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Sample records for chromosomal integration sites

  1. Integration sites of Epstein-Barr virus genome on chromosomes of human lymphoblastoid cell lines

    SciTech Connect

    Wuu, K.D.; Chen, Y.J.; Wang-Wuu, S.

    1994-09-01

    Epstein-Barr virus (EBV) is the pathogen of infectious mononucleosis. The viral genome is present in more than 95% of the African cases of Burkitt lymphoma and it is usually maintained in episomal form in the tumor cells. Viral integration has been described only for Nanalwa which is a Burkitt lymphoma cell line lacking episomes. In order to examine the role of EBV in the immortalization of human Blymphocytes, we investigated whether the EBV integration into the human genome is essential. If the integration does occur, we would like to know whether the integration is randomly distributed or whether the viral DNA integrates preferentially at certain sites. Fourteen in vitro immortalized human lymphoblastoid cell lines (LCLs) were examined by fluorescence in situ hybridization (FISH) with a biotinylated EBV BamHI w DNA fragment as probe. The episomal form of EBV DNA was found in all cells of these cell lines, while only about 65% of the cells have the integrated viral DNA. This might suggest that integration is not a pre-requisite for cell immortalization. Although all chromosomes, except Y, have been found with integrated viral genome, chromsomes 1 and 5 are the most frequent EBV DNA carrier (p<0.05). Nine chromosome bands, namely, 1p31, 1q31, 2q32, 3q13, 3q26, 5q14, 6q24, 7q31 and 12q21, are preferential targets for EBV integration (p<0.001). Eighty percent of the total 938 EBV hybridization signals were found to be at G-band-positive area. This suggests that the mechanism of EBV integration might be different from that of the retroviruses, which specifically integrate to G-band-negative areas. Thus, we conclude that the integration of EBV to host genome is non-random and it may have something to do with the structure of chromosome and DNA sequences.

  2. 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

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

    PubMed

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

    1991-04-01

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

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

    PubMed Central

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

    2014-01-01

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

  5. 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.

  6. 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.

  7. Improved recombinant antibody production by CHO cells using a production enhancer DNA element with repeated transgene integration at a predetermined chromosomal site.

    PubMed

    Kawabe, Yoshinori; Inao, Takanori; Komatsu, Shodai; Huang, Guan; Ito, Akira; Omasa, Takeshi; Kamihira, Masamichi

    2017-03-01

    Chinese hamster ovary (CHO) cells are one of the most useful host cell lines for the production of biopharmaceutical proteins. Although a series of production processes have been refined to improve protein productivity and cost performance, establishing producer cells is still time-consuming and labor-intensive. Recombinase-mediated site-specific gene integration into a predetermined chromosomal locus may enable predictable protein expression, reducing the laborious process of cell screening. We previously developed an accumulative site-specific gene integration system (AGIS) using Cre recombinase and mutated loxP sites for transgene integration and amplification in the CHO cell genome. Epigenetic modifier elements such as insulators are effective DNA cis-regulatory elements for stabilizing transgene expression. Here, we attempted to enhance transgene expression in recombinant CHO cells generated by AGIS using a production enhancer DNA element (PE) derived from the CHO genome. The PE was introduced into an expression unit for a recombinant scFv-Fc antibody. The effect on scFv-Fc productivity of PE position and orientation within the transgene was evaluated, while keeping the background chromosomal structure constant. For the optimal PE arrangement, scFv-Fc productivity was enhanced 2.6-fold compared with an expression unit without a PE. The enhancing effect of the PE on transgene expression was also observed when two or three PE-flanked expression units were inserted as tandem repeats. These results indicate that AGIS using the PE-flanked expression unit is a promising approach for establishing producer cell lines for biopharmaceutical protein production.

  8. 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.

  9. 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

    2016-01-15

    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.

  10. Retroviral integration: Site matters

    PubMed Central

    Demeulemeester, Jonas; De Rijck, Jan

    2015-01-01

    Here, we review genomic target site selection during retroviral integration as a multistep process in which specific biases are introduced at each level. The first asymmetries are introduced when the virus takes a specific route into the nucleus. Next, by co‐opting distinct host cofactors, the integration machinery is guided to particular chromatin contexts. As the viral integrase captures a local target nucleosome, specific contacts introduce fine‐grained biases in the integration site distribution. In vivo, the established population of proviruses is subject to both positive and negative selection, thereby continuously reshaping the integration site distribution. By affecting stochastic proviral expression as well as the mutagenic potential of the virus, integration site choice may be an inherent part of the evolutionary strategies used by different retroviruses to maximise reproductive success. PMID:26293289

  11. Transcription termination maintains chromosome integrity.

    PubMed

    Washburn, Robert S; Gottesman, Max E

    2011-01-11

    DNA replication fork movement is impeded by collisions with transcription elongation complexes (TEC). We propose that a critical function of transcription termination factors is to prevent TEC from blocking DNA replication and inducing replication fork arrest, one consequence of which is DNA double-strand breaks. We show that inhibition of Rho-dependent transcription termination by bicyclomycin in Escherichia coli induced double-strand breaks. Cells deleted for Rho-cofactors nusA and nusG were hypersensitive to bicyclomycin, and had extensive chromosome fragmentation even in the absence of the drug. An RNA polymerase mutation that destabilizes TEC (rpoB*35) increased bicyclomycin resistance >40-fold. Double-strand break formation depended on DNA replication, and can be explained by replication fork collapse. Deleting recombination genes required for replication fork repair (recB and ruvC) increased sensitivity to bicyclomycin, as did loss of the replication fork reloading helicases rep and priA. We propose that Rho responds to a translocating replisome by releasing obstructing TEC.

  12. Transgene integration and chromosome alterations in two transgenic lines of tritordeum.

    PubMed

    Barro, F; Martín, A; Cabrera, A

    2003-01-01

    Plants from two transgenic lines of tritordeum (an amphiploid between Triticum turgidum cv. durum and Hordeumn chilense) have been analyzed by fluorescence in-situ hybridization (FISH) to characterize the transgene integration sites and chromosome rearrangements. Transgenic lines were transformed in two different events with the genes encoding for the high-molecular-weight glutenin subunits (HMW-GS), 1Ax1 and/or 1Dx5. Three integration sites and four translocations were detected. All three integration sites were located on chromosome segments of Hordeum chilense translocated into wheat chromosomes. No translocations from wheat into H. chilense chromosomes were observed. Both HMW-GS transgenes were expressed at high levels in the endosperm of transgenic plants. The analysis by FISH of transgenic plants allowed the early detection of homozygous and heterozygous plants. The consequences and implications of translocations on breeding are discussed.

  13. Structural comparison of three types of staphylococcal cassette chromosome mec integrated in the chromosome in methicillin-resistant Staphylococcus aureus.

    PubMed

    Ito, T; Katayama, Y; Asada, K; Mori, N; Tsutsumimoto, K; Tiensasitorn, C; Hiramatsu, K

    2001-05-01

    The beta-lactam resistance gene mecA of Staphylococcus aureus is carried by a novel mobile genetic element, designated staphylococcal cassette chromosome mec (SCCmec), identified in the chromosome of a Japanese methicillin-resistant S. aureus (MRSA) strain. We now report identification of two additional types of mecA-carrying genetic elements found in the MRSA strains isolated in other countries of the world. There were substantial differences in the size and nucleotide sequences between the elements and the SCCmec. However, new elements shared the chromosomal integration site with the SCCmec. Structural analysis of the new elements revealed that they possessed all of the salient features of the SCCmec: conserved terminal inverted repeats and direct repeats at the integration junction points, conserved genetic organization around the mecA gene, and the presence of cassette chromosome recombinase (ccr) genes responsible for the movements of SCCmec. The elements, therefore, were considered to comprise the SCCmec family of staphylococcal mobile genetic elements together with the previously identified SCCmec. Among 38 epidemic MRSA strains isolated in 20 countries, 34 were shown to possess one of the three typical SCCmec elements on the chromosome. Our findings indicated that there are at least three distinct MRSA clones in the world with different types of SCCmec in their chromosome.

  14. 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.

  15. Deciphering the Code for Retroviral Integration Target Site Selection

    PubMed Central

    Santoni, Federico Andrea; Hartley, Oliver; Luban, Jeremy

    2010-01-01

    Upon cell invasion, retroviruses generate a DNA copy of their RNA genome and integrate retroviral cDNA within host chromosomal DNA. Integration occurs throughout the host cell genome, but target site selection is not random. Each subgroup of retrovirus is distinguished from the others by attraction to particular features on chromosomes. Despite extensive efforts to identify host factors that interact with retrovirion components or chromosome features predictive of integration, little is known about how integration sites are selected. We attempted to identify markers predictive of retroviral integration by exploiting Precision-Recall methods for extracting information from highly skewed datasets to derive robust and discriminating measures of association. ChIPSeq datasets for more than 60 factors were compared with 14 retroviral integration datasets. When compared with MLV, PERV or XMRV integration sites, strong association was observed with STAT1, acetylation of H3 and H4 at several positions, and methylation of H2AZ, H3K4, and K9. By combining peaks from ChIPSeq datasets, a supermarker was identified that localized within 2 kB of 75% of MLV proviruses and detected differences in integration preferences among different cell types. The supermarker predicted the likelihood of integration within specific chromosomal regions in a cell-type specific manner, yielding probabilities for integration into proto-oncogene LMO2 identical to experimentally determined values. The supermarker thus identifies chromosomal features highly favored for retroviral integration, provides clues to the mechanism by which retrovirus integration sites are selected, and offers a tool for predicting cell-type specific proto-oncogene activation by retroviruses. PMID:21124862

  16. DNA Secondary Structure at Chromosomal Fragile Sites in Human Disease

    PubMed Central

    Thys, Ryan G; Lehman, Christine E; Pierce, Levi C. T; Wang, Yuh-Hwa

    2015-01-01

    DNA has the ability to form a variety of secondary structures that can interfere with normal cellular processes, and many of these structures have been associated with neurological diseases and cancer. Secondary structure-forming sequences are often found at chromosomal fragile sites, which are hotspots for sister chromatid exchange, chromosomal translocations, and deletions. Structures formed at fragile sites can lead to instability by disrupting normal cellular processes such as DNA replication and transcription. The instability caused by disruption of replication and transcription can lead to DNA breakage, resulting in gene rearrangements and deletions that cause disease. In this review, we discuss the role of DNA secondary structure at fragile sites in human disease. PMID:25937814

  17. Structure of the chromosomal insertion site for pSAM2: functional analysis in Escherichia coli.

    PubMed

    Raynal, A; Tuphile, K; Gerbaud, C; Luther, T; Guérineau, M; Pernodet, J L

    1998-04-01

    The element pSAM2 from Streptomyces ambofaciens integrates into the chromosome through site-specific recombination between the element (attP) and the chromosomal (attB) sites. These regions share an identity segment of 58bp extending from the anti-codon loop through the 3' end of a tRNA(Pro) gene. To facilitate the study of the attB site, the int and xis genes, expressed from an inducible promoter, and attP from pSAM2 were cloned on plasmids in Escherichia coil. Compatible plasmids carrying the different attB regions to be tested were introduced in these E. coli strains. Under these conditions, Int alone could promote site-specific integration; Int and Xis were both required for site-specific excision. This experimental system was used to study the sequences required in attB for efficient site-specific recombination. A 26 bp sequence, centred on the anti-codon loop region and not completely included in the identity segment, retained all the functionality of attB; shorter sequences allowed integration with lower efficiencies. By comparing the 26-bp-long attB with attP, according to the Lambda model, we propose that B and B', C and C' core-type Int binding sites consist of 9 bp imperfect inverted repeats separated by a 5 bp overlap region.

  18. 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.

  19. 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

  20. Chromosomal fragile site, FRA16A: Implications for fragile site genesis

    SciTech Connect

    Richards, R.I.; Nancarrow, J.K.; Mangelsdorf, M.

    1994-09-01

    Fragile sites are chemically induced non-staining gaps in chromosomes. Different fragile sites vary in frequency in the population and in the chemistry of their induction. The fragile sites sequenced to date (FRAXA and FRAXE) are rare, folate sensitive sites located on the X chromosomes. They have similar DNA sequence composition of a p(CCG)n repeat adjacent to a methylatable CpG island. Individuals expressing the fragile site have an unstable expanded repeat and methylation of the adjacent CpG island. FRAXA is associated with the most common form of familial mental retardation, Fragile X Syndrome. In order to further understand the relationship between the DNA sequence composition, position in the genome, and the chemistry of induction of fragile sites, we have characterized the rare, folate sensitive fragile site on human chromosome 16 referred to as FRA16A. The molecular basis of FRA16A was found to be expansion of a normally polymorphic p(CCG)n repeat. This repeat was adjacent to a CpG island that was methylated in fragile-site-expressing individuals. The FRA16A locus in individuals who do not express the fragile site is not a site of DNA methylation (imprinting) which suggests that the methylation associated with fragile sites may be a consequence and not a cause of their genesis. We have analyzed the normal repeat copy numbers for the fragile site p(CCG)n repeats in European, Japanese and Indian populations. While the FRAXA and FRAXE repeats show similar distributions of copy numbers, the FRA16A p(CCG)n repeat in Europeans has a greater range and number of alleles (23.7% have n>25) than its Japanese and Indian counterparts. In conjunction with our previous data demonstrating linkage disequilibrium (founder chromosomes) at the FRAXA locus, these data suggest that certain p(CCG)n repeats are inherently unstable.

  1. Meiotic recombination cold spots in chromosomal cohesion sites.

    PubMed

    Ito, Masaru; Kugou, Kazuto; Fawcett, Jeffrey A; Mura, Sachiko; Ikeda, Sho; Innan, Hideki; Ohta, Kunihiro

    2014-05-01

    Meiotic chromosome architecture called 'axis-loop structures' and histone modifications have been shown to regulate the Spo11-dependent formation of DNA double-strand breaks (DSBs) that trigger meiotic recombination. Using genome-wide chromatin immunoprecipitation (ChIP) analyses followed by deep sequencing, we compared the genome-wide distribution of the axis protein Rec8 (the kleisin subunit of meiotic cohesin) with that of oligomeric DNA covalently bound to Spo11, indicative of DSB sites. The frequency of DSB sites is overall constant between Rec8 binding sites. However, DSB cold spots are observed in regions spanning ±0.8 kb around Rec8 binding sites. The axis-associated cold spots are not due to the exclusion of Spo11 localization from the axis, because ChIP experiments showed that substantial Spo11 persists at Rec8 binding sites during DSB formation. Spo11 fused with Gal4 DNA binding domain (Gal4BD-Spo11) tethered in close proximity (≤0.8 kb) to Rec8 binding sites hardly forms meiotic DSBs, in contrast with other regions. In addition, H3K4 trimethylation (H3K4me3) remarkably decreases at Rec8 binding sites. These results suggest that reduced histone H3K4me3 in combination with inactivation of Spo11 activity on the axis discourages DSB hot spot formation.

  2. Identification and Validation of Novel Chromosomal Integration and Expression Loci in Escherichia coli Flagellar Region 1

    PubMed Central

    Juhas, Mario; Ajioka, James W.

    2015-01-01

    Escherichia coli is used as a chassis for a number of Synthetic Biology applications. The lack of suitable chromosomal integration and expression loci is among the main hurdles of the E. coli engineering efforts. We identified and validated chromosomal integration and expression target sites within E. coli K12 MG1655 flagellar region 1. We analyzed five open reading frames of the flagellar region 1, flgA, flgF, flgG, flgI, and flgJ, that are well-conserved among commonly-used E. coli strains, such as MG1655, W3110, DH10B and BL21-DE3. The efficiency of the integration into the E. coli chromosome and the expression of the introduced genetic circuit at the investigated loci varied significantly. The integrations did not have a negative impact on growth; however, they completely abolished motility. From the investigated E. coli K12 MG1655 flagellar region 1, flgA and flgG are the most suitable chromosomal integration and expression loci. PMID:25816013

  3. Dual roles for the telomeric repeats in chromosomally integrated human herpesvirus-6.

    PubMed

    Ohye, Tamae; Inagaki, Hidehito; Ihira, Masaru; Higashimoto, Yuki; Kato, Koji; Oikawa, Junko; Yagasaki, Hiroshi; Niizuma, Takahiro; Takahashi, Yoshiyuki; Kojima, Seiji; Yoshikawa, Tetsushi; Kurahashi, Hiroki

    2014-04-02

    Approximately 1 percent of healthy individuals carry human herpesvirus-6 within a host chromosome. This is referred to as chromosomally integrated herpesvirus-6 (CIHHV-6). In this study, we investigated the chromosomal integration site in six individuals harboring CIHHV-6B. Using FISH, we found that HHV-6B signals are consistently located at the telomeric region. The proximal endpoints of the integrated virus were mapped at one of two telomere-repeat-like sequences (TRSs) within the DR-R in all cases. In two cases, we isolated junction fragments between the viral TRS and human telomere repeats. The distal endpoints were mapped at the distal TRS in all cases. The size of the distal TRS was found to be ~5 kb which is sufficient to fulfill cellular telomeric functions. We conclude that the viral TRS in the DR regions fulfill dual functions for CIHHV-6: homology-mediated integration into the telomeric region of the chromosome and neo-telomere formation that is then stably transmitted.

  4. Integration of common bean ( Phaseolus vulgaris L.) linkage and chromosomal maps.

    PubMed

    Pedrosa, A; Vallejos, C E; Bachmair, A; Schweizer, D

    2003-01-01

    Fluorescent in situ hybridisation of pooled, closely linked RFLP markers was used to integrate the genetic linkage map and the mitotic chromosome map of the common bean. Pooled RFLP probes showed clear and reproducible signals and allowed the assignment of all linkage groups to the chromosomes of two Phaseolus vulgaris cultivars, Saxa and Calima. Low extension values for signals originating from clustered RFLPs suggest that these clones are physically close to each other and that clusters in the genetic map are not a result of suppression of recombination due to the occurrence of chromosome rearrangements. For linkage group K, clustering of markers could be associated with proximity to centromeres. High variation in the number of 45S rDNA loci was observed among cultivars, suggesting that these terminal sites are highly recombinogenic in common bean.

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

    SciTech Connect

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

    1990-05-01

    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{sup {minus}}, G6PD{sup +} 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.

  6. [Development of genetically stable recombinant Saccharomyces cerevisiae strains using combinational chromosomal integration].

    PubMed

    Zuo, Qi; Zhao, Xinqing; Liu, Haijun; Hu, Shiyang; Ma, Zhongyi; Bai, Fengwu

    2014-04-01

    Chromosomal integration enables stable phenotype and therefore has become an important strategy for breeding of industrial Saccharomyces cerevisiae strains. pAUR135 is a plasmid that enables recycling use of antibiotic selection marker, and once attached with designated homologous sequences, integration vector for stable expression can be constructed. Development of S. cerevisiae strains by metabolic engineering normally demands overexpression of multiple genes, and employing pAUR135 plasmid, it is possible to construct S. cerevisiae strains by combinational integration of multiple genes in multiple sites, which results in different ratios of expressions of these genes. Xylose utilization pathway was taken as an example, with three pAUR135-based plasmids carrying three xylose assimilation genes constructed in this study. The three genes were sequentially integrated on the chromosome of S. cerevisiae by combinational integration. Xylose utilization rate was improved 24.4%-35.5% in the combinational integration strain comparing with that of the control strain with all the three genes integrated in one location. Strain improvement achieved by combinational integration is a novel method to manipulate multiple genes for genetic engineering of S. cerevisiae, and the recombinant strains are free of foreign sequences and selection markers. In addition, stable phenotype can be maintained, which is important for breeding of industrial strains. Therefore, combinational integration employing pAUR135 is a novel method for metabolic engineering of industrial S. cerevisiae strains.

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

    PubMed

    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.

  8. 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.

  9. Insertion of transformation vector DNA into different chromosomal sites of Dictyostelium discoideum as determined by pulse field electrophoresis.

    PubMed

    Cole, R A; Williams, K L

    1988-06-10

    Chromosomes of the cellular slime mold Dictyostelium discoideum were fractionated on three pulse field gel electrophoresis systems (pulse field, orthogonal field and C.H.E.F. (Contour-clamped Homogeneous Electric Fields] into a series of 13 bands ranging from 0.1 Mb to over 2 Mb in size. Since this organism has only seven chromosomes (estimated to be 1-10 Mb), and -90 copies of an 88-kilobase linear ribosomal DNA molecule (14% of genome), it was apparent that not all of these bands were whole chromosomes. However these bands were reproducibly obtained with the cell preparation used. They fell into three categories: i) four large poorly resolved DNA molecules (-2 Mb in size) which represent very large fragments or intact chromosomes, ii) eight faint bands ranging from 0.1 Mb to 2 Mb, iii) a prominent band in the apparent size range of about 0.15 Mb. Cloned Fragment V of an EcoR1 digest of the ribosomal DNA, hybridized to the 0.15 Mb band indicating it contained the linear ribosomal DNA. This chromosomal banding pattern was used to examine the stability and location of vector DNA in 16 transformed strains of D. discoideum. Each transformed strain was initially selected on the basis of G418 resistance with an integrating vector containing pBR322 sequences. Eleven transformants still carried pBR322 sequences after more than 60 generations of growth without selection on G418. All four strains transformed with constructs containing regions of the D. discoideum plasmid Ddp1 had lost their pBR322 insert, indicating that integration of Dictyostelium plasmid DNA into chromosomes leads to instability. Orthogonal field electrophoresis of the eleven strains still carrying pBR322 sequences revealed at least seven different integrating sites for the transforming DNA. We conclude that these vectors have many possible sites of integration in the D. discoideum genome.

  10. Chromosome

    MedlinePlus

    Chromosomes are structures found in the center (nucleus) of cells that carry long pieces of DNA. DNA ... is the building block of the human body. Chromosomes also contain proteins that help DNA exist in ...

  11. Chromosomally integrated human herpesvirus-6 in transplant recipients.

    PubMed

    Lee, S-O; Brown, R A; Razonable, R R

    2012-08-01

    Human herpesvirus-6 (HHV-6) is unique among human herpesviruses because of its ability to integrate into chromosomes. This entity, termed chromosomally integrated HHV-6 (CIHHV-6), is often mistaken for active infection and treated unnecessarily. The clinical significance of CIHHV-6 in transplant recipients is not defined. Herein, the clinical characteristics of 7 liver transplant patients with CIHHV-6 from our recent study, together with 14 other published cases of CIHHV-6 were reviewed. Of the 21 cases, CIHHV-6B was reported most commonly among solid organ transplant recipients, while CIHHV-6A was mostly seen in allogeneic hematopoietic stem cell recipients. None of the 21 patients developed clinical symptoms related to HHV-6 after transplantation. However, antiviral therapy was administered to 5 asymptomatic patients mistaken to have HHV-6 infection because of their very high HHV-6 DNA levels, 3 who developed symptomatic cytomegalovirus disease, and 1 with graft-versus-host disease that was mistaken for HHV-6 infection. In patients who received antiviral therapy, there was no apparent decline in HHV-6 DNA load, although change in viral kinetics is difficult to discern in the setting of high baseline HHV-6 DNA load. Clinicians should be aware of this entity of CIHHV-6 so that antiviral therapy can be considered in the proper clinical context.

  12. Inherited Chromosomally Integrated Human Herpesvirus 6 and Breast Cancer.

    PubMed

    Gravel, Annie; Dubuc, Isabelle; Brooks-Wilson, Angela; Aronson, Kristan J; Simard, Jacques; Velásquez-García, Héctor A; Spinelli, John J; Flamand, Louis

    2017-03-01

    Background: Inherited chromosomally integrated human herpesvirus 6 (iciHHV-6) is a condition observed in approximately 1% of the population. Whether such a genetic alteration predisposes to cancer development in currently unknown. Two studies were conducted to determine whether iciHHV-6 is associated with cancer development.Methods: First, a screen of 19,597 people from the province of Quebec (Canada) was conducted. A replication test, using data from a population-based case-control study of 1,090 women with incident breast cancer and 1,053 controls from British Columbia and Ontario (Canada) was conducted. DNA samples were analyzed by qPCR and droplet digital PCR to identify iciHHV-6(+) carriers.Results: In the initial study, a potential association between iciHHV-6 positivity and breast cancer was identified [OR = 2.66; 95% confidence interval (CI), 0.95-7.44]. In the replication dataset, no association was found between iciHHV-6 positivity in women and breast cancer (OR = 0.87; 95% CI, 0.35-2.15).Conclusions: We found no statistically significant associations between inherited chromosomally integrated HHV-6 and breast cancer in women.Impact: These results do not provide evidence to suggest that iciHHV-6 is a risk factor for breast cancer. Cancer Epidemiol Biomarkers Prev; 26(3); 425-7. ©2016 AACR.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. Integrated microfluidics for chromosome engineering--preparation, transportation and manipulation.

    PubMed

    Inoue, Takahito; Takahashi, Katsunori; Yokoyama, Hiroshi

    2002-12-01

    The design and fabrication process of microfluidics for chromosome transportation and manipulation are described. Micro-channels for fluid networks could be built on single-crystal silicon substrates by anisotropic wet etching. After injection of a chromosome-containing buffer solution into the channels by a mechanical pump, chromosome electrophoresis was carried out for its precise transportation. The behavior of chromosomes placed in direct (DC) and alternating (AC) electric fields is explained in detail. The frequency and size dependent amplitude of the chromosome observed in this study showed the existence of inertial hydrodynamic effects in an oscillatory motion. Using a rotating AC electric field, chromosomes could be rotated due to the induced polarization of surface charge and electric double layer of the chromosomes. This study thus shows the possibility of chromosome engineering on microchips, which is expected to enable the development of new devices for chromosome research.

  18. 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.

  19. 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.

  20. FISH-Based Analysis of Clonally Derived CHO Cell Populations Reveals High Probability for Transgene Integration in a Terminal Region of Chromosome 1 (1q13)

    PubMed Central

    Li, Shengwei; Gao, Xiaoping; Peng, Rui; Zhang, Sheng; Fu, Wei

    2016-01-01

    A basic goal in the development of recombinant proteins is the generation of cell lines that express the desired protein stably over many generations. Here, we constructed engineered Chinese hamster ovary cell lines (CHO-S) with a pCHO-hVR1 vector that carried an extracellular domain of a VEGF receptor (VR) fusion gene. Forty-five clones with high hVR1 expression were selected for karyotype analysis. Using fluorescence in situ hybridization (FISH) and G-banding, we found that pCHO-hVR1 was integrated into three chromosomes, including chromosomes 1, Z3 and Z4. Four clones were selected to evaluate their productivity under non-fed, non-optimized shake flask conditions. The results showed that clones 1 and 2 with integration sites on chromosome 1 revealed high levels of hVR1 products (shake flask of approximately 800 mg/L), whereas clones 3 and 4 with integration sites on chromosomes Z3 or Z4 had lower levels of hVR1 products. Furthermore, clones 1 and 2 maintained their productivity stabilities over a continuous period of 80 generations, and clones 3 and 4 showed significant declines in their productivities in the presence of selection pressure. Finally, pCHO-hVR1 localized to the same region at chromosome 1q13, the telomere region of normal chromosome 1. In this study, these results demonstrate that the integration of exogenous hVR1 gene on chromosome 1, band q13, may create a high protein-producing CHO-S cell line, suggesting that chromosome 1q13 may contain a useful target site for the high expression of exogenous protein. This study shows that the integration into the target site of chromosome 1q13 may avoid the problems of random integration that cause gene silencing or also overcome position effects, facilitating exogenous gene expression in CHO-S cells. PMID:27684722

  1. Beyond the Chromosome: The Prevalence of Unique Extra-Chromosomal Bacteriophages with Integrated Virulence Genes in Pathogenic Staphylococcus aureus

    PubMed Central

    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

  2. PionX sites mark the X chromosome for dosage compensation.

    PubMed

    Villa, Raffaella; Schauer, Tamas; Smialowski, Pawel; Straub, Tobias; Becker, Peter B

    2016-09-08

    The rules defining which small fraction of related DNA sequences can be selectively bound by a transcription factor are poorly understood. One of the most challenging tasks in DNA recognition is posed by dosage compensation systems that require the distinction between sex chromosomes and autosomes. In Drosophila melanogaster, the male-specific lethal dosage compensation complex (MSL-DCC) doubles the level of transcription from the single male X chromosome, but the nature of this selectivity is not known. Previous efforts to identify X-chromosome-specific target sequences were unsuccessful as the identified MSL recognition elements lacked discriminative power. Therefore, additional determinants such as co-factors, chromatin features, RNA and chromosome conformation have been proposed to refine targeting further. Here, using an in vitro genome-wide DNA binding assay, we show that recognition of the X chromosome is an intrinsic feature of the MSL-DCC. MSL2, the male-specific organizer of the complex, uses two distinct DNA interaction surfaces-the CXC and proline/basic-residue-rich domains-to identify complex DNA elements on the X chromosome. Specificity is provided by the CXC domain, which binds a novel motif defined by DNA sequence and shape. This motif characterizes a subclass of MSL2-binding sites, which we name PionX (pioneering sites on the X) as they appeared early during the recent evolution of an X chromosome in D. miranda and are the first chromosomal sites to be bound during de novo MSL-DCC assembly. Our data provide the first, to our knowledge, documented molecular mechanism through which the dosage compensation machinery distinguishes the X chromosome from an autosome. They highlight fundamental principles in the recognition of complex DNA elements by protein that will have a strong impact on many aspects of chromosome biology.

  3. 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

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

    PubMed

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

    2017-01-01

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

  5. 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

  6. [Homologue pairing: initiation sites and effects on crossing over and chromosome disjunction in Drosophila melanogaster].

    PubMed

    Chubykin, V L

    1996-01-01

    The role of homologue pairing and chromocentral association of chromosomes in recombination and segregation during cell division is discussed. Peculiarities of mitotic and meiotic chromosome pairing in Drosophila males and females are considered. On the basis of our own and published data, the presence and localization of sites of homologue pairing initiation in euchromatin are substantiated. The effects of transfer of initiation sites along a chromosome (exemplified by inversions) on chromosome pairing (asynapsis), crossing over (intrachromosomal, interchromosomal, and centromeric effects), and segregation are discussed. To record the effects of pairing sites on crossing over, a method of comparing crossing-over frequencies in an inverted region with those in a region of the same size and position with regard to the centromere on cytological maps was proposed. Chromosomes orient toward opposite division poles during paracentromeric heterochromatin pairing. This occurs after successful euchromatin pairing, during which the chromocentral circular structure is reorganized. If heterochromatin pairing is disrupted because of structural or locus mutations, nonexchange bivalents segregate randomly. In this case, chromosome coordination may occur due to proximal chiasmata or chromocentral associations between homologues.

  7. Chromosomally integrated human herpesvirus 6: questions and answers.

    PubMed

    Pellett, Philip E; Ablashi, Dharam V; Ambros, Peter F; Agut, Henri; Caserta, Mary T; Descamps, Vincent; Flamand, Louis; Gautheret-Dejean, Agnès; Hall, Caroline B; Kamble, Rammurti T; Kuehl, Uwe; Lassner, Dirk; Lautenschlager, Irmeli; Loomis, Kristin S; Luppi, Mario; Lusso, Paolo; Medveczky, Peter G; Montoya, Jose G; Mori, Yasuko; Ogata, Masao; Pritchett, Joshua C; Rogez, Sylvie; Seto, Edward; Ward, Katherine N; Yoshikawa, Tetsushi; Razonable, Raymund R

    2012-05-01

    Chromosomally integrated human herpesvirus 6 (ciHHV-6) is a condition in which the complete HHV-6 genome is integrated into the host germ line genome and is vertically transmitted in a Mendelian manner. The condition is found in less than 1% of controls in the USA and UK, but has been found at a somewhat higher prevalence in transplant recipients and other patient populations in several small studies. HHV-6 levels in whole blood that exceed 5.5 log10 copies/ml are strongly suggestive of ciHHV-6. Monitoring DNA load in plasma and serum is unreliable, both for identifying and for monitoring subjects with ciHHV-6 due to cell lysis and release of cellular DNA. High HHV-6 DNA loads associated with ciHHV-6 can lead to erroneous diagnosis of active infection. Transplant recipients with ciHHV-6 may be at increased risk for bacterial infection and graft rejection. ciHHV-6 can be induced to a state of active viral replication in vitro. It is not known whether ciHHV-6 individuals are put at clinical risk by the use of drugs that have been associated with HHV-6 reactivation in vivo or in vitro. Nonetheless, we urge careful observation when use of such drugs is indicated in individuals known to have ciHHV-6. Little is known about whether individuals with ciHHV-6 develop immune tolerance for viral proteins. Further research is needed to determine the role of ciHHV-6 in disease.

  8. Chromosomally integrated human herpesvirus 6: questions and answers

    PubMed Central

    Pellett, Philip E; Ablashi, Dharam V; Ambros, Peter F; Agut, Henri; Caserta, Mary T; Descamps, Vincent; Flamand, Louis; Gautheret-Dejean, Agnès; Hall, Caroline B; Kamble, Rammurti T; Kuehl, Uwe; Lassner, Dirk; Lautenschlager, Irmeli; Loomis, Kristin S; Luppi, Mario; Lusso, Paolo; Medveczky, Peter G; Montoya, Jose G; Mori, Yasuko; Ogata, Masao; Pritchett, Joshua C; Rogez, Sylvie; Seto, Edward; Ward, Katherine N; Yoshikawa, Tetsushi; Razonable, Raymund R

    2012-01-01

    SUMMARY Chromosomally integrated human herpesvirus 6 (ciHHV-6) is a condition in which the complete HHV-6 genome is integrated into the host germ line genome and is vertically transmitted in a Mendelian manner. The condition is found in less than 1% of controls in the USA and UK, but has been found at a somewhat higher prevalence in transplant recipients and other patient populations in several small studies. HHV-6 levels in whole blood that exceed 5.5 log10 copies/ml are strongly suggestive of ciHHV-6. Monitoring DNA load in plasma and serum is unreliable, both for identifying and for monitoring subjects with ciHHV-6 due to cell lysis and release of cellular DNA. High HHV-6 DNA loads associated with ciHHV-6 can lead to erroneous diagnosis of active infection. Transplant recipients with ciHHV-6 may be at increased risk for bacterial infection and graft rejection. ciHHV-6 can be induced to a state of active viral replication in vitro. It is not known whether ciHHV-6 individuals are put at clinical risk by the use of drugs that have been associated with HHV-6 reactivation in vivo or in vitro. Nonetheless, we urge careful observation when use of such drugs is indicated in individuals known to have ciHHV-6. Little is known about whether individuals with ciHHV-6 develop immune tolerance for viral proteins. Further research is needed to determine the role of ciHHV-6 in disease. Copyright © 2011 John Wiley & Sons, Ltd. PMID:22052666

  9. Molecular mechanisms of retroviral integration site selection

    PubMed Central

    Kvaratskhelia, Mamuka; Sharma, Amit; Larue, Ross C.; Serrao, Erik; Engelman, Alan

    2014-01-01

    Retroviral replication proceeds through an obligate integrated DNA provirus, making retroviral vectors attractive vehicles for human gene-therapy. Though most of the host cell genome is available for integration, the process of integration site selection is not random. Retroviruses differ in their choice of chromatin-associated features and also prefer particular nucleotide sequences at the point of insertion. Lentiviruses including HIV-1 preferentially integrate within the bodies of active genes, whereas the prototypical gammaretrovirus Moloney murine leukemia virus (MoMLV) favors strong enhancers and active gene promoter regions. Integration is catalyzed by the viral integrase protein, and recent research has demonstrated that HIV-1 and MoMLV targeting preferences are in large part guided by integrase-interacting host factors (LEDGF/p75 for HIV-1 and BET proteins for MoMLV) that tether viral intasomes to chromatin. In each case, the selectivity of epigenetic marks on histones recognized by the protein tether helps to determine the integration distribution. In contrast, nucleotide preferences at integration sites seem to be governed by the ability for the integrase protein to locally bend the DNA duplex for pairwise insertion of the viral DNA ends. We discuss approaches to alter integration site selection that could potentially improve the safety of retroviral vectors in the clinic. PMID:25147212

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

    PubMed Central

    Glassing, Angela; Lewis, Thomas A.

    2015-01-01

    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 E. 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

  11. A genome-wide analysis of common fragile sites: What features determine chromosomal instability in the human genome?

    PubMed Central

    Fungtammasan, Arkarachai; Walsh, Erin; Chiaromonte, Francesca; Eckert, Kristin A.; Makova, Kateryna D.

    2012-01-01

    Chromosomal common fragile sites (CFSs) are unstable genomic regions that break under replication stress and are involved in structural variation. They frequently are sites of chromosomal rearrangements in cancer and of viral integration. However, CFSs are undercharacterized at the molecular level and thus difficult to predict computationally. Newly available genome-wide profiling studies provide us with an unprecedented opportunity to associate CFSs with features of their local genomic contexts. Here, we contrasted the genomic landscape of cytogenetically defined aphidicolin-induced CFSs (aCFSs) to that of nonfragile sites, using multiple logistic regression. We also analyzed aCFS breakage frequencies as a function of their genomic landscape, using standard multiple regression. We show that local genomic features are effective predictors both of regions harboring aCFSs (explaining ∼77% of the deviance in logistic regression models) and of aCFS breakage frequencies (explaining ∼45% of the variance in standard regression models). In our optimal models (having highest explanatory power), aCFSs are predominantly located in G-negative chromosomal bands and away from centromeres, are enriched in Alu repeats, and have high DNA flexibility. In alternative models, CpG island density, transcription start site density, H3K4me1 coverage, and mononucleotide microsatellite coverage are significant predictors. Also, aCFSs have high fragility when colocated with evolutionarily conserved chromosomal breakpoints. Our models are predictive of the fragility of aCFSs mapped at a higher resolution. Importantly, the genomic features we identified here as significant predictors of fragility allow us to draw valuable inferences on the molecular mechanisms underlying aCFSs. PMID:22456607

  12. Distribution of 5S and 45S rDNA sites in plants with holokinetic chromosomes and the "chromosome field" hypothesis.

    PubMed

    Sousa, A; Barros e Silva, A E; Cuadrado, A; Loarce, Y; Alves, M V; Guerra, M

    2011-08-01

    Secondary constrictions or 45S rDNA sites are commonly reported to be located mainly in the terminal regions of the chromosomes. This distribution has been assumed to be related to the existence of a "chromosome field" lying between the centromere and the telomere, an area in which certain cytogenetic events may predominantly occur. If this hypothesis is true this distribution should not be observed in holokinetic chromosomes, as they do not have a localized centromere. In order to evaluate this hypothesis, a comparative study was made of the distributions of 5S and 45S rDNA sites using fluorescence in situ hybridization in representatives of the genera Eleocharis, Diplacrum, Fimbristylis, Kyllinga and Rhynchospora, all of which belong to the family Cyperaceae. The numbers of sites per diploid chromosome complement varied from 2 to ∼10 for 5S rDNA, and from 2 to ∼45 for 45S rDNA. All of the 11 species analyzed had terminally located 45S rDNA sites on the chromosomes whereas the 5S rDNA sites also generally had terminal distributions, except for the Rhynchospora species, where their position was almost always interstitial. These results, together with other previously published data, suggest that the variation in the number and position of the rDNA sites in species with holokinetic chromosomes is non-random and similar to that reported for species with monocentric chromosomes. Therefore, the predominant terminal position of the 45S rDNA sites does not appear to be influenced by the centromere-telomere polarization as suggested by the "chromosome field" hypothesis. Additionally, the hybridization of 5S and 45S rDNA sites provides interesting markers to distinguish several chromosomes on the rather symmetrical karyotypes of Cyperaceae.

  13. 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.

  14. Casposon integration shows strong target site preference and recapitulates protospacer integration by CRISPR-Cas systems.

    PubMed

    Béguin, Pierre; Charpin, Nicole; Koonin, Eugene V; Forterre, Patrick; Krupovic, Mart

    2016-12-01

    Casposons are a recently discovered group of large DNA transposons present in diverse bacterial and archaeal genomes. For integration into the host chromosome, casposons employ an endonuclease that is homologous to the Cas1 protein involved in protospacer integration by the CRISPR-Cas adaptive immune system. Here we describe the site-preference of integration by the Cas1 integrase (casposase) encoded by the casposon of the archaeon Aciduliprofundum boonei Oligonucleotide duplexes derived from the terminal inverted repeats (TIR) of the A. boonei casposon as well as mini-casposons flanked by the TIR inserted preferentially at a site reconstituting the original A. boonei target site. As in the A. boonei genome, the insertion was accompanied by a 15-bp direct target site duplication (TSD). The minimal functional target consisted of the 15-bp TSD segment and the adjacent 18-bp sequence which comprises the 3' end of the tRNA-Pro gene corresponding to the TΨC loop. The functional casposase target site bears clear resemblance to the leader sequence-repeat junction which is the target for protospacer integration catalyzed by the Cas1-Cas2 adaptation module of CRISPR-Cas. These findings reinforce the mechanistic similarities and evolutionary connection between the casposons and the adaptation module of the prokaryotic adaptive immunity systems.

  15. Casposon integration shows strong target site preference and recapitulates protospacer integration by CRISPR-Cas systems

    PubMed Central

    Béguin, Pierre; Charpin, Nicole; Koonin, Eugene V.; Forterre, Patrick; Krupovic, Mart

    2016-01-01

    Casposons are a recently discovered group of large DNA transposons present in diverse bacterial and archaeal genomes. For integration into the host chromosome, casposons employ an endonuclease that is homologous to the Cas1 protein involved in protospacer integration by the CRISPR-Cas adaptive immune system. Here we describe the site-preference of integration by the Cas1 integrase (casposase) encoded by the casposon of the archaeon Aciduliprofundum boonei. Oligonucleotide duplexes derived from the terminal inverted repeats (TIR) of the A. boonei casposon as well as mini-casposons flanked by the TIR inserted preferentially at a site reconstituting the original A. boonei target site. As in the A. boonei genome, the insertion was accompanied by a 15-bp direct target site duplication (TSD). The minimal functional target consisted of the 15-bp TSD segment and the adjacent 18-bp sequence which comprises the 3′ end of the tRNA-Pro gene corresponding to the TΨC loop. The functional casposase target site bears clear resemblance to the leader sequence-repeat junction which is the target for protospacer integration catalyzed by the Cas1–Cas2 adaptation module of CRISPR-Cas. These findings reinforce the mechanistic similarities and evolutionary connection between the casposons and the adaptation module of the prokaryotic adaptive immunity systems. PMID:27655632

  16. 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...

  17. 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.

  18. 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.

  19. Correction of chromosomal mutation and random integration in embryonic stem cells with helper-dependent adenoviral vectors.

    PubMed

    Ohbayashi, Fumi; Balamotis, Michael A; Kishimoto, Atsuhiro; Aizawa, Emi; Diaz, Arturo; Hasty, Paul; Graham, Frank L; Caskey, C Thomas; Mitani, Kohnosuke

    2005-09-20

    For gene therapy of inherited diseases, targeted integration/gene repair through homologous recombination (HR) between exogenous and chromosomal DNA would be an ideal strategy to avoid potentially serious problems of random integration such as cellular transformation and gene silencing. Efficient sequence-specific modification of chromosomes by HR would also advance both biological studies and therapeutic applications of a variety of stem cells. Toward these goals, we developed an improved strategy of adenoviral vector (AdV)-mediated HR and examined its ability to correct an insertional mutation in the hypoxanthine phosphoribosyl transferase (Hprt) locus in male mouse ES cells. The efficiency of HR was compared between four types of AdVs that contained various lengths of homologies at the Hprt locus and with various multiplicities of infections. The frequency of HR with helper-dependent AdVs (HD AdVs) with an 18.6-kb homology reached 0.2% per transduced cell at a multiplicity of infection of 10 genomes per cell. Detection of random integration at DNA levels by PCR revealed extremely high efficiency of 5% per cell. We also isolated and characterized chromosomal sites where HD AdVs integrated in a random manner. In contrast to retroviral, lentiviral, and adeno-associated viral vectors, which tend to integrate into genes, the integration sites of AdV was distributed randomly inside and outside genes. These findings suggest that HR mediated by HD AdVs is efficient and relatively safe and might be a new viable option for ex vivo gene therapy as well as a tool for chromosomal manipulation of a variety of stem cells.

  20. 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

  1. Characterization of CpG sites that escape methylation on the inactive human X-chromosome.

    PubMed

    Moen, Erika L; Litwin, Edward; Arnovitz, Stephen; Zhang, Xu; Zhang, Wei; Dolan, M Eileen; Godley, Lucy A

    2015-01-01

    In many whole genome studies of gene expression or modified cytosines, data from probes localized to the X-chromosome are removed from analyses due to gender bias. Previously, we observed population differences in cytosine modifications between Caucasian and African lymphoblastoid cell lines (LCLs) on the autosomes using whole genome arrays to measure modified cytosines. DNA methylation plays a critical role in establishment and maintenance of X-chromosome inactivation in females. Therefore, we reasoned that by investigating cytosine modification patterns specifically on the X-chromosome, we could obtain valuable information about a chromosome that is often disregarded in genome-wide analyses. We investigated population differences in cytosine modification patterns along the X-chromosome between Caucasian and African LCLs and identified novel sites that escape methylation on the inactive X-chromosome (Xi) in females. We characterized the chromatin state of these loci by incorporating the extensive histone modification ChIP-seq data generated by ENCODE. To explore the relationship between DNA and histone modifications further, we hypothesized that BRD4, a protein that binds acetylated histones, could be preventing some sites from becoming de novo methylated. To test this, we treated 4 female LCLs with JQ1, a small molecule inhibitor of BRD4, but found that JQ1 treatment induced minor changes in cytosine modification levels, and the majority of sites escaping methylation on the Xi remained unmethylated. This suggests that other epigenetic mechanisms or transcription factors are likely playing a larger role in protecting these sites from de novo methylation on the Xi.

  2. Cre recombinase-mediated site-specific recombination between plant chromosomes.

    PubMed Central

    Qin, M; Bayley, C; Stockton, T; Ow, D W

    1994-01-01

    We report the use of the bacteriophage P1 Cre-lox system for generating conservative site-specific recombination between tobacco chromosomes. Two constructs, one containing a promoterless hygromycin-resistance gene preceded by a lox site (lox-hpt) and the other containing a cauliflower mosaic virus 35S promoter linked to a lox sequence and the cre coding region (35S-lox-cre), were introduced separately into tobacco plants. Crosses between plants harboring either construct produced plants with the two constructs situated on different chromosomes. Plants with recombination events were identified by selecting for hygromycin resistance, a phenotype expressed upon recombination. Molecular analysis showed that these recombination events occurred specifically at the lox sites and resulted in the reciprocal exchange of flanking host DNA. Progenies of these plants showed 67-100% cotransmission of the new transgenes, 35S-lox-hpt and lox-cre, consistent with the preferential cosegregation of translocated chromosomes. These results illustrate that site-specific recombination systems can be useful tools for the large-scale manipulation of eukaryotic chromosomes in vivo. Images PMID:8127869

  3. Progress towards integrated physical and genetic maps of chromosome 22

    SciTech Connect

    Bell, C.J.; Barnoski, B.; Budarf, M.L.

    1994-09-01

    Our immediate objective is to identify and characterize a set of overlapping YAC clones spanning the long arm of chromosome 22 by STS content mapping of the CEPH YAC libraries. STSs are assigned to bins defined by a 25 interval hybrid mapping panel and the YAC libraries are screened by amplification of hierarchical pools of yeast DNAs. Thus far, 382 probes and STSs have been assigned to bins and 251 probes and STSs have been used to identify a total 659 YACs, which is nearly 5X coverage of the chromosome. We have assembled more than twenty putative contigs using simulated annealing, split and merge, and backtracking optimization algorithms. The largest contig contains 82 STSs linked with 250 YACs, connects bin 12 to 15, and contains approximately one third of the long arm. The proximal region also contains two large contigs in bins 1-2 and bin 9, between them containing 26 STSs and 62 YACs. The DiGeorge (DGCR) region, encompassing bins 3-5, is currently 80% covered in a contig of cosmids. The distal portion (bins 17-22) is the least represented in YACs, due to a paucity of markers and a lower hit-rate of YACs per STS. We are targeting this region with new STSs from inter-Alu PCR plasmid libraries derived from radiation hybrid cell lines that retain relevant portions of the chromosome. A concurrent objective of the center is to construct a robust multi-point linkage map of the chromosome. STRPs (simple tandem repeat polymorphisms) were assembled into a 20-point skeletal map and a 29-point framework map; 15 of the markers in the skeletal map have been unequivocally assigned to single intervals in the hybrid panel with identical linkage and bin orders. This genetic map, combined with markers ordered by pulsed field gel maps covering large portions of the chromosome, provides a robust framework for ordering YACs within contigs and ordering contigs along the chromosome.

  4. Twenty-one polymorphic markers from human chromosome 12 for integration of genetic and physical maps

    SciTech Connect

    LeBlanc-Straceski, J.M.; Kissel, H.; Murtaugh, L.; Kucherlapati, R.; Montogmery, K.T.; Krauter, K.S. ); Tsai, P.; Ward, D.C. )

    1994-01-15

    Twenty-one physically mapped, polymorphic markers have been developed from a chromosome 12-specific cosmid library. The markers consist of CA repeat-containing sequence-tagged sites (STSs) derived from cosmid clones mapped by fluorescence in situ hybridization (FISH). Three methods for determining the sequence flanking CA microsatellites were used, including one using degenerate primer sets for direct sequence analysis. Oligonucleotide primer pairs suitable for use in polymerase chain reaction (PCR) were selected from the sequences flanking the CA microsatellite and were tested for their ability to generate unique PCR products. The informativeness of these STSs as genetic markers was determined by typing 10 unrelated individuals who are part of the Centre d'Etude du Polymorphisme Humaine (EPH) pedigrees. Eleven of the 21 FISH-mapped, polymorphic STSs are heterozygous in 7 or more of the individuals tested. Since these markers are derived from physically mapped cosmids, genetic linkage analysis with them will facilitate the integration of the developing physical and genetic maps of chromosome 12. 29 refs., 4 figs., 2 tabs.

  5. Retroviral integration: Site matters: Mechanisms and consequences of retroviral integration site selection.

    PubMed

    Demeulemeester, Jonas; De Rijck, Jan; Gijsbers, Rik; Debyser, Zeger

    2015-11-01

    Here, we review genomic target site selection during retroviral integration as a multistep process in which specific biases are introduced at each level. The first asymmetries are introduced when the virus takes a specific route into the nucleus. Next, by co-opting distinct host cofactors, the integration machinery is guided to particular chromatin contexts. As the viral integrase captures a local target nucleosome, specific contacts introduce fine-grained biases in the integration site distribution. In vivo, the established population of proviruses is subject to both positive and negative selection, thereby continuously reshaping the integration site distribution. By affecting stochastic proviral expression as well as the mutagenic potential of the virus, integration site choice may be an inherent part of the evolutionary strategies used by different retroviruses to maximise reproductive success.

  6. A standard vector for the chromosomal integration and characterization of BioBrick™ parts in Escherichia coli

    PubMed Central

    2013-01-01

    Background The chromosomal integration of biological parts in the host genome enables the engineering of plasmid-free stable strains with single-copy insertions of the desired gene networks. Although different integrative vectors were proposed, no standard pre-assembled genetic tool is available to carry out this task. Synthetic biology concepts can contribute to the development of standardized and user friendly solutions to easily produce engineered strains and to rapidly characterize the desired genetic parts in single-copy context. Results In this work we report the design of a novel integrative vector that allows the genomic integration of biological parts compatible with the RFC10, RFC23 and RFC12 BioBrick™ standards in Escherichia coli. It can also be specialized by using BioBrick™ parts to target the desired integration site in the host genome. The usefulness of this vector has been demonstrated by integrating a set of BioBrick™ devices in two different loci of the E. coli chromosome and by characterizing their activity in single-copy. Construct stability has also been evaluated and compared with plasmid-borne solutions. Conclusions Physical modularity of biological parts has been successfully applied to construct a ready-to-engineer BioBrick™ vector, suitable for a stable chromosomal insertion of standard parts via the desired recombination method, i.e. the bacteriophage integration mechanism or homologous recombination. In contrast with previously proposed solutions, it is a pre-assembled vector containing properly-placed restriction sites for the direct transfer of various formats of BioBrick™ parts. This vector can facilitate the characterization of parts avoiding copy number artefacts and the construction of antibiotic resistance-free engineered microbes, suitable for industrial use. PMID:23663425

  7. Implications of FRA 16A structure for the mechanism of chromosomal fragile site genesis

    SciTech Connect

    Nancarrow, J.K.; Kremer, E.; Holman, K.; Eyre, H.; Callen, D.F.; Sutherland, G.R.; Richards, R.I. ); Doggett, N.A. ); Paslier, D.Le )

    1994-06-24

    Fragile sites are chemically induced nonstaining gaps in chromosomes. Different fragile sites vary in frequency in the population and in the chemistry of their induction. DNA sequences encompassing and including the rare, autosomal, folate-sensitive fragile site, FRA16A, were isolated by positional cloning. The molecular basis of FRA16A was found to be expansion of a normally polymorphic p(CCG)[sub n] repeat. This repeat was adjacent to a CpG island that was methylated in fragile site-expressing individuals. The FRA16A locus in individuals who do not express the fragile site is not a site of DNA methylation (imprinting), which suggests that the methylation associated with fragile sites may be a consequence and not a cause of their genesis.

  8. Launch site integration of Liquid Rocket Boosters

    NASA Technical Reports Server (NTRS)

    Scott, Leland P.; Dickinson, William J.

    1989-01-01

    The impacts of introducing Liquid Rocket Boosters (LRB) into the STS/KSC launch environment are identified and evaluated. Proposed ground systems configurations are presented along with a launch site requirements summary. Pre-launch processing scenarios are described and the required facility modifications and new facility requirements are analyzed. Flight vehicle design recommendations to enhance launch processing are discussed. Processing approaches to integrate LRB with existing STS launch operations are evaluated. The key features and significance of launch site transition to a new STS configuration in parallel with ongoing launch activities are enumerated.

  9. Random transposon vectors pUTTns for the markerless integration of exogenous genes into gram-negative eubacteria chromosomes.

    PubMed

    Li, Rong; Wang, Guangli; Shen, Bin; Wang, Rong; Song, Yao; Li, Shunpeng; Jiang, Jiandong

    2009-11-01

    A set of random transposon vectors pUTTns that facilitates the markerless integration of new functions into the chromosome of gram-negative bacteria has been developed. The vectors, which are derived from mini-Tn5 transposons, are located on a R6K-based suicide delivery plasmid that provides the IS50(R) transposase tnp gene in cis, but they are external to the mobile element. The vectors' conjugal transfer to recipients is mediated by RP4 mobilization functions in the donor. Internal to the mini-Tn5 element is a cassette that contains a selectable antibiotic resistance marker (kanamycin, chloramphenicol, or tetracycline resistance gene), a counter-selectable marker (sacB), a 430-bp repeat of the sacB gene 3' end acted as the directly-repeated (DR) sequence, and modified multiple cloning sites (MCS). After two total rounds of transposon integration and recombination between the two DRs, only the exogenous DNA inserted into the MCS (passenger genes) and a single 430-bp scar sacBDR fragment remained in the chromosome after excision. The utility of these vectors was demonstrated by integrating the organophosphorus insecticide hydrolase gene (mpd) into the chromosome of Escherichia, Pseudomonas, Sphingomonas, and Paracoccus species. Sequential integration of another organophosphorus insecticide hydrolase gene (oph) into the previously engineered bacteria, without bringing any selectable markers, was also successful. These engineered bacteria were relatively stable. Cell viability and original degrading characteristics were not affected compared with the original recipients. This shows that the developed system is very useful for the markerless integration of exogenous genes into the chromosome of gram-negative eubacteria.

  10. Chromosome segregation impacts on cell growth and division site selection in Corynebacterium glutamicum.

    PubMed

    Donovan, Catriona; Schauss, Astrid; Krämer, Reinhard; Bramkamp, Marc

    2013-01-01

    Spatial and temporal regulation of bacterial cell division is imperative for the production of viable offspring. In many rod-shaped bacteria, regulatory systems such as the Min system and nucleoid occlusion ensure the high fidelity of midcell divisome positioning. However, regulation of division site selection in bacteria lacking recognizable Min and nucleoid occlusion remains less well understood. Here, we describe one such rod-shaped organism, Corynebacterium glutamicum, which does not always place the division septum precisely at midcell. Here we now show at single cell level that cell growth and division site selection are spatially and temporally regulated by chromosome segregation. Mutants defective in chromosome segregation have more variable cell growth and aberrant placement of the division site. In these mutants, division septa constrict over and often guillotine the nucleoid, leading to nonviable, DNA-free cells. Our results suggest that chromosome segregation or some nucleoid associated factor influences growth and division site selection in C. glutamicum. Understanding growth and regulation of C. glutamicum cells will also be of importance to develop strains for industrial production of biomolecules, such as amino acids.

  11. Chromosome Segregation Impacts on Cell Growth and Division Site Selection in Corynebacterium glutamicum

    PubMed Central

    Donovan, Catriona; Schauss, Astrid; Krämer, Reinhard; Bramkamp, Marc

    2013-01-01

    Spatial and temporal regulation of bacterial cell division is imperative for the production of viable offspring. In many rod-shaped bacteria, regulatory systems such as the Min system and nucleoid occlusion ensure the high fidelity of midcell divisome positioning. However, regulation of division site selection in bacteria lacking recognizable Min and nucleoid occlusion remains less well understood. Here, we describe one such rod-shaped organism, Corynebacterium glutamicum, which does not always place the division septum precisely at midcell. Here we now show at single cell level that cell growth and division site selection are spatially and temporally regulated by chromosome segregation. Mutants defective in chromosome segregation have more variable cell growth and aberrant placement of the division site. In these mutants, division septa constrict over and often guillotine the nucleoid, leading to nonviable, DNA-free cells. Our results suggest that chromosome segregation or some nucleoid associated factor influences growth and division site selection in C. glutamicum. Understanding growth and regulation of C. glutamicum cells will also be of importance to develop strains for industrial production of biomolecules, such as amino acids. PMID:23405112

  12. 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.

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

    PubMed Central

    Xu, Ruiping; Ke, Yang

    2016-01-01

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

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

    PubMed

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

    2016-02-02

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

  15. 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

  16. 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

  17. Bacteriophage P1 pac sites inserted into the chromosome greatly increase packaging and transduction of Escherichia coli genomic DNA.

    PubMed

    Huang, Haomin; Masters, Millicent

    2014-11-01

    The Escherichia coli bacteriophage P1 packages host chromosome separately from phage DNA, and transfers it to recipient cells at low frequency in a process called generalized transduction. Phage genomes are packaged from concatemers beginning at a specific site, pac. To increase transduction rate, we have inserted pac into the chromosome at up to five equally spaced positions; at least this many are fully tolerated in the absence of P1 infection. A single chromosomal pac greatly increases transduction of downstream markers without decreasing phage yields; 3.5 × as much total chromosomal DNA is packaged. Additional insertions decrease phage yield by > 90% and also decrease phage DNA synthesis, although less dramatically. Packaging of chromosomal markers near to and downstream of each inserted pac site is, at the same time, increased by greater than 10 fold. Transduction of markers near an inserted pac site can be increased by over 1000-fold, potentially allowing identification of such transductants by screening.

  18. 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

  19. 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.

  20. Detection and chromosomal assignment of SV40-DNA integration in Chinese hamster cell lines by chromosome sorting and dot blot hybridization.

    PubMed

    Hutter, K J; Klefenz, H; Goerttler, K

    1990-01-01

    A combination of cytometric (chromosome sorting), molecular (dot blot hybridization using radio-active and/or biotinylated DNA probes) and cytogenetic (G-banding) evaluation is described which allows the rapid identification of single copy and repetitive viral integrates and their assignment to chromosome groups or even individual chromosomes. In the case of Chinese hamster cell line CO 631 it could be demonstrated that SV40 DNA was solely integrated into a submetacentric marker chromosome. Such a cytometric/molecular/cytogenetic "identogram" may prove to be a useful tool in many areas of cell and tumor biology. Furthermore, amounts of chromosomes sufficient for analysis as well as subsequent cloning experiments can be accumulated.

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

    PubMed

    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

    2015-06-15

    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.

  2. 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

  3. Comparative fitness assessment of Anopheles stephensi transgenic lines receptive to site-specific integration.

    PubMed

    Amenya, D A; Bonizzoni, M; Isaacs, A T; Jasinskiene, N; Chen, H; Marinotti, O; Yan, G; James, A A

    2010-04-01

    Genetically modified mosquitoes that are unable to transmit pathogens offer opportunities for controlling vector-borne diseases such as malaria and dengue. Site-specific gene recombination technologies are advantageous in the development of these insects because antipathogen effector genes can be inserted at integration sites in the genome that cause the least alteration in mosquito fitness. Here we describe Anopheles stephensi transgenic lines containing phi C31 attP'docking' sites linked to a fluorescent marker gene. Chromosomal insertion sites were determined and life-table parameters were assessed for transgenic mosquitoes of each line. No significant differences in fitness between the transgenic and nontransgenic mosquitoes were detected in this study. These transgenic lines are suitable for future site-specific integrations of antiparasite transgenes into the attP sites.

  4. Identification of missing proteins in the neXtProt database and unregistered phosphopeptides in the PhosphoSitePlus database as part of the Chromosome-centric Human Proteome Project.

    PubMed

    Shiromizu, Takashi; Adachi, Jun; Watanabe, Shio; Murakami, Tatsuo; Kuga, Takahisa; Muraoka, Satoshi; Tomonaga, Takeshi

    2013-06-07

    The Chromosome-Centric Human Proteome Project (C-HPP) is an international effort for creating an annotated proteomic catalog for each chromosome. The first step of the C-HPP project is to find evidence of expression of all proteins encoded on each chromosome. C-HPP also prioritizes particular protein subsets, such as those with post-translational modifications (PTMs) and those found in low abundance. As participants in C-HPP, we integrated proteomic and phosphoproteomic analysis results from chromosome-independent biomarker discovery research to create a chromosome-based list of proteins and phosphorylation sites. Data were integrated from five independent colorectal cancer (CRC) samples (three types of clinical tissue and two types of cell lines) and lead to the identification of 11,278 proteins, including 8,305 phosphoproteins and 28,205 phosphorylation sites; all of these were categorized on a chromosome-by-chromosome basis. In total, 3,033 "missing proteins", i.e., proteins that currently lack evidence by mass spectrometry, in the neXtProt database and 12,852 unknown phosphorylation sites not registered in the PhosphoSitePlus database were identified. Our in-depth phosphoproteomic study represents a significant contribution to C-HPP. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD000089.

  5. 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.

  6. Identification of specific DNA methylation sites on the Y-chromosome as biomarker in prostate cancer

    PubMed Central

    Du, Fengxia; Zhu, Yasheng; Yu, Hui; Zhang, Chenyu; Li, Xiaohua; Yang, Caiyun; Liu, Huixian; Wang, Dong; Meng, Hao; Chang, Shuang; Han, Xiao; Sun, Yinghao; Sun, Yingli

    2015-01-01

    As a diagnostic biomarker, prostate special antigen (PSA) tests always generate false positive results and lead to unnecessary and/or repeat biopsies. Therefore, there is an urgent need for developing more sensitive, specific diagnostic biomarkers. We epigenotyped methylated sites in cancer tissues and adjacent normal tissues from 66 patients. In comparation with normal adjacent tissues, we observed that there were 6 aberrant methylation sites in prostate cancer tissues on the Y-chromosome. We further performed pyrosequencing using urine of PCa patients and we identified one methylated site (cg05163709) as a potential biomarker. We evaluated the predictive capacity of the aberrant methylated sites using the area under receiver operating characteristic (ROC) curve (AUC). The ROC analysis showed a higher AUC for cg05163709 (0.915) than prostate-specific antigen (PSA, 0.769). These results indicated that aberrant DNA methylation of cg05163709 on the Y-chromosome could serve as a potential diagnostic biomarker with high sensitivity and specificity. PMID:26485765

  7. 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.

  8. 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.

  9. Clonal expansion of Escherichia coli ST38 carrying a chromosomally integrated OXA-48 carbapenemase gene.

    PubMed

    Turton, Jane F; Doumith, Michel; Hopkins, Katie L; Perry, Claire; Meunier, Daniele; Woodford, Neil

    2016-06-01

    Many isolates of Escherichia coli carrying blaOXA-48 referred to Public Health England's national reference laboratory during 2014 and 2015 shared similar pulsed-field gel electrophoresis (PFGE) profiles, despite coming from patients in multiple different hospitals and regions. Whole genome sequencing on an Illumina platform revealed that these belonged to sequence type (ST) 38. The OXA-48 gene is usually carried on a 62 kb IncL/M plasmid (pOXA48a), but those belonging to this ST appeared either to lack plasmid elements or to have only a partial complement. Two isolates, one belonging to a main cluster sharing identical PFGE profiles and the other having a distinct profile, were further sequenced on a minION. The long reads provided by the nanopore sequencing technology facilitated assembly of a much larger contig around the blaOXA-48 region, showing that both isolates shared a similar arrangement, with a plasmid fragment containing blaOXA-48 flanked by IS1R elements integrated into the chromosome, although the length of the plasmid fragment and the insertion site differed between the two isolates. That belonging to the main cluster contained a 21.9 kb Tn6237 insert, as previously described in E. coli EC-15 from Lebanon, but in a different insertion site. PCR mapping indicated that a further 14/31 representatives of this cluster also contained this insert in the same insertion site, with most of the remainder differing only by having additional E. coli sequence on one side of the insertion. This sub-cluster of ST38 was found from 25 different hospital laboratories, suggesting widespread distribution of a successful type.

  10. 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.

  11. Integration Preferences of Wildtype AAV-2 for Consensus Rep-Binding Sites at Numerous Loci in the Human Genome

    PubMed Central

    Hüser, Daniela; Gogol-Döring, Andreas; Lutter, Timo; Weger, Stefan; Winter, Kerstin; Hammer, Eva-Maria; Cathomen, Toni; Reinert, Knut; Heilbronn, Regine

    2010-01-01

    Adeno-associated virus type 2 (AAV) is known to establish latency by preferential integration in human chromosome 19q13.42. The AAV non-structural protein Rep appears to target a site called AAVS1 by simultaneously binding to Rep-binding sites (RBS) present on the AAV genome and within AAVS1. In the absence of Rep, as is the case with AAV vectors, chromosomal integration is rare and random. For a genome-wide survey of wildtype AAV integration a linker-selection-mediated (LSM)-PCR strategy was designed to retrieve AAV-chromosomal junctions. DNA sequence determination revealed wildtype AAV integration sites scattered over the entire human genome. The bioinformatic analysis of these integration sites compared to those of rep-deficient AAV vectors revealed a highly significant overrepresentation of integration events near to consensus RBS. Integration hotspots included AAVS1 with 10% of total events. Novel hotspots near consensus RBS were identified on chromosome 5p13.3 denoted AAVS2 and on chromsome 3p24.3 denoted AAVS3. AAVS2 displayed seven independent junctions clustered within only 14 bp of a consensus RBS which proved to bind Rep in vitro similar to the RBS in AAVS3. Expression of Rep in the presence of rep-deficient AAV vectors shifted targeting preferences from random integration back to the neighbourhood of consensus RBS at hotspots and numerous additional sites in the human genome. In summary, targeted AAV integration is not as specific for AAVS1 as previously assumed. Rather, Rep targets AAV to integrate into open chromatin regions in the reach of various, consensus RBS homologues in the human genome. PMID:20628575

  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. 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.

  14. 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.

  15. Improvement of Fibrinolytic Activity of Bacillus subtilis 168 by Integration of a Fibrinolytic Gene into the Chromosome.

    PubMed

    Jeong, Seon-Ju; Park, Ji Yeong; Lee, Jae Yong; Lee, Kang Wook; Cho, Kye Man; Kim, Gyoung Min; Shin, Jung-Hye; Kim, Jong-Sang; Kim, Jeong Hwan

    2015-11-01

    Fibrinolytic enzyme genes (aprE2, aprE176, and aprE179) were introduced into the Bacillus subtilis 168 chromosome without any antibiotic resistance gene. An integration vector, pDG1662, was used to deliver the genes into the amyE site of B. subtilis 168. Integrants, SJ3-5nc, SJ176nc, and SJ179nc, were obtained after two successive homologous recombinations. The integration of each fibrinolytic gene into the middle of the amyE site was confirmed by phenotypes (Amy(-), Spec(S)) and colony PCR results for these strains. The fibrinolytic activities of the integrants were higher than that of B. subtilis 168 by at least 3.2-fold when grown in LB broth. Cheonggukjang was prepared by inoculating each of B. subtilis 168, SJ3-5nc, SJ176nc, and SJ179nc, and the fibrinolytic activity of cheonggukjang was 4.6 ± 0.7, 10.8 ± 0.9, 7.0 ± 0.6, and 8.0 ± 0.2 (U/g of cheonggukjang), respectively at 72 h. These results showed that construction of B. subtilis strains with enhanced fibrinolytic activities is possible by integration of a strong fibrinolytic gene via a marker-free manner.

  16. 3Disease Browser: A Web server for integrating 3D genome and disease-associated chromosome rearrangement data

    PubMed Central

    Li, Ruifeng; Liu, Yifang; Li, Tingting; Li, Cheng

    2016-01-01

    Chromosomal rearrangement (CR) events have been implicated in many tumor and non-tumor human diseases. CR events lead to their associated diseases by disrupting gene and protein structures. Also, they can lead to diseases through changes in chromosomal 3D structure and gene expression. In this study, we search for CR-associated diseases potentially caused by chromosomal 3D structure alteration by integrating Hi-C and ChIP-seq data. Our algorithm rediscovers experimentally verified disease-associated CRs (polydactyly diseases) that alter gene expression by disrupting chromosome 3D structure. Interestingly, we find that intellectual disability may be a candidate disease caused by 3D chromosome structure alteration. We also develop a Web server (3Disease Browser, http://3dgb.cbi.pku.edu.cn/disease/) for integrating and visualizing disease-associated CR events and chromosomal 3D structure. PMID:27734896

  17. Reactivation of chromosomally integrated human herpesvirus-6 by telomeric circle formation.

    PubMed

    Prusty, Bhupesh K; Krohne, George; Rudel, Thomas

    2013-01-01

    More than 95% of the human population is infected with human herpesvirus-6 (HHV-6) during early childhood and maintains latent HHV-6 genomes either in an extra-chromosomal form or as a chromosomally integrated HHV-6 (ciHHV-6). In addition, approximately 1% of humans are born with an inheritable form of ciHHV-6 integrated into the telomeres of chromosomes. Immunosuppression and stress conditions can reactivate latent HHV-6 replication, which is associated with clinical complications and even death. We have previously shown that Chlamydia trachomatis infection reactivates ciHHV-6 and induces the formation of extra-chromosomal viral DNA in ciHHV-6 cells. Here, we propose a model and provide experimental evidence for the mechanism of ciHHV-6 reactivation. Infection with Chlamydia induced a transient shortening of telomeric ends, which subsequently led to increased telomeric circle (t-circle) formation and incomplete reconstitution of circular viral genomes containing single viral direct repeat (DR). Correspondingly, short t-circles containing parts of the HHV-6 DR were detected in cells from individuals with genetically inherited ciHHV-6. Furthermore, telomere shortening induced in the absence of Chlamydia infection also caused circularization of ciHHV-6, supporting a t-circle based mechanism for ciHHV-6 reactivation.

  18. 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.

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

    DOE PAGES

    Guss, Adam M.; Rother, Michael; Zhang, Jun Kai; ...

    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

  20. 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.

  1. Toward a bacterial genome technology: integration of the Escherichia coli prophage lambda genome into the Bacillus subtilis 168 chromosome.

    PubMed

    Itaya, M

    1995-07-22

    A novel approach to the cloning large DNAs in the Bacillus subtilis chromosome was examined. An Escherichia coli prophage lambda DNA (48.5 kb) was assembled in the chromosome of B. subtilis. The lambda DNA was first subcloned in four segments, having partially overlapping regions. Assembly of the complete prophage was achieved by successive transformation using three discrete DNA integration modes: overlap-elongation, Campbell-type integration, and gap-filling. In the B. subtilis chromosome, DNA was elongated, using contiguous DNA segments, via overlap-elongation. Jumping from one end of a contiguous DNA stretch to another segment was achieved by Campbell-type integration. The remaining gap was sealed by gap-filling. The incorporated lambda DNA thus assembled was stably replicated as part of the 4188 kb B. subtilis chromosome under non-selective conditions. The present method can be used to accommodate larger DNAs in the B. subtilis chromosome and possible applications of this technique are discussed.

  2. Molecular cytogenetic characterization of chromosome site-specific repetitive sequences in the Arctic lamprey (Lethenteron camtschaticum, Petromyzontidae)

    PubMed Central

    Ishijima, Junko; Uno, Yoshinobu; Nunome, Mitsuo; Nishida, Chizuko; Kuraku, Shigehiro

    2017-01-01

    Abstract All extant lamprey karyotypes are characterized by almost all dot-shaped microchromosomes. To understand the molecular basis of chromosome structure in lampreys, we performed chromosome C-banding and silver staining and chromosome mapping of the 18S–28S and 5S ribosomal RNA (rRNA) genes and telomeric TTAGGG repeats in the Arctic lamprey (Lethenteron camtschaticum). In addition, we cloned chromosome site-specific repetitive DNA sequences and characterized them by nucleotide sequencing, chromosome in situ hybridization, and filter hybridization. Three types of repetitive sequences were detected; a 200-bp AT-rich repetitive sequence, LCA-EcoRIa that co-localized with the 18S–28S rRNA gene clusters of 3 chromosomal pairs; a 364-bp AT-rich LCA-EcoRIb sequence that showed homology to the EcoRI sequence family from the sea lamprey (Petromyzon marinus), which contains short repeats as centromeric motifs; and a GC-rich 702-bp LCA-ApaI sequence that was distributed on nearly all chromosomes and showed significant homology with the integrase-coding region of a Ty3/Gypsy family long terminal repeat (LTR) retrotransposon. All three repetitive sequences are highly conserved within the Petromyzontidae or within Petromyzontidae and Mordaciidae. Molecular cytogenetic characterization of these site-specific repeats showed that they may be correlated with programed genome rearrangement (LCA-EcoRIa), centromere structure and function (LCA-EcoRIb), and site-specific amplification of LTR retroelements through homogenization between non-homologous chromosomes (LCA-ApaI). PMID:28025319

  3. Virological analysis of inherited chromosomally integrated human herpesvirus-6 in three hematopoietic stem cell transplant patients.

    PubMed

    Miura, H; Kawamura, Y; Kudo, K; Ihira, M; Ohye, T; Kurahashi, H; Kawashima, N; Miyamura, K; Yoshida, N; Kato, K; Takahashi, Y; Kojima, S; Yoshikawa, T

    2015-10-01

    We analyzed 3 hematopoietic stem cell transplant (HSCT) recipients with inherited chromosomally integrated human herpesvirus-6 (inherited CIHHV-6). Cases 1 (inherited CIHHV-6A) and 2 (inherited CIHHV-6B) were inherited CIHHV-6 recipients. Case 3 received bone marrow from a donor with inherited CIHHV-6B. Following HSCT, HHV-6B was isolated from Case 1. HHV-6A and -6B messenger RNAs were detected in Cases 1 and 3.

  4. Chromosomal Integration of the Klebsiella pneumoniae Carbapenemase Gene, blaKPC, in Klebsiella Species Is Elusive but Not Rare.

    PubMed

    Mathers, Amy J; Stoesser, Nicole; Chai, Weidong; Carroll, Joanne; Barry, Katie; Cherunvanky, Anita; Sebra, Robert; Kasarskis, Andrew; Peto, Tim E; Walker, A Sarah; Sifri, Costi D; Crook, Derrick W; Sheppard, Anna E

    2017-03-01

    Carbapenemase genes in Enterobacteriaceae are mostly described as being plasmid associated. However, the genetic context of carbapenemase genes is not always confirmed in epidemiological surveys, and the frequency of their chromosomal integration therefore is unknown. A previously sequenced collection of blaKPC-positive Enterobacteriaceae from a single U.S. institution (2007 to 2012; n = 281 isolates from 182 patients) was analyzed to identify chromosomal insertions of Tn4401, the transposon most frequently harboring blaKPC Using a combination of short- and long-read sequencing, we confirmed five independent chromosomal integration events from 6/182 (3%) patients, corresponding to 15/281 (5%) isolates. Three patients had isolates identified by perirectal screening, and three had infections which were all successfully treated. When a single copy of blaKPC was in the chromosome, one or both of the phenotypic carbapenemase tests were negative. All chromosomally integrated blaKPC genes were from Klebsiella spp., predominantly K. pneumoniae clonal group 258 (CG258), even though these represented only a small proportion of the isolates. Integration occurred via IS15-ΔI-mediated transposition of a larger, composite region encompassing Tn4401 at one locus of chromosomal integration, seen in the same strain (K. pneumoniae ST340) in two patients. In summary, we identified five independent chromosomal integrations of blaKPC in a large outbreak, demonstrating that this is not a rare event. blaKPC was more frequently integrated into the chromosome of epidemic CG258 K. pneumoniae lineages (ST11, ST258, and ST340) and was more difficult to detect by routine phenotypic methods in this context. The presence of chromosomally integrated blaKPC within successful, globally disseminated K. pneumoniae strains therefore is likely underestimated.

  5. Chromosomal Integration of the Klebsiella pneumoniae Carbapenemase Gene, blaKPC, in Klebsiella Species Is Elusive but Not Rare

    PubMed Central

    Chai, Weidong; Carroll, Joanne; Barry, Katie; Cherunvanky, Anita; Sebra, Robert; Kasarskis, Andrew; Peto, Tim E.; Walker, A. Sarah; Sifri, Costi D.; Crook, Derrick W.; Sheppard, Anna E.

    2016-01-01

    ABSTRACT Carbapenemase genes in Enterobacteriaceae are mostly described as being plasmid associated. However, the genetic context of carbapenemase genes is not always confirmed in epidemiological surveys, and the frequency of their chromosomal integration therefore is unknown. A previously sequenced collection of blaKPC-positive Enterobacteriaceae from a single U.S. institution (2007 to 2012; n = 281 isolates from 182 patients) was analyzed to identify chromosomal insertions of Tn4401, the transposon most frequently harboring blaKPC. Using a combination of short- and long-read sequencing, we confirmed five independent chromosomal integration events from 6/182 (3%) patients, corresponding to 15/281 (5%) isolates. Three patients had isolates identified by perirectal screening, and three had infections which were all successfully treated. When a single copy of blaKPC was in the chromosome, one or both of the phenotypic carbapenemase tests were negative. All chromosomally integrated blaKPC genes were from Klebsiella spp., predominantly K. pneumoniae clonal group 258 (CG258), even though these represented only a small proportion of the isolates. Integration occurred via IS15-ΔI-mediated transposition of a larger, composite region encompassing Tn4401 at one locus of chromosomal integration, seen in the same strain (K. pneumoniae ST340) in two patients. In summary, we identified five independent chromosomal integrations of blaKPC in a large outbreak, demonstrating that this is not a rare event. blaKPC was more frequently integrated into the chromosome of epidemic CG258 K. pneumoniae lineages (ST11, ST258, and ST340) and was more difficult to detect by routine phenotypic methods in this context. The presence of chromosomally integrated blaKPC within successful, globally disseminated K. pneumoniae strains therefore is likely underestimated. PMID:28031204

  6. 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.

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

    PubMed

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

    2016-07-01

    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.

  8. Fragile sites, telomeric DNA sequences, B chromosomes, and DNA content in raccoon dogs, Nyctereutes procyonoides, with comparative notes on foxes, coyote, wolf, and raccoon.

    PubMed

    Wurster-Hill, D H; Ward, O G; Davis, B H; Park, J P; Moyzis, R K; Meyne, J

    1988-01-01

    Earlier studies of the genus Nyctereutes disclosed two subspecies of differing chromosome numbers accompanied by B chromosomes. To further define the relationship of these subspecies to each other, and to other carnivores, and to learn more about the structure and function of their chromosomes, we characterized and compared the genomes in terms of DNA content by flow cytometry, fragile sites induced by aphidicolin, and telomere sequences using biotinylated DNA probes detected with fluorescence. We also characterized the B chromosomes of these two subspecies.

  9. Spo11-accessory proteins link double-strand break sites to the chromosome axis in early meiotic recombination.

    PubMed

    Panizza, Silvia; Mendoza, Marco A; Berlinger, Marc; Huang, Lingzhi; Nicolas, Alain; Shirahige, Katsuhiko; Klein, Franz

    2011-08-05

    Meiotic recombination between homologous chromosomes initiates via programmed DNA double-strand breaks (DSBs), generated by complexes comprising Spo11 transesterase plus accessory proteins. DSBs arise concomitantly with the development of axial chromosome structures, where the coalescence of axis sites produces linear arrays of chromatin loops. Recombining DNA sequences map to loops, but are ultimately tethered to the underlying axis. How and when such tethering occurs is currently unclear. Using ChIPchip in yeast, we show that Spo11-accessory proteins Rec114, Mer2, and Mei4 stably interact with chromosome axis sequences, upon phosphorylation of Mer2 by S phase Cdk. This axis tethering requires meiotic axis components (Red1/Hop1) and is modulated in a domain-specific fashion by cohesin. Loss of Rec114, Mer2, and Mei4 binding correlates with loss of DSBs. Our results strongly suggest that hotspot sequences become tethered to axis sites by the DSB machinery prior to DSB formation.

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

    PubMed Central

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

    2012-01-01

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

  11. A rapid and reliable strategy for chromosomal integration of gene(s) with multiple copies

    PubMed Central

    Gu, Pengfei; Yang, Fan; Su, Tianyuan; Wang, Qian; Liang, Quanfeng; Qi, Qingsheng

    2015-01-01

    Direct optimization of the metabolic pathways on the chromosome requires tools that can fine tune the overexpression of a desired gene or optimize the combination of multiple genes. Although plasmid-dependent overexpression has been used for this task, fundamental issues concerning its genetic stability and operational repeatability have not been addressed. Here, we describe a rapid and reliable strategy for chromosomal integration of gene(s) with multiple copies (CIGMC), which uses the flippase from the yeast 2-μm plasmid. Using green fluorescence protein as a model, we verified that the fluorescent intensity was in accordance with the integration copy number of the target gene. When a narrow-host-range replicon, R6K, was used in the integrative plasmid, the maximum integrated copy number of Escherichia coli reached 15. Applying the CIGMC method to optimize the overexpression of single or multiple genes in amino acid biosynthesis, we successfully improved the product yield and stability of the production. As a flexible strategy, CIGMC can be used in various microorganisms other than E. coli. PMID:25851494

  12. A highly efficient single-step, markerless strategy for multi-copy chromosomal integration of large biochemical pathways in Saccharomyces cerevisiae.

    PubMed

    Shi, Shuobo; Liang, Youyun; Zhang, Mingzi M; Ang, Ee Lui; Zhao, Huimin

    2016-01-01

    Despite recent advances in genome editing capabilities for the model organism Saccharomyces cerevisiae, the chromosomal integration of large biochemical pathways for stable industrial production remains challenging. In this work, we developed a simple platform for high-efficiency, single-step, markerless, multi-copy chromosomal integration of full biochemical pathways in Saccharomyces cerevisiae. In this Di-CRISPR (delta integration CRISPR-Cas) platform based on the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated systems (Cas), we specifically designed guide RNA sequences to target multiple delta sites in the yeast genome. The generation of double stranded breaks at the delta sites allowed simultaneous integration of multiple copies of linearized donor DNA containing large biochemical pathways. With our newly developed Di-CRISPR platform, we were able to attain highly efficient and markerless integration of large biochemical pathways and achieve an unprecedented 18-copy genomic integration of a 24 kb combined xylose utilization and (R,R)-2,3-butanediol (BDO) production pathway in a single step, thus generating a strain that was able to produce BDO directly from xylose. The simplicity and high efficiency of the Di-CRISPR platform could provide a superior alternative to high copy plasmids and would render this platform an invaluable tool for genome editing and metabolic engineering in S. cerevisiae.

  13. 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

  14. Site-specific initiation of DNA replication in metazoan chromosomes and the role of nuclear organization.

    PubMed

    Gilbert, D M; Miyazawa, H; Nallaseth, F S; Ortega, J M; Blow, J J; DePamphilis, M L

    1993-01-01

    We have asked whether or not Xenopus eggs or egg extracts, which have previously been shown to replicate essentially any DNA molecule, will preferentially utilize a known mammalian OBR. Our results reveal that Xenopus egg extracts can preferentially initiate DNA replication at sites chosen in vivo by the hamster cell, provided that the DNA substrate is presented to the extract in the form of a nucleus rather than bare DNA. Thus, site-specific initiation of DNA replication in metazoan cell chromosomes appears to be determined by nuclear organization as well as DNA sequence. We have also considered whether or not BPV, which was previously reported to regulate its copy number through negative as well as positive cis-acting sequences, provides a suitable paradigm for cellular origins. The BPV genome was found to contain cis-acting sequences that can suppress DNA replication driven by a lytic virus such as PyV. However, this suppression did not require any BPV protein, did not limit PyV origin activity to one initiation event per S phase, and did not affect BPV origin activity. These results, together with data from other laboratories, strongly suggest that BPV is simply a slow-replicating version of SV40 and PyV and therefore is not an appropriate model to explain how initiation of cellular DNA replication is limited to once per cell cycle.

  15. 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

    2013-11-06

    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".

  16. An immunocompetent child with chromosomally integrated human herpesvirus 6B accidentally identified during the care of Mycoplasma pneumoniae infection.

    PubMed

    Oikawa, Junko; Tanaka, Junko; Yoshikawa, Tetsushi; Morita, Yoshinori; Hishiki, Haruka; Ishiwada, Naruhiko; Ohye, Tamae; Kurahashi, Hiroki; Kohno, Yoichi

    2014-01-01

    Human herpesvirus 6 (HHV-6) is the only virus known to integrate into human chromosomes and be transmitted from parents to offspring. Less than 1% of the population carries integrated HHV-6 in their genomes. Here, we report the case of a 9-year-old Japanese girl with an extraordinarily high copy number of HHV-6B in her genome. The integrated virus genome was detected by real-time polymerase chain reaction (PCR) in cerebrospinal fluid and serum during the treatment of meningoencephalitis and pneumonia caused by Mycoplasma pneumoniae infection. Furthermore, the HHV-6B genome was detected in hair follicle, plasma, and whole blood in the patient and her mother, but not in the patient's father. Fluorescence in situ hybridization revealed that the viral genome was integrated into chromosome 22. Therefore, these results emphasize the importance of screening for chromosomally integrated HHV-6 prior to starting unnecessary antiviral therapies, particularly for patients harboring HHV-6 with a high copy number.

  17. 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.

  18. The human chromosomal fragile sites more often involved in constitutional deletions and duplications - A genetic and statistical assessment

    NASA Astrophysics Data System (ADS)

    Gomes, Dora Prata; Sequeira, Inês J.; Figueiredo, Carlos; Rueff, José; Brás, Aldina

    2016-12-01

    Human chromosomal fragile sites (CFSs) are heritable loci or regions of the human chromosomes prone to exhibit gaps, breaks and rearrangements. Determining the frequency of deletions and duplications in CFSs may contribute to explain the occurrence of human disease due to those rearrangements. In this study we analyzed the frequency of deletions and duplications in each human CFS. Statistical methods, namely data display, descriptive statistics and linear regression analysis were applied to analyze this dataset. We found that FRA15C, FRA16A and FRAXB are the most frequently involved CFSs in deletions and duplications occurring in the human genome.

  19. 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

  20. Fertilizability and chromosomal integrity of frozen-thawed Bryde's whale (Balaenoptera edeni) spermatozoa intracytoplasmically injected into mouse oocytes.

    PubMed

    Watanabe, H; Tateno, H; Kusakabe, H; Matsuoka, T; Kamiguchi, Y; Fujise, Y; Ishikawa, H; Ohsumi, S; Fukui, Y

    2007-02-01

    Prior to attempting the in vitro production of embryos in the Bryde's whale (Balaenoputera edeni), we investigated whether spermatozoa can retain the capacity for oocyte activation and pronucleus formation as well as chromosomal integrity under cryopreservation by using intracytoplasmic sperm injection (ICSI) into mouse oocytes. Regardless of motility and viability, whale spermatozoa efficiently led to the activation of mouse oocytes (90.3-97.4%), and sperm nuclei successfully transformed into male pronucleus within activated ooplasm (87.2-93.6%). Chromosome analysis at the first cleavage metaphase (M) of the hybrid zygotes revealed that a majority (95.2%) of motile spermatozoa had the normal chromosome complement, while the percentage of chromosomal normality was significantly reduced to 63.5% in immotile spermatozoa and 50.0% in dead spermatozoa due to the increase in structural chromosome aberrations. This is the first report showing that motile Bryde's whale spermatozoa are competent to support embryonic development.

  1. 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.

  2. Ku Binding on Telomeres Occurs at Sites Distal from the Physical Chromosome Ends

    PubMed Central

    Pasquier, Emeline

    2016-01-01

    The Ku complex binds non-specifically to DNA breaks and ensures repair via NHEJ. However, Ku is also known to bind directly to telomeric DNA ends and its presence there is associated with telomere capping, but avoiding NHEJ. How the complex discriminates between a DNA break and a telomeric extremity remains unknown. Our results using a tagged Ku complex, or a chromosome end capturing method, in budding yeast show that yKu association with telomeres can occur at sites distant from the physical end, on sub-telomeric elements, as well as on interstitial telomeric repeats. Consistent with previous studies, our results also show that yKu associates with telomeres in two distinct and independent ways: either via protein-protein interactions between Yku80 and Sir4 or via direct DNA binding. Importantly, yKu associates with the new sites reported here via both modes. Therefore, in sir4Δ cells, telomere bound yKu molecules must have loaded from a DNA-end near the transition of non-telomeric to telomeric repeat sequences. Such ends may have been one sided DNA breaks that occur as a consequence of stalled replication forks on or near telomeric repeat DNA. Altogether, the results predict a new model for yKu function at telomeres that involves yKu binding at one-sided DNA breaks caused by replication stalling. On telomere proximal chromatin, this binding is not followed by initiation of non-homologous end-joining, but rather by break-induced replication or repeat elongation by telomerase. After repair, the yKu-distal portion of telomeres is bound by Rap1, which in turn reduces the potential for yKu to mediate NHEJ. These results thus propose a solution to a long-standing conundrum, namely how to accommodate the apparently conflicting functions of Ku on telomeres. PMID:27930670

  3. NOR sites detected by Ag-dAPI staining of an unusual autosome chromosome of Bradysia hygida (Diptera:Sciaridae) colocalize with C-banded heterochromatic region.

    PubMed

    Gaspar, Vanessa Pinatto; Borges, Alex Rodrigues; Fernandez, Maria Aparecida

    2002-01-01

    The study of chromosomes in insects is a good tool in mitotic process analysis, zoographic localization and evolution investigation. Among them, the Sciaridae offers a karyotype with a small number of chromosomes, where the heterochromatin and nucleolar organizer region, NOR, are easily analyzed in metaphase chromosomes obtained from cerebral ganglia squashes. In this work, the heterochromatic regions on Bradysia hygida mitotic chromosomes, revealed by C-banding, were identified as centromeric blocks on A and C chromosomes and as dark interstitial region in B and X chromosomes. By Ag-DAPI staining, active nucleolus organizer region, NOR, was revealed associated to the constitutive heterochromatin in the end of the C autosome chromosome. The C-band regions and the unusual ribosomal site localization are discussed.

  4. Genetic characterization of site-specific integration functions of phi AAU2 infecting "Arthrobacter aureus" C70.

    PubMed

    Le Marrec, C; Moreau, S; Loury, S; Blanco, C; Trautwetter, A

    1996-04-01

    All the essential genetic determinants for site-specific integration of corynephage phi AAU2 are contained within a 1,756-bp DNA fragment, carried on the integrative plasmid p5510, and are shown to be functional in Escherichia coli. One open reading frame, ORF4, encoding a protein of 266 amino acids was shown to represent the phi AAU2 integrase. The nucleotide sequence of the phi AAU2 attachment site, attP, and the attB, attL, and attR sequences in the host "Arthrobacter aureus" C70 were determined. Identical nucleotide sequences were shown to be responsible for the integration of p5510 in the chromosomes of Corynebacterium glutamicum, Brevibacterium divaricatum, and B. lactofermentum, and a sequence almost identical to attB was found to be present in these three strains. In contrast to other phage site-specific recombination systems, a plasmid encompassing only int-attP failed to integrate into the host chromosome. This led to the identification of an 800-bp noncoding region, immediately upstream of int, absolutely required for site-specific integration of p5510.

  5. Genetic characterization of site-specific integration functions of phi AAU2 infecting "Arthrobacter aureus" C70.

    PubMed Central

    Le Marrec, C; Moreau, S; Loury, S; Blanco, C; Trautwetter, A

    1996-01-01

    All the essential genetic determinants for site-specific integration of corynephage phi AAU2 are contained within a 1,756-bp DNA fragment, carried on the integrative plasmid p5510, and are shown to be functional in Escherichia coli. One open reading frame, ORF4, encoding a protein of 266 amino acids was shown to represent the phi AAU2 integrase. The nucleotide sequence of the phi AAU2 attachment site, attP, and the attB, attL, and attR sequences in the host "Arthrobacter aureus" C70 were determined. Identical nucleotide sequences were shown to be responsible for the integration of p5510 in the chromosomes of Corynebacterium glutamicum, Brevibacterium divaricatum, and B. lactofermentum, and a sequence almost identical to attB was found to be present in these three strains. In contrast to other phage site-specific recombination systems, a plasmid encompassing only int-attP failed to integrate into the host chromosome. This led to the identification of an 800-bp noncoding region, immediately upstream of int, absolutely required for site-specific integration of p5510. PMID:8606175

  6. High-Resolution Mapping of the Drosophila Fourth Chromosome Using Site-Directed Terminal Deficiencies

    PubMed Central

    Sousa-Neves, Rui; Lukacsovich, Tamas; Mizutani, Claudia Mieko; Locke, John; Podemski, Lynn; Marsh, J. Lawrence

    2005-01-01

    For more than 80 years, the euchromatic right arm of the Drosophila fourth chromosome (101F-102F) has been one of the least genetically accessible regions of the fly genome despite the fact that many important genes reside there. To improve the mapping of genes on the fourth chromosome, we describe a strategy to generate targeted deficiencies and we describe 13 deficiencies that subdivide the 300 kb between the cytological coordinates 102A6 and 102C1 into five discrete regions plus a 200-kb region from 102C1 to 102D6. Together these deficiencies substantially improve the mapping capabilities for mutant loci on the fourth chromosome. PMID:15466427

  7. Cascade of chromosomal rearrangements caused by a heterogeneous T-DNA integration supports the double-strand break repair model for T-DNA integration.

    PubMed

    Hu, Yufei; Chen, Zhiyu; Zhuang, Chuxiong; Huang, Jilei

    2017-02-28

    Transferred DNA (T-DNA) from Agrobacterium tumefaciens can be integrated into the plant genome. The double-strand break repair (DSBR) pathway is a major model for T-DNA integration. From this model, we expect that two ends of a T-DNA molecule would invade into a single DNA double-strand break (DSB) or independent DSBs in the plant genome. We call the later phenomenon a heterogeneous T-DNA integration which has never been observed. In this work, we demonstrated it in an Arabidopsis T-DNA insertion mutant seb19. To resolve the chromosomal structural changes caused by T-DNA integration at both the nucleotide and chromosome levels, we performed inverse PCR, genome resequencing, fluorescence in situ hybridization and linkage analysis. We found, in seb19, a single T-DNA connected two different chromosomal loci and caused complex chromosomal rearrangements. The specific break-junction pattern in seb19 is consistent with the result of heterogeneous T-DNA integration but not of recombination between two T-DNA insertions. We demonstrated that, in seb19, heterogeneous T-DNA integration evoked a cascade of incorrect repair of seven DSBs on chromosome 4 and 5, and then produced translocation, inversion, duplication and deletion. Heterogeneous T-DNA integration supports the DSBR model and suggests that two ends of a T-DNA molecule could be integrated into the plant genome independently. Our results also show a new origin of chromosomal abnormalities. This article is protected by copyright. All rights reserved.

  8. 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

  9. The actinophage RP3 DNA integrates site-specifically into the putative tRNA(Arg)(AGG) gene of Streptomyces rimosus.

    PubMed Central

    Gabriel, K; Schmid, H; Schmidt, U; Rausch, H

    1995-01-01

    The temperate actinophage RP3 integrates site-specifically into the chromosome of Streptomyces rimosus R6-554. The phage attachment site attP and the hybrid attachment sites of the integrated prophage--attL and attR--were cloned and sequenced. The 54nt core sequence, common to all RP3 related attachment sites, comprises the 3' terminal end of a putative tRNA(Arg)(AGG) gene. AttB bears the complete tRNA gene which is restored in attL after integration. A 7.5kb HindIII fragment, bearing attP, was used to construct an integrative plasmid to simulate the integration process in vivo and to localize the phage genes necessary for site specific integration. The int and xis genes were sequenced and compared to other recombination genes. PMID:7870591

  10. Targeted induction of meiotic double-strand breaks reveals chromosomal domain-dependent regulation of Spo11 and interactions among potential sites of meiotic recombination.

    PubMed

    Fukuda, Tomoyuki; Kugou, Kazuto; Sasanuma, Hiroyuki; Shibata, Takehiko; Ohta, Kunihiro

    2008-02-01

    Meiotic recombination is initiated by programmed DNA double-strand break (DSB) formation mediated by Spo11. DSBs occur with frequency in chromosomal regions called hot domains but are seldom seen in cold domains. To obtain insights into the determinants of the distribution of meiotic DSBs, we examined the effects of inducing targeted DSBs during yeast meiosis using a UAS-directed form of Spo11 (Gal4BD-Spo11) and a meiosis-specific endonuclease, VDE (PI-SceI). Gal4BD-Spo11 cleaved its target sequence (UAS) integrated in hot domains but rarely in cold domains. However, Gal4BD-Spo11 did bind to UAS and VDE efficiently cleaved its recognition sequence in either context, suggesting that a cold domain is not a region of inaccessible or uncleavable chromosome structure. Importantly, self-association of Spo11 occurred at UAS in a hot domain but not in a cold domain, raising the possibility that Spo11 remains in an inactive intermediate state in cold domains. Integration of UAS adjacent to known DSB hotspots allowed us to detect competitive interactions among hotspots for activation. Moreover, the presence of VDE-introduced DSB repressed proximal hotspot activity, implicating DSBs themselves in interactions among hotspots. Thus, potential sites for Spo11-mediated DSB are subject to domain-specific and local competitive regulations during and after DSB formation.

  11. Converting Maturing Nuclear Sites to Integrated Power Production Islands

    DOE PAGES

    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

  12. Characterization of the attP site of the integrative element pSAM2 from Streptomyces ambofaciens.

    PubMed

    Raynal, Alain; Friedmann, Annick; Tuphile, Karine; Guerineau, Michel; Pernodet, Jean-Luc

    2002-01-01

    pSAM2 is integrated into the Streptomyces ambofaciens chromosome through site-specific recombination between the element (attP) and the chromosomal (attB) site. The 43 kDa integrase protein encoded by pSAM2 catalyses this recombination event. Tools have been developed to study site-specific recombination in Escherichia coli. In vivo studies showed that a 360 bp fragment of attP is required for efficient site-specific recombination and that int can be provided in trans. pSAM2 integrase was purified and overexpressed in E. coli and Int binding at the attP site was studied. DNaseI footprinting revealed two sites that bind integrase strongly and appear to be symmetrical with regard to the core site. These two P1/P2 arm-type sites both contain a 17 bp motif that is identical except at one position, GTCACGCAG(A/T)TAGACAC. P1 and P2 are essential for site-specific recombination.

  13. Identification of Host-Chromosome Binding Sites and Candidate Gene Targets for Kaposi's Sarcoma-Associated Herpesvirus LANA

    PubMed Central

    Lu, Fang; Tsai, Kevin; Chen, Horng-Shen; Wikramasinghe, Priyankara; Davuluri, Ramana V.; Showe, Louise; Domsic, John; Marmorstein, Ronen

    2012-01-01

    LANA is essential for tethering the Kaposi's sarcoma-associated herpesvirus (KSHV) genome to metaphase chromosomes and for modulating host-cell gene expression, but the binding sites in the host-chromosome remain unknown. Here, we use LANA-specific chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) to identify LANA binding sites in the viral and host-cell genomes of a latently infected pleural effusion lymphoma cell line BCBL1. LANA bound with high occupancy to the KSHV genome terminal repeats (TR) and to a few minor binding sites in the KSHV genome, including the LANA promoter region. We identified 256 putative LANA binding site peaks with P < 0.01 and overlap in two independent ChIP-Seq experiments. We validated several of the high-occupancy binding sites by conventional ChIP assays and quantitative PCR. Candidate cellular LANA binding motifs were identified and assayed for binding to purified recombinant LANA protein in vitro but bound with low affinity compared to the viral TR binding site. More than half of the LANA binding sites (170/256) could be mapped to within 2.5 kb of a cellular gene transcript. Pathways and Gene Ontogeny (GO) analysis revealed that LANA binds to genes within the p53 and tumor necrosis factor (TNF) regulatory network. Further analysis revealed partial overlap of LANA and STAT1 binding sites in several gamma interferon (IFN-γ)-regulated genes. We show that ectopic expression of LANA can downmodulate IFN-γ-mediated activation of a subset of genes, including the TAP1 peptide transporter and proteasome subunit beta type 9 (PSMB9), both of which are required for class I antigen presentation. Our data provide a potential mechanism through which LANA may regulate several host cell pathways by direct binding to gene regulatory elements. PMID:22419807

  14. 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...

  15. Structural analysis of loci involved in pSAM2 site-specific integration in Streptomyces.

    PubMed

    Boccard, F; Smokvina, T; Pernodet, J L; Friedmann, A; Guérineau, M

    1989-01-01

    pSAM2 is an 11-kb plasmid integrated in the Streptomyces ambofaciens ATCC23877 and ATCC15154 genomes and found additionally as a free replicon in an uv derivative. After transfer into S. ambofaciens DSM40697 (devoid of pSAM2) or into Streptomyces lividans, specific integration of pSAM2 occurred very efficiently. A 58-bp sequence (att) present in both pSAM2 (attP) and S. ambofaciens strain DSM40697 (attB) attachment regions is found at the boundaries (attL and attR) of integrated pSAM2 in S. ambofaciens strain ATCC23877. The S. lividans chromosomal integration zone contained an imperfectly conserved att sequence (attB), and the integration event of pSAM2 was located within a 49-bp sequence of attB. Only one primary functional attB sequence was present in the S. lividans or S. ambofaciens DSM40697 total DNA. The integration zone of S. lividans hybridized with the integration zone of S. ambofaciens DSM40697. The two integration zones were homologous only to the right side of the att sequence. The conserved region contained an open reading frame (ORF A) with a stop codon located 99 bp from the attB sequence in both strains. S. ambofaciens DSM40697 contained DNA sequences related to pSAM2 on the left side of the att site. The att sequence was included in a region conserved in Streptomyces antibioticus, Streptomyces actuosus, Streptomyces bikiniensis, Streptomyces coelicolor, Streptomyces glaucescens, and Streptomyces parvulus. Site-specific integration of a pSAM2 derivative was characterized in another unrelated strain, Streptomyces griseofuscus. This strain contained an imperfectly conserved 58-bp attB sequence, and the integration event took place within a 45-bp sequence of attB. Site-specific integration of pSAM2 in three nonrelated Streptomyces strains suggests the wide host range of pSAM2 integration in Streptomyces.

  16. 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

  17. 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...

  18. Marker-free site-specific gene integration in plants.

    PubMed

    Srivastava, Vibha; Ow, David W

    2004-12-01

    For nearly 15 years, the use of site-specific recombination systems in plants has focused on the deletion or integration of DNA. Each of these applications offers practical solutions to two problems in biotechnology: the presence of unneeded DNA in the transgene locus and variation in the locus structure among independent transgenic lines. Given that each of these separate applications is becoming more practical for commercial use, it is time to consider combining them into an integrated technology. Here we propose a strategy for a "combined step" method that makes use of two site-specific recombination systems: one for integrating the DNA and a second for removing sequences that are not needed after DNA transfer. This strategy is based on published data and exemplifies the use of the Cre-lox, FLP-FRT and R-RS inducible systems.

  19. Modification site localization scoring integrated into a search engine.

    PubMed

    Baker, Peter R; Trinidad, Jonathan C; Chalkley, Robert J

    2011-07-01

    Large proteomic data sets identifying hundreds or thousands of modified peptides are becoming increasingly common in the literature. Several methods for assessing the reliability of peptide identifications both at the individual peptide or data set level have become established. However, tools for measuring the confidence of modification site assignments are sparse and are not often employed. A few tools for estimating phosphorylation site assignment reliabilities have been developed, but these are not integral to a search engine, so require a particular search engine output for a second step of processing. They may also require use of a particular fragmentation method and are mostly only applicable for phosphorylation analysis, rather than post-translational modifications analysis in general. In this study, we present the performance of site assignment scoring that is directly integrated into the search engine Protein Prospector, which allows site assignment reliability to be automatically reported for all modifications present in an identified peptide. It clearly indicates when a site assignment is ambiguous (and if so, between which residues), and reports an assignment score that can be translated into a reliability measure for individual site assignments.

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

    PubMed Central

    Choo, Amanda; Lee, Cheng Shoou; Dayan, Sonia; O’Keefe, Louise

    2015-01-01

    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. Development of a Site-Directed Integration Plasmid for Heterologous Gene Expression in Mycoplasma gallisepticum

    PubMed Central

    Indikova, Ivana; Szostak, Michael P.

    2013-01-01

    Deciphering the molecular basis of the interactions between the parasite Mycoplasma gallisepticum and its avian hosts suffers from the lack of genetic tools available for the pathogen. In the absence of well established methods for targeted disruption of relevant M. gallisepticum genes, we started to develop suicide vectors and equipped them with a short fragment of M. gallisepticum origin or replication (oriCMG). We failed to create a disruption vector, although by adding a further short fragment of the M. gallisepticum tufB upstream region we created a “Trojan horse” plasmid. This is fully integrated into the genomic DNA of M. gallisepticum, always at the same site, oriCMG, and is able to carry and express any gene of interest in the genetic background of M. gallisepticum. Successful expression of a heterologous gene was shown with the lacZ gene of E. coli. When used for gene complementation or expression of hybrid genes in M. gallisepticum, a site-specific combined integration/expression vector constitutes an improvement on randomly integrating transposons, which might have unexpected effects on the expression of chromosomal genes. PMID:24278444

  2. Hanford Site waste treatment/storage/disposal integration

    SciTech Connect

    MCDONALD, K.M.

    1999-02-24

    In 1998 Waste Management Federal Services of Hanford, Inc. began the integration of all low-level waste, mixed waste, and TRU waste-generating activities across the Hanford site. With seven contractors, dozens of generating units, and hundreds of waste streams, integration was necessary to provide acute waste forecasting and planning for future treatment activities. This integration effort provides disposition maps that account for waste from generation, through processing, treatment and final waste disposal. The integration effort covers generating facilities from the present through the life-cycle, including transition and deactivation. The effort is patterned after the very successful DOE Complex EM Integration effort. Although still in the preliminary stages, the comprehensive onsite integration effort has already reaped benefits. These include identifying significant waste streams that had not been forecast, identifying opportunities for consolidating activities and services to accelerate schedule or save money; and identifying waste streams which currently have no path forward in the planning baseline. Consolidation/integration of planned activities may also provide opportunities for pollution prevention and/or avoidance of secondary waste generation. A workshop was held to review the waste disposition maps, and to identify opportunities with potential cost or schedule savings. Another workshop may be held to follow up on some of the long-term integration opportunities. A change to the Hanford waste forecast data call would help to align the Solid Waste Forecast with the new disposition maps.

  3. Construction of a yeast artificial chromosome contig spanning the pseudoautosomal region and isolation of 25 new sequence-tagged sites

    SciTech Connect

    Slim, R. Laboratoire de Cytogenetique et Genetique Oncologiques, Villejuif ); Le Paslier, D.; Ougen, P.; Billault, A.; Cohen, D. ); Compain, S.; Levilliers, J.; Mintz, L.; Weissenbach, J.; Petit, C. )

    1993-06-01

    Thirty-one yeast artificial chromosomes (YACs) from the human pseudoautosomal region were identified by a combination of sequence-tagged site (STS) screenings and colony hybridizations, using a subtelomeric interspersed repetitive element mapping predominantly to the pseudoautosomal region. Twenty-five new pseudoautosomal STSs were generated, of which 4 detected restriction fragment length polymorphisms. A total of 33 STSs were used to assemble the 31 YACs into a single contiguous set of overlapping DNA fragments spanning at least 2.3 megabases of the pseudoautosomal region. In addition, four pseudoautosomal genes including hydroxyindole O-methyltransferase have been positioned on this set of fragments. 48 refs., 1 fig., 3 tabs.

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

    PubMed Central

    Valens, Michèle; Thiel, Axel

    2016-01-01

    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

  5. 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...

  6. Analyses of germline, chromosomally integrated human herpesvirus 6A and B genomes indicate emergent infection and new inflammatory mediators.

    PubMed

    Tweedy, J; Spyrou, M A; Hubacek, P; Kuhl, U; Lassner, D; Gompels, U A

    2015-02-01

    Human herpesvirus-6A (HHV-6A) is rarer than HHV-6B in many infant populations. However, they are similarly prevalent as germline, chromosomally integrated genomes (ciHHV-6A/B). This integrated form affects 0.1-1 % of the human population, where potentially virus gene expression could be in every cell, although virus relationships and health effects are not clear. In a Czech/German patient cohort ciHHV-6A was more common and diverse than ciHHV-6B. Quantitative PCR, nucleotide sequencing and telomeric integration site amplification characterized ciHHV-6 in 44 German myocarditis/cardiomyopathy and Czech malignancy/inflammatory disease (MI) patients plus donors. Comparisons were made to sequences from global virus reference strains, and blood DNA from childhood-infections from Zambia (HHV-6A mainly) and Japan (HHV-6B). The MI cohort were 86 % (18/21) ciHHV-6A, the cardiac cohort 65 % (13/20) ciHHV-6B, suggesting different disease links. Reactivation was supported by findings of 1) recombination between ciHHV-6A and HHV-6B genes in 20 % (4/21) of the MI cohort; 2) expression in a patient subset, of early/late transcripts from the inflammatory mediator genes chemokine receptor U51 and chemokine U83, both identical to ciHHV-6A DNA sequences; and 3) superinfection shown by deep sequencing identifying minor virus-variants only in ciHHV-6A, which expressed transcripts, indicating virus infection reactivates latent ciHHV-6A. Half the MI cohort had more than two copies per cell, median 5.2, indicative of reactivation. Remarkably, the integrated genomes encoded the secreted-active form of virus chemokines, rare in virus from childhood-infections. This shows integrated virus genomes can contribute new human genes with links to inflammatory pathology and supports ciHHV-6A reactivation as a source for emergent infection.

  7. Additional copies of the proteolipid protein gene causing Pelizaeus-Merzbacher disease arise by separate integration into the X chromosome.

    PubMed

    Hodes, M E; Woodward, K; Spinner, N B; Emanuel, B S; Enrico-Simon, A; Kamholz, J; Stambolian, D; Zackai, E H; Pratt, V M; Thomas, I T; Crandall, K; Dlouhy, S R; Malcolm, S

    2000-07-01

    The proteolipid protein gene (PLP) is normally present at chromosome Xq22. Mutations and duplications of this gene are associated with Pelizaeus-Merzbacher disease (PMD). Here we describe two new families in which males affected with PMD were found to have a copy of PLP on the short arm of the X chromosome, in addition to a normal copy on Xq22. In the first family, the extra copy was first detected by the presence of heterozygosity of the AhaII dimorphism within the PLP gene. The results of FISH analysis showed an additional copy of PLP in Xp22.1, although no chromosomal rearrangements could be detected by standard karyotype analysis. Another three affected males from the family had similar findings. In a second unrelated family with signs of PMD, cytogenetic analysis showed a pericentric inversion of the X chromosome. In the inv(X) carried by several affected family members, FISH showed PLP signals at Xp11.4 and Xq22. A third family has previously been reported, in which affected members had an extra copy of the PLP gene detected at Xq26 in a chromosome with an otherwise normal banding pattern. The identification of three separate families in which PLP is duplicated at a noncontiguous site suggests that such duplications could be a relatively common but previously undetected cause of genetic disorders.

  8. 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.

  9. Fully efficient chromosome dimer resolution in Escherichia coli cells lacking the integral membrane domain of FtsK

    PubMed Central

    Dubarry, Nelly; Barre, François-Xavier

    2010-01-01

    In bacteria, septum formation frequently initiates before the last steps of chromosome segregation. This is notably the case when chromosome dimers are formed by homologous recombination. Chromosome segregation then requires the activity of a double-stranded DNA transporter anchored at the septum by an integral membrane domain, FtsK. It was proposed that the transmembrane segments of proteins of the FtsK family form pores across lipid bilayers for the transport of DNA. Here, we show that truncated Escherichia coli FtsK proteins lacking all of the FtsK transmembrane segments allow for the efficient resolution of chromosome dimers if they are connected to a septal targeting peptide through a sufficiently long linker. These results indicate that FtsK does not need to transport DNA through a pore formed by its integral membrane domain. We propose therefore that FtsK transports DNA before membrane fusion, at a time when there is still an opening in the constricted septum. PMID:20033058

  10. Jumping translocation in acute monocytic leukemia (M5b) with alternative breakpoint sites in the long arm of donor chromosome 3.

    PubMed

    McGrattan, Peter; Logan, Amy; Humphreys, Mervyn; Bowers, Margaret

    2010-09-01

    An 86-year-old man presented with acute hepatic failure, worsening thrombocytopenia, and anemia having been diagnosed and managed expectantly with cytogenetically normal RAEB-1. After 20 months a diagnosis of disease transformation to acute monocytic leukemia (M5b) was made. Conventional G-banded analysis of unstimulated bone marrow cultures demonstrated a jumping translocation (JT) involving proximal and distal breakpoints on donor chromosome 3 at bands 3q1?2 and 3q21, respectively. Recipient chromosomes included the long-arm telomeric regions of chromosomes 5, 10, 14, 16, and 19. A low-level trisomy 8 clone was also found in association with both proximal and distal JT clones. Conventional G-banded analysis of unstimulated peripheral blood cultures detected the proximal 3q1?2 JT clone involving recipient chromosome 10 several weeks after transformation to acute monocytic leukemia. Interestingly, JTs involving recipient chromosomes 5, 14, 16, and 19 were not detected in this peripheral blood sample. Palliative care was administered until his demise 2.2 months after disease transformation. There have been fewer than 70 cases of acquired JTs reported in the literature, including one myeloproliferative neoplasm and five acute myeloid leukemias involving a single breakpoint site on donor chromosome 3. Our case is unique as it is the first acquired case to demonstrate a JT involving alternative pericentromeric breakpoint sites on a single donor chromosome consisting of a proximal breakpoint at 3q1?2 and a more distal breakpoint at 3q21.

  11. 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

    2016-03-07

    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.

  12. 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

  13. [The effect of heavy metal ions and peptide bioregulators on the expression of chromosome fragile sites in the individuals of different age groups and breast cancer patients].

    PubMed

    Dzhokhadze, T A; Ganozishvili, M N; Lezhava, T A

    2008-09-01

    Expression rates of chromosome fragile sites in peripheral blood lymphocytes have been studied in clinically healthy individuals of different age groups (20-38 yrs and 75-86 yrs) and breast cancer patients (8 cases). In individuals with a normal check-up of different age groups the heavy metal (nickel, zinc and cobalt) ions were also examined on their influence on the expression of the fragile sites and the peptide bioregulators (Livagen and Epithalon) were tested on their ability to correct the pattern of expression. Short-term lymphocyte cultures were used as tested material. The analysis showed that the chromosomes of people from young and old age groups differ from each other by the expression pattern of fragile sites - the chromosomes of young individuals were found to be more active by spontaneous formation of fragile sites. They were also sensitive to their induction by heavy metals. Both tested bioregulators lessen heavy metals effect that was statistically reliable only for the young people group. As for the patients with breast cancer general elevated fragility of chromosomes and specific distribution of the fragile sites along the chromosomes were revealed.

  14. 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

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

    PubMed Central

    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

  16. Marek's disease herpesvirus vaccines integrate into chicken host chromosomes yet lack a virus-host phenotype associated with oncogenic transformation.

    PubMed

    McPherson, Marla C; Cheng, Hans H; Delany, Mary E

    2016-11-04

    Marek's disease (MD) is a lymphotropic and oncogenic disease of chickens that can lead to death in susceptible and unvaccinated host birds. The causative pathogen, MD virus (MDV), a highly oncogenic alphaherpesvirus, integrates into host genome near the telomeres. MD occurrence is controlled across the globe by biosecurity, selective breeding for enhanced MD genetic resistance, and widespread vaccination of flocks using attenuated serotype 1 MDV or other serotypes. Despite over 40 years of usage, the specific mechanism(s) of MD vaccine-related immunity and anti-tumor effects are not known. Here we investigated the cytogenetic interactions of commonly used MD vaccine strains of all three serotypes (HVT, SB-1, and Rispens) with the host to determine if all were equally capable of host genome integration. We also studied the dynamic profiles of chromosomal association and integration of the three vaccine strains, a first for MD vaccine research. Our cytogenetic data provide evidence that all three MD vaccine strains tested integrate in the chicken host genome as early as 1 day after vaccination similar to oncogenic strains. However, a specific, transformation-associated virus-host phenotype observed for oncogenic viruses is not established. Our results collectively provide an updated model of MD vaccine-host genome interaction and an improved understanding of the possible mechanisms of vaccinal immunity. Physical integration of the oncogenic MDV genome into host chromosomes along with cessation of viral replication appears to have joint signification in MDV's ability to induce oncogenic transformation. Whereas for MD vaccine serotypes, a sustained viral replication stage and lack of the chromosome-integrated only stage were shared traits during early infection.

  17. 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...

  18. The site of integration of the herpes simplex virus type 1 thymidine kinase gene in human cells transformed by an HSV-1 DNA fragment.

    PubMed

    Kit, S; Hazen, M; Otsuka, H; Qavi, H; Trkula, D; Dubbs, D R

    1981-12-01

    To analyze the site of integration of the herpes simplex virus type I (HSV-I) thymidine kinase (TK) gene in biochemically transformed human cells, TK-HeLa-(BU25) cells were transformed to the TK+ phenotype by a cloned, 2 kbp Pvull fragment of HSV-I DNA. The transformed cells [HeLa(BU25)/TF pAGO PP3] were fused with mouse LM(TK-) cells, and human-mouse somatic cell hybrid clones (LH PP3 clones 1, 2, 3, 5 and 6) were isolated in HATG-ouabain selective medium. The HeLa(BU25)/TF pAGO PP3 cells and the LH PP3 hybrid clones expressed HSV-I specific TK activity and a herpesvirus-associated nuclear antigen, and contained herpesvirus nucleotide sequences. Molecular hybridization experiments were carried out to map the HSV-I and flanking cellular nucleotide sequences in the biochemically transformed cells. These experiments demonstrated that the HSV-I nucleotide sequences were integrated at a single site, and that the same cellular nucleotide sequences flanked the viral DNA in transformed HeLa(BU25)/TF pAGO PP3 and LH PP3 clone 5 cells. TK- revertant subclones isolated by growing the LH PP3 clone 5 cells in BrdUrd (and diphtheria toxin) failed to form colonies in HATG medium, but retained HSV-I nucleotide sequences. Isozyme analyses on 21 gene-enzyme systems representing 21 human chromosomes revealed that all of the LH PP3 clonal lines expressed human hexosaminidase B, which has been assigned to chromosome 5, and all were sensitive to diphtheria toxin, which is also a marker for chromosome 5. Chromosome analyses showed that chromosome 5 was the nly human chromosome present in mitoses of LH PP3 clone 5 cells and that human chromosome 5 was present in most of the mitoses of LH PP3 clone 1, 2, 3, and 6 cells. The latter clones also contained 1 or 2 additional human chromosomes in some of the cells. As expected from the molecular hybridization analyses, TK- revertants of LH PP3 clone 5 cells retained portions of chromosome 5 and expressed human hexosaminidase B. The results

  19. 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.

  20. Hanford Site waste management and environmental restoration integration plan

    SciTech Connect

    Merrick, D.L.

    1990-04-30

    The Hanford Site Waste Management and Environmental Restoration Integration Plan'' describes major actions leading to waste disposal and site remediation. The primary purpose of this document is to provide a management tool for use by executives who need to quickly comprehend the waste management and environmental restoration programs. The Waste Management and Environmental Restoration Programs have been divided into missions. Waste Management consists of five missions: double-shell tank (DST) wastes; single-shell tank (SST) wastes (surveillance and interim storage, stabilization, and isolation); encapsulated cesium and strontium; solid wastes; and liquid effluents. Environmental Restoration consists of two missions: past practice units (PPU) (including characterization and assessment of SST wastes) and surplus facilities. For convenience, both aspects of SST wastes are discussed in one place. A general category of supporting activities is also included. 20 refs., 14 figs., 7 tabs.

  1. 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.

  2. Genome-organizing factors Top2 and Hmo1 prevent chromosome fragility at sites of S phase transcription.

    PubMed

    Bermejo, Rodrigo; Capra, Thelma; Gonzalez-Huici, Victor; Fachinetti, Daniele; Cocito, Andrea; Natoli, Gioacchino; Katou, Yuki; Mori, Hiroshi; Kurokawa, Ken; Shirahige, Katsuhiko; Foiani, Marco

    2009-09-04

    Specialized topoisomerases solve the topological constraints arising when replication forks encounter transcription. We have investigated the contribution of Top2 in S phase transcription. Specifically in S phase, Top2 binds intergenic regions close to transcribed genes. The Top2-bound loci exhibit low nucleosome density and accumulate gammaH2A when Top2 is defective. These intergenic loci associate with the HMG protein Hmo1 throughout the cell cycle and are refractory to the histone variant Htz1. In top2 mutants, Hmo1 is deleterious and accumulates at pericentromeric regions in G2/M. Our data indicate that Top2 is dispensable for transcription and that Hmo1 and Top2 bind in the proximity of genes transcribed in S phase suppressing chromosome fragility at the M-G1 transition. We propose that an Hmo1-dependent epigenetic signature together with Top2 mediate an S phase architectural pathway to preserve genome integrity.

  3. Integration of genetic and physical maps of the Primula vulgaris S locus and localization by chromosome in situ hybridization.

    PubMed

    Li, Jinhong; Webster, Margaret A; Wright, Jonathan; Cocker, Jonathan M; Smith, Matthew C; Badakshi, Farah; Heslop-Harrison, Pat; Gilmartin, Philip M

    2015-10-01

    Heteromorphic flower development in Primula is controlled by the S locus. The S locus genes, which control anther position, pistil length and pollen size in pin and thrum flowers, have not yet been characterized. We have integrated S-linked genes, marker sequences and mutant phenotypes to create a map of the P. vulgaris S locus region that will facilitate the identification of key S locus genes. We have generated, sequenced and annotated BAC sequences spanning the S locus, and identified its chromosomal location. We have employed a combination of classical genetics and three-point crosses with molecular genetic analysis of recombinants to generate the map. We have characterized this region by Illumina sequencing and bioinformatic analysis, together with chromosome in situ hybridization. We present an integrated genetic and physical map across the P. vulgaris S locus flanked by phenotypic and DNA sequence markers. BAC contigs encompass a 1.5-Mb genomic region with 1 Mb of sequence containing 82 S-linked genes anchored to overlapping BACs. The S locus is located close to the centromere of the largest metacentric chromosome pair. These data will facilitate the identification of the genes that orchestrate heterostyly in Primula and enable evolutionary analyses of the S locus.

  4. 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

  5. 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.

  6. 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)

  7. 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.

  8. 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.

  9. 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.

  10. Integration of high-resolution physical and genetic map reveals differential recombination frequency between chromosomes and the genome assembling quality in cucumber.

    PubMed

    Lou, Qunfeng; He, Yuhua; 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.

  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.

  12. Chromosomal polymorphism in two species of Hypancistrus (Siluriformes: Loricariidae): an integrative approach for understanding their biodiversity.

    PubMed

    da Silva, Maelin; Ribeiro, Emanuell D; Matoso, Daniele A; Sousa, Leandro M; Hrbek, Tomas; Py-Daniel, Lucia Rapp; Feldberg, Eliana

    2014-04-01

    Structural chromosome changes are widely described in different vertebrate groups and generate genetic, phenotypic and behavioral diversity. During the evolution of loricariids, several rearrangements (fissions, fusions, inversions) seem to have occurred. Hypancistrus, tribe Ancistrini, are highly demanded for fishkeeping around the world. In this tribe, the diploid chromosome number 2n = 52 is considered a synapomorphy, and paracentric-type inversions appear to be involved in the chromosomal evolution of the tribe. The present study investigated the karyotypes of H. zebra and H. cf. debilittera using cytogenetic, classical and molecular tools, as well as DNA barcoding. Data reveal that, although diploid number in both species corroborates the proposed synapomorphy for the tribe, there is a complex karyotype dynamics, reflected in the intense chromosomal polymorphism, resulting from rearrangements involving ribosomal regions (5S and 18S rDNA), which are suggested to be paracentric inversions. Besides, DNA barcode confirms reciprocal monophyletism between the species, validating the existence of two species, only. This scenario, coupled with genomic instability caused by exogenous sequences such as Rex-3 retrotransposons and the species' sedentary lifestyle, which helps the fast polymorphism fixation, may reflect different phenotypic color patterns in natural populations, as observed in H. cf. debilittera.

  13. High-affinity sites form an interaction network to facilitate spreading of the MSL complex across the X chromosome in Drosophila

    PubMed Central

    Ramirez, Fidel; Lingg, Thomas; Toscano, Sarah; Lam, Kin Chung; Georgiev, Plamen; Chung, Ho-Ryun; Lajoie, Bryan; de Wit, Elzo; Zhan, Ye; de Laat, Wouter; Dekker, Job; Manke, Thomas; Akhtar, Asifa

    2016-01-01

    Summary Dosage compensation mechanisms provide a paradigm to study the contribution of chromosomal conformation towards targeting and spreading of epigenetic regulators over a specific chromosome. By using Hi-C and 4C analyses we show that high-affinity sites (HAS), landing platforms of the male-specific lethal (MSL) complex, are enriched around topologically associating domain (TAD) boundaries on the X chromosome and harbor more long-range contacts in a sex-independent manner. Ectopically expressed roX1 and roX2 RNA target HAS on the X chromosome in trans and, via spatial proximity, induce spreading of the MSL complex in cis, leading to increased expression of neighboring autosomal genes. We show that the MSL complex regulates nucleosome positioning at HAS, thus acting locally rather than influencing the overall chromosomal architecture. We propose that sex-independent three-dimensional conformation of the X chromosome poises it for exploitation by the MSL complex, thereby facilitating spreading in males. PMID:26431028

  14. 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.

  15. Novel method to rescue a lethal phenotype through integration of target gene onto the X-chromosome

    PubMed Central

    Sakata, Kazuya; Araki, Kimi; Nakano, Hiroyasu; Nishina, Takashi; Komazawa-Sakon, Sachiko; Murai, Shin; Lee, Grace E.; Hashimoto, Daisuke; Suzuki, Chigure; Uchiyama, Yasuo; Notohara, Kenji; Gukovskaya, Anna S.; Gukovsky, Ilya; Yamamura, Ken-ichi; Baba, Hideo; Ohmuraya, Masaki

    2016-01-01

    The loss-of-function mutations of serine protease inhibitor, Kazal type 1 (SPINK1) gene are associated with human chronic pancreatitis, but the underlying mechanisms remain unknown. We previously reported that mice lacking Spink3, the murine homologue of human SPINK1, die perinatally due to massive pancreatic acinar cell death, precluding investigation of the effects of SPINK1 deficiency. To circumvent perinatal lethality, we have developed a novel method to integrate human SPINK1 gene on the X chromosome using Cre-loxP technology and thus generated transgenic mice termed “X-SPINK1“. Consistent with the fact that one of the two X chromosomes is randomly inactivated, X-SPINK1 mice exhibit mosaic pattern of SPINK1 expression. Crossing of X-SPINK1 mice with Spink3+/− mice rescued perinatal lethality, but the resulting Spink3−/−;XXSPINK1 mice developed spontaneous pancreatitis characterized by chronic inflammation and fibrosis. The results show that mice lacking a gene essential for cell survival can be rescued by expressing this gene on the X chromosome. The Spink3−/−;XXSPINK1 mice, in which this method has been applied to partially restore SPINK1 function, present a novel genetic model of chronic pancreatitis. PMID:27845447

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

    PubMed Central

    Walz, C; Schlehofer, J R

    1992-01-01

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

  17. Prevalence of chromosomally integrated human herpesvirus 6 in patients with human herpesvirus 6-central nervous system dysfunction.

    PubMed

    Hill, Joshua A; Sedlak, Ruth Hall; Zerr, Danielle M; Huang, Meei-Li; Yeung, Cecilia; Myerson, David; Jerome, Keith R; Boeckh, Michael J

    2015-02-01

    We identified 37 hematopoietic cell transplantation recipients with human herpesvirus 6 (HHV-6) central nervous system dysfunction and tested donor-recipient pairs for chromosomally integrated HHV-6 (ciHHV-6). One patient had ciHHV-6A with possible HHV-6A reactivation and encephalitis. There was no ciHHV-6 enrichment in this group, but larger studies are needed to determine if patients with ciHHV-6 are at increased risk for HHV-6-associated diseases or other complications.

  18. 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.

  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. 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

  1. Participation of translesion synthesis DNA polymerases in the maintenance of chromosome integrity in yeast Saccharomyces cerevisiae.

    PubMed

    Kochenova, O V; Soshkina, J V; Stepchenkova, E I; Inge-Vechtomov, S G; Shcherbakova, P V

    2011-01-01

    We employed a genetic assay based on illegitimate hybridization of heterothallic Saccharomyces cerevisiae strains (the α-test) to analyze the consequences for genome stability of inactivating translesion synthesis (TLS) DNA polymerases. The α-test is the only assay that measures the frequency of different types of mutational changes (point mutations, recombination, chromosome or chromosome arm loss) and temporary changes in genetic material simultaneously. All these events are manifested as illegitimate hybridization and can be distinguished by genetic analysis of the hybrids and cytoductants. We studied the effect of Polζ, Polη, and Rev1 deficiency on the genome stability in the absence of genotoxic treatment and in UV-irradiated cells. We show that, in spite of the increased percent of accurately repaired primary lesions, chromosome fragility, rearrangements, and loss occur in the absence of Polζ and Polη. Our findings contribute to further refinement of the current models of translesion synthesis and the organization of eukaryotic replication fork.

  2. An A-T linker adapter polymerase chain reaction method for chromosome walking without restriction site cloning bias.

    PubMed

    Trinh, Quoclinh; Xu, Wentao; Shi, Hui; Luo, Yunbo; Huang, Kunlun

    2012-06-01

    A-T linker adapter polymerase chain reaction (PCR) was modified and employed for the isolation of genomic fragments adjacent to a known DNA sequence. The improvements in the method focus on two points. The first is the modification of the PO(4) and NH(2) groups in the adapter to inhibit the self-ligation of the adapter or the generation of nonspecific products. The second improvement is the use of the capacity of rTaq DNA polymerase to add an adenosine overhang at the 3' ends of digested DNA to suppress self-ligation in the digested DNA and simultaneously resolve restriction site clone bias. The combination of modifications in the adapter and in the digested DNA leads to T/A-specific ligation, which enhances the flexibility of this method and makes it feasible to use many different restriction enzymes with a single adapter. This novel A-T linker adapter PCR overcomes the inherent limitations of the original ligation-mediated PCR method such as low specificity and a lack of restriction enzyme choice. Moreover, this method also offers higher amplification efficiency, greater flexibility, and easier manipulation compared with other PCR methods for chromosome walking. Experimental results from 143 Arabidopsis mutants illustrate that this method is reliable and efficient in high-throughput experiments.

  3. Human expressed tagged sites on the X chromosome: A mapping resource for heritable sex-linked chorioretinal disorders

    SciTech Connect

    MacDonald, I.M.; Nesslinger, N.; Wong, P.

    1994-09-01

    We have isolated a bank of human X-specific genomic clones which harbor chorioretinal expressed sequences using library to library cross-screening. The steps included (1) the creation of a {lambda}gt-10 library of human chorioretinal cDNA, (2) the creation of a human X-specific EMBL-3 genomic library from a somatic cell hybrid (82082a) containing the X chromosome as its only human component and lacking the hamster X, and (3) a PCR-based cross-screen to identify 78 clones expressed in choroid and retina. The characterization of one human X-specific EMBL-3 clone (XEH.8; DXS542) has provided a clear illustration of the feasibility of this approach. FISH mapping confirms the regional localization of XEH.8 to Xp11. Localization of additional clones, XEH.1, XEH.34, XEH.41, and XEH.52 will be presented along with partial sequencing and characterization. Our approach has focused on the search for expressed sequences which can serve as expressed tagged sites (ESTs) in mapping or candidate genes for heritable eye disorders.

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

    PubMed Central

    Olthof, Nadine C.; Huebbers, Christian U.; Kolligs, Jutta; Henfling, Mieke; Ramaekers, Frans C.S.; Cornet, Iris; van Lent-Albrechts, Josefa A.; Stegmann, Sander 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-Peter; Speel, Ernst-Jan M.

    2017-01-01

    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 analysed 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

  5. 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.

  6. 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

  7. Characterization of genetic elements required for site-specific integration of Lactobacillus delbrueckii subsp. bulgaricus bacteriophage mv4 and construction of an integration-proficient vector for Lactobacillus plantarum.

    PubMed Central

    Dupont, L; Boizet-Bonhoure, B; Coddeville, M; Auvray, F; Ritzenthaler, P

    1995-01-01

    Temperate phage mv4 integrates its DNA into the chromosome of Lactobacillus delbrueckii subsp. bulgaricus strains via site-specific recombination. Nucleotide sequencing of a 2.2-kb attP-containing phage fragment revealed the presence of four open reading frames. The larger open reading frame, close to the attP site, encoded a 427-amino-acid polypeptide with similarity in its C-terminal domain to site-specific recombinases of the integrase family. Comparison of the sequences of attP, bacterial attachment site attB, and host-phage junctions attL and attR identified a 17-bp common core sequence, where strand exchange occurs during recombination. Analysis of the attB sequence indicated that the core region overlaps the 3' end of a tRNA(Ser) gene. Phage mv4 DNA integration into the tRNA(Ser) gene preserved an intact tRNA(Ser) gene at the attL site. An integration vector based on the mv4 attP site and int gene was constructed. This vector transforms a heterologous host, L. plantarum, through site-specific integration into the tRNA(Ser) gene of the genome and will be useful for development of an efficient integration system for a number of additional bacterial species in which an identical tRNA gene is present. PMID:7836291

  8. Human Herpesvirus 6 DNA Levels in Cerebrospinal Fluid Due to Primary Infection Differ from Those Due to Chromosomal Viral Integration and Have Implications for Diagnosis of Encephalitis▿

    PubMed Central

    Ward, Katherine N.; Leong, Hoe Nam; Thiruchelvam, Anton D.; Atkinson, Claire E.; Clark, Duncan A.

    2007-01-01

    The prevalence and concentration of human herpesvirus 6 (HHV-6) DNA in the cerebrospinal fluid (CSF) of the immunocompetent in primary infection was compared with that in viral chromosomal integration. Samples from 510 individuals with suspected encephalitis, 200 young children and 310 older children and/or adults, and 12 other patients were tested. HHV-6 DNA concentration (log10 copies/ml) was measured in CSF, serum, and whole blood using PCR. Serum HHV-6 immunoglobulin G antibody was measured by indirect immunofluorescence. Primary infection was defined by antibody seroconversion and/or a low concentration of HHV-6 DNA (<3.0 log10 copies/ml) in a seronegative serum. Chromosomal integration was defined by a high concentration of viral DNA in serum (≥3.5 log10 copies/ml) or whole blood (≥6.0 log10 copies/ml). The prevalences of CSF HHV-6 DNA in primary infection and chromosomal integration were 2.5% and 2.0%, respectively, in the young children (<2 years) and 0% and 1.3%, respectively, in the older children and/or adults. The mean concentration of CSF HHV-6 DNA in 9 children with primary infection (2.4 log10 copies/ml) was significantly lower than that of 21 patients with viral chromosomal integration (4.0 log10 copies/ml). Only HHV-6B DNA was found in primary infection, whereas in viral integration, 4 patients had HHV-6A and 17 patients HHV-6B. Apart from primary infection, chromosomal integration is the most likely cause of HHV-6 DNA in the CSF of the immunocompetent. Our results show that any diagnosis of HHV-6 encephalitis or other type of active central nervous system infection should not be made without first excluding chromosomal HHV-6 integration by measuring DNA load in CSF, serum, and/or whole blood. PMID:17229866

  9. 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

  10. Identification of a novel common proviral integration site, flit-1, in feline leukemia virus induced thymic lymphoma.

    PubMed

    Fujino, Yasuhito; Liao, Chun-Peng; Zhao, Yan Shi; Pan, Judong; Mathes, Lawrence E; Hayes, Kathleen A; Ohno, Koichi; Tsujimoto, Hajime; Roy-Burman, Pradip

    2009-03-30

    A new proviral integration site for feline leukemia virus (FeLV), termed flit-1, was identified from feline thymic lymphoma. Among 35 FeLV-related tumors examined, 5 of 25 thymic lymphomas demonstrated proviral insertion within flit-1 locus whereas none of four alimentary and five multicentric lymphomas and one T-lymphoid leukemia examined had rearrangement in this region. Extensive sequence analysis has shown that flit-1, which is noncoding, is conserved on human chromosome 12 and mouse chromosome 15. The human and murine homologs of flit-1 are positioned approximately 30-kb upstream to activin-A receptor type II-like 1 (ACVRL1/ALK1) gene. Expression of ACVRL1 mRNA was examined in two of five lymphomas with flit-1 rearrangement and detected in both of the two whereas normal thymuses and seven lymphoid tumors without flit-1 rearrangement had no detectable expression. Therefore, flit-1 appears to represent a novel FeLV proviral common integration domain that may influence lymphomagenesis as insertional mutagenesis.

  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. Complete nucleotide sequence of a new filamentous phage, Xf109, which integrates its genome into the chromosomal DNA of Xanthomonas oryzae.

    PubMed

    Yeh, Ting Y

    2017-02-01

    Unlike Ff-like coliphages, certain filamentous Inoviridae phages integrate their genomes into the host chromosome and enter a prophage state in their infectious cycle. This lysogenic life cycle was first reported for Xanthomonas citri Cf phage. However, except for the X. citri phages Cf and XacF1, complete genome sequence information about lysogenic Xanthomonas phages is not available to date. A proviral sequence of Xf109 phage was identified in the genome of Xanthomonas oryzae, the rice bacterial blight pathogen, and revived as infectious virions to lysogenize its host de novo. The genome of Xf109 phage is 7190 nucleotides in size and contains 12 predicted open reading frames in an organization similar to that of the Cf phage genome. Seven of the Xf109 proteins show significant sequence similarity to Cf and XacF1 phage proteins, while its ORF4 shares 92 % identity with the major coat protein of X. phage oryzae Xf. Integration of Xf109 phage DNA into the host genome is site-specific, and the attP/attB sequence contains the dif core sequence 5'-TATACATTATGCGAA-3', which is identical to that of Cf, XacF1, and Xanthomonas campestris phage ϕLf. To my knowledge, this is the first complete genome sequence of a filamentous bacteriophage that infects X. oryzae.

  14. 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.

  15. 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.

  16. Karyotype characterization reveals active 45S rDNA sites located on chromosome termini in Smilax rufescens (Smilacaceae).

    PubMed

    Pizzaia, D; Oliveira, V M; Martins, A R; Appezzato-da-Glória, B; Forni-Martins, E; Aguiar-Perecin, M L R

    2013-04-25

    The genus Smilax (Smilacaceae) includes species of medicinal interest; consequently, their identification is important for the control of raw material used in the manufacture of phytotherapeutic products. We investigated the karyotype of Smilax rufescens in order to look for patterns that would be useful for comparative studies of this genus. To accomplish this, we developed procedures to grow plants and optimize root pretreatment with mitotic fuse inhibitors to obtain metaphase spreads showing clear chromosome morphology. The karyotype, analyzed in Feulgen-stained preparations, was asymmetric, with N = 16 chromosomes gradually decreasing in size; the larger ones were subtelocentric and the smaller chromosomes were submetacentric or metacentric. Nearly terminal secondary constrictions were visualized on the short arm of chromosome pairs 7, 11, and 14, but they were clearly detected only in one of the homologues of each pair. The nucleolus organizer regions (NORs) were mapped by silver staining and fluorescent in situ hybridization of 45S rDNA probes. Silver signals (Ag-NORs) colocalized with rDNA loci were detected at the termini of the short arm of 6 chromosomes. The secondary constriction heteromorphism observed in Feulgen-stained metaphases suggests that differential rRNA gene expression between homologous rDNA loci can occur, resulting in different degrees of chromatin decondensation. In addition, a heteromorphic chromosome pair was identified and was interpreted as being a sex chromosome pair in this dioecious species.

  17. Cloning of Frankia species putative tRNA(Pro) genes and their efficacy for pSAM2 site-specific integration in Streptomyces lividans.

    PubMed

    Alegre, M T; Cournoyer, B; Mesas, J M; Guérineau, M; Normand, P; Pernodet, J L

    1994-12-01

    pSAM2 is a conjugative Streptomyces ambofaciens mobile genetic element that can transfer and integrate site specifically in the genome. The chromosomal attachment site (attB) for pSAM2 site-specific recombination for two Frankia species was analyzed. It overlaps putative proline tRNA genes having a 3'-terminal CCA sequence, an uncommon feature among actinomycetes. pSAM2 is able to integrate into a cloned Frankia attB site harbored in Streptomyces lividans. The integration event removes the 3'-terminal CCA sequence and introduces a single nucleotide difference in the T psi C loop of the putative Frankia tRNA(Pro) gene. Major differences between the attP sequence from pSAM2 and the Frankia attB sequence restrict the identity segment to a 43-bp-long region. Only one mismatch is found between these well-conserved att segments. This nucleotide substitution makes a BstBI recognition site in Frankia attB and was used to localize the recombination site in a 25-bp region going from the anticodon to the T psi C loop of the tRNA(Pro) sequence. Integration of pSAM2 into the Frankia attB site is the first step toward introduction of pSAM2 derivatives into Frankia spp.

  18. Cloning of Frankia species putative tRNA(Pro) genes and their efficacy for pSAM2 site-specific integration in Streptomyces lividans.

    PubMed Central

    Alegre, M T; Cournoyer, B; Mesas, J M; Guérineau, M; Normand, P; Pernodet, J L

    1994-01-01

    pSAM2 is a conjugative Streptomyces ambofaciens mobile genetic element that can transfer and integrate site specifically in the genome. The chromosomal attachment site (attB) for pSAM2 site-specific recombination for two Frankia species was analyzed. It overlaps putative proline tRNA genes having a 3'-terminal CCA sequence, an uncommon feature among actinomycetes. pSAM2 is able to integrate into a cloned Frankia attB site harbored in Streptomyces lividans. The integration event removes the 3'-terminal CCA sequence and introduces a single nucleotide difference in the T psi C loop of the putative Frankia tRNA(Pro) gene. Major differences between the attP sequence from pSAM2 and the Frankia attB sequence restrict the identity segment to a 43-bp-long region. Only one mismatch is found between these well-conserved att segments. This nucleotide substitution makes a BstBI recognition site in Frankia attB and was used to localize the recombination site in a 25-bp region going from the anticodon to the T psi C loop of the tRNA(Pro) sequence. Integration of pSAM2 into the Frankia attB site is the first step toward introduction of pSAM2 derivatives into Frankia spp. PMID:7811067

  19. A new generation of retroviral producer cells: predictable and stable virus production by Flp-mediated site-specific integration of retroviral vectors.

    PubMed

    Schucht, R; Coroadinha, A S; Zanta-Boussif, M A; Verhoeyen, E; Carrondo, M J T; Hauser, H; Wirth, Dagmar

    2006-08-01

    We developed a new strategy that provides well-defined high-titer producer cells for recombinant retroviruses in a minimum amount of time. The strategy involves the targeted integration of the retroviral vector into a chromosomal locus with favorable properties. For proof of concept we established a novel HEK293-based retroviral producer cell line, called Flp293A, with a single-copy retroviral vector integrated at a selected chromosomal locus. The vector was flanked by noninteracting Flp-recombinase recognition sites and was exchanged for different retroviral vectors via Flp-mediated cassette exchange. All analyzed cell clones showed correct integration and identical titers for each of the vectors, confirming that the expression characteristics from the parental cell were preserved. Titers up to 2.5 x 10(7) infectious particles/10(6) cells were obtained. Also, high-titer producer cells for a therapeutic vector that encodes the 8.9-kb collagen VII cDNA in a marker-free cassette were obtained within 3 weeks without screening. Thus, we provide evidence that the precise integration of viral vectors into a favorable chromosomal locus leads to high and predictable virus production. This method is compatible with other retroviral vectors, including self-inactivating vectors and marker-free vectors. Further, it provides a tool for evaluation of different retroviral vector designs.

  20. 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

  1. Human MLH1 suppresses the insertion of telomeric sequences at intra-chromosomal sites in telomerase-expressing cells.

    PubMed

    Jia, Pingping; Chastain, Megan; Zou, Ying; Her, Chengtao; Chai, Weihang

    2016-11-29

    Aberrant formation of interstitial telomeric sequences (ITSs) promotes genome instabilities. However, it is unclear how aberrant ITS formation is suppressed in human cells. Here, we report that MLH1, a key protein involved in mismatch repair (MMR), suppresses telomeric sequence insertion (TSI) at intra-chromosomal regions. The frequency of TSI can be elevated by double-strand break (DSB) inducer and abolished by ATM/ATR inhibition. Suppression of TSI requires MLH1 recruitment to DSBs, indicating that MLH1's role in DSB response/repair is important for suppressing TSI. Moreover, TSI requires telomerase activity but is independent of the functional status of p53 and Rb. Lastly, we show that TSI is associated with chromosome instabilities including chromosome loss, micronuclei formation and chromosome breakage that are further elevated by replication stress. Our studies uncover a novel link between MLH1, telomerase, telomere and genome stability.

  2. 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

  3. Retrovirus-like vectors for Saccharomyces cerevisiae: integration of foreign genes controlled by efficient promoters into yeast chromosomal DNA.

    PubMed

    Jacobs, E; Dewerchin, M; Boeke, J D

    1988-07-30

    Using modified Saccharomyces cerevisiae Ty1 elements located on a 2 mu plasmid, reverse-transcriptase-mediated transposition into yeast chromosomes of expression cassettes containing a foreign gene can be induced. These expression cassettes consist of the yeast ARG3 and CUP1 promoter sequences fused to the Escherichia coli galK structural gene. Expression cassettes as large as 2 kb can be inserted into Ty elements and transposed efficiently to various sites in the yeast genome. A third yeast promoter (from the yeast CAR1 gene) seems to be unsuitable for use in the expression cassette. This may be because it does not allow the transcription run-through necessary for Ty1 transposition. Ways of improving this vector system are discussed, as are its advantages over episomal vector systems.

  4. Effect of pH value of freeze-drying solution on the chromosome integrity and developmental ability of mouse spermatozoa.

    PubMed

    Kaneko, Takehito; Whittingham, David G; Yanagimachi, Ryuzo

    2003-01-01

    The nuclei of freeze-dried mouse spermatozoa are able to retain their chromosome integrity and developmental potential. To optimize the conditions of freeze-drying, we examined whether pH values of the freeze-drying solution affect the chromosome integrity and developmental potential of sperm nuclei. The sperm freeze-drying solution we used contained a high concentration (50 mM) of calcium-chelating EGTA. Sperm chromosomes were examined at the metaphase of the first mitosis after injection of freeze-dried spermatozoa into matured oocytes. The developmental potential of sperm nuclei was assessed by examining the development of fetuses in midgestation. The results showed that both sperm chromosomes and sperm developmental potential are maintained better when the freeze-drying solution was slightly alkaline (pH 8.0) rather than near neutral or acidic (pH 7.4-6.0). The data indicated that the chromosome integrity and developmental ability of mouse spermatozoa are affected by the pH value of freeze-drying solution.

  5. migS, a cis-acting site that affects bipolar positioning of oriC on the Escherichia coli chromosome

    PubMed Central

    Yamaichi, Yoshiharu; Niki, Hironori

    2004-01-01

    During replication of the Escherichia coli chromosome, the replicated Ori domains migrate towards opposite cell poles, suggesting that a cis-acting site for bipolar migration is located in this region. To identify this cis-acting site, a series of mutants was constructed by splitting subchromosomes from the original chromosome. One mutant, containing a 720 kb subchromosome, was found to be defective in the bipolar positioning of oriC. The creation of deletion mutants allowed the identification of migS, a 25 bp sequence, as the cis-acting site for the bipolar positioning of oriC. When migS was located at the replication terminus, the chromosomal segment showed bipolar positioning. migS was able to rescue bipolar migration of plasmid DNA containing a mutation in the SopABC partitioning system. Interestingly, multiple copies of the migS sequence on a plasmid in trans inhibited the bipolar positioning of oriC. Taken together, these findings indicate that migS plays a crucial role in the bipolar positioning of oriC. In addition, real-time analysis of the dynamic morphological changes of nucleoids in wild-type and migS mutants suggests that bipolar positioning of the replicated oriC contributes to nucleoid organization. PMID:14685268

  6. Integrative genomics analysis of chromosome 5p gain in cervical cancer reveals target over-expressed genes, including Drosha

    PubMed Central

    Scotto, Luigi; Narayan, Gopeshwar; Nandula, Subhadra V; Subramaniyam, Shivakumar; Kaufmann, Andreas M; Wright, Jason D; Pothuri, Bhavana; Mansukhani, Mahesh; Schneider, Achim; Arias-Pulido, Hugo; Murty, Vundavalli V

    2008-01-01

    Background Copy number gains and amplifications are characteristic feature of cervical cancer (CC) genomes for which the underlying mechanisms are unclear. These changes may possess oncogenic properties by deregulating tumor-related genes. Gain of short arm of chromosome 5 (5p) is the most frequent karyotypic change in CC. Methods To examine the role of 5p gain, we performed a combination of single nucleotide polymorphism (SNP) array, fluorescence in situ hybridization (FISH), and gene expression analyses on invasive cancer and in various stages of CC progression. Results The SNP and FISH analyses revealed copy number increase (CNI) of 5p in 63% of invasive CC, which arises at later stages of precancerous lesions in CC development. We integrated chromosome 5 genomic copy number and gene expression data to identify key target over expressed genes as a consequence of 5p gain. One of the candidates identified was Drosha (RNASEN), a gene that is required in the first step of microRNA (miRNA) processing in the nucleus. Other 5p genes identified as targets of CNI play a role in DNA repair and cell cycle regulation (BASP1, TARS, PAIP1, BRD9, RAD1, SKP2, and POLS), signal transduction (OSMR), and mitochondrial oxidative phosphorylation (NNT, SDHA, and NDUFS6), suggesting that disruption of pathways involving these genes may contribute to CC progression. Conclusion Taken together, we demonstrate the power of integrating genomics data with expression data in deciphering tumor-related targets of CNI. Identification of 5p gene targets in CC denotes an important step towards biomarker development and forms a framework for testing as molecular therapeutic targets. PMID:18559093

  7. Insertional inactivation of the Staphylococcus aureus beta-toxin by bacteriophage phi 13 occurs by site- and orientation-specific integration of the phi 13 genome.

    PubMed

    Coleman, D; Knights, J; Russell, R; Shanley, D; Birkbeck, T H; Dougan, G; Charles, I

    1991-04-01

    Lysogenization of Staphylococcus aureus by the serotype F converting bacteriophage phi 13 results in loss of beta-toxin expression. Sequence analysis of the S. aureus beta-toxin gene (hlb), the attachment site (attP)-containing region of phi 13 DNA and the chromosome/bacteriophage DNA junctions of a phi 13 lysogen, revealed that the molecular mechanism of loss of beta-toxin expression was due to insertion of the phi 13 genome into the 5' end of hlb. The insertion site (attB) within hlb contained a 14 base pair core sequence in common with attP and both ends of the integrated linear prophage genome of a phi 13 lysogen. These findings indicate that integration of the phi 13 genome into hlb is site- and orientation-specific.

  8. Short Synthetic Terminators for Assembly of Transcription Units in Vitro and Stable Chromosomal Integration in Yeast S. cerevisiae.

    PubMed

    MacPherson, Murray; Saka, Yasushi

    2017-01-20

    Assembly of synthetic genetic circuits is central to synthetic biology. Yeast S. cerevisiae, in particular, has proven to be an ideal chassis for synthetic genome assemblies by exploiting its efficient homologous recombination. However, this property of efficient homologous recombination poses a problem for multigene assemblies in yeast, since repeated usage of standard parts, such as transcriptional terminators, can lead to rearrangements of the repeats in assembled DNA constructs in vivo. To address this issue in developing a library of orthogonal genetic components for yeast, we designed a set of short synthetic terminators based on a consensus sequence with random linkers to avoid repetitive sequences. We constructed a series of expression vectors with these synthetic terminators for efficient assembly of synthetic genes using Gateway recombination reactions. We also constructed two BAC (bacterial artificial chromosome) vectors for assembling multiple transcription units with the synthetic terminators in vitro and their integration in the yeast genome. The tandem array of synthetic genes integrated in the genome by this method is highly stable because there are few homologous segments in the synthetic constructs. Using this system of assembly and genomic integration of transcription units, we tested the synthetic terminators and their influence on the proximal transcription units. Although all the synthetic terminators have the common consensus with the identical length, they showed different activities and impacts on the neighboring transcription units.

  9. Fusion primer and nested integrated PCR (FPNI-PCR): a new high-efficiency strategy for rapid chromosome walking or flanking sequence cloning

    PubMed Central

    2011-01-01

    Background The advent of genomics-based technologies has revolutionized many fields of biological enquiry. However, chromosome walking or flanking sequence cloning is still a necessary and important procedure to determining gene structure. Such methods are used to identify T-DNA insertion sites and so are especially relevant for organisms where large T-DNA insertion libraries have been created, such as rice and Arabidopsis. The currently available methods for flanking sequence cloning, including the popular TAIL-PCR technique, are relatively laborious and slow. Results Here, we report a simple and effective fusion primer and nested integrated PCR method (FPNI-PCR) for the identification and cloning of unknown genomic regions flanked known sequences. In brief, a set of universal primers was designed that consisted of various 15-16 base arbitrary degenerate oligonucleotides. These arbitrary degenerate primers were fused to the 3' end of an adaptor oligonucleotide which provided a known sequence without degenerate nucleotides, thereby forming the fusion primers (FPs). These fusion primers are employed in the first step of an integrated nested PCR strategy which defines the overall FPNI-PCR protocol. In order to demonstrate the efficacy of this novel strategy, we have successfully used it to isolate multiple genomic sequences namely, 21 orthologs of genes in various species of Rosaceace, 4 MYB genes of Rosa rugosa, 3 promoters of transcription factors of Petunia hybrida, and 4 flanking sequences of T-DNA insertion sites in transgenic tobacco lines and 6 specific genes from sequenced genome of rice and Arabidopsis. Conclusions The successful amplification of target products through FPNI-PCR verified that this novel strategy is an effective, low cost and simple procedure. Furthermore, FPNI-PCR represents a more sensitive, rapid and accurate technique than the established TAIL-PCR and hiTAIL-PCR procedures. PMID:22093809

  10. [Integration of plasmid pPL 7065 into chromosome of Bac. pumilis].

    PubMed

    Lysenko, A M; Koz'mipa, L M; Abromova, M A; Lukin, A A

    1980-01-01

    Hybridization of tritium-labelled plasmid 7065 with total DNA of several Bac. pumilis strains differing in the degree of spore-formation showed that strain 7065-k contains the plasmid in an integral state.

  11. 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

  12. Origin and evolution of B chromosomes in the cichlid fish Astatotilapia latifasciata based on integrated genomic analyses.

    PubMed

    Valente, Guilherme T; Conte, Matthew A; Fantinatti, Bruno E A; Cabral-de-Mello, Diogo C; Carvalho, Robson F; Vicari, Marcelo R; Kocher, Thomas D; Martins, Cesar

    2014-08-01

    Approximately 15% of eukaryotes contain supernumerary B chromosomes. When present, B chromosomes frequently represent as much as 5% of the genome. Despite thousands of reports describing the distribution of supernumeraries in various taxa, a comprehensive theory for the origin, maintenance, and evolution of B chromosomes has not emerged. Here, we sequence the complete genomes of individual cichlid fish (Astatotilapia latifasciata) with and without B chromosomes, as well as microdissected B chromosomes, to identify DNA sequences on the B. B sequences were further analyzed through quantitative polymerase chain reaction and in situ hybridization. We find that the B chromosome contains thousands of sequences duplicated from essentially every chromosome in the ancestral karyotype. Although most genes on the B chromosome are fragmented, a few are largely intact, and we detect evidence that at least three of them are transcriptionally active. We propose a model in which the B chromosome originated early in the evolutionary history of Lake Victoria cichlids from a small fragment of one autosome. DNA sequences originating from several autosomes, including protein-coding genes and transposable elements, subsequently inserted into this proto-B. We propose that intact B chromosome genes involved with microtubule organization, kinetochore structure, recombination and progression through the cell cycle may play a role in driving the transmission of the B chromosome. Furthermore, our work suggests that karyotyping is an essential step prior to genome sequencing to avoid problems in genome assembly and analytical biases created by the presence of high copy number sequences on the B chromosome.

  13. Chromosomal localization of 5S and 18S-5.8S-25S ribosomal DNA sites in five Asian pines using fluorescence in situ hybridization.

    PubMed

    Liu, Z-L; Zhang, D; Hong, D-Y; Wang, X-R

    2003-01-01

    Fluorescence in situ hybridization (FISH) was employed on mitotic metaphase chromosome preparations of five Asian Pinus species: Pinus tabuliformis, Pinus yunnanensis, Pinus densata, Pinus massoniana and Pinus merkusii, using simultaneously DNA probes of the 18S rRNA gene and the 5S rRNA gene including the non-transcribed spacer sequences. The number and location of 18S rDNA sites varied markedly (5-10 pairs of strong signals) among the five pines. A maximum of 20 major 18S rDNA sites was observed in the diploid genome (2n = 24) of P. massoniana. The 5S rDNA FISH pattern was less variable, with one major site and one minor site commonly observed in each species. The differentiation of rDNA sites on chromosomes among the five pines correlates well with their phylogenic positions in Pinus as reconstructed from other molecular data. P. densata, a species of hybrid origin, resembles its parents ( P. tabuliformis and P. yunnanensis), including some components characteristic of each parent in its pattern. However, the species is unique, showing new features resulting possibly from recombination and genome reorganization.

  14. 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

  15. 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.

  16. Wolbachia genome integrated in an insect chromosome: Evolution and fate of laterally transferred endosymbiont genes

    PubMed Central

    Nikoh, Naruo; Tanaka, Kohjiro; Shibata, Fukashi; Kondo, Natsuko; Hizume, Masahiro; Shimada, Masakazu; Fukatsu, Takema

    2008-01-01

    Recent accumulation of microbial genome data has demonstrated that lateral gene transfers constitute an important and universal evolutionary process in prokaryotes, while those in multicellular eukaryotes are still regarded as unusual, except for endosymbiotic gene transfers from mitochondria and plastids. Here we thoroughly investigated the bacterial genes derived from a Wolbachia endosymbiont on the nuclear genome of the beetle Callosobruchus chinensis. Exhaustive PCR detection and Southern blot analysis suggested that ∼30% of Wolbachia genes, in terms of the gene repertoire of wMel, are present on the insect nuclear genome. Fluorescent in situ hybridization located the transferred genes on the proximal region of the basal short arm of the X chromosome. Molecular evolutionary and other lines of evidence indicated that the transferred genes are probably derived from a single lateral transfer event. The transferred genes were, for the length examined, structurally disrupted, freed from functional constraints, and transcriptionally inactive. Hence, most, if not all, of the transferred genes have been pseudogenized. Notwithstanding this, the transferred genes were ubiquitously detected from Japanese and Taiwanese populations of C. chinensis, while the number of the transferred genes detected differed between the populations. The transferred genes were not detected from congenic beetle species, indicating that the transfer event occurred after speciation of C. chinensis, which was estimated to be one or several million years ago. These features of the laterally transferred endosymbiont genes are compared with the evolutionary patterns of mitochondrial and plastid genome fragments acquired by nuclear genomes through recent endosymbiotic gene transfers. PMID:18073380

  17. Localization of preferential sites of rearrangement within the BCR gene in Philadelphia chromosome-positive acute lymphoblastic leukemia

    SciTech Connect

    Denny, C.T.; Shah, N.P.; Ogden, S.; Willman, C.; McConnell, T.; Crist, W.; Carroll, A.; Witte, O.N. )

    1989-06-01

    The Philadelphia chromosome associated with acute lymphoblastic leukemia (ALL) has been linked to a hybrid BCR/ABL protein product that differs from that found in chronic myelogenous leukemia. This implies that the molecular structures of the two chromosomal translocations also differ. Localization of translocation breakpoints in Philadelphia chromosome-positive ALL has been impeded due to the only partial characterization of the BCR locus. The authors have isolated the entire 130-kilobase BCR genomic locus from a human cosmid library. They have demonstrated that these breakpoints are all located at the 3{prime} end of the intron around an unusual restriction fragment length polymorphism caused by deletion of a 1-kilobase fragment containing Alu family reiterated sequences. This clustering is unexpected in light of previous theories of rearrangement in Philadelphia chromosome-positive chronic myelogenous leukemia that would have predicted a random dispersion of breakpoints in the first intron in Philadelphia chromosome-positive ALL. The proximity of the translocation breakpoints to this constitutive deletion may indicate shared mechanisms of rearrangement or that such polymorphisms mark areas of the genome prone to recombination.

  18. Molecular and virological evidence of viral activation from chromosomally integrated human herpesvirus 6A in a patient with X-linked severe combined immunodeficiency.

    PubMed

    Endo, Akifumi; Watanabe, Ken; Ohye, Tamae; Suzuki, Kyoko; Matsubara, Tomoyo; Shimizu, Norio; Kurahashi, Hiroki; Yoshikawa, Tetsushi; Katano, Harutaka; Inoue, Naoki; Imai, Kohsuke; Takagi, Masatoshi; Morio, Tomohiro; Mizutani, Shuki

    2014-08-15

    It has been unclear whether chromosomally integrated human herpesvirus 6 (ciHHV-6) can be activated with pathogenic effects on the human body. We present molecular and virological evidence of ciHHV-6A activation in a patient with X-linked severe combined immunodeficiency. These findings have significant implications for the management of patients with ciHHV-6.

  19. Air Vehicle Technology Integration Program (AVTIP). Delivery Order 0054: Opportune Landing Site (OLS) Critical Experiment

    DTIC Science & Technology

    2008-04-01

    AFRL-RB-WP-TR-2009-3118 AIR VEHICLE TECHNOLOGY INTEGRATION PROGRAM (AVTIP) Delivery Order 0054: Opportune Landing Site (OLS) Critical...VEHICLE TECHNOLOGY INTEGRATION PROGRAM (AVTIP) Delivery Order 0054: Opportune Landing Site (OLS) Critical Experiment 5a. CONTRACT NUMBER F33615-00

  20. Estimating Diversity of Black Flies in the Simulium ignescens and Simulium tunja Complexes in Colombia: Chromosomal Rearrangements as the Core of Integrative Taxonomy.

    PubMed

    Colorado-Garzón, Fredy A; Adler, Peter H; García, Luis F; Muñoz de Hoyos, Paulina; Bueno, Marta L; Matta, Nubia E

    2017-01-01

    Black flies (Diptera: Simuliidae) are distributed throughout the world, with more than 2200 formally described species. The family is renowned for its high frequency of cryptic species, offering an opportunity for integrative taxonomy, based on morphological, chromosomal, and molecular approaches. The biodiversity within Simulium (Psilopelmia) ignescens and S. (Psilopelmia) tunja in Colombia was estimated from the larval stage; 10 morphoforms were recognized based on 7 structural characters. This remarkable morphological variation was evaluated through 23 markers on the polytene chromosomes. We established 1 new cytoform in each nominal species. The congruence of the morphological and chromosomal assignments was evaluated using the mitochondrial marker Cytochrome Oxidase subunit I (COI) for each morphoform. The molecular data supported the chromosomal recognition of cytoforms (i.e., cryptic species). We also established the suitability of the COI marker for linking the pupal stage with each cytoform. Our results reveal the presence of hidden biodiversity in S. ignescens and S. tunja and demonstrate the power of polytene chromosomes as a tool for evaluating simuliid diversity, while illustrating the importance of integrated analyses in modern taxonomy.

  1. Tyramide-FISH mapping of single genes for development of an integrated recombination and cytogenetic map of chromosome 5 of Allium cepa.

    PubMed

    Romanov, Dmitry; Divashuk, Mikhail; Havey, Michael J; Khrustaleva, Ludmila

    2015-03-01

    Chromosome 5 of onion carries major quantitative trait loci (QTL) that control dry-matter content, pungency and storability of bulbs, amounts and types of epicuticular waxes, and resistances to abiotic factors, all of which are of interest to breeders. SNPs, SSRs, and RFLPs in expressed regions of the onion genome have been genetically mapped, and we used these clones and sequences from the NCBI database to develop DNA probes for in situ hybridization to integrate the genetic and physical maps of onion chromosome 5. We produced genomic amplicons from expressed regions of the onion genome that carried both exons and introns in order to increase the hybridization specificity of the probes and to enlarge the target DNA sizes. Tyramide-FISH technique was used to increase the detection sensitivity of relatively short target DNA regions, which range from 950 to 2100 bp. Through the integration of genetic and chromosomal maps, we were able to estimate the distribution of recombination events along onion chromosome 5. We demonstrated the efficiency of chromosomal in situ mapping of exon-intron genomic clones for the extremely large genome of onion.

  2. Site-specific gene integration in rice genome mediated by the FLP-FRT recombination system.

    PubMed

    Nandy, Soumen; Srivastava, Vibha

    2011-08-01

    Plant transformation based on random integration of foreign DNA often generates complex integration structures. Precision in the integration process is necessary to ensure the formation of full-length, single-copy integration. Site-specific recombination systems are versatile tools for precise genomic manipulations such as DNA excision, inversion or integration. The yeast FLP-FRT recombination system has been widely used for DNA excision in higher plants. Here, we report the use of FLP-FRT system for efficient targeting of foreign gene into the engineered genomic site in rice. The transgene vector containing a pair of directly oriented FRT sites was introduced by particle bombardment into the cells containing the target locus. FLP activity generated by the co-bombarded FLP gene efficiently separated the transgene construct from the vector-backbone and integrated the backbone-free construct into the target site. Strong FLP activity, derived from the enhanced FLP protein, FLPe, was important for the successful site-specific integration (SSI). The majority of the transgenic events contained a precise integration and expressed the transgene. Interestingly, each transgenic event lacked the co-bombarded FLPe gene, suggesting reversion of the integration structure in the presence of the constitutive FLPe expression. Progeny of the precise transgenic lines inherited the stable SSI locus and expressed the transgene. This work demonstrates the application of FLP-FRT system for site-specific gene integration in plants using rice as a model.

  3. 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

  4. 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. ...

  5. 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

  6. 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.

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

    PubMed

    Huang, Yan; Hidalgo-Bravo, Alberto; Zhang, Enjie; Cotton, Victoria E; Mendez-Bermudez, Aaron; Wig, Gunjan; Medina-Calzada, Zahara; Neumann, Rita; Jeffreys, Alec J; Winney, Bruce; Wilson, James F; Clark, Duncan A; Dyer, Martin J; Royle, Nicola J

    2014-01-01

    Linear chromosomes are stabilized by telomeres, but the presence of short dysfunctional telomeres triggers cellular senescence in human somatic tissues, thus contributing to ageing. Approximately 1% of the population inherits a chromosomally integrated copy of human herpesvirus 6 (CI-HHV-6), but the consequences of integration for the virus and for the telomere with the insertion are unknown. Here we show that the telomere on the distal end of the integrated virus is frequently the shortest measured in somatic cells but not the germline. The telomere carrying the CI-HHV-6 is also prone to truncations that result in the formation of a short telomere at a novel location within the viral genome. We detected extra-chromosomal circular HHV-6 molecules, some surprisingly comprising the entire viral genome with a single fully reconstituted direct repeat region (DR) with both terminal cleavage and packaging elements (PAC1 and PAC2). Truncated CI-HHV-6 and extra-chromosomal circular molecules are likely reciprocal products that arise through excision of a telomere-loop (t-loop) formed within the CI-HHV-6 genome. In summary, we show that the CI-HHV-6 genome disrupts stability of the associated telomere and this facilitates the release of viral sequences as circular molecules, some of which have the potential to become fully functioning viruses.

  8. Structural Insights into the Assembly of the Adeno-associated Virus Type 2 Rep68 Protein on the Integration Site AAVS1.

    PubMed

    Musayev, Faik N; Zarate-Perez, Francisco; Bishop, Clayton; Burgner, John W; Escalante, Carlos R

    2015-11-13

    Adeno-associated virus (AAV) is the only eukaryotic virus with the property of establishing latency by integrating site-specifically into the human genome. The integration site known as AAVS1 is located in chromosome 19 and contains multiple GCTC repeats that are recognized by the AAV non-structural Rep proteins. These proteins are multifunctional, with an N-terminal origin-binding domain (OBD) and a helicase domain joined together by a short linker. As a first step to understand the process of site-specific integration, we proceeded to characterize the recognition and assembly of Rep68 onto the AAVS1 site. We first determined the x-ray structure of AAV-2 Rep68 OBD in complex with the AAVS1 DNA site. Specificity is achieved through the interaction of a glycine-rich loop that binds the major groove and an α-helix that interacts with a downstream minor groove on the same face of the DNA. Although the structure shows a complex with three OBD molecules bound to the AAVS1 site, we show by using analytical centrifugation and electron microscopy that the full-length Rep68 forms a heptameric complex. Moreover, we determined that a minimum of two direct repeats is required to form a stable complex and to melt DNA. Finally, we show that although the individual domains bind DNA poorly, complex assembly requires oligomerization and cooperation between its OBD, helicase, and the linker domains.

  9. Structural Insights into the Assembly of the Adeno-associated Virus Type 2 Rep68 Protein on the Integration Site AAVS1*

    PubMed Central

    Musayev, Faik N.; Zarate-Perez, Francisco; Bishop, Clayton; Burgner, John W.; Escalante, Carlos R.

    2015-01-01

    Adeno-associated virus (AAV) is the only eukaryotic virus with the property of establishing latency by integrating site-specifically into the human genome. The integration site known as AAVS1 is located in chromosome 19 and contains multiple GCTC repeats that are recognized by the AAV non-structural Rep proteins. These proteins are multifunctional, with an N-terminal origin-binding domain (OBD) and a helicase domain joined together by a short linker. As a first step to understand the process of site-specific integration, we proceeded to characterize the recognition and assembly of Rep68 onto the AAVS1 site. We first determined the x-ray structure of AAV-2 Rep68 OBD in complex with the AAVS1 DNA site. Specificity is achieved through the interaction of a glycine-rich loop that binds the major groove and an α-helix that interacts with a downstream minor groove on the same face of the DNA. Although the structure shows a complex with three OBD molecules bound to the AAVS1 site, we show by using analytical centrifugation and electron microscopy that the full-length Rep68 forms a heptameric complex. Moreover, we determined that a minimum of two direct repeats is required to form a stable complex and to melt DNA. Finally, we show that although the individual domains bind DNA poorly, complex assembly requires oligomerization and cooperation between its OBD, helicase, and the linker domains. PMID:26370092

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

    PubMed

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

    2015-06-30

    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.

  11. Viral Determinants of Integration Site Preferences of Simian Immunodeficiency Virus-Based Vectors

    PubMed Central

    Monse, Hella; Laufs, Stephanie; Kuate, Seraphin; Zeller, W. Jens; Fruehauf, Stefan; Überla, Klaus

    2006-01-01

    Preferential integration into transcriptionally active regions of genomes has been observed for retroviral vectors based on gamma-retroviruses and lentiviruses. However, differences in the integration site preferences were detected, which might be explained by differences in viral components of the preintegration complexes. Viral determinants of integration site preferences have not been defined. Therefore, integration sites of simian immunodeficiency virus (SIV)-based vectors produced in the absence of accessory genes or lacking promoter and enhancer elements were compared. Similar integration patterns for the different SIV vectors indicate that vif, vpr, vpx, nef, env, and promoter or enhancer elements are not required for preferential integration of SIV into transcriptionally active regions of genomes. PMID:16873270

  12. Pseudo attP sites in favor of transgene integration and expression in cultured porcine cells identified by streptomyces phage phiC31 integrase

    PubMed Central

    2013-01-01

    Phage PhiC31 integrase integrates attB-containing plasmid into pseudo attP site in eukaryotic genomes in a unidirectional site-specific manner and maintains robust transgene expression. Few studies, however, explore its potential in livestock. This study aims to discover the molecular basis of PhiC31 integrase-mediated site-specific recombination in pig cells. We show that PhiC31 integrase can mediate site-specific transgene integration into the genome of pig kidney PK15 cells. Intramolecular recombination in pig PK15 cell line occurred at maximum frequency of 82% with transiently transfected attB- and attP-containing plasmids. An optimal molar ratio of pCMV-Int to pEGFP-N1-attB at 5:1 was observed for maximum number of cell clones under drug selection. Four candidate pseudo attP sites were identified by TAIL-PCR from those cell clones with single-copy transgene integration. Two of them gave rise to higher integration frequency occurred at 33%. 5′ and 3′ junction PCR showed that transgene integration mediated by PhiC31 integrase was mono-allelic. Micro- deletion and insertion were observed by sequencing the integration border, indicating that double strand break was induced by the recombination. We then constructed rescue reporter plasmids by ABI-REC cloning of the four pseudo attP sites into pBCPB + plasmid. Transfection of these rescue plasmids and pCMV-Int resulted in expected intramolecular recombination between attB and pseudo attP sites. This proved that the endogenous pseudo attP sites were functional substrates for PhiC31 integrase-mediated site-specific recombination. Two pseudo attP sites maintained robust extracellular and intracellular EGFP expression. Alamar blue assay showed that transgene integration into these specific sites had little effect on cell proliferation. This is the first report to document the potential use of PhiC31 integrase to mediate site-specific recombination in pig cells. Our work established an ideal model to study the

  13. 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 ...

  14. Collaborating sites for community-oriented integrated care and health promotion

    PubMed Central

    Thomas, Paul

    2017-01-01

    London Journal of Primary Care wishes to develop a network of collaborating sites to better understand how to achieve community-oriented integrated care and health promotion in different contexts. A collaborating site can do more than submit papers. It can develop its own domain on the LJPC website, contribute to the development of LJPC policy, and stimulate discussions with other collaborating sites. At any time a collaborating site can opt out. In addition to securing papers for publication, a site might nurture a network of supporters, teach people to use multiple research and quality improvement methods, develop a system of governance for locally led inquiries, develop case studies of community-oriented integrated care and health promotion and facilitate within-site and between-site learning and change. PMID:28356918

  15. Refined physical map of the human PAX2/HOX11/NFKB2 cancer gene region at 10q24 and relocalization of the HPV6AI1 viral integration site to 14q13.3-q21.1

    PubMed Central

    Gough, Sheryl M; McDonald, Margaret; Chen, Xiao-Ning; Korenberg, Julie R; Neri, Antonino; Kahn, Tomas; Eccles, Michael R; Morris, Christine M

    2003-01-01

    Background Chromosome band 10q24 is a gene-rich domain and host to a number of cancer, developmental, and neurological genes. Recurring translocations, deletions and mutations involving this chromosome band have been observed in different human cancers and other disease conditions, but the precise identification of breakpoint sites, and detailed characterization of the genetic basis and mechanisms which underlie many of these rearrangements has yet to be resolved. Towards this end it is vital to establish a definitive genetic map of this region, which to date has shown considerable volatility through time in published works of scientific journals, within different builds of the same international genomic database, and across the differently constructed databases. Results Using a combination of chromosome and interphase fluorescent in situ hybridization (FISH), BAC end-sequencing and genomic database analysis we present a physical map showing that the order and chromosomal orientation of selected genes within 10q24 is CEN-CYP2C9-PAX2-HOX11-NFKB2-TEL. Our analysis has resolved the orientation of an otherwise dynamically evolving assembly of larger contigs upstream of this region, and in so doing verifies the order and orientation of a further 9 cancer-related genes and GOT1. This study further shows that the previously reported human papillomavirus type 6a DNA integration site HPV6AI1 does not map to 10q24, but that it maps at the interface of chromosome bands 14q13.3-q21.1. Conclusions This revised map will allow more precise localization of chromosome rearrangements involving chromosome band 10q24, and will serve as a useful baseline to better understand the molecular aetiology of chromosomal instability in this region. In particular, the relocation of HPV6AI1 is important to report because this HPV6a integration site, originally isolated from a tonsillar carcinoma, was shown to be rearranged in other HPV6a-related malignancies, including 2 of 25 genital condylomas

  16. Selection against Robertsonian fusions involving housekeeping genes in the house mouse: integrating data from gene expression arrays and chromosome evolution.

    PubMed

    Ruiz-Herrera, Aurora; Farré, Marta; Ponsà, Montserrat; Robinson, Terence J

    2010-11-01

    Monobrachial homology resulting from Robertsonian (Rb) fusions is thought to contribute to chromosomal speciation through underdominance. Given the karyotypic diversity characterizing wild house mouse populations [Mus musculus domesticus, (MMU)], variation that results almost exclusively from Rb fusions (diploid numbers range from 22 to 40) and possibly whole arm reciprocal translocations (WARTs), this organism represents an excellent model for testing hypotheses of chromosomal evolution. Previous studies of chromosome size and recombination rates have failed to explain the bias for certain chromosomes to be involved more frequently than others in these rearrangements. Here, we show that the pericentromeric region of one such chromosome, MMU19, which is infrequently encountered as a fusion partner in wild populations, is significantly enriched for housekeeping genes when compared to other chromosomes in the genome. These data suggest that there is selection against breakpoints in the pericentromeric region and provide new insights into factors that constrain chromosomal reorganizations in house mice. Given the anticipated increase in vertebrate whole genome sequences, the examination of gene content and expression profiles of the pericentromeric regions of other mammalian lineages characterized by Rb fusions (i.e., other rodents, bats, and bovids, among others) is both achievable and crucial to developing broadly applicable models of chromosome evolution.

  17. Prolonged Integration Site Selection of a Lentiviral Vector in the Genome of Human Keratinocytes

    PubMed Central

    Qian, Wei; Wang, Yong; Li, Rui-fu; Zhou, Xin; Liu, Jing; Peng, Dai-zhi

    2017-01-01

    Background Lentiviral vectors have been successfully used for human skin cell gene transfer studies. Defining the selection of integration sites for retroviral vectors in the host genome is crucial in risk assessment analysis of gene therapy. However, genome-wide analyses of lentiviral integration sites in human keratinocytes, especially after prolonged growth, are poorly understood. Material/Methods In this study, 874 unique lentiviral vector integration sites in human HaCaT keratinocytes after long-term culture were identified and analyzed with the online tool GTSG-QuickMap and SPSS software. Results The data indicated that lentiviral vectors showed integration site preferences for genes and gene-rich regions. Conclusions This study will likely assist in determining the relative risks of the lentiviral vector system and in the design of a safe lentiviral vector system in the gene therapy of skin diseases. PMID:28255155

  18. 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

  19. 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.

  20. 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.

  1. The diversity of the structure and genomic integration sites of HTLV-1 provirus in MT-2 cell lines.

    PubMed

    Hashikura, Yuuki; Umeki, Kazumi; Umekita, Kunihiko; Nomura, Hajime; Yamamoto, Ikuo; Hasegawa, Hiroo; Yanagihara, Katsunori; Okayama, Akihiko

    2016-07-01

    A human T-lymphotropic virus Type 1 (HTLV-1) positive cell line, MT-2, derived from human cord leukocytes co-culturing with adult T cell leukemia/lymphoma (ATL) cells is commonly used in HTLV-1 research; however, the details of provirus integrated in MT-2 genome have not yet been characterized. In this study, five types of HTLV-1 proviral sequences were detected in 11 different sites of the genome in a reference MT-2 cell line. The five types of HTLV-1 proviral sequences were one complete proviral genome, two types of proviruses with deletion of large internal viral sequences (5.3 and 3.9 kB), one provirus with a large deletion (6.2 kB) from 5'LTR to position 6257, and one provirus of LTR only. The provirus with identical deletion of large internal viral sequence (5.3 kB) was found to be integrated into six different sites (chromosomes). A complete provirus and three of four types of defective provirus were consistently detected in two other MT-2 cell lines cultured in different laboratories. Not only Tax/Rex RNA and HBZ RNA, but also the transcriptional product for a specific defective provirus, were detectable in all three MT-2 cell lines. Because it has been reported that defective provirus is frequently detected in ATL cells, these results may be important in understanding the mechanism of HTLV-1 proviral polymorphism, which may be related to leukemogenesis. In addition, the large variation in integrated HTLV-1 proviruses makes it important for researchers to exercise caution in their assessment and interpretation of results using MT-2 cell lines.

  2. 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.

  3. 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

  4. Genomic structure of the EWS gene and its relationship to EWSR1, a site of tumor-associated chromosome translocation

    SciTech Connect

    Plougastel, B.; Zucman, J.; Peter, M.; Thomas, G.; Delattre, O. )

    1993-12-01

    The EWS gene has been identified based on its location at the chromosome 22 breakpoint of the t(11;22)(q24;q12) translocation that characterizes Ewing sarcoma and related neuroectodermal tumors. The EWS gene spans about 40 kb of DNA and is encoded by 17 exons. The nucleotide sequence of the exons is identical to that of the previously described cDNA. The first 7 exons encode the N-terminal domain of EWS, which consists of a repeated degenerated polypeptide of 7 to 12 residues rich in tyrosine, serine, threonine, glycine, and glutamine. Exons 11, 12, and 13 encode the putative RNA binding domain. The three glycine- and arginine-rich motifs of the gene are mainly encoded by exons 8-9, 14, and 16. The DNA sequence in the 5[prime] region of the gene has features of a CpG-rich island and lacks canonical promoter elements, such as TATA and CCAAT consensus sequences. Positions of the chromosome 22 breakpoints were determined for 19 Ewing tumors. They were localized in introns 7 or 8 in 18 cases and in intron 10 in 1 case. 26 refs., 5 figs.

  5. 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

  6. 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.

  7. Marker chromosomes.

    PubMed

    Rao, Kiran Prabhaker; Belogolovkin, Victoria

    2013-04-01

    Marker chromosomes are a morphologically heterogeneous group of structurally abnormal chromosomes that pose a significant challenge in prenatal diagnosis. Phenotypes associated with marker chromosomes are highly variable and range from normal to severely abnormal. Clinical outcomes are very difficult to predict when marker chromosomes are detected prenatally. In this review, we outline the classification, etiology, cytogenetic characterization, and clinical consequences of marker chromosomes, as well as practical approaches to prenatal diagnosis and genetic counseling.

  8. The Drosophila Ash1 Gene Product, Which Is Localized at Specific Sites on Polytene Chromosomes, Contains a Set Domain and a Phd Finger

    PubMed Central

    Tripoulas, N.; LaJeunesse, D.; Gildea, J.; Shearn, A.

    1996-01-01

    The determined state of Drosophila imaginal discs depends on stable patterns of homeotic gene expression. The stability of these patterns requires the function of the ash1 gene, a member of the trithorax group. The primary translation product of the 7.5-kb ash1 transcript is predicted to be a basic protein of 2144 amino acids. The ASH1 protein contains a SET domain and a PHD finger. Both of these motifs are found in the products of some trithorax group and Polycomb group genes. We have determined the nucleotide sequence alterations in 10 ash1 mutant alleles and have examined their mutant phenotype. The best candidate for a null allele is ash1(22). The truncated protein product of this mutant allele is predicted to contain only 47 amino acids. The ASH1 protein is localized on polytene chromosomes of larval salivary glands at >100 sites. The chromosomal localization of ASH1 implies that it functions at the transcriptional level to maintain the expression pattern of homeotic selector genes. PMID:8725238

  9. 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.

  10. Does transcription play a role in creating a condensin binding site?

    PubMed

    Bernard, Pascal; Vanoosthuyse, Vincent

    2015-01-01

    The highly conserved condensin complex is essential for the condensation and integrity of chromosomes through cell division. Published data argue that high levels of transcription contribute to specify some condensin-binding sites on chromosomes but the exact role of transcription in this process remains elusive. Here we discuss our recent data addressing the role of transcription in establishing a condensin-binding site.

  11. 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

  12. 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.

  13. Mice expressing an error-prone DNA polymerase in mitochondria display elevated replication pausing and chromosomal breakage at fragile sites of mitochondrial DNA

    PubMed Central

    Bailey, Laura J.; Cluett, Tricia J.; Reyes, Aurelio; Prolla, Tom A.; Poulton, Joanna; Leeuwenburgh, Christiaan; Holt, Ian J.

    2009-01-01

    Expression of a proof-reading deficient form of mitochondrial DNA (mtDNA) polymerase γ, POLG, causes early death accompanied by features of premature ageing in mouse. However, the mechanism of cellular senescence remains unresolved. In addition to high levels of point mutations of mtDNA, the POLG mutator mouse harbours linear mtDNAs. Using one- and two-dimensional agarose gel electrophoresis, we show that the linear mtDNAs derive from replication intermediates and are indicative of replication pausing and chromosomal breakage at the accompanying fragile sites. Replication fork arrest is not random but occurs at specific sites close to two cis-elements known as OH and OL. Pausing at these sites may be enhanced in the case of exonuclease-deficient POLG owing to delayed resumption of DNA replication, or replisome instability. In either case, the mtDNA replication cycle is perturbed and this might explain the progeroid features of the POLG mutator mouse. PMID:19244310

  14. Acute myeloid leukemia with monosomy 7, ectopic virus integration site-1 overexpression and central diabetes insipidus: A case report.

    PubMed

    Ma, Hongbing; Yang, Jing; Xiang, Bing; Jia, Yongqian

    2015-06-01

    Central diabetes insipidus (DI) is a rare complication in patients with acute myeloid leukemia (AML), typically occurring in patients with abnormalities of chromosomes 3 or 7. The association between AML with monosomy 7 and DI has been described in a number of studies; however, DI has been rarely reported in cases of ectopic virus integration site-1 (EVI1)-positive AML with monosomy 7. The current study reports a case of AML with monosomy 7 and EVI1 overexpression, with central DI as the initial symptom. The patient was an 18-year-old female who presented with polyuria and polydipsia. Bone marrow aspiration revealed 83.5% myeloperoxidase-positive blasts without trilineage myelodysplasia. The karyotype was 45,XX,-7, and the patient presented monosomy 7 and EVI1 overexpression (-7/EVI1(+)) without 3q aberration. Treatment with induction therapy was unsuccessful. To the best of our knowledge, this is the second case of DI-AML with -7/EVI1(+) and without a 3q aberration. The possible mechanisms associated with EVI1, monosomy 7 and DI were investigated.

  15. 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.

  16. 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.

  17. 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."

  18. New ΦBT1 site-specific integrative vectors with neutral phenotype in Streptomyces.

    PubMed

    Gonzalez-Quiñonez, Nathaly; López-García, María Teresa; Yagüe, Paula; Rioseras, Beatriz; Pisciotta, Annalisa; Alduina, Rosa; Manteca, Ángel

    2016-03-01

    Integrative plasmids are one of the best options to introduce genes in low copy and in a stable form into bacteria. The ΦC31-derived plasmids constitute the most common integrative vectors used in Streptomyces. They integrate at different positions (attB and pseudo-attB sites) generating different mutations. The less common ΦBT1-derived vectors integrate at the unique attB site localized in the SCO4848 gene (S. coelicolor genome) or their orthologues in other streptomycetes. This work demonstrates that disruption of SCO4848 generates a delay in spore germination. SCO4848 is co-transcribed with SCO4849, and the spore germination phenotype is complemented by SCO4849. Plasmids pNG1-4 were created by modifying the ΦBT1 integrative vector pMS82 by introducing a copy of SCO4849 under the control of the promoter region of SCO4848. pNG2 and pNG4 also included a copy of the P ermE * in order to facilitate gene overexpression. pNG3 and pNG4 harboured a copy of the bla gene (ampicillin resistance) to facilitate selection in E. coli. pNG1-4 are the only integrative vectors designed to produce a neutral phenotype when they are integrated into the Streptomyces genome. The experimental approach developed in this work can be applied to create phenotypically neutral integrative plasmids in other bacteria.

  19. 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.

  20. 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.

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

    PubMed Central

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

    1993-01-01

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

  2. Chromosomal imbalances exclusively detected in invasive front area are associated with poor outcome in laryngeal carcinomas from different anatomical sites.

    PubMed

    Ambrosio, Eliane Papa; Silveira, Cássia Gisele Terrassani; Drigo, Sandra Aparecida; Sacomano, Vivian de Souza; Molck, Miriam Coelho; Rocha, Rafael Malagoli; Domingues, Maria Aparecida Custódio; Soares, Fernando Augusto; Kowalski, Luiz Paulo; Rogatto, Silvia Regina

    2013-10-01

    Laryngeal squamous cell carcinoma (LSCC) is a malignant neoplasm exhibiting aggressive phenotype, high recurrence rate, and risk of developing second primary tumors. Current evidence suggests that cells in the invasive front of carcinomas have different molecular profiles compared to those in superficial areas. This study aimed to identify candidate genes in the invasive front and superficial cells from laryngeal carcinomas that would be useful as molecular markers. Invasive front and tumor surface cells of 32 LSCC were evaluated by high-resolution comparative genomic hybridization. Both CCND1 copy number gains and cyclin D1 protein expression were evaluated to confirm gains of 11q13.3. Losses of 3q26.2-q29 and 18q23 were confirmed by loss of heterozygosity analysis. The most frequent chromosomal alterations observed only in invasive front cells involved gains of 1p, 4q, and 9p and losses of 3p, 11p, 12p, 13q, 17q, 18p, 19q, 20q, 21q, and Xp. Gains of 11q13 were detected in both components from glottis and supraglottis but only in invasive front cells from transglottic tumors. Fluorescence in situ hybridization confirmed gains of CCND1/CPE11 in a subset of cases. In supraglottic tumors, cyclin D1 positivity was associated with distant metastasis (P = 0.0018) and with decreased disease-free survival (P = 0.042). Loss of heterozygosity at 3q26.2 and 18q23 were associated with lymph node involvement (P = 0.055) and worsened prognosis, respectively. In conclusion, this study revealed regions that could be targeted in the search for molecular markers in LSCC. Cyclin D1 may be useful as a prognostic marker in supraglottic tumors.

  3. 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

    SciTech Connect

    White, A.; Tomfohrde, J.; Barnes, R. ); Stewart, E.; Cavalli-Sforza, L. ); Le Paslier, D. ); Weissenbach, J. ); Farrer, L. ); Bowcock, A. Eugene McDermott Center of Human Growth and Development, Dallas, TX )

    1993-11-15

    The authors 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 D12S31 and D13S59. Here they describe the construction of a contig of [approx]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-D13S227-D13S228-AFM238vc3-D13S133-AFM084xc5-D13S137-D13S169, D13S155-D13S59-telomere. The recombination/physical distance ratio varies from [approx] 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.

  4. Nicotine-induced Disturbances of Meiotic Maturation in Cultured Mouse Oocytes: Alterations of Spindle Integrity and Chromosome Alignment.

    PubMed

    Zenzes, Maria Teresa; Bielecki, Ryszard

    2004-09-15

    We investigated whether nicotine exposure in vitro of mouse oocytes affects spindle and chromosome function during meiotic maturation (M-I and M-II). Oocytes in germinal vesicle (GV) stage were cultured in nicotine for 8 h or for 16 h, to assess effects in M-I and in metaphase II (M-II). The latter culture setting used the three protocols: 8 h nicotine then 8 h medium (8N + 8M); 16 h nicotine (16N); 8 h medium then 8 h nicotine (8M + 8N). Non-toxic concentrations of nicotine at 1.0, 2.5, 5.0 and 10.0 mmol/L were used. Spindle-chromosome configurations were analyzed with wide-field optical sectioning microscopy. In 8 h cultures, nicotine exposure resulted in dose-related increased proportions of M-I oocytes with defective spindle-chromosome configurations. A dose-related delayed entry into anaphase I was also detected. In 16 h cultures, nicotine exposure for the first 8 h (8N + 8M), or for 16 h (16N), resulted in dose- and time-related increased proportions of oocytes arrested in M-I (10 mmol/L; 8 h: 53.2%, controls 9.6%; 16 h: 87.6%, controls 8.5%). Defects in M-I spindles and chromosomes caused M-I arrest leading to dose-related decreased proportions of oocytes that reached metaphase-II (10 mmol/L 8 h: 46.8%, controls 90.4%;16 h: 12.4%, controls 91.5%). A delayed anaphase-I affected the normal timing of M-II, leading to abnormal oocytes with dispersed chromosomes, or with double spindles and no polar body. Nicotine exposure during the second 8 h (8M + 8N) resulted in dose-related, increased proportions of M-II oocytes with defective spindles and chromosomes (10 mmol/L: 42.9%, controls 2.0%). Nicotine has no adverse effects on GV break down, but induces spindle and chromosome defects compromising oocyte meiotic maturation and development.

  5. Chromosomal Integration of Retinoic Acid Response Elements Prevents Cooperative Transcriptional Activation by Retinoic Acid Receptor and Retinoid X Receptor

    PubMed Central

    Lefebvre, Bruno; Brand, Céline; Lefebvre, Philippe; Ozato, Keiko

    2002-01-01

    All-trans-retinoic acid receptors (RAR) and 9-cis-retinoic acid receptors (RXR) are nuclear receptors known to cooperatively activate transcription from retinoid-regulated promoters. By comparing the transactivating properties of RAR and RXR in P19 cells using either plasmid or chromosomal reporter genes containing the mRARβ2 gene promoter, we found contrasting patterns of transcriptional regulation in each setting. Cooperativity between RXR and RAR occurred at all times with transiently introduced promoters, but was restricted to a very early stage (<3 h) for chromosomal promoters. This time-dependent loss of cooperativity was specific for chromosomal templates containing two copies of a retinoid-responsive element (RARE) and was not influenced by the spacing between the two RAREs. This loss of cooperativity suggested a delayed acquisition of RAR full transcriptional competence because (i) cooperativity was maintained at RAR ligand subsaturating concentrations, (ii) overexpression of SRC-1 led to loss of cooperativity and even to strong repression of chromosomal templates activity, and (iii) loss of cooperativity was observed when additional cis-acting response elements were activated. Surprisingly, histone deacetylase inhibitors counteracted this loss of cooperativity by repressing partially RAR-mediated activation of chromosomal promoters. Loss of cooperativity was not correlated to local histone hyperacetylation or to alteration of constitutive RNA polymerase II (RNAP) loading at the promoter region. Unexpectedly, RNAP binding to transcribed regions was correlated to the RAR activation state as well as to acetylation levels of histones H3 and H4, suggesting that RAR acts at the mRARβ promoter by triggering the switch from an RNA elongation-incompetent RNAP form towards an RNA elongation-competent RNAP. PMID:11839811

  6. Integrating Risk Analyses and Tools at the U.S. Department of Energy Hanford Site

    SciTech Connect

    Lober, R. W.; Yasek, R. M.; Morse, J. G.; Buck, J. W.; Henderson, C. C.; Sams, T. L.; Bunn, A. L.; Vaughn, P.

    2002-02-26

    Risk assessment and environmental impact analysis at the U.S. Department of Energy (DOE) Hanford Site in Washington State has made significant progress in refining the strategy for using risk analysis to support closing of several hundred waste sites plus 149 single-shell tanks at the Hanford Site. A Single-Shell Tank System Closure Work Plan outlines the current basis for closing the single-shell tank systems. An analogous site approach has been developed to address closure of aggregated groups of similar waste sites. Because of the complexity, decision time frames, proximity of non-tank farm waste sites to tank farms, scale, and regulatory considerations, various projects are providing integrated assessments to support risk analyses and decision-making. This paper will describe the approach for using risk assessment to support waste site and tank closure decisions, the tools being developed, and how integration of these risk assessments and analyses are being performed to ad dress near-term and longterm decisions.

  7. 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.

  8. How Do I Do That? Integrating Web Sites into the Gifted Education Classroom

    ERIC Educational Resources Information Center

    Besnoy, Kevin

    2006-01-01

    The purpose of this article is twofold: (1) to provide teachers of the gifted with a reference for adopting IT pedagogical standards, strategies, and tools, specifically Web sites, into the gifted education curriculum; and (2) to show gifted educators the need for research-based standards regarding the integration of websites into the gifted…

  9. Integration of aerial imaging and variable-rate technology for site-specific aerial herbicide application

    Technology Transfer Automated Retrieval System (TEKTRAN)

    As remote sensing and variable rate technology are becoming more available for aerial applicators, practical methodologies on effective integration of these technologies are needed for site-specific aerial applications of crop production and protection materials. The objectives of this study were to...

  10. 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...

  11. Development of the integrated programme for the cleanup of the Sellafield (site)

    SciTech Connect

    Wylie, P.

    2007-07-01

    The Sellafield site in the United Kingdom is highly complex and integrated site. It has two operating reprocessing plants, a fuel fabrication facility and significant processing facilities for the conditioning of high level, intermediate level and low level wastes. It plays a major role in supporting the UK nuclear generation and dealing with the legacy from the early nuclear programmes. Sellafield also supports a number of international nuclear programmes. The UK nuclear industry is undergoing an intense programme of change with many nuclear facilities coming to the end of their lives, new reactors being discussed and a different organisational structure for running the UK nuclear programme. Common to all of this is increased determination to decommission and cleanup historic nuclear wastes. The challenge for the site has been how to put together a cleanup programme for the site whilst recognising the ongoing international strategic value of many of the facilities on the site. The work to produce an integrated plan for Sellafield is now complete and extends from the detailed consideration of facilities and processes through to socio economic impact on the local community. Extensive use has been made of operational research tools and strategic models that cover costs, resources and technical aspects for over 300 active facilities, and over 1000 waste streams. Key strategic decisions facing the site have been analysed and decision calendars produced. These illustrate the options available for each strategic decision and the latest date by which decisions can be taken. Many of the most significant decisions affecting the site need to be progressed within the next few years. The paper describes the process by which the integrated plan for Sellafield has been produced, some of the tools and techniques used in analysing them and the key decisions facing the site. (authors)

  12. 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.

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

    PubMed Central

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

    1992-01-01

    The human androgen-receptor gene (HUMARA; GenBank) contains a highly polymorphic trinucleotide repeat in the first exon. We have found that the methylation of HpaII and HhaI sites less than 100 bp 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, we examined X inactivation patterns in the B lymphocytes of potential and obligate carriers of X-linked agammaglobulinemia. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:1281384

  14. Chromosome-wide mechanisms to decouple gene expression from gene dose during sex-chromosome evolution

    PubMed Central

    Wheeler, Bayly S; Anderson, Erika; Frøkjær-Jensen, Christian; Bian, Qian; Jorgensen, Erik; Meyer, Barbara J

    2016-01-01

    Changes in chromosome number impair fitness by disrupting the balance of gene expression. Here we analyze mechanisms to compensate for changes in gene dose that accompanied the evolution of sex chromosomes from autosomes. Using single-copy transgenes integrated throughout the Caenorhabditis elegans genome, we show that expression of all X-linked transgenes is balanced between XX hermaphrodites and XO males. However, proximity of a dosage compensation complex (DCC) binding site (rex site) is neither necessary to repress X-linked transgenes nor sufficient to repress transgenes on autosomes. Thus, X is broadly permissive for dosage compensation, and the DCC acts via a chromosome-wide mechanism to balance transcription between sexes. In contrast, no analogous X-chromosome-wide mechanism balances transcription between X and autosomes: expression of compensated hermaphrodite X-linked transgenes is half that of autosomal transgenes. Furthermore, our results argue against an X-chromosome dosage compensation model contingent upon rex-directed positioning of X relative to the nuclear periphery. DOI: http://dx.doi.org/10.7554/eLife.17365.001 PMID:27572259

  15. 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.

  16. An integrated CRISPR Bombyx mori genome editing system with improved efficiency and expanded target sites.

    PubMed

    Ma, Sanyuan; Liu, Yue; Liu, Yuanyuan; Chang, Jiasong; Zhang, Tong; Wang, Xiaogang; Shi, Run; Lu, Wei; Xia, Xiaojuan; Zhao, Ping; Xia, Qingyou

    2017-02-09

    Genome editing enabled unprecedented new opportunities for targeted genomic engineering of a wide variety of organisms ranging from microbes, plants, animals and even human embryos. The serial establishing and rapid applications of genome editing tools significantly accelerated Bombyx mori (B. mori) research during the past years. However, the only CRISPR system in B. mori was the commonly used SpCas9, which only recognize target sites containing NGG PAM sequence. In the present study, we first improve the efficiency of our previous established SpCas9 system by 3.5 folds. The improved high efficiency was also observed at several loci in both BmNs cells and B. mori embryos. Then to expand the target sites, we showed that two newly discovered CRISPR system, SaCas9 and AsCpf1, could also induce highly efficient site-specific genome editing in BmNs cells, and constructed an integrated CRISPR system. Genome-wide analysis of targetable sites was further conducted and showed that the integrated system cover 69,144,399 sites in B. mori genome, and one site could be found in every 6.5 bp. The efficiency and resolution of this CRISPR platform will probably accelerate both fundamental researches and applicable studies in B. mori, and perhaps other insects.

  17. 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.

  18. Heat recovery subsystem and overall system integration of fuel cell on-site integrated energy systems

    NASA Astrophysics Data System (ADS)

    Mougin, L. J.

    1983-07-01

    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.

  19. An integrated YAC clone contig for the WAGR region on human chromosome 11p13-p14.1

    SciTech Connect

    Gawin, B.; Klamt, B.; Koenig, A.; Thaete, C.

    1995-11-01

    The WAGR syndrome (Wilms tumor, aniridia, genito-urinary anomalies, and mental retardation) deletion region on chromosome 11p13 has been extensively characterized by deletion analysis and long-range restriction mapping. A dense probe set is available for this genomic region, which harbors a number of disease gene loci, some of which still are not cloned. The identification of candidates for these genes would be greatly facilitated by a complete gene map for this chromosomal segment. As an initial step toward this goal, we have isolated the entire region in 58 overlapping YAC clones. The contig spanning 8 Mb from RAG1 to KCNA4 has been assembled by STS and probe content mapping for 76 loci with an average spacing of about 100 kb. A subset of clones has been analyzed by PFG analysis to position these within the known physical map. Common microsatellite markers permit an alignment of the YAC contig with the genetic and radiation hybrid maps of chromosome 11. Ten known genes, some with much more refined map positions, are placed in the contig. The severalfold coverage of 11p13-p14.1 provides a reliable resource for the future development of a complete gene map of this region. 34 refs., 1 fig., 2 tabs.

  20. Fourth international workshop on human chromosome 5. Final progress report

    SciTech Connect

    McPherson, J.D.

    1996-12-31

    The Fourth International Workshop on Human Chromosome 5 was held in Manchester, UK on November 9--10, 1996 and was hosted by the University of Manchester. The major goals of the workshop were: (1) to collate the various genetic, cytogenetic and physical maps of human chromosome 5; (2) to integrate these maps and identify/correct discrepancies between them wherever possible; (3) to catalogue the sequence-ready contigs of the chromosome; (4) to co-ordinate the various sequencing efforts to avoid future duplication; (5) to establish the first (to the author`s knowledge) web site for the human chromosome 5 community which contains the above information in a readily accessible form.

  1. Synchronization of chromosome dynamics and cell division in bacteria.

    PubMed

    Thanbichler, Martin

    2010-01-01

    Bacterial cells have evolved a variety of regulatory circuits that tightly synchronize their chromosome replication and cell division cycles, thereby ensuring faithful transmission of genetic information to their offspring. Complex multicomponent signaling cascades are used to monitor the progress of cytokinesis and couple replication initiation to the separation of the two daughter cells. Moreover, the cell-division apparatus actively participates in chromosome partitioning and, particularly, in the resolution of topological problems that impede the segregation process, thus coordinating chromosome dynamics with cell constriction. Finally, bacteria have developed mechanisms that harness the cell-cycle-dependent positioning of individual chromosomal loci or the nucleoid to define the cell-division site and control the timing of divisome assembly. Each of these systems manages to integrate a complex set of spatial and temporal cues to regulate and execute critical steps in the bacterial cell cycle.

  2. Hice1, a Novel Microtubule-Associated Protein Required for Maintenance of Spindle Integrity and Chromosomal Stability in Human Cells▿ †

    PubMed Central

    Wu, Guikai; Lin, Yi-Tzu; Wei, Randy; Chen, Yumay; Shan, Zhiyin; Lee, Wen-Hwa

    2008-01-01

    Spindle integrity is critical for efficient mitotic progression and accurate chromosome segregation. Deregulation of spindles often leads to structural and functional aberrations, ultimately promoting segregation errors and aneuploidy, a hallmark of most human cancers. Here we report the characterization of a previously identified human sarcoma antigen (gene located at 19p13.11), Hice1, an evolutionarily nonconserved 46-kDa coiled-coil protein. Hice1 shows distinct cytoplasmic localization and associates with interphase centrosomes and mitotic spindles, preferentially at the spindle pole vicinity. Depletion of Hice1 by RNA interference resulted in abnormal and unstable spindle configurations, mitotic delay at prometaphase and metaphase, and elevated aneuploidy. Conversely, loss of Hice1 had minimal effects on interphase centrosome duplication. We also found that both full-length Hice1 and Hice1-N1, which is composed of 149 amino acids of the N-terminal region, but not the mutant lacking the N-terminal region, exhibited activities of microtubule bundling and stabilization at a near-physiological concentration. Consistently, overexpression of Hice1 rendered microtubule bundles in cells resistant to nocodazole- or cold-treatment-induced depolymerization. These results demonstrate that Hice1 is a novel microtubule-associated protein important for maintaining spindle integrity and chromosomal stability, in part by virtue of its ability to bind, bundle, and stabilize microtubules. PMID:18362163

  3. Enterobacter cloacae complex isolates harboring blaNMC-A or blaIMI-type class A carbapenemase genes on novel chromosomal integrative elements and plasmids.

    PubMed

    Boyd, David A; Mataseje, Laura F; Davidson, Ross; Delport, Johannes A; Fuller, Jeff; Hoang, Linda; Lefebvre, Brigitte; Levett, Paul; Roscoe, Diane L; Willey, Barbara M; Mulvey, Michael R

    2017-02-21

    Carbapenem-resistant Enterobacter cloacae complex isolates submitted to a reference laboratory from 2010 to 2015 were screened by PCR for seven common carbapenemase gene groups, namely, KPC, NDM, OXA-48, VIM, IMP, GES, and NMC-A/IMI. Nineteen of the submitted isolates (1.7%) were found to harbor Ambler class A blaNMC-A or blaIMI-type carbapenemases. All 19 isolates were resistant to at least one carbapenem but susceptible to aminoglycosides, trimethoprim-sulfamethoxazole, tigecycline, and ciprofloxacin. Most isolates (17/19) gave positive results with the Carba-NP test for phenotypic carbapenemase detection. Isolates were genetically diverse by pulsed-field gel electrophoresis macrorestriction analysis, multilocus sequence typing, and hsp60 gene analysis. The genes were found in various Enterobacter cloacae complex species, however blaNMC-A was highly associated with Enterobacter ludwiggii Whole genome sequencing and bioinformatics analysis revealed that all NMC-A (n=10), IMI-1 (n=5), and IMI-9 (n=2) producers harbored the carbapenemase gene on EludIMEX-1-like integrative mobile elements, EcloIMEXs, located in the identical chromosomal locus. Two novel genes, blaIMI-5 and blaIMI-6, were harbored on different IncFII-type plasmids. Enterobacter cloacae complex isolates harboring blaNMC-A/IMI-type carbapenemases are relatively rare in Canada. Though mostly found integrated into the chromosome, some variants are located on plasmids that may enhance their mobility potential.

  4. 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.

  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.

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. Sal-Site: integrating new and existing ambystomatid salamander research and informational resources.

    PubMed

    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

    2005-12-16

    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 http://www.ambystoma.org.

  11. Chromosome evolution in Eulipotyphla.

    PubMed

    Biltueva, L; Vorobieva, N

    2012-01-01

    We integrated chromosome painting information on 5 core-insectivora species available in the literature with new Zoo-FISH data for Iberian shrew (Sorex granarius) and Altai mole (Talpa altaica). Our analysis of these 7 species allowed us to determine the chromosomal features of Eulipotyphla genomes and to update the previously proposed ancestral karyotype for 2 main groups of the Sorex genus. The chromosome painting evidence with human painting probes (HSA) reveals the presence of the 2 unique associations HSA4/5 and 1/10p/12/22b, which support Eulipotyphla. There are a series of synapomorphies both for Erinaceidae (HSA3/1/5, 3/17, 11/15 and 10/20) and for Soricinae (HSA5/9, 6/7/16, 8/3/21 and 11/12/22). We found associations that link Talpidae/Erinaceidae (HSA7/8, 1/5 and 1/19p), Talpidae/Soricidae (HSA1/8/4) and Erinaceidae/Soricidae (HSA4/20 and 2/13). Genome conservation in Eulipotyphla was estimated on the basis of the number of evolutionary breaks in the ancestral mammalian chromosomes. In total, 7 chromosomes of the boreo-eutherian ancestor (BEA8 or 10, 9, 17, 18, 20-22) were retained in all eulipotyphlans studied; among them moles show the highest level of chromosome conservation. The integration of sequence data into the chromosome painting information allowed us to further examine the chromosomal syntenies within a phylogenetic perspective. Based on our analysis we offer the most parsimonious reconstruction of phylogenetic relationships in Eulipotyphla. The cytogenetic reconstructions based on these data do not conflict with molecular phylogenies supporting basal position of Talpidae in the order.

  12. 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.

  13. HPV integration detection in CaSki and SiHa using detection of integrated papillomavirus sequences and restriction-site PCR.

    PubMed

    Raybould, Rachel; Fiander, Alison; Wilkinson, Gavin W G; Hibbitts, Sam

    2014-09-01

    Human Papillomavirus (HPV) infection is the primary cause of cervical neoplasia. HPV DNA is integrated into the human genome in the majority of cervical cancers. The nature of integration may differ with integration incorporating a single copy of HPV or occurring in concatenated form. Our understanding of HPV tumorigenesis is largely based on studies using characterised cell lines with defined integration sites; these cell lines provide an invaluable standard for validation of diagnostic assays. Cell lines also further understanding of integration mechanisms in clinical samples. The objective of this study was to explore integration assays and to investigate integration events in cell lines where HPV is integrated in concatenated form. Restriction site PCR and detection of integrated papillomavirus sequences were performed on DNA from SiHa and CaSki. A novel integration site on Xq27.3 and HPV genome rearrangements were detected in CaSki DNA. However, where integration was previously detected by FISH in CaSki, and reported to be integrated in concatenated form, integration was not detected by DIPS or RS-PCR. The data presented illustrate that HPV copy number can hinder integration detection; this needs consideration when interpreting results from tests applied to clinical samples.

  14. 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.

  15. 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.

  16. Engineering of Systematic Elimination of a Targeted Chromosome in Human Cells

    PubMed Central

    Kato, Hiroki; Nguyen, Huong Thi Nguyen; Pham, Thanh Thi Mai; Han, Xu; Hirofuji, Yuta; Nonaka, Kazuaki

    2017-01-01

    Embryonic trisomy leads to abortion or congenital genetic disorders in humans. The most common autosomal chromosome abnormalities are trisomy of chromosomes 13, 18, and 21. Although alteration of gene dosage is thought to contribute to disorders caused by extra copies of chromosomes, genes associated with specific disease phenotypes remain unclear. To generate a normal cell from a trisomic cell as a means of etiological analysis or candidate therapy for trisomy syndromes, we developed a system to eliminate a targeted chromosome from human cells. Chromosome 21 was targeted by integration of a DNA cassette in HeLa cells that harbored three copies of chromosome 21. The DNA cassette included two inverted loxP sites and a herpes simplex virus thymidine kinase (HSV-tk) gene. This system causes missegregation of chromosome 21 after expression of Cre recombinase and subsequently enables the selection of cells lacking the chromosome by culturing in a medium that includes ganciclovir (GCV). Cells harboring only two copies of chromosome 21 were efficiently induced by transfection of a Cre expression vector, indicating that this approach is useful for eliminating a targeted chromosome.

  17. Location of 45S Ribosomal Genes in Mitotic and Meiotic Chromosomes of Buthid Scorpions.

    PubMed

    Mattos, Viviane Fagundes; Carvalho, Leonardo Sousa; Cella, Doralice Maria; Schneider, Marielle Cristina

    2014-09-01

    Buthid scorpions exhibit a high variability in diploid number within genera and even within species. Cytogenetically, Buthidae differs from other families of Scorpiones based on its low diploid numbers, holocentric chromosomes, and complex chromosomal chains, which form during meiosis. In this study, we analyzed the distribution of the 45S ribosomal DNA (rDNA) genes in the mitotic and meiotic chromosomes of seven buthid species belonging to the genera Rhopalurus and Tityus with the ultimate goal of elucidating the chromosome organization in these scorpions. The chromosome number ranged from 2n=6 to 2n=28. Despite the high variance in diploid number, all species examined carried their 45S rDNA sites in the terminal region of exactly two chromosomes. Analyses of meiotic cells revealed 45S rDNA clusters in the chromosomal chains of Rhopalurus agamemnon, Tityus bahiensis, Tityus confluens, and Tityus martinpaechi, or in bivalent-like configuration in Rhopalurus rochai, Tityus bahiensis, Tityus confluens, Tityus fasciolatus, and Tityus paraguayensis. In the species examined, the 45S rDNA sites colocalized with constitutive heterochromatin regions. In light of the high chromosome variability and maintenance of number and terminal position of 45S rDNA sites in buthids, the heterochromatin may act to conserve the integrity of the ribosomal genes.

  18. 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.

  19. Site-Specific Integration of Exogenous Genes Using Genome Editing Technologies in Zebrafish.

    PubMed

    Kawahara, Atsuo; Hisano, Yu; Ota, Satoshi; Taimatsu, Kiyohito

    2016-05-13

    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.

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. A plasmid from the methylotrophic actinomycete Amycolatopsis methanolica capable of site-specific integration.

    PubMed Central

    Vrijbloed, J W; Madoń, J; Dijkhuizen, L

    1994-01-01

    Amycolatopsis methanolica contains a 13.3-kb plasmid (pMEA300) which is present both in the free state and integrated at a unique genomic location. A 2.1-kb pMEA300 DNA fragment was sequenced, revealing the putative attP site and two open reading frames, xis and int, showing similarity to genes encoding excisionases and integrases, respectively. Images PMID:7961475

  6. Weak Palindromic Consensus Sequences Are a Common Feature Found at the Integration Target Sites of Many Retroviruses

    PubMed Central

    Wu, Xiaolin; Li, Yuan; Crise, Bruce; Burgess, Shawn M.; Munroe, David J.

    2005-01-01

    Integration into the host genome is one of the hallmarks of the retroviral life cycle and is catalyzed by virus-encoded integrases. While integrase has strict sequence requirements for the viral DNA ends, target site sequences have been shown to be very diverse. We carefully examined a large number of integration target site sequences from several retroviruses, including human immunodeficiency virus type 1, simian immunodeficiency virus, murine leukemia virus, and avian sarcoma-leukosis virus, and found that a statistical palindromic consensus, centered on the virus-specific duplicated target site sequence, was a common feature at integration target sites for these retroviruses. PMID:15795304

  7. Evi-2, a common integration site involved in murine myeloid leukemogenesis.

    PubMed Central

    Buchberg, A M; Bedigian, H G; Jenkins, N A; Copeland, N G

    1990-01-01

    BXH-2 mice have the highest incidence of spontaneous retrovirally induced myeloid leukemia of any known inbred strain and, as such, represent a valuable model system for identifying cellular proto-oncogenes involved in myeloid disease. Chronic murine leukemia viruses often induce disease by insertional activation or mutation of cellular proto-oncogenes. These loci are identified as common viral integration sites in tumor DNAs. Here we report on the characterization of a novel common viral integration site in BXH-2 myeloid leukemias, designated Evi-2. Within the cluster of viral integration sites that define Evi-2, we identified a gene that has the potential for encoding a novel protein of 223 amino acids. This putative proto-oncogene possesses all of the structural features of a transmembrane protein. Within the transmembrane domain is a "leucine zipper," suggesting that Evi-2 is involved in either homopolymer or heteropolymer formation, which may play an important role in the normal functioning of Evi-2. Interestingly, the human homolog of Evi-2 has recently been shown to be tightly linked to the von Recklinghausen neurofibromatosis locus, suggesting a role for Evi-2 in human disease as well. Images PMID:2167436

  8. Subsurface Temperature Modeling using Integrated Modeling for Nuclear Reactor Site Assessment in Volcanic Zone

    NASA Astrophysics Data System (ADS)

    Nurhandoko, Bagus Endar B.; Kurniadi, Rizal; Fatiah, Elfa; Rizal Abda, Muhammad; Martha, Rio; Widowati, Sri

    2017-01-01

    Indonesia has giant vulcanic arc that almost the largest vulcanic arc in the world. Therefore, one of the main risk for nuclear site plan is the vulcanic area. Therefore to reduce the risk, one of most safety nuclear site plant is old vulcanic area. In this paper, we propose to predict subsurface temperature profile to ensure the condition of subsurface of vulcanic zone. Geothermal heat flow is important parameter in modeling of subsurface temperature. The subsurface temperature is one of vulcanic activity parameter which very important for nuclear site plant risk assesment. The integrated modeling for predicting subsurface temperature profile is carried out by combining geothermal heat flow and subsurface profiles resulted from either seismic or gravity measurement. The finite difference of Fourier’s law is applied to surface temperature, temperature gradient, geothermal heat flow and thermal conductivity profile for producing subsurface temperature distribution accurately. This subsurface temperature profile is essential to characterize the vulcanic zone whether it is still active or inactive. Characterization of vulcanic activity is very useful to ensure or to minimize the risk of nuclear site plant in vulcanic zone. One of interesting case study of nuclear site plan in Indonesia is mount Muriah site plan, this method is useful to ensure whether mount Muriah is still active or inactive now

  9. 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…

  10. Modeling Chromosomes

    ERIC Educational Resources Information Center

    Robertson, Carol

    2016-01-01

    Learning about chromosomes is standard fare in biology classrooms today. However, students may find it difficult to understand the relationships among the "genome", "chromosomes", "genes", a "gene locus", and "alleles". In the simple activity described in this article, which follows the 5E approach…

  11. Chromosome I controls chromosome II replication in Vibrio cholerae.

    PubMed

    Baek, Jong Hwan; Chattoraj, Dhruba K

    2014-02-01

    Control of chromosome replication involves a common set of regulators in eukaryotes, whereas bacteria with divided genomes use chromosome-specific regulators. How bacterial chromosomes might communicate for replication is not known. In Vibrio cholerae, which has two chromosomes (chrI and chrII), replication initiation is controlled by DnaA in chrI and by RctB in chrII. DnaA has binding sites at the chrI origin of replication as well as outside the origin. RctB likewise binds at the chrII origin and, as shown here, to external sites. The binding to the external sites in chrII inhibits chrII replication. A new kind of site was found in chrI that enhances chrII replication. Consistent with its enhancing activity, the chrI site increased RctB binding to those chrII origin sites that stimulate replication and decreased binding to other sites that inhibit replication. The differential effect on binding suggests that the new site remodels RctB. The chaperone-like activity of the site is supported by the finding that it could relieve the dependence of chrII replication on chaperone proteins DnaJ and DnaK. The presence of a site in chrI that specifically controls chrII replication suggests a mechanism for communication between the two chromosomes for replication.

  12. Site Selection for Ground Based Free Electron Laser Technology Integration Experiment (GBFEL-TIE), Volume 3: Preferred Site Evaluation

    DTIC Science & Technology

    1987-04-01

    established that the GBFEL-TIE could be accomplished at any of the three candidate sites. However, based on differences identified during site investigations...construction and operation of the GBFEL-TIE, including environmental mitigation costs, differed only slightly among the three sites, with the Orogrande site...the differences in the findings that would affect site selection, and established a relative ranking of the three sites under consideration. The

  13. Analysis of XMRV integration sites from human prostate cancer tissues suggests PCR contamination rather than genuine human infection.

    PubMed

    Garson, Jeremy A; Kellam, Paul; Towers, Greg J

    2011-02-25

    XMRV is a gammaretrovirus associated in some studies with human prostate cancer and chronic fatigue syndrome. Central to the hypothesis of XMRV as a human pathogen is the description of integration sites in DNA from prostate tumour tissues. Here we demonstrate that 2 of 14 patient-derived sites are identical to sites cloned in the same laboratory from experimentally infected DU145 cells. Identical integration sites have never previously been described in any retrovirus infection. We propose that the patient-derived sites are the result of PCR contamination. This observation further undermines the notion that XMRV is a genuine human pathogen.

  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. Spp1, a member of the Set1 Complex, promotes meiotic DSB formation in promoters by tethering histone H3K4 methylation sites to chromosome axes.

    PubMed

    Sommermeyer, Vérane; Béneut, Claire; Chaplais, Emmanuel; Serrentino, Maria Elisabetta; Borde, Valérie

    2013-01-10

    Meiotic chromosomes are organized into arrays of loops that are anchored to the chromosome axis structure. Programmed DNA double-strand breaks (DSBs) that initiate meiotic recombination, catalyzed by Spo11 and accessory DSB proteins, form in loop sequences in promoters, whereas the DSB proteins are located on chromosome axes. Mechanisms bridging these two chromosomal regions for DSB formation have remained elusive. Here we show that Spp1, a conserved member of the histone H3K4 methyltransferase Set1 complex, is required for normal levels of DSB formation and is associated with chromosome axes during meiosis, where it physically interacts with the Mer2 DSB protein. The PHD finger module of Spp1, which reads H3K4 methylation close to promoters, promotes DSB formation by tethering these regions to chromosome axes and activating cleavage by the DSB proteins. This paper provides the molecular mechanism linking DSB sequences to chromosome axes and explains why H3K4 methylation is important for meiotic recombination.

  16. 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

  17. A machine-learning approach for predicting palmitoylation sites from integrated sequence-based features.

    PubMed

    Li, Liqi; Luo, Qifa; Xiao, Weidong; Li, Jinhui; Zhou, Shiwen; Li, Yongsheng; Zheng, Xiaoqi; Yang, Hua

    2017-02-01

    Palmitoylation is the covalent attachment of lipids to amino acid residues in proteins. As an important form of protein posttranslational modification, it increases the hydrophobicity of proteins, which contributes to the protein transportation, organelle localization, and functions, therefore plays an important role in a variety of cell biological processes. Identification of palmitoylation sites is necessary for understanding protein-protein interaction, protein stability, and activity. Since conventional experimental techniques to determine palmitoylation sites in proteins are both labor intensive and costly, a fast and accurate computational approach to predict palmitoylation sites from protein sequences is in urgent need. In this study, a support vector machine (SVM)-based method was proposed through integrating PSI-BLAST profile, physicochemical properties, [Formula: see text]-mer amino acid compositions (AACs), and [Formula: see text]-mer pseudo AACs into the principal feature vector. A recursive feature selection scheme was subsequently implemented to single out the most discriminative features. Finally, an SVM method was implemented to predict palmitoylation sites in proteins based on the optimal features. The proposed method achieved an accuracy of 99.41% and Matthews Correlation Coefficient of 0.9773 for a benchmark dataset. The result indicates the efficiency and accuracy of our method in prediction of palmitoylation sites based on protein sequences.

  18. 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.

  19. Integration of Proteomics and Transcriptomics Data Sets for the Analysis of a Lymphoma B-Cell Line in the Context of the Chromosome-Centric Human Proteome Project.

    PubMed

    Díez, Paula; Droste, Conrad; Dégano, Rosa M; González-Muñoz, María; Ibarrola, Nieves; Pérez-Andrés, Martín; Garin-Muga, Alba; Segura, Víctor; Marko-Varga, Gyorgy; LaBaer, Joshua; Orfao, Alberto; Corrales, Fernando J; De Las Rivas, Javier; Fuentes, Manuel

    2015-09-04

    A comprehensive study of the molecular active landscape of human cells can be undertaken to integrate two different but complementary perspectives: transcriptomics, and proteomics. After the genome era, proteomics has emerged as a powerful tool to simultaneously identify and characterize the compendium of thousands of different proteins active in a cell. Thus, the Chromosome-centric Human Proteome Project (C-HPP) is promoting a full characterization of the human proteome combining high-throughput proteomics with the data derived from genome-wide expression profiling of protein-coding genes. Here we present a full proteomic profiling of a human lymphoma B-cell line (Ramos) performed using a nanoUPLC-LTQ-Orbitrap Velos proteomic platform, combined to an in-depth transcriptomic profiling of the same cell type. Data are available via ProteomeXchange with identifier PXD001933. Integration of the proteomic and transcriptomic data sets revealed a 94% overlap in the proteins identified by both -omics approaches. Moreover, functional enrichment analysis of the proteomic profiles showed an enrichment of several functions directly related to the biological and morphological characteristics of B-cells. In turn, about 30% of all protein-coding genes present in the whole human genome were identified as being expressed by the Ramos cells (stable average of 30% genes along all the chromosomes), revealing the size of the protein expression-set present in one specific human cell type. Additionally, the identification of missing proteins in our data sets has been reported, highlighting the power of the approach. Also, a comparison between neXtProt and UniProt database searches has been performed. In summary, our transcriptomic and proteomic experimental profiling provided a high coverage report of the expressed proteome from a human lymphoma B-cell type with a clear insight into the biological processes that characterized these cells. In this way, we demonstrated the usefulness of

  20. PROSPER: An Integrated Feature-Based Tool for Predicting Protease Substrate Cleavage Sites

    PubMed Central

    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

  1. Expression of a chromosomally integrated, single-copy GFP gene in Candida albicans, and its use as a reporter of gene regulation.

    PubMed

    Morschhäuser, J; Michel, S; Hacker, J

    1998-02-01

    Genetically engineered versions of the GFP gene, which encodes the green fluorescent protein of Aequorea victoria, were placed under the control of the constitutively active Candida albicans ACT1 promoter and integrated in single copy into the genome of this pathogenic yeast. Integrative transformants in which one of the two ACT1 alleles had been replaced by a GFP gene exhibited a homogeneous, constitutive fluorescent phenotype. Cells expressing GFP with the wild-type chromophore exhibited very weak fluorescence compared to those GFP proteins with the S65T or S65A, V68L, S72A (GFPmut2) chromophore mutations. Substitution of the CTG codon, which specifies serine instead of leucine in C. albicans, by TTG was absolutely necessary for GFP expression. Although GFP mRNA levels in cells containing a GFP gene with the CTG codon were comparable to those of transformants containing GFP with the TTG substitution, only the latter produced GFP protein, as detected by Western blotting, suggesting that the frequent failure to express heterologous genes in C. albicans is principally due to the noncanonical codon usage. Transformants expressing the modified GFP gene from the promoter of the SAP2 gene, which encodes one of the secreted acid proteinases of C. albicans, showed fluorescence only under conditions which promote proteinase expression, thereby demonstrating the utility of stable, chromosomally integrated GFP reporter genes for the study of gene activation in C. albicans.

  2. 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.

  3. Evidence that Plasmodium falciparum chromosome end clusters are cross-linked by protein and are the sites of both virulence gene silencing and activation.

    PubMed

    Marty, Allison J; Thompson, Jennifer K; Duffy, Michael F; Voss, Till S; Cowman, Alan F; Crabb, Brendan S

    2006-10-01

    The malaria parasite Plasmodium falciparum undergoes antigenic variation through allelic exclusion and variant expression of surface proteins encoded by the var gene family. Regulation of var genes is under epigenetic control and involves reversible silencing and activation that requires the physical repositioning of a var locus into a transcriptionally permissive zone of the nuclear periphery. P. falciparum chromosome ends appear to aggregate into large perinuclear clusters which house both subtelomeric and chromosome central var genes. In this study we further define the composition of telomeric clusters using fluorescent in situ hybridization, and provide evidence that chromosome end clusters are formed by cross-linking protein. In addition, we demonstrate that a subtelomeric reporter gene and a var gene remain within clusters regardless of their transcriptional status. Our findings support a model whereby a highly localized structure dedicated to the activation of a single var gene can be housed within a gene dense chromosome end cluster that is otherwise transcriptionally silent.

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

    PubMed

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

    2014-01-01

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

  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

    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.

  6. 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").

  7. 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.

  8. 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.

  9. The explosion sites of nearby supernovae seen with integral field spectroscopy

    NASA Astrophysics Data System (ADS)

    Kuncarayakti, Hanindyo

    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 via the spectrum. 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.

  10. 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.

  11. INTEGRATED PROCESS GAS MODELING FOR TRITIUM SYSTEMS AT THE SAVANNAH RIVER SITE

    SciTech Connect

    Hang, T; Anita Poore, A

    2007-08-30

    Significant savings are being realized from the consolidated tritium gas-processing operations at the Savannah River Site. However, the trade-off is some reduction of operational flexibility due to decreased storage capacity for process and waste gases. Savannah River National Laboratory researchers are developing an integrated process gas model for tritium processing using Aspen Custom Modeler{trademark} (ACM) software. The modeling involves fully characterizing process flow streams (gas composition, quantity), frequency of batch transfers, and availability of equipment in the flow stream. The model provides a valuable engineering tool to identify flow bottlenecks, thereby enabling adjustments to be made to improve process operations.

  12. Current DOT research on the effect of multiple site damage on structural integrity

    NASA Astrophysics Data System (ADS)

    Tong, P.; Arin, Kemal; Jeong, David Y.; Greif, R.; Brewer, John C.; Bobo, Stephan N.; Sampath, Sam N.

    1992-07-01

    Multiple site damage (MSD) is a type of cracking that may be found in aging airplanes and which may adversely affect their continuing airworthiness. The Volpe National Transportation Systems Center has supported the Federal Aviation Administration Technical Center on structural integrity research for the past two and half years. The work has focused on understanding the behavior of MSD, detection of MSD during airframe inspection, and the avoidance of MSD in future designs. These three elements of the MSD problem are addressed and a summary of the completed work, the current status, and requirements for future research is provided.

  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. 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

  15. 2001 Columbia River Recreation Survey -- Implications for Hanford Site Integrated Assessment

    SciTech Connect

    Anderson, Dave M.; Scott, Michael J.; Bunn, Amoret L.; Fowler, Richard A.; Prendergast, Ellen L.; Miley, Terri B.; Eschbach, Tara O.; Jaksch, John A.

    2002-05-16

    This report presents the results from the Columbia River Recreation Survey conducted in the summer of 2001. The survey combined on-site personal interviews with parties engaged in river recreation with on-site field observations to develop a picture of summer river recreation on the Columbia. The study area stretched from just below Priest Rapids Dam in the north to McNary Dam in the south, and was divided into four "Areas" that correspond to the river areas used by the Groundwater/Vadose Zone Integration Project. This study is part of the Groundwater/Vadose Zone Integration Project and was commissioned specifically to document the current recreation use levels in these areas of the river, and to elicit recreation-related expenditure information from visitors. This information informs economic and environmental models used to measure the economic risk posed by possible, but unlikely, releases of contaminants from the Hanford site into the Columbia River. During the study period, researchers collected 256 survey responses and 396 field observations from recreation sites up and down both shores of the river in the study area. Results presented include analysis of trip duration by river activity, trip frequency, and visitor place of origin. Economics-related results include trip expenditure profiles by activity and place of origin. Data also were collected on fishing effort. Visitors also were asked to indicate what activity or destination substitution they would choose in the hypothetical example that the river could not be accessed. The report also highlights some limitations in the approach. Principally, by doing this research in the summer, the recreational use of the river in the other seasons was not documented. The report provides data that suggest the significance of the other seasons - particularly spring and fall - for salmon and steelhead fishing. This stretch of the Columbia is also well known for waterfowl hunting.

  16. Development of a genomic site for gene integration and expression in Enterococcus faecalis

    PubMed Central

    DebRoy, Sruti; van der Hoeven, Ransome; Singh, Kavindra V.; Gao, Peng; Harvey, Barrett R.; Murray, Barbara E.; Garsin, Danielle A.

    2012-01-01

    Enterococcus faecalis, a gram-positive opportunistic pathogen, has become one of the leading causes of nosocomial infections. Normally a resident of the gastrointestinal tract, extensive use of antibiotics has resulted in the rise of E. faecalis strains that are resistant to multiple antibiotics. This, compounded with the ability to easily exchange antibiotic determinants with other bacteria, has made certain E. faecalis infections difficult to treat medically. The genetic toolbox for the study of E. faecalis has expanded greatly in recent years, but has lacked methodology to stably introduce a gene in single copy in a non-disruptive manner for complementation or expression of non-native genes. In this study, we identified a specific site in the genome of E. faecalis OG1RF that can serve as an expression site for a gene of interest. This site is well conserved in most of the sequenced E. faecalis genomes. A vector has also been developed to integrate genes into this site by allelic exchange. Using this system, we complemented an in-frame deletion in eutV, demonstrating that the mutation does not cause polar effects. We also generated an E. faecalis OG1RF strain that stably expresses the green fluorescent protein and is comparable to the parent strain in terms of in vitro growth and pathogenicity in C. elegans and mice. Another major advantage of this new methodology is the ability to express integrated genes without the need for maintaining antibiotic selection, making this an ideal tool for functional studies of genes in infection models and co-culture systems. PMID:22542850

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

    PubMed

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

    2014-08-26

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

  18. 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).

  19. Prediction of phosphorylation sites based on the integration of multiple classifiers.

    PubMed

    Han, R Z; Wang, D; Chen, Y H; Dong, L K; Fan, Y L

    2017-02-23

    Phosphorylation is an important part of post-translational modifications of proteins, and is essential for many biological activities. Phosphorylation and dephosphorylation can regulate signal transduction, gene expression, and cell cycle regulation in many cellular processes. Phosphorylation is extremely important for both basic research and drug discovery to rapidly and correctly identify the attributes of a new protein phosphorylation sites. Moreover, abnormal phosphorylation can be used as a key medical feature related to a disease in some cases. The using of computational methods could improve the accuracy of detection of phosphorylation sites, which can provide predictive guidance for the prevention of the occurrence and/or the best course of treatment for certain diseases. Furthermore, this approach can effectively reduce the costs of biological experiments. In this study, a flexible neural tree (FNT), particle swarm optimization, and support vector machine algorithms were used to classify data with secondary encoding according to the physical and chemical properties of amino acids for feature extraction. Comparison of the classification results obtained from the three classifiers showed that the classification of the FNT was the best. The three classifiers were then integrated in the form of a minority subordinate to the majority vote to obtain the results. The performance of the integrated model showed improvement in sensitivity (87.41%), specificity (87.60%), and accuracy (87.50%).

  20. 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

  1. Human ficolin: cDNA cloning, demonstration of peripheral blood leucocytes as the major site of synthesis and assignment of the gene to chromosome 9.

    PubMed Central

    Lu, J; Tay, P N; Kon, O L; Reid, K B

    1996-01-01

    Pig ficolins and a number of other proteins contain sequences that are homologous to the C-terminal halves of fibrinogen beta- and gamma-chains. To clone the cDNA for human ficolin, two degenerate oligonucleotide primers were synthesized, based on two stretches of protein sequence that were highly conserved among those proteins, and used for PCR with cDNA from a human uterus lambda gt11 library as a template. A PCR product with a predicted size of 300 bp was obtained and this was used to screen a uterus cDNA library. Of the positive clones isolated, two (U1 and U2), containing inserts of 1.7 and 1.1 kb respectively, were found to encode human ficolin. The cDNA-derived amino acid sequence of human ficolin has approx. 75% identity with, and a similar domain organization to, the two pig ficolin sequences, which are characterized by the presence of a leader peptide, a short N-terminal segment followed by a collagen-like region and then by a C-terminal fibrinogen-like domain. The 1.1 kb insert of clone U2 was used in Northern-blot analysis, and a very strong signal for a 1.4 kb mRNA species was detected in mRNA from human peripheral blood leucocytes. This showed that, despite the initial characterization of pig ficolin as a putative receptor on uterine cells for transforming growth factor beta 1, blood leucocytes are probably the major site of human ficolin synthesis. Much weaker signals of the same size were also detected in spleen, lung and thymus and may be due to the presence of tissue macrophages or trapped blood in these tissues. An mRNA species of approx. 1.3 kb in human liver also weakly hybridized to the U2 probe, indicating the presence of a sequence that was distinct from, but related to, ficolin. The gene for human ficolin has been mapped to chromosome 9. PMID:8573080

  2. Collaboration for the integration of HIV prevention at Title X family planning service delivery sites.

    PubMed

    Tran, Nhan T; Hallerdin, Jule M; Flowers-Maple, Charon; Moskosky, Susan B

    2010-01-01

    Since 2001, the U.S. Department of Health and Human Services' Office of Family Planning (OFP), in collaboration with the Minority AIDS Initiative, has provided supplemental grant funds to Title X-funded family planning service delivery sites to expand the availability of human immunodeficiency virus (HIV) prevention services. This work has resulted in three major outcomes: (1) increased institutional capacity for the delivery of HIV-prevention services at Title X family planning service delivery sites, (2) the successful implementation of HIV-prevention services at these sites, and (3) the identification of HIV-positive individuals who were referred to care services. These efforts resulted in a total of 539,667 unduplicated individuals being tested for HIV. These tests resulted in the identification of 1,692 HIV-positive individuals who otherwise may not have been tested for HIV. More than 85% of the HIV-positive cases were detected among clients who self-identified as members of racial/ethnic minority groups. The integration of HIV-prevention services is a feasible and effective strategy for detecting HIV infection among women, including women in racial/ethnic minority groups.

  3. 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/.

  4. The Cherenkov Telescope array on-site integral sensitivity: observing the Crab

    NASA Astrophysics Data System (ADS)

    Fioretti, Valentina; Bulgarelli, Andrea; Schüssler, Fabian

    2016-07-01

    The Cherenkov Telescope Array (CTA) is the future large observatory in the very high energy (VHE) domain. Operating from 20 GeV to 300 TeV, it will be composed of tens of Imaging Air Cherenkov Telescopes (IACTs) displaced in a large area of a few square kilometers in both the southern and northern hemispheres. Thanks to the wide energy coverage and the tremendous boost in effective area (10 times better than the current IACTs), for the first time a VHE observatory will be able to detect transient phenomena in short exposures. The CTA/DATA On-Site Analysis (OSA) is the system devoted to the development of dedicated pipelines and algorithms to be used at the CTA site for the reconstruction, data quality monitoring, science monitoring and realtime science alerting during observations. The minimum exposure required to issue a science alert is not a general requirement of the observatory but is a function of the astrophysical object under study, because the ability to detect a given source is determined by the integral sensitivity which, in addition to the CTA Monte Carlo simulations, providing the energy-dependent instrument response (e.g. the effective area and the background rate), requires the spectral distribution of the science target. The OSA integral sensitivity is computed here for the most studied source at Gamma-rays, the Crab Nebula, for a set of exposures ranging from 1000 seconds to 50 hours, using the full CTA Southern array. The reason for the Crab Nebula selection as the first example of OSA integral sensitivity is twofold: (i) this source is characterized by a broad spectrum covering the entire CTA energy range; (ii) it represents, at the time of writing, the standard candle in VHE and it is often used as unit for the IACTs sensitivity. The effect of different Crab Nebula emission models on the CTA integral sensitivity is evaluated, to emphasize the need for representative spectra of the CTA science targets in the evaluation of the OSA use cases. Using

  5. Site-specific integration and tailoring of cassette design for sustainable gene transfer.

    PubMed

    Lombardo, Angelo; Cesana, Daniela; Genovese, Pietro; Di Stefano, Bruno; Provasi, Elena; Colombo, Daniele F; Neri, Margherita; Magnani, Zulma; Cantore, Alessio; Lo Riso, Pietro; Damo, Martina; Pello, Oscar M; Holmes, Michael C; Gregory, Philip D; Gritti, Angela; Broccoli, Vania; Bonini, Chiara; Naldini, Luigi

    2011-08-21

    Integrative gene transfer methods are limited by variable transgene expression and by the consequences of random insertional mutagenesis that confound interpretation in gene-function studies and may cause adverse events in gene therapy. Site-specific integration may overcome these hurdles. Toward this goal, we studied the transcriptional and epigenetic impact of different transgene expression cassettes, targeted by engineered zinc-finger nucleases to the CCR5 and AAVS1 genomic loci of human cells. Analyses performed before and after integration defined features of the locus and cassette design that together allow robust transgene expression without detectable transcriptional perturbation of the targeted locus and its flanking genes in many cell types, including primary human lymphocytes. We thus provide a framework for sustainable gene transfer in AAVS1 that can be used for dependable genetic manipulation, neutral marking of the cell and improved safety of therapeutic applications, and demonstrate its feasibility by rapidly generating human lymphocytes and stem cells carrying targeted and benign transgene insertions.

  6. 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.

  7. Chromosomal rearrangements associated with LINE elements in the mouse genome.

    PubMed Central

    Shyman, S; Weaver, S

    1985-01-01

    Two segments of DNA that have apparently inserted in the interval between the two adult beta-globin genes in BALB/c (Hbbd haplotype) but not in C57B1/10 (Hbbs haplotype) mouse strains have been described (1). These putative insertions, each about 1000 bp in length, mapped near a repetitive element. To determine the precise position of these alleged insertions, their target sites, and the nature of their boundaries, we cloned and sequenced the appropriate regions of both chromosomes. One of the two segments is not an insertion but rather a region between two independently integrated L1 repetitive elements (LINEs) (2), one in Hbbd and the other in the Hbbs chromosome. The other segment is an insertion of 940 bp which is located within the L1 element in the Hbbd chromosome. This insert is unusual in that it exists in only one copy in the BALB/c genome. PMID:2991852

  8. 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.

  9. Integration of genotoxicity and population genetic analyses in kangaroo rats (Dipodomys merriami) exposed to radionuclide contamination at the Nevada Test Site, USA.

    PubMed

    Theodorakis, C W; Bickham, J W; Lamb, T; Medica, P A; Lyne, T B

    2001-02-01

    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-haplotye 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.

  10. 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.

  11. 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.

  12. An Integrated Approach to Quantify Groundwater - Surface Water Interactions: the Norman Research Site, Oklahoma

    NASA Astrophysics Data System (ADS)

    Mendoza-Sanchez, I.; Phanikumar, M. S.; Masoner, J.; Cozzarelli, I.; McGuire, J. T.

    2009-12-01

    An intensive investigation of hydrogeologic and biogeochemical processes controlling contaminant migration and attenuation is in progress at the Norman Landfill Research Site in Oklahoma. The site involves a wetland that overlies a landfill leachate plume. The wetland-aquifer system actively exchanges contaminants and nutrients. These chemicals move from the wetland to the aquifer and vice versa depending on the groundwater-surface water (GW-SW) exchange rate and flow direction. The GW-SW flux has to be quantified to better understand the fate and transport of contaminants and nutrients. Different types of data have been collected at the site over a period of ten years including meteorological data, isotopic composition of water samples, water levels, pan evaporation rates, and seepage fluxes. This paper reports the development and application of a process-based water balance model based on long-term climate, soil, vegetation and hydrological dynamics of the system to determine the GW-SW flow rates. Our integrated approach involved model evaluation by means of the following independent measurements: (a) groundwater inflow calculation using stable isotopes of oxygen and hydrogen ( ) (b) seepage flux measurements in the wetland hyporheic sediment and c) pan evaporation measurements on land and in the wetland. The approach was found to be useful for identifying the dominant hydrological processes at the site, including recharge and subsurface flows. Recharge results from the model compared well with estimates obtained using isotope methods from previous studies and allowed us to identify specific annual signatures of this important process during the period of study. Results indicate that subsurface flow components in the system are seasonal and readily respond to rainfall events. The wetland water balance is dominated by local groundwater inputs and regional groundwater flow contributes little to the overall water balance.

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

    PubMed

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

    2015-11-10

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

  14. High-throughput genomic mapping of vector integration sites in gene therapy studies.

    PubMed

    Beard, Brian C; Adair, Jennifer E; Trobridge, Grant D; Kiem, Hans-Peter

    2014-01-01

    Gene therapy has enormous potential to treat a variety of infectious and genetic diseases. To date hundreds of patients worldwide have received hematopoietic cell products that have been gene-modified with retrovirus vectors carrying therapeutic transgenes, and many patients have been cured or demonstrated disease stabilization as a result (Adair et al., Sci Transl Med 4:133ra57, 2012; Biffi et al., Science 341:1233158, 2013; Aiuti et al., Science 341:1233151, 2013; Fischer et al., Gene 525:170-173, 2013). Unfortunately, for some patients the provirus integration dysregulated the expression of nearby genes leading to clonal outgrowth and, in some cases, cancer. Thus, the unwanted side effect of insertional mutagenesis has become a major concern for retrovirus gene therapy. The careful study of retrovirus integration sites (RIS) and the contribution of individual gene-modified clones to hematopoietic repopulating cells is of crucial importance for all gene therapy studies. Supporting this, the US Food and Drug Administration (FDA) has mandated the careful monitoring of RIS in all clinical trials of gene therapy. An invaluable method was developed: linear amplification mediated-polymerase chain reaction (LAM-PCR) capable of analyzing in vitro and complex in vivo samples, capturing valuable genomic information directly flanking the site of provirus integration. Linking this method and similar methods to high-throughput sequencing has now made possible an unprecedented understanding of the integration profile of various retrovirus vectors, and allows for sensitive monitoring of their safety. It also allows for a detailed comparison of improved safety-enhanced gene therapy vectors. An important readout of safety is the relative contribution of individual gene-modified repopulating clones. One limitation of LAM-PCR is that the ability to capture the relative contribution of individual clones is compromised because of the initial linear PCR common to all current methods

  15. 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.

  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. Chromosomal evolution in Rodentia.

    PubMed

    Romanenko, S A; Perelman, P L; Trifonov, V A; Graphodatsky, A S

    2012-01-01

    Rodentia is the most species-rich mammalian order and includes several important laboratory model species. The amount of new information on karyotypic and phylogenetic relations within and among rodent taxa is rapidly increasing, but a synthesis of these data is currently lacking. Here, we have integrated information drawn from conventional banding studies, recent comparative painting investigations and molecular phylogenetic reconstructions of different rodent taxa. This permitted a revision of several ancestral karyotypic reconstructions, and a more accurate depiction of rodent chromosomal evolution.

  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. Human artificial chromosomes for gene delivery and the development of animal models.

    PubMed

    Kazuki, Yasuhiro; Oshimura, Mitsuo

    2011-09-01

    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.

  20. High-definition mapping of retroviral integration sites defines the fate of allogeneic T cells after donor lymphocyte infusion.

    PubMed

    Cattoglio, Claudia; Maruggi, Giulietta; Bartholomae, Cynthia; Malani, Nirav; Pellin, Danilo; Cocchiarella, Fabienne; Magnani, Zulma; Ciceri, Fabio; Ambrosi, Alessandro; von Kalle, Christof; Bushman, Frederic D; Bonini, Chiara; Schmidt, Manfred; Mavilio, Fulvio; Recchia, Alessandra

    2010-12-22

    The infusion of donor lymphocytes transduced with a retroviral vector expressing the HSV-TK suicide gene in patients undergoing hematopoietic stem cell transplantation for leukemia/lymphoma promotes immune reconstitution and prevents infections and graft-versus-host disease. Analysis of the clonal dynamics of genetically modified lymphocytes in vivo is of crucial importance to understand the potential genotoxic risk of this therapeutic approach. We used linear amplification-mediated PCR and pyrosequencing to build a genome-wide, high-definition map of retroviral integration sites in the genome of peripheral blood T cells from two different donors and used gene expression profiling and bioinformatics to associate integration clusters to transcriptional activity and to genetic and epigenetic features of the T cell genome. Comparison with matched random controls and with integrations obtained from CD34(+) hematopoietic stem/progenitor cells showed that integration clusters occur within chromatin regions bearing epigenetic marks associated with active promoters and regulatory elements in a cell-specific fashion. Analysis of integration sites in T cells obtained ex vivo two months after infusion showed no evidence of integration-related clonal expansion or dominance, but rather loss of cells harboring integration events interfering with RNA post-transcriptional processing. The study shows that high-definition maps of retroviral integration sites are a powerful tool to analyze the fate of genetically modified T cells in patients and the biological consequences of retroviral transduction.

  1. 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

  2. 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.

  3. 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.

  4. Genome-wide characterization of transcriptional start sites in humans by integrative transcriptome analysis

    PubMed Central

    Yamashita, Riu; Sathira, Nuankanya P.; Kanai, Akinori; Tanimoto, Kousuke; Arauchi, Takako; Tanaka, Yoshiaki; Hashimoto, Shin-ichi; Sugano, Sumio; Nakai, Kenta; Suzuki, Yutaka

    2011-01-01

    We performed a genome-wide analysis of transcriptional start sites (TSSs) in human genes by multifaceted use of a massively parallel sequencer. By analyzing 800 million sequences that were obtained from various types of transcriptome analyses, we characterized 140 million TSS tags in 12 human cell types. Despite the large number of TSS clusters (TSCs), the number of TSCs was observed to decrease sharply with increasing expression levels. Highly expressed TSCs exhibited several characteristic features: Nucleosome-seq analysis revealed highly ordered nucleosome structures, ChIP-seq analysis detected clear RNA polymerase II binding signals in their surrounding regions, evaluations of previously sequenced and newly shotgun-sequenced complete cDNA sequences showed that they encode preferable transcripts for protein translation, and RNA-seq analysis of polysome-incorporated RNAs yielded direct evidence that those transcripts are actually translated into proteins. We also demonstrate that integrative interpretation of transcriptome data is essential for the selection of putative alternative promoter TSCs, two of which also have protein consequences. Furthermore, discriminative chromatin features that separate TSCs at different expression levels were found for both genic TSCs and intergenic TSCs. The collected integrative information should provide a useful basis for future biological characterization of TSCs. PMID:21372179

  5. 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.

  6. 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.

  7. Study of component technologies for fuel cell on-site integrated energy systems

    NASA Astrophysics Data System (ADS)

    Lee, W. D.; Mathias, S.

    1980-12-01

    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.

  8. 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.

  9. Integration of Pig-a, micronucleus, chromosome aberration and comet assay endpoints in a 28-day rodent toxicity study with urethane

    PubMed Central

    Stankowski, Leon F.; Aardema, Marilyn J.; Lawlor, Timothy E.; Pant, Kamala; Roy, Shambhu; Xu, Yong; Elbekai, Reem

    2015-01-01

    As part of the international Pig-a validation trials, we examined the induction of Pig-a mutant reticulocytes and red blood cells (RETCD59− and RBCCD59−, respectively) in peripheral blood of male Sprague Dawley® rats treated with urethane (25, 100 and 250mg/kg/day) or saline by oral gavage for 29 days. Additional endpoints integrated into this study were: micronucleated reticulocytes (MN-RET) in peripheral blood; chromosome aberrations (CAb) and DNA damage (%tail intensity via the comet assay) in peripheral blood lymphocytes (PBL); micronucleated polychromatic erythrocytes (MN-PCE) in bone marrow; and DNA damage (comet) in various organs at termination (the 29th dose was added for the comet endpoint at sacrifice). Ethyl methanesulfonate (EMS; 200mg/kg/day on Days 3, 4, 13, 14, 15, 27, 28 and 29) was evaluated as the concurrent positive control (PC). All animals survived to termination and none exhibited overt toxicity, but there were significant differences in body weight and body weight gain in the 250-mg/kg/day urethane group, as compared with the saline control animals. Statistically significant, dose-dependent increases were observed for urethane for: RETCD59− and RBCCD59− (on Days 15 and 29); MN-RET (on Days 4, 15 and 29); and MN-PCE (on Day 29). The comet assay yielded positive results in PBL (Day 15) and liver (Day 29), but negative results for PBL (Days 4 and 29) and brain, kidney and lung (Day 29). No significant increases in PBL CAb were observed at any sample time. Except for PBL CAb (likely due to excessive cytotoxicity), EMS-induced significant increases in all endpoints/tissues. These results compare favorably with earlier in vivo observations and demonstrate the utility and sensitivity of the Pig-a in vivo gene mutation assay, and its ability to be easily integrated, along with other standard genotoxicity endpoints, into 28-day rodent toxicity studies. PMID:25934985

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

    PubMed Central

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

    2014-01-01

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

  11. 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

  12. Fitness cost implications of PhiC31-mediated site-specific integrations in target-site strains of the Mexican fruit fly, Anastrepha ludens (Diptera: Tephritidae).

    PubMed

    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.

  13. Chromosome-specific segmentation revealed by structural analysis of individually isolated chromosomes.

    PubMed

    Kitada, Kunio; Taima, Akira; Ogasawara, Kiyomoto; Metsugi, Shouichi; Aikawa, Satoko

    2011-04-01

    Analysis of structural rearrangements at the individual chromosomal level is still technologically challenging. Here we optimized a chromosome isolation method using fluorescent marker-assisted laser-capture and laser-beam microdissection and applied it to structural analysis of two aberrant chromosomes found in a lung cancer cell line. A high-density array-comparative genomic hybridization (array-CGH) analysis of DNA samples prepared from each of the chromosomes revealed that these two chromosomes contained 296 and 263 segments, respectively, ranging from 1.5 kb to 784.3 kb in size, derived from different portions of chromosome 8. Among these segments, 242 were common in both aberrant chromosomes, but 75 were found to be chromosome-specific. Sequences of 263 junction sites connecting the ends of segments were determined using a PCR/Sanger-sequencing procedure. Overlapping microhomologies were found at 169 junction sites. Junction partners came from various portions of chromosome 8 and no biased pattern in the positional distribution of junction partners was detected. These structural characteristics suggested the occurrence of random fragmentation of the entire chromosome 8 followed by random rejoining of these fragments. Based on that, we proposed a model to explain how these aberrant chromosomes are formed. Through these structural analyses, it was demonstrated that the optimized chromosome isolation method described here can provide high-quality chromosomal DNA for high resolution array-CGH analysis and probably for massively parallel sequencing analysis.

  14. 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.

  15. 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

  16. Integrated Methods for Site Characterization and Conceptual Model Development for a Contaminated Fractured-Bedrock Aquifer

    NASA Astrophysics Data System (ADS)

    Johnson, C. D.; Kastrinos, J. R.; Haeni, F. P.

    2005-12-01

    A multi-disciplined and team-based approach was used to integrate geophysical, hydrologic, and chemical data to characterize lithology, fractures, and hydraulic properties of fractured crystalline bedrock and to determine the nature and extent of ground-water contamination from a landfill and former chemical-waste disposal pits at the University of Connecticut. Detection of volatile organic compounds (VOCs) in domestic bedrock wells in the mid-1980s led to this investigation, in which a team comprised of hydrologists, engineers, geophysicists, geologists, chemists, toxicologists, and community-involvement personnel collected, analyzed, and evaluated data; developed and refined a conceptual model of the ground-water flow and contaminant distribution at the site; and evaluated alternatives and implemented a final remediation plan. The characterization phase began in 1999 and the remediation phase is currently ongoing. An integrated and iterative approach of using multiple methods in phases was important for corroborating the interpretation of individual methods and essential for guiding the design and implementation of additional testing at the site. The use of geophysical data early in the investigation allowed the study team to obtain detailed subsurface information using a minimum of boreholes. Surface geophysical methods were used to target potential discharge of contaminants from the landfill for further investigation. Borehole geophysical methods were used to investigate the anomalies identified by surface geophysical methods, the location and orientation of fractures that intersect and surround each well, the direction and magnitude of ambient flow in the wells, and the transmissive fractures that could provide pathways for contaminant migration. Borehole geophysical and hydraulic data were used to design discrete-zone monitoring systems for the collection of hydraulic head and chemical data and to prevent cross contamination through the boreholes. The results

  17. Integrated Geophysical and Archaeological investigations to study the site of Aquinum (Frosinone, Italy)

    NASA Astrophysics Data System (ADS)

    Piro, Salvatore; Ceraudo, Giuseppe; Zamuner, Daniela

    2010-05-01

    To enhance the knowledge finalised to the location and conservation of the unknown buried structures below the actual studied levels, in the territory of the Ancient Aquinum (Frosinone, Italy) a scientific collaboration, inside the "Ager Aquinas Project" between the University of Salento (Department of Cultural Heritage - Laboratory of Ancient Topography and Photogrammetry) and the Institute of Technologies Applied to Cultural Heritage (ITABC-C.N.R.) has been developed, during 2008-2009 and it is still in progress. The site which is the subject of this paper had been identified in the past through air photo interpretation of vertical historical coverage and field - walking surveys. Ancient Aquinum is characterised by two main aspects: the first depends by the presence of a very big defence-system with mighty walls and large ditch; the second characteristic is the presence or regular but not orthogonal road - system of the town, bordered by an unusual parallelogram shape of the blocks. With the results obtained after the elaborations of the first aerial data sets and field surveys, has been possible to map the main town - planning, drawing the main road system inside and outside the town. Although the analysis of the air photo evidence allowed the global interpretation of the site, it was not possible to reconstruct the archaeological evidences in the central portion of the town. Therefore the Project, during 2008, started with new acquisition and elaboration of aerial photos, field-walking surveys and GPR surveys with the aim to better define the urban plan of the central portion of the ancient town. The location, depth, and size of the buried buildings were effectively estimated from non-destructive remote sensing with a gradiometric and ground-penetrating radar systems. Recent archaeological excavations made (by Prof. Giuseppe Ceraudo - University of Salento, Lecce) during the summer 2009, have confirmed the structures individuated with the geophysical methods

  18. Physicochemical and mineralogical characterization of uranium-contaminated soils from the Fernald Integrated Demonstration Site

    SciTech Connect

    Elless, M.P.; Lee, S.Y.; Timpson, M.E.

    1994-09-01

    An integrated approach that utilizes various characterization technologies has been developed for the Uranium Soil Integrated Demonstration program. The Fernald Environmental Restoration Management Corporation site near Cincinnati, Ohio, was selected as the host facility for this demonstration. Characterization of background, untreated contaminated, and treated contaminated soils was performed to assess the contamination and the effect of treatment efforts to remove uranium from these soils. Carbonate minerals were present in the contaminated soils (added for erosion control) but were absent in the nearby background soils. Because of the importance of the carbonate anion to uranium solubility, the occurrence of carbonate minerals in these soils will be an important factor in the development of a successful remediation technology. Uranium partitioning data among several particle-size fractions indicate that conventional soil washing will be ineffective for remediation of these soils and that chemical extraction will be necessary to lower the uranium concentration to the target level (52 mg/kg). Carbonate-based (sodium carbonate/bicarbonate) and acid-based (sulfuric and citric acids) lixiviants were employed for the selective removal of uranium from these soils. Characterization results have identified uranium phosphate minerals as the predominant uranium mineral form in both the untreated and treated soils. The low solubility associated with phosphate minerals is primarily responsible for their occurrence in the posttreated soils. Artificial weathering of the treated soils caused by the treatments, particularly acid-based lixiviants, was documented by their detrimental effects on several physicochemical characteristics of these soils (e.g., soil pH, particle-size distribution, and mineralogy).

  19. 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.

  20. Analysis of chromosome 21 yeast artificial chromosome (YAC) clones.

    PubMed Central

    Tassone, F; Cheng, S; Gardiner, K

    1992-01-01

    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. Images Figure 2 Figure 1 PMID:1463009

  1. 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)

  2. 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.

  3. 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

  4. Significance of the quantitative measurement of the chr16: 51320015 integration site in hepatocytes of patients with chronic hepatitis B.

    PubMed

    Ruan, Peng; Dai, Xiufang; Sun, Zequn; Zhou, Chunfang; Yang, Fan

    2015-11-01

    The present study reported the presence of a hepatitis B virus (HBV) major integration site (MIS) chr16: 51320015 and discussed the significance of quantitative measurement of this site. A total of 30 hepatitis B e antigen (HBeAg) positive (+) and 30 HBeAg negative (‑) patients with chronic hepatitis B (CHB) were enrolled in the present study, and the levels of intrahepatic (IH) covalently closed circular DNA (cccDNA), serum HBV DNA and hepatitis B surface antigen (HBsAg) were detected. Conventional reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and Sanger sequencing were designed to verify the chr16: 51320015 integration site, and the copy numbers of this site were measured using molecular clone and SYBR Green I RT‑qPCR. This site was found to be present in the hepatocytes of all the enrolled patients, and the average number of copies was 1.46x10‑2 ± 4.94x10‑2 copies/cell (3.48x10‑5‑0.212 copies/cell). No significant difference in the copy numbers of this site were observed between the HBeAg (+) (1.43 ± 9.79x10‑1 copies/cell) and HBeAg (‑) patients (6.58x10‑2 ± 2.47x10‑2 copies/cell; P>0.05), which were positively correlated with the levels of serum HBsAg (P=0.0038), but were not correlated with the levels of IH cccDNA (P=0.7785). In conclusion, the chr16:51320015 integration site may be a novel site, which persists in a several patients with HBV infection, and may accumulate in the hepatocytes due to clonal expansion. The diagnostic and therapeutic values of this site require further investigation.

  5. From Chaos to Content: An Integrated Approach to Government Web Sites

    SciTech Connect

    Demuth, Nora H.; Knudson, Christa K.

    2005-01-03

    The web development team of the Environmental Technology Directorate (ETD) at the U.S. Department of Energy’s Pacific Northwest National Laboratory (PNNL) redesigned the ETD website as a database-driven system, powered by the newly designed ETD Common Information System (ETD-CIS). The ETD website was redesigned in response to an analysis that showed the previous ETD websites were inefficient, costly, and lacking in a consistent focus. Redesigned and newly created websites based on a new ETD template provide a consistent image, meet or exceed accessibility standards, and are linked through a common database. The protocols used in developing the ETD website support integration of further organizational sites and facilitate internal use by staff and training on ETD website development and maintenance. Other PNNL organizations have approached the ETD web development team with an interest in applying the methods established by the ETD system. The ETD system protocol could potentially be used by other DOE laboratories to improve their website efficiency and content focus. “The tools by which we share science information must be as extraordinary as the information itself.[ ]” – DOE Science Director Raymond Orbach

  6. Dietary vitamin A regulates wingless-related MMTV integration site signaling to alter the hair cycle.

    PubMed

    Suo, Liye; Sundberg, John P; Everts, Helen B

    2015-05-01

    Alopecia areata (AA) 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 AA in the graft-induced C3H/HeJ mouse model of AA. In this study, we also found that dietary vitamin A, in a dose dependent manner, activated the hair follicle stem cells (SCs) 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 wingless-type Mouse Mammary Tumor Virus integration site family (WNT) signaling) and levels of WNT7A 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 SCs.

  7. 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

  8. The C1 area of rostral ventrolateral medulla: a central site integrating autonomic responses to hemorrhage.

    PubMed

    Reis, D J; Ruggiero, D A; Morrison, S F

    1989-12-01

    Activation of the sympathetic neurons and release of adrenomedullary catecholamines are the principal early reflex responses to hemorrhage. These are initiated by arterial baro- and chemoreceptors, from other cardiopulmonary receptors, and by intracerebral receptors responding to ischemia. A principal gateway for integrating the autonomic responses are a small collection of neurons in a region of the rostral ventrolateral medulla (RVL), containing a cluster of neurons of the C1 adrenergic cell group, the C1 area. Neurons in the C1 area of RVL project exclusively to autonomic nuclei of the spinal cord, are tonically active, and fire with a rhythm linked to the cardiac cycle. They are essential for maintaining resting discharge of sympathetic nerves and, consequently, arterial pressure (AP) and heart rate. They also are critical for reflex changes in AP in the baro- and chemoreceptor, somato-sympathetic (pain), and cerebral ischemic reflexes. Neurons of the C1 area are under tonic excitatory and inhibitory control by pathways from other autonomic centers. They are controlled by a range of neurotransmitters including, gamma aminobutyric acid (GABA), acetylcholine, catecholamines, enkephalin, and several neuropeptides. They also serve as a site of action for the hypotensive actions of several clinically important neurotransmitters. The C1-area of RVL may play a critical role in the autonomic responses to hemorrhage and may be an important target for drugs seeking to treat hemorrhagic shock.

  9. 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

  10. 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...

  11. 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.

  12. Lampbrush chromosomes enable study of cohesin dynamics.

    PubMed

    Austin, Christopher; Novikova, Natalya; Guacci, Vincent; Bellini, Michel

    2009-01-01

    The lampbrush chromosomes present in the nuclei of amphibian oocytes offer unique biological approaches for study of the mechanisms that regulate chromatin structure with high spatial resolution. We discuss fundamental aspects of the remarkable organization and plasticity exhibited by lampbrush chromosomes. We then utilize lampbrush chromosomes to characterize the chromosomal distribution and dynamics of cohesin, the four-protein complex (RAD21/MCD1/SCC1, SMC1, SMC3, SCC3/SA2) responsible for sister chromatid cohesion. We find that endogenous SMC3 and newly expressed hRAD21 co-localize on chromosomal axes, sites where sister chromatids are tightly paired. We present evidence suggesting that hRAD21 recruitment to lampbrush chromosomes is modulated by chromosomal SMC1 and SMC3. Notably, using a technique for de novo chromosome assembly, we demonstrate that both SMC3 and hRAD21 are recruited to single, unreplicated lampbrush chromatids. Finally, we used our novel method of analyzing the oocyte nucleus under oil combined with fluorescence recovery after photobleaching, to provide direct evidence that cohesin is highly dynamic at discrete, condensed chromosomal regions. Collectively, these data demonstrate that lampbrush chromosomes provide a unique and powerful tool for combining biochemical and cytological analyses for dissection of complex chromosomal processes.

  13. Improved production of genetically modified fetuses with homogeneous transgene expression after transgene integration site analysis and recloning in cattle.

    PubMed

    Bressan, Fabiana Fernandes; Dos Santos Miranda, Moyses; Perecin, Felipe; De Bem, Tiago Henrique; Pereira, Flavia Thomaz Verechia; Russo-Carbolante, Elisa Maria; Alves, Daiani; Strauss, Bryan; Bajgelman, Marcio; Krieger, José Eduardo; Binelli, Mario; Meirelles, Flavio Vieira

    2011-02-01

    Animal cloning by nuclear transfer (NT) has made the production of transgenic animals using genetically modified donor cells possible and ensures the presence of the gene construct in the offspring. The identification of transgene insertion sites in donor cells before cloning may avoid the production of animals that carry undesirable characteristics due to positional effects. This article compares blastocyst development and competence to establish pregnancies of bovine cloned embryos reconstructed with lentivirus-mediated transgenic fibroblasts containing either random integration of a transgene (random integration group) or nuclear transfer derived transgenic fibroblasts with known transgene insertion sites submitted to recloning (recloned group). In the random integration group, eGFP-expressing bovine fetal fibroblasts were selected by fluorescence activated cell sorting (FACS) and used as nuclei donor cells for NT. In the recloned group, a fibroblast cell line derived from a transgenic cloned fetus was characterized regarding transgene insertion and submitted to recloning. The recloned group had higher blastocyst production (25.38 vs. 14.42%) and higher percentage of 30-day pregnancies (14.29 vs. 2.56%) when compared to the random integration group. Relative eGFP expression analysis in fibroblasts derived from each cloned embryo revealed more homogeneous expression in the recloned group. In conclusion, the use of cell lines recovered from transgenic fetuses after identification of the transgene integration site allowed for the production of cells and fetuses with stable transgene expression, and recloning may improve transgenic animal yields.

  14. 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

  15. The map of chromosome 20.

    PubMed Central

    Simpson, N E

    1988-01-01

    The number of gene assignments to human chromosome 20 has increased slowly until recently. Only seven genes and one fragile site were confirmed assignments to chromosome 20 at the Ninth Human Gene Mapping Workshop in September 1987 (HGM9). One fragile site, 13 additional genes, and 10 DNA sequences that identify restriction fragment length polymorphisms (RFLPs), however, were provisionally added to the map at HGM9. Five mutated genes on chromosome 20 have a relation to disease: a mutation in the adenosine deaminase gene results in a deficiency of the enzyme and severe combined immune deficiency; mutations in the gene for the growth hormone releasing factor result in some forms of dwarfism; mutations in the closely linked genes for the hormones arginine vasopressin and oxytocin and their neurophysins are probably responsible for some diabetes insipidus; and mutations in the gene that regulates both alpha-neuraminidase and beta-galactosidase activities determine galactosialidosis. The gene for the prion protein is on chromosome 20; it is related to the infectious agent of kuru, Creutzfeld-Jacob disease, and Gertsmann-Straussler syndrome, although the nature of the relationship is not completely understood. Two genes that code for tyrosine kinases are on the chromosome, SRC1 the proto-oncogene and a gene (HCK) coding for haemopoietic kinase (an src-like kinase), but no direct relation to cancer has been shown for either of these kinases. The significance of non-random loss of chromosome 20 in the malignant diseases non-lymphocytic leukaemia and polycythaemia vera is not understood. Twenty-four additional loci are assigned to the chromosome: five genes that code for binding proteins, one for a light chain of ferritin, genes for three enzymes (inosine triphosphatase, s-adenosylhomocysteine hydrolase, and sterol delta 24-reductase), one for each of a secretory protein and an opiate neuropeptide, a cell surface antigen, two fragile sites, and 10 DNA sequences (one

  16. Use of an integrated flow model to estimate ecologically relevant hydrologic characteristics at stream biomonitoring sites

    USGS Publications Warehouse

    Kennen, J.G.; Kauffman, L.J.; Ayers, M.A.; Wolock, D.M.; Colarullo, S.J.

    2008-01-01

    We developed an integrated hydroecological model to provide a comprehensive set of hydrologic variables representing five major components of the flow regime at 856 aquatic-invertebrate monitoring sites in New Jersey. The hydroecological model simulates streamflow by routing water that moves overland and through the subsurface from atmospheric delivery to the watershed outlet. Snow accumulation and melt, evapotranspiration, precipitation, withdrawals, discharges, pervious- and impervious-area runoff, and lake storage were accounted for in the water balance. We generated more than 78 flow variables, which describe the frequency, magnitude, duration, rate of change, and timing of flow events. Highly correlated variables were filtered by principal component analysis to obtain a non-redundant subset of variables that explain the majority of the variation in the complete set. This subset of variables was used to evaluate the effect of changes in the flow regime on aquatic-invertebrate assemblage structure at 856 biomonitoring sites. We used non-metric multidimensional scaling (NMS) to evaluate variation in aquatic-invertebrate assemblage structure across a disturbance gradient. We employed multiple linear regression (MLR) analysis to build a series of MLR models that identify the most important environmental and hydrologic variables driving the differences in the aquatic-invertebrate assemblages across the disturbance gradient. The first axis of NMS ordination was significantly related to many hydrologic, habitat, and land-use/land-cover variables, including the average number of annual storms producing runoff, ratio of 25-75% exceedance flow (flashiness), diversity of natural stream substrate, and the percentage of forested land near the stream channel (forest buffer). Modifications in the hydrologic regime as the result of changes in watershed land use appear to promote the retention of highly tolerant aquatic species; in contrast, species that are sensitive to

  17. 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

  18. Male Mouse Recombination Maps for Each Autosome Identified by Chromosome Painting

    PubMed Central

    Froenicke, Lutz; Anderson, Lorinda K.; Wienberg, Johannes; Ashley, Terry

    2002-01-01

    Linkage maps constructed from genetic analysis of gene order and crossover frequency provide few clues to the basis of genomewide distribution of meiotic recombination, such as chromosome structure, that influences meiotic recombination. To bridge this gap, we have generated the first cytological recombination map that identifies individual autosomes in the male mouse. We prepared meiotic chromosome (synaptonemal complex [SC]) spreads from 110 mouse spermatocytes, identified each autosome by multicolor fluorescence in situ hybridization of chromosome-specific DNA libraries, and mapped >2,000 sites of recombination along individual autosomes, using immunolocalization of MLH1, a mismatch repair protein that marks crossover sites. We show that SC length is strongly correlated with crossover frequency and distribution. Although the length of most SCs corresponds to that predicted from their mitotic chromosome length rank, several SCs are longer or shorter than expected, with corresponding increases and decreases in MLH1 frequency. Although all bivalents share certain general recombination features, such as few crossovers near the centromeres and a high rate of distal recombination, individual bivalents have unique patterns of crossover distribution along their length. In addition to SC length, other, as-yet-unidentified, factors influence crossover distribution leading to hot regions on individual chromosomes, with recombination frequencies as much as six times higher than average, as well as cold spots with no recombination. By reprobing the SC spreads with genetically mapped BACs, we demonstrate a robust strategy for integrating genetic linkage and physical contig maps with mitotic and meiotic chromosome structure. PMID:12432495

  19. 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

  20. 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

  1. Discovery of supernumerary B chromosomes in Drosophila melanogaster.

    PubMed

    Bauerly, Elisabeth; Hughes, Stacie E; Vietti, Dana R; Miller, Danny E; McDowell, William; Hawley, R Scott

    2014-04-01

    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 4(th) 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 4(th) chromosome heterochromatin, the B chromosomes lack most, if not all, 4(th) 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.

  2. Genome architecture: domain organization of interphase chromosomes.

    PubMed

    Bickmore, Wendy A; van Steensel, Bas

    2013-03-14

    The architecture of interphase chromosomes is important for the regulation of gene expression and genome maintenance. Chromosomes are linearly segmented into hundreds of domains with different protein compositions. Furthermore, the spatial organization of chromosomes is nonrandom and is characterized by many local and long-range contacts among genes and other sequence elements. A variety of genome-wide mapping techniques have made it possible to chart these properties at high resolution. Combined with microscopy and computational modeling, the results begin to yield a more coherent picture that integrates linear and three-dimensional (3D) views of chromosome organization in relation to gene regulation and other nuclear functions.

  3. 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.

  4. 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.

  5. Simulating dissolved organic carbon dynamics at the swedish integrated monitoring sites with the integrated catchments model for carbon, INCA-C.

    PubMed

    Futter, M N; Löfgren, S; Köhler, S J; Lundin, L; Moldan, F; Bringmark, L

    2011-12-01

    Surface water concentrations of dissolved organic carbon ([DOC]) are changing throughout the northern hemisphere due to changes in climate, land use and acid deposition. However, the relative importance of these drivers is unclear. Here, we use the Integrated Catchments model for Carbon (INCA-C) to simulate long-term (1996-2008) streamwater [DOC] at the four Swedish integrated monitoring (IM) sites. These are unmanaged headwater catchments with old-growth forests and no major changes in land use. Daily, seasonal and long-term variations in streamwater [DOC] driven by runoff, seasonal temperature and atmospheric sulfate (SO₄(2-)) deposition were observed at all sites. Using INCA-C, it was possible to reproduce observed patterns of variability in streamwater [DOC] at the four IM sites. Runoff was found to be the main short-term control on [DOC]. Seasonal patterns in [DOC] were controlled primarily by soil temperature. Measured SO₄(2-) deposition explained some of the long-term [DOC] variability at all sites.

  6. 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.

  7. 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

  8. 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.

  9. An integration strategy for the NEPA and RCRA/CERCLA programs at the Savannah River Site

    SciTech Connect

    Shedrow, C.B.; Gaughan, B.W.; Moore-Shedrow, D.B.

    1993-10-01

    Savannah River Site (SRS) environmental remediation activities are conducted according to applicable environmental laws and regulations, including the Resource Conservation and Recovery Act (RCRA), the Comprehensive Environmental Response, Compensation and Liability Act (CERCLA) and the National Environmental Policy Act (NEPA). Waste unit cleanups are accomplished by evaluating RCRA and CERCLA requirements at the sites, then selecting and implementing the appropriate cleanup measures. All State and Federal regulations, including the NEPA, are considered for applicability to each waste site. This strategy is discussed.

  10. Genetics Home Reference: Y chromosome infertility

    MedlinePlus

    ... deletions" of the human Y chromosome and their relationship with male infertility. J Genet Genomics. 2008 Apr; ... healthcare professional . About Genetics Home Reference Site Map Customer Support Selection Criteria for Links USA.gov Copyright ...

  11. Mechanisms of Chromosome Congression during Mitosis.

    PubMed

    Maiato, Helder; Gomes, Ana Margarida; Sousa, Filipe; Barisic, Marin

    2017-02-17

    means of tubulin post-translational modifications. This so-called "tubulin code" might work as a navigation system that selectively guides kinetochore motors with opposite polarities along specific spindle microtubule populations, ultimately leading to the congression of peripheral chromosomes. We propose an integrated model of chromosome congression in mammalian cells that depends essentially on the following parameters: (1) chromosome position relative to the spindle poles after nuclear envelope breakdown; (2) establishment of stable end-on kinetochore-microtubule attachments and bi-orientation; (3) coordination between kinetochore- and arm-associated motors; and (4) spatial signatures associated with post-translational modifications of specific spindle microtubule populations. The physiological consequences of abnormal chromosome congression, as well as the therapeutic potential of inhibiting chromosome congression are also discussed.

  12. Mechanisms of Chromosome Congression during Mitosis

    PubMed Central

    Maiato, Helder; Gomes, Ana Margarida; Sousa, Filipe; Barisic, Marin

    2017-01-01

    diversity by means of tubulin post-translational modifications. This so-called “tubulin code” might work as a navigation system that selectively guides kinetochore motors with opposite polarities along specific spindle microtubule populations, ultimately leading to the congression of peripheral chromosomes. We propose an integrated model of chromosome congression in mammalian cells that depends essentially on the following parameters: (1) chromosome position relative to the spindle poles after nuclear envelope breakdown; (2) establishment of stable end-on kinetochore-microtubule attachments and bi-orientation; (3) coordination between kinetochore- and arm-associated motors; and (4) spatial signatures associated with post-translational modifications of specific spindle microtubule populations. The physiological consequences of abnormal chromosome congression, as well as the therapeutic potential of inhibiting chromosome congression are also discussed. PMID:28218637

  13. 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.

  14. 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...

  15. Adeno-Associated Virus Rep Represses the Human Integration Site Promoter by Two Pathways That Are Similar to Those Required for the Regulation of the Viral p5 Promoter

    PubMed Central

    Dutheil, Nathalie; Smith, Sarah C.; Agúndez, Leticia; Vincent-Mistiaen, Zoé I.; Escalante, Carlos R.; Linden, R. Michael

    2014-01-01

    ABSTRACT Adeno-associated virus serotype 2 (AAV2) can efficiently replicate in cells that have been infected with helper viruses, such as adenovirus or herpesvirus. However, in the absence of helper virus infection, AAV2 establishes latency by integrating its genome site specifically into PPP1R12C, a gene located on chromosome 19. This integration target site falls into one of the most gene-dense regions of the human genome, thus inviting the question as to whether the virus has evolved mechanisms to control this complex transcriptional environment in order to facilitate integration, maintain an apparently innocuous latency, and/or establish conditions that are conducive to the rescue of the integrated viral genome. The viral replication (Rep) proteins control and direct every known aspect of the viral life cycle and have been shown to tightly control all AAV2 promoters. In addition, a number of heterologous promoters are repressed by the AAV2 Rep proteins. Here, we demonstrate that Rep proteins efficiently repress expression from the target site PPP1R12C promoter. We find evidence that this repression employs mechanisms similar to those described for Rep-mediated AAV2 p5 promoter regulation. Furthermore, we show that the repression of the cellular target site promoter is based on two distinct mechanisms, one relying on the presence of a functional Rep binding motif within the 5′ untranslated region (UTR) of PPP1R12C, whereas the second pathway requires only an intact nucleoside triphosphate (NTP) binding site within the Rep proteins, indicating the possible reliance of this pathway on interactions of the Rep proteins with cellular proteins that mediate or regulate cellular transcription. IMPORTANCE The observation that repression of transcription from the adeno-associated virus serotype 2 (AAV2) p5 and integration target site promoters is mediated by shared mechanisms highlights the possible coevolution of virus and host and could lead to the identification of

  16. 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.

  17. Characterization of a Novel Putative Xer-Dependent Integrative Mobile Element Carrying the bla(NMC-A) Carbapenemase Gene, Inserted into the Chromosome of Members of the Enterobacter cloacae Complex.

    PubMed

    Antonelli, Alberto; D'Andrea, Marco Maria; Di Pilato, Vincenzo; Viaggi, Bruno; Torricelli, Francesca; Rossolini, Gian Maria

    2015-10-01

    An Enterobacter ludwigii strain was isolated during routine screening of a Japanese patient for carriage of carbapenem-resistant Enterobacteriaceae. PCR analysis revealed the blaNMC-A carbapenemase gene. Whole-genome sequencing revealed that blaNMC-A was inserted in the chromosome and associated with a novel 29.1-kb putative Xer-dependent integrative mobile element, named EludIMEX-1. Bioinformatic analysis identified similar elements in the genomes of an Enterobacter asburiae strain and of other Enterobacter cloacae complex strains, confirming the mobile nature of this element.

  18. Characterization of a Novel Putative Xer-Dependent Integrative Mobile Element Carrying the blaNMC-A Carbapenemase Gene, Inserted into the Chromosome of Members of the Enterobacter cloacae Complex

    PubMed Central

    Antonelli, Alberto; D'Andrea, Marco Maria; Di Pilato, Vincenzo; Viaggi, Bruno; Torricelli, Francesca

    2015-01-01

    An Enterobacter ludwigii strain was isolated during routine screening of a Japanese patient for carriage of carbapenem-resistant Enterobacteriaceae. PCR analysis revealed the blaNMC-A carbapenemase gene. Whole-genome sequencing revealed that blaNMC-A was inserted in the chromosome and associated with a novel 29.1-kb putative Xer-dependent integrative mobile element, named EludIMEX-1. Bioinformatic analysis identified similar elements in the genomes of an Enterobacter asburiae strain and of other Enterobacter cloacae complex strains, confirming the mobile nature of this element. PMID:26248383

  19. 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.

  20. TTP AL921102: An integrated geophysics program for non-intrusive characterization of mixed-Waste landfill sites. Final report

    SciTech Connect

    Hasbrouck, J.C.

    1993-09-01

    This Technical Task conducted for the US Department of Energy Office of Technology Development demonstrates the effectiveness of integrating several surface geophysical techniques to nonintrusively characterize mixed-waste landfill sites. An integrated approach enables an area to be characterized faster and cheaper because repeated access is not necessary and offers data and interpretations not attainable by a single technique. Field demonstrations using the complex galvanic resistivity, spontaneous potential (SP), ground-penetrating radar (GPR), time-domain electromagnetic (TDEM), shear-wave (S-wave) seismic and compressional-wave (P-wave) seismic geophysical techniques were conducted at the Mixed-Waste Landfill Integrated Demonstration (MWLID) test site at the Sandia National Laboratories/New Mexico in Albuquerque. Data were acquired in two areas that have both known and unknown attributes. Although data from numerous profiles were analyzed, three lines were chosen as representative of the landfill site: Line 20E that crosses both the known Chromic Acid and Organics Pits, Line 60E that transectes an essentially barren area, and Line 125E located in an area with unknown subsurface conditions.

  1. Digital Reconstruction of AN Archaeological Site Based on the Integration of 3d Data and Historical Sources

    NASA Astrophysics Data System (ADS)

    Guidi, G.; Russo, M.; Angheleddu, D.

    2013-02-01

    The methodology proposed in this paper in based on an integrated approach for creating a 3D digital reconstruction of an archaeological site, using extensively the 3D documentation of the site in its current state, followed by an iterative interaction between archaeologists and digital modelers, leading to a progressive refinement of the reconstructive hypotheses. The starting point of the method is the reality-based model, which, together with ancient drawings and documents, is used for generating the first reconstructive step. Such rough approximation of a possible architectural structure can be annotated through archaeological considerations that has to be confronted with geometrical constraints, producing a reduction of the reconstructive hypotheses to a limited set, each one to be archaeologically evaluated. This refinement loop on the reconstructive choices is iterated until the result become convincing by both points of view, integrating in the best way all the available sources. The proposed method has been verified on the ruins of five temples in the My Son site, a wide archaeological area located in central Vietnam. The integration of 3D surveyed data and historical documentation has allowed to support a digital reconstruction of not existing architectures, developing their three-dimensional digital models step by step, from rough shapes to highly sophisticate virtual prototypes.

  2. Human chromosome 8.

    PubMed Central

    Wood, S

    1988-01-01

    The role of human chromosome 8 in genetic disease together with the current status of the genetic linkage map for this chromosome is reviewed. Both hereditary genetic disease attributed to mutant alleles at gene loci on chromosome 8 and neoplastic disease owing to somatic mutation, particularly chromosomal translocations, are discussed. PMID:3070042

  3. 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

  4. Application of service examinations to transuranic waste container integrity at the Hanford Site. Revision 1

    SciTech Connect

    Duncan, D.R.; Burbank, D.A. Jr.; Anderson, B.C.; Demiter, J.A.

    1993-09-01

    Transuranic waste containers in retrievable storage trenches at the Hanford Site and their storage environment are described. The containers are of various types, predominantly steel 0.21-m{sup 3} (55-gal) drums and boxes of many different sizes and materials. The storage environment is direct soil burial and aboveground storage under plastic tarps with earth on top of the tarps. Available data from several transuranic waste storage sites are summarized and degradation rates are projected for containers in storage at the Hanford Site.

  5. 8-Methoxypsoralen induced mutations are highly targeted at crosslinkable sites of photoaddition on the non-transcribed strand of a mammalian chromosomal gene.

    PubMed Central

    Sage, E; Drobetsky, E A; Moustacchi, E

    1993-01-01

    We have determined the mutational specificity of 8-methoxypsoralen photoaddition at the endogenous adenine phosphoribosyltransferase gene of Chinese hamster ovary cells hemizygous for this locus. In addition, the distribution of 8-methoxypsoralen photo-adducts was resolved in vitro at the DNA sequence level, and compared with the observed site specificity for mutation. Among 27 mutants characterized, all were single base changes at AT base pairs: 16 A:T-->T:A, six A:T-->C:G, four A:T-->G:C and one -T frameshift. All these vents were targeted to potential sites of photoaddition. The vast majority of these sites were also detectable in vitro, suggesting that 8-methoxypsoralen plus UVA-induced mutational hotspots may be damage hotspots. Furthermore 26/27 mutations occurred at crosslinkable 5'TpA sites, supporting the notion that 8-methoxypsoralen biadducts rather than monoadducts are major premutagenic lesions in mammalian cells. Since 90% of our mutation collection could have resulted from damage on the non-transcribed strand, it appears that photoadducted thymine residues on the transcribed strand of the adenine phosphoribosyltransferase gene may be preferentially repaired. We therefore suggest a model for mutagenesis, induced by psoralen biadducts, based on the preferential incision of biadducts followed by translesion synthesis past modified T bases persisting on the non-transcribed strand. Images PMID:8440233

  6. Development of Site-Specific Soil Design Basis Earthquake (DBE) Parameters for the Integrated Waste Treatment Unit (IWTU)

    SciTech Connect

    Payne, Suzette

    2008-08-01

    Horizontal and vertical PC 3 (2,500 yr) Soil Design Basis Earthquake (DBE) 5% damped spectra, corresponding time histories, and strain-compatible soil properties were developed for the Integrated Waste Treatment Unit (IWTU). The IWTU is located at the Idaho Nuclear Technology and Engineering Center (INTEC) at the Idaho National Laboratory (INL). Mean and 84th percentile horizontal DBE spectra derived from site-specific site response analyses were evaluated for the IWTU. The horizontal and vertical PC 3 (2,500 yr) Soil DBE 5% damped spectra at the 84th percentile were selected for Soil Structure Interaction (SSI) analyses at IWTU. The site response analyses were performed consistent with applicable Department of Energy (DOE) Standards, recommended guidance of the Nuclear Regulatory Commission (NRC), American Society of Civil Engineers (ASCE) Standards, and recommendations of the Blue Ribbon Panel (BRP) and Defense Nuclear Facilities Safety Board (DNFSB).

  7. Development of an Integrated Natural Barrier Database System for Site Evaluation of a Deep Geologic Repository in Korea - 13527

    SciTech Connect

    Jung, Haeryong; Lee, Eunyong; Jeong, YiYeong; Lee, Jeong-Hwan

    2013-07-01

    Korea Radioactive-waste Management Corporation (KRMC) established in 2009 has started a new project to collect information on long-term stability of deep geological environments on the Korean Peninsula. The information has been built up in the integrated natural barrier database system available on web (www.deepgeodisposal.kr). The database system also includes socially and economically important information, such as land use, mining area, natural conservation area, population density, and industrial complex, because some of this information is used as exclusionary criteria during the site selection process for a deep geological repository for safe and secure containment and isolation of spent nuclear fuel and other long-lived radioactive waste in Korea. Although the official site selection process has not been started yet in Korea, current integrated natural barrier database system and socio-economic database is believed that the database system will be effectively utilized to narrow down the number of sites where future investigation is most promising in the site selection process for a deep geological repository and to enhance public acceptance by providing readily-available relevant scientific information on deep geological environments in Korea. (authors)

  8. Distribution and integrated assessment of lead in an abandoned lead-acid battery site in Southwest China before redevelopment.

    PubMed

    Wang, Mei; Zhang, Chao; Zhang, Zhuo; Li, Fasheng; Guo, Guanlin

    2016-06-01

    Lead-acid battery sites have contributed enormous amounts of lead to the environment, significantly affecting its global biogeochemical cycle and leaving the potential risks to human health. An abandoned lead-acid battery site prepared for redevelopment was selected in order to study the distribution of lead in soils, plants, rhizosphere soils and soil solutions. In total, 197 samples from 77 boreholes were collected and analyzed. Single extractions by acetic acid (HOAc) were conducted to assess the bioavailability and speciation of lead in soils for comparison with the parts of the plants that are aboveground. Health risks for future residential development were evaluated by the integrated exposure uptake biokinetic (IEUBK) model. The results indicated that lead concentrations in 83% of the soil samples exceeded the Chinese Environmental Quality Standard for soil (350 mg/kg for Pb) and mainly occurred at depths between 0 and 1.5 m while accumulating at the surface of demolished construction waste and miscellaneous fill. Lead concentrations in soil solutions and HOAc extraction leachates were linked closely to the contents of aboveground Broussonetia papyrifera and Artemisia annua, two main types of local plants that were found at the site. The probability density of lead in blood (PbB) in excess of 10 µg/dL could overtake the 99% mark in the residential scenario. The findings provided a relatively integrated method to illustrate the onsite investigations and assessment for similar sites before remediation and future development from more comprehensive aspects.

  9. Mitotic chromosome structure

    SciTech Connect

    Heermann, Dieter W.

    2012-07-15

    Mounting evidence is compiling linking the physical organizational structure of chromosomes and the nuclear structure to biological function. At the base of the physical organizational structure of both is the concept of loop formation. This implies that physical proximity within chromosomes is provided for otherwise distal genomic regions and thus hierarchically organizing the chromosomes. Together with entropy many experimental observations can be explained with these two concepts. Among the observations that can be explained are the measured physical extent of the chromosomes, their shape, mechanical behavior, the segregation into territories (chromosomal and territories within chromosomes), the results from chromosome conformation capture experiments, as well as linking gene expression to structural organization.

  10. Two mutations in the locus control region hypersensitivity site-2 (5' HS-2) of haplotype 19 beta s chromosomes alter binding of trans-acting factors.

    PubMed

    Morgan, J C; Scott, D F; Lanclos, K D

    1996-01-01

    There are five major haplotypes associated with sickle cell anemia (SS). Individuals homozygous for haplotypes 3 (Senegal) and 31 (Saudi Arabian) have high fetal hemoglobin (HbF) levels (15 to 30% of total hemoglobin) whereas individuals homozygous for haplotypes 17 (Cameroon), 19 (Benin), and 20 (Bantu) have low HbF levels (1 to 10%). We previously identified several point mutations in the LCR 5'HS-2 that were specific for haplotype 19 beta s chromosomes (compared to the GenBank HUMHBB reference sequence, T-->G at position 8580, A-->G at position 8598, and A-->T at position 9114). We postulated that one or more of these mutations may alter the binding of specific trans-acting factors and ultimately affect the expression of HbF in these sickle cell patients. We performed gel mobility shift assays using 32P-end-labeled double-stranded 19mers corresponding to each of the LCR 5'HS-2 normal (GenBank) and mutant sequences. Nuclear extracts prepared from HeLa and HEL cells were used in our experiments and neither the normal nor mutant sequence at position 8580 bound trans-acting factors in either nuclear extract. The 8598 mutant increased binding of Sp1; using purified protein and both nuclear extracts. HEL extracts were used to quantify the increase in Sp1 binding to the 8598 mutation and we found an increase in binding of 66 and 47%, respectively, in two shifted bands. The 9114 mutation sharply decreased binding of an unknown trans-acting factor by 74%. This factor was present in both HeLa and HEL nuclear extracts.

  11. Green Remediation Best Management Practices: Integrating Renewable Energy into Site Cleanup

    EPA Pesticide Factsheets

    The U.S. Environmental Protection Agency (EPA) Principles for Greener Cleanups outline the Agency's policy for evaluating and minimizing the environmental 'footprint' of activities undertaken when cleaning up a contaminated site.

  12. Integration of Thirty Years of Hydrogeological Investigations at the Waste Isolation Pilot Plant Site

    NASA Astrophysics Data System (ADS)

    Beauheim, R. L.; Domski, P. S.; Holt, R. M.; Powers, D. W.

    2008-12-01

    strength CaSO4 waters, thought to represent relatively recent recharge through gypsum karst, to brines contaminated with potash-processing effluent. Geophysical logs from an abundance of oil and gas wells around the WIPP site have been used to map facies boundaries within other Rustler members that can be related to Culebra hydrology. The results of these three decades of study have been integrated into a conceptual model for Culebra hydrology. Some of these studies have been carried out in collaboration with university researchers, and all of the data from these investigations are freely available. *Sandia is a multiprogram laboratory operated by Sandia Corporation, a Lockheed Martin Company, for the United States Department of Energy's National Nuclear Security Administration under contract DE-AC04- 94AL85000. This research is funded by WIPP programs administered by the Office of Environmental Management (EM) of the U.S DOE.

  13. Functional integrity of the interrenal tissue of yellow perch from contaminated sites tested in vivo

    SciTech Connect

    Girard, C.; Brodeur, J.C.; Hontela, A.

    1995-12-31

    The normal activation of the hypothalamo-pituitary-interrenal axis (HPI axis) in response to capture is disrupted in fish subjected to life-long exposure to heavy metals, PCBs and PAHs. The ability to increase plasma cortisol in yellow perch (Perca flavescens) from sites contaminated by heavy metals and organic compounds, and from a reference site was assessed by the Capture stress test and by the ACTH Challenge test, a new standardized in vivo method designed for field studies. The effects of seasonal factors, such as temperature and gonadal maturity on these tests were investigated. Measures of liver and muscle glycogen and histopathology were made to further characterize the biochemical and structural changes that may occur along with hormonal changes. The Capture stress test showed that an acute source of stress induced a lower cortisol response in fish from the highly contaminated site compared to the reference site, revealing a functional impairment of the HPI axis. The ACTH Challenge test showed that the hormonal responsiveness of the cortisol-secreting interrenal tissue, stimulated by a standard dose of ACTH injected i.p., was lower in fish from the highly contaminated site than the reference site. Spring is the season during which the impairment was the most evident. The possibility of using the reduced capacity of feral fish to respond to a standardized ACTH Challenge as an early bioindicator of toxic stress is discussed.

  14. The gravity of pollination: integrating at-site features into spatial analysis of contemporary pollen movement.

    PubMed

    DiLeo, Michelle F; Siu, Jenna C; Rhodes, Matthew K; López-Villalobos, Adriana; Redwine, Angela; Ksiazek, Kelly; Dyer, Rodney J

    2014-08-01

    Pollen-mediated gene flow is a major driver of spatial genetic structure in plant populations. Both individual plant characteristics and site-specific features of the landscape can modify the perceived attractiveness of plants to their pollinators and thus play an important role in shaping spatial genetic variation. Most studies of landscape-level genetic connectivity in plants have focused on the effects of interindividual distance using spatial and increasingly ecological separation, yet have not incorporated individual plant characteristics or other at-site ecological variables. Using spatially explicit simulations, we first tested the extent to which the inclusion of at-site variables influencing local pollination success improved the statistical characterization of genetic connectivity based upon examination of pollen pool genetic structure. The addition of at-site characteristics provided better models than those that only considered interindividual spatial distance (e.g. IBD). Models parameterized using conditional genetic covariance (e.g. population graphs) also outperformed those assuming panmixia. In a natural population of Cornus florida L. (Cornaceae), we showed that the addition of at-site characteristics (clumping of primary canopy opening above each maternal tree and maternal tree floral output) provided significantly better models describing gene flow than models including only between-site spatial (IBD) and ecological (isolation by resistance) variables. Overall, our results show that including interindividual and local ecological variation greatly aids in characterizing landscape-level measures of contemporary gene flow.

  15. 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

    ... must operators of collection sites take to protect the security and integrity of urine collections? (a) Collectors and operators of collection sites must take the steps listed in this section to prevent..., cleaning agents, or other possible adulterants are present; (4) Inspect the site to ensure that no...

  16. 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

    ... must operators of collection sites take to protect the security and integrity of urine collections? (a) Collectors and operators of collection sites must take the steps listed in this section to prevent..., cleaning agents, or other possible adulterants are present; (4) Inspect the site to ensure that no...

  17. 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.

  18. Identification of Multiresistance Gene cfr in Methicillin-Resistant Staphylococcus aureus from Pigs: Plasmid Location and Integration into a Staphylococcal Cassette Chromosome mec Complex

    PubMed Central

    Li, Dexi; Wu, Congming; Wang, Yang; Fan, Run

    2015-01-01

    The multiresistance gene cfr was found in 8/231 porcine methicillin-resistant Staphylococcus aureus isolates. They were characterized by multilocus sequence typing, spa typing, dru typing, and staphylococcal cassette chromosome mec (SCCmec) typing as ST627-t002-dt12w-IVb, ST6-t304-dt12w-IVb, ST9-t899-dt12w-IVb, ST9-t899-dt12ae-IVb, or ST63-t899-dt12v-IVb. Different cfr gene regions were detected on plasmids of ca. 35 kb in seven isolates. For the first time, an ISEnfa4-cfr-IS256 fragment was found to be inserted upstream of the ccr genes in a chromosomal SCCmec IVb element of the remaining isolate. PMID:25824234

  19. Mechanisms for Complex Chromosomal Insertions

    PubMed Central

    Szafranski, Przemyslaw; Akdemir, Zeynep Coban; Yuan, Bo; Cooper, Mitchell L.; Magriñá, Maria A.; Bacino, Carlos A.; Lalani, Seema R.; Patel, Ankita; Song, Rodger H.; Bi, Weimin; Cheung, Sau Wai; Carvalho, Claudia M. B.; Lupski, James R.

    2016-01-01

    Chromosomal insertions are genomic rearrangements with a chromosome segment inserted into a non-homologous chromosome or a non-adjacent locus on the same chromosome or the other homologue, constituting ~2% of nonrecurrent copy-number gains. Little is known about the molecular mechanisms of their formation. We identified 16 individuals with complex insertions among 56,000 individuals tested at Baylor Genetics using clinical array comparative genomic hybridization (aCGH) and fluorescence in situ hybridization (FISH). Custom high-density aCGH was performed on 10 individuals with available DNA, and breakpoint junctions were fine-mapped at nucleotide resolution by long-range PCR and DNA sequencing in 6 individuals to glean insights into potential mechanisms of formation. We observed microhomologies and templated insertions at the breakpoint junctions, resembling the breakpoint junction signatures found in complex genomic rearrangements generated by replication-based mechanism(s) with iterative template switches. In addition, we analyzed 5 families with apparently balanced insertion in one parent detected by FISH analysis and found that 3 parents had additional small copy-number variants (CNVs) at one or both sides of the inserting fragments as well as at the inserted sites. We propose that replicative repair can result in interchromosomal complex insertions generated through chromothripsis-like chromoanasynthesis involving two or three chromosomes, and cause a significant fraction of apparently balanced insertions harboring small flanking CNVs. PMID:27880765

  20. An integration of aeromagnetic and electrical resistivity methods in dam site investigation

    SciTech Connect

    Aina, A.; Olorunfemi, M.O.; Ojo, J.S.

    1996-03-01

    Aeromagnetic map and electrical resistivity sounding data obtained along eight traverses were examined at two sites across the Katsina-Ala River. The principal goals of this exercise were to define depths to the bedrock, bedrock relief, geologic structures, define the nature of the superficial deposit, and select probable minor and major axes for hydroelectric power dams. The aeromagnetic map shows that the basement rocks trend roughly northeast-southwest, which correlates with the strike of foliation measurements made on rock outcrops along the river channel. A network of cross cutting lineaments, suspected to be faults/fractures that trend approximately northeast/southwest and northwest/southeast, was also delineated from the magnetic map. The depths to the bedrock estimated from resistivity depth sounding data at site 1 generally vary from 1--53.1 m. Depths to the bedrock estimated at site 2 range from 1.9--19.5 m. The superficial deposit varies from clay to sandy clay, to clayey sand (with boulders in places), and to sand and laterite. The bedrock relief is relatively flat and gently undulates along most of the traverses, with an overall dip towards the river channel. Traverses E-F or I-J at site 1 and K-L at site 2 are probable dame axes. These traverses are characterized by relatively thin overburden thicknesses and rock heads dipping toward the river channel, thereby reducing the likelihood of water seepages from the flanks of the proposed dam axes.

  1. Identifying 2'-O-methylationation sites by integrating nucleotide chemical properties and nucleotide compositions.

    PubMed

    Chen, Wei; Feng, Pengmian; Tang, Hua; Ding, Hui; Lin, Hao

    2016-06-01

    2'-O-methylationation is an important post-transcriptional modification and plays important roles in many biological processes. Although experimental technologies have been proposed to detect 2'-O-methylationation sites, they are cost-ineffective. As complements to experimental techniques, computational methods will facilitate the identification of 2'-O-methylationation sites. In the present study, we proposed a support vector machine-based method to identify 2'-O-methylationation sites. In this method, RNA sequences were formulated by nucleotide chemical properties and nucleotide compositions. In the jackknife cross-validation test, the proposed method obtained an accuracy of 95.58% for identifying 2'-O-methylationation sites in the human genome. Moreover, the model was also validated by identifying 2'-O-methylation sites in the Mus musculus and Saccharomyces cerevisiae genomes, and the obtained accuracies are also satisfactory. These results indicate that the proposed method will become a useful tool for the research on 2'-O-methylation.

  2. Meiotic chromosome abnormalities in human spermatogenesis.

    PubMed

    Martin, Renée H

    2006-08-01

    The last few years have witnessed an explosion in the information about chromosome abnormalities in human sperm and the meiotic events that predispose to these abnormalities. We have determined that all chromosomes are susceptible to nondisjunction, but chromosomes 21 and 22 and, especially, the sex chromosomes have an increased frequency of aneuploidy. Studies are just beginning on the effects of potential mutagens on the chromosomal constitution of human sperm. The effects of pesticides and cancer therapeutic agents have been reviewed. In the last decade, there has been a great impetus to study chromosome abnormalities in sperm from infertile men because the advent of intracytoplasmic sperm injection (ICSI) made it possible for these men to father pregnancies. A large number of studies have demonstrated that infertile men have an increased frequency of chromosomally abnormal sperm and children, even when they have a normal somatic karyotype. Meiotic studies on the pachytene stage of spermatogenesis have demonstrated that infertile men have impaired chromosome synapsis, a significantly decreased frequency of recombination, and an increased frequency of chromosomes completely lacking a recombination site. Such errors make these cells susceptible to meiotic arrest and the production of aneuploid gametes.

  3. Integrating knowledge-based multi-criteria evaluation techniques with GIS for landfill site selection: A case study using AHP

    NASA Astrophysics Data System (ADS)

    Fagbohun, B. J.; Aladejana, O. O.

    2016-09-01

    A major challenge in most growing urban areas of developing countries, without a pre-existing land use plan is the sustainable and efficient management of solid wastes. Siting a landfill is a complicated task because of several environmental regulations. This challenge gives birth to the need to develop efficient strategies for the selection of proper waste disposal sites in accordance with all existing environmental regulations. This paper presents a knowledge-based multi-criteria decision analysis using GIS for the selection of suitable landfill site in Ado-Ekiti, Nigeria. In order to identify suitable sites for landfill, seven factors - land use/cover, geology, river, soil, slope, lineament and roads - were taken into consideration. Each factor was classified and ranked based on prior knowledge about the area and existing guidelines. Weights for each factor were determined through pair-wise comparison using Saaty's 9 point scale and AHP. The integration of factors according to their weights using weighted index overlay analysis revealed that 39.23 km2 within the area was suitable to site a landfill. The resulting suitable area was classified as high suitability covering 6.47 km2 (16.49%), moderate suitability 25.48 km2 (64.95%) and low suitability 7.28 km2 (18.56%) based on their overall weights.

  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. B Chromosomes - A Matter of Chromosome Drive.

    PubMed

    Houben, Andreas

    2017-01-01

    B chromosomes are supernumerary chromosomes which are often preferentially inherited, deviating from usual Mendelian segregation. The balance between the so-called chromosome drive and the negative effects that the presence of Bs applies on the fitness of their host determines the frequency of Bs in a particular population. Drive is the key for understanding most B chromosomes. Drive occurs in many ways at pre-meiotic, meiotic or post-meiotic divisions, but the molecular mechanism remains unclear. The cellular mechanism of drive is reviewed based on the findings obtained for the B chromosomes of rye, maize and other species. How novel analytical tools will expand our ability to uncover the biology of B chromosome drive is discussed.

  6. Integration of site-specific health information: Agency for Toxic Substances and Disease Registry health assessments

    SciTech Connect

    Lesperance, A.M.; Siegel, M.R.

    1990-12-01

    The Agency for Toxic Substances and Disease Registry is required to conduct a health assessment of any site that is listed on or proposed for the US Environmental Protection Agency's National Priorities List. Sixteen US Department of Energy (DOE) sites currently fall into this category. Health assessments contain a qualitative description of impacts to public health and the environment from hazardous waste sites, as well as recommendations for actions to mitigate or eliminate risk. Because these recommendations may have major impacts on compliance activities at DOE facilities, the health assessments are an important source of information for the monitoring activities of DOE's Office of Environmental Compliance (OEC). This report provides an overview of the activities involved in preparing the health assessment, its role in environmental management, and its key elements.

  7. Integrative systems and synthetic biology of cell-matrix adhesion sites

    PubMed Central

    Zamir, Eli

    2016-01-01

    ABSTRACT The complexity of cell-matrix adhesion convolves its roles in the development and functioning of multicellular organisms and their evolutionary tinkering. Cell-matrix adhesion is mediated by sites along the plasma membrane that anchor the actin cytoskeleton to the matrix via a large number of proteins, collectively called the integrin adhesome. Fundamental challenges for understanding how cell-matrix adhesion sites assemble and function arise from their multi-functionality, rapid dynamics, large number of components and molecular diversity. Systems biology faces these challenges in its strive to understand how the integrin adhesome gives rise to functional adhesion sites. Synthetic biology enables engineering intracellular modules and circuits with properties of interest. In this review I discuss some of the fundamental questions in systems biology of cell-matrix adhesion and how synthetic biology can help addressing them. PMID:26853318

  8. Repair of I-SceI induced DSB at a specific site of chromosome in human cells: influence of low-dose, low-dose-rate gamma-rays.

    PubMed

    Yatagai, Fumio; Suzuki, Masao; Ishioka, Noriaki; Ohmori, Hitoshi; Honma, Masamitsu

    2008-11-01

    We investigated the influence of low-dose, low-dose-rate gamma-ray irradiation on DNA double strand break (DSB) repair in human lymphoblastoid TK6 cells. A single DSB was introduced at intron 4 of the TK+ allele (chromosome 17) by transfection with the I-SceI expression vector pCBASce. We assessed for DSB repair due to non-homologous end-joining (NHEJ) by determining the generation of TK-deficient mutants in the TK6 derivative TSCE5 (TK +/-) carrying an I-SceI recognition site. We similarly estimated DSB repair via homologous recombination (HR) at the same site in the derived compound heterozygote (TK-/-) cell line TSCER2 that carries an additional point mutation in exon 5. The NHEJ repair of DSB was barely influenced by pre-irradiation of the cells with 30 mGy gamma-rays at 1.2 mGy h(-1). DSB repair by HR, in contrast, was enhanced by approximately 50% after pre-irradiation of the cells under these conditions. Furthermore, when I-SceI digestion was followed by irradiation at a dose of 8.5 mGy, delivered at a dose rate of only 0.125 mGy h(-1), HR repair efficiency was enhanced by approximately 80%. This experimental approach can be applied to characterize DSB repair in the low-dose region of ionizing radiation.

  9. Genome-wide mapping of histone H3K9me2 in acute myeloid leukemia reveals large chromosomal domains associated with massive gene silencing and sites of genome instability

    PubMed Central

    Popova, Evgenya Y.; Keasey, Nikki; Loughran, Thomas P.; Claxton, David F.

    2017-01-01

    A facultative heterochromatin mark, histone H3 lysine 9 dimethylation (H3K9me2), which is mediated by histone methyltransferases G9a/GLP (EHMT2/1), undergoes dramatic rearrangements during myeloid cell differentiation as observed by chromatin imaging. To determine whether these structural transitions also involve genomic repositioning of H3K9me2, we used ChIP-sequencing to map genome-wide topography of H3K9me2 in normal human granulocytes, normal CD34+ hematopoietic progenitors, primary myeloblasts from acute myeloid leukemia (AML) patients, and a model leukemia cell line K562. We observe that H3K9me2 naturally repositions from the previously designated “repressed” chromatin state in hematopoietic progenitors to predominant association with heterochromatin regions in granulocytes. In contrast, AML cells accumulate H3K9me2 on previously undefined large (> 100 Kb) genomic blocks that are enriched with AML-specific single nucleotide variants, sites of chromosomal translocations, and genes downregulated in AML. Specifically, the AML-specific H3K9me2 blocks are enriched with genes regulated by the proto-oncogene ERG that promotes stem cell characteristics. The AML-enriched H3K9me2 blocks (in contrast to the heterochromatin-associated H3K9me2 blocks enriched in granulocytes) are reduced by pharmacological inhibition of the histone methyltransferase G9a/GLP in K562 cells concomitantly with transcriptional activation of ERG and ETS1 oncogenes. Our data suggest that G9a/GLP mediate formation of transient H3K9me2 blocks that are preserved in AML myeloblasts and may lead to an increased rate of AML-specific mutagenesis and chromosomal translocations. PMID:28301528

  10. Simplifying documentation while approaching site closure: integrated health & safety plans as documented safety analysis

    SciTech Connect

    Brown, Tulanda

    2003-06-01

    At the Fernald Closure Project (FCP) near Cincinnati, Ohio, environmental restoration activities are supported by Documented Safety Analyses (DSAs) that combine the required project-specific Health and Safety Plans, Safety Basis Requirements (SBRs), and Process Requirements (PRs) into single Integrated Health and Safety Plans (I-HASPs). By isolating any remediation activities that deal with Enriched Restricted Materials, the SBRs and PRs assure that the hazard categories of former nuclear facilities undergoing remediation remain less than Nuclear. These integrated DSAs employ Integrated Safety Management methodology in support of simplified restoration and remediation activities that, so far, have resulted in the decontamination and demolition (D&D) of over 150 structures, including six major nuclear production plants. This paper presents the FCP method for maintaining safety basis documentation, using the D&D I-HASP as an example.

  11. Supporting Fernald Site Closure with Integrated Health and Safety Plans as Documented Safety Analyses

    SciTech Connect

    Kohler, S.; Brown, T.; Fisk, P.; Krach, F.; Klein, B.

    2004-03-01

    At the Fernald Closure Project (FCP) near Cincinnati, Ohio, environmental restoration activities are supported by Documented Safety Analyses (DSAs) that combine the required project-specific Health and Safety Plans, Safety Basis Requirements (SBRs), and Process Requirements (PRs) into single Integrated Health and Safety Plans (I-HASPs). These integrated DSAs employ Integrated Safety Management methodology in support of simplified restoration and remediation activities that, so far, have resulted in the decontamination and demolition (D&D) of over 200 structures, including eight major nuclear production plants. There is one of twelve nuclear facilities still remaining (Silos containing uranium ore residues) with its own safety basis documentation. This paper presents the status of the FCP's safety basis documentation program, illustrating that all of the former nuclear facilities and activities have now replaced. Basis of Interim Operations (BIOs) with I-HASPs as their safety basis during the closure process.

  12. Integration of Rooftop Photovoltaic Systems in St. Paul Ford Site's Redevelopment Plans

    SciTech Connect

    Olis, D.; Mosey, G.

    2015-03-01

    The purpose of this analysis is to estimate how much electricity the redeveloped Ford Motor Company assembly plant site in St. Paul, Minnesota, might consume under different development scenarios and how much rooftop photovoltaic (PV) generation might be possible at the site. Because the current development scenarios are high-level, preliminary sketches that describe mixes of residential, retail, commercial, and industrial spaces, electricity consumption and available rooftop area for PV under each scenario can only be grossly estimated. These results are only indicative and should be used for estimating purposes only and to help inform development goals and requirements moving forward.

  13. Fuel cell on-site integrated energy system parametric analysis of a residential complex

    NASA Technical Reports Server (NTRS)

    Simons, S. N.

    1977-01-01

    The use of phosphoric acid fuel cell powerplant to provide all the electricity required by an 81-unit garden apartment complex is studied. Byproduct heat is recovered and provides some of the heat required by the complex. The onsite integrated energy system contains energy conversion equipment including combinations of compression and absorption chillers, heat pumps, electric resistance heaters, and thermal storage. The annual fuel requirement for several onsite integrated energy systems as well as the fuel cell breakeven cost for one specific system were calculated. It is found that electrical efficiency cannot be traded off against thermal efficiency without paying a penalty in system efficiency.

  14. Scientific Opportunities for Monitoring at Environmental Remediation Sites (SOMERS): Integrated Systems-Based Approaches to Monitoring

    SciTech Connect

    Bunn, Amoret L.; Wellman, Dawn M.; Deeb, Rula A.; Hawley, Elizabeth L.; Truex, Michael J.; Peterson, Mark; Freshley, Mark D.; Pierce, Eric M.; McCord, John; Young, Michael H.; Gilmore, Tyler J.; Miller, Rick; Miracle, Ann L.; Kaback, Dawn; Eddy-Dilek, Carol; Rossabi, Joe; Lee, Michelle H.; Bush, Richard P.; Beam , Paul; Chamberlain, G. M.; Marble, Justin; Whitehurst, Latrincy; Gerdes, Kurt D.; Collazo, Yvette

    2012-05-15

    Through an inter-disciplinary effort, DOE is addressing a need to advance monitoring approaches from sole reliance on cost- and labor-intensive point-source monitoring to integrated systems-based approaches such as flux-based approaches and the use of early indicator parameters. Key objectives include identifying current scientific, technical and implementation opportunities and challenges, prioritizing science and technology strategies to meet current needs within the DOE complex for the most challenging environments, and developing an integrated and risk-informed monitoring framework.

  15. 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

  16. 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

  17. Localization of chromosomal protein HMG-1 in polytene chromosomes of Chironomus thummi

    PubMed Central

    1981-01-01

    The distribution of accessible antigenic sites in the chromosomal protein high mobility group one (HMG-1) in Chironomus thummi polytene chromosomes is visualized by immunofluorescence. The results indicate that (a) HMG-1 is distributed in a distinct banding pattern along the entire length of the chromosomes; (b) the banding pattern obtained with fluorescent antibody does not strictly correspond to that observed by phase-contrast microscopy; and (c) the amount of HMG-1 increases, and the fluorescent banding pattern changes, during the development of the organism. Our findings suggest that the protein may be involved in the modulation of the structure of selected loci in the chromosome. PMID:6453126

  18. Integrated mild gasification processing at the Homer City Electric Power Generating Station site. Final report, July 1989--June 1993

    SciTech Connect

    Battista, J.J.; Zawadzki, E.A.

    1993-07-01

    A new process for the production of commercial grade coke, char, and carbon products has been evaluated by Penelec/NYSEG. The process, developed by Coal Technology Corporation, CTC, utilizes a unique screw reactor to produce a devolatilized char from a wide variety of coals for the production of commercial grade coke for use in blast furnaces, foundries, and other processes requiring high quality coke. This process is called the CTC Mild Gasification Process (MGP). The process economics are significantly enhanced by integrating the new technology into an existing power generating complex. Cost savings are realized by the coke producer, the coke user, and the electric utility company. Site specific economic studies involving the Homer City Generating Station site in Western Pennsylvania, confirmed that an integrated MGP at the Homer City site, using coal fines produced at the Homer City Coal Preparation Plant, would reduce capital and operating costs significantly and would enable the HC Owners to eliminate thermal dryers, obtain low cost fuel in the form of combustible gases and liquids, and obtain lower cost replacement coal on the spot market. A previous report, identified as the Interim Report on the Project, details the technical and economic studies.

  19. TAO1 kinase maintains chromosomal stability by facilitating proper congression of chromosomes.

    PubMed

    Shrestha, Roshan L; Tamura, Naoka; Fries, Anna; Levin, Nicolas; Clark, Joanna; Draviam, Viji M

    2014-06-01

    Chromosomal instability can arise from defects in chromosome-microtubule attachment. Using a variety of drug treatments, we show that TAO1 kinase is required for ensuring the normal congression of chromosomes. Depletion of TAO1 reduces the density of growing interphase and mitotic microtubules in human cells, showing TAO1's role in controlling microtubule dynamics. We demonstrate the aneugenic nature of chromosome-microtubule attachment defects in TAO1-depleted cells using an error-correction assay. Our model further strengthens the emerging paradigm that microtubule regulatory pathways are important for resolving erroneous kinetochore-microtubule attachments and maintaining the integrity of the genome, regardless of the spindle checkpoint status.

  20. An Integrated Decision Model for Evaluating Educational Web Sites from the Fuzzy Subjective and Objective Perspectives

    ERIC Educational Resources Information Center

    Huang, Tony Cheng-Kui; Huang, Chih-Hong

    2010-01-01

    With advances in information and network technologies, lots of data have been digitized to reveal information for users by the construction of Web sites. Unfortunately, they are both overloading and overlapping in Internet so that users cannot distinguish their quality. To address this issue in education, Hwang, Huang, and Tseng proposed a group…

  1. UAS Integration in the NAS Project Test Site Kick-off Meeting

    NASA Technical Reports Server (NTRS)

    Kopardekar, Parimal; Witzberger, Kevin; Hackenberg, Davis L.; Murphy, Jim

    2015-01-01

    This briefing was presented during the Test Site Kick Off Meeting to discuss the contract awards for Task 1 and Task 2. This briefing covered a high level overview for contract deliverables, Task 1 - UAS Traffic Management and Task 2, Live Virtual Constructive Distributed Environment.

  2. Reference site selection for wetland condition assessments: Integrating best professional judgement and objective selection criteria

    EPA Science Inventory

    The National Wetlands Condition Assessment (NWCA), one of a series of water assessments being conducted by states, tribes, the U.S. Environmental Protection Agency (EPA), and ot