%) coincide with the position at which non-random gaps and breaks appear in the MDA-MB-436 cells. Forty-nine (44%) of the 112 integrations appeared to be at position of known fragile sites, 46 (41%) were at the non-random chromosomal sites not previously described as "true" fragile sites. It is possible, however, that these non-random instabilities signal of genomic regions equivalent to fragile sites, that either have not previously been detected due to low level expression or that are activated in a tissue- or cell-type-specific manner. Collectively, our results show a preferential integration of exogenous DNA into fragile sites and other non-random regions of high genomic instability in MDA

  9. A Graph Based Framework to Model Virus Integration Sites.

    PubMed

    Fronza, Raffaele; Vasciaveo, Alessandro; Benso, Alfredo; Schmidt, Manfred

    2016-01-01

    With next generation sequencing thousands of virus and viral vector integration genome targets are now under investigation to uncover specific integration preferences and to define clusters of integration, termed common integration sites (CIS), that may allow to assess gene therapy safety or to detect disease related genomic features such as oncogenes. Here, we addressed the challenge to: 1) define the notion of CIS on graph models, 2) demonstrate that the structure of CIS enters in the category of scale-free networks and 3) show that our network approach analyzes CIS dynamically in an integrated systems biology framework using the Retroviral Transposon Tagged Cancer Gene Database (RTCGD) as a testing dataset. PMID:27257470

  10. Integration of HIV in the Human Genome: Which Sites Are Preferential? A Genetic and Statistical Assessment

    PubMed Central

    Gonçalves, Juliana; Moreira, Elsa; Sequeira, Inês J.; Rodrigues, António S.; Rueff, José; Brás, Aldina

    2016-01-01

    Chromosomal fragile sites (FSs) are loci where gaps and breaks may occur and are preferential integration targets for some viruses, for example, Hepatitis B, Epstein-Barr virus, HPV16, HPV18, and MLV vectors. However, the integration of the human immunodeficiency virus (HIV) in Giemsa bands and in FSs is not yet completely clear. This study aimed to assess the integration preferences of HIV in FSs and in Giemsa bands using an in silico study. HIV integration positions from Jurkat cells were used and two nonparametric tests were applied to compare HIV integration in dark versus light bands and in FS versus non-FS (NFSs). The results show that light bands are preferential targets for integration of HIV-1 in Jurkat cells and also that it integrates with equal intensity in FSs and in NFSs. The data indicates that HIV displays different preferences for FSs compared to other viruses. The aim was to develop and apply an approach to predict the conditions and constraints of HIV insertion in the human genome which seems to adequately complement empirical data. PMID:27294106

  11. Stress induced by premature chromatin condensation triggers chromosome shattering and chromothripsis at DNA sites still replicating in micronuclei or multinucleate cells when primary nuclei enter mitosis.

    PubMed

    Terzoudi, Georgia I; Karakosta, Maria; Pantelias, Antonio; Hatzi, Vasiliki I; Karachristou, Ioanna; Pantelias, Gabriel

    2015-11-01

    Combination of next-generation DNA sequencing, single nucleotide polymorphism array analyses and bioinformatics has revealed the striking phenomenon of chromothripsis, described as complex genomic rearrangements acquired in a single catastrophic event affecting one or a few chromosomes. Via an unproven mechanism, it is postulated that mechanical stress causes chromosome shattering into small lengths of DNA, which are then randomly reassembled by DNA repair machinery. Chromothripsis is currently examined as an alternative mechanism of oncogenesis, in contrast to the present paradigm that considers a stepwise development of cancer. While evidence for the mechanism(s) underlying chromosome shattering during cancer development remains elusive, a number of hypotheses have been proposed to explain chromothripsis, including ionizing radiation, DNA replication stress, breakage-fusion-bridge cycles, micronuclei formation and premature chromosome compaction. In the present work, we provide experimental evidence on the mechanistic basis of chromothripsis and on how chromosomes can get locally shattered in a single catastrophic event. Considering the dynamic nature of chromatin nucleoprotein complex, capable of rapid unfolding, disassembling, assembling and refolding, we first show that chromatin condensation at repairing or replicating DNA sites induces the mechanical stress needed for chromosome shattering to ensue. Premature chromosome condensation is then used to visualize the dynamic nature of interphase chromatin and demonstrate that such mechanical stress and chromosome shattering can also occur in chromosomes within micronuclei or asynchronous multinucleate cells when primary nuclei enter mitosis. Following an aberrant mitosis, chromosomes could find themselves in the wrong place at the wrong time so that they may undergo massive DNA breakage and rearrangement in a single catastrophic event. Specifically, our results support the hypothesis that premature chromosome

  12. Vertical integration of cosmid and YAC resources for interval mapping on the X-chromosome

    SciTech Connect

    Holland, J.; Coffey, A.J.; Giannelli, F.; Bentley, D.R. )

    1993-02-01

    The vertical integration of cosmid and yeast artificial chromosome (YAC) resources is of particular importance in the development of high-resolution maps of selected regions of the human genome. A resource of approximately 95,000 cosmids constructed using DNA from primary fibroblasts of karyotype 49,XXXXX was validated by detailed characterization of a 200-kb cosmid contig spanning exons 8-20 of the dystrophin gene. This resource was used to construct contigs in 0.65 Mb of Xq26 by hybridization of gel-purified YAC DNA to high-density gridded arrays of the cosmid library; positive cosmids were overlapped by fingerprinting. Contigs were oriented and ordered relative to existing YACs in the region using cross-hybridization. The overlaps between a representative set of cosmids define 54 intervals of 5-20 kb and were used to construct a high-resolution cosmid interval map of the region, locating markers, dinucleotide repeats, and candidate CpG islands. This approach can be applied rapidly to large regions of the genome and without recourse to subcloning of individual YACs. 49 refs., 5 figs.

  13. Integration-Free iPS Cells Engineered Using Human Artificial Chromosome Vectors

    PubMed Central

    Hiratsuka, Masaharu; Uno, Narumi; Ueda, Kana; Kurosaki, Hajime; Imaoka, Natsuko; Kazuki, Kanako; Ueno, Etsuya; Akakura, Yutaro; Katoh, Motonobu; Osaki, Mitsuhiko; Kazuki, Yasuhiro; Nakagawa, Masato; Yamanaka, Shinya; Oshimura, Mitsuo

    2011-01-01

    Human artificial chromosomes (HACs) have unique characteristics as gene-delivery vectors, including episomal transmission and transfer of multiple, large transgenes. Here, we demonstrate the advantages of HAC vectors for reprogramming mouse embryonic fibroblasts (MEFs) into induced pluripotent stem (iPS) cells. Two HAC vectors (iHAC1 and iHAC2) were constructed. Both carried four reprogramming factors, and iHAC2 also encoded a p53-knockdown cassette. iHAC1 partially reprogrammed MEFs, and iHAC2 efficiently reprogrammed MEFs. Global gene expression patterns showed that the iHACs, unlike other vectors, generated relatively uniform iPS cells. Under non-selecting conditions, we established iHAC-free iPS cells by isolating cells that spontaneously lost iHAC2. Analyses of pluripotent markers, teratomas and chimeras confirmed that these iHAC-free iPS cells were pluripotent. Moreover, iHAC-free iPS cells with a re-introduced HAC encoding Herpes Simplex virus thymidine kinase were eliminated by ganciclovir treatment, indicating that the HAC safeguard system functioned in iPS cells. Thus, the HAC vector could generate uniform, integration-free iPS cells with a built-in safeguard system. PMID:21998730

  14. Fermentation of crystalline cellulose to ethanol by Klebsiella oxytoca containing chromosomally integrated zymomonas mobilis genes

    SciTech Connect

    Doran, J.B.; Ingram, L.O.

    1993-09-01

    Complete enzymatic hydrolysis of cellulose to glucose is generally required for efficient fermentation to ethanol. This hydrolysis requires endoglucanase, exoglucanase, and cellobiase. The Gram-negative bacterium, Klebsiella oxytoca, contains the native ability to transport and metabolize cellobiose, minimizing the need for extracellular cellobiase. Strain P2 is a recombinant derivative in which the Zymomonas mobilis pdc and adhB genes have been integrated into the chromosome and expressed, directing the metabolism of pyruvate to ethanol. This organism has been evaluated in simultaneous saccharification and fermentation (SSF) experiments to determine optimal conditions and limits of performance. The temperature was varied between 32 and 40{degree}C over a pH range of 5.0-5.8 with 100 g/L crystalline cellulose (Sigmacell 50, Sigma Chemical Company, St. Louis, MO) as the substrate and commercial cellulase (Spezyme CE, South San Francisco, CA). A broad optimum for SSF was observed, with a pH of 5.2-5.5 and temperatures of 32-35{degree}C, which allowed the production of over 44 g of ethanol/L (81-86% of the maximum theoretical yield). Although the rate of ethanol production increased with cellulase, diminishing improvements were observed at enzyme loadings above 10 filter paper units/g of cellulose. 34 refs., 5 figs., 2 tabs.

  15. Comprehensive Integrated Planning Process for the Oak Ridge Operations Sites

    SciTech Connect

    Bechtel Jacobs Company LLC; Lockheed Martin Energy Research Corporation; Lockheed Martin Energy Systems, Inc.

    1999-09-01

    This plan is intended to assist the U.S. Department of Energy (DOE) and contractor personnel in implementing a comprehensive integrated planning process consistent with DOE Order 430.1A, "Life Cycle Asset Management," and Oak Ridge Operations (ORO) Order 430 on sites under the jurisdiction of DOE-ORO. Those sites are the Oak Ridge Reservation, in Oak Ridge, Tennessee; the Paducah Gaseous Diffusion Plant, in Paducah, Kentucky; and the Portsmouth Gaseous Diffusion Plant, in Piketon, Ohio. DOE contractors at these sites are charged with developing and producing this plan, which is referred to as simply the Comprehensive Integrated Plan.

  16. HHV-8-unrelated primary effusion-like lymphoma associated with clonal loss of inherited chromosomally-integrated human herpesvirus-6A from the telomere of chromosome 19q.

    PubMed

    Zhang, Enjie; Cotton, Victoria E; Hidalgo-Bravo, Alberto; Huang, Yan; Bell, Adam J; Jarrett, Ruth F; Wilkie, Gavin S; Davison, Andrew J; Nacheva, Ellie P; Siebert, Reiner; Majid, Aneela; Kelpanides, Inga; Jayne, Sandrine; Dyer, Martin J; Royle, Nicola J

    2016-01-01

    Primary effusion lymphomas (PEL) are associated with human herpesvirus-8 (HHV-8) and usually occur in immunocompromised individuals. However, there are numerous reports of HHV-8-unrelated PEL-like lymphomas with unknown aetiology. Here we characterize an HHV-8-unrelated PEL-like lymphoma in an elderly woman who was negative for human immunodeficiency viruses 1 and 2, and hepatitis B and C. The woman was, however, a carrier of an inherited-chromosomally-integrated human herpesvirus-6A (iciHHV-6A) genome in one 19q telomere. The iciHHV-6A genome was complete in blood DNA, encoding a full set of protein-coding genes. Interestingly, the entire iciHHV-6A genome was absent from the HHV-8-unrelated-PEL-like lymphoma cells despite retention of both copies of chromosome 19. The somatic loss of the 19q-iciHHV-6A genome occurred very early during lymphoma development and we propose it occurred via telomere-loop formation and excision to release a circular viral genome that was subsequently lost. Whether release of the HHV-6A genome from the telomere contributed to lymphomagenesis, or was coincidental, remains unclear but this event may have deregulated the expression of HHV-6A or 19q genes or else disrupted telomere function. To establish the frequency and importance of iciHHV-6 loss from telomeres, the HHV-6 copy number should be assessed in tumours that arise in iciHHV-6 carriers. PMID:26947392

  17. HHV-8-unrelated primary effusion-like lymphoma associated with clonal loss of inherited chromosomally-integrated human herpesvirus-6A from the telomere of chromosome 19q

    PubMed Central

    Zhang, Enjie; Cotton, Victoria E.; Hidalgo-Bravo, Alberto; Huang, Yan; J. Bell, Adam; F. Jarrett, Ruth; Wilkie, Gavin S.; Davison, Andrew J.; P. Nacheva, Ellie; Siebert, Reiner; Majid, Aneela; Kelpanides, Inga; Jayne, Sandrine; Dyer, Martin J.; Royle, Nicola J.

    2016-01-01

    Primary effusion lymphomas (PEL) are associated with human herpesvirus-8 (HHV-8) and usually occur in immunocompromised individuals. However, there are numerous reports of HHV-8-unrelated PEL-like lymphomas with unknown aetiology. Here we characterize an HHV-8-unrelated PEL-like lymphoma in an elderly woman who was negative for human immunodeficiency viruses 1 and 2, and hepatitis B and C. The woman was, however, a carrier of an inherited-chromosomally-integrated human herpesvirus-6A (iciHHV-6A) genome in one 19q telomere. The iciHHV-6A genome was complete in blood DNA, encoding a full set of protein-coding genes. Interestingly, the entire iciHHV-6A genome was absent from the HHV-8-unrelated-PEL-like lymphoma cells despite retention of both copies of chromosome 19. The somatic loss of the 19q-iciHHV-6A genome occurred very early during lymphoma development and we propose it occurred via telomere-loop formation and excision to release a circular viral genome that was subsequently lost. Whether release of the HHV-6A genome from the telomere contributed to lymphomagenesis, or was coincidental, remains unclear but this event may have deregulated the expression of HHV-6A or 19q genes or else disrupted telomere function. To establish the frequency and importance of iciHHV-6 loss from telomeres, the HHV-6 copy number should be assessed in tumours that arise in iciHHV-6 carriers. PMID:26947392

  18. The MaoP/maoS Site-Specific System Organizes the Ori Region of the E. coli Chromosome into a Macrodomain.

    PubMed

    Valens, Michèle; Thiel, Axel; Boccard, Frédéric

    2016-09-01

    The Ori region of bacterial genomes is segregated early in the replication cycle of bacterial chromosomes. Consequently, Ori region positioning plays a pivotal role in chromosome dynamics. The Ori region of the E. coli chromosome is organized as a macrodomain with specific properties concerning DNA mobility, segregation of loci and long distance DNA interactions. Here, by using strains with chromosome rearrangements and DNA mobility as a read-out, we have identified the MaoP/maoS system responsible for constraining DNA mobility in the Ori region and limiting long distance DNA interactions with other regions of the chromosome. MaoP belongs to a group of proteins conserved in the Enterobacteria that coevolved with Dam methylase including SeqA, MukBEF and MatP that are all involved in the control of chromosome conformation and segregation. Analysis of DNA rings excised from the chromosome demonstrated that the single maoS site is required in cis on the chromosome to exert its effect while MaoP can act both in cis and in trans. The position of markers in the Ori region was affected by inactivating maoP. However, the MaoP/maoS system was not sufficient for positioning the Ori region at the ¼-¾ regions of the cell. We also demonstrate that the replication and the resulting expansion of bulk DNA are localized centrally in the cell. Implications of these results for chromosome positioning and segregation in E. coli are discussed. PMID:27627105

  19. Karyotyping and in situ chromosomal localization of rDNA sites in black cumin Bunium persicum (Boiss) B. Fedtsch,1915 (Apiaceae)

    PubMed Central

    Chahota, R. K.; Mukai, Y.; Chaudhary, H.K.; Kishore, Naval; Sharma, T.R.

    2011-01-01

    Abstract The fluorescent in situ hybridization (FISH) technique has been applied to somatic chromosomes in the medicinally important species, Bunium persicum, to elucidate its karyotypes. The bicolour FISH technique involving 18S-5.8S-26S and 5S ribosomal RNA genes as probes was used to assign physical localization and measurement of rDNA sites on homologous pairs of chromosomes. The two 18S-5.8S-26S rRNA gene sites were at the terminal regions of the short arms of the chromosomes 1 and 2 involving NOR region of chromosome 1. The 5S rDNA sites were found on subtelomeric region of the long arm of the chromosome number 5 and at interstitial regions of the short arm of chromosome 7. Based on direct visual analysis of chromosome length, morphology and position of FISH signals, a pioneer attempt has been made to construct metaphase karyotype in Bunium persicum, an endangered medicinal plant of North Western Himalayas. PMID:24260640

  20. WWOX, the chromosomal fragile site FRA16D spanning gene: its role in metabolism and contribution to cancer.

    PubMed

    Richards, Robert I; Choo, Amanda; Lee, Cheng Shoou; Dayan, Sonia; O'Keefe, Louise

    2015-03-01

    The WWOX gene spans the common chromosomal fragile site FRA16D that is located within a massive (780 kb) intron. The WWOX gene is very long, at 1.1 Mb, which may contribute to the very low abundance of the full-length 1.4 kb mRNA. Alternative splicing also accounts for a variety of aberrant transcripts, most of which are devoid of C-terminal sequences required for WWOX to act as an oxidoreductase. The mouse WWOX gene also spans a chromosomal fragile site implying some sort of functional relationship that confers a selective advantage. The encoded protein domains of WWOX are conserved through evolution (between humans and Drosophila melanogaster) and include WW domains, an NAD -binding site, short-chain dehydrogenase/reductase enzyme and nuclear compartmentalization signals. This homology has enabled functional analyses in D. melanogaster that demonstrate roles for WWOX in reactive oxygen species regulation and metabolism. Indeed the human WWOX gene is also responsive to altered metabolism. Cancer cells typically exhibit altered metabolism (Warburg effect). Many cancers exhibit FRA16D DNA instability that results in aberrant WWOX expression and is associated with poor prognosis for these cancers. It is therefore thought that aberrant WWOX expression contributes to the altered metabolism in cancer. In addition, others have found that a specific (low-expression) allele of WWOX genotype contributes to cancer predisposition. PMID:25595186

  1. A cloning vector for creation of Escherichia coli lacZ translational fusions and generation of linear template for chromosomal integration.

    PubMed

    Uhlich, Gaylen A; Chen, Chin-Yi

    2012-05-01

    A novel cloning vector to aid in the construction of single copy β-galactosidase reporter systems for gene expression studies in lactose metabolizing Escherichia coli strains, including STEC, is described. The plasmid allows construction of translational fusions of cloned gene promoters to a short segment of E. coli lacZ. A selectable spectinomycin resistance marker flanked by a short lacI segment is positioned 5' to the cloning site. PCR amplification using opposing primers complementary to the upstream lacI fragment and the downstream lacZ fragment generates a linear template suitable for integration using pRedET recombination. Integration of linear template derived from the recombinant plasmid into host strains replaces the entire native lacZ promoter and fuses the promoter of interest in-frame with the lacZ gene, thus simultaneously producing a single-copy, chromosomal reporter system and eliminating background lacZ expression. Studies comparing ahpC expression from a chromosomal fusion in the lac open with that on a plasmid in E. coli strain EDL933 are shown. PMID:22197962

  2. Converting Maturing Nuclear Sites to Integrated Power Production Islands

    DOE PAGESBeta

    Solbrig, Charles W.

    2011-01-01

    Nuclear islands, which are integrated power production sites, could effectively sequester and safeguard the US stockpile of plutonium. A nuclear island, an evolution of the integral fast reactor, utilizes all the Transuranics (Pu plus minor actinides) produced in power production, and it eliminates all spent fuel shipments to and from the site. This latter attribute requires that fuel reprocessing occur on each site and that fast reactors be built on-site to utilize the TRU. All commercial spent fuel shipments could be eliminated by converting all LWR nuclear power sites to nuclear islands. Existing LWR sites have the added advantage ofmore » already possessing a license to produce nuclear power. Each could contribute to an increase in the nuclear power production by adding one or more fast reactors. Both the TRU and the depleted uranium obtained in reprocessing would be used on-site for fast fuel manufacture. Only fission products would be shipped to a repository for storage. The nuclear island concept could be used to alleviate the strain of LWR plant sites currently approaching or exceeding their spent fuel pool storage capacity. Fast reactor breeding ratio could be designed to convert existing sites to all fast reactors, or keep the majority thermal.« less

  3. An integrated physical map of 210 markers assigned to the short arm of human chromosome 11

    SciTech Connect

    Redeker, E.; Hoovers, J.M.N.; Alders, M.

    1994-06-01

    Using a panel of patient cell lines with chromosomal breakpoints, the authors constructed a physical map for the short arm of human chromosome 11. They focused on 11p15, a chromosome band harboring at least 25 known genes and associated with the Beckwith-Wiedemann syndrome, several childhood tumors, and genomic imprinting. This underlines the need for a physical map for this region. They divided the short arm of chromosome 11 into 18 breakpoint regions, and a large series of new and previously described genes and markers was mapped within these intervals using fluorescence in situ hybridization. Cosmid fingerprint analysis showed that 19 of these markers were included in cosmid contigs. A detailed 10-Mb pulsed-field physical map of the region 11p15.3-pter was constructed. These three different approaches enabled the high-resolution mapping of 210 markers, including 22 known genes. 64 refs., 6 figs., 3 tabs.

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

    EPA Science Inventory

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

  5. Induced and natural break sites in the chromosomes of Hawaiian Drosophila

    SciTech Connect

    Tonzetich, J.; Lyttle, T.W.; Carson, H.L.

    1988-03-01

    Gamma-irradiation of a laboratory strain of the Hawaiian species of Drosophila heteroneura yielded 310 breaks in the five major acrocentric polytene chromosomes. Their map positions conform to the Poisson distribution, unlike most of the 436 natural breaks mapped in 105 closely related species endemic to Hawaii. Genome element E is longer and has more induced breaks than the others. Both in Hawaiian and related species groups, this element shows increased polymorphism and fixation of naturally occurring inversions. The X chromosome (element A) also accumulates many natural breaks; the majority of the resulting aberrations become fixed rather than remain as polymorphisms. Although size may play a small role in initial break distribution, the major effects relative to the establishment of a rearrangement in natural populations are ascribed to the interaction of selection and drift. Nonconformance of the natural breaks to the Poisson distribution appears to be due to the tendency for breaks to accumulate both in the proximal euchromatic portion of each arm and in heterochromatic regions that are not replicated in the polytene chromosomes.

  6. Integration and validation of the physical map of chromosome 21 using a high resolution somatic cell hybrid panel, STS mapping, and rare cleavage

    SciTech Connect

    Patterson, D.; Graw, S.; Gardiner, K.

    1994-09-01

    We are constructing a minimal tiling path of chromosome 21 based on the 810 YAC set described by Chumakov et al., the YACs isolated by the Chromosome 21 Joint YAC Screening Effort, and several additional YAC, cosmid, and P1 libraries. We are integrating the STS and YAC contig data with the high resolution somatic cell hybrid map of chromosome 21 to validate that the YACs chosen as part of the tilling path accurately represent the chromosomal region from which they are derived, to resolve problems of homology between chromosome 21 and other acrocentric chromosomes, to help resolve STS order, to integrate additional markers, especially genes, into the map, and to help in registration of the STS and YAC maps with the cytogenetic and genetic linkage maps. The physical boundaries of 23 different somatic cell hybrids have been determined. This appears to require reordering of some of the STSs on chromosome 21 and suggests the necessity of isolation of additional DNA markers and clones. A remarkable clustering of chromosome 21 breakpoints is observed. Rec-A Assisted Restriction Endonuclease (RARE) cleavage is being used to assess whether YACs accurately reflect this genomic region of origin and to assess the extent of overlap of YACs. This information will aid in the most efficient generation of high quality reagents for sequencing chromosome 21.

  7. Introgression maintains the genetic integrity of the mating-type determining chromosome of the fungus Neurospora tetrasperma

    PubMed Central

    Corcoran, Pádraic; Anderson, Jennifer L.; Jacobson, David J.; Sun, Yu; Ni, Peixiang; Lascoux, Martin; Johannesson, Hanna

    2016-01-01

    Genome evolution is driven by a complex interplay of factors, including selection, recombination, and introgression. The regions determining sexual identity are particularly dynamic parts of eukaryotic genomes that are prone to molecular degeneration associated with suppressed recombination. In the fungus Neurospora tetrasperma, it has been proposed that this molecular degeneration is counteracted by the introgression of nondegenerated DNA from closely related species. In this study, we used comparative and population genomic analyses of 92 genomes from eight phylogenetically and reproductively isolated lineages of N. tetrasperma, and its three closest relatives, to investigate the factors shaping the evolutionary history of the genomes. We found that suppressed recombination extends across at least 6 Mbp (∼63%) of the mating-type (mat) chromosome in N. tetrasperma and is associated with decreased genetic diversity, which is likely the result primarily of selection at linked sites. Furthermore, analyses of molecular evolution revealed an increased mutational load in this region, relative to recombining regions. However, comparative genomic and phylogenetic analyses indicate that the mat chromosomes are temporarily regenerated via introgression from sister species; six of eight lineages show introgression into one of their mat chromosomes, with multiple Neurospora species acting as donors. The introgressed tracts have been fixed within lineages, suggesting that they confer an adaptive advantage in natural populations, and our analyses support the presence of selective sweeps in at least one lineage. Thus, these data strongly support the previously hypothesized role of introgression as a mechanism for the maintenance of mating-type determining chromosomal regions. PMID:26893460

  8. H-NS Facilitates Sequence Diversification of Horizontally Transferred DNAs during Their Integration in Host Chromosomes.

    PubMed

    Higashi, Koichi; Tobe, Toru; Kanai, Akinori; Uyar, Ebru; Ishikawa, Shu; Suzuki, Yutaka; Ogasawara, Naotake; Kurokawa, Ken; Oshima, Taku

    2016-01-01

    Bacteria can acquire new traits through horizontal gene transfer. Inappropriate expression of transferred genes, however, can disrupt the physiology of the host bacteria. To reduce this risk, Escherichia coli expresses the nucleoid-associated protein, H-NS, which preferentially binds to horizontally transferred genes to control their expression. Once expression is optimized, the horizontally transferred genes may actually contribute to E. coli survival in new habitats. Therefore, we investigated whether and how H-NS contributes to this optimization process. A comparison of H-NS binding profiles on common chromosomal segments of three E. coli strains belonging to different phylogenetic groups indicated that the positions of H-NS-bound regions have been conserved in E. coli strains. The sequences of the H-NS-bound regions appear to have diverged more so than H-NS-unbound regions only when H-NS-bound regions are located upstream or in coding regions of genes. Because these regions generally contain regulatory elements for gene expression, sequence divergence in these regions may be associated with alteration of gene expression. Indeed, nucleotide substitutions in H-NS-bound regions of the ybdO promoter and coding regions have diversified the potential for H-NS-independent negative regulation among E. coli strains. The ybdO expression in these strains was still negatively regulated by H-NS, which reduced the effect of H-NS-independent regulation under normal growth conditions. Hence, we propose that, during E. coli evolution, the conservation of H-NS binding sites resulted in the diversification of the regulation of horizontally transferred genes, which may have facilitated E. coli adaptation to new ecological niches. PMID:26789284

  9. Heritable fragile sites on human chromosomes. V. A new class of fragile site requiring BrdU for expression.

    PubMed Central

    Sutherland, G R; Baker, E; Seshadri, R S

    1980-01-01

    A new fragile site at 10q25 is described, representing a new class of fragile site that requires bromodeoxyuridine (BrdU) in the culture medium for expression. This new fragile site is present in approximately one in 30 of the Australian population; it has only been observed in heterozygotes. Images Fig. 1 PMID:7395866

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

    PubMed Central

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

    1987-01-01

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

  11. CTCF Recruits Centromeric Protein CENP-E to the Pericentromeric/Centromeric Regions of Chromosomes through Unusual CTCF Binding Sites

    PubMed Central

    Xiao, Tiaojiang; Wongtrakoongate, Patompon; Trainor, Cecelia

    2015-01-01

    SUMMARY The role of CTCF in stabilizing long range interactions between chromatin sites essential for maintaining nuclear architecture is well established. Most of these interactions involve recruitment of the cohesin complex to chromatin via CTCF. We find that CTCF also interacts with the centromeric protein CENP-E both in vitro and in vivo. We identified CTCF sites in pericentric/centromeric DNA and found that early in mitosis CTCF binds and recruits CENP-E to these sites. Unlike most known CTCF genomic sites, the CTCF binding sites in the pericentric/centromeric regions interact strongly with the C-terminal fingers of CTCF. Over-expression of a small CENP-E fragment, targeted to these CTCF sites, results in delay in alignment of some chromosomes during mitosis, suggesting that the recruitment of CENP-E by CTCF is physiologically important. We conclude that CTCF helps recruit CENP-E to the centromere during mitosis, and may do so through a structure stabilized by the CTCF/CENP-E complex. PMID:26321640

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

    PubMed Central

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

    1996-01-01

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

  13. Identification of recognition sites for myc/max/mxd network proteins by a whole human chromosome 19 selection strategy.

    PubMed

    Akopov, S B; Chernov, I P; Wahlström, T; Kostina, M B; Klein, G; Henriksson, M; Nikolaev, L G

    2008-11-01

    In this study, we have identified 20 human sequences containing Myc network binding sites in a library from the whole human chromosome 19. We demonstrated binding of the Max protein to these sequences both in vitro and in vivo. The majority of the identified sequences contained one or several CACGTG or CATGTG E-boxes. Several of these sites were located within introns or in their vicinity and the corresponding genes were found to be up- or down-regulated in differentiating HL-60 cells. Our data show the proof of principle for using this strategy in identification of Max target genes, and this method can also be applied for other transcription factors. PMID:19120031

  14. Engineering of chromosomal wax ester synthase integrated Saccharomyces cerevisiae mutants for improved biosynthesis of fatty acid ethyl esters.

    PubMed

    Shi, Shuobo; Valle-Rodríguez, Juan Octavio; Siewers, Verena; Nielsen, Jens

    2014-09-01

    In recent years, significant advances have been made to engineer robust microbes for overproducing biochemical products from renewable resources. These accomplishments have to a large extend been based on plasmid based methods. However, plasmid maintenance may cause a metabolic burden on the host cell and plasmid-based overexpression of genes can result in genetically unstable strains, which contributes to loss in productivity. Here, a chromosome engineering method based on delta integration was applied in Saccharomyces cerevisiae for the production of fatty acid ethyl esters (FAEEs), which can be directly used as biodiesel and would be a possible substitute for conventional petroleum-based diesel. An integration construct was designed and integrated into chromosomal delta sequences by repetitive transformation, which resulted in 1-6 copies of the integration construct per genome. The corresponding FAEE production increased up to 34 mg/L, which is an about sixfold increase compared to the equivalent plasmid-based producer. The integrated cassette in the yeast genome was stably maintained in nonselective medium after deletion of RAD52 which is essential for efficient homologous recombination. To obtain a further increase of FAEE production, genes encoding endogenous acyl-CoA binding protein (ACB1) and a bacterial NADP(+)-dependent glyceraldehyde-3-phosphate dehydrogenase (gapN) were overexpressed in the final integration strain, which resulted in another 40% percent increase in FAEE production. Our integration strategy enables easy engineering of strains with adjustable gene copy numbers integrated into the genome and this allows for an easy evaluation of the effect of the gene copy number on pathway flux. It therefore represents a valuable tool for introducing and expressing a heterologous pathway in yeast. PMID:24752598

  15. TECHNOLOGY INTEGRATION FOR CONTAMINATED SITE REMEDIATION: CLEANUP GOALS & PERFORMANCE CRITERIA

    EPA Science Inventory

    There is a need to develop and field-test integrated remediation technologies that operate in a synergistic manner for cost-effective treatment of contaminated sites to achieve risk-based and rational endpoints. Aggressive technologies designed for rapid source-zone remediation m...

  16. Site-specific recombinases as tools for heterologous gene integration.

    PubMed

    Hirano, Nobutaka; Muroi, Tetsurou; Takahashi, Hideo; Haruki, Mitsuru

    2011-10-01

    Site-specific recombinases are the enzymes that catalyze site-specific recombination between two specific DNA sequences to mediate DNA integration, excision, resolution, or inversion and that play a pivotal role in the life cycles of many microorganisms including bacteria and bacteriophages. These enzymes are classified as tyrosine-type or serine-type recombinases based on whether a tyrosine or serine residue mediates catalysis. All known tyrosine-type recombinases catalyze the formation of a Holliday junction intermediate, whereas the catalytic mechanism of all known serine-type recombinases includes the 180° rotation and rejoining of cleaved substrate DNAs. Both recombinase families are further subdivided into two families; the tyrosine-type recombinases are subdivided by the recombination directionality, and the serine-type recombinases are subdivided by the protein size. Over more than two decades, many different site-specific recombinases have been applied to in vivo genome engineering, and some of them have been used successfully to mediate integration, deletion, or inversion in a wide variety of heterologous genomes, including those from bacteria to higher eukaryotes. Here, we review the recombination mechanisms of the best characterized recombinases in each site-specific recombinase family and recent advances in the application of these recombinases to genomic manipulation, especially manipulations involving site-specific gene integration into heterologous genomes. PMID:21822899

  17. Directed chromosomal integration and expression of the reporter gene gusA3 in Lactobacillus acidophilus NCFM.

    PubMed

    Douglas, Grace L; Klaenhammer, Todd R

    2011-10-01

    Lactobacillus acidophilus NCFM is a probiotic microbe that survives passage through the human gastrointestinal tract and interacts with the host epithelium and mucosal immune cells. The potential for L. acidophilus to express antigens at mucosal surfaces has been investigated with various antigens and plasmid expression vectors. Plasmid instability and antibiotic selection complicate the possibility of testing these constructs in human clinical trials. Integrating antigen encoding genes into the chromosome for expression is expected to eliminate selection requirements and provide genetic stability. In this work, a reporter gene encoding a β-glucuronidase (GusA3) was integrated into four intergenic chromosomal locations. The integrants were tested for genetic stability and GusA3 activity. Two locations were selected for insertion downstream of constitutively highly expressed genes, one downstream of slpA (LBA0169), encoding a highly expressed surface-layer protein, and one downstream of phosphopyruvate hydratase (LBA0889), a highly expressed gene with homologs in other lactic acid bacteria. An inducible location was selected downstream of lacZ (LBA1462), encoding a β-galactosidase. A fourth location was selected in a low-expression region. The expression of gusA3 was evaluated from each location by measuring GusA3 activity on 4-methyl-umbelliferyl-β-d-glucuronide (MUG). GusA3 activity from both highly expressed loci was more than three logs higher than the gusA3-negative parent, L. acidophilus NCK1909. GusA3 activity from the lacZ locus was one log higher in cells grown in lactose than in glucose. The differences in expression levels between integration locations highlights the importance of rational targeting with gene cassettes intended for chromosomal expression. PMID:21873486

  18. Integration of High-Resolution Physical and Genetic Map Reveals Differential Recombination Frequency between Chromosomes and the Genome Assembling Quality in Cucumber

    PubMed Central

    Cheng, Chunyan; Zhang, Zhonghua; Li, Ji; Huang, Sanwen; Chen, Jinfeng

    2013-01-01

    Cucumber is an important model crop and the first species sequenced in Cucurbitaceae family. Compared to the fast increasing genetic and genomics resources, the molecular cytogenetic researches in cucumber are still very limited, which results in directly the shortage of relation between plenty of physical sequences or genetic data and chromosome structure. We mapped twenty-three fosmids anchored by SSR markers from LG-3, the longest linkage group, and LG-4, the shortest linkage group on pachytene chromosomes 3 and 4, using uorescence in situ hybridization (FISH). Integrated molecular cytogenetic maps of chromosomes 3 and 4 were constructed. Except for three SSR markers located on heterochromatin region, the cytological order of markers was concordant with those on the linkage maps. Distinct structural differences between chromosomes 3 and 4 were revealed by the high resolution pachytene chromosomes. The extreme difference of genetic length between LG-3 and LG-4 was mainly attributed to the difference of overall recombination frequency. The significant differentiation of heterochromatin contents in chromosomes 3 and 4 might have a direct correlation with recombination frequency. Meanwhile, the uneven distribution of recombination frequency along chromosome 4 was observed, and recombination frequency of the long arm was nearly 3.5 times higher than that of the short arm. The severe suppression of recombination was exhibited in centromeric and heterochromatin domains of chromosome 4. Whereas a close correlation between the gene density and recombination frequency was observed in chromosome 4, no significant correlation was observed between them along chromosome 3. The comparison between cytogenetic and sequence maps revealed a large gap on the pericentromeric heterochromatin region of sequence map of chromosome 4. These results showed that integrated molecular cytogenetic maps can provide important information for the study of genetic and genomics in cucumber. PMID

  19. In vivo interaction of the Escherichia coli integration host factor with its specific binding sites.

    PubMed

    Engelhorn, M; Boccard, F; Murtin, C; Prentki, P; Geiselmann, J

    1995-08-11

    The histone-like protein integration host factor (IHF) of Escherichia coli binds to specific binding sites on the chromosome or on mobile genetic elements, and is involved in many cellular processes. We have analyzed the interaction of IHF with five different binding sites in vitro and in vivo using UV laser footprinting, a technique that probes the immediate environment and conformation of a segment of DNA. Using this generally applicable technique we can directly compare the binding modes and interaction strengths of a DNA binding protein in its physiological environment within the cell to measurements performed in vitro. We conclude that the interactions between IHF and its specific binding sites are identical in vitro and in vivo. The footprinting signal is consistent with the model of IHF-binding to DNA proposed by Yang and Nash (1989). The occupancy of binding sites varies with the concentration of IHF in the cell and allows to estimate the concentration of free IHF protein in the cell. PMID:7659518

  20. In vivo interaction of the Escherichia coli integration host factor with its specific binding sites.

    PubMed

    Engelhorn, M; Boccard, F; Murtin, C; Prentki, P; Geiselmann, J

    1995-09-11

    The histone-like protein integration host factor (IHF) of Escherichia coli binds to specific binding sites on the chromosome or on mobile genetic elements, and is involved in many cellular processes. We have analyzed the interaction of IHF with five different binding sites in vitro and in vivo using UV laser footprinting, a technique that probes the immediate environment and conformation of a segment of DNA. Using this generally applicable technique we can directly compare the binding modes and interaction strengths of a DNA binding protein in its physiological environment within the cell to measurements performed in vitro. We conclude that the interactions between IHF and its specific binding sites are identical in vitro and in vivo. The footprinting signal is consistent with the model of IHF-binding to DNA proposed by Yang and Nash (1989). The occupancy of binding sites varies with the concentration of IHF in the cell and allows to estimate the concentration of free IHF protein in the cell. PMID:7567442

  1. Differentially Expressed Genes Distributed Over Chromosomes and Implicated in Certain Biological Processes for Site Insertion Genetically Modified Rice Kemingdao

    PubMed Central

    Liu, Zhi; Li, Yunhe; Zhao, Jie; Chen, Xiuping; Jian, Guiliang; Peng, Yufa; Qi, Fangjun

    2012-01-01

    Release of genetically modified (GM) plants has sparked off intensive debates worldwide partly because of concerns about potential adverse unintended effects of GM plants to the agro system and the safety of foods. In this study, with the aim of revealing the molecular basis for unintended effects of a single site insertion GM Kemingdao (KMD) rice transformed with a synthetic cry1Ab gene, and bridging unintended effects of KMD rice through clues of differentially expressed genes, comparative transcriptome analyses were performed for GM KMD rice and its parent rice of Xiushui11 (XS11). The results showed that 680 differentially expressed transcripts were identified from 30-day old seedlings of GM KMD rice. The absolute majority of these changed expression transcripts dispersed and located over all rice chromosomes, and existed physical distance on chromosome from the insertion site, while only two transcripts were found to be differentially expressed within the 21 genes located within 100 kb up and down-stream of the insertion site. Pathway and biology function analyses further revealed that differentially expressed transcripts of KMD rice were involved in certain biological processes, and mainly implicated in two types of pathways. One type was pathways implicated in plant stress/defense responses, which were considerably in coordination with the reported unintended effects of KMD rice, which were more susceptible to rice diseases compared to its parent rice XS11; the other type was pathways associated with amino acids metabolism. With this clue, new unintended effects for changes in amino acids synthesis of KMD rice leaves were successfully revealed. Such that an actual case was firstly provided for identification of unintended effects in GM plants by comparative transciptome analysis. PMID:22811617

  2. Integrated grassland observation sites and integrated cropland observation sites at El Reno, Oklahoma

    Technology Transfer Automated Retrieval System (TEKTRAN)

    With the financial support from the National Science Foundation and the USDA National Institute of Food and Agriculture, a team of researchers from the University of Oklahoma and the USDA ARS Grazinglands Research Laboratory have worked together and established two Integrated Grassland Observation s...

  3. Significant differences in integration sites of Moloney murine leukemia virus/Moloney murine sarcoma virus retroviral vector carrying recombinant coagulation factor IX in two human cell lines.

    PubMed

    Castilho-Fernandes, Andrielle; Fontes, Aparecida Maria; Abraham, Kuruvilla Joseph; de Freitas, Marcela Cristina Corrêa; da Rosa, Nathalia Gonsales; Picanço-Castro, Virginia; de Sousa Russo-Carbolante, Elisa Maria; Covas, Dimas Tadeu

    2015-05-01

    Ligation-mediated-PCR was performed followed by the mapping of 177 and 150 integration sites from HepG2 and Hek293 transduced with chimera vector carrying recombinant human Factor IX (rhFIX) cDNA, respectively. The sequences were analyzed for chromosome preference, CpG, transcription start site (TSS), repetitive elements, fragile sites and target genes. In HepG2, rhFIX was had an increased preference for chromosomes 6 and 17; the median distance to the nearest CpG islands was 15,240 base pairs and 37 % of the integrations occurred in RefSeq genes. In Hek293, rhFIX had an increased preference for chromosome 5; the median distance to the nearest CpG islands was 209,100 base pairs and 74 % of the integrations occurred in RefSeq genes. The integrations in both cell lines were distant from the TSS. The integration patterns associated with this vector are different in each cell line. PMID:25650340

  4. A sugar beet (Beta vulgaris L.) reference FISH karyotype for chromosome and chromosome-arm identification, integration of genetic linkage groups and analysis of major repeat family distribution.

    PubMed

    Paesold, Susanne; Borchardt, Dietrich; Schmidt, Thomas; Dechyeva, Daryna

    2012-11-01

    We developed a reference karyotype for B. vulgaris which is applicable to all beet cultivars and provides a consistent numbering of chromosomes and genetic linkage groups. Linkage groups of sugar beet were assigned to physical chromosome arms by FISH (fluorescent in situ hybridization) using a set of 18 genetically anchored BAC (bacterial artificial chromosome) markers. Genetic maps of sugar beet were correlated to chromosome arms, and North-South orientation of linkage groups was established. The FISH karyotype provides a technical platform for genome studies and can be applied for numbering and identification of chromosomes in related wild beet species. The discrimination of all nine chromosomes by BAC probes enabled the study of chromosome-specific distribution of the major repetitive components of sugar beet genome comprising pericentromeric, intercalary and subtelomeric satellites and 18S-5.8S-25S and 5S rRNA gene arrays. We developed a multicolor FISH procedure allowing the identification of all nine sugar beet chromosome pairs in a single hybridization using a pool of satellite DNA probes. Fiber-FISH was applied to analyse five chromosome arms in which the furthermost genetic marker of the linkage group was mapped adjacently to terminal repetitive sequences on pachytene chromosomes. Only on two arms telomere arrays and the markers are physically linked, hence these linkage groups can be considered as terminally closed making the further identification of distal informative markers difficult. The results support genetic mapping by marker localization, the anchoring of contigs and scaffolds for the annotation of the sugar beet genome sequence and the analysis of the chromosomal distribution patterns of major families of repetitive DNA. PMID:22775355

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

    PubMed Central

    Walz, C; Schlehofer, J R

    1992-01-01

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

  6. Integration of Environmental Compliance at the Savannah River Site - 13024

    SciTech Connect

    Hoel, David; Griffith, Michael

    2013-07-01

    The Savannah River Site (SRS) is a large federal installation hosting diverse missions and multiple organizations with competing regulatory needs. Accordingly, there was a need to integrate environmental compliance strategies to ensure the consistent flow of information between Department of Energy-Savannah River (DOE-SR), the regulatory agencies and other interested parties. In order to meet this objective, DOE and major SRS contractors and tenants have committed to a strategy of collaboratively working together to ensure that a consistent, integrated, and fully coordinated approach to environmental compliance and regulator relationships is maintained. DOE-SR and Savannah River Nuclear Solutions, LLC, the SRS management and operations contractor, have established an environmental compliance integration process that provides for the consistent flow down of requirements to projects, facilities, SRS contractors, and subcontractors as well as the upward flow of information to assist in the early identification and resolution of environmental regulatory issues and enhancement of compliance opportunities. In addition, this process strongly fosters teamwork to collaboratively resolve complex regulatory challenges, promote pollution prevention and waste minimization opportunities to advance site missions in a manner that balances near-term actions with the long-term site vision, while being protective of human health and the environment. Communication tools are being utilized, some with enhancements, to ensure appropriate information is communicated to all levels with environmental responsibility at SRS. SRS internal regulatory integration is accomplished through a variety of informational exchange forums (e.g., Challenges, Opportunities and Resolution (COR) Team, DOE's Joint Site Regulatory Integration Team, and the Senior Environmental Managers Council (SEMC)). SRS communications and problem-solving with the regulatory agencies have been enhanced through formation of an

  7. Dr.VIS v2.0: an updated database of human disease-related viral integration sites in the era of high-throughput deep sequencing.

    PubMed

    Yang, Xiaobo; Li, Ming; Liu, Qi; Zhang, Yabing; Qian, Junyan; Wan, Xueshuai; Wang, Anqiang; Zhang, Haohai; Zhu, Chengpei; Lu, Xin; Mao, Yilei; Sang, Xinting; Zhao, Haitao; Zhao, Yi; Zhang, Xiaoyan

    2015-01-01

    Dr.VIS is a database of human disease-related viral integration sites (VIS). The number of VIS has grown rapidly since Dr.VIS was first released in 2011, and there is growing recognition of the important role that viral integration plays in the development of malignancies. The updated database version, Dr.VIS v2.0 (http://www.bioinfo.org/drvis or bminfor.tongji.edu.cn/drvis_v2), represents 25 diseases, covers 3340 integration sites of eight oncogenic viruses in human chromosomes and provides more accurate information about VIS from high-throughput deep sequencing results obtained mainly after 2012. Data of VISes for three newly identified oncogenic viruses for 14 related diseases have been added to this 2015 update, which has a 5-fold increase of VISes compared to Dr.VIS v1.0. Dr.VIS v2.0 has 2244 precise integration sites, 867 integration regions and 551 junction sequences. A total of 2295 integration sites are located near 1730 involved genes. Of the VISes, 1153 are detected in the exons or introns of genes, with 294 located up to 5 kb and a further 112 located up to 10 kb away. As viral integration may alter chromosome stability and gene expression levels, characterizing VISes will contribute toward the discovery of novel oncogenes, tumor suppressor genes and tumor-associated pathways. PMID:25355513

  8. Adeno-associated virus (AAV) Rep proteins mediate complex formation between AAV DNA and its integration site in human DNA.

    PubMed Central

    Weitzman, M D; Kyöstiö, S R; Kotin, R M; Owens, R A

    1994-01-01

    AAV is unique among eukaryotic viruses in the ability of its DNA to integrate preferentially into a specific region of the human genome. Understanding AAV integration may aid in developing gene therapy systems with predictable integration sites. Using a gel mobility-shift assay, we have identified a DNA sequence within the AAV integration locus on human chromosome 19 which is specifically bound by the AAV Rep78 and Rep68 proteins. This Rep recognition sequence is a GCTC repeating motif very similar to sequences within the inverted terminal repeats of the AAV genome which are also bound by Rep78 and Rep68. Cloned oligonucleotides containing the recognition sequence can direct specific binding by Rep proteins. Binding assays with mutant Rep proteins show that the amino-terminal portion of Rep78 and Rep68 can direct binding to either the AAV terminal repeat hairpin DNA or chromosome 19. This human genomic DNA can be complexed with AAV DNA by Rep proteins as demonstrated by a dual-label (32P/biotin) assay. These results suggest a role for Rep in targeting viral integration. Images PMID:8016070

  9. A molecular site-site integral equation that yields the dielectric constant

    NASA Astrophysics Data System (ADS)

    Dyer, Kippi M.; Perkyns, John S.; Stell, George; Pettitt, B. Montgomery

    2008-09-01

    Our recent derivation [K. M. Dyer et al., J. Chem. Phys. 127, 194506 (2007)] of a diagrammatically proper, site-site, integral equation theory using molecular angular expansions is extended to polar fluids. With the addition of atomic site charges we take advantage of the formal long-ranged potential field cancellations before renormalization to generate a set of numerically stable equations. Results for calculations in a minimal (spherical) angular basis set are presented for the radial distribution function, the first dipolar (110) projection, and the dielectric constant for two model diatomic systems. All results, when compared to experiment and simulation, are a significant quantitative and qualitative improvement over previous site-site theories. More importantly, the dielectric constant is not trivial and close to simulation and experiment.

  10. Characterization of the lactococcal temperate phage TP901-1 and its site-specific integration.

    PubMed Central

    Christiansen, B; Johnsen, M G; Stenby, E; Vogensen, F K; Hammer, K

    1994-01-01

    The temperate lactococcal phage TP901-1, induced by UV light from Lactococcus lactis subsp. cremoris 901-1, was characterized. The restriction map was found to be circular, and the packaging of TP901-1 DNA was concluded to occur by a headful mechanism. The pac region was localized on the 38.4-kb phage genome. TP901-1 belongs to the class of P335 phages (V. Braun, S. Hertwig, H. Neve, A. Geis, and M. Teuber, J. Gen. Microbiol. 135:2551-2560, 1989). Evidence is presented that the phages TP936-1 (V. Braun, S. Hertwig, H. Neve, A. Geis, and M. Teuber, J. Gen. Microbiol. 135:2551-2560, 1989) and C3-T1 (A. W. Jarvis, V. R. Parker, and M. B. Bianchin, Can. J. Microbiol. 38:398-404, 1992) are very closely related to or are identical to TP901-1. The lytically propagated TP901-1 phages were able to lysogenize both indicator strains Lactococcus cremoris 3107 and Wg2. Lysogenization resulted in site-specific integration of the phage genome into the bacterial chromosome. Only one chromosomal attB site was found in 20 independent lysogens. The attP region of TP901-1 and the attL and attR regions were cloned and sequenced. The results showed a core region of only 5 bp, in which the recombination occurs, followed after a 1-bp mismatch by a 7-bp identical region, TCAAT(T/C)AAGGTAA. This result was further verified by sequencing of the attB region obtained by PCR. An integration vector was constructed with the 6.5-kb EcoRI fragment from TP901-1 containing attP. This vector also functions in the plasmid-free strains, MG1363 and LM0230 with only one specific attB site, strongly indicating a more general use of the TP901-1-based integration vector in lactococci. Images PMID:8106318

  11. Systems Thinking Versus Population Thinking: Genotype Integration and Chromosomal Organization 1930s-1950s.

    PubMed

    Lamm, Ehud

    2015-11-01

    This article describes how empirical discoveries in the 1930s-1950s regarding population variation for chromosomal inversions affected Theodosius Dobzhansky and Richard Goldschmidt. A significant fraction of the empirical work I discuss was done by Dobzhansky and his coworkers; Goldschmidt was an astute interpreter, with strong and unusual commitments. I argue that both belong to a mechanistic tradition in genetics, concerned with the effects of chromosomal organization and systems on the inheritance patterns of species. Their different trajectories illustrate how scientists' commitments affect how they interpret new evidence and adjust to it. Dobzhansky was moved to revised views about selection, while Goldschmidt moved his attention to different genetic phenomena. However different, there are significant connections between the two that enrich our understanding of their views. I focus on two: the role of developmental considerations in Dobzhansky's thought and the role of neutrality and drift in Goldschmidt's evolutionary account. Dobzhansky's struggle with chromosomal variation is not solely about competing schools of thought within the selectionist camp, as insightfully articulated by John Beatty, but also a story of competition between selectionist thinking and developmental perspectives. In contraposition, Goldschmidt emphasized the role of low penetrance mutations that spread neutrally and pointed out that drift could result from developmental canalization. This account adds to the dominant story about Goldschmidt's resistance to the splitting of development from genetics, as told by Garland Allen and Michael Dietrich. The story I tell illustrates how developmental thinking and genetic thinking conflicted and influenced researchers with different convictions about the significance of chromosomal organization. PMID:25708087

  12. Integration ofhup cosmid pHU52 into the chromosomal DNA ofCicer-Rhizobium using Tn5 as an homologous sequence.

    PubMed

    Kunnimalaiyaan, M; Lodha, M L; Sreekumar, K R

    1992-11-01

    Cosmid pHU52, which carrieshup genes ofBradyrhizobium japonicum, has been integrated into theCicer-Rhizobium G36-84 genome via Tn5-mediated homologous recombination. Tn5 was inserted into both the cosmid pHU52 and the chromosome ofCicer-Rhizobium to provide a region of DNA homology, without affecting the expression of necessary genes. An incompatible plasmid, pPH1JI, was used to select those few cells that had undergone recombination. The integration of the cosmid was demonstrated by Southern blot analysis. Chromosomal integration of thehup genes maximized stability and minimized the potential for their horizontal transfer to other bacterial species. The integratedhup genes were found to expressex planta as well in nodules. The method described illustrates how a given gene can be stably integrated into the chromosome. PMID:24425601

  13. Non integrative strategy decreases chromosome instability and improves endogenous pluripotency genes reactivation in porcine induced pluripotent-like stem cells

    PubMed Central

    Congras, Annabelle; Barasc, Harmonie; Canale-Tabet, Kamila; Plisson-Petit, Florence; Delcros, Chantal; Feraud, Olivier; Oudrhiri, Noufissa; Hadadi, Eva; Griscelli, Franck; Bennaceur-Griscelli, Annelise; Turhan, Ali; Afanassieff, Marielle; Ferchaud, Stéphane; Pinton, Alain; Yerle-Bouissou, Martine; Acloque, Hervé

    2016-01-01

    The pig is an emerging animal model, complementary to rodents for basic research and for biomedical and agronomical purposes. However despite the progress made on mouse and rat models to produce genuine pluripotent cells, it remains impossible to produce porcine pluripotent cell lines with germline transmission. Reprogramming of pig somatic cells using conventional integrative strategies remains also unsatisfactory. In the present study, we compared the outcome of both integrative and non-integrative reprogramming strategies on pluripotency and chromosome stability during pig somatic cell reprogramming. The porcine cell lines produced with integrative strategies express several pluripotency genes but they do not silence the integrated exogenes and present a high genomic instability upon passaging. In contrast, pig induced pluripotent-like stem cells produced with non-integrative reprogramming system (NI-iPSLCs) exhibit a normal karyotype after more than 12 months in culture and reactivate endogenous pluripotency markers. Despite the persistent expression of exogenous OCT4 and MYC, these cells can differentiate into derivatives expressing markers of the three embryonic germ layers and we propose that these NI-iPSLCs can be used as a model to bring new insights into the molecular factors controlling and maintaining pluripotency in the pig and other non-rodent mammalians. PMID:27245508

  14. Non integrative strategy decreases chromosome instability and improves endogenous pluripotency genes reactivation in porcine induced pluripotent-like stem cells.

    PubMed

    Congras, Annabelle; Barasc, Harmonie; Canale-Tabet, Kamila; Plisson-Petit, Florence; Delcros, Chantal; Feraud, Olivier; Oudrhiri, Noufissa; Hadadi, Eva; Griscelli, Franck; Bennaceur-Griscelli, Annelise; Turhan, Ali; Afanassieff, Marielle; Ferchaud, Stéphane; Pinton, Alain; Yerle-Bouissou, Martine; Acloque, Hervé

    2016-01-01

    The pig is an emerging animal model, complementary to rodents for basic research and for biomedical and agronomical purposes. However despite the progress made on mouse and rat models to produce genuine pluripotent cells, it remains impossible to produce porcine pluripotent cell lines with germline transmission. Reprogramming of pig somatic cells using conventional integrative strategies remains also unsatisfactory. In the present study, we compared the outcome of both integrative and non-integrative reprogramming strategies on pluripotency and chromosome stability during pig somatic cell reprogramming. The porcine cell lines produced with integrative strategies express several pluripotency genes but they do not silence the integrated exogenes and present a high genomic instability upon passaging. In contrast, pig induced pluripotent-like stem cells produced with non-integrative reprogramming system (NI-iPSLCs) exhibit a normal karyotype after more than 12 months in culture and reactivate endogenous pluripotency markers. Despite the persistent expression of exogenous OCT4 and MYC, these cells can differentiate into derivatives expressing markers of the three embryonic germ layers and we propose that these NI-iPSLCs can be used as a model to bring new insights into the molecular factors controlling and maintaining pluripotency in the pig and other non-rodent mammalians. PMID:27245508

  15. Integrated Patient Education on U.S. Hospital Web Sites.

    PubMed

    Huang, Edgar; Wu, Kerong; Edwards, Kelsey

    2016-01-01

    Based on a census of the 2015 Most Wired Hospitals, this content analysis aimed to find out how patient education has been integrated on these best IT hospitals' Web sites to serve the purposes of marketing and meeting online visitors' needs. This study will help hospitals to understand where the weaknesses are in their interactive patient education implementation and come up with a smart integration strategy. The study found that 70% of these hospitals had adopted interactive patient education contents, 76.6% of such contents were from a third-party developer, and only 20% of the hospitals linked their patient education contents to one or more of the hospital's resources while 26% cross-references such contents. The authors concluded that more hospitals should take advantage of modern information communication technology to cross-reference their patient education contents and to integrate such contents into their overall online marketing strategy to benefit patients and themselves. PMID:27139406

  16. Fragile sites of 45S rDNA of Lolium multiflorum are not hotspots for chromosomal breakages induced by X-ray.

    PubMed

    Rocha, Laiane Corsini; Mittelmann, Andrea; Houben, Andreas; Techio, Vânia Helena

    2016-07-01

    Sites of 45S rDNA of Lolium are regions denominated fragile sites (FSs), constituting regions slightly stained with DAPI due to increased DNA unpacking in metaphasic chromosomes. Considered to be fragile regions in the genome, the FSs might be more responsive to induced breaks and result in chromosomal fragments and rearrangements, unless repairing mechanisms such as recombination or de novo telomere formation play a role at the break site of the DNA. Thus, this study aimed at investigating if SFs from Lolium are hotspots for the occurrence of breakages induced by X-ray and if they are regions favorable to synthesize new telomeres, using Hordeum vulgare as a comparative model. Lolium multiflorum and H. vulgare seedlings were irradiated with 20 and 50 Gy X-ray and evaluated one day following the irradiation and at 7-days intervals for a 28-days period, using FISH technique with 45S rDNA and Arabidopsis-type telomere probes in order to investigate the presence of chromosomal breakages and new telomere formation. H. vulgare did not survive after a few days of irradiation due to the increased rate of abnormalities. L. multiflorum also exhibited chromosomal abnormalities following the exposure, yet over the 28-days trial it had a decrease in the chromosomal damage rate and formation of de novo telomere has not been detected along this time. Despite being considered to be fragile regions in the genome, the 45S rDNA sites of Lolium are not hotspots to chromosomal breakages after the induction of breakages. PMID:27174104

  17. Discriminatory profile of rDNA sites and trend for acrocentric chromosome formation in the genus Trachinotus Lacépède, 1801 (Perciformes, Carangidae).

    PubMed

    Jacobina, Uedson Pereira; Vicari, Marcelo Ricardo; Bertollo, Luiz Antonio Carlos; Molina, Wagner Franco

    2012-01-01

    Chromosomal traits have provided valuable information for phylogeny and taxonomy of several fish groups. Three Atlantic Carangidae species of the genus Trachinotus Lacépède, 1801 (Trachinotus goodei Jordan et Evermann, 1896, Trachinotus carolinus (Linnaeus, 1766)and Trachinotus falcatus (Linnaeus, 1758)) were investigated, having 2n=48 chromosomes but different chromosomal arms (FN number), i.e., 52, 56 and 58, respectively, in view of the different number of two-armed chromosomes found in their karyotypes. Thus, Trachinotus goodei, Trachinotus carolinus and Trachinotus falcatus present a progressive distancefrom the probable basal karyotype proposed for Perciformes (2n=48 acrocentrics, FN=48). At first sight, these findings do not agree with the phylogenetic hypothesis based on mitochondrial sequences, where Trachinotus goodei appear as the most derived species, followed by Trachinotus falcatus and Trachinotus carolinus, respectively. However, the chromosomal mapping of ribosomal DNAs was informative for clarifying this apparent conflict. Indeed, the multiple 5S and 18S rDNA sites found in Trachinotus goodei corroborate the most derived condition for this species. In this sense, the occurrence of the unexpected number of two-armed chromosomes and FN value for this species, as well as for Trachinotus carolinus, must be due to additional rounds of acrocentric formation in these species, modifying the macrostructure of their karyotypes. PMID:24260676

  18. Discriminatory profile of rDNA sites and trend for acrocentric chromosome formation in the genus Trachinotus Lacépède, 1801 (Perciformes, Carangidae)

    PubMed Central

    Jacobina, Uedson Pereira; Vicari, Marcelo Ricardo; Bertollo, Luiz Antonio Carlos; Molina, Wagner Franco

    2012-01-01

    Abstract Chromosomal traits have provided valuable information for phylogeny and taxonomy of several fish groups. Three Atlantic Carangidae species of the genus Trachinotus Lacépède, 1801 (Trachinotus goodei Jordan et Evermann, 1896, Trachinotus carolinus (Linnaeus, 1766)and Trachinotus falcatus (Linnaeus, 1758)) were investigated, having 2n=48 chromosomes but different chromosomal arms (FN number), i.e., 52, 56 and 58, respectively, in view of the different number of two-armed chromosomes found in their karyotypes. Thus, Trachinotus goodei, Trachinotus carolinus and Trachinotus falcatus present a progressive distancefrom the probable basal karyotype proposed for Perciformes (2n=48 acrocentrics, FN=48). At first sight, these findings do not agree with the phylogenetic hypothesis based on mitochondrial sequences, where Trachinotus goodei appear as the most derived species, followed by Trachinotus falcatus and Trachinotus carolinus, respectively. However, the chromosomal mapping of ribosomal DNAs was informative for clarifying this apparent conflict. Indeed, the multiple 5S and 18S rDNA sites found in Trachinotus goodei corroborate the most derived condition for this species. In this sense, the occurrence of the unexpected number of two-armed chromosomes and FN value for this species, as well as for Trachinotus carolinus, must be due to additional rounds of acrocentric formation in these species, modifying the macrostructure of their karyotypes. PMID:24260676

  19. A potentially critical Hpa II site of the X chromosome-linked PGK1 gene is unmethylated prior to the onset of meiosis of human oogenic cells

    SciTech Connect

    Singer-Sam, J.; Dai, A.; Riggs, A.D. ); Goldstein, L.; Gartler, S.M. )

    1992-02-15

    Hpa II site H8 is in the CpG-rich 5{prime} untranslated region of the human X chromosome-linked gene for phosphoglycerate kinase 1 (PGK1). It is the only Hpa II site in the CpG island' whose methylation pattern is perfectly correlated with transcriptional silence of this gene. The authors measured DNA methylation at site H8 in fetal oogonia and oocytes and found, using a quantitative assay based on the polymerase chain reaction, that purified germ cells isolated by micromanipulation were unmethylated in 47-day to 110-day fetuses, whereas ovaries depleted of germ cells and non-ovary tissues were methylated. They conclude that site H8 is the unmethylated in germ cells prior to the onset of meiosis and reactivation of the X chromosome.

  20. Site wide integration of the Rocky Flats closure project

    SciTech Connect

    Burdge, L.F.; Golan, P.

    1998-06-01

    The prime contractor for the Rocky Flats Closure Project (RFCP), Kaiser-Hill, in concert with the Department of Energy--Rocky Flats Field Office (DOE-RFFO) has applied a fully integrated, life-cycle, critical path schedule and work planning system to manage the work that is required to close the Site. The closure of the Site is complex, in that it houses over 700 facilities, 19,600 kilograms of Special Nuclear Material (Plutonium and Uranium), and over 160,000 cubic meters of Transuranic, Low Level, and Hazardous Waste. The deactivation, decommissioning, decontaminating, and demolition of this large number of facilities, while at the same time accommodating difficult on-going activities, significantly increases the sophistication required in the planning process. The Rocky Flats team has overcome these difficulties by establishing a money oriented critical path process, to provide a least-cost avenue to supporting on-going activities and a line-of-balance process for production oriented activities. These processes, when integrated with a typical activity-based project planning system, guide the way to the shortest and most cost-effective course for the closure of the Rocky Flats Site.

  1. An Integrated Site-Wide Assessment of Nuclear Wastes to Remain at the Hanford Site, Washington

    SciTech Connect

    Morse, J.G.; Bryce, R.W.; Hildebrand, R.D.; Kincaid, C.T.

    2004-10-06

    Since its creation in 1943 until 1988, the Hanford Site, a facility in the U.S. Department of Energy (DOE) nuclear weapons complex was dedicated to the production of weapons grade plutonium and other special nuclear materials. The Hanford Site is located in eastern Washington State and is bordered on the north and east by the Columbia River. Decades of creating fuel, irradiating it in reactors, and processing it to recover nuclear material left numerous waste sites that involved the discharge of contaminated liquids and the disposal of contaminated solid waste. Today, the primary mission of the Hanford Site is to safely cleanup and manage the site's legacy waste. A site-wide risk assessment methodology has been developed to assist the DOE, as well as state and federal regulatory agencies, in making decisions regarding needed remedial actions at past waste sites, and safe disposal of future wastes. The methodology, referred to as the System Assessment Capability (SAC), utilizes an integrated set of models that track potential contaminants from inventory through vadose zone, groundwater, Columbia River and air pathways to human and ecological receptors.

  2. Tn4351 transposes in Bacteroides spp. and mediates the integration of plasmid R751 into the Bacteroides chromosome

    SciTech Connect

    Shoemaker, N.B.; Getty, C.; Gardner, J.F.; Salyers, A.A.

    1986-03-01

    The gene for resistance to erythromycin and clindamycin, which is carried on the conjugative Bacteroides plasmid, pBF4, has been shown previously to be part of an element (Tn4351) that transposes in Escherichia coli. The authors have now introduced Tn4351 into Bacteroides uniformis 0061 on the following two suicide vectors: (i) the broad-host-range IncP plasmid R751 (R751::Tn4351) and (ii) pSS-2, a chimeric plasmid which contains 33 kilobases of pBF4 (including Tn4351) cloned into the IncQ plasmid RSF1010 and which is mobilized by R751. When E. coli HB101, carrying either R751::Tn4351 or R751 and pSS-2, was mated with B. uniformis under aerobic conditions, Em/sup r/ transconjugants were detected at a frequency of 10 /sup -6/ to 10/sup -5/ (R751::Tn4351) or 10/sup -8/ to 10/sup -6/ (R751 and pSS-2). In matings involving pSS-2, all Em/sup r/ transconjugants contained simple insertions of Tn4351 in the chromosome, whereas in matings involving R751::Tn4351, about half of the Em/sup r/ transconjugants had R751 cointegrated with Tn4351 in the chromosome. Of the Em/sup r/ transconjugants, 13% were auxotrophs. Bacteroides spp. which had R751 cointegrated with Tn4351 in the chromosome did not transfer R751 or Tn4351 to E. coli HB101 or to isogenic B. uniformis, nor did the integrated R751 mobilize pE5-2, an E. coli-Bacteroides shuttle vector that contains a transfer origin that is recognized by R751.

  3. Integrating Y-chromosome, mitochondrial, and autosomal data to analyze the origin of pig breeds.

    PubMed

    Ramírez, O; Ojeda, A; Tomàs, A; Gallardo, D; Huang, L S; Folch, J M; Clop, A; Sánchez, A; Badaoui, B; Hanotte, O; Galman-Omitogun, O; Makuza, S M; Soto, H; Cadillo, J; Kelly, L; Cho, I C; Yeghoyan, S; Pérez-Enciso, M; Amills, M

    2009-09-01

    We have investigated the origin of swine breeds through the joint analysis of mitochondrial, microsatellite, and Y-chromosome polymorphisms in a sample of pigs and wild boars with a worldwide distribution. Genetic differentiation between pigs and wild boars was remarkably weak, likely as a consequence of a sustained gene flow between both populations. The analysis of nuclear markers evidenced the existence of a close genetic relationship between Near Eastern and European wild boars making it difficult to infer their relative contributions to the gene pool of modern European breeds. Moreover, we have shown that European and Far Eastern pig populations have contributed maternal and paternal lineages to the foundation of African and South American breeds. Although West African pigs from Nigeria and Benin exclusively harbored European alleles, Far Eastern and European genetic signatures of similar intensity were detected in swine breeds from Eastern Africa. This region seems to have been a major point of entry of livestock species in the African continent as a result of the Indian Ocean trade. Finally, South American creole breeds had essentially a European ancestry although Asian Y-chromosome and mitochondrial haplotypes were found in a few Nicaraguan pigs. The existence of Spanish and Portuguese commercial routes linking Asia with America might have favored the introduction of Far Eastern breeds into this continent. PMID:19535739

  4. ANKRD53 interacts with DDA3 and regulates chromosome integrity during mitosis.

    PubMed

    Kim, Seul; Jang, Chang-Young

    2016-02-12

    Spindle dynamics drives chromosome movement and mitotic progression during mitosis. Microtubule (MT)-associated proteins (MAPs) regulate MT stabilization/destabilization and MT polymerization/depolymerization for congression of sister chromatids at the mitotic equator and subsequent segregation toward the spindle poles. Here, we identified ANKRD53 as a novel DDA3-interacting protein through proteomic analysis. Based on expression profiles, ANKRD53 is phosphorylated by mitotic kinases during mitosis. In ANKRD53-depleted HeLa cells, the progression of mitosis was delayed and the number of unaligned chromosomes increased substantially. In addition, spindle MT polymerization decreased and the spindle assembly checkpoint (SAC) was concomitantly activated by the decreased spindle dynamics in ANKRD53-depleted cells. Although ANKRD53 is recruited to the mitotic spindle by DDA3, it counteracts the activity of DDA3 for spindle MT polymerization. Furthermore, ANKRD53 depletion increased the number of bi-nuclei and polylobed nuclei. Thus, ANKRD53 is recruited to the mitotic spindle by DDA3 and acts as a regulator of spindle dynamics and cytokinesis. PMID:26820536

  5. Characterization of the chromosomal integration of Saccharopolyspora plasmid pCM32 and its application to improve production of spinosyn in Saccharopolyspora spinosa.

    PubMed

    Chen, Jian; Xia, Haiyang; Dang, Fujun; Xu, Qingyu; Li, Wenjun; Qin, Zhongjun

    2015-12-01

    Saccharopolyspora spinosa produces tetra-cyclic macrolide spinosyns, a group of highly efficient pesticidal agents. However, this species lacks efficient vectors for genetic manipulation. In this study, the circular plasmid pCM32 was newly isolated from Saccharopolyspora endophytica YIM 61095. The complete nucleotide sequence of pCM32 consists of 14,611 bp and is predicted to encode 17 open reading frames (ORFs). Interestingly, a putative int gene in pCM32 was predicted by homologous alignment to encode an integrase belonging to the tyrosine family of integrases/recombinases. Plasmid pCM238 containing this int locus derived from pCM32 could be transferred by conjugation from Escherichia coli into Sa. spinosa at a high frequency. Integration of pCM238 in the host chromosome was demonstrated as site-specific recombination (at the tRNA (Ser) gene) via a 56-bp core sequence within the attP/attB sites. Plasmid pCM265, a shuttle vector containing the int and attP sequences of pCM32, was constructed to introduce foreign genes into Sa. spinosa. The production of spinosad approximately doubled in Sa. spinosa NRRL18395 after introducing pCM265-derived plasmids carrying the genes for phosphofructokinase (PFK) or anthranilate synthase. These results indicate that plasmid pCM32 is an actinomycete integrative and conjugative element (AICE) and that its derived integrative vectors are useful for efficiently introducing foreign DNA into Sa. spinosa. PMID:26260388

  6. What Integration Sites Tell Us about HIV Persistence.

    PubMed

    Hughes, Stephen H; Coffin, John M

    2016-05-11

    Advances in technology have made it possible to analyze integration sites in cells from HIV-infected patients. A significant fraction of infected cells in patients on long-term therapy are clonally expanded; in some cases the integrated viral DNA contributes to the clonal expansion of the infected cells. Although the large majority (>95%) of the HIV proviruses in treated patients are defective, expanded clones can carry replication-competent proviruses, and cells from these clones can release infectious virus. As discussed in this Perspective, it is likely that cells that produce virus are strongly selected against in vivo, and cells with replication competent proviruses expand and survive because only a small fraction of the cells produce virus. These findings have implications for strategies that are intended to eliminate the reservoir of infected cells that has made it almost impossible to cure HIV-infected patients. PMID:27173927

  7. Opportunities for Launch Site Integrated System Health Engineering and Management

    NASA Technical Reports Server (NTRS)

    Waterman, Robert D.; Langwost, Patricia E.; Waterman, Susan J.

    2005-01-01

    The launch site processing flow involves operations such as functional verification, preflight servicing and launch. These operations often include hazards that must be controlled to protect human life and critical space hardware assets. Existing command and control capabilities are limited to simple limit checking durig automated monitoring. Contingency actions are highly dependent on human recognition, decision making, and execution. Many opportunities for Integrated System Health Engineering and Management (ISHEM) exist throughout the processing flow. This paper will present the current human-centered approach to health management as performed today for the shuttle and space station programs. In addition, it will address some of the more critical ISHEM needs, and provide recommendations for future implementation of ISHEM at the launch site.

  8. Characteristics of the volatile organic compounds -- Arid Integrated Demonstration Site

    SciTech Connect

    Last, G.V.; Lenhard, R.J.; Bjornstad, B.N.; Evans, J.C.; Roberson, K.R.; Spane, F.A.; Amonette, J.E.; Rockhold, M.L.

    1991-10-01

    The Volatile Organic Compounds -- Arid Integrated Demonstration Program (VOC-Arid ID) is targeted at demonstration and testing of technologies for the evaluation and cleanup of volatile organic compounds and associated contaminants at arid DOE sites. The initial demonstration site is an area of carbon tetrachloride (CCl{sub 4}) contamination located near the center of the Hanford Site. The movement of CCl{sub 4} and other volatile organic contaminants in the subsurface is very complex. The problem at the Hanford Site is further complicated by the concurrent discharge of other waste constituents including acids, lard oil, organic phosphates, and transuranic radionuclides. In addition, the subsurface environment is very complex, with large spatial variabilities in hydraulic properties. A thorough understanding of the problem is essential to the selection of appropriate containment, retrieval, and/or in situ remedial technologies. The effectiveness of remedial technologies depends on knowing where the contaminants are, how they are held up in a given physical and chemical subsurface environment; and knowing the physical, chemical, and microbiological changes that are induced by the various remedial technologies.

  9. AnaLysis of Expression on human chromosome 21, ALE-HSA21: a pilot integrated web resource.

    PubMed

    Scarpato, Margherita; Esposito, Roberta; Evangelista, Daniela; Aprile, Marianna; Ambrosio, Maria Rosaria; Angelini, Claudia; Ciccodicola, Alfredo; Costa, Valerio

    2014-01-01

    Transcriptome studies have shown the pervasive nature of transcription, demonstrating almost all the genes undergo alternative splicing. Accurately annotating all transcripts of a gene is crucial. It is needed to understand the impact of mutations on phenotypes, to shed light on genetic and epigenetic regulation of mRNAs and more generally to widen our knowledge about cell functionality and tissue diversity. RNA-sequencing (RNA-Seq), and the other applications of the next-generation sequencing, provides precious data to improve annotations' accuracy, simultaneously creating issues related to the variety, complexity and the size of produced data. In this 'scenario', the lack of user-friendly resources, easily accessible to researchers with low skills in bioinformatics, makes difficult to retrieve complete information about one or few genes without browsing a jungle of databases. Concordantly, the increasing amount of data from 'omics' technologies imposes to develop integrated databases merging different data formats coming from distinct but complementary sources. In light of these considerations, and given the wide interest in studying Down syndrome-a genetic condition due to the trisomy of human chromosome 21 (HSA21)-we developed an integrated relational database and a web interface, named ALE-HSA21 (AnaLysis of Expression on HSA21), accessible at http://bioinfo.na.iac.cnr.it/ALE-HSA21. This comprehensive and user-friendly web resource integrates-for all coding and noncoding transcripts of chromosome 21-existing gene annotations and transcripts identified de novo through RNA-Seq analysis with predictive computational analysis of regulatory sequences. Given the role of noncoding RNAs and untranslated regions of coding genes in key regulatory mechanisms, ALE-HSA21 is also an interesting web-based platform to investigate such processes. The 'transcript-centric' and easily-accessible nature of ALE-HSA21 makes this resource a valuable tool to rapidly retrieve data at

  10. Characterization of a new Lactobacillus salivarius strain engineered to express IBV multi-epitope antigens by chromosomal integration.

    PubMed

    Ma, Bing-cun; Yang, Xin; Wang, Hong-ning; Cao, Hai-peng; Xu, Peng-wei; Ding, Meng-die; Liu, Hui

    2016-01-01

    To obtain adhesive and safe lactic acid bacteria (LAB) strains for expressing heterologous antigens, we screened LAB inhabitants in intestine of Tibetan chickens by analyzing their adhesion and safety properties and the selected LAB was engineered to express heterologous antigen (UTEpi C-A) based on chromosomal integration strategy. We demonstrated that a new Lactobacillu salivarius TCMM17 strain is strongly adhesive to chicken intestinal epithelial cells, contains no endogenous plasmids, is susceptible to tested antimicrobials, and shows no toxicities. In order to examine the potential of TCMM17 strain as heterogenous antigen delivering vehicle, we introduced a UTEpi C-A expression cassette in its chromosome by constructing a non-replicative plasmid (pORI280-UUTEpi C-AD). The recombinant TCMM17 strain (∆TCMM17) stably was found to keep the gene cassette through 50 generations, and successfully displayed EpiC encoded by the cassette on its surface. This work provides a universal platform for development of novel oral vaccines and expression of further antigens of avian pathogens. PMID:26618736

  11. Transumbilical laparoendoscopic single-site gonadectomy for Turner's syndrome with Y-chromosome mosaicism.

    PubMed

    Mizuno, Kentaro; Kojima, Yoshiyuki; Nishio, Hidenori; Tozawa, Keiichi; Mizuno, Haruo; Kohri, Kenjiro; Hayashi, Yutaro

    2012-08-01

    Laparoendoscopic single-site surgery (LESS), a minimally invasive procedure, is gaining widespread acknowledgment in pediatric urology. We report the case of a 7-year-old girl with Turner's syndrome with 45,XO/46,XY mosaicism, for whom bilateral prophylactic gonadectomy using LESS was performed. Histopathological findings revealed bilateral streak gonads. The surgical and cosmetic outcome was excellent. Diagnostic and therapeutic laparoscopy is essential for accurate clinical management of patients with disorders of sex development. Although this is only a single case report, it supports the theory that LESS is an exceedingly practical and superior technique for such children, since it provides excellent magnification, as well as allowing normal psychological development owing to the concealed scar. Further studies and long-term follow-up are required to evaluate the benefits and limitations of applying LESS in pediatrics. PMID:22417680

  12. Hanford and Savannah River Site Programmatic and Technical Integration

    SciTech Connect

    Ramsey, William Gene

    2013-08-15

    Abstract only. The Hanford Site and the Savannah River Site (SRS) were the primary plutonium production facilities within the U.S. nuclear weapons complex. Radioactive wastes were generated as part of these missions and are stored in similar fashion. The majority of radioactivity maintained by the two sites is located in underground carbon steel tanks in the physical form of supernatant, saltcake, or sludge. Disposition of SRS tank waste is ongoing by converting it into glass (pathway for sludge and radionuclides separated from supernatant or dissolved saltcake) or cement (pathway for the decontaminated supernatant and dissolved saltcake). Tank closure activity has also begun at SRS and will continue for the duration of mission. The Hanford tank waste inventory is roughly 2/3rds larger than SRS's by volume- but nominally half the radioactivity. The baseline disposition path includes high-level and low-activity waste vitrification with separate disposition of contact-handled transuranic tank waste. Retrieval of tank waste from aging single­ shell tanks (SSTs) into double-shell tanks (DSTs) is currently ongoing. As vitrification commences later this decade, Hanford will be in a similar operations mode as SRS. Site integration is increasing as the missions align. The ongoing integration is centered on key issues that impact both sites- regardless of mission timeframe. Three recent workshop exchanges have been held to improve communication with the primary intent of improving operations and technical work organization. The topics of these workshops are as follows: DST space utilization, optimization, and closure; Waste Feed Qualification; and, Cementitious Waste Forms. Key goals for these and future exchanges include aligning research and technology, preparing for joint initiatives (to maximize budgetary value for the customer), and reviewing lessons learned. Each site has played a leading role in the development of technology and operational practices that can be

  13. Integration and expression of alpha-amylase and endoglucanase genes in the Lactobacillus plantarum chromosome.

    PubMed Central

    Scheirlinck, T; Mahillon, J; Joos, H; Dhaese, P; Michiels, F

    1989-01-01

    A commercial grass silage starter strain of Lactobacillus plantarum was transformed by high-frequency electroporation with plasmids containing an alpha-amylase gene from Bacillus stearothermophilus and an endoglucanase gene from Clostridium thermocellum. Both genes were expressed from their native regulatory signals, and active enzymes were found in the supernatant. However, the segregational stability of the transforming plasmids was rather low. Therefore, the transforming genes were inserted in the L. plantarum chromosome by means of single homologous recombination. In the majority of the transformants, this led to extremely stable segregation and expression of the transforming genes, without generating secondary mutations in the host. Increased selective pressure led to tandem amplification of the transforming DNA. The transformed strains demonstrated the ability of L. plantarum to express heterologous gene products; they can be used to detect the inoculum in silage ecology studies; and they demonstrate the feasibility of engineering truly cellulolytic silage starter bacteria. Images PMID:2679379

  14. The Metabolic Enzyme ManA Reveals a Link between Cell Wall Integrity and Chromosome Morphology

    PubMed Central

    Elbaz, Maya; Ben-Yehuda, Sigal

    2010-01-01

    Synchronizing cell growth, division and DNA replication is an essential property of all living cells. Accurate coordination of these cellular events is especially crucial for bacteria, which can grow rapidly and undergo multifork replication. Here we show that the metabolic protein ManA, which is a component of mannose phosphotransferase system, participates in cell wall construction of the rod shaped bacterium Bacillus subtilis. When growing rapidly, cells lacking ManA exhibit aberrant cell wall architecture, polyploidy and abnormal chromosome morphologies. We demonstrate that these cellular defects are derived from the role played by ManA in cell wall formation. Furthermore, we show that ManA is required for maintaining the proper carbohydrate composition of the cell wall, particularly of teichoic acid constituents. This perturbed cell wall synthesis causes asynchrony between cell wall elongation, division and nucleoid segregation. PMID:20862359

  15. BRIT1 regulates early DNA damage response, chromosomal integrity,and cancer

    SciTech Connect

    Rai, Rekha; Dai, Hui; Multani, Asha S.; Li, Kaiyi; Chin, Koei; Gray, Joe; Lahad, John P.; Liang, Jiyong; Mills, Gordon B.; Meric-Bernstam, Funda; Lin, Shiaw-Yih

    2006-08-24

    BRIT1, initially identified as an hTERT repressor, hasadditional functions at DNA damage checkpoints. Here, we demonstratedthat BRIT1 formed nuclear foci minutes after irradiation. The foci ofBRIT1 co-localized with 53BP1, MDC1, NBS1, ATM, RPA, and ATR. BRIT1 wasrequired for activation of these elements, indicating that BRIT1 is aproximal factor in the DNA damage response pathway. Depletion of BRIT1increased the accumulation of chromosomal aberrations. In addition,decreased levels of BRIT1 were detected in several types of human cancerwith BRIT1 expression being inversely correlated with genomic instabilityand metastasis. These results identify BRIT1 as a crucial DNA damageregulator in the ATM/ATR pathways and suggest that it functions as atumor suppressor gene.

  16. Integrated fate and toxicity assessment for site contaminants

    SciTech Connect

    MacDonell, Margaret; Peterson, John; Finster, Molly; Douglas, R.

    2007-07-01

    Understanding the fate and toxicity of environmental contaminants is essential to framing practical management decisions. Forms and bioavailable concentrations often change over time due to natural physical, chemical, and biological processes. For some sites, hundreds of contaminants may be of initial interest, and even small projects can involve a substantial number of contaminants. With multiple assessments common, attention to effectiveness and efficiency is important, and integrating fate and toxicity information provides a valuable way to focus the analyses. Fate assessments help identify what forms may be present where and when, while toxicity information indicates what health effects could result if people were exposed. The integration process is illustrated by an application for the Hanford site, to support long-term management decisions for the cesium and strontium capsules. Fate data, health-based benchmarks, and related toxicity information were effectively combined to indicate performance targets for chemicals and radionuclides identified for capsule leachate that could migrate to groundwater. More than 50 relevant benchmarks and toxicity context were identified for 15 of the 17 study contaminants; values for chronic drinking water exposure provided the common basis for selected indicators. For two chemicals, toxicity information was identified from the scientific literature to guide the performance targets. (authors)

  17. A system for multi-locus chromosomal integration and transformation-free selection marker rescue

    PubMed Central

    Siddiqui, Michael S.; Choksi, Atri; Smolke, Christina D.

    2014-01-01

    Yeast integrating plasmids (YIPs) are a versatile tool for stable integration in Saccharomyces cerevisiae. However, current YIP systems necessitate time- and labor-intensive methods for cloning and selection marker rescue. Here we describe the design, construction, and validation of a new YIP system capable of accelerating the stable integration of multiple expression constructs into different loci in the yeast S. cerevisiae. These “directed pop-out” plasmids enable a simple, two-step integration protocol that results in a scarless integration alongside a complete rescue of the selection marker. These plasmids combine three key features: a dedicated “YIPout” fragment directs a recombination event that rescues the selection marker while avoiding undesired excision of the target DNA sequence, a multi-fragment modular DNA assembly system simplifies cloning, and a new set of counterselectable markers enables serial integration followed by a transformation-free marker rescue event. We constructed and tested directed pop-out YIPs for integration of fluorescent reporter genes into four yeast loci. We validated our new YIP design by integrating three reporter genes into three different loci with transformation-free rescue of selection markers. These new YIP designs will facilitate the construction of yeast strains that express complex heterologous metabolic pathways. PMID:25226817

  18. Taz1-Shelterin Promotes Facultative Heterochromatin Assembly at Chromosome-Internal Sites Containing Late Replication Origins.

    PubMed

    Zofall, Martin; Smith, Deborah R; Mizuguchi, Takeshi; Dhakshnamoorthy, Jothy; Grewal, Shiv I S

    2016-06-16

    Facultative heterochromatin regulates gene expression, but its assembly is poorly understood. Previously, we identified facultative heterochromatin islands in the fission yeast genome and found that RNA elimination machinery promotes island assembly at meiotic genes. Here, we report that Taz1, a component of the telomere protection complex Shelterin, is required to assemble heterochromatin islands at regions corresponding to late replication origins that are sites of double-strand break formation during meiosis. The loss of Taz1 or other Shelterin subunits, including Ccq1 that interacts with Clr4/Suv39h, abolishes heterochromatin at late origins and causes derepression of associated genes. Moreover, the late-origin regulator Rif1 affects heterochromatin at Taz1-dependent islands and subtelomeric regions. We explore the connection between facultative heterochromatin and replication control and show that heterochromatin machinery affects replication timing. These analyses reveal the role of Shelterin in facultative heterochromatin assembly at late origins, which has important implications for genome stability and gene regulation. PMID:27264871

  19. Characterization of X Chromosome Inactivation Using Integrated Analysis of Whole-Exome and mRNA Sequencing

    PubMed Central

    Szelinger, Szabolcs; Malenica, Ivana; Corneveaux, Jason J.; Siniard, Ashley L.; Kurdoglu, Ahmet A.; Ramsey, Keri M.; Schrauwen, Isabelle; Trent, Jeffrey M.; Narayanan, Vinodh; Huentelman, Matthew J.; Craig, David W.

    2014-01-01

    In females, X chromosome inactivation (XCI) is an epigenetic, gene dosage compensatory mechanism by inactivation of one copy of X in cells. Random XCI of one of the parental chromosomes results in an approximately equal proportion of cells expressing alleles from either the maternally or paternally inherited active X, and is defined by the XCI ratio. Skewed XCI ratio is suggestive of non-random inactivation, which can play an important role in X-linked genetic conditions. Current methods rely on indirect, semi-quantitative DNA methylation-based assay to estimate XCI ratio. Here we report a direct approach to estimate XCI ratio by integrated, family-trio based whole-exome and mRNA sequencing using phase-by-transmission of alleles coupled with allele-specific expression analysis. We applied this method to in silico data and to a clinical patient with mild cognitive impairment but no clear diagnosis or understanding molecular mechanism underlying the phenotype. Simulation showed that phased and unphased heterozygous allele expression can be used to estimate XCI ratio. Segregation analysis of the patient's exome uncovered a de novo, interstitial, 1.7 Mb deletion on Xp22.31 that originated on the paternally inherited X and previously been associated with heterogeneous, neurological phenotype. Phased, allelic expression data suggested an 83∶20 moderately skewed XCI that favored the expression of the maternally inherited, cytogenetically normal X and suggested that the deleterious affect of the de novo event on the paternal copy may be offset by skewed XCI that favors expression of the wild-type X. This study shows the utility of integrated sequencing approach in XCI ratio estimation. PMID:25503791

  20. Expansion of GA Dinucleotide Repeats Increases the Density of CLAMP Binding Sites on the X-Chromosome to Promote Drosophila Dosage Compensation

    PubMed Central

    Chery, Jessica; Siggers, Trevor; Boor, Sonia; Bliss, Jacob; Liu, Wei; Jogl, Gerwald; Rohs, Remo; Singh, Nadia D.; Bulyk, Martha L.; Tolstorukov, Michael Y.; Larschan, Erica

    2016-01-01

    Dosage compensation is an essential process that equalizes transcript levels of X-linked genes between sexes by forming a domain of coordinated gene expression. Throughout the evolution of Diptera, many different X-chromosomes acquired the ability to be dosage compensated. Once each newly evolved X-chromosome is targeted for dosage compensation in XY males, its active genes are upregulated two-fold to equalize gene expression with XX females. In Drosophila melanogaster, the CLAMP zinc finger protein links the dosage compensation complex to the X-chromosome. However, the mechanism for X-chromosome identification has remained unknown. Here, we combine biochemical, genomic and evolutionary approaches to reveal that expansion of GA-dinucleotide repeats likely accumulated on the X-chromosome over evolutionary time to increase the density of CLAMP binding sites, thereby driving the evolution of dosage compensation. Overall, we present new insight into how subtle changes in genomic architecture, such as expansions of a simple sequence repeat, promote the evolution of coordinated gene expression. PMID:27414415

  1. Integrated Geophysical Analysis at a Legacy Test Site

    NASA Astrophysics Data System (ADS)

    Yang, X.; Mellors, R. J.; Sweeney, J. J.; Sussman, A. J.

    2015-12-01

    We integrate magnetic, electromagnetic (EM), gravity, and seismic data to develop a unified and consistent model of the subsurface at the U20ak site on Pahute Mesa at the Nevada National Nuclear Security Site (NNSS). The 1985 test, conducted in tuff at a depth of approximately 600 m did not collapse to the surface or produce a crater. The purpose of the geophysical measurements is to characterize the subsurface above and around the presumed explosion cavity. The magnetic data are used to locate steel borehole casings and pipes and are correlated with surface observations. The EM data show variation in lithology at depth and clear signatures from borehole casings and surface cables. The gravity survey detects a clear gravity low in the area of the explosion. The seismic data indicates shallow low velocity zone and indications of a deeper low velocity zones. In this study, we conduct 2D inversion of EM data for better characterization of site geology and use a common 3D density model to jointly interpret both the seismic and gravity data along with constraints on lithology boundaries from the EM. The integration of disparate geophysical datasets allows improved understanding of the non-prompt physical signatures of an underground nuclear explosion (UNE). LLNL Release Number: LLNL-ABS-675677. The authors express their gratitude to the National Nuclear Security Administration, Defense Nuclear Nonproliferation Research and Development, and the Comprehensive Inspection Technologies and UNESE working group, a multi-institutional and interdisciplinary group of scientists and engineers. This work was performed by Lawrence Livermore National Laboratory and Los Alamos National Laboratory under award number DE-AC52-06NA25946.

  2. AnaLysis of Expression on human chromosome 21, ALE-HSA21: a pilot integrated web resource

    PubMed Central

    Scarpato, Margherita; Esposito, Roberta; Evangelista, Daniela; Aprile, Marianna; Ambrosio, Maria Rosaria; Angelini, Claudia; Ciccodicola, Alfredo; Costa, Valerio

    2014-01-01

    Transcriptome studies have shown the pervasive nature of transcription, demonstrating almost all the genes undergo alternative splicing. Accurately annotating all transcripts of a gene is crucial. It is needed to understand the impact of mutations on phenotypes, to shed light on genetic and epigenetic regulation of mRNAs and more generally to widen our knowledge about cell functionality and tissue diversity. RNA-sequencing (RNA-Seq), and the other applications of the next-generation sequencing, provides precious data to improve annotations' accuracy, simultaneously creating issues related to the variety, complexity and the size of produced data. In this ‘scenario’, the lack of user-friendly resources, easily accessible to researchers with low skills in bioinformatics, makes difficult to retrieve complete information about one or few genes without browsing a jungle of databases. Concordantly, the increasing amount of data from ‘omics’ technologies imposes to develop integrated databases merging different data formats coming from distinct but complementary sources. In light of these considerations, and given the wide interest in studying Down syndrome—a genetic condition due to the trisomy of human chromosome 21 (HSA21)—we developed an integrated relational database and a web interface, named ALE-HSA21 (AnaLysis of Expression on HSA21), accessible at http://bioinfo.na.iac.cnr.it/ALE-HSA21. This comprehensive and user-friendly web resource integrates—for all coding and noncoding transcripts of chromosome 21—existing gene annotations and transcripts identified de novo through RNA-Seq analysis with predictive computational analysis of regulatory sequences. Given the role of noncoding RNAs and untranslated regions of coding genes in key regulatory mechanisms, ALE-HSA21 is also an interesting web-based platform to investigate such processes. The ‘transcript-centric’ and easily-accessible nature of ALE-HSA21 makes this resource a valuable tool to

  3. Commercial integration and partnering at Savannah River Site

    SciTech Connect

    Steele, J.R.; Babione, R.A.; Shikashio, L.A.; Wacaster, A.J.; Paterson, A.D.

    1994-06-01

    Savannah River Site (SRS), particularly the Savannah River Technology Center (SRTC) with the experience from the first successful Integrated Technology Demonstration, can provide an excellent foundation for meeting DOE-EM`s objectives with the new DOE-EM five focus area approach. With this in mind, SRTC established an activity to pursue full commercialization of environmental technologies. This report is an assessment of the status of commercialization at SRS and provides recommendations for enhancement as well as some tools critical to implementation. A review was made of the current situation at SRS with regards to taking technology development to commercial fruition. This was done from the perspective of comparing it to known commercialization models and processes. It was found that SRTC already works through many of the steps in these processes. With integration and action-oriented efforts of the inclusion of business and market factors, SRTC could become an aggressive, successful developer of commercialized technologies. Commercial success criteria tools were developed with regards to integrating them with SRTC selection criteria to ensure that all critical factors are covered in technology commercialization project evaluations. Private investors are very clear that their interest lies in funding commercial enterprises, not merely technologies. Mobilizing private capital is critical to real job growth and long-term economic development. Also, potential industry partners were identified that are willing to be involved with SRS` technology applications and regional development efforts. As another important component to success, regional support organizations were reviewed and evaluated.

  4. The piggyBac transposon displays local and distant reintegration preferences and can cause mutations at noncanonical integration sites.

    PubMed

    Li, Meng Amy; Pettitt, Stephen J; Eckert, Sabine; Ning, Zemin; Rice, Stephen; Cadiñanos, Juan; Yusa, Kosuke; Conte, Nathalie; Bradley, Allan

    2013-04-01

    The DNA transposon piggyBac is widely used as a tool in mammalian experimental systems for transgenesis, mutagenesis, and genome engineering. We have characterized genome-wide insertion site preferences of piggyBac by sequencing a large set of integration sites arising from transposition from two separate genomic loci and a plasmid donor in mouse embryonic stem cells. We found that piggyBac preferentially integrates locally to the excision site when mobilized from a chromosomal location and identified other nonlocal regions of the genome with elevated insertion frequencies. piggyBac insertions were associated with expressed genes and markers of open chromatin structure and were excluded from heterochromatin. At the nucleotide level, piggyBac prefers to insert into TA-rich regions within a broader GC-rich context. We also found that piggyBac can insert into sites other than its known TTAA insertion site at a low frequency (2%). Such insertions introduce mismatches that are repaired with signatures of host cell repair pathways. Transposons could be mobilized from plasmids with the observed noncanonical flanking regions, indicating that piggyBac could generate point mutations in the genome. PMID:23358416

  5. The piggyBac Transposon Displays Local and Distant Reintegration Preferences and Can Cause Mutations at Noncanonical Integration Sites

    PubMed Central

    Li, Meng Amy; Pettitt, Stephen J.; Eckert, Sabine; Ning, Zemin; Rice, Stephen; Cadiñanos, Juan; Yusa, Kosuke; Conte, Nathalie

    2013-01-01

    The DNA transposon piggyBac is widely used as a tool in mammalian experimental systems for transgenesis, mutagenesis, and genome engineering. We have characterized genome-wide insertion site preferences of piggyBac by sequencing a large set of integration sites arising from transposition from two separate genomic loci and a plasmid donor in mouse embryonic stem cells. We found that piggyBac preferentially integrates locally to the excision site when mobilized from a chromosomal location and identified other nonlocal regions of the genome with elevated insertion frequencies. piggyBac insertions were associated with expressed genes and markers of open chromatin structure and were excluded from heterochromatin. At the nucleotide level, piggyBac prefers to insert into TA-rich regions within a broader GC-rich context. We also found that piggyBac can insert into sites other than its known TTAA insertion site at a low frequency (2%). Such insertions introduce mismatches that are repaired with signatures of host cell repair pathways. Transposons could be mobilized from plasmids with the observed noncanonical flanking regions, indicating that piggyBac could generate point mutations in the genome. PMID:23358416

  6. Arid sites stakeholder participation in evaluating innovative technologies: VOC-Arid Site Integrated Demonstration

    SciTech Connect

    Peterson, T.S.; McCabe, G.H.; Brockbank, B.R.

    1995-05-01

    Developing and deploying innovative environmental cleanup technologies is an important goal for the U.S. Department of Energy (DOE), which faces challenging remediation problems at contaminated sites throughout the United States. Achieving meaningful, constructive stakeholder involvement in cleanup programs, with the aim of ultimate acceptance of remediation decisions, is critical to meeting those challenges. DOE`s Office of Technology Development sponsors research and demonstration of new technologies, including, in the past, the Volatile Organic Compounds Arid Site Integrated Demonstration (VOC-Arid ID), hosted at the Hanford Site in Washington State. The purpose of the VOC-Arid ID has been to develop and demonstrate new technologies for remediating carbon tetrachloride and other VOC contamination in soils and ground water. In October 1994 the VOC-Arid ID became a part of the Contaminant Plume Containment and Remediation Focus Area (Plume Focus Area). The VOC Arid ID`s purpose of involving stakeholders in evaluating innovative technologies will now be carried on in the Plume Focus Area in cooperation with Site Technology Coordination Groups and Site Specific Advisory Boards. DOE`s goal is to demonstrate promising technologies once and deploy those that are successful across the DOE complex. Achieving that goal requires that the technologies be acceptable to the groups and individuals with a stake in DOE facility cleanup. Such stakeholders include groups and individuals with an interest in cleanup, including regulatory agencies, Native American tribes, environmental and civic interest groups, public officials, environmental technology users, and private citizens. This report documents the results of the stakeholder involvement program, which is an integral part of the VOC-Arid ID.

  7. Chromosomal Integration and Expression of Two Bacterial α-Acetolactate Decarboxylase Genes in Brewer's Yeast

    PubMed Central

    Blomqvist, K.; Suihko, M.-L.; Knowles, J.; Penttilä, M.

    1991-01-01

    A bacterial gene encoding α-acetolactate decarboxylase, isolated from Klebsiella terrigena or Enterobacter aerogenes, was expressed in brewer's yeast. The genes were expressed under either the yeast phosphoglycerokinase (PGK1) or the alcohol dehydrogenase (ADH1) promoter and were integrated by gene replacement by using cotransformation into the PGK1 or ADH1 locus, respectively, of a brewer's yeast. The expression level of the α-acetolactate decarboxylase gene of the PGK1 integrant strains was higher than that of the ADH1 integrants. Under pilot-scale brewing conditions, the α-acetolactate decarboxylase activity of the PGK1 integrant strains was sufficient to reduce the formation of diacetyl below the taste threshold value, and no lagering was needed. The brewing properties of the recombinant yeast strains were otherwise unaltered, and the quality (most importantly, the flavor) of the trial beers produced was as good as that of the control beer. Images PMID:16348559

  8. Site-Wide Integrated Water Monitoring - Defining and Implementing Sampling Objectives to Support Site Closure - 13060

    SciTech Connect

    Wilborn, Bill; Knapp, Kathryn; Farnham, Irene; Marutzky, Sam

    2013-07-01

    The Underground Test Area (UGTA) activity is responsible for assessing and evaluating the effects of the underground nuclear weapons tests on groundwater at the Nevada National Security Site (NNSS), formerly the Nevada Test Site (NTS), and implementing a corrective action closure strategy. The UGTA strategy is based on a combination of characterization, modeling studies, monitoring, and institutional controls (i.e., monitored natural attenuation). The closure strategy verifies through appropriate monitoring activities that contaminants of concern do not exceed the SDWA at the regulatory boundary and that adequate institutional controls are established and administered to ensure protection of the public. Other programs conducted at the NNSS supporting the environmental mission include the Routine Radiological Environmental Monitoring Program (RREMP), Waste Management, and the Infrastructure Program. Given the current programmatic and operational demands for various water-monitoring activities at the same locations, and the ever-increasing resource challenges, cooperative and collaborative approaches to conducting the work are necessary. For this reason, an integrated sampling plan is being developed by the UGTA activity to define sampling and analysis objectives, reduce duplication, eliminate unnecessary activities, and minimize costs. The sampling plan will ensure the right data sets are developed to support closure and efficient transition to long-term monitoring. The plan will include an integrated reporting mechanism for communicating results and integrating process improvements within the UGTA activity as well as between other U.S. Department of Energy (DOE) Programs. (authors)

  9. Site-Wide Integrated Water Monitoring -- Defining and Implementing Sampling Objectives to Support Site Closure

    SciTech Connect

    Wilborn, Bill; Marutzky, Sam; Knapp, Kathryn

    2013-02-24

    The Underground Test Area (UGTA) activity is responsible for assessing and evaluating the effects of the underground nuclear weapons tests on groundwater at the Nevada National Security Site (NNSS), formerly the Nevada Test Site (NTS), and implementing a corrective action closure strategy. The UGTA strategy is based on a combination of characterization, modeling studies, monitoring, and institutional controls (i.e., monitored natural attenuation). The closure strategy verifies through appropriate monitoring activities that contaminants of concern do not exceed the SDWA at the regulatory boundary and that adequate institutional controls are established and administered to ensure protection of the public. Other programs conducted at the NNSS supporting the environmental mission include the Routine Radiological Environmental Monitoring Program (RREMP), Waste Management, and the Infrastructure Program. Given the current programmatic and operational demands for various water-monitoring activities at the same locations, and the ever-increasing resource challenges, cooperative and collaborative approaches to conducting the work are necessary. For this reason, an integrated sampling plan is being developed by the UGTA activity to define sampling and analysis objectives, reduce duplication, eliminate unnecessary activities, and minimize costs. The sampling plan will ensure the right data sets are developed to support closure and efficient transition to long-term monitoring. The plan will include an integrated reporting mechanism for communicating results and integrating process improvements within the UGTA activity as well as between other U.S. Department of Energy (DOE) Programs.

  10. Demonstration of Eastman Christensen horizontal drilling system -- Integrated Demonstration Site, Savannah River Site

    SciTech Connect

    Not Available

    1992-12-01

    An innovative horizontal drilling system was used to install two horizontal wells as part of an integrated demonstration project at the Savannah River Site (SRS), Aiken, South Carolina. The SRS is located in south-central South Carolina in the upper Coastal Plain physiographic province. The demonstration site is located near the A/M Area, and is currently known as the Integated Demonstration Site. The Department of Energy's Office of Technology Development initiated an integrated demonstration of innovative technologies for cleanup of volatile organic compounds (VOCS) in soils and groundwater at the SRS in 1989. The overall goal of the program is to demonstrate, at a single location, multiple technologies in the fields of drilling, characterization, monitoring, and remediation. Innovative technologies are compared to one another and to baseline technologies in terms of technical performance and cost effectiveness. Transfer of successfully demonstrated technologies and systems to DOE environmental restoration organizations, to other government agencies, and to industry is a critical part of the program.

  11. Demonstration of Eastman Christensen horizontal drilling system -- Integrated Demonstration Site, Savannah River Site

    SciTech Connect

    Not Available

    1992-12-01

    An innovative horizontal drilling system was used to install two horizontal wells as part of an integrated demonstration project at the Savannah River Site (SRS), Aiken, South Carolina. The SRS is located in south-central South Carolina in the upper Coastal Plain physiographic province. The demonstration site is located near the A/M Area, and is currently known as the Integated Demonstration Site. The Department of Energy`s Office of Technology Development initiated an integrated demonstration of innovative technologies for cleanup of volatile organic compounds (VOCS) in soils and groundwater at the SRS in 1989. The overall goal of the program is to demonstrate, at a single location, multiple technologies in the fields of drilling, characterization, monitoring, and remediation. Innovative technologies are compared to one another and to baseline technologies in terms of technical performance and cost effectiveness. Transfer of successfully demonstrated technologies and systems to DOE environmental restoration organizations, to other government agencies, and to industry is a critical part of the program.

  12. Chromosomal Integrity after UV Irradiation Requires FANCD2-Mediated Repair of Double Strand Breaks

    PubMed Central

    Federico, María Belén; Vallerga, María Belén; Radl, Analía; Paviolo, Natalia Soledad; Bocco, José Luis; Di Giorgio, Marina; Soria, Gastón; Gottifredi, Vanesa

    2016-01-01

    Fanconi Anemia (FA) is a rare autosomal recessive disorder characterized by hypersensitivity to inter-strand crosslinks (ICLs). FANCD2, a central factor of the FA pathway, is essential for the repair of double strand breaks (DSBs) generated during fork collapse at ICLs. While lesions different from ICLs can also trigger fork collapse, the contribution of FANCD2 to the resolution of replication-coupled DSBs generated independently from ICLs is unknown. Intriguingly, FANCD2 is readily activated after UV irradiation, a DNA-damaging agent that generates predominantly intra-strand crosslinks but not ICLs. Hence, UV irradiation is an ideal tool to explore the contribution of FANCD2 to the DNA damage response triggered by DNA lesions other than ICL repair. Here we show that, in contrast to ICL-causing agents, UV radiation compromises cell survival independently from FANCD2. In agreement, FANCD2 depletion does not increase the amount of DSBs generated during the replication of UV-damaged DNA and is dispensable for UV-induced checkpoint activation. Remarkably however, FANCD2 protects UV-dependent, replication-coupled DSBs from aberrant processing by non-homologous end joining, preventing the accumulation of micronuclei and chromatid aberrations including non-homologous chromatid exchanges. Hence, while dispensable for cell survival, FANCD2 selectively safeguards chromosomal stability after UV-triggered replication stress. PMID:26765540

  13. Chromosomal Integrity after UV Irradiation Requires FANCD2-Mediated Repair of Double Strand Breaks.

    PubMed

    Federico, María Belén; Vallerga, María Belén; Radl, Analía; Paviolo, Natalia Soledad; Bocco, José Luis; Di Giorgio, Marina; Soria, Gastón; Gottifredi, Vanesa

    2016-01-01

    Fanconi Anemia (FA) is a rare autosomal recessive disorder characterized by hypersensitivity to inter-strand crosslinks (ICLs). FANCD2, a central factor of the FA pathway, is essential for the repair of double strand breaks (DSBs) generated during fork collapse at ICLs. While lesions different from ICLs can also trigger fork collapse, the contribution of FANCD2 to the resolution of replication-coupled DSBs generated independently from ICLs is unknown. Intriguingly, FANCD2 is readily activated after UV irradiation, a DNA-damaging agent that generates predominantly intra-strand crosslinks but not ICLs. Hence, UV irradiation is an ideal tool to explore the contribution of FANCD2 to the DNA damage response triggered by DNA lesions other than ICL repair. Here we show that, in contrast to ICL-causing agents, UV radiation compromises cell survival independently from FANCD2. In agreement, FANCD2 depletion does not increase the amount of DSBs generated during the replication of UV-damaged DNA and is dispensable for UV-induced checkpoint activation. Remarkably however, FANCD2 protects UV-dependent, replication-coupled DSBs from aberrant processing by non-homologous end joining, preventing the accumulation of micronuclei and chromatid aberrations including non-homologous chromatid exchanges. Hence, while dispensable for cell survival, FANCD2 selectively safeguards chromosomal stability after UV-triggered replication stress. PMID:26765540

  14. Chromosome Microarray.

    PubMed

    Anderson, Sharon

    2016-01-01

    Over the last half century, knowledge about genetics, genetic testing, and its complexity has flourished. Completion of the Human Genome Project provided a foundation upon which the accuracy of genetics, genomics, and integration of bioinformatics knowledge and testing has grown exponentially. What is lagging, however, are efforts to reach and engage nurses about this rapidly changing field. The purpose of this article is to familiarize nurses with several frequently ordered genetic tests including chromosomes and fluorescence in situ hybridization followed by a comprehensive review of chromosome microarray. It shares the complexity of microarray including how testing is performed and results analyzed. A case report demonstrates how this technology is applied in clinical practice and reveals benefits and limitations of this scientific and bioinformatics genetic technology. Clinical implications for maternal-child nurses across practice levels are discussed. PMID:27276104

  15. Effects of a Balanced Translocation between Chromosomes 1 and 11 Disrupting the DISC1 Locus on White Matter Integrity

    PubMed Central

    Whalley, Heather C.; Dimitrova, Rali; Sprooten, Emma; Dauvermann, Maria R.; Romaniuk, Liana; Duff, Barbara; Watson, Andrew R.; Moorhead, Bill; Bastin, Mark; Semple, Scott I.; Giles, Stephen; Hall, Jeremy; Thomson, Pippa; Roberts, Neil; Hughes, Zoe A.; Brandon, Nick J.; Dunlop, John; Whitcher, Brandon; Blackwood, Douglas H. R.; McIntosh, Andrew M.; Lawrie, Stephen M.

    2015-01-01

    Objective Individuals carrying rare, but biologically informative genetic variants provide a unique opportunity to model major mental illness and inform understanding of disease mechanisms. The rarity of such variations means that their study involves small group numbers, however they are amongst the strongest known genetic risk factors for major mental illness and are likely to have large neural effects. DISC1 (Disrupted in Schizophrenia 1) is a gene containing one such risk variant, identified in a single Scottish family through its disruption by a balanced translocation of chromosomes 1 and 11; t(1;11) (q42.1;q14.3). Method Within the original pedigree, we examined the effects of the t(1;11) translocation on white matter integrity, measured by fractional anisotropy (FA). This included family members with (n = 7) and without (n = 13) the translocation, along with a clinical control sample of patients with psychosis (n = 34), and a group of healthy controls (n = 33). Results We report decreased white matter integrity in five clusters in the genu of the corpus callosum, the right inferior fronto-occipital fasciculus, acoustic radiation and fornix. Analysis of the mixed psychosis group also demonstrated decreased white matter integrity in the above regions. FA values within the corpus callosum correlated significantly with positive psychotic symptom severity. Conclusions We demonstrate that the t(1;11) translocation is associated with reduced white matter integrity in frontal commissural and association fibre tracts. These findings overlap with those shown in affected patients with psychosis and in DISC1 animal models and highlight the value of rare but biologically informative mutations in modeling psychosis. PMID:26102360

  16. Integration of Mutation and Chromosomal Damage Endpoints into 28-Day Repeat Dose Toxicology Studies

    PubMed Central

    Dertinger, Stephen D.; Phonethepswath, Souk; Franklin, Dean; Weller, Pamela; Torous, Dorothea K.; Bryce, Steven M.; Avlasevich, Svetlana; Bemis, Jeffrey C.; Hyrien, Ollivier; Palis, James; MacGregor, James T.

    2010-01-01

    Two endpoints of genetic toxicity, mutation at the X-linked Pig-a gene and chromosomal damage in the form of micronucleated reticulocytes (MN-RETs), were evaluated in blood samples obtained from 28-day repeat-dosing studies typical of those employed in toxicity evaluations. Male Wistar Han rats were treated at 24-h intervals on days 1 through 28 with one of five prototypical genotoxicants: N-ethyl-N-nitrosourea, 7,12-dimethyl-12-benz[a]anthracene, 4-nitroquinoline-1-oxide (4NQO), benzo(a)pyrene, and N-methyl-N-nitrosourea. Flow cytometric scoring of CD59-negative erythrocytes (indicative of glycosylphosphatidylinositol anchor deficiency and hence Pig-a mutation) was performed using blood specimens obtained on days −1, 15, 29, and 56. Blood specimens collected on days 4 and 29 were evaluated for MN-RET frequency using flow cytometry–based MicroFlow Kits. With the exception of 4NQO, each chemical induced significant increases in the frequency of MN-RETs on days 4 and 29. All five agents increased the frequency of mutant phenotype (CD59 negative) reticulocytes (RETs) and erythrocytes. Mutation responses in RETs occurred earlier than in erythrocytes and tended to peak, or nearly peak, at day 29. In contrast, the mutant phenotype erythrocyte responses were modest on day 29 and required additional time to reach their maximal value. The observed kinetics were expected based on the known turnover of RETs and erythrocytes. The data show that RETs can serve as an appropriate indicator cell population for 28-day studies. Collectively, these data suggest that blood-based genotoxicity endpoints can be effectively incorporated into routine toxicology studies, a strategy that would reduce animal usage while providing valuable genetic toxicity information within the context of other toxicological endpoints. PMID:20202993

  17. Metabolic pathway engineering for fatty acid ethyl ester production in Saccharomyces cerevisiae using stable chromosomal integration.

    PubMed

    de Jong, Bouke Wim; Shi, Shuobo; Valle-Rodríguez, Juan Octavio; Siewers, Verena; Nielsen, Jens

    2015-03-01

    Fatty acid ethyl esters are fatty acid derived molecules similar to first generation biodiesel (fatty acid methyl esters; FAMEs) which can be produced in a microbial cell factory. Saccharomyces cerevisiae is a suitable candidate for microbial large scale and long term cultivations, which is the typical industrial production setting for biofuels. It is crucial to conserve the metabolic design of the cell factory during industrial cultivation conditions that require extensive propagation. Genetic modifications therefore have to be introduced in a stable manner. Here, several metabolic engineering strategies for improved production of fatty acid ethyl esters in S. cerevisiae were combined and the genes were stably expressed from the organisms' chromosomes. A wax ester synthase (ws2) was expressed in different yeast strains with an engineered acetyl-CoA and fatty acid metabolism. Thus, we compared expression of ws2 with and without overexpression of alcohol dehydrogenase (ADH2), acetaldehyde dehydrogenase (ALD6) and acetyl-CoA synthetase (acs SE (L641P) ) and further evaluated additional overexpression of a mutant version of acetyl-CoA decarboxylase (ACC1 (S1157A,S659A) ) and the acyl-CoA binding protein (ACB1). The combined engineering efforts of the implementation of ws2, ADH2, ALD6 and acs SE (L641P) , ACC1 (S1157A,S659A) and ACB1 in a S. cerevisiae strain lacking storage lipid formation (are1Δ, are2Δ, dga1Δ and lro1Δ) and β-oxidation (pox1Δ) resulted in a 4.1-fold improvement compared with sole expression of ws2 in S. cerevisiae. PMID:25422103

  18. A Single parS Sequence from the Cluster of Four Sites Closest to oriC Is Necessary and Sufficient for Proper Chromosome Segregation in Pseudomonas aeruginosa

    PubMed Central

    Jecz, Paulina; Bartosik, Aneta A.; Glabski, Krzysztof; Jagura-Burdzy, Grazyna

    2015-01-01

    Among the mechanisms that control chromosome segregation in bacteria are highly-conserved partitioning systems comprising three components: ParA protein (a deviant Walker-type ATPase), ParB protein (a DNA-binding element) and multiple cis-acting palindromic centromere-like sequences, designated parS. Ten putative parS sites have been identified in the P. aeruginosa PAO1 genome, four localized in close proximity of oriC and six, diverged by more than one nucleotide from a perfect palindromic sequence, dispersed along the chromosome. Here, we constructed and analyzed P. aeruginosa mutants deprived of each single parS sequence and their different combinations. The analysis included evaluation of a set of phenotypic features, chromosome segregation, and ParB localization in the cells. It was found that ParB binds specifically to all ten parS sites, although with different affinities. The P. aeruginosa parS mutant with all ten parS sites modified (parSnull) is viable however it demonstrates the phenotype characteristic for parAnull or parBnull mutants: slightly slower growth rate, high frequency of anucleate cells, and defects in motility. The genomic position and sequence of parS determine its role in P. aeruginosa biology. It transpired that any one of the four parS sites proximal to oriC (parS1 to parS4), which are bound by ParB with the highest affinity, is necessary and sufficient for the parABS role in chromosome partitioning. When all these four sites are mutated simultaneously, the strain shows the parSnull phenotype, which indicates that none of the remaining six parS sites can substitute for these four oriC-proximal sites in this function. A single ectopic parS2 (inserted opposite oriC in the parSnull mutant) facilitates ParB organization into regularly spaced condensed foci and reverses some of the mutant phenotypes but is not sufficient for accurate chromosome segregation. PMID:25794281

  19. Nevada National Security Site Integrated Groundwater Sampling Plan, Revision 0

    SciTech Connect

    Marutzky, Sam; Farnham, Irene

    2014-10-01

    The purpose of the Nevada National Security Site (NNSS) Integrated Sampling Plan (referred to herein as the Plan) is to provide a comprehensive, integrated approach for collecting and analyzing groundwater samples to meet the needs and objectives of the U.S. Department of Energy (DOE), National Nuclear Security Administration Nevada Field Office (NNSA/NFO) Underground Test Area (UGTA) Activity. Implementation of this Plan will provide high-quality data required by the UGTA Activity for ensuring public protection in an efficient and cost-effective manner. The Plan is designed to ensure compliance with the UGTA Quality Assurance Plan (QAP). The Plan’s scope comprises sample collection and analysis requirements relevant to assessing the extent of groundwater contamination from underground nuclear testing. This Plan identifies locations to be sampled by corrective action unit (CAU) and location type, sampling frequencies, sample collection methodologies, and the constituents to be analyzed. In addition, the Plan defines data collection criteria such as well-purging requirements, detection levels, and accuracy requirements; identifies reporting and data management requirements; and provides a process to ensure coordination between NNSS groundwater sampling programs for sampling of interest to UGTA. This Plan does not address compliance with requirements for wells that supply the NNSS public water system or wells involved in a permitted activity.

  20. Viral load, gene expression and mapping of viral integration sites in HPV16-associated HNSCC cell lines.

    PubMed

    Olthof, Nadine C; Huebbers, Christian U; Kolligs, Jutta; Henfling, Mieke; Ramaekers, Frans C S; Cornet, Iris; van Lent-Albrechts, Josefa A; Stegmann, Alexander P A; Silling, Steffi; Wieland, Ulrike; Carey, Thomas E; Walline, Heather M; Gollin, Susanne M; Hoffmann, Thomas K; de Winter, Johan; Kremer, Bernd; Klussmann, Jens P; Speel, Ernst-Jan M

    2015-03-01

    HPV-related HNSCC generally have a better prognosis than HPV-negative HNSCC. However, a subgroup of HPV-positive tumors with poor prognosis has been recognized, particularly related to smoking, EGFR overexpression and chromosomal instability. Viral integration into the host genome might contribute to carcinogenesis, as is shown for cervical carcinomas. Therefore, all HPV16-positive HNSCC cell lines currently available have been carefully analyzed for viral and host genome parameters. The viral integration status, viral load, viral gene expression and the presence of aneusomies was evaluated in the cell lines UD-SCC-2, UM-SCC-047, UM-SCC-104, UPCI:SCC090, UPCI:SCC152, UPCI:SCC154 and 93VU147T. HPV integration was examined using FISH, APOT-PCR and DIPS-PCR. Viral load and the expression of the viral genes E2, E6 and E7 were determined via quantitative PCR. All cell lines showed integration-specific staining patterns and signals indicating transcriptional activity using FISH. APOT- and DIPS-PCR identified integration-derived fusion products in six cell lines and only episomal products for UM-SCC-104. Despite the observed differences in viral load and the number of viral integration sites, this did not relate to the identified viral oncogene expression. Furthermore, cell lines exhibited EGFR expression and aneusomy (except UPCI:SCC154). In conclusion, all HPV16-positive HNSCC cell lines showed integrated and/or episomal viral DNA that is transcriptionally active, although viral oncogene expression was independent of viral copy number and the number of viral integration sites. Because these cell lines also contain EGFR expression and aneusomy, which are parameters of poor prognosis, they should be considered suitable model systems for the development of new antiviral therapies. PMID:25082736

  1. Vector-mediated chromosomal integration of the glutamate decarboxylase gene in streptococcus thermophilus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The integrative vector pINTRS was used to transfer glutamate decarboxylase (GAD) activity to Streptococcus thermophilus ST128, thus allowing for the production of '-aminobutyric acid (GABA). In pINTRS, the gene encoding glutamate decarboxylase, gadB, was flanked by DNA fragments homologous to a S. ...

  2. Chromosome-wide haplotype sharing: a measure integrating recombination information to reconstruct the phylogeny of human populations.

    PubMed

    Xu, Shuhua; Jin, Li

    2011-11-01

    The vast amount of recombination information in the human genome has long been ignored or deliberately avoided in studies on human population genetic relationships. One reason is that estimation of the recombination parameter from genotyping data is computationally challenging and practically difficult. Here we propose chromosome-wide haplotype sharing (CHS) as a measure of genetic similarity between human populations, which is an indirect approach to integrate recombination information. We showed in both empirical and simulated data that recombination differences and genetic differences between human populations are strongly correlated, indicating that recombination events in different human populations are evolutionarily related. We further demonstrated that CHS can be used to reconstruct reliable phylogenies of human populations and the majority of the variation in CHS matrix can be attributed to recombination. However, for distantly related populations, the utility of CHS to reconstruct correct phylogeny is limited, suggesting that the linear correlation of CHS and population divergence could have been disturbed by recurrent recombination events over a large time scale. The CHS we proposed in this study is a practical approach without involving computationally challenging and time-consuming estimation of recombination parameter. The advantage of CHS is rooted in its integration of both drift and recombination information, therefore providing additional resolution especially for populations separated recently. PMID:21972961

  3. Novel Y-chromosomal microdeletions associated with non-obstructive azoospermia uncovered by high throughput sequencing of sequence-tagged sites (STSs)

    PubMed Central

    Liu, Xiao; Li, Zesong; Su, Zheng; Zhang, Junjie; Li, Honggang; Xie, Jun; Xu, Hanshi; Jiang, Tao; Luo, Liya; Zhang, Ruifang; Zeng, Xiaojing; Xu, Huaiqian; Huang, Yi; Mou, Lisha; Hu, Jingchu; Qian, Weiping; Zeng, Yong; Zhang, Xiuqing; Xiong, Chengliang; Yang, Huanming; Kristiansen, Karsten; Cai, Zhiming; Wang, Jun; Gui, Yaoting

    2016-01-01

    Y-chromosomal microdeletion (YCM) serves as an important genetic factor in non-obstructive azoospermia (NOA). Multiplex polymerase chain reaction (PCR) is routinely used to detect YCMs by tracing sequence-tagged sites (STSs) in the Y chromosome. Here we introduce a novel methodology in which we sequence 1,787 (post-filtering) STSs distributed across the entire male-specific Y chromosome (MSY) in parallel to uncover known and novel YCMs. We validated this approach with 766 Chinese men with NOA and 683 ethnically matched healthy individuals and detected 481 and 98 STSs that were deleted in the NOA and control group, representing a substantial portion of novel YCMs which significantly influenced the functions of spermatogenic genes. The NOA patients tended to carry more and rarer deletions that were enriched in nearby intragenic regions. Haplogroup O2* was revealed to be a protective lineage for NOA, in which the enrichment of b1/b3 deletion in haplogroup C was also observed. In summary, our work provides a new high-resolution portrait of deletions in the Y chromosome. PMID:26907467

  4. Novel Y-chromosomal microdeletions associated with non-obstructive azoospermia uncovered by high throughput sequencing of sequence-tagged sites (STSs).

    PubMed

    Liu, Xiao; Li, Zesong; Su, Zheng; Zhang, Junjie; Li, Honggang; Xie, Jun; Xu, Hanshi; Jiang, Tao; Luo, Liya; Zhang, Ruifang; Zeng, Xiaojing; Xu, Huaiqian; Huang, Yi; Mou, Lisha; Hu, Jingchu; Qian, Weiping; Zeng, Yong; Zhang, Xiuqing; Xiong, Chengliang; Yang, Huanming; Kristiansen, Karsten; Cai, Zhiming; Wang, Jun; Gui, Yaoting

    2016-01-01

    Y-chromosomal microdeletion (YCM) serves as an important genetic factor in non-obstructive azoospermia (NOA). Multiplex polymerase chain reaction (PCR) is routinely used to detect YCMs by tracing sequence-tagged sites (STSs) in the Y chromosome. Here we introduce a novel methodology in which we sequence 1,787 (post-filtering) STSs distributed across the entire male-specific Y chromosome (MSY) in parallel to uncover known and novel YCMs. We validated this approach with 766 Chinese men with NOA and 683 ethnically matched healthy individuals and detected 481 and 98 STSs that were deleted in the NOA and control group, representing a substantial portion of novel YCMs which significantly influenced the functions of spermatogenic genes. The NOA patients tended to carry more and rarer deletions that were enriched in nearby intragenic regions. Haplogroup O2* was revealed to be a protective lineage for NOA, in which the enrichment of b1/b3 deletion in haplogroup C was also observed. In summary, our work provides a new high-resolution portrait of deletions in the Y chromosome. PMID:26907467

  5. AB039. Novel Y-chromosomal microdeletions associated with non-obstructive azoospermia uncovered by high throughput sequencing of sequence-tagged sites (STSs)

    PubMed Central

    Li, Zesong

    2016-01-01

    Y-chromosomal microdeletion (YCM) serves as an important genetic factor in non-obstructive azoospermia (NOA). Multiplex polymerase chain reaction (PCR) is routinely used to detect YCMs by tracing sequence-tagged sites (STSs) in the Y chromosome. Here we introduce a novel methodology in which we sequence 1,787 (post-filtering) STSs distributed across the entire male-specific Y chromosome (MSY) in parallel to uncover known and novel YCMs. We validated this approach with 766 Chinese men with NOA and 683 ethnically matched healthy individuals and detected 481 and 98 STSs that were deleted in the NOA and control group, representing a substantial portion of novel YCMs which significantly influenced the functions of spermatogenic genes. The NOA patients tended to carry more and rarer deletions that were enriched in nearby intragenic regions. Haplogroup O2* was revealed to be a protective lineage for NOA, in which the enrichment of b1/b3 deletion in haplogroup C was also observed. In summary, our work provides a new high-resolution portrait of deletions in the Y chromosome.

  6. Chromosomal Conditions

    MedlinePlus

    ... 150 babies is born with a chromosomal condition. Down syndrome is an example of a chromosomal condition. Because ... all pregnant women be offered prenatal tests for Down syndrome and other chromosomal conditions. A screening test is ...

  7. Inherited chromosomally integrated human herpesvirus 6 as a predisposing risk factor for the development of angina pectoris

    PubMed Central

    Gravel, Annie; Dubuc, Isabelle; Morissette, Guillaume; Sedlak, Ruth H.; Jerome, Keith R.; Flamand, Louis

    2015-01-01

    Inherited chromosomally integrated human herpesvirus-6 (iciHHV-6) results in the germ-line transmission of the HHV-6 genome. Every somatic cell of iciHHV-6+ individuals contains the HHV-6 genome integrated in the telomere of chromosomes. Whether having iciHHV-6 predisposes humans to diseases remains undefined. DNA from 19,597 participants between 40 and 69 years of age were analyzed by quantitative PCR (qPCR) for the presence of iciHHV-6. Telomere lengths were determined by qPCR. Medical records, hematological, biochemical, and anthropometric measurements and telomere lengths were compared between iciHHV-6+ and iciHHV-6− subjects. The prevalence of iciHHV-6 was 0.58%. Two-way ANOVA with a Holm–Bonferroni correction was used to determine the effects of iciHHV6, sex, and their interaction on continuous outcomes. Two-way logistic regression with a Holm–Bonferroni correction was used to determine the effects of iciHHV6, sex, and their interaction on disease prevalence. Of 50 diseases monitored, a single one, angina pectoris, is significantly elevated (3.3×) in iciHHV-6+ individuals relative to iciHHV-6− subjects (P = 0.017; 95% CI, 1.73–6.35). When adjusted for potential confounding factors (age, body mass index, percent body fat, and systolic blood pressure), the prevalence of angina remained three times greater in iciHHV-6+ subjects (P = 0.015; 95%CI, 1.23–7.15). Analyses of telomere lengths between iciHHV-6− without angina, iciHHV-6− with angina, and iciHHV-6+ with angina indicate that iciHHV-6+ with angina have shorter telomeres than age-matched iciHHV-6− subjects (P = 0.006). Our study represents, to our knowledge, the first large-scale analysis of disease association with iciHHV-6. Our results are consistent with iciHHV-6 representing a risk factor for the development of angina. PMID:26080419

  8. BIOTIC INTEGRITY OF STREAMS IN THE SAVANNAH RIVER SITE INTEGRATOR OPERABLE UNITS, 1996 TO 2003

    SciTech Connect

    Paller, M; Susan Dyer, S

    2004-11-08

    The Savannah River Site (SRS) has been divided into six Integrator Operable Units (IOUs) that correspond to the watersheds of the five major streams on the SRS (Upper Three Runs, Fourmile Branch, Pen Branch, Steel Creek, and Lower Three Runs) and the portions of the Savannah River and Savannah River Swamp associated with the SRS. The streams are the primary integrators within each IOU because they potentially receive, through surface or subsurface drainage, soluble contaminants from all waste sites within their watersheds. If these contaminants reach biologically significant levels, they would be expected to effect the numbers, types, and health of stream organisms. In this study, biological sampling was conducted within each IOU as a measure of the cumulative ecological effects of the waste sites within the IOUs. The use of information from biological sampling to assess environmental quality is often termed bioassessment. The IOU bioassessment program included 38 sites in SRS streams and nine sites in the Savannah River. Sampling was conducted in 1996 to 1998, 2000, and 2003. Four bioassessment methods were used to evaluate ecological conditions in the IOU streams: the Index of Biotic Integrity, the Fish Health Assessment Index, measurement of fish tissue contaminant levels, and two benthic macroinvertebrate indices. The Index of Biotic Integrity (IBI) is an EPA supported method based on comparison of ecologically important and sensitive fish assemblage variables between potentially disturbed and reference (i.e., undisturbed) sites. It is designed to assess the ability of a stream to support a self-sustaining biological community and ecological processes typical of undisturbed, natural conditions. Since many types of contaminants can bioaccumulate, fish tissue contaminant data were used to determine the types of chemicals fish were exposed to and their relative magnitudes among IOUs. The Fish Health Assessment Index (HAI) is an EPA supported method for assessing

  9. The Eastern Lau Basin Integrated Studies Site (ISS)

    NASA Astrophysics Data System (ADS)

    Wiens, D.

    2003-12-01

    A new venue for Ridge 2000 (R2K) Integrated Studies, the Eastern Lau Spreading Center (ELSC) adds the element of a spreading center in a back-arc basin to the R2K program. The ELSC, located in the western Pacific near Tonga, is a 390 km-long first-order ridge segment that displays a broad range of effects of the back-arc environment. At its southern end it is only 40 km from the Tonga arc volcanic front and is propagating southward into a back-arc rift. At its northern end it is 100 km from the volcanic front and terminates at a large, non-transform offset. The ELSC displays substantial and systematic changes in multiple parameters: spreading rate, magma source and lava chemistry, axial depth and morphology, melt lens characteristics, and crustal thickness and structure. A main focus of the work at the ELSC is to determine how changes in these forcing functions affect key parameters such as magma source composition, crustal structure, and characteristics of hydrothermal venting such as temperature, chemistry, and faunal composition and abundance. Prior reconnaissance investigation shows that these hydrothermal field characteristics also vary within the southernmost segments, and show distinct differences compared with well-studied mid-ocean ridge sites. Four R2K cruises are planned for the ELSC in 2004. The first cruise (PI: Martinez) will investigate interrelationships among crustal structure, volcanism, and hydrothermal activity using deep tow, CTD's, and Tow-Yo. A second cruise (PI: Langmuir) will focus on petrological and water column properties using dredging, CTD's and ABE. The next cruise (PI: Tivey) will provide an initial characterization of vent fields, fluid chemistry, mineralogy, and biodiversity using Jason II and net tows, and a final cruise (PI: Childress) will investigate community ecology using Jason II. An additional study (PI: Thurnherr) will deploy autonomous floats to investigate hydrothermal plume circulation and dispersal . An overview of the

  10. Gene therapy for hemophilia B with liver-specific element mediated by Rep-RBE site-specific integration system.

    PubMed

    Xu, Zhengxin; Ye, Juan; Zhang, Amin; Xie, Linjun; Shen, Qi; Xue, Jinglun; Chen, Jinzhong

    2015-02-01

    Adeno-associated virus (AAV) is a nonpathogenic virus capable of targeting human chromosome 19 for integration at AAVS1 site, and a 16 bp Rep binding element (RBE) sequence of AAV was sufficient for mediating this specific integration in the presence of AAV regulation proteins (Rep). Previously, we cotransduced 2 plasmids, pRBE-CMV-hFIX and pRC, into the AAVS1 transgenic mice by hydrodynamic injection, and a long-term expression of human coagulation Factor IX (hFIX) was observed. The corresponding AAVS1 locus site-specific integrations were verified by nested polymerase chain reaction. In this study, we established a novel hFIX expression plasmid, pRBE-HCR-hAAT-hFIX, driven by a liver-specific promoter by replacing the CMV promoter of pRBE-CMV-hFIX with a humanized promoter consisting of HCR-hAAT. The expression of hFIX in vitro was almost the same in transient transfection of pRBE-CMV-hFIX or pRBE-HCR-hAAT-hFIX. AAVS1-specific integrations were identified both in mice transfected with pRC/pRBE-CMV-hFIX cocktail and pRC/pRBE-HCR-hAAT-hFIX cocktail. However, the expression of hFIX of pRBE-HCR-hAAT-hFIX mice was higher and persisted longer. It achieved more than 1% of normal plasma hFIX concentration and maintained for 240 days. The result suggested that RBE-HCR-hAAT element could improve the expression of hFIX and present potential usage of Rep-RBE site-specific integration in gene therapy for hemophilia B. PMID:25295466

  11. Repeat Hydrography at the Endeavour Integrated Study Site, 2004 - 2006

    NASA Astrophysics Data System (ADS)

    Kellogg, J. P.; McDuff, R. E.; Thomson, R. E.; Stahr, F. R.

    2006-12-01

    Significant differences exist between hydrographic transects made in the summers from 2004 to 2006 at the Endeavour Segment Integrated Study Site on the Juan de Fuca Ridge. Along and across axis sections describe the hydrographic conditions above the segment in three dimensions. The resulting sections allow for rapid evaluation of the characteristics of the neutrally buoyant plume over each of the vent fields and its location relative to the ridge axis. Results indicate heat content over the northern vent fields, Salty Dawg and Sasquatch, significantly increased between the summers of 2004 and 2005. In 2004, the plumes over these vent fields were barely discernable while in 2005 prominent plumes existed with potential temperature anomalies over 0.1°C. At the time of a rapid response cruise in March 2005, no significant change in the heat content of the water column was detected. By July 2005, dramatic changes had occurred in the overlying water column structure. The potential temperature anomaly section from 2005 is indicative of a thicker (about 75 m) neutrally buoyant plume with and substantially more heat at the north end of the valley. In 2004, the shallowest plume depth was 1900 m contrasted with 1830 m in 2005. Vent data being obtained by other RIDGE 2000 and UW Keck investigators will help constrain the underlying causes of these changes. New hydrography will be collected in August September 2006.

  12. Repeat Hydrography at the Endeavour Integrated Study Site, 2004 - 2005

    NASA Astrophysics Data System (ADS)

    Kellogg, J. P.; McDuff, R. E.; Thomson, R. E.; Stahr, F. R.

    2005-12-01

    Significant differences exist between hydrographic transects made in 2004 and 2005 at the Endeavour Segment Integrated Study Site on the Juan de Fuca Ridge. Sections that describe the conditions above the segment utilize twenty-one nearly uniformly spaced hydrographic stations from south of Mothra to north of the Sasquatch hydrothermal vent fields. Criteria used in choosing station locations included depth, ~500 m spacing from other stations, and being centrally located in the valley. The resulting sections allow for rapid evaluation of the characteristics of the neutrally buoyant plume over each of the vent fields. Preliminary results indicate heat content over the northern vent fields, Salty Dawg and Sasquatch, significantly increased between the summers of 2004 and 2005. In 2004, the plumes over these vent fields were barely discernable while in 2005 prominent plumes existed with potential temperature anomalies over 0.1°C. Vent data being obtained by other RIDGE 2000 and UW Keck investigators will help constrain the underlying causes of these changes. Isopycnals in the 2005 sections are also elevated along the entire length of the transect by approximately 50 m or more. The potential temperature anomaly section from 2005 is indicative of a thicker (about 75 m) neutrally buoyant plume and substantially more heat at the north end of the valley. In 2004, the shallowest plume depth was 1900 m contrasted with 1830 m in 2005.

  13. The role of HTLV-1 clonality, proviral structure, and genomic integration site in adult T-cell leukemia/lymphoma

    PubMed Central

    Cook, Lucy B.; Melamed, Anat; Niederer, Heather; Valganon, Mikel; Laydon, Daniel; Foroni, Letizia; Taylor, Graham P.; Matsuoka, Masao; Bangham, Charles R. M.

    2014-01-01

    Adult T-cell leukemia/lymphoma (ATL) occurs in ∼5% of human T-lymphotropic virus type 1 (HTLV-1)–infected individuals and is conventionally thought to be a monoclonal disease in which a single HTLV-1+ T-cell clone progressively outcompetes others and undergoes malignant transformation. Here, using a sensitive high-throughput method, we quantified clonality in 197 ATL cases, identified genomic characteristics of the proviral integration sites in malignant and nonmalignant clones, and investigated the proviral features (genomic structure and 5′ long terminal repeat methylation) that determine its capacity to express the HTLV-1 oncoprotein Tax. Of the dominant, presumed malignant clones, 91% contained a single provirus. The genomic characteristics of the integration sites in the ATL clones resembled those of the frequent low-abundance clones (present in both ATL cases and carriers) and not those of the intermediate-abundance clones observed in 24% of ATL cases, suggesting that oligoclonal proliferation per se does not cause malignant transformation. Gene ontology analysis revealed an association in 6% of cases between ATL and integration near host genes in 3 functional categories, including genes previously implicated in hematologic malignancies. In all cases of HTLV-1 infection, regardless of ATL, there was evidence of preferential survival of the provirus in vivo in acrocentric chromosomes (13, 14, 15, 21, and 22). PMID:24735963

  14. Integration of biological data by kernels on graph nodes allows prediction of new genes involved in mitotic chromosome condensation.

    PubMed

    Hériché, Jean-Karim; Lees, Jon G; Morilla, Ian; Walter, Thomas; Petrova, Boryana; Roberti, M Julia; Hossain, M Julius; Adler, Priit; Fernández, José M; Krallinger, Martin; Haering, Christian H; Vilo, Jaak; Valencia, Alfonso; Ranea, Juan A; Orengo, Christine; Ellenberg, Jan

    2014-08-15

    The advent of genome-wide RNA interference (RNAi)-based screens puts us in the position to identify genes for all functions human cells carry out. However, for many functions, assay complexity and cost make genome-scale knockdown experiments impossible. Methods to predict genes required for cell functions are therefore needed to focus RNAi screens from the whole genome on the most likely candidates. Although different bioinformatics tools for gene function prediction exist, they lack experimental validation and are therefore rarely used by experimentalists. To address this, we developed an effective computational gene selection strategy that represents public data about genes as graphs and then analyzes these graphs using kernels on graph nodes to predict functional relationships. To demonstrate its performance, we predicted human genes required for a poorly understood cellular function-mitotic chromosome condensation-and experimentally validated the top 100 candidates with a focused RNAi screen by automated microscopy. Quantitative analysis of the images demonstrated that the candidates were indeed strongly enriched in condensation genes, including the discovery of several new factors. By combining bioinformatics prediction with experimental validation, our study shows that kernels on graph nodes are powerful tools to integrate public biological data and predict genes involved in cellular functions of interest. PMID:24943848

  15. Structural analysis of a hepatitis B virus genome integrated into chromosome 17p of a human hepatocellular carcinoma

    SciTech Connect

    Zhou, Y.Z.; Slagle, B.L.; Donehower, L.A.; van Tuinen, P.; Ledbetter, D.H.; Butel, J.S.

    1988-11-01

    Hepatitis B virus (HBV) is clearly a factor in the development of hepatocellular carcinoma, but its mechanism of action remains obscure. One possibility is that the HBV integration event alters the expression of a nearby growth-regulatory cellular gene. A 9-kilobase (kb) DNA fragment containing an HBV insert plus flanking cellular sequences was cloned from a hepatoma specimen from Shanghai, People's Republic of China. Restriction mapping of the insert revealed a large inverted repeat structure consisting of both viral sequences (encompassing all of the core and pre-S regions and portions of the X and S genes) and at least 3 kb of unique cellular sequences. The virus-cell junction mapped 11 nucleotides from the DRI region, in a position within the HBV X gene and included in the cohesive overlap region. A probe generated from 1.0 kb of the flanking cellular DNA mapped the viral insert to chromosome 17 in the region designated 17p11.2-17p12, which is near the human proto-oncogene p53. Sequence data from a portion of the flanking cellular DNA revealed a stretch of approximately 70 base pairs that showed highly significant homology with a conserved region of a number of functional mammalian DNA, including the human autonomously replicating sequence 1 (ASRI).

  16. Structural analysis of a hepatitis B virus genome integrated into chromosome 17p of a human hepatocellular carcinoma.

    PubMed Central

    Zhou, Y Z; Slagle, B L; Donehower, L A; vanTuinen, P; Ledbetter, D H; Butel, J S

    1988-01-01

    Hepatitis B virus (HBV) is clearly a factor in the development of hepatocellular carcinoma, but its mechanism of action remains obscure. One possibility is that the HBV integration event alters the expression of a nearby growth-regulatory cellular gene. A 9-kilobase (kb) DNA fragment containing an HBV insert plus flanking cellular sequences was cloned from a hepatoma specimen from Shanghai, People's Republic of China. Restriction mapping of the insert revealed a large inverted repeat structure consisting of both viral sequences (encompassing all of the core and pre-S regions and portions of the X and S genes) and at least 3 kb of unique cellular sequences. The virus-cell junction mapped 11 nucleotides from the DR1 region, in a position within the HBV X gene and included in the cohesive overlap region. A probe generated from 1.0 kb of the flanking cellular DNA mapped the viral insert to chromosome 17 in the region designated 17p11.2-17p12, which is near the human proto-oncogene p53. Sequence data from a portion of the flanking cellular DNA revealed a stretch of approximately 70 base pairs that showed highly significant homology with a conserved region of a number of functional mammalian DNAs, including the human autonomously replicating sequence 1 (ARS1). Images PMID:2845134

  17. Integration of biological data by kernels on graph nodes allows prediction of new genes involved in mitotic chromosome condensation

    PubMed Central

    Hériché, Jean-Karim; Lees, Jon G.; Morilla, Ian; Walter, Thomas; Petrova, Boryana; Roberti, M. Julia; Hossain, M. Julius; Adler, Priit; Fernández, José M.; Krallinger, Martin; Haering, Christian H.; Vilo, Jaak; Valencia, Alfonso; Ranea, Juan A.; Orengo, Christine; Ellenberg, Jan

    2014-01-01

    The advent of genome-wide RNA interference (RNAi)–based screens puts us in the position to identify genes for all functions human cells carry out. However, for many functions, assay complexity and cost make genome-scale knockdown experiments impossible. Methods to predict genes required for cell functions are therefore needed to focus RNAi screens from the whole genome on the most likely candidates. Although different bioinformatics tools for gene function prediction exist, they lack experimental validation and are therefore rarely used by experimentalists. To address this, we developed an effective computational gene selection strategy that represents public data about genes as graphs and then analyzes these graphs using kernels on graph nodes to predict functional relationships. To demonstrate its performance, we predicted human genes required for a poorly understood cellular function—mitotic chromosome condensation—and experimentally validated the top 100 candidates with a focused RNAi screen by automated microscopy. Quantitative analysis of the images demonstrated that the candidates were indeed strongly enriched in condensation genes, including the discovery of several new factors. By combining bioinformatics prediction with experimental validation, our study shows that kernels on graph nodes are powerful tools to integrate public biological data and predict genes involved in cellular functions of interest. PMID:24943848

  18. 'Ca. Liberibacter asiaticus' carries an excision plasmid prophage and a chromosomally integrated prophage that becomes lytic in plant infections.

    PubMed

    Zhang, Shujian; Flores-Cruz, Zomary; Zhou, Lijuan; Kang, Byung-Ho; Fleites, Laura A; Gooch, Mark D; Wulff, Nelson A; Davis, Michael J; Duan, Yong-Ping; Gabriel, Dean W

    2011-04-01

    Huanglongbing (HLB), also known as citrus greening, is a lethal disease of citrus caused by several species of 'Candidatus Liberibacter', a psyllid-transmitted, phloem-limited, alpha proteobacteria. 'Ca. Liberibacter asiaticus' is widespread in Florida citrus. The recently published 'Ca. L. asiaticus' psy62 genome, derived from a psyllid, revealed a prophage-like region of DNA in the genome, but phage have not been associated with 'Ca. L. asiaticus' to date. In the present study, shotgun sequencing and a fosmid DNA library of curated 'Ca. L. asiaticus' UF506, originally derived from citrus symptomatic for HLB, revealed two largely homologous, circular phage genomes, SC1 and SC2. SC2 encoded putative adhesin and peroxidase genes that had not previously been identified in 'Ca. L. asiaticus' and which may be involved in lysogenic conversion. SC2 also appeared to lack lytic cycle genes and replicated as a prophage excision plasmid, in addition to being found integrated in tandem with SC1 in the UF506 chromosome. By contrast, SC1 carried suspected lytic cycle genes and was found in nonintegrated, lytic cycle forms only in planta. Phage particles associated with 'Ca. L. asiaticus' were found in the phloem of infected periwinkles by transmission electron microscopy. In psyllids, both SC1 and SC2 were found only as prophage. PMID:21190436

  19. Disrupted fornix integrity in children with chromosome 22q11.2 deletion syndrome

    PubMed Central

    Deng, Yi; Goodrich-Hunsaker, Naomi J.; Cabaral, Margarita; Amaral, David G.; Buonocore, Michael H.; Harvey, Danielle; Kalish, Kristopher; Carmichael, Owen; Schumann, Cynthia M.; Lee, Aaron; Dougherty, Robert F.; Perry, Lee M.; Wandell, Brian A.; Simon, Tony J.

    2015-01-01

    The fornix is the primary subcortical output fiber system of the hippocampal formation. In children with 22q11.2 deletion syndrome (22q11.2DS), hippocampal volume reduction has been commonly reported, but few studies as yet have evaluated the integrity of the fornix. Therefore, we investigated the fornix of 45 school-aged children with 22q11.2DS and 38 matched typically developing (TD) children. Probabilistic diffusion tensor imaging (DTI) tractography was used to reconstruct the body of fornix in each child's brain native space. Compared with children, significantly lower fractional anisotropy (FA) and higher radial diffusivity (RD) was observed bilaterally in the body of the fornix in children with 22q11.2DS. Irregularities were especially prominent in the posterior aspect of the fornix where it emerges from the hippocampus. Smaller volumes of hippocampal formations were also found in the 22q11.2DS group. The reduced hippocampal volumes were correlated with fornix lower FA and higher RD in the right hemisphere. Our findings provide neuroanatomical evidence of disrupted hippocampal connectivity in children with 22q11.2DS, which may help to further understand the biological basis for spatial impairments, affective regulation, and other factors related to the ultra-high risk for schizophrenia in this population. PMID:25748884

  20. Microsatellite and single nucleotide polymorphisms in the β-globin locus control region-hypersensitive Site 2: SPECIFICITY of Tunisian βs chromosomes.

    PubMed

    Ben Mustapha, Maha; Moumni, Imen; Zorai, Amine; Douzi, Kaïs; Ghanem, Abderraouf; Abbes, Salem

    2012-01-01

    The diversity of sickle cell disease severity is attributed to several cis acting factors, among them the single nucleotide polymorphisms (SNPs) and (AT) rich region in the β-locus control region (β-LCR). This contains five DNase I hypersensitive sites (HS) located 6 to 22 kb upstream to the ϵ gene. The most important of these is the HS2 (5' β-LCR-HS2), characterized by the presence of three different SNPs and a microsatellite region known to be in association with β(S) chromosomes in various populations. The aim of this study was to present the molecular investigation of the 5' β-LCR-HS2 site in normal and sickle cell disease individuals in order to determine if there is any correlation or specificity between these molecular markers, the β(S) Tunisian chromosomes and phenotypical expression of sickle cell disease. One hundred and twenty-four chromosomes from Tunisian individuals (49 β(S) carriers and 13 normal individuals) were screened by polymerase chain reaction (PCR) and sequencing for the polymorphic short tandem microsatellite repeats (AT)(X)N(12)(AT)(Y) and the three SNPs (rs7119428, rs9736333 and rs60240093) of the 5' β-LCR-HS2. Twelve configurations of the microsatellite motif were found with an ancestral configuration elaborated by ClustalW software. Normal and mutated alleles were observed at the homozygous and heterozygous states for the three SNPs. Correlation between microsatellites and SNPs suggests that mutant SNP alleles were mainly associated, in the homozygous sickle cell disease phenotype, with the (AT)(8)N(12)GT(AT)(7) configuration, whereas, normal SNP alleles were associated with the (AT)(X)N(12)(AT)(11) configurations in normal β(A) chromosomes. The correlation of these various configurations with Hb F expression was also investigated. The principal component analysis (PCA) showed the correlation between the homozygous sickle cell disease phenotype, mutated SNP alleles and the Benin microsatellite configuration (AT)(8)N(12)GT

  1. Integrated Research Methods for Applied Urban Hydrogeology of Karst Sites

    NASA Astrophysics Data System (ADS)

    Epting, J.; Romanov, D. K.; Kaufmann, G.; Huggenberger, P.

    2008-12-01

    Integrated and adaptive surface- and groundwater monitoring and management in urban areas require innovative process-oriented approaches. To accomplish this, it is necessary to develop and combine interdisciplinary instruments that facilitate adequately quantifying cumulative effects on groundwater flow regimes. While the characterization and modeling of flow in heterogeneous and fractured media has been investigated intensively, there are no well-developed long-term hydrogeological research sites for gypsum karst. Considering that infrastructures in karst regions, particularly in gypsum, are prone to subsidence, severe problems can arise in urban areas. In the 1880's, a river dam was constructed on gypsum-containing rock, Southeast of Basel, Switzerland. Over the last 30 years, subsidence of the dam and an adjacent highway has been observed. Surface water infiltrates upstream of the dam, circulates in the gravel deposits and in the weathered bedrock around and beneath the dam and exfiltrates downstream into the river. These processes enhance karstification processes in the soluble units of the gypsum. As a result an extended weathering zone within the bedrock and the development of preferential flow paths within voids and conduits can be observed. To prevent further subsidence, construction measures were conducted in two major project phases in 2006 and 2007. The highway was supported by a large number of pillars embedded in the non- weathered rock and by a sealing pile wall, to prevent infiltrating river water circulating around the dam and beneath the foundation of the highway. To safeguard surface and subsurface water resources during the construction measures, an extensive observation network was set up. Protection schemes and geotechnical investigations that are necessary for engineering projects often provide "windows of opportunity", bearing the possibility to change perceptions concerning the sustainable development of water resources and coordinate future

  2. Current Research at the Endeavour Ridge 2000 Integrated Studies Site

    NASA Astrophysics Data System (ADS)

    Butterfield, D. A.; Kelley, D. S.; Ridge 2000 Community, R.

    2004-12-01

    Integrated geophysical, geological, chemical, and biological studies are being conducted on the Endeavour segment with primary support from NSF, the W.M. Keck Foundation, and NSERC (Canada). The research includes a seismic network, physical and chemical sensors, high-precision mapping and time-series sampling. Several research expeditions have taken place at the Endeavour ISS in the past year. In June 2003, an NSF-sponsored cruise with R.V. al T.G.Thompson/ROV al Jason2 installed microbial incubators in drill-holes in the sides of active sulfide chimneys and sampled rocks, fluids, and microbes in the Mothra and Main Endeavour Field (MEF). In July 2003, with al Thompson/Jason2, an NSF-LEXEN project at Baby Bare on Endeavour east flank conducted sampling through seafloor-penetrating probes, plus time-series sampling of fluids, microbes, and rocks at the MEF. In September 2003, with al Thompson/ROV al ROPOS, the Keck Proto-Neptune project installed a seismic network consisting of 1 broadband and 7 short-period seismometers, installation of chemical/physical sensors and time-series samplers for chemistry and microbiology in the MEF and Clam Bed sites, collection of rocks, fluids, animals, and microbes. In May/June 2004, an NSF-sponsored al Atlantis/Alvin cruise recovered sulfide incubators installed in 2003, redeployed a sulfide incubator, mapped MEF and Mothra vent fields with high-resolution Imagenix sonar, sampled fluids from MEF, Mothra, and Clam Bed, recovered year-long time-series fluid and microbial samplers from MEF and Clam Bed, recovered and installed hot vent temperature-resistivity monitors, cleaned up the MEF and deployed new markers at major sulfide structures. In August 2004, there were two MBARI/Keck-sponsored cruises with R.V. al Western Flyer/ROV al Tiburon. The first cruise completed the seismic network with addition of two more broadband seismometers and serviced all 7 short-period seismometers. al Tiburon then performed microbial and chemical

  3. Integrated Mapping and Imaging at a Legacy Test Site (Invited)

    NASA Astrophysics Data System (ADS)

    Sussman, A. J.; Schultz-Fellenz, E. S.; Kelley, R. E.; Sweeney, J. J.; Vigil, S.; DiBenedetto, J.; Chipman, V.

    2013-12-01

    A team of multi-disciplinary geoscientists was tasked to characterize and evaluate a legacy nuclear detonation site in order to develop research locations with the long-term goal of improving treaty monitoring, verification, and other national security applications. There was a test at the site of interest that was detonated on June 12, 1985 in a vertical emplacement borehole at a depth of 608m below the surface in rhyolites. With announced yield of 20-150 kt, the event did not collapse to the surface and form a crater, but rather experienced a subsurface collapse with more subtle surface expressions of deformation. This result provides the team with an opportunity to evaluate a number of surface and subsurface inspection technologies in a broad context. The team collected ground-based visual observation, ground penetrating radar, electromagnetic, ground-based and airborne LiDAR, ground-based and airborne hyperspectral, gravity and magnetics, dc and induction electrical methods, and active seismic data during field campaigns in the summers of 2012 and 2013. Detection of features was performed using various approaches that were assessed for accuracy, efficiency and diversity of target features. For example, whereas the primary target of the ground-based visual observation survey was to map the surface features, the target of the gravity survey was to attempt the detection of a possible subsurface collapse zone which might be located as little as 200 meters below the surface. The datasets from surveys described above are integrated into a geographical information system (GIS) database for analysis and visualization. Other presentations during this session provide further details as to some of the work conducted. Work by Los Alamos National Laboratory and Lawrence Livermore National Laboratory was sponsored by the National Nuclear Security Administration Award No. DE-AC52-06NA25946/NST10-NCNS-PD00. Work by National Security Technologies, LLC, was performed under

  4. Organization of the Bacillus subtilis 168 chromosome between kdg and the attachment site of the SP beta prophage: use of Long Accurate PCR and yeast artificial chromosomes for sequencing.

    PubMed

    Capuano, V; Galleron, N; Pujic, P; Sorokin, A; Ehrlich, S D

    1996-11-01

    Within the Bacillus subtilis genome sequencing project, the region between lysA and ilvA was assigned to our laboratory. In this report we present the sequence of the last 36 kb of this region, between the kdg operon and the attachment site of the SP beta prophage. A two-step strategy was used for the sequencing. In the first step, total chromosomal DNA was cloned in phage M13-based vectors and the clones carrying inserts from the target region were identified by hybridization with a cognate yeast artificial chromosome (YAC) from our collection. Sequencing of the clones allowed us to establish a number of contigs. In the second step the contigs were mapped by Long Accurate (LA) PCR and the remaining gaps closed by sequencing of the PCR products. The level of sequence inaccuracy due to LA PCR errors appeared to be about 1 in 10,000, which does not affect significantly the final sequence quality. This two-step strategy is efficient and we suggest that it can be applied to sequencing of longer chromosomal regions. The 36 kb sequence contains 38 coding sequences (CDSs), 19 of which encode unknown proteins. Seven genetic loci already mapped in this region, xpt, metB, ilvA, ilvD, thyB, dfrA and degR were identified. Eleven CDSs were found to display significant similarities to known proteins from the data banks, suggesting possible functions for some of the novel genes: cspD may encode a cold shock protein; bcsA, the first bacterial homologue of chalcone synthase; exol, a 5' to 3' exonuclease, similar to that of DNA polymerase I of Escherichia coli; and bsaA, a stress-response-associated protein. The protein encoded by yplP has homology with the transcriptional NifA-like regulators. The arrangement of the genes relative to possible promoters and terminators suggests 19 potential transcription units. PMID:8969496

  5. Mapping strategies: Chromosome 16 workshop. Final technical report

    SciTech Connect

    Not Available

    1989-12-31

    The following topics from a workshop on chromosome 16 are briefly discussed: genetic map of chromosome 16; chromosome breakpoint map of chromosome 16; integrated physical/genetic map of chromosome 16; pulsed field map of the 16p13.2--p13.3 region (3 sheets); and a report of the HGM10 chromosome 16 committee.

  6. CRISPR-PCS: a powerful new approach to inducing multiple chromosome splitting in Saccharomyces cerevisiae.

    PubMed

    Sasano, Yu; Nagasawa, Koki; Kaboli, Saeed; Sugiyama, Minetaka; Harashima, Satoshi

    2016-01-01

    PCR-mediated chromosome splitting (PCS) was developed in the yeast Saccharomyces cerevisiae. It is based on homologous recombination and enables division of a chromosome at any point to form two derived and functional chromosomes. However, because of low homologous recombination activity, PCS is limited to a single site at a time, which makes the splitting of multiple loci laborious and time-consuming. Here we have developed a highly efficient and versatile chromosome engineering technology named CRISPR-PCS that integrates PCS with the novel genome editing CRISPR/Cas9 system. This integration allows PCS to utilize induced double strand breaks to activate homologous recombination. CRISPR-PCS enhances the efficiency of chromosome splitting approximately 200-fold and enables generation of simultaneous multiple chromosome splits. We propose that CRISPR-PCS will be a powerful tool for breeding novel yeast strains with desirable traits for specific industrial applications and for investigating genome function. PMID:27530680

  7. CRISPR-PCS: a powerful new approach to inducing multiple chromosome splitting in Saccharomyces cerevisiae

    PubMed Central

    Sasano, Yu; Nagasawa, Koki; Kaboli, Saeed; Sugiyama, Minetaka; Harashima, Satoshi

    2016-01-01

    PCR-mediated chromosome splitting (PCS) was developed in the yeast Saccharomyces cerevisiae. It is based on homologous recombination and enables division of a chromosome at any point to form two derived and functional chromosomes. However, because of low homologous recombination activity, PCS is limited to a single site at a time, which makes the splitting of multiple loci laborious and time-consuming. Here we have developed a highly efficient and versatile chromosome engineering technology named CRISPR-PCS that integrates PCS with the novel genome editing CRISPR/Cas9 system. This integration allows PCS to utilize induced double strand breaks to activate homologous recombination. CRISPR-PCS enhances the efficiency of chromosome splitting approximately 200-fold and enables generation of simultaneous multiple chromosome splits. We propose that CRISPR-PCS will be a powerful tool for breeding novel yeast strains with desirable traits for specific industrial applications and for investigating genome function. PMID:27530680

  8. Construction of Escherichia coli strains with chromosomally integrated expression cassettes for the synthesis of 2′-fucosyllactose

    PubMed Central

    2013-01-01

    Background The trisaccharide 2′-fucosyllactose (2′-FL) is one of the most abundant oligosaccharides found in human milk. Due to its prebiotic and anti-infective properties, 2′-FL is discussed as nutritional additive for infant formula. Besides chemical synthesis and extraction from human milk, 2′-FL can be produced enzymatically in vitro and in vivo. The most promising approach for a large-scale formation of 2′-FL is the whole cell biosynthesis in Escherichia coli by intracellular synthesis of GDP-L-fucose and subsequent fucosylation of lactose with an appropriate α1,2-fucosyltransferase. Even though whole cell approaches have been demonstrated for the synthesis of 2′-FL, further improvements of the engineered E. coli host are required to increase product yields. Furthermore, an antibiotic-free method of whole cell synthesis of 2′-FL is desirable to simplify product purification and to avoid traces of antibiotics in a product with nutritional purpose. Results Here we report the construction of the first selection marker-free E. coli strain that produces 2′-FL from lactose and glycerol. To construct this strain, recombinant genes of the de novo synthesis pathway for GDP-L-fucose as well as the gene for the H. pylori fucosyltransferase futC were integrated into the chromosome of E. coli JM109 by using the λ-Red recombineering technique. Strains carrying additional copies of the futC gene and/or the gene fkp (from Bacteroides fragilis) for an additional salvage pathway for GDP-L-fucose production were used and shown to further improve production of 2′-FL in shake flask experiments. An increase of the intracellular GDP-L-fucose concentration by expression of fkp gene as well as an additional copy of the futC gene lead to an enhanced formation of 2′-FL. Using an improved production strain, feasibility of large scale 2′-FL production was demonstrated in an antibiotic-free fed-batch fermentation (13 l) with a final 2′-FL concentration of 20.28

  9. An Integrated Chromosome Map of Microsatellite Markers and Inversion Breakpoints for an Asian Malaria Mosquito, Anopheles stephensi

    PubMed Central

    Kamali, Maryam; Sharakhova, Maria V.; Baricheva, Elina; Karagodin, Dmitrii; Tu, Zhijian

    2011-01-01

    Anopheles stephensi is one of the major vectors of malaria in the Middle East and Indo-Pakistan subcontinent. Understanding the population genetic structure of malaria mosquitoes is important for developing adequate and successful vector control strategies. Commonly used markers for inferring anopheline taxonomic and population status include microsatellites and chromosomal inversions. Knowledge about chromosomal locations of microsatellite markers with respect to polymorphic inversions could be useful for better understanding a genetic structure of natural populations. However, fragments with microsatellites used in population genetic studies are usually too short for successful labeling and hybridization with chromosomes. We designed new primers for amplification of microsatellite loci identified in the A. stephensi genome sequenced with next-generation technologies. Twelve microsatellites were mapped to polytene chromosomes from ovarian nurse cells of A. stephensi using fluorescent in situ hybridization. All microsatellites hybridized to unique locations on autosomes, and 7 of them localized to the largest arm 2R. Ten microsatellites were mapped inside the previously described polymorphic chromosomal inversions, including 4 loci located inside the widespread inversion 2Rb. We analyzed microsatellite-based population genetic data available for A. stephensi in light of our mapping results. This study demonstrates that the chromosomal position of microsatellites may affect estimates of population genetic parameters and highlights the importance of developing physical maps for nonmodel organisms. PMID:21810771

  10. vanG Element Insertions within a Conserved Chromosomal Site Conferring Vancomycin Resistance to Streptococcus agalactiae and Streptococcus anginosus

    PubMed Central

    Srinivasan, Velusamy; Metcalf, Benjamin J.; Knipe, Kristen M.; Ouattara, Mahamoudou; McGee, Lesley; Shewmaker, Patricia L.; Glennen, Anita; Nichols, Megin; Harris, Carol; Brimmage, Mary; Ostrowsky, Belinda; Park, Connie J.; Schrag, Stephanie J.; Frace, Michael A.; Sammons, Scott A.

    2014-01-01

    ABSTRACT Three vancomycin-resistant streptococcal strains carrying vanG elements (two invasive Streptococcus agalactiae isolates [GBS-NY and GBS-NM, both serotype II and multilocus sequence type 22] and one Streptococcus anginosus [Sa]) were examined. The 45,585-bp elements found within Sa and GBS-NY were nearly identical (together designated vanG-1) and shared near-identity over an ~15-kb overlap with a previously described vanG element from Enterococcus faecalis. Unexpectedly, vanG-1 shared much less homology with the 49,321-bp vanG-2 element from GBS-NM, with widely different levels (50% to 99%) of sequence identity shared among 44 related open reading frames. Immediately adjacent to both vanG-1 and vanG-2 were 44,670-bp and 44,680-bp integrative conjugative element (ICE)-like sequences, designated ICE-r, that were nearly identical in the two group B streptococcal (GBS) strains. The dual vanG and ICE-r elements from both GBS strains were inserted at the same position, between bases 1328 and 1329, within the identical RNA methyltransferase (rumA) genes. A GenBank search revealed that although most GBS strains contained insertions within this specific site, only sequence type 22 (ST22) GBS strains contained highly related ICE-r derivatives. The vanG-1 element in Sa was also inserted within this position corresponding to its rumA homolog adjacent to an ICE-r derivative. vanG-1 insertions were previously reported within the same relative position in the E. faecalis rumA homolog. An ICE-r sequence perfectly conserved with respect to its counterpart in GBS-NY was apparent within the same site of the rumA homolog of a Streptococcus dysgalactiae subsp. equisimilis strain. Additionally, homologous vanG-like elements within the conserved rumA target site were evident in Roseburia intestinalis. PMID:25053786

  11. Capturing Chromosome Conformation

    NASA Astrophysics Data System (ADS)

    Dekker, Job; Rippe, Karsten; Dekker, Martijn; Kleckner, Nancy

    2002-02-01

    We describe an approach to detect the frequency of interaction between any two genomic loci. Generation of a matrix of interaction frequencies between sites on the same or different chromosomes reveals their relative spatial disposition and provides information about the physical properties of the chromatin fiber. This methodology can be applied to the spatial organization of entire genomes in organisms from bacteria to human. Using the yeast Saccharomyces cerevisiae, we could confirm known qualitative features of chromosome organization within the nucleus and dynamic changes in that organization during meiosis. We also analyzed yeast chromosome III at the G1 stage of the cell cycle. We found that chromatin is highly flexible throughout. Furthermore, functionally distinct AT- and GC-rich domains were found to exhibit different conformations, and a population-average 3D model of chromosome III could be determined. Chromosome III emerges as a contorted ring.

  12. A triallelic genetic male sterility locus in Brassica napus: an integrative strategy for its physical mapping and possible local chromosome evolution around it

    PubMed Central

    Lu, Wei; Liu, Jun; Xin, Qiang; Wan, Lili; Hong, Dengfeng; Yang, Guangsheng

    2013-01-01

    Background and Aims Spontaneous male sterility is an advantageous trait for both constructing efficient pollination control systems and for understanding the developmental process of the male reproductive unit in many crops. A triallelic genetic male-sterile locus (BnMs5) has been identified in Brassica napus; however, its complicated genome structure has greatly hampered the isolation of this locus. The aim of this study was to physically map BnMs5 through an integrated map-based cloning strategy and analyse the local chromosomal evolution around BnMs5. Methods A large F2 population was used to integrate the existing genetic maps around BnMs5. A map-based cloning strategy in combination with comparative mapping among B. napus, Arabidopsis, Brassica rapa and Brassica oleracea was employed to facilitate the identification of a target bacterial artificial chromosome (BAC) clone covering the BnMs5 locus. The genomic sequences from the Brassica species were analysed to reveal the regional chromosomal evolution around BnMs5. Key Results BnMs5 was finally delimited to a 0·3-cM genetic fragment from an integrated local genetic map, and was anchored on the B. napus A8 chromosome. Screening of a B. napus BAC clone library and identification of the positive clones validated that JBnB034L06 was the target BAC clone. The closest flanking markers restrict BnMs5 to a 21-kb region on JBnB034L06 containing six predicted functional genes. Good collinearity relationship around BnMs5 between several Brassica species was observed, while violent chromosomal evolutionary events including insertions/deletions, duplications and single nucleotide mutations were also found to have extensively occurred during their divergence. Conclusions This work represents major progress towards the molecular cloning of BnMs5, as well as presenting a powerful, integrative method to mapping loci in plants with complex genomic architecture, such as the amphidiploid B. napus. PMID:23243189

  13. Prioritizing functional phosphorylation sites based on multiple feature integration

    PubMed Central

    Xiao, Qingyu; Miao, Benpeng; Bi, Jie; Wang, Zhen; Li, Yixue

    2016-01-01

    Protein phosphorylation is an important type of post-translational modification that is involved in a variety of biological activities. Most phosphorylation events occur on serine, threonine and tyrosine residues in eukaryotes. In recent years, many phosphorylation sites have been identified as a result of advances in mass-spectrometric techniques. However, a large percentage of phosphorylation sites may be non-functional. Systematically prioritizing functional sites from a large number of phosphorylation sites will be increasingly important for the study of their biological roles. This study focused on exploring the intrinsic features of functional phosphorylation sites to predict whether a phosphosite is likely to be functional. We found significant differences in the distribution of evolutionary conservation, kinase association, disorder score, and secondary structure between known functional and background phosphorylation datasets. We built four different types of classifiers based on the most representative features and found that their performances were similar. We also prioritized 213,837 human phosphorylation sites from a variety of phosphorylation databases, which will be helpful for subsequent functional studies. All predicted results are available for query and download on our website (Predict Functional Phosphosites, PFP, http://pfp.biosino.org/). PMID:27090940

  14. HIV integration site distributions in resting and activated CD4+ T cells infected in culture

    PubMed Central

    Brady, Troy; Agosto, Luis M.; Malani, Nirav; Berry, Charles C.; O'Doherty, Una; Bushman, Frederic

    2010-01-01

    Objective The goal of this study was to investigate whether the location of HIV integration differs in resting versus activated T cells, a feature that could contribute to the formation of latent viral reservoirs via effects on integration targeting. Design Primary resting or activated CD4+ T cells were infected with purified X4-tropic HIV in the presence and absence of nucleoside triphosphates and genomic locations of integrated provirus determined. Methods We sequenced and analyzed a total of 2661 HIV integration sites using linker-mediated PCR and 454 sequencing. Integration site data sets were then compared to each other and to computationally generated random distributions. Results HIV integration was favored in active transcription units in both cell types, but integration sites from activated cells were found more often in genomic regions that were dense in genes, dense in CpG islands, and enriched in G/C bases. Integration sites from activated cells were also more strongly correlated with histone methylation patterns associated with active genes. Conclusion These data indicate that integration site distributions show modest but significant differences between resting and activated CD4+ T cells, and that integration in resting cells occurs more often in regions that may be suboptimal for proviral gene expression. PMID:19550285

  15. Stella preserves maternal chromosome integrity by inhibiting 5hmC-induced γH2AX accumulation

    PubMed Central

    Nakatani, Tsunetoshi; Yamagata, Kazuo; Kimura, Tohru; Oda, Masaaki; Nakashima, Hiroyuki; Hori, Mayuko; Sekita, Yoichi; Arakawa, Tatsuhiko; Nakamura, Toshinobu; Nakano, Toru

    2015-01-01

    In the mouse zygote, Stella/PGC7 protects 5-methylcytosine (5mC) of the maternal genome from Tet3-mediated oxidation to 5-hydroxymethylcytosine (5hmC). Although ablation of Stella causes early embryonic lethality, the underlying molecular mechanisms remain unknown. In this study, we report impaired DNA replication and abnormal chromosome segregation (ACS) of maternal chromosomes in Stella-null embryos. In addition, phosphorylation of H2AX (γH2AX), which has been reported to inhibit DNA replication, accumulates in the maternal chromatin of Stella-null zygotes in a Tet3-dependent manner. Cell culture assays verified that ectopic appearance of 5hmC induces abnormal accumulation of γH2AX and subsequent growth retardation. Thus, Stella protects maternal chromosomes from aberrant epigenetic modifications to ensure early embryogenesis. PMID:25694116

  16. Site-specific in situ amplification of the integrated polyomavirus genome: a case for a context-specific over-replication model of gene amplification.

    PubMed

    Syu, L J; Fluck, M M

    1997-08-01

    The fate of the genome of the polyoma (Py) tumor virus following integration in the chromosomes of transformed rat FR3T3 cells was re-examined. The viral sequences were integrated at a single transformant-specific chromosomal site in each of 22 transformants tested. In situ amplification of the viral sequences was observed in 24 of 34 transformants analyzed. Large T antigen, the unique viral function involved in initiating DNA replication from the viral origin, was essential for the amplification process. There was an absolute requirement for a reiteration of viral sequences and the extent of the reiteration affected the degree of amplification. The reiteration may be important for homologous recombination-mediated resolution of in situ amplified sequences. Among 11 transformants harboring a 1 to 2 kb repeat, the degree of amplification was transformant-specific and varied over a wide range. At the high end of the spectrum, the genome copy number increased 1300-fold at steady state, while at the low end, amplification was below twofold. Some aspect of the host chromatin at the site integration that affected viral gene expression, also directly or indirectly modulated the amplification. Use of high-resolution electrophoresis for the analysis of the integrated amplified sequences revealed a recurring novel pattern, consisting of a ladder with numerous bands separated by a constant distance approximately the size of the Py genome. We suggest that this pattern was generated by conversion of the amplified viral genomes to head to tail linear arrays with cell to cell variations in the number of genome repeats at single, transformant-specific, chromosomal sites. In light of the known "out of schedule" firing of the Py origin, we propose an "onion skin" structure intermediate and present a homologous recombination model for the conversion from onion skins to linear arrays. The relevance of the in situ amplification of the Py genome to cellular gene amplification is

  17. Genome structure and primitive sex chromosome revealed in Populus

    SciTech Connect

    Tuskan, Gerald A; Yin, Tongming; Gunter, Lee E; Blaudez, D

    2008-01-01

    We constructed a comprehensive genetic map for Populus and ordered 332 Mb of sequence scaffolds along the 19 haploid chromosomes in order to compare chromosomal regions among diverse members of the genus. These efforts lead us to conclude that chromosome XIX in Populus is evolving into a sex chromosome. Consistent segregation distortion in favor of the sub-genera Tacamahaca alleles provided evidence of divergent selection among species, particularly at the proximal end of chromosome XIX. A large microsatellite marker (SSR) cluster was detected in the distorted region even though the genome-wide distribute SSR sites was uniform across the physical map. The differences between the genetic map and physical sequence data suggested recombination suppression was occurring in the distorted region. A gender-determination locus and an overabundance of NBS-LRR genes were also co-located to the distorted region and were put forth as the cause for divergent selection and recombination suppression. This hypothesis was verified by using fine-scale mapping of an integrated scaffold in the vicinity of the gender-determination locus. As such it appears that chromosome XIX in Populus is in the process of evolving from an autosome into a sex chromosome and that NBS-LRR genes may play important role in the chromosomal diversification process in Populus.

  18. VISPA: a computational pipeline for the identification and analysis of genomic vector integration sites.

    PubMed

    Calabria, Andrea; Leo, Simone; Benedicenti, Fabrizio; Cesana, Daniela; Spinozzi, Giulio; Orsini, Massimilano; Merella, Stefania; Stupka, Elia; Zanetti, Gianluigi; Montini, Eugenio

    2014-01-01

    The analysis of the genomic distribution of viral vector genomic integration sites is a key step in hematopoietic stem cell-based gene therapy applications, allowing to assess both the safety and the efficacy of the treatment and to study the basic aspects of hematopoiesis and stem cell biology. Identifying vector integration sites requires ad-hoc bioinformatics tools with stringent requirements in terms of computational efficiency, flexibility, and usability. We developed VISPA (Vector Integration Site Parallel Analysis), a pipeline for automated integration site identification and annotation based on a distributed environment with a simple Galaxy web interface. VISPA was successfully used for the bioinformatics analysis of the follow-up of two lentiviral vector-based hematopoietic stem-cell gene therapy clinical trials. Our pipeline provides a reliable and efficient tool to assess the safety and efficacy of integrating vectors in clinical settings. PMID:25342980

  19. Enhancing Students' Thinking Skills: Exploring Model Technology-Integration Sites.

    ERIC Educational Resources Information Center

    Moersch, Christopher

    1998-01-01

    Examines ways to integrate technology into social studies, science, mathematics, and language arts. Describes model elementary and middle-school classrooms in which technology is used to investigate the concept of property, study soil porosity and the water cycle, run a student store, and promote environmental activism. (PEN)

  20. INTEGRATING FUEL CELL TECHNOLOGY AND AFO/CAFO WASTE SITES

    EPA Science Inventory

    This project is an effort to evaluate animal feeding operation and confined animal feeding operation sites for energy-production on a state-wide level. The project will determine the potential energy and environmental benefits of methane utilization in hydrogen fuel cells in the...

  1. A high-resolution integrated physical, cytogenetic, and genetic map of human chromosome 11: distal p13 to proximal p15.1.

    PubMed

    Fantes, J A; Oghene, K; Boyle, S; Danes, S; Fletcher, J M; Bruford, E A; Williamson, K; Seawright, A; Schedl, A; Hanson, I

    1995-01-20

    We describe a detailed physical map of human chromosome 11, extending from the distal part of p13 through the entirety of p14 to proximal p15.1. The primary level of mapping is based on chromosome breakpoints that divide the region into 20 intervals. At higher resolution YACs cover approximately 12 Mb of the region, and in many places overlapping cosmids are ordered in contiguous arrays. The map incorporates 18 known genes, including precise localization of the GTF2H1 gene encoding the 62-kDa subunit of TFIIH. We have also localized four expressed sequences of unknown function. The physical map incorporates genetic markers that allow relationships between physical and genetic distance to be examined, and similarly includes markers from a radiation hybrid map of 11. The cytogenetic location of cosmids has been examined on high-resolution banded chromosomes by fluorescence in situ hybridization, and FLpter values have been determined. The map therefore fully integrates physical, genic, genetic, and cytogenetic information and should provide a robust framework for the rapid and accurate assignment of new markers at a high level of resolution in this region of 11p. PMID:7789978

  2. Human Chromosomal Translocations at CpG Sites and a Theoretical Basis for their Lineage and Stage Specificity

    PubMed Central

    Tsai, Albert G.; Lu, Haihui; Raghavan, Sathees C.; Muschen, Markus; Hsieh, Chih-Lin; Lieber, Michael R.

    2008-01-01

    SUMMARY We have assembled, annotated, and analyzed a database of over 1700 breakpoints from the most common chromosomal rearrangements in human leukemias and lymphomas. Using this database, we show that although the CpG dinucleotide constitutes only 1% of the human genome, it accounts for 40–70% of breakpoints at proB/pre-B stage translocation regions – specifically, those near the bcl-2, bcl-1, and E2A genes. We do not observe CpG hotspots in rearrangements involving lymphoid-myeloid progenitors, mature B cells, or T cells. The stage-specificity, lineage-specificity, CpG targeting, and unique breakpoint distributions at these cluster regions may be explained by a lesion-specific double-strand breakage mechanism involving the RAG complex acting at AID-deaminated methyl-CpGs. PMID:19070581

  3. Chromosomal Integration of the Extended-Spectrum β-Lactamase Gene blaCTX-M-15 in Salmonella enterica Serotype Concord Isolates from Internationally Adopted Children▿

    PubMed Central

    Fabre, Laëtitia; Delauné, Aurélia; Espié, Emmanuelle; Nygard, Karin; Pardos, Maria; Polomack, Lucette; Guesnier, Françoise; Galimand, Marc; Lassen, Jørgen; Weill, François-Xavier

    2009-01-01

    We report the emergence of Salmonella enterica isolates of serotype Concord (and its monophasic variant 6,7:l,v:-) producing the extended-spectrum β-lactamases (ESBLs) SHV-12 and CTX-M-15 in France and Norway between 2001 and 2006 (43 in France and 26 in Norway). The majority of these isolates were from adopted children from Ethiopia, most of whom were healthy carriers. Several symptomatic secondary cases were found in the adoptive families and health care facilities in France. Serotype Concord isolates collected before 2003 produced SHV-12 encoded on a 340-kb conjugative plasmid of replicon IncI1. Isolates collected after 2003 produced CTX-M-15. We detected two conjugative plasmids carrying blaCTX-M-15. One plasmid, approximately 300 kb in size, was positive for the IncHI2 replicon and the plasmid-mediated quinolone resistance gene qnrA1. The other plasmid, from one of the earliest CTX-M-15-producing isolates collected, was a fusion plasmid with IncY and IncA/C2 replicons and was 200 kb in size. However, we showed, using Southern hybridization of I-CeuI-digested chromosomal DNA and S1 nuclease analysis of plasmid DNA, that most isolates had a blaCTX-M-15 gene located on chromosomal DNA. Analysis of the flanking regions of the chromosomally located blaCTX-M-15 gene by cloning revealed an ISEcp1 truncated by an intact IS26 upstream from the blaCTX-M-15 gene and a truncated orf477 gene downstream from blaCTX-M-15. We found regions beyond the IS26 and the orf477 genes that were derived from IncA/C2 plasmids, suggesting the chromosomal integration of part of the blaCTX-M-15-carrying IncY and IncA/C2 fusion plasmid from early CTX-M-15-producing isolates. PMID:19273688

  4. Chromosome rearrangements and transposable elements.

    PubMed

    Lonnig, Wolf-Ekkehard; Saedler, Heinz

    2002-01-01

    There has been limited corroboration to date for McClintock's vision of gene regulation by transposable elements (TEs), although her proposition on the origin of species by TE-induced complex chromosome reorganizations in combination with gene mutations, i.e., the involvement of both factors in relatively sudden formations of species in many plant and animal genera, has been more promising. Moreover, resolution is in sight for several seemingly contradictory phenomena such as the endless reshuffling of chromosome structures and gene sequences versus synteny and the constancy of living fossils (or stasis in general). Recent wide-ranging investigations have confirmed and enlarged the number of earlier cases of TE target site selection (hot spots for TE integration), implying preestablished rather than accidental chromosome rearrangements for nonhomologous recombination of host DNA. The possibility of a partly predetermined generation of biodiversity and new species is discussed. The views of several leading transposon experts on the rather abrupt origin of new species have not been synthesized into the macroevolutionary theory of the punctuated equilibrium school of paleontology inferred from thoroughly consistent features of the fossil record. PMID:12429698

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

    PubMed Central

    Serrao, Erik; Engelman, Alan N.

    2016-01-01

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

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

    PubMed

    Serrao, Erik; Engelman, Alan N

    2016-01-01

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

  7. The Bacteroides mobilizable transposon Tn4555 integrates by a site-specific recombination mechanism similar to that of the gram-positive bacterial element Tn916.

    PubMed Central

    Tribble, G D; Parker, A C; Smith, C J

    1997-01-01

    The Bacteroides mobilizable transposon Tn4555 is a 12.2-kb molecule that encodes resistance to cefoxitin. Conjugal transposition is hypothesized to occur via a circular intermediate and is stimulated by coresident tetracycline resistance elements and low levels of tetracycline. In this work, the ends of the transposon were identified and found to consist of 12-bp imperfect inverted repeats, with an extra base at one end. In the circular form, the ends were separated by a 6-bp "coupling sequence" which was associated with either the left or the right transposon terminus when the transposon was inserted into the chromosome. Tn4555 does not duplicate its target site upon insertion. Using a conjugation-based transposition assay, we showed that the coupling sequence originated from 6 bases of genomic DNA flanking either side of the transposon prior to excision. Tn4555 preferentially transposed into a 589-bp genomic locus containing a 207-bp direct repeat. Integration occurred before or after the repeated sequence, with one integration site between the two repeats. These observations are consistent with a transposition model based on site-specific recombination. In the bacteriophage lambda model for site-specific recombination, the bacteriophage recombines with the Escherichia coli chromosome via a 7-bp "crossover" region. We propose that the coupling sequence of Tn4555 is analogous in function to the crossover region of lambda but that unlike the situation in lambda, recombination occurs between regions of nonhomologous DNA. This ability to recombine into divergent target sites is also a feature of the gram-positive bacterial transposon Tn916. PMID:9098073

  8. An integrated remote sensing approach for identifying ecological range sites. [parker mountain

    NASA Technical Reports Server (NTRS)

    Jaynes, R. A.

    1983-01-01

    A model approach for identifying ecological range sites was applied to high elevation sagebrush-dominated rangelands on Parker Mountain, in south-central Utah. The approach utilizes map information derived from both high altitude color infrared photography and LANDSAT digital data, integrated with soils, geological, and precipitation maps. Identification of the ecological range site for a given area requires an evaluation of all relevant environmental factors which combine to give that site the potential to produce characteristic types and amounts of vegetation. A table is presented which allows the user to determine ecological range site based upon an integrated use of the maps which were prepared. The advantages of identifying ecological range sites through an integrated photo interpretation/LANDSAT analysis are discussed.

  9. Complex reorganization and predominant non-homologous repair following chromosomal breakage in karyotypically balanced germline rearrangements and transgenic integration

    PubMed Central

    Chiang, Colby; Jacobsen, Jessie C.; Ernst, Carl; Hanscom, Carrie; Heilbut, Adrian; Blumenthal, Ian; Mills, Ryan E.; Kirby, Andrew; Lindgren, Amelia M.; Rudiger, Skye R.; McLaughlan, Clive J.; Bawden, C. Simon; Reid, Suzanne J.; Faull, Richard L. M.; Snell, Russell G.; Hall, Ira M.; Shen, Yiping; Ohsumi, Toshiro K.; Borowsky, Mark L.; Daly, Mark J.; Lee, Charles; Morton, Cynthia C.; MacDonald, Marcy E.; Gusella, James F.; Talkowski, Michael E.

    2012-01-01

    We defined the genetic landscape of balanced chromosomal rearrangements at nucleotide resolution by sequencing 141 breakpoints from cytogenetically-interpreted translocations and inversions. We confirm that the recently described phenomenon of “chromothripsis” (massive chromosomal shattering and reorganization) is not unique to cancer cells but also occurs in the germline where it can resolve to a karyotypically balanced state with frequent inversions. We detected a high incidence of complex rearrangements (19.2%) and substantially less reliance on microhomology (31%) than previously observed in benign CNVs. We compared these results to experimentally-generated DNA breakage-repair by sequencing seven transgenic animals, and revealed extensive rearrangement of the transgene and host genome with similar complexity to human germline alterations. Inversion is the most common rearrangement, suggesting that a combined mechanism involving template switching and non-homologous repair mediates the formation of balanced complex rearrangements that are viable, stably replicated and transmitted unaltered to subsequent generations. PMID:22388000

  10. Genomic characterization of viral integration sites in HPV-related cancers.

    PubMed

    Bodelon, Clara; Untereiner, Michael E; Machiela, Mitchell J; Vinokurova, Svetlana; Wentzensen, Nicolas

    2016-11-01

    Persistent infection with carcinogenic human papillomaviruses (HPV) causes the majority of anogenital cancers and a subset of head and neck cancers. The HPV genome is frequently found integrated into the host genome of invasive cancers. The mechanisms of how it may promote disease progression are not well understood. Thoroughly characterizing integration events can provide insights into HPV carcinogenesis. Individual studies have reported limited number of integration sites in cell lines and human samples. We performed a systematic review of published integration sites in HPV-related cancers and conducted a pooled analysis to formally test for integration hotspots and genomic features enriched in integration events using data from the Encyclopedia of DNA Elements (ENCODE). Over 1,500 integration sites were reported in the literature, of which 90.8% (N = 1,407) were in human tissues. We found 10 cytobands enriched for integration events, three previously reported ones (3q28, 8q24.21 and 13q22.1) and seven additional ones (2q22.3, 3p14.2, 8q24.22, 14q24.1, 17p11.1, 17q23.1 and 17q23.2). Cervical infections with HPV18 were more likely to have breakpoints in 8q24.21 (p = 7.68 × 10(-4) ) than those with HPV16. Overall, integration sites were more likely to be in gene regions than expected by chance (p = 6.93 × 10(-9) ). They were also significantly closer to CpG regions, fragile sites, transcriptionally active regions and enhancers. Few integration events occurred within 50 Kb of known cervical cancer driver genes. This suggests that HPV integrates in accessible regions of the genome, preferentially genes and enhancers, which may affect the expression of target genes. PMID:27343048

  11. INTEGRAL RCS : Launch Site Operations and In-orbit Behavior

    NASA Astrophysics Data System (ADS)

    Pessana, M.; Ravera, F.; Poidomani, G.; Gitins, M.; Lafranconi, R.

    2004-10-01

    The INTEGRAL (INTErnational Gamma-Ray Astrophysics Laboratory) Spacecraft was launched from Baikonur on October the 17th 2002. The Spacecraft Propulsion (RCS) is a monopropellant system, using anhydrous hydrazine and 20 N thrusters. It was loaded and pressurised at the Baikonur filling facilities prior the launch. The outcomes of this activity together with a correlation with the tanks loading procedure are given. During LEOP the behaviour of the RCS was recorded via the telemetry by the three Pressure Transducers, the TCS thermistors and by the thrusters thermocouples. The above parameters allow determining the proper status of the system and its functional behaviour. During the LEOP an anomalous temperature evolution was detected on a line spot downstream the tanks.

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

    PubMed

    Wei, Yunzhou; Terns, Michael P

    2016-06-16

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

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

    PubMed Central

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

    2013-01-01

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

  14. Escherichia coli integration host factor bends the DNA at the ends of IS1 and in an insertion hotspot with multiple IHF binding sites.

    PubMed Central

    Prentki, P; Chandler, M; Galas, D J

    1987-01-01

    The integration host factor of Escherichia coli (IHF) is a small, histone-like protein which participates in the integration of bacteriophage lambda into the E. coli chromosome and in a number of regulatory processes. Our recent footprinting analysis has shown that IHF binds specifically to the ends of the transposable element IS1, as well as to several sites within a short segment of the plasmid pBR322. We have extended our studies of the binding of the IHF molecule to these sites in vitro using a gel retardation assay. We report here that IHF bends the DNA upon binding, as judged from the strong cyclic dependence of the protein-induced mobility shift on the position of the binding site. Using cloned, synthetic ends of IS1 as substrates, we have found that some mutations within the conserved bases of the IHF consensus binding sequence abolish binding, and that alterations of the flanking sequences can greatly reduce IHF binding. The presence of multiple IHF sites on a single DNA fragment increases binding very little, indicating that IHF does not bind cooperatively in this complex. We discuss the possibility that DNA bending is related to the role IHF plays in forming and stabilizing nucleoprotein complexes, and suggest that bending at the IHF sites may be important to its diverse effects in the cell. Images Fig. 3. Fig. 4. Fig. 5. PMID:2822395

  15. Fragile X expression and X inactivation. I. The expression of the fragile site at Xq27.3 is not suppressed on inactive X chromosomes separated from the active homologue.

    PubMed

    Wöhrle, D; Fryns, J P; Steinbach, P

    1990-10-01

    The inactive fragile X chromosomes of a 47,fra(X),fra(X),Y male with a typical fragile X phenotype were successfully separated from the active homologues by means of somatic cell hybridization. It was shown by FUdR-induction and caffein-posttreatment that the separated inactive X chromosomes expressed their fragile sites and that the presence of an active mutated X chromosome was not a prerequisite for fragile X expression. The fragility seems to be an intrinsic property of the individual fragile site. This result is in favour of the classical concept that the fragile site at Xq27.3 has a primary pathogenetic function in this syndrome, although the fragility itself could represent a secondary phenomenon related to an unknown alteration of the DNA in this chromosome region. It is also concluded that inactivation of the fragile X chromosome in females is not responsible for either false negative fragile X findings or the observation of fragile X negative colonies isolated from fragile X positive fibroblasts in heterozygotes. PMID:2227957

  16. Characterization of the Hansenula polymorpha PUR7 gene and its use as selectable marker for targeted chromosomal integration.

    PubMed

    Haan, Gert Jan; van Dijk, Ralf; Kiel, Jan A K W; Veenhuis, Marten

    2002-03-01

    The Hansenula polymorpha genes encoding the putative functional homologs of the enzymes involved in the seventh and eighth step in purine biosynthesis, HpPUR7 and HpPUR8, were cloned and sequenced. An overexpression vector designated pHIPA4 was constructed, which contains the HpPUR7 gene as selectable marker and allows expression of genes of interest via the strong, inducible alcohol oxidase promoter. An ade11 auxotrophic mutant that is affected in the activity of the HpPUR7 gene product was used to construct strain NCYC495 ade11.1 leu1.1 ura3. This strain grew on methanol at wild-type rates (doubling time of approximately 4 h) and is suitable for independent introduction of four expression cassettes, each using one of the markers for selection, in addition to the zeocin resistance marker. It was subsequently used as a host for overproduction of two endogenous peroxisomal matrix proteins, amine oxidase and catalase. Efficient site-specific integration of pHIPA4 and overproduction of amine oxidase and catalase is demonstrated. The expression cassette appeared to be pre-eminently suited to mediate moderate protein production levels. The advantages of pHIPA4 and the new triple auxotrophic strain in relation to the use of H. polymorpha as a versatile cell factory or as a model organism for fundamental studies on the principles of peroxisome homeostasis is discussed. PMID:12702317

  17. Characterization of a chromosomally integrated luxCDABE marker for investigation of shiga toxin-producing Escherichia coli O91:H21 shedding in cattle

    NASA Astrophysics Data System (ADS)

    Hong, Yingying; Mathew, Alan G.

    2011-06-01

    Shiga toxin-producing Escherichia coli (STEC) O91:H21 has been recognized as a potential life-threatening foodborne pathogen and is commonly involved in human infections in European countries. Fecal shedding of the organism by cattle is considered to be the ultimate source for contaminations. Studies examining STEC shedding patterns often include inoculation of strains carrying antibiotic resistance makers for identifiable recovery. However, indigenous intestinal microflora exhibiting similar antibiotic resistance patterns can confound such studies. Such was the case in a study by our group when attempting to characterize shedding patterns of O91:H21 in calves. A chromosomally integrated bioluminescence marker using a luxCDABE cassette from Photorhabdus luminescens was developed in O91:H21 to overcome such shortcomings of antibiotic resistance markers during animal challenge experiment. The marker was validated in various aspects and was shown to have no impact on metabolic reactions, isotype virulence gene patterns, cost to growth, and additionally demonstrated high in vitro stability. Together, the results indicated that a chromosomally integrated luxCDABE based marker may be a superior system for the study of STEC colonization and shedding in cattle.

  18. A 4.5-megabase yeast artificial chromosome contig from human chromosome 13q14.3 ordering 9 polymorphic microsatellites (22 sequence-tagged sites) tightly linked to the Wilson disease locus.

    PubMed Central

    White, A; Tomfohrde, J; Stewart, E; Barnes, R; Le Paslier, D; Weissenbach, J; Cavalli-Sforza, L; Farrer, L; Bowcock, A

    1993-01-01

    We have previously performed a genetic analysis of multiply affected families to map a locus responsible for Wilson disease (WND) to a 0.3-centimorgan (cM) region within chromosome 13q14.3, between D13S31 and D13S59. Here we describe the construction of a contig of approximately 4.5 Mb, which spans this region and extends from D13S25 to D13S59. This contig consists of 28 genomic yeast artificial chromosome (YAC) clones. Five critical crossover events have been defined in this interval in two unaffected (Centre d'Etudes du Polymorphisme Humain) and three WND families. The combination of sequence tagged site content mapping of YACs with both polymorphic and nonpolymorphic markers and recombination breakpoint mapping resulted in the following order of polymorphic markers: centromere-RB1-D13S25-AFM205vh2-D13S31-D13S22 7-D13S228-AFM238vc3-D13S133- AFM084xc5-D13S137-D13S169, D13S155-D13S59-telomere. The recombination/physical distance ratio varies from approximately 3000 kb per cM in the region between D13S31 and D13S25 to 6000 kb per cM in the region between D13S31 and D13S59. Three WND families exhibiting recombination between the disease locus and D13S31 or D13S59 were genotyped for additional markers in this region and further refined the location of the WND gene to between D13S155 and D13S133. Nine of the markers in this region of < 1 cM are polymorphic microsatellites (seven have observed heterozygosities of 70% or above) that will be extremely useful in prenatal and preclinical diagnosis of this disease. This physical map is an essential step in the isolation of the WND gene and is a framework for the identification of candidate genes. PMID:8234264

  19. DNFSB Recommendation 94-1 Hanford site integrated stabilization management plan, volumes 1 and 2

    SciTech Connect

    Gerber, E.W.

    1996-03-15

    This document comprises the Hanford Site Integrated Stabilization Management Plan (SISMP). This document describes the DOE`s plans at the Hanford Site to address concerns identified in Defense Nuclear Facilites Safety Board (DNFSB) Recommendation 94-1. This document also identifies plans for other spent nuclear fuel (SNF) inventories at the Hanford Site which are not within the scope of DNFSB Recommendation 94-1 for reference purposes because of their interrelationship with plans for SNF within the scope of DNFSB Recommendation 94-1. The SISMP was also developed to assist DOE in initial formulation of the Research and Development Plan and the Integrated Facilities Plan.

  20. Common chromosomal fragile site FRA16D sequence: identification of the FOR gene spanning FRA16D and homozygous deletions and translocation breakpoints in cancer cells.

    PubMed

    Ried, K; Finnis, M; Hobson, L; Mangelsdorf, M; Dayan, S; Nancarrow, J K; Woollatt, E; Kremmidiotis, G; Gardner, A; Venter, D; Baker, E; Richards, R I

    2000-07-01

    Fluorescence in situ hybridization of a tile path of DNA subclones has previously enabled the cyto-genetic definition of the minimal DNA sequence which spans the FRA16D common chromosomal fragile site, located at 16q23.2. Homozygous deletion of the FRA16D locus has been reported in adenocarcinomas of stomach, colon, lung and ovary. We have sequenced the 270 kb containing the FRA16D fragile site and the minimal homozygously deleted region in tumour cells. This sequence enabled localization of some of the tumour cell breakpoints to regions which contain AT-rich secondary structures similar to those associated with the FRA10B and FRA16B rare fragile sites. The FRA16D DNA sequence also led to the identification of an alternatively spliced gene, named FOR (fragile site FRA16D oxidoreductase), exons of which span both the fragile site and the minimal region of homozygous deletion. In addition, the complete DNA sequence of the FRA16D-containing FOR intron reveals no evidence of additional authentic transcripts. Alternatively spliced FOR transcripts (FOR I, FOR II and FOR III) encode proteins which share N-terminal WW domains and differ at their C-terminus, with FOR III having a truncated oxidoreductase domain. FRA16D-associated deletions selectively affect the FOR gene transcripts. Three out of five previously mapped translocation breakpoints in multiple myeloma are also located within the FOR gene. FOR is therefore the principle genetic target for DNA instability at 16q23.2 and perturbation of FOR function is likely to contribute to the biological consequences of DNA instability at FRA16D in cancer cells. PMID:10861292

  1. Organics in soils and groundwater at non-arid sites (A-1) integrated demonstration

    SciTech Connect

    Steele, J.L.; Kaback, D.S.; Looney, B.B.

    1994-06-01

    One of the most common environmental problems in the United States is soils and groundwater contaminated with volatile chemical solvents classified as Volatile Organic Compounds (VOCs), which were used as degreasers and cleaning agents. Leakage of solvents (trichloroethylene and tetrachloroethylene) from an underground process sewer line has contaminated soils and underlying groundwaters at SRS. This site was chosen for DOE-OTD`s integrated demonstration program to demonstrate innovative technologies for cleanup of soils and groundwater contaminated with VOCs. The Savannah River Site was especially well suited as the test bed for this integrated demonstration project due to the presence of a pre-existing line source of soil and groundwater-based contamination, on-going environmental remediation efforts at the site, and full cooperation from the concerned environmental regulatory agencies. The Integrated Demonstration (ID) at the Savannah River Site has demonstrated systems of technologies and evaluated them with respect to performance, safety and cost effectiveness.

  2. Chromosomal Flexibility

    ERIC Educational Resources Information Center

    Journal of College Science Teaching, 2005

    2005-01-01

    Scientists have shown that a genetic element on one chromosome may direct gene activity on another. Howard Hughes Medical Institute (HHMI) researchers report that a multitasking master-control region appears to over-see both a set of its own genes and a related gene on a nearby chromosome. The findings reinforce the growing importance of location…

  3. Integration of geophysics within the Argonne expedited site characterization Program at a site in the southern High Plains

    SciTech Connect

    Hastings, B.; Hildebrandt, G.; Meyer, T.; Saunders, W.; Burton, J.C.

    1995-05-01

    An Argonne National Laboratory Expedited Site Characterization (ESC) program was carried out at a site in the central United States. The Argonne ESC process emphasizes an interdisciplinary approach in which all available information is integrated to produce as complete a picture as possible of the geologic and hydrologic controls on contaminant distribution and transport. As part of this process, all pertinent data that have been collected from previous investigations are thoroughly analyzed before a decision is made to collect additional information. A seismic reflection program recently concluded at the site had produced inconclusive results. Before we decided whether another acquisition program was warranted, we examined the existing data set to evaluate the quality of the raw data, the appropriateness of the processing sequence, and the integrity of the interpretation. We decided that the field data were of sufficient quality to warrant reprocessing and reinterpretation. The main thrust of the reprocessing effort was to enhance the continuity of a shallow, low-frequency reflection identified as a perching horizon within the Ogallala formation. The reinterpreted seismic data were used to locate the boundaries of the perched aquifer, which helped to guide the Argonne ESC drilling and sampling program. In addition, digitized geophysical well log data from previous drilling programs were reinterpreted and integrated into the geologic and hydrogeologic model.

  4. A Site-Specific Integrative Plasmid Found in Pseudomonas aeruginosa Clinical Isolate HS87 along with A Plasmid Carrying an Aminoglycoside-Resistant Gene.

    PubMed

    Bi, Dexi; Xie, Yingzhou; Tai, Cui; Jiang, Xiaofei; Zhang, Jie; Harrison, Ewan M; Jia, Shiru; Deng, Zixin; Rajakumar, Kumar; Ou, Hong-Yu

    2016-01-01

    Plasmids play critical roles in bacterial fitness and evolution of Pseudomonas aeruginosa. Here two plasmids found in a drug-resistant P. aeruginosa clinical isolate HS87 were completely sequenced. The pHS87b plasmid (11.2 kb) carries phage-related genes and function-unknown genes. Notably, pHS87b encodes an integrase and has an adjacent tRNAThr-associated attachment site. A corresponding integrated form of pHS87b at the tRNAThr locus was identified on the chromosome of P. aeruginosa, showing that pHS87b is able to site-specifically integrate into the 3'-end of the tRNAThr gene. The pHS87a plasmid (26.8 kb) displays a plastic structure containing a putative replication module, stability factors and a variable region. The RepA of pHS87a shows significant similarity to the replication proteins of pPT23A-family plasmids. pHS87a carries a transposon Tn6049, a truncated insertion sequence ΔIS1071 and a Tn402-like class 1 integron which contains an aacA4 cassette that may confer aminoglycoside resistance. Thus, pHS87b is a site-specific integrative plasmid whereas pHS87a is a plastic antibiotic resistance plasmid. The two native plasmids may promote the fitness and evolution of P. aeruginosa. PMID:26841043

  5. A Site-Specific Integrative Plasmid Found in Pseudomonas aeruginosa Clinical Isolate HS87 along with A Plasmid Carrying an Aminoglycoside-Resistant Gene

    PubMed Central

    Tai, Cui; Jiang, Xiaofei; Zhang, Jie; Harrison, Ewan M.; Jia, Shiru; Deng, Zixin; Rajakumar, Kumar; Ou, Hong-Yu

    2016-01-01

    Plasmids play critical roles in bacterial fitness and evolution of Pseudomonas aeruginosa. Here two plasmids found in a drug-resistant P. aeruginosa clinical isolate HS87 were completely sequenced. The pHS87b plasmid (11.2 kb) carries phage-related genes and function-unknown genes. Notably, pHS87b encodes an integrase and has an adjacent tRNAThr-associated attachment site. A corresponding integrated form of pHS87b at the tRNAThr locus was identified on the chromosome of P. aeruginosa, showing that pHS87b is able to site-specifically integrate into the 3’-end of the tRNAThr gene. The pHS87a plasmid (26.8 kb) displays a plastic structure containing a putative replication module, stability factors and a variable region. The RepA of pHS87a shows significant similarity to the replication proteins of pPT23A-family plasmids. pHS87a carries a transposon Tn6049, a truncated insertion sequence ΔIS1071 and a Tn402-like class 1 integron which contains an aacA4 cassette that may confer aminoglycoside resistance. Thus, pHS87b is a site-specific integrative plasmid whereas pHS87a is a plastic antibiotic resistance plasmid. The two native plasmids may promote the fitness and evolution of P. aeruginosa. PMID:26841043

  6. The genes encoding the peripheral cannabinoid receptor and alpha-L-fucosidase are located near a newly identified common virus integration site, Evi11.

    PubMed Central

    Valk, P J; Hol, S; Vankan, Y; Ihle, J N; Askew, D; Jenkins, N A; Gilbert, D J; Copeland, N G; de Both, N J; Löwenberg, B; Delwel, R

    1997-01-01

    A new common region of virus integration, Evi11, has been identified in two retrovirally induced murine myeloid leukemia cell lines, NFS107 and NFS78. By interspecific backcross analysis, it was shown that Evi11 is located at the distal end of mouse chromosome 4, in a region that shows homology with human 1p36. The genes encoding the peripheral cannabinoid receptor (Cnr2) and alpha-L-fucosidase (Fuca1) were identified near the integration site by using a novel exon trapping system. Cnr2 is suggested to be the target gene for viral interference in Evi11, since proviruses are integrated in the first intron of Cnr2 and retroviral integrations alter mRNA expression of Cnr2 in NFS107 and NFS78. In addition, proviral integrations were demonstrated within the 3' untranslated region of Cnr2 in five independent newly derived CasBrM-MuLV (mouse murine leukemia virus) tumors, CSL13, CSL14, CSL16, CSL27, and CSL97. The Cnr2 gene encodes a seven-transmembrane G-protein-coupled receptor which is normally expressed in hematopoietic tissues. Our data suggest that the peripheral cannabinoid receptor gene might be involved in leukemogenesis as a result of aberrant expression of Cnr2 due to retroviral integration in Evi11. PMID:9261404

  7. The characterisation of Abelson Helper Integration Site-1 in skeletal muscle and its links to the metabolic syndrome

    PubMed Central

    Prior, Matthew J.; Foletta, Victoria C.; Jowett, Jeremy B.; Segal, David H.; Carless, Melanie A.; Curran, Joanne E.; Dyer, Tom D.; Moses, Eric K.; McAinch, Andrew J.; Konstantopoulos, Nicky; Bozaoglu, Kiymet; Collier, Greg R.; Cameron-Smith, David; Blangero, John; Walder, Ken R.

    2011-01-01

    The human Abelson helper integration site-1 (AHI1) gene is associated with both neurological and haematological disorders; however, it is also located in a chromosomal region linked to metabolic syndrome phenotypes and was identified as a type 2 diabetes susceptibility gene from a genome-wide association study. To further define a possible role in type 2 diabetes development, AHI1 mRNA expression levels were investigated in a range of tissues and found to be highly expressed in skeletal muscle as well as displaying elevated levels in brain regions and gonad tissues. Further analysis in a rodent, polygenic animal model of obesity and type 2 diabetes identified increased Ahi-1 mRNA levels in red gastrocnemius muscle from fasted impaired glucose tolerant and diabetic rodents compared with normal animals (p<0.002). Moreover, elevated gene expression levels were confirmed in skeletal muscle from fasted obese and type 2 diabetic human subjects (p≤0.02). RNAi-mediated suppression of Ahi-1 resulted in increased glucose transport in rat L6 myotubes in both the basal and insulin-stimulated states (p<0.01). Finally, SNP association studies identified two novel AHI1 genetic variants linked with fasting blood glucose levels in Mexican American subjects (p<0.037). These findings indicate a novel role for AHI1 in skeletal muscle and identify additional genetic links with metabolic syndrome phenotypes suggesting an involvement of AHI1 in the maintenance of glucose homeostasis and type 2 diabetes progression. PMID:20045148

  8. DNFSB recommendation 94-1 Hanford site integrated stabilization management plan - VOLUMES 1-3

    SciTech Connect

    Gerber, E.W.

    1996-09-23

    The US Department of Energy (DOE) has developed an Integrated Program Plan (IPP) to address concerns identified in Defense Nuclear Facilities Safety Board (DNFSB) Recommendation 94-1. The IPP describes the actions that DOE plans to implement at its various sites to convert excess fissile materials to forms or conditions suitable for safe interim storage. The baseline IPP was issued as DOE's DNFSB Recommendation 94-1 Implementation Plan (IP), which was transmitted to the DNFSB on February 28, 1995. The IPP was subsequently supplemented with an Integrated Facilities Plan and a Research and Development Plan, which further develop complex-wide research and development and long-range facility requirements and plans. These additions to the baseline IPP were developed based on a systems engineering approach that integrated facilities and capabilities at the various DOE sites and focused on attaining safe interim storage with minimum safety risks and environmental impacts. Each affected DOE site has developed a Site Integrated Stabilization Management Plan (SISMP) to identify individual site plans to implement the DNFSB Recommendation 94-1 IPP. The SISMPs were developed based on the objectives, requirements, and commitments identified in the DNFSB Recommendation 94-1 IP. The SISMPs supported formulation of the initial versions of the Integrated Facilities Plan and the Research and Development Plan. The SISMPs are periodically updated to reflect improved integration between DOE sites as identified during the IPP systems engineering evaluations. This document constitutes the Hanford SISMP. This document includes the planned work scope, costs and schedules for activities at the Hanford site to implement the DNFSB Recommendation 94-1 IPP.

  9. Methylation of HpaII and HhaI sites near the polymorphic CAG repeat in the human androgen-receptor gene correlates with X chromosome inactivation

    SciTech Connect

    Allen, R.C.; Zoghbi, H.Y.; Moseley, A.B.; Rosenblatt, H.M.; Belmont, J.W. )

    1992-12-01

    The human androgen-receptor gene (HUMARA; GenBank) contains a highly polymorphic trinucleotide repeat in the first exon. The authors have found that the methylation of HpaII and HhaI sites less than 100 pb away from this polymorphic short tandem repeat (STR) correlates with X inactivation. The close proximity of the restriction-enzyme sites to the STR allows the development of a PCR assay that distinguishes between the maternal and paternal alleles and identifies their methylation status. The accuracy of this assay was tested on (a) DNA from hamster/human hybrid cell lines containing either an active or inactive human X chromosome; (b) DNA from normal males and females; and (c) DNA from females showing nonrandom patterns of X inactivation. Data obtained using this assay correlated substantially with those obtained using the PGK, HPRT, and M27[beta] probes, which detect X inactivation patterns by Southern blot analysis. In order to demonstrate one application of this assay, the authors examined X inactivation patterns in the B lymphocytes of potential and obligate carriers of X-linked agammaglobulinemia. 42 refs., 5 figs., 1 tab.

  10. Rapid and efficient introduction of a foreign gene into bacterial artificial chromosome-cloned varicella vaccine by Tn7-mediated site-specific transposition

    SciTech Connect

    Somboonthum, Pranee; Koshizuka, Tetsuo; Okamoto, Shigefumi; Matsuura, Masaaki; Gomi, Yasuyuki; Takahashi, Michiaki; Yamanishi, Koichi; Mori, Yasuko

    2010-06-20

    Using a rapid and reliable system based on Tn7-mediated site-specific transposition, we have successfully constructed a recombinant Oka varicella vaccine (vOka) expressing the mumps virus (MuV) fusion protein (F). The backbone of the vector was our previously reported vOka-BAC (bacterial artificial chromosome) genome. We inserted the transposon Tn7 attachment sequence, LacZ{alpha}-mini-attTn7, into the region between ORF12 and ORF13 to generate a vOka-BAC-Tn genome. The MuV-F expressing cassette was transposed into the vOka-BAC genome at the mini-attTn7 transposition site. MuV-F protein was expressed in recombinant virus, rvOka-F infected cells. In addition, the MuV-F protein was cleaved in the rvOka-F infected cells as in MuV-infected cells. The growth of rvOka-F was similar to that of the original recombinant vOka without the F gene. Thus, we show that Tn7-mediated transposition is an efficient method for introducing a foreign gene expression cassette into the vOka-BAC genome as a live virus vector.

  11. DNFSB Recommendation 94-1 Hanford Site Integrated Stabilization Management Plan. Volume 1

    SciTech Connect

    Gerber, E.W.

    1995-10-01

    The US Department of Energy (DOE) has developed an Integrated Program Plan (IPP) to address concerns identified in Defense Nuclear Facilities Safety Board Recommendation 94-1. The IPP describes the actions that DOE plans to implement at its various sites to convert excess fissile materials to forms or conditions suitable for safe interim storage. The baseline IPP was issued as DOE`s Defense Nuclear Facilities Safety Board (DNFSB) Recommendation 94-1 Implementation Plan (IP), which was transmitted to the DNFSB on February 28, 1995. The IPP is being further developed to include complex-wide requirements for research and development and a long-range facility requirements section. The planned additions to the baseline IPP are being developed based on a systems engineering approach that integrates facilities and capabilities at the various DOE sites and focuses on attaining safe interim storage with minimum safety risks and environmental impacts. Each affected DOE site has developed a Site Integrated Stabilization Management Plan (SISMP) to identify individual site plans to implement the DNFSB Recommendation 94-1 and to provide a basis for formulating planned additions to the IPP. The SISMPs were developed based on the objectives, requirements, and commitments identified in the baseline DNFSB Recommendation 94-1 IPP. The SISMPs will be periodically updated to reflect improved integration between DOE sites as identified during the IPP systems engineering evaluations.

  12. Common Viral Integration Sites Identified in Avian Leukosis Virus-Induced B-Cell Lymphomas

    PubMed Central

    Justice, James F.; Morgan, Robin W.

    2015-01-01

    ABSTRACT Avian leukosis virus (ALV) induces B-cell lymphoma and other neoplasms in chickens by integrating within or near cancer genes and perturbing their expression. Four genes—MYC, MYB, Mir-155, and TERT—have previously been identified as common integration sites in these virus-induced lymphomas and are thought to play a causal role in tumorigenesis. In this study, we employ high-throughput sequencing to identify additional genes driving tumorigenesis in ALV-induced B-cell lymphomas. In addition to the four genes implicated previously, we identify other genes as common integration sites, including TNFRSF1A, MEF2C, CTDSPL, TAB2, RUNX1, MLL5, CXorf57, and BACH2. We also analyze the genome-wide ALV integration landscape in vivo and find increased frequency of ALV integration near transcriptional start sites and within transcripts. Previous work has shown ALV prefers a weak consensus sequence for integration in cultured human cells. We confirm this consensus sequence for ALV integration in vivo in the chicken genome. PMID:26670384

  13. Chromosomal Integration of tcb Chlorocatechol Degradation Pathway Genes as a Means of Expanding the Growth Substrate Range of Bacteria To Include Haloaromatics

    PubMed Central

    Klemba, Michael; Jakobs, Barbara; Wittich, Rolf-Michael; Pieper, Dietmar

    2000-01-01

    The tcbR-tcbCDEF gene cluster, coding for the chlorocatechol ortho-cleavage pathway in Pseudomonas sp. strain P51, has been cloned into a Tn5-based minitransposon. The minitransposon carrying the tcb gene cluster and a kanamycin resistance gene was transferred to Pseudomonas putida KT2442, and chromosomal integration was monitored by selection either for growth on 3-chlorobenzoate or for kanamycin resistance. Transconjugants able to utilize 3-chlorobenzoate as a sole carbon source were obtained, although at a >100-fold lower frequency than kanamycin-resistant transconjugants. The vast majority of kanamycin-resistant transconjugants were not capable of growth on 3-chlorobenzoate. Southern blot analysis revealed that many transconjugants selected directly on 3-chlorobenzoate contained multiple chromosomal copies of the tcb gene cluster, whereas those selected for kanamycin resistance possessed a single copy. Subsequent selection of kanamycin resistance-selected single-copy transconjugants for growth on 3-chlorobenzoate yielded colonies capable of utilizing this carbon source, but no amplification of the tcb gene cluster was apparent. Introduction of two copies of the tcb gene cluster without prior 3-chlorobenzoate selection resulted in transconjugants able to grow on this carbon source. Expression of the tcb chlorocatechol catabolic operon in P. putida thus represents a useful model system for analysis of the relationship among gene dosage, enzyme expression level, and growth on chloroaromatic substrates. PMID:10919778

  14. AGU Launches Web Site for New Scientific Integrity and Professional Ethics Policy

    NASA Astrophysics Data System (ADS)

    Townsend, Randy

    2013-03-01

    AGU's Scientific Integrity and Professional Ethics policy, approved by the AGU Board of Directors and Council in December 2012, is now available online on a new Web site, http://ethics.agu.org. As the Web site states, the policy embodies a "set of guidelines for scientific integrity and professional ethics for the actions of the members and the governance of the Union in its internal activities; in its public persona; and most importantly, in the research and peer review processes of its scientific publications, its communications and outreach, and its scientific meetings."

  15. Archaeological site protection: An integral component of the Exxon Valdez shoreline cleanup

    SciTech Connect

    Wooley, C.B.; Haggarty, J.C.

    1995-12-31

    A major cultural site identification and protection program in Prince William Sound and the Gulf of Alaska was conducted as part of the Exxon Valdez spill response. In cooperation with state and federal agencies and Native corporations with historic preservation mandates, the four-year program was designed to identify archaeological sites in the area of the spill, determine the effect of planned cleanup on them, and mitigate impacts to sites during cleanup. Archaeological site protection constraints, augmented by an extensive cultural resource training program, were an integral part of each shoreline-specific cleanup plan. As a result, impacts attributable to the cleanup were limited to minor disturbances and two vandalism incidents. Impacts from oiling were minimal largely because most intertidal cultural sites had lost their fragile constituents and contextual integrity as a result of prespill erosion. State and federal studies confirmed the efficacy of the site identification and protection program, finding negligible impacts attributable to either direct oiling or the cleanup at intact sites. The Cultural Resource Program also developed innovative management strategies with implications for future emergency responses involving complex land management and site protection issues. The program greatly enhanced the knowledge of the area`s history by collecting and synthesizing considerable new archaeological information. 27 refs., 5 figs., 1 tab.

  16. Demonstration of innovative monitoring technologies at the Savannah River Integrated Demonstration Site

    SciTech Connect

    Rossabi, J.; Jenkins, R.A.; Wise, M.B.

    1993-12-31

    The Department of Energy`s Office of Technology Development initiated an Integrated Demonstration Program at the Savannah River Site in 1989. The objective of this program is to develop, demonstrate, and evaluate innovative technologies that can improve present-day environmental restoration methods. The Integrated Demonstration Program at SRS is entitled ``Cleanup of Organics in Soils and Groundwater at Non-Arid Sites.`` New technologies in the areas of drilling, characterization, monitoring, and remediation are being demonstrated and evaluated for their technical performance and cost effectiveness in comparison with baseline technologies. Present site characterization and monitoring methods are costly, time-consuming, overly invasive, and often imprecise. Better technologies are required to accurately describe the subsurface geophysical and geochemical features of a site and the nature and extent of contamination. More efficient, nonintrusive characterization and monitoring techniques are necessary for understanding and predicting subsurface transport. More reliable procedures are also needed for interpreting monitoring and characterization data. Site characterization and monitoring are key elements in preventing, identifying, and restoring contaminated sites. The remediation of a site cannot be determined without characterization data, and monitoring may be required for 30 years after site closure.

  17. Retroviral vector performance in defined chromosomal Loci of modular packaging cell lines.

    PubMed

    Gama-Norton, L; Herrmann, S; Schucht, R; Coroadinha, A S; Löw, R; Alves, P M; Bartholomae, C C; Schmidt, M; Baum, C; Schambach, A; Hauser, H; Wirth, D

    2010-08-01

    The improvement of safety and titer of retroviral vectors produced in standard retroviral packaging cell lines is hampered because production relies on uncontrollable vector integration events. The influences of chromosomal surroundings make it difficult to dissect the performance of a specific vector from the chromosomal surroundings of the respective integration site. Taking advantage of a technology that relies on the use of packaging cell lines with predefined integration sites, we have systematically evaluated the performance of several retroviral vectors. In two previously established modular packaging cell lines (Flp293A and 293 FLEX) with single, defined chromosomal integration sites, retroviral vectors were integrated by means of Flp-mediated site-specific recombination. Vectors that are distinguished by different long terminal repeat promoters were introduced in either the sense or reverse orientation. The results show that the promoter, viral vector orientation, and integration site are the main determinants of the titer. Furthermore, we exploited the viral production systems to evaluate read-through activity. Read-through is thought to be caused by inefficient termination of vector transcription and is inherent to the nature of retroviral vectors. We assessed the frequency of transduction of sequences flanking the retroviral vectors from both integration sites. The approach presented here provides a platform for systematic design and evaluation of the efficiency and safety of retroviral vectors optimized for a given producer cell line. PMID:20222806

  18. Detection of Human Herpesvirus 6B (HHV-6B) Reactivation in Hematopoietic Cell Transplant Recipients with Inherited Chromosomally Integrated HHV-6A by Droplet Digital PCR.

    PubMed

    Sedlak, Ruth Hall; Hill, Joshua A; Nguyen, Thuy; Cho, Michelle; Levin, Greg; Cook, Linda; Huang, Meei-Li; Flamand, Louis; Zerr, Danielle M; Boeckh, Michael; Jerome, Keith R

    2016-05-01

    The presence of inherited chromosomally integrated human herpesvirus 6 (ciHHV-6) in hematopoietic cell transplant (HCT) donors or recipients confounds molecular testing for HHV-6 reactivation, which occurs in 30 to 50% of transplants. Here we describe a multiplex droplet digital PCR clinical diagnostic assay that concurrently distinguishes between HHV-6 species (A or B) and identifies inherited ciHHV-6. By applying this assay to recipient post-HCT plasma and serum samples, we demonstrated reactivation of HHV-6B in 25% (4/16 recipients) of HCT recipients with donor- or recipient-derived inherited ciHHV-6A, underscoring the need for diagnostic testing for HHV-6 infection even in the presence of ciHHV-6. PMID:26888901

  19. Individuals with inherited chromosomally integrated human herpes virus 6 (ciHHV-6) have functionally active HHV-6 specific T-cell immunity.

    PubMed

    Strenger, V; Kayser, S; Witte, K-E; Lassner, D; Schwinger, W; Jahn, G; Urban, C; Feuchtinger, T

    2016-02-01

    To evaluate the human herpes virus 6 (HHV-6) -specific immune response in individuals with chromosomally integrated HHV-6 (ciHHV-6), we measured HHV-6-antigen-specific cytokine responses (interferon-γ, interleukin-2, tumour necrosis factor-α) in T cells by flow cytometry in 12 and 16 individuals with and without ciHHV-6, respectively. All individuals with ciHHV-6 showed HHV-6-specific T cells with higher frequencies of HHV-6-specific CD8(+) cells (0.03-14.93, median 2.15% of CD8(+) cells) compared with non-ciHHV-6 (0.0-10.67, median 0.36%, p 0.026). The observed increased HHV-6-specific functionally active responses in individuals with ciHHV-6 clearly disprove speculations on immune tolerance in ciHHV-6 and indicate clinical and immunological implications of ciHHV-6. PMID:26482270

  20. Chromosome Abnormalities

    MedlinePlus

    ... decade, newer techniques have been developed that allow scientists and doctors to screen for chromosomal abnormalities without using a microscope. These newer methods compare the patient's DNA to a normal DNA ...

  1. Using the Choquet integral for screening geological CO2 storage sites

    SciTech Connect

    Zhang, Y.

    2011-03-01

    For geological CO{sub 2} storage site selection, it is desirable to reduce the number of candidate sites through a screening process before detailed site characterization is performed. Screening generally involves defining a number of criteria which then need to be evaluated for each site. The importance of each criterion to the final evaluation will generally be different. Weights reflecting the relative importance of these criteria can be provided by experts. To evaluate a site, each criterion must be evaluated and scored, and then aggregated, taking into account the importance of the criteria. We propose the use of the Choquet integral for aggregating the scores. The Choquet integral considers the interactions among criteria, i.e. whether they are independent, complementary to each other, or partially repetitive. We also evaluate the Shapley index, which demonstrates how the importance of a given piece of information may change if it is considered by itself or together with other available information. An illustrative example demonstrates how the Choquet integral properly accounts for the presence of redundancy in two site-evaluation criteria, making the screening process more defensible than the standard weighted-average approach.

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

    PubMed

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

    2016-01-01

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

  3. Schizophrenia: A genome scan targets chromosomes 3p and 8p as potential sites of susceptibility genes

    SciTech Connect

    Pulver, A.E.; Lasseter, V.K.; Kasch, L.

    1995-06-19

    Using a systematically ascertained sample of 57 families, each having 2 or more members with a consensus diagnosis of schizophrenia (DSM-III-R criteria), we have carried out linkage studies of 520 loci, covering approximately 70% of the genome for susceptibility loci for schizophrenia. A two-stage strategy based on lod score thresholds from simulation studies of our sample identified regions for further exploration. In each region, a dense map of highly informative dinucleotide repeat polymorphisms (heterozygosity greater than .70) was analyzed using dominant, recessive, and {open_quotes}affected only{close_quotes} models and nonparametric sib pair identity-by-descent methods. For one region, 8p22-p21, affected sib-pair analyses gave a P value = .0001, corresponding to a lod score approximately equal to 3.00. For 8p22-p21, the maximum two-point lod score occurred using the {open_quotes}affected only{close_quotes} recessive model (Z{sub max} = 2.35; {theta}{sub M} = {theta}{sub F}); allowing for a constant sex difference in recombination fractions found in reference pedigrees, Z{sub max} = 2.78 ({theta}{sub M}/{theta}{sub F} = 3). For a second region, 3p26-p24, the maximum two-point lod score was 2.34 ({open_quotes}affected only{close_quotes} dominant model), and the affected sib-pair P value was .01. These two regions are worthy of further exploration as potential sites of susceptibility genes for schizophrenia. 59 refs., 2 figs., 4 tabs.

  4. Technology development for phosphoric acid fuel cell powerplant (phase 2). [on site integrated energy systems

    NASA Technical Reports Server (NTRS)

    Christner, L.

    1980-01-01

    Progress is reported in the development of material, cell components, and reformers for on site integrated energy systems. Internal resistance and contact resistance were improved. Dissolved gases (O2, N2, and CO2) were found to have no effect on the electrochemical corrosion of phenolic composites. Stack performance was increased by 100 mV over the average 1979 level.

  5. Multiethnic Neighbourhoods as Sites of Social Capital Formation: Examining Social to Political "Integration" in Schools

    ERIC Educational Resources Information Center

    Basu, Ranu

    2006-01-01

    In an "ideal" democratic society, publicly funded schools serve many purposes. Aside from its educational mandate, schools are places for neighbourhood integration, social capital formation and the fostering of civil society. For newly arrived immigrants, especially those with young children, schools are important sites of settlement experiences.…

  6. INTEGRATION OF BUILDING AND ENERGY TECHNOLOGY WITH ON-SITE WASTE MANAGEMENT IN THE YEAR 2000

    EPA Science Inventory

    During this study the potential feasibility of integrating waste management, water supply and on-site energy generation was examined with the objective of improving the objective of improving the overall resource efficiency of the typical residential unit. This report can be used...

  7. Integrated decision support, sensor networks and adaptive control for wireless site-specific sprinkler irrigation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The development of site-specific sprinkler irrigation water management systems will be a major factor in future efforts to improve the various efficiencies of water-use and to support a sustainable irrigated environment. The challenge is to develop fully integrated management systems with supporting...

  8. Integrated Decision Support, Sensor Networks and Adaptive Control for Wireless Site-specific Sprinkler Irrigation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The development of site-specific sprinkler irrigation water management systems will be a major factor in future efforts to improve the various efficiencies of water-use and to support a sustainable irrigated environment. The challenge is to develop fully integrated management systems with supporting...

  9. TECHNOLOGY INTEGRATION FOR CONTAMINATED SITE REMEDIATION: CLEANUP GOALS AND PERFORMANCE CRITERIA

    EPA Science Inventory

    There is a need to develop and field-test integrated remediation technologies that operate in a synergistic manner for cost-effective treatment of contaminated sites to achieve risk-based and rational endpoints. Aggressive technologies designed for rapid source-zone remediation m...

  10. A novel integral membrane protein is differentially expressed in the chick growth plate and maps to chromosome 1.

    PubMed

    Lester, D H; Farquharson, C; Paton, I R; Seawright, E; Jefferies, D; Whitehead, C C; Burt, D W; Houston, B

    1999-08-01

    The growth plate is a specialised region of cartilage located at the growing ends of long bones in higher vertebrates. It is responsible for longitudinal bone growth and is under the control of many local and systemic factors. The growth plate consists of an orderly arrangement of small proliferative and larger mature hypertrophic chondrocytes. This paper describes the isolation by differential display of a 988-bp cDNA fragment derived from a transcript that is more highly expressed in proliferating rather than hypertrophic chondrocytes of the chick growth plate. Using 3' RACE, a further 939 bp of cDNA sequence was obtained. The 1.9 kb sequence contains a 924-bp open reading frame encoding an unknown 308 amino acid protein. This protein has a putative transmembrane domain near its N-terminus and three dileucine motifs at its carboxy tail. This gene was expressed in all other tissues examined. A polymorphism was identified by SSCP analysis and the gene was mapped to the centromeric region of the short arm of chicken chromosome 1, close to the locus for autosomal dwarfism. PMID:10467705

  11. The chromosome periphery during mitosis.

    PubMed

    Hernandez-Verdun, D; Gautier, T

    1994-03-01

    A complex structure, visible by electron microscopy, surrounds each chromosome during mitosis. The organization of this structure is distinct from that of the chromosomes and the cytoplasm. It forms a perichromosomal layer that can be isolated together with the chromosomes. This layer covers the chromosomes except in centromeric regions. The perichromosomal layer includes nuclear and nucleolar proteins as well as ribonucleoproteins (RNPs). The list of proteins and RNAs identified includes nuclear matrix proteins (perichromin, peripherin), nucleolar proteins (perichro-monucleolin, Ki-67 antigen, B23 protein, fibrillarin, p103, p52), ribosomal proteins (S1) and snRNAs (U3 RNAs). Only limited information is available about how and when the perichromosomal layer is formed. During early prophase, the proteins extend from the nucleoli towards the periphery of the nucleus. Thin cordon-like structures reach the nuclear envelope delimiting areas in which chromosomes condense. At telophase, the proteins are associated with the part of the chromosomes remaining condensed and accumulate in newly formed nucleoli in regions where chromatin is already decondensed. The perichromosomal layer contains several different classes of proteins and RNPs and it has been attributed various roles: (1) in chromosome organization, (2) as a barrier around the chromosomes, (3) involvement in compartmentation of the cells in prophase and telophase and (4) a binding site for chromosomal passenger proteins necessary to the early process of nuclear assembly. PMID:8166671

  12. An Assay to Detect In Vivo Y Chromosome Loss in Drosophila Wing Disc Cells

    PubMed Central

    Szabad, Janos; Bellen, Hugo J.; Venken, Koen J. T.

    2012-01-01

    Loss of the Y chromosome in Drosophila has no impact on cell viability and therefore allows us to assay the impact of environmental agents and genetic alterations on chromosomal loss. To detect in vivo chromosome loss in cells of the developing Drosophila wing primordia, we first engineered a Y chromosome with an attP docking site. By making use of the ΦC31 integrase system, we site-specifically integrated a genomic transgene encompassing the multiple wing hair (mwh) locus into this attP site, leading to a mwh+Y chromosome. This chromosome fully rescues the mwh mutant phenotype, an excellent recessive wing cell marker mutation. Loss of this mwh+Y chromosome in wing primordial cells then leads to manifestation of the mwh mutant phenotype in mwh-homozygous cells. The forming mwh clones permit us to quantify the effect of agents and genetic alterations by assaying frequency and size of the mwh mosaic spots. To illustrate the use of the mwh+Y loss system, the effects of four known mutagens (X-rays, colchicine, ethyl methanesulfonate, and formaldehyde) and two genetic conditions (loss- and gain-of-function lodestar mutant alleles) are documented. The procedure is simple, sensitive, and inexpensive. PMID:22973547

  13. DNFSB Recommendation 94-1 Hanford Site Integrated Stabilization Management Plan. Volume 2

    SciTech Connect

    McCormack, R.L.

    1995-08-01

    The Hanford Site Integrated Stabilization Management Plan (SISMP) is being developed in support of the US Department of Energy`s (DOE) Defense Nuclear Facilities Safety Board (DNFSB) Recommendation 94-1 Integrated Program Plan (IPP). Volume 1 of the SISMP identifies the technical scope and costs associated with Hanford Site plans to resolve concerns identified in DNFSB Recommendation 94-1. Volume 2 of the SISMP provides the Resource Loaded Integrated Schedules for Spent Nuclear Fuel Project and Plutonium Finishing Plant activities identified in Volume 1 of the SISMP. Appendix A provides the schedules and progress curves related to spent nuclear fuel management. Appendix B provides the schedules and progress curves related to plutoniumbearing material management. Appendix C provides programmatic logic diagrams that were referenced in Volume 1 of the SISMP.

  14. Heat recovery subsystem and overall system integration of fuel cell on-site integrated energy systems

    NASA Technical Reports Server (NTRS)

    Mougin, L. J.

    1983-01-01

    The best HVAC (heating, ventilating and air conditioning) subsystem to interface with the Engelhard fuel cell system for application in commercial buildings was determined. To accomplish this objective, the effects of several system and site specific parameters on the economic feasibility of fuel cell/HVAC systems were investigated. An energy flow diagram of a fuel cell/HVAC system is shown. The fuel cell system provides electricity for an electric water chiller and for domestic electric needs. Supplemental electricity is purchased from the utility if needed. An excess of electricity generated by the fuel cell system can be sold to the utility. The fuel cell system also provides thermal energy which can be used for absorption cooling, space heating and domestic hot water. Thermal storage can be incorporated into the system. Thermal energy is also provided by an auxiliary boiler if needed to supplement the fuel cell system output. Fuel cell/HVAC systems were analyzed with the TRACE computer program.

  15. Biosafety Assessment of Site-directed Transgene Integration in Human Umbilical Cord–lining Cells

    PubMed Central

    Sivalingam, Jaichandran; Krishnan, Shruti; Ng, Wai Har; Lee, Sze Sing; Phan, Toan Thang; Kon, Oi Lian

    2010-01-01

    Biosafety and efficacy considerations that impede clinical application of gene therapy could be addressed by nonviral ex vivo cell therapy, utilizing transgenic cells that have been comprehensively pre-evaluated for genotoxic potential and transgene expression. We evaluated the genotoxic potential of phiC31 bacteriophage integrase-mediated transgene integration in cord-lining epithelial cells (CLECs) readily cultured from the outer membrane of human umbilical cords, by sequencing and mapping integration sites, spectral karyotyping, high-resolution genome copy number, transcriptome, and transgene copy number analyses and in vivo tumorigenicity. Of 44 independent integration events, <5% were exonic and 85% of modified cells had integrated ≤2 transgene(s). Expression of 95.6% of genes was unaltered in modified cells. Only three small regions showed genome copy number changes that did not correlate with altered gene expression or integration sites. Spectral karyotyping revealed rare nonrecurrent occurrence of three different translocations. Integrase-modified cells were not tumorigenic in immunocompromised mice for at least 4 months. Stable integration of a human factor VIII (FVIII) construct conferred durable FVIII secretion in vitro. Xenoimplantation of FVIII-secreting CLECs in immunocompetent hemophilic mice achieved significant phenotypic correction. Pre-evaluated clonal populations of phiC31 integrase–modified CLECs could be useful as bioimplants for monogenic diseases such as hemophilia. PMID:20424600

  16. Integrated test plan for crosswell compressional and shear wave seismic tomography for site characterization at the VOC Arid Site

    SciTech Connect

    Elbring, G.J.; Narbutovskih, S.M.

    1994-02-01

    This integrated test plan describes the demonstration of the crosswell acoustic tomography technique as part of the Volatile Organic Compounds-Arid Integrated Demonstration (VOC-Arid ID). The purpose of this demonstration is to image the subsurface seismic velocity structure and to relate the resulting velocity model to lithology and saturation. In fiscal year (FY) 1994 an initial fielding will test three different downhole sources at two different sites at the Hanford US Department of Energy facility to identify which sources will provide the energy required to propagate between existing steel-cased wells at these two sites. Once this has been established, a second fielding will perform a full compressional and shear wave tomographic survey at the most favorable site. Data reduction, analysis, and interpretation of this full data set will be completed by the end of this fiscal year. Data collection for a second survey will be completed by the end of the fiscal year, and data reduction for this data set will be completed in FY 1995. The specific need is detailed subsurface characterization with minimum intrusion. This technique also has applications for long term vadose zone monitoring for both Resource Conservation and Recovery Act (RCRA) waste storage facilities and for remediation monitoring. Images produced are continuous between boreholes. This is a significant improvement over the single point data derived solely from core information. Saturation changes, either naturally occurring (e.g., perched water tables) or remediation induced (e.g., water table mounding from injection wells or during inwell air sparging) could be imaged. These crosswell data allow optimal borehole placement for groundwater remediation, associated monitoring wells and possibly evaluation of the effective influence of a particular remediation technique.

  17. Site-specific integration of adeno-associated virus involves partial duplication of the target locus

    PubMed Central

    Henckaerts, Els; Dutheil, Nathalie; Zeltner, Nadja; Kattman, Steven; Kohlbrenner, Erik; Ward, Peter; Clément, Nathalie; Rebollo, Patricia; Kennedy, Marion; Keller, Gordon M.; Linden, R. Michael

    2009-01-01

    A variety of viruses establish latency by integrating their genome into the host genome. The integration event generally occurs in a nonspecific manner, precluding the prediction of functional consequences from resulting disruptions of affected host genes. The nonpathogenic adeno-associated virus (AAV) is unique in its ability to stably integrate in a site-specific manner into the human MBS85 gene. To gain a better understanding of the integration mechanism and the consequences of MBS85 disruption, we analyzed the molecular structure of AAV integrants in various latently infected human cell lines. Our study led to the observation that AAV integration causes an extensive but partial duplication of the target gene. Intriguingly, the molecular organization of the integrant leaves the possibility that a functional copy of the disrupted target gene could potentially be preserved despite the resulting rearrangements. A latently infected, Mbs85-targeted mouse ES cell line was generated to study the functional consequences of the observed duplication-based integration mechanism. AAV-modified ES cell lines continued to self-renew, maintained their multilineage differentiation potential and contributed successfully to mouse development when injected into blastocysts. Thus, our study reveals a viral strategy for targeted genome addition with the apparent absence of functional consequences. PMID:19372372

  18. Synthetic chromosomes.

    PubMed

    Schindler, Daniel; Waldminghaus, Torsten

    2015-11-01

    What a living organism looks like and how it works and what are its components-all this is encoded on DNA, the genetic blueprint. Consequently, the way to change an organism is to change its genetic information. Since the first pieces of recombinant DNA have been used to transform cells in the 1970s, this approach has been enormously extended. Bigger and bigger parts of the genetic information have been exchanged or added over the years. Now we are at a point where the construction of entire chromosomes becomes a reachable goal and first examples appear. This development leads to fundamental new questions, for example, about what is possible and desirable to build or what construction rules one needs to follow when building synthetic chromosomes. Here we review the recent progress in the field, discuss current challenges and speculate on the appearance of future synthetic chromosomes. PMID:26111960

  19. Chromosome and cell genetics

    SciTech Connect

    Sharma, A.K.; Sharma, A.

    1985-01-01

    This book contains 11 chapters. Some of the titles are: Chromosomes in differentiation; Chromosome axis; Nuclear and organelle split genes; Chemical mutagenesis; and Chromosome architecture and additional elements.

  20. Small ubiquitin-related modifier ligase activity of Mms21 is required for maintenance of chromosome integrity during the unperturbed mitotic cell division cycle in Saccharomyces cerevisiae.

    PubMed

    Rai, Ragini; Varma, Satya P M V; Shinde, Nikhil; Ghosh, Shilpa; Kumaran, Srikala P; Skariah, Geena; Laloraya, Shikha

    2011-04-22

    The SUMO ligase activity of Mms21/Nse2, a conserved member of the Smc5/6 complex, is required for resisting extrinsically induced genotoxic stress. We report that the Mms21 SUMO ligase activity is also required during the unchallenged mitotic cell cycle in Saccharomyces cerevisiae. SUMO ligase-defective cells were slow growing and spontaneously incurred DNA damage. These cells required caffeine-sensitive Mec1 kinase-dependent checkpoint signaling for survival even in the absence of extrinsically induced genotoxic stress. SUMO ligase-defective cells were sensitive to replication stress and displayed synthetic growth defects with DNA damage checkpoint-defective mutants such as mec1, rad9, and rad24. MMS21 SUMO ligase and mediator of replication checkpoint 1 gene (MRC1) were epistatic with respect to hydroxyurea-induced replication stress or methyl methanesulfonate-induced DNA damage sensitivity. Subjecting Mms21 SUMO ligase-deficient cells to transient replication stress resulted in enhancement of cell cycle progression defects such as mitotic delay and accumulation of hyperploid cells. Consistent with the spontaneous activation of the DNA damage checkpoint pathway observed in the Mms21-mediated sumoylation-deficient cells, enhanced frequency of chromosome breakage and loss was detected in these mutant cells. A mutation in the conserved cysteine 221 that is engaged in coordination of the zinc ion in Loop 2 of the Mms21 SPL-RING E3 ligase catalytic domain resulted in strong replication stress sensitivity and also conferred slow growth and Mec1 dependence to unchallenged mitotically dividing cells. Our findings establish Mms21-mediated sumoylation as a determinant of cell cycle progression and maintenance of chromosome integrity during the unperturbed mitotic cell division cycle in budding yeast. PMID:21324902

  1. GENOME-WIDE LINKAGE ANALYSIS TO IDENTIFY CHROMOSOMAL REGIONS AFFECTING PHENOTYPIC TRAITS IN THE CHICKEN. IV. SKELETAL INTEGRITY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two unique chicken F2 populations generated from a broiler breeder male line and two genetically distinct inbred (greater than 99%) chicken lines (Leghom and Fayoumi), were used for whole genome QTL analysis. Twelve phenotypic skeletal integrity traits (6 absolute and 6 relative traits) were measure...

  2. An integrated genetic and physical map of homoeologous chromosomes 12 and 26 in Upland cotton (G. hirsutum L.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Upland cotton (G. hirsutum L.) is the leading fiber crop worldwide. Genetic improvement of fiber quality and yield is facilitated by a variety of genomics tools. An integrated genetic and physical map is needed to better characterize quantitative trait loci and to allow for the positional cloning ...

  3. Genetic improvement of Escherichia coli for ethanol production: Chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II

    SciTech Connect

    Ohta, Kazuyoshi; Beall, D.S.; Mejia, J.P.; Shanmugam, K.T.; Ingram, L.O. )

    1991-04-01

    Zymomonas mobilis genes for pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) were integrated into the Escherichia coli chromosome within or near the pyruvate formate-lyase gene (pfl). Integration improved the stability of the Z. mobilis genes in E. coli, but further selection was required to increase expression. Spontaneous mutants were selected for resistance to high levels of chloramphenicol that also expressed high levels of the Z. mobilis genes. Analogous mutants were selected for increased expression of alcohol dehydrogenase on aldehyde indicator plates. These mutants were functionally equivalent to the previous plasmid-based strains for the fermentation of xylose and glucose to ethanol. Ethanol concentrations of 54.4 and 41.6 g/liter were obtained from 10% glucose and 8% xylose, respectively. The efficiency of conversion exceeded theoretical limits (0.51 g of ethanol/g of sugar) on the basis of added sugars because of the additional production of ethanol from the catabolism of complex nutrients. Further mutations were introduced to inactivate succinate production (frd) and to block homologous recombination (recA).

  4. Rearrangement of a common cellular DNA domain on chromosome 4 in human primary liver tumors

    SciTech Connect

    Pasquinelli, C.; Garreau, F.; Bougueleret, L.; Cariani, E.; Thiers, V.; Croissant, O.; Hadchouel, M.; Tiollais, P.; Brechot, C. ); Grzeschik, K.H. )

    1988-02-01

    Hepatitis B virus (HBV) DNA integration has been shown to occur frequently in human hepatocellular carcinomas. The authors have investigated whether common cellular DNA domains might be rearranged, possibly by HBV integration, in human primary liver tumors. Unique cellular DNA sequences adjacent to an HBV integration site were isolated from a patient with hepatitis B surface antigen-positive hepatocellular carcinoma. These probes detected rearrangement of this cellular region of chromosomal DNA in 3 of 50 additional primary liver tumors studied. Of these three tumor samples, two contained HBV DNA, without an apparent link between the viral DNA and the rearranged allele; HBV DNA sequences were not detected in the third tumor sample. By use of a panel of somatic cell hybrids, these unique cellular DNA sequences were shown to be located on chromosome 4. Therefore, this region of chromosomal DNA might be implicated in the formation of different tumors at one step of liver cell transformation, possible related to HBV integration.

  5. A dynamic multimedia fuzzy-stochastic integrated environmental risk assessment approach for contaminated sites management.

    PubMed

    Hu, Yan; Wen, Jing-Ya; Li, Xiao-Li; Wang, Da-Zhou; Li, Yu

    2013-10-15

    A dynamic multimedia fuzzy-stochastic integrated environmental risk assessment approach was developed for contaminated sites management. The contaminant concentrations were simulated by a validated interval dynamic multimedia fugacity model, and different guideline values for the same contaminant were represented as a fuzzy environmental guideline. Then, the probability of violating environmental guideline (Pv) can be determined by comparison between the modeled concentrations and the fuzzy environmental guideline, and the constructed relationship between the Pvs and environmental risk levels was used to assess the environmental risk level. The developed approach was applied to assess the integrated environmental risk at a case study site in China, simulated from 1985 to 2020. Four scenarios were analyzed, including "residential land" and "industrial land" environmental guidelines under "strict" and "loose" strictness. It was found that PAH concentrations will increase steadily over time, with soil found to be the dominant sink. Source emission in soil was the leading input and atmospheric sedimentation was the dominant transfer process. The integrated environmental risks primarily resulted from petroleum spills and coke ovens, while the soil environmental risks came from coal combustion. The developed approach offers an effective tool for quantifying variability and uncertainty in the dynamic multimedia integrated environmental risk assessment and the contaminated site management. PMID:23995555

  6. Implementation of an integrated CERCLA-NEPA environmental process at a FUSRAP site

    SciTech Connect

    Devgun, J.S.; Beskid, N.J.; Robertson, R.C.; Atkin, R.G.

    1989-01-01

    The Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA) of 1980 and its amendment in 1986 through the Superfund Amendments and Reauthorization Act (SARA) have significantly affected the environmental compliance process. Remedial actions at contaminated sites now must satisfy the requirements of not only the National Environmental Policy Act (NEPA) but also CERCLA, as amended. For planning and conducting remedial actions under the Formerly Utilized Sites Remedial Action Program (FUSRAP), the US Department of Energy has developed and implemented an integrated process aimed at satisfying the requirements of both NEPA and CERCLA, as amended. The integrated approach involves a single, comprehensive environmental process that results in a single set of documentation. The process is streamlined, efficient, and cost effective and it minimizes confusion to the public. This paper discusses the need for, and the elements of, the integrated process as applied to FUSRAP sites. The implementation of the process is illustrated through a case study of FUSRAP sites in Tonawanda, New York. 5 figs.

  7. Comprehensive profiling of retroviral integration sites using target enrichment methods from historical koala samples without an assembled reference genome.

    PubMed

    Cui, Pin; Löber, Ulrike; Alquezar-Planas, David E; Ishida, Yasuko; Courtiol, Alexandre; Timms, Peter; Johnson, Rebecca N; Lenz, Dorina; Helgen, Kristofer M; Roca, Alfred L; Hartman, Stefanie; Greenwood, Alex D

    2016-01-01

    Background. Retroviral integration into the host germline results in permanent viral colonization of vertebrate genomes. The koala retrovirus (KoRV) is currently invading the germline of the koala (Phascolarctos cinereus) and provides a unique opportunity for studying retroviral endogenization. Previous analysis of KoRV integration patterns in modern koalas demonstrate that they share integration sites primarily if they are related, indicating that the process is currently driven by vertical transmission rather than infection. However, due to methodological challenges, KoRV integrations have not been comprehensively characterized. Results. To overcome these challenges, we applied and compared three target enrichment techniques coupled with next generation sequencing (NGS) and a newly customized sequence-clustering based computational pipeline to determine the integration sites for 10 museum Queensland and New South Wales (NSW) koala samples collected between the 1870s and late 1980s. A secondary aim of this study sought to identify common integration sites across modern and historical specimens by comparing our dataset to previously published studies. Several million sequences were processed, and the KoRV integration sites in each koala were characterized. Conclusions. Although the three enrichment methods each exhibited bias in integration site retrieval, a combination of two methods, Primer Extension Capture and hybridization capture is recommended for future studies on historical samples. Moreover, identification of integration sites shows that the proportion of integration sites shared between any two koalas is quite small. PMID:27069793

  8. Comprehensive profiling of retroviral integration sites using target enrichment methods from historical koala samples without an assembled reference genome

    PubMed Central

    Alquezar-Planas, David E.; Ishida, Yasuko; Courtiol, Alexandre; Timms, Peter; Johnson, Rebecca N.; Lenz, Dorina; Helgen, Kristofer M.; Roca, Alfred L.; Hartman, Stefanie

    2016-01-01

    Background. Retroviral integration into the host germline results in permanent viral colonization of vertebrate genomes. The koala retrovirus (KoRV) is currently invading the germline of the koala (Phascolarctos cinereus) and provides a unique opportunity for studying retroviral endogenization. Previous analysis of KoRV integration patterns in modern koalas demonstrate that they share integration sites primarily if they are related, indicating that the process is currently driven by vertical transmission rather than infection. However, due to methodological challenges, KoRV integrations have not been comprehensively characterized. Results. To overcome these challenges, we applied and compared three target enrichment techniques coupled with next generation sequencing (NGS) and a newly customized sequence-clustering based computational pipeline to determine the integration sites for 10 museum Queensland and New South Wales (NSW) koala samples collected between the 1870s and late 1980s. A secondary aim of this study sought to identify common integration sites across modern and historical specimens by comparing our dataset to previously published studies. Several million sequences were processed, and the KoRV integration sites in each koala were characterized. Conclusions. Although the three enrichment methods each exhibited bias in integration site retrieval, a combination of two methods, Primer Extension Capture and hybridization capture is recommended for future studies on historical samples. Moreover, identification of integration sites shows that the proportion of integration sites shared between any two koalas is quite small. PMID:27069793

  9. The BET family of proteins targets Moloney Murine Leukemia Virus integration near transcription start sites

    PubMed Central

    De Rijck, Jan; de Kogel, Christine; Demeulemeester, Jonas; Vets, Sofie; Ashkar, Sara El; Malani, Nirav; Bushman, Frederic D; Landuyt, Bart; Husson, Steven J.; Busschots, Katrien; Gijsbers, Rik; Debyser, Zeger

    2014-01-01

    Summary A hallmark of retroviral replication is integration of the viral genome in the host cell DNA. This characteristic makes retrovirus-based vectors attractive delivery vehicles for gene therapy. However, adverse events in gene therapeutic trials, caused by activation of proto-oncogenes due to Murine Leukemia Virus (MLV)-derived vector integration, hamper their application. Here we show that bromodomain and extraterminal (BET) proteins (BRD2, BRD3 and BRD4) and MLV integrase specifically interact and co-localize within the nucleus of the cell. Inhibition of the BET proteins chromatin interaction via specific bromodomain inhibitors blocks MLV virus replication at the integration step. MLV integration site distribution parallels the chromatin binding profile of BET proteins, and expression of an artificial fusion protein of the BET integrase binding domain with the chromatin interaction domain of the lentiviral targeting factor LEDGF/p75, retargets MLV integration away from TSS and into the body of actively transcribed genes, conform to the Human Immunodeficiency Virus (HIV) integration pattern. Together these data validate BET proteins as MLV integration targeting factors. PMID:24183673

  10. Oak Ridge National Laboratory Management & Integration Perspective Subcontractors as Partners in Site Restoration

    SciTech Connect

    Brill, A.; Eidam, G.

    2002-02-26

    In 1997, the U. S. Department of Energy (DOE) Oak Ridge Operations (ORO) Office awarded the Management and Integration (M&I) contract for all five of their Oak Ridge Operations facilities to Bechtel Jacobs Company, LLC (BJC). This paper will focus on the success and challenges of several of the M&I projects at the Oak Ridge National Laboratory (ORNL). The initial goals for BJC were to transition up to 93% of their staff to the subcontract community as they moved away from operations to ''integration.'' The perspectives of BJC and one of their Remedial Action/Decontamination & Decommissioning (RADD) subcontractors will be combined in this paper to share with others how ''partnering'' together was essential for success. Projects completed by Safety and Ecology Corporation (SEC) under their RADD subcontract will be used to illustrate the process and the challenges/successes to completion. These projects will include pond remediation, tank remediation, and building cleanup for reuse. All these projects were ''fixed price'' with defined milestones keyed into award fee for BJC and regulatory milestones for DOE. By working together to form integrated teams focused on site remediation without sacrificing safety, all milestones were met. This paper will discuss the following items associated with the M&I environmental restoration projects at ORNL: overview of the M&I Contract; challenges in transitioning from ''operations'' to ''integration''; subcontracting strategies; subcontractor pre-qualification process; overview of ORNL Projects; and integrated team effort required to achieve site restoration goals.

  11. Erace--an integrated system for treating organic-contaminated sites

    SciTech Connect

    Caley, S.M.; Heath, W.O.; Bergsman, T.M.; Gauglitz, P.A.; Pillay, C.; Moss, R.W.; Shah, R.R.; Goheen, S.C.; Camiaoni, D.M.

    1994-11-01

    The U.S. Department of Energy`s (DOE) Pacific Northwest Laboratory (PNL) is developing a suite of electrical technologies for treating sites contaminated with hazardous organic compounds. These include: (1) Six-Phase Soil Heating (SPSH) to remove volatile and semi-volatile organic compounds from soils; (2) In Situ Corona (ISC) to decompose nonvolatile and bound organic contaminants in soils; (3) High-Energy Corona (HEC) to treat contaminated off-gases; and (4) Liquid Corona (LC) to treat contaminated liquids. These four technologies comprise ERACE (Electrical Remediation at Contaminated Environments), an integrated system for accomplishing site remediation with little or no secondary wastes produced that would require off-site treatment or disposal. Each ERACE technology can be employed individually as a stand-alone treatment process, or combined as a system for total site remediation. For example, an ERACE system for treating sites contaminated with volatile organics would integrate SPSH to remove the contaminants from the soil, LC to continuously treat an aqueous stream condensed out of the soil off-gas, and HEC to treat non-condensibles remaining in the off-gas, before atmospheric release.

  12. Integrated environmental site characterization involving geochemistry, geophysics, and geology: A shortcut to remediation

    SciTech Connect

    Viellenave, J.; Slatten, M.; Church, G.; Anderson, M.

    1996-11-01

    Environmental site characterization processes have evolved from simple drill-and-sample routines into more sophisticated evaluations of increasingly complex problems involving a variety of contaminants. Strategic integration of several geoscience tools into a more holistic approach benefits the site owner/operator by developing a synoptic perspective of the site at the earliest possible time, allowing for more selective and focused use of the expensive and invasive technologies. The ultimate effect is a better site characterization, including attention to difficult PRP issues, lower liability, fewer risks of bypassing potentially hazardous contaminant accumulations, and a result that is more targeted to environmental and human health risks. An integrated site investigation system requires good geology and hydrology, but is properly augmented by use of modem and sophisticated geochemical and geophysical tools. Establishing characterization objectives is critical in deciding what geoscience tool(s) to deploy in any given situation. For each tool, critical criteria are identified that will enable the user to best decide which to use for what purposes.

  13. Sal-Site: Integrating new and existing ambystomatid salamander research and informational resources

    PubMed Central

    Smith, Jeramiah J; Putta, Srikrishna; Walker, John A; Kump, D Kevin; Samuels, Amy K; Monaghan, James R; Weisrock, David W; Staben, Chuck; Voss, S Randal

    2005-01-01

    Salamanders of the genus Ambystoma are a unique model organism system because they enable natural history and biomedical research in the laboratory or field. We developed Sal-Site to integrate new and existing ambystomatid salamander research resources in support of this model system. Sal-Site hosts six important resources: 1) Salamander Genome Project: an information-based web-site describing progress in genome resource development, 2) Ambystoma EST Database: a database of manually edited and analyzed contigs assembled from ESTs that were collected from A. tigrinum tigrinum and A. mexicanum, 3) Ambystoma Gene Collection: a database containing full-length protein-coding sequences, 4) Ambystoma Map and Marker Collection: an image and database resource that shows the location of mapped markers on linkage groups, provides information about markers, and provides integrating links to Ambystoma EST Database and Ambystoma Gene Collection databases, 5) Ambystoma Genetic Stock Center: a website and collection of databases that describe an NSF funded salamander rearing facility that generates and distributes biological materials to researchers and educators throughout the world, and 6) Ambystoma Research Coordination Network: a web-site detailing current research projects and activities involving an international group of researchers. Sal-Site is accessible at . PMID:16359543

  14. X-Chromosome dosage compensation.

    PubMed

    Meyer, Barbara J

    2005-01-01

    In mammals, flies, and worms, sex is determined by distinctive regulatory mechanisms that cause males (XO or XY) and females (XX) to differ in their dose of X chromosomes. In each species, an essential X chromosome-wide process called dosage compensation ensures that somatic cells of either sex express equal levels of X-linked gene products. The strategies used to achieve dosage compensation are diverse, but in all cases, specialized complexes are targeted specifically to the X chromosome(s) of only one sex to regulate transcript levels. In C. elegans, this sex-specific targeting of the dosage compensation complex (DCC) is controlled by the same developmental signal that establishes sex, the ratio of X chromosomes to sets of autosomes (X:A signal). Molecular components of this chromosome counting process have been defined. Following a common step of regulation, sex determination and dosage compensation are controlled by distinct genetic pathways. C. elegans dosage compensation is implemented by a protein complex that binds both X chromosomes of hermaphrodites to reduce transcript levels by one-half. The dosage compensation complex resembles the conserved 13S condensin complex required for both mitotic and meiotic chromosome resolution and condensation, implying the recruitment of ancient proteins to the new task of regulating gene expression. Within each C. elegans somatic cell, one of the DCC components also participates in the separate mitotic/meiotic condensin complex. Other DCC components play pivotal roles in regulating the number and distribution of crossovers during meiosis. The strategy by which C. elegans X chromosomes attract the condensin-like DCC is known. Small, well-dispersed X-recognition elements act as entry sites to recruit the dosage compensation complex and to nucleate spreading of the complex to X regions that lack recruitment sites. In this manner, a repressed chromatin state is spread in cis over short or long distances, thus establishing the

  15. Integrative Analysis of CRISPR/Cas9 Target Sites in the Human HBB Gene.

    PubMed

    Luo, Yumei; Zhu, Detu; Zhang, Zhizhuo; Chen, Yaoyong; Sun, Xiaofang

    2015-01-01

    Recently, the clustered regularly interspaced short palindromic repeats (CRISPR) system has emerged as a powerful customizable artificial nuclease to facilitate precise genetic correction for tissue regeneration and isogenic disease modeling. However, previous studies reported substantial off-target activities of CRISPR system in human cells, and the enormous putative off-target sites are labor-intensive to be validated experimentally, thus motivating bioinformatics methods for rational design of CRISPR system and prediction of its potential off-target effects. Here, we describe an integrative analytical process to identify specific CRISPR target sites in the human β-globin gene (HBB) and predict their off-target effects. Our method includes off-target analysis in both coding and noncoding regions, which was neglected by previous studies. It was found that the CRISPR target sites in the introns have fewer off-target sites in the coding regions than those in the exons. Remarkably, target sites containing certain transcriptional factor motif have enriched binding sites of relevant transcriptional factor in their off-target sets. We also found that the intron sites have fewer SNPs, which leads to less variation of CRISPR efficiency in different individuals during clinical applications. Our studies provide a standard analytical procedure to select specific CRISPR targets for genetic correction. PMID:25918715

  16. Integrative Analysis of CRISPR/Cas9 Target Sites in the Human HBB Gene

    PubMed Central

    Luo, Yumei; Zhang, Zhizhuo; Chen, Yaoyong; Sun, Xiaofang

    2015-01-01

    Recently, the clustered regularly interspaced short palindromic repeats (CRISPR) system has emerged as a powerful customizable artificial nuclease to facilitate precise genetic correction for tissue regeneration and isogenic disease modeling. However, previous studies reported substantial off-target activities of CRISPR system in human cells, and the enormous putative off-target sites are labor-intensive to be validated experimentally, thus motivating bioinformatics methods for rational design of CRISPR system and prediction of its potential off-target effects. Here, we describe an integrative analytical process to identify specific CRISPR target sites in the human β-globin gene (HBB) and predict their off-target effects. Our method includes off-target analysis in both coding and noncoding regions, which was neglected by previous studies. It was found that the CRISPR target sites in the introns have fewer off-target sites in the coding regions than those in the exons. Remarkably, target sites containing certain transcriptional factor motif have enriched binding sites of relevant transcriptional factor in their off-target sets. We also found that the intron sites have fewer SNPs, which leads to less variation of CRISPR efficiency in different individuals during clinical applications. Our studies provide a standard analytical procedure to select specific CRISPR targets for genetic correction. PMID:25918715

  17. Identifying Human Kinase-Specific Protein Phosphorylation Sites by Integrating Heterogeneous Information from Various Sources

    PubMed Central

    Li, Tingting; Du, Pufeng; Xu, Nanfang

    2010-01-01

    Phosphorylation is an important type of protein post-translational modification. Identification of possible phosphorylation sites of a protein is important for understanding its functions. Unbiased screening for phosphorylation sites by in vitro or in vivo experiments is time consuming and expensive; in silico prediction can provide functional candidates and help narrow down the experimental efforts. Most of the existing prediction algorithms take only the polypeptide sequence around the phosphorylation sites into consideration. However, protein phosphorylation is a very complex biological process in vivo. The polypeptide sequences around the potential sites are not sufficient to determine the phosphorylation status of those residues. In the current work, we integrated various data sources such as protein functional domains, protein subcellular location and protein-protein interactions, along with the polypeptide sequences to predict protein phosphorylation sites. The heterogeneous information significantly boosted the prediction accuracy for some kinase families. To demonstrate potential application of our method, we scanned a set of human proteins and predicted putative phosphorylation sites for Cyclin-dependent kinases, Casein kinase 2, Glycogen synthase kinase 3, Mitogen-activated protein kinases, protein kinase A, and protein kinase C families (avaiable at http://cmbi.bjmu.edu.cn/huphospho). The predicted phosphorylation sites can serve as candidates for further experimental validation. Our strategy may also be applicable for the in silico identification of other post-translational modification substrates. PMID:21085571

  18. Study of component technologies for fuel cell on-site integrated energy system. Volume 2: Appendices

    NASA Technical Reports Server (NTRS)

    Lee, W. D.; Mathias, S.

    1980-01-01

    This data base catalogue was compiled in order to facilitate the analysis of various on site integrated energy system with fuel cell power plants. The catalogue is divided into two sections. The first characterizes individual components in terms of their performance profiles as a function of design parameters. The second characterizes total heating and cooling systems in terms of energy output as a function of input and control variables. The integrated fuel cell systems diagrams and the computer analysis of systems are included as well as the cash flows series for baseline systems.

  19. Background Information for the Nevada National Security Site Integrated Sampling Plan, Revision 0

    SciTech Connect

    Farnham, Irene; Marutzky, Sam

    2014-12-01

    This document describes the process followed to develop the Nevada National Security Site (NNSS) Integrated Sampling Plan (referred to herein as the Plan). It provides the Plan’s purpose and objectives, and briefly describes the Underground Test Area (UGTA) Activity, including the conceptual model and regulatory requirements as they pertain to groundwater sampling. Background information on other NNSS groundwater monitoring programs—the Routine Radiological Environmental Monitoring Plan (RREMP) and Community Environmental Monitoring Program (CEMP)—and their integration with the Plan are presented. Descriptions of the evaluations, comments, and responses of two Sampling Plan topical committees are also included.

  20. Total integrated slidable and valveless solid phase extraction-polymerase chain reaction-capillary electrophoresis microdevice for mini Y chromosome short tandem repeat genotyping.

    PubMed

    Kim, Yong Tae; Lee, Dohwan; Heo, Hyun Young; Sim, Jeong Eun; Woo, Kwang Man; Kim, Do Hyun; Im, Sung Gap; Seo, Tae Seok

    2016-04-15

    A fully integrated slidable and valveless microsystem, which performs solid phase DNA extraction (SPE), micro-polymerase chain reaction (μPCR) and micro-capillary electrophoresis (μCE) coupled with a portable genetic analyser, has been developed for forensic genotyping. The use of a slidable chip, in which a 1 μL-volume of the PCR chamber was patterned at the center, does not necessitate any microvalves and tubing systems for fluidic control. The functional micro-units of SPE, μPCR, and μCE were fabricated on a single glass wafer by conventional photolithography, and the integrated microdevice consists of three layers: from top to bottom, a slidable chip, a channel wafer in which a SPE chamber, a mixing microchannel, and a CE microchannel were fabricated, and a Ti/Pt resistance temperature detector (RTD) wafer. The channel glass wafer and the RTD glass wafer were thermally bonded, and the slidable chip was placed on the designated functional unit. The entire process from the DNA extraction using whole human blood sample to identification of target Y chromosomal short tandem repeat (STR) loci was serially carried out with simply sliding the slidable chamber from one to another functional unit. Monoplex and multiplex detection of amelogenin and mini Y STR loci were successfully analysed on the integrated slidable SPE-μPCR-μCE microdevice by using 1 μL whole human blood within 60 min. The proposed advanced genetic analysis microsystem is capable of point-of-care DNA testing with sample-in-answer-out capability, more importantly, without use of complicated microvalves and microtubing systems for liquid transfer. PMID:26657593

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

    PubMed Central

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

    1995-01-01

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

  2. Integrated system for gathering, processing, and reporting data relating to site contamination

    DOEpatents

    Long, Delmar D.; Goldberg, Mitchell S.; Baker, Lorie A.

    1997-01-01

    An integrated screening system comprises an intrusive sampling subsystem, a field mobile laboratory subsystem, a computer assisted design/geographical information subsystem, and a telecommunication linkup subsystem, all integrated to provide synergistically improved data relating to the extent of site soil/groundwater contamination. According to the present invention, data samples related to the soil, groundwater or other contamination of the subsurface material are gathered and analyzed to measure contaminants. Based on the location of origin of the samples in three-dimensional space, the analyzed data are transmitted to a location display. The data from analyzing samples and the data from the locating the origin are managed to project the next probable sample location. The next probable sample location is then forwarded for use as a guide in the placement of ensuing sample location, whereby the number of samples needed to accurately characterize the site is minimized.

  3. Integrated system for gathering, processing, and reporting data relating to site contamination

    DOEpatents

    Long, D.D.; Goldberg, M.S.; Baker, L.A.

    1997-11-11

    An integrated screening system comprises an intrusive sampling subsystem, a field mobile laboratory subsystem, a computer assisted design/geographical information subsystem, and a telecommunication linkup subsystem, all integrated to provide synergistically improved data relating to the extent of site soil/groundwater contamination. According to the present invention, data samples related to the soil, groundwater or other contamination of the subsurface material are gathered and analyzed to measure contaminants. Based on the location of origin of the samples in three-dimensional space, the analyzed data are transmitted to a location display. The data from analyzing samples and the data from the locating the origin are managed to project the next probable sample location. The next probable sample location is then forwarded for use as a guide in the placement of ensuing sample location, whereby the number of samples needed to accurately characterize the site is minimized. 10 figs.

  4. Site-Specific Integration of Exogenous Genes Using Genome Editing Technologies in Zebrafish.

    PubMed

    Kawahara, Atsuo; Hisano, Yu; Ota, Satoshi; Taimatsu, Kiyohito

    2016-01-01

    The zebrafish (Danio rerio) is an ideal vertebrate model to investigate the developmental molecular mechanism of organogenesis and regeneration. Recent innovation in genome editing technologies, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system, have allowed researchers to generate diverse genomic modifications in whole animals and in cultured cells. The CRISPR/Cas9 and TALEN techniques frequently induce DNA double-strand breaks (DSBs) at the targeted gene, resulting in frameshift-mediated gene disruption. As a useful application of genome editing technology, several groups have recently reported efficient site-specific integration of exogenous genes into targeted genomic loci. In this review, we provide an overview of TALEN- and CRISPR/Cas9-mediated site-specific integration of exogenous genes in zebrafish. PMID:27187373

  5. Site-Specific Integration of Exogenous Genes Using Genome Editing Technologies in Zebrafish

    PubMed Central

    Kawahara, Atsuo; Hisano, Yu; Ota, Satoshi; Taimatsu, Kiyohito

    2016-01-01

    The zebrafish (Danio rerio) is an ideal vertebrate model to investigate the developmental molecular mechanism of organogenesis and regeneration. Recent innovation in genome editing technologies, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system, have allowed researchers to generate diverse genomic modifications in whole animals and in cultured cells. The CRISPR/Cas9 and TALEN techniques frequently induce DNA double-strand breaks (DSBs) at the targeted gene, resulting in frameshift-mediated gene disruption. As a useful application of genome editing technology, several groups have recently reported efficient site-specific integration of exogenous genes into targeted genomic loci. In this review, we provide an overview of TALEN- and CRISPR/Cas9-mediated site-specific integration of exogenous genes in zebrafish. PMID:27187373

  6. DNFSB recommendation 94-1 Hanford site integrated stabilization management plan

    SciTech Connect

    McCormack, R.L.

    1997-05-07

    In May 1994, the Defense Nuclear Facilities Safety Board (DNFSB) issued DNFSB Recommendation 94-1 (Conway 1994), which identified concerns related to US Department of Energy (DOE) management of legacy fissile materials remaining from past defense production activities. The DNFSB expressed concern about the existing storage conditions for these materials and the slow pace at which the conditions were being remediated. The DNFSB also expressed its belief that additional delays in stabilizing these fissile materials would be accompanied by further deterioration of safety and unnecessary increased risks to workers and the public. In February 1995, DOE issued the DNFSB Recommendation 94-1 Implementation Plan (O`Leary 1995) to address the concerns identified in DNFSB Recommendation 94-1. The Implementation Plan (IP) identifies several DOE commitments to achieve safe interim storage for the legacy fissile materials, and constitutes DOE`s baseline DNFSB Recommendation 94-1 Integrated Program Plan (IPP). The IPP describes the actions DOE plans to implement within the DOE complex to convert its excess fissile materials to forms or conditions suitable for safe interim storage. The IPP was subsequently supplemented with an Integrated Facilities Plan and a Research and Development Plan, which further develop complex-wide research and development and long-range facility requirements and plans. The additions to the baseline IPP were developed based on a systems engineering approach that integrated facilities and capabilities at the various DOE sites and focused on attaining safe interim storage with minimum safety risks and environmental impacts. Each affected DOE site has developed a Site Integrated Stabilization Management Plan (SISMP) to identify individual site plans to implement the DNFSB Recommendation 94-1 IPP. The SISMPs were developed based on the objectives, requirements, and commitments identified in the DNFSB Recommendation 94-1 IP. The SISMPs also supported

  7. Chromosomal Mapping of Repetitive DNA Sequences in Five Species of Astyanax (Characiformes, Characidae) Reveals Independent Location of U1 and U2 snRNA Sites and Association of U1 snRNA and 5S rDNA.

    PubMed

    Silva, Duilio M Z A; Utsunomia, Ricardo; Pansonato-Alves, José C; Oliveira, Cláudio; Foresti, Fausto

    2015-01-01

    Astyanax is a genus of Characidae fishes currently composed of 155 valid species. Previous cytogenetic studies revealed high chromosomal diversification among them, and several studies have been performed using traditional cytogenetic techniques to investigate karyotypes and chromosomal locations of 18S and 5S rDNA genes. However, only a few studies are currently available about other repetitive sequences. Here, the chromosomal location of small nuclear RNA genes, identified as U1 and U2 snRNA clusters, was established and compared to the distribution of 5S rDNA and histone clusters in 5 Astyanax species (A. paranae, A. fasciatus, A. bockmanni, A. altiparanae, and A. jordani) using FISH. The cytogenetic mapping of U1 and U2 snRNA demonstrated a conserved pattern in the number of sites per genome independent of the location in Astyanax species. The location of the U1 snRNA gene was frequently associated with 5S rDNA sequences, indicating a possible interaction between the distinct repetitive DNA families. Finally, comparisons involving the location of U1 and U2 snRNA clusters in the chromosomes of Astyanax species revealed a very diverse pattern, suggesting that many rearrangements have occurred during the diversification process of this group. PMID:26329975

  8. Develop and test fuel cell powered on-site integrated total energy system

    NASA Technical Reports Server (NTRS)

    Kaufman, A.; Feigenbaum, H.; Wang, C. L.; Werth, J.; Whelan, J. A.

    1983-01-01

    Test results are presented for a 24 cell, two sq ft (4kW) stack. This stack is a precursor to a 25kW stack that is a key milestone. Results are discussed in terms of cell performance, electrolyte management, thermal management, and reactant gas manifolding. The results obtained in preliminary testing of a 50kW methanol processing subsystem are discussed. Subcontracting activities involving application analysis for fuel cell on site integrated energy systems are updated.

  9. Develop and test fuel cell powered on-site integrated total energy system

    NASA Technical Reports Server (NTRS)

    1983-01-01

    Test results are given for a 5 kW stack and initial results for an integrated, grid connected system operating from methanol fuel. Site selection criteria are presented for future demonstration of a 50 or 100 kW OS/IES. Preliminary results are also given with approximate internal rates of return to the building owner. Progress in development and construction of a 50 kW modular methanol/steam reformer is reported.

  10. Human Artificial Chromosomes for Gene Delivery and the Development of Animal Models

    PubMed Central

    Kazuki, Yasuhiro; Oshimura, Mitsuo

    2011-01-01

    Random integration of conventional gene delivery vectors such as viruses, plasmids, P1 phage-derived artificial chromosomes, bacterial artificial chromosomes and yeast artificial chromosomes can be associated with transgene silencing. Furthermore, integrated viral sequences can activate oncogenes adjacent to the insertion site resulting in cancer. Various human artificial chromosomes (HACs) exhibit several potential characteristics desired for an ideal gene delivery vector, including stable episomal maintenance and the capacity to carry large genomic loci with their regulatory elements, thus allowing the physiological regulation of the introduced gene in a manner similar to that of native chromosomes. HACs have been generated mainly using either a “top-down approach” (engineered chromosomes), or a “bottom-up approach” (de novo artificial chromosomes). The recent emergence of stem cell–based tissue engineering has opened up new avenues for gene and cell therapies. This review describes the lessons learned and prospects identified mainly from studies in the construction of HACs and HAC-mediated gene expression systems in cultured cells, as well as in animals. PMID:21750534

  11. Cloning and sequencing of viral integration site in human fibroblasts immortalized by simian virus 40.

    PubMed

    Yano, O; Hirano, H; Karasaki, Y; Higashi, K; Nakamura, H; Akiya, S; Gotoh, S

    1991-02-01

    We have analyzed cellular DNA sequences at the viral genome integration site in a human fibroblast cell line VA13 immortalized by simian virus 40 (SV40). The computer analysis of the junctional cellular DNA sequences did not show any homology to the DNA sequences previously reported. This suggests that immortalization by SV40 was not induced by the destruction of any known oncogene or anti-oncogene at the integration site. We did not find the precise substantial sequence homology at the junctional site between the cellular DNA and SV40 DNA, indicating that the recombination mechanism involved does not require precise sequence homology and therefore, SV40 genome was probably not integrated by homologous recombination. Short direct and inverted repeats of 5 to 29 nucleotides were found in the junctional cellular and SV40 DNA. Cellular DNA abutting SV40 DNA was found by the Northern blot analysis to be expressed in diploid human fibroblasts and SV40-transformed cells. The nature of this RNA is now under study. PMID:1851675

  12. Integration Site and Clonal Expansion in Human Chronic Retroviral Infection and Gene Therapy

    PubMed Central

    Niederer, Heather A.; Bangham, Charles R. M.

    2014-01-01

    Retroviral vectors have been successfully used therapeutically to restore expression of genes in a range of single-gene diseases, including several primary immunodeficiency disorders. Although clinical trials have shown remarkable results, there have also been a number of severe adverse events involving malignant outgrowth of a transformed clonal population. This clonal expansion is influenced by the integration site profile of the viral integrase, the transgene expressed, and the effect of the viral promoters on the neighbouring host genome. Infection with the pathogenic human retrovirus HTLV-1 also causes clonal expansion of cells containing an integrated HTLV-1 provirus. Although the majority of HTLV-1-infected people remain asymptomatic, up to 5% develop an aggressive T cell malignancy. In this review we discuss recent findings on the role of the genomic integration site in determining the clonality and the potential for malignant transformation of cells carrying integrated HTLV-1 or gene therapy vectors, and how these results have contributed to the understanding of HTLV-1 pathogenesis and to improvements in gene therapy vector safety. PMID:25365582

  13. Technology needs for remediation: Hanford and other DOE sites. Buried Waste Integrated Demonstration Program

    SciTech Connect

    Stapp, D.C.

    1993-01-01

    Technologies are being developed under the Buried Waste Integrated Demonstration (BWID) program to facilitate remediation of the US Department of Energy`s (DOE) buried and stored low-level radioactive, transuranic (TRU), and mixed radioactive and hazardous buried wastes. The BWID program is being coordinated by the Idaho National Engineering Laboratory (INEL) in southeastern Idaho, a DOE site that has large volumes of buried radioactive wastes. The program is currently focusing its efforts on the problems at INEL`s Subsurface Disposal Area (SDA) of the Radioactive Waste Management Complex (RWMC). As specific technologies are successfully demonstrated, they will be available for transfer to applications at other DOE buried waste sites. The purpose of this study is to present buried waste technology needs that have been identified for DOE sites other than INEL.

  14. Integrated Weed Control for Land Stewardship at Legacy Management's Rocky Flats Site in Colorado - 13086

    SciTech Connect

    Nelson, Jody K.

    2013-07-01

    Land stewardship is one of nine sustainability programs in the U.S. Department of Energy's Environmental Management System. Land stewardship includes maintaining and improving ecosystem health. At the Rocky Flats Site near Westminster, Colorado, land stewardship is an integral component of the Office of Legacy Management's post-closure monitoring and management at the site. Nearly 263 hectares (650 acres) were disturbed and re-vegetated during site cleanup and closure operations. Proactive management of revegetation areas is critical to the successful reestablishment of native grasslands, wetlands, and riparian communities. The undisturbed native plant communities that occur at the site also require active management to maintain the high-quality wetlands and other habitats that are home to numerous species of birds and other wildlife such as elk and deer, rare plant communities, and the federally listed threatened Preble's meadow jumping mouse. Over the past several decades, an increase of Noxious weeds has impacted much of Colorado's Front Range. As a result, weed control is a key component of the land stewardship program at Rocky Flats. Thirty-three species of state-listed Noxious weeds are known to occur in the Central and Peripheral Operable Units at Rocky Flats, along with another five species that are considered invasive at the site. Early detection and rapid response to control new invasive species is crucial to the program. An integrated weed control/vegetation management approach is key to maintaining healthy, sustainable plant communities that are able to resist Noxious weed invasions. Weed mapping, field surveys, and field-staff training sessions (to learn how to identify new potential problem species) are conducted to help detect and prevent new weed problems. The integrated approach at Rocky Flats includes administrative and cultural techniques (prevention), mechanical controls, biological controls, and chemical controls. Several species of biocontrol

  15. Chromosome 5 allelic losses are early events in tumours of the papilla of Vater and occur at sites similar to those of gastric cancer.

    PubMed Central

    Achille, A.; Baron, A.; Zamboni, G.; Di Pace, C.; Orlandini, S.; Scarpa, A.

    1998-01-01

    During our studies of DNA fingerprinting of tumours of the pancreas and papilla (ampulla) of Vater, using arbitrarily primed polymerase chain reaction (AP-PCR), we noticed two bands showing a decreased intensity in six of ten ampullary tumours with respect to matched normal tissues. Those bands were both assigned to chromosome 5. Such a finding was somewhat in contrast with the reportedly low frequency of APC gene mutations in ampullary cancers, located at chromosome 5q21, and suggested that loci different from that of APC might be the target of chromosome 5 allelic losses (LOH) in these tumours. Therefore, we analysed chromosome 5 LOH in a panel of 27 ampullary tumours, including eight adenomas, four early- and 15 advanced-stage cancers, using 16 PCR-amplified CA microsatellite polymorphic markers spanning the entire chromosome. Nineteen cases (70%) showed LOH, and the interstitial deletions found in these tumours described two smallest common deleted regions, in which putative suppressor genes might reside. They were at 5q13.3-q14 and at 5q23-q31 respectively, which correspond to those found in gastric tumours. In addition, the presence of 5q LOH in six of eight adenomas and in three of four early-stage cancers suggests that such phenomena occur at early stages of neoplastic progression of the ampullary epithelium. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:9862579

  16. Chromosome Analysis

    NASA Technical Reports Server (NTRS)

    1998-01-01

    Perceptive Scientific Instruments, Inc., provides the foundation for the Powergene line of chromosome analysis and molecular genetic instrumentation. This product employs image processing technology from NASA's Jet Propulsion Laboratory and image enhancement techniques from Johnson Space Center. Originally developed to send pictures back to earth from space probes, digital imaging techniques have been developed and refined for use in a variety of medical applications, including diagnosis of disease.

  17. Molecular cloning of a highly conserved mouse and human integral membrane protein (Itm1) and genetic mapping to mouse chromosome 9

    SciTech Connect

    Hong, Guizhu; Tylzanowski, P.; Deleersnijder, W.

    1996-02-01

    We have isolated and characterized a novel cDNA coding for a highly hydrophobic protein (B5) from a fetal mouse mandibular condyle cDNA library. The full-length mouse B5 cDNA is 3095 nucleotides long and contains a potential open reading frame coding for a protein of 705 amino acids with a calculated molecular weight of 80.5 kDa. The B5 mRNA is differentially polyadenylated, with the most abundant transcript having a length of 2.7 kb. The human homolog of B5 was isolated from a cDNA testis library. The predicted amino acid sequence of the human B5 is 98.5% identical to that of mouse. The most striking feature of the B5 protein is the presence of numerous (10-14) potential transmembrane domains, characteristic of an integral membrane protein. Similarity searches in public databanks reveal that B5 is 58% similar to the T12A2.2 gene of Caenorhabditis elegans and 60% similar to the STT3 gene of Saccharomyces cerevisiae. Futhermore, the report of an EST sequence (Accession No. Z13858) related to the human B5, but identical to the STT3 gene, indicates that B5 belongs to a larger gene family coding for novel putative transmembrane proteins. This family exhibits a remarkable degree of conservation in different species. The gene for B5, designated Itm1 (Integral membrane protein 1), is located on mouse chromosome 9. 28 refs., 4 figs.

  18. Lipofection of Purified Adeno-Associated Virus Rep68 Protein: toward a Chromosome-Targeting Nonviral Particle

    PubMed Central

    Lamartina, Stefania; Roscilli, Giuseppe; Rinaudo, Daniela; Delmastro, Paola; Toniatti, Carlo

    1998-01-01

    Adeno-associated virus (AAV) integrates very efficiently into a specific site (AAVS1) of human chromosome 19. Two elements of the AAV genome are sufficient: the inverted terminal repeats (ITRs) and the Rep78 or Rep68 protein. The incorporation of the AAV integration machinery in nonviral delivery systems is of great interest for gene therapy. We demonstrate that purified recombinant Rep68 protein is functionally active when directly delivered into human cells by using the polycationic liposome Lipofectamine, promoting the rescue-replication of a codelivered ITR-flanked cassette in adenovirus-infected cells and its site-specific integration in noninfected cells. The sequencing of cloned virus-host DNA junctions confirmed that lipofected Rep68 protein triggers site-specific integration at the same sites in chromosome 19 already characterized in cells latently infected with AAV. PMID:9696870

  19. Different integrated geophysical approaches to investigate archaeological sites in urban and suburban area.

    NASA Astrophysics Data System (ADS)

    Piro, Salvatore; Papale, Enrico; Zamuner, Daniela

    2016-04-01

    Geophysical methods are frequently used in archaeological prospection in order to provide detailed information about the presence of structures in the subsurface as well as their position and their geometrical reconstruction, by measuring variations of some physical properties. Often, due to the limited size and depth of an archaeological structure, it may be rather difficult to single out its position and extent because of the generally low signal-to-noise ratio. This problem can be overcome by improving data acquisition, processing techniques and by integrating different geophysical methods. In this work, two sites of archaeological interest, were investigated employing several methods (Ground Penetrating Radar (GPR), Electrical Resistivity Tomography (ERT), Fluxgate Differential Magnetic) to obtain precise and detailed maps of subsurface bodies. The first site, situated in a suburban area between Itri and Fondi, in the Aurunci Natural Regional Park (Central Italy), is characterized by the presence of remains of past human activity dating from the third century B.C. The second site, is instead situated in an urban area in the city of Rome (Basilica di Santa Balbina), where historical evidence is also present. The methods employed, allowed to determine the position and the geometry of some structures in the subsurface related to this past human activity. To have a better understanding of the subsurface, we then performed a qualitative and quantitative integration of this data, which consists in fusing the data from all the methods used, to have a complete visualization of the investigated area. Qualitative integration consists in graphically overlaying the maps obtained by the single methods; this method yields only images, not new data that may be subsequently analyzed. Quantitative integration is instead performed by mathematical and statistical solutions, which allows to have a more accurate reconstruction of the subsurface and generates new data with high

  20. Understanding Transcription Factor Regulation by Integrating Gene Expression and DNase I Hypersensitive Sites

    PubMed Central

    Wang, Guohua; Wang, Fang; Huang, Qian; Li, Yu; Liu, Yunlong; Wang, Yadong

    2015-01-01

    Transcription factors are proteins that bind to DNA sequences to regulate gene transcription. The transcription factor binding sites are short DNA sequences (5–20 bp long) specifically bound by one or more transcription factors. The identification of transcription factor binding sites and prediction of their function continue to be challenging problems in computational biology. In this study, by integrating the DNase I hypersensitive sites with known position weight matrices in the TRANSFAC database, the transcription factor binding sites in gene regulatory region are identified. Based on the global gene expression patterns in cervical cancer HeLaS3 cell and HelaS3-ifnα4h cell (interferon treatment on HeLaS3 cell for 4 hours), we present a model-based computational approach to predict a set of transcription factors that potentially cause such differential gene expression. Significantly, 6 out 10 predicted functional factors, including IRF, IRF-2, IRF-9, IRF-1 and IRF-3, ICSBP, belong to interferon regulatory factor family and upregulate the gene expression levels responding to the interferon treatment. Another factor, ISGF-3, is also a transcriptional activator induced by interferon alpha. Using the different transcription factor binding sites selected criteria, the prediction result of our model is consistent. Our model demonstrated the potential to computationally identify the functional transcription factors in gene regulation. PMID:26425553

  1. Hormone dependency of chromosome aberrations induced by 7,12-dimethylbenz(a)anthracene in rat bone marrow cells: site-specific increase by erythropoietin

    SciTech Connect

    Ueda, N.; Suglyama, T.; Chattopadhyay, S.C.; Goto-Mimura, K.; Maeda, S.

    1981-08-01

    The frequency of chromosome aberrations (CA) 6 hours after iv injection of 50 mg 7,12-dimethylbenz(a)anthracene (DMBA0/kg was studied in bone marrow cells of the noninbred Long-Evans rat under various hematopoietic conditions. The percentage of metaphase cells with CA was enhanced by anemia and suppressed by polycythemia. The low incidence of CA in polycythemic rats was reversed by 6 U of sheep erythropoietin (EP) injected at the time of DMBA treatment. The interchromosomal and intrachromosomal distribution of CA indicated that hematopoietic stimuli, more specifically EP, greatly enhanced DMBA-induced CA in specific chromosomal regions.

  2. PROSPER: an integrated feature-based tool for predicting protease substrate cleavage sites.

    PubMed

    Song, Jiangning; Tan, Hao; Perry, Andrew J; Akutsu, Tatsuya; Webb, Geoffrey I; Whisstock, James C; Pike, Robert N

    2012-01-01

    The ability to catalytically cleave protein substrates after synthesis is fundamental for all forms of life. Accordingly, site-specific proteolysis is one of the most important post-translational modifications. The key to understanding the physiological role of a protease is to identify its natural substrate(s). Knowledge of the substrate specificity of a protease can dramatically improve our ability to predict its target protein substrates, but this information must be utilized in an effective manner in order to efficiently identify protein substrates by in silico approaches. To address this problem, we present PROSPER, an integrated feature-based server for in silico identification of protease substrates and their cleavage sites for twenty-four different proteases. PROSPER utilizes established specificity information for these proteases (derived from the MEROPS database) with a machine learning approach to predict protease cleavage sites by using different, but complementary sequence and structure characteristics. Features used by PROSPER include local amino acid sequence profile, predicted secondary structure, solvent accessibility and predicted native disorder. Thus, for proteases with known amino acid specificity, PROSPER provides a convenient, pre-prepared tool for use in identifying protein substrates for the enzymes. Systematic prediction analysis for the twenty-four proteases thus far included in the database revealed that the features we have included in the tool strongly improve performance in terms of cleavage site prediction, as evidenced by their contribution to performance improvement in terms of identifying known cleavage sites in substrates for these enzymes. In comparison with two state-of-the-art prediction tools, PoPS and SitePrediction, PROSPER achieves greater accuracy and coverage. To our knowledge, PROSPER is the first comprehensive server capable of predicting cleavage sites of multiple proteases within a single substrate sequence using

  3. Mapping and ordered cloning of the human X chromosome

    SciTech Connect

    Caskey, C.T.; Nelson, D.L.

    1992-12-01

    Progress is reported on gathering X chromosome specific libraries and integrating those with the library produced in this project. Further studies on understanding Fragile X Syndrome and other hereditary diseases related to the X chromosome are described. (DT)

  4. Analysis of chromosome 21 yeast artificial chromosome (YAC) clones

    SciTech Connect

    Tassone, F. A. Gemelli School of Medicine, Rome ); Cheng, S.; Gardiner, K. )

    1992-12-01

    Chromosome 21 contains genes relevant to several important diseases. Yeast artificial chromosome (YAC) clones, because they span >100 kbp, will provide attractive material for initiating searches for such genes. Twenty-two YAC clones, each of which maps to a region of potential relevance either to aspects of the Down syndrome phenotype or to one of the other chromosome 21-associated genetic diseases, have been analyzed in detail. Clones total [approximately]6,000 kb and derive from all parts of the long arm. Rare restriction-site maps have been constructed for each clone and have been used to determine regional variations in clonability, methylation frequency, CpG island density, and CpG island frequency versus gene density. This information will be useful for the isolation and mapping of new genes to chromosome 21 and for walking in YAC libraries. 48 refs., 3 figs., 4 tabs.

  5. Hanford Integrated Planning Process: 1993 Hanford Site-specific science and technology plan

    SciTech Connect

    Not Available

    1993-12-01

    This document is the FY 1993 report on Hanford Site-specific science and technology (S&T) needs for cleanup of the Site as developed via the Hanford Integrated Planning Process (HIPP). It identifies cleanup problems that lack demonstrated technology solutions and technologies that require additional development. Recommendations are provided regarding allocation of funding to address Hanford`s highest-priority technology improvement needs, technology development needs, and scientific research needs, all compiled from a Sitewide perspective. In the past, the S&T agenda for Hanford Site cleanup was sometimes driven by scientists and technologists, with minimal input from the ``problem owners`` (i.e., Westinghouse Hanford Company [WHC] staff who are responsible for cleanup activities). At other times, the problem-owners made decisions to proceed with cleanup without adequate scientific and technological inputs. Under both of these scenarios, there was no significant stakeholder involvement in the decision-making process. One of the key objectives of HIPP is to develop an understanding of the integrated S&T requirements to support the cleanup mission, (a) as defined by the needs of the problem owners, the values of the stakeholders, and the technology development expertise that exists at Hanford and elsewhere. This requires a periodic, systematic assessment of these needs and values to appropriately define a comprehensive technology development program and a complementary scientific research program. Basic to our success is a methodology that is defensible from a technical perspective and acceptable to the stakeholders.

  6. Transient Microgeographic Clines during B Chromosome Invasion.

    PubMed

    Camacho, Juan Pedro M; Shaw, Michael W; Cabrero, Josefa; Bakkali, Mohammed; Ruíz-Estévez, Mercedes; Ruíz-Ruano, Francisco J; Martín-Blázquez, Rubén; López-León, María Dolores

    2015-11-01

    The near-neutral model of B chromosome evolution predicts that the invasion of a new population should last some tens of generations, but the details on how it proceeds in real populations are mostly unknown. Trying to fill this gap, we analyze here a natural population of the grasshopper Eyprepocnemis plorans at three time points during the last 35 years. Our results show that B chromosome frequency increased significantly during this period and that a cline observed in 1992 had disappeared in 2012 once B chromosome frequency reached an upper limit at all sites sampled. This indicates that, during B chromosome invasion, transient clines for B chromosome frequency are formed at the invasion front on a microgeographic scale. Computer simulation experiments showed that the pattern of change observed for genotypic frequencies is consistent with the existence of B chromosome drive through females and selection against individuals with a high number of B chromosomes. PMID:26655780

  7. Chromosome 19 International Workshop

    SciTech Connect

    Pericak-Vance, M.A. . Medical Center); Ropers, H.H. . Dept. of Human Genetics); Carrano, A.J. )

    1993-01-04

    The Second International Workshop on Human Chromosome 19 was hosted on January 25 and 26, 1992, by the Department of Human Genetics, University Hospital Nijmegen, The Netherlands, at the 'Meerdal Conference Center'. The workshop was supported by a grant from the European Community obtained through HUGO, the Dutch Research Organization (NWO) and the Muscular Dystrophy Association (MDA). Travel support for American participants was provided by the Department of Energy. The goals of this workshop were to produce genetic, physical and integrated maps of chromosome 19, to identify inconsistencies and gaps, and to discuss and exchange resources and techniques available for the completion of these maps. The second day of the meeting was largely devoted to region or disease specific efforts. In particular, the meeting served as a platform for assessing and discussing the recent progress made into the molecular elucidation of myotonic dystrophy.

  8. Icebreaker-3 Drill Integration and Testing at Two Mars-Analog Sites

    NASA Technical Reports Server (NTRS)

    Glass, B.; Bergman, D.; Yaggi, B.; Dave, A.; Zacny, K.

    2016-01-01

    A decade of evolutionary development of integrated automated drilling and sample handling at analog sites and in test chambers has made it possible to go 1 meter through hard rocks and ice layers on Mars. The latest Icebreaker-3 drill has been field tested in 2014 at the Haughton Crater Marsanalog site in the Arctic and in 2015 with a Mars lander mockup in Rio Tinto, Spain, (with sample transfer arm and with a prototype life-detection instrument). Tests in Rio Tinto in 2015 successfully demonstrated that the drill sample (cuttings) was handed-off from the drill to the sample transfer arm and thence to the on-deck instrument inlet where it was taken in and analyzed ("dirt-to-data").

  9. 300 Area Integrated Field-Scale Subsurface Research Challenge (IFRC) Field Site Management Plan

    SciTech Connect

    Freshley, Mark D.

    2008-12-31

    Pacific Northwest National Laboratory (PNNL) has established the 300 Area Integrated Field-Scale Subsurface Research Challenge (300 Area IFRC) on the Hanford Site in southeastern Washington State for the U.S. Department of Energy’s (DOE) Office of Biological and Environmental Research (BER) within the Office of Science. The project is funded by the Environmental Remediation Sciences Division (ERSD). The purpose of the project is to conduct research at the 300 IFRC to investigate multi-scale mass transfer processes associated with a subsurface uranium plume impacting both the vadose zone and groundwater. The management approach for the 300 Area IFRC requires that a Field Site Management Plan be developed. This is an update of the plan to reflect the installation of the well network and other changes.

  10. MAC Europe 1991 campaign: AIRSAR/AVIRIS data integration for agricultural test site classification

    NASA Technical Reports Server (NTRS)

    Sangiovanni, S.; Buongiorno, M. F.; Ferrarini, M.; Fiumara, A.

    1993-01-01

    During summer 1991, multi-sensor data were acquired over the Italian test site 'Otrepo Pavese', an agricultural flat area in Northern Italy. This area has been the Telespazio pilot test site for experimental activities related to agriculture applications. The aim of the investigation described in the following paper is to assess the amount of information contained in the AIRSAR (Airborne Synthetic Aperture Radar) and AVIRIS (Airborne Visible/Infrared Imaging Spectrometer) data, and to evaluate classification results obtained from each sensor data separately and from the combined dataset. All classifications are examined by means of the resulting confusion matrices and Khat coefficients. Improvements of the classification results obtained by using the integrated dataset are finally evaluated.

  11. Analysis of a fuel cell on-site integrated energy system for a residential complex

    NASA Technical Reports Server (NTRS)

    Simons, S. N.; Maag, W. L.

    1979-01-01

    Declining supplies of domestic oil and gas and the increased cost of energy resulted in a renewed emphasis in utilizing available resources in the most efficient manner possible. This, in turn, brought about a reassessment of a number of methods for converting fossil fuels to end uses at the highest practical efficiency. One of these is the on-site integrated energy system (OS/IES). This system provides electric power from an on-site power plant and recovers heat from the power plant that would normally be rejected to the environment. An OS/IES is potentially useful in any application that requires both electricity and heat. Several OS/IES are analyzed for a residential complex. The paper is divided into two sections; the first compares three energy supply systems, the second compares various designs for fuel cell OS/IES.

  12. Radionuclide disequilibria studies for investigating the integrity of potential nuclear waste disposal sites: subseabed studies.

    SciTech Connect

    Laul, J.C.; Thomas, C.W.; Petersen, M.R.; Perkins, R.W.

    1981-09-01

    This study of subseabed sediments indicates that natural radionuclides can be employed to define past long-term migration rates and thereby evaluate the integrity of potential disposal sites in ocean sediments. The study revealed the following conclusions: (1) the sedimentation rate of both the long and short cores collected in the North Pacific is 2.5 mm/1000 yr or 2.5 m/m.yr in the upper 3 meters; (2) the sedimentation rate has been rather constant over the last one million years; and (3) slow diffusive processes dominate within the sediment. Reworking of the sediment by physical processes or organisms is not observed.

  13. Site-controlled Ag nanocrystals grown by molecular beam epitaxy-Towards plasmonic integration technology

    SciTech Connect

    Urbanczyk, Adam; Noetzel, Richard

    2012-12-15

    We demonstrate site-controlled growth of epitaxial Ag nanocrystals on patterned GaAs substrates by molecular beam epitaxy with high degree of long-range uniformity. The alignment is based on lithographically defined holes in which position controlled InAs quantum dots are grown. The Ag nanocrystals self-align preferentially on top of the InAs quantum dots. No such ordering is observed in the absence of InAs quantum dots, proving that the ordering is strain-driven. The presented technique facilitates the placement of active plasmonic nanostructures at arbitrarily defined positions enabling their integration into complex devices and plasmonic circuits.

  14. D Integrated Methodologies for the Documentation and the Virtual Reconstruction of AN Archaeological Site

    NASA Astrophysics Data System (ADS)

    Balletti, C.; Guerra, F.; Scocca, V.; Gottardi, C.

    2015-02-01

    Highly accurate documentation and 3D reconstructions are fundamental for analyses and further interpretations in archaeology. In the last years the integrated digital survey (ground-based survey methods and UAV photogrammetry) has confirmed its main role in the documentation and comprehension of excavation contexts, thanks to instrumental and methodological development concerning the on site data acquisition. The specific aim of the project, reported in this paper and realized by the Laboratory of Photogrammetry of the IUAV University of Venice, is to check different acquisition systems and their effectiveness test, considering each methodology individually or integrated. This research focuses on the awareness that the integration of different survey's methodologies can as a matter of fact increase the representative efficacy of the final representations; these are based on a wider and verified set of georeferenced metric data. Particularly the methods' integration allows reducing or neutralizing issues related to composite and complex objects' survey, since the most appropriate tools and techniques can be chosen considering the characteristics of each part of an archaeological site (i.e. urban structures, architectural monuments, small findings). This paper describes the experience in several sites of the municipality of Sepino (Molise, Italy), where the 3d digital acquisition of cities and structure of monuments, sometimes hard to reach, was realized using active and passive techniques (rage-based and image based methods). This acquisition was planned in order to obtain not only the basic support for interpretation analysis, but also to achieve models of the actual state of conservation of the site on which some reconstructive hypotheses can be based on. Laser scanning data were merged with Structure from Motion techniques' clouds into the same reference system, given by a topographical and GPS survey. These 3d models are not only the final results of the metric

  15. Fuel cell on-site integrated energy system parametric analysis of a residential complex

    NASA Technical Reports Server (NTRS)

    Simons, S. N.

    1977-01-01

    A parametric energy-use analysis was performed for a large apartment complex served by a fuel cell on-site integrated energy system (OS/IES). The variables parameterized include operating characteristics for four phosphoric acid fuel cells, eight OS/IES energy recovery systems, and four climatic locations. The annual fuel consumption for selected parametric combinations are presented and a breakeven economic analysis is presented for one parametric combination. The results show fuel cell electrical efficiency and system component choice have the greatest effect on annual fuel consumption; fuel cell thermal efficiency and geographic location have less of an effect.

  16. Effects of chromosomal gene copy number and locations on polyhydroxyalkanoate synthesis by Escherichia coli and Halomonas sp.

    PubMed

    Yin, Jin; Wang, Huan; Fu, Xiao-Zhi; Gao, Xue; Wu, Qiong; Chen, Guo-Qiang

    2015-07-01

    Chromosomal integration and expression of heterologous gene(s) are favored in industrial biotechnology due to the inheriting expression stability. Yet, chromosomal expression is commonly weaker than plasmid one. The effect on gene expression level at 13 chromosomal locations in Escherichia coli was investigated using the polyhydroxybutyrate (PHB) synthesis pathway encoded by a phaCAB operon as a reporter. When 11 copies of phaCAB were randomly integrated into 11 of the 13 chromosomal locations, respectively, 5.2 wt% of PHB was produced. PHB (34.1 wt%) was accumulated by a recombinant E. coli inserted chromosomally with 50 copies of phaCAB in the active asnB site using a Cre-loxP recombination method. This PHB accumulation level was equivalent to a medium-copy-number plasmid expression system, suggesting the importance of chromosomal gene copy number for PHB production by E. coli. This result was used to manipulate a Halomonas strain. One copy of genes scpAB encoding methylmalonyl-CoA mutase and methylmalonyl-CoA decarboxylase was inserted into the strongest expression site porin in the chromosome of the 2-methylcitrate synthase (prpC) deleted mutant Halomonas TD08, leading to the synthesis of poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) from glucose as the sole carbon source. The chromosome-engineered strain produced PHBV consisting of 5-12 mol% 3-hydroxyvalerate (3HV) stably compared with unstable fluctuation of 7-25 mol% 3HV by a medium-copy-number plasmid system. These results demonstrated that chromosome engineering based on active transcriptional site and gene copy number is more feasible for polyhydroxyalkanoate (PHA) synthesis in Halomonas TD08 compared with in E. coli. PMID:25758961

  17. The EuroSITES network: Integrating and enhancing fixed-point open ocean observatories around Europe

    NASA Astrophysics Data System (ADS)

    Lampitt, Richard S.; Larkin, Kate E.; EuroSITES Consortium

    2010-05-01

    EuroSITES is a 3 year (2008-2011) EU collaborative project (3.5MEuro) with the objective to integrate and enhance the nine existing open ocean fixed point observatories around Europe (www.eurosites.info). These observatories are primarily composed of full depth moorings and make multidisciplinary in situ observations within the water column as the European contribution to the global array OceanSITES (www.oceansites.org). In the first 18 months, all 9 observatories have been active and integration has been significant through the maintenance and enhancement of observatory hardware. Highlights include the enhancement of observatories with sensors to measure O2, pCO2, chlorophyll, and nitrate in near real-time from the upper 1000 m. In addition, some seafloor missions are also actively supported. These include seafloor platforms currently deployed in the Mediterranean, one for tsunami detection and one to monitor fluid flow related to seismic activity and slope stability. Upcoming seafloor science missions in 2010 include monitoring benthic biological communities and associated biogeochemistry as indicators of climate change in both the Northeast Atlantic and Mediterranean. EuroSITES also promotes the development of innovative sensors and samplers in order to progress capability to measure climate-relevant properties of the ocean. These include further developing current technologies for autonomous long-term monitoring of oxygen consumption in the mesopelagic, pH and mesozooplankton abundance. Many of these science missions are directly related to complementary activities in other European projects such as EPOCA, HYPOX and ESONET. In 2010 a direct collaboration including in situ field work will take place between ESONET and EuroSITES. The demonstration mission MODOO (funded by ESONET) will be implemented in 2010 at the EuroSITES PAP observatory. Field work will include deployment of a seafloor lander system with various sensors which will send data to shore in real

  18. Integrated observation and analysis of pre-earthquake related signals over major geohazard sites

    NASA Astrophysics Data System (ADS)

    Ouzounov, Dimitar; Pulinets, Sergey; Tramutoli, Valerio; Lee, Lou; Liu, Tiger; Hayakawa, Masashi; Hattori, Katsumi; Kafatos, Menas; Taylor, Patrick

    2013-04-01

    We are conducting an integrated study involving multi-parameter observations in our investigation of phenomena proceeding major earthquakes. Our approach is based on a systematic analysis of several parameters namely: foreshock seismic activities; gas discharge; thermal infrared radiation; outgoing radiation flux; ionospheric electron density; VLF sub-ionospheric propagation anomalies; and atmospheric temperature and humidity which we propose are associated with earthquakes, For the first time we intend to have a similar set of integrated geophysical measurements (in-situ and satellite) over regions of active earthquakes and volcanoes. We are in the process of establishing an iSITES (Integrated sites) framework for coordinate measurements, data sharing and validation. So far we include six regions: Southern California (USA), Eastern Honshu (Japan), Central and Southern Italy, Taiwan (ROC), Corinth rift (Greece), Kamchatka and Sakhalin (Russia). We are continuing the same approach of near real-time information sharing established under the EU-FP7 PRE-EARTHQUAKE project. In the future we may well coordinate this effort with GEOSS supersite site project. This provides a new opportunity to cross validate our results with the dense networks of in-situ and space measurements. Initially we tested the iSITES observational data in two different tectonic regions, first with recent large earthquakes, viz.- Tohoku-oki (M9, 2011, Japan) and Emilia (M5.9, 2012, N. Italy), and L'Aquia (2009, Central Italy) regions. Our retrospective analyses of these satellite data have shown the presence of anomalies in the atmosphere. Second, we did a retrospective analysis to check the re-occurrence of similar anomalous behavior over Taiwan, Japan and Kamchatka, which include 40 major earthquakes (M>5.9) for the period of 2005-2009. We found anomalous behavior before all of these events with no false negatives; false positives were less then 20%. Our initial results suggest that iSITES

  19. Use of homologous expression-secretion signals and vector-free stable chromosomal integration in engineering of Lactobacillus plantarum for alpha-amylase and levanase expression.

    PubMed Central

    Hols, P; Ferain, T; Garmyn, D; Bernard, N; Delcour, J

    1994-01-01

    The genuine alpha-amylase gene from Bacillus licheniformis (amyL) is not expressed in Lactobacillus plantarum, but replacement of the amyL promoter by a strong L. plantarum promoter leads to efficient expression of the gene and secretion of more than 90% of the alpha-amylase into the culture supernatant. A series of L. plantarum genetic cassettes (transcription and translation with or without secretion) were cloned by translation fusion of random DNA fragments to the silent amyL coding frame in the pGIP212 probe vector (P. Hols, A. Baulard, D. Garmyn, B. Delplace, S. Hogan, and J. Delcour, Gene 118:21-30, 1992). Five different cassettes were sequenced and found to harbor genetic signals similar to those of other gram-positive bacteria. The functions of the cloned cassettes and the cassettes isolated previously from Enterococcus faecalis were compared in E. faecalis and L. plantarum, respectively. All signals were well recognized in L. plantarum, but cassettes isolated from L. plantarum led to a low level of amylase production in E. faecalis, suggesting that the L. plantarum signals are more species specific. Six transcriptional or translational fusions were constructed to express the Bacillus subtilis levanase gene (sacC) in L. plantarum. All of these constructions were capable of inducing levanase production and secretion in the culture supernatant, and, furthermore, L. plantarum strains harboring the most efficient fusions could grow in MRS medium containing inulin as the major carbon source. Finally, a two-step chromosomal integration procedure was used to achieve efficient stabilization of an amylase construction without any residual resistance marker or vector sequence. Images PMID:8017927

  20. Integrating care for high-risk patients in England using the virtual ward model: lessons in the process of care integration from three case sites

    PubMed Central

    Lewis, Geraint; Vaithianathan, Rhema; Wright, Lorraine; Brice, Mary R; Lovell, Paul; Rankin, Seth; Bardsley, Martin

    2013-01-01

    Background Patients at high risk of emergency hospitalisation are particularly likely to experience fragmentation in care. The virtual ward model attempts to integrate health and social care by offering multidisciplinary case management to people at high predicted risk of unplanned hospitalisation. Objective To describe the care practice in three virtual ward sites in England and to explore how well each site had achieved meaningful integration. Method Case studies conducted in Croydon, Devon and Wandsworth during 2011–2012, consisting of semi-structured interviews, workshops, and site visits. Results Different versions of the virtual wards intervention had been implemented in each site. In Croydon, multidisciplinary care had reverted back to one-to-one case management. Conclusions To integrate successfully, virtual ward projects should safeguard the multidisciplinary nature of the intervention, ensure the active involvement of General Practitioners, and establish feedback processes to monitor performance such as the number of professions represented at each team meeting. PMID:24250284

  1. Integration of genotoxicity and population genetic analyses in kangaroo rats (Dipodomys merriami) exposed to radionuclide contamination at the Nevada Test Site, USA

    USGS Publications Warehouse

    Theodorakis, Christopher W.; Bickham, John W.; Lamb, Trip; Medica, Philip A.; Lyne, T. Barrett

    2001-01-01

    We examined effects of radionuclide exposure at two atomic blast sites on kangaroo rats (Dipodomys merriami) at the Nevada Test Site, Nevada, USA, using genotoxicity and population genetic analyses. We assessed chromosome damage by micronucleus and flow cytometric assays and genetic variation by randomly amplified polymorphic DNA (RAPD) and mitochondrial DNA (mtDNA) analyses. The RAPD analysis showed no population structure, but mtDNA exhibited differentiation among and within populations. Genotoxicity effects were not observed when all individuals were analyzed. However, individuals with mtDNA haplotypes unique to the contaminated sites had greater chromosomal damage than contaminated-site individuals with haplotypes shared with reference sites. When interpopulation comparisons used individuals with unique haplotypes, one contaminated site had greater levels of chromosome damage than one or both of the reference sites. We hypothesize that shared-haplotype individuals are potential migrants and that unique-haplotype individuals are potential long-term residents. A parsimony approach was used to estimate the minimum number of migration events necessary to explain the haplotype distributions on a phylogenetic tree. The observed predominance of migration events into the contaminated sites supported our migration hypothesis. We conclude the atomic blast sites are ecological sinks and that immigration masks the genotoxic effects of radiation on the resident populations.

  2. Deciphering evolutionary strata on plant sex chromosomes and fungal mating-type chromosomes through compositional segmentation.

    PubMed

    Pandey, Ravi S; Azad, Rajeev K

    2016-03-01

    Sex chromosomes have evolved from a pair of homologous autosomes which differentiated into sex determination systems, such as XY or ZW system, as a consequence of successive recombination suppression between the gametologous chromosomes. Identifying the regions of recombination suppression, namely, the "evolutionary strata", is central to understanding the history and dynamics of sex chromosome evolution. Evolution of sex chromosomes as a consequence of serial recombination suppressions is well-studied for mammals and birds, but not for plants, although 48 dioecious plants have already been reported. Only two plants Silene latifolia and papaya have been studied until now for the presence of evolutionary strata on their X chromosomes, made possible by the sequencing of sex-linked genes on both the X and Y chromosomes, which is a requirement of all current methods that determine stratum structure based on the comparison of gametologous sex chromosomes. To circumvent this limitation and detect strata even if only the sequence of sex chromosome in the homogametic sex (i.e. X or Z chromosome) is available, we have developed an integrated segmentation and clustering method. In application to gene sequences on the papaya X chromosome and protein-coding sequences on the S. latifolia X chromosome, our method could decipher all known evolutionary strata, as reported by previous studies. Our method, after validating on known strata on the papaya and S. latifolia X chromosome, was applied to the chromosome 19 of Populus trichocarpa, an incipient sex chromosome, deciphering two, yet unknown, evolutionary strata. In addition, we applied this approach to the recently sequenced sex chromosome V of the brown alga Ectocarpus sp. that has a haploid sex determination system (UV system) recovering the sex determining and pseudoautosomal regions, and then to the mating-type chromosomes of an anther-smut fungus Microbotryum lychnidis-dioicae predicting five strata in the non

  3. HIM-8 Binds to the X Chromosome Pairing Center and Mediates Chromosome-Specific Meiotic Synapsis

    PubMed Central

    Phillips, Carolyn M.; Wong, Chihunt; Bhalla, Needhi; Carlton, Peter M.; Weiser, Pinky; Meneely, Philip M.; Dernburg, Abby F.

    2015-01-01

    SUMMARY The him-8 gene is essential for proper meiotic segregation of the X chromosomes in C. elegans. Here we show that loss of him-8 function causes profound X chromosome-specific defects in homolog pairing and synapsis. him-8 encodes a C2H2 zinc-finger protein that is expressed during meiosis and concentrates at a site on the X chromosome known as the meiotic pairing center (PC). A role for HIM-8 in PC function is supported by genetic interactions between PC lesions and him-8 mutations. HIM-8 bound chromosome sites associate with the nuclear envelope (NE) throughout meiotic prophase. Surprisingly, a point mutation in him-8 that retains both chromosome binding and NE localization fails to stabilize pairing or promote synapsis. These observations indicate that stabilization of homolog pairing is an active process in which the tethering of chromosome sites to the NE may be necessary but is not sufficient. PMID:16360035

  4. Development of an enhanced chromosomal expression system based on porin synthesis operon for halophile Halomonas sp.

    PubMed

    Yin, Jin; Fu, Xiao-Zhi; Wu, Qiong; Chen, Jin-Chun; Chen, Guo-Qiang

    2014-11-01

    Since halophile Halomonas spp. can grow contamination free in seawater under unsterile and continuous conditions, it holds great promise for industrial biotechnology to produce low-cost chemicals in an economic way. Yet, metabolic engineering methods are urgently needed for Halomonas spp. It is commonly known that chromosomal expression is more stable yet weaker than plasmid one is. To overcome this challenge, a novel chromosomal expression method was developed for halophile Halomonas TD01 and its derivatives based on a strongly expressed porin gene as a site for external gene integration. The gene of interest was inserted downstream the porin gene, forming an artificial operon porin-inserted gene. This chromosome expression system was proven functional by some examples: First, chromosomal expression of heterologous polyhydroxybutyrate (PHB) synthase gene phaC Re from Ralstonia eutropha completely restored the PHB accumulation level in endogenous phaC knockout mutant of Halomonas TD01. The integrated phaC Re was expressed at the highest level when inserted at the locus of porin compared with insertions in other chromosome locations. Second, an inducible expression system was constructed in phaC-deleted Halomonas TD01 by integrating the lac repressor gene (lacI) into the porin site in the host chromosome. The native porin promoter was inserted with the key 21 bp DNA of lac operator (lacO) sequence to become an inducible promoter encoded in a plasmid. This inducible system allowed on-off switch of gene expression in Halomonas TD strains. Thus, the stable and strong chromosomal expression method in Halomonas TD spp. was established. PMID:25070598

  5. Integrated Geophysycal Prospecting in Late Antiquity and Early Medieval Sites in Italy

    NASA Astrophysics Data System (ADS)

    Giannotta, Maria Teresa; Leucci, Giovanni; De Giorgi, Lara; Matera, Loredana; Persico, Raffaele; Muci, Giuseppe

    2016-04-01

    In this contribution, the results of some integrated geophysical prospecting (magnetometric and GPR) are exposed. This work has been performed in collaboration between archaeologists and geophysicists within the research project "History and Global Archaeology of the Rural Landascapes in Italy, between Late Antiquity and Medieval period. Integrated systems of sources, methodologies, and technologies for a sustainable development", financed by the Italian Ministry for Instruction, University and Research MIUR. In particular, the archaeological sites of Badia and San Giovanni in Malcantone, both in the Apulia Region (eastern-southern Italy) have been prospect. The sites have been identified on the basis of available documents, archaeological surveys and testimonies. In particular, we know that in Badia [1] it was probable the presence of an ancient roman villa of the late ancient period (strongly damaged by the subsequent ploughing activities). Whereas in San Giovanni there is still, today, a small chapel (deconsecrated) that was likely to be part of a previous larger church (probably a basilica of the early Christian period) restricted in the subsequent centuries (probably in more phases). The Saracen raids of the XVI centuries made the site ruined and abandoned. In both sites integrated prospecting have been performed [2-6] with a the integration of archaeological, magnetometer and a GPR data have provided some interesting results, allowing to overcome the difficulties relative to an extensive GPR prospecting, that could not be performed because of the intrinsic superficial roughness and/or the intensive ploughing activities. The prospecting activities, in particular, have added elements that seem to confirm the main archaeological hypothesis that motivate their performing, as it will be show at the conference. References [1] M. T, Giannotta, G. Leucci, R. Persico, M. Leo Imperiale, The archaeological site of Badia in terra d'Otranto: contribution of the

  6. LASAGNA-Search: an integrated web tool for transcription factor binding site search and visualization.

    PubMed

    Lee, Chic; Huang, Chun-Hsi

    2013-03-01

    The release of ChIP-seq data from the ENCyclopedia Of DNA Elements (ENCODE) and Model Organism ENCyclopedia Of DNA Elements (modENCODE) projects has significantly increased the amount of transcription factor (TF) binding affinity information available to researchers. However, scientists still routinely use TF binding site (TFBS) search tools to scan unannotated sequences for TFBSs, particularly when searching for lesser-known TFs or TFs in organisms for which ChIP-seq data are unavailable. The sequence analysis often involves multiple steps such as TF model collection, promoter sequence retrieval, and visualization; thus, several different tools are required. We have developed a novel integrated web tool named LASAGNA-Search that allows users to perform TFBS searches without leaving the web site. LASAGNA-Search uses the LASAGNA (Length-Aware Site Alignment Guided by Nucleotide Association) algorithm for TFBS alignment. Important features of LASAGNA-Search include (i) acceptance of unaligned variable-length TFBSs, (ii) a collection of 1726 TF models, (iii) automatic promoter sequence retrieval, (iv) visualization in the UCSC Genome Browser, and (v) gene regulatory network inference and visualization based on binding specificities. LASAGNA-Search is freely available at http://biogrid.engr.uconn.edu/lasagna_search/. PMID:23599922

  7. Research plan for integrated ecosystem and pollutant monitoring at remote wilderness study sites

    SciTech Connect

    Bruns, D.A.; Wiersma, G.B.

    1988-03-01

    This research plan outlines an approach to the measurement of pollutants and ecosystem parameters at remote, high-elevation, wilderness study sites. A multimedia, systems approach to environmental monitoring is emphasized. The primary purpose of the research is to apply and field test a technical report entitled ''Guidelines for measuring the physical, chemical, and biological condition of wilderness ecosystems.'' This document intended to provide Federal Land Managers with information to establish environmental monitoring programs in wilderness areas. To date, this monitoring document has yet to be evaluated under rigorous field conditions at a remote, high-elevation Rocky Mountain site. For the purpose of field testing approaches to monitoring of pollutants and ecosystems in remote, wilderness areas, evaluation criteria were developed. These include useability, cost-effectiveness, data variability, alternative approaches, ecosystems conceptual approach, and quality assurance. Both the Forest Service and INEL environmental monitoring techniques will be evaluated with these criteria. Another objective of this research plan is to obtain an integrated data base on pollutants and ecosystem structure and function at a remote study site. The methods tested in this project will be used to acquire these data from a systems approach. This includes multimedia monitoring of air and water quality, soils, and forest, stream, and lake ecosystems. 71 refs., 1 fig., 9 tabs.

  8. Discovery of Supernumerary B Chromosomes in Drosophila melanogaster

    PubMed Central

    Bauerly, Elisabeth; Hughes, Stacie E.; Vietti, Dana R.; Miller, Danny E.; McDowell, William; Hawley, R. Scott

    2014-01-01

    B chromosomes are small, heterochromatic chromosomes that are transmitted in a non-Mendelian manner. We have identified a stock of Drosophila melanogaster that recently (within the last decade) acquired an average of 10 B chromosomes per fly. These B chromosomes are transmitted by both males and females and can be maintained for multiple generations in a wild-type genetic background despite the fact that they cause high levels of 4th chromosome meiotic nondisjunction in females. Most curiously, these B chromosomes are mitotically unstable, suggesting either the absence of critical chromosomal sites or the inability of the meiotic or mitotic systems to cope with many additional chromosomes. These B chromosomes also contain centromeres and are primarily composed of the heterochromatic AATAT satellite sequence. Although the AATAT sequence comprises the majority of the 4th chromosome heterochromatin, the B chromosomes lack most, if not all, 4th chromosome euchromatin. Presumably as a consequence of their heterochromatic content, these B chromosomes significantly modify position-effect variegation in two separate reporter systems, acting as enhancers of variegation in one case and suppressors in the other. The identification of B chromosomes in a genetically tractable organism like D. melanogaster will facilitate studies of chromosome evolution and the analysis of the mechanisms by which meiotic and mitotic processes cope with additional chromosomes. PMID:24478336

  9. Computational model for chromosomal instabilty

    NASA Astrophysics Data System (ADS)

    Zapperi, Stefano; Bertalan, Zsolt; Budrikis, Zoe; La Porta, Caterina

    2015-03-01

    Faithful segregation of genetic material during cell division requires alignment of the chromosomes between the spindle poles and attachment of their kinetochores to each of the poles. Failure of these complex dynamical processes leads to chromosomal instability (CIN), a characteristic feature of several diseases including cancer. While a multitude of biological factors regulating chromosome congression and bi-orientation have been identified, it is still unclear how they are integrated into a coherent picture. Here we address this issue by a three dimensional computational model of motor-driven chromosome congression and bi-orientation. Our model reveals that successful cell division requires control of the total number of microtubules: if this number is too small bi-orientation fails, while if it is too large not all the chromosomes are able to congress. The optimal number of microtubules predicted by our model compares well with early observations in mammalian cell spindles. Our results shed new light on the origin of several pathological conditions related to chromosomal instability.

  10. Chromosome Architecture and Genome Organization

    PubMed Central

    Bernardi, Giorgio

    2015-01-01

    How the same DNA sequences can function in the three-dimensional architecture of interphase nucleus, fold in the very compact structure of metaphase chromosomes and go precisely back to the original interphase architecture in the following cell cycle remains an unresolved question to this day. The strategy used to address this issue was to analyze the correlations between chromosome architecture and the compositional patterns of DNA sequences spanning a size range from a few hundreds to a few thousands Kilobases. This is a critical range that encompasses isochores, interphase chromatin domains and boundaries, and chromosomal bands. The solution rests on the following key points: 1) the transition from the looped domains and sub-domains of interphase chromatin to the 30-nm fiber loops of early prophase chromosomes goes through the unfolding into an extended chromatin structure (probably a 10-nm “beads-on-a-string” structure); 2) the architectural proteins of interphase chromatin, such as CTCF and cohesin sub-units, are retained in mitosis and are part of the discontinuous protein scaffold of mitotic chromosomes; 3) the conservation of the link between architectural proteins and their binding sites on DNA through the cell cycle explains the “mitotic memory” of interphase architecture and the reversibility of the interphase to mitosis process. The results presented here also lead to a general conclusion which concerns the existence of correlations between the isochore organization of the genome and the architecture of chromosomes from interphase to metaphase. PMID:26619076

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

    PubMed Central

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

    2015-01-01

    Oncoviruses cause tremendous global cancer burden. For several DNA tumor viruses, human genome integration is consistently associated with cancer development. However, genomic features associated with tumor viral integration are poorly understood. We sought to define genomic determinants for 1897 loci prone to hosting human papillomavirus (HPV), hepatitis B virus (HBV) or Merkel cell polyomavirus (MCPyV). These were compared to HIV, whose enzyme-mediated integration is well understood. A comprehensive catalog of integration sites was constructed from the literature and experimentally-determined HPV integration sites. Features were scored in eight categories (genes, expression, open chromatin, histone modifications, methylation, protein binding, chromatin segmentation and repeats) and compared to random loci. Random forest models determined loci classification and feature selection. HPV and HBV integrants were not fragile site associated. MCPyV preferred integration near sensory perception genes. Unique signatures of integration-associated predictive genomic features were detected. Importantly, repeats, actively-transcribed regions and histone modifications were common tumor viral integration signatures. PMID:26569308

  12. Relationships between chromosome structure and chromosomal aberrations

    NASA Astrophysics Data System (ADS)

    Eidelman, Yuri; Andreev, Sergey

    An interphase nucleus of human lymphocyte was simulated by the novel Monte Carlo tech-nique. The main features of interphase chromosome structure and packaging were taken into account: different levels of chromatin organisation; nonrandom localisation of chromosomes within a nucleus; chromosome loci dynamics. All chromosomes in a nucleus were modelled as polymer globules. A dynamic pattern of intra/interchromosomal contacts was simulated. The detailed information about chromosomal contacts, such as distribution of intrachromoso-mal contacts over the length of each chromosome and dependence of contact probability on genomic separation between chromosome loci, were calculated and compared to the new exper-imental data obtained by the Hi-C technique. Types and frequencies of simple and complex radiation-induced chromosomal exchange aberrations (CA) induced by X-rays were predicted with taking formation and decay of chromosomal contacts into account. Distance dependence of exchange formation probability was calculated directly. mFISH data for human lymphocytes were analysed. The calculated frequencies of simple CA agreed with the experimental data. Complex CA were underestimated despite the dense packaging of chromosome territories within a nucleus. Possible influence of chromosome-nucleus structural organisation on the frequency and spectrum of radiation-induced chromosome aberrations is discussed.

  13. Retroviral Insertions in Evi12, a Novel Common Virus Integration Site Upstream of Tra1/Grp94, Frequently Coincide with Insertions in the Gene Encoding the Peripheral Cannabinoid Receptor Cnr2

    PubMed Central

    Valk, Peter J. M.; Vankan, Yolanda; Joosten, Marieke; Jenkins, Nancy A.; Copeland, Neal G.; Löwenberg, Bob; Delwel, Ruud

    1999-01-01

    The common virus integration site (VIS) Evi11 was recently identified within the gene encoding the hematopoietic G-protein-coupled peripheral cannabinoid receptor Cnr2 (also referred to as Cb2). Here we show that Cnr2 is a frequent target (12%) for insertion of Cas-Br-M murine leukemia virus (MuLV) in primary tumors in NIH/Swiss mice. Multiple provirus insertions in Evi11 were cloned and shown to be located within the 3′ untranslated region of the candidate proto-oncogene Cnr2. These results suggest that proviral insertion in the Cnr2 gene is an important step in Cas-Br-M MuLV-induced leukemogenesis in NIH/Swiss mice. To isolate Evi11/Cnr2 collaborating proto-oncogenes, we searched for novel common VISs in the Cas-Br-M MuLV-induced primary tumors and identified a novel frequent common VIS, Evi12 (14%). Interestingly, 54% of the Evi11/Cnr2-rearranged primary tumors contained insertions in Evi12 as well, which suggests cooperative action of the target genes in these two common VISs in leukemogenesis. By interspecific backcross analysis it was shown that Evi12 resides on mouse chromosome 10 in a region that shares homology with human chromosomes 12q and 19p. Sequence analysis demonstrated that Evi12 is located upstream of the gene encoding the molecular chaperone Tra1/Grp94, which was previously mapped to mouse chromosome 10 and human chromosome 12q22–24. Thus, Tra1/Grp94 is a candidate target gene for retroviral activation or inactivation in Evi12. However, Northern and Western blot analyses did not provide evidence that proviral insertion had altered the expression of Tra1/Grp94. Additional studies are required to determine whether Tra1/Grp94 or another candidate proto-oncogene in Evi12 is involved in leukemogenesis. PMID:10196250

  14. Chromosome Compaction by Active Loop Extrusion.

    PubMed

    Goloborodko, Anton; Marko, John F; Mirny, Leonid A

    2016-05-24

    During cell division, chromosomes are compacted in length by more than a 100-fold. A wide range of experiments demonstrated that in their compacted state, mammalian chromosomes form arrays of closely stacked consecutive ∼100 kb loops. The mechanism underlying the active process of chromosome compaction into a stack of loops is unknown. Here we test the hypothesis that chromosomes are compacted by enzymatic machines that actively extrude chromatin loops. When such loop-extruding factors (LEF) bind to chromosomes, they progressively bridge sites that are further away along the chromosome, thus extruding a loop. We demonstrate that collective action of LEFs leads to formation of a dynamic array of consecutive loops. Simulations and an analytically solved model identify two distinct steady states: a sparse state, where loops are highly dynamic but provide little compaction; and a dense state, where there are more stable loops and dramatic chromosome compaction. We find that human chromosomes operate at the border of the dense steady state. Our analysis also shows how the macroscopic characteristics of the loop array are determined by the microscopic properties of LEFs and their abundance. When the number of LEFs are used that match experimentally based estimates, the model can quantitatively reproduce the average loop length, the degree of compaction, and the general loop-array morphology of compact human chromosomes. Our study demonstrates that efficient chromosome compaction can be achieved solely by an active loop-extrusion process. PMID:27224481

  15. Frequent site-specific deletion of coliphage lambda murine sarcoma virus recombinants and its use in the identification of a retrovirus integration site.

    PubMed Central

    McClements, W L; Enquist, L W; Oskarsson, M; Sullivan, M; Vande Woude, G F

    1980-01-01

    Stocks of hybrid lambda phages carrying the complete integrated provirus of either m1 or HT1 Moloney murine sarcoma virus, as well as flanking host sequences, frequently contain significant numbers of phages carrying a specific deletion. This deletion arises from a recombination event between the terminally repeated sequences in the provirus that deletes the unique Moloney murine sarcoma virus sequences bracketed by the terminally repeated sequences. Physical mapping has shown that the deletion phage retains one complete copy of the terminally repeated sequence and the flanking mink host sequences. One such deletion, lambdaHT1r+, was used to characterize a mink genomic DNA sequence that contains an HT1 Moloney murine sarcoma virus integration site. This integration site sequence from normal mink cells was also cloned into phage lambda. An analysis of the heteroduplexes between the integration site and the lambdaHT1r+ deletion indicated that no major rearrangement of host sequences occurred upon integration of the Moloney murine sarcoma provirus. Images PMID:6255187

  16. Added value for on-site fuel cells through equipment and application integration

    SciTech Connect

    Whitaker, R.

    1996-12-31

    On-site fuel cell power plants are not an exciting new electricity generating technology. They are an economic and beneficial addition to the operating systems of commercial buildings and industrial facilities. ONSI Corporation is part of International Fuel Cells Corporation and is jointly owned by United Technologies Corporation, Toshiba, and Ansaldo. ONSI has proven in the last three years that initial demand for packaged fuel cell power plants, like our 200 kW PC25{trademark} fuel cell shown in Figure 1, comes from the commercial building sector. However, this sector and the companies which service it are only tangentially interested in fuel cells as an emerging electricity generating technology. What they are most interested in is how the PC25 can integrate into their building`s system; how it can deliver energy efficient dollars to the bottom line, and how it can deliver operating benefits to their business.

  17. Interim Status of the Accelerated Site Technology Deployment Integrated Decontamination and Decommissioning Project

    SciTech Connect

    A. M Smith; G. E. Matthern; R. H. Meservey

    1998-11-01

    The Idaho National Engineering and Environmental Laboratory (INEEL), Fernald Environmental Management Project (FEMP), and Argonne National Laboratory - East (ANL-E) teamed to establish the Accelerated Site Technology Deployment (ASTD) Integrated Decontamination and Decommissioning (ID&D) project to increase the use of improved technologies in D&D operations. The project is making the technologies more readily available, providing training, putting the technologies to use, and spreading information about improved performance. The improved technologies are expected to reduce cost, schedule, radiation exposure, or waste volume over currently used baseline methods. They include some of the most successful technologies proven in the large-scale demonstrations and in private industry. The selected technologies are the Pipe Explorer, the GammaCam, the Decontamination Decommissioning and Remediation Optimal Planning System (DDROPS), the BROKK Demolition Robot, the Personal Ice Cooling System (PICS), the Oxy-Gasoline Torch, the Track-Mounted Shear, and the Hand-Held Shear.

  18. A Two-site Randomized Clinical Trial of Integrated Psychosocial Treatment for ADHD-Inattentive Type

    PubMed Central

    Pfiffner, Linda J.; Hinshaw, Stephen P.; Owens, Elizabeth; Zalecki, Christine; Kaiser, Nina M.; Villodas, Miguel; McBurnett, Keith

    2014-01-01

    Objective This study evaluated the efficacy of the Child Life and Attention Skills (CLAS) program, a behavioral psychosocial treatment integrated across home and school, for youth with Attention Deficit Hyperactivity Disorder-Inattentive Type (ADHD-I). Method In a two-site randomized controlled trial, 199 children (ages 7-11) were randomized to CLAS (N=74), parent-focused treatment (PFT, N=74), or treatment as usual (TAU, N=51). We compared groups on parent and teacher ratings of inattention symptoms, organizational skills, social skills, and global improvement at post-treatment, and also at follow-up during the subsequent school year. Results CLAS resulted in greater improvements in teacher-reported inattention, organizational skills, social skills, and global functioning relative to both PFT and TAU at post-treatment. Parents of children in CLAS reported greater improvement in organizational skills than PFT and greater improvements on all outcomes relative to TAU at post-treatment. Differences between CLAS and TAU were maintained at follow-up for most parent-reported measures but were not significant for teacher-reported outcomes. Conclusions These findings extend support for CLAS across two study sites, revealing that integrating parent, teacher, and child treatment components, specifically adapted for ADHD-I, is superior to parent training alone and to usual care. Direct involvement of teachers and children in CLAS appears to amplify effects at school and home and underscores the importance of coordinating parent, teacher, and child treatment components for cross-setting effects on symptoms and impairment associated with ADHD-I. PMID:24865871

  19. CPP-603 Underwater Fuel Storage Facility Site Integrated Stabilization Management Plan (SISMP), Volume I

    SciTech Connect

    Denney, R.D.

    1995-10-01

    The CPP-603 Underwater Fuel Storage Facility (UFSF) Site Integrated Stabilization Management Plan (SISMP) has been constructed to describe the activities required for the relocation of spent nuclear fuel (SNF) from the CPP-603 facility. These activities are the only Idaho National Engineering Laboratory (INEL) actions identified in the Implementation Plan developed to meet the requirements of the Defense Nuclear Facilities Safety Board (DNFSB) Recommendation 94-1 to the Secretary of Energy regarding an improved schedule for remediation in the Defense Nuclear Facilities Complex. As described in the DNFSB Recommendation 94-1 Implementation Plan, issued February 28, 1995, an INEL Spent Nuclear Fuel Management Plan is currently under development to direct the placement of SNF currently in existing INEL facilities into interim storage, and to address the coordination of intrasite SNF movements with new receipts and intersite transfers that were identified in the DOE SNF Programmatic and INEL Environmental Restoration and Waste Management Environmental Impact Statement Record, of Decision. This SISMP will be a subset of the INEL Spent Nuclear Fuel Management Plan and the activities described are being coordinated with other INEL SNF management activities. The CPP-603 relocation activities have been assigned a high priority so that established milestones will be meet, but there will be some cases where other activities will take precedence in utilization of available resources. The Draft INEL Site Integrated Stabilization Management Plan (SISMP), INEL-94/0279, Draft Rev. 2, dated March 10, 1995, is being superseded by the INEL Spent Nuclear Fuel Management Plan and this CPP-603 specific SISMP.

  20. Integrated Closure and Monitoring Plan for the Area 3 and Area 5 Radioactive Waste Management Sites at the Nevada Test Site

    SciTech Connect

    S. E. Rawlinson

    2001-09-01

    Bechtel Nevada (BN) manages two low-level Radioactive Waste Management Sites (RWMSs) (one site is in Area 3 and the other is in Area 5) at the Nevada Test Site (NTS) for the U.S. Department of Energy's (DOE's) National Nuclear Security Administration Nevada Operations Office (NNSA/NV). The current DOE Order governing management of radioactive waste is 435.1. Associated with DOE Order 435.1 is a Manual (DOE M 435.1-1) and Guidance (DOE G 435.1-1). The Manual and Guidance specify that preliminary closure and monitoring plans for a low-level waste (LLW) management facility be developed and initially submitted with the Performance Assessment (PA) and Composite Analysis (CA) for that facility. The Manual and Guidance, and the Disposal Authorization Statement (DAS) issued for the Area 3 RWMS further specify that the preliminary closure and monitoring plans be updated within one year following issuance of a DAS. This Integrated Closure and Monitoring Plan (ICMP) fulfills both requirements. Additional updates will be conducted every third year hereafter. This document is an integrated plan for closing and monitoring both RWMSs, and is based on guidance issued in 1999 by the DOE for developing closure plans. The plan does not follow the format suggested by the DOE guidance in order to better accommodate differences between the two RWMSs, especially in terms of operations and site characteristics. The modification reduces redundancy and provides a smoother progression of the discussion. The closure and monitoring plans were integrated because much of the information that would be included in individual plans is the same, and integration provides efficient presentation and program management. The ICMP identifies the regulatory requirements, describes the disposal sites and the physical environment where they are located, and defines the approach and schedule for both closing and monitoring the sites.

  1. Large-scale discovery of insertion hotspots and preferential integration sites of human transposed elements

    PubMed Central

    Levy, Asaf; Schwartz, Schraga; Ast, Gil

    2010-01-01

    Throughout evolution, eukaryotic genomes have been invaded by transposable elements (TEs). Little is known about the factors leading to genomic proliferation of TEs, their preferred integration sites and the molecular mechanisms underlying their insertion. We analyzed hundreds of thousands nested TEs in the human genome, i.e. insertions of TEs into existing ones. We first discovered that most TEs insert within specific ‘hotspots’ along the targeted TE. In particular, retrotransposed Alu elements contain a non-canonical single nucleotide hotspot for insertion of other Alu sequences. We next devised a method for identification of integration sequence motifs of inserted TEs that are conserved within the targeted TEs. This method revealed novel sequences motifs characterizing insertions of various important TE families: Alu, hAT, ERV1 and MaLR. Finally, we performed a global assessment to determine the extent to which young TEs tend to nest within older transposed elements and identified a 4-fold higher tendency of TEs to insert into existing TEs than to insert within non-TE intergenic regions. Our analysis demonstrates that TEs are highly biased to insert within certain TEs, in specific orientations and within specific targeted TE positions. TE nesting events also reveal new characteristics of the molecular mechanisms underlying transposition. PMID:20008508

  2. Space Borne and Ground Based InSAR Data Integration: The Åknes Test Site

    NASA Astrophysics Data System (ADS)

    Bardi, Federica; Raspini, Federico; Ciampalini, Andrea; Kristensen, Lene; Rouyet, Line; Rune Lauknes, Tom; Frauenfelder, Regula; Casagli, Nicola

    2016-04-01

    This work concerns a proposal of integration between InSAR (Interferometric Synthetic Aperture Radar) data acquired by ground based (GB) and satellite platforms. The selected test site is the Åknes rockslide, which affects the western Norwegian coast; the availability of GB-InSAR and satellite InSAR data, and the accessibility of a wide literature make the landslide suitable for testing the proposed procedure. The first step consists in the organization of a geodatabase, performed in GIS environment, containing all the available data. The second step concerns the analysis of satellite and GB-InSAR data, separately. Two datasets, acquired by RADARSAT-2 (related to a period between October 2008 and August 2013) and by a combination of TerraSAR-X and TanDEM-X (acquired between July 2010 and October 2012), both of them in ascending orbit, processed applying SBAS (Small BAseline Subset), are available. GB-InSAR data related to 5 different campaigns of measurements, referred to the summer seasons of 2006, 2008, 2009, 2010 and 2012 are available too. The third step relies on data integration, performed firstly on a qualitative point of view and lately on a semi-quantitative point of view. The results of the proposed procedure have been validated by comparing them with GPS (Global Positioning System) data.

  3. Study of component technologies for fuel cell on-site integrated energy systems

    NASA Technical Reports Server (NTRS)

    Lee, W. D.; Mathias, S.

    1980-01-01

    Heating, ventilation and air conditioning equipment are integrated with three types of fuel cells. System design and computer simulations are developed to utilize the thermal energy discharge of the fuel in the most cost effective manner. The fuel provides all of the electric needs and a loss of load probability analysis is used to ensure adequate power plant reliability. Equipment cost is estimated for each of the systems analyzed. A levelized annual cost reflecting owning and operating costs including the cost of money was used to select the most promising integrated system configurations. Cash flows are presented for the most promising 16 systems. Several systems for the 96 unit apartment complex (a retail store was also studied) were cost competitive with both gas and electric based conventional systems. Thermal storage is shown to be beneficial and the optimum absorption chiller sizing (waste heat recovery) in connection with electric chillers are developed. Battery storage was analyzed since the system is not electric grid connected. Advanced absorption chillers were analyzed as well. Recommendations covering financing, technical development, and policy issues are given to accelerate the commercialization of the fuel cell for on-site power generation in buildings.

  4. OC ToGo: bed site image integration into OpenClinica with mobile devices

    NASA Astrophysics Data System (ADS)

    Haak, Daniel; Gehlen, Johan; Jonas, Stephan; Deserno, Thomas M.

    2014-03-01

    Imaging and image-based measurements nowadays play an essential role in controlled clinical trials, but electronic data capture (EDC) systems insufficiently support integration of captured images by mobile devices (e.g. smartphones and tablets). The web application OpenClinica has established as one of the world's leading EDC systems and is used to collect, manage and store data of clinical trials in electronic case report forms (eCRFs). In this paper, we present a mobile application for instantaneous integration of images into OpenClinica directly during examination on patient's bed site. The communication between the Android application and OpenClinica is based on the simple object access protocol (SOAP) and representational state transfer (REST) web services for metadata, and secure file transfer protocol (SFTP) for image transfer, respectively. OpenClinica's web services are used to query context information (e.g. existing studies, events and subjects) and to import data into the eCRF, as well as export of eCRF metadata and structural information. A stable image transfer is ensured and progress information (e.g. remaining time) visualized to the user. The workflow is demonstrated for a European multi-center registry, where patients with calciphylaxis disease are included. Our approach improves the EDC workflow, saves time, and reduces costs. Furthermore, data privacy is enhanced, since storage of private health data on the imaging devices becomes obsolete.

  5. Analysis of a fuel cell on-site integrated energy system for a residential complex

    NASA Technical Reports Server (NTRS)

    Simons, S. N.; Maag, W. L.

    1979-01-01

    The energy use and costs of the on-site integrated energy system (OS/IES) which provides electric power from an on-site power plant and recovers heat that would normally be rejected to the environment is compared to a conventional system purchasing electricity from a utility and a phosphoric acid fuel cell powered system. The analysis showed that for a 500-unit apartment complex a fuel OS/IES would be about 10% more energy conservative in terms of total coal consumption than a diesel OS/IES system or a conventional system. The fuel cell OS/IES capital costs could be 30 to 55% greater than the diesel OS/IES capital costs for the same life cycle costs. The life cycle cost of a fuel cell OS/IES would be lower than that for a conventional system as long as the cost of electricity is greater than $0.05 to $0.065/kWh. An analysis of several parametric combinations of fuel cell power plant and state-of-art energy recovery systems and annual fuel requirement calculations for four locations were made. It was shown that OS/IES component choices are a major factor in fuel consumption, with the least efficient system using 25% more fuel than the most efficient. Central air conditioning and heat pumps result in minimum fuel consumption while individual air conditioning units increase it, and in general the fuel cell of highest electrical efficiency has the lowest fuel consumption.

  6. Moving target indicating radar applications in an integrated site security suite

    SciTech Connect

    Appenzeller, R.C. )

    1991-01-01

    The integration of a small, lightweight, low power consumption radar into a site security sensor suite can provide several key advantages in the ability to detect vehicles and personnel over large ground areas. This paper presents rationale for the inclusion of a man-portable Moving Target Indicator (MTI) radar in several security scenarios and outlines the technical specifics of a candidate radar. The Department of Energy (DOE) is currently investigating the effectiveness of a combination of optical sensors in concert with a scanning narrow beam radar at the Nevada Test Site in Mercury, Nevada. Demonstration results from these previous test activities are included herein. Of particular interest is the complimentary nature of this sensor suite where the large field of view achievable with radar allows the optical sensors to be used as pinpoint target classification devices. The inclusion of a radar minimizes operator fatigue caused by watching cameras scanning in azimuth and elevation. Advances in the areas of nuisance alarm rejection and improved range detection against single personnel targets were made in 1990 and this capability is included in the current production version.

  7. The explosion sites of nearby supernovae seen with integral field spectroscopy

    NASA Astrophysics Data System (ADS)

    Kuncarayakti, Hanindyo

    2015-08-01

    The progenitor stars of supernovae are still not very well constrained, despite numerous efforts in studying these objects directly or indirectly. There have been detections of the progenitor candidates in pre-explosion Hubble Space Telescope images, but these are rare and it is difficult to increase the statistics due to the limited availability of usable pre-explosion images. Alternatively, one may perform statistical studies on the supernova environments to derive useful constraints on the SN progenitor star. Integral field spectroscopy of nearby supernova sites within ~30 Mpc have been obtained using multiple IFU spectrographs in Hawaii and Chile. This technique enables both spatial and spectral information of the explosion sites to be acquired simultaneously, thus providing the identification of the parent stellar population of the supernova progenitor and the estimates for its physical parameters including age and metallicity. While this work has mainly been done in the optical wavelengths using instruments such as VIMOS, GMOS, and MUSE, a near-infrared approach has also been carried out using the AO-assisted SINFONI. By studying the supernova parent stellar population, we aim to characterize the mass and metallicity of the progenitors of different types of supernovae.

  8. The InterPro database, an integrated documentation resource for protein families, domains and functional sites.

    PubMed

    Apweiler, R; Attwood, T K; Bairoch, A; Bateman, A; Birney, E; Biswas, M; Bucher, P; Cerutti, L; Corpet, F; Croning, M D; Durbin, R; Falquet, L; Fleischmann, W; Gouzy, J; Hermjakob, H; Hulo, N; Jonassen, I; Kahn, D; Kanapin, A; Karavidopoulou, Y; Lopez, R; Marx, B; Mulder, N J; Oinn, T M; Pagni, M; Servant, F; Sigrist, C J; Zdobnov, E M

    2001-01-01

    Signature databases are vital tools for identifying distant relationships in novel sequences and hence for inferring protein function. InterPro is an integrated documentation resource for protein families, domains and functional sites, which amalgamates the efforts of the PROSITE, PRINTS, Pfam and ProDom database projects. Each InterPro entry includes a functional description, annotation, literature references and links back to the relevant member database(s). Release 2.0 of InterPro (October 2000) contains over 3000 entries, representing families, domains, repeats and sites of post-translational modification encoded by a total of 6804 different regular expressions, profiles, fingerprints and Hidden Markov Models. Each InterPro entry lists all the matches against SWISS-PROT and TrEMBL (more than 1,000,000 hits from 462,500 proteins in SWISS-PROT and TrEMBL). The database is accessible for text- and sequence-based searches at http://www.ebi.ac.uk/interpro/. Questions can be emailed to interhelp@ebi.ac.uk. PMID:11125043

  9. InterPro: an integrated documentation resource for protein families, domains and functional sites.

    PubMed

    Mulder, Nicola J; Apweiler, Rolf; Attwood, Terri K; Bairoch, Amos; Bateman, Alex; Binns, David; Biswas, Margaret; Bradley, Paul; Bork, Peer; Bucher, Phillip; Copley, Richard; Courcelle, Emmanuel; Durbin, Richard; Falquet, Laurent; Fleischmann, Wolfgang; Gouzy, Jerome; Griffith-Jones, Sam; Haft, Daniel; Hermjakob, Henning; Hulo, Nicolas; Kahn, Daniel; Kanapin, Alexander; Krestyaninova, Maria; Lopez, Rodrigo; Letunic, Ivica; Orchard, Sandra; Pagni, Marco; Peyruc, David; Ponting, Chris P; Servant, Florence; Sigrist, Christian J A

    2002-09-01

    The exponential increase in the submission of nucleotide sequences to the nucleotide sequence database by genome sequencing centres has resulted in a need for rapid, automatic methods for classification of the resulting protein sequences. There are several signature and sequence cluster-based methods for protein classification, each resource having distinct areas of optimum application owing to the differences in the underlying analysis methods. In recognition of this, InterPro was developed as an integrated documentation resource for protein families, domains and functional sites, to rationalise the complementary efforts of the individual protein signature database projects. The member databases - PRINTS, PROSITE, Pfam, ProDom, SMART and TIGRFAMs - form the InterPro core. Related signatures from each member database are unified into single InterPro entries. Each InterPro entry includes a unique accession number, functional descriptions and literature references, and links are made back to the relevant member database(s). Release 4.0 of InterPro (November 2001) contains 4,691 entries, representing 3,532 families, 1,068 domains, 74 repeats and 15 sites of post-translational modification (PTMs) encoded by different regular expressions, profiles, fingerprints and hidden Markov models (HMMs). Each InterPro entry lists all the matches against SWISS-PROT and TrEMBL (2,141,621 InterPro hits from 586,124 SWISS-PROT and TrEMBL protein sequences). The database is freely accessible for text- and sequence-based searches. PMID:12230031

  10. The InterPro database, an integrated documentation resource for protein families, domains and functional sites

    PubMed Central

    Apweiler, R.; Attwood, T. K.; Bairoch, A.; Bateman, A.; Birney, E.; Biswas, M.; Bucher, P.; Cerutti, L.; Corpet, F.; Croning, M. D. R.; Durbin, R.; Falquet, L.; Fleischmann, W.; Gouzy, J.; Hermjakob, H.; Hulo, N.; Jonassen, I.; Kahn, D.; Kanapin, A.; Karavidopoulou, Y.; Lopez, R.; Marx, B.; Mulder, N. J.; Oinn, T. M.; Pagni, M.; Servant, F.; Sigrist, C. J. A.; Zdobnov, E. M.

    2001-01-01

    Signature databases are vital tools for identifying distant relationships in novel sequences and hence for inferring protein function. InterPro is an integrated documentation resource for protein families, domains and functional sites, which amalgamates the efforts of the PROSITE, PRINTS, Pfam and ProDom database projects. Each InterPro entry includes a functional description, annotation, literature references and links back to the relevant member database(s). Release 2.0 of InterPro (October 2000) contains over 3000 entries, representing families, domains, repeats and sites of post-translational modification encoded by a total of 6804 different regular expressions, profiles, fingerprints and Hidden Markov Models. Each InterPro entry lists all the matches against SWISS-PROT and TrEMBL (more than 1 000 000 hits from 462 500 proteins in SWISS-PROT and TrEMBL). The database is accessible for text- and sequence-based searches at http://www.ebi.ac.uk/interpro/. Questions can be emailed to interhelp@ebi.ac.uk. PMID:11125043

  11. Structural Integrity of the B24 Site in Human Insulin Is Important for Hormone Functionality*

    PubMed Central

    Žáková, Lenka; Kletvíková, Emília; Veverka, Václav; Lepšík, Martin; Watson, Christopher J.; Turkenburg, Johan P.; Jiráček, Jiří; Brzozowski, Andrzej M.

    2013-01-01

    Despite the recent first structural insight into the insulin-insulin receptor complex, the role of the C terminus of the B-chain of insulin in this assembly remains unresolved. Previous studies have suggested that this part of insulin must rearrange to reveal amino acids crucial for interaction with the receptor. The role of the invariant PheB24, one of the key residues of the hormone, in this process remains unclear. For example, the B24 site functionally tolerates substitutions to d-amino acids but not to l-amino acids. Here, we prepared and characterized a series of B24-modified insulin analogues, also determining the structures of [d-HisB24]-insulin and [HisB24]-insulin. The inactive [HisB24]-insulin molecule is remarkably rigid due to a tight accommodation of the l-His side chain in the B24 binding pocket that results in the stronger tethering of B25-B28 residues to the protein core. In contrast, the highly active [d-HisB24]-insulin is more flexible, and the reverse chirality of the B24Cα atom swayed the d-HisB24 side chain into the solvent. Furthermore, the pocket vacated by PheB24 is filled by PheB25, which mimics the PheB24 side and main chains. The B25→B24 downshift results in a subsequent downshift of TyrB26 into the B25 site and the departure of B26-B30 residues away from the insulin core. Our data indicate the importance of the aromatic l-amino acid at the B24 site and the structural invariance/integrity of this position for an effective binding of insulin to its receptor. Moreover, they also suggest limited, B25-B30 only, unfolding of the C terminus of the B-chain upon insulin activation. PMID:23447530

  12. A simple biophysical model emulates budding yeast chromosome condensation.

    PubMed

    Cheng, Tammy M K; Heeger, Sebastian; Chaleil, Raphaël A G; Matthews, Nik; Stewart, Aengus; Wright, Jon; Lim, Carmay; Bates, Paul A; Uhlmann, Frank

    2015-01-01

    Mitotic chromosomes were one of the first cell biological structures to be described, yet their molecular architecture remains poorly understood. We have devised a simple biophysical model of a 300 kb-long nucleosome chain, the size of a budding yeast chromosome, constrained by interactions between binding sites of the chromosomal condensin complex, a key component of interphase and mitotic chromosomes. Comparisons of computational and experimental (4C) interaction maps, and other biophysical features, allow us to predict a mode of condensin action. Stochastic condensin-mediated pairwise interactions along the nucleosome chain generate native-like chromosome features and recapitulate chromosome compaction and individualization during mitotic condensation. Higher order interactions between condensin binding sites explain the data less well. Our results suggest that basic assumptions about chromatin behavior go a long way to explain chromosome architecture and are able to generate a molecular model of what the inside of a chromosome is likely to look like. PMID:25922992

  13. Tyramide-FISH mapping of single genes for development of an integrated recombination and cytogenetic map of chromosome 5 of Allium cepa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chromosome 5 of onion carries major quantitative trait loci (QTL) of interest to breeders that control dry-matter content, pungency and storability of bulbs, amounts and types of epicuticular waxes, and resistances to abiotic factors. SNPs, SSRs and RFLPs in expressed regions of the onion genome hav...

  14. Chromosomal destabilization during gene amplification.

    PubMed Central

    Ruiz, J C; Wahl, G M

    1990-01-01

    Acentric extrachromosomal elements, such as submicroscopic autonomously replicating circular molecules (episomes) and double minute chromosomes, are common early, and in some cases initial, intermediates of gene amplification in many drug-resistant and tumor cell lines. In order to gain a more complete understanding of the amplification process, we investigated the molecular mechanisms by which such extrachromosomal elements are generated and we traced the fate of these amplification intermediates over time. The model system consists of a Chinese hamster cell line (L46) created by gene transfer in which the initial amplification product was shown previously to be an unstable extrachromosomal element containing an inverted duplication spanning more than 160 kilobases (J. C. Ruiz and G. M. Wahl, Mol. Cell. Biol. 8:4302-4313, 1988). In this study, we show that these molecules were formed by a process involving chromosomal deletion. Fluorescence in situ hybridization was performed at multiple time points on cells with amplified sequences. These studies reveal that the extrachromosomal molecules rapidly integrate into chromosomes, often near or at telomeres, and once integrated, the amplified sequences are themselves unstable. These data provide a molecular and cytogenetic chronology for gene amplification in this model system; an early event involves deletion to generate extrachromosomal elements, and subsequent integration of these elements precipitates a cascade of chromosome instability. Images PMID:2188107

  15. Patterns of Neutral Genetic Variation on Recombining Sex Chromosomes

    PubMed Central

    Kirkpatrick, Mark; Guerrero, Rafael F.; Scarpino, Samuel V.

    2010-01-01

    Many animals and plants have sex chromosomes that recombine over much of their length. Here we develop coalescent models for neutral sites on these chromosomes. The emphasis is on expected coalescence times (proportional to the expected amount of neutral genetic polymorphism), but we also derive some results for linkage disequilibria between neutral sites. We analyze the standard neutral model, a model with polymorphic Y chromosomes under balancing selection, and the invasion of a neo-Y chromosome. The results may be useful for testing hypotheses regarding how new sex chromosomes originate and how selection acts upon them. PMID:20124026

  16. Integrating intrusive and nonintrusive characterization methods to achieve a conceptual site model for the SLDA FUSRAP site - 8265.

    SciTech Connect

    Durham, L. A.; Peterson, J. M.; Frothingham, D. G.; Frederick, W. T.; Lenart, W.; Environmental Science Division; U. S. Army Corps of Engineers, Pittsburg District; U. S. Army Corps of Engineers, Buffalo District

    2008-01-01

    The US Army Corps of Engineers (USACE) is addressing radiological contamination following Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA) requirements at the Shallow Land Disposal Area (SLDA) site, which is a radiologically contaminated property that is part of the Formerly utilized Sites Remedial Action Program (FUSRAP). The SLDA is an 18-hectare (44-acre) site in Parks township, Armstrong County, Pennsylvania, about 37 kilometers (23 miles) east-northeast of Pittsburgh. According to historical record, radioactive wastes were disposed of at the SLDA in a series of trenches by the Nuclear Materials and Equipment Company (NUMEC) in the 1960s. The wastes originated from the nearby Apollo nuclear fuel fabrication facility, which began operations under NUMEC in the late 1950s and fabricated enriched uranium into naval reactor fuel elements. It is believed that the waste materials were buried in a series of pits constructed adjacent to one another in accordance with an Atomic Energy Commission (AEC) regulation that has since been rescinded. A CERCLA remedial investigation/feasibility study (RI/FS) process was completed for the SLDA site, and the results of the human health risk assessment indicated that the radiologically contaminated wastes could pose a risk to human health in the future. There are no historical records that provide the exact location of these pits. However, based on geophysical survey results conducted in the 1980s, these pits were defined by geophysical anomalies and were depicted on historical site drawings as trenches. At the SLDA site, a combination of investigative methods and tools was used in the RI/FS and site characterization activities. The SLDA site provides an excellent example of how historical documents and data, historical aerial photo analysis, physical sampling, and nonintrusive geophysical and gamma walkover surveys were used in combination to reduce the uncertainty in the location of the trenches. The

  17. The centromere: epigenetic control of chromosome segregation during mitosis.

    PubMed

    Westhorpe, Frederick G; Straight, Aaron F

    2015-01-01

    A fundamental challenge for the survival of all organisms is maintaining the integrity of the genome in all cells. Cells must therefore segregate their replicated genome equally during each cell division. Eukaryotic organisms package their genome into a number of physically distinct chromosomes, which replicate during S phase and condense during prophase of mitosis to form paired sister chromatids. During mitosis, cells form a physical connection between each sister chromatid and microtubules of the mitotic spindle, which segregate one copy of each chromatid to each new daughter cell. The centromere is the DNA locus on each chromosome that creates the site of this connection. In this review, we present a brief history of centromere research and discuss our current knowledge of centromere establishment, maintenance, composition, structure, and function in mitosis. PMID:25414369

  18. Fitness Cost Implications of PhiC31-Mediated Site-Specific Integrations in Target-Site Strains of the Mexican Fruit Fly, Anastrepha ludens (Diptera: Tephritidae)

    PubMed Central

    Meza, José S.; Díaz-Fleischer, Francisco; Sánchez-Velásquez, Lázaro R.; Zepeda-Cisneros, Cristina Silvia; Handler, Alfred M.; Schetelig, Marc F.

    2014-01-01

    Site-specific recombination technologies are powerful new tools for the manipulation of genomic DNA in insects that can improve transgenesis strategies such as targeting transgene insertions, allowing transgene cassette exchange and DNA mobilization for transgene stabilization. However, understanding the fitness cost implications of these manipulations for transgenic strain applications is critical. In this study independent piggyBac-mediated attP target-sites marked with DsRed were created in several genomic positions in the Mexican fruit fly, Anastrepha ludens. Two of these strains, one having an autosomal (attP_F7) and the other a Y-linked (attP_2-M6y) integration, exhibited fitness parameters (dynamic demography and sexual competitiveness) similar to wild type flies. These strains were thus selected for targeted insertion using, for the first time in mexfly, the phiC31-integrase recombination system to insert an additional EGFP-marked transgene to determine its effect on host strain fitness. Fitness tests showed that the integration event in the int_2-M6y recombinant strain had no significant effect, while the int_F7 recombinant strain exhibited significantly lower fitness relative to the original attP_F7 target-site host strain. These results indicate that while targeted transgene integrations can be achieved without an additional fitness cost, at some genomic positions insertion of additional DNA into a previously integrated transgene can have a significant negative effect. Thus, for targeted transgene insertions fitness costs must be evaluated both previous to and subsequent to new site-specific insertions in the target-site strain. PMID:25303238

  19. The Skogaryd Research Site - Integration of terrestrial and freshwater greenhouse gas sources and sinks

    NASA Astrophysics Data System (ADS)

    Klemedtsson, L.

    2012-04-01

    atmosphere. The budgets have ignored dissolved carbon (C) and nitrogen (N) transport in water to streams and lakes and the subsequent exchange between the atmosphere and surface waters. Aquatic habitats can be significant net sources of CO2 and methane (CH4) and potential hot spots for N2O release, all important for natural greenhouse gas (GHG) emissions. Inland waters need to be included in the C and GHG balances for terrestrial landscapes. This project aims to quantify GHG balances at the landscape scale in forested regions that include land-atmosphere, land-water, and water-atmosphere exchange of CO2, CH4 and N2O. Different terrestrial and aquatic ecosystems will be linked holistically, using site specific techniques at different scales, from aircraft (km2) to chambers (m2) to develop integrated models that can be used to quantify net GHG flux for management strategies. The LAGGE project involves six Swedish universities and site is open for more cooperations.

  20. Integrated bio-magnetostratigraphy of ODP Site 709 (equatorial Indian Ocean).

    NASA Astrophysics Data System (ADS)

    Villa, Giuliana; Fioroni, Chiara; Florindo, Fabio

    2015-04-01

    Over the last decade, calcareous nannofossil biostratigraphy of the lower Eocene-Oligocene sediments has shown great potential, through identification of several new nannofossil species and bioevents (e.g. Fornaciari et al., 2010; Bown and Dunkley Jones, 2012; Toffanin et al., 2013). These studies formed the basis for higher biostratigraphic resolution leading to definition of a new nannofossil biozonation (Agnini et al., 2014). In this study, we investigate the middle Eocene-lower Oligocene sediments from ODP Hole 709C (ODP Leg 115) by means of calcareous nannofossils and magnetostratigraphy. Ocean Drilling Program (ODP) Site 709 was located in the equatorial Indian Ocean and biostratigraphy has been investigated in the nineties (Okada, 1990; Fornaciari et al., 1990) while paleomagnetic data from the Initial Report provided only a poorly constrained magnetostratigraphic interpretation, thus the cored succession was dated only by means of biostratigraphy. Our goal is to test the reliability in the Indian Ocean of the biohorizons recently identified at Site 711 (Fioroni et al., in press), by means of high resolution sampling, new taxonomic updates, quantitative analyses on calcareous nannofossils allowed to increase the number of useful bioevents and to compare their reliability and synchroneity. The new magnetostratigraphic analyses and integrated stratigraphy allow also to achieve an accurate biochronology of the time interval spanning Chrons C20 (middle Eocene) and C12 (early Oligocene). In addition, this equatorial site represents an opportunity to study the carbonate accumulation history and the large fluctuations of the carbonate compensation depth (CCD) during the Eocene (e.g. Pälike et al., 2012). The investigated interval encompasses the Middle Eocene Climatic Optimum (MECO), and the long cooling trend that leads to the Oligocene glacial state. By means of our new bio-magnetostratigraphic data and paleoecological results we provide further insights on

  1. Monocyte-derived macrophages exhibit distinct and more restricted HIV-1 integration site repertoire than CD4(+) T cells.

    PubMed

    Kok, Yik Lim; Vongrad, Valentina; Shilaih, Mohaned; Di Giallonardo, Francesca; Kuster, Herbert; Kouyos, Roger; Günthard, Huldrych F; Metzner, Karin J

    2016-01-01

    The host genetic landscape surrounding integrated HIV-1 has an impact on the fate of the provirus. Studies analysing HIV-1 integration sites in macrophages are scarce. We studied HIV-1 integration site patterns in monocyte-derived macrophages (MDMs) and activated CD4(+) T cells derived from seven antiretroviral therapy (ART)-treated HIV-1-infected individuals whose cells were infected ex vivo with autologous HIV-1 isolated during the acute phase of infection. A total of 1,484 unique HIV-1 integration sites were analysed. Their distribution in the human genome and genetic features, and the effects of HIV-1 integrase polymorphisms on the nucleotide selection specificity at these sites were indistinguishable between the two cell types, and among HIV-1 isolates. However, the repertoires of HIV-1-hosting gene clusters overlapped to a higher extent in MDMs than in CD4(+) T cells. The frequencies of HIV-1 integration events in genes encoding HIV-1-interacting proteins were also different between the two cell types. Lastly, HIV-1-hosting genes linked to clonal expansion of latently HIV-1-infected CD4(+) T cells were over-represented in gene hotspots identified in CD4(+) T cells but not in those identified in MDMs. Taken together, the repertoire of genes targeted by HIV-1 in MDMs is distinct from and more restricted than that of CD4(+) T cells. PMID:27067385

  2. Monocyte-derived macrophages exhibit distinct and more restricted HIV-1 integration site repertoire than CD4+ T cells

    PubMed Central

    Kok, Yik Lim; Vongrad, Valentina; Shilaih, Mohaned; Di Giallonardo, Francesca; Kuster, Herbert; Kouyos, Roger; Günthard, Huldrych F.; Metzner, Karin J.

    2016-01-01

    The host genetic landscape surrounding integrated HIV-1 has an impact on the fate of the provirus. Studies analysing HIV-1 integration sites in macrophages are scarce. We studied HIV-1 integration site patterns in monocyte-derived macrophages (MDMs) and activated CD4+ T cells derived from seven antiretroviral therapy (ART)-treated HIV-1-infected individuals whose cells were infected ex vivo with autologous HIV-1 isolated during the acute phase of infection. A total of 1,484 unique HIV-1 integration sites were analysed. Their distribution in the human genome and genetic features, and the effects of HIV-1 integrase polymorphisms on the nucleotide selection specificity at these sites were indistinguishable between the two cell types, and among HIV-1 isolates. However, the repertoires of HIV-1-hosting gene clusters overlapped to a higher extent in MDMs than in CD4+ T cells. The frequencies of HIV-1 integration events in genes encoding HIV-1-interacting proteins were also different between the two cell types. Lastly, HIV-1-hosting genes linked to clonal expansion of latently HIV-1-infected CD4+ T cells were over-represented in gene hotspots identified in CD4+ T cells but not in those identified in MDMs. Taken together, the repertoire of genes targeted by HIV-1 in MDMs is distinct from and more restricted than that of CD4+ T cells. PMID:27067385

  3. Seismic aftershock monitoring for on-site inspection purposes. Experience from Integrated Field Exercise 2008.

    NASA Astrophysics Data System (ADS)

    Labak, P.; Arndt, R.; Villagran, M.

    2009-04-01

    One of the sub-goals of the Integrated Field Experiment in 2008 (IFE08) in Kazakhstan was testing the prototype elements of the Seismic aftershock monitoring system (SAMS) for on-site inspection purposes. The task of the SAMS is to collect the facts, which should help to clarify nature of the triggering event. Therefore the SAMS has to be capable to detect and identify events as small as magnitude -2 in the inspection area size up to 1000 km2. Equipment for 30 mini-arrays and 10 3-component stations represented the field equipment of the SAMS. Each mini-array consisted of a central 3-component seismometer and 3 vertical seismometers at the distance about 100 m from the central seismometer. The mini-arrays covered approximately 80% of surrogate inspection area (IA) on the territory of former Semipalatinsk test site. Most of the stations were installed during the first four days of field operations by the seismic sub-team, which consisted of 10 seismologists. SAMS data center comprised 2 IBM Blade centers and 8 working places for data archiving, detection list production and event analysis. A prototype of SAMS software was tested. Average daily amount of collected raw data was 15-30 GB and increased according to the amount of stations entering operation. Routine manual data screening and data analyses were performed by 2-6 subteam members. Automatic screening was used for selected time intervals. Screening was performed using the Sonoview program in frequency domain and using the Geotool and Hypolines programs for screening in time domain. The screening results were merged into the master event list. The master event list served as a basis of detailed analysis of unclear events and events identified to be potentially in the IA. Detailed analysis of events to be potentially in the IA was performed by the Hypoline and Geotool programs. In addition, the Hyposimplex and Hypocenter programs were also used for localization of events. The results of analysis were integrated

  4. Correlation Between Interphase Chromatin Structure and - and High-Let Radiation-Induced - and Intra-Chromosome Exchange Hotspots

    NASA Astrophysics Data System (ADS)

    Zhang, Ye; Wu, Honglu; Mangala, Lingegowda; Asaithamby, Aroumougame; Chen, David

    2012-07-01

    CORRELATION BETWEEN INTERPHASE CHROMATIN STRUCTURE AND LOW- AND HIGH-LET RADIATION-INDUCED INTER- AND INTRA-CHROMOSOME EXCHANGE HOTSPOTS Ye Zhang1,2, Lingegowda S. Mangala1,3, Aroumougame Asaithamby4, David J. Chen4, and Honglu Wu1 1 NASA Johnson Space Center, Houston, Texas, USA 2 Wyle Integrated Science and Engineering Group, Houston, Texas, USA 3 University of Houston Clear Lake, Houston, Texas, USA 4 University of Texas, Southwestern Medical Center, Dallas, Texas, USA To investigate the relationship between chromosome aberrations induced by low- and high-LET radiation and chromatin folding, we reconstructed the three dimensional structure of chromosome 3 and measured the physical distances between different regions of this chromosome. Previously, we investigated the location of breaks involved in inter- and intrachromosomal type exchange events in chromosome 3 of human epithelial cells, using the multicolor banding in situ hybridization (mBAND) technique. After exposure to both low- and high-LET radiations in vitro, intra-chromosome exchanges occurred preferentially between a break in the 3p21 and one in the 3q11 regions, and the breaks involved in inter-chromosome exchanges occurred in two regions near the telomeres of the chromosome. In this study, human epithelial cells were fixed in G1 phase and interphase chromosomes hybridized with an mBAND probe for chromosome 3 were captured with a laser scanning confocal microscope. The 3-dimensional structure of interphase chromosome 3 with different colored regions was reconstructed, and the distance between different regions was measured. We show that, in most of the G1 cells, the regions containing 3p21 and 3q11 are colocalized in the center of the chromosome domain, whereas, the regions towards the telomeres of the chromosome are located in the peripherals of the chromosome domain. Our results demonstrate that the distribution of breaks involved in radiation-induced inter and intra-chromosome aberrations depends

  5. A novel donor splice site in intron 11 of the CFTR gene, created by mutation 1811 + 1.6kbA {yields} G, produces a new exon: High frequency in spanish cystic fibrosis chromosomes and association with severe phenotype

    SciTech Connect

    Chillon, M.; Casals, T.; Gimenez, J.; Ramos, D.; Nunes, V.; Estivill, X.; Doerk, T.; Will, K.; Fonknechten, N.

    1995-03-01

    mRNA analysis of the cystic fibrosis transmembrane regulator (CFTR) gene in tissues of cystic fibrosis (CF) patients has allowed us to detect a cryptic exon. The new exon involves 49 base pairs between exons 11 and 12 and is due to a point mutation (1811+1.6bA{yields}G) that creates a new donor splice site in intron 11. Semiquantitative mRNA analysis showed that 1811+1.6kbA{r_arrow}G-mRNA was 5-10-fold less abundant than {triangle}F508 mRNA. Mutations 1811+1.6kbA{yields}G was found in 21 Spanish and 1 German CF chromosome(s), making it the fourth-most-frequent mutation (2%) in the Spanish population. Individuals with genotype {triangle}F508/1811+1.6kbA{yields}G have only 1%-3% of normal CFTR mRNA. This loss of 97% of normal CFTR mRNA must be responsible for the pancreatic insufficiency and for the severe CF phenotype in these patients. 30 refs., 3 figs., 2 tabs.

  6. The Precarious Prokaryotic Chromosome

    PubMed Central

    2014-01-01

    Evolutionary selection for optimal genome preservation, replication, and expression should yield similar chromosome organizations in any type of cells. And yet, the chromosome organization is surprisingly different between eukaryotes and prokaryotes. The nuclear versus cytoplasmic accommodation of genetic material accounts for the distinct eukaryotic and prokaryotic modes of genome evolution, but it falls short of explaining the differences in the chromosome organization. I propose that the two distinct ways to organize chromosomes are driven by the differences between the global-consecutive chromosome cycle of eukaryotes and the local-concurrent chromosome cycle of prokaryotes. Specifically, progressive chromosome segregation in prokaryotes demands a single duplicon per chromosome, while other “precarious” features of the prokaryotic chromosomes can be viewed as compensations for this severe restriction. PMID:24633873

  7. The precarious prokaryotic chromosome.

    PubMed

    Kuzminov, Andrei

    2014-05-01

    Evolutionary selection for optimal genome preservation, replication, and expression should yield similar chromosome organizations in any type of cells. And yet, the chromosome organization is surprisingly different between eukaryotes and prokaryotes. The nuclear versus cytoplasmic accommodation of genetic material accounts for the distinct eukaryotic and prokaryotic modes of genome evolution, but it falls short of explaining the differences in the chromosome organization. I propose that the two distinct ways to organize chromosomes are driven by the differences between the global-consecutive chromosome cycle of eukaryotes and the local-concurrent chromosome cycle of prokaryotes. Specifically, progressive chromosome segregation in prokaryotes demands a single duplicon per chromosome, while other "precarious" features of the prokaryotic chromosomes can be viewed as compensations for this severe restriction. PMID:24633873

  8. B-chromosome evolution.

    PubMed Central

    Camacho, J P; Sharbel, T F; Beukeboom, L W

    2000-01-01

    B chromosomes are extra chromosomes to the standard complement that occur in many organisms. They can originate in a number of ways including derivation from autosomes and sex chromosomes in intra- and interspecies crosses. Their subsequent molecular evolution resembles that of univalent sex chromosomes, which involves gene silencing, heterochromatinization and the accumulation of repetitive DNA and transposons. B-chromosome frequencies in populations result from a balance between their transmission rates and their effects on host fitness. Their long-term evolution is considered to be the outcome of selection on the host genome to eliminate B chromosomes or suppress their effects and on the B chromosome's ability to escape through the generation of new variants. Because B chromosomes interact with the standard chromosomes, they can play an important role in genome evolution and may be useful for studying molecular evolutionary processes. PMID:10724453

  9. Visual exploratory analysis of integrated chromosome 19 proteomic data derived from glioma cancer stem-cell lines based on novel nonlinear dimensional data reduction techniques

    NASA Astrophysics Data System (ADS)

    Lespinats, Sylvain; Pinker-Domenig, Katja; Meyer-Bäse, Uwe; Meyer-Bäse, Anke

    2015-05-01

    Chromosome 19 is known to be linked to neurodegeneration and many cancers. Glioma-derived cancer stem cells (GSCs) are tumor-initiating cells and may be refractory to radiation and chemotherapy and thus have important implications for tumor biology and therapeutics. The analysis and interpretation of large proteomic data sets requires the development of new data mining and visualization approaches. Traditional techniques are insufficient to interpret and visualize these resulting experimental data. The emphasis of this paper lies in the presentation of novel approaches for the visualization, clustering and projection representation to unveil hidden data structures relevant for the accurate interpretation of biological experiments. These qualitative and quantitative methods are applied to the proteomic analysis of data sets derived from the GSCs. The achieved clustering and visualization results provide a more detailed insight into the expression patterns for chromosome 19 proteins.

  10. Papillomavirus sequences integrate near cellular oncogenes in some cervical carcinomas

    SciTech Connect

    Duerst, M.; Croce, C.M.; Gissmann, L.; Schwarz, E.; Huebner, K.

    1987-02-01

    The chromosomal locations of cellular sequences flanking integrated papillomavirus DNA in four cervical cell lines and a primary cervical carcinoma have been determined. The two human papillomavirus (HPV) 16 flanking sequences derived from the tumor were localized to chromosomes regions 20pter..-->..20q13 and 3p25..-->..3qter, regions that also contain the protooncogenes c-src-1 and c-raf-1, respectively. The HPV 16 integration site in the SiHa cervical carcinoma-derived cell line is in chromosome region 13q14..-->..13q32. The HPV 18 integration site in SW756 cervical carcinoma cells is in chromosome 12 but is not closely linked to the Ki-ras2 gene. Finally, in two cervical carcinoma cell lines, HeLa and C4-I, HPV 18 DNA is integrated in chromosome 8, 5' of the c-myc gene. The HeLaHPV 18 integration site is within 40 kilobases 5' of the c-myc gene, inside the HL60 amplification unit surrounding and including the c-myc gene. Additionally, steady-state levels of c-myc mRNA are elevated in HeLa and C4-I cells relative to other cervical carcinoma cell lines. Thus, in at least some genital tumors, cis-activation of cellular oncogenes by HPV may be involved in malignant transformation of cervical cells.

  11. Heritable fragile sites on human chromosomes. VII. Children homozygous for the BrdU-requiring fra(10)q25 are phenotypically normal

    PubMed Central

    Sutherland, Grant R.

    1981-01-01

    A brother and sister have been detected who are homozygous for the bromodeoxyuridine (BrdU)-requiring fragile site at 10q25. The children are phenotypically normal, indicating that homozygosity for this fragile site is harmless, at least during childhood. ImagesFig. 2 PMID:7325157

  12. Endeavour Segment, Juan de Fuca Ridge, Integrated Studies Site (ISS) Update and Opportunities

    NASA Astrophysics Data System (ADS)

    Butterfield, D.; Ridge Community

    2003-12-01

    The Ridge 2000 (R2K) Integrated Studies bull's eye on the Juan de Fuca Ridge is focused on the Main Endeavour hydrothermal field, located on the central portion of the Endeavour Segment. This vent field is one of the most vigorously venting systems along the global mid-ocean ridge spreading network, hosting at least 18 large sulfide structures that contains more than100 smokers. Prior to a magmatic event in 2000 some of the edifices had been venting 380C, volatile-rich fluids with extremely low chlorinities for a decade. In addition to the Main Endeavour Field there are four other known high temperature vent fields spaced approximately 2 kilometers apart along the segment (with hints of more) and abundant areas of diffuse flow, both nearby and distal to the high temperature venting. Diffuse flow from the structures and from a variety of basaltic-hosted sites provides rich habitats abundant with microbial and macrofaunal communities. There are well-developed gradients in volatile concentrations along axis that may reflect influence from a sedimentary source to the north, and high chlorinity fluids vent from the most southern (Mothra) and northern fields (Sasquatch). Twenty years of research have laid a firm base for the 5-year plans of R2K at this site, which include examining the response of this segment to perturbations induced by tectonic and magmatic events, identification of the reservoirs, fluxes, and feedbacks of mass and energy at this site, and predictive modeling coupled with field observations. Since designation as an IS site, high-resolution bathymetric mapping (EM300) and an extensive multi-channel seismic survey have been conducted along the entire segment. Smaller focused areas have also been mapped at meter resolution by SM2000 sonar. Intense field programs in 2003 established the first in-situ seismic array along a mid-ocean ridge, which includes installation of a buried broadband seismometer and 7 short-period seismometers emplaced within basaltic

  13. Reducing the risk of surgical site infection using a multidisciplinary approach: an integrative review

    PubMed Central

    Gillespie, Brigid M; Kang, Evelyn; Roberts, Shelley; Lin, Frances; Morley, Nicola; Finigan, Tracey; Homer, Allison; Chaboyer, Wendy

    2015-01-01

    Purpose To identify and describe the strategies and processes used by multidisciplinary teams of health care professionals to reduce surgical site infections (SSIs). Materials and methods An integrative review of the research literature was undertaken. Searches were conducted in April 2015. Following review of the included studies, data were abstracted using summary tables and the methodological quality of each study assessed using the Standards for Quality Improvement Reporting Excellence guidelines by two reviewers. Discrepancies were dealt with through consensus. Inductive content analysis was used to identify and describe the strategies/processes used by multidisciplinary health care teams to prevent SSI. Results and discussion In total, 13 studies met the inclusion criteria. Of these, 12 studies used quantitative methods, while a single study used qualitative interviews. The majority of the studies were conducted in North America. All quantitative studies evaluated multifaceted quality-improvement interventions aimed at preventing SSI in patients undergoing surgery. Across the 13 studies reviewed, the following multidisciplinary team-based approaches were enacted: using a bundled approach, sharing responsibility, and, adhering to best practice. The majority of studies described team collaborations that were circumscribed by role. None of the reviewed studies used strategies that included the input of allied health professionals or patient participation in SSI prevention. Conclusion Patient-centered interventions aimed at increasing patient participation in SSI prevention and evaluating the contributions of allied health professionals in team-based SSI prevention requires future research. PMID:26508870

  14. Dietary vitamin A regulates wingless-related MMTV integration site signaling to alter the hair cycle

    PubMed Central

    Suo, Liye; Sundberg, John P; Everts, Helen B.

    2016-01-01

    Alopecia areata is an autoimmune hair loss disease caused by a cell mediated immune attack of the lower portion of the cycling hair follicle. Feeding mice 3–7 times the recommended level of dietary vitamin A accelerated the progression of alopecia areata in the graft-induced C3H/HeJ mouse model of alopecia areata. In this study we also found that dietary vitamin A, in a dose dependent manner, activated the hair follicle stem cells to induce the development and growth phase of the hair cycle (anagen), which may have made the hair follicle more susceptible to autoimmune attack. Our purpose here is to determine the mechanism by which dietary vitamin A regulates the hair cycle. We found that vitamin A in a dose dependent manner increased nuclear localized beta-catenin (CTNNB1; a marker of canonical WNT signaling) and levels of WNT7A (wingless-related MMTV integration site 7A) within the hair follicle bulge in these C3H/HeJ mice. These findings suggest that feeding mice high levels of dietary vitamin A increases WNT signaling to activate hair follicle stem cells. PMID:25361771

  15. Myeloid ecotropic viral integration site 1 (MEIS) 1 involvement in embryonic implantation

    PubMed Central

    Xu, Bei; Geerts, Dirk; Qian, Kun; Zhang, Hanwang; Zhu, Guijin

    2008-01-01

    BACKGROUND The HOXA10 homeobox gene controls embryonic uterine development and adult endometrial receptivity. The three-amino-acid loop extension (TALE) family homeobox genes like myeloid ecotropic viral integration site 1 (MEIS) provide enhanced target gene activation and specificity in HOX-regulated cellular processes by acting as HOX cofactors. METHODS AND RESULTS Analysis of an Affymetrix data set in the public domain showed high expression of MEIS1 in human endometrium. MEIS1 expression was confirmed during the human menstrual cycle by RT–PCR and in situ hybridization and was increased during the secretory compared with proliferative phase of the cycle (P = 0.0001), the time of implantation. To assess the importance of maternal Meis1 expression in a mouse model, the uteri of Day 2 pregnant mice were injected with Meis1 over-expression or small interfering RNA (siRNA) constructs. Blocking Meis1 expression by siRNA before implantation significantly reduced average implantation rates (P = 0.00001). Increased or decreased Meis1 expression significantly increased or decreased the expression of integrin β3, a transcriptional target of HOXA10 and an important factor in early embryo-endometrium interactions (P = 0.006). Manipulating Meis1 expression before implantation also dramatically affected the number of pinopodes, uterine endometrial epithelial projections that develop at the time of endometrial receptivity. CONCLUSIONS The results suggest that in mouse, meis1 contributes to regulating endometrial development during the menstrual cycle and establishing the conditions necessary for implantation. PMID:18408019

  16. Assessing Hydrologic Impacts of Land Configuration Changes Using an Integrated Hydrologic Model at the Rocky Flats Environmental Technology Site, Colorado

    NASA Astrophysics Data System (ADS)

    Prucha, R. H.; Dayton, C. S.; Hawley, C. M.

    2002-12-01

    The Rocky Flats Environmental Technology Site (RFETS) in Golden, Colorado, a former Department of Energy nuclear weapons manufacturing facility, is currently undergoing closure. The natural semi-arid interaction between surface and subsurface flow at RFETS is complex and complicated by the industrial modifications to the flow system. Using a substantial site data set, a distributed parameter, fully-integrated hydrologic model was developed to assess the hydrologic impact of different hypothetical site closure configurations on the current flow system and to better understand the integrated hydrologic behavior of the system. An integrated model with this level of detail has not been previously developed in a semi-arid area, and a unique, but comprehensive, approach was required to calibrate and validate the model. Several hypothetical scenarios were developed to simulate hydrologic effects of modifying different aspects of the site. For example, some of the simulated modifications included regrading the current land surface, changing the existing surface channel network, removing subsurface trenches and gravity drain flow systems, installing a slurry wall and geotechnical cover, changing the current vegetative cover, and converting existing buildings and pavement to permeable soil areas. The integrated flow model was developed using a rigorous physically-based code so that realistic design parameters can simulate these changes. This code also permitted evaluation of changes to complex integrated hydrologic system responses that included channelized and overland flow, pond levels, unsaturated zone storage, groundwater heads and flow directions, and integrated water balances for key areas. Results generally show that channel flow offsite decreases substantially for different scenarios, while groundwater heads generally increase within the reconfigured industrial area most of which is then discharged as evapotranspiration. These changes have significant implications to

  17. Chromosome aberrations in Japanese fishermen exposed to fallout radiation 420-1200 km distant from the nuclear explosion test site at Bikini Atoll: report 60 years after the incident.

    PubMed

    Tanaka, Kimio; Ohtaki, Megu; Hoshi, Masaharu

    2016-08-01

    During the period from March to May, 1954, the USA conducted six nuclear weapon tests at the "Bravo" detonation sites at the Bikini and Enewetak Atolls, Marshall Islands. At that time, the crew of tuna fishing boats and cargo ships that were operating approximately 150-1200 km away from the test sites were exposed to radioactive fallout. The crew of the fishing boats and those on cargo ships except the "5th Fukuryu-maru" did not undergo any health examinations at the time of the incident. In the present study, chromosome aberrations in peripheral blood lymphocytes were examined in detail by the G-banding method in 17 crew members from 8 fishing boats and 2 from one cargo ship, 60 years after the tests. None of the subjects examined had suffered from cancer. The percentages of both stable-type aberrations such as translocation, inversion and deletion, and unstable-type aberrations such as dicentric and centric ring in the study group were significantly higher (1.4- and 2.3-fold, respectively) than those in nine age-matched controls. In the exposed and control groups, the percentages of stable-type aberrations were 3.35 % and 2.45 %, respectively, and the numbers of dicentric and centric ring chromosomes per 100 cells were 0.35 and 0.15, respectively. Small clones were observed in three members of the exposed group. These results suggest that the crews were exposed to slightly higher levels of fallout than had hitherto been assumed. PMID:27017218

  18. Low Impact Development Design—Integrating Suitability Analysis and Site Planning for Reduction of Post-Development Stormwater Quantity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A land-suitability analysis (LSA) was integrated with open-space conservation principles, based on watershed physiographic and soil characteristics, to derive a low-impact development (LID) residential plan for a three hectare site in Coshocton OH, USA. The curve number method was used to estimate t...

  19. Low-Impact Development Design—Integrating Suitability Analysis and Site Planning For Reduction Of Post-Development Stormwater Quantity

    EPA Science Inventory

    A land-suitability analysis (LSA) was integrated with open-space conservation principles, based on watershed physiographic and soil characteristics, to derive a low-impact development (LID) residential plan for a three hectare site in Coshocton OH, USA. The curve number method wa...

  20. Control of cell identity genes occurs in insulated neighborhoods in mammalian chromosomes.

    PubMed

    Dowen, Jill M; Fan, Zi Peng; Hnisz, Denes; Ren, Gang; Abraham, Brian J; Zhang, Lyndon N; Weintraub, Abraham S; Schuijers, Jurian; Lee, Tong Ihn; Zhao, Keji; Young, Richard A

    2014-10-01

    The pluripotent state of embryonic stem cells (ESCs) is produced by active transcription of genes that control cell identity and repression of genes encoding lineage-specifying developmental regulators. Here, we use ESC cohesin ChIA-PET data to identify the local chromosomal structures at both active and repressed genes across the genome. The results produce a map of enhancer-promoter interactions and reveal that super-enhancer-driven genes generally occur within chromosome structures that are formed by the looping of two interacting CTCF sites co-occupied by cohesin. These looped structures form insulated neighborhoods whose integrity is important for proper expression of local genes. We also find that repressed genes encoding lineage-specifying developmental regulators occur within insulated neighborhoods. These results provide insights into the relationship between transcriptional control of cell identity genes and control of local chromosome structure. PMID:25303531

  1. Using remote sensing data for exploitation of integrated renewable energy at coastal site in South Italy

    NASA Astrophysics Data System (ADS)

    Calaudi, Rosamaria; Lo Feudo, Teresa; Calidonna, Claudia Roberta; Sempreviva, Anna Maria

    2016-04-01

    Renewable energy sources are major components of the strategy to reduce harmful emissions and to replace depleting fossil energy resources. Data from Remote Sensing can provide detailed information for analysis for sources of renewable energy and to determine the potential energy and socially acceptability of suggested location. Coastal sites of Southern Italy have the advantage of favorable climatic conditions to use renewable energy, such us cloud free days and local breeze phenomena. Many ports are located where they have opportunities for exploitation of renewable energy, by using existing port area and by taking advantage of their coastal locations. Policies of European-Committee and Global-Navigation-PIANC for a better use of energy and an efficient supply from renewable sources are also focused on the construction of port facilities in zero emissions. Using data from Remote Sensing, can reduce the financial resources currently required for finding and assessing suitable areas, we defined an integrated methodology for potential wind and solar energy in harbor areas. In this study we compared the hourly solar power energy using MSG-SEVIRI (Meteosat Second Generation Spinning Enhanced Visible and Infrared) data products DSSF (Down-welling Surface Short-wave-Flux), and PV-Plant measurements with Nominal Power Peak of 19,85 kWp. The PV Plant is situated at a coastal site in Calabrian region, located near Vibo Valentia harbor area. We estimate potential energy by using input solar radiation of Satellite data, with same characteristics of the PV-plant. The RMSE and BIAS for hourly averaged solar electrical reproducibility are estimated including clear and sky conditions. Comparison between energy reproducibility by using DSSF product and PV-plant measurements, made over the period October 2013-June 2014, showed a good agreement in our costal site and generally overestimate (RMSE(35W/m2) and BIAS(4W/m2)) electrical reproducibility from a PV-plant. For wind resource

  2. Spontaneous Formation of Compound X Chromosomes in Drosophila Melanogaster

    PubMed Central

    Morrison, R. J.; Raymond, J. D.; Zunt, J. R.; Lim, J. K.; Simmons, M. J.

    1988-01-01

    Males carrying different X chromosomes were tested for the ability to produce daughters with attached-X chromosomes. This ability is characteristic of males carrying an X chromosome derived from 59b-z, a multiply marked X chromosome, and is especially pronounced in males carrying the unstable 59b-z chromosomes Uc and Uc-l(r). Recombination experiments with one of the Uc-l(r) chromosomes showed that the formation of compound chromosomes depends on two widely separated segments. One of these is proximal to the forked locus and is probably proximal to the carnation locus. This segment may contain the actual site of chromosome attachment. The other essential segment lies between the crossveinless and vermilion loci and may contain multiple factors that influence the attachment process. PMID:3135238

  3. Marker chromosomes lacking {alpha}-satellite DNA: A new intriguing class of abnormalities

    SciTech Connect

    Becker, L.A.; Zinn, A.B.; Stallard, J.R.

    1994-09-01

    Recent studies have implicated {alpha}-satellite DNA as an integral part of the centromere and important for the normal segregation of chromosomes. We analyzed four supernumerary marker chromosomes in which fluorescence in situ hybridization (FISH) could detect neither pancentromeric or chromosome specific {alpha}-satellite DNA. Mosaicism of the markers existed, but each was present in the majority of cells indicating that they segregated normally. FISH with chromosome-specific libraries identified the origins of these markers as chromosomes 13 (1 case) and 15 (3 cases). High resolution analysis, combined with hybridization of a series of cosmid probes, revealed that each marker was a symmetrical duplication of the terminal long arm of the parent chromosome. Telomeric sequences were detected by FISH indicating linear structures. Breakpoint heterogeneity, as defined by cosmid probes, was demonstrated in the three cases involving chromosome 15. No pericentromeric satellite III DNA could be detected on three markers. Studies with anti-centromere antibodies are in progress to assay for centromeric antigens on the markers, as expected at functional centromeric sites. Our results demonstrate that the precise structural identification and heterogeneity of these markers can be easily elucidated using FISH with unique sequence cosmid probes. We conclude from our studies and others in the literature: (1) there is a newly defined class of markers lacking {alpha}-satellite DNA and containing duplications of terminal sequences; (2)neither {alpha}-satellite nor satellite III DNA at levels detectable by FISH is necessary for fidelity in the normal segregation of chromosomes; and (3) these markers were most likely formed by recombination of the long arms during meiosis.

  4. Verification of geomechanical integrity and prediction of long-term mineral trapping for the Ketzin CO2 storage pilot site

    NASA Astrophysics Data System (ADS)

    Kempka, Thomas; De Lucia, Marco; Kühn, Michael

    2014-05-01

    Static and dynamic numerical modelling generally accompany the entire CO2 storage site life cycle. Thereto, it is required to match the employed models with field observations on a regular basis in order to predict future site behaviour. We investigated the coupled processes at the Ketzin CO2 storage pilot site [1] using a model coupling concept focusing on the temporal relevance of processes involved (hydraulic, chemical and mechanical) at given time-scales (site operation, abandonment and long-term stabilization). For that purpose, long-term dynamic multi-phase flow simulations [2], [3] established the basis for all simulations discussed in the following. Hereby, pressure changes resulting in geomechanical effects are largest during site operation, whereas geochemical reactions are governed by slow kinetics resulting in a long-term stabilization. To account for mechanical integrity, which may be mainly affected during site operation, we incorporated a regional-scale coupled hydro-mechanical model. Our simulation results show maximum ground surface displacements of about 4 mm, whereas shear and tensile failure are not observed. Consequently, the CO2 storage operation at the Ketzin pilot site does not compromise reservoir, caprock and fault integrity. Chemical processes responsible for mineral trapping are expected to mainly occur during long-term stabilization at the Ketzin pilot site [4]. Hence, our previous assessment [3] was extended by integrating two long-term mineral trapping scenarios. Thereby, mineral trapping contributes to the trapping mechanisms with 11.7 % after 16,000 years of simulation in our conservative and with 30.9 % in our maximum reactivity scenarios. Dynamic flow simulations indicate that only 0.2 % of the CO2 injected (about 67,270 t CO2 in total) is in gaseous state, but structurally trapped after 16,000 years. Depending on the studied long-term scenario, CO2 dissolution is the dominating trapping mechanism with 68.9 % and 88

  5. Combining microseismic and geomechanical observations to interpret storage integrity at the In Salah CCS site

    NASA Astrophysics Data System (ADS)

    Goertz-Allmann, Bettina P.; Kühn, Daniela; Oye, Volker; Bohloli, Bahman; Aker, Eyvind

    2014-07-01

    We present results from microseismic monitoring and geomechanical analysis obtained at the industrial-scale CO2 sequestration site at the In Salah gas development project in Algeria. More than 5000 microseismic events have been detected at a pilot monitoring well using a master event cross-correlation method. The microseismic activity occurs in four distinct clusters and thereof three clearly correlate with injection rates and wellhead pressures. These event clusters are consistent with a location within the reservoir interval. However, due to insufficient network geometry there are large uncertainties on event location. We estimate a fracture pressure of 155 bar (at the wellhead) from the comparison of injection pressure and injection rate and conclude that reservoir fracture pressure of the injection horizon has most likely been exceeded occasionally, accompanied by increased microseismic activity. Our analysis of 3-D ray tracing for direct and converted phases suggests that one of the event clusters is located at a shallower depth than the reservoir injection interval. However, this event cluster is most likely unrelated to changes in the injection activity at a single well, as the event times do not correlate with the wellhead pressures. Furthermore, this event cluster shows b-values close to one, indicating re-activated natural or tectonic seismicity on pre-existing weakness zones rather than injection induced seismicity. Analysis of event azimuths and significant shear wave splitting of up to 5 per cent provide further valuable insight into fluid migration and fracture orientation at the reservoir level. Although only one geophone was available during the critical injection period, the microseismic monitoring of CO2 injection at In Salah is capable of addressing some of the most relevant questions about fluid migration and reservoir integrity. An improved monitoring array with larger aperture and higher sensitivity is highly recommended, as it could greatly

  6. Methodology and software to detect viral integration site hot-spots

    PubMed Central

    2011-01-01

    Background Modern gene therapy methods have limited control over where a therapeutic viral vector inserts into the host genome. Vector integration can activate local gene expression, which can cause cancer if the vector inserts near an oncogene. Viral integration hot-spots or 'common insertion sites' (CIS) are scrutinized to evaluate and predict patient safety. CIS are typically defined by a minimum density of insertions (such as 2-4 within a 30-100 kb region), which unfortunately depends on the total number of observed VIS. This is problematic for comparing hot-spot distributions across data sets and patients, where the VIS numbers may vary. Results We develop two new methods for defining hot-spots that are relatively independent of data set size. Both methods operate on distributions of VIS across consecutive 1 Mb 'bins' of the genome. The first method 'z-threshold' tallies the number of VIS per bin, converts these counts to z-scores, and applies a threshold to define high density bins. The second method 'BCP' applies a Bayesian change-point model to the z-scores to define hot-spots. The novel hot-spot methods are compared with a conventional CIS method using simulated data sets and data sets from five published human studies, including the X-linked ALD (adrenoleukodystrophy), CGD (chronic granulomatous disease) and SCID-X1 (X-linked severe combined immunodeficiency) trials. The BCP analysis of the human X-linked ALD data for two patients separately (774 and 1627 VIS) and combined (2401 VIS) resulted in 5-6 hot-spots covering 0.17-0.251% of the genome and containing 5.56-7.74% of the total VIS. In comparison, the CIS analysis resulted in 12-110 hot-spots covering 0.018-0.246% of the genome and containing 5.81-22.7% of the VIS, corresponding to a greater number of hot-spots as the data set size increased. Our hot-spot methods enable one to evaluate the extent of VIS clustering, and formally compare data sets in terms of hot-spot overlap. Finally, we show that the

  7. Abnormal human sex chromosome constitutions

    SciTech Connect

    1993-12-31

    Chapter 22, discusses abnormal human sex chromosome constitution. Aneuploidy of X chromosomes with a female phenotype, sex chromosome aneuploidy with a male phenotype, and various abnormalities in X chromosome behavior are described. 31 refs., 2 figs.

  8. Chromosomal Disorders and Autism.

    ERIC Educational Resources Information Center

    Gillberg, Christopher

    1998-01-01

    This paper reviews the literature on chromosomal aberrations in autism, especially possible gene markers. It notes that Chromosome 15 and numerical and structural abnormalities of the sex chromosomes have been most frequently reported as related to the genesis of autism. (Author/DB)

  9. Chromosomal development of cancer

    SciTech Connect

    1993-12-31

    Chapter 30, describes the chromosomal development of cancer. It has been established through cytological research that the number of chromosomes in cancer cells often deviates greatly from the usual number in healthy cells of the host organism. This chapter includes discussions on chromosome studies in ascites tumors, stemline and tumor development, mitotic aberrations in cancer, and selection and tumor progression. 25 refs., 2 figs.

  10. Azimuthal Resistivity Investigation of an Unconfined Aquifer at the Hanford Integrated Field Research Challenge Site

    NASA Astrophysics Data System (ADS)

    Greenwood, W. J.; Ward, A. L.; Versteeg, R. J.; Johnson, T. C.; Draper, K.

    2009-12-01

    Developing a robust large-scale groundwater contaminate transport model requires quantifying the effect of heterogeneity and anisotropy on solute transport. Here we investigated the feasibility of using surface azimuthal resistivity methods to characterize near-surface anisotropy and heterogeneity in order to improve the conceptual model for uranium transport through unconsolidated sediment at the Integrated Field Research Challenge Site (IFRC) which borders the Columbia River. A generalized azimuthal resistivity array was constructed with seven telescoping radii and 15° rotations between each electrode. Azimuthal array data were acquired by multiplexing with the MPT-DAS1 system connected to 172 surface electrodes. Array geometries included the square array, arrow array, offset wenner and equatorial dipole-dipole. Effective depths of exploration ranged between 5 and 57 m. Results from the upper 5m of exploration depth exhibit an isotropic resistivity which is consistent with the excavation and homogonous fill depth of the waste ponds at the IFRC. Exploration depths beyond 5 m are influenced by the Hanford and Ringold Formations. These formations exhibit a strong anisotropic resistivity which increases with depth. Assuming that the response is entirely controlled by hydrologic anisotropy, these azimuthal resistivity data suggest a preferential path with a mean azimuth between 150° and 170°. This azimuthal resistivity trend coincides with an incision feature in the Ringold formation measured in a suite of core logs and is consistent with the trajectory of a tracer plume from an injection test conducted in March 2009. Surface azimuthal resistivity methods may also have application in characterizing localized anisotropy and heterogeneity within shallow alluvial deposits at Hanford allowing for the optimal placement of tracer injections and borehole electrodes.

  11. Petrophysical properties of saprolites from the Oak Ridge Integrated Field Research Challenge site, Tennessee

    SciTech Connect

    Revil, André; Skold, Magnus; Hubbard, Susan S.; Wu, Yuxin; Watson, David B.; Karaoulis, Marios

    2013-01-01

    At the Oak Ridge Integrated Field Research Challenge site, near Oak Ridge, Tennessee, the shallow saprolitic aquifer is contaminated by nitric acid, uranium, and metals originating from the former S3 settling ponds. To interpret low-frequency geophysical methods used to image contaminant plumes, we have characterized the petrophysical properties of three representative saprolite core samples. Their hydraulic conductivity ranges from to in agreement with field data. Complex conductivity measurements, in the frequency range of 1 mHz to 45 kHz, were performed with NaCl solutions with electrical conductivities in the range to , a range representative of field conditions. The electrical conductivity data were well reproduced with a simple linear conductivity model between the saprolite conductivity and the pore water conductivity. The conductivity plots were used to estimate the formation factor (the cementation exponent was about ) and the surface conductivity ( ). The magnitude of the surface conductivity depended on the degree of weathering and therefore on the amount of smectite and mixed layer (illite-smectite) clays present in the saprolite. The chargeability of the core samples was in the range of and is strongly dependent on the salinity. We also performed streaming potential measurements with the same pore fluid composition as that used for the complex conductivity measurements. We found an excess of movable electrical charges on the order of 100 to in agreement with previous investigations connecting the movable excess charge density to permeability. The zeta potential was in the range of to independent on the salinity. The electrical measurements were consistent with an average cation exchange capacity in the range of 1.4 to and a specific surface area on the order of 4000 to about 30,000 . Read More: http://library.seg.org/doi/abs/10.1190/geo2012-0176.1

  12. Integration of surface and upper air measurements for determining evapotranspiration at a high mountain site

    NASA Astrophysics Data System (ADS)

    Connell, Bernadette Helen

    An integrated approach using surface and upper air measurements was used to estimate evapotranspiration in a high mountain area. The first part of this work focused on identifying potential errors introduced into the results due to the characteristics of the sonde instrument and the dynamic environment it was used in. The second part of this work focused on estimating the evapotranspiration. Sources of error were reviewed for radiosonde measurements in the Atmospheric Boundary Layer and focusing on two radiosonde models manufactured by Atmospheric Instrumentation Research, Inc. Temperature and humidity lag errors and wind errors were analyzed. Errors in measurement of azimuth and elevation angles and pressure over short time intervals and at higher altitudes introduced wind vector errors greater than 5 m s-1. Mean temperature and humidity lag errors were small, but individual large lag errors occurred with dramatic changes in the environment, such as near the surface or at the top of the boundary layer. Dual sonde flights showed mean instrument error comparable to lag error. Vertical profiles of potential temperature and specific humidity measured by radiosondes within the surface boundary layer were used to estimate Bowen Ratio at a high elevation alpine site for 2 cases representing moist/windy and dry/calm conditions. The Bowen Ratio was combined with surface global radiation measurements to estimate evaporation (ET) through an energy balance approach. Weather modified vegetation characteristics and their spatial distribution reflect the large local differences in the latent (ET) and sensible heat fluxes measured a 2 surface stations close by. The sonde estimates correlated well (R2 = 0.90) with a weighted average of the surface measured ET in both an undisturbed high pressure ridge system characterized by a dry deep convective boundary layer and during the passage of a weak frontal system characterized by a shallower mechanically mixed moist boundary layer.

  13. INTEGRATIVE ASSESSMENT OF BENTHIC EFFECTS FROM REMEDIAL ACTIVITIES AT SUPERFUND SITES

    EPA Science Inventory

    At contaminated sediment sites, the Superfund program must decide whether to leave the site alone or select a remedial option. These decisions are based in part on the relative risks to the environment and health posed by each alternative. Whatever decision is made at a site, the...

  14. Identifying targets of selection in mosaic genomes with machine learning: applications in Anopheles gambiae for detecting sites within locally adapted chromosomal inversions.

    PubMed

    He, Qixin; Knowles, L Lacey

    2016-05-01

    Chromosomal inversions are important structural changes that may facilitate divergent selection when they capture co-adaptive loci in the face of gene flow. However, identifying selection targets within inversions can be challenging. The high degrees of differentiation between heterokaryotypes, as well as the differences in demographic histories of collinear regions compared with inverted ones, reduce the power of traditional outlier analyses for detecting selected loci. Here, we develop a new approach that uses discriminant functions informed from inversion-specific expectations to classify loci that are under selection (or drift). Analysis of RAD sequencing data we collected in a classic dipteran species with polymorphic inversion clines-Anopheles gambiae, a malaria vector species from sub-Saharan Africa-demonstrates the benefits of the approach compared with traditional outlier analyses. We focus specifically on two polymorphic inversions, the 2La and 2Rb arrangements that predominate in dry habitats and the 2L+(a) and 2R+(b) arrangements in wet habitats, which contrast with the minimal geographic structure of SNPs from collinear regions. With our approach, we identify two strongly selected regions within 2La associated with dry habitat. Moreover, we also show that the prevalence of selection is greater in the arrangement 2L+(a) that is associated with wet habitat (unlike presumed importance of selective divergence associated with the shift of the mosquitoes into dry habitats). We discuss the implications of these results with respect to studies of rapid adaptation in these malaria vectors, and in particular, the insights our newly developed approach offers for identifying not only potential targets of selection, but also the population that has undergone adaptive change. PMID:26994406

  15. A chromosome-centric human proteome project (C-HPP) to characterize the sets of proteins encoded in chromosome 17.

    PubMed

    Liu, Suli; Im, Hogune; Bairoch, Amos; Cristofanilli, Massimo; Chen, Rui; Deutsch, Eric W; Dalton, Stephen; Fenyo, David; Fanayan, Susan; Gates, Chris; Gaudet, Pascale; Hincapie, Marina; Hanash, Samir; Kim, Hoguen; Jeong, Seul-Ki; Lundberg, Emma; Mias, George; Menon, Rajasree; Mu, Zhaomei; Nice, Edouard; Paik, Young-Ki; Uhlen, Mathias; Wells, Lance; Wu, Shiaw-Lin; Yan, Fangfei; Zhang, Fan; Zhang, Yue; Snyder, Michael; Omenn, Gilbert S; Beavis, Ronald C; Hancock, William S

    2013-01-01

    We report progress assembling the parts list for chromosome 17 and illustrate the various processes that we have developed to integrate available data from diverse genomic and proteomic knowledge bases. As primary resources, we have used GPMDB, neXtProt, PeptideAtlas, Human Protein Atlas (HPA), and GeneCards. All sites share the common resource of Ensembl for the genome modeling information. We have defined the chromosome 17 parts list with the following information: 1169 protein-coding genes, the numbers of proteins confidently identified by various experimental approaches as documented in GPMDB, neXtProt, PeptideAtlas, and HPA, examples of typical data sets obtained by RNASeq and proteomic studies of epithelial derived tumor cell lines (disease proteome) and a normal proteome (peripheral mononuclear cells), reported evidence of post-translational modifications, and examples of alternative splice variants (ASVs). We have constructed a list of the 59 "missing" proteins as well as 201 proteins that have inconclusive mass spectrometric (MS) identifications. In this report we have defined a process to establish a baseline for the incorporation of new evidence on protein identification and characterization as well as related information from transcriptome analyses. This initial list of "missing" proteins that will guide the selection of appropriate samples for discovery studies as well as antibody reagents. Also we have illustrated the significant diversity of protein variants (including post-translational modifications, PTMs) using regions on chromosome 17 that contain important oncogenes. We emphasize the need for mandated deposition of proteomics data in public databases, the further development of improved PTM, ASV, and single nucleotide variant (SNV) databases, and the construction of Web sites that can integrate and regularly update such information. In addition, we describe the distribution of both clustered and scattered sets of protein families on the

  16. Long-Range Chromosome Interactions Mediated by Cohesin Shape Circadian Gene Expression

    PubMed Central

    Xu, Yichi; Guo, Weimin; Li, Ping; Zhang, Yan; Zhao, Meng; Fan, Zenghua; Zhao, Zhihu; Yan, Jun

    2016-01-01

    Mammalian circadian rhythm is established by the negative feedback loops consisting of a set of clock genes, which lead to the circadian expression of thousands of downstream genes in vivo. As genome-wide transcription is organized under the high-order chromosome structure, it is largely uncharted how circadian gene expression is influenced by chromosome architecture. We focus on the function of chromatin structure proteins cohesin as well as CTCF (CCCTC-binding factor) in circadian rhythm. Using circular chromosome conformation capture sequencing, we systematically examined the interacting loci of a Bmal1-bound super-enhancer upstream of a clock gene Nr1d1 in mouse liver. These interactions are largely stable in the circadian cycle and cohesin binding sites are enriched in the interactome. Global analysis showed that cohesin-CTCF co-binding sites tend to insulate the phases of circadian oscillating genes while cohesin-non-CTCF sites are associated with high circadian rhythmicity of transcription. A model integrating the effects of cohesin and CTCF markedly improved the mechanistic understanding of circadian gene expression. Further experiments in cohesin knockout cells demonstrated that cohesin is required at least in part for driving the circadian gene expression by facilitating the enhancer-promoter looping. This study provided a novel insight into the relationship between circadian transcriptome and the high-order chromosome structure. PMID:27135601

  17. Human T-Cell Leukemia Virus Type 1 Integration Target Sites in the Human Genome: Comparison with Those of Other Retroviruses▿ ‡

    PubMed Central

    Derse, David; Crise, Bruce; Li, Yuan; Princler, Gerald; Lum, Nicole; Stewart, Claudia; McGrath, Connor F.; Hughes, Stephen H.; Munroe, David J.; Wu, Xiaolin

    2007-01-01

    Retroviral integration into the host genome is not entirely random, and integration site preferences vary among different retroviruses. Human immunodeficiency virus (HIV) prefers to integrate within active genes, whereas murine leukemia virus (MLV) prefers to integrate near transcription start sites and CpG islands. On the other hand, integration of avian sarcoma-leukosis virus (ASLV) shows little preference either for genes, transcription start sites, or CpG islands. While host cellular factors play important roles in target site selection, the viral integrase is probably the major viral determinant. It is reasonable to hypothesize that retroviruses with similar integrases have similar preferences for target site selection. Although integration profiles are well defined for members of the lentivirus, spumaretrovirus, alpharetrovirus, and gammaretrovirus genera, no members of the deltaretroviruses, for example, human T-cell leukemia virus type 1 (HTLV-1), have been evaluated. We have mapped 541 HTLV-1 integration sites in human HeLa cells and show that HTLV-1, like ASLV, does not specifically target transcription units and transcription start sites. Comparing the integration sites of HTLV-1 with those of ASLV, HIV, simian immunodeficiency virus, MLV, and foamy virus, we show that global and local integration site preferences correlate with the sequence/structure of virus-encoded integrases, supporting the idea that integrase is the major determinant of retroviral integration site selection. Our results suggest that the global integration profiles of other retroviruses could be predicted from phylogenetic comparisons of the integrase proteins. Our results show that retroviruses that engender different insertional mutagenesis risks can have similar integration profiles. PMID:17409138

  18. Integral Equations in the Study of Polar and Ionic Interaction Site Fluids

    NASA Astrophysics Data System (ADS)

    Howard, Jesse J.; Pettitt, B. Montgomery

    2011-10-01

    We consider some of the current integral equation approaches and application to model polar liquid mixtures. We show the use of multidimensional integral equations and in particular progress on the theory and applications of three dimensional integral equations. The IEs we consider may be derived from equilibrium statistical mechanical expressions incorporating a classical Hamiltonian description of the system. We give example including salt solutions, inhomogeneous solutions and systems including proteins and nucleic acids.

  19. Integral equations in the study of polar and ionic interaction site fluids

    PubMed Central

    Howard, Jesse J.

    2011-01-01

    In this review article we consider some of the current integral equation approaches and application to model polar liquid mixtures. We consider the use of multidimensional integral equations and in particular progress on the theory and applications of three dimensional integral equations. The IEs we consider may be derived from equilibrium statistical mechanical expressions incorporating a classical Hamiltonian description of the system. We give example including salt solutions, inhomogeneous solutions and systems including proteins and nucleic acids. PMID:22383857

  20. Using integrated geospatial mapping and conceptual site models to guide risk-based environmental clean-up decisions.

    PubMed

    Mayer, Henry J; Greenberg, Michael R; Burger, Joanna; Gochfield, Michael; Powers, Charles; Kosson, David; Keren, Roger; Danis, Christine; Vyas, Vikram

    2005-04-01

    Government and private sector organizations are increasingly turning to the use of maps and other visual models to provide a depiction of environmental hazards and the potential risks they represent to humans and ecosystems. Frequently, the graphic presentation is tailored to address a specific contaminant, its location and possible exposure pathways, and potential receptors. Its format is usually driven by the data available, choice of graphics technology, and the audience being served. A format that is effective for displaying one contaminant at one scale at one site, however, may be ineffective in accurately portraying the circumstances surrounding a different contaminant at the same site, or the same contaminant at a different site, because of limitations in available data or the graphics technology being used. This is the daunting challenge facing the U.S. Department of Energy (DOE), which is responsible for the nation's legacy wastes from nuclear weapons research, testing, and production at over 100 sites in the United States. In this article, we discuss the development and use of integrated geospatial mapping and conceptual site models to identify hazards and evaluate alternative long-term environmental clean-up strategies at DOE sites located across the United States. While the DOE probably has the greatest need for such information, the Department of Defense and other public and private responsible parties for many large and controversial National Priority List or Superfund sites would benefit from a similar approach. PMID:15876215

  1. Novel complex integrating mitochondria and the microtubular cytoskeleton with chromosome remodeling and tumor suppressor RASSF1 deduced by in silico homology analysis, interaction cloning in yeast, and colocalization in cultured cells.

    PubMed

    Liu, Leyuan; Amy, Vo; Liu, Guoqin; McKeehan, Wallace L

    2002-01-01

    Availability of the complete sequence of the human genome and sequence homology analysis has accelerated new protein discovery and clues to protein function. Protein-protein interaction cloning suggests multisubunit complexes and pathways. Here, we combine these molecular approaches with cultured cell colocalization analysis to suggest a novel complex and a pathway that integrate the mitochondrial location and the microtubular cytoskeleton with chromosome remodeling, apoptosis, and tumor suppression based on a novel leucine-rich pentatricopeptide repeat-motif-containing protein (LRPPRC) that copurified with the fibroblast growth factor receptor complex. One round of interaction cloning and sequence homology analysis defined a primary LRPPRC complex with novel subunits cat eye syndrome chromosome region candidate 2 (CECR2), ubiquitously expressed transcript (UXT), and chromosome 19 open reading frames 5 (C19ORF5) but still of unknown function. Immuno, deoxyribonucleic acid (DNA), and green fluorescent protein (GFP) tag colocalization analyses revealed that LRPPRC appears in both cytosol and nuclei of cultured cells, colocalizes with mitochondria and beta-tubulin rather than with alpha-actin in the cytosol of interphase cells, and exhibits phase-dependent organization around separating chromosomes in mitotic cells. GFP-tagged CECR2B was strictly nuclear and colocalized with condensed DNA in apoptotic cells. GFP-tagged UXT and GFP-tagged C19ORF5 appeared in both cytosol and nuclei and colocalized with LRPPRC and beta-tubulin. Cells exhibiting nuclear C19ORF5 were apoptotic. Screening for interactive substrates with the primary LRPPRC substrates in the human liver complementary DNA library revealed that CECR2B interacted with chromatin-associated TFIID-associated protein TAFII30 and ribonucleic acid splicing factor SRP40, UXT bridged to CBP/p300-binding factor CITED2 and kinetochore-associated factor BUB3, and C19ORF5 complexed with mitochondria-associated NADH

  2. The Eastern Lau Basin Integrated Studies Site (ISS): Recent Progress and Future Plans

    NASA Astrophysics Data System (ADS)

    Wiens, D.; Martinez, F.; Langmuir, C.; Tivey, M.; Childress, J.; Fisher, C.; Wheat, G.; Perfit, M.; Blackman, D.; Kim, S.

    2004-12-01

    Rapid progress is being made in understanding the Eastern Lau Spreading Center (ELSC) Integrated Studies Site, which is a new focus area in the Ridge 2000 Program. The ELSC, located in the western Pacific near Tonga, is a 390 km-long first-order ridge segment that displays a broad range of effects of the back-arc environment. Its southern end, at only 40 km from the Tonga arc volcanic front, is propagating southward into a back-arc rift. Its northern end is 100 km from the volcanic front and terminates at a large, nontransform offset. The ELSC undergoes substantial and systematic changes in primary parameters affecting crustal accretion including spreading rate and mantle source composition. As a consequence it displays large changes along its length in lava chemistry, axial depth and morphology, melt lens characteristics, and crustal thickness and structure. A focus of the work at the ELSC is to understand how changes in these forcing functions affect crustal accretion, hydrothermal venting, and faunal composition and abundance. A geophysical/hydrothermal study of the entire ELSC during an initial R2K cruise in April-May 2004 (PI: Martinez) disclosed a surprisingly high level of hydrothermal activity along the ELSC. This survey involved shipboard multibeam, two deep-towed sonars (DSL120A and IMI30), and concurrent MAPR/Chemical Scanner, CTD/rosette tow-yos, and vertical casts. Hydrothermal activity as indicated by water column plumes increases toward the north, even though magmatic robustness decreases. An ancillary study (PI: Thurnherr) also deployed autonomous floats during this cruise to investigate deep circulation patterns that affect hydrothermal plumes and faunal dispersal. A second cruise in September 2004 (PI: Langmuir) focused on locating vent sources using ABE, petrological sampling, and determining water column properties. Three additional cruises are scheduled during 2005. The first (PI: Tivey) will provide an initial characterization of vent fields

  3. Chromosomes, conflict, and epigenetics: chromosomal speciation revisited.

    PubMed

    Brown, Judith D; O'Neill, Rachel J

    2010-01-01

    Since Darwin first noted that the process of speciation was indeed the "mystery of mysteries," scientists have tried to develop testable models for the development of reproductive incompatibilities-the first step in the formation of a new species. Early theorists proposed that chromosome rearrangements were implicated in the process of reproductive isolation; however, the chromosomal speciation model has recently been questioned. In addition, recent data from hybrid model systems indicates that simple epistatic interactions, the Dobzhansky-Muller incompatibilities, are more complex. In fact, incompatibilities are quite broad, including interactions among heterochromatin, small RNAs, and distinct, epigenetically defined genomic regions such as the centromere. In this review, we will examine both classical and current models of chromosomal speciation and describe the "evolving" theory of genetic conflict, epigenetics, and chromosomal speciation. PMID:20438362

  4. 49 CFR 40.43 - What steps must operators of collection sites take to protect the security and integrity of urine...

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... to protect the security and integrity of urine collections? 40.43 Section 40.43 Transportation Office... PROGRAMS Collection Sites, Forms, Equipment and Supplies Used in DOT Urine Collections § 40.43 What steps must operators of collection sites take to protect the security and integrity of urine collections?...

  5. 49 CFR 40.43 - What steps must operators of collection sites take to protect the security and integrity of urine...

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... to protect the security and integrity of urine collections? 40.43 Section 40.43 Transportation Office... PROGRAMS Collection Sites, Forms, Equipment and Supplies Used in DOT Urine Collections § 40.43 What steps must operators of collection sites take to protect the security and integrity of urine collections?...

  6. An integrated multi criteria approach for landfill siting in a conflicting environmental, economical and socio-cultural area.

    PubMed

    Eskandari, Mahnaz; Homaee, Mehdi; Mahmodi, Shahla

    2012-08-01

    Landfill site selection is a complicated multi criteria land use planning that should convince all related stakeholders with different insights. This paper addresses an integrating approach for landfill siting based on conflicting opinions among environmental, economical and socio-cultural expertise. In order to gain optimized siting decision, the issue was investigated in different viewpoints. At first step based on opinion sampling and questionnaire results of 35 experts familiar with local situations, the national environmental legislations and international practices, 13 constraints and 15 factors were built in hierarchical structure. Factors divided into three environmental, economical and socio-cultural groups. In the next step, the GIS-database was developed based on the designated criteria. In the third stage, the criteria standardization and criteria weighting were accomplished. The relative importance weights of criteria and subcriteria were estimated, respectively, using analytical hierarchy process and rank ordering methods based on different experts opinions. Thereafter, by using simple additive weighting method, the suitability maps for landfill siting in Marvdasht, Iran, was evaluated in environmental, economical and socio-cultural visions. The importance of each group of criteria in its own vision was assigned to be higher than two other groups. In the fourth stage, the final suitability map was obtained after crossing three resulted maps in different visions and reported in five suitability classes for landfill construction. This map indicated that almost 1224 ha of the study area can be considered as best suitable class for landfill siting considering all visions. In the last stage, a comprehensive field visit was performed to verify the selected site obtained from the proposed model. This field inspection has confirmed the proposed integrating approach for the landfill siting. PMID:22503155

  7. Model coupling methodology for thermo-hydro-mechanical-chemical numerical simulations in integrated assessment of long-term site behaviour

    NASA Astrophysics Data System (ADS)

    Kempka, Thomas; De Lucia, Marco; Kühn, Michael

    2015-04-01

    The integrated assessment of long-term site behaviour taking into account a high spatial resolution at reservoir scale requires a sophisticated methodology to represent coupled thermal, hydraulic, mechanical and chemical processes of relevance. Our coupling methodology considers the time-dependent occurrence and significance of multi-phase flow processes, mechanical effects and geochemical reactions (Kempka et al., 2014). Hereby, a simplified hydro-chemical coupling procedure was developed (Klein et al., 2013) and validated against fully coupled hydro-chemical simulations (De Lucia et al., 2015). The numerical simulation results elaborated for the pilot site Ketzin demonstrate that mechanical reservoir, caprock and fault integrity are maintained during the time of operation and that after 10,000 years CO2 dissolution is the dominating trapping mechanism and mineralization occurs on the order of 10 % to 25 % with negligible changes to porosity and permeability. De Lucia, M., Kempka, T., Kühn, M. A coupling alternative to reactive transport simulations for long-term prediction of chemical reactions in heterogeneous CO2 storage systems (2014) Geosci Model Dev Discuss 7:6217-6261. doi:10.5194/gmdd-7-6217-2014. Kempka, T., De Lucia, M., Kühn, M. Geomechanical integrity verification and mineral trapping quantification for the Ketzin CO2 storage pilot site by coupled numerical simulations (2014) Energy Procedia 63:3330-3338, doi:10.1016/j.egypro.2014.11.361. Klein E, De Lucia M, Kempka T, Kühn M. Evaluation of longterm mineral trapping at the Ketzin pilot site for CO2 storage: an integrative approach using geo-chemical modelling and reservoir simulation. Int J Greenh Gas Con 2013; 19:720-730. doi:10.1016/j.ijggc.2013.05.014.

  8. EBNA2 Drives Formation of New Chromosome Binding Sites and Target Genes for B-Cell Master Regulatory Transcription Factors RBP-jκ and EBF1

    PubMed Central

    Lu, Fang; Chen, Horng-Shen; Kossenkov, Andrew V.; DeWispeleare, Karen; Won, Kyoung-Jae; Lieberman, Paul M.

    2016-01-01

    Epstein-Barr Virus (EBV) transforms resting B-lymphocytes into proliferating lymphoblasts to establish latent infections that can give rise to malignancies. We show here that EBV-encoded transcriptional regulator EBNA2 drives the cooperative and combinatorial genome-wide binding of two master regulators of B-cell fate, namely EBF1 and RBP-jκ. Previous studies suggest that these B-cell factors are statically bound to target gene promoters. In contrast, we found that EBNA2 induces the formation of new binding for both RBP-jκ and EBF1, many of which are in close physical proximity in the cellular and viral genome. These newly induced binding sites co-occupied by EBNA2-EBF1-RBP-jκ correlate strongly with transcriptional activation of linked genes that are important for B-lymphoblast function. Conditional expression or repression of EBNA2 leads to a rapid alteration in RBP-jκ and EBF1 binding. Biochemical and shRNA depletion studies provide evidence for cooperative assembly at co-occupied sites. These findings reveal that EBNA2 facilitate combinatorial interactions to induce new patterns of transcription factor occupancy and gene programming necessary to drive B-lymphoblast growth and survival. PMID:26752713

  9. EBNA2 Drives Formation of New Chromosome Binding Sites and Target Genes for B-Cell Master Regulatory Transcription Factors RBP-jκ and EBF1.

    PubMed

    Lu, Fang; Chen, Horng-Shen; Kossenkov, Andrew V; DeWispeleare, Karen; Won, Kyoung-Jae; Lieberman, Paul M

    2016-01-01

    Epstein-Barr Virus (EBV) transforms resting B-lymphocytes into proliferating lymphoblasts to establish latent infections that can give rise to malignancies. We show here that EBV-encoded transcriptional regulator EBNA2 drives the cooperative and combinatorial genome-wide binding of two master regulators of B-cell fate, namely EBF1 and RBP-jκ. Previous studies suggest that these B-cell factors are statically bound to target gene promoters. In contrast, we found that EBNA2 induces the formation of new binding for both RBP-jκ and EBF1, many of which are in close physical proximity in the cellular and viral genome. These newly induced binding sites co-occupied by EBNA2-EBF1-RBP-jκ correlate strongly with transcriptional activation of linked genes that are important for B-lymphoblast function. Conditional expression or repression of EBNA2 leads to a rapid alteration in RBP-jκ and EBF1 binding. Biochemical and shRNA depletion studies provide evidence for cooperative assembly at co-occupied sites. These findings reveal that EBNA2 facilitate combinatorial interactions to induce new patterns of transcription factor occupancy and gene programming necessary to drive B-lymphoblast growth and survival. PMID:26752713

  10. Mapping and ordered cloning of the human X chromosome. Progress report, September 1991--November 1992

    SciTech Connect

    Caskey, C.T.; Nelson, D.L.

    1992-12-01

    Progress is reported on gathering X chromosome specific libraries and integrating those with the library produced in this project. Further studies on understanding Fragile X Syndrome and other hereditary diseases related to the X chromosome are described. (DT)

  11. Using radiation hybrids to generate region-specific markers for human chromosome 9

    SciTech Connect

    Britt, D.E.; Mark, H.F.L.; Nebres, M.

    1994-09-01

    The production of sequence tagged sites and polymorphic markers is an important step in generating a physical map of the human genome and identifying loci involved in genetic diseases. Our work involves the physical mapping of the short arm of human chromosome 9, the site of at least one tumor supressor gene, as well as the locus involved in cartilage hair hypoplasia. Our goal is to increase the number of markers available for 9p, using a panel of radiation hybrids we have constructed and characterized. The hybrids were generated from a monochromosomal hybrid that contains human chromosome 9 marked with a retroviral vector. Radiation hybrids were produced that contain overlapping regions of the chromosome surrounding the site of retroviral integration. In order to generate markers specific for the short arm, Alu-PCR products from a radiation hybrid containing only 9p were cloned. Clones were mapped back to a subpanel of hybrids and grouped into intervals. By using a hybrid subpanel containing overlapping portions 9p, we are able to identify clones from defined regions. DNA from the clones was sequenced and this information used to generate sequence tagged sites. We have also developed a number of new polymorphic markers, taking advantage of the high degree of polymorphism of the 3{prime} end of each Alu sequence. For each polymorphic marker, a specific primer was designed from the cloned DNA and then paired with an Alu primer. These primer pairs were used to amplify DNA from unrelated individuals, in order to identify primer sets that detect useful polymorphisms. Both the STS and polymorphic markers will be extremely useful in the construction of a physical map of chromosome 9, and in the identification of genes on the short arm of the chromosome.

  12. Plasmid integration in a wide range of bacteria mediated by the integrase of Lactobacillus delbrueckii bacteriophage mv4.

    PubMed Central

    Auvray, F; Coddeville, M; Ritzenthaler, P; Dupont, L

    1997-01-01

    Bacteriophage mv4 is a temperate phage infecting Lactobacillus delbrueckii subsp. bulgaricus. During lysogenization, the phage integrates its genome into the host chromosome at the 3' end of a tRNA(Ser) gene through a site-specific recombination process (L. Dupont et al., J. Bacteriol., 177:586-595, 1995). A nonreplicative vector (pMC1) based on the mv