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Sample records for clone library analyses

  1. Succession of microbial communities during a biostimulation process as evaluated by DGGE and clone library analyses

    International Nuclear Information System (INIS)

    Ogino, A.; Nakahara, T.

    2001-01-01

    Aims: The objective of this study was to investigate the changes in the indigenous bacterial community structure for assessing the impact of biostimulation on spilled oil. Methods and Results: Changes in the bacterial community structure were monitored by denaturing gradient gel electrophoresis (DGGE) and clone library methods based on 16S rRNA gene (rDNA) sequences. The results of DGGE, coupled with the use of the Shannon index and principal component analysis (PCA) and clone library analyses, were consistent. In the treated (fertilized) area, one operational taxonomic unit (OTU) became dominant during the fertilization period, and it was most closely related to Pseudomonas putida. Conclusions: The bacterial community structure in the treated area was markedly different from that in the control (non-fertilized) area during the fertilization period, but in the two areas it became similar at 14 weeks after the end of fertilization. Significance and Impact of the Study: The results suggest that the bacterial community structure was disrupted by the biostimulation treatment, but that it recovered immediately after the end of fertilization. (Author)

  2. Hybrid sequencing approach applied to human fecal metagenomic clone libraries revealed clones with potential biotechnological applications.

    Science.gov (United States)

    Džunková, Mária; D'Auria, Giuseppe; Pérez-Villarroya, David; Moya, Andrés

    2012-01-01

    Natural environments represent an incredible source of microbial genetic diversity. Discovery of novel biomolecules involves biotechnological methods that often require the design and implementation of biochemical assays to screen clone libraries. However, when an assay is applied to thousands of clones, one may eventually end up with very few positive clones which, in most of the cases, have to be "domesticated" for downstream characterization and application, and this makes screening both laborious and expensive. The negative clones, which are not considered by the selected assay, may also have biotechnological potential; however, unfortunately they would remain unexplored. Knowledge of the clone sequences provides important clues about potential biotechnological application of the clones in the library; however, the sequencing of clones one-by-one would be very time-consuming and expensive. In this study, we characterized the first metagenomic clone library from the feces of a healthy human volunteer, using a method based on 454 pyrosequencing coupled with a clone-by-clone Sanger end-sequencing. Instead of whole individual clone sequencing, we sequenced 358 clones in a pool. The medium-large insert (7-15 kb) cloning strategy allowed us to assemble these clones correctly, and to assign the clone ends to maintain the link between the position of a living clone in the library and the annotated contig from the 454 assembly. Finally, we found several open reading frames (ORFs) with previously described potential medical application. The proposed approach allows planning ad-hoc biochemical assays for the clones of interest, and the appropriate sub-cloning strategy for gene expression in suitable vectors/hosts.

  3. Hybrid sequencing approach applied to human fecal metagenomic clone libraries revealed clones with potential biotechnological applications.

    Directory of Open Access Journals (Sweden)

    Mária Džunková

    Full Text Available Natural environments represent an incredible source of microbial genetic diversity. Discovery of novel biomolecules involves biotechnological methods that often require the design and implementation of biochemical assays to screen clone libraries. However, when an assay is applied to thousands of clones, one may eventually end up with very few positive clones which, in most of the cases, have to be "domesticated" for downstream characterization and application, and this makes screening both laborious and expensive. The negative clones, which are not considered by the selected assay, may also have biotechnological potential; however, unfortunately they would remain unexplored. Knowledge of the clone sequences provides important clues about potential biotechnological application of the clones in the library; however, the sequencing of clones one-by-one would be very time-consuming and expensive. In this study, we characterized the first metagenomic clone library from the feces of a healthy human volunteer, using a method based on 454 pyrosequencing coupled with a clone-by-clone Sanger end-sequencing. Instead of whole individual clone sequencing, we sequenced 358 clones in a pool. The medium-large insert (7-15 kb cloning strategy allowed us to assemble these clones correctly, and to assign the clone ends to maintain the link between the position of a living clone in the library and the annotated contig from the 454 assembly. Finally, we found several open reading frames (ORFs with previously described potential medical application. The proposed approach allows planning ad-hoc biochemical assays for the clones of interest, and the appropriate sub-cloning strategy for gene expression in suitable vectors/hosts.

  4. Efficient preparation of shuffled DNA libraries through recombination (Gateway) cloning.

    Science.gov (United States)

    Lehtonen, Soili I; Taskinen, Barbara; Ojala, Elina; Kukkurainen, Sampo; Rahikainen, Rolle; Riihimäki, Tiina A; Laitinen, Olli H; Kulomaa, Markku S; Hytönen, Vesa P

    2015-01-01

    Efficient and robust subcloning is essential for the construction of high-diversity DNA libraries in the field of directed evolution. We have developed a more efficient method for the subcloning of DNA-shuffled libraries by employing recombination cloning (Gateway). The Gateway cloning procedure was performed directly after the gene reassembly reaction, without additional purification and amplification steps, thus simplifying the conventional DNA shuffling protocols. Recombination-based cloning, directly from the heterologous reassembly reaction, conserved the high quality of the library and reduced the time required for the library construction. The described method is generally compatible for the construction of DNA-shuffled gene libraries. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  5. The first report: An analysis of bacterial flora of the first voided urine specimens of patients with male urethritis using the 16S ribosomal RNA gene-based clone library method.

    Science.gov (United States)

    You, Chunlin; Hamasuna, Ryoichi; Ogawa, Midori; Fukuda, Kazumasa; Hachisuga, Toru; Matsumoto, Tetsuro; Taniguchi, Hatsumi

    2016-06-01

    To analyse the bacterial flora of urine from patients with male urethritis using the clone library method. Urine specimens from patients with urethritis were used. The bacterial flora was analysed according to the 16S ribosomal RNA gene-based clone library method. In addition, Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma urealyticum or Ureaplasma parvum were detected by the conventional PCR methods (TMA or real-time PCR) and data from the clone library and conventional PCR methods were compared. Among 58 urine specimens, 38 were successfully analysed using the clone library method. From the specimens, 2427 clones were evaluated and 95 bacterial phylotypes were detected. N. gonorrhoeae was detected from 6 specimens and as the predominant bacterial species in 5 specimens. M. genitalium was detected from 5 specimens and as the predominant bacterial species in 3 specimens. C. trachomatis was detected from 15 specimens using the TMA method, but was detected from only 1 specimen using the clone library method. U. parvum was detected from only 2 specimens using the clone library method. In addition, Haemophilus influenzae and Neisseria meningitidis were also detected in 8 and 1 specimens, respectively. Gardnerella vaginalis, which is a potential pathogen for bacterial vaginitis in women, was detected in 10 specimens. The clone library method can detect the occupancy rate of each bacteria species among the bacterial flora and may be a new method for bacterial analyses in male urethritis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Comparative evaluation of prokaryotic 16S rDNA clone libraries and SSCP in groundwater samples.

    Science.gov (United States)

    Larentis, Michael; Alfreider, Albin

    2011-06-01

    A comparison of ribosomal RNA sequence analysis methods based on clone libraries and single-strand conformational polymorphism technique (SSCP) was performed with groundwater samples obtained between 523-555 meters below surface. The coverage of analyzed clones by phylotype-richness estimates was between 88-100%, confirming that the clone libraries were adequately examined. Analysis of individual bands retrieved from SSCP gels identified 1-6 different taxonomic units per band, suggesting that a single SSCP band does often represent more than one single prokaryotic species. The prokaryotic diversity obtained by both methods showed an overall difference of 42-80%. In comparison to SSCP, clone libraries underestimated the phylogenetic diversity and only 36-66% of the phylotypes observed with SSCP were also detected with the clone libraries. An exception was a sample where the SSCP analysis of Archaea identified only half of the phylotypes retrieved by the clone library. Overall, this study suggests that the clone library and the SSCP approach do not provide an identical picture of the prokaryotic diversity in groundwater samples. The results clearly show that the SSCP method, although this approach is prone to generate methodological artifacts, was able to detect significantly more phylotypes than microbial community analysis based on clone libraries. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Strong spurious transcription likely contributes to DNA insert bias in typical metagenomic clone libraries.

    Science.gov (United States)

    Lam, Kathy N; Charles, Trevor C

    2015-01-01

    Clone libraries provide researchers with a powerful resource to study nucleic acid from diverse sources. Metagenomic clone libraries in particular have aided in studies of microbial biodiversity and function, and allowed the mining of novel enzymes. Libraries are often constructed by cloning large inserts into cosmid or fosmid vectors. Recently, there have been reports of GC bias in fosmid metagenomic libraries, and it was speculated to be a result of fragmentation and loss of AT-rich sequences during cloning. However, evidence in the literature suggests that transcriptional activity or gene product toxicity may play a role. To explore possible mechanisms responsible for sequence bias in clone libraries, we constructed a cosmid library from a human microbiome sample and sequenced DNA from different steps during library construction: crude extract DNA, size-selected DNA, and cosmid library DNA. We confirmed a GC bias in the final cosmid library, and we provide evidence that the bias is not due to fragmentation and loss of AT-rich sequences but is likely occurring after DNA is introduced into Escherichia coli. To investigate the influence of strong constitutive transcription, we searched the sequence data for promoters and found that rpoD/σ(70) promoter sequences were underrepresented in the cosmid library. Furthermore, when we examined the genomes of taxa that were differentially abundant in the cosmid library relative to the original sample, we found the bias to be more correlated with the number of rpoD/σ(70) consensus sequences in the genome than with simple GC content. The GC bias of metagenomic libraries does not appear to be due to DNA fragmentation. Rather, analysis of promoter sequences provides support for the hypothesis that strong constitutive transcription from sequences recognized as rpoD/σ(70) consensus-like in E. coli may lead to instability, causing loss of the plasmid or loss of the insert DNA that gives rise to the transcription. Despite

  8. Systematic cloning of human minisatellites from ordered array charomid libraries.

    Science.gov (United States)

    Armour, J A; Povey, S; Jeremiah, S; Jeffreys, A J

    1990-11-01

    We present a rapid and efficient method for the isolation of minisatellite loci from human DNA. The method combines cloning a size-selected fraction of human MboI DNA fragments in a charomid vector with hybridization screening of the library in ordered array. Size-selection of large MboI fragments enriches for the longer, more variable minisatellites and reduces the size of the library required. The library was screened with a series of multi-locus probes known to detect a large number of hypervariable loci in human DNA. The gridded library allowed both the rapid processing of positive clones and the comparative evaluation of the different multi-locus probes used, in terms of both the relative success in detecting hypervariable loci and the degree of overlap between the sets of loci detected. We report 23 new human minisatellite loci isolated by this method, which map to 14 autosomes and the sex chromosomes.

  9. Corruption of phage-display libraries by target-unrelated clones: Diagnosis and countermeasures

    Science.gov (United States)

    Thomas, William D.; Golomb, Miriam; Smith, George P.

    2010-01-01

    Phage display is used to discover peptides or proteins with a desired target property—most often, affinity for a target selector molecule. Libraries of phage clones displaying diverse surface peptides are subject to a selection process designed to enrich for the target behavior, and subsequently propagated to restore phage numbers. A recurrent problem is enrichment of clones, called target-unrelated phage (TUPs), that lack the target behavior. Many TUPs are propagation-related; they have mutations conferring a growth advantage, and are enriched during the propagations accompanying selection. Unlike other filamentous phage libraries, fd-tet-based libraries are relatively resistant to propagation-related TUP corruption. Their minus strand origin is disrupted by a large cassette that simultaneously confers resistance to tetracycline and imposes a rate-limiting growth defect that cannot be bypassed with simple mutations. Nonetheless, a new type of propagation-related TUP emerged in the output of in vivo selections from an fd-tet library. The founding clone had a complex rearrangement that restored the minus strand origin while retaining tetracycline resistance. The rearrangement involved two recombination events, one with a contaminant having a wild-type minus strand origin. The founder’s infectivity advantage spread by simple recombination to clones displaying different peptides. We propose measures for minimizing TUP corruption. PMID:20692225

  10. Analysis of a diverse assemblage of diazotrophic bacteria from Spartina alterniflora using DGGE and clone library screening.

    Science.gov (United States)

    Lovell, Charles R; Decker, Peter V; Bagwell, Christopher E; Thompson, Shelly; Matsui, George Y

    2008-05-01

    Methods to assess the diversity of the diazotroph assemblage in the rhizosphere of the salt marsh cordgrass, Spartina alterniflora were examined. The effectiveness of nifH PCR-denaturing gradient gel electrophoresis (DGGE) was compared to that of nifH clone library analysis. Seventeen DGGE gel bands were sequenced and yielded 58 nonidentical nifH sequences from a total of 67 sequences determined. A clone library constructed using the GC-clamp nifH primers that were employed in the PCR-DGGE (designated the GC-Library) yielded 83 nonidentical sequences from a total of 257 nifH sequences. A second library constructed using an alternate set of nifH primers (N-Library) yielded 83 nonidentical sequences from a total of 138 nifH sequences. Rarefaction curves for the libraries did not reach saturation, although the GC-Library curve was substantially dampened and appeared to be closer to saturation than the N-Library curve. Phylogenetic analyses showed that DGGE gel band sequencing recovered nifH sequences that were frequently sampled in the GC-Library, as well as sequences that were infrequently sampled, and provided a species composition assessment that was robust, efficient, and relatively inexpensive to obtain. Further, the DGGE method permits a large number of samples to be examined for differences in banding patterns, after which bands of interest can be sampled for sequence determination.

  11. Corruption of phage display libraries by target-unrelated clones: diagnosis and countermeasures.

    Science.gov (United States)

    Thomas, William D; Golomb, Miriam; Smith, George P

    2010-12-15

    Phage display is used to discover peptides or proteins with a desired target property-most often, affinity for a target selector molecule. Libraries of phage clones displaying diverse surface peptides are subject to a selection process designed to enrich for the target behavior and subsequently propagated to restore phage numbers. A recurrent problem is enrichment of clones, called target-unrelated phages or peptides (TUPs), that lack the target behavior. Many TUPs are propagation related; they have mutations conferring a growth advantage and are enriched during the propagations accompanying selection. Unlike other filamentous phage libraries, fd-tet-based libraries are relatively resistant to propagation-related TUP corruption. Their minus-strand origin is disrupted by a large cassette that simultaneously confers resistance to tetracycline and imposes a rate-limiting growth defect that cannot be bypassed with simple mutations. Nonetheless, a new type of propagation-related TUP emerged in the output of in vivo selections from an fd-tet library. The founding clone had a complex rearrangement that restored the minus-strand origin while retaining tetracycline resistance. The rearrangement involved two recombination events, one with a contaminant having a wild-type minus-strand origin. The founder's infectivity advantage spread by simple recombination to clones displaying different peptides. We propose measures for minimizing TUP corruption. Copyright © 2010 Elsevier Inc. All rights reserved.

  12. Cloning of low dose radiation induced gene RIG1 by RACE based on non-cloned cDNA library

    International Nuclear Information System (INIS)

    Luo Ying; Sui Jianli; Tie Yi; Zhang Yuanping; Zhou Pingkun; Sun Zhixian

    2001-01-01

    Objective: To obtain full-length cDNA of radiation induced new gene RIG1 based on its EST fragment. Methods: Based on non-cloned cDNA library, enhanced nested RACE PCR and biotin-avidin labelled probe for magnetic bead purification was used to obtain full-length cDNA of RIG1. Results: About 1 kb of 3' end of RIG1 gene was successfully cloned by this set of methods and cloning of RIG1 5' end is proceeding well. Conclusion: The result is consistent with the design of experiment. This set of protocol is useful for cloning of full-length gene based on EST fragment

  13. T-cell libraries allow simple parallel generation of multiple peptide-specific human T-cell clones.

    Science.gov (United States)

    Theaker, Sarah M; Rius, Cristina; Greenshields-Watson, Alexander; Lloyd, Angharad; Trimby, Andrew; Fuller, Anna; Miles, John J; Cole, David K; Peakman, Mark; Sewell, Andrew K; Dolton, Garry

    2016-03-01

    Isolation of peptide-specific T-cell clones is highly desirable for determining the role of T-cells in human disease, as well as for the development of therapies and diagnostics. However, generation of monoclonal T-cells with the required specificity is challenging and time-consuming. Here we describe a library-based strategy for the simple parallel detection and isolation of multiple peptide-specific human T-cell clones from CD8(+) or CD4(+) polyclonal T-cell populations. T-cells were first amplified by CD3/CD28 microbeads in a 96U-well library format, prior to screening for desired peptide recognition. T-cells from peptide-reactive wells were then subjected to cytokine-mediated enrichment followed by single-cell cloning, with the entire process from sample to validated clone taking as little as 6 weeks. Overall, T-cell libraries represent an efficient and relatively rapid tool for the generation of peptide-specific T-cell clones, with applications shown here in infectious disease (Epstein-Barr virus, influenza A, and Ebola virus), autoimmunity (type 1 diabetes) and cancer. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  14. Flow Cytometry-Assisted Cloning of Specific Sequence Motifs from Complex 16S rRNA Gene Libraries

    DEFF Research Database (Denmark)

    Nielsen, Jeppe Lund; Schramm, Andreas; Bernhard, Anne E.

    2004-01-01

    for Systems Biology,3 Seattle, Washington, and Department of Ecological Microbiology, University of Bayreuth, Bayreuth, Germany2 A flow cytometry method was developed for rapid screening and recovery of cloned DNA containing common sequence motifs. This approach, termed fluorescence-activated cell sorting......  FLOW CYTOMETRY-ASSISTED CLONING OF SPECIFIC SEQUENCE MOTIFS FROM COMPLEX 16S RRNA GENE LIBRARIES Jeppe L. Nielsen,1 Andreas Schramm,1,2 Anne E. Bernhard,1 Gerrit J. van den Engh,3 and David A. Stahl1* Department of Civil and Environmental Engineering, University of Washington,1 and Institute......-assisted cloning, was used to recover sequences affiliated with a unique lineage within the Bacteroidetes not abundant in a clone library of environmental 16S rRNA genes.  ...

  15. Phylogenetic characterization of a biogas plant microbial community integrating clone library 16S-rDNA sequences and metagenome sequence data obtained by 454-pyrosequencing.

    Science.gov (United States)

    Kröber, Magdalena; Bekel, Thomas; Diaz, Naryttza N; Goesmann, Alexander; Jaenicke, Sebastian; Krause, Lutz; Miller, Dimitri; Runte, Kai J; Viehöver, Prisca; Pühler, Alfred; Schlüter, Andreas

    2009-06-01

    The phylogenetic structure of the microbial community residing in a fermentation sample from a production-scale biogas plant fed with maize silage, green rye and liquid manure was analysed by an integrated approach using clone library sequences and metagenome sequence data obtained by 454-pyrosequencing. Sequencing of 109 clones from a bacterial and an archaeal 16S-rDNA amplicon library revealed that the obtained nucleotide sequences are similar but not identical to 16S-rDNA database sequences derived from different anaerobic environments including digestors and bioreactors. Most of the bacterial 16S-rDNA sequences could be assigned to the phylum Firmicutes with the most abundant class Clostridia and to the class Bacteroidetes, whereas most archaeal 16S-rDNA sequences cluster close to the methanogen Methanoculleus bourgensis. Further sequences of the archaeal library most probably represent so far non-characterised species within the genus Methanoculleus. A similar result derived from phylogenetic analysis of mcrA clone sequences. The mcrA gene product encodes the alpha-subunit of methyl-coenzyme-M reductase involved in the final step of methanogenesis. BLASTn analysis applying stringent settings resulted in assignment of 16S-rDNA metagenome sequence reads to 62 16S-rDNA amplicon sequences thus enabling frequency of abundance estimations for 16S-rDNA clone library sequences. Ribosomal Database Project (RDP) Classifier processing of metagenome 16S-rDNA reads revealed abundance of the phyla Firmicutes, Bacteroidetes and Euryarchaeota and the orders Clostridiales, Bacteroidales and Methanomicrobiales. Moreover, a large fraction of 16S-rDNA metagenome reads could not be assigned to lower taxonomic ranks, demonstrating that numerous microorganisms in the analysed fermentation sample of the biogas plant are still unclassified or unknown.

  16. Creating libraries for commercial yeast strains through miniaturization of cloning and transformations using the BioRAPTR FRD Microfluidic workstation

    Science.gov (United States)

    The ability to miniaturize molecular reactions can lead to significant cost savings when creating libraries of thousands of clones. For this application Beckman Coulter partnered with the USDA to provide a low-volume automated solution for library cloning for use in the development of yeast strains...

  17. Bacterial community composition in different sediments from the Eastern Mediterranean Sea: a comparison of four 16S ribosomal DNA clone libraries.

    Science.gov (United States)

    Polymenakou, Paraskevi N; Bertilsson, Stefan; Tselepides, Anastasios; Stephanou, Euripides G

    2005-10-01

    The regional variability of sediment bacterial community composition and diversity was studied by comparative analysis of four large 16S ribosomal DNA (rDNA) clone libraries from sediments in different regions of the Eastern Mediterranean Sea (Thermaikos Gulf, Cretan Sea, and South lonian Sea). Amplified rDNA restriction analysis of 664 clones from the libraries indicate that the rDNA richness and evenness was high: for example, a near-1:1 relationship among screened clones and number of unique restriction patterns when up to 190 clones were screened for each library. Phylogenetic analysis of 207 bacterial 16S rDNA sequences from the sediment libraries demonstrated that Gamma-, Delta-, and Alphaproteobacteria, Holophaga/Acidobacteria, Planctomycetales, Actinobacteria, Bacteroidetes, and Verrucomicrobia were represented in all four libraries. A few clones also grouped with the Betaproteobacteria, Nitrospirae, Spirochaetales, Chlamydiae, Firmicutes, and candidate division OPl 1. The abundance of sequences affiliated with Gammaproteobacteria was higher in libraries from shallow sediments in the Thermaikos Gulf (30 m) and the Cretan Sea (100 m) compared to the deeper South Ionian station (2790 m). Most sequences in the four sediment libraries clustered with uncultured 16S rDNA phylotypes from marine habitats, and many of the closest matches were clones from hydrocarbon seeps, benzene-mineralizing consortia, sulfate reducers, sulk oxidizers, and ammonia oxidizers. LIBSHUFF statistics of 16S rDNA gene sequences from the four libraries revealed major differences, indicating either a very high richness in the sediment bacterial communities or considerable variability in bacterial community composition among regions, or both.

  18. Changes in the Composition of Drinking Water Bacterial Clone Libraries Introduced by Using Two Different 16S rRNA Gene PCR Primers

    Science.gov (United States)

    Sequence analysis of 16S rRNA gene clone libraries is a popular tool used to describe the composition of natural microbial communities. Commonly, clone libraries are developed by direct cloning of 16S rRNA gene PCR products. Different primers are often employed in the initial amp...

  19. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

    Science.gov (United States)

    The bacterial composition of chlorinated drinking water was analyzed using 16S rRNA gene clone libraries derived from DNA extracts of 12 samples and compared to clone libraries previously generated using RNA extracts from the same samples. Phylogenetic analysis of 761 DNA-based ...

  20. Clone DB: an integrated NCBI resource for clone-associated data

    Science.gov (United States)

    Schneider, Valerie A.; Chen, Hsiu-Chuan; Clausen, Cliff; Meric, Peter A.; Zhou, Zhigang; Bouk, Nathan; Husain, Nora; Maglott, Donna R.; Church, Deanna M.

    2013-01-01

    The National Center for Biotechnology Information (NCBI) Clone DB (http://www.ncbi.nlm.nih.gov/clone/) is an integrated resource providing information about and facilitating access to clones, which serve as valuable research reagents in many fields, including genome sequencing and variation analysis. Clone DB represents an expansion and replacement of the former NCBI Clone Registry and has records for genomic and cell-based libraries and clones representing more than 100 different eukaryotic taxa. Records provide details of library construction, associated sequences, map positions and information about resource distribution. Clone DB is indexed in the NCBI Entrez system and can be queried by fields that include organism, clone name, gene name and sequence identifier. Whenever possible, genomic clones are mapped to reference assemblies and their map positions provided in clone records. Clones mapping to specific genomic regions can also be searched for using the NCBI Clone Finder tool, which accepts queries based on sequence coordinates or features such as gene or transcript names. Clone DB makes reports of library, clone and placement data on its FTP site available for download. With Clone DB, users now have available to them a centralized resource that provides them with the tools they will need to make use of these important research reagents. PMID:23193260

  1. Isolation of Specific Clones from Nonarrayed BAC Libraries through Homologous Recombination

    Directory of Open Access Journals (Sweden)

    Mikhail Nefedov

    2011-01-01

    Full Text Available We have developed a new approach to screen bacterial artificial chromosome (BAC libraries by recombination selection. To test this method, we constructed an orangutan BAC library using an E. coli strain (DY380 with temperature inducible homologous recombination (HR capability. We amplified one library segment, induced HR at 42∘C to make it recombination proficient, and prepared electrocompetent cells for transformation with a kanamycin cassette to target sequences in the orangutan genome through terminal recombineering homologies. Kanamycin-resistant colonies were tested for the presence of BACs containing the targeted genes by the use of a PCR-assay to confirm the presence of the kanamycin insertion. The results indicate that this is an effective approach for screening clones. The advantage of recombination screening is that it avoids the high costs associated with the preparation, screening, and archival storage of arrayed BAC libraries. In addition, the screening can be conceivably combined with genetic engineering to create knockout and reporter constructs for functional studies.

  2. Application of clone library analysis and real-time PCR for comparison of microbial communities in a low-grade copper sulfide ore bioheap leachate.

    Science.gov (United States)

    Bowei, Chen; Xingyu, Liu; Wenyan, Liu; Jiankang, Wen

    2009-11-01

    The microbial communities of leachate from a bioleaching heap located in China were analyzed using the 16S rRNA gene clone library and real-time quantitative PCR. Both methods showed that Leptospirillum spp. were the dominant bacteria, and Ferroplasma acidiphilum were the only archaea detected in the leachate. Clone library results indicated that nine operational taxonomic units (OTUs) were obtained, which fell into four divisions, the Nitrospirae (74%), the gamma-Proteobacteria (14%), the Actinobacteria (6%) and the Euryarchaeota (6%). The results obtained by real-time PCR in some ways were the same as clone library analysis. Furthermore, Sulfobacillus spp., detected only by real-time PCR, suggests that real-time PCR was a reliable technology to study the microbial communities in bioleaching environments. It is a useful tool to assist clone library analysis, to further understand microbial consortia and to have comprehensive and exact microbiological information about bioleaching environments. Finally, the interactions among the microorganisms detected in the leachate were summarized according to the characteristics of these species.

  3. Differential distribution and abundance of diazotrophic bacterial communities across different soil niches using a gene-targeted clone library approach.

    Science.gov (United States)

    Yousuf, Basit; Kumar, Raghawendra; Mishra, Avinash; Jha, Bhavanath

    2014-11-01

    Diazotrophs are key players of the globally important biogeochemical nitrogen cycle, having a significant role in maintaining ecosystem sustainability. Saline soils are pristine and unexplored habitats representing intriguing ecosystems expected to harbour potential diazotrophs capable of adapting in extreme conditions, and these implicated organisms are largely obscure. Differential occurrence of diazotrophs was studied by the nifH gene-targeted clone library approach. Four nifH gene clone libraries were constructed from different soil niches, that is saline soils (low and high salinity; EC 3.8 and 7.1 ds m(-1) ), and agricultural and rhizosphere soil. Additionally, the abundance of diazotrophic community members was assessed using quantitative PCR. Results showed environment-dependent metabolic versatility and the presence of nitrogen-fixing bacteria affiliated with a range of taxa, encompassing members of the Alphaproteobacteria, Betaproteobacteria, Deltaproteobacteria, Gammaproteobacteria, Cyanobacteria and Firmicutes. The analyses unveiled the dominance of Alphaproteobacteria and Gammaproteobacteria (Pseudomonas, Halorhodospira, Ectothiorhodospira, Bradyrhizobium, Agrobacterium, Amorphomonas) as nitrogen fixers in coastal-saline soil ecosystems, and Alphaproteobacteria and Betaproteobacteria (Bradyrhizobium, Azohydromonas, Azospirillum, Ideonella) in agricultural/rhizosphere ecosystems. The results revealed a repertoire of novel nitrogen-fixing bacterial guilds particularly in saline soil ecosystems. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  4. A Polymerase Chain Reaction-Based Method for Isolating Clones from a Complimentary DNA Library in Sheep

    Science.gov (United States)

    Friis, Thor Einar; Stephenson, Sally; Xiao, Yin; Whitehead, Jon

    2014-01-01

    The sheep (Ovis aries) is favored by many musculoskeletal tissue engineering groups as a large animal model because of its docile temperament and ease of husbandry. The size and weight of sheep are comparable to humans, which allows for the use of implants and fixation devices used in human clinical practice. The construction of a complimentary DNA (cDNA) library can capture the expression of genes in both a tissue- and time-specific manner. cDNA libraries have been a consistent source of gene discovery ever since the technology became commonplace more than three decades ago. Here, we describe the construction of a cDNA library using cells derived from sheep bones based on the pBluescript cDNA kit. Thirty clones were picked at random and sequenced. This led to the identification of a novel gene, C12orf29, which our initial experiments indicate is involved in skeletal biology. We also describe a polymerase chain reaction-based cDNA clone isolation method that allows the isolation of genes of interest from a cDNA library pool. The techniques outlined here can be applied in-house by smaller tissue engineering groups to generate tools for biomolecular research for large preclinical animal studies and highlights the power of standard cDNA library protocols to uncover novel genes. PMID:24447069

  5. Evaluation of a pooled strategy for high-throughput sequencing of cosmid clones from metagenomic libraries.

    Science.gov (United States)

    Lam, Kathy N; Hall, Michael W; Engel, Katja; Vey, Gregory; Cheng, Jiujun; Neufeld, Josh D; Charles, Trevor C

    2014-01-01

    High-throughput sequencing methods have been instrumental in the growing field of metagenomics, with technological improvements enabling greater throughput at decreased costs. Nonetheless, the economy of high-throughput sequencing cannot be fully leveraged in the subdiscipline of functional metagenomics. In this area of research, environmental DNA is typically cloned to generate large-insert libraries from which individual clones are isolated, based on specific activities of interest. Sequence data are required for complete characterization of such clones, but the sequencing of a large set of clones requires individual barcode-based sample preparation; this can become costly, as the cost of clone barcoding scales linearly with the number of clones processed, and thus sequencing a large number of metagenomic clones often remains cost-prohibitive. We investigated a hybrid Sanger/Illumina pooled sequencing strategy that omits barcoding altogether, and we evaluated this strategy by comparing the pooled sequencing results to reference sequence data obtained from traditional barcode-based sequencing of the same set of clones. Using identity and coverage metrics in our evaluation, we show that pooled sequencing can generate high-quality sequence data, without producing problematic chimeras. Though caveats of a pooled strategy exist and further optimization of the method is required to improve recovery of complete clone sequences and to avoid circumstances that generate unrecoverable clone sequences, our results demonstrate that pooled sequencing represents an effective and low-cost alternative for sequencing large sets of metagenomic clones.

  6. Complementation of radiation-sensitive Ataxia telangiectasia cells after transfection of cDNA expression libraries and cosmid clones from wildtype cells

    International Nuclear Information System (INIS)

    Fritz, E.

    1994-06-01

    In this Ph.D.-thesis, phenotypic complementation of AT-cells (AT5BIVA) by transfection of cDNA-expression-libraries was adressed: After stable transfection of cDNA-expression-libraries G418 resistant clones were selected for enhanced radioresistance by a fractionated X-ray selection. One surviving transfectant clone (clone 514) exhibited enhanced radiation resistance in dose-response experiments and further X-ray selections. Cell cycle analysis revealed complementation of untreated and irradiated 514-cells in cell cycle progression. The rate of DNA synthesis, however, is not diminished after irradiation but shows the reverse effect. A transfected cDNA-fragment (AT500-cDNA) was isolated from the genomic DNA of 514-cells and proved to be an unknown DNA sequence. A homologous sequence could be detected in genomic DNA from human cell lines, but not in DNA from other species. The cDNA-sequence could be localized to human chromosome 11. In human cells the cDNA sequence is part of two large mRNAs. 4 different cosmid clones containing high molecular genomic DNA from normal human cells could be isolated from a library, each hybridizing to the AT500-cDNA. After stable transfection into AT-cells, one cosmid-clone was able to confer enhanced radiation resistance both in X-ray selections and dose-response experiments. The results indicate that the cloned cDNA-fragment is based on an unknown gene from human chromosome 11 which partially complements the radiosensitivity and the defective cell cycle progression in AT5BIVA cells. (orig.) [de

  7. IDENTIFICATION OF ACTIVE BACTERIAL COMMUNITIES IN A MODEL DRINKING WATER BIOFILM SYSTEM USING 16S RRNA-BASED CLONE LIBRARIES

    Science.gov (United States)

    Recent phylogenetic studies have used DNA as the target molecule for the development of environmental 16S rDNA clone libraries. As DNA may persist in the environment, DNA-based libraries cannot be used to identify metabolically active bacteria in water systems. In this study, a...

  8. The first insight into the salvia (lamiaceae) genome via bac library construction and high-throughput sequencing of target bac clones

    International Nuclear Information System (INIS)

    Hao, D.C.; Vautrin, S.; Berges, H.; Chen, S.L.

    2015-01-01

    Salvia is a representative genus of Lamiaceae, a eudicot family with significant species diversity and population adaptibility. One of the key goals of Salvia genomics research is to identify genes of adaptive significance. This information may help to improve the conservation of adaptive genetic variation and the management of medicinal plants to increase their health and productivity. Large-insert genomic libraries are a fundamental tool for achieving this purpose. We report herein the construction, characterization and screening of a gridded BAC library for Salvia officinalis (sage). The S. officinalis BAC library consists of 17,764 clones and the average insert size is 107 Kb, corresponding to 3 haploid genome equivalents. Seventeen positive clones (average insert size 115 Kb) containing five terpene synthase (TPS) genes were screened out by PCR and 12 of them were subject to Illumina HiSeq 2000 sequencing, which yielded 28,097,480 90-bp raw reads (2.53 Gb). Scaffolds containing sabinene synthase (Sab), a Sab homolog, TPS3 (kaurene synthase-like 2), copalyl diphosphate synthase 2 and one cytochrome P450 gene were retrieved via de novo assembly and annotation, which also have flanking noncoding sequences, including predicted promoters and repeat sequences. Among 2,638 repeat sequences, there are 330 amplifiable microsatellites. This BAC library provides a new resource for Lamiaceae genomic studies, including microsatellite marker development, physical mapping, comparative genomics and genome sequencing. Characterization of positive clones provided insights into the structure of the Salvia genome. These sequences will be used in the assembly of a future genome sequence for S. officinalis. (author)

  9. Construction of BAC Libraries from Flow-Sorted Chromosomes.

    Science.gov (United States)

    Šafář, Jan; Šimková, Hana; Doležel, Jaroslav

    2016-01-01

    Cloned DNA libraries in bacterial artificial chromosome (BAC) are the most widely used form of large-insert DNA libraries. BAC libraries are typically represented by ordered clones derived from genomic DNA of a particular organism. In the case of large eukaryotic genomes, whole-genome libraries consist of a hundred thousand to a million clones, which make their handling and screening a daunting task. The labor and cost of working with whole-genome libraries can be greatly reduced by constructing a library derived from a smaller part of the genome. Here we describe construction of BAC libraries from mitotic chromosomes purified by flow cytometric sorting. Chromosome-specific BAC libraries facilitate positional gene cloning, physical mapping, and sequencing in complex plant genomes.

  10. DNA cloning: a practical approach. Volume 1

    Energy Technology Data Exchange (ETDEWEB)

    Glover, D M [ed.

    1985-01-01

    This book is written for the advanced molecular biologist who needs a detailed discussion of cloning technology. Topics of discussion include: genomic library cloning (size of a genomic library, screening methods, chromosome walking, host cell genetics, and general features of bacteriophage Iambda); use of gt10 and gt11 cDNA lambda vectors and general cDNA cloning; RNase H-Pol I cDNA synthesis; method of detecting fusion proteins produced in bacteria; pEMBL family of double-stranded plasmid vectors that can be used to generate single strands; Escherichia coli transformation; production of mutations in cloned sequences; and cloning in gram negative bacteria.

  11. Surviving extreme polar winters by desiccation: clues from Arctic springtail (Onychiurus arcticus EST libraries

    Directory of Open Access Journals (Sweden)

    Kube Michael

    2007-12-01

    Full Text Available Abstract Background Ice, snow and temperatures of -14°C are conditions which most animals would find difficult, if not impossible, to survive in. However this exactly describes the Arctic winter, and the Arctic springtail Onychiurus arcticus regularly survives these extreme conditions and re-emerges in the spring. It is able to do this by reducing the amount of water in its body to almost zero: a process that is called "protective dehydration". The aim of this project was to generate clones and sequence data in the form of ESTs to provide a platform for the future molecular characterisation of the processes involved in protective dehydration. Results Five normalised libraries were produced from both desiccating and rehydrating populations of O. arcticus from stages that had previously been defined as potentially informative for molecular analyses. A total of 16,379 EST clones were generated and analysed using Blast and GO annotation. 40% of the clones produced significant matches against the Swissprot and trembl databases and these were further analysed using GO annotation. Extraction and analysis of GO annotations proved an extremely effective method for identifying generic processes associated with biochemical pathways, proving more efficient than solely analysing Blast data output. A number of genes were identified, which have previously been shown to be involved in water transport and desiccation such as members of the aquaporin family. Identification of these clones in specific libraries associated with desiccation validates the computational analysis by library rather than producing a global overview of all libraries combined. Conclusion This paper describes for the first time EST data from the arctic springtail (O. arcticus. This significantly enhances the number of Collembolan ESTs in the public databases, providing useful comparative data within this phylum. The use of GO annotation for analysis has facilitated the identification of a

  12. Analysis of microbiota associated with peri-implantitis using 16S rRNA gene clone library

    Directory of Open Access Journals (Sweden)

    Tatsuro Koyanagi

    2010-05-01

    Full Text Available Background: Peri-implantitis (PI is an inflammatory disease which leads to the destruction of soft and hard tissues around osseointegrated implants. The subgingival microbiota appears to be responsible for peri-implant lesions and although the complexity of the microbiota has been reported in PI, the microbiota responsible for PI has not been identified. Objective: The purpose of this study was to identify the microbiota in subjects who have PI, clinically healthy implants, and periodontitis-affected teeth using 16S rRNA gene clone library analysis to clarify the microbial differences. Design: Three subjects participated in this study. The conditions around the teeth and implants were evaluated based on clinical and radiographic examinations and diseased implants, clinically healthy implants, and periodontally diseased teeth were selected. Subgingival plaque samples were taken from the deepest pockets using sterile paper points. Prevalence and identity of bacteria was analyzed using a 16S rRNA gene clone library technique. Results: A total of 112 different species were identified from 335 clones sequenced. Among the 112 species, 51 (46% were uncultivated phylotypes, of which 22 were novel phylotypes. The numbers of bacterial species identified at the sites of PI, periodontitis, and periodontally healthy implants were 77, 57, and 12, respectively. Microbiota in PI mainly included Gram-negative species and the composition was more diverse when compared to that of the healthy implant and periodontitis. The phyla Chloroflexi, Tenericutes, and Synergistetes were only detected at PI sites, as were Parvimonas micra, Peptostreptococcus stomatis, Pseudoramibacter alactolyticus, and Solobacterium moorei. Low levels of periodontopathic bacteria, such as Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, were seen in peri-implant lesions. Conclusions: The biofilm in PI showed a more complex microbiota when compared to periodontitis and

  13. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development - Poster

    Science.gov (United States)

    We examined the bacterial composition of chlorinated drinking water using 16S rRNA gene clone libraries derived from RNA and DNA extracted from twelve water samples collected in three different months (June, August, and September of 2007). Phylogenetic analysis of 1234 and 1117 ...

  14. Identification and Preliminary Analysis of Several Centromere-associated Bacterial Artificial Chromosome Clones from a Diploid Wheat Library

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Although the centromeres of some plants have been investigated previously, our knowledge of the wheat centromere is still very limited. To understand the structure and function of the wheat centromere, we used two centromeric repeats (RCS1 and CCS1-5ab) to obtain some centromere-associated bacterial artificial chromosome (BAC) clones in 32 RCS1-related BAC clones that had been screened out from a diploid wheat (Triticum boeoticum Boiss.; 2n=2x=14) BAC library. Southern hybridization results indicated that, of the 32 candidates,there were 28 RCS1-positive clones. Based on gel blot patterns, the frequency of RCS1 was approximately one copy every 69.4 kb in these 28 RCS1-positive BAC clones. More bands were detected when the same filter was probed with CCS1-5ab. Furthermore, the CCS1 bands covered all the bands detected by RCS1, which suggests that some CCS1 repeats were distributed together with RCS1. The frequency of CCS1 families was once every 35.8 kb, nearly twice that of RCS1. Fluorescence in situ hybridization (FISH) analysis indicated that the five BAC clones containing RCS1 and CCS1 sequences all detected signals at the centromeric regions in hexaploid wheat, but the signal intensities on the A-genome chromosomes were stronger than those on the B- and/or D-genome chromosomes. The FISH analysis among nine Triticeae cereals indicated that there were A-genomespecific (or rich) sequences dispersing on chromosome arms in the BAC clone TbBAC5. In addition, at the interphase cells, the centromeres of diploid species usually clustered at one pole and formed a ring-like allocation in the period before metaphase.

  15. Construction of bacterial artificial chromosome libraries for Zhikong Scallop Chlamys farreri

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yang; ZHANG Xiaojun; Chantel F.SCHEURING; ZHANG Hongbin; LI Fuhua; XIANG Jianhai

    2008-01-01

    Two Large-insert genomic bacterial artificial chromosome (BAC) libraries of Zhikong scallop Chlamys farreri were constructed to promote our genetic and genomic research.High-quality megabase-sized DNA was isolated from the adductor muscle of the scallop and partially digested by BamH I and Mbo I,respectively.The BamH I library consisted of 53760 clones while the Mbo I library consisted of 7680 clones.Approximately 96% of the clones in BamH I library contained nuclear DNA inserts in average size of 100 kb,providing a coverage of 5.3 haploid genome equivalents.Similarly,the Mbo I library with an average insert of 145 kb and no insert-empty clones,thus providing a genome coverage of 1.1 haploid genome equivalents.

  16. High-Throughput Cloning and Expression Library Creation for Functional Proteomics

    Science.gov (United States)

    Festa, Fernanda; Steel, Jason; Bian, Xiaofang; Labaer, Joshua

    2013-01-01

    The study of protein function usually requires the use of a cloned version of the gene for protein expression and functional assays. This strategy is particular important when the information available regarding function is limited. The functional characterization of the thousands of newly identified proteins revealed by genomics requires faster methods than traditional single gene experiments, creating the need for fast, flexible and reliable cloning systems. These collections of open reading frame (ORF) clones can be coupled with high-throughput proteomics platforms, such as protein microarrays and cell-based assays, to answer biological questions. In this tutorial we provide the background for DNA cloning, discuss the major high-throughput cloning systems (Gateway® Technology, Flexi® Vector Systems, and Creator™ DNA Cloning System) and compare them side-by-side. We also report an example of high-throughput cloning study and its application in functional proteomics. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP12). Details can be found at http://www.proteomicstutorials.org. PMID:23457047

  17. Bioproduction and characterization of extracellular melanin-like pigment from industrially polluted metagenomic library equipped Escherichia coli.

    Science.gov (United States)

    Amin, Shivani; Rastogi, Rajesh P; Sonani, Ravi R; Ray, Arabinda; Sharma, Rakesh; Madamwar, Datta

    2018-04-15

    To explore the potential genes from the industrially polluted Amlakhadi canal, located in Ankleshwar, Gujarat, India, its community genome was extracted and cloned into E. coli EPI300™-T1 R using a fosmid vector (pCC2 FOS™) generating a library of 3,92,000 clones with average size of 40kb of DNA-insert. From this library, the clone DM1 producing brown colored melanin-like pigment was isolated and characterized. For over expression of the pigment, further sub-cloning of the clone DM1 was done. Sub-clone containing 10kb of the insert was sequenced for gene identification. The amino acids sequence of a protein 4-Hydroxyphenylpyruvate dioxygenase (HPPD), which is know to be involved in melanin biosynthesis was obtained from the gene sequence. The sequence-homology based 3D structure model of HPPD was constructed and analyzed. The physico-chemical nature of pigment was further analysed using 1 H and 13 C NMR, LC-MS, FTIR and UV-visible spectroscopy. The pigment was readily soluble in DMSO with an absorption maximum around 290nm. Based on the genetic and chemical characterization, the compound was confirmed as melanin-like pigment. The present results indicate that the metagenomic library from industrially polluted environment generated a microbial tool for the production of melanin-like pigment. Copyright © 2018 Elsevier B.V. All rights reserved.

  18. Molecular cloning of chicken metallothionein. Deduction of the complete amino acid sequence and analysis of expression using cloned cDNA

    Energy Technology Data Exchange (ETDEWEB)

    Wei, D; Andrews, G K

    1988-01-25

    A cDNA library was constructed using RNA isolated from the livers of chickens which had been treated with zinc. This library was screened with a RNA probe complementary to mouse metallothionein-I (MT), and eight chicken MT cDNA clones were obtained. All of the cDNA clones contained nucleotide sequences homologous to regions of the longest (375 bp) cDNA clone. The latter contained an open reading frame of 189 bp, and the deduced amino acid sequence indicates a protein of 63 amino acids of which 20 are cysteine residues. Amino acid composition and partial amino acid sequence analyses of purified chicken MT protein agreed with the amino acid composition and sequence deduced from the cloned cDNA. Amino acid sequence comparison establish that chicken MT shares extensive homology with mammalian MTs. Southern blot analysis of chicken DNA indicates that the chicken MT gene is not a part of a large family of related sequences, but rather is likely to be a unique gene sequence. In the chicken liver, levels of chicken MT mRNA were rapidly induced by metals (Cd/sup 2 +/, Zn/sup 2 +/, Cu/sup 2 +/), glucocorticoids and lipopolysaccharide. MT mRNA was present in low levels in embryonic liver and increased to high levels during the first week after hatching before decreasing again to the basal levels found in adult liver. The results of this study establish that MT is highly conserved between birds and mammals and is regulated in the chicken by agents which also regulate expression of mammalian MT genes. However, in contrast to the mammals, the results suggest the existence of a single isoform of MT in the chicken.

  19. Isolation of BAC Clones Containing Conserved Genes from Libraries of Three Distantly Related Moths: A Useful Resource for Comparative Genomics of Lepidoptera

    Directory of Open Access Journals (Sweden)

    Yuji Yasukochi

    2011-01-01

    Full Text Available Lepidoptera, butterflies and moths, is the second largest animal order and includes numerous agricultural pests. To facilitate comparative genomics in Lepidoptera, we isolated BAC clones containing conserved and putative single-copy genes from libraries of three pests, Heliothis virescens, Ostrinia nubilalis, and Plutella xylostella, harboring the haploid chromosome number, =31, which are not closely related with each other or with the silkworm, Bombyx mori, (=28, the sequenced model lepidopteran. A total of 108–184 clones representing 101–182 conserved genes were isolated for each species. For 79 genes, clones were isolated from more than two species, which will be useful as common markers for analysis using fluorescence in situ hybridization (FISH, as well as for comparison of genome sequence among multiple species. The PCR-based clone isolation method presented here is applicable to species which lack a sequenced genome but have a significant collection of cDNA or EST sequences.

  20. Practical Tips for Construction of Custom Peptide Libraries and Affinity Selection by Using Commercially Available Phage Display Cloning Systems

    Directory of Open Access Journals (Sweden)

    Keisuke Fukunaga

    2012-01-01

    Full Text Available Phage display technology is undoubtedly a powerful tool for affinity selection of target-specific peptide. Commercially available premade phage libraries allow us to take screening in the easiest way. On the other hand, construction of a custom phage library seems to be inaccessible, because several practical tips are absent in instructions. This paper focuses on what should be born in mind for beginners using commercially available cloning kits (Ph.D. with type 3 vector and T7Select systems for M13 and T7 phage, respectively. In the M13 system, Pro or a basic amino acid (especially, Arg should be avoided at the N-terminus of peptide fused to gp3. In both systems, peptides containing odd number(s of Cys should be designed with caution. Also, DNA sequencing of a constructed library before biopanning is highly recommended for finding unexpected bias.

  1. Determining the Possible Etiology of Hospital-Acquired Pneumonia Using a Clone Library Analysis in Japan.

    Science.gov (United States)

    Yatera, Kazuhiro; Noguchi, Shingo; Yamasaki, Kei; Kawanami, Toshinori; Fukuda, Kazumasa; Naito, Keisuke; Akata, Kentaro; Kido, Takashi; Ishimoto, Hiroshi; Sakamoto, Noriho; Taniguchi, Hatsumi; Mukae, Hiroshi

    2017-05-01

    Obtaining precise etiological information regarding causative bacteria is important for the proper use of antimicrobials in hospital-acquired pneumonia (HAP), which is associated with a high rate of mortality. The aim of this study was to comparatively investigate the bacterial diversity in bronchoalveolar lavage fluid (BALF) in Japanese patients with HAP by the clone library method using the 16S rRNA gene. This study included Japanese patients with HAP who were treated at our hospital and referring hospitals. BALF specimens were obtained from pneumonia lesions identified on chest radiographs and/or computed tomography. Sputum specimens were also evaluated in patients with sputum production. Sixty-eight patients were ultimately enrolled. BALF cultivation revealed bacterial positivity in 53 of 68 (77.9%) patients, and Staphylococcus aureus (30.9%) was the most frequently isolated, followed by Pseudomonas aeruginosa (16.2%), and Escherichia coli (10.3%). In contrast, the clone library analysis identified the presence of some bacterial phenotype in 65 of 68 (95.6%) patients, and streptococci (16.2%), Corynebacterium species (11.8%), anaerobes (10.3%) were frequently detected as the predominant phylotypes. Both methods tended to detect S. aureus, Klebsiella pneumoniae, and E. coli in patients with late-onset pneumonia. In addition, the cases that phylotypes of S. aureus and P. aeruginosa were found to account for > 5% of the bacterial flora of each case were 42.9% and 72.7%, respectively. These results indicate that attention should be paid to the roles of gram-positive bacilli such as streptococci, Corynebacterium species and anaerobes, in addition to Gram-negative bacilli, in the pathogenesis of HAP.

  2. Third-Generation Sequencing and Analysis of Four Complete Pig Liver Esterase Gene Sequences in Clones Identified by Screening BAC Library.

    Science.gov (United States)

    Zhou, Qiongqiong; Sun, Wenjuan; Liu, Xiyan; Wang, Xiliang; Xiao, Yuncai; Bi, Dingren; Yin, Jingdong; Shi, Deshi

    2016-01-01

    Pig liver carboxylesterase (PLE) gene sequences in GenBank are incomplete, which has led to difficulties in studying the genetic structure and regulation mechanisms of gene expression of PLE family genes. The aim of this study was to obtain and analysis of complete gene sequences of PLE family by screening from a Rongchang pig BAC library and third-generation PacBio gene sequencing. After a number of existing incomplete PLE isoform gene sequences were analysed, primers were designed based on conserved regions in PLE exons, and the whole pig genome used as a template for Polymerase chain reaction (PCR) amplification. Specific primers were then selected based on the PCR amplification results. A three-step PCR screening method was used to identify PLE-positive clones by screening a Rongchang pig BAC library and PacBio third-generation sequencing was performed. BLAST comparisons and other bioinformatics methods were applied for sequence analysis. Five PLE-positive BAC clones, designated BAC-10, BAC-70, BAC-75, BAC-119 and BAC-206, were identified. Sequence analysis yielded the complete sequences of four PLE genes, PLE1, PLE-B9, PLE-C4, and PLE-G2. Complete PLE gene sequences were defined as those containing regulatory sequences, exons, and introns. It was found that, not only did the PLE exon sequences of the four genes show a high degree of homology, but also that the intron sequences were highly similar. Additionally, the regulatory region of the genes contained two 720bps reverse complement sequences that may have an important function in the regulation of PLE gene expression. This is the first report to confirm the complete sequences of four PLE genes. In addition, the study demonstrates that each PLE isoform is encoded by a single gene and that the various genes exhibit a high degree of sequence homology, suggesting that the PLE family evolved from a single ancestral gene. Obtaining the complete sequences of these PLE genes provides the necessary foundation for

  3. Cloning and study of the pectate lyase gene of Erwinia carotovora

    International Nuclear Information System (INIS)

    Bukanov, N.O.; Fonshtein, M.Yu.; Evtushenkov, A.N.; Syarinskii, M.A.; Strel'chenko, P.P.; Yankovski, N.K.; Alikhanyan, S.I.; Fomichev, Yu.K.; Debabov, V.G.

    1986-01-01

    The cloning of the gene of a secretable protein of Erwinia carotovora, pectate lyase, in Escherichia coli was described. Primary cloning was conducted using the phage vector λ 47.1. In the gene library of E. carotovora obtained, eight phages carrying the gene sought were identified according to the appearance of enzymatic activity of the gene product, pectate lyase, in situ. The BamHI fragment of DNA, common to all these phages, was recloned on the plasmid pUC19. It was shown that the cloned pectate lyase gene is represented on the E. carotovora chromosome in one copy. Methods of production of representative gene libraries on phage vectors from no less than 1 μg of cloned DNA even for the genomes of eukaryotes have now been developed. Vectors have been created, for example, λ 47.1, permitting the selection only of hybrid molecules. A number of methods have been developed for the search for a required gene in the library, depending on whether the cloned gene can be expressed or not, and if it can, what properties it will impart to the hybrid clone containing it

  4. C4P cross-section libraries for safety analyses with SIMMER and related studies

    International Nuclear Information System (INIS)

    Rineiski, A.; Sinitsa, V.; Gabrielli, F.; Maschek, W.

    2011-01-01

    A code and data system, C 4 P, is under development at KIT. It includes fine-group master libraries and tools for generating problem-oriented cross-section libraries, primarily for safety studies with the SIMMER code and related analyses. In the paper, the 560-group master library and problem oriented 40-group and 72-group cross-section libraries, for thermal and fast systems, respectively, are described and their performances are investigated. (author)

  5. Construction and Identification of Bacterial Artificial Chromosome Library for 0-613-2R in Upland Cotton

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    A bacterial artificial chromosome (BAC) library containing a large genomic DNA insert is an important tool for genome physical mapping, map-based cloning, and genome sequencing. To isolate genes via a map-based cloning strategy and to perform physical mapping of the cotton genome, a high-quality BAC library containing large cotton DNA inserts is needed. We have developed a BAC library of the restoring line 0-613-2R for isolating the fertility restorer (Rf1) gene and genomic research in cotton (Gossypium hirsutum L.). The BAC library contains 97 825 clones stored in 255 pieces of a 384-well microtiter plate. Random samples of BACs digested with the Notl enzyme indicated that the average insert size is approximately 130 kb, with a range of 80-275 kb,and 95.7% of the BAC clones in the library have an average insert size larger than 100 kb. Based on a cotton genome size of 2 250 Mb, library coverage is 5.7 x haploid genome equivalents. Four clones were selected randomly from the library to determine the stability of the BAC clones. There were no different fingerprints for 0 and 100 generations of each clone digested with Notl and Hindlll enzymes. Thus, the stability of a single BAC clone can be sustained at least for 100 generations. Eight simple sequence repeat (SSR) markers flanking the Rf1 gene were chosen to screen the BAC library by pool using PCR method and 25 positive clones were identified with 3.1 positive clones per SSR marker.

  6. Cloning of observables

    International Nuclear Information System (INIS)

    Ferraro, Alessandro; Galbiati, Matteo; Paris, Matteo G A

    2006-01-01

    We introduce the concept of cloning for classes of observables and classify cloning machines for qubit systems according to the number of parameters needed to describe the class under investigation. A no-cloning theorem for observables is derived and the connections between cloning of observables and joint measurements of noncommuting observables are elucidated. Relationships with cloning of states and non-demolition measurements are also analysed. (letter to the editor)

  7. Analyses of criticality and reactivity for TRACY experiments based on JENDL-3.3 data library

    International Nuclear Information System (INIS)

    Sono, Hiroki; Miyoshi, Yoshinori; Nakajima, Ken

    2003-01-01

    The parameters on criticality and reactivity employed for computational simulations of the TRACY supercritical experiments were analyzed using a recently revised nuclear data library, JENDL-3.3. The parameters based on the JENDL-3.3 library were compared to those based on two former-used libraries, JENDL-3.2 and ENDF/B-VI. In the analyses computational codes, MVP, MCNP version 4C and TWOTRAN, were used. The following conclusions were obtained from the analyses: (1) The computational biases of the effective neutron multiplication factor attributable to the nuclear data libraries and to the computational codes do not depend the TRACY experimental conditions such as fuel conditions. (2) The fractional discrepancies in the kinetic parameters and coefficients of reactivity are within ∼5% between the three libraries. By comparison between calculations and measurements of the parameters, the JENDL-3.3 library is expected to give closer values to the measurements than the JENDL-3.2 and ENDF/B-VI libraries. (3) While the reactivity worth of transient rods expressed in the $ unit shows ∼5% discrepancy between the three libraries according to their respective β eff values, there is little discrepancy in that expressed in the Δk/k unit. (author)

  8. Construction of High-Quality Camel Immune Antibody Libraries.

    Science.gov (United States)

    Romão, Ema; Poignavent, Vianney; Vincke, Cécile; Ritzenthaler, Christophe; Muyldermans, Serge; Monsion, Baptiste

    2018-01-01

    Single-domain antibodies libraries of heavy-chain only immunoglobulins from camelids or shark are enriched for high-affinity antigen-specific binders by a short in vivo immunization. Thus, potent binders are readily retrieved from relatively small-sized libraries of 10 7 -10 8 individual transformants, mostly after phage display and panning on a purified target. However, the remaining drawback of this strategy arises from the need to generate a dedicated library, for nearly every envisaged target. Therefore, all the procedures that shorten and facilitate the construction of an immune library of best possible quality are definitely a step forward. In this chapter, we provide the protocol to generate a high-quality immune VHH library using the Golden Gate Cloning strategy employing an adapted phage display vector where a lethal ccdB gene has to be substituted by the VHH gene. With this procedure, the construction of the library can be shortened to less than a week starting from bleeding the animal. Our libraries exceed 10 8 individual transformants and close to 100% of the clones harbor a phage display vector having an insert with the length of a VHH gene. These libraries are also more economic to make than previous standard approaches using classical restriction enzymes and ligations. The quality of the Nanobodies that are retrieved from immune libraries obtained by Golden Gate Cloning is identical to those from immune libraries made according to the classical procedure.

  9. An optimized lentiviral vector system for conditional RNAi and efficient cloning of microRNA embedded short hairpin RNA libraries.

    Science.gov (United States)

    Adams, Felix F; Heckl, Dirk; Hoffmann, Thomas; Talbot, Steven R; Kloos, Arnold; Thol, Felicitas; Heuser, Michael; Zuber, Johannes; Schambach, Axel; Schwarzer, Adrian

    2017-09-01

    RNA interference (RNAi) and CRISPR-Cas9-based screening systems have emerged as powerful and complementary tools to unravel genetic dependencies through systematic gain- and loss-of-function studies. In recent years, a series of technical advances helped to enhance the performance of virally delivered RNAi. For instance, the incorporation of short hairpin RNAs (shRNAs) into endogenous microRNA contexts (shRNAmiRs) allows the use of Tet-regulated promoters for synchronous onset of gene knockdown and precise interrogation of gene dosage effects. However, remaining challenges include lack of efficient cloning strategies, inconsistent knockdown potencies and leaky expression. Here, we present a simple, one-step cloning approach for rapid and efficient cloning of miR-30 shRNAmiR libraries. We combined a human miR-30 backbone retaining native flanking sequences with an optimized all-in-one lentiviral vector system for conditional RNAi to generate a versatile toolbox characterized by higher doxycycline sensitivity, reduced leakiness and enhanced titer. Furthermore, refinement of existing shRNA design rules resulted in substantially improved prediction of powerful shRNAs. Our approach was validated by accurate quantification of the knockdown potency of over 250 single shRNAmiRs. To facilitate access and use by the scientific community, an online tool was developed for the automated design of refined shRNA-coding oligonucleotides ready for cloning into our system. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Human Chromosome 21: Mapping of the chromosomes and cloning of cDNAs

    Energy Technology Data Exchange (ETDEWEB)

    Antonarakis, S.E.

    1991-09-01

    The objective of the research funded by DOE grant DE-FG02-89ER60857 from 6/15/89 to 8/31/91 was to contribute to the physical mapping of human chromosome 21 (HC21) by cloning large fragments of DNA into Yeast Artificial Chromosomes (YACs) and identify YACs that map on HC21. A total of 54 sequence tagged sites (STS) have been developed and mapped in our laboratory to HC21 and can be used as initial reference points for YAC identification and construction of overlapping clones. A small YAC library was constructed which is HC21 specific. DNA from somatic cell hybrid WAV17 or from flow-sorted HC21 was partially digested with EcoRI, ligated into vectors PJS97, PJS98, and YACs have been obtained with average size insert of more than 300 kb. This library has been deposited in D. Patterson's lab for the Joint YAC screening effort. Additional YAC libraries from ICI Pharmaceuticals or from Los Alamos National Laboratories have been screened with several STS and positive YACs have been identified. Work in progress includes screening of YAC libraries in order to construct overlapping clones, characterization of the cloning ends of YACs, characterization of additional STS and cloning of HC21 specific cDNAs. 15 refs., 2 figs., 5 tabs.

  11. [cDNA library construction from panicle meristem of finger millet].

    Science.gov (United States)

    Radchuk, V; Pirko, Ia V; Isaenkov, S V; Emets, A I; Blium, Ia B

    2014-01-01

    The protocol for production of full-size cDNA using SuperScript Full-Length cDNA Library Construction Kit II (Invitrogen) was tested and high quality cDNA library from meristematic tissue of finger millet panicle (Eleusine coracana (L.) Gaertn) was created. The titer of obtained cDNA library comprised 3.01 x 10(5) CFU/ml in avarage. In average the length of cDNA insertion consisted about 1070 base pairs, the effectivity of cDNA fragment insertions--99.5%. The selective sequencing of cDNA clones from created library was performed. The sequences of cDNA clones were identified with usage of BLAST-search. The results of cDNA library analysis and selective sequencing represents prove good functionality and full length character of inserted cDNA clones. Obtained cDNA library from meristematic tissue of finger millet panicle represents good and valuable source for isolation and identification of key genes regulating metabolism and meristematic development and for mining of new molecular markers to conduct out high quality genetic investigations and molecular breeding as well.

  12. Seasonal diversity of planktonic protists in Southwestern Alberta rivers over a 1-year period as revealed by terminal restriction fragment length polymorphism and 18S rRNA gene library analyses.

    Science.gov (United States)

    Thomas, Matthew C; Selinger, L Brent; Inglis, G Douglas

    2012-08-01

    The temporal dynamics of planktonic protists in river water have received limited attention despite their ecological significance and recent studies linking phagotrophic protists to the persistence of human-pathogenic bacteria. Using molecular-based techniques targeting the 18S rRNA gene, we studied the seasonal diversity of planktonic protists in Southwestern Alberta rivers (Oldman River Basin) over a 1-year period. Nonmetric multidimensional scaling analysis of terminal restriction fragment length polymorphism (T-RFLP) data revealed distinct shifts in protistan community profiles that corresponded to season rather than geographical location. Community structures were examined by using clone library analysis; HaeIII restriction profiles of 18S rRNA gene amplicons were used to remove prevalent solanaceous plant clones prior to sequencing. Sanger sequencing of the V1-to-V3 region of the 18S rRNA gene libraries from spring, summer, fall, and winter supported the T-RFLP results and showed marked seasonal differences in the protistan community structure. The spring library was dominated by Chloroplastidae (29.8%), Centrohelida (28.1%), and Alveolata (25.5%), while the summer and fall libraries contained primarily fungal clones (83.0% and 88.0%, respectively). Alveolata (35.6%), Euglenozoa (24.4%), Chloroplastida (15.6%), and Fungi (15.6%) dominated the winter library. These data demonstrate that planktonic protists, including protozoa, are abundant in river water in Southwestern Alberta and that conspicuous seasonal shifts occur in the community structure.

  13. [Construction of fetal mesenchymal stem cell cDNA subtractive library].

    Science.gov (United States)

    Yang, Li; Wang, Dong-Mei; Li, Liang; Bai, Ci-Xian; Cao, Hua; Li, Ting-Yu; Pei, Xue-Tao

    2002-04-01

    To identify differentially expressed genes between fetal mesenchymal stem cell (MSC) and adult MSC, especially specified genes expressed in fetal MSC, a cDNA subtractive library of fetal MSC was constructed using suppression subtractive hybridization (SSH) technique. At first, total RNA was isolated from fetal and adult MSC. Using SMART PCR synthesis method, single-strand and double-strand cDNAs were synthesized. After Rsa I digestion, fetal MSC cDNAs were divided into two groups and ligated to adaptor 1 and adaptor 2 respectively. Results showed that the amplified library contains 890 clones. Analysis of 890 clones with PCR demonstrated that 768 clones were positive. The positive rate is 86.3%. The size of inserted fragments in these positive clones was between 0.2 - 1 kb, with an average of 400 - 600 bp. SSH is a convenient and effective method for screening differentially expressed genes. The constructed cDNA subtractive library of fetal MSC cDNA lays solid foundation for screening and cloning new and specific function related genes of fetal MSC.

  14. A new set of ESTs and cDNA clones from full-length and normalized libraries for gene discovery and functional characterization in citrus

    Directory of Open Access Journals (Sweden)

    Alamar Santiago

    2009-09-01

    Full Text Available Abstract Background Interpretation of ever-increasing raw sequence information generated by modern genome sequencing technologies faces multiple challenges, such as gene function analysis and genome annotation. Indeed, nearly 40% of genes in plants encode proteins of unknown function. Functional characterization of these genes is one of the main challenges in modern biology. In this regard, the availability of full-length cDNA clones may fill in the gap created between sequence information and biological knowledge. Full-length cDNA clones facilitate functional analysis of the corresponding genes enabling manipulation of their expression in heterologous systems and the generation of a variety of tagged versions of the native protein. In addition, the development of full-length cDNA sequences has the power to improve the quality of genome annotation. Results We developed an integrated method to generate a new normalized EST collection enriched in full-length and rare transcripts of different citrus species from multiple tissues and developmental stages. We constructed a total of 15 cDNA libraries, from which we isolated 10,898 high-quality ESTs representing 6142 different genes. Percentages of redundancy and proportion of full-length clones range from 8 to 33, and 67 to 85, respectively, indicating good efficiency of the approach employed. The new EST collection adds 2113 new citrus ESTs, representing 1831 unigenes, to the collection of citrus genes available in the public databases. To facilitate functional analysis, cDNAs were introduced in a Gateway-based cloning vector for high-throughput functional analysis of genes in planta. Herein, we describe the technical methods used in the library construction, sequence analysis of clones and the overexpression of CitrSEP, a citrus homolog to the Arabidopsis SEP3 gene, in Arabidopsis as an example of a practical application of the engineered Gateway vector for functional analysis. Conclusion The new

  15. A new set of ESTs and cDNA clones from full-length and normalized libraries for gene discovery and functional characterization in citrus

    Science.gov (United States)

    Marques, M Carmen; Alonso-Cantabrana, Hugo; Forment, Javier; Arribas, Raquel; Alamar, Santiago; Conejero, Vicente; Perez-Amador, Miguel A

    2009-01-01

    Background Interpretation of ever-increasing raw sequence information generated by modern genome sequencing technologies faces multiple challenges, such as gene function analysis and genome annotation. Indeed, nearly 40% of genes in plants encode proteins of unknown function. Functional characterization of these genes is one of the main challenges in modern biology. In this regard, the availability of full-length cDNA clones may fill in the gap created between sequence information and biological knowledge. Full-length cDNA clones facilitate functional analysis of the corresponding genes enabling manipulation of their expression in heterologous systems and the generation of a variety of tagged versions of the native protein. In addition, the development of full-length cDNA sequences has the power to improve the quality of genome annotation. Results We developed an integrated method to generate a new normalized EST collection enriched in full-length and rare transcripts of different citrus species from multiple tissues and developmental stages. We constructed a total of 15 cDNA libraries, from which we isolated 10,898 high-quality ESTs representing 6142 different genes. Percentages of redundancy and proportion of full-length clones range from 8 to 33, and 67 to 85, respectively, indicating good efficiency of the approach employed. The new EST collection adds 2113 new citrus ESTs, representing 1831 unigenes, to the collection of citrus genes available in the public databases. To facilitate functional analysis, cDNAs were introduced in a Gateway-based cloning vector for high-throughput functional analysis of genes in planta. Herein, we describe the technical methods used in the library construction, sequence analysis of clones and the overexpression of CitrSEP, a citrus homolog to the Arabidopsis SEP3 gene, in Arabidopsis as an example of a practical application of the engineered Gateway vector for functional analysis. Conclusion The new EST collection denotes an

  16. CitEST libraries

    Directory of Open Access Journals (Sweden)

    Maria Luísa P. Natividade Targon

    2007-01-01

    Full Text Available In order to obtain a better understanding of what is citrus, 33 cDNA libraries were constructed from different citrus species and genera. Total RNA was extracted from fruits, leaves, flowers, bark, seeds and roots, and subjected or not to different biotic and abiotic stresses (pathogens and drought and at several developmental stages. To identify putative promoter sequences, as well as molecular markers that could be useful for breeding programs, one shotgun library was prepared from sweet orange (Citrus sinensis var. Olimpia. In addition, EST libraries were also constructed for a citrus pathogen, the oomycete Phythophthora parasitica in either virulent or avirulent form. A total of 286,559 cDNA clones from citrus were sequenced from their 5’ end, generating 242,790 valid reads of citrus. A total of 9,504 sequences were produced in the shotgun library and the valid reads were assembled using CAP3. In this procedure, we obtained 1,131 contigs and 4,083 singletons. A total of 19,200 cDNA clones from P. parasitica were sequenced, resulting in 16,400 valid reads. The number of ESTs generated in this project is, to our knowledge, the largest citrus sequence database in the world.

  17. Microbial community in persistent apical periodontitis: a 16S rRNA gene clone library analysis.

    Science.gov (United States)

    Zakaria, M N; Takeshita, T; Shibata, Y; Maeda, H; Wada, N; Akamine, A; Yamashita, Y

    2015-08-01

    To characterize the microbial composition of persistent periapical lesions of root filled teeth using a molecular genetics approach. Apical lesion samples were collected from 12 patients (23-80 years old) who visited the Kyushu University Hospital for apicectomy with persistent periapical lesions associated with root filled teeth. DNA was directly extracted from each sample and the microbial composition was comprehensively analysed using clone library analysis of the 16S rRNA gene. Enterococcus faecalis, Candida albicans and specific fimA genotypes of Porphyromonas gingivalis were confirmed using polymerase chain reaction (PCR) analysis with specific primers. Bacteria were detected in all samples, and the dominant findings were P. gingivalis (19.9%), Fusobacterium nucleatum (11.2%) and Propionibacterium acnes (9%). Bacterial diversity was greater in symptomatic lesions than in asymptomatic ones. In addition, the following bacteria or bacterial combinations were characteristic to symptomatic lesions: Prevotella spp., Treponema spp., Peptostreptococcaceae sp. HOT-113, Olsenella uli, Slackia exigua, Selemonas infelix, P. gingivalis with type IV fimA, and a combination of P. gingivalis, F. nucleatum, and Peptostreptococcaceae sp. HOT-113 and predominance of Streptococcus spp. On the other hand, neither Enterococcus faecalis nor C. albicans were detected in any of the samples. Whilst a diverse bacterial species were observed in the persistent apical lesions, some characteristic patterns of bacterial community were found in the symptomatic lesions. The diverse variation of community indicates that bacterial combinations as a community may cause persistent inflammation in periapical tissues rather than specific bacterial species. © 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd.

  18. Distribution and uses of legume DNA clone resources

    International Nuclear Information System (INIS)

    Young, N.D.

    2001-01-01

    Since 1990, my lab has developed and distributed various DNA clone resources for the legumes. In the first several years, the focus was on members of the tropical genus, Vigna, including the widely cultivated species, mungbean (V. radiata) and cowpea (V. unguiculata). Both of these grain legumes play key roles in agriculture in developing countries of Asia (mungbean) and Africa (cowpea). Moreover, because there is substantial genome conservation among legumes, these genetic resources have also been utilized by a wide range of researchers in other crop species. In 1997, my lab began to focus on the development and distribution of a new generation of DNA clone resources; Bacterial Artificial Chromosomes (BAC). A library of these clones was constructed in soybean (Glycine max) the most important legume species worldwide in terms of economic value. Again, the library has become a valuable resource for the legume research community and has been widely used in studies of legume genomics. (author)

  19. Bibliometric analyses on repository contents as a library service for the evaluation of research

    NARCIS (Netherlands)

    Veller, van M.G.P.; Gerritsma, W.

    2010-01-01

    Since the last two decennia, the library of Wageningen University and Research (or Wageningen UR) has been involved in several bibliometric analyses for the evaluation of scientific output of staff, chair groups, research institutes and graduate schools. In these bibliometric analyses several

  20. Speeding cis-trans regulation discovery by phylogenomic analyses coupled with screenings of an arrayed library of Arabidopsis transcription factors.

    Directory of Open Access Journals (Sweden)

    Gabriel Castrillo

    Full Text Available Transcriptional regulation is an important mechanism underlying gene expression and has played a crucial role in evolution. The number, position and interactions between cis-elements and transcription factors (TFs determine the expression pattern of a gene. To identify functionally relevant cis-elements in gene promoters, a phylogenetic shadowing approach with a lipase gene (LIP1 was used. As a proof of concept, in silico analyses of several Brassicaceae LIP1 promoters identified a highly conserved sequence (LIP1 element that is sufficient to drive strong expression of a reporter gene in planta. A collection of ca. 1,200 Arabidopsis thaliana TF open reading frames (ORFs was arrayed in a 96-well format (RR library and a convenient mating based yeast one hybrid (Y1H screening procedure was established. We constructed an episomal plasmid (pTUY1H to clone the LIP1 element and used it as bait for Y1H screenings. A novel interaction with an HD-ZIP (AtML1 TF was identified and abolished by a 2 bp mutation in the LIP1 element. A role of this interaction in transcriptional regulation was confirmed in planta. In addition, we validated our strategy by reproducing the previously reported interaction between a MYB-CC (PHR1 TF, a central regulator of phosphate starvation responses, with a conserved promoter fragment (IPS1 element containing its cognate binding sequence. Finally, we established that the LIP1 and IPS1 elements were differentially bound by HD-ZIP and MYB-CC family members in agreement with their genetic redundancy in planta. In conclusion, combining in silico analyses of orthologous gene promoters with Y1H screening of the RR library represents a powerful approach to decipher cis- and trans-regulatory codes.

  1. Horse cDNA clones encoding two MHC class I genes

    Energy Technology Data Exchange (ETDEWEB)

    Barbis, D.P.; Maher, J.K.; Stanek, J.; Klaunberg, B.A.; Antczak, D.F.

    1994-12-31

    Two full-length clones encoding MHC class I genes were isolated by screening a horse cDNA library, using a probe encoding in human HLA-A2.2Y allele. The library was made in the pcDNA1 vector (Invitrogen, San Diego, CA), using mRNA from peripheral blood lymphocytes obtained from a Thoroughbred stallion (No. 0834) homozygous for a common horse MHC haplotype (ELA-A2, -B2, -D2; Antczak et al. 1984; Donaldson et al. 1988). The clones were sequenced, using SP6 and T7 universal primers and horse-specific oligonucleotides designed to extend previously determined sequences.

  2. Complementation of the UV-sensitive phenotype of a xeroderma pigmentosum human cell line by transfection with a cDNA clone library

    International Nuclear Information System (INIS)

    Teitz, T.; Naiman, T.; Avissar, S.S.; Bar, S.; Okayama, H.; Canaani, D.

    1987-01-01

    In previous work, a xeroderma pigmentosum cell line belonging to complementation group C was established by transformation with origin-defective simian virus 40. We now report the complementation of the UV sensitivity of this cell line by gene transfer. A human cDNA clone library constructed in a mammalian expression vector, and itself incorporated in a lambda phage vector, was introduced into the cells as a calcium phosphate precipitate. Following selection to G418 resistance, provided by the neo gene of the vector, transformants were selected for UV resistance. Twenty-one cell clones were obtained with UV-resistance levels typical of normal human fibroblasts. All transformants contained vector DNA sequences in their nuclei. Upon further propagation in the absence of selection for G418 resistance, about half of the primary transformants remained UV-resistant. Secondary transformants were generated by transfection with a partial digest of total chromosomal DNA from one of these stable transformants. This resulted in 15 G418-resistant clones, 2 of which exhibited a UV-resistant phenotype. The other primary clones lost UV resistance rapidly when subcultured in the absence of G418. Importantly, several retained UV resistance under G418 selection pressure. The acquisition of UV resistance by secondary transformants derived by transfection of DNA from a stable primary transformant, and the linkage between G418 and UV resistances in the unstable primary transformants, strongly suggests that the transformants acquired UV resistance through DNA-mediated gene transfer and not by reversion

  3. Analysing malaria drug trials on a per-individual or per-clone basis: a comparison of methods.

    Science.gov (United States)

    Jaki, Thomas; Parry, Alice; Winter, Katherine; Hastings, Ian

    2013-07-30

    There are a variety of methods used to estimate the effectiveness of antimalarial drugs in clinical trials, invariably on a per-person basis. A person, however, may have more than one malaria infection present at the time of treatment. We evaluate currently used methods for analysing malaria trials on a per-individual basis and introduce a novel method to estimate the cure rate on a per-infection (clone) basis. We used simulated and real data to highlight the differences of the various methods. We give special attention to classifying outcomes as cured, recrudescent (infections that never fully cleared) or ambiguous on the basis of genetic markers at three loci. To estimate cure rates on a per-clone basis, we used the genetic information within an individual before treatment to determine the number of clones present. We used the genetic information obtained at the time of treatment failure to classify clones as recrudescence or new infections. On the per-individual level, we find that the most accurate methods of classification label an individual as newly infected if all alleles are different at the beginning and at the time of failure and as a recrudescence if all or some alleles were the same. The most appropriate analysis method is survival analysis or alternatively for complete data/per-protocol analysis a proportion estimate that treats new infections as successes. We show that the analysis of drug effectiveness on a per-clone basis estimates the cure rate accurately and allows more detailed evaluation of the performance of the treatment. Copyright © 2012 John Wiley & Sons, Ltd.

  4. Cdna cloning and expression analyses of the isoflavone reductase-like gene of dendrobium officinale

    International Nuclear Information System (INIS)

    Qian, X.; Xu, S.Z.

    2015-01-01

    The full length of the isoflavone reductase-like gene (IRL) cDNA of Dendrobium officinale was cloned by using reverse transcription (RT) PCR combined with cDNA library, the IRL function was identified by Bioinformatics and prokaryotic expression analyses, and the IRL expression levels in the organs and tissues of D. officinale plants with different ages were determined by using real-time quantitative PCR (RT-qPCR). The results indicated that the full length of the cDNA of D. officinale IRL, DoIRL, was 1238 bp (accession no. KJ661023). Its open reading frame (ORF) was 930 bp which encoded 309 amino acids with a predicted molecular mass of 34 kDa, the 5 untranslated region (UTR) was 61 bp and the 3 UTR containing a poly (A) tail was 247 bp. The deduced amino acid sequence of DoIRL, DoIRL, was forecast to contain a NAD(P)H-binding motif (GGTGYIG) in the N-terminal region, two conserved N-glycosylation sites, a conserved nitrogen metabolite repression regulator (NmrA) domain and a phenylcoumaran benzylic ether reductase (PCBER) domain, to hold the nearest phylogenetic relationship with the PCBER of Striga asiatica, and to share both 73% identity with the isoflavone reductases-like (IRLs) of Cucumis sativus and Striga asiatica. In Escherichia coli 'BL21' cells, the DoIRL cDNA expression produced a protein band holding the predicted molecular mass of 34 kDa. DoIRL expressed in all organs and tissues of D. officinale plants with different ages at comparatively low levels, and the expression level in the leaves of the two-year-old plants was the highest. (author)

  5. RTA, a candidate G protein-coupled receptor: Cloning, sequencing, and tissue distribution

    International Nuclear Information System (INIS)

    Ross, P.C.; Figler, R.A.; Corjay, M.H.; Barber, C.M.; Adam, N.; Harcus, D.R.; Lynch, K.R.

    1990-01-01

    Genomic and cDNA clones, encoding a protein that is a member of the guanine nucleotide-binding regulatory protein (G protein)-coupled receptor superfamily, were isolated by screening rat genomic and thoracic aorta cDNA libraries with an oligonucleotide encoding a highly conserved region of the M 1 muscarinic acetylcholine receptor. Sequence analyses of these clones showed that they encode a 343-amino acid protein (named RTA). The RTA gene is single copy, as demonstrated by restriction mapping and Southern blotting of genomic clones and rat genomic DNA. RTA RNA sequences are relatively abundant throughout the gut, vas deferens, uterus, and aorta but are only barely detectable (on Northern blots) in liver, kidney, lung, and salivary gland. In the rat brain, RTA sequences are markedly abundant in the cerebellum. TRA is most closely related to the mas oncogene (34% identity), which has been suggested to be a forebrain angiotensin receptor. They conclude that RTA is not an angiotensin receptor; to date, they have been unable to identify its ligand

  6. A universal phage display system for the seamless construction of Fab libraries.

    Science.gov (United States)

    Nelson, Renae S; Valadon, Philippe

    2017-11-01

    The construction of Fab phage libraries requires the cloning of domains from both the light and the heavy chain of antibodies. Despite the advent of powerful strategies such as splicing-by-overlap extension PCR, obtaining high quality libraries with excellent coverage remains challenging. Here, we explored the use of type IIS restriction enzymes for the seamless cloning of Fab libraries. We analyzed human, murine and rabbit germline antibody repertoires and identified combinations of restriction enzymes that exhibit very few or no recognition sites in the antibody sequences. We describe three phagemid vectors, pUP-22Hb, pUP-22Mc and pUP-22Rc, which were employed for cloning the Fab repertoire of these hosts using BsmBI and SapI (human) or SapI alone (mouse and rabbit). Using human serum albumin as a model immunization, we built a mouse/human chimeric Fab library and a mouse Fab library in a single step ligation and successfully panned multiple cognate antibodies. The overall process is highly scalable and faster than PCR-based techniques, with a Fab insertion success rate of around 80%. By using carefully chosen overhangs on each end of the antibody domains, this approach paves the way to the universal, sequence- and vector-independent cloning and reformatting of antibody libraries. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. GenMapDB: a database of mapped human BAC clones

    OpenAIRE

    Morley, Michael; Arcaro, Melissa; Burdick, Joshua; Yonescu, Raluca; Reid, Thomas; Kirsch, Ilan R.; Cheung, Vivian G.

    2001-01-01

    GenMapDB (http://genomics.med.upenn.edu/genmapdb) is a repository of human bacterial artificial chromosome (BAC) clones mapped by our laboratory to sequence-tagged site markers. Currently, GenMapDB contains over 3000 mapped clones that span 19 chromosomes, chromosomes 2, 4, 5, 9–22, X and Y. This database provides positional information about human BAC clones from the RPCI-11 human male BAC library. It also contains restriction fragment analysis data and end sequen...

  8. A rapid method for screening arrayed plasmid cDNA library by PCR

    International Nuclear Information System (INIS)

    Hu Yingchun; Zhang Kaitai; Wu Dechang; Li Gang; Xiang Xiaoqiong

    1999-01-01

    Objective: To develop a PCR-based method for rapid and effective screening of arrayed plasmid cDNA library. Methods: The plasmid cDNA library was arrayed and screened by PCR with a particular set of primers. Results: Four positive clones were obtained through about one week. Conclusion: This method can be applied to screening not only normal cDNA clones, but also cDNA clones-containing small size fragments. This method offers significant advantages over traditional screening method in terms of sensitivity, specificity and efficiency

  9. Whitefly (Bemisia tabaci genome project: analysis of sequenced clones from egg, instar, and adult (viruliferous and non-viruliferous cDNA libraries

    Directory of Open Access Journals (Sweden)

    Czosnek Henryk

    2006-04-01

    Full Text Available Abstract Background The past three decades have witnessed a dramatic increase in interest in the whitefly Bemisia tabaci, owing to its nature as a taxonomically cryptic species, the damage it causes to a large number of herbaceous plants because of its specialized feeding in the phloem, and to its ability to serve as a vector of plant viruses. Among the most important plant viruses to be transmitted by B. tabaci are those in the genus Begomovirus (family, Geminiviridae. Surprisingly, little is known about the genome of this whitefly. The haploid genome size for male B. tabaci has been estimated to be approximately one billion bp by flow cytometry analysis, about five times the size of the fruitfly Drosophila melanogaster. The genes involved in whitefly development, in host range plasticity, and in begomovirus vector specificity and competency, are unknown. Results To address this general shortage of genomic sequence information, we have constructed three cDNA libraries from non-viruliferous whiteflies (eggs, immature instars, and adults and two from adult insects that fed on tomato plants infected by two geminiviruses: Tomato yellow leaf curl virus (TYLCV and Tomato mottle virus (ToMoV. In total, the sequence of 18,976 clones was determined. After quality control, and removal of 5,542 clones of mitochondrial origin 9,110 sequences remained which included 3,843 singletons and 1,017 contigs. Comparisons with public databases indicated that the libraries contained genes involved in cellular and developmental processes. In addition, approximately 1,000 bases aligned with the genome of the B. tabaci endosymbiotic bacterium Candidatus Portiera aleyrodidarum, originating primarily from the egg and instar libraries. Apart from the mitochondrial sequences, the longest and most abundant sequence encodes vitellogenin, which originated from whitefly adult libraries, indicating that much of the gene expression in this insect is directed toward the production

  10. Description of Transmutation Library for Fuel Cycle System Analyses

    International Nuclear Information System (INIS)

    Piet, Steven J.; Bays, Samuel E.; Hoffman, Edward A.

    2010-01-01

    This report documents the Transmutation Library that is used in Fuel Cycle System Analyses. This version replaces the 2008 version.(Piet2008) The Transmutation Library has the following objectives: (1) Assemble past and future transmutation cases for system analyses. (2) For each case, assemble descriptive information such as where the case was documented, the purpose of the calculation, the codes used, source of feed material, transmutation parameters, and the name of files that contain raw or source data. (3) Group chemical elements so that masses in separation and waste processes as calculated in dynamic simulations or spreadsheets reflect current thinking of those processes. For example, the CsSr waste form option actually includes all Group 1A and 2A elements. (4) Provide mass fractions at input (charge) and output (discharge) for each case. (5) Eliminate the need for either ''fission product other'' or ''actinide other'' while conserving mass. Assessments of waste and separation cannot use ''fission product other'' or ''actinide other'' as their chemical behavior is undefined. (6) Catalog other isotope-specific information in one place, e.g., heat and dose conversion factors for individual isotopes. (7) Describe the correlations for how input and output compositions change as a function of UOX burnup (for LWR UOX fuel) or fast reactor (FR) transuranic (TRU) conversion ratio (CR) for either FR-metal or FR-oxide. This document therefore includes the following sections: (1) Explanation of the data set information, i.e., the data that describes each case. In no case are all of the data presented in the Library included in previous documents. In assembling the Library, we return to raw data files to extract the case and isotopic data, into the specified format. (2) Explanation of which isotopes and elements are tracked. For example, the transition metals are tracked via the following: two Zr isotopes, Zr-other, Tc99, Tc-other, two Mo-Ru-Rh-Pd isotopes, Mo

  11. Libraries of Synthetic TALE-Activated Promoters: Methods and Applications.

    Science.gov (United States)

    Schreiber, T; Tissier, A

    2016-01-01

    The discovery of proteins with programmable DNA-binding specificities triggered a whole array of applications in synthetic biology, including genome editing, regulation of transcription, and epigenetic modifications. Among those, transcription activator-like effectors (TALEs) due to their natural function as transcription regulators, are especially well-suited for the development of orthogonal systems for the control of gene expression. We describe here the construction and testing of libraries of synthetic TALE-activated promoters which are under the control of a single TALE with a given DNA-binding specificity. These libraries consist of a fixed DNA-binding element for the TALE, a TATA box, and variable sequences of 19 bases upstream and 43 bases downstream of the DNA-binding element. These libraries were cloned using a Golden Gate cloning strategy making them usable as standard parts in a modular cloning system. The broad range of promoter activities detected and the versatility of these promoter libraries make them valuable tools for applications in the fine-tuning of expression in metabolic engineering projects or in the design and implementation of regulatory circuits. © 2016 Elsevier Inc. All rights reserved.

  12. Construction of a nurse shark (Ginglymostoma cirratum bacterial artificial chromosome (BAC library and a preliminary genome survey

    Directory of Open Access Journals (Sweden)

    Inoko Hidetoshi

    2006-05-01

    Full Text Available Abstract Background Sharks are members of the taxonomic class Chondrichthyes, the oldest living jawed vertebrates. Genomic studies of this group, in comparison to representative species in other vertebrate taxa, will allow us to theorize about the fundamental genetic, developmental, and functional characteristics in the common ancestor of all jawed vertebrates. Aims In order to obtain mapping and sequencing data for comparative genomics, we constructed a bacterial artificial chromosome (BAC library for the nurse shark, Ginglymostoma cirratum. Results The BAC library consists of 313,344 clones with an average insert size of 144 kb, covering ~4.5 × 1010 bp and thus providing an 11-fold coverage of the haploid genome. BAC end sequence analyses revealed, in addition to LINEs and SINEs commonly found in other animal and plant genomes, two new groups of nurse shark-specific repetitive elements, NSRE1 and NSRE2 that seem to be major components of the nurse shark genome. Screening the library with single-copy or multi-copy gene probes showed 6–28 primary positive clones per probe of which 50–90% were true positives, demonstrating that the BAC library is representative of the different regions of the nurse shark genome. Furthermore, some BAC clones contained multiple genes, making physical mapping feasible. Conclusion We have constructed a deep-coverage, high-quality, large insert, and publicly available BAC library for a cartilaginous fish. It will be very useful to the scientific community interested in shark genomic structure, comparative genomics, and functional studies. We found two new groups of repetitive elements specific to the nurse shark genome, which may contribute to the architecture and evolution of the nurse shark genome.

  13. Construction of a nurse shark (Ginglymostoma cirratum) bacterial artificial chromosome (BAC) library and a preliminary genome survey.

    Science.gov (United States)

    Luo, Meizhong; Kim, Hyeran; Kudrna, Dave; Sisneros, Nicholas B; Lee, So-Jeong; Mueller, Christopher; Collura, Kristi; Zuccolo, Andrea; Buckingham, E Bryan; Grim, Suzanne M; Yanagiya, Kazuyo; Inoko, Hidetoshi; Shiina, Takashi; Flajnik, Martin F; Wing, Rod A; Ohta, Yuko

    2006-05-03

    Sharks are members of the taxonomic class Chondrichthyes, the oldest living jawed vertebrates. Genomic studies of this group, in comparison to representative species in other vertebrate taxa, will allow us to theorize about the fundamental genetic, developmental, and functional characteristics in the common ancestor of all jawed vertebrates. In order to obtain mapping and sequencing data for comparative genomics, we constructed a bacterial artificial chromosome (BAC) library for the nurse shark, Ginglymostoma cirratum. The BAC library consists of 313,344 clones with an average insert size of 144 kb, covering ~4.5 x 1010 bp and thus providing an 11-fold coverage of the haploid genome. BAC end sequence analyses revealed, in addition to LINEs and SINEs commonly found in other animal and plant genomes, two new groups of nurse shark-specific repetitive elements, NSRE1 and NSRE2 that seem to be major components of the nurse shark genome. Screening the library with single-copy or multi-copy gene probes showed 6-28 primary positive clones per probe of which 50-90% were true positives, demonstrating that the BAC library is representative of the different regions of the nurse shark genome. Furthermore, some BAC clones contained multiple genes, making physical mapping feasible. We have constructed a deep-coverage, high-quality, large insert, and publicly available BAC library for a cartilaginous fish. It will be very useful to the scientific community interested in shark genomic structure, comparative genomics, and functional studies. We found two new groups of repetitive elements specific to the nurse shark genome, which may contribute to the architecture and evolution of the nurse shark genome.

  14. Creation of BAC genomic resources for cocoa ( Theobroma cacao L.) for physical mapping of RGA containing BAC clones.

    Science.gov (United States)

    Clément, D; Lanaud, C; Sabau, X; Fouet, O; Le Cunff, L; Ruiz, E; Risterucci, A M; Glaszmann, J C; Piffanelli, P

    2004-05-01

    We have constructed and validated the first cocoa ( Theobroma cacao L.) BAC library, with the aim of developing molecular resources to study the structure and evolution of the genome of this perennial crop. This library contains 36,864 clones with an average insert size of 120 kb, representing approximately ten haploid genome equivalents. It was constructed from the genotype Scavina-6 (Sca-6), a Forastero clone highly resistant to cocoa pathogens and a parent of existing mapping populations. Validation of the BAC library was carried out with a set of 13 genetically-anchored single copy and one duplicated markers. An average of nine BAC clones per probe was identified, giving an initial experimental estimation of the genome coverage represented in the library. Screening of the library with a set of resistance gene analogues (RGAs), previously mapped in cocoa and co-localizing with QTL for resistance to Phytophthora traits, confirmed at the physical level the tight clustering of RGAs in the cocoa genome and provided the first insights into the relationships between genetic and physical distances in the cocoa genome. This library represents an available BAC resource for structural genomic studies or map-based cloning of genes corresponding to important QTLs for agronomic traits such as resistance genes to major cocoa pathogens like Phytophthora spp ( palmivora and megakarya), Crinipellis perniciosa and Moniliophthora roreri.

  15. Development of high-throughput phenotyping of metagenomic clones from the human gut microbiome for modulation of eukaryotic cell growth.

    Science.gov (United States)

    Gloux, Karine; Leclerc, Marion; Iliozer, Harout; L'Haridon, René; Manichanh, Chaysavanh; Corthier, Gérard; Nalin, Renaud; Blottière, Hervé M; Doré, Joël

    2007-06-01

    Metagenomic libraries derived from human intestinal microbiota (20,725 clones) were screened for epithelial cell growth modulation. Modulatory clones belonging to the four phyla represented among the metagenomic libraries were identified (hit rate, 0.04 to 8.7% depending on the screening cutoff). Several candidate loci were identified by transposon mutagenesis and subcloning.

  16. Chromosome microdissection and cloning in human genome and genetic disease analysis

    International Nuclear Information System (INIS)

    Kao, Faten; Yu, Jingwei

    1991-01-01

    A procedure has been described for microdissection and microcloning of human chromosomal DNA sequences in which universal amplification of the dissected fragments by Mbo I linker adaptor and polymerase chain reaction is used. A very large library comprising 700,000 recombinant plasmid microclones from 30 dissected chromosomes of human chromosome 21 was constructed. Colony hybridization showed that 42% of the clones contained repetitive sequences and 58% contained single or low-copy sequences. The insert sizes generated by complete Mbo I cleavage ranged from 50 to 1,100 base pairs with a mean of 416 base pairs. Southern blot analysis of microclones from the library confirmed their human origin and chromosome 21 specificity. Some of these clones have also been regionally mapped to specific sites of chromosome 21 by using a regional mapping panel of cell hybrids. This chromosome microtechnology can generate large numbers of microclones with unique sequences from defined chromosomal regions and can be used for processes such as (i) isolating corresponding yeast artificial chromosome clones with large inserts, (ii) screening various cDNA libraries for isolating expressed sequences, and (iii) constructing region-specific libraries of the entire human genome. The studies described here demonstrate the power of this technology for high-resolution genome analysis and explicate their use in an efficient search for disease-associated genes localized to specific chromosomal regions

  17. Identification of cDNA clones expressing immunodiagnostic antigens from Trichinella spiralis

    International Nuclear Information System (INIS)

    Zarlenga, D.; Gamble, H.R.

    1987-01-01

    A cDNA expression library was built in lambda gt11 phage using poly A mRNA isolated from Trichinella spiralis muscle stage larvae. This library was screened with rabbit antibodies to parasite excretory-secretory (ES) products and greater than 180 clones were isolated. Thirteen clones producing highly immunogenic protein antigens were plaque purified and rescreened with pig antisera to T.spiralis, Trichuris suis or Ascaris suum to identify clones producing epitopes specific to T.spiralis ES products, only. Two clones, TsAc-2 and TsAc-8, which displayed strong interactions with pig antisera to T. spiralis were lysogenized in E. coli Y1089 and the protein extracted. Western blots of the crude fusion proteins revealed molecular weights of 133 kD and 129 kD, respectively. Northern blot analysis of total RNA with 32 P labelled cDNA:lambda gt11 probes indicated single RNA transcripts for each clone with molecular sizes corresponding to 800-850 nucleotides. dscDNA inserts were estimated by southern blot analysis to be 500 bp and 340 bp, respectively, with no cross-hybridization observed between the cloned sequences. Dot blots using pig sera to screen crude fusion protein preparations, total bacterial protein (negative controls) and crude worm extract or ES products from T.spiralis, T.suis and A.suum (positive controls) corroborated the specificity and sensitivity of these clones as potential diagnostic antigens for swine trichinellosis

  18. Construction of the BAC Library of Small Abalone (Haliotis diversicolor) for Gene Screening and Genome Characterization.

    Science.gov (United States)

    Jiang, Likun; You, Weiwei; Zhang, Xiaojun; Xu, Jian; Jiang, Yanliang; Wang, Kai; Zhao, Zixia; Chen, Baohua; Zhao, Yunfeng; Mahboob, Shahid; Al-Ghanim, Khalid A; Ke, Caihuan; Xu, Peng

    2016-02-01

    The small abalone (Haliotis diversicolor) is one of the most important aquaculture species in East Asia. To facilitate gene cloning and characterization, genome analysis, and genetic breeding of it, we constructed a large-insert bacterial artificial chromosome (BAC) library, which is an important genetic tool for advanced genetics and genomics research. The small abalone BAC library includes 92,610 clones with an average insert size of 120 Kb, equivalent to approximately 7.6× of the small abalone genome. We set up three-dimensional pools and super pools of 18,432 BAC clones for target gene screening using PCR method. To assess the approach, we screened 12 target genes in these 18,432 BAC clones and identified 16 positive BAC clones. Eight positive BAC clones were then sequenced and assembled with the next generation sequencing platform. The assembled contigs representing these 8 BAC clones spanned 928 Kb of the small abalone genome, providing the first batch of genome sequences for genome evaluation and characterization. The average GC content of small abalone genome was estimated as 40.33%. A total of 21 protein-coding genes, including 7 target genes, were annotated into the 8 BACs, which proved the feasibility of PCR screening approach with three-dimensional pools in small abalone BAC library. One hundred fifty microsatellite loci were also identified from the sequences for marker development in the future. The BAC library and clone pools provided valuable resources and tools for genetic breeding and conservation of H. diversicolor.

  19. Construction and Analysis of Siberian Tiger Bacterial Artificial Chromosome Library with Approximately 6.5-Fold Genome Equivalent Coverage

    Science.gov (United States)

    Liu, Changqing; Bai, Chunyu; Guo, Yu; Liu, Dan; Lu, Taofeng; Li, Xiangchen; Ma, Jianzhang; Ma, Yuehui; Guan, Weijun

    2014-01-01

    Bacterial artificial chromosome (BAC) libraries are extremely valuable for the genome-wide genetic dissection of complex organisms. The Siberian tiger, one of the most well-known wild primitive carnivores in China, is an endangered animal. In order to promote research on its genome, a high-redundancy BAC library of the Siberian tiger was constructed and characterized. The library is divided into two sub-libraries prepared from blood cells and two sub-libraries prepared from fibroblasts. This BAC library contains 153,600 individually archived clones; for PCR-based screening of the library, BACs were placed into 40 superpools of 10 × 384-deep well microplates. The average insert size of BAC clones was estimated to be 116.5 kb, representing approximately 6.46 genome equivalents of the haploid genome and affording a 98.86% statistical probability of obtaining at least one clone containing a unique DNA sequence. Screening the library with 19 microsatellite markers and a SRY sequence revealed that each of these markers were present in the library; the average number of positive clones per marker was 6.74 (range 2 to 12), consistent with 6.46 coverage of the tiger genome. Additionally, we identified 72 microsatellite markers that could potentially be used as genetic markers. This BAC library will serve as a valuable resource for physical mapping, comparative genomic study and large-scale genome sequencing in the tiger. PMID:24608928

  20. Chapter 7. Cloning and analysis of natural product pathways.

    Science.gov (United States)

    Gust, Bertolt

    2009-01-01

    The identification of gene clusters of natural products has lead to an enormous wealth of information about their biosynthesis and its regulation, and about self-resistance mechanisms. Well-established routine techniques are now available for the cloning and sequencing of gene clusters. The subsequent functional analysis of the complex biosynthetic machinery requires efficient genetic tools for manipulation. Until recently, techniques for the introduction of defined changes into Streptomyces chromosomes were very time-consuming. In particular, manipulation of large DNA fragments has been challenging due to the absence of suitable restriction sites for restriction- and ligation-based techniques. The homologous recombination approach called recombineering (referred to as Red/ET-mediated recombination in this chapter) has greatly facilitated targeted genetic modifications of complex biosynthetic pathways from actinomycetes by eliminating many of the time-consuming and labor-intensive steps. This chapter describes techniques for the cloning and identification of biosynthetic gene clusters, for the generation of gene replacements within such clusters, for the construction of integrative library clones and their expression in heterologous hosts, and for the assembly of entire biosynthetic gene clusters from the inserts of individual library clones. A systematic approach toward insertional mutation of a complete Streptomyces genome is shown by the use of an in vitro transposon mutagenesis procedure.

  1. [Construction of human phage antibody library and screening for human monoclonal antibodies of amylin].

    Science.gov (United States)

    Gong, Qian; Li, Chang-ying; Chang, Ji-wu; Zhu, Tie-hong

    2012-06-01

    To screen monoclonal antibodies to amylin from a constructed human phage antibody library and identify their antigenic specificity and combining activities. The heavy chain Fd fragment and light chain of human immunoglobulin genes were amplified from peripheral blood lymphocytes of healthy donors using RT-PCR, and then inserted into phagemid pComb3XSS to generate a human phage antibody library. The insertion of light chain or heavy chain Fd genes were identified by PCR after the digestion of Sac I, Xba I, Xho Iand Spe I. One of positive clones was analyzed by DNA sequencing. The specific anti-amylin clones were screened from antibody library against human amylin antigens and then the positive clones were determined by Phage-ELISA analysis. A Fab phage antibody library with 0.8×10(8); members was constructed with the efficacy of about 70%. DNA sequence analysis indicated V(H); gene belonged to V(H);3 gene family and V(λ); gene belonged to the V(λ); gene family. Using human amylin as panning antigen, specific anti-amylin Fab antibodies were enriched by screening the library for three times. Phage-ELISA assay showed the positive clones had very good specificity to amylin antigen. The successful construction of a phage antibody library and the identification of anti-amylin Fab antibodies provide a basis for further study and preparation of human anti-amylin antibodies.

  2. cDNA library construction of two human Demodexspecies.

    Science.gov (United States)

    Niu, DongLing; Wang, RuiLing; Zhao, YaE; Yang, Rui; Hu, Li; Lei, YuYang; Dan, WeiChao

    2017-06-01

    The research of Demodex, a type of pathogen causing various dermatoses in animals and human beings, is lacking at RNA level. This study aims at extracting RNA and constructing cDNA library for Demodex. First, P. cuniculiand D. farinaewere mixed to establish homogenization method for RNA extraction. Second, D. folliculorumand D. breviswere collected and preserved in Trizol, which were mixed with D. farinaerespectively to extract RNA. Finally, cDNA library was constructed and its quality was assessed. The results indicated that for D. folliculorum& D. farinae, the recombination rate of cDNA library was 90.67% and the library titer was 7.50 × 104 pfu/ml. 17 of the 59 positive clones were predicted to be of D. folliculorum; For D. brevis& D. farinae, the recombination rate was 90.96% and the library titer was 7.85 x104 pfu/ml. 40 of the 59 positive clones were predicted to be of D. brevis. Further detection by specific primers demonstrated that mtDNA cox1, cox3and ATP6 detected from cDNA libraries had 96.52%-99.73% identities with the corresponding sequences in GenBank. In conclusion, the cDNA libraries constructed for Demodexmixed with D. farinaewere successful and could satisfy the requirements for functional genes detection.

  3. Primer sets for cloning the human repertoire of T cell Receptor Variable regions.

    Science.gov (United States)

    Boria, Ilenia; Cotella, Diego; Dianzani, Irma; Santoro, Claudio; Sblattero, Daniele

    2008-08-29

    Amplification and cloning of naïve T cell Receptor (TR) repertoires or antigen-specific TR is crucial to shape immune response and to develop immuno-based therapies. TR variable (V) regions are encoded by several genes that recombine during T cell development. The cloning of expressed genes as large diverse libraries from natural sources relies upon the availability of primers able to amplify as many V genes as possible. Here, we present a list of primers computationally designed on all functional TR V and J genes listed in the IMGT, the ImMunoGeneTics information system. The list consists of unambiguous or degenerate primers suitable to theoretically amplify and clone the entire TR repertoire. We show that it is possible to selectively amplify and clone expressed TR V genes in one single RT-PCR step and from as little as 1000 cells. This new primer set will facilitate the creation of more diverse TR libraries than has been possible using currently available primer sets.

  4. Mining olive genome through library sequencing and bioinformatics ...

    African Journals Online (AJOL)

    As one of the initial steps of olive (Olea europaea L.) genome analysis, a small insert genomic DNA library was constructed (digesting olive genomic DNA with SmaI and cloning the digestion products into pUC19 vector) and randomly picked 83 colonies were sequenced. Analysis of the insert sequences revealed 12 clones ...

  5. Cloning and sequencing of the peroxisomal amine oxidase gene from Hansenula polymorpha

    NARCIS (Netherlands)

    Bruinenberg, P. G.; Evers, M.; Waterham, H. R.; Kuipers, J.; Arnberg, A. C.; AB, G.

    1989-01-01

    We have cloned the AMO gene, encoding the microbody matrix enzyme amine oxidase (EC 1.4.3.6) from the yeast Hansenula polymorpha. The gene was isolated by differential screening of a cDNA library, immunoselection, and subsequent screening of a H. polymorpha genomic library. The nucleotide sequence

  6. Cloning of transgenic tobacco BY-2 cells; an efficient method to analyse and reduce high natural heterogeneity of transgene expression.

    Science.gov (United States)

    Nocarova, Eva; Fischer, Lukas

    2009-04-22

    Phenotypic characterization of transgenic cell lines, frequently used in plant biology studies, is complicated because transgene expression in individual cells is often heterogeneous and unstable. To identify the sources and to reduce this heterogeneity, we transformed tobacco (Nicotiana tabacum L.) BY-2 cells with a gene encoding green fluorescent protein (GFP) using Agrobacterium tumefaciens, and then introduced a simple cloning procedure to generate cell lines derived from the individual transformed cells. Expression of the transgene was monitored by analysing GFP fluorescence in the cloned lines and also in lines obtained directly after transformation. The majority ( approximately 90%) of suspension culture lines derived from calli that were obtained directly from transformation consisted of cells with various levels of GFP fluorescence. In contrast, nearly 50% of lines generated by cloning cells from the primary heterogeneous suspensions consisted of cells with homogenous GFP fluorescence. The rest of the lines exhibited "permanent heterogeneity" that could not be resolved by cloning. The extent of fluorescence heterogeneity often varied, even among genetically identical clones derived from the primary transformed lines. In contrast, the offspring of subsequent cloning of the cloned lines was uniform, showing GFP fluorescence intensity and heterogeneity that corresponded to the original clone. The results demonstrate that, besides genetic heterogeneity detected in some lines, the primary lines often contained a mixture of epigenetically different cells that could be separated by cloning. This indicates that a single integration event frequently results in various heritable expression patterns, which are probably accidental and become stabilized in the offspring of the primary transformed cells early after the integration event. Because heterogeneity in transgene expression has proven to be a serious problem, it is highly advisable to use transgenes tagged with

  7. Cost-effective sequencing of full-length cDNA clones powered by a de novo-reference hybrid assembly.

    Science.gov (United States)

    Kuroshu, Reginaldo M; Watanabe, Junichi; Sugano, Sumio; Morishita, Shinichi; Suzuki, Yutaka; Kasahara, Masahiro

    2010-05-07

    Sequencing full-length cDNA clones is important to determine gene structures including alternative splice forms, and provides valuable resources for experimental analyses to reveal the biological functions of coded proteins. However, previous approaches for sequencing cDNA clones were expensive or time-consuming, and therefore, a fast and efficient sequencing approach was demanded. We developed a program, MuSICA 2, that assembles millions of short (36-nucleotide) reads collected from a single flow cell lane of Illumina Genome Analyzer to shotgun-sequence approximately 800 human full-length cDNA clones. MuSICA 2 performs a hybrid assembly in which an external de novo assembler is run first and the result is then improved by reference alignment of shotgun reads. We compared the MuSICA 2 assembly with 200 pooled full-length cDNA clones finished independently by the conventional primer-walking using Sanger sequencers. The exon-intron structure of the coding sequence was correct for more than 95% of the clones with coding sequence annotation when we excluded cDNA clones insufficiently represented in the shotgun library due to PCR failure (42 out of 200 clones excluded), and the nucleotide-level accuracy of coding sequences of those correct clones was over 99.99%. We also applied MuSICA 2 to full-length cDNA clones from Toxoplasma gondii, to confirm that its ability was competent even for non-human species. The entire sequencing and shotgun assembly takes less than 1 week and the consumables cost only approximately US$3 per clone, demonstrating a significant advantage over previous approaches.

  8. Construction of a Bacterial Artificial Chromosome Library of TM-1, a Standard Line for Genetics and Genomics in Upland Cotton

    Institute of Scientific and Technical Information of China (English)

    Yan Hu; Wang-Zhen Guo; Tian-Zhen Zhang

    2009-01-01

    A bacterial artificial chromosome (BAC) library was constructed for Gossyplum hirsutum acc. TM-1, a genetic and genomic standard line for Upland cotton. The library consists of 147 456 clones with an average insert size of 122.8 kb ranging from 97 to 240 kb. About 96.0% of the clones have inserts over 100 kb. Therefore, this library represents theoretically 7.4 haploid genome equivalents based on an AD genome size of 2 425 Mb. Clones were stored in 384 384- well plates and arrayed into multiplex pools for rapid and reliable library screening. BAC screening was carded out by four-round polymerase chain reactions using 23 simple sequence repeats (SSR) markers, three sequence-related amplified polymorphism markers and one pair of pdmere for a gene associated with fiber development to test the quality of the library. Correspondingly, in total 92 positive BAC clones were Identified with an average four positive clones per SSR marker, ranging from one to eight hits. Additionally, since these SSR markers have been localized to chromosome 12 (A12) and 26 (D12) according to the genetic map, these BAC clonee are expected to serve as seeds for the physical mapping of these two homologous chromosomes, sequentially map-based cloning of quantitative trait loci or genes associated with Important agronomic traits.

  9. Anchoring a Defined Sequence to the 55' Ends of mRNAs : The Bolt to Clone Rare Full Length mRNAs and Generate cDNA Libraries porn a Few Cells.

    Science.gov (United States)

    Baptiste, J; Milne Edwards, D; Delort, J; Mallet, J

    1993-01-01

    Among numerous applications, the polymerase chain reaction (PCR) (1,2) provides a convenient means to clone 5' ends of rare mRNAs and to generate cDNA libraries from tissue available in amounts too low to be processed by conventional methods. Basically, the amplification of cDNAs by the PCR requires the availability of the sequences of two stretches of the molecule to be amplified. A sequence can easily be imposed at the 5' end of the first-strand cDNAs (corresponding to the 3' end of the mRNAs) by priming the reverse transcription with a specific primer (for cloning the 5' end of rare messenger) or with an oligonucleotide tailored with a poly (dT) stretch (for cDNA library construction), taking advantage of the poly (A) sequence that is located at the 3' end of mRNAs. Several strategies have been devised to tag the 3' end of the ss-cDNAs (corresponding to the 55' end of the mRNAs). We (3) and others have described strategies based on the addition of a homopolymeric dG (4,5) or dA (6,7) tail using terminal deoxyribonucleotide transferase (TdT) ("anchor-PCR" [4]). However, this strategy has important limitations. The TdT reaction is difficult to control and has a low efficiency (unpublished observations). But most importantly, the return primers containing a homopolymeric (dC or dT) tail generate nonspecific amplifications, a phenomenon that prevents the isolation of low abundance mRNA species and/or interferes with the relative abundance of primary clones in the library. To circumvent these drawbacks, we have used two approaches. First, we devised a strategy based on a cRNA enrichment procedure, which has been useful to eliminate nonspecific-PCR products and to allow detection and cloning of cDNAs of low abundance (3). More recently, to avoid the nonspecific amplification resulting from the annealing of the homopolymeric tail oligonucleotide, we have developed a novel anchoring strategy that is based on the ligation of an oligonucleotide to the 35' end of ss

  10. Construction and Analysis of Siberian Tiger Bacterial Artificial Chromosome Library with Approximately 6.5-Fold Genome Equivalent Coverage

    Directory of Open Access Journals (Sweden)

    Changqing Liu

    2014-03-01

    Full Text Available Bacterial artificial chromosome (BAC libraries are extremely valuable for the genome-wide genetic dissection of complex organisms. The Siberian tiger, one of the most well-known wild primitive carnivores in China, is an endangered animal. In order to promote research on its genome, a high-redundancy BAC library of the Siberian tiger was constructed and characterized. The library is divided into two sub-libraries prepared from blood cells and two sub-libraries prepared from fibroblasts. This BAC library contains 153,600 individually archived clones; for PCR-based screening of the library, BACs were placed into 40 superpools of 10 × 384-deep well microplates. The average insert size of BAC clones was estimated to be 116.5 kb, representing approximately 6.46 genome equivalents of the haploid genome and affording a 98.86% statistical probability of obtaining at least one clone containing a unique DNA sequence. Screening the library with 19 microsatellite markers and a SRY sequence revealed that each of these markers were present in the library; the average number of positive clones per marker was 6.74 (range 2 to 12, consistent with 6.46 coverage of the tiger genome. Additionally, we identified 72 microsatellite markers that could potentially be used as genetic markers. This BAC library will serve as a valuable resource for physical mapping, comparative genomic study and large-scale genome sequencing in the tiger.

  11. Functional cloning using pFB retroviral cDNA expression libraries.

    Science.gov (United States)

    Felts, Katherine A; Chen, Keith; Zaharee, Kim; Sundar, Latha; Limjoco, Jamie; Miller, Anna; Vaillancourt, Peter

    2002-09-01

    Retroviral cDNA expression libraries allow the efficient introduction of complex cDNA libraries into virtually any mitotic cell type for screening based on gene function. The cDNA copy number per cell can be easily controlled by adjusting the multiplicity of infection, thus cell populations may be generated in which >90% of infected cells contain one to three cDNAs. We describe the isolation of two known oncogenes and one cell-surface receptor from a human Burkitt's lymphoma (Daudi) cDNA library inserted into the high-titer retroviral vector pFB.

  12. Cloning and functional expression of a human pancreatic islet glucose-transporter cDNA

    International Nuclear Information System (INIS)

    Permutt, M.A.; Koranyi, L.; Keller, K.; Lacy, P.E.; Scharp, D.W.; Mueckler, M.

    1989-01-01

    Previous studies have suggested that pancreatic islet glucose transport is mediated by a high-K m , low-affinity facilitated transporter similar to that expressed in liver. To determine the relationship between islet and liver glucose transporters, liver-type glucose-transporter cDNA clones were isolated from a human liver cDNA library. The liver-type glucose-transporter cDNA clone hybridized to mRNA transcripts of the same size in human liver and pancreatic islet RNA. A cDNA library was prepared from purified human pancreatic islet tissue and screened with human liver-type glucose-transporter cDNA. The authors isolated two overlapping cDNA clones encompassing 2600 base pairs, which encode a pancreatic islet protein identical in sequence to that of the putative liver-type glucose-transporter protein. Xenopus oocytes injected with synthetic mRNA transcribed from a full-length cDNA construct exhibited increased uptake of 2-deoxyglucose, confirming the functional identity of the clone. These cDNA clones can now be used to study regulation of expression of the gene and to assess the role of inherited defects in this gene as a candidate for inherited susceptibility to non-insulin-dependent diabetes mellitus

  13. Application of the IEAF-2001 activation data library to activation analyses of the IFMIF high flux test module

    International Nuclear Information System (INIS)

    Fischer, U.; Wilson, P.P.H.; Leichtle, D.; Simakov, S.P.; Moellendorff, U. von; Konobeev, A.; Korovin, Yu.; Pereslavtsev, P.; Schmuck, I.

    2002-01-01

    A complete activation data library IEAF-2001 (intermediate energy activation file) has been developed in standard ENDF-6 format with neutron-induced activation cross sections for 679 target nuclides from Z=1 (hydrogen) to Z=84 (polonium) and incident neutron energies up to 150 MeV. Using the NJOY processing code, an IEAF-2001 working library has been prepared in a 256 energy group structure for enabling activation analyses of the International Fusion Material Irradiation Facility (IFMIF) D-Li neutron source. This library was applied to the activation analysis of the IFMIF high flux test module using the recent Analytical and Laplacian Adaptive Radioactivity Analysis activation code which is capable of handling the variety of reaction channels open in the energy domain above 20 MeV. The IEAF-2001 activation library was thus shown to be suitable for activation analyses in fusion technology and intermediate energy applications such as the IFMIF D-Li neutron source

  14. Cloning of a Gene Whose Expression is Increased in Scrapie and in Senile Plaques in Human Brain

    Science.gov (United States)

    Wietgrefe, S.; Zupancic, M.; Haase, A.; Chesebro, B.; Race, R.; Frey, W.; Rustan, T.; Friedman, R. L.

    1985-12-01

    A complementary DNA library was constructed from messenger RNA's extracted from the brains of mice infected with the scrapie agent. The library was differentially screened with the objectives of finding clones that might be used as markers of infection and finding clones of genes whose increased expression might be correlated with the pathological changes common to scrapie and Alzheimer's disease. A gene was identified whose expression is increased in scrapie. The complementary DNA corresponding to this gene hybridized preferentially and focally to cells in the brains of scrapie-infected animals. The cloned DNA also hybridized to the neuritic plaques found with increased frequency in brains of patients with Alzheimer's disease.

  15. Molecular cloning and characterization of a novel human testis ...

    Indian Academy of Sciences (India)

    Molecular cloning and characterization of a novel human testis-specific gene by use of ... pared against 70 other libraries, and the hits showing >10- fold differences .... proteins or testis-development-related proteins such as TSP-. NY, TPX1 ...

  16. Cloning and characterization of a Candida albicans maltase gene involved in sucrose utilization.

    Science.gov (United States)

    Geber, A; Williamson, P R; Rex, J H; Sweeney, E C; Bennett, J E

    1992-01-01

    In order to isolate the structural gene involved in sucrose utilization, we screened a sucrose-induced Candida albicans cDNA library for clones expressing alpha-glucosidase activity. The C. albicans maltase structural gene (CAMAL2) was isolated. No other clones expressing alpha-glucosidase activity. were detected. A genomic CAMAL2 clone was obtained by screening a size-selected genomic library with the cDNA clone. DNA sequence analysis reveals that CAMAL2 encodes a 570-amino-acid protein which shares 50% identity with the maltase structural gene (MAL62) of Saccharomyces carlsbergensis. The substrate specificity of the recombinant protein purified from Escherichia coli identifies the enzyme as a maltase. Northern (RNA) analysis reveals that transcription of CAMAL2 is induced by maltose and sucrose and repressed by glucose. These results suggest that assimilation of sucrose in C. albicans relies on an inducible maltase enzyme. The family of genes controlling sucrose utilization in C. albicans shares similarities with the MAL gene family of Saccharomyces cerevisiae and provides a model system for studying gene regulation in this pathogenic yeast. Images PMID:1400249

  17. Primer sets for cloning the human repertoire of T cell Receptor Variable regions

    Directory of Open Access Journals (Sweden)

    Santoro Claudio

    2008-08-01

    Full Text Available Abstract Background Amplification and cloning of naïve T cell Receptor (TR repertoires or antigen-specific TR is crucial to shape immune response and to develop immuno-based therapies. TR variable (V regions are encoded by several genes that recombine during T cell development. The cloning of expressed genes as large diverse libraries from natural sources relies upon the availability of primers able to amplify as many V genes as possible. Results Here, we present a list of primers computationally designed on all functional TR V and J genes listed in the IMGT®, the ImMunoGeneTics information system®. The list consists of unambiguous or degenerate primers suitable to theoretically amplify and clone the entire TR repertoire. We show that it is possible to selectively amplify and clone expressed TR V genes in one single RT-PCR step and from as little as 1000 cells. Conclusion This new primer set will facilitate the creation of more diverse TR libraries than has been possible using currently available primer sets.

  18. Giant panda BAC library construction and assembly of a 650-kb contig spanning major histocompatibility complex class II region

    Directory of Open Access Journals (Sweden)

    Pan Hui-Juan

    2007-09-01

    Full Text Available Abstract Background Giant panda is rare and endangered species endemic to China. The low rates of reproductive success and infectious disease resistance have severely hampered the development of captive and wild populations of the giant panda. The major histocompatibility complex (MHC plays important roles in immune response and reproductive system such as mate choice and mother-fetus bio-compatibility. It is thus essential to understand genetic details of the giant panda MHC. Construction of a bacterial artificial chromosome (BAC library will provide a new tool for panda genome physical mapping and thus facilitate understanding of panda MHC genes. Results A giant panda BAC library consisting of 205,800 clones has been constructed. The average insert size was calculated to be 97 kb based on the examination of 174 randomly selected clones, indicating that the giant panda library contained 6.8-fold genome equivalents. Screening of the library with 16 giant panda PCR primer pairs revealed 6.4 positive clones per locus, in good agreement with an expected 6.8-fold genomic coverage of the library. Based on this BAC library, we constructed a contig map of the giant panda MHC class II region from BTNL2 to DAXX spanning about 650 kb by a three-step method: (1 PCR-based screening of the BAC library with primers from homologous MHC class II gene loci, end sequences and BAC clone shotgun sequences, (2 DNA sequencing validation of positive clones, and (3 restriction digest fingerprinting verification of inter-clone overlapping. Conclusion The identifications of genes and genomic regions of interest are greatly favored by the availability of this giant panda BAC library. The giant panda BAC library thus provides a useful platform for physical mapping, genome sequencing or complex analysis of targeted genomic regions. The 650 kb sequence-ready BAC contig map of the giant panda MHC class II region from BTNL2 to DAXX, verified by the three-step method, offers a

  19. Cloning and sequencing of the bovine gastrin gene

    DEFF Research Database (Denmark)

    Lund, T; Rehfeld, J F; Olsen, Jørgen

    1989-01-01

    In order to deduce the primary structure of bovine preprogastrin we therefore sequenced a gastrin DNA clone isolated from a bovine liver cosmid library. Bovine preprogastrin comprises 104 amino acids and consists of a signal peptide, a 37 amino acid spacer-sequence, the gastrin-34 sequence followed...

  20. Rapid isolation of IgNAR variable single-domain antibody fragments from a shark synthetic library.

    Science.gov (United States)

    Shao, Cui-Ying; Secombes, Chris J; Porter, Andrew J

    2007-01-01

    The immunoglobulin isotype IgNAR (Novel Antigen Receptor) was discovered in the serum of the nurse shark (Ginglymostoma cirratum) and wobbegong shark (Orectolobus maculates) as a homodimer of two protein chains, each composed of a single variable domain (V) domain and five constant domains. The IgNAR variable domain contains an intact antigen-binding site and functions as an independent domain able to react to antigen with both high specificity and affinity. Here we describe the successful construction of a synthetic phage-displayed library based upon a single anti-lysozyme clone HEL-5A7 scaffold, which was previously selected from an immune IgNAR variable domain library. The complementarity-determining region 3 (CDR3) loop of this clone was varied in both length and composition and the derived library was used to pan against two model proteins, lysozyme and leptin. A single anti-lysozyme clone (Ly-X20) and anti-leptin clone (Lep-12E1) were selected for further study. Both clones were shown to be functionally expressed in Escherichia coli, extremely thermostable and bind to corresponding antigens specifically. The results here demonstrate that a synthetic IgNAR variable domain library based on a single framework scaffold can be used as a route to generate antigen binders quickly, easily and without the need of immunization.

  1. A library of IUE white dwarf spectra for stellar population analyses.

    Science.gov (United States)

    Bica, E.; Bonatto, C.; Giovannini, O.

    1996-10-01

    We present high Signal to Noise ratio IUE spectra of different classes of white dwarfs, to be used as templates for stellar population analyses in the ultraviolet region. We present average stellar parameters associated to each group. The library contains 6 groups for DA's, 2 for DO's and 5 for DB's. We also present equivalent widths of spectral features, and continuum measurements. We call attention to the spectral characteristics which are promising indicators of the presence of white dwarfs in the spectra of composite stellar populations.

  2. Efficient pooling designs for library screening

    OpenAIRE

    Bruno, William J.; Knill, Emanuel; Balding, David J.; Bruce, D. C.; Doggett, N. A.; Sawhill, W. W.; Stallings, R. L.; Whittaker, Craig C.; Torney, David C.

    1994-01-01

    We describe efficient methods for screening clone libraries, based on pooling schemes which we call ``random $k$-sets designs''. In these designs, the pools in which any clone occurs are equally likely to be any possible selection of $k$ from the $v$ pools. The values of $k$ and $v$ can be chosen to optimize desirable properties. Random $k$-sets designs have substantial advantages over alternative pooling schemes: they are efficient, flexible, easy to specify, require fewer pools, and have er...

  3. Clone and characterization of photolyase-gene from soybean

    International Nuclear Information System (INIS)

    Najrana, T.; Hirouchi, T.; Yamamoto, K.

    2003-01-01

    Full text: Cyclobutane pyrimidine dimer (CPD) and pyrimidine [6-4] pyrimidone photoproduct (6-4pp) are the major products of UV-radiation. Both CPD and 6-4pp posses lethal as well as mutagenic property. Excision repair and photoreactivation are involved as major pathways in repairing those photoproducts. To repair those products plant uses photoreactivation as a major pathway. In photoreactivation process photolyase (enzyme encoded by PHR-gene) catalyzes the splitting of the dimer into a monomer under blue light. Photolyase is specific for damage CPD or 6-4pp. The CPD and 6-4pp photolyases are responsible for repairing CPD and 6-4pp lesions respectively. Several investigators reported that removal of CPD lesion is necessary for survival in higher plants in the early development. Thus one should realize the importance of clone and characterization of CPD-photolyase gene from plants especially from those are lying in the list of foods such as wheat, corn, soybean etc. cDNA library (pSPORT-P) of soybean was amplified using the primers that designated as common for CPD-photolyase gene for plants. These primers gave the desire size of PCR product. Desirable PCR product inserted into TA-cloning vector and sequenced. Amino acid sequence revealed considerable homology with CPD-photolyases of rice, arabidopsis thaliana. Then using dilution-PCR amplification method (Hirouchi et al., MGG in press) I have identified the true clone from cDNA library of soybean that containing the full length of CPD-photolyase gene. Full length of cloned gene is about 1698 bps long and exist start and stop codon. Amino acid sequence of the cloned gene shows more than 70% homology with rice, arabidopsis thaliana. Cloned gene enables to complement the E. coli ( phr-uvrA-recA-) system that is completely defective in photoreactivation. The size of CPD-photolyase of soybean is about 56 KDa as identified by 12% SDS PAGE

  4. 454 sequencing of pooled BAC clones on chromosome 3H of barley

    Directory of Open Access Journals (Sweden)

    Yamaji Nami

    2011-05-01

    Full Text Available Abstract Background Genome sequencing of barley has been delayed due to its large genome size (ca. 5,000Mbp. Among the fast sequencing systems, 454 liquid phase pyrosequencing provides the longest reads and is the most promising method for BAC clones. Here we report the results of pooled sequencing of BAC clones selected with ESTs genetically mapped to chromosome 3H. Results We sequenced pooled barley BAC clones using a 454 parallel genome sequencer. A PCR screening system based on primer sets derived from genetically mapped ESTs on chromosome 3H was used for clone selection in a BAC library developed from cultivar "Haruna Nijo". The DNA samples of 10 or 20 BAC clones were pooled and used for shotgun library development. The homology between contig sequences generated in each pooled library and mapped EST sequences was studied. The number of contigs assigned on chromosome 3H was 372. Their lengths ranged from 1,230 bp to 58,322 bp with an average 14,891 bp. Of these contigs, 240 showed homology and colinearity with the genome sequence of rice chromosome 1. A contig annotation browser supplemented with query search by unique sequence or genetic map position was developed. The identified contigs can be annotated with barley cDNAs and reference sequences on the browser. Homology analysis of these contigs with rice genes indicated that 1,239 rice genes can be assigned to barley contigs by the simple comparison of sequence lengths in both species. Of these genes, 492 are assigned to rice chromosome 1. Conclusions We demonstrate the efficiency of sequencing gene rich regions from barley chromosome 3H, with special reference to syntenic relationships with rice chromosome 1.

  5. MOLECULAR CLONING OF OVINE cDNA LEPTIN GENE

    Directory of Open Access Journals (Sweden)

    CLAUDIA TEREZIA SOCOL

    2008-05-01

    Full Text Available An efficient bacterial transformation system suitable for cloning the coding sequence of the ovine leptin gene in E. coli DH5α host cells using the pGEMT easy vector it is described in this paper. The necessity of producing leptin is based on the fact that the role of this molecule in the animal and human organism is still unknown, leptin not existing as commercial product on the Romanian market. The results obtained in the bacterial transformation, cloning, recombinant clones selection, control of the insertion experiments and DNA computational analysis represent the first steps in further genetic engineering experiments such as production of DNA libraries, DNA sequencing, protein expression, etc., for a further contribution in elucidating the role of leptin in the animal and human organism.

  6. Construction of a bacterial artificial chromosome library of S-type CMS maize mitochondria

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    In order to isolate mitochondrial genes easily, we have developed a new method to construct S-type CMS maize mitochondrial gene library by means of embedding mitochondria and enzymatic digesting mitochondria in situ, preparing mtDNA by electrophoresis, digesting LMP agarose with β-agarase, using BAC vector and electroporation. About 2 500 white clones of Mo17 CMS-J mitochondrial gene library were obtained with the average size of 18.24 kb, ranging from 5 to 40 kb, 63.6% inserts came from mitochondrial genome and represented 48 ′ mitochondrial genome equivalents. All the probes had detected the positive clones in the gene library. It is helpful to elucidating the maize mitochondrial genome structure and mechanism of S-type CMS, and may give some valuable reference to the construction of other plant mitochondrial genome library.

  7. Cloning the enterotoxin gene from Clostridium perfringens type A

    OpenAIRE

    Iwanejko, Lesley Ann.

    1991-01-01

    A C. perfringens type A genomic library was constructed in E. coli by banking overlapping 6-10 kbp Hind III fragments of chromosomal DNA from the enterotoxin (CPE) positive strain NCTC 8239 into the pUC derived vector pHG165. The library was screened by colony hybridization with a degenerate 26 bp oligonucleotide probe, derived from the amino acid sequence CPE9_17A. complex mixture of plasmid DNA was isolated from the only hybridization positive clone. A second round of screening picked out a...

  8. Cloning of the human androgen receptor cDNA

    International Nuclear Information System (INIS)

    Govindan, M.V.; Burelle, M.; Cantin, C.; Kabrie, C.; Labrie, F.; Lachance, Y.; Leblanc, G.; Lefebvre, C.; Patel, P.; Simard, J.

    1988-01-01

    The authors discuss how in order to define the functional domains of the human androgen receptor, complementary DNA (cDNA) clones encoding the human androgen receptor (hAR) have been isolated from a human testis λgtll cDNA library using synthetic oligonnucleotide probes, homologous to segments of the human glucocorticoid, estradiol and progesterone receptors. The cDNA clones corresponding to the human glucocorticoid, estradiol and progesterone receptors were eliminated after cross-hybridization with their respective cDNA probes and/or after restriction mapping of the cDNA clones. The remaining cDNA clones were classified into different groups after analysis by restriction digestion and cross-hybridization. Two of the largest cDNA clones from each group were inserted into an expression vector in both orientations. The linearized plasmids were used as templates in in vitro transcription with T7 RNA polymerase. Subsequent in vitro translation of the purified transcripts in rabbit reticulocyte lysate followed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) permitted the characterization of the encoded polyeptides. The expressed proteins larger than 30,000 Da were analyzed for their ability to bind tritium-labelled dihydrotestosterone ([ 3 H] DHT) with high affinity and specificity

  9. Bacterial diversity analysis of Huanglongbing pathogen-infected citrus, using PhyloChip and 16S rRNA gene clone library sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Shankar Sagaram, U.; DeAngelis, K.M.; Trivedi, P.; Andersen, G.L.; Lu, S.-E.; Wang, N.

    2009-03-01

    The bacterial diversity associated with citrus leaf midribs was characterized 1 from citrus groves that contained the Huanglongbing (HLB) pathogen, which has yet to be cultivated in vitro. We employed a combination of high-density phylogenetic 16S rDNA microarray and 16S rDNA clone library sequencing to determine the microbial community composition of symptomatic and asymptomatic citrus midribs. Our results revealed that citrus leaf midribs can support a diversity of microbes. PhyloChip analysis indicated that 47 orders of bacteria from 15 phyla were present in the citrus leaf midribs while 20 orders from phyla were observed with the cloning and sequencing method. PhyloChip arrays indicated that nine taxa were significantly more abundant in symptomatic midribs compared to asymptomatic midribs. Candidatus Liberibacter asiaticus (Las) was detected at a very low level in asymptomatic plants, but was over 200 times more abundant in symptomatic plants. The PhyloChip analysis was further verified by sequencing 16S rDNA clone libraries, which indicated the dominance of Las in symptomatic leaves. These data implicate Las as the pathogen responsible for HLB disease. Citrus is the most important commercial fruit crop in Florida. In recent years, citrus Huanglongbing (HLB), also called citrus greening, has severely affected Florida's citrus production and hence has drawn an enormous amount of attention. HLB is one of the most devastating diseases of citrus (6,13), characterized by blotchy mottling with green islands on leaves, as well as stunting, fruit decline, and small, lopsided fruits with poor coloration. The disease tends to be associated with a phloem-limited fastidious {alpha}-proteobacterium given a provisional Candidatus status (Candidatus Liberobacter spp. later changed to Candidatus Liberibacter spp.) in nomenclature (18,25,34). Previous studies indicate that HLB infection causes disorder in the phloem and severely impairs the translocation of assimilates in

  10. A novel esterase gene cloned from a metagenomic library from neritic sediments of the South China Sea

    Science.gov (United States)

    2011-01-01

    Background Marine microbes are a large and diverse group, which are exposed to a wide variety of pressure, temperature, salinity, nutrient availability and other environmental conditions. They provide a huge potential source of novel enzymes with unique properties that may be useful in industry and biotechnology. To explore the lipolytic genetic resources in the South China Sea, 23 sediment samples were collected in the depth South China Sea sediments assemblage in plasmid vector containing about 194 Mb of community DNA was prepared. Screening of a part of the unamplified library resulted in isolation of 15 unique lipolytic clones with the ability to hydrolyze tributyrin. A positive recombinant clone (pNLE1), containing a novel esterase (Est_p1), was successfully expressed in E. coli and purified. In a series of assays, Est_p1 displayed maximal activity at pH 8.57, 40°C, with ρ-Nitrophenyl butyrate (C4) as substrate. Compared to other metagenomic esterases, Est_p1 played a notable role in specificity for substrate C4 (kcat/Km value 11,500 S-1m M-1) and showed no inhibited by phenylmethylsulfonyl fluoride, suggested that the substrate binding pocket was suitable for substrate C4 and the serine active-site residue was buried at the bottom of substrate binding pocket which sheltered by a lid structure. Conclusions Esterase, which specificity towards short chain fatty acids, especially butanoic acid, is commercially available as potent flavoring tools. According the outstanding activity and specificity for substrate C4, Est_p1 has potential application in flavor industries requiring hydrolysis of short chain esters. PMID:22067554

  11. Isolation of full-length putative rat lysophospholipase cDNA using improved methods for mRNA isolation and cDNA cloning

    International Nuclear Information System (INIS)

    Han, J.H.; Stratowa, C.; Rutter, W.J.

    1987-01-01

    The authors have cloned a full-length putative rat pancreatic lysophospholipase cDNA by an improved mRNA isolation method and cDNA cloning strategy using [ 32 P]-labelled nucleotides. These new methods allow the construction of a cDNA library from the adult rat pancreas in which the majority of recombinant clones contained complete sequences for the corresponding mRNAs. A previously recognized but unidentified long and relatively rare cDNA clone containing the entire sequence from the cap site at the 5' end to the poly(A) tail at the 3' end of the mRNA was isolated by single-step screening of the library. The size, amino acid composition, and the activity of the protein expressed in heterologous cells strongly suggest this mRNA codes for lysophospholipase

  12. Molecular cloning of cDNAs of human liver and placenta NADH-cytochrome b5 reductase

    International Nuclear Information System (INIS)

    Yubisui, T.; Naitoh, Y.; Zenno, S.; Tamura, M.; Takeshita, M.; Sakaki, Y.

    1987-01-01

    A cDNA coding for human liver NADH-cytochrome b 5 reductase was cloned from a human liver cDNA library constructed in phage λgt11. The library was screened by using an affinity-purified rabbit antibody against NADH-cytochrome b 5 reductase of human erythrocytes. A cDNA about 1.3 kilobase pairs long was isolated. By using the cDNA as a probe, another cDNA (pb 5 R141) of 1817 base pairs was isolated that hybridized with a synthetic oligonucleotide encoding Pro-Asp-Ile-Lys-Tyr-Pro, derived from the amino acid sequence at the amino-terminal region of the enzyme from human erythrocytes. Furthermore, by using the pb 5 R141 as a probe, cDNA clones having more 5' sequence were isolated from a human placenta cDNA library. The amino acid sequences deduced from the nucleotide sequences of these cDNA clones overlapped each other and consisted of a sequence that completely coincides with that of human erythrocytes and a sequence of 19 amino acid residues extended at the amino-terminal side. The latter sequence closely resembles that of the membrane-binding domain of steer liver microsomal enzyme

  13. Constructing and detecting a cDNA library for mites.

    Science.gov (United States)

    Hu, Li; Zhao, YaE; Cheng, Juan; Yang, YuanJun; Li, Chen; Lu, ZhaoHui

    2015-10-01

    RNA extraction and construction of complementary DNA (cDNA) library for mites have been quite challenging due to difficulties in acquiring tiny living mites and breaking their hard chitin. The present study is to explore a better method to construct cDNA library for mites that will lay the foundation on transcriptome and molecular pathogenesis research. We selected Psoroptes cuniculi as an experimental subject and took the following steps to construct and verify cDNA library. First, we combined liquid nitrogen grinding with TRIzol for total RNA extraction. Then, switching mechanism at 5' end of the RNA transcript (SMART) technique was used to construct full-length cDNA library. To evaluate the quality of cDNA library, the library titer and recombination rate were calculated. The reliability of cDNA library was detected by sequencing and analyzing positive clones and genes amplified by specific primers. The results showed that the RNA concentration was 836 ng/μl and the absorbance ratio at 260/280 nm was 1.82. The library titer was 5.31 × 10(5) plaque-forming unit (PFU)/ml and the recombination rate was 98.21%, indicating that the library was of good quality. In the 33 expressed sequence tags (ESTs) of P. cuniculi, two clones of 1656 and 1658 bp were almost identical with only three variable sites detected, which had an identity of 99.63% with that of Psoroptes ovis, indicating that the cDNA library was reliable. Further detection by specific primers demonstrated that the 553-bp Pso c II gene sequences of P. cuniculi had an identity of 98.56% with those of P. ovis, confirming that the cDNA library was not only reliable but also feasible.

  14. Cloning, expression, and chromosome mapping of human galectin-7

    DEFF Research Database (Denmark)

    Madsen, Peder; Rasmussen, H H; Flint, T

    1995-01-01

    The galectins are a family of beta-galactoside-binding proteins implicated in modulating cell-cell and cell-matrix interactions. Here we report the cloning and expression of a novel member of this family (galectin-7) that correspond to IEF (isoelectric focusing) 17 (12,700 Da; pI, 7.6) in the human...... keratinocyte protein data base, and that is strikingly down-regulated in SV40 transformed keratinocytes (K14). The cDNA was cloned from a lambda gt11 cDNA expression library using degenerated oligodeoxyribonucleotides back-translated from an IEF 17 peptide sequence. The protein encoded by the galectin-7 clone......14 keratinocytes imply a role in cell-cell and/or cell-matrix interactions necessary for normal growth control. The galectin-7 gene was mapped to chromosome 19. Udgivelsesdato: 1995-Mar-17...

  15. Library 3.0 intelligent libraries and apomediation

    CERN Document Server

    Kwanya, Tom; Underwood, Peter

    2015-01-01

    The emerging generation of research and academic library users expect the delivery of user-centered information services. 'Apomediation' refers to the supporting role librarians can give users by stepping in when users need help. Library 3.0 explores the ongoing debates on the "point oh” phenomenon and its impact on service delivery in libraries. This title analyses Library 3.0 and its potential in creating intelligent libraries capable of meeting contemporary needs, and the growing role of librarians as apomediators. Library 3.0 is divided into four chapters. The first chapter introduces and places the topic in context. The second chapter considers "point oh” libraries. The third chapter covers library 3.0 librarianship, while the final chapter explores ways libraries can move towards '3.0'.

  16. Study on the Mitochondrial Genome of Sea Island Cotton (Gossypium barbadense) by BAC Library Screening

    Institute of Scientific and Technical Information of China (English)

    SU Ai-guo; LI Shuang-shuang; LIU Guo-zheng; LEI Bin-bin; KANG Ding-ming; LI Zhao-hu; MA Zhi-ying; HUA Jin-ping

    2014-01-01

    The plant mitochondrial genome displays complex features, particularly in terms of cytoplasmic male sterility (CMS). Therefore, research on the cotton mitochondrial genome may provide important information for analyzing genome evolution and exploring the molecular mechanism of CMS. In this paper, we present a preliminary study on the mitochondrial genome of sea island cotton (Gossypium barbadense) based on positive clones from the bacterial artiifcial chromosome (BAC) library. Thirty-ifve primers designed with the conserved sequences of functional genes and exons of mitochondria were used to screen positive clones in the genome library of the sea island cotton variety called Pima 90-53. Ten BAC clones were obtained and veriifed for further study. A contig was obtained based on six overlapping clones and subsequently laid out primarily on the mitochondrial genome. One BAC clone, clone 6 harbored with the inserter of approximate 115 kb mtDNA sequence, in which more than 10 primers fragments could be ampliifed, was sequenced and assembled using the Solexa strategy. Fifteen mitochondrial functional genes were revealed in clone 6 by gene annotation. The characteristics of the syntenic gene/exon of the sequences and RNA editing were preliminarily predicted.

  17. A highly redundant BAC library of Atlantic salmon (Salmo salar: an important tool for salmon projects

    Directory of Open Access Journals (Sweden)

    Koop Ben F

    2005-04-01

    Full Text Available Abstract Background As farming of Atlantic salmon is growing as an aquaculture enterprise, the need to identify the genomic mechanisms for specific traits is becoming more important in breeding and management of the animal. Traits of importance might be related to growth, disease resistance, food conversion efficiency, color or taste. To identify genomic regions responsible for specific traits, genomic large insert libraries have previously proven to be of crucial importance. These large insert libraries can be screened using gene or genetic markers in order to identify and map regions of interest. Furthermore, large-scale mapping can utilize highly redundant libraries in genome projects, and hence provide valuable data on the genome structure. Results Here we report the construction and characterization of a highly redundant bacterial artificial chromosome (BAC library constructed from a Norwegian aquaculture strain male of Atlantic salmon (Salmo salar. The library consists of a total number of 305 557 clones, in which approximately 299 000 are recombinants. The average insert size of the library is 188 kbp, representing 18-fold genome coverage. High-density filters each consisting of 18 432 clones spotted in duplicates have been produced for hybridization screening, and are publicly available 1. To characterize the library, 15 expressed sequence tags (ESTs derived overgos and 12 oligo sequences derived from microsatellite markers were used in hybridization screening of the complete BAC library. Secondary hybridizations with individual probes were performed for the clones detected. The BACs positive for the EST probes were fingerprinted and mapped into contigs, yielding an average of 3 contigs for each probe. Clones identified using genomic probes were PCR verified using microsatellite specific primers. Conclusion Identification of genes and genomic regions of interest is greatly aided by the availability of the CHORI-214 Atlantic salmon BAC

  18. Cloning and characterization of the gsk gene encoding guanosine kinase of Escherichia coli

    DEFF Research Database (Denmark)

    Harlow, Kenneth W.; Nygaard, Per; Hove-Jensen, Bjarne

    1995-01-01

    The Escherichia coli gsk gene encoding guanosine kinase was cloned from the Kohara gene library by complementation of the E. coli gsk-1 mutant allele. The cloned DNA fragment was sequenced and shown to encode a putative polypeptide of 433 amino acids with a molecular mass of 48,113 Da. Minicell...

  19. Towards an understanding of British public attitudes concerning human cloning.

    Science.gov (United States)

    Shepherd, Richard; Barnett, Julie; Cooper, Helen; Coyle, Adrian; Moran-Ellis, Jo; Senior, Victoria; Walton, Chris

    2007-07-01

    The ability of scientists to apply cloning technology to humans has provoked public discussion and media coverage. The present paper reports on a series of studies examining public attitudes to human cloning in the UK, bringing together a range of quantitative and qualitative methods to address this question. These included a nationally representative survey, an experimental vignette study, focus groups and analyses of media coverage. Overall the research presents a complex picture of attitude to and constructions of human cloning. In all of the analyses, therapeutic cloning was viewed more favourably than reproductive cloning. However, while participants in the focus groups were generally negative about both forms of cloning, and this was also reflected in the media analyses, quantitative results showed more positive responses. In the quantitative research, therapeutic cloning was generally accepted when the benefits of such procedures were clear, and although reproductive cloning was less accepted there was still substantial support. Participants in the focus groups only differentiated between therapeutic and reproductive cloning after the issue of therapeutic cloning was explicitly raised; initially they saw cloning as being reproductive cloning and saw no real benefits. Attitudes were shown to be associated with underlying values associated with scientific progress rather than with age, gender or education, and although there were a few differences in the quantitative data based on religious affiliation, these tended to be small effects. Likewise in the focus groups there was little direct appeal to religion, but the main themes were 'interfering with nature' and the 'status of the embryo', with the latter being used more effectively to try to close down further discussion. In general there was a close correspondence between the media analysis and focus group responses, possibly demonstrating the importance of media as a resource, or that the media reflect

  20. Cloning and heterologous expression of a gene encoding lycopene ...

    African Journals Online (AJOL)

    This report describes the cloning and expression of a gene lycopene epsilon cyclase, (LCYE) from Camellia sinensis var assamica which is a precursor of the carotenoid lutein in tea. The 1982 bp cDNA sequence with 1599 bp open reading frame of LCYE was identified from an SSH library constructed for quality trait in tea.

  1. Molecular cloning of transcripts induced by UV-radiation in rodent cells

    International Nuclear Information System (INIS)

    Fornace, A.J. Jr.; Mitchell, J.B.

    1987-01-01

    Several inducible DNA repair genes have been well characterized in bacteria. In eukaryotes including mammalian cells, there is increasing evidence that similar events may occur. Recently, the authors have shown that hybridization subtraction can be used to enrich for sequences induced only several fold by a particular cell treatment such as heat shock. Chinese hamster V79 cells were UV-irradiated with 17 Jm/sup -2/ and cDNA was synthesized from the polyadenylated (poly A) RNA. This ''UV'' cDNA was hybridized with a 3 fold excess of polyA RNA from unirradiated cells and the nonhybridizing cDNA was isolated. With this approach, UV-induced sequences were enriched over 20 fold. This enriched cDNA was cloned into a high copy number plasmid and a cDNA library was constructed. By RNA dot blot and northern analysis, 42 clones from this library were found to represent transcripts induced 3 to 25 fold by UV. The most common isolates were found to be metallothionein transcripts by DNA sequencing. The metallothionein transcripts were found to be induced 10 to 25 fold by UV with maximum induction at 4-8 h after 10 Jm/sup -2/. A similar approach was also used with a Chinese hamster ovary line which does not express metallothionein and multiple clones were isolated which represented transcripts induced 3-15 fold by UV. Except for the metallothionein clones, the other Chinese hamster cDNA clones have not been identified, but it is probable that the protein products of at least some of these transcripts play a role in the cellular response to UV damage

  2. Chicken microsatellite markers isolated from libraries enriched for simple tandem repeats.

    Science.gov (United States)

    Gibbs, M; Dawson, D A; McCamley, C; Wardle, A F; Armour, J A; Burke, T

    1997-12-01

    The total number of microsatellite loci is considered to be at least 10-fold lower in avian species than in mammalian species. Therefore, efficient large-scale cloning of chicken microsatellites, as required for the construction of a high-resolution linkage map, is facilitated by the construction of libraries using an enrichment strategy. In this study, a plasmid library enriched for tandem repeats was constructed from chicken genomic DNA by hybridization selection. Using this technique the proportion of recombinant clones that cross-hybridized to probes containing simple tandem repeats was raised to 16%, compared with < 0.1% in a non-enriched library. Primers were designed from 121 different sequences. Polymerase chain reaction (PCR) analysis of two chicken reference pedigrees enabled 72 loci to be localized within the collaborative chicken genetic map, and at least 30 of the remaining loci have been shown to be informative in these or other crosses.

  3. Construction of an American mink Bacterial Artificial Chromosome (BAC library and sequencing candidate genes important for the fur industry

    Directory of Open Access Journals (Sweden)

    Christensen Knud

    2011-07-01

    Full Text Available Abstract Background Bacterial artificial chromosome (BAC libraries continue to be invaluable tools for the genomic analysis of complex organisms. Complemented by the newly and fast growing deep sequencing technologies, they provide an excellent source of information in genomics projects. Results Here, we report the construction and characterization of the CHORI-231 BAC library constructed from a Danish-farmed, male American mink (Neovison vison. The library contains approximately 165,888 clones with an average insert size of 170 kb, representing approximately 10-fold coverage. High-density filters, each consisting of 18,432 clones spotted in duplicate, have been produced for hybridization screening and are publicly available. Overgo probes derived from expressed sequence tags (ESTs, representing 21 candidate genes for traits important for the mink industry, were used to screen the BAC library. These included candidate genes for coat coloring, hair growth and length, coarseness, and some receptors potentially involved in viral diseases in mink. The extensive screening yielded positive results for 19 of these genes. Thirty-five clones corresponding to 19 genes were sequenced using 454 Roche, and large contigs (184 kb in average were assembled. Knowing the complete sequences of these candidate genes will enable confirmation of the association with a phenotype and the finding of causative mutations for the targeted phenotypes. Additionally, 1577 BAC clones were end sequenced; 2505 BAC end sequences (80% of BACs were obtained. An excess of 2 Mb has been analyzed, thus giving a snapshot of the mink genome. Conclusions The availability of the CHORI-321 American mink BAC library will aid in identification of genes and genomic regions of interest. We have demonstrated how the library can be used to identify specific genes of interest, develop genetic markers, and for BAC end sequencing and deep sequencing of selected clones. To our knowledge, this is the

  4. Direct cloning from enrichment cultures, a reliable strategy for isolation of complete operons and genes from microbial consortia.

    Science.gov (United States)

    Entcheva, P; Liebl, W; Johann, A; Hartsch, T; Streit, W R

    2001-01-01

    Enrichment cultures of microbial consortia enable the diverse metabolic and catabolic activities of these populations to be studied on a molecular level and to be explored as potential sources for biotechnology processes. We have used a combined approach of enrichment culture and direct cloning to construct cosmid libraries with large (>30-kb) inserts from microbial consortia. Enrichment cultures were inoculated with samples from five environments, and high amounts of avidin were added to the cultures to favor growth of biotin-producing microbes. DNA was extracted from three of these enrichment cultures and used to construct cosmid libraries; each library consisted of between 6,000 and 35,000 clones, with an average insert size of 30 to 40 kb. The inserts contained a diverse population of genomic DNA fragments isolated from the consortia organisms. These three libraries were used to complement the Escherichia coli biotin auxotrophic strain ATCC 33767 Delta(bio-uvrB). Initial screens resulted in the isolation of seven different complementing cosmid clones, carrying biotin biosynthesis operons. Biotin biosynthesis capabilities and growth under defined conditions of four of these clones were studied. Biotin measured in the different culture supernatants ranged from 42 to 3,800 pg/ml/optical density unit. Sequencing the identified biotin synthesis genes revealed high similarities to bio operons from gram-negative bacteria. In addition, random sequencing identified other interesting open reading frames, as well as two operons, the histidine utilization operon (hut), and the cluster of genes involved in biosynthesis of molybdopterin cofactors in bacteria (moaABCDE).

  5. Cloning and sequence analysis of serine proteinase of Gloydius ussuriensis venom gland

    International Nuclear Information System (INIS)

    Sun Dejun; Liu Shanshan; Yang Chunwei; Zhao Yizhuo; Chang Shufang; Yan Weiqun

    2005-01-01

    Objective: To construct a cDNA library by using mRNA from Gloydius ussuriensis (G. Ussuriensis) venom gland, to clone and analyze serine proteinase gene from the cDNA library. Methods: Total RNA was isolated from venom gland of G. ussuriensis, mRNA was purified by using mRNA isolation Kit. The whole length cDNA was synthesized by means of smart cDNA synthesis strategy, and amplified by long distance PCR procedure, lately cDAN was cloned into vector pBluescrip-sk. The recombinant cDNA was transformed into E. coli DH5α. The cDNA of serine proteinase gene in the venom gland of G. ussuriensis was detected and amplified using the in situ hybridization. The cDNA fragment was inserted into pGEMT vector, cloned and its nucleotide sequence was determined. Results: The capacity of cDNA library of venom gland was above 2.3 x 10 6 . Its open reading frame was composed of 702 nucleotides and coded a protein pre-zymogen of 234 amino acids. It contained 12 cysteine residues. The sequence analysis indicated that the deduced amino acid sequence of the cDNA fragment shared high identity with the thrombin-like enzyme genes of other snakes in the GenBank. the query sequence exhibited strong amino acid sequence homology of 85% to the serine proteas of T. gramineus, thrombin-like serine proteinase I of D. acutus and serine protease catroxase II of C. atrox respectively. Based on the amino acid sequences of other thrombin-like enzymes, the catalytic residues and disulfide bridges of this thrombin-like enzyme were deduced as follows: catalytic residues, His 41 , Asp 86 , Ser 180 ; and six disulfide bridges Cys 7 -Cys 139 , Cys 26 -Cys 42 , Cys 74 -Cys 232 , Cys 118 -Cys 186 , Cys 150 -Cys 165 , Cys 176 -Cys 201 . Conclusion: The capacity of cDNA library of venom gland is above 2.3 x 10 6 , overtop the level of 10 5 capicity. The constructed cDNA library of G. ussuriensis venom gland would be helpful platform to detect new target genes and further gene manipulate. The cloned serine

  6. Isolation and characterization of cDNA clones for carrot extensin and a proline-rich 33-kDa protein

    International Nuclear Information System (INIS)

    Chen, J.; Varner, J.E.

    1985-01-01

    Extensins are hydroxyproline-rich glycoproteins associated with most dicotyledonous plant cell walls. To isolate cDNA clones encoding extensin, the authors started by isolating poly(A) + RNA from carrot root tissue, and then translating the RNA in vitro, in the presence of tritiated leucine or proline. A 33-kDa peptide was identified in the translation products as a putative extensin precursor. From a cDNA library constructed with poly(A) + RNA from wounded carrots, one cDNA clone (pDC5) was identified that specifically hybridized to poly(A) + RNA encoding this 33-kDa peptide. They isolated three cDNA clones (pDC11, pDC12, and pDC16) from another cDNA library using pCD5 as a probe. DNA sequence data, RNA hybridization analysis, and hybrid released in vitro translation indicate that the cDNA clones pDC11 encodes extensin and that cDNA clones pDC12 and pDC16 encode the 33-kDa peptide, which as yet has an unknown identity and function. The assumption that the 33-kDa peptide was an extensin precursor was invalid. RNA hybridization analysis showed that RNA encoded by both clone types is accumulated upon wounding

  7. Construction and characterization of a yeast artificial chromosome library containing seven haploid human genome equivalents

    International Nuclear Information System (INIS)

    Albertsen, H.M.; Abderrahim, H.; Cann, H.M.; Dausset, J.; Le Paslier, D.; Cohen, D.

    1990-01-01

    Prior to constructing a library of yeast artificial chromosomes (YACs) containing very large human DNA fragments, the authors performed a series of preliminary experiments aimed at developing a suitable protocol. They found an inverse relationship between YAC insert size and transformation efficiency. Evidence of occasional rearrangement within YAC inserts was found resulting in clonally stable internal deletions or clonally unstable size variations. A protocol was developed for preparative electrophoretic enrichment of high molecular mass human DNA fragments from partial restriction digests and ligation with the YAC vector in agarose. A YAC library has been constructed from large fragments of DNA from an Epstein-Barr virus-transformed human lymphoblastoid cell line. The library presently contains 50,000 clones, 95% of which are greater than 250 kilobase pairs in size. The mean YAC size of the library, calculated from 132 randomly isolated clones, is 430 kilobase pairs. The library thus contains the equivalent of approximately seven haploid human genomes

  8. Rapid approach for cloning bacterial single-genes directly from soils ...

    African Journals Online (AJOL)

    Obtaining functional genes of bacteria from environmental samples usually depends on library-based approach which is not favored as its large amount of work with small possibility of positive clones. A kind of bacterial single-gene encoding glutamine synthetase (GS) was selected as example to detect the efficiency of ...

  9. Sequence of a cloned cDNA encoding human ribosomal protein S11

    Energy Technology Data Exchange (ETDEWEB)

    Lott, J B; Mackie, G A

    1988-02-11

    The authors have isolated a cloned cDNA that encodes human ribosomal protein (rp) S11 by screening a human fibroblast cDNA library with a labelled 204 bp DNA fragment encompassing residues 212-416 of pRS11, a rat rp Sll cDNA clone. The human rp S11 cloned cDNA consists of 15 residues of the 5' leader, the entire coding sequence and all 51 residues of the 3' untranslated region. The predicted amino acid sequence of 158 residues is identical to rat rpS11. The nucleotide sequence in the coding region differs, however, from that in rat in the first position in two codons and in the third position in 44 codons.

  10. Cloning of cDNA encoding steroid 11β-hydroxylase (P450c11)

    International Nuclear Information System (INIS)

    Chua, S.C.; Szabo, P.; Vitek, A.; Grzeschik, K.H.; John, M.; White, P.C.

    1987-01-01

    The authors have isolated bovine and human adrenal cDNA clones encoding the adrenal cytochrome P-450 specific for 11β-hydroxylation (P450c11). A bovine adrenal cDNA library constructed in the bacteriophage λ vector gt10 was probed with a previously isolated cDNA clone corresponding to part of the 3' untranslated region of the 4.2-kilobase (kb) mRNA encoding P450c11. Several clones with 3.2-kb cDNA inserts were isolated. Sequence analysis showed that they overlapped the original probe by 300 base pairs (bp). Combined cDNA and RNA sequence data demonstrated a continuous open reading frame of 1509 bases. P450c11 is predicted to contain 479 amino acid residues in the mature protein in addition to a 24-residue amino-terminal mitochondrial signal sequence. A bovine clone was used to isolate a homologous clone with a 3.5-kb insert from a human adrenal cDNA library. A region of 1100 bp was 81% homologous to 769 bp of the coding sequence of the bovine cDNA except for a 400-bp segment presumed to be an unprocessed intron. Hybridization of the human cDNA to DNA from a panel of human-rodent somatic cell hybrid lines and in situ hybridization to metaphase spreads of human chromosomes localized the gene to the middle of the long arm of chromosome 8. These data should be useful in developing reagents for heterozygote detection and prenatal diagnosis of 11β-hydroxylase deficiency, the second most frequent cause of congenital adrenal hyperplasia

  11. Cloning of the cDNA for human 12-lipoxygenase

    International Nuclear Information System (INIS)

    Izumi, T.; Hoshiko, S.; Radmark, O.; Samuelsson, B.

    1990-01-01

    A full-length cDNA clone encoding 12-lipoxygenase was isolated from a human platelet cDNA library by using a cDNA for human reticulocyte 15-lipoxygenase as probe for the initial screening. The cDNA had an open reading frame encoding 662 amino acid residues with a calculated molecular weight of 75,590. Three independent clones revealed minor heterogeneities in their DNA sequences. Thus, in three positions of the deduced amino acid sequence, there is a choice between two different amino acids. The deduced sequence from the clone plT3 showed 65% identity with human reticulocyte 15-lipoxygenase and 42% identity with human leukocyte 5-lipoxygenase. The 12-lipoxygenase cDNA recognized a 3.0-kilobase mRNA species in platelets and human erythroleukemia cells (HEL cells). Phorbol 12-tetradecanoyl 13-acetate induced megakaryocytic differentiation of HEL cells and 12-lipoxygenase activity and increased mRNA for 12-lipoxygenase. The identity of the cloned 12-lipoxygenase was assured by expression in a mammalian cell line (COS cells). Human platelet 12-lipoxygenase has been difficult to purify to homogeneity. The cloning of this cDNA will increase the possibilities to elucidate the structure and function of this enzyme

  12. Phenol emulsion-enhanced DNA-driven subtractive cDNA cloning: isolation of low-abundance monkey cortex-specific mRNAs

    International Nuclear Information System (INIS)

    Travis, G.H.; Sutcliffe, J.G.

    1988-01-01

    To isolate cDNA clones of low-abundance mRNAs expressed in monkey cerebral cortex but absent from cerebellum, the authors developed an improved subtractive cDNA cloning procedure that requires only modest quantities of mRNA. Plasmid DNA from a monkey cerebellum cDNA library was hybridized in large excess to radiolabeled monkey cortex cDNA in a phenol emulsion-enhanced reaction. The unhybridized cortex cDNA was isolated by chromatography on hydroxyapatite and used to probe colonies from a monkey cortex cDNA library. Of 60,000 colonies screened, 163 clones were isolated and confirmed by colony hybridization or RNA blotting to represent mRNAs, ranging from 0.001% to 0.1% abundance, specific to or highly enriched in cerebral cortex relative to cerebellum. Clones of one medium-abundance mRNA were recovered almost quantitatively. Two of the lower-abundance mRNAs were expressed at levels reduced by a factor of 10 in Alzheimer disease relative to normal human cortex. One of these was identified as the monkey preprosomatostatin I mRNA

  13. Enhanced specificity in immunoscreening of expression cDNA clones using radiolabeled antigen overlay

    International Nuclear Information System (INIS)

    Chao, S.; Chao, L.; Chao, J.

    1989-01-01

    A highly sensitive and specific method has been developed for immunoscreening clones from an expression cDNA library. The procedures utilize a radiolabeled antigen detection method described originally for the immunoblotting of plasma proteins. Screening of rat alpha 1-antitrypsin clones was used. Comparison between Western blots of alpha 1-antitrypsin using both labeled antigen and protein A detection methods showed that the former yielded lower background and greater sensitivity than the latter. Further, this technique was shown to have a lower detection limit of less than 20 ng through Western blot analysis of varying concentrations of alpha 1-antitrypsin. The procedures are based on the expression of the protein by cDNA clones containing the DNA inserts in the correct reading frame. Following the transfer of phage proteins to nitrocellulose membranes, the bivalent antibodies bind monovalently to both nitrocellulose-bound-antigen in the phage lysates and radiolabeled antigen. The radiolabeled antigen overlay method is superior to the protein A detection method in sensitivity, specificity and reproducibility. This improved method can be applied in general for screening expression cDNA libraries, provided that the specific antiserum and radiolabeled antigen are available

  14. Construction of a BAC library and identification of Dmrt1 gene of the rice field eel, Monopterus albus

    International Nuclear Information System (INIS)

    Jang Songhun; Zhou Fang; Xia Laixin; Zhao Wei; Cheng Hanhua; Zhou Rongjia

    2006-01-01

    A bacterial artificial chromosome (BAC) library was constructed using nuclear DNA from the rice field eel (Monopterus albus). The BAC library consists of a total of 33,000 clones with an average insert size of 115 kb. Based on the rice field eel haploid genome size of 600 Mb, the BAC library is estimated to contain approximately 6.3 genome equivalents and represents 99.8% of the genome of the rice field eel. This is first BAC library constructed from this species. To estimate the possibility of isolating a specific clone, high-density colony hybridization-based library screening was performed using Dmrt1 cDNA of the rice field eel as a probe. Both library screening and PCR identification results revealed three positive BAC clones which were overlapped, and formed a contig covering the Dmrt1 gene of 195 kb. By sequence comparisons with the Dmrt1 cDNA and sequencing of first four intron-exon junctions, Dmrt1 gene of the rice field eel was predicted to contain four introns and five exons. The sizes of first and second intron are 1.5 and 2.6 kb, respectively, and the sizes of last two introns were predicted to be about 20 kb. The Dmrt1 gene structure was conserved in evolution. These results also indicate that the BAC library is a useful resource for BAC contig construction and molecular isolation of functional genes

  15. End Sequencing and Finger Printing of Human & Mouse BAC Libraries

    Energy Technology Data Exchange (ETDEWEB)

    Fraser, C

    2005-09-27

    This project provided for continued end sequencing of existing and new BAC libraries constructed to support human sequencing as well as to initiate BAC end sequencing from the mouse BAC libraries constructed to support mouse sequencing. The clones, the sequences, and the fingerprints are now an available resource for the community at large. Research and development of new metaodologies for BAC end sequencing have reduced costs and increase throughput.

  16. [Human cloning and the protection of women's interests].

    Science.gov (United States)

    Canabes, Marcela Ahumada

    2008-01-01

    The Human Cloning, both therapeutic and full birth cloning, involves and affects women in a special way. The United Nation's Declaration on the Cloning of Human Beings includes a special clause referred to them. Also the Spanish law does it. This works pretend to analyse the meaning of the inclusion of women's interests in this document. At the same time, I will consider the foundations and the importance of the reference to the women.

  17. A feruloyl esterase derived from a leachate metagenome library

    CSIR Research Space (South Africa)

    Rashamuse, K

    2012-01-01

    Full Text Available A feruloyl esterase encoding gene (designated fae6), derived from a leachate metagenomic library, was cloned and the nucleotide sequence of the insert DNA determined. Translational analysis revealed that fae6 consists of a 515 amino acid polypeptide...

  18. Chorioallantoic placenta defects in cloned mice

    International Nuclear Information System (INIS)

    Wakisaka-Saito, Noriko; Kohda, Takashi; Inoue, Kimiko; Ogonuki, Narumi; Miki, Hiromi; Hikichi, Takafusa; Mizutani, Eiji; Wakayama, Teruhiko; Kaneko-Ishino, Tomoko; Ogura, Atsuo; Ishino, Fumitoshi

    2006-01-01

    Somatic cell nuclear transfer technology has been applied to produce live clones successfully in several mammalian species, but the success rates are very low. In mice, about half of the nuclear transfer embryos undergo implantation, but very few survive to term. We undertook detailed histological analyses of placentas from cloned mouse embryos generated from cumulus cells at 10.5 dpc of pregnancy, by which stage most clones have terminated their development. At 10.5 dpc, the extraembryonic tissues displayed several defined histological patterns, each reflecting their stage of developmental arrest. The most notable abnormality was the poor development of the spongiotrophoblast layer of diploid cells. This is in contrast to the placental hyperplasia frequently observed in somatic clones at 12.5 dpc or later stages. A variety of structural abnormalities were also observed in the embryos. Both placental and embryonic defects likely contribute to the low success rate of the mouse clones

  19. Binding proteins for the regulatory subunit (RII-B) of brain cAMP-dependent protein kinase II: isolation and initial characterization of cDNA clones

    International Nuclear Information System (INIS)

    Bregman, D.B.; Hu, E.; Rubin, C.S.

    1987-01-01

    In mammalian brain several proteins bind RII-B with high affinity. An example is P75, which co-purifies with RII-B and also complexes Ca 2+ -calmodulin. Thus, RII-B binding proteins (RBPs) might play a role in integrating the Ca 2+ and cAMP signalling pathways in the CNS. In order to study the structure and function of these polypeptides they have isolated cloned cDNAs for RBPs by screening brain λgt11 expression libraries using a functional assay: the binding of 32 P-labeled RII to fusion proteins produced by recombinants expressing RII binding domains. Inserts from rat brain recombinant clones λ7B and λ10B both hybridize to a brain mRNA of 7000 nucleotides. Northern gel analyses indicate that the putative RBP mRNA is also expressed in lung, but not in several other tissues. The λ7B insert was subcloned into the expression plasmid pINIA. A 50 kDa high affinity RII-B binding polypeptide accumulated in E. coli transformed with pINIA-7B. Two RBP cDNAs (λ77, λ100A) have been retrieved from a bovine λgt 11 library using a monoclonal antibody directed against P75 and the binding assay respectively. On Southern blots the insert from λ100A hybridizes to the cDNA insert from clones λ77, suggesting that λ 77 cDNA might contain sequences coding for both an RII binding domain and a P75 epitope. The bovine λ100A insert also hybridizes with the rat λ7B clone indicating that an RII binding domain is conserved in the two species

  20. Construction and characterization of cDNA library for IRM-2 mice

    International Nuclear Information System (INIS)

    Wang Qin; Li Jin; Song Li; Liu Qiang; Yue Jingyin; Mu Chuanjie; Tang Weisheng; Fan Feiyue

    2010-01-01

    Objective: To screen and isolate the radioresistance related genes of IRM-2 mice. Methods: cDNA library of IRM-2 mice was constructed by SMART technique. Total RNA was isolated from spleens of IRM-2 male mice. The first-strand cDNA was synthesized by using PowerScript reverse transcriptase, and double-strand cDNA was synthesized and amplified by long PCR. The PCR products were purified, digested with restriction enzyme Sfi I. The ds-cDNA fragment less than 500 bp was fractionated and ligated to the Sfi I-digested pDNR-LIB vector. The ligation mixture was transformed into E. coil DH5 α by electroporation transformation to generate the unamplified cDNA library. The quality of cDNA library was identified by PCR technique. 130 clones from cDNA library were sequenced and compared with GenBank database. Results: The cDNA library contained 2.25 x 10 6 independent clones with an average insert size of 1.2 kb. The ratio of recombination and full-length was 95% and 55%, respectively. 21 pieces of EST sequences from cDNA library were not the same as the known mice genes and registered into GenBank EST database, with registered number DW474856-DW474876. Conclusions: cDNA library of IRM-2 mice has been constructed successfully. 21 pieces of EST implies that radioresistance correlative genes may be in IRM-2 mice, which will lay a foundation for isolating and identifying radioresistance related genes in further study. (authors)

  1. Versatile P(acman) BAC Libraries for Transgenesis Studies in Drosophila melanogaster

    Energy Technology Data Exchange (ETDEWEB)

    Venken, Koen J.T.; Carlson, Joseph W.; Schulze, Karen L.; Pan, Hongling; He, Yuchun; Spokony, Rebecca; Wan, Kenneth H.; Koriabine, Maxim; de Jong, Pieter J.; White, Kevin P.; Bellen, Hugo J.; Hoskins, Roger A.

    2009-04-21

    We constructed Drosophila melanogaster BAC libraries with 21-kb and 83-kb inserts in the P(acman) system. Clones representing 12-fold coverage and encompassing more than 95percent of annotated genes were mapped onto the reference genome. These clones can be integrated into predetermined attP sites in the genome using Phi C31 integrase to rescue mutations. They can be modified through recombineering, for example to incorporate protein tags and assess expression patterns.

  2. Cloning and identification of the gene coding for the 140-kd subunit of Drosophila RNA polymerase II

    OpenAIRE

    Faust, Daniela M.; Renkawitz-Pohl, Renate; Falkenburg, Dieter; Gasch, Alexander; Bialojan, Siegfried; Young, Richard A.; Bautz, Ekkehard K. F.

    1986-01-01

    Genomic clones of Drosophila melanogaster were isolated from a λ library by cross-hybridization with the yeast gene coding for the 150-kd subunit of RNA polymerase II. Clones containing a region of ∼2.0 kb with strong homology to the yeast gene were shown to code for a 3.9-kb poly(A)+-RNA. Part of the coding region was cloned into an expression vector. A fusion protein was obtained which reacted with an antibody directed against RNA polymerase II of Drosophila. Peptide mapping of the fusion p...

  3. Heterogeneity of rat tropoelastin mRNA revealed by cDNA cloning

    International Nuclear Information System (INIS)

    Pierce, R.A.; Deak, S.B.; Stolle, C.A.; Boyd, C.D.

    1990-01-01

    A λgt11 library constructed from poly(A+) RNA isolated from aortic tissue of neonatal rats was screened for rat tropoelastin cDNAs. The first, screen, utilizing a human tropoelastin cDNA clone, provided rat tropoelastin cDNAs spanning 2.3 kb of carboxy-terminal coding sequence and extended into the 3'-untranslated region. A subsequent screen using a 5' rat tropoelastin cDNA clone yielded clones extending into the amino-terminal signal sequence coding region. Sequence analysis of these clones has provided the complete derived amino acid sequence of rat tropoelastin and allowed alignment and comparison with published bovine cDNA sequence. While the overall structure of rat tropoelastin is similar to bovine sequence, numerous substitutions, deletions, and insertions demonstrated considerable heterogeneity between species. In particular, the pentapeptide repeat VPGVG, characteristic of all tropoelastins analyzed to date, is replaced in rat tropoelastin by a repeating pentapeptide, IPGVG. The hexapeptide repeat VGVAPG, the bovine elastin receptor binding peptide, is not encoded by rat tropoelastin cDNAs. Variations in coding sequence between rat tropoelastin CDNA clones were also found which may represent mRNA heterogeneity produced by alternative splicing of the rat tropoelastin pre-mRNA

  4. Identification of eukaryotic open reading frames in metagenomic cDNA libraries made from environmental samples.

    Science.gov (United States)

    Grant, Susan; Grant, William D; Cowan, Don A; Jones, Brian E; Ma, Yanhe; Ventosa, Antonio; Heaphy, Shaun

    2006-01-01

    Here we describe the application of metagenomic technologies to construct cDNA libraries from RNA isolated from environmental samples. RNAlater (Ambion) was shown to stabilize RNA in environmental samples for periods of at least 3 months at -20 degrees C. Protocols for library construction were established on total RNA extracted from Acanthamoeba polyphaga trophozoites. The methodology was then used on algal mats from geothermal hot springs in Tengchong county, Yunnan Province, People's Republic of China, and activated sludge from a sewage treatment plant in Leicestershire, United Kingdom. The Tenchong libraries were dominated by RNA from prokaryotes, reflecting the mainly prokaryote microbial composition. The majority of these clones resulted from rRNA; only a few appeared to be derived from mRNA. In contrast, many clones from the activated sludge library had significant similarity to eukaryote mRNA-encoded protein sequences. A library was also made using polyadenylated RNA isolated from total RNA from activated sludge; many more clones in this library were related to eukaryotic mRNA sequences and proteins. Open reading frames (ORFs) up to 378 amino acids in size could be identified. Some resembled known proteins over their full length, e.g., 36% match to cystatin, 49% match to ribosomal protein L32, 63% match to ribosomal protein S16, 70% to CPC2 protein. The methodology described here permits the polyadenylated transcriptome to be isolated from environmental samples with no knowledge of the identity of the microorganisms in the sample or the necessity to culture them. It has many uses, including the identification of novel eukaryotic ORFs encoding proteins and enzymes.

  5. Construction of an Americn mink Bacterial Artificial Chromosome (BAC) library and sequencing candidate genes important for the fur industry

    DEFF Research Database (Denmark)

    Anistoroaei, Razvan Marian; Hallers, Boudewijn ten; Nefedov, Michael

    2011-01-01

    BACKGROUND: Bacterial artificial chromosome (BAC) libraries continue to be invaluable tools for the genomic analysis of complex organisms. Complemented by the newly and fast growing deep sequencing technologies, they provide an excellent source of information in genomics projects. RESULTS: Here, we...... report the construction and characterization of the CHORI-231 BAC library constructed from a Danish-farmed, male American mink (Neovison vison). The library contains approximately 165,888 clones with an average insert size of 170 kb, representing approximately 10-fold coverage. High-density filters, each...... consisting of 18,432 clones spotted in duplicate, have been produced for hybridization screening and are publicly available. Overgo probes derived from expressed sequence tags (ESTs), representing 21 candidate genes for traits important for the mink industry, were used to screen the BAC library...

  6. Cloning and sequencing of Lol pI, the major allergenic protein of rye-grass pollen.

    Science.gov (United States)

    Griffith, I J; Smith, P M; Pollock, J; Theerakulpisut, P; Avjioglu, A; Davies, S; Hough, T; Singh, M B; Simpson, R J; Ward, L D

    1991-02-25

    We have isolated a full length cDNA clone encoding the major glycoprotein allergen Lol pI. The clone was selected using a combination of immunological screening of a cDNA expression library and PCR amplification of Lol pI-specific transcripts. Lol pI expressed in bacteria as a fusion protein shows recognition by specific IgE antibodies present in sera of grass pollen-allergic subjects. Northern analysis has shown that the Lol pI transcripts are expressed only in pollen of rye-grass. Molecular cloning of Lol pI provides a molecular genetic approach to study the structure-function relationship of allergens.

  7. Strategic marketing planning in library

    Directory of Open Access Journals (Sweden)

    Karmen Štular-Sotošek

    2000-01-01

    Full Text Available The article is based on the idea that every library can design instruments for creating events and managing the important resources of today's world, especially to manage the changes. This process can only be successful if libraries use adequate marketing methods. Strategic marketing planning starts with the analysis of library's mission, its objectives, goals and corporate culture. By analysing the public environment, the competitive environment and the macro environment, libraries recognise their opportunities and threats. These analyses are the foundations for library definitions: What does the library represent?, What does it aspire to? Which goals does it want to reach? What kind of marketing strategy will it use for its target market?

  8. Molecular cloning of lupin leghemoglobin cDNA

    DEFF Research Database (Denmark)

    Konieczny, A; Jensen, E O; Marcker, K A

    1987-01-01

    Poly(A)+ RNA isolated from root nodules of yellow lupin (Lupinus luteus, var. Ventus) has been used as a template for the construction of a cDNA library. The ds cDNA was synthesized and inserted into the Hind III site of plasmid pBR 322 using synthetic Hind III linkers. Clones containing sequences...... specific for nodules were selected by differential colony hybridization using 32P-labeled cDNA synthesized either from nodule poly(A)+ RNA or from poly(A)+ RNA of uninfected root as probes. Among the recombinant plasmids, the cDNA gene for leghemoglobin was identified. The protein structure derived from...... its nucleotide sequence was consistent with known amino acid sequence of lupin Lb II. The cloned lupin Lb cDNA hybridized to poly(A)+ RNA from nodules only, which is in accordance with the general concept, that leghemoglobin is expressed exclusively in nodules. Udgivelsesdato: 1987-null...

  9. Error Analysis of Deep Sequencing of Phage Libraries: Peptides Censored in Sequencing

    Directory of Open Access Journals (Sweden)

    Wadim L. Matochko

    2013-01-01

    Full Text Available Next-generation sequencing techniques empower selection of ligands from phage-display libraries because they can detect low abundant clones and quantify changes in the copy numbers of clones without excessive selection rounds. Identification of errors in deep sequencing data is the most critical step in this process because these techniques have error rates >1%. Mechanisms that yield errors in Illumina and other techniques have been proposed, but no reports to date describe error analysis in phage libraries. Our paper focuses on error analysis of 7-mer peptide libraries sequenced by Illumina method. Low theoretical complexity of this phage library, as compared to complexity of long genetic reads and genomes, allowed us to describe this library using convenient linear vector and operator framework. We describe a phage library as N×1 frequency vector n=ni, where ni is the copy number of the ith sequence and N is the theoretical diversity, that is, the total number of all possible sequences. Any manipulation to the library is an operator acting on n. Selection, amplification, or sequencing could be described as a product of a N×N matrix and a stochastic sampling operator (Sa. The latter is a random diagonal matrix that describes sampling of a library. In this paper, we focus on the properties of Sa and use them to define the sequencing operator (Seq. Sequencing without any bias and errors is Seq=Sa IN, where IN is a N×N unity matrix. Any bias in sequencing changes IN to a nonunity matrix. We identified a diagonal censorship matrix (CEN, which describes elimination or statistically significant downsampling, of specific reads during the sequencing process.

  10. The Shiite Pluralistic Position on Human Cloning

    Directory of Open Access Journals (Sweden)

    Sayyid Hasan Islami Ardekani

    2012-01-01

    Full Text Available With regard to human cloning or artificial human reproduction – and contrary to the opinions of Sunni scholars - Shiite thinkers have not held a unified position. After having surveyed a number of Shiite fatwas and analyses on the subject, this essay will classify them into four groups. The first group states that we are granted absolute permission to engage in human cloning; while the second group believes that there is limited permission; the third group argues that cloning as such is primarily permitted but because of its consequences and secondary grounds it is prohibited and unlawful; and the fourth group is of the view that cloning as such and by itself is prohibited and unlawful. In what follows, the author has examined these four views, ending in support of the permission theory.

  11. New vectors in fission yeast: application for cloning the his2 gene

    DEFF Research Database (Denmark)

    Weilguny, D; Praetorius, M; Carr, Alan

    1991-01-01

    of transforming Sc. pombe ura4 strains, as well as ura 3 strains of the distantly related budding yeast Saccharomyces cerevisiae. We have used pON163 for the construction of two fission yeast genomic libraries. From these gene banks clones were isolated that were able to complement fission yeast his2 mutants...

  12. A novel lentiviral scFv display library for rapid optimization and selection of high affinity antibodies.

    Science.gov (United States)

    Qudsia, Sehar; Merugu, Siva B; Mangukiya, Hitesh B; Hema, Negi; Wu, Zhenghua; Li, Dawei

    2018-04-30

    Antibody display libraries have become a popular technique to screen monoclonal antibodies for therapeutic purposes. An important aspect of display technology is to generate an optimization library by changing antibody affinity to antigen through mutagenesis and screening the high affinity antibody. In this study, we report a novel lentivirus display based optimization library antibody in which Agtuzumab scFv is displayed on cell membrane of HEK-293T cells. To generate an optimization library, hotspot mutagenesis was performed to achieve diverse antibody library. Based on sequence analysis of randomly selected clones, library size was estimated approximately to be 1.6 × 10 6 . Lentivirus display vector was used to display scFv antibody on cell surface and flow cytometery was performed to check the antibody affinity to antigen. Membrane bound scFv antibodies were then converted to secreted antibody through cre/loxP recombination. One of the mutant clones, M8 showed higher affinity to antigen in flow cytometery analysis. Further characterization of cellular and secreted scFv through western blot showed that antibody affinity was increased by three fold after mutagenesis. This study shows successful construction of a novel antibody library and suggests that hotspot mutagenesis could prove a useful and rapid optimization tool to generate similar libraries with various degree of antigen affinity. Copyright © 2018 Elsevier Inc. All rights reserved.

  13. [Construction of forward and reverse subtracted cDNA libraries between muscle tissue of Meishan and Landrace pigs].

    Science.gov (United States)

    Xu, De-Quan; Zhang, Yi-Bing; Xiong, Yuan-Zhu; Gui, Jian-Fang; Jiang, Si-Wen; Su, Yu-Hong

    2003-07-01

    Using suppression subtractive hybridization (SSH) technique, forward and reverse subtracted cDNA libraries were constructed between Longissimus muscles from Meishan and Landrace pigs. A housekeeping gene, G3PDH, was used to estimate the efficiency of subtractive cDNA. In two cDNA libraries, G3PDH was subtracted very efficiently at appropriate 2(10) and 2(5) folds, respectively, indicating that some differentially expressed genes were also enriched at the same folds and the two subtractive cDNA libraries were very successful. A total of 709 and 673 positive clones were isolated from forward and reverse subtracted cDNA libraries, respectively. Analysis of PCR showed that most of all plasmids in the clones contained 150-750 bp inserts. The construction of subtractive cDNA libraries between muscle tissue from different pig breeds laid solid foundations for isolating and identifying the genes determining muscle growth and meat quality, which will be important to understand the mechanism of muscle growth, determination of meat quality and practice of molecular breeding.

  14. A novel heavy domain antibody library with functionally optimized complementarity determining regions.

    Directory of Open Access Journals (Sweden)

    Ole Aalund Mandrup

    Full Text Available Today a number of synthetic antibody libraries of different formats have been created and used for the selection of a large number of recombinant antibodies. One of the determining factors for successful isolation of recombinant antibodies from libraries lies in the quality of the libraries i.e. the number of correctly folded, functional antibodies contained in the library. Here, we describe the construction of a novel, high quality, synthetic single domain antibody library dubbed Predator. The library is based on the HEL4 domain antibody with the addition of recently reported mutations concerning the amino acid composition at positions critical for the folding characteristics and aggregation propensities of domain antibodies. As a unique feature, the CDR3 of the library was designed to mimic the natural human immune response by designating amino acids known to be prevalent in functional antibodies to the diversity in CDR3. CDR randomizations were performed using trinucleotide synthesis to avoid the presence of stop codons. Furthermore a novel cycle free elongation method was used for the conversion of the synthesized single stranded DNA containing the randomized CDRs into double stranded DNA of the library. In addition a modular approach has been adopted for the scaffold in which each CDR region is flanked by unique restrictions sites, allowing easy affinity maturation of selected clones by CDR shuffling. To validate the quality of the library, one round phage display selections were performed on purified antigens and highly complex antigen mixtures such as cultured eukaryotic cells resulting in several specific binders. The further characterization of some of the selected clones, however, indicates a reduction in thermodynamic stability caused by the inclusion the additional mutations to the HEL4 scaffold.

  15. Quantifying and resolving multiple vector transformants in S. cerevisiae plasmid libraries.

    Science.gov (United States)

    Scanlon, Thomas C; Gray, Elizabeth C; Griswold, Karl E

    2009-11-20

    In addition to providing the molecular machinery for transcription and translation, recombinant microbial expression hosts maintain the critical genotype-phenotype link that is essential for high throughput screening and recovery of proteins encoded by plasmid libraries. It is known that Escherichia coli cells can be simultaneously transformed with multiple unique plasmids and thusly complicate recombinant library screening experiments. As a result of their potential to yield misleading results, bacterial multiple vector transformants have been thoroughly characterized in previous model studies. In contrast to bacterial systems, there is little quantitative information available regarding multiple vector transformants in yeast. Saccharomyces cerevisiae is the most widely used eukaryotic platform for cell surface display, combinatorial protein engineering, and other recombinant library screens. In order to characterize the extent and nature of multiple vector transformants in this important host, plasmid-born gene libraries constructed by yeast homologous recombination were analyzed by DNA sequencing. It was found that up to 90% of clones in yeast homologous recombination libraries may be multiple vector transformants, that on average these clones bear four or more unique mutant genes, and that these multiple vector cells persist as a significant proportion of library populations for greater than 24 hours during liquid outgrowth. Both vector concentration and vector to insert ratio influenced the library proportion of multiple vector transformants, but their population frequency was independent of transformation efficiency. Interestingly, the average number of plasmids born by multiple vector transformants did not vary with their library population proportion. These results highlight the potential for multiple vector transformants to dominate yeast libraries constructed by homologous recombination. The previously unrecognized prevalence and persistence of multiply

  16. Quantifying and resolving multiple vector transformants in S. cerevisiae plasmid libraries

    Directory of Open Access Journals (Sweden)

    Gray Elizabeth C

    2009-11-01

    Full Text Available Abstract Background In addition to providing the molecular machinery for transcription and translation, recombinant microbial expression hosts maintain the critical genotype-phenotype link that is essential for high throughput screening and recovery of proteins encoded by plasmid libraries. It is known that Escherichia coli cells can be simultaneously transformed with multiple unique plasmids and thusly complicate recombinant library screening experiments. As a result of their potential to yield misleading results, bacterial multiple vector transformants have been thoroughly characterized in previous model studies. In contrast to bacterial systems, there is little quantitative information available regarding multiple vector transformants in yeast. Saccharomyces cerevisiae is the most widely used eukaryotic platform for cell surface display, combinatorial protein engineering, and other recombinant library screens. In order to characterize the extent and nature of multiple vector transformants in this important host, plasmid-born gene libraries constructed by yeast homologous recombination were analyzed by DNA sequencing. Results It was found that up to 90% of clones in yeast homologous recombination libraries may be multiple vector transformants, that on average these clones bear four or more unique mutant genes, and that these multiple vector cells persist as a significant proportion of library populations for greater than 24 hours during liquid outgrowth. Both vector concentration and vector to insert ratio influenced the library proportion of multiple vector transformants, but their population frequency was independent of transformation efficiency. Interestingly, the average number of plasmids born by multiple vector transformants did not vary with their library population proportion. Conclusion These results highlight the potential for multiple vector transformants to dominate yeast libraries constructed by homologous recombination. The

  17. Advancing Eucalyptus genomics: identification and sequencing of lignin biosynthesis genes from deep-coverage BAC libraries

    Directory of Open Access Journals (Sweden)

    Kudrna David

    2011-03-01

    Full Text Available Abstract Background Eucalyptus species are among the most planted hardwoods in the world because of their rapid growth, adaptability and valuable wood properties. The development and integration of genomic resources into breeding practice will be increasingly important in the decades to come. Bacterial artificial chromosome (BAC libraries are key genomic tools that enable positional cloning of important traits, synteny evaluation, and the development of genome framework physical maps for genetic linkage and genome sequencing. Results We describe the construction and characterization of two deep-coverage BAC libraries EG_Ba and EG_Bb obtained from nuclear DNA fragments of E. grandis (clone BRASUZ1 digested with HindIII and BstYI, respectively. Genome coverages of 17 and 15 haploid genome equivalents were estimated for EG_Ba and EG_Bb, respectively. Both libraries contained large inserts, with average sizes ranging from 135 Kb (Eg_Bb to 157 Kb (Eg_Ba, very low extra-nuclear genome contamination providing a probability of finding a single copy gene ≥ 99.99%. Libraries were screened for the presence of several genes of interest via hybridizations to high-density BAC filters followed by PCR validation. Five selected BAC clones were sequenced and assembled using the Roche GS FLX technology providing the whole sequence of the E. grandis chloroplast genome, and complete genomic sequences of important lignin biosynthesis genes. Conclusions The two E. grandis BAC libraries described in this study represent an important milestone for the advancement of Eucalyptus genomics and forest tree research. These BAC resources have a highly redundant genome coverage (> 15×, contain large average inserts and have a very low percentage of clones with organellar DNA or empty vectors. These publicly available BAC libraries are thus suitable for a broad range of applications in genetic and genomic research in Eucalyptus and possibly in related species of Myrtaceae

  18. Cloning of the cDNA and gene for a human D2 dopamine receptor

    International Nuclear Information System (INIS)

    Grady, D.K.; Makam, H.; Stofko, R.E.; Bunzow, J.R.; Civelli, O.; Marchionni, M.A.; Alfano, M.; Frothingham, L.; Fischer, J.B.; Burke-Howie, K.J.; Server, A.C.

    1989-01-01

    A clone encoding a human D 2 dopamine receptor was isolated from a pituitary cDNA library and sequenced. The deduced protein sequence is 96% identical with that of the cloned rat receptor with one major difference: the human receptor contains an additional 29 amino acids in its putative third cytoplasmic loop. Southern blotting demonstrated the presence of only one human D 2 receptor gene. Two overlapping phage containing the gene were isolated and characterized. DNA sequence analysis of these clones showed that the coding sequence is interrupted by six introns and that the additional amino acids present in the human pituitary receptor are encoded by a single exon of 87 base pairs. The involvement of this sequence in alternative splicing and its biological significance are discussed

  19. Cloning

    Science.gov (United States)

    Cloning describes the processes used to create an exact genetic replica of another cell, tissue or organism. ... named Dolly. There are three different types of cloning: Gene cloning, which creates copies of genes or ...

  20. What is Cloning?

    Science.gov (United States)

    Donate Home Cloning What is Cloning What is Cloning Clones are organisms that are exact genetic copies. ... clones made through modern cloning technologies. How Is Cloning Done? Many people first heard of cloning when ...

  1. CDNA cloning, characterization and expression of an endosperm-specific barley peroxidase

    DEFF Research Database (Denmark)

    Rasmussen, Søren Kjærsgård; Welinder, K.G.; Hejgaard, J.

    1991-01-01

    A barley peroxidase (BP 1) of pI ca. 8.5 and M(r) 37000 has been purified from mature barley grains. Using antibodies towards peroxidase BP 1, a cDNA clone (pcR7) was isolated from cDNA expression library. The nucleotide sequence of pcR7 gave a derived amino acid sequence identical to the 158 C...

  2. Toward a molecular cytogenetic map for cultivated sunflower (Helianthus annuus L.) by landed BAC/BIBAC clones.

    Science.gov (United States)

    Feng, Jiuhuan; Liu, Zhao; Cai, Xiwen; Jan, Chao-Chien

    2013-01-01

    Conventional karyotypes and various genetic linkage maps have been established in sunflower (Helianthus annuus L., 2n = 34). However, the relationship between linkage groups and individual chromosomes of sunflower remains unknown and has considerable relevance for the sunflower research community. Recently, a set of linkage group-specific bacterial /binary bacterial artificial chromosome (BAC/BIBAC) clones was identified from two complementary BAC and BIBAC libraries constructed for cultivated sunflower cv. HA89. In the present study, we used these linkage group-specific clones (~100 kb in size) as probes to in situ hybridize to HA89 mitotic chromosomes at metaphase using the BAC-fluorescence in situ hybridization (FISH) technique. Because a characteristic of the sunflower genome is the abundance of repetitive DNA sequences, a high ratio of blocking DNA to probe DNA was applied to hybridization reactions to minimize the background noise. As a result, all sunflower chromosomes were anchored by one or two BAC/BIBAC clones with specific FISH signals. FISH analysis based on tandem repetitive sequences, such as rRNA genes, has been previously reported; however, the BAC-FISH technique developed here using restriction fragment length polymorphism (RFLP)-derived BAC/BIBAC clones as probes to apply genome-wide analysis is new for sunflower. As chromosome-specific cytogenetic markers, the selected BAC/BIBAC clones that encompass the 17 linkage groups provide a valuable tool for identifying sunflower cytogenetic stocks (such as trisomics) and tracking alien chromosomes in interspecific crosses. This work also demonstrates the potential of using a large-insert DNA library for the development of molecular cytogenetic resources.

  3. Cloning and sequencing of cDNA encoding human DNA topoisomerase II and localization of the gene to chromosome region 17q21-22

    International Nuclear Information System (INIS)

    Tsai-Pflugfelder, M.; Liu, L.F.; Liu, A.A.; Tewey, K.M.; Whang-Peng, J.; Knutsen, T.; Huebner, K.; Croce, C.M.; Wang, J.C.

    1988-01-01

    Two overlapping cDNA clones encoding human DNA topoisomerase II were identified by two independent methods. In one, a human cDNA library in phage λ was screened by hybridization with a mixed oligonucleotide probe encoding a stretch of seven amino acids found in yeast and Drosophila DNA topoisomerase II; in the other, a different human cDNA library in a λgt11 expression vector was screened for the expression of antigenic determinants that are recognized by rabbit antibodies specific to human DNA topoisomerase II. The entire coding sequences of the human DNA topoisomerase II gene were determined from these and several additional clones, identified through the use of the cloned human TOP2 gene sequences as probes. Hybridization between the cloned sequences and mRNA and genomic DNA indicates that the human enzyme is encoded by a single-copy gene. The location of the gene was mapped to chromosome 17q21-22 by in situ hybridization of a cloned fragment to metaphase chromosomes and by hybridization analysis with a panel of mouse-human hybrid cell lines, each retaining a subset of human chromosomes

  4. Construction of a 7-fold BAC library and cytogenetic mapping of 10 genes in the giant panda (Ailuropoda melanoleuca

    Directory of Open Access Journals (Sweden)

    Zhang Ying

    2006-11-01

    Full Text Available Abstract Background The giant panda, one of the most primitive carnivores, is an endangered animal. Although it has been the subject of many interesting studies during recent years, little is known about its genome. In order to promote research on this genome, a bacterial artificial chromosome (BAC library of the giant panda was constructed in this study. Results This BAC library contains 198,844 clones with an average insert size of 108 kb, which represents approximately seven equivalents of the giant panda haploid genome. Screening the library with 15 genes and 8 microsatellite markers demonstrates that it is representative and has good genome coverage. Furthermore, ten BAC clones harbouring AGXT, GHR, FSHR, IRBP, SOX14, TTR, BDNF, NT-4, LH and ZFX1 were mapped to 8 pairs of giant panda chromosomes by fluorescence in situ hybridization (FISH. Conclusion This is the first large-insert genomic DNA library for the giant panda, and will contribute to understanding this endangered species in the areas of genome sequencing, physical mapping, gene cloning and comparative genomic studies. We also identified the physical locations of ten genes on their relative chromosomes by FISH, providing a preliminary framework for further development of a high resolution cytogenetic map of the giant panda.

  5. Molecular cloning and characterization of recA-like gene from Schizosaccharomyces pombe

    International Nuclear Information System (INIS)

    Lee, J.S.; Kang, J.K.; Yoon, S.M.; Park, Y.; Yang, Y.K.; Kim, S.W.; Park, J.K.; Park, J.G.; Hong, S.H.; Park, S.D.

    1996-01-01

    We have previously purified and characterized a RecA-like protein from Schizosaccharomyces pombe (S. pombe). In the present study, we have cloned a gene encoding the RecA-like protein. The S. pombe recA-like gene was isolated by immunological screening of the expression library of S. pombe using anti-Escherichia coli (E. coli) RecA antibody as a probe. From 10(6) plaques screened, 6 putative clones were finally isolated. Five of the clones screened contained the same kinds of DNA inserts, as determined by crosshybridization analysis. Among the clones, TC-2 was selected for further studies. The pGEM3Zf(-)Delta 17 vector harboring the 4.3 kb DNA insert of TC-2 clone was capable of producing abeta-gal/RecA-like fusion protein, suggesting that the cloned gene encodes the RecA-like protein of S. pombe. It was also revealed by Southern hybridization analysis that the same DNA sequence as the cloned recA-like gene is located within the S. pombe chromosomal DNA. In addition, the cloned recA-like gene was transcribed into a 3.0 kb RNA transcript, as judged by Northern blot analysis. The level of the RNA transcript of recA-like gene was increased approximately 1.6 to 2.4-fold upon treatment with DNA damaging agents such as ultraviolet (UV)-light, methyl methanesulfonate (MMS), and mitomycin-C (MMC). This data suggests that the cloned S. pombe recA-like gene is slightly inducible to DNAdamage as in E. coli recA gene. These results suggest that an inducible repair mechanism analogous to that of E. coli may exist in fission yeast S. pombe

  6. A novel salt-tolerant chitobiosidase discovered by genetic screening of a metagenomic library derived from chitin-amended agricultural soil

    NARCIS (Netherlands)

    Cretoiu, Mariana Silvia; Berini, Francesca; Kielak, Anna Maria; Marinelli, Flavia; van Elsas, Jan Dirk

    2015-01-01

    Here, we report on the construction of a metagenomic library from a chitin-amended disease-suppressive agricultural soil and its screening for genes that encode novel chitinolytic enzymes. The library, constructed in fosmids in an Escherichia coli host, comprised 145,000 clones containing inserts of

  7. Construction of high quality Gateway™ entry libraries and their application to yeast two-hybrid for the monocot model plant Brachypodium distachyon

    Directory of Open Access Journals (Sweden)

    Kumimoto Roderick W

    2011-05-01

    Full Text Available Abstract Background Monocots, especially the temperate grasses, represent some of the most agriculturally important crops for both current food needs and future biofuel development. Because most of the agriculturally important grass species are difficult to study (e.g., they often have large, repetitive genomes and can be difficult to grow in laboratory settings, developing genetically tractable model systems is essential. Brachypodium distachyon (hereafter Brachypodium is an emerging model system for the temperate grasses. To fully realize the potential of this model system, publicly accessible discovery tools are essential. High quality cDNA libraries that can be readily adapted for multiple downstream purposes are a needed resource. Additionally, yeast two-hybrid (Y2H libraries are an important discovery tool for protein-protein interactions and are not currently available for Brachypodium. Results We describe the creation of two high quality, publicly available Gateway™ cDNA entry libraries and their derived Y2H libraries for Brachypodium. The first entry library represents cloned cDNA populations from both short day (SD, 8/16-h light/dark and long day (LD, 20/4-h light/dark grown plants, while the second library was generated from hormone treated tissues. Both libraries have extensive genome coverage (~5 × 107 primary clones each and average clone lengths of ~1.5 Kb. These entry libraries were then used to create two recombination-derived Y2H libraries. Initial proof-of-concept screens demonstrated that a protein with known interaction partners could readily re-isolate those partners, as well as novel interactors. Conclusions Accessible community resources are a hallmark of successful biological model systems. Brachypodium has the potential to be a broadly useful model system for the grasses, but still requires many of these resources. The Gateway™ compatible entry libraries created here will facilitate studies for multiple user

  8. Construction and characterization of two BAC libraries representing a deep-coverage of the genome of chicory (Cichorium intybus L., Asteraceae

    Directory of Open Access Journals (Sweden)

    Gonthier Lucy

    2010-08-01

    Full Text Available Abstract Background The Asteraceae represents an important plant family with respect to the numbers of species present in the wild and used by man. Nonetheless, genomic resources for Asteraceae species are relatively underdeveloped, hampering within species genetic studies as well as comparative genomics studies at the family level. So far, six BAC libraries have been described for the main crops of the family, i.e. lettuce and sunflower. Here we present the characterization of BAC libraries of chicory (Cichorium intybus L. constructed from two genotypes differing in traits related to sexual and vegetative reproduction. Resolving the molecular mechanisms underlying traits controlling the reproductive system of chicory is a key determinant for hybrid development, and more generally will provide new insights into these traits, which are poorly investigated so far at the molecular level in Asteraceae. Findings Two bacterial artificial chromosome (BAC libraries, CinS2S2 and CinS1S4, were constructed from HindIII-digested high molecular weight DNA of the contrasting genotypes C15 and C30.01, respectively. C15 was hermaphrodite, non-embryogenic, and S2S2 for the S-locus implicated in self-incompatibility, whereas C30.01 was male sterile, embryogenic, and S1S4. The CinS2S2 and CinS1S4 libraries contain 89,088 and 81,408 clones. Mean insert sizes of the CinS2S2 and CinS1S4 clones are 90 and 120 kb, respectively, and provide together a coverage of 12.3 haploid genome equivalents. Contamination with mitochondrial and chloroplast DNA sequences was evaluated with four mitochondrial and four chloroplast specific probes, and was estimated to be 0.024% and 1.00% for the CinS2S2 library, and 0.028% and 2.35% for the CinS1S4 library. Using two single copy genes putatively implicated in somatic embryogenesis, screening of both libraries resulted in detection of 12 and 13 positive clones for each gene, in accordance with expected numbers. Conclusions This

  9. Construction and characterization of two BAC libraries representing a deep-coverage of the genome of chicory (Cichorium intybus L., Asteraceae).

    Science.gov (United States)

    Gonthier, Lucy; Bellec, Arnaud; Blassiau, Christelle; Prat, Elisa; Helmstetter, Nicolas; Rambaud, Caroline; Huss, Brigitte; Hendriks, Theo; Bergès, Hélène; Quillet, Marie-Christine

    2010-08-11

    The Asteraceae represents an important plant family with respect to the numbers of species present in the wild and used by man. Nonetheless, genomic resources for Asteraceae species are relatively underdeveloped, hampering within species genetic studies as well as comparative genomics studies at the family level. So far, six BAC libraries have been described for the main crops of the family, i.e. lettuce and sunflower. Here we present the characterization of BAC libraries of chicory (Cichorium intybus L.) constructed from two genotypes differing in traits related to sexual and vegetative reproduction. Resolving the molecular mechanisms underlying traits controlling the reproductive system of chicory is a key determinant for hybrid development, and more generally will provide new insights into these traits, which are poorly investigated so far at the molecular level in Asteraceae. Two bacterial artificial chromosome (BAC) libraries, CinS2S2 and CinS1S4, were constructed from HindIII-digested high molecular weight DNA of the contrasting genotypes C15 and C30.01, respectively. C15 was hermaphrodite, non-embryogenic, and S2S2 for the S-locus implicated in self-incompatibility, whereas C30.01 was male sterile, embryogenic, and S1S4. The CinS2S2 and CinS1S4 libraries contain 89,088 and 81,408 clones. Mean insert sizes of the CinS2S2 and CinS1S4 clones are 90 and 120 kb, respectively, and provide together a coverage of 12.3 haploid genome equivalents. Contamination with mitochondrial and chloroplast DNA sequences was evaluated with four mitochondrial and four chloroplast specific probes, and was estimated to be 0.024% and 1.00% for the CinS2S2 library, and 0.028% and 2.35% for the CinS1S4 library. Using two single copy genes putatively implicated in somatic embryogenesis, screening of both libraries resulted in detection of 12 and 13 positive clones for each gene, in accordance with expected numbers. This indicated that both BAC libraries are valuable tools for molecular

  10. Cloning of precursors for two MIH/VIH-related peptides in the prawn, Macrobrachium rosenbergii.

    Science.gov (United States)

    Yang, W J; Rao, K R

    2001-11-30

    Two cDNA clones (634 and 1366 bp) encoding MIH/VIH (molt-inhibiting hormone/vitellogenesis-inhibiting hormone)-related peptides were isolated and sequenced from a Macrobrachium rosenbergii eyestalk ganglia cDNA library. The clones contain a 360 and 339 bp open-reading frame, and their conceptually translated peptides consist of a 41 and 34 amino acid signal peptide, respectively, and a 78 amino acid residue mature peptide hormone. The amino acid sequences of the peptides exhibit higher identities with other known MIHs and VIH (44-69%) than with CHHs (28-33%). This is the first report describing the cloning and sequencing of two MIH/VIH-related peptides in a single crustacean species. Transcription of these mRNAs was detected in the eyestalk ganglia, but not in the thoracic ganglia, hepatopancreas, gut, gill, heart, or muscle.

  11. Cloning of Plasmodium falciparum by single-cell sorting.

    Science.gov (United States)

    Miao, Jun; Li, Xiaolian; Cui, Liwang

    2010-10-01

    Malaria parasite cloning is traditionally carried out mainly by using the limiting dilution method, which is laborious, imprecise, and unable to distinguish multiply-infected RBCs. In this study, we used a parasite engineered to express green fluorescent protein (GFP) to evaluate a single-cell sorting method for rapidly cloning Plasmodium falciparum. By dividing a two-dimensional scattergram from a cell sorter into 17 gates, we determined the parameters for isolating singly-infected erythrocytes and sorted them into individual cultures. Pre-gating of the engineered parasites for GFP allowed the isolation of almost 100% GFP-positive clones. Compared with the limiting dilution method, the number of parasite clones obtained by single-cell sorting was much higher. Molecular analyses showed that parasite isolates obtained by single-cell sorting were highly homogenous. This highly efficient single-cell sorting method should prove very useful for cloning both P. falciparum laboratory populations from genetic manipulation experiments and clinical samples. Copyright 2010 Elsevier Inc. All rights reserved.

  12. Cloning of Plasmodium falciparum by single-cell sorting

    Science.gov (United States)

    Miao, Jun; Li, Xiaolian; Cui, Liwang

    2010-01-01

    Malaria parasite cloning is traditionally carried out mainly by using the limiting dilution method, which is laborious, imprecise, and unable to distinguish multiply-infected RBCs. In this study, we used a parasite engineered to express green fluorescent protein (GFP) to evaluate a single-cell sorting method for rapidly cloning Plasmodium falciparum. By dividing a two dimensional scattergram from a cell sorter into 17 gates, we determined the parameters for isolating singly-infected erythrocytes and sorted them into individual cultures. Pre-gating of the engineered parasites for GFP allowed the isolation of almost 100% GFP-positive clones. Compared with the limiting dilution method, the number of parasite clones obtained by single-cell sorting was much higher. Molecular analyses showed that parasite isolates obtained by single-cell sorting were highly homogenous. This highly efficient single-cell sorting method should prove very useful for cloning both P. falciparum laboratory populations from genetic manipulation experiments and clinical samples. PMID:20435038

  13. library use instruction and the pattern of utilization of library services ...

    African Journals Online (AJOL)

    Global Journal

    The data collected was analysed using descriptive statistics (simple percentage %). ... semesters and it should be an independent credit carrying course under the General Studies ... large and well stocked a library is, if the ..... Online. LIBRARY USE INSTRUCTION AND THE PATTERN OF UTILIZATION OF LIBRARY ...

  14. Cloning and expression of a cDNA coding for a human monocyte-derived plasminogen activator inhibitor.

    OpenAIRE

    Antalis, T M; Clark, M A; Barnes, T; Lehrbach, P R; Devine, P L; Schevzov, G; Goss, N H; Stephens, R W; Tolstoshev, P

    1988-01-01

    Human monocyte-derived plasminogen activator inhibitor (mPAI-2) was purified to homogeneity from the U937 cell line and partially sequenced. Oligonucleotide probes derived from this sequence were used to screen a cDNA library prepared from U937 cells. One positive clone was sequenced and contained most of the coding sequence as well as a long incomplete 3' untranslated region (1112 base pairs). This cDNA sequence was shown to encode mPAI-2 by hybrid-select translation. A cDNA clone encoding t...

  15. Analyses of an expressed sequence tag library from Taenia solium, Cysticerca.

    Directory of Open Access Journals (Sweden)

    Jonas Lundström

    Full Text Available BACKGROUND: Neurocysticercosis is a disease caused by the oral ingestion of eggs from the human parasitic worm Taenia solium. Although drugs are available they are controversial because of the side effects and poor efficiency. An expressed sequence tag (EST library is a method used to describe the gene expression profile and sequence of mRNA from a specific organism and stage. Such information can be used in order to find new targets for the development of drugs and to get a better understanding of the parasite biology. METHODS AND FINDINGS: Here an EST library consisting of 5760 sequences from the pig cysticerca stage has been constructed. In the library 1650 unique sequences were found and of these, 845 sequences (52% were novel to T. solium and not identified within other EST libraries. Furthermore, 918 sequences (55% were of unknown function. Amongst the 25 most frequently expressed sequences 6 had no relevant similarity to other sequences found in the Genbank NR DNA database. A prediction of putative signal peptides was also performed and 4 among the 25 were found to be predicted with a signal peptide. Proposed vaccine and diagnostic targets T24, Tsol18/HP6 and Tso31d could also be identified among the 25 most frequently expressed. CONCLUSIONS: An EST library has been produced from pig cysticerca and analyzed. More than half of the different ESTs sequenced contained a sequence with no suggested function and 845 novel EST sequences have been identified. The library increases the knowledge about what genes are expressed and to what level. It can also be used to study different areas of research such as drug and diagnostic development together with parasite fitness via e.g. immune modulation.

  16. Public libraries in the library regions in the year 2009

    Directory of Open Access Journals (Sweden)

    Milena Bon

    2011-01-01

    Full Text Available Purpose: Regional public libraries were initiated in 2003 to connect professional activities of libraries within regional networks and to ensure coordinated library development in a region in cooperation with the Library System Development Centre at the National and University Library performing a coordinating role. The article analyses the performance of public libraries and their integration in regional library networks in order to find out the level of development of conditions of performance of public libraries.Methodology/approach: Statistical data for the year 2009 were the basis for the overview of library activities of ten library regions with regard to applicable legislation and library standards. The level of regional library activities is compared to the socio-economic situation of statistical regions thus representing a new approach to the presentation of Slovenian’s public libraries’ development.Results: Absolute values indicate better development of nine libraries in the central Slovenia region while relative values offer a totally different picture. Four libraries in the region of Nova Gorica prove the highest level of development.Research limitation: Research is limited to the year 2009 and basic statistical analysis.Originality/practical implications: Findings of the analysis are useful for public libraries to plan their development strategy within a region and for financial bodies to provide for adequate financing for library activities in a specific region. The basic condition for successful public library performance is the even and harmonized development of conditions of performance as recommended by library standards.

  17. Adhesive polypeptides of Staphylococcus aureus identified using a novel secretion library technique in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Holm Liisa

    2011-05-01

    Full Text Available Abstract Background Bacterial adhesive proteins, called adhesins, are frequently the decisive factor in initiation of a bacterial infection. Characterization of such molecules is crucial for the understanding of bacterial pathogenesis, design of vaccines and development of antibacterial drugs. Because adhesins are frequently difficult to express, their characterization has often been hampered. Alternative expression methods developed for the analysis of adhesins, e.g. surface display techniques, suffer from various drawbacks and reports on high-level extracellular secretion of heterologous proteins in Gram-negative bacteria are scarce. These expression techniques are currently a field of active research. The purpose of the current study was to construct a convenient, new technique for identification of unknown bacterial adhesive polypeptides directly from the growth medium of the Escherichia coli host and to identify novel proteinaceous adhesins of the model organism Staphylococcus aureus. Results Randomly fragmented chromosomal DNA of S. aureus was cloned into a unique restriction site of our expression vector, which facilitates secretion of foreign FLAG-tagged polypeptides into the growth medium of E. coli ΔfliCΔfliD, to generate a library of 1663 clones expressing FLAG-tagged polypeptides. Sequence and bioinformatics analyses showed that in our example, the library covered approximately 32% of the S. aureus proteome. Polypeptides from the growth medium of the library clones were screened for binding to a selection of S. aureus target molecules and adhesive fragments of known staphylococcal adhesins (e.g coagulase and fibronectin-binding protein A as well as polypeptides of novel function (e.g. a universal stress protein and phosphoribosylamino-imidazole carboxylase ATPase subunit were detected. The results were further validated using purified His-tagged recombinant proteins of the corresponding fragments in enzyme-linked immunoassay and

  18. Recombinational Cloning Using Gateway and In-Fusion Cloning Schemes

    Science.gov (United States)

    Throop, Andrea L.; LaBaer, Joshua

    2015-01-01

    The comprehensive study of protein structure and function, or proteomics, depends on the obtainability of full-length cDNAs in species-specific expression vectors and subsequent functional analysis of the expressed protein. Recombinational cloning is a universal cloning technique based on site-specific recombination that is independent of the insert DNA sequence of interest, which differentiates this method from the classical restriction enzyme-based cloning methods. Recombinational cloning enables rapid and efficient parallel transfer of DNA inserts into multiple expression systems. This unit summarizes strategies for generating expression-ready clones using the most popular recombinational cloning technologies, including the commercially available Gateway® (Life Technologies) and In-Fusion® (Clontech) cloning technologies. PMID:25827088

  19. Reliability analysis of the Ahringer Caenorhabditis elegans RNAi feeding library: a guide for genome-wide screens

    Directory of Open Access Journals (Sweden)

    Lu Yiming

    2011-03-01

    Full Text Available Abstract Background The Ahringer C. elegans RNAi feeding library prepared by cloning genomic DNA fragments has been widely used in genome-wide analysis of gene function. However, the library has not been thoroughly validated by direct sequencing, and there are potential errors, including: 1 mis-annotation (the clone with the retired gene name should be remapped to the actual target gene; 2 nonspecific PCR amplification; 3 cross-RNAi; 4 mis-operation such as sample loading error, etc. Results Here we performed a reliability analysis on the Ahringer C. elegans RNAi feeding library, which contains 16,256 bacterial strains, using a bioinformatics approach. Results demonstrated that most (98.3% of the bacterial strains in the library are reliable. However, we also found that 2,851 (17.54% bacterial strains need to be re-annotated even they are reliable. Most of these bacterial strains are the clones having the retired gene names. Besides, 28 strains are grouped into unreliable category and 226 strains are marginal because of probably expressing unrelated double-stranded RNAs (dsRNAs. The accuracy of the prediction was further confirmed by direct sequencing analysis of 496 bacterial strains. Finally, a freely accessible database named CelRNAi (http://biocompute.bmi.ac.cn/CelRNAi/ was developed as a valuable complement resource for the feeding RNAi library by providing the predicted information on all bacterial strains. Moreover, submission of the direct sequencing result or any other annotations for the bacterial strains to the database are allowed and will be integrated into the CelRNAi database to improve the accuracy of the library. In addition, we provide five candidate primer sets for each of the unreliable and marginal bacterial strains for users to construct an alternative vector for their own RNAi studies. Conclusions Because of the potential unreliability of the Ahringer C. elegans RNAi feeding library, we strongly suggest the user examine

  20. Cloning and expression of the Legionella micdadei "common antigen" in Escherichia coli

    DEFF Research Database (Denmark)

    Bangsborg, Jette Marie; Collins, M T; Høiby, N

    1989-01-01

    To study individual Legionella antigens, a Legionella micdadei genomic library in Escherichia coli SC181 was established. Partially Sau3A digested L. micdadei DNA fragments (15-25 kilobase pairs (kb] were cloned into the tetracycline resistance gene of the cosmid vector pHC79. Four thousand...... ampicillin resistant recombinants were obtained; seven hundred were screened for expression of Legionella antigens in Western blot analysis with a polyspecific E. coli-absorbed anti-L. micdadei rabbit antibody. One of the positive clones expressed a 60 kilodalton (K) antigen, which reacted strongly...... will provide important information with respect to genetic vs. antigenic relatedness among Legionellae and other Gram-negative species, as well as to CA structure and possible function....

  1. Evaluation of flooding tolerance in cuttings of Populus clones used ...

    African Journals Online (AJOL)

    We analysed the responses to flooding of 14 poplar clones used for forestation at the Paraná River Delta, Argentina. Some are commercial clones planted in the area, and others belong to a poplar breeding program from the National Institute of Agricultural Technology (INTA) in Argentina. Potted plants of 60 cm high ...

  2. Cloning of a novel gene, Cymg1, related to family 2 cystatins and ...

    Indian Academy of Sciences (India)

    We have cloned a novel gene, Cymg1 (GenBank accession number AY600990), from a mouse testis cDNA library. Cymg1 is located in 2G3 of mouse chromosome 2. The cDNA includes an open reading frame that encodes 141 amino acid residues. The encoded polypeptide has a cysteine protease inhibitor domain found ...

  3. Molecular cloning, functional expression, and tissue distribution of a novel human gap junction-forming protein, connexin-31.9. Interaction with zona occludens protein-1

    NARCIS (Netherlands)

    Nielsen, Peter A; Beahm, Derek L; Giepmans, Ben N G; Baruch, Amos; Hall, James E; Kumar, Nalin M

    2002-01-01

    A novel human connexin gene (GJA11) was cloned from a genomic library. The open reading frame encoded a hypothetical protein of 294 amino acid residues with a predicted molecular mass of 31,933, hence referred to as connexin-31.9 (Cx31.9) or alpha 11 connexin. A clone in GenBank containing the

  4. Unified Approach to Universal Cloning and Phase-Covariant Cloning

    OpenAIRE

    Hu, Jia-Zhong; Yu, Zong-Wen; Wang, Xiang-Bin

    2008-01-01

    We analyze the problem of approximate quantum cloning when the quantum state is between two latitudes on the Bloch's sphere. We present an analytical formula for the optimized 1-to-2 cloning. The formula unifies the universal quantum cloning (UQCM) and the phase covariant quantum cloning.

  5. Human terminal deoxyribonucleotidyltransferase: molecular cloning and structural analysis of the gene and 5' flanking region

    International Nuclear Information System (INIS)

    Riley, L.K.; Morrow, J.K.; Danton, M.J.; Coleman, M.S.

    1988-01-01

    Human terminal deoxyribonucleotidyltransferase cDNA contains an open reading frame of 1530 base pairs (bp) corresponding to a protein containing 510 amino acids. The encoded protein is a template-independent DNA polymerase found only in a restricted population of normal and malignant prelymphocytes. To begin to investigate the genetic elements responsible for the tissue-specific expression of terminal deoxyribonucleotidyltransferase, genomic clones, containing the entire human gene were isolated and characterized. Initially, cDNA clones were isolated from a library generated from the human lymphoblastoid cell line, MOLT-4R. A cDNA clone containing the entire coding region of the protein was used to isolate a series of overlapping clones from two human genomic libraries. The gene comprises 11 exons and 10 introns and spans 49.4 kilobases. The 5' flanking region (709 bp) including exon 1 was sequenced. Several putative transcription initiation sites were mapped. Within 500 nucleotides of the translation start site, a series of promoter elements was detected. TATA and CAAT sequences, respectively, were found to start at nucleotides -185 and -204, -328 and -370, and -465 and -505. Start sites were found for a cyclic AMP-dependent promoter analog at nucleotide -121, an eight-base sequence corresponding to the IgG promoter enhancer (cd) at nucleotide -455, and an analog of the IgG promoter (pd) at nucleotide -159. These findings suggest that transcripts coding for terminal deoxyribonucleotidyltransferase may be variable in length and that transcription may be influenced by a variety of genetic elements

  6. THE TERMINOLOGY OF LIBRARY SCIENCE

    OpenAIRE

    Љиљана Матић

    2014-01-01

    The master’s thesis entitled The Terminology of Library Science presents the general state of the terminology of library science in the Serbian language and analyses the terminological system which was formed in the last couple of decades in relation to library and information science. The terminology of library science is seen as a characteristic of professional language. The research is conducted on a corpus which excludes sources relating extremely to either library science or information ...

  7. Brain cDNA clone for human cholinesterase

    International Nuclear Information System (INIS)

    McTiernan, C.; Adkins, S.; Chatonnet, A.; Vaughan, T.A.; Bartels, C.F.; Kott, M.; Rosenberry, T.L.; La Du, B.N.; Lockridge, O.

    1987-01-01

    A cDNA library from human basal ganglia was screened with oligonucleotide probes corresponding to portions of the amino acid sequence of human serum cholinesterase. Five overlapping clones, representing 2.4 kilobases, were isolated. The sequenced cDNA contained 207 base pairs of coding sequence 5' to the amino terminus of the mature protein in which there were four ATG translation start sites in the same reading frame as the protein. Only the ATG coding for Met-(-28) lay within a favorable consensus sequence for functional initiators. There were 1722 base pairs of coding sequence corresponding to the protein found circulating in human serum. The amino acid sequence deduced from the cDNA exactly matched the 574 amino acid sequence of human serum cholinesterase, as previously determined by Edman degradation. Therefore, our clones represented cholinesterase rather than acetylcholinesterase. It was concluded that the amino acid sequences of cholinesterase from two different tissues, human brain and human serum, were identical. Hybridization of genomic DNA blots suggested that a single gene, or very few genes coded for cholinesterase

  8. Construction and characterization of a full-length cDNA library for the wheat stripe rust pathogen (Puccinia striiformis f. sp. tritici

    Directory of Open Access Journals (Sweden)

    Chen Xianming

    2007-06-01

    Full Text Available Abstract Background Puccinia striiformis is a plant pathogenic fungus causing stripe rust, one of the most important diseases on cereal crops and grasses worldwide. However, little is know about its genome and genes involved in the biology and pathogenicity of the pathogen. We initiated the functional genomic research of the fungus by constructing a full-length cDNA and determined functions of the first group of genes by sequence comparison of cDNA clones to genes reported in other fungi. Results A full-length cDNA library, consisting of 42,240 clones with an average cDNA insert of 1.9 kb, was constructed using urediniospores of race PST-78 of P. striiformis f. sp. tritici. From 196 sequenced cDNA clones, we determined functions of 73 clones (37.2%. In addition, 36 clones (18.4% had significant homology to hypothetical proteins, 37 clones (18.9% had some homology to genes in other fungi, and the remaining 50 clones (25.5% did not produce any hits. From the 73 clones with functions, we identified 51 different genes encoding protein products that are involved in amino acid metabolism, cell defense, cell cycle, cell signaling, cell structure and growth, energy cycle, lipid and nucleotide metabolism, protein modification, ribosomal protein complex, sugar metabolism, transcription factor, transport metabolism, and virulence/infection. Conclusion The full-length cDNA library is useful in identifying functional genes of P. striiformis.

  9. Cloning and properties of the Salmonella typhimurium tricarboxylate transport operon in Escherichia coli

    International Nuclear Information System (INIS)

    Widenhorn, K.A.; Boos, W.; Somers, J.M.; Kay, W.W.

    1988-01-01

    The tricarboxylate transport operon (tctI) was cloned in Escherichia coli as a 12-kilobase (kb) fragment from an EcoRI library of the Salmonella typhimurium chromosome in λgtWES. It was further subcloned as a 12-kb fragment into pACYC184 and as an 8-kb fragment into pBR322. By insertional mutagenesis mediated by λTn5, restriction mapping, and phenotypic testing, the tctI operon was localized to a 4.5-kb region. The tctC gene which encodes a periplasmic binding protein (C-protein) was located near the center of the insert. E. coli/tctI clones on either multicopy or single-copy vectors grew on the same tricarboxylates as S. typhimurium, although unusually long growth lags were observed. E. coli/tctI clones exhibited similar [ 14 C] fluorocitrate transport kinetics to those of S. typhimurium, whereas E. coli alone was virtually impermeable to [ 14 C] fluorocitrate. The periplasmic C proteins (C1 and C2 isoelectric forms) were produced in prodigious quantities from the cloned strains. Motile E. coli/tctI clones were not chemotactic toward citrate, whereas tctI deletion mutants of S. typhimurium were. Taken together, these observations indicate that tctI is not an operon involved in chemotaxis

  10. Genomic clone encoding the α chain of the OKM1, LFA-1, and platelet glycoprotein IIb-IIIa molecules

    International Nuclear Information System (INIS)

    Cosgrove, L.J.; Sandrin, M.S.; Rajasekariah, P.; McKenzie, I.F.C.

    1986-01-01

    LFA-1, an antigen involved in cytolytic T lymphocyte-mediated killing, and Mac-1, the receptor for complement component C3bi, constitute a family of structurally and functionally related cell surface glycoproteins involved in cellular interactions. In both mouse and man, Mac-1 (OKM1) and LFA-1 share a common 95-kDa β subunit but are distinguished by their α chains, which have different cellular distributions, apparent molecular masses (165 and 177 kDa, respectively), and peptide maps. The authors report the isolation of a genomic clone from a human genomic library that on transfection into mouse fibroblasts produced a molecule(s) reactive with monoclonal antibodies to OKM1, to LFA-1, and to platelet glycoprotein IIb-IIIa. This gene was cloned by several cycles of transfection of L cells with a human genomic library cloned in λ phase Charon 4A and subsequent rescue of the λ phage. Transfection with the purified recombinant λ DNA yielded a transfectant that expressed the three human α chains of OKM1, LFA-1, and glycoprotein IIb-IIIa, presumably in association with the murine β chain

  11. WebPrInSeS: automated full-length clone sequence identification and verification using high-throughput sequencing data.

    Science.gov (United States)

    Massouras, Andreas; Decouttere, Frederik; Hens, Korneel; Deplancke, Bart

    2010-07-01

    High-throughput sequencing (HTS) is revolutionizing our ability to obtain cheap, fast and reliable sequence information. Many experimental approaches are expected to benefit from the incorporation of such sequencing features in their pipeline. Consequently, software tools that facilitate such an incorporation should be of great interest. In this context, we developed WebPrInSeS, a web server tool allowing automated full-length clone sequence identification and verification using HTS data. WebPrInSeS encompasses two separate software applications. The first is WebPrInSeS-C which performs automated sequence verification of user-defined open-reading frame (ORF) clone libraries. The second is WebPrInSeS-E, which identifies positive hits in cDNA or ORF-based library screening experiments such as yeast one- or two-hybrid assays. Both tools perform de novo assembly using HTS data from any of the three major sequencing platforms. Thus, WebPrInSeS provides a highly integrated, cost-effective and efficient way to sequence-verify or identify clones of interest. WebPrInSeS is available at http://webprinses.epfl.ch/ and is open to all users.

  12. Molecular cloning and expression of bovine kappa-casein in Escherichia coli

    International Nuclear Information System (INIS)

    Kang, Y.C.; Richardson, T.

    1988-01-01

    A cDNA library was constructed using poly(A) + RNA from bovine mammary gland. This cDNA library of 6000 clones was screened employing colony hybridization using 32 P-labelled oligonucleotide probes and restriction endonuclease mapping. The cDNA from the selected plasmid, pKR76, was sequenced using the dideoxy-chain termination method. The cDNA insert of pKR76 carries the full-length sequence, which codes for mature kappa-casein protein. The amino acid sequence deduced from the cDNA sequence fits the published amino acid sequence with three exceptions; the reported pyroglutamic acid at position 1, tyrosine at position 35, and aspartic acid at position 81 are, respectively, a glutamine, a histidine, and an asparagine in the clone containing pKR76. The MspI-, NlaIV-cleaved fragment (630 base pair) from the kappa-casein cDNA insert has been subcloned into expression vectors pUC18 and pKK233-2, which contain a lac promoter and a trc promoter, respectively. Escherichia coli cells carrying the recombinant expression plasmids were shown to produce kappa-casein protein having the expected mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and being recognized by specific antibodies raised against natural bovine kappa-casein

  13. Identification of genes and pathways related to phenol degradation in metagenomic libraries from petroleum refinery wastewater.

    Directory of Open Access Journals (Sweden)

    Cynthia C Silva

    Full Text Available Two fosmid libraries, totaling 13,200 clones, were obtained from bioreactor sludge of petroleum refinery wastewater treatment system. The library screening based on PCR and biological activity assays revealed more than 400 positive clones for phenol degradation. From these, 100 clones were randomly selected for pyrosequencing in order to evaluate the genetic potential of the microorganisms present in wastewater treatment plant for biodegradation, focusing mainly on novel genes and pathways of phenol and aromatic compound degradation. The sequence analysis of selected clones yielded 129,635 reads at an estimated 17-fold coverage. The phylogenetic analysis showed Burkholderiales and Rhodocyclales as the most abundant orders among the selected fosmid clones. The MG-RAST analysis revealed a broad metabolic profile with important functions for wastewater treatment, including metabolism of aromatic compounds, nitrogen, sulphur and phosphorus. The predicted 2,276 proteins included phenol hydroxylases and cathecol 2,3- dioxygenases, involved in the catabolism of aromatic compounds, such as phenol, byphenol, benzoate and phenylpropanoid. The sequencing of one fosmid insert of 33 kb unraveled the gene that permitted the host, Escherichia coli EPI300, to grow in the presence of aromatic compounds. Additionally, the comparison of the whole fosmid sequence against bacterial genomes deposited in GenBank showed that about 90% of sequence showed no identity to known sequences of Proteobacteria deposited in the NCBI database. This study surveyed the functional potential of fosmid clones for aromatic compound degradation and contributed to our knowledge of the biodegradative capacity and pathways of microbial assemblages present in refinery wastewater treatment system.

  14. cDNA cloning and mRNA expression of heat shock protein 70 gene ...

    African Journals Online (AJOL)

    In this study, the full-length heat shock protein 70 of Tegillarca granosa was cloned from cDNA library by rapid amplification of cDNA end (RACE). The open reading frame (ORF) of heat shock protein 70 was 1968 bp, and it encoded a protein of 655 amino acids with a predicted molecular weight of 71.48 kDa and an ...

  15. Personality consistency analysis in cloned quarantine dog candidates

    Directory of Open Access Journals (Sweden)

    Jin Choi

    2017-01-01

    Full Text Available In recent research, personality consistency has become an important characteristic. Diverse traits and human-animal interactions, in particular, are studied in the field of personality consistency in dogs. Here, we investigated the consistency of dominant behaviours in cloned and control groups followed by the modified Puppy Aptitude Test, which consists of ten subtests to ascertain the influence of genetic identity. In this test, puppies are exposed to stranger, restraint, prey-like object, noise, startling object, etc. Six cloned and four control puppies participated and the consistency of responses at ages 7–10 and 16 weeks in the two groups was compared. The two groups showed different consistencies in the subtests. While the average scores of the cloned group were consistent (P = 0.7991, those of the control group were not (P = 0.0089. Scores of Pack Drive and Fight or Flight Drive were consistent in the cloned group, however, those of the control group were not. Scores of Prey Drive were not consistent in either the cloned or the control group. Therefore, it is suggested that consistency of dominant behaviour is affected by genetic identity and some behaviours can be influenced more than others. Our results suggest that cloned dogs could show more consistent traits than non-cloned. This study implies that personality consistency could be one of the ways to analyse traits of puppies.

  16. Chromosome region-specific libraries for human genome analysis. Final progress report, 1 March 1991--28 February 1994

    Energy Technology Data Exchange (ETDEWEB)

    Kao, F.T.

    1994-04-01

    The objectives of this grant proposal include (1) development of a chromosome microdissection and PCR-mediated microcloning technology, (2) application of this microtechnology to the construction of region-specific libraries for human genome analysis. During this grant period, the authors have successfully developed this microtechnology and have applied it to the construction of microdissection libraries for the following chromosome regions: a whole chromosome 21 (21E), 2 region-specific libraries for the long arm of chromosome 2, 2q35-q37 (2Q1) and 2q33-q35 (2Q2), and 4 region-specific libraries for the entire short arm of chromosome 2, 2p23-p25 (2P1), 2p21-p23 (2P2), 2p14-p16 (wP3) and 2p11-p13 (2P4). In addition, 20--40 unique sequence microclones have been isolated and characterized for genomic studies. These region-specific libraries and the single-copy microclones from the library have been used as valuable resources for (1) isolating microsatellite probes in linkage analysis to further refine the disease locus; (2) isolating corresponding clones with large inserts, e.g. YAC, BAC, P1, cosmid and phage, to facilitate construction of contigs for high resolution physical mapping; and (3) isolating region-specific cDNA clones for use as candidate genes. These libraries are being deposited in the American Type Culture Collection (ATCC) for general distribution.

  17. The identification of specific cDNA clones from tall and dwarf rice plants

    International Nuclear Information System (INIS)

    Youssefian, S.; Kamada, I.; Sano, H.

    1990-01-01

    Full text: The use of dwarfing genes in rice breeding has proceeded for several years without a clear understanding of the genetic, hormonal and physiological mechanisms involved. This issue was addressed by focussing on the isolation of specific clones from tall- and dwarf-derived cDNA libraries. The materials used include near-isogenic lines of the tall rice cultivar 'Shiokari', differing at the DGWG or 'Tanginbozu' dwarfing gene loci. Also used were tall and dwarf 'Ginbozu' rice, the latter having been induced by treatment with 5-azacytidine, a potent demethylating agent. Subtractive and differential hybridisation have, to date, identified several candidate tall- and dwarf-specific clones. Their further characterisation is currently underway. (author)

  18. Enzyme free cloning for high throughput gene cloning and expression

    NARCIS (Netherlands)

    de Jong, R.N.; Daniëls, M.; Kaptein, R.; Folkers, G.E.

    2006-01-01

    Structural and functional genomics initiatives significantly improved cloning methods over the past few years. Although recombinational cloning is highly efficient, its costs urged us to search for an alternative high throughput (HTP) cloning method. We implemented a modified Enzyme Free Cloning

  19. Cloning and expression of clt genes encoding milk-clotting proteases from Myxococcus xanthus 422.

    Science.gov (United States)

    Poza, M; Prieto-Alcedo, M; Sieiro, C; Villa, T G

    2004-10-01

    The screening of a gene library of the milk-clotting strain Myxococcus xanthus 422 constructed in Escherichia coli allowed the description of eight positive clones containing 26 open reading frames. Only three of them (cltA, cltB, and cltC) encoded proteins that exhibited intracellular milk-clotting ability in E. coli, Saccharomyces cerevisiae, and Pichia pastoris expression systems.

  20. The Virtual Library in Action

    International Nuclear Information System (INIS)

    Addis, Louise

    1999-01-01

    The SLAC Library has for many years provided SPIRES-HEP, a 300,000 record bibliographic database, to the world particle physics community via the Internet as well as through clone sites in Europe and Japan. The 1991 introduction of the e-print archives at LANL coupled with the World-Wide-Web (WWW) from CERN suddenly made it possible to provide easy linkage between bibliographic database records and the actual full-text of papers. The SLAC Library has turned this possibility into reality by converting hundreds of TeX source documents each month into viewable postscript complete with figures. These (now more than 20,000) postscript files are linked to the HEP database, and the full-text is rendered universally visible via WWW. We discuss the project, the collaboration of physicists and librarians, what is easy, what is hard, and our vision for the future

  1. The Virtual Library in Action

    Energy Technology Data Exchange (ETDEWEB)

    Addis, Louise

    1999-07-02

    The SLAC Library has for many years provided SPIRES-HEP, a 300,000 record bibliographic database, to the world particle physics community via the Internet as well as through clone sites in Europe and Japan. The 1991 introduction of the e-print archives at LANL coupled with the World-Wide-Web (WWW) from CERN suddenly made it possible to provide easy linkage between bibliographic database records and the actual full-text of papers. The SLAC Library has turned this possibility into reality by converting hundreds of TeX source documents each month into viewable postscript complete with figures. These (now more than 20,000) postscript files are linked to the HEP database, and the full-text is rendered universally visible via WWW. We discuss the project, the collaboration of physicists and librarians, what is easy, what is hard, and our vision for the future.

  2. The participatory public library

    DEFF Research Database (Denmark)

    Rasmussen, Casper Hvenegaard

    2016-01-01

    of theoretical approaches and practical examples to obtain a varied understanding of user participation in public libraries. Research fields outside library and information science have developed a wide range of theoretical approaches on user participation. Examples from cultural policy, museum studies......Purpose From collection to connection has been a buzzword in the library world for more than a decade. This catchy phrase indicates that users are seen not only as borrowers, but as active participants. The aim of this paper is to investigate and analyse three questions in relation to user...... participation in public libraries in a Nordic perspective. How can participation in public libraries be characterised? Why should libraries deal with user participation? What kinds of different user participation can be identified in public libraries? Design/methodology/approach The paper uses a selection...

  3. A Seminar on Human Cloning: Cloning in Reproductive Medicine

    OpenAIRE

    Illmensee, Karl

    2001-01-01

    This review article summarizes the historical development of mammalian cloning, presents current advances and presumed risk factors in the field of reproductive cloning, discusses possible clinical applications of therapeutic and diagnostic cloning and outlines prospective commercial trends in pharmacytical cloning. Predictable progress in biotechnology and stem cell engineering should prove to be advantageous for patients' health and for novel benefits in reproductive and regenerative medicine.

  4. Quantitative discrimination of Aggregatibacter actinomycetemcomitans highly leukotoxic JP2 clone from non-JP2 clones in diagnosis of aggressive periodontitis.

    Science.gov (United States)

    Yoshida, Akihiro; Ennibi, Oum-Keltoum; Miyazaki, Hideo; Hoshino, Tomonori; Hayashida, Hideaki; Nishihara, Tatsuji; Awano, Shuji; Ansai, Toshihiro

    2012-10-11

    Aggregatibacter actinomycetemcomitans is the etiological agent of periodontitis, and there is a strong association between clone JP2 and aggressive periodontitis in adolescents of African descent. The JP2 clone has an approximately 530-bp deletion (∆530) in the promoter region of the lkt/ltx gene, which encodes leukotoxin, and this clone has high leukotoxic activity. Therefore, this clone is very important in aggressive periodontitis. To diagnose this disease, culture methods and conventional PCR techniques are used. However, quantitative detection based on qPCR for the JP2 clone has not been developed due to genetic difficulties. In this study, we developed a qPCR-based quantification method specific to the JP2 clone. Based on our analysis of the DNA sequence of the lkt/ltx gene and its flanking region, we designed a reverse primer specific for the ∆530 deletion border sequence and developed a JP2-specific PCR-based quantification method using this primer. We also analyzed the DNA sequence of the ∆530 locus and found it to be highly conserved (97-100%) among 17 non-JP2 strains. Using the ∆530 locus, we designed a qPCR primer-probe set specific to non-JP2 clones. Next, we determined the numbers of JP2 and non-JP2 clone cells in the periodontal pockets of patients with aggressive periodontitis. The JP2-specific primers specifically amplified the genomic DNA of the A. actinomycetemcomitans JP2 clone and did not react with other bacterial DNA, whereas the non-JP2 specific primers reacted only with A. actinomycetemcomitans non-JP2 clones. Samples from the 88 periodontal sites in the 11 patients with aggressive periodontitis were analyzed. The bacterial cell numbers in 88 periodontal sites ranged from 0 to 4.8 × 10(8) (mean 1.28 × 10(7)) for JP2 clones and from 0 to 1.6 × 10(6) for non-JP2 clones (mean 1.84 × 10(5)). There were significant differences in the JP2 cell number between a clinical attachment level (CAL) ≤6 mm and a level ≥7 mm (p clones. This

  5. cDNA cloning and immunological characterization of the rye grass allergen Lol p I.

    Science.gov (United States)

    Perez, M; Ishioka, G Y; Walker, L E; Chesnut, R W

    1990-09-25

    The complete amino acid sequence of two "isoallergenic" forms of Lol p I, the major rye grass (Lolium perenne) pollen allergen, was deduced from cDNA sequence analysis. cDNA clones isolated from a Lolium perenne pollen library contained an open reading frame coding for a 240-amino acid protein. Comparison of the nucleotide and deduced amino acid sequence of two of these clones revealed four changes at the amino acid level and numerous nucleotide differences. Both clones contained one possible asparagine-linked glycosylation site. Northern blot analysis shows one RNA species of 1.2 kilobases. Based on the complete amino acid sequence of Lol p I, overlapping peptides covering the entire molecule were synthesized. Utilizing these peptides we have identified a determinant within the Lol p I molecule that is recognized by human leukocyte antigen class II-restricted T cells obtained from persons allergic to rye grass pollen.

  6. Cloning and expression in Escherichia coli of cellulases genes from Clostridium IBUN 22A

    Directory of Open Access Journals (Sweden)

    Lucy Carolina Vargas Pabón

    2002-01-01

    Full Text Available Genomic library of the native strain Clostridium IBUN 22A was constructed, using plasmid pBluescriptlI® KS+/ - as cloning vector and its expression in Escherichia coli was evaluated. Eight recombination clones with enzymatic activity were detected by enzymatic screening and using the red-Congo test with three substrates: cellobiose, carboxymethyl cellulose (CMC and cellulose powder (native. Restriction analysis of three recombination plasmids, representative of each enzymatic activity showed the inserted size (1600, 13000 and 11000bp approximately for pBS68, pBS25 and pBS57 respectively. More studies of protein expression and enzymatic characterization will allow theses enzymes and other typical parameters to be defined. In the same way the fragment sequence cloned will lead to a more detailed analysis and definition of the biotechnological potential of this strain regarding solvent production using cellulosic substrates for fermentation.

  7. cDNA cloning of rat and human medium chain acyl-CoA dehydrogenase (MCAD)

    International Nuclear Information System (INIS)

    Matsubara, Y.; Kraus, J.P.; Rosenberg, L.E.; Tanaka, K.

    1986-01-01

    MCAD is one of three mitochondrial flavoenzymes which catalyze the first step in the β-oxidation of straight chain fatty acids. It is a tetramer with a subunit Mr of 45 kDa. MCAD is synthesized in the cytosol as a 49 kDa precursor polypeptide (pMCAD), imported into mitochondria, and cleaved to the mature form. Genetic deficiency of MCAD causes recurrent episodes of hypoglycemic coma accompanied by medium chain dicarboxylic aciduria. Employing a novel approach, the authors now report isolation of partial rat and human cDNA clones encoding pMCAD. mRNA encoding pMCAD was purified to near homogeneity by polysome immunoadsorption using polyclonal monospecific antibody. Single-stranded [ 32 P]labeled cDNA probe was synthesized using the enriched mRNA as template, and was used to screen directly 16,000 colonies from a total rat liver cDNA library constructed in pBR322. One clone (600 bp) was detected by in situ hybridization. Hybrid-selected translation with this cDNA yielded a 49 kDa polypeptide indistinguishable in size from rat pMCAD and immunoprecipitable with anti-MCAD antibody. Using the rat cDNA as probe, 43,000 colonies from a human liver cDNA library were screened. Four identical positive clones (400 bp) were isolated and positively identified by hybrid-selected translation and immunoprecipitation. The sizes of rat and human mRNAs encoding pMCAD were 2.2 kb and 2.4 kb, respectively, as determined by Northern blotting

  8. Cloning of the relative genes of endocrine exophthalmos

    International Nuclear Information System (INIS)

    Zheng, JG

    2004-01-01

    Aim: In order to clarify the pathogenesis of endocrine exophthalmos, and lay foundations for finding the new functions of its relative genes, the cloning of its relative genes was carried out. Methods: The thyroid tissues of 10 hyperthyroidism patients, 5 of them with endocrine exophthalmos and 5 without that, were obtained. Their mRNA were collected respectively by using Quick Prep Micro mRNA purification kit. Then the same amount of the mRNA from 5 patients with endocrine exophthalmos was added into an eppendorf tube to form a mRNA pool. And that of the 5 patients without endocrine exophthalmos was also prepared as the other pool. As a model, the pool was used to synthesize the single and double chains of cDNA through SMART Tm PCR cDNA Synthesis Kit. The double chains cDNA from the endocrine exophthalmos patients, being used as tester, and that from the patients without endocrine exophthalmos, being used as driver, were digested by restriction endonucleases Hae III to get the fragments which was less than 500 bases. The tester cDNA was ligated with adapt or 1 or 2 respectively. Then the subtractive suppressive hybridization was performed between tester and driver cDNA. And the efficacies of subtraction were measured. The differential genes between the thyroid tissues of endocrine exophthalmos and the thyroid tissues without endocrine exophthalmos were obtained through two cycles of subtractive hybridization and two cycles PCR. The differential genes were cloned into the vector of pT-Adv, and then transformed into E.coliDH5a. 48 white clonies were selected to build the subtractive suppressive library of the relative genes of endocrine exophthalmos. The primer 2 was applied for the colony PCR of the relative genes. The amplified genes were obtained and purified by using Quaqwich Spine PCR Purification Kit. According to the principle of random primer, the double chains cDNA from the thyroid tissues with or without endocrine exophthalmos were digested by Hae III

  9. A function-based screen for seeking RubisCO active clones from metagenomes: novel enzymes influencing RubisCO activity.

    Science.gov (United States)

    Böhnke, Stefanie; Perner, Mirjam

    2015-03-01

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) is a key enzyme of the Calvin cycle, which is responsible for most of Earth's primary production. Although research on RubisCO genes and enzymes in plants, cyanobacteria and bacteria has been ongoing for years, still little is understood about its regulation and activation in bacteria. Even more so, hardly any information exists about the function of metagenomic RubisCOs and the role of the enzymes encoded on the flanking DNA owing to the lack of available function-based screens for seeking active RubisCOs from the environment. Here we present the first solely activity-based approach for identifying RubisCO active fosmid clones from a metagenomic library. We constructed a metagenomic library from hydrothermal vent fluids and screened 1056 fosmid clones. Twelve clones exhibited RubisCO activity and the metagenomic fragments resembled genes from Thiomicrospira crunogena. One of these clones was further analyzed. It contained a 35.2 kb metagenomic insert carrying the RubisCO gene cluster and flanking DNA regions. Knockouts of twelve genes and two intergenic regions on this metagenomic fragment demonstrated that the RubisCO activity was significantly impaired and was attributed to deletions in genes encoding putative transcriptional regulators and those believed to be vital for RubisCO activation. Our new technique revealed a novel link between a poorly characterized gene and RubisCO activity. This screen opens the door to directly investigating RubisCO genes and respective enzymes from environmental samples.

  10. Analysis of an epigenetic argument against human reproductive cloning.

    Science.gov (United States)

    Nordgren, Anders

    2006-08-01

    Human reproductive cloning is a much disputed ethical issue. This technology is often condemned as being contrary to human dignity. However, there are also risk arguments. An ethical argument that is often put forward by scientists but seldom developed in more detail focuses on health risks in animal cloning. There is a high risk that animal clones exhibit abnormalities and these are increasingly believed to be due to errors in epigenetic reprogramming. The argument is that human reproductive cloning should not be carried out because human clones are also likely to exhibit abnormalities due to inappropriate epigenetic reprogramming. Different versions of this epigenetic argument are analysed, a categorical version and a non-categorical. The non-categorical version is suggested to be more well-considered. With regard to policy making on human reproductive cloning, the categorical version can be used to prescribe a permanent ban, while the non-categorical version can be used to prescribe a temporary ban. The implications of the precautionary principle--as interpreted in the European Union--are investigated. The conclusion is that it seems possible to support a temporary ban by reference to this principle.

  11. Novel β-lactamase-random peptide fusion libraries for phage display selection of cancer cell-targeting agents suitable for enzyme prodrug therapy

    Science.gov (United States)

    Shukla, Girja S.; Krag, David N.

    2010-01-01

    Novel phage-displayed random linear dodecapeptide (X12) and cysteine-constrained decapeptide (CX10C) libraries constructed in fusion to the amino-terminus of P99 β-lactamase molecules were used for identifying β-lactamase-linked cancer cell-specific ligands. The size and quality of both libraries were comparable to the standards of other reported phage display systems. Using the single-round panning method based on phage DNA recovery, we identified severalβ-lactamase fusion peptides that specifically bind to live human breast cancer MDA-MB-361 cells. The β-lactamase fusion to the peptides helped in conducting the enzyme activity-based clone normalization and cell-binding screening in a very time- and cost-efficient manner. The methods were suitable for 96-well readout as well as microscopic imaging. The success of the biopanning was indicated by the presence of ~40% cancer cell-specific clones among recovered phages. One of the binding clones appeared multiple times. The cancer cell-binding fusion peptides also shared several significant motifs. This opens a new way of preparing and selecting phage display libraries. The cancer cell-specific β-lactamase-linked affinity reagents selected from these libraries can be used for any application that requires a reporter for tracking the ligand molecules. Furthermore, these affinity reagents have also a potential for their direct use in the targeted enzyme prodrug therapy of cancer. PMID:19751096

  12. Construction of full-length cDNA library of white flower Salvia ...

    African Journals Online (AJOL)

    In order to screen and isolate secondary metabolite biosynthesis related gene, we construct a cDNA library of white flower Salvia miltiorrhiza bge. f.alba. High quality of total RNA was successfully isolated from roots of white flower S. miltiorrhiza using modified CTAB method. Double strand cDNA was cloned into pDNR-LIB ...

  13. Cloning of observables

    OpenAIRE

    Ferraro, Alessandro; Galbiati, Matteo; Paris, Matteo G. A.

    2005-01-01

    We introduce the concept of cloning for classes of observables and classify cloning machines for qubit systems according to the number of parameters needed to describe the class under investigation. A no-cloning theorem for observables is derived and the connections between cloning of observables and joint measurements of noncommuting observables are elucidated. Relationships with cloning of states and non-demolition measurements are also analyzed.

  14. Cloning of oleosin, a putative new hazelnut allergen, using a hazelnut cDNA library

    NARCIS (Netherlands)

    Akkerdaas, Jaap H.; Schocker, Frauke; Vieths, Stefan; Versteeg, Serge; Zuidmeer, Laurian; Hefle, Sue L.; Aalberse, Rob C.; Richter, Klaus; Ferreira, Fatima; van Ree, Ronald

    2006-01-01

    The clinical presentation of non-pollen related allergy to hazelnut can be severe and systemic. So far, only a limited number of non-pollen related hazelnut allergens have been identified and characterized. The aim of this study was to identify and clone new hazelnut allergens. A lambda ZAP cDNA

  15. Using Partial Genomic Fosmid Libraries for Sequencing CompleteOrganellar Genomes

    Energy Technology Data Exchange (ETDEWEB)

    McNeal, Joel R.; Leebens-Mack, James H.; Arumuganathan, K.; Kuehl, Jennifer V.; Boore, Jeffrey L.; dePamphilis, Claude W.

    2005-08-26

    Organellar genome sequences provide numerous phylogenetic markers and yield insight into organellar function and molecular evolution. These genomes are much smaller in size than their nuclear counterparts; thus, their complete sequencing is much less expensive than total nuclear genome sequencing, making broader phylogenetic sampling feasible. However, for some organisms it is challenging to isolate plastid DNA for sequencing using standard methods. To overcome these difficulties, we constructed partial genomic libraries from total DNA preparations of two heterotrophic and two autotrophic angiosperm species using fosmid vectors. We then used macroarray screening to isolate clones containing large fragments of plastid DNA. A minimum tiling path of clones comprising the entire genome sequence of each plastid was selected, and these clones were shotgun-sequenced and assembled into complete genomes. Although this method worked well for both heterotrophic and autotrophic plants, nuclear genome size had a dramatic effect on the proportion of screened clones containing plastid DNA and, consequently, the overall number of clones that must be screened to ensure full plastid genome coverage. This technique makes it possible to determine complete plastid genome sequences for organisms that defy other available organellar genome sequencing methods, especially those for which limited amounts of tissue are available.

  16. [Primary culture of cat intestinal epithelial cell and construction of its cDNA library].

    Science.gov (United States)

    Ye, L; Gui-Hua, Z; Kun, Y; Hong-Fa, W; Ting, X; Gong-Zhen, L; Wei-Xia, Z; Yong, C

    2017-04-12

    Objective To establish the primary cat intestinal epithelial cells (IECs) culture methods and construct the cDNA library for the following yeast two-hybrid experiment, so as to screen the virulence interaction factors among the final host. Methods The primary cat IECs were cultured by the tissue cultivation and combined digestion with collagenase XI and dispase I separately. Then the cat IECs cultured was identified with the morphological observation and cyto-keratin detection, by using goat anti-cyto-keratin monoclonal antibodies. The mRNA of cat IECs was isolated and used as the template to synthesize the first strand cDNA by SMART™ technology, and then the double-strand cDNAs were acquired by LD-PCR, which were subsequently cloned into the plasmid PGADT7-Rec to construct yeast two-hybrid cDNA library in the yeast strain Y187 by homologous recombination. Matchmaker™ Insert Check PCR was used to detect the size distribution of cDNA fragments after the capacity calculation of the cDNA library. Results The comparison of the two cultivation methods indicated that the combined digestion of collagenase XI and dispase I was more effective than the tissue cultivation. The cat IECs system of continuous culture was established and the cat IECs with high purity were harvested for constructing the yeast two-hybrid cDNA library. The library contained 1.1×10 6 independent clones. The titer was 2.8×10 9 cfu/ml. The size of inserted fragments was among 0.5-2.0 kb. Conclusion The yeast two-hybrid cDNA library of cat IECs meets the requirements of further screen research, and this study lays the foundation of screening the Toxoplasma gondii virulence interaction factors among the cDNA libraries of its final hosts.

  17. Cloning of the DNA repair gene, uvsF, by transformation of Aspergillus nidulans.

    Science.gov (United States)

    Oza, K; Käfer, E

    1990-06-01

    As a first step in the cloning of the DNA repair gene uvsF of Aspergillus nidulans, uvsF pyrG double mutant strains were transformed with a genomic library which carried the complementing Neurospora pyr-4 gene in the vector. Rare pyr+ uvs+ cotransformants were obtained on media lacking pyrimidines, overlayed with MMS (methyl-methane sulfonate) to which uvsF is hypersensitive. Among MMS-resistant transformants, Southerns revealed two types which showed single bands of different sizes when BglII-digested genomic DNA was probed with the vector. Both types produced uvsF- recombinants without vector sequences in homozygous crosses, but only those with the larger band also produced haploid uvs+ progeny. Using BglII-digested genomic DNA to transform Escherichia coli, plasmids of the corresponding two sizes could be rescued. Their inserts had a short internal region in common, giving evidence of rearrangement(s). In secondary transformation of uvsF mutants, only the plasmids with the larger insert showed complementation and these were used to screen Aspergillus libraries. Three types of genomic and two overlapping cDNA clones were identified. The cDNAs hybridized not only to each other, but also to the common region of the rescued plasmids. Therefore, cDNA subclones were used to map the putative uvsF sequences to a short segment in one genomic clone. In Northerns, the complementing large plasmid hybridized to three mRNAs, while the cDNA subclone identified one of these as the probable uvsF message.

  18. Construction and identification of subtracted cDNA library in bone marrow cells of radon-exposed mice

    International Nuclear Information System (INIS)

    Li Jianxiang; Nie Jihua; Tong Jian; Fu Chunling; Zhou Jianwei

    2008-01-01

    Objective: To construct and identify subtracted cDNA library in bone marrow cells of mice exposed to radon inhalation. Methods: Adult male BALB/c mice, weighing 18-22 g, were placed in a multi- functional radon chamber. One group of mice was exposed to radon up to the accumulative dose of 105 work level month (WLM). The control group of mice was housed in a room with an accumulative dose of 1 WLM. To construct a subtracted cDNA library enriched with differentially expressed genes, the SMART technique and the suppression subtractive hybridization were performed. The obtained forward and reverse cDNA fragments were directly inserted into pMD18-T vector and transformed into E. coli JM109. The inserting cDNA fragments were screened by the blue-and-white blot screening and nested PCR of bacterium liquid. Results: The 244 of 285 white bacteria clones obtained randomly were positive clones contained 100-1100 bp inserted cDNA fragments. Conclusions: The forward and reverse subtracted cDNA library in bone marrow cells of mice exposed to radon inhalation is successfully constructed. (authors)

  19. The LAW library

    International Nuclear Information System (INIS)

    Green, N.M.; Parks, C.V.; Arwood, J.W.

    1989-01-01

    The 238 group LAW library is a new multigroup library based on ENDF/B-V data. It contains data for 302 materials and will be distributed by the Radiation Shielding Information Center, located at Oak Ridge National Laboratory. It was generated for use in neutronics calculations required in radioactive waste analyses, though it has equal utility in any study requiring multigroup neutron cross sections

  20. Effects of field-grown genetically modified Zoysia grass on bacterial community structure.

    Science.gov (United States)

    Lee, Yong-Eok; Yang, Sang-Hwan; Bae, Tae-Woong; Kang, Hong-Gyu; Lim, Pyung-Ok; Lee, Hyo-Yeon

    2011-04-01

    Herbicide-tolerant Zoysia grass has been previously developed through Agrobacterium-mediated transformation. We investigated the effects of genetically modified (GM) Zoysia grass and the associated herbicide application on bacterial community structure by using culture-independent approaches. To assess the possible horizontal gene transfer (HGT) of transgenic DNA to soil microorganisms, total soil DNAs were amplified by PCR with two primer sets for the bar and hpt genes, which were introduced into the GM Zoysia grass by a callus-type transformation. The transgenic genes were not detected from the total genomic DNAs extracted from 1.5 g of each rhizosphere soils of GM and non-GM Zoysia grasses. The structures and diversities of the bacterial communities in rhizosphere soils of GM and non-GM Zoysia grasses were investigated by constructing 16S rDNA clone libraries. Classifier, provided in the RDP II, assigned 100 clones in the 16S rRNA gene sequences library into 11 bacterial phyla. The most abundant phyla in both clone libraries were Acidobacteria and Proteobacteria. The bacterial diversity of the GM clone library was lower than that of the non- GM library. The former contained four phyla, whereas the latter had seven phyla. Phylogenetic trees were constructed to confirm these results. Phylogenetic analyses of the two clone libraries revealed considerable difference from each other. The significance of difference between clone libraries was examined with LIBSHUFF statistics. LIBSHUFF analysis revealed that the two clone libraries differed significantly (P〈0.025), suggesting alterations in the composition of the microbial community associated with GM Zoysia grass.

  1. Metagenome Analyses of Corroded Concrete Wastewater Pipe Biofilms Reveals a Complex Microbial System

    Science.gov (United States)

    Analysis of whole-metagenome pyrosequencing data and 16S rRNA gene clone libraries was used to determine microbial composition and functional genes associated with biomass harvested from crown (top) and invert (bottom) sections of a corroded wastewater pipe. Taxonomic and functio...

  2. Construction of naïve camelids VHH repertoire in phage display-based library.

    Science.gov (United States)

    Sabir, Jamal S M; Atef, Ahmed; El-Domyati, Fotouh M; Edris, Sherif; Hajrah, Nahid; Alzohairy, Ahmed M; Bahieldin, Ahmed

    2014-04-01

    Camelids have unique antibodies, namely HCAbs (VHH) or commercially named Nanobodies(®) (Nb) that are composed only of a heavy-chain homodimer. As libraries based on immunized camelids are time-consuming, costly and likely redundant for certain antigens, we describe the construction of a naïve camelid VHHs library from blood serum of non-immunized camelids with affinity in the subnanomolar range and suitable for standard immune applications. This approach is rapid and recovers VHH repertoire with the advantages of being more diverse, non-specific and devoid of subpopulations of specific antibodies, which allows the identification of binders for any potential antigen (or pathogen). RNAs from a number of camelids from Saudi Arabia were isolated and cDNAs of the diverse vhh gene were amplified; the resulting amplicons were cloned in the phage display pSEX81 vector. The size of the library was found to be within the required range (10(7)) suitable for subsequent applications in disease diagnosis and treatment. Two hundred clones were randomly selected and the inserted gene library was either estimated for redundancy or sequenced and aligned to the reference camelid vhh gene (acc. No. ADE99145). Results indicated complete non-specificity of this small library in which no single event of redundancy was detected. These results indicate the efficacy of following this approach in order to yield a large and diverse enough gene library to secure the presence of the required version encoding the required antibodies for any target antigen. This work is a first step towards the construction of phage display-based biosensors useful in disease (e.g., TB or tuberculosis) diagnosis and treatment. Copyright © 2014 Académie des sciences. Published by Elsevier SAS. All rights reserved.

  3. Molecular cloning and expression of Corynebacterium glutamicum genes for amino acid synthesis in Escherichia coli cells

    International Nuclear Information System (INIS)

    Beskrovnaya, O.Yu.; Fonshtein, M.Yu.; Kolibaba, L.G.; Yankovskii, N.K.; Debabov, V.G.

    1989-01-01

    Molecular cloning of Corynebacterium glutamicum genes for threonine and lysine synthesis has been done in Escherichia coli cells. The clonal library of EcoRI fragments of chromosomal DNA of C. glutamicum was constructed on the plasmid vector λpSL5. The genes for threonine and lysine synthesis were identified by complementation of E. coli mutations in thrB and lysA genes, respectively. Recombinant plasmids, isolated from independent ThrB + clone have a common 4.1-kb long EcoRI DNA fragment. Hybrid plasmids isolated from LysA + transductants of E. coli have common 2.2 and 3.3 kb long EcoRI fragments of C. glutamicum DNA. The hybrid plasmids consistently transduced the markers thrB + and lysA + . The Southern hybridization analysis showed that the cloned DNA fragments hybridized with the fragments of identical length in C. glutamicum chromosomes

  4. Preliminary assessment of the grading of Eucalyptus clones using carbon isotope discrimination

    International Nuclear Information System (INIS)

    Bond, W.J.; Stock, W.D.

    1990-01-01

    Stable carbon isotopes were analysed in leaf material of nine Eucalyptus clones grown in field trials in the eastern Transvaal. Carbon isotope ratios, measured as d 13 C, differed within tree canopies, between replicate trees and between clones. Values from both north and south canopy positions were correlated with tree height after 13 months. Unexplained variation in the correlation may be interpreted, theoretically, as an indication that some clones use less water for the same level of productivity. With further testing, the method may have promise for early screening of clones in genotype/environment interaction trials and in selecting water-efficient trees. 3 figs., 3 tabs., 7 refs

  5. Construction of a cDNA library from human retinal pigment epithelial cells challenged with rod outer segments.

    Science.gov (United States)

    Cavaney, D M; Rakoczy, P E; Constable, I J

    1995-05-01

    To study genes expressed by retinal pigment epithelial (RPE) cells during phagocytosis and digestion of rod outer segments (ROS), a complementary (c)DNA library was produced using an in-vitro model. The cDNA library can be used to study molecular changes which contribute to the development of diseases due to a failure in outer segment phagocytosis and digestion by RPE cells. Here we demonstrate a way to study genes and their functions using a molecular biological approach and describing the first step involved in this process, the construction of a cDNA library. Human RPE cells obtained from the eyes of a seven-year-old donor were cultured and challenged with bovine ROS. The culture was harvested and total RNA was extracted. Complementary DNA was transcribed from the messenger (m)RNA and was directionally cloned into the LambdaGEM-4 bacteriophage vector successfully. Some clones were picked and the DNA extracted, to determine the size of the inserts as a measure of the quality of the library. Molecular biology and cell culture are important tools to be used in eye research, especially in areas where tissue is limiting and animal models are not available. We now have a ROS challenged RPE cDNA library which will be used to identify genes responsible for degrading phagocytosed debris within the retinal pigment epithelium.

  6. Quantitative discrimination of Aggregatibacter actinomycetemcomitans highly leukotoxic JP2 clone from non-JP2 clones in diagnosis of aggressive periodontitis

    Directory of Open Access Journals (Sweden)

    Yoshida Akihiro

    2012-10-01

    (CAL ≤6 mm and a level ≥7 mm (p A. actinomycetemcomitans. Conclusions We successfully developed a quantitative and discriminative PCR-based method for the detection of A. actinomycetemcomitans JP2 and non-JP2 clones. This technique will contribute to future analyses of the quantitative relationship between this organism and aggressive periodontitis.

  7. Molecular comparison of bacterial communities within iron-containing flocculent mats associated with submarine volcanoes along the Kermadec Arc.

    Science.gov (United States)

    Hodges, Tyler W; Olson, Julie B

    2009-03-01

    Iron oxide sheaths and filaments are commonly found in hydrothermal environments and have been shown to have a biogenic origin. These structures were seen in the flocculent material associated with two submarine volcanoes along the Kermadec Arc north of New Zealand. Molecular characterization of the bacterial communities associated with the flocculent samples indicated that no known Fe-oxidizing bacteria dominated the recovered clone libraries. However, clones related to the recently described Fe-oxidizing bacterium Mariprofundus ferrooxydans were obtained from both the iron-containing flocculent (Fe-floc) and sediment samples, and peaks corresponding to Mariprofundus ferrooxydans, as well as the related clones, were observed in several of our terminal restriction fragment length polymorphism profiles. A large group of epsilonproteobacterial sequences, for which there is no cultured representative, dominated clones from the Fe-floc libraries and were less prevalent in the sediment sample. Phylogenetic analyses indicated that several operational taxonomic units appeared to be site specific, and statistical analyses of the clone libraries found that all samples were significantly different from each other. Thus, the bacterial communities in the Fe-floc samples were not more closely related to each other than to the sediment communities.

  8. The Clone Factory

    Science.gov (United States)

    Stoddard, Beryl

    2005-01-01

    Have humans been cloned? Is it possible? Immediate interest is sparked when students are asked these questions. In response to their curiosity, the clone factory activity was developed to help them understand the process of cloning. In this activity, students reenact the cloning process, in a very simplified simulation. After completing the…

  9. 3G vector-primer plasmid for constructing full-length-enriched cDNA libraries.

    Science.gov (United States)

    Zheng, Dong; Zhou, Yanna; Zhang, Zidong; Li, Zaiyu; Liu, Xuedong

    2008-09-01

    We designed a 3G vector-primer plasmid for the generation of full-length-enriched complementary DNA (cDNA) libraries. By employing the terminal transferase activity of reverse transcriptase and the modified strand replacement method, this plasmid (assembled with a polydT end and a deoxyguanosine [dG] end) combines priming full-length cDNA strand synthesis and directional cDNA cloning. As a result, the number of steps involved in cDNA library preparation is decreased while simplifying downstream gene manipulation, sequencing, and subcloning. The 3G vector-primer plasmid method yields fully represented plasmid primed libraries that are equivalent to those made by the SMART (switching mechanism at 5' end of RNA transcript) approach.

  10. A Blumeria graminis f.sp. hordei BAC library - contig building and microsynteny studies

    DEFF Research Database (Denmark)

    Pedersen, C.; Wu, B.; Giese, H.

    2002-01-01

    A bacterial artificial chromosome (BAC) library of Blumeria graminis f.sp. hordei, containing 12,000 clones with an average insert size of 41 kb, was constructed. The library represents about three genome equivalents and BAC-end sequencing showed a high content of repetitive sequences, making...... contigs, at or close to avirulence loci, were constructed. Single nucleotide polymorphism (SNP) markers were developed from BAC-end sequences to link the contigs to the genetic maps. Two other BAC contigs were used to study microsynteny between B. graminis and two other ascomycetes, Neurospora crassa...

  11. Construction of high-quality Caco-2 three-frame cDNA library and its application to yeast two-hybrid for the human astrovirus protein-protein interaction.

    Science.gov (United States)

    Zhao, Wei; Li, Xin; Liu, Wen-Hui; Zhao, Jian; Jin, Yi-Ming; Sui, Ting-Ting

    2014-09-01

    Human epithelial colorectal adenocarcinoma (Caco-2) cells are widely used as an in vitro model of the human small intestinal mucosa. Caco-2 cells are host cells of the human astrovirus (HAstV) and other enteroviruses. High quality cDNA libraries are pertinent resources and critical tools for protein-protein interaction research, but are currently unavailable for Caco-2 cells. To construct a three-open reading frame, full length-expression cDNA library from the Caco-2 cell line for application to HAstV protein-protein interaction screening, total RNA was extracted from Caco-2 cells. The switching mechanism at the 5' end of the RNA transcript technique was used for cDNA synthesis. Double-stranded cDNA was digested by Sfi I and ligated to reconstruct a pGADT7-Sfi I three-frame vector. The ligation mixture was transformed into Escherichia coli HST08 premium electro cells by electroporation to construct the primary cDNA library. The library capacity was 1.0×10(6)clones. Gel electrophoresis results indicated that the fragments ranged from 0.5kb to 4.2kb. Randomly picked clones show that the recombination rate was 100%. The three-frame primary cDNA library plasmid mixture (5×10(5)cfu) was also transformed into E. coli HST08 premium electro cells, and all clones were harvested to amplify the cDNA library. To detect the sufficiency of the cDNA library, HAstV capsid protein as bait was screened and tested against the Caco-2 cDNA library by a yeast two-hybrid (Y2H) system. A total of 20 proteins were found to interact with the capsid protein. These results showed that a high-quality three-frame cDNA library from Caco-2 cells was successfully constructed. This library was efficient for the application to the Y2H system, and could be used for future research. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. A new Python library to analyse skeleton images confirms malaria parasite remodelling of the red blood cell membrane skeleton

    Directory of Open Access Journals (Sweden)

    Juan Nunez-Iglesias

    2018-02-01

    Full Text Available We present Skan (Skeleton analysis, a Python library for the analysis of the skeleton structures of objects. It was inspired by the “analyse skeletons” plugin for the Fiji image analysis software, but its extensive Application Programming Interface (API allows users to examine and manipulate any intermediate data structures produced during the analysis. Further, its use of common Python data structures such as SciPy sparse matrices and pandas data frames opens the results to analysis within the extensive ecosystem of scientific libraries available in Python. We demonstrate the validity of Skan’s measurements by comparing its output to the established Analyze Skeletons Fiji plugin, and, with a new scanning electron microscopy (SEM-based method, we confirm that the malaria parasite Plasmodium falciparum remodels the host red blood cell cytoskeleton, increasing the average distance between spectrin-actin junctions.

  13. A new Python library to analyse skeleton images confirms malaria parasite remodelling of the red blood cell membrane skeleton.

    Science.gov (United States)

    Nunez-Iglesias, Juan; Blanch, Adam J; Looker, Oliver; Dixon, Matthew W; Tilley, Leann

    2018-01-01

    We present Skan (Skeleton analysis), a Python library for the analysis of the skeleton structures of objects. It was inspired by the "analyse skeletons" plugin for the Fiji image analysis software, but its extensive Application Programming Interface (API) allows users to examine and manipulate any intermediate data structures produced during the analysis. Further, its use of common Python data structures such as SciPy sparse matrices and pandas data frames opens the results to analysis within the extensive ecosystem of scientific libraries available in Python. We demonstrate the validity of Skan's measurements by comparing its output to the established Analyze Skeletons Fiji plugin, and, with a new scanning electron microscopy (SEM)-based method, we confirm that the malaria parasite Plasmodium falciparum remodels the host red blood cell cytoskeleton, increasing the average distance between spectrin-actin junctions.

  14. Properties of promoters cloned randomly from the Saccharomyces cerevisiae genome.

    Science.gov (United States)

    Santangelo, G M; Tornow, J; McLaughlin, C S; Moldave, K

    1988-01-01

    Promoters were isolated at random from the genome of Saccharomyces cerevisiae by using a plasmid that contains a divergently arrayed pair of promoterless reporter genes. A comprehensive library was constructed by inserting random (DNase I-generated) fragments into the intergenic region upstream from the reporter genes. Simple in vivo assays for either reporter gene product (alcohol dehydrogenase or beta-galactosidase) allowed the rapid identification of promoters from among these random fragments. Poly(dA-dT) homopolymer tracts were present in three of five randomly cloned promoters. With two exceptions, each RNA start site detected was 40 to 100 base pairs downstream from a TATA element. All of the randomly cloned promoters were capable of activating reporter gene transcription bidirectionally. Interestingly, one of the promoter fragments originated in a region of the S. cerevisiae rDNA spacer; regulated divergent transcription (presumably by RNA polymerase II) initiated in the same region. Images PMID:2847031

  15. Bengal Bay clone ST772-MRSA-V outbreak: conserved clone causes investigation challenges.

    Science.gov (United States)

    Blomfeldt, A; Larssen, K W; Moghen, A; Haugum, K; Steen, T W; Jørgensen, S B; Aamot, H V

    2017-03-01

    The Bengal Bay clone, ST772-MRSA-V, associated with multi-drug resistance, Panton-Valentine leukocidin (PVL) and skin and soft tissue infections, is emerging worldwide. In Norway, a country with low prevalence of meticillin-resistant Staphylococcus aureus (MRSA), increased occurrence of ST772-MRSA-V has also caused hospital outbreaks. The conserved nature of this clone challenged the outbreak investigations. To evaluate the usefulness of S. aureus protein A (spa) typing, multiple-locus variable number tandem repeat fingerprinting/analysis (MLVF/MLVA) and pulsed-field gel electrophoresis (PFGE) when investigating outbreaks with a conserved MRSA clone. A panel of 25 MRSA isolates collected in 2004-2014, consisting of six hospital outbreak isolates and 19 sporadic isolates, were analysed using spa typing, polymerase chain reaction detection of genes encoding PVL, MLVF/MLVA and PFGE. All isolates were ST772-MRSA-V-t657 and resistant to erythromycin, gentamicin and norfloxacin, and 88% were PVL positive. PFGE could not discriminate between the isolates (≥85% similarity). MLVF resolved five types [Simpson's index of diversity (SID)=0.56], MLVA resolved six types (SID=0.66), and both methods separated the hospital isolates into two defined outbreaks. MLVF/MLVA could not discriminate all epidemiologically unlinked cases and identical genotypes originated from a timespan of 10 years. MLVA was regarded as most suitable due to its higher discriminatory power and ability to provide unambiguous profiles. However, the Bengal Bay clone may require higher resolution methods for exact demarcation of outbreaks due to low diversity among isolates. Copyright © 2016 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

  16. Molecular cloning of L-methylmalonyl-CoA mutase: Gene transfer and analysis of mut cell lines

    International Nuclear Information System (INIS)

    Ledley, F.D.; Lumetta, M.; Nguyen, P.N.; Kolhouse, J.F.; Allen, R.H.

    1988-01-01

    L-Methylmalonyl-CoA mutase (MCM, EC 5.4.99.2) is a mitochondrial adenosylcobalamin-requiring enzyme that catalyzes the isomerization of L-methylmalonyl-CoA to succinyl-CoA. This enzyme is deficient in methylmalonic acidemia, an often fatal disorder of organic acid metabolism. Antibody against human placental MCM was used to screen human placenta and liver cDNA expression libraries for MCM cDNA clones. One clone expressed epitopes that could affinity-purify antibodies against MCM. A cDNA corresponding in length to the mRNA was obtained and introduced into COS cells by DNA-mediated gene transfer. Cells transformed with this clone expressed increased levels of MCM enzymatic activity. RNA blot analysis of cells genetically deficient in MCM indicates that several deficient cell lines have a specific decrease in the amount of hybridizable mRNA. These data confirm the authenticity of the MCM cDNA clone, establish the feasibility of constituting MCM activity by gene transfer for biochemical analysis and gene therapy, and provide a preliminary picture of the genotypic spectrum underlying MCM deficiency

  17. Cloning and expression of a widely expressed receptor tyrosine phosphatase

    DEFF Research Database (Denmark)

    Sap, J; D'Eustachio, P; Givol, D

    1990-01-01

    We describe the identification of a widely expressed receptor-type (transmembrane) protein tyrosine phosphatase (PTPase; EC 3.1.3.48). Screening of a mouse brain cDNA library under low-stringency conditions with a probe encompassing the intracellular (phosphatase) domain of the CD45 lymphocyte...... antigen yielded cDNA clones coding for a 794-amino acid transmembrane protein [hereafter referred to as receptor protein tyrosine phosphatase alpha (R-PTP-alpha)] with an intracellular domain displaying clear homology to the catalytic domains of CD45 and LAR (45% and 53%, respectively). The 142-amino acid...

  18. B-G cDNA clones have multiple small repeats and hybridize to both chicken MHC regions

    DEFF Research Database (Denmark)

    Kaufman, J; Salomonsen, J; Skjødt, K

    1989-01-01

    We used rabbit antisera to the chicken MHC erythrocyte molecule B-G and to the class I alpha chain (B-F) to screen lambda gt11 cDNA expression libraries made with RNA selected by oligo-dT from bone marrow cells of anemic B19 homozygous chickens. Eight clones were found to encode B-G molecules which...

  19. Construction and characterization of the alpha form of a cardiac myosin heavy chain cDNA clone and its developmental expression in the Syrian hamster.

    OpenAIRE

    Liew, C C; Jandreski, M A

    1986-01-01

    A cDNA clone, pVHC1, was isolated from a Syrian hamster heart cDNA library and was compared to the rat alpha (pCMHC21) and beta (pCMHC5) ventricular myosin heavy chain cDNA clones. The DNA sequence and amino acid sequence deducted from the DNA show more homology with pCMHC21 than pCMHC5. This indicates that pVHC1 is an alpha ventricular myosin heavy chain cDNA clone. However, even though pVHC1 shows a high degree of nucleotide and amino acid conservation with the rat myosin heavy chain sequen...

  20. Cloning of the DNA Repair Gene, Uvsf, by Transformation of Aspergillus Nidulans

    OpenAIRE

    Oza, K.; Kafer, E.

    1990-01-01

    As a first step in the cloning of the DNA repair gene uvsF of Aspergillus nidulans, uvsF pyrG double mutant strains were transformed with a genomic library which carried the complementing Neurospora pyr-4 gene in the vector. Rare pyr(+) uvs(+) cotransformants were obtained on media lacking pyrimidines, overlayed with MMS (methyl-methane sulfonate) to which uvsF is hypersensitive. Among MMS-resistant transformants, Southerns revealed two types which showed single bands of different sizes when ...

  1. Factors Affecting the Full Use of Library and Information Management Systems by Library Personnel

    Science.gov (United States)

    Skretas, Georgios

    2005-01-01

    Purpose: To provide a general list of factors that affects and determines the full use of library information management systems (LIMS) by library staff. Design/methodology/approach: The factors, which were identified mainly during participation in the implementation of automation projects in Greece, are listed and briefly analysed in categories…

  2. Statistical inference for classification of RRIM clone series using near IR reflectance properties

    Science.gov (United States)

    Ismail, Faridatul Aima; Madzhi, Nina Korlina; Hashim, Hadzli; Abdullah, Noor Ezan; Khairuzzaman, Noor Aishah; Azmi, Azrie Faris Mohd; Sampian, Ahmad Faiz Mohd; Harun, Muhammad Hafiz

    2015-08-01

    RRIM clone is a rubber breeding series produced by RRIM (Rubber Research Institute of Malaysia) through "rubber breeding program" to improve latex yield and producing clones attractive to farmers. The objective of this work is to analyse measurement of optical sensing device on latex of selected clone series. The device using transmitting NIR properties and its reflectance is converted in terms of voltage. The obtained reflectance index value via voltage was analyzed using statistical technique in order to find out the discrimination among the clones. From the statistical results using error plots and one-way ANOVA test, there is an overwhelming evidence showing discrimination of RRIM 2002, RRIM 2007 and RRIM 3001 clone series with p value = 0.000. RRIM 2008 cannot be discriminated with RRIM 2014; however both of these groups are distinct from the other clones.

  3. Assessment of S(α, β) libraries for criticality safety evaluations of wet storage pools by refined trend analyses

    International Nuclear Information System (INIS)

    Kolbe, E.; Vasiliev, A.; Ferroukhi, H.

    2009-01-01

    In a recent criticality safety evaluation (CSE) of a commercial wet storage pool applying MCNPX-2.5.0 in combination with the ENDF/B-VII.0 and JEFF-3.1 continuous energy cross section libraries, the maximum permissible initial fuel-enrichment limit for water reflected configurations was found to be dependant upon the applied neutron cross section library. More detailed investigations indicated that the difference is mainly caused by different sub-libraries for thermal neutron scattering based on parameterizations of the S(α, β) scattering matrix. Hence an analysis of trends was done with respect to the low energy neutron flux in order to assess the S(α, β) data sets. First, when performing the trend analysis based on the full set of 149 benchmarks that were employed for the validation, significant trends could not be found. But by analyzing a selected subset of benchmarks clear trends with respect to the low energy neutron flux could be detected. The results presented in this paper demonstrate the sensitivity of specific configurations to the parameterizations of the S(α, β) scattering matrix and thus may help to improve CSE of wet storage pools. Finally, in addition to the low energy neutron flux, we also refined the trend analyses with respect to other key (spectrum-related) parameters by performing them with various selected subsets of the full suite of 149 benchmarks. The corresponding outcome using MCNPX 2.5.0 in combination with the ENDF/B-VII.0, ENDF/B-VI.8, JEFF-3.1, JEF-2.2, and JENDL-3.3 neutron cross section libraries are presented and discussed. (authors)

  4. CLONING AND SEQUENCING OF THE GENE FOR A LACTOCOCCAL ENDOPEPTIDASE, AN ENZYME WITH SEQUENCE SIMILARITY TO MAMMALIAN ENKEPHALINASE

    NARCIS (Netherlands)

    Mierau, Igor; Tan, Paris S.T.; Haandrikman, Alfred J.; Kok, Jan; Leenhouts, Kees J.; Konings, Wil N.; Venema, Gerard

    The gene specifying an endopeptidase of Lactococcus lactis, named pepO, was cloned from a genomic library of L. lactis subsp. cremoris P8-247 in lambdaEMBL3 and was subsequently sequenced. pepO is probably the last gene of an operon encoding the binding-protein-dependent oligopeptide transport

  5. Towards Culture 3.0 - Performative space in the public library

    DEFF Research Database (Denmark)

    Jochumsen, Henrik; Skot-Hansen, Dorte; Rasmussen, Casper Hvenegaard

    2015-01-01

    The aim of this article is to analyse and discuss the development of performative spaces in public libraries from a cultural policy perspective. First, a framework of three concepts of culture, 1.0–3.0, is used as a tool to analyse the overall development of public libraries. Against...... this background, we introduce the notion of performative spaces in public libraries by highlighting Nordic examples. The tendency can also be seen on a broader level in European and North American libraries, where a ‘performative turn’ can be seen as the relationship between the library and its users, especially...... the younger ‘digital natives’. The rationales behind the emergence of performative spaces in public libraries are analysed and discussed: democratisation, empowerment and economic impact. This article concludes that the performative spaces are legitimized by multiple rationales in the same way as cultural...

  6. Targeting mammalian organelles with internalizing phage (iPhage) libraries

    Science.gov (United States)

    Rangel, Roberto; Dobroff, Andrey S.; Guzman-Rojas, Liliana; Salmeron, Carolina C.; Gelovani, Juri G.; Sidman, Richard L.; Pasqualini, Renata; Arap, Wadih

    2015-01-01

    Techniques largely used for protein interaction studies and discovery of intracellular receptors, such as affinity capture complex purification and yeast two-hybrid, may produce inaccurate datasets due to protein insolubility, transient or weak protein interactions, or irrelevant intracellular context. A versatile tool to overcome these limitations as well as to potentially create vaccines and engineer peptides and antibodies as targeted diagnostic and therapeutic agents, is the phage display technique. We have recently developed a new technology for screening internalizing phage (iPhage) vectors and libraries utilizing a ligand/receptor-independent mechanism to penetrate eukaryotic cells. iPhage particles provide a unique discovery platform for combinatorial intracellular targeting of organelle ligands along with their corresponding receptors and to fingerprint functional protein domains in living cells. Here we explain the design, cloning, construction, and production of iPhage-based vectors and libraries, along with basic ligand-receptor identification and validation methodologies for organelle receptors. An iPhage library screening can be performed in ~8 weeks. PMID:24030441

  7. Parallel Sequencing of Expressed Sequence Tags from Two Complementary DNA Libraries for High and Low Phosphorus Adaptation in Common Beans

    Directory of Open Access Journals (Sweden)

    Matthew W. Blair

    2011-11-01

    Full Text Available Expressed sequence tags (ESTs have proven useful for gene discovery in many crops. In this work, our objective was to construct complementary DNA (cDNA libraries from root tissues of common beans ( L. grown under low and high P hydroponic conditions and to conduct EST sequencing and comparative analyses of the libraries. Expressed sequence tag analysis of 3648 clones identified 2372 unigenes, of which 1591 were annotated as known genes while a total of 465 unigenes were not associated with any known gene. Unigenes with hits were categorized according to biological processes, molecular function, and cellular compartmentalization. Given the young tissue used to make the root libraries, genes for catalytic activity and binding were highly expressed. Comparisons with previous root EST sequencing and between the two libraries made here resulted in a set of genes to study further for differential gene expression and adaptation to low P, such as a 14 kDa praline-rich protein, a metallopeptidase, tonoplast intrinsic protein, adenosine triphosphate (ATP citrate synthase, and cell proliferation genes expressed in the low P treated plants. Given that common beans are often grown on acid soils of the tropics and subtropics that are usually low in P these genes and the two parallel libraries will be useful for selection for better uptake of this essential macronutrient. The importance of EST generation for common bean root tissues under low P and other abiotic soil stresses is also discussed.

  8. Construction and characterization of a plant transformation-competent BIBAC library of the black Sigatoka-resistant banana Musa acuminata cv. Tuu Gia (AA).

    Science.gov (United States)

    Ortiz-Vázquez, E; Kaemmer, D; Zhang, H-B; Muth, J; Rodríguez-Mendiola, M; Arias-Castro, C; James, Andrew

    2005-02-01

    A plant transformation-competent binary bacterial artificial chromosome (BIBAC) library was constructed from Musa acuminata cv. Tuu Gia (AA), a black Sigatoka-resistant diploid banana. After digestion of high-molecular-weight banana DNA by HindIII, several methods of DNA size selection were tested, followed by ligation, using a vector/insert molar ratio of 4:1. The library consists of 30,700 clones stored in 80 384-well microtiter plates. The mean insert size was estimated to be 100 kb, and the frequency of inserts with internal NotI sites was 61%. The majority of insert sizes fell into the range of 100+/-20 kb, making them suitable for Agrobacterium-mediated transformation. Only 1% and 0.9% of the clones contain chloroplast and mitochondrial DNA, respectively. This is the first BIBAC library for banana, estimated to represent five times its haploid genome (600 Mbp). It was demonstrated by hybridization that the library contains typical members of resistance gene and defense gene families that can be used for transformation of disease susceptible banana cultivars for banana genetic improvement.

  9. Multipartite asymmetric quantum cloning

    International Nuclear Information System (INIS)

    Iblisdir, S.; Gisin, N.; Acin, A.; Cerf, N.J.; Filip, R.; Fiurasek, J.

    2005-01-01

    We investigate the optimal distribution of quantum information over multipartite systems in asymmetric settings. We introduce cloning transformations that take N identical replicas of a pure state in any dimension as input and yield a collection of clones with nonidentical fidelities. As an example, if the clones are partitioned into a set of M A clones with fidelity F A and another set of M B clones with fidelity F B , the trade-off between these fidelities is analyzed, and particular cases of optimal N→M A +M B cloning machines are exhibited. We also present an optimal 1→1+1+1 cloning machine, which is an example of a tripartite fully asymmetric cloner. Finally, it is shown how these cloning machines can be optically realized

  10. [Cloning and characterization of genes differentially expressed in human dental pulp cells and gingival fibroblasts].

    Science.gov (United States)

    Wang, Zhong-dong; Wu, Ji-nan; Zhou, Lin; Ling, Jun-qi; Guo, Xi-min; Xiao, Ming-zhen; Zhu, Feng; Pu, Qin; Chai, Yu-bo; Zhao, Zhong-liang

    2007-02-01

    To study the biological properties of human dental pulp cells (HDPC) by cloning and analysis of genes differentially expressed in HDPC in comparison with human gingival fibroblasts (HGF). HDPC and HGF were cultured and identified by immunocytochemistry. HPDC and HGF subtractive cDNA library was established by PCR-based modified subtractive hybridization, genes differentially expressed by HPDC were cloned, sequenced and compared to find homogeneous sequence in GenBank by BLAST. Cloning and sequencing analysis indicate 12 genes differentially expressed were obtained, in which two were unknown genes. Among the 10 known genes, 4 were related to signal transduction, 2 were related to trans-membrane transportation (both cell membrane and nuclear membrane), and 2 were related to RNA splicing mechanisms. The biological properties of HPDC are determined by the differential expression of some genes and the growth and differentiation of HPDC are associated to the dynamic protein synthesis and secretion activities of the cell.

  11. Project management in the library workplace

    CERN Document Server

    Daugherty, Alice

    2018-01-01

    This volume of Advances in Library Administration and Organization attempts to put project management into the toolboxes of library administrators through overviews of concepts, analyses of experiences, and forecasts for the use of project management within the profession.

  12. Isolation of a 97-kb minimal essential MHC B locus from a new reverse-4D BAC library of the golden pheasant.

    Directory of Open Access Journals (Sweden)

    Qing Ye

    Full Text Available The bacterial artificial chromosome (BAC system is widely used in isolation of large genomic fragments of interest. Construction of a routine BAC library requires several months for picking clones and arraying BACs into superpools in order to employ 4D-PCR to screen positive BACs, which might be time-consuming and laborious. The major histocompatibility complex (MHC is a cluster of genes involved in the vertebrate immune system, and the classical avian MHC-B locus is a minimal essential one, occupying a 100-kb genomic region. In this study, we constructed a more effective reverse-4D BAC library for the golden pheasant, which first creates sub-libraries and then only picks clones of positive sub-libraries, and identified several MHC clones within thirty days. The full sequencing of a 97-kb reverse-4D BAC demonstrated that the golden pheasant MHC-B locus contained 20 genes and showed good synteny with that of the chicken. The notable differences between these two species were the numbers of class II B loci and NK genes and the inversions of the TAPBP gene and the TAP1-TAP2 region. Furthermore, the inverse TAP2-TAP1 was unique in the golden pheasant in comparison with that of chicken, turkey, and quail. The newly defined genomic structure of the golden pheasant MHC will give an insight into the evolutionary history of the avian MHC.

  13. Usage Analysis of Web 2.0 and Library 2.0 Tools by Librarians in Kwara State Academic Libraries

    Science.gov (United States)

    Tella, Adeyinka; Soluoku, Taofeeqat

    2016-01-01

    This study analysed the usage of Web 2.0 and Library 2.0 tools by librarians in Kwara State academic libraries. A sample of 40 librarians was surveyed through total enumeration sampling technique from four different tertiary education institutions libraries in Kwara State, Nigeria. Questionnaire was used for the collection of data. The collected…

  14. Selection of diethylstilbestrol-specific single-chain antibodies from a non-immunized mouse ribosome display library.

    Directory of Open Access Journals (Sweden)

    Yanan Sun

    Full Text Available Single chain variable fragments (scFvs against diethylstilbestrol (DES were selected from the splenocytes of non-immunized mice by ribosome display technology. A naive library was constructed and engineered to allow in vitro transcription and translation using an E. coli lysate system. Alternating selection in solution and immobilization in microtiter wells was used to pan mRNA-ribosome-antibody (ARM complexes. After seven rounds of ribosome display, the expression vector pTIG-TRX containing the selected specific scFv DNAs were transformed into Escherichia coli BL21 (DE3 for expression. Twenty-six positive clones were screened and five clones had high antibody affinity and specificity to DES as evidenced by indirect competitive ELISA. Sequence analysis showed that these five DES-specific scFvs had different amino acid sequences, but the CDRs were highly similar. Surface plasmon resonance (SPR analysis was used to determine binding kinetics of one clone (30-1. The measured K(D was 3.79 µM. These results indicate that ribosome display technology can be used to efficiently isolate hapten-specific antibody (Ab fragments from a naive library; this study provides a methodological framework for the development of novel immunoassays for multiple environmental pollutants with low molecular weight detection using recombinant antibodies.

  15. Cloning the human lysozyme cDNA: Inverted Alu repeat in the mRNA and in situ hybridization for macrophages and Paneth cells

    International Nuclear Information System (INIS)

    Chung, L.P.; Keshav, S.; Gordon, S.

    1988-01-01

    Lysozyme is a major secretory product of human and rodent macrophages and a useful marker for myelomonocytic cells. Based on the known human lysozyme amino acid sequence, oligonucleotides were synthesized and used as probes to screen a phorbol 12-myristate 13-acetate-treated U937 cDNA library. A full-length human lysozyme cDNA clone, pHL-2, was obtained and characterized. Sequence analysis shows that human lysozyme, like chicken lysozyme, has in 18-amino-acid-long signal peptide, but unlike the chicken lysozyme cDNA, the human lysozyme cDNA has a >1-kilobase-long 3' nontranslated sequence. Interestingly, within this 3' region, an inverted repeat of the Alu family of repetitive sequences was discovered. In RNA blot analyses, DNA probes prepared from pHL-2 can be used to detect lysozyme mRNA not only from human but also from mouse and rat. Moreover, by in situ hybridization, complementary RNA transcripts have been used as probes to detect lysozyme mRNA in mouse macrophages and Paneth cells. This human lysozyme cDNA clone is therefore likely to be a useful molecular probe for studying macrophage distribution and gene expression

  16. cDNA cloning of human DNA topoisomerase I. Catalytic activity of a 67.7-kDa carboxyl-terminal fragment

    International Nuclear Information System (INIS)

    D'Arpa, P.; Machlin, P.S.; Ratrie, H. III; Rothfield, N.F.; Cleveland, D.W.; Earnshaw, W.C.

    1988-01-01

    cDNA clones encoding human topoisomerase I were isolated from an expression vector library (λgt11) screened with autoimmune anti-topoisomerase I serum. One of these clones has been expressed as a fusion protein comprised of a 32-kDa fragment of the bacterial TrpE protein linked to 67.7 kDa of protein encoded by the cDNA. Three lines of evidence indicate that the cloned cDNA encodes topoisomerase I. (i) Proteolysis maps of the fusion protein and human nuclear topoisomerase I are essentially identical. (ii) The fusion protein relaxes supercoiled DNA, an activity that can be immunoprecipitated by anti-topoisomerase I serum. (iii) Sequence analysis has revealed that the longest cDNA clone (3645 base pairs) encodes a protein of 765 amino acids that shares 42% identity with Saccharomyces cerevisiae topoisomerase I. The sequence data also show that the catalytically active 67.7-kDa fragment is comprised of the carboxyl terminus

  17. Comparative Transcriptome Analysis of Latex Reveals Molecular Mechanisms Underlying Increased Rubber Yield in Hevea brasiliensis Self-Rooting Juvenile Clones.

    Science.gov (United States)

    Li, Hui-Liang; Guo, Dong; Zhu, Jia-Hong; Wang, Ying; Chen, Xiong-Ting; Peng, Shi-Qing

    2016-01-01

    Rubber tree (Hevea brasiliensis) self-rooting juvenile clones (JCs) are promising planting materials for rubber production. In a comparative trial between self-rooting JCs and donor clones (DCs), self-rooting JCs exhibited better performance in rubber yield. To study the molecular mechanism associated with higher rubber yield in self-rooting JCs, we sequenced and comparatively analyzed the latex of rubber tree self-rooting JCs and DCs at the transcriptome level. Total raw reads of 34,632,012 and 35,913,020 bp were obtained from the library of self-rooting JCs and DCs, respectively, by using Illumina HiSeq 2000 sequencing technology. De novo assemblies yielded 54689 unigenes from the library of self-rooting JCs and DCs. Among 54689 genes, 1716 genes were identified as differentially expressed between self-rooting JCs and DCs via comparative transcript profiling. Functional analysis showed that the genes related to the mass of categories were differentially enriched between the two clones. Several genes involved in carbohydrate metabolism, hormone metabolism and reactive oxygen species scavenging were up-regulated in self-rooting JCs, suggesting that the self-rooting JCs provide sufficient molecular basis for the increased rubber yielding, especially in the aspects of improved latex metabolisms and latex flow. Some genes encoding epigenetic modification enzymes were also differentially expressed between self-rooting JCs and DCs. Epigenetic modifications may lead to gene differential expression between self-rooting JCs and DCs. These data will provide new cues to understand the molecular mechanism underlying the improved rubber yield of H. brasiliensis self-rooting clones.

  18. Methylation-sensitive linking libraries enhance gene-enriched sequencing of complex genomes and map DNA methylation domains

    Directory of Open Access Journals (Sweden)

    Bharti Arvind K

    2008-12-01

    Full Text Available Abstract Background Many plant genomes are resistant to whole-genome assembly due to an abundance of repetitive sequence, leading to the development of gene-rich sequencing techniques. Two such techniques are hypomethylated partial restriction (HMPR and methylation spanning linker libraries (MSLL. These libraries differ from other gene-rich datasets in having larger insert sizes, and the MSLL clones are designed to provide reads localized to "epigenetic boundaries" where methylation begins or ends. Results A large-scale study in maize generated 40,299 HMPR sequences and 80,723 MSLL sequences, including MSLL clones exceeding 100 kb. The paired end reads of MSLL and HMPR clones were shown to be effective in linking existing gene-rich sequences into scaffolds. In addition, it was shown that the MSLL clones can be used for anchoring these scaffolds to a BAC-based physical map. The MSLL end reads effectively identified epigenetic boundaries, as indicated by their preferential alignment to regions upstream and downstream from annotated genes. The ability to precisely map long stretches of fully methylated DNA sequence is a unique outcome of MSLL analysis, and was also shown to provide evidence for errors in gene identification. MSLL clones were observed to be significantly more repeat-rich in their interiors than in their end reads, confirming the correlation between methylation and retroelement content. Both MSLL and HMPR reads were found to be substantially gene-enriched, with the SalI MSLL libraries being the most highly enriched (31% align to an EST contig, while the HMPR clones exhibited exceptional depletion of repetitive DNA (to ~11%. These two techniques were compared with other gene-enrichment methods, and shown to be complementary. Conclusion MSLL technology provides an unparalleled approach for mapping the epigenetic status of repetitive blocks and for identifying sequences mis-identified as genes. Although the types and natures of

  19. Identification of immune protective genes of Eimeria maxima through cDNA expression library screening.

    Science.gov (United States)

    Yang, XinChao; Li, MengHui; Liu, JianHua; Ji, YiHong; Li, XiangRui; Xu, LiXin; Yan, RuoFeng; Song, XiaoKai

    2017-02-16

    Eimeria maxima is one of the most prevalent Eimeria species causing avian coccidiosis, and results in huge economic loss to the global poultry industry. Current control strategies, such as anti-coccidial medication and live vaccines have been limited because of their drawbacks. The third generation anticoccidial vaccines including the recombinant vaccines as well as DNA vaccines have been suggested as a promising alternative strategy. To date, only a few protective antigens of E. maxima have been reported. Hence, there is an urgent need to identify novel protective antigens of E. maxima for the development of neotype anticoccidial vaccines. With the aim of identifying novel protective genes of E. maxima, a cDNA expression library of E. maxima sporozoites was constructed using Gateway technology. Subsequently, the cDNA expression library was divided into 15 sub-libraries for cDNA expression library immunization (cDELI) using parasite challenged model in chickens. Protective sub-libraries were selected for the next round of screening until individual protective clones were obtained, which were further sequenced and analyzed. Adopting the Gateway technology, a high-quality entry library was constructed, containing 9.2 × 10 6 clones with an average inserted fragments length of 1.63 kb. The expression library capacity was 2.32 × 10 7 colony-forming units (cfu) with an average inserted fragments length of 1.64 Kb. The expression library was screened using parasite challenged model in chickens. The screening yielded 6 immune protective genes including four novel protective genes of EmJS-1, EmRP, EmHP-1 and EmHP-2, and two known protective genes of EmSAG and EmCKRS. EmJS-1 is the selR domain-containing protein of E. maxima whose function is unknown. EmHP-1 and EmHP-2 are the hypothetical proteins of E. maxima. EmRP and EmSAG are rhomboid-like protein and surface antigen glycoproteins of E. maxima respectively, and involved in invasion of the parasite. Our

  20. Peripheral blood and intrathyroidal T cell clones from patients with thyroid autoimmune diseases.

    Science.gov (United States)

    Massart, C; Caroff, G; Maugendre, D; Genetet, N; Gibassier, J

    1999-01-01

    For a better understanding of the pathogenesis of thyroid autoimmune diseases, we have studied morphological and functional properties of T clones from peripheral blood lymphocytes (PBL) and from intrathyroidal lymphocytes (ITL) obtained from 3 patients with Graves' disease or 1 Hashimoto's thyroiditis. Investigations were carried out on clones cultured alone or cocultured with autologous thyrocytes. Clonage efficiency ranged from 30% to 33% for PBL and 10% to 36% for ITL. A predominance of CD4-positive clones was observed whatever the origin of the lymphocytes or the autoimmune pathology. Gamma interferon (IFN-gamma) was detected in the majority (17/19) of the clones tested. Intracytoplasmic interleukin (IL-4) was secreted in 7/19 clones and both cytokines were produced in 5/19 clones. In coculture a proliferative response and tumour necrosis factor (TNF-alpha) production were observed with 6 clones (4 from Graves thyrocytes and 2 from thyroiditis). No cytotoxic clone was derived from Graves or thyroiditis tissues. These data demonstrate that the large majority of T clones are principally CD4-T cells; all the clones secreted TNF-alpha and a large majority produced IFN-gamma. Only a few clones produced IL-4 alone or associated with IFN-gamma. Six T clones induced proliferative response and of TNF-alpha secretion in coculture. Further investigations must be performed on these antigen-reactive T clones to analyse their role in the pathogenesis of the human thyroid autoimmune diseases.

  1. Molecular cloning and characterization of pathogenesis-related ...

    African Journals Online (AJOL)

    We described the cloning and characterization of pathogenesis-related protein 5 gene in maize, named ZmPR5 (GenBank Accession Number: HM230665). Molecular and bioinformatic analyses of ZmPR5 revealed an open reading frame (ORF) of 525 bp, encoding a protein of 175 amino acids (aa) and a deduced ...

  2. The Impact of the Economic Downturn on Libraries: With Special Reference to University Libraries

    Science.gov (United States)

    Nicholas, David; Rowlands, Ian; Jubb, Michael; Jamali, Hamid R.

    2010-01-01

    Evidence is presented of the extent to which libraries from around the world are experiencing financial hardship as a result of the world-wide economic downturn. Comparative analyses are provides on the grounds of country, sector and size of institution. The article concentrates on the situation of UK and US university libraries and is based on…

  3. Escherichia coli rpiA gene encoding ribose phosphate isomerase A

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Maigaard, Marianne

    1993-01-01

    The rpiA gene encoding ribose phosphate isomerase A was cloned from phage 1A2(471) of the Kohara gene library. Subcloning, restriction, and complementation analyses revealed an 1,800-bp SspI-generated DNA fragment that contained the entire control and coding sequences. This DNA fragment was seque......The rpiA gene encoding ribose phosphate isomerase A was cloned from phage 1A2(471) of the Kohara gene library. Subcloning, restriction, and complementation analyses revealed an 1,800-bp SspI-generated DNA fragment that contained the entire control and coding sequences. This DNA fragment...

  4. Transformational Leadership and Stakeholder Management in Library Change

    Directory of Open Access Journals (Sweden)

    Dolores Sucozhañay

    2014-11-01

    Full Text Available This article aims to analyse the role of library managers as change agent when implementing Library 2.0, using transformational leadership and stakeholder management approaches. To do so, a case study in a Latin American academic library was performed. The experiences acquired for a period of six years were analysed, during which three library managers were involved in managing change. Qualitative data from documents, interviews, and observations were collected, and qualitative analysis methods were used to obtain in-depth understanding of the change process. Results show that lack of transformational leadership and stakeholder management contribute to delayed implementation and limited adoption of innovations. Although library managers recognized the importance of different stakeholders to implement changes, they did not apply systematic and proactive strategies to define and manage them. All in all, library managers should be trained as change agents, with emphasis on transformational leadership and stakeholder management skills.

  5. Novel Zn2+-chelating peptides selected from a fimbria-displayed random peptide library

    DEFF Research Database (Denmark)

    Kjærgaard, Kristian; Schembri, Mark; Klemm, Per

    2001-01-01

    The display of peptide sequences on the surface of bacteria is a technology that offers exciting applications in biotechnology and medical research. Type 1 fimbriae are surface organelles of Escherichia coli which mediate D-mannose-sensitive binding to different host surfaces by virtue of the Fim......H adhesin. FimH is a component of the fimbrial organelle that can accommodate and display a diverse range of peptide sequences on the E. coli cell surface. In this study we have constructed a random peptide library in FimH. The library, consisting of similar to 40 million individual clones, was screened...

  6. Security issues and service provision in Laz Otti Memorial Library ...

    African Journals Online (AJOL)

    Lagos Journal of Library and Information Science ... Data was analysed with the aid of Statistical Package for Social Sciences (SPSS. ... To curb this menace, library management must educate users on use of library resources ... Keywords: Library security, Theft, Mutilation, Vandalism, Library resources; Laz Otti Memorial ...

  7. Definition of natural T cell antigens with mimicry epitopes obtained from dedicated synthetic peptide libraries.

    Science.gov (United States)

    Hiemstra, H S; van Veelen, P A; Schloot, N C; Geluk, A; van Meijgaarden, K E; Willemen, S J; Leunissen, J A; Benckhuijsen, W E; Amons, R; de Vries, R R; Roep, B O; Ottenhoff, T H; Drijfhout, J W

    1998-10-15

    Progress has recently been made in the use of synthetic peptide libraries for the identification of T cell-stimulating ligands. T cell epitopes identified from synthetic libraries are mimics of natural epitopes. Here we show how the mimicry epitopes obtained from synthetic peptide libraries enable unambiguous identification of natural T cell Ags. Synthetic peptide libraries were screened with Mycobacterium tuberculosis-reactive and -autoreactive T cell clones. In two cases, database homology searches with mimicry epitopes isolated from a dedicated synthetic peptide library allowed immediate identification of the natural antigenic protein. In two other cases, an amino acid pattern that reflected the epitope requirements of the T cell was determined by substitution and omission mixture analysis. Subsequently, the natural Ag was identified from databases using this refined pattern. This approach opens new perspectives for rapid and reliable Ag definition, representing a feasible alternative to the biochemical and genetic approaches described thus far.

  8. Coupled high-throughput functional screening and next generation sequencing for identification of plant polymer decomposing enzymes in metagenomic libraries

    Directory of Open Access Journals (Sweden)

    Mari eNyyssönen

    2013-09-01

    Full Text Available Recent advances in sequencing technologies generate new predictions and hypotheses about the functional roles of environmental microorganisms. Yet, until we can test these predictions at a scale that matches our ability to generate them, most of them will remain as hypotheses. Function-based mining of metagenomic libraries can provide direct linkages between genes, metabolic traits and microbial taxa and thus bridge this gap between sequence data generation and functional predictions. Here we developed high-throughput screening assays for function-based characterization of activities involved in plant polymer decomposition from environmental metagenomic libraries. The multiplexed assays use fluorogenic and chromogenic substrates, combine automated liquid handling and use a genetically modified expression host to enable simultaneous screening of 12,160 clones for 14 activities in a total of 170,240 reactions. Using this platform we identified 374 (0.26 % cellulose, hemicellulose, chitin, starch, phosphate and protein hydrolyzing clones from fosmid libraries prepared from decomposing leaf litter. Sequencing on the Illumina MiSeq platform, followed by assembly and gene prediction of a subset of 95 fosmid clones, identified a broad range of bacterial phyla, including Actinobacteria, Bacteroidetes, multiple Proteobacteria sub-phyla in addition to some Fungi. Carbohydrate-active enzyme genes from 20 different glycoside hydrolase families were detected. Using tetranucleotide frequency binning of fosmid sequences, multiple enzyme activities from distinct fosmids were linked, demonstrating how biochemically-confirmed functional traits in environmental metagenomes may be attributed to groups of specific organisms. Overall, our results demonstrate how functional screening of metagenomic libraries can be used to connect microbial functionality to community composition and, as a result, complement large-scale metagenomic sequencing efforts.

  9. Screening and Identification of Peptides Specifically Targeted to Gastric Cancer Cells from a Phage Display Peptide Library

    Science.gov (United States)

    Sahin, Deniz; Taflan, Sevket Onur; Yartas, Gizem; Ashktorab, Hassan; Smoot, Duane T

    2018-04-25

    Background: Gastric cancer is the second most common cancer among the malign cancer types. Inefficiency of traditional techniques both in diagnosis and therapy of the disease makes the development of alternative and novel techniques indispensable. As an alternative to traditional methods, tumor specific targeting small peptides can be used to increase the efficiency of the treatment and reduce the side effects related to traditional techniques. The aim of this study is screening and identification of individual peptides specifically targeted to human gastric cancer cells using a phage-displayed peptide library and designing specific peptide sequences by using experimentally-eluted peptide sequences. Methods: Here, MKN-45 human gastric cancer cells and HFE-145 human normal gastric epithelial cells were used as the target and control cells, respectively. 5 rounds of biopannning with a phage display 12-peptide library were applied following subtraction biopanning with HFE-145 control cells. The selected phage clones were established by enzyme-linked immunosorbent assay and immunofluorescence detection. We first obtain random phage clones after five biopanning rounds, determine the binding levels of each individual clone. Then, we analyze the frequencies of each amino acid in best binding clones to determine positively overexpressed amino acids for designing novel peptide sequences. Results: DE532 (VETSQYFRGTLS) phage clone was screened positive, showing specific binding on MKN-45 gastric cancer cells. DE-Obs (HNDLFPSWYHNY) peptide, which was designed by using amino acid frequencies of experimentally selected peptides in the 5th round of biopanning, showed specific binding in MKN-45 cells. Conclusion: Selection and characterization of individual clones may give us specifically binding peptides, but more importantly, data extracted from eluted phage clones may be used to design theoretical peptides with better binding properties than even experimentally selected ones

  10. Stochasticity or the fatal `imperfection' of cloning

    Indian Academy of Sciences (India)

    2005-01-07

    Jan 7, 2005 ... The concept of clone is analysed with the aim of exploring the limits to which a phenotype can be said to be determined geneticaly. First of all, mutations that result from the replication, topological manipulation or lesion of DNA introduce a source of heritable variation in an otherwise identical genetic ...

  11. Measuring Effectiveness in a Virtual Library

    Science.gov (United States)

    Finch, Jannette L.

    2010-01-01

    Measuring quality of service in academic libraries traditionally includes quantifiable data such as collection size, staff counts, circulation numbers, reference service statistics, qualitative analyses of customer satisfaction, shelving accuracy, and building comfort. In the libraries of the third millennium, virtual worlds, Web content and…

  12. Functional Screening of Antibiotic Resistance Genes from a Representative Metagenomic Library of Food Fermenting Microbiota

    Directory of Open Access Journals (Sweden)

    Chiara Devirgiliis

    2014-01-01

    Full Text Available Lactic acid bacteria (LAB represent the predominant microbiota in fermented foods. Foodborne LAB have received increasing attention as potential reservoir of antibiotic resistance (AR determinants, which may be horizontally transferred to opportunistic pathogens. We have previously reported isolation of AR LAB from the raw ingredients of a fermented cheese, while AR genes could be detected in the final, marketed product only by PCR amplification, thus pointing at the need for more sensitive microbial isolation techniques. We turned therefore to construction of a metagenomic library containing microbial DNA extracted directly from the food matrix. To maximize yield and purity and to ensure that genomic complexity of the library was representative of the original bacterial population, we defined a suitable protocol for total DNA extraction from cheese which can also be applied to other lipid-rich foods. Functional library screening on different antibiotics allowed recovery of ampicillin and kanamycin resistant clones originating from Streptococcus salivarius subsp. thermophilus and Lactobacillus helveticus genomes. We report molecular characterization of the cloned inserts, which were fully sequenced and shown to confer AR phenotype to recipient bacteria. We also show that metagenomics can be applied to food microbiota to identify underrepresented species carrying specific genes of interest.

  13. Bovine viral diarrhea virus: molecular cloning of genomic RNA and its diagnostic application

    International Nuclear Information System (INIS)

    Brock, K.V.

    1987-01-01

    Molecular cloning of a field isolate of bovine viral diarrhea virus (BVDV) strain 72 RNA was done in this study. The sensitivity and specificity of cloned cDNA sequences in hybridization assays with various BVDV strains were determined. cDNA was synthesized from polyadenylated BVDV RNA templates with oligo-dT primers, reverse transcriptase, and DNA polymerase I. The newly synthesized double-stranded BVDV cDNA was C-tailed with terminal deoxytransferase and annealed into G-tailed, Pst-1-cut pUC9 plasmid. Escherichia coli was transformed with the recombinant plasmids and a library of approximately 200 BVDV specific cDNA clones varying in length from 0.5 to 2.6 kilobases were isolated. The sensitivity and specificity of hybridization between the labelled cDNA and BVDV target sequences were determined. Cloned BVDV sequences were isolated from pUC9 plasmid DNA and labelled with 32 P by nick translation. The detection limit by dot blot hybridization assay was 20 pg of purified genomic BVDV RNA. cDNA hybridization probes were specific for all strains of BVDV tested, regardless of whether they were noncytopathic and cytopathic, but did not hybridize with heterologous bovine viruses tested. Probes did not hybridize with uninfected cell culture or cellular RNA. Hybridization probes were at least as sensitive as infectivity assays in detecting homologous virus

  14. Human therapeutic cloning (NTSC): applying research from mammalian reproductive cloning.

    Science.gov (United States)

    French, Andrew J; Wood, Samuel H; Trounson, Alan O

    2006-01-01

    Human therapeutic cloning or nuclear transfer stem cells (NTSC) to produce patient-specific stem cells, holds considerable promise in the field of regenerative medicine. The recent withdrawal of the only scientific publications claiming the successful generation of NTSC lines afford an opportunity to review the available research in mammalian reproductive somatic cell nuclear transfer (SCNT) with the goal of progressing human NTSC. The process of SCNT is prone to epigenetic abnormalities that contribute to very low success rates. Although there are high mortality rates in some species of cloned animals, most surviving clones have been shown to have normal phenotypic and physiological characteristics and to produce healthy offspring. This technology has been applied to an increasing number of mammals for utility in research, agriculture, conservation, and biomedicine. In contrast, attempts at SCNT to produce human embryonic stem cells (hESCs) have been disappointing. Only one group has published reliable evidence of success in deriving a cloned human blastocyst, using an undifferentiated hESC donor cell, and it failed to develop into a hESC line. When optimal conditions are present, it appears that in vitro development of cloned and parthenogenetic embryos, both of which may be utilized to produce hESCs, may be similar to in vitro fertilized embryos. The derivation of ESC lines from cloned embryos is substantially more efficient than the production of viable offspring. This review summarizes developments in mammalian reproductive cloning, cell-to-cell fusion alternatives, and strategies for oocyte procurement that may provide important clues facilitating progress in human therapeutic cloning leading to the successful application of cell-based therapies utilizing autologous hESC lines.

  15. Cloning and characterization of the rec2 gene of Ustilago maydis

    International Nuclear Information System (INIS)

    Bauchwitz, R.P.; Holloman, W.K.

    1989-01-01

    The authors are exploring the molecular basis for genetic recombination using the corn smut fungus Ustilago maydis, from which the first two eucaryotic DNA repair and recombination mutants, rec1 and rec2, were described. Cells mutant at the rec2 locus are unable to repair lethal damage to their DNA from UV and X irradiation or from chemical alkylating agents such as N-methyl-nitrosoguanidine. Rec2 mutants retain only a residual level of DNA-damage inducible mitotic recombination, and are unable to complete meiosis. Using an autonomously replicating plasmid vector for Ustilago, they established the first nonintegrating plasmid library of the Ustilago genome. The rec2 locus was cloned by complementation of the rec2 mutation in vivo. One clone was found to restore all of the deficient activities. Although this rec2 complementing clone is present on a multicopy plasmid, the authors observed that it fully restored but did not further increase the fifty-fold inducibility of heteroallelic recombination at the nitrate reductase and inositol loci of rec2 or wild type cells. Northern blot analysis using the rec2 complementing clone revealed three UV inducible transcripts, one of which is absent in a rec2 mutant strain. This transcript organization resembles that of the yeast rad10 and the human ERCC-1 genes (MCB 9:1794), but sequence obtained to date from rec2 does not show homology with these genes. They have also observed that the rec2 mutation may alter the level of homologous integration of transformed DNA markers. Integration of a Leu1 complementing plasmid by Scott Fotheringham of the lab has shown that while much of plasmid integration in wild type Ustilago is nonhomologous, integration in at least some rec2 strains is entirely homologous. They are using the cloned rec2 gene to confirm that rec2 is indeed involved in altering the level of homologous integration in Ustilago, and if so, they plan to clone a mammalian analogue of rec2

  16. Designing and Implementing an Assay for the Detection of Rare and Divergent NRPS and PKS Clones in European, Antarctic and Cuban Soils.

    Directory of Open Access Journals (Sweden)

    Gregory C A Amos

    Full Text Available The ever increasing microbial resistome means there is an urgent need for new antibiotics. Metagenomics is an underexploited tool in the field of drug discovery. In this study we aimed to produce a new updated assay for the discovery of biosynthetic gene clusters encoding bioactive secondary metabolites. PCR assays targeting the polyketide synthases (PKS and non-ribosomal peptide synthetases (NRPS were developed. A range of European soils were tested for their biosynthetic potential using clone libraries developed from metagenomic DNA. Results revealed a surprising number of NRPS and PKS clones with similarity to rare Actinomycetes. Many of the clones tested were phylogenetically divergent suggesting they were fragments from novel NRPS and PKS gene clusters. Soils did not appear to cluster by location but did represent NRPS and PKS clones of diverse taxonomic origin. Fosmid libraries were constructed from Cuban and Antarctic soil samples; 17 fosmids were positive for NRPS domains suggesting a hit rate of less than 1 in 10 genomes. NRPS hits had low similarities to both rare Actinobacteria and Proteobacteria; they also clustered with known antibiotic producers suggesting they may encode for pathways producing novel bioactive compounds. In conclusion we designed an assay capable of detecting divergent NRPS and PKS gene clusters from the rare biosphere; when tested on soil samples results suggest the majority of NRPS and PKS pathways and hence bioactive metabolites are yet to be discovered.

  17. Entamoeba Clone-Recognition Experiments: Morphometrics, Aggregative Behavior, and Cell-Signaling Characterization.

    Science.gov (United States)

    Espinosa, Avelina; Paz-Y-Miño-C, Guillermo; Hackey, Meagan; Rutherford, Scott

    2016-05-01

    Studies on clone- and kin-discrimination in protists have proliferated during the past decade. We report clone-recognition experiments in seven Entamoeba lineages (E. invadens IP-1, E. invadens VK-1:NS, E. terrapinae, E. moshkovskii Laredo, E. moshkovskii Snake, E. histolytica HM-1:IMSS and E. dispar). First, we characterized morphometrically each clone (length, width, and cell-surface area) and documented how they differed statistically from one another (as per single-variable or canonical-discriminant analyses). Second, we demonstrated that amebas themselves could discriminate self (clone) from different (themselves vs. other clones). In mix-cell-line cultures between closely-related (E. invadens IP-1 vs. E. invadens VK-1:NS) or distant-phylogenetic clones (E. terrapinae vs. E. moshkovskii Laredo), amebas consistently aggregated with same-clone members. Third, we identified six putative cell-signals secreted by the amebas (RasGap/Ankyrin, coronin-WD40, actin, protein kinases, heat shock 70, and ubiquitin) and which known functions in Entamoeba spp. included: cell proliferation, cell adhesion, cell movement, and stress-induced encystation. To our knowledge, this is the first multi-clone characterization of Entamoeba spp. morphometrics, aggregative behavior, and cell-signaling secretion in the context of clone-recognition. Protists allow us to study cell-cell recognition from ecological and evolutionary perspectives. Modern protistan lineages can be central to studies about the origins and evolution of multicellularity. © 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists.

  18. Analysing and Navigating Natural Products Space for Generating Small, Diverse, But Representative Chemical Libraries.

    Science.gov (United States)

    O'Hagan, Steve; Kell, Douglas B

    2018-01-01

    Armed with the digital availability of two natural products libraries, amounting to some 195 885 molecular entities, we ask the question of how we can best sample from them to maximize their "representativeness" in smaller and more usable libraries of 96, 384, 1152, and 1920 molecules. The term "representativeness" is intended to include diversity, but for numerical reasons (and the likelihood of being able to perform a QSAR) it is necessary to focus on areas of chemical space that are more highly populated. Encoding chemical structures as fingerprints using the RDKit "patterned" algorithm, we first assess the granularity of the natural products space using a simple clustering algorithm, showing that there are major regions of "denseness" but also a great many very sparsely populated areas. We then apply a "hybrid" hierarchical K-means clustering algorithm to the data to produce more statistically robust clusters from which representative and appropriate numbers of samples may be chosen. There is necessarily again a trade-off between cluster size and cluster number, but within these constraints, libraries containing 384 or 1152 molecules can be found that come from clusters that represent some 18 and 30% of the whole chemical space, with cluster sizes of, respectively, 50 and 27 or above, just about sufficient to perform a QSAR. By using the online availability of molecules via the Molport system (www.molport.com), we are also able to construct (and, for the first time, provide the contents of) a small virtual library of available molecules that provided effective coverage of the chemical space described. Consistent with this, the average molecular similarities of the contents of the libraries developed is considerably smaller than is that of the original libraries. The suggested libraries may have use in molecular or phenotypic screening, including for determining possible transporter substrates. © 2017 The Authors. Biotechnology Journal Published by Wiley

  19. Caracterização isoenzimática e morfológica de clones e introduções de alho Morphological and electrophoretic characterization of garlic clones

    Directory of Open Access Journals (Sweden)

    Walter José Siqueira

    1985-01-01

    Full Text Available Em virtude do grande número de denominações locais para clones de alho, nem sempre correspondentes a materiais distintos, conduziu-se o presente estudo objetivando a caracterização e classificação de 72 clones e introduções de alho (Allium sativum L., e um clone de alho-rei (A. ampeloprasum L.. Isso foi feito analisando as isoenzimas alcooldesidrogenase (ADH, esterase (EST, peroxidase (PRX e fosfoglucoisomerase (PGI através da técnica de eletroforese horizontal em gel de amido hidrolisado de batata. Verificou-se que os clones nacionais e introduzidos se enquadram nos grupos aqui denominados DIKA ou CJLB, respectivamente para os padrões de ADH, EST, PRX e PGI. Entretanto, os padrões CILB, CJKB e CIKB foram observados em alguns clones estrangeiros, sugerindo sua maior variabilidade em relação aos nacionais. O alho-rei apresentou padrões diferentes dos encontrados na espécie A. sativum L. A associação dos resultados da técnica de eletroforese de isoenzinas com a caracterização morfológica da parte aérea, bulbos, bulbilhos, coloração externa dos bulbos e bulbilhos e ciclo cultural, permitiu a classificação dos clones nacionais de alho em 19 grupos distintos.Since there exist different local names for the same garlic (Allium sativum L. clones, it was made an attempt to distinguish them by the morphology, cycle and isozyme electrophoresis. The isozyme analysis of alcoholdehydrogenase, esterase, peroxydase and phosphoglucoisomerase separated the Brazilian clones in two groups. The foreign clones had different band patterns adding other three more groups. Morphology of bulbs and clones allowed the separation of clones into eight groups; top morphology into ten and cycle length into three. Morphology, cycle and electrophoresis together characterized the seventy two analysed clones into nineteen distinct groups.

  20. Construction of a llama bacterial artificial chromosome library with approximately 9-fold genome equivalent coverage.

    Science.gov (United States)

    Airmet, K W; Hinckley, J D; Tree, L T; Moss, M; Blumell, S; Ulicny, K; Gustafson, A K; Weed, M; Theodosis, R; Lehnardt, M; Genho, J; Stevens, M R; Kooyman, D L

    2012-01-01

    The Ilama is an important agricultural livestock in much of South America. The llama is increasing in popularity in the United States as a companion animal. Little work has been done to improve llama production using modern technology. A paucity of information is available regarding the llama genome. We report the construction of a llama bacterial artificial chromosome (BAC) library of about 196,224 clones in the vector pECBAC1. Using flow cytometry and bovine, human, mouse, and chicken as controls, we determined the llama genome size to be 2.4 × 10⁹ bp. The average insert size of the library is 137.8 kb corresponding to approximately 9-fold genome coverage. Further studies are needed to further characterize the library and llama genome. We anticipate that this new library will help facilitate future genomic studies in the llama.

  1. Coding sequence of human rho cDNAs clone 6 and clone 9

    Energy Technology Data Exchange (ETDEWEB)

    Chardin, P; Madaule, P; Tavitian, A

    1988-03-25

    The authors have isolated human cDNAs including the complete coding sequence for two rho proteins corresponding to the incomplete isolates previously described as clone 6 and clone 9. The deduced a.a. sequences, when compared to the a.a. sequence deduced from clone 12 cDNA, show that there are in human at least three highly homologous rho genes. They suggest that clone 12 be named rhoA, clone 6 : rhoB and clone 9 : rhoC. RhoA, B and C proteins display approx. 30% a.a. identity with ras proteins,. mainly clustered in four highly homologous internal regions corresponding to the GTP binding site; however at least one significant difference is found; the 3 rho proteins have an Alanine in position corresponding to ras Glycine 13, suggesting that rho and ras proteins might have slightly different biochemical properties.

  2. Ethical issues in animal cloning.

    Science.gov (United States)

    Fiester, Autumn

    2005-01-01

    The issue of human reproductive cloning has recently received a great deal attention in public discourse. Bioethicists, policy makers, and the media have been quick to identify the key ethical issues involved in human reproductive cloning and to argue, almost unanimously, for an international ban on such attempts. Meanwhile, scientists have proceeded with extensive research agendas in the cloning of animals. Despite this research, there has been little public discussion of the ethical issues raised by animal cloning projects. Polling data show that the public is decidedly against the cloning of animals. To understand the public's reaction and fill the void of reasoned debate about the issue, we need to review the possible objections to animal cloning and assess the merits of the anti-animal cloning stance. Some objections to animal cloning (e.g., the impact of cloning on the population of unwanted animals) can be easily addressed, while others (e.g., the health of cloned animals) require more serious attention by the public and policy makers.

  3. Optimally cloned binary coherent states

    DEFF Research Database (Denmark)

    Mueller, C. R.; Leuchs, G.; Marquardt, Ch

    2017-01-01

    their quantum-optimal clones. We analyze the Wigner function and the cumulants of the clones, and we conclude that optimal cloning of binary coherent states requires a nonlinearity above second order. We propose several practical and near-optimal cloning schemes and compare their cloning fidelity to the optimal...

  4. Molecular cloning and functional characterization of a rainbow trout liver Oatp

    Energy Technology Data Exchange (ETDEWEB)

    Steiner, Konstanze, E-mail: konstanze.steiner@uni-konstanz.de [University of Konstanz, Human- and Environmental Toxicology, 78464 Konstanz (Germany); Hagenbuch, Bruno, E-mail: bhagenbuch@kumc.edu [Pharmacology, Toxicology and Therapeutics, The University of Kansas Medical Center, Kansas City 66160, KS (United States); Dietrich, Daniel R., E-mail: daniel.dietrich@uni-konstanz.de [University of Konstanz, Human- and Environmental Toxicology, 78464 Konstanz (Germany)

    2014-11-01

    Cyanobacterial blooms have an impact on the aquatic ecosystem due to the production of toxins (e.g. microcystins, MCs), which constrain fish health or even cause fish death. However the toxicokinetics of the most abundant toxin, microcystin-LR (MC-LR), are not yet fully understood. To investigate the uptake mechanism, the novel Oatp1d1 in rainbow trout (rtOatp1d1) was cloned, identified and characterized. The cDNA isolated from a clone library consisted of 2772 bp containing a 2115 bp open reading frame coding for a 705 aa protein with an approximate molecular mass of 80 kDa. This fish specific transporter belongs to the OATP1 family and has most likely evolved from a common ancestor of OATP1C1. Real time PCR analysis showed that rtOatp1d1 is predominantly expressed in the liver, followed by the brain while expression in other organs was not detectable. Transient transfection in HEK293 cells was used for further characterization. Like its human homologues OATP1A1, OATP1B1 and OATP1B3, rtOatp1d1 displayed multi-specific transport including endogenous and xenobiotic substrates. Kinetic analyses revealed a K{sub m} value of 13.9 μM and 13.4 μM for estrone-3-sulfate and methotrexate, respectively and a rather low affinity for taurocholate with a K{sub m} value of 103 μM. Furthermore, it was confirmed that rtOatp1d1 is a MC-LR transporter and therefore most likely plays a key role in the susceptibility of rainbow trout to MC intoxications. - Highlights: • A new Oatp1d1 in rainbow trout (rtOatp1d1) was cloned, identified and characterized. • rtOatp1d1 is predominantly expressed in the liver. • rtOatp1d1 displays multi-specific transport of endogenous and xenobiotic substrates. • rtOatp1d1 is a homologue of the OATP1A1, OATP1B1 and OATP1B3. • rtOatp1d1 is a microcystin (MC) transporter.

  5. Molecular cloning and functional characterization of a rainbow trout liver Oatp

    International Nuclear Information System (INIS)

    Steiner, Konstanze; Hagenbuch, Bruno; Dietrich, Daniel R.

    2014-01-01

    Cyanobacterial blooms have an impact on the aquatic ecosystem due to the production of toxins (e.g. microcystins, MCs), which constrain fish health or even cause fish death. However the toxicokinetics of the most abundant toxin, microcystin-LR (MC-LR), are not yet fully understood. To investigate the uptake mechanism, the novel Oatp1d1 in rainbow trout (rtOatp1d1) was cloned, identified and characterized. The cDNA isolated from a clone library consisted of 2772 bp containing a 2115 bp open reading frame coding for a 705 aa protein with an approximate molecular mass of 80 kDa. This fish specific transporter belongs to the OATP1 family and has most likely evolved from a common ancestor of OATP1C1. Real time PCR analysis showed that rtOatp1d1 is predominantly expressed in the liver, followed by the brain while expression in other organs was not detectable. Transient transfection in HEK293 cells was used for further characterization. Like its human homologues OATP1A1, OATP1B1 and OATP1B3, rtOatp1d1 displayed multi-specific transport including endogenous and xenobiotic substrates. Kinetic analyses revealed a K m value of 13.9 μM and 13.4 μM for estrone-3-sulfate and methotrexate, respectively and a rather low affinity for taurocholate with a K m value of 103 μM. Furthermore, it was confirmed that rtOatp1d1 is a MC-LR transporter and therefore most likely plays a key role in the susceptibility of rainbow trout to MC intoxications. - Highlights: • A new Oatp1d1 in rainbow trout (rtOatp1d1) was cloned, identified and characterized. • rtOatp1d1 is predominantly expressed in the liver. • rtOatp1d1 displays multi-specific transport of endogenous and xenobiotic substrates. • rtOatp1d1 is a homologue of the OATP1A1, OATP1B1 and OATP1B3. • rtOatp1d1 is a microcystin (MC) transporter

  6. Biomathematical Description of Synthetic Peptide Libraries

    Science.gov (United States)

    Trepel, Martin

    2015-01-01

    Libraries of randomised peptides displayed on phages or viral particles are essential tools in a wide spectrum of applications. However, there is only limited understanding of a library's fundamental dynamics and the influences of encoding schemes and sizes on their quality. Numeric properties of libraries, such as the expected number of different peptides and the library's coverage, have long been in use as measures of a library's quality. Here, we present a graphical framework of these measures together with a library's relative efficiency to help to describe libraries in enough detail for researchers to plan new experiments in a more informed manner. In particular, these values allow us to answer-in a probabilistic fashion-the question of whether a specific library does indeed contain one of the "best" possible peptides. The framework is implemented in a web-interface based on two packages, discreteRV and peptider, to the statistical software environment R. We further provide a user-friendly web-interface called PeLiCa (Peptide Library Calculator, http://www.pelica.org), allowing scientists to plan and analyse their peptide libraries. PMID:26042419

  7. Directed Selection of Recombinant Human Monoclonal Antibodies to Herpes Simplex Virus Glycoproteins from Phage Display Libraries

    Science.gov (United States)

    Sanna, Pietro Paolo; Williamson, R. Anthony; de Logu, Alessandro; Bloom, Floyd E.; Burton, Dennis R.

    1995-07-01

    Human monoclonal antibodies have considerable potential in the prophylaxis and treatment of viral disease. However, only a few such antibodies suitable for clinical use have been produced to date. We have previously shown that large panels of human recombinant monoclonal antibodies against a plethora of infectious agents, including herpes simplex virus types 1 and 2, can be established from phage display libraries. Here we demonstrate that facile cloning of recombinant Fab fragments against specific viral proteins in their native conformation can be accomplished by panning phage display libraries against viral glycoproteins "captured" from infected cell extracts by specific monoclonal antibodies immobilized on ELISA plates. We have tested this strategy by isolating six neutralizing recombinant antibodies specific for herpes simplex glycoprotein gD or gB, some of which are against conformationally sensitive epitopes. By using defined monoclonal antibodies for the antigen-capture step, this method can be used for the isolation of antibodies to specific regions and epitopes within the target viral protein. For instance, monoclonal antibodies to a nonneutralizing epitope can be used in the capture step to clone antibodies to neutralizing epitopes, or antibodies to a neutralizing epitope can be used to clone antibodies to a different neutralizing epitope. Furthermore, by using capturing antibodies to more immunodominant epitopes, one can direct the cloning to less immunogenic ones. This method should be of value in generating antibodies to be used both in the prophylaxis and treatment of viral infections and in the characterization of the mechanisms of antibody protective actions at the molecular level.

  8. Molecular cloning, expression, and characterization of four novel thermo-alkaliphilic enzymes retrieved from a metagenomic library.

    Science.gov (United States)

    Maruthamuthu, Mukil; van Elsas, Jan Dirk

    2017-01-01

    Enzyme discovery is a promising approach to aid in the deconstruction of recalcitrant plant biomass in an industrial process. Novel enzymes can be readily discovered by applying metagenomics on whole microbiomes. Our goal was to select, examine, and characterize eight novel glycoside hydrolases that were previously detected in metagenomic libraries, to serve biotechnological applications with high performance. Here, eight glycosyl hydrolase family candidate genes were selected from metagenomes of wheat straw-degrading microbial consortia using molecular cloning and subsequent gene expression studies in Escherichia coli. Four of the eight enzymes had significant activities on either p NP-β-d-galactopyranoside, p NP-β-d-xylopyranoside, p NP-α-l-arabinopyranoside or p NP-α-d-glucopyranoside. These proteins, denoted as proteins 1, 2, 5 and 6, were his-tag purified and their nature and activities further characterized using molecular and activity screens with the p NP-labeled substrates. Proteins 1 and 2 showed high homologies with (1) a β-galactosidase (74%) and (2) a β-xylosidase (84%), whereas the remaining two (5 and 6) were homologous with proteins reported as a diguanylate cyclase and an aquaporin, respectively. The β-galactosidase- and β-xylosidase-like proteins 1 and 2 were confirmed as being responsible for previously found thermo-alkaliphilic glycosidase activities of extracts of E. coli carrying the respective source fosmids. Remarkably, the β-xylosidase-like protein 2 showed activities with both p NP-Xyl and p NP-Ara in the temperature range 40-50 °C and pH range 8.0-10.0. Moreover, proteins 5 and 6 showed thermotolerant α-glucosidase activity at pH 10.0. In silico structure prediction of protein 5 revealed the presence of a potential "GGDEF" catalytic site, encoding α-glucosidase activity, whereas that of protein 6 showed a "GDSL" site, encoding a 'new family' α-glucosidase activity. Using a rational screening approach, we identified and

  9. Influence of menstruation on the microbiota of healthy women's labia minora as analyzed using a 16S rRNA gene-based clone library method.

    Science.gov (United States)

    Shiraishi, Tsukasa; Fukuda, Kazumasa; Morotomi, Nobuo; Imamura, Yuri; Mishima, Junko; Imai, Shigeo; Miyazawa, Kiyoshi; Taniguchi, Hatsumi

    2011-01-01

    The aim of this study was to determine the influence of menstruation on the bacterial population of healthy Japanese women's vulvas, especially the labia minora. Labia minora swabs were obtained from 10 premenopausal, nonpregnant Japanese women at premenstruation and on day 2 of menstruation. Vaginal swabs were also obtained from 3 out of the 10 women. No significant difference was found in the average bacterial cell count between the menstruation and premenstruation samples. Molecular analysis using a 16S rRNA gene-based clone library method detected 22 genera from the labia minora swabs (total 20), with the genus Lactobacillus being predominant at both premenstruation and during menstruation in 7 out of the 10 women. Of the other 3 women, 2 showed various kinds of bacterial species, including oral and fecal bacteria, with Atopobium vaginae and Gardnerella vaginalis predominating in the remaining woman's vulva in both conditions. In total, 6 out of 10 cases (60%) showed significantly different microbiota of the labia minora between the two conditions. These results imply that menstruation may promote a distortion of the bacterial flora around the vulva, although it causes no significant increase of the bacterial count.

  10. The topsy-turvy cloning law.

    Science.gov (United States)

    Brassington, Iain; Oultram, Stuart

    2011-03-01

    In debates about human cloning, a distinction is frequently drawn between therapeutic and reproductive uses of the technology. Naturally enough, this distinction influences the way that the law is framed. The general consensus is that therapeutic cloning is less morally problematic than reproductive cloning--one can hold this position while holding that both are morally unacceptable--and the law frequently leaves the way open for some cloning for the sake of research into new therapeutic techniques while banning it for reproductive purposes. We claim that the position adopted by the law has things the wrong way around: if we accept a moral distinction between therapeutic and reproductive cloning, there are actually more reasons to be morally worried about therapeutic cloning than about reproductive cloning. If cloning is the proper object of legal scrutiny, then, we ought to make sure that we are scrutinising the right kind of clone.

  11. Alternative splicing of human elastin mRNA indicated by sequence analysis of cloned genomic and complementary DNA

    International Nuclear Information System (INIS)

    Indik, Z.; Yeh, H.; Ornstein-goldstein, N.; Sheppard, P.; Anderson, N.; Rosenbloom, J.C.; Peltonen, L.; Rosenbloom, J.

    1987-01-01

    Poly(A) + RNA, isolated from a single 7-mo fetal human aorta, was used to synthesize cDNA by the RNase H method, and the cDNA was inserted into λgt10. Recombinant phage containing elastin sequences were identified by hybridization with cloned, exon-containing fragments of the human elastin gene. Three clones containing inserts of 3.3, 2.7, and 2.3 kilobases were selected for further analysis. Three overlapping clones containing 17.8 kilobases of the human elastin gene were also isolated from genomic libraries. Complete sequence analysis of the six clones demonstrated that: (i) the cDNA encompassed the entire translated portion of the mRNA encoding 786 amino acids, including several unusual hydrophilic amino acid sequences not previously identified in porcine tropoelastin, (ii) exons encoding either hydrophobic or crosslinking domains in the protein alternated in the gene, and (iii) a great abundance of Alu repetitive sequences occurred throughout the introns. The data also indicated substantial alternative splicing of the mRNA. These results suggest the potential for significant variation in the precise molecular structure of the elastic fiber in the human population

  12. USGS Digital Spectral Library splib06a

    Science.gov (United States)

    Clark, Roger N.; Swayze, Gregg A.; Wise, Richard A.; Livo, K. Eric; Hoefen, Todd M.; Kokaly, Raymond F.; Sutley, Stephen J.

    2007-01-01

    ), one simply needs a diagnostic absorption band. The mapping system uses continuum-removed reference spectral features fitted to features in observed spectra. Spectral features for such algorithms can be obtained from a spectrum of a sample containing large amounts of contaminants, including those that add other spectral features, as long as the shape of the diagnostic feature of interest is not modified. If, however, the data are needed for radiative transfer models to derive mineral abundances from reflectance spectra, then completely uncontaminated spectra are required. This library contains spectra that span a range of quality, with purity indicators to flag spectra for (or against) particular uses. Acquiring spectral measurements and performing sample characterizations for this library has taken about 15 person-years of effort. Software to manage the library and provide scientific analysis capability is provided (Clark, 1980, 1993). A personal computer (PC) reader for the library is also available (Livo and others, 1993). The program reads specpr binary files (Clark, 1980, 1993) and plots spectra. Another program that reads the specpr format is written in IDL (Kokaly, 2005). In our view, an ideal spectral library consists of samples covering a very wide range of materials, has large wavelength range with very high precision, and has enough sample analyses and documentation to establish the quality of the spectra. Time and available resources limit what can be achieved. Ideally, for each mineral, the sample analysis would include X-ray diffraction (XRD), electron microprobe (EM) or X-ray fluorescence (XRF), and petrographic microscopic analyses. For some minerals, such as iron oxides, additional analyses such as Mossbauer would be helpful. We have found that to make the basic spectral measurements, provide XRD, EM or XRF analyses, and microscopic analyses, document the results, and complete an entry of one spectral library sample, all takes about

  13. Phosphoribosylpyrophosphate synthetase of Bacillus subtilis. Cloning, characterization and chromosomal mapping of the prs gene

    DEFF Research Database (Denmark)

    Nilsson, Dan; Hove-Jensen, Bjarne

    1987-01-01

    The gene (prs) encoding phosphoribosylpyrophosphate (PRPP) synthetase has been cloned from a library of Bacillus subtilis DNA by complementation of an Escherichia coli prs mutation. Flanking DNA sequences were pruned away by restriction endonuclease and exonuclease BAL 31 digestions, resulting...... in a DNA fragment of approx. 1.8 kb complementing the E. coli prs mutation. Minicell experiments revealed that this DNA fragment coded for a polypeptide, shown to be the PRPP synthetase subunit, with an Mr of approx. 40,000. B. subtilis strains harbouring the prs gene in a multicopy plasmid contained up...... to nine-fold increased PRPP synthetase activity. The prs gene was cloned in an integration vector and the resulting hybrid plasmid inserted into the B. subtilis chromosome by homologous recombination. The integration site was mapped by transduction and the gene order established as purA-guaA-prs-cysA....

  14. Broilers fed dietary vitamins harbor higher diversity of cecal bacteria and higher ratio of Clostridium, Faecalibacterium, and Lactobacillus than broilers with no dietary vitamins revealed by 16S rRNA gene clone libraries.

    Science.gov (United States)

    Luo, Yu-heng; Peng, Huan-wei; Wright, André-Denis G; Bai, Shi-ping; Ding, Xue-mei; Zeng, Qiu-feng; Li, Hua; Zheng, Ping; Su, Zhuo-wei; Cui, Ren-yong; Zhang, Ke-ying

    2013-09-01

    Research on the interaction between dietary vitamins and intestinal bacteria is poorly understood. To investigate the effect of dietary vitamins on the cecal bacterial communities, 2 bacterial 16S rRNA gene clone libraries were constructed from pooled PCR products obtained from the cecal digesta of 28-d broilers fed diets with vitamins (V) at the NRC level or with no vitamins (NV). The results showed that BW gain and average feed intake of V broilers was significantly higher (P vitamins can increase the ratio of facultative pathogenic bacteria and decrease the diversity of bacteria in the cecum of broilers. Our results provide new leads for further investigations on the interaction between dietary vitamin additives and the gut health of broilers.

  15. Academic Cloning.

    Science.gov (United States)

    Sikula, John P.; Sikula, Andrew F.

    1980-01-01

    The authors define "cloning" as an integral feature of all educational systems, citing teaching practices which reward students for closely reproducing the teacher's thoughts and/or behaviors and administrative systems which tend to promote like-minded subordinates. They insist, however, that "academic cloning" is not a totally…

  16. An Engineered Virus Library as a Resource for the Spectrum-wide Exploration of Virus and Vector Diversity

    Directory of Open Access Journals (Sweden)

    Wenli Zhang

    2017-05-01

    Full Text Available Adenoviruses (Ads are large human-pathogenic double-stranded DNA (dsDNA viruses presenting an enormous natural diversity associated with a broad variety of diseases. However, only a small fraction of adenoviruses has been explored in basic virology and biomedical research, highlighting the need to develop robust and adaptable methodologies and resources. We developed a method for high-throughput direct cloning and engineering of adenoviral genomes from different sources utilizing advanced linear-linear homologous recombination (LLHR and linear-circular homologous recombination (LCHR. We describe 34 cloned adenoviral genomes originating from clinical samples, which were characterized by next-generation sequencing (NGS. We anticipate that this recombineering strategy and the engineered adenovirus library will provide an approach to study basic and clinical virology. High-throughput screening (HTS of the reporter-tagged Ad library in a panel of cell lines including osteosarcoma disease-specific cell lines revealed alternative virus types with enhanced transduction and oncolysis efficiencies. This highlights the usefulness of this resource.

  17. Rabies virus cross-reactive murine T cell clones: analysis of helper and delayed-type hypersensitivity function.

    NARCIS (Netherlands)

    H. Bunschoten; B. Dietzschold; I.J.Th.M. Claassen (Ivo); R. Klapmuts; F. UytdeHaag; A.D.M.E. Osterhaus (Albert)

    1990-01-01

    textabstractThree T cell clones derived from rabies virus-immunized BALB/c mice were analysed for specificity and function. The clones proved to be broadly cross-reactive by responding to different rabies virus isolates (PM, ERA, CVS, HEP) and other representatives of the genus Lyssavirus, like the

  18. Literature promotion in Public Libraries

    DEFF Research Database (Denmark)

    Kann-Christensen, Nanna; Balling, Gitte

    2011-01-01

    This article discusses a model that can be used in order to analyse notions on literature promotion in public libraries. The model integrates different issues which interact with how literature promotion is understood and thought of in public libraries. Besides cultural policy we regard the logics...... of new public management (NPM) and professional logics in the field of public libraries. Cultural policy along with the identification of underlying logics present among politicians, government officials, managers and librarians/promoters of literature, play an important part in creating an understanding...... of literature promotion in Danish libraries. Thus the basic premise for the development of the model is that cultural policy (Policy) has an important influence on notions on literature promotion and other activities in public libraries, but that cultural policy must be seen in some kind of interaction...

  19. Construction of a 7-fold BAC library and cytogenetic mapping of 10 genes in the giant panda (Ailuropoda melanoleuca)

    OpenAIRE

    Liu, Wei; Zhao, Yonghui; Liu, Zhaoliang; Zhang, Ying; Lian, Zhengxing; Li, Ning

    2006-01-01

    Abstract Background The giant panda, one of the most primitive carnivores, is an endangered animal. Although it has been the subject of many interesting studies during recent years, little is known about its genome. In order to promote research on this genome, a bacterial artificial chromosome (BAC) library of the giant panda was constructed in this study. Results This BAC library contains 198,844 clones with an average insert size of 108 kb, which represents approximately seven equivalents o...

  20. A multi-substrate approach for functional metagenomics-based screening for (hemi)cellulases in two wheat straw-degrading microbial consortia unveils novel thermoalkaliphilic enzymes.

    Science.gov (United States)

    Maruthamuthu, Mukil; Jiménez, Diego Javier; Stevens, Patricia; van Elsas, Jan Dirk

    2016-01-28

    Functional metagenomics is a promising strategy for the exploration of the biocatalytic potential of microbiomes in order to uncover novel enzymes for industrial processes (e.g. biorefining or bleaching pulp). Most current methodologies used to screen for enzymes involved in plant biomass degradation are based on the use of single substrates. Moreover, highly diverse environments are used as metagenomic sources. However, such methods suffer from low hit rates of positive clones and hence the discovery of novel enzymatic activities from metagenomes has been hampered. Here, we constructed fosmid libraries from two wheat straw-degrading microbial consortia, denoted RWS (bred on untreated wheat straw) and TWS (bred on heat-treated wheat straw). Approximately 22,000 clones from each library were screened for (hemi)cellulose-degrading enzymes using a multi-chromogenic substrate approach. The screens yielded 71 positive clones for both libraries, giving hit rates of 1:440 and 1:1,047 for RWS and TWS, respectively. Seven clones (NT2-2, T5-5, NT18-17, T4-1, 10BT, NT18-21 and T17-2) were selected for sequence analyses. Their inserts revealed the presence of 18 genes encoding enzymes belonging to twelve different glycosyl hydrolase families (GH2, GH3, GH13, GH17, GH20, GH27, GH32, GH39, GH53, GH58, GH65 and GH109). These encompassed several carbohydrate-active gene clusters traceable mainly to Klebsiella related species. Detailed functional analyses showed that clone NT2-2 (containing a beta-galactosidase of ~116 kDa) had highest enzymatic activity at 55 °C and pH 9.0. Additionally, clone T5-5 (containing a beta-xylosidase of ~86 kDa) showed > 90% of enzymatic activity at 55 °C and pH 10.0. This study employed a high-throughput method for rapid screening of fosmid metagenomic libraries for (hemi)cellulose-degrading enzymes. The approach, consisting of screens on multi-substrates coupled to further analyses, revealed high hit rates, as compared with recent other studies. Two

  1. A single-copy galK promoter cloning vector suitable for cloning strong promoters

    DEFF Research Database (Denmark)

    Dandanell, Gert; Court, Donald L.; Hammer, Karin

    1986-01-01

    We report the construction of lambda galK promoter cloning vectors for cloning and characterization of strong promoters. This phage, which contains a unique HindIII cloning site, was applied to the cloning and analysis of transcription initiations of the regulatory region of the deo-operon of...

  2. Biological activity evaluation of cloned and expressed caprine growth hormone from local Pakistani goat breed beetal

    International Nuclear Information System (INIS)

    Butt, H.I.; Shahzad, M.I.; Bashir, Q.

    2011-01-01

    Growth hormone cDNA of local goat breed-beetal was amplified by RT PCR and gene including leader sequence was cloned in pTZR57 cloning vector. The cGH-pTZR57 clone was confirmed by restriction digestion and sequence analyses before finally sub-cloning the gene in pND- a mammalian expression vector. The clones were again confirmed by restriction digestion and PCR analyses. Highly purified, supercoiled cGH-pND construct was used to transfect Vero cell lines for expression studies. The in vitro expression of cGH was checked by dot-ELISA technique. After confirming its in vitro cell line based expression, the construct was injected to 4 weeks old balb/c mice intramuscularly. Two animals were euthanized per week till four weeks to monitor the in vivo biological activity by evaluating the tibia epiphyseal width and body weight gain assays. Significant increase in tibia epiphyseal width and gain in body weight was observed from vaccinated animals. The study supports the concept that DNA based therapeutics are an efficient and cost effective method for gene delivery and in vivo transgene expressions. (author)

  3. Growth hormone and prolactin in Andrias davidianus: cDNA cloning, tissue distribution and phylogenetic analysis.

    Science.gov (United States)

    Yang, Liping; Meng, Zining; Liu, Yun; Zhang, Yong; Liu, Xiaochun; Lu, Danqi; Huang, Junhai; Lin, Haoran

    2010-01-15

    The Chinese giant salamander (Andrias davidianus) is one of the largest and 'living fossil' species of amphibian. To obtain genetic information for this species, the cDNAs encoding growth hormone (adGH) and prolactin (adPRL) were cloned from a pituitary cDNA library. The isolated adGH cDNA consisted of 864 bp and encoded a propeptide of 215 amino acids, while the cDNA of adPRL was 1106 bp in length and encoded a putative peptide of 229 amino acids. Expression of the GH and PRL mRNA was only detected in the pituitary. Phylogenetic analyses were performed based on the isolated pituitary hormone sequences using maximum parsimony and neighbor-joining algorithms. The clustering results are similar to that based on the morphological characteristics or the rRNA genes, which indicate that the two orders (Anura and Caudata) of amphibian were monophyletic, and that A. davidianus was diverged early in the Caudate clade. These results indicated that both the GH and PRL sequence might be useful to study the phylogenies of relatively moderate evolved groups.

  4. Critical experiments analyses by using 70 energy group library based on ENDF/B-VI

    Energy Technology Data Exchange (ETDEWEB)

    Tahara, Yoshihisa; Matsumoto, Hideki [Mitsubishi Heavy Industries Ltd., Yokohama (Japan). Nuclear Energy Systems Engineering Center; Huria, H.C.; Ouisloumen, M.

    1998-03-01

    The newly developed 70-group library has been validated by comparing kinf from a continuous energy Monte-Carlo code MCNP and two dimensional spectrum calculation code PHOENIX-CP. The code employs Discrete Angular Flux Method based on Collision Probability. The library has been also validated against a large number of critical experiments and numerical benchmarks for assemblies with MOX and Gd fuels. (author)

  5. Characterization of a pollen-specific cDNA clone from Nicotiana tabacum expressed during microgametogenesis and germination.

    Science.gov (United States)

    Weterings, K; Reijnen, W; van Aarssen, R; Kortstee, A; Spijkers, J; van Herpen, M; Schrauwen, J; Wullems, G

    1992-04-01

    This report describes the isolation and characterization of a cDNA clone representing a gene specifically expressed in pollen. A cDNA library was constructed against mRNA from mature pollen of Nicotiana tabacum. It was screened differentially against cDNA from mRNA of leaf and of pollen. One clone, NTPc303, was further characterized. On northern blot this clone hybridizes to a transcript 2100 nucleotides in length. NTPc303 is abundant in pollen. Expression of the corresponding gene is restricted to pollen, because no other generative or vegetative tissue contains transcripts hybridizing to NTPc303. Expression of NTP303 is evolutionarily conserved: homologous transcripts are present in pollen from various plant species. The first NTP303 transcripts are detectable on northern blot at the early bi-nucleate stage and accumulate until the pollen has reached maturity. During germination and pollen tube growth in vitro new NTP303 transcripts appear. This transcription has been proved by northern blots as well as by pulse labelling experiments. Nucleotide sequence analysis revealed that NTPc303 has an open reading frame coding for a predicted protein of 62 kDa. This protein shares homology to ascorbate oxidase and other members of the blue copper oxidase family. A possible function for this clone during pollen germination is discussed.

  6. Changes in the gut microbiota of cloned and non-cloned control pigs during development of obesity: gut microbiota during development of obesity in cloned pigs.

    Science.gov (United States)

    Pedersen, Rebecca; Andersen, Anders Daniel; Mølbak, Lars; Stagsted, Jan; Boye, Mette

    2013-02-07

    Obesity induced by a high-caloric diet has previously been associated with changes in the gut microbiota in mice and in humans. In this study, pigs were cloned to minimize genetic and biological variation among the animals with the aim of developing a controlled metabolomic model suitable for a diet-intervention study. Cloning of pigs may be an attractive way to reduce genetic influences when investigating the effect of diet and obesity on different physiological sites. The aim of this study was to assess and compare the changes in the composition of the gut microbiota of cloned vs. non-cloned pigs during development of obesity by a high-fat/high-caloric diet. Furthermore, we investigated the association between diet-induced obesity and the relative abundance of the phyla Firmicutes and Bacteroidetes in the fecal-microbiota. The fecal microbiota from obese cloned (n = 5) and non-cloned control pigs (n= 6) was investigated biweekly over a period of 136 days, by terminal restriction fragment length polymorphism (T-RFLP) and quantitative real time PCR (qPCR). A positive correlation was observed between body-weight at endpoint and percent body-fat in cloned (r=0.9, Pmicrobiota between the cloned pigs or between cloned and non-cloned control pigs. Body-weight correlated positively with the relative abundance of Firmicutes in both cloned (r=0.37; Pgut microbiota in neither the obese nor the lean state. Diet-induced obesity was associated with an increase in the relative abundance of Firmicutes over time. Our results suggest that cloned pigs are not a more suitable animal model for gut microbiota-obesity related studies than non-cloned pigs. This study is the first to evaluate if cloned pigs provide a better animal model than conventional pigs in diet-intervention, obesity and gut microbiota research.

  7. Cloning and cDNA sequence of the dihydrolipoamide dehydrogenase component of human α-ketoacid dehydrogenase complexes

    International Nuclear Information System (INIS)

    Pons, G.; Raefsky-Estrin, C.; Carothers, D.J.; Pepin, R.A.; Javed, A.A.; Jesse, B.W.; Ganapathi, M.K.; Samols, D.; Patel, M.S.

    1988-01-01

    cDNA clones comprising the entire coding region for human dihydrolipoamide dehydrogenase have been isolated from a human liver cDNA library. The cDNA sequence of the largest clone consisted of 2082 base pairs and contained a 1527-base open reading frame that encodes a precursor dihydrolipoamide dehydrogenase of 509 amino acid residues. The first 35-amino acid residues of the open reading frame probably correspond to a typical mitochondrial import leader sequence. The predicted amino acid sequence of the mature protein, starting at the residue number 36 of the open reading frame, is almost identical (>98% homology) with the known partial amino acid sequence of the pig heart dihydrolipoamide dehydrogenase. The cDNA clone also contains a 3' untranslated region of 505 bases with an unusual polyadenylylation signal (TATAAA) and a short poly(A) track. By blot-hybridization analysis with the cDNA as probe, two mRNAs, 2.2 and 2.4 kilobases in size, have been detected in human tissues and fibroblasts, whereas only one mRNA (2.4 kilobases) was detected in rat tissues

  8. Expression cloning of camelid nanobodies specific for Xenopus embryonic antigens.

    Directory of Open Access Journals (Sweden)

    Keiji Itoh

    Full Text Available Developmental biology relies heavily on the use of conventional antibodies, but their production and maintenance involves significant effort. Here we use an expression cloning approach to identify variable regions of llama single domain antibodies (known as nanobodies, which recognize specific embryonic antigens. A nanobody cDNA library was prepared from lymphocytes of a llama immunized with Xenopus embryo lysates. Pools of bacterially expressed cDNAs were sib-selected for the ability to produce specific staining patterns in gastrula embryos. Three different nanobodies were isolated: NbP1 and NbP3 stained yolk granules, while the reactivity of NbP7 was predominantly restricted to the cytoplasm and the cortex. The isolated nanobodies recognized specific protein bands in immunoblot analysis. A reverse proteomic approach identified NbP1 target antigen as EP45/Seryp, a serine protease inhibitor. Given the unique stability of nanobodies and the ease of their expression in diverse systems, we propose that nanobody cDNA libraries represent a promising resource for molecular markers for developmental biology.

  9. Generation of human Fab antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences.

    Science.gov (United States)

    Andris-Widhopf, Jennifer; Steinberger, Peter; Fuller, Roberta; Rader, Christoph; Barbas, Carlos F

    2011-09-01

    The development of therapeutic antibodies for use in the treatment of human diseases has long been a goal for many researchers in the antibody field. One way to obtain these antibodies is through phage-display libraries constructed from human lymphocytes. This protocol describes the construction of human Fab (fragment antigen binding) antibody libraries. In this method, the individual rearranged heavy- and light-chain variable regions are amplified separately and are linked through a series of overlap polymerase chain reaction (PCR) steps to give the final Fab products that are used for cloning.

  10. Quantum cloning machines and the applications

    Energy Technology Data Exchange (ETDEWEB)

    Fan, Heng, E-mail: hfan@iphy.ac.cn [Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190 (China); Collaborative Innovation Center of Quantum Matter, Beijing 100190 (China); Wang, Yi-Nan; Jing, Li [School of Physics, Peking University, Beijing 100871 (China); Yue, Jie-Dong [Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190 (China); Shi, Han-Duo; Zhang, Yong-Liang; Mu, Liang-Zhu [School of Physics, Peking University, Beijing 100871 (China)

    2014-11-20

    No-cloning theorem is fundamental for quantum mechanics and for quantum information science that states an unknown quantum state cannot be cloned perfectly. However, we can try to clone a quantum state approximately with the optimal fidelity, or instead, we can try to clone it perfectly with the largest probability. Thus various quantum cloning machines have been designed for different quantum information protocols. Specifically, quantum cloning machines can be designed to analyze the security of quantum key distribution protocols such as BB84 protocol, six-state protocol, B92 protocol and their generalizations. Some well-known quantum cloning machines include universal quantum cloning machine, phase-covariant cloning machine, the asymmetric quantum cloning machine and the probabilistic quantum cloning machine. In the past years, much progress has been made in studying quantum cloning machines and their applications and implementations, both theoretically and experimentally. In this review, we will give a complete description of those important developments about quantum cloning and some related topics. On the other hand, this review is self-consistent, and in particular, we try to present some detailed formulations so that further study can be taken based on those results.

  11. Quantum cloning machines and the applications

    International Nuclear Information System (INIS)

    Fan, Heng; Wang, Yi-Nan; Jing, Li; Yue, Jie-Dong; Shi, Han-Duo; Zhang, Yong-Liang; Mu, Liang-Zhu

    2014-01-01

    No-cloning theorem is fundamental for quantum mechanics and for quantum information science that states an unknown quantum state cannot be cloned perfectly. However, we can try to clone a quantum state approximately with the optimal fidelity, or instead, we can try to clone it perfectly with the largest probability. Thus various quantum cloning machines have been designed for different quantum information protocols. Specifically, quantum cloning machines can be designed to analyze the security of quantum key distribution protocols such as BB84 protocol, six-state protocol, B92 protocol and their generalizations. Some well-known quantum cloning machines include universal quantum cloning machine, phase-covariant cloning machine, the asymmetric quantum cloning machine and the probabilistic quantum cloning machine. In the past years, much progress has been made in studying quantum cloning machines and their applications and implementations, both theoretically and experimentally. In this review, we will give a complete description of those important developments about quantum cloning and some related topics. On the other hand, this review is self-consistent, and in particular, we try to present some detailed formulations so that further study can be taken based on those results

  12. Cloning and characterization of profilin (Pru du 4), a cross-reactive almond (Prunus dulcis) allergen.

    Science.gov (United States)

    Tawde, Pallavi; Venkatesh, Yeldur P; Wang, Fang; Teuber, Suzanne S; Sathe, Shridhar K; Roux, Kenneth H

    2006-10-01

    The identity of allergenic almond proteins is incomplete. Our objective was to characterize patient IgE reactivity to a recombinant and corresponding native almond allergen. An almond cDNA library was screened with sera from patients with allergy for IgE binding proteins. Two reactive clones were sequenced, and 1 was expressed. The expressed recombinant allergen and its native counterpart (purified from unprocessed almond flour) were assayed by 1-dimensional and 2-dimensional gel electrophoresis, dot blot, and ELISA, and screened for cross-reactivity with grass profilin. The 2 selected clones encoded profilin (designated Pru du 4) sequences that differed by 2 silent mutations. By dot-blot analyses, 6 of 18 patient sera (33%) reacted with the recombinant Pru du 4 protein, and 8 of 18 (44%) reacted with the native form. ELISA results were similar. Almond and ryegrass profilins were mutually inhibitable. Two-dimensional immunoblotting revealed the presence of more than 1 native almond profilin isoform. The strength of reactivity of some patients' serum IgE differed markedly between assays and between native and recombinant profilins. Almond nut profilin is an IgE-binding food protein that is cross-reactive with grass pollen profilin and is susceptible to denaturation, resulting in variable reactivity between assay types and between patients. Serum IgE of nearly half of the tested patients with almond allergy reacts with almond nut profilin. Because most patients also had pollinosis, the well-known cross-reactivity between pollen and food profilins could account for this pattern of reactivity.

  13. Identification of a new geographically widespread multiresistant Acinetobacter baumannii clone from European hospitals.

    Science.gov (United States)

    van Dessel, Helke; Dijkshoorn, Lenie; van der Reijden, Tanny; Bakker, Nancy; Paauw, Armand; van den Broek, Peterhans; Verhoef, Jan; Brisse, Sylvain

    2004-03-01

    The aim of the study was to investigate the genetic diversity of Acinetobacter baumannii clinical strains that had previously been allocated to three major groups based on automated ribotyping. Forty-seven isolates from European hospitals and one isolate from a South African hospital, geographically representative of the three ribogroups (ribogroups 1, 2 and 3 with 10, 23 and 15 isolates, respectively), were analysed using the highly discriminatory fingerprinting methods AFLP and pulsed-field gel electrophoresis (PFGE). Based on AFLP data, the isolates clustered into three main groups, each corresponding to one ribogroup. Inclusion of reference strains of the previously described clones I and II, responsible for outbreaks in northwestern European hospitals, showed that ribogroups 1 and 2 correspond to clones I and II, respectively, whereas ribogroup 3 apparently represents a new clone. This clone III was found in France, The Netherlands, Italy and Spain. Clones I and II were not limited to northwestern European countries, as they were also recovered from Spain, South Africa, Poland and Italy (clone I) and from Spain, Portugal, South Africa, France, Greece and Turkey (clone II). Combined AFLP and PFGE data showed intraclonal diversity and led to the distinction of 23 different genotypes. Three genotypes, two of them belonging to clone II and one to clone III, were found in different hospitals and may correspond to subsets of isolates with a more recent clonal relationship, which emphasizes the epidemic potential of these organisms.

  14. Journal of Genetics | Indian Academy of Sciences

    Indian Academy of Sciences (India)

    The female spinach genome was taken as blocker and cDNA library specifically expressed in Y chromosome was constructed. Moreover, expressed sequence tag (EST) sequences in cDNA library were cloned, sequenced and bioinformatics was analysed. There were 63 valid EST sequences obtained in this study.

  15. Molecular cloning of cDNA for rat brain metallothionein-2 and regulation of its gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Saijoh, Kiyofumi; Sumino, Kimiaki [Department of Public Health, Kobe University School of Medicine (Japan); Kuno, Takayoshi; Shuntoh, Hisato; Tanaka, Chikako [Department of Pharmacology, Kobe University of Medicine (Japan)

    1989-01-01

    A rat brain metallothionein-II (MT-II) complementary DNA (cDNA) clone was isolated from a cDNA plasmid library, which was prepared from non-treated rat brain mRNA, by a colony screening procedure using /sup 32/P-labeled synthetic oligonucleotide probes. It is deduced that the clone encodes for a protein of 61 amino acids comprising 20 cysteines, which is highly homologous to MT-IIs in other species. Northern blot analysis demonstrated major mRNA species in the brain, liver and kidneys (approximately 350 b in size), which is induced in response to dexamethasone, zinc, cadmium and mercury but not to methyl mercury. These findings confirm that MT-II genes are expressed and regulated both by steroid and heavy metals in the brain as well as in peripheral organs. (author).

  16. Molecular cloning of cDNA for rat brain metallothionein-2 and regulation of its gene expression

    International Nuclear Information System (INIS)

    Saijoh, Kiyofumi; Sumino, Kimiaki; Kuno, Takayoshi; Shuntoh, Hisato; Tanaka, Chikako

    1989-01-01

    A rat brain metallothionein-II (MT-II) complementary DNA (cDNA) clone was isolated from a cDNA plasmid library, which was prepared from non-treated rat brain mRNA, by a colony screening procedure using 32 P-labeled synthetic oligonucleotide probes. It is deduced that the clone encodes for a protein of 61 amino acids comprising 20 cysteines, which is highly homologous to MT-IIs in other species. Northern blot analysis demonstrated major mRNA species in the brain, liver and kidneys (approximately 350 b in size), which is induced in response to dexamethasone, zinc, cadmium and mercury but not to methyl mercury. These findings confirm that MT-II genes are expressed and regulated both by steroid and heavy metals in the brain as well as in peripheral organs. (author)

  17. [Nuclear transfer and therapeutic cloning].

    Science.gov (United States)

    Xu, Xiao-Ming; Lei, An-Min; Hua, Jin-Lian; Dou, Zhong-Ying

    2005-03-01

    Nuclear transfer and therapeutic cloning have widespread and attractive prospects in animal agriculture and biomedical applications. We reviewed that the quality of oocytes and nuclear reprogramming of somatic donor cells were the main reasons of the common abnormalities in cloned animals and the low efficiency of cloning and showed the problems and outlets in therapeutic cloning, such as some basic problems in nuclear transfer affected clinical applications of therapeutic cloning. Study on isolation and culture of nuclear transfer embryonic stem (ntES) cells and specific differentiation of ntES cells into important functional cells should be emphasized and could enhance the efficiency. Adult stem cells could help to cure some great diseases, but could not replace therapeutic cloning. Ethics also impeded the development of therapeutic cloning. It is necessary to improve many techniques and reinforce the research of some basic theories, then somatic nuclear transfer and therapeutic cloning may apply to agriculture reproduction and benefit to human life better.

  18. Software problems in library automation in India

    OpenAIRE

    Francis, A. T.

    1998-01-01

    Important software problems faced by the library professionals in India are analysed and points out various compatibility and suitability issues in the selection of a library software. The paper also hints that these problems has affected the progress of computerisation of libraries. Upto date and detailed information on softwares available in India can prevent several issues that may arise in the course of computerisation. An agency/mechanism to continuously evaluate the softwares may be ...

  19. Ruminal metagenomic libraries as a source of relevant hemicellulolytic enzymes for biofuel production.

    Science.gov (United States)

    Duque, Estrella; Daddaoua, Abdelali; Cordero, Baldo F; Udaondo, Zulema; Molina-Santiago, Carlos; Roca, Amalia; Solano, Jennifer; Molina-Alcaide, Eduarda; Segura, Ana; Ramos, Juan-Luis

    2018-04-17

    The success of second-generation (2G) ethanol technology relies on the efficient transformation of hemicellulose into monosaccharides and, particularly, on the full conversion of xylans into xylose for over 18% of fermentable sugars. We sought new hemicellulases using ruminal liquid, after enrichment of microbes with industrial lignocellulosic substrates and preparation of metagenomic libraries. Among 150 000 fosmid clones tested, we identified 22 clones with endoxylanase activity and 125 with β-xylosidase activity. These positive clones were sequenced en masse, and the analysis revealed open reading frames with a low degree of similarity with known glycosyl hydrolases families. Among them, we searched for enzymes that were thermostable (activity at > 50°C) and that operate at high rate at pH around 5. Upon a wide series of assays, the clones exhibiting the highest endoxylanase and β-xylosidase activities were identified. The fosmids were sequenced, and the corresponding genes cloned, expressed and proteins purified. We found that the activity of the most active β-xylosidase was at least 10-fold higher than that in commercial enzymatic fungal cocktails. Endoxylanase activity was in the range of fungal enzymes. Fungal enzymatic cocktails supplemented with the bacterial hemicellulases exhibited enhanced release of sugars from pretreated sugar cane straw, a relevant agricultural residue. © 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  20. Probabilistic cloning of equidistant states

    International Nuclear Information System (INIS)

    Jimenez, O.; Roa, Luis; Delgado, A.

    2010-01-01

    We study the probabilistic cloning of equidistant states. These states are such that the inner product between them is a complex constant or its conjugate. Thereby, it is possible to study their cloning in a simple way. In particular, we are interested in the behavior of the cloning probability as a function of the phase of the overlap among the involved states. We show that for certain families of equidistant states Duan and Guo's cloning machine leads to cloning probabilities lower than the optimal unambiguous discrimination probability of equidistant states. We propose an alternative cloning machine whose cloning probability is higher than or equal to the optimal unambiguous discrimination probability for any family of equidistant states. Both machines achieve the same probability for equidistant states whose inner product is a positive real number.

  1. GALARIO: a GPU accelerated library for analysing radio interferometer observations

    Science.gov (United States)

    Tazzari, Marco; Beaujean, Frederik; Testi, Leonardo

    2018-06-01

    We present GALARIO, a computational library that exploits the power of modern graphical processing units (GPUs) to accelerate the analysis of observations from radio interferometers like Atacama Large Millimeter and sub-millimeter Array or the Karl G. Jansky Very Large Array. GALARIO speeds up the computation of synthetic visibilities from a generic 2D model image or a radial brightness profile (for axisymmetric sources). On a GPU, GALARIO is 150 faster than standard PYTHON and 10 times faster than serial C++ code on a CPU. Highly modular, easy to use, and to adopt in existing code, GALARIO comes as two compiled libraries, one for Nvidia GPUs and one for multicore CPUs, where both have the same functions with identical interfaces. GALARIO comes with PYTHON bindings but can also be directly used in C or C++. The versatility and the speed of GALARIO open new analysis pathways that otherwise would be prohibitively time consuming, e.g. fitting high-resolution observations of large number of objects, or entire spectral cubes of molecular gas emission. It is a general tool that can be applied to any field that uses radio interferometer observations. The source code is available online at http://github.com/mtazzari/galario under the open source GNU Lesser General Public License v3.

  2. Quantum cloning and signaling

    International Nuclear Information System (INIS)

    Simon, C.; Weihs, G.; Zeilinger, A.

    1999-01-01

    We discuss the close connections between cloning of quantum states and superluminal signaling. We present an optimal universal cloning machine based on stimulated emission recently proposed by the authors. As an instructive example, we show how a scheme for superluminal communication based on this cloning machine fails. (Authors)

  3. Cloning and expression of the receptor for human urokinase plasminogen activator, a central molecule in cell surface, plasmin dependent proteolysis

    DEFF Research Database (Denmark)

    Roldan, A.L.; Cubellis, M.V.; Masucci, M.T.

    1990-01-01

    , and therefore the capacity of cells to migrate and invade neighboring tissues. We have isolated a 1.4 kb cDNA clone coding for the entire human uPAR. An oligonucleotide synthesized on the basis of the N-terminal sequence of the purified protein was used to screen a cDNA library made from SV40 transformed human......, a size very close to that of the cloned cDNA. Expression of the uPAR cDNA in mouse cells confirms that the clone is complete and expresses a functional uPA binding protein, located on the cell surface and with properties similar to the human uPAR. Caseinolytic plaque assay, immunofluorescence analysis......The surface receptor for urokinase plasminogen activator (uPAR) has been recognized in recent years as a key molecule in regulating plasminogen mediated extracellular proteolysis. Surface plasminogen activation controls the connections between cells, basement membrane and extracellular matrix...

  4. Recombination-assisted megaprimer (RAM) cloning

    Science.gov (United States)

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

    2014-01-01

    No molecular cloning technique is considered universally reliable, and many suffer from being too laborious, complex, or expensive. Restriction-free cloning is among the simplest, most rapid, and cost-effective methods, but does not always provide successful results. We modified this method to enhance its success rate through the use of exponential amplification coupled with homologous end-joining. This new method, recombination-assisted megaprimer (RAM) cloning, significantly extends the application of restriction-free cloning, and allows efficient vector construction with much less time and effort when restriction-free cloning fails to provide satisfactory results. The following modifications were made to the protocol:•Limited number of PCR cycles for both megaprimer synthesis and the cloning reaction to reduce error propagation.•Elimination of phosphorylation and ligation steps previously reported for cloning methods that used exponential amplification, through the inclusion of a reverse primer in the cloning reaction with a 20 base pair region of homology to the forward primer.•The inclusion of 1 M betaine to enhance both reaction specificity and yield. PMID:26150930

  5. Generation and analyses of human synthetic antibody libraries and their application for protein microarrays

    DEFF Research Database (Denmark)

    Säll, Anna; Walle, Maria; Wingren, Christer

    2016-01-01

    in a high-throughput manner. To address this we designed and constructed two human synthetic antibody fragment (scFv) libraries denoted HelL-11 and HelL-13. By the use of phage display technology, in total 466 unique scFv antibodies specific for 114 different antigens were generated. The specificities......Antibody-based proteomics offers distinct advantages in the analysis of complex samples for discovery and validation of biomarkers associated with disease. However, its large-scale implementation requires tools and technologies that allow development of suitable antibody or antibody fragments...... for diagnostics and therapeutics. In addition, this work provides a great example on how a synthetic approach can be used to optimize library designs. By having precise control of the diversity introduced into the antigen-binding sites, synthetic libraries offer increased understanding of how different diversity...

  6. Molecular cloning and complete nucleotide sequence of a human ventricular myosin light chain 1

    Energy Technology Data Exchange (ETDEWEB)

    Hoffmann, E; Shi, Q W; Floroff, M; Mickle, D A.G.; Wu, T W; Olley, P M; Jackowski, G

    1988-03-25

    Human ventricular plasmid library was constructed. The library was screened with the oligonucleotide probe (17-mer) corresponding to a conserve region of myosin light chain 1 near the carboxy terminal. Full length cDNA recombinant plasmid containing 1100 bp insert was isolated. RNA blot hybridization with this insert detected a message of approximately 1500 bp corresponding to the size of VLCl and mRNA. Complete nucleotide sequence of the coding region was determined in M13 subclones using dideoxy chain termination method. With the isolation of this clone (pCD HLVCl), the publication of the complete nucleotide sequence of HVLCl and the predicted secondary structure of this protein will aid in understanding of the biochemistry of myosin and its function in contraction, the evolution of myosin light genes and the genetic, developmental and physiological regulation of myosin genes.

  7. Human cloning and child welfare.

    Science.gov (United States)

    Burley, J; Harris, J

    1999-01-01

    In this paper we discuss an objection to human cloning which appeals to the welfare of the child. This objection varies according to the sort of harm it is expected the clone will suffer. The three formulations of it that we will consider are: 1. Clones will be harmed by the fearful or prejudicial attitudes people may have about or towards them (H1); 2. Clones will be harmed by the demands and expectations of parents or genotype donors (H2); 3. Clones will be harmed by their own awareness of their origins, for example the knowledge that the genetic donor is a stranger (H3). We will show why these three versions of the child welfare objection do not necessarily supply compelling reasons to ban human reproductive cloning. The claim that we will develop and defend in the course of our discussion is that even if it is the case that a cloned child will suffer harms of the type H1-H3, it is none the less permissible to conceive by cloning so long as these cloning-induced welfare deficits are not such as to blight the existence of the resultant child, whoever this may be. PMID:10226914

  8. Cloning of a novel gene from Penicillium oxalicum I1 which in Escherichia coli enhances the secretion of acetic acid

    Directory of Open Access Journals (Sweden)

    Xue, L.

    2018-01-01

    Full Text Available Description of the subject. Organic acids play an important role in the conversion of insoluble ions into soluble ones in soil. Heterologous overexpression of a single gene in a cell is the optimal strategy for increasing the secretion of organic acids solubilizing phosphate. Objectives. In this study, we constructed a primary cDNA library of Penicillium oxalicum I1, and screened clones that can solubilize P in tricalcium phosphate (TCP medium. We aimed to obtain the gene expressed in Escherichia coli, which can enhance organic acid secretion. Method. A primary cDNA library of Penicillium oxalicum I1 was constructed using the switching mechanism at the 5'-end of RNA transcription. The organic acid secretion ability of E. coli DH5α™ with overexpressed P. oxalicum I1gene was tested in TCP medium where glucose is the sole carbon source. Afterwards, pyruvic acid, citric acid, α-ketoglutaric acid, succinic acid, fumaric acid, and malic acid were used as sole carbon source substitutes for glucose in the TCP medium to test the organic acid secretion ability of the transformed E. coli DH5α™. Results. A total of 106 clones showed halos in TCP medium, among which clone I-2 displayed clear halo. The full-length cDNA of clone I-2 was 1,151 bp, with a complete open reading frame of 702 bp, which encoded a hypothetical protein of 233 amino acids. The cDNA sequence showed 68% identity and 73% query cover with other fungal gene sequences of which the function remains unknown. Escherichia coli containing the cloned gene secreted up to 567 mg·l-1 acetic acid within 48 h. The use of glucose, pyruvic acid, α-ketoglutaric acid, and malic acid improved the acetic acid secretion of the E. coli DH5α™ clone I-2. By contrast, the use of citric acid, succinic acid, and fumaric acid did not improve the acetic acid secretion of clone I-2 compared to a control E. coli DH5α™ strain bearing only the cloning vector without any insert. Conclusions. We obtained a

  9. Successful pod infections by Moniliophthora roreri result in differential Theobroma cacao gene expression depending on the clone's level of tolerance.

    Science.gov (United States)

    Ali, Shahin S; Melnick, Rachel L; Crozier, Jayne; Phillips-Mora, Wilberth; Strem, Mary D; Shao, Jonathan; Zhang, Dapeng; Sicher, Richard; Meinhardt, Lyndel; Bailey, Bryan A

    2014-09-01

    An understanding of the tolerance mechanisms of Theobroma cacao used against Moniliophthora roreri, the causal agent of frosty pod rot, is important for the generation of stable disease-tolerant clones. A comparative view was obtained of transcript populations of infected pods from two susceptible and two tolerant clones using RNA sequence (RNA-Seq) analysis. A total of 3009 transcripts showed differential expression among clones. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis of differentially expressed genes indicated shifts in 152 different metabolic pathways between the tolerant and susceptible clones. Real-time quantitative reverse transcription polymerase chain reaction (real-time qRT-PCR) analyses of 36 genes verified the differential expression. Regression analysis validated a uniform progression in gene expression in association with infection levels and fungal loads in the susceptible clones. Expression patterns observed in the susceptible clones diverged in tolerant clones, with many genes showing higher expression at a low level of infection and fungal load. Principal coordinate analyses of real-time qRT-PCR data separated the gene expression patterns between susceptible and tolerant clones for pods showing malformation. Although some genes were constitutively differentially expressed between clones, most results suggested that defence responses were induced at low fungal load in the tolerant clones. Several elicitor-responsive genes were highly expressed in tolerant clones, suggesting rapid recognition of the pathogen and induction of defence genes. Expression patterns suggested that the jasmonic acid-ethylene- and/or salicylic acid-mediated defence pathways were activated in the tolerant clones, being enhanced by reduced brassinosteroid (BR) biosynthesis and catabolic inactivation of both BR and abscisic acids. Finally, several genes associated with hypersensitive response-like cell death were also induced in tolerant clones. © 2014

  10. Social behavior and kin discrimination in a mixed group of cloned and non cloned heifers (Bos taurus).

    Science.gov (United States)

    Coulon, M; Baudoin, C; Abdi, H; Heyman, Y; Deputte, B L

    2010-12-01

    For more than ten years, reproductive biotechnologies using somatic cell nuclear transfer have made possible the production of cloned animals in various domestic and laboratory species. The influence of the cloning process on offspring characteristics has been studied in various developmental aspects, however, it has not yet been documented in detail for behavioral traits. Behavioral studies of cloned animals have failed to show clear inter-individual differences associated with the cloning process. Preliminary results showed that clones favor each other's company. Preferential social interactions were observed among cloned heifers from the same donor in a mixed herd that also included cloned heifers and control heifers produced by artificial insemination (AI). These results suggest behavioral differences between cloned and non-cloned animals and similarities between clones from the same donor. The aim of the present study was to replicate and to extend these previous results and to study behavioral and cognitive mechanisms of this preferential grouping. We studied a group composed of five cloned heifers derived from the same donor cow, two cloned heifers derived from another donor cow, and AI heifers. Cloned heifers from the same donor were more spatially associated and interacted more between themselves than with heifers derived from another donor or with the AI individuals. This pattern indicates a possible kin discrimination in clones. To study this process, we performed an experiment (using an instrumental conditioning procedure with food reward) of visual discrimination between images of heads of familiar heifers, either related to the subjects or not. The results showed that all subjects (AI and cloned heifers) discriminated between images of familiar cloned heifers produced from the same donor and images of familiar unrelated heifers. Cattle discriminated well between images and used morphological similarities characteristic of cloned related heifers. Our

  11. Construction of a DNA library representing 15q11-13 by subtraction of two flow sorted marker chromosome-specific libraries

    Energy Technology Data Exchange (ETDEWEB)

    Blennow, E.; Werelius, B.; Nordenskjoeld, M. [Karolinska Hospital, Stockholm (Sweden)] [and others

    1994-09-01

    Constitutional extra {open_quotes}marker chromosomes{close_quotes} are found in {approx}0.5/1000 of newborns. Of these, 50% are inverted duplications of the pericentromeric region of chromosome 15, including two variants; (1) inv dup(15)(pter{yields}q11:q11{yields}pter) and (2) inv dup(15) (pter{yields}q12-13::q12-13{yields}pter). Variant (1) is found in phenotypically normal individuals, whereas variant (2) will produce a typical clinical picture including mental retardation, autism, hyperactivity and discrete dysmorphic features. Fluorescence in situ hybridization (FISH) using single copy probes from the Prader-Willi region confirms these observations as well as chromosome painting using a flow-sorted marker chromosome-specific library from a variant (1) marker, hybridized to the chromosomes of a patient with a variant (2) marker chromosome. Followingly, a flow-sorted biotinylated variant (1) library was subtracted from a non-labeled variant (2) library using magnetic beads and subsequent amplification by degenerate oligonucleotide-primed PCR (DOP-PCR). The successful result was demonstrated by using the amplified material for chromosome painting on chromosome slides from variant (1) and variant (2) patients. We have constructed a library from 15q11-13. This region contains genes producing a specific abnormal phenotype when found in a tri- or tetrasomic state. The region also contains the genes responsible for the Prader-Willi and Angelman syndromes when the paternal/maternal copy is missing, respectively. It is therefore a region where parental imprinting plays an important role. The isolated library may be used to isolate single copy clones which will allow further investigations of this region.

  12. Optimally cloned binary coherent states

    Science.gov (United States)

    Müller, C. R.; Leuchs, G.; Marquardt, Ch.; Andersen, U. L.

    2017-10-01

    Binary coherent state alphabets can be represented in a two-dimensional Hilbert space. We capitalize this formal connection between the otherwise distinct domains of qubits and continuous variable states to map binary phase-shift keyed coherent states onto the Bloch sphere and to derive their quantum-optimal clones. We analyze the Wigner function and the cumulants of the clones, and we conclude that optimal cloning of binary coherent states requires a nonlinearity above second order. We propose several practical and near-optimal cloning schemes and compare their cloning fidelity to the optimal cloner.

  13. Human placental Na+, K+-ATPase α subunit: cDNA cloning, tissue expression, DNA polymorphism, and chromosomal localization

    International Nuclear Information System (INIS)

    Chehab, F.F.; Kan, Y.W.; Law, M.L.; Hartz, J.; Kao, F.T.; Blostein, R.

    1987-01-01

    A 2.2-kilobase clone comprising a major portion of the coding sequence of the Na + , K + -ATPase α subunit was cloned from human placenta and its sequence was identical to that encoding the α subunit of human kidney and HeLa cells. Transfer blot analysis of the mRNA products of the Na + , K + -ATPase gene from various human tissues and cell lines revealed only one band (≅ 4.7 kilobases) under low and high stringency washing conditions. The levels of expression in the tissues were intestine > placenta > liver > pancreas, and in the cell lines the levels were human erythroleukemia > butyrate-induced colon > colon > brain > HeLa cells. mRNA was undetectable in reticulocytes, consistent with the authors failure to detect positive clones in a size-selected ( > 2 kilobases) λgt11 reticulocyte cDNA library. DNA analysis revealed by a polymorphic EcoRI band and chromosome localization by flow sorting and in situ hybridization showed that the α subunit is on the short is on the short arm (band p11-p13) of chromosome 1

  14. Construction and characterization of a forward subtracted library of blue mussels Mytilus edulis for the identification of gene transcription signatures and biomarkers of styrene exposure

    International Nuclear Information System (INIS)

    Diaz de Cerio, O.; Hands, E.; Humble, J.; Cajaraville, M.P.; Craft, J.A.; Cancio, I.

    2013-01-01

    Highlights: ► Transcription responses in blue mussels exposed to styrene have been studied by SSH. ► 1440 Clones were obtained from which 287 were sequenced. ► Immune system, cancer-related and ribosomal genes identified as upregulated genes. ► Chitin and β-1-3-glucan metabolism genes highly represented in subtracted library. -- Abstract: Transcriptional profiling can elucidate adaptive/toxicity pathways participating in achieving homeostasis or leading to pathogenesis in marine biota exposed to chemical substances. With the aim of analyzing transcriptional responses in the mussel Mytilus edulis exposed to the corrosive and putatively carcinogenic hydrocarbon styrene (3–5 ppm, 3 days), a forward subtracted (SSH) cDNA library was produced. Female mussels were selected and digestive gland mRNA was isolated. A library with 1440 clones was produced and a total of 287 clones were sequenced, 53% being identified through BlastN analysis against Mytibase and DeepSeaVent databases. Those genes included GO terms such as ‘response to drugs’, ‘immune defense’ and ‘cell proliferation’. Furthermore, sequences related to chitin and beta-1-3-glucan metabolism were also up-regulated by styrene. Many of the obtained sequences could not be annotated constituting new mussel sequences. In conclusion, this SSH study reveals novel sequences useful to generate molecular biomarkers of styrene exposure in mussels

  15. Three concepts of cloning in human beings.

    Science.gov (United States)

    Cui, Ke-Hui

    2005-07-01

    Human cloning, organ cloning and tissue cloning are various types of cloning that occur at different levels with different methodologies. According to three standards of terminology for an embryo (fertilization through germ cells, development in the uterus and having the potential to produce a human life), tissue cloning and type I organ cloning will not produce an embryo. In contrast, human cloning and type II organ cloning will produce an embryo. Thus, only non-germinal tissue cloning and type I organ cloning are beyond the ethical question and will not change human beings as a species. Using cloned tissues to make new tissues or organs is promising for the future of medicine.

  16. Molecular cloning of the α subunit of human and guinea pig leukocyte adhesion glycoprotein Mo1: Chromosomal localization and homology to the α subunits of integrins

    International Nuclear Information System (INIS)

    Arnaout, M.A.; Remold-O'Donnell, E.; Pierce, M.W.; Harris, P.; Tenen, D.G.

    1988-01-01

    The cell surface-glycoprotein Mo1 is a member of the family of leukocyte cell adhesion molecules (Leu-CAMs) that includes lymphocyte function-associated antigen 1 (LFA-1) and p150,95. Each Leu-CAM is a heterodimer with a distinct α subunit noncovalently associated with a common β subunit. The authors describe the isolation and analysis of two partial cDNA clones encoding the α subunit of the Leu-CAM Mo1 in humans and guinea pigs. A monoclonal antibody directed against an epitope in the carboxyl-terminal portion of the guinea pig α chain was used for immunoscreening a λgt11 expression library. The sequence of a 378-base-pair insert from one immunoreactive clone revealed a single continuous open reading frame encoding 126 amino acids including a 26-amino acid tryptic peptide isolated from the purified guinea pig α subunit. A cDNA clone of identical size was isolated from a human monocyte/lymphocyte cDNA library by using the guinea pig clone as a probe. The human clone also encoded a 126-amino acid peptide including the sequence of an additional tryptic peptide present in purified human Mo1α chain. Southern analysis of DNA from hamster-human hybrids localized the human Mo1α chain to chromosome 16, which has been shown to contain the gene for the α chain of lymphocyte function-associated antigen 1. These data suggest that the α subunits of Leu-CAMs evolved by gene duplication from a common ancestral gene and strengthen the hypothesis that the α subunits of these heterodimeric cell adhesion molecules on myeloid and lymphoid cells, platelets, and fibroblasts are evolutionary related

  17. Local cloning of CAT states

    International Nuclear Information System (INIS)

    Rahaman, Ramij

    2011-01-01

    In this Letter we analyze the (im)possibility of the exact cloning of orthogonal three-qubit CAT states under local operation and classical communication (LOCC) with the help of a restricted entangled state. We also classify the three-qubit CAT states that can (not) be cloned under LOCC restrictions and extend the results to the n-qubit case. -- Highlights: → We analyze the (im)possibility of exact cloning of orthogonal CAT states under LOCC. → We also classify the set of CAT states that can(not) be cloned by LOCC. → No set of orthogonal CAT states can be cloned by LOCC with help of similar CAT state. → Any two orthogonal n-qubit GHZ-states can be cloned by LOCC with help of a GHZ state.

  18. Construction and partial sequencing of a subtractive library in Calcutta 4 (Musa AA in early stage of infection with Mycosphaerella fijiensis Morelet

    Directory of Open Access Journals (Sweden)

    Milady Mendoza-Rodríguez

    2006-10-01

    Full Text Available The study of genes involved in plant defense response against pathogen attack, is one of most important steps leading to the elucidation of disease resistance molecular mechanisms. The generation of subtracted deoxyribonucleic acid libraries (cDNA, by means of suppression subtractive hybridization technique (SSH, has been used for this purpose. A subtractive hybridization was made between a cDNA population obtained from ‘Calcutta 4’ inoculated leaves with M. fijiensis (CCIBP-Pf83 and a mixture of cDNA from ‘Calcutta 4’ non inoculated leaves and mycelium. Leaves samples were taken at 6, 10 and 12 days after inoculation. The subtracted library was obtained by cloning and transformation of subtracted products and as a result, 600 recombinants clones were obtained. Sequence analysis of sixty nine clones, revealed redundancy of the expressed sequence tags and most of them showed no homology with reported sequences at databases and only 13 % had a high homology with metalothioneins. The results constitute a step in advance in the molecular study of Musa-Mycosphaerella fijiensis interaction. Key words: Banana-Mycosphaerella fijiensis interaction, BlackSigatoka, Musa spp., suppression subtractive hybridization

  19. cDNA cloning and expression analyses of phytoene synthase 1, phytoene desaturase and ζ-carotene desaturase genes from Solanum lycopersicum KKU-T34003

    Directory of Open Access Journals (Sweden)

    Krittaya Supathaweewat

    2013-10-01

    Full Text Available We report on the cloning of Psy1, Pds and Zds cDNAs encoding the enzymes responsible for lycopene biosynthesis,namely phytoene synthase 1 (PSY1, phytoene desaturase (PDS and -carotene desaturase (ZDS, respectively, from high-lycopene tomato cultivar, Solanum lycopersicum KKU-T34003. DNA sequence analyses showed that the complete openreading frames of Psy1, Pds and Zds cDNAs were 1,239, 1,752 and 1,767 base pairs in length and encoded proteins of 412,583 and 588 amino acids, respectively. Phylogenetic and the conserved domain analyses suggest that PSY1, PDS and ZDSfrom S. lycopersicum KKU-T34003 potentially have similar structures and biological functions to the corresponding proteinsfrom other plants. Gene expression studies showed that Psy1 was expressed only in the petal and the breaker fruit, whereasthe expressions of Pds and Zds were observed in the petal, the breaker fruit and the leaf. The highest expression level for allgenes was detected in the breaker-stage fruit, suggesting that carotenoid accumulation was developmentally regulated inthe chromoplast-containing tissues.

  20. Combustion quality of poplar and willow clones grown as SRC at four sites in Brandenburg, Germany

    DEFF Research Database (Denmark)

    Liu, Na; Ugilt Larsen, Søren; Jørgensen, Uffe

    2017-01-01

    The fuel quality was assessed for nine poplar clones (AF2, Androscoggin, Max1, Max3, Max4, Monviso, Muhle-Larsen, NE42, Weser6) and one willow clone (Inger) grown as short rotation coppice (SRC) on four sites in the Brandenburg area in Germany. Fuel quality was analysed in 3-year old shoots in te...

  1. Effects of donor fibroblast cell type and transferred cloned embryo number on the efficiency of pig cloning.

    Science.gov (United States)

    Li, Zicong; Shi, Junsong; Liu, Dewu; Zhou, Rong; Zeng, Haiyu; Zhou, Xiu; Mai, Ranbiao; Zeng, Shaofen; Luo, Lvhua; Yu, Wanxian; Zhang, Shouquan; Wu, Zhenfang

    2013-02-01

    Currently, cloning efficiency in pigs is very low. Donor cell type and number of cloned embryos transferred to an individual surrogate are two major factors that affect the successful rate of somatic cell nuclear transfer (SCNT) in pigs. This study aimed to compare the influence of different donor fibroblast cell types and different transferred embryo numbers on recipients' pregnancy rate and delivery rate, the average number of total clones born, clones born alive and clones born healthy per litter, and the birth rate of healthy clones (=total number of healthy cloned piglets born /total number of transferred cloned embryos). Three types of donor fibroblasts were tested in large-scale production of cloned pigs, including fetal fibroblasts (FFBs) from four genetically similar Western swine breeds of Pietrain (P), Duroc (D), Landrace (L), and Yorkshire (Y), which are referred to as P,D,LY-FFBs, adult fibroblasts (AFBs) from the same four breeds, which are designated P,D,L,Y-AFBs, and AFBs from a Chinese pig breed of Laiwu (LW), which is referred to as LW-AFBs. Within each donor fibroblast cell type group, five transferred cloned embryo number groups were tested. In each embryo number group, 150-199, 200-249, 250-299, 300-349, or 350-450 cloned embryos were transferred to each individual recipient sow. For the entire experiment, 92,005 cloned embryos were generated from nearly 115,000 matured oocytes and transferred to 328 recipients; in total, 488 cloned piglets were produced. The results showed that the mean clones born healthy per litter resulted from transfer of embryos cloned from LW-AFBs (2.53 ± 0.34) was similar with that associated with P,D,L,Y-FFBs (2.72 ± 0.29), but was significantly higher than that resulted from P,D,L,Y-AFBs (1.47 ± 0.18). Use of LW-AFBs as donor cells for SCNT resulted in a significantly higher pregnancy rate (72.00% vs. 59.30% and 48.11%) and delivery rate (60.00% vs. 45.93% and 35.85%) for cloned embryo recipients, and a

  2. Selection of cholera toxin specific IgNAR single-domain antibodies from a naïve shark library.

    Science.gov (United States)

    Liu, Jinny L; Anderson, George P; Delehanty, James B; Baumann, Richard; Hayhurst, Andrew; Goldman, Ellen R

    2007-03-01

    Shark immunoglobulin new antigen receptor (IgNAR, also referred to as NAR) variable domains (Vs) are single-domain antibody (sdAb) fragments containing only two hypervariable loop structures forming 3D topologies for a wide range of antigen recognition and binding. Their small size ( approximately 12kDa) and high solubility, thermostability and binding specificity make IgNARs an exceptional alternative source of engineered antibodies for sensor applications. Here, two new shark NAR V display libraries containing >10(7) unique clones from non-immunized (naïve) adult spiny dogfish (Squalus acanthias) and smooth dogfish (Mustelus canis) sharks were constructed. The most conserved consensus sequences derived from random clone sequence were compared with published nurse shark (Ginglymostoma cirratum) sequences. Cholera toxin (CT) was chosen for panning one of the naïve display libraries due to its severe pathogenicity and commercial availability. Three very similar CT binders were selected and purified soluble monomeric anti-CT sdAbs were characterized using Luminex(100) and traditional ELISA assays. These novel anti-CT sdAbs selected from our newly constructed shark NAR V sdAb library specifically bound to soluble antigen, without cross reacting with other irrelevant antigens. They also showed superior heat stability, exhibiting slow loss of activity over the course of one hour at high temperature (95 degrees C), while conventional antibodies lost all activity in the first 5-10min. The successful isolation of target specific sdAbs from one of our non-biased NAR libraries, demonstrate their ability to provide binders against an unacquainted antigen of interest.

  3. Cloning and Identification of Recombinant Argonaute-Bound Small RNAs Using Next-Generation Sequencing.

    Science.gov (United States)

    Gangras, Pooja; Dayeh, Daniel M; Mabin, Justin W; Nakanishi, Kotaro; Singh, Guramrit

    2018-01-01

    Argonaute proteins (AGOs) are loaded with small RNAs as guides to recognize target mRNAs. Since the target specificity heavily depends on the base complementarity between two strands, it is important to identify small guide and long target RNAs bound to AGOs. For this purpose, next-generation sequencing (NGS) technologies have extended our appreciation truly to the nucleotide level. However, the identification of RNAs via NGS from scarce RNA samples remains a challenge. Further, most commercial and published methods are compatible with either small RNAs or long RNAs, but are not equally applicable to both. Therefore, a single method that yields quantitative, bias-free NGS libraries to identify small and long RNAs from low levels of input will be of wide interest. Here, we introduce such a procedure that is based on several modifications of two published protocols and allows robust, sensitive, and reproducible cloning and sequencing of small amounts of RNAs of variable lengths. The method was applied to the identification of small RNAs bound to a purified eukaryotic AGO. Following ligation of a DNA adapter to RNA 3'-end, the key feature of this method is to use the adapter for priming reverse transcription (RT) wherein biotinylated deoxyribonucleotides specifically incorporated into the extended complementary DNA. Such RT products are enriched on streptavidin beads, circularized while immobilized on beads and directly used for PCR amplification. We provide a stepwise guide to generate RNA-Seq libraries, their purification, quantification, validation, and preparation for next-generation sequencing. We also provide basic steps in post-NGS data analyses using Galaxy, an open-source, web-based platform.

  4. Generation and analyses of human synthetic antibody libraries and their application for protein microarrays.

    Science.gov (United States)

    Säll, Anna; Walle, Maria; Wingren, Christer; Müller, Susanne; Nyman, Tomas; Vala, Andrea; Ohlin, Mats; Borrebaeck, Carl A K; Persson, Helena

    2016-10-01

    Antibody-based proteomics offers distinct advantages in the analysis of complex samples for discovery and validation of biomarkers associated with disease. However, its large-scale implementation requires tools and technologies that allow development of suitable antibody or antibody fragments in a high-throughput manner. To address this we designed and constructed two human synthetic antibody fragment (scFv) libraries denoted HelL-11 and HelL-13. By the use of phage display technology, in total 466 unique scFv antibodies specific for 114 different antigens were generated. The specificities of these antibodies were analyzed in a variety of immunochemical assays and a subset was further evaluated for functionality in protein microarray applications. This high-throughput approach demonstrates the ability to rapidly generate a wealth of reagents not only for proteome research, but potentially also for diagnostics and therapeutics. In addition, this work provides a great example on how a synthetic approach can be used to optimize library designs. By having precise control of the diversity introduced into the antigen-binding sites, synthetic libraries offer increased understanding of how different diversity contributes to antibody binding reactivity and stability, thereby providing the key to future library optimization. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  5. Tetranectin, a plasminogen kringle 4-binding protein. Cloning and gene expression pattern in human colon cancer

    DEFF Research Database (Denmark)

    Wewer, U M; Albrechtsen, R

    1992-01-01

    BACKGROUND: Tetranectin is a recently discovered protein that binds to kringle 4 region of plasminogen (Clemmensen I, Petersen LC, Kluft C. Eur J Biochem 1986; 156:327. EXPERIMENTAL DESIGN: The mRNA encoding human tetranectin was cloned by using degenerate primers in a reverse transcriptase...... reaction followed by polymerase chain reaction amplification. The resulting polymerase chain reaction product was examined by DNA sequencing and subsequently used as probe for screening a human placental cDNA library. A full length cDNA clone (TET-1) was isolated, characterized, and used for Northern blot...... prominent in the lungs and spleen. No hybridization signal was detected in three carcinoma cell lines examined in parallel. Northern blot analysis of poly A+ RNA isolated from solid tumors revealed a tetranectin specific mRNA band. In situ hybridizations on tissue sections of colon carcinomas and normal...

  6. Lessons learned from cloning dogs.

    Science.gov (United States)

    Kim, M J; Oh, H J; Kim, G A; Park, J E; Park, E J; Jang, G; Ra, J C; Kang, S K; Lee, B C

    2012-08-01

    The aim of this article is to review dog cloning research and to suggest its applications based on a discussion about the normality of cloned dogs. Somatic cell nuclear transfer was successfully used for production of viable cloned puppies despite limited understanding of in vitro dog embryo production. Cloned dogs have similar growth characteristics to those born from natural fertilization, with no evidence of serious adverse effects. The offspring of cloned dogs also have similar growth performance and health to those of naturally bred puppies. Therefore, cloning in domestic dogs can be applied as an assisted reproductive technique to conserve endangered species, to treat sterile canids or aged dogs, to improve reproductive performance of valuable individuals and to generate disease model animals. © 2012 Blackwell Verlag GmbH.

  7. Consumers' attitudes toward consumption of cloned beef. The impact of exposure to technological information about animal cloning.

    Science.gov (United States)

    Aizaki, Hideo; Sawada, Manabu; Sato, Kazuo

    2011-10-01

    Novel food technologies, such as cloning, have been introduced into the meat production sector; however, their use is not widely supported by many consumers. This study was designed to assess whether Japanese consumers' attitudes toward consumption of cloned beef (specifically, beef derived from bovine embryo and somatic cell-cloned cattle) would change after they were provided with technological information on animal cloning through a web-based survey. The results revealed that most respondents did not discriminate between their attitudes toward the consumption of the two types of cloned beef, and that most respondents did not change their attitudes toward cloned beef after receiving the technological information. The respondents' individual characteristics, including their knowledge about the food safety of cloned beef and their basic knowledge about animal cloning, influenced the likelihood of a change in their attitudes after they received the information. In conclusion, some consumers might become less uncomfortable about the consumption of cloned beef by the straightforward provision of technological information about animal cloning; however, most consumers are likely to maintain their attitudes. Copyright © 2011 Elsevier Ltd. All rights reserved.

  8. Isolation and characterization of full-length cDNA clones coding for cholinesterase from fetal human tissues

    International Nuclear Information System (INIS)

    Prody, C.A.; Zevin-Sonkin, D.; Gnatt, A.; Goldberg, O.; Soreq, H.

    1987-01-01

    To study the primary structure and regulation of human cholinesterases, oligodeoxynucleotide probes were prepared according to a consensus peptide sequence present in the active site of both human serum pseudocholinesterase and Torpedo electric organ true acetylcholinesterase. Using these probes, the authors isolated several cDNA clones from λgt10 libraries of fetal brain and liver origins. These include 2.4-kilobase cDNA clones that code for a polypeptide containing a putative signal peptide and the N-terminal, active site, and C-terminal peptides of human BtChoEase, suggesting that they code either for BtChoEase itself or for a very similar but distinct fetal form of cholinesterase. In RNA blots of poly(A) + RNA from the cholinesterase-producing fetal brain and liver, these cDNAs hybridized with a single 2.5-kilobase band. Blot hybridization to human genomic DNA revealed that these fetal BtChoEase cDNA clones hybridize with DNA fragments of the total length of 17.5 kilobases, and signal intensities indicated that these sequences are not present in many copies. Both the cDNA-encoded protein and its nucleotide sequence display striking homology to parallel sequences published for Torpedo AcChoEase. These finding demonstrate extensive homologies between the fetal BtChoEase encoded by these clones and other cholinesterases of various forms and species

  9. Development of in vitro transposon assisted signal sequence trapping and its use in screening Bacillus halodurans C125 and Sulfolobus solfataricus P2 gene libraries

    DEFF Research Database (Denmark)

    Becker, F.; Schnorr, K.; Wilting, R.

    2004-01-01

    To identify genes encoding extracytosolic proteins, a minitransposon, TnSig, containing a signal-less beta-lactamase ('bla) as reporter gene, was constructed and used for in vitro transposition of genomic libraries made in Escherichia coli. The 'bla gene was cloned into a bacteriophage MU...... minitransposon enabling translational fusions between 'bla and target genes. Fusion of TnSig in the correct reading frame to a protein carrying transmembrane domains or signal peptides resulted in ampicillin resistance of the corresponding clone. Prokaryotic gene libraries from the alkaliphilic bacterium...... Bacillus halodurans C125 and the hyperthermophilic archaeon Sulfolobus solfataricus P2 were tagged with TnSig. The genomic sequences, which are publicly available (EMBL BA000004 and EMBL AE006641), were used for rapid open reading frame (ORF) identification and prediction of protein localisation...

  10. A Bac Library and Paired-PCR Approach to Mapping and Completing the Genome Sequence of Sulfolobus Solfataricus P2

    DEFF Research Database (Denmark)

    She, Qunxin; Confalonieri, F.; Zivanovic, Y.

    2000-01-01

    The original strategy used in the Sulfolobus solfatnricus genome project was to sequence non overlapping, or minimally overlapping, cosmid or lambda inserts without constructing a physical map. However, after only about two thirds of the genome sequence was completed, this approach became counter......-productive because there was a high sequence bias in the cosmid and lambda libraries. Therefore, a new approach was devised for linking the sequenced regions which may be generally applicable. BAC libraries were constructed and terminal sequences of the clones were determined and used for both end mapping and PCR...

  11. A binary plasmid system for shuffling combinatorial antibody libraries.

    Science.gov (United States)

    Collet, T A; Roben, P; O'Kennedy, R; Barbas, C F; Burton, D R; Lerner, R A

    1992-11-01

    We have used a binary system of replicon-compatible plasmids to test the potential for promiscuous recombination of heavy and light chains within sets of human Fab fragments isolated from combinatorial antibody libraries. Antibody molecules showed a surprising amount of promiscuity in that a particular heavy chain could recombine with multiple light chains with retention of binding to a protein antigen. The degree to which a given heavy chain productively paired with any light chain to bind antigen varied from 43% to 100% and depended strongly on the heavy-chain sequence. Such productive crosses resulted in a set of Fab fragments of similar apparent binding constants, which seemed to differ mainly in the amount of active Fab fragment produced in the bacterial cell. The dominance of the heavy chain in the antibody-antigen interaction was further explored in a set of directed crosses, in which heavy and light chains derived from antigen-specific clones were crossed with nonrelated heavy and light chains. In these crosses, an Fab fragment retained antigen binding only if it contained a heavy chain from an antigen-specific clone. In no case did the light chain confer detectable affinity when paired with indifferent heavy chains. The surprising promiscuity of heavy chains has ramifications for the evaluation of the diversity of combinatorial libraries made against protein antigens and should allow the combination of one such promiscuous heavy chain with an engineered light chain to form an Fab fragment carrying synthetic cofactors to assist in antibody catalysis.

  12. Production of a Human Antibody Library in the Phage-Display Vector pSEX81.

    Science.gov (United States)

    Welschof, M; Little, M; Dörsam, H

    1998-01-01

    Human monoclonal antibodies (MAbs) are more suitable than MAbs of animal origin for clinical applications because of lower hypersensitivity reactions, less formation of circulating immune complexes and lower anti-immunoglobulin responses The classical production of human MAbs via the hybridoma technique or Epstein-Barr virus (EBV) transformation is limited by the instability of cell lines, low antibody production, and the problems of imununizing humans with certain antigens (1,2). A promising alternative 1s the production of human recombinant antibodies (3). Recombinant DNA technology has made it possible to clone human antibody genes in vectors and to generate antibody expression libraries (4-7). One approach has been to amplify and recombine the IgG repertoire of an "immunized" donor. This has been used to isolate several antibodies related to diseases (8,9). In order to obtain more universal antibody libraries the naive IgM repertoire of several "unimmunized" donors were pooled (10,12). The complexity of the combinatorial libraries has been further increased by creating the so-called "semisynthetic" antibody libraries (22-14).

  13. Molecular cloning, sequence analysis and phylogeny of first caudata g-type lysozyme in axolotl (Ambystoma mexicanum).

    Science.gov (United States)

    Yu, Haining; Gao, Jiuxiang; Lu, Yiling; Guang, Huijuan; Cai, Shasha; Zhang, Songyan; Wang, Yipeng

    2013-11-01

    Lysozymes are key proteins that play important roles in innate immune defense in many animal phyla by breaking down the bacterial cell-walls. In this study, we report the molecular cloning, sequence analysis and phylogeny of the first caudate amphibian g-lysozyme: a full-length spleen cDNA library from axolotl (Ambystoma mexicanum). A goose-type (g-lysozyme) EST was identified and the full-length cDNA was obtained using RACE-PCR. The axolotl g-lysozyme sequence represents an open reading frame for a putative signal peptide and the mature protein composed of 184 amino acids. The calculated molecular mass and the theoretical isoelectric point (pl) of this mature protein are 21523.0 Da and 4.37, respectively. Expression of g-lysozyme mRNA is predominantly found in skin, with lower levels in spleen, liver, muscle, and lung. Phylogenetic analysis revealed that caudate amphibian g-lysozyme had distinct evolution pattern for being juxtaposed with not only anura amphibian, but also with the fish, bird and mammal. Although the first complete cDNA sequence for caudate amphibian g-lysozyme is reported in the present study, clones encoding axolotl's other functional immune molecules in the full-length cDNA library will have to be further sequenced to gain insight into the fundamental aspects of antibacterial mechanisms in caudate.

  14. Molecular cloning and functional characterization of borneol dehydrogenase from the glandular trichomes of Lavandula x intermedia.

    Science.gov (United States)

    Sarker, Lukman S; Galata, Mariana; Demissie, Zerihun A; Mahmoud, Soheil S

    2012-12-15

    Several varieties of Lavandula x intermedia (lavandins) are cultivated for their essential oils (EOs) for use in cosmetic, hygiene and personal care products. These EOs are mainly constituted of monoterpenes including camphor, which contributes an off odor reducing the olfactory appeal of the oil. We have recently constructed a cDNA library from the glandular trichomes (the sites of EO synthesis) of L. x intermedia plants. Here, we describe the cloning of a borneol dehydrogenase cDNA (LiBDH) from this library. The 780 bp open reading frame of the cDNA encoded a 259 amino acid short chain alcohol dehydrogenase with a predicted molecular mass of ca. 27.5 kDa. The recombinant LiBDH was expressed in Escherichia coli, purified by Ni-NTA agarose affinity chromatography, and functionally characterized in vitro. The bacterially produced enzyme specifically converted borneol to camphor as the only product with K(m) and k(cat) values of 53 μM and 4.0 × 10(-4) s(-1), respectively. The LiBDH transcripts were specifically expressed in glandular trichomes of mature flowers indicating that like other Lavandula monoterpene synthases the expression of this gene is regulated in a tissue-specific manner. The cloning of LiBDH has far reaching implications in improving the quality of Lavandula EOs through metabolic engineering. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Selection of new clones of linalool chemotype from genetic recombination in Lippia alba

    Directory of Open Access Journals (Sweden)

    Elcio Rodrigo Rufino

    2012-01-01

    Full Text Available The aromatic and medicinal species Lippia alba is vigorous and rugged native to the South America (Atlantic Rainforest. Because it is an allogamous and self-incompatible species, natural populations have high morphological and chemical variability. This work had as objective to conduct a preliminary screening to identify new promising clones from a novel (recombinant base population of Lippia alba with regard to its agronomic and phytochemical traits, using the linalool oil or chemotype as model. The two superior linalool clones, obtained by collection, were used as controls. Traits evaluated included: dry mass of leaves (DML, oil yield percentage (EOY%, oil production per plant (OP, and linalool percentage (LN%. Forty linalool chemotype clones were evaluated in three experiments, in a random block design with four replicates and four cuttings (clones per plot. Besides means comparisons, multivariate analysis was used in order to aid in the preliminary selection of clones. There were positive correlations from moderate to strong for DML vs. EOY%, OP vs. EOY% and DML vs. OP. Linalool clones superior or similar to both controls were identified for the DML, EOY%, OP, and LN% traits (univariate analyses, aimed at further validating experimentation. Five distinct groups were defined in the cluster analysis (UPGMA, each containing subgroups as well.

  16. Sustracted library obtained from mutant sugarcane variety B 4362 resistant to rust

    Directory of Open Access Journals (Sweden)

    María I. Oloriz

    2002-07-01

    Full Text Available The hypersensitive response is one of the most powerful mechanisms for which the plants resist pathogen attack. Mutations carried out previously on the variety B4362, of sugarcane, originated five mutants that express this mechanism towards the attack of rust (Puccinia melanocephala Syd.. By means of a subtractive hybridization among the cDNA obtained starting from the resistant clone inoculated with rust and a pool of cDNA of the susceptible variety (B4362 inoculated and of the resistant clone not inoculated, it was possible to reduce the number of genes expressed during the infection with the fungus. A subtractive library was carried out where we hope that most of the genes are involved in the hypersensitive response that present these mutants towards the infection of the pathogen. Key words: Subtractive hybridization, hypersensitive response, Puccinia melanocephala Syd.

  17. FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method

    Directory of Open Access Journals (Sweden)

    Lu Jia

    2011-10-01

    Full Text Available Abstract Background Although a variety of methods and expensive kits are available, molecular cloning can be a time-consuming and frustrating process. Results Here we report a highly simplified, reliable, and efficient PCR-based cloning technique to insert any DNA fragment into a plasmid vector or into a gene (cDNA in a vector at any desired position. With this method, the vector and insert are PCR amplified separately, with only 18 cycles, using a high fidelity DNA polymerase. The amplified insert has the ends with ~16-base overlapping with the ends of the amplified vector. After DpnI digestion of the mixture of the amplified vector and insert to eliminate the DNA templates used in PCR reactions, the mixture is directly transformed into competent E. coli cells to obtain the desired clones. This technique has many advantages over other cloning methods. First, it does not need gel purification of the PCR product or linearized vector. Second, there is no need of any cloning kit or specialized enzyme for cloning. Furthermore, with reduced number of PCR cycles, it also decreases the chance of random mutations. In addition, this method is highly effective and reproducible. Finally, since this cloning method is also sequence independent, we demonstrated that it can be used for chimera construction, insertion, and multiple mutations spanning a stretch of DNA up to 120 bp. Conclusion Our FastCloning technique provides a very simple, effective, reliable, and versatile tool for molecular cloning, chimera construction, insertion of any DNA sequences of interest and also for multiple mutations in a short stretch of a cDNA.

  18. Cyclodipeptides from metagenomic library of a japanese marine sponge

    Energy Technology Data Exchange (ETDEWEB)

    He, Rui; Wang, Bochu; Zhub, Liancai, E-mail: wangbc2000@126.com [Bioengineering College, Chongqing University, Chongqing, (China); Wang, Manyuan [School of Traditional Chinese Medicine, Capital University of Medical Sciences, Beijing (China); Wakimoto, Toshiyuki; Abe, Ikuro, E-mail: abei@mol.f.u-tokyo.ac.jp [Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo (Japan)

    2013-12-01

    Culture-independent metagenomics is an attractive and promising approach to explore unique bioactive small molecules from marine sponges harboring uncultured symbiotic microbes. Therefore, we conducted functional screening of the metagenomic library constructed from the Japanese marine sponge Discodermia calyx. Bioassay-guided fractionation of plate culture extract of antibacterial clone pDC113 afforded eleven cyclodipeptides: Cyclo(l-Thr-l-Leu) (1), Cyclo(l-Val-d-Pro) (2), Cyclo(l-Ile-d-Pro) (3), Cyclo(l-Leu-l-Pro) (4), Cyclo(l-Val-l-Leu) (5), Cyclo(l-Leu-l-Ile) (6), Cyclo(l-Leu-l-Leu) (7), Cyclo(l-Phe-l-Tyr) (8), Cyclo(l-Trp-l-Pro) (9), Cyclo(l-Val-l-Trp) (10) and Cyclo(l-Ile-l-Trp) (11). To the best of our knowledge, these are first cyclodepeptides isolated from metagenomic library. Sequence analysis suggested that isolated cyclodipeptides were not synthesized by nonribosomal peptide synthetases and there was no significant indication of cyclodipeptide synthetases. (author)

  19. Cyclodipeptides from metagenomic library of a japanese marine sponge

    International Nuclear Information System (INIS)

    He, Rui; Wang, Bochu; Zhub, Liancai; Wang, Manyuan; Wakimoto, Toshiyuki; Abe, Ikuro

    2013-01-01

    Culture-independent metagenomics is an attractive and promising approach to explore unique bioactive small molecules from marine sponges harboring uncultured symbiotic microbes. Therefore, we conducted functional screening of the metagenomic library constructed from the Japanese marine sponge Discodermia calyx. Bioassay-guided fractionation of plate culture extract of antibacterial clone pDC113 afforded eleven cyclodipeptides: Cyclo(l-Thr-l-Leu) (1), Cyclo(l-Val-d-Pro) (2), Cyclo(l-Ile-d-Pro) (3), Cyclo(l-Leu-l-Pro) (4), Cyclo(l-Val-l-Leu) (5), Cyclo(l-Leu-l-Ile) (6), Cyclo(l-Leu-l-Leu) (7), Cyclo(l-Phe-l-Tyr) (8), Cyclo(l-Trp-l-Pro) (9), Cyclo(l-Val-l-Trp) (10) and Cyclo(l-Ile-l-Trp) (11). To the best of our knowledge, these are first cyclodepeptides isolated from metagenomic library. Sequence analysis suggested that isolated cyclodipeptides were not synthesized by nonribosomal peptide synthetases and there was no significant indication of cyclodipeptide synthetases. (author)

  20. The LAW Library -- A multigroup cross-section library for use in radioactive waste analysis calculations

    International Nuclear Information System (INIS)

    Greene, N.M.; Arwood, J.W.; Wright, R.Q.; Parks, C.V.

    1994-08-01

    The 238-group LAW Library is a new multigroup neutron cross-section library based on ENDF/B-V data, with five sets of data taken from ENDF/B-VI ( 14 N 7 , 15 N 7 , 16 O 8 , 154Eu 63 , and 155 Eu 63 ). These five nuclides are included because the new evaluations are thought to be superior to those in Version 5. The LAW Library contains data for over 300 materials and will be distributed by the Radiation Shielding Information Center, located at Oak Ridge National Laboratory. It was generated for use in neutronics calculations required in radioactive waste analyses, although it has equal utility in any study requiring multigroup neutron cross sections

  1. Cloning, killing, and identity.

    Science.gov (United States)

    McMahan, J

    1999-01-01

    One potentially valuable use of cloning is to provide a source of tissues or organs for transplantation. The most important objection to this use of cloning is that a human clone would be the sort of entity that it would be seriously wrong to kill. I argue that entities of the sort that you and I essentially are do not begin to exist until around the seventh month of fetal gestation. Therefore to kill a clone prior to that would not be to kill someone like you or me but would be only to prevent one of us from existing. And even after one of us begins to exist, the objections to killing it remain comparatively weak until its psychological capacities reach a certain level of maturation. These claims support the permissibility of killing a clone during the early stages of its development in order to use its organs for transplantation. PMID:10226909

  2. Cloning-free CRISPR

    NARCIS (Netherlands)

    Arbab, Mandana; Srinivasan, Sharanya; Hashimoto, Tatsunori; Geijsen, Niels; Sherwood, Richard I.

    2015-01-01

    We present self-cloning CRISPR/Cas9 (scCRISPR), a technology that allows for CRISPR/Cas9-mediated genomic mutation and site-specific knockin transgene creation within several hours by circumventing the need to clone a site-specific single-guide RNA (sgRNA) or knockin homology construct for each

  3. Animal cloning: problems and prospects.

    Science.gov (United States)

    Wells, D N

    2005-04-01

    An efficient animal cloning technology would provide many new opportunities for livestock agriculture, human medicine, and animal conservation. Nuclear cloning involves the production of animals that are genetically identical to the donor cells used in a technique known as nuclear transfer (NT). However, at present it is an inefficient process: in cattle, only around 6% of the embryos transferred to the reproductive tracts of recipient cows result in healthy, longterm surviving clones. Of concern are the high losses throughout gestation, during birth and in the post-natal period through to adulthood. Many of the pregnancy losses relate to failure of the placenta to develop and function correctly. Placental dysfunction may also have an adverse influence on postnatal health. These anomalies are probably due to incorrect epigenetic reprogramming of the donor genome following NT, leading to inappropriate patterns of gene expression during the development of clones. Whilst some physiological tests on surviving clones suggest normality, other reports indicate a variety of post-natal clone-associated abnormalities. This variability in outcome may reflect species-specific and/or cloning methodological differences. Importantly, to date it appears that these clone-associated phenotypes are not transmitted to offspring following sexual reproduction. This indicates that they represent epigenetic errors, rather than genetic errors, which are corrected during gametogenesis. Whilst this needs confirmation at the molecular level, it provides initial confidence in the first application of NT in agriculture, namely, the production of small numbers of cloned sires from genetically elite bulls, for natural mating, to effectively disseminate genetic gain. In addition to the animal welfare concerns with the technology, the underlying health of the animals and the consequential effect on food safety are critical aspects that require investigation to gain regulatory and consumer

  4. Phylogenetic and functional diversity of metagenomic libraries of phenol degrading sludge from petroleum refinery wastewater treatment system.

    Science.gov (United States)

    Silva, Cynthia C; Hayden, Helen; Sawbridge, Tim; Mele, Pauline; Kruger, Ricardo H; Rodrigues, Marili Vn; Costa, Gustavo Gl; Vidal, Ramon O; Sousa, Maíra P; Torres, Ana Paula R; Santiago, Vânia Mj; Oliveira, Valéria M

    2012-03-27

    In petrochemical refinery wastewater treatment plants (WWTP), different concentrations of pollutant compounds are received daily in the influent stream, including significant amounts of phenolic compounds, creating propitious conditions for the development of particular microorganisms that can rapidly adapt to such environment. In the present work, the microbial sludge from a refinery WWTP was enriched for phenol, cloned into fosmid vectors and pyrosequenced. The fosmid libraries yielded 13,200 clones and a comprehensive bioinformatic analysis of the sequence data set revealed a complex and diverse bacterial community in the phenol degrading sludge. The phylogenetic analyses using MEGAN in combination with RDP classifier showed a massive predominance of Proteobacteria, represented mostly by the genera Diaphorobacter, Pseudomonas, Thauera and Comamonas. The functional classification of phenol degrading sludge sequence data set generated by MG-RAST showed the wide metabolic diversity of the microbial sludge, with a high percentage of genes involved in the aerobic and anaerobic degradation of phenol and derivatives. In addition, genes related to the metabolism of many other organic and xenobiotic compounds, such as toluene, biphenyl, naphthalene and benzoate, were found. Results gathered herein demonstrated that the phenol degrading sludge has complex phylogenetic and functional diversities, showing the potential of such community to degrade several pollutant compounds. This microbiota is likely to represent a rich resource of versatile and unknown enzymes which may be exploited for biotechnological processes such as bioremediation.

  5. Reversibility of continuous-variable quantum cloning

    International Nuclear Information System (INIS)

    Filip, Radim; Marek, Petr; Fiurasek, Jaromir

    2004-01-01

    We analyze a reversibility of optimal Gaussian 1→2 quantum cloning of a coherent state using only local operations on the clones and classical communication between them and propose a feasible experimental test of this feature. Performing Bell-type homodyne measurement on one clone and anticlone, an arbitrary unknown input state (not only a coherent state) can be restored in the other clone by applying appropriate local unitary displacement operation. We generalize this concept to a partial reversal of the cloning using only local operations and classical communication (LOCC) and we show that this procedure converts the symmetric cloner to an asymmetric cloner. Further, we discuss a distributed LOCC reversal in optimal 1→M Gaussian cloning of coherent states which transforms it to optimal 1→M ' cloning for M ' < M. Assuming the quantum cloning as a possible eavesdropping attack on quantum communication link, the reversibility can be utilized to improve the security of the link even after the attack

  6. [Cloning, Expression and Immunodiagnostic Evaluation of the Fasciola gigantica Thioredoxin Peroxidase].

    Science.gov (United States)

    Wang, Yue-qi; Zhou, Yan; Cheng, Na; Chen, Mu-xin; Ai, Lin; Liu, Yu-hua; Zhang, Jian-guo; Luo, Jia-jun; Xu, Xue-nian

    2015-04-01

    To immunoscreen the gene encoding thioredoxin peroxidase (TPx) from a cDNA library made from adult Fasciola gigantica worms, clone and express the gene, and evaluate the immunodiagnostic value of TPx recombinant protein. The A ZAP cDNA library was immunoscreened with pooled serum of fascioliasis gigantica patients. The obtained positive clones were sequenced and analyzed by multiple sequence alignment. The full-length (rFgTPx) and N-termianal truncated (rFgTPx_nt) sequence of FgTPx was subcloned into prokaryotic plasmid pET28a(+) with a non-fusion expression technique, respectively. The recombinant proteins of rFgTPx and rFgTPx_nt were purified by His-bind affinity column (Ni-NTA). rFgTPx and rFgTPx_nt were used in indirect ELISA to test the antibody response of the serum samples. Sera of 27 fascioliasis gigantica patients, 15 patients with schistosomaisis japonica, 15 clonorchiasis sinensis patients, and 32 healthy donors were tested by using the recombinant protein based ELISA. The TPx recombinant proteins were obtained through expression, purification and renaturation, the relative molecular mass of rFgTPx and rFgTPx_nt were Mr 30,000 and Mr 26,000, respectively. The total diagnostic coincidence rate, sensitivity and specificity of rFgTPx_nt-based ELISA was 87.6% (78/89), 66.7% (18/27), and 96.8% (60/62), respectively. The cross reaction with Schistosoma japonicum and Clonorchis sinensis was 0 and 1/15 for rFgTPx_nt, respectively. Before and after treatment, A450 value of the serum samples from fascioliasis patients was 0.233 ± 0.088 and 0.129 ± 0.072, respectively (t = 4.27, P Fasciola gigantica infection.

  7. Human cloning. Fact or fiction

    International Nuclear Information System (INIS)

    Abushama, Mandy D.; Ahmed, Badreldeen I.

    2003-01-01

    Cloning is the production of one or more individual plants or animals that are genetically identical to other plant, animal or human. Scientists even demonstrated that they were able to clone frog tadpoles from frog embryonic cells using nuclear transfer.Many animals have been cloned from adult cells using nuclear transfer. Somatic cell nuclear transfer which refers to the transfer of the nucleous from a somatic cell to an egg cell. Article further deals with benefits and misuses of human cloning

  8. Local cloning of two product states

    International Nuclear Information System (INIS)

    Ji Zhengfeng; Feng Yuan; Ying Mingsheng

    2005-01-01

    Local quantum operations and classical communication (LOCC) put considerable constraints on many quantum information processing tasks such as cloning and discrimination. Surprisingly, however, discrimination of any two pure states survives such constraints in some sense. We show that cloning is not that lucky; namely, probabilistic LOCC cloning of two product states is strictly less efficient than global cloning. We prove our result by giving explicitly the efficiency formula of local cloning of any two product states

  9. Microaspiration of esophageal gland cells and cDNA library construction for identifying parasitism genes of plant-parasitic nematodes.

    Science.gov (United States)

    Hussey, Richard S; Huang, Guozhong; Allen, Rex

    2011-01-01

    Identifying parasitism genes encoding proteins secreted from a plant-parasitic nematode's esophageal gland cells and injected through its stylet into plant tissue is the key to understanding the molecular basis of nematode parasitism of plants. Parasitism genes have been cloned by directly microaspirating the cytoplasm from the esophageal gland cells of different parasitic stages of cyst or root-knot nematodes to provide mRNA to create a gland cell-specific cDNA library by long-distance reverse-transcriptase polymerase chain reaction. cDNA clones are sequenced and deduced protein sequences with a signal peptide for secretion are identified for high-throughput in situ hybridization to confirm gland-specific expression.

  10. Structured Review of Code Clone Literature

    NARCIS (Netherlands)

    Hordijk, W.T.B.; Ponisio, Laura; Wieringa, Roelf J.

    2008-01-01

    This report presents the results of a structured review of code clone literature. The aim of the review is to assemble a conceptual model of clone-related concepts which helps us to reason about clones. This conceptual model unifies clone concepts from a wide range of literature, so that findings

  11. Molecular analysis of bacterial populations in water samples from two Uranium mill tailings by using RISA retrieval

    International Nuclear Information System (INIS)

    Selenska-Pobell, S.; Radeva, G.

    2004-01-01

    Ribosomal intergenic spacer amplification (RISA) retrieval was applied to analyse the natural bacterial communities in drain waters of two uranium mill tailings - Gittersee/Coschuetz in Germany and Shiprock in the USA. About 35% of the clones from RISA library constructed for the samples of the German tailings represented a microdiverse population of Planctomycetales. The rest of the clones were affiliated with rather diverse bacterial groups including γ- and δ-Proteobacteria, Cytophaga/Flavobacterium/Bacteroides (CFB), Nitrospira, Verrucomicrobia and Actinobacteria. 8% of the cloned sequences represented a novel bacterial lineage from the recently described division NC3. Bacterial diversity in the Shiprock mill tailings was found to be significantly lower. RISA library constructed for those samples contained only two larger groups of clones representing β-proteobacterial species and one small group which was affiliated with δ-Proteobacteria. (authors)

  12. Molecular analysis of bacterial populations in water samples from two Uranium mill tailings by using RISA retrieval

    Energy Technology Data Exchange (ETDEWEB)

    Selenska-Pobell, S [Forschungszentrum Rossendorf, Institute of Radiochemistry, Dresden (Germany); Radeva, G [Bulgarian Academy of Sciences, Institute of Molecular Biology, Sofia (Bulgaria)

    2004-07-01

    Ribosomal intergenic spacer amplification (RISA) retrieval was applied to analyse the natural bacterial communities in drain waters of two uranium mill tailings - Gittersee/Coschuetz in Germany and Shiprock in the USA. About 35% of the clones from RISA library constructed for the samples of the German tailings represented a microdiverse population of Planctomycetales. The rest of the clones were affiliated with rather diverse bacterial groups including {gamma}- and {delta}-Proteobacteria, Cytophaga/Flavobacterium/Bacteroides (CFB), Nitrospira, Verrucomicrobia and Actinobacteria. 8% of the cloned sequences represented a novel bacterial lineage from the recently described division NC3. Bacterial diversity in the Shiprock mill tailings was found to be significantly lower. RISA library constructed for those samples contained only two larger groups of clones representing {beta}-proteobacterial species and one small group which was affiliated with {delta}-Proteobacteria. (authors)

  13. Clones, Drones and Dragons: Ongoing Uncertainties around School Leader Development

    Science.gov (United States)

    Walker, Allan

    2015-01-01

    This article examines a number of key issues around successful school leadership and leader development. Three metaphors are used to frame, track and analyse recent research and commentary in the area--these are clones, drones and dragons. Although development mechanisms rarely fall neatly within one category, the metaphors provide a useful way to…

  14. MPO cDNA clone identifies an RFLP with PstI

    Energy Technology Data Exchange (ETDEWEB)

    Miki, T; Weil, S C; Rosner, G L; Reid, M S; Kidd, K K

    1988-02-25

    A myeloperoxidase (MPO) cDNA clone (pHMP7: 270 base pair insert in the vector pGEM-1reverse arrow was isolated from a library created from human promyelocytic (HL-60) cell mRNA. PstI (CTGCA/G) (New England Biolabs) identifies a simple two-allele polymorphism with bands at either 2.2 kb (Al) or 2.0 kb (A2). There are three constant bands at 2.8 kb, 0.95 kb and 0.6 kb. Preliminary family data show evidence of linkage to several markers in proximal 17q, with MPO closest to the Growth Hormone cluster at 17q22-q24. Autosomal condominant segregation was observed in four large reference pedigrees with several informative matings.

  15. The role of international institutions in the formation of international bioethical law: UNESCO and the United Nations General Assembly attempt to govern human cloning.

    Science.gov (United States)

    Kuppuswamy, Chamundeeswari

    2007-01-01

    This article analyses the international governance of human reproductive cloning. Noting that bioethics is a new field of engagement for international lawyers, it recounts some of the institutional developments in bioethical law making. The role of UNESCO and the United Nations General Assembly is scrutinized and the author discusses the relative merits of the institutions' governance of human reproductive cloning. The author suggests that some international institutions and mechanisms are better suited than others for bioethical law making. The 2005 General Assembly resolution on human cloning is analysed in this context.

  16. Human cloning: can it be made safe?

    Science.gov (United States)

    Rhind, Susan M; Taylor, Jane E; De Sousa, Paul A; King, Tim J; McGarry, Michelle; Wilmut, Ian

    2003-11-01

    There are continued claims of attempts to clone humans using nuclear transfer, despite the serious problems that have been encountered in cloning other mammals. It is known that epigenetic and genetic mechanisms are involved in clone failure, but we still do not know exactly how. Human reproductive cloning is unethical, but the production of cells from cloned embryos could offer many potential benefits. So, can human cloning be made safe?

  17. Probabilistic cloning and deleting of quantum states

    International Nuclear Information System (INIS)

    Feng Yuan; Zhang Shengyu; Ying Mingsheng

    2002-01-01

    We construct a probabilistic cloning and deleting machine which, taking several copies of an input quantum state, can output a linear superposition of multiple cloning and deleting states. Since the machine can perform cloning and deleting in a single unitary evolution, the probabilistic cloning and other cloning machines proposed in the previous literature can be thought of as special cases of our machine. A sufficient and necessary condition for successful cloning and deleting is presented, and it requires that the copies of an arbitrarily presumed number of the input states are linearly independent. This simply generalizes some results for cloning. We also derive an upper bound for the success probability of the cloning and deleting machine

  18. Cloning and expression of 130-kd mosquito-larvicidal delta-endotoxin gene of Bacillus thuringiensis var. Israelensis in Escherichia coli.

    Science.gov (United States)

    Angsuthanasombat, C; Chungjatupornchai, W; Kertbundit, S; Luxananil, P; Settasatian, C; Wilairat, P; Panyim, S

    1987-07-01

    Five recombinant E. coli clones exhibiting toxicity to Aedes aegypti larvae were obtained from a library of 800 clones containing XbaI DNA fragments of 110 kb plasmid from B. thuringiensis var. israelensis. All the five clones (pMU 14/258/303/388/679) had the same 3.8-kb insert and encoded a major protein of 130 kDa which was highly toxic to A. aegypti larvae. Three clones (pMU 258/303/388) transcribed the 130 kD a gene in the same direction as that of lac Z promoter of pUC12 vector whereas the transcription of the other two (pMU 14/679) was in the opposite direction. A 1.9-kb fragment of the 3.8 kb insert coded for a protein of 65 kDa. Partial DNA sequence of the 3.8 kb insert, corresponding to the 5'-terminal of the 130 kDa gene, revealed a continuous reading frame, a Shine-Dalgarno sequence and a tentative 5'-regulatory region. These results demonstrated that the 3.8 kb insert is a minimal DNA fragment containing a regulatory region plus the coding sequence of the 130 kDa protein that is highly toxic to mosquito larvae.

  19. Asymmetric quantum cloning machines

    International Nuclear Information System (INIS)

    Cerf, N.J.

    1998-01-01

    A family of asymmetric cloning machines for quantum bits and N-dimensional quantum states is introduced. These machines produce two approximate copies of a single quantum state that emerge from two distinct channels. In particular, an asymmetric Pauli cloning machine is defined that makes two imperfect copies of a quantum bit, while the overall input-to-output operation for each copy is a Pauli channel. A no-cloning inequality is derived, characterizing the impossibility of copying imposed by quantum mechanics. If p and p ' are the probabilities of the depolarizing channels associated with the two outputs, the domain in (√p,√p ' )-space located inside a particular ellipse representing close-to-perfect cloning is forbidden. This ellipse tends to a circle when copying an N-dimensional state with N→∞, which has a simple semi-classical interpretation. The symmetric Pauli cloning machines are then used to provide an upper bound on the quantum capacity of the Pauli channel of probabilities p x , p y and p z . The capacity is proven to be vanishing if (√p x , √p y , √p z ) lies outside an ellipsoid whose pole coincides with the depolarizing channel that underlies the universal cloning machine. Finally, the tradeoff between the quality of the two copies is shown to result from a complementarity akin to Heisenberg uncertainty principle. (author)

  20. Effective and efficient model clone detection

    DEFF Research Database (Denmark)

    Störrle, Harald

    2015-01-01

    Code clones are a major source of software defects. Thus, it is likely that model clones (i.e., duplicate fragments of models) have a significant negative impact on model quality, and thus, on any software created based on those models, irrespective of whether the software is generated fully...... automatically (“MDD-style”) or hand-crafted following the blueprint defined by the model (“MBSD-style”). Unfortunately, however, model clones are much less well studied than code clones. In this paper, we present a clone detection algorithm for UML domain models. Our approach covers a much greater variety...... of model types than existing approaches while providing high clone detection rates at high speed....

  1. Cloning of a cDNA encoding a surface antigen of Schistosoma mansoni schistosomula recognized by sera of vassinated mice

    International Nuclear Information System (INIS)

    Dalton, J.P.; Tom, T.D.; Strand, M.

    1987-01-01

    Spleen cells of mice vaccinated with radiation-attenuated Schistosoma mansoni cercariae were used to produce monoclonal antibodies directed against newly transformed schistosomular surface antigens. One of these monoclonal antibodies recognized a polypeptide of 18 kDa. Binding was measured by radioimmunoassay. This glycoprotein was purified by monoclonal antibody immunoaffinity chromatography and a polyclonal antiserum was prepared against it. Immunofluorescence assays showed that the polyclonal antiserum bound to the surface of newly transformed schistosomula and lung-stage organisms but not to the surface of liver-stage and adult worms. Using this polyclonal antiserum we isolated recombinant clones from an adult worm cDNA expression library constructed in λgt11. Clone 654.2 contained an insert of 0.52 kilobase and hybridized to a 1.2-kilobase mRNA species from adult worms. Most importantly, clone 654.2 produced a fusion protein of 125 kDa that was reactive with sera of vaccinated mice that are capable of transferring resistance. This result encourages future vaccination trials with the fusion protein

  2. Wildlife conservation and reproductive cloning.

    Science.gov (United States)

    Holt, William V; Pickard, Amanda R; Prather, Randall S

    2004-03-01

    Reproductive cloning, or the production of offspring by nuclear transfer, is often regarded as having potential for conserving endangered species of wildlife. Currently, however, low success rates for reproductive cloning limit the practical application of this technique to experimental use and proof of principle investigations. In this review, we consider how cloning may contribute to wildlife conservation strategies. The cloning of endangered mammals presents practical problems, many of which stem from the paucity of knowledge about their basic reproductive biology. However, situations may arise where resources could be targeted at recovering lost or under-represented genetic lines; these could then contribute to the future fitness of the population. Approaches of this type would be preferable to the indiscriminate generation of large numbers of identical individuals. Applying cloning technology to non-mammalian vertebrates may be more practical than attempting to use conventional reproductive technologies. As the scientific background to cloning technology was pioneered using amphibians, it may be possible to breed imminently threatened amphibians, or even restore extinct amphibian species, by the use of cloning. In this respect species with external embryonic development may have an advantage over mammals as developmental abnormalities associated with inappropriate embryonic reprogramming would not be relevant.

  3. Cloning and expression of calmodulin gene in Scoparia dulcis.

    Science.gov (United States)

    Saitoh, Daisuke; Asakura, Yuki; Nkembo, Marguerite Kasidimoko; Shite, Masato; Sugiyama, Ryuji; Lee, Jung-Bum; Hayashi, Toshimitsu; Kurosaki, Fumiya

    2007-06-01

    A homology-based cloning strategy yielded a cDNA clone, designated Sd-cam, encoding calmodulin protein from Scoparia dulcis. The restriction digests of genomic DNA of S. dulcis showed a single hybridized signal when probed with the fragment of this gene in Southern blot analyses, suggesting that Sd-cam occurs as a sole gene encoding calmodulin in the plant. The reverse-transcription polymerase chain reaction analysis revealed that Sd-cam was appreciably expressed in leaf, root and stem tissues. It appeared that transcription of this gene increased transiently when the leaf cultures of S. dulcis were treated with methyl jasmonate and calcium ionophore A23187. These results suggest that transcriptional activation of Sd-cam is one of the early cellular events of the methyl jasmonate-induced responses of S. dulcis.

  4. [Construction and screening of phage antibody libraries against epidermal growth factor receptor and soluble expression of single chain Fv].

    Science.gov (United States)

    Sheng, Wei-Jin; Miao, Qing-Fang; Zhen, Yong-Su

    2009-06-01

    Recent studies have shown that epidermal growth factor receptor (EGFR) is an important target for cancer therapy. The present study prepared single chain Fv (scFv) directed against EGFR. Balb/c mice were immunized by human carcinoma A431 cells, and total RNA of the splenic cells was extracted. VH and VL gene fragments were amplified by RT-PCR and further joined into scFv gene with a linker, then scFv gene fragments were ligated into the phagemid vector pCANTAB 5E. The phagemid containing scFv were transformed into electro-competent E. coli TG1 cells. The recombinant phage antibody library was constructed through rescuing the transformed cells with help phage M13K07. The specified recombinant phages were enriched through 5 rounds of affinity panning and the anti-EGFR phage scFv clones were screened and identified with ELISA. A total of 48 clones from the library were selected randomly and 45 clones were identified positive. After infecting E. coli HB2151 cells with one positive clone, soluble recombinant antibodies about 27 kD were produced and located in the periplasm and the supernatant. The result of sequencing showed that the scFv gene was 768 bp, which encoded 256 amino acid residues. VH and VL including 3 CDRs and 4 FRs, respectively, were all homologous to mouse Ig. The soluble scFv showed the specific binding activity to purified EGFR and EGFR located in carcinoma cell membrane. The successful preparation of anti-EGFR scFv will provide an EGFR targeted molecule for the development of antibody-based drugs and biological therapy of cancer.

  5. Functional study and regional mapping of 44 hormono-regulated genes isolated from a porcine granulosa cell library

    Directory of Open Access Journals (Sweden)

    Hatey François

    2001-01-01

    Full Text Available Abstract cDNA clones from a pig granulosa cell cDNA library were isolated by differential hybridisation for follicle stimulating hormone (FSH regulation in granulosa cells in a previous study. The clones that did not match any known sequence were studied for their expression in granulosa cells (treated or not by FSH and in fresh isolated ovarian follicles mainly by comparative RT-PCR analysis. These results give functional data on genes that may be implicated in follicular growing. These ESTs have been localised on the porcine genome, using a somatic cell hybrid panel, providing new type I markers on the porcine map and information on the comparative map between humans and pigs.

  6. Comparative analyses of amplicon migration behavior in differing denaturing gradient gel electrophoresis (DGGE) systems

    Science.gov (United States)

    Thornhill, D. J.; Kemp, D. W.; Sampayo, E. M.; Schmidt, G. W.

    2010-03-01

    Denaturing gradient gel electrophoresis (DGGE) is commonly utilized to identify and quantify microbial diversity, but the conditions required for different electrophoretic systems to yield equivalent results and optimal resolution have not been assessed. Herein, the influence of different DGGE system configuration parameters on microbial diversity estimates was tested using Symbiodinium, a group of marine eukaryotic microbes that are important constituents of coral reef ecosystems. To accomplish this, bacterial clone libraries were constructed and sequenced from cultured isolates of Symbiodinium for the ribosomal DNA internal transcribed spacer 2 (ITS2) region. From these, 15 clones were subjected to PCR with a GC clamped primer set for DGGE analyses. Migration behaviors of the resulting amplicons were analyzed using a range of conditions, including variation in the composition of the denaturing gradient, electrophoresis time, and applied voltage. All tests were conducted in parallel on two commercial DGGE systems, a C.B.S. Scientific DGGE-2001, and the Bio-Rad DCode system. In this context, identical nucleotide fragments exhibited differing migration behaviors depending on the model of apparatus utilized, with fragments denaturing at a lower gradient concentration and applied voltage on the Bio-Rad DCode system than on the C.B.S. Scientific DGGE-2001 system. Although equivalent PCR-DGGE profiles could be achieved with both brands of DGGE system, the composition of the denaturing gradient and application of electrophoresis time × voltage must be appropriately optimized to achieve congruent results across platforms.

  7. A plasmid toolkit for cloning chimeric cDNAs encoding customized fusion proteins into any Gateway destination expression vector

    Science.gov (United States)

    2013-01-01

    Background Valuable clone collections encoding the complete ORFeomes for some model organisms have been constructed following the completion of their genome sequencing projects. These libraries are based on Gateway cloning technology, which facilitates the study of protein function by simplifying the subcloning of open reading frames (ORF) into any suitable destination vector. The expression of proteins of interest as fusions with functional modules is a frequent approach in their initial functional characterization. A limited number of Gateway destination expression vectors allow the construction of fusion proteins from ORFeome-derived sequences, but they are restricted to the possibilities offered by their inbuilt functional modules and their pre-defined model organism-specificity. Thus, the availability of cloning systems that overcome these limitations would be highly advantageous. Results We present a versatile cloning toolkit for constructing fully-customizable three-part fusion proteins based on the MultiSite Gateway cloning system. The fusion protein components are encoded in the three plasmids integral to the kit. These can recombine with any purposely-engineered destination vector that uses a heterologous promoter external to the Gateway cassette, leading to the in-frame cloning of an ORF of interest flanked by two functional modules. In contrast to previous systems, a third part becomes available for peptide-encoding as it no longer needs to contain a promoter, resulting in an increased number of possible fusion combinations. We have constructed the kit’s component plasmids and demonstrate its functionality by providing proof-of-principle data on the expression of prototype fluorescent fusions in transiently-transfected cells. Conclusions We have developed a toolkit for creating fusion proteins with customized N- and C-term modules from Gateway entry clones encoding ORFs of interest. Importantly, our method allows entry clones obtained from ORFeome

  8. Cloning and molecular analyses of a gibberellin 20-oxidase gene expressed specifically in developing seeds of watermelon.

    Science.gov (United States)

    Kang, H G; Jun, S H; Kim, J; Kawaide, H; Kamiya, Y; An, G

    1999-10-01

    To understand the biosynthesis and functional role of gibberellins (GAs) in developing seeds, we isolated Cv20ox, a cDNA clone from watermelon (Citrullus lanatus) that shows significant amino acid homology with GA 20-oxidases. The complementary DNA clone was expressed in Escherichia coli as a fusion protein, which oxidized GA(12) at C-20 to the C(19) compound GA(9), a precursor of bioactive GAs. RNA-blot analysis showed that the Cv20ox gene was expressed specifically in developing seeds. The gene was strongly expressed in the integument tissues, and it was also expressed weakly in inner seed tissues. In parthenocarpic fruits induced by 1-(2-chloro-4-pyridyl)-3-phenylurea treatment, the expression pattern of Cv20ox did not change, indicating that the GA 20-oxidase gene is expressed primarily in the maternal cells of developing seeds. The promoter of Cv20ox was isolated and fused to the beta-glucuronidase (GUS) gene. In a transient expression system, beta-glucuronidase staining was detectable only in the integument tissues of developing watermelon seeds.

  9. Construction of a plant-transformation-competent BIBAC library and genome sequence analysis of polyploid Upland cotton (Gossypium hirsutum L.).

    Science.gov (United States)

    Lee, Mi-Kyung; Zhang, Yang; Zhang, Meiping; Goebel, Mark; Kim, Hee Jin; Triplett, Barbara A; Stelly, David M; Zhang, Hong-Bin

    2013-03-28

    Cotton, one of the world's leading crops, is important to the world's textile and energy industries, and is a model species for studies of plant polyploidization, cellulose biosynthesis and cell wall biogenesis. Here, we report the construction of a plant-transformation-competent binary bacterial artificial chromosome (BIBAC) library and comparative genome sequence analysis of polyploid Upland cotton (Gossypium hirsutum L.) with one of its diploid putative progenitor species, G. raimondii Ulbr. We constructed the cotton BIBAC library in a vector competent for high-molecular-weight DNA transformation in different plant species through either Agrobacterium or particle bombardment. The library contains 76,800 clones with an average insert size of 135 kb, providing an approximate 99% probability of obtaining at least one positive clone from the library using a single-copy probe. The quality and utility of the library were verified by identifying BIBACs containing genes important for fiber development, fiber cellulose biosynthesis, seed fatty acid metabolism, cotton-nematode interaction, and bacterial blight resistance. In order to gain an insight into the Upland cotton genome and its relationship with G. raimondii, we sequenced nearly 10,000 BIBAC ends (BESs) randomly selected from the library, generating approximately one BES for every 250 kb along the Upland cotton genome. The retroelement Gypsy/DIRS1 family predominates in the Upland cotton genome, accounting for over 77% of all transposable elements. From the BESs, we identified 1,269 simple sequence repeats (SSRs), of which 1,006 were new, thus providing additional markers for cotton genome research. Surprisingly, comparative sequence analysis showed that Upland cotton is much more diverged from G. raimondii at the genomic sequence level than expected. There seems to be no significant difference between the relationships of the Upland cotton D- and A-subgenomes with the G. raimondii genome, even though G

  10. Islamic perspectives on human cloning.

    Science.gov (United States)

    Sadeghi, Mahmoud

    2007-01-01

    The present paper seeks to assess various views from Islamic jurists relating to human cloning, which is one of the controversial topics in the recent past. Taking Islamic jurisprudence principles, such as the rule of necessity for self preservation and respect for human beings, the rule of la darar wa la dirar ('the necessity to refrain from causing harm to oneself and others') and the rule of usr wa haraj, one may indicate that if human cloning could not be prohibited, as such, it could still be opposed because it gives way to various harmful consequences, which include family disorder, chaos in the clone's family relationships, physical and mental diseases for clones and suffering of egg donors and surrogate mothers. However with due attention to the fact that the reasons behind the prohibition of abortion only restrict the destruction of human embryos in their post-implantation stages, human cloning for biomedical research and exploitation of stem cells from cloned embryos at the blastocyst stage for therapeutic purposes would be acceptable.

  11. Quantum cloning machines for equatorial qubits

    International Nuclear Information System (INIS)

    Fan Heng; Matsumoto, Keiji; Wang Xiangbin; Wadati, Miki

    2002-01-01

    Quantum cloning machines for equatorial qubits are studied. For the case of a one to two phase-covariant quantum cloning machine, we present the networks consisting of quantum gates to realize the quantum cloning transformations. The copied equatorial qubits are shown to be separable by using Peres-Horodecki criterion. The optimal one to M phase-covariant quantum cloning transformations are given

  12. Isolation and characterization of human cDNA clones encoding the α and the α' subunits of casein kinase II

    International Nuclear Information System (INIS)

    Lozeman, F.J.; Litchfield, D.W.; Piening, C.; Takio, Koji; Walsh, K.A.; Krebs, E.G.

    1990-01-01

    Casein kinase II is a widely distributed protein serine/threonine kinase. The holoenzyme appears to be a tetramer, containing two α or α' subunits (or one of each) and two β subunits. Complementary DNA clones encoding the subunits of casein kinase II were isolated from a human T-cell λgt 10 library using cDNA clones isolated from Drosophila melanogasten. One of the human cDNA clones (hT4.1) was 2.2 kb long, including a coding region of 1176 bp preceded by 156 bp (5' untranslated region) and followed by 871 bp (3' untranslated region). The hT4.1 close was nearly identical in size and sequence with a cDNA clone from HepG2 human hepatoma cultured cells. Another of the human T-cell cDNA clones (hT9.1) was 1.8 kb long, containing a coding region of 1053 bp preceded by 171 by (5' untranslated region) and followed by 550 bp (3' untranslated region). Amino acid sequences deduced from these two cDNA clones were about 85% identical. Most of the difference between the two encoded polypeptides was in the carboxy-terminal region, but heterogeneity was distributed throughout the molecules. Partial amino acid sequence was determined in a mixture of α and α' subunits from bovine lung casein kinase II. The bovine sequences aligned with the 2 human cDNA-encoded polypeptides with only 2 discrepancies out of 535 amino acid positions. This confirmed that the two human T-cell cDNA clones encoded the α and α' subunits of casein kinase II. These studies show that there are two distinct catalytic subunits for casein II (α and α') and that the sequence of these subunits is largely conserved between the bovine and the human

  13. A Gateway MultiSite recombination cloning toolkit.

    Directory of Open Access Journals (Sweden)

    Lena K Petersen

    Full Text Available The generation of DNA constructs is often a rate-limiting step in conducting biological experiments. Recombination cloning of single DNA fragments using the Gateway system provided an advance over traditional restriction enzyme cloning due to increases in efficiency and reliability. Here we introduce a series of entry clones and a destination vector for use in two, three, and four fragment Gateway MultiSite recombination cloning whose advantages include increased flexibility and versatility. In contrast to Gateway single-fragment cloning approaches where variations are typically incorporated into model system-specific destination vectors, our Gateway MultiSite cloning strategy incorporates variations in easily generated entry clones that are model system-independent. In particular, we present entry clones containing insertions of GAL4, QF, UAS, QUAS, eGFP, and mCherry, among others, and demonstrate their in vivo functionality in Drosophila by using them to generate expression clones including GAL4 and QF drivers for various trp ion channel family members, UAS and QUAS excitatory and inhibitory light-gated ion channels, and QUAS red and green fluorescent synaptic vesicle markers. We thus establish a starter toolkit of modular Gateway MultiSite entry clones potentially adaptable to any model system. An inventory of entry clones and destination vectors for Gateway MultiSite cloning has also been established (www.gatewaymultisite.org.

  14. Update on the First Cloned Dog and Outlook for Canine Cloning.

    Science.gov (United States)

    Jang, Goo; Lee, ByeongChun

    2015-10-01

    As man's best friend, dogs have an important position in human society. Ten years ago, we reported the first cloned dog, and his birth has raised various scientific issues, such as those related to health, reproduction, and life span. He has developed without any unique health issues. In this article, we summarize and present perspectives on canine cloning.

  15. Neutron cross section libraries for analysis of fusion neutronics experiments

    International Nuclear Information System (INIS)

    Kosako, Kazuaki; Oyama, Yukio; Maekawa, Hiroshi; Nakamura, Tomoo

    1988-03-01

    We have prepared two computer code systems producing neutron cross section libraries to analyse fusion neutronics experiments. First system produces the neutron cross section library in ANISN format, i.e., the multi-group constants in group independent format. This library can be obtained by using the multi-group constant processing code system MACS-N and the ANISN format cross section compiling code CROKAS. Second system is for the continuous energy cross section library for the MCNP code. This library can be obtained by the nuclear data processing system NJOY which generates pointwise energy cross sections and the cross section compiling code MACROS for the MCNP library. In this report, we describe the production procedures for both types of the cross section libraries, and show six libraries with different conditions in ANISN format and a library for the MCNP code. (author)

  16. Development of a T7 Phage Display Library to Detect Sarcoidosis and Tuberculosis by a Panel of Novel Antigens

    Directory of Open Access Journals (Sweden)

    Harvinder Talwar

    2015-04-01

    Full Text Available Sarcoidosis is a granulomatous inflammatory disease, diagnosed through tissue biopsy of involved organs in the absence of other causes such as tuberculosis (TB. No specific serologic test is available to diagnose and differentiate sarcoidosis from TB. Using a high throughput method, we developed a T7 phage display cDNA library derived from mRNA isolated from bronchoalveolar lavage (BAL cells and leukocytes of sarcoidosis patients. This complex cDNA library was biopanned to obtain 1152 potential sarcoidosis antigens and a microarray was constructed to immunoscreen two different sets of sera from healthy controls and sarcoidosis. Meta-analysis identified 259 discriminating sarcoidosis antigens, and multivariate analysis identified 32 antigens with a sensitivity of 89% and a specificity of 83% to classify sarcoidosis from healthy controls. Additionally, interrogating the same microarray platform with sera from subjects with TB, we identified 50 clones that distinguish between TB, sarcoidosis and healthy controls. The top 10 sarcoidosis and TB specific clones were sequenced and homologies were searched in the public database revealing unique epitopes and mimotopes in each group. Here, we show for the first time that immunoscreenings of a library derived from sarcoidosis tissue differentiates between sarcoidosis and tuberculosis antigens. These novel biomarkers can improve diagnosis of sarcoidosis and TB, and may aid to develop or evaluate a TB vaccine.

  17. Enhancing a Core Journal Collection for Digital Libraries

    Science.gov (United States)

    Kovacevic, Ana; Devedzic, Vladan; Pocajt, Viktor

    2010-01-01

    Purpose: This paper aims to address the problem of enhancing the selection of titles offered by a digital library, by analysing the differences in these titles when they are cited by local authors in their publications and when they are listed in the digital library offer. Design/methodology/approach: Text mining techniques were used to identify…

  18. Water use by Eucalypt Clones Growing at Kongowe, Kibaha, Tanzania

    African Journals Online (AJOL)

    A study was carried out between April and October 2014 to investigate the amount of water used by Eucalypt clones grown at Kongowe, Tanzania. Sap flow sensors using heat pulse velocity were deployed across GC 167, GC 15 and GC 940 in wet and dry seasons. Data on water use were analysed using sap flow software ...

  19. Human catechol-O-methyltransferase: Cloning and expression of the membrane-associated form

    International Nuclear Information System (INIS)

    Bertocci, B.; Miggiano, V.; Da Prada, M.; Dembic, Z.; Lahm, H.W.; Malherbe, P.

    1991-01-01

    A cDNA clone for human catechol-O-methyltransferase was isolated from a human hepatoma cell line (Hep G2) cDNA library by hybridization screening with a porcine cDNA probe. The cDNA clone was sequenced and found to have an insert of 1226 nucleotides. The deduced primary structure of hCOMT is composed of 271 amino acid residues with the predicted molecular mass of 30 kDa. At its N terminus it has a hydrophobic segment of 21 amino acid residues that may be responsible for insertion of hCOMT into the endoplasmic reticulum membrane. The primary structure of hCOMT exhibits high homology to the porcine partial cDNA sequence (93%). The deduced amino acid sequence contains two tryptic peptide sequences (T-22, T-33) found in porcine liver catechol-O-methyltransferase (CEMT). The coding region of hCOMT cDNA was placed under the control of the cytomegalovirus promoter to transfect human kidney 293 cells. The recombinant hCOMT was shown by immunoblot analysis to be mainly associated with the membrane fraction. RNA blot analysis revealed one COMT mRNA transcript of 1.4 kilobases in Hep G2 poly(A) + RNA

  20. Cloning, functional expression, and characterization of a PKA-activated gastric Cl- channel.

    Science.gov (United States)

    Malinowska, D H; Kupert, E Y; Bahinski, A; Sherry, A M; Cuppoletti, J

    1995-01-01

    cDNA encoding a Cl- channel was isolated from a rabbit gastric library, sequenced, and expressed in Xenopus oocytes. The predicted protein (898 amino acids, relative molecular mass 98,433 Da) was overall 93% similar to the rat brain ClC-2 Cl- channel. However, a 151-amino acid stretch toward the COOH-terminus was 74% similar to ClC-2 with six amino acids deleted. Two new potential protein kinase A (PKA) phosphorylation sites (also protein kinase C phosphorylation sites) were introduced. cRNA-injected Xenopus oocytes expressed a Cl- channel that was active at pHtrans 3 and had a linear current-voltage (I-V) curve and a slope conductance of 29 +/- 1 pS at 800 mM CsCl. A fivefold Cl- gradient caused a rightward shift in the I-V curve with a reversal potential of +30 +/- 3 mV, indicating anion selectivity. The selectivity was I- > Cl- > NO3-. The native and recombinant Cl- channel were both activated in vitro by PKA catalytic subunit and ATP. The electrophysiological and regulatory properties of the cloned and the native channel were similar. The cloned protein may be the Cl- channel involved in gastric HCl secretion.

  1. Bacterial diversity in the active stage of a bioremediation system for mineral oil hydrocarbon-contaminated soils.

    Science.gov (United States)

    Popp, Nicole; Schlömann, Michael; Mau, Margit

    2006-11-01

    Soils contaminated with mineral oil hydrocarbons are often cleaned in off-site bioremediation systems. In order to find out which bacteria are active during the degradation phase in such systems, the diversity of the active microflora in a degrading soil remediation system was investigated by small-subunit (SSU) rRNA analysis. Two sequential RNA extracts from one soil sample were generated by a procedure incorporating bead beating. Both extracts were analysed separately by generating individual SSU rDNA clone libraries from cDNA of the two extracts. The sequencing results showed moderate diversity. The two clone libraries were dominated by Gammaproteobacteria, especially Pseudomonas spp. Alphaproteobacteria and Betaproteobacteria were two other large groups in the clone libraries. Actinobacteria, Firmicutes, Bacteroidetes and Epsilonproteobacteria were detected in lower numbers. The obtained sequences were predominantly related to genera for which cultivated representatives have been described, but were often clustered together in the phylogenetic tree, and the sequences that were most similar were originally obtained from soils and not from pure cultures. Most of the dominant genera in the clone libraries, e.g. Pseudomonas, Acinetobacter, Sphingomonas, Acidovorax and Thiobacillus, had already been detected in (mineral oil hydrocarbon) contaminated environmental samples. The occurrence of the genera Zymomonas and Rhodoferax was novel in mineral oil hydrocarbon-contaminated soil.

  2. Knowledge Organisation Systems in North American Digital Library Collections

    Science.gov (United States)

    Shiri, Ali; Chase-Kruszewski, Sarah

    2009-01-01

    Purpose: The purpose of this paper is to report an investigation into the types of knowledge organisation systems (KOSs) utilised in North American digital library collections. Design/methodology/approach: The paper identifies, analyses and deep scans online North American hosted digital libraries. It reviews the literature related to the…

  3. Therapeutic and reproductive cloning: a critique.

    Science.gov (United States)

    Bowring, Finn

    2004-01-01

    This article is a critical examination of the science and ethics of human cloning. It summarises the key scientific milestones in the development of nuclear transplantation, explains the importance of cloning to research into the medical potential of embryonic stem cells, and discusses the well-worn distinction between 'therapeutic' and 'reproductive' cloning. Suggesting that this distinction will be impossible to police, it goes on to consider the ethics of full human cloning. It is concluded that it represents an unacceptable form of parental despotism, and that the genetic engineering and cloning of future human beings will fracture the foundations of modern humanism.

  4. About the Library - Betty Petersen Memorial Library

    Science.gov (United States)

    branch library of the NOAA Central Library. The library serves the NOAA Science Center in Camp Springs , Maryland. History and Mission: Betty Petersen Memorial Library began as a reading room in the NOAA Science Science Center staff and advises the library on all aspects of the library program. Library Newsletters

  5. Novel cloning machine with supplementary information

    International Nuclear Information System (INIS)

    Qiu Daowen

    2006-01-01

    Probabilistic cloning was first proposed by Duan and Guo. Then Pati established a novel cloning machine (NCM) for copying superposition of multiple clones simultaneously. In this paper, we deal with the novel cloning machine with supplementary information (NCMSI). For the case of cloning two states, we demonstrate that the optimal efficiency of the NCMSI in which the original party and the supplementary party can perform quantum communication equals that achieved by a two-step cloning protocol wherein classical communication is only allowed between the original and the supplementary parties. From this equivalence, it follows that NCMSI may increase the success probabilities for copying. Also, an upper bound on the unambiguous discrimination of two nonorthogonal pure product states is derived. Our investigation generalizes and completes the results in the literature

  6. GENETIC DIVERGENCE AND MORPHO - AGRONOMIC PERFORMANCE OF JATROPHA CURCAS L. CLONES FOR SELECTION OF CLONAL VARIETIES

    Directory of Open Access Journals (Sweden)

    ADRIANA QUEIROZ DE ALMEIDA

    2016-01-01

    Full Text Available The knowledge about genetic diversity of jatropha crop is important for genetic conservation resources and breeding of this species. The aim of this study was to evaluate the genetic diversity and performance of jatropha clones through morphological characterization to selection of clonal varieties for biofuels production. The clones were obtained through shoot cuttings from previous selection in a population of half - sibs progenies. The morphoagronomic analyses of clones was carried out at 180 days after transplantation and were evaluated plant height, stem diameter, number of primary branches and number of secondary branches, number of bunches and number of fruits per plant. Evaluating clones performance, significant results were found for the number of secondary branches. About analysis of genetic diversity, the measures of dissimilarity genetic varied from 0.62 to 13.11, this way, the UFRBPR14 and UFRBPR15 clones were more divergent. The Tocher method was efficient to verify formation of four groups. The characteristics that most contributed to the divergence among clones were branches number, height and number of bunches, and, stem diameter had lower contribution. The jatropha clones differed only in the secondary branches number and multivariate analysis showed divergence among the jatropha clones with formation of four groups. Also, branches number, plant height and number of bunches were characteristic that contributed to genetic divergence.

  7. Construction and screening of marine metagenomic libraries.

    Science.gov (United States)

    Weiland, Nancy; Löscher, Carolin; Metzger, Rebekka; Schmitz, Ruth

    2010-01-01

    Marine microbial communities are highly diverse and have evolved during extended evolutionary processes of physiological adaptations under the influence of a variety of ecological conditions and selection pressures. They harbor an enormous diversity of microbes with still unknown and probably new physiological characteristics. Besides, the surfaces of marine multicellular organisms are typically covered by a consortium of epibiotic bacteria and act as barriers, where diverse interactions between microorganisms and hosts take place. Thus, microbial diversity in the water column of the oceans and the microbial consortia on marine tissues of multicellular organisms are rich sources for isolating novel bioactive compounds and genes. Here we describe the sampling, construction of large-insert metagenomic libraries from marine habitats and exemplarily one function based screen of metagenomic clones.

  8. Purification, cDNA Cloning, and Developmental Expression of the Nodule-Specific Uricase from Phaseolus vulgaris L. 1

    Science.gov (United States)

    Sánchez, Federico; Campos, Francisco; Padilla, Jaime; Bonneville, Jean-Marc; Enríquez, Consuelo; Caput, Daniel

    1987-01-01

    Nodule-specific uricase (uricase II) from Phaseolus vulgaris L. was purified to homogeneity by chromatographic methods. Purification data indicated that uricase II is approximately 2% of the total soluble protein from mature nodules. Specific antiserum was raised and used to determine the developmental expression and for immunoselection of polysomes. Uricase II was antigenically detected early in nodule development, 2 to 3 days before nitrogen fixation. Uricase-encoding cDNA clones were isolated by hybridizing a nodule-specific pUC9 cDNA library with labeled mRNA from immunoselected polysomes and a 35,000 molecular weight uricase II-encoding cDNA from soybean. An homologous clone (pNF-UR07) was used to assess the expression pattern of the specific transcript during development. Northern-blot analysis indicated that uricase II mRNA is exclusively expressed in nodule tissue. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:16665575

  9. Building Library Collections: The Horse Is Riding Us!

    Directory of Open Access Journals (Sweden)

    Milton T. Wolf

    2003-06-01

    Full Text Available Unsound use of automation and digital idolatry are reducing access to research information.Technology should be the means not the end to better libraries. By understanding how digital information has been captured by multinational corporations,one must thread one s way through the labyrinth of mis-information clones and data corruption to a more wholesome collection of diverse points of view. Collection building will always be labor intensive, and therefore time-consuming,because intellectual judgments are involved. Giving away selection to vendors and publishers only furthers the degradation of research and ultimately threatens not only intellectual investigation but also national security. The way forward is backward to the hard work of selecting.

  10. America's Star Libraries, 2010: Top-Rated Libraries

    Science.gov (United States)

    Lyons, Ray; Lance, Keith Curry

    2010-01-01

    The "LJ" Index of Public Library Service 2010, "Library Journal"'s national rating of public libraries, identifies 258 "star" libraries. Created by Ray Lyons and Keith Curry Lance, and based on 2008 data from the IMLS, it rates 7,407 public libraries. The top libraries in each group get five, four, or three stars. All included libraries, stars or…

  11. Artificial cloning of domestic animals.

    Science.gov (United States)

    Keefer, Carol L

    2015-07-21

    Domestic animals can be cloned using techniques such as embryo splitting and nuclear transfer to produce genetically identical individuals. Although embryo splitting is limited to the production of only a few identical individuals, nuclear transfer of donor nuclei into recipient oocytes, whose own nuclear DNA has been removed, can result in large numbers of identical individuals. Moreover, clones can be produced using donor cells from sterile animals, such as steers and geldings, and, unlike their genetic source, these clones are fertile. In reality, due to low efficiencies and the high costs of cloning domestic species, only a limited number of identical individuals are generally produced, and these clones are primarily used as breed stock. In addition to providing a means of rescuing and propagating valuable genetics, somatic cell nuclear transfer (SCNT) research has contributed knowledge that has led to the direct reprogramming of cells (e.g., to induce pluripotent stem cells) and a better understanding of epigenetic regulation during embryonic development. In this review, I provide a broad overview of the historical development of cloning in domestic animals, of its application to the propagation of livestock and transgenic animal production, and of its scientific promise for advancing basic research.

  12. Cloning and characterization of a mitochondrial glyoxalase II from Brassica juncea that is upregulated by NaCl, Zn, and ABA

    International Nuclear Information System (INIS)

    Saxena, Mukesh; Bisht, Rekha; Roy, Suchandra Deb; Sopory, S.K.; Bhalla-Sarin, Neera

    2005-01-01

    A cDNA (1061 bp) Bj glyII was cloned from a mannitol induced library of Brassica juncea. It encoded a protein of 335 amino acids with a molecular weight of 36.52 kDa. The deduced amino acid sequence of the clone showed 92% and 56% identity with Pennisetum and rice glyoxalase II, respectively, and 30% identity was observed with the human glyoxalase II. Search for the identical residues revealed the presence of highly conserved THHHXDH domain which is involved in zinc binding. p-NN and pSORT analysis of this sequence revealed a N-terminal mitochondrial target peptide. The cDNA was cloned in pMAL and a fusion protein with MBP (78 kDa) was expressed in Escherichia coli. The recombinant protein was purified approximately sixfold by affinity purification on amylose column and showed its pH optima at 7.0. The K m was determined to be 120 μM using S-D-lactoylglutathione as substrate. The expression of Bj glyII under various abiotic stress conditions showed that it is upregulated by salinity, heavy metal stress, and ABA

  13. [Scientific ethics of human cloning].

    Science.gov (United States)

    Valenzuela, Carlos Y

    2005-01-01

    True cloning is fission, budding or other types of asexual reproduction. In humans it occurs in monozygote twinning. This type of cloning is ethically and religiously good. Human cloning can be performed by twinning (TWClo) or nuclear transfer (NTClo). Both methods need a zygote or a nuclear transferred cell, obtained in vitro (IVTec). They are under the IVTec ethics. IVTecs use humans (zygotes, embryos) as drugs or things; increase the risk of malformations; increase development and size of abnormalities and may cause long-term changes. Cloning for preserving extinct (or almost extinct) animals or humans when sexual reproduction is not possible is ethically valid. The previous selection of a phenotype in human cloning violates some ethical principles. NTClo for reproductive or therapeutic purposes is dangerous since it increases the risk for nucleotide or chromosome mutations, de-programming or re-programming errors, aging or malignancy of the embryo cells thus obtained.

  14. Cloning and expression of a human kidney cDNA for an α2-adrenergic receptor subtype

    International Nuclear Information System (INIS)

    Regan, J.W.; Kobilka, T.S.; Yang-Feng, T.L.; Caron, M.G.; Lefkowitz, R.J.; Kobilka, B.K.

    1988-01-01

    An α 2 -adrenergic receptor subtype has been cloned from a human kidney cDNA library using the gene for the human platelet α 2 -adrenergic receptor as a probe. The deduced amino acid sequence resembles the human platelet α 2 -adrenergic receptor and is consistent with the structure of other members of he family of guanine nucleotide-binding protein-coupled receptors. The cDNA was expressed in a mammalian cell line (COS-7), and the α 2 -adrenergic ligand [ 3 H]rauwolscine was bound. Competition curve analysis with a variety of adrenergic ligands suggests that this cDNA clone represents the α 2 B-adrenergic receptor. The gene for this receptor is on human chromosome 4, whereas the gene for the human platelet α 2 -adrenergic receptor (α 2 A) lies on chromosome 10. This ability to express the receptor in mammalian cells, free of other adrenergic receptor subtypes, should help in developing more selective α-adrenergic ligands

  15. cDNA cloning of porcine brain prolyl endopeptidase and identification of the active-site seryl residue

    Energy Technology Data Exchange (ETDEWEB)

    Rennex, D.; Hemmings, B.A.; Hofsteenge, J.; Stone, S.R. (Friedrich Miescher-Institut, Basel (Switzerland))

    1991-02-26

    Prolyl endopeptidase is a cytoplasmic serine protease. The enzyme was purified from porcine kidney, and oligonucleotides based on peptide sequences from this protein were used to isolate a cDNA clone from a porcine brain library. This clone contained the complete coding sequence of prolyl endopeptidase and encoded a polypeptide with a molecular mass of 80751 Da. The deduced amino acid sequence of prolyl endopeptidase showed no sequence homology with other known serine proteases. ({sup 3}H)Diisopropyl fluorophosphate was used to identify the active-site serine of prolyl endopeptidase. One labeled peptide was isolated and sequenced. The sequence surrounding the active-site serine was Asn-Gly-Gly-Ser-Asn-Gly-Gly. This sequence is different from the active-site sequences of other known serine proteases. This difference and the lack of overall homology with the known families of serine proteases suggest that prolyl endopeptidase represents a new type of serine protease.

  16. The ethics of human reproductive cloning.

    Science.gov (United States)

    Strong, Carson

    2005-03-01

    This article addresses the question of whether human reproductive cloning could be ethically justifiable in at least some cases involving infertile couples who would choose cloning as a way to have a genetically related child. At present, the risk of congenital anomalies constitutes a compelling argument against human reproductive cloning. The article explores whether reproductive cloning could be ethically justifiable if, at some future time, cloning becomes possible without an elevated risk of anomalies. It is argued that freedom to use cloning is a form of procreative freedom and, as such, deserves respect. All of the objections that have been raised against human reproductive cloning fall under three main categories: those that appeal to the interests of the child, those based on consequences for society, and those arising from teleological views. Objections that appeal to the child's interests are, in turn, of two main kinds: consequentialist and deontological. All of these types of objections are examined, and it is found that each involves serious problems that prevent it from being a reasonable objection in the context of the infertility cases considered. It is concluded that human reproductive cloning would be ethically justifiable in at least some cases involving infertile couples, provided that it could be performed without an elevated risk of anomalies.

  17. Featured Library: Parrish Library

    OpenAIRE

    Kirkwood, Hal P, Jr

    2015-01-01

    The Roland G. Parrish Library of Management & Economics is located within the Krannert School of Management at Purdue University. Between 2005 - 2007 work was completed on a white paper that focused on a student-centered vision for the Management & Economics Library. The next step was a massive collection reduction and a re-envisioning of both the services and space of the library. Thus began a 3 phase renovation from a 2 floor standard, collection-focused library into a single floor, 18,000s...

  18. Therapeutic cloning: The ethical limits

    International Nuclear Information System (INIS)

    Whittaker, Peter A.

    2005-01-01

    A brief outline of stem cells, stem cell therapy and therapeutic cloning is given. The position of therapeutic cloning with regard to other embryonic manipulations - IVF-based reproduction, embryonic stem formation from IVF embryos and reproductive cloning - is indicated. The main ethically challenging stages in therapeutic cloning are considered to be the nuclear transfer process including the source of eggs for this and the destruction of an embryo to provide stem cells for therapeutic use. The extremely polarised nature of the debate regarding the status of an early human embryo is noted, and some potential alternative strategies for preparing immunocompatible pluripotent stem cells are indicated

  19. Economical quantum cloning in any dimension

    International Nuclear Information System (INIS)

    Durt, Thomas; Fiurasek, Jaromir; Cerf, Nicolas J.

    2005-01-01

    The possibility of cloning a d-dimensional quantum system without an ancilla is explored, extending on the economical phase-covariant cloning machine for qubits found in Phys. Rev. A 60, 2764 (1999). We prove the impossibility of constructing an economical version of the optimal universal 1→2 cloning machine in any dimension. We also show, using an ansatz on the generic form of cloning machines, that the d-dimensional 1→2 phase-covariant cloner, which optimally clones all balanced superpositions with arbitrary phases, can be realized economically only in dimension d=2. The used ansatz is supported by numerical evidence up to d=7. An economical phase-covariant cloner can nevertheless be constructed for d>2, albeit with a slightly lower fidelity than that of the optimal cloner requiring an ancilla. Finally, using again an ansatz on cloning machines, we show that an economical version of the 1→2 Fourier-covariant cloner, which optimally clones the computational basis and its Fourier transform, is also possible only in dimension d=2

  20. No-cloning theorem on quantum logics

    International Nuclear Information System (INIS)

    Miyadera, Takayuki; Imai, Hideki

    2009-01-01

    This paper discusses the no-cloning theorem in a logicoalgebraic approach. In this approach, an orthoalgebra is considered as a general structure for propositions in a physical theory. We proved that an orthoalgebra admits cloning operation if and only if it is a Boolean algebra. That is, only classical theory admits the cloning of states. If unsharp propositions are to be included in the theory, then a notion of effect algebra is considered. We proved that an atomic Archimedean effect algebra admitting cloning operation is a Boolean algebra. This paper also presents a partial result, indicating a relation between the cloning on effect algebras and hidden variables.

  1. Generation of human scFv antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences.

    Science.gov (United States)

    Andris-Widhopf, Jennifer; Steinberger, Peter; Fuller, Roberta; Rader, Christoph; Barbas, Carlos F

    2011-09-01

    The development of therapeutic antibodies for use in the treatment of human diseases has long been a goal for many researchers in the antibody field. One way to obtain these antibodies is through phage-display libraries constructed from human lymphocytes. This protocol describes the construction of human scFv (single chain antibody fragment) libraries using a short linker (GGSSRSS) or a long linker (GGSSRSSSSGGGGSGGGG). In this method, the individual rearranged heavy- and light-chain variable regions are amplified separately and are linked through a series of overlap polymerase chain reaction (PCR) steps to give the final scFv products that are used for cloning.

  2. Phase-covariant quantum cloning of qudits

    International Nuclear Information System (INIS)

    Fan Heng; Imai, Hiroshi; Matsumoto, Keiji; Wang, Xiang-Bin

    2003-01-01

    We study the phase-covariant quantum cloning machine for qudits, i.e., the input states in a d-level quantum system have complex coefficients with arbitrary phase but constant module. A cloning unitary transformation is proposed. After optimizing the fidelity between input state and single qudit reduced density operator of output state, we obtain the optimal fidelity for 1 to 2 phase-covariant quantum cloning of qudits and the corresponding cloning transformation

  3. Cloning and Expression of Nano Body Gene against Enterotoxin B of Staphylococcus Aureus

    Directory of Open Access Journals (Sweden)

    Zahra Tavassoli

    2017-02-01

    Full Text Available Background & Objectives: Staphylococcus aureus bacteria causes many different diseases by secretion of various enterotoxins. Therefore, it is necessary to develop ways that facilitate the detection of enterotoxins. Nowadays, immunochemical methods which are based on monoclonal antibody technology are used. The heavy chain antibodies that are called VHH or Nano body were found in blood serum of the Camelidae family. The unique properties of this antibody such as their binding to small molecules like toxins make them attractive candidates for the development of immunodiagnostic tests. The present study was done to achieve a VHH molecules against Staphylococcus enterotoxin B. Materials & Methods: Freighting phage library for isolate private Nano bodies against enterotoxin B was done in previous works. Next, pCANTAB 5E vector that consists VHH, extracted from E.coli bacteria strain xl1blue, and after doing PCR process with relative primers, sub cloning in pET21a(+ as an expression vector with cut sites NdeI and XhoI was done. Transformation in E.coli bacteria strain BL21(DE3 was done. Then, the cells effected with IPTG and producing time, and other terms were optimized. Finally, the expression of the protein with SDS-PAGE and western blot techniques was evaluated. Result: For proving cloning of nano body gene in pET21a (+ vector, nucleotide sequence of gene was analyzed, and transforming to E.coli bacteria strain BL21(DE3 was successful. After inspiration, active protein in cell was seen by SDS-PAGE technique and proved by western blot. Conclusion: cloning, sub cloning, and nonabody expression were surveyed in this research. Production of this protein can help to develop new therapeutic methods and produce vaccine against enterotoxin B of Staphylococcus aureus

  4. International benchmark tests of the FENDL-1 Nuclear Data Library

    International Nuclear Information System (INIS)

    Fischer, U.

    1997-01-01

    An international benchmark validation task has been conducted to validate the fusion evaluated nuclear data library FENDL-1 through data tests against integral 14 MeV neutron experiments. The main objective of this task was to qualify the FENDL-1 working libraries for fusion applications and to elaborate recommendations for further data improvements. Several laboratories and institutions from the European Union, Japan, the Russian Federation and US have contributed to the benchmark task. A large variety of existing integral 14 MeV benchmark experiments was analysed with the FENDL-1 working libraries for continuous energy Monte Carlo and multigroup discrete ordinate calculations. Results of the benchmark analyses have been collected, discussed and evaluated. The major findings, conclusions and recommendations are presented in this paper. With regard to the data quality, it is summarised that fusion nuclear data have reached a high confidence level with the available FENDL-1 data library. With few exceptions this holds for the materials of highest importance for fusion reactor applications. As a result of the performed benchmark analyses, some existing deficiencies and discrepancies have been identified that are recommended for removal in theforthcoming FENDL-2 data file. (orig.)

  5. Synthesis and evaluation of a series of 6-chloro-4-methylumbelliferyl glycosides as fluorogenic reagents for screening metagenomic libraries for glycosidase activity.

    Science.gov (United States)

    Chen, Hong-Ming; Armstrong, Zachary; Hallam, Steven J; Withers, Stephen G

    2016-02-08

    Screening of large enzyme libraries such as those derived from metagenomic sources requires sensitive substrates. Fluorogenic glycosides typically offer the best sensitivity but typically must be used in a stopped format to generate good signal. Use of fluorescent phenols of pKa libraries yielded a "hit rate" of 1 in 60. Hits were then readily deconvoluted with the individual substrates in a single plate to identify specific activities within each clone. The use of such a collection of substrates greatly accelerates the screening process. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. The impact of R1and R3a genes on tuber resistance to late blight of the potato breeding clones

    Directory of Open Access Journals (Sweden)

    Zoteyeva Nadezhda

    2016-04-01

    Full Text Available Potato breeding clones were evaluated for resistance to late blight (agent Phytophthora infestans using tuber inoculation tests and for presence of the resistance alleles of R1 and R3a genes in polymerase chain reaction tests. Among clones tested those expressing high, moderate and low resistance were identified. The data were analysed for the impact of R1 and R3a genes on tuber resistance to late blight in tested plant material. In previous evaluations performed on smaller amount of clones the tuber resistance levels significantly depended on presence/absence of the resistance allele of R3a gene and did not depend on presence of R1 gene allele. In the current study the statistical analyses did not prove the significant difference in resistance levels depending on presence of the resistance alleles, neither of R1 gene, nor of R3a gene. Tuber resistant clones bearing R3a gene resistance alleles still noticeably prevailed over the clones bearing the alleles of R1 gene as well as over the clones bearing the no resistance alleles of both genes. In several cases the resistance of clones with detected resistance allele of R1 gene was higher compared to those derived from the same crosses and showing amplification of the allele of R3a gene or those with no resistance alleles. Clones accumulating the resistance alleles of both (R1 and R3a genes expressed high tuber resistance accompanied by necrotic reaction.

  7. Cloning and analysis of an HMG gene from the lamprey Lampetra fluviatilis

    DEFF Research Database (Denmark)

    Sharman, A C; Hay-Schmidt, Anders; Holland, P W

    1997-01-01

    Evolution has shaped the organisation of vertebrate genomes, including the human genome. To shed further light on genome history, we have cloned and analysed an HMG gene from lamprey, representing one of the earliest vertebrate lineages. Genes of the HMG1/2 family encode chromosomal proteins...

  8. Eye tracking in Library and Information Science

    DEFF Research Database (Denmark)

    Lund, Haakon

    2016-01-01

    Purpose The purpose of this paper is to present a systematic literature review of the application of eye-tracking technology within the field of library and information science. Eye-tracking technology has now reached a level of maturity, which makes the use of the technology more accessible....... Subsequently, a growing interest in employing eye tracking as a methodology within library and information science research must be anticipated. Design/methodology/approach The review follows the guidelines set in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses recommendations. Two...... reference databases are searched for relevant references: Library and Information Science Abstracts and Library, Information Science and Technology Abstracts. The main selection criteria are peer-reviewed literature that describes the experimental setting, including which eye-tracking equipment was used...

  9. Single-tube library preparation for degraded DNA

    DEFF Research Database (Denmark)

    Carøe, Christian; Gopalakrishnan, Shyam; Vinner, Lasse

    2018-01-01

    these obstacles and enable higher throughput are therefore of interest to researchers working with degraded DNA. 2.In this study, we compare four Illumina library preparation protocols, including two “single-tube” methods developed for this study with the explicit aim of improving data quality and reducing...... of chemically damaged and highly fragmented DNA molecules. In particular, the enzymatic reactions and DNA purification steps during library preparation can result in DNA template loss and sequencing biases, affecting downstream analyses. The development of library preparation methods that circumvent...... preparation time and expenses. The methods are tested on grey wolf (Canis lupus) museum specimens. 3.We found single-tube protocols increase library complexity, yield more reads that map uniquely to the reference genome, reduce processing time, and may decrease laboratory costs by 90%. 4.Given the advantages...

  10. Amino acid sequence of bovine muzzle epithelial desmocollin derived from cloned cDNA: a novel subtype of desmosomal cadherins.

    Science.gov (United States)

    Koch, P J; Goldschmidt, M D; Walsh, M J; Zimbelmann, R; Schmelz, M; Franke, W W

    1991-05-01

    Desmosomes are cell-type-specific intercellular junctions found in epithelium, myocardium and certain other tissues. They consist of assemblies of molecules involved in the adhesion of specific cell types and in the anchorage of cell-type-specific cytoskeletal elements, the intermediate-size filaments, to the plasma membrane. To explore the individual desmosomal components and their functions we have isolated DNA clones encoding the desmosomal glycoprotein, desmocollin, using antibodies and a cDNA expression library from bovine muzzle epithelium. The cDNA-deduced amino-acid sequence of desmocollin (presently we cannot decide to which of the two desmocollins, DC I or DC II, this clone relates) defines a polypeptide with a calculated molecular weight of 85,000, with a single candidate sequence of 24 amino acids sufficiently long for a transmembrane arrangement, and an extracellular aminoterminal portion of 561 amino acid residues, compared to a cytoplasmic part of only 176 amino acids. Amino acid sequence comparisons have revealed that desmocollin is highly homologous to members of the cadherin family of cell adhesion molecules, including the previously sequenced desmoglein, another desmosome-specific cadherin. Using riboprobes derived from cDNAs for Northern-blot analyses, we have identified an mRNA of approximately 6 kb in stratified epithelia such as muzzle epithelium and tongue mucosa but not in two epithelial cell culture lines containing desmosomes and desmoplakins. The difference may indicate drastic differences in mRNA concentration or the existence of cell-type-specific desmocollin subforms. The molecular topology of desmocollin(s) is discussed in relation to possible functions of the individual molecular domains.

  11. Effect of TH-lines and clones on the growth and differentiation of B cell clones in microculture.

    Science.gov (United States)

    Kotloff, D B; Cebra, J J

    1988-02-01

    Antibody isotype expression by B cell clones was analyzed using in vitro microcultures containing low numbers of hapten-gelatin-enriched B cells and higher numbers of hemocyanin-specific helper T cell lines or clones. Twenty-eight to sixty-three percent of clones grown in microculture with haptenated hemocyanin and T cells from established lines expressed IgG and/or IgA isotypes in random mixtures, almost always accompanied by IgM. Helper T cells from hemocyanin-specific clones also supported the expression of non-IgM isotypes by the B cell clones, suggesting that a single specificity of T cell can provide sufficient growth and differentiation factors for the display of isotype switching. A positive correlation between the antibody output of clones and the expression of non-IgM isotypes indicated that the switching process may be associated with cell division. Although memory B cells that give clones expressing IgG and/or IgA in the absence of IgM are also enriched on haptenated gelatin, they are not stimulable under conditions of this microculture assay.

  12. Multigroup cross section library; WIMS library

    International Nuclear Information System (INIS)

    Kannan, Umasankari

    2000-01-01

    The WIMS library has been extensively used in thermal reactor calculations. This multigroup constants library was originally developed from the UKNDL in the late 60's and has been updated in 1986. This library has been distributed with the WIMS-D code by NEA data bank. The references to WIMS library in literature are the 'old' which is the original as developed by the AEA Winfrith and the 'new' which is the current 1986 WIMS library. IAEA has organised a CRP where a new and fully updated WIMS library will soon be available. This paper gives an overview of the definitions of the group constants that go into any basic nuclear data library used for reactor calculations. This paper also outlines the contents of the WIMS library and some of its shortcomings

  13. Comparative Genome Analyses of Streptococcus suis Isolates from Endocarditis Demonstrate Persistence of Dual Phenotypic Clones.

    Science.gov (United States)

    Tohya, Mari; Watanabe, Takayasu; Maruyama, Fumito; Arai, Sakura; Ota, Atsushi; Athey, Taryn B T; Fittipaldi, Nahuel; Nakagawa, Ichiro; Sekizaki, Tsutomu

    2016-01-01

    Many bacterial species coexist in the same niche as heterogeneous clones with different phenotypes; however, understanding of infectious diseases by polyphenotypic bacteria is still limited. In the present study, encapsulation in isolates of the porcine pathogen Streptococcus suis from persistent endocarditis lesions was examined. Coexistence of both encapsulated and unencapsulated S. suis isolates was found in 26 out of 59 endocarditis samples. The isolates were serotype 2, and belonged to two different sequence types (STs), ST1 and ST28. The genomes of each of the 26 pairs of encapsulated and unencapsulated isolates from the 26 samples were sequenced. The data showed that each pair of isolates had one or more unique nonsynonymous mutations in the cps gene, and the encapsulated and unencapsulated isolates from the same samples were closest to each other. Pairwise comparisons of the sequences of cps genes in 7 pairs of encapsulated and unencapsulated isolates identified insertion/deletions (indels) ranging from one to 104 bp in different cps genes of unencapsulated isolates. Capsule expression was restored in a subset of unencapsulated isolates by complementation in trans with cps expression vectors. Examination of gene content common to isolates indicated that mutation frequency was higher in ST28 pairs than in ST1 pairs. Genes within mobile genetic elements were mutation hot spots among ST28 isolates. Taken all together, our results demonstrate the coexistence of dual phenotype (encapsulated and unencapsulated) bacterial clones and suggest that the dual phenotypes arose independently in each farm by means of spontaneous mutations in cps genes.

  14. Comparative Genome Analyses of Streptococcus suis Isolates from Endocarditis Demonstrate Persistence of Dual Phenotypic Clones.

    Directory of Open Access Journals (Sweden)

    Mari Tohya

    Full Text Available Many bacterial species coexist in the same niche as heterogeneous clones with different phenotypes; however, understanding of infectious diseases by polyphenotypic bacteria is still limited. In the present study, encapsulation in isolates of the porcine pathogen Streptococcus suis from persistent endocarditis lesions was examined. Coexistence of both encapsulated and unencapsulated S. suis isolates was found in 26 out of 59 endocarditis samples. The isolates were serotype 2, and belonged to two different sequence types (STs, ST1 and ST28. The genomes of each of the 26 pairs of encapsulated and unencapsulated isolates from the 26 samples were sequenced. The data showed that each pair of isolates had one or more unique nonsynonymous mutations in the cps gene, and the encapsulated and unencapsulated isolates from the same samples were closest to each other. Pairwise comparisons of the sequences of cps genes in 7 pairs of encapsulated and unencapsulated isolates identified insertion/deletions (indels ranging from one to 104 bp in different cps genes of unencapsulated isolates. Capsule expression was restored in a subset of unencapsulated isolates by complementation in trans with cps expression vectors. Examination of gene content common to isolates indicated that mutation frequency was higher in ST28 pairs than in ST1 pairs. Genes within mobile genetic elements were mutation hot spots among ST28 isolates. Taken all together, our results demonstrate the coexistence of dual phenotype (encapsulated and unencapsulated bacterial clones and suggest that the dual phenotypes arose independently in each farm by means of spontaneous mutations in cps genes.

  15. Comparative Genome Analyses of Streptococcus suis Isolates from Endocarditis Demonstrate Persistence of Dual Phenotypic Clones

    Science.gov (United States)

    Tohya, Mari; Watanabe, Takayasu; Maruyama, Fumito; Arai, Sakura; Ota, Atsushi; Athey, Taryn B. T.; Fittipaldi, Nahuel; Nakagawa, Ichiro; Sekizaki, Tsutomu

    2016-01-01

    Many bacterial species coexist in the same niche as heterogeneous clones with different phenotypes; however, understanding of infectious diseases by polyphenotypic bacteria is still limited. In the present study, encapsulation in isolates of the porcine pathogen Streptococcus suis from persistent endocarditis lesions was examined. Coexistence of both encapsulated and unencapsulated S. suis isolates was found in 26 out of 59 endocarditis samples. The isolates were serotype 2, and belonged to two different sequence types (STs), ST1 and ST28. The genomes of each of the 26 pairs of encapsulated and unencapsulated isolates from the 26 samples were sequenced. The data showed that each pair of isolates had one or more unique nonsynonymous mutations in the cps gene, and the encapsulated and unencapsulated isolates from the same samples were closest to each other. Pairwise comparisons of the sequences of cps genes in 7 pairs of encapsulated and unencapsulated isolates identified insertion/deletions (indels) ranging from one to 104 bp in different cps genes of unencapsulated isolates. Capsule expression was restored in a subset of unencapsulated isolates by complementation in trans with cps expression vectors. Examination of gene content common to isolates indicated that mutation frequency was higher in ST28 pairs than in ST1 pairs. Genes within mobile genetic elements were mutation hot spots among ST28 isolates. Taken all together, our results demonstrate the coexistence of dual phenotype (encapsulated and unencapsulated) bacterial clones and suggest that the dual phenotypes arose independently in each farm by means of spontaneous mutations in cps genes. PMID:27433935

  16. Library Web Sites in Pakistan: An Analysis of Content

    Science.gov (United States)

    Qutab, Saima; Mahmood, Khalid

    2009-01-01

    Purpose: The purpose of this paper is to investigate library web sites in Pakistan, to analyse their content and navigational strengths and weaknesses and to give recommendations for developing better web sites and quality assessment studies. Design/methodology/approach: Survey of web sites of 52 academic, special, public and national libraries in…

  17. Human reproductive cloning: a conflict of liberties.

    Science.gov (United States)

    Havstad, Joyce C

    2010-02-01

    Proponents of human reproductive cloning do not dispute that cloning may lead to violations of clones' right to self-determination, or that these violations could cause psychological harms. But they proceed with their endorsement of human reproductive cloning by dismissing these psychological harms, mainly in two ways. The first tactic is to point out that to commit the genetic fallacy is indeed a mistake; the second is to invoke Parfit's non-identity problem. The argument of this paper is that neither approach succeeds in removing our moral responsibility to consider and to prevent psychological harms to cloned individuals. In fact, the same commitment to personal liberty that generates the right to reproduce by means of cloning also creates the need to limit that right appropriately. Discussion of human reproductive cloning ought to involve a careful and balanced consideration of both the relevant aspects of personal liberty - the parents' right to reproductive freedom and the cloned child's right to self-determination.

  18. Changes in the gut microbiota of cloned and non-cloned control pigs during development of obesity: gut microbiota during development of obesity in cloned pigs

    DEFF Research Database (Denmark)

    Pedersen, Rebecca; Andersen, Anders Daniel; Mølbak, Lars

    2013-01-01

    Background Obesity induced by a high-caloric diet has previously been associated with changes in the gut microbiota in mice and in humans. In this study, pigs were cloned to minimize genetic and biological variation among the animals with the aim of developing a controlled metabolomic model...... suitable for a diet-intervention study. Cloning of pigs may be an attractive way to reduce genetic influences when investigating the effect of diet and obesity on different physiological sites. The aim of this study was to assess and compare the changes in the composition of the gut microbiota of cloned vs....... non-cloned pigs during development of obesity by a high-fat/high-caloric diet. Furthermore, we investigated the association between diet-induced obesity and the relative abundance of the phyla Firmicutes and Bacteroidetes in the fecal-microbiota. The fecal microbiota from obese cloned (n = 5) and non...

  19. Probabilistic cloning of three symmetric states

    International Nuclear Information System (INIS)

    Jimenez, O.; Bergou, J.; Delgado, A.

    2010-01-01

    We study the probabilistic cloning of three symmetric states. These states are defined by a single complex quantity, the inner product among them. We show that three different probabilistic cloning machines are necessary to optimally clone all possible families of three symmetric states. We also show that the optimal cloning probability of generating M copies out of one original can be cast as the quotient between the success probability of unambiguously discriminating one and M copies of symmetric states.

  20. Microdissection and molecular manipulation of single chromosomes in woody fruit trees with small chromosomes using pomelo (Citrus grandis) as a model. I. Construction of single chromosomal DNA libraries.

    Science.gov (United States)

    Huang, D; Wu, W; Zhou, Y; Hu, Z; Lu, L

    2004-05-01

    Construction of single chromosomal DNA libraries by means of chromosome microdissection and microcloning will be useful for genomic research, especially for those species that have not been extensively studied genetically. Application of the technology of microdissection and microcloning to woody fruit plants has not been reported hitherto, largely due to the generally small sizes of metaphase chromosomes and the difficulty of chromosome preparation. The present study was performed to establish a method for single chromosome microdissection and microcloning in woody fruit species using pomelo as a model. The standard karyotype of a pomelo cultivar ( Citrus grandis cv. Guanxi) was established based on 20 prometaphase photomicrographs. According to the standard karyotype, chromosome 1 was identified and isolated with fine glass microneedles controlled by a micromanipulator. DNA fragments ranging from 0.3 kb to 2 kb were acquired from the isolated single chromosome 1 via two rounds of PCR mediated by Sau3A linker adaptors and then cloned into T-easy vectors to generate a DNA library of chromosome 1. Approximately 30,000 recombinant clones were obtained. Evaluation based on 108 randomly selected clones showed that the sizes of the cloned inserts varied from 0.5 kb to 1.5 kb with an average of 860 bp. Our research suggests that microdissection and microcloning of single small chromosomes in woody plants is feasible.

  1. Immune TB Antibody Phage Display Library as a Tool To Study B Cell Immunity in TB Infections.

    Science.gov (United States)

    Hamidon, Nurul Hamizah; Suraiya, Siti; Sarmiento, Maria E; Acosta, Armando; Norazmi, Mohd Nor; Lim, Theam Soon

    2018-03-01

    B cells and in particular antibodies has always played second fiddle to cellular immunity in regard to tuberculosis (TB). However, recent studies has helped position humoral immunity especially antibodies back into the foray in relation to TB immunity. Therefore, the ability to correlate the natural antibody responses of infected individuals toward TB antigens would help strengthen this concept. Phage display is an intriguing approach that can be utilized to study antibody-mediated responses against a particular infection via harvesting the B cell repertoire from infected individuals. The development of disease-specific antibody libraries or immune libraries is useful to better understand antibody-mediated immune responses against specific disease antigens. This study describes the generation of an immune single-chain variable fragment (scFv) library derived from TB-infected individuals. The immune library with an estimated diversity of 10 9 independent clones was then applied for the identification of monoclonal antibodies against Mycobacterium tuberculosis α-crystalline as a model antigen. Biopanning of the library isolated three monoclonal antibodies with unique gene usage. This strengthens the role of antibodies in TB immunity in addition to the role played by cellular immunity. The developed library can be applied against other TB antigens and aid antibody-derived TB immunity studies in the future.

  2. Discussion on construction of scientific and technological digital library in nuclear industry

    International Nuclear Information System (INIS)

    Yin Huilan

    2010-01-01

    With the rapid development of digital and network technology, traditional libraries have been unable to meet the needs of the times. Digital libraries will gradually take the place of traditional libraries. Under the circumstances, how will the libraries of the enterprises in nuclear industry face this transformation? This paper gives the brief descriptions and comparative analyses in the four aspects: the definition of the digital library, the meaning of nuclear scientific and technological digital library, the characteristics of the digital library, and major problems in the construction of nuclear scientific and technological digital library that should be solved. Therefore, setting up the digital library is very important. At the same time, it's very necessary and urgent for the libraries of the enterprises in nuclear industry to establish nuclear scientific and technological digital library. (author)

  3. BIOETHICS AND HUMAN CLONING

    Directory of Open Access Journals (Sweden)

    Željko Kaluđerović

    2011-12-01

    Full Text Available In this paper the authors analyze the process of negotiating and beginning of the United Nations Declaration on Human Cloning as well as the paragraphs of the very Declaration. The negotiation was originally conceived as a clear bioethical debate that should have led to a general agreement to ban human cloning. However, more often it had been discussed about human rights, cultural, civil and religious differences between people and about priorities in case of eventual conflicts between different value systems. In the end, a non-binding Declaration on Human Cloning had been adopted, full of numerous compromises and ambiguous formulations, that relativized the original intention of proposer states. According to authors, it would have been better if bioethical discussion and eventual regulations on cloning mentioned in the following text had been left over to certain professional bodies, and only after the public had been fully informed about it should relevant supranational organizations have taken that into consideration.

  4. Isolation and partial characterization of peripheral blood CD4+ T cell clones expressing γδT cell receptors

    International Nuclear Information System (INIS)

    Kyoizumi, Seishi; Akiyama, Mitoshi; Hirai, Yuko; Kusunoki, Yoichiro.

    1990-06-01

    Rare T cell clones bearing both CD4 and T cell receptors (TCRγ and TCRδ) were obtained from human peripheral blood by cell sorting using anti-CD4 and anti-TCRδ1 antibodies. All the clones established were reactive with anti-TCRγδ1 antibody, whereas only about 20 % of the clones showed reactivity with anti-δTCS1 antibody. Unlike CD4 + T cells bearing TCRαβ, all the clones tested were lectin-dependent and showed CD3 antibody-redirected cytolytic activity. About 60 % exhibited natural killer cell-like activity. Immunoprecipitation analysis of TCRγδ showed that each clone expressed either a disulfide-linked or nondisulfide-linked heterodimer consisting of 37-44 kilodalton TCRγ and TCRδ chains. Southern blot analyses of TCRγ and TCRδ genes revealed some identical rearrangement patterns, suggesting the limited heterogeneity of CD4 + TCRγδ + T cells in peripheral blood. (author)

  5. Cloning and characterization of a cDNA encoding topoisomerase II in pea and analysis of its expression in relation to cell proliferation.

    Science.gov (United States)

    Reddy, M K; Nair, S; Tewari, K K; Mudgil, Y; Yadav, B S; Sopory, S K

    1999-09-01

    We have isolated and sequenced four overlapping cDNA clones to identify the full-length cDNA for topoisomerase II (PsTopII) from pea. Using degenerate primers, based on the conserved amino acid sequences of other eukaryotic type II topoisomerases, a 680 bp fragment was PCR-amplified with pea cDNA as template. This fragment was used as a probe to screen an oligo-dT-primed pea cDNA library. A partial cDNA clone was isolated that was truncated at the 3' end. RACE-PCR was employed to isolate the remaining portion of the gene. The total size of PsTopII is 4639 bp with an open reading frame of 4392 bp. The deduced amino acid sequence shows a strong homology to other eukaryotic topoisomerase II (topo II) at the N-terminus end. The topo II transcript was abundant in proliferative tissues. We also show that the level of topo II transcripts could be stimulated by exogenous application of growth factors that induced proliferation in vitro cultures. Light irradiation to etiolated tissue strongly stimulated the expression of topo II. These results suggest that topo II gene expression is up-regulated in response to light and hormones and correlates with cell proliferation. Besides, we have also isolated and analysed the 5'-flanking region of the pea TopII gene. This is first report on the isolation of a putative promoter for topoisomerase II from plants.

  6. Pennsylvania Academic Libraries and Student Retention and Graduation: A Preliminary Investigation with Confusing Results

    Directory of Open Access Journals (Sweden)

    Gregory A. Crawford

    2014-11-01

    Full Text Available This study examined the relationships between specific institutional financial variables and two library-related variables on graduation and retention rates for colleges and universities through correlations and multiple regression analysis. The analyses used data for Pennsylvania colleges and universities that were extracted from the Integrated Postsecondary Educational Data System (IPEDS and the Academic Libraries Survey (ALS.  All analyses were run using IBM SPSS software. The correlations showed that both library expenses per student and library use per student were significantly correlated with both graduation and retention rates. In contrast, the multiple regression results showed that neither library budgets nor library use had significant effects on either graduation rates or retention rates. As would be expected, instructional expenses per student had the highest correlation with both graduation and retention and also yielded the strongest coefficient in the resulting regression equations.

  7. Construction and characterization of a bacterial artificial chromosome library of the causal agent of Black Sigatoka fungal leaf spot disease of banana and plantain, Mycosphaerella fijiensis.

    Science.gov (United States)

    Canto-Canché, Blondy; Guillén-Maldonado, Diana Karina; Peraza-Echeverría, Leticia; Conde-Ferráez, Laura; James-Kay, Andrew

    2007-05-01

    A bacterial artificial chromosome library of the causal agent of the Black Sigatoka leaf spot disease of banana and plantain, Mycosphaerella fijiensis, has been constructed using a non-sphaeroplasting technique and characterized using both homologous and heterologous probes. After first and a second size selection of PFGE-fractionated DNA, a ligation was obtained using a 1:4 molar ratio (insert:vector). One hundred random clones were analyzed, and the mean insert size was estimated to be 90 kb. The range of the insert sizes was between 40 and 160 kb. The highest percentage of inserts belonged to the range between 80 and 100 kb; 32% of the inserts had 2 or 3 internal NotI sites. This library consists of 1920 clones, if the genomic size is at least 35 Mb, then this represents 4.9 x genome equivalents, which was supported by hybridization results with homologous and heterologous probes.

  8. cDNA Cloning, expression and characterization of an allergenic 60s ribosomal protein of almond (prunus dulcis).

    Science.gov (United States)

    Abolhassani, Mohsen; Roux, Kenneth H

    2009-06-01

    Tree nuts, including almond (prunus dulcis) are a source of food allergens often associated with life-threatening allergic reactions in susceptible individuals. Although the proteins in almonds have been biochemically characterized, relatively little has been reported regarding the identity of the allergens involved in almond sensitivity. The present study was undertaken to identify the allergens of the almond by cDNA library approach. cDNA library of almond seeds was constructed in Uni-Zap XR lamda vector and expressed in E. coli XL-1 blue. Plaques were immunoscreened with pooled sera of allergic patients. The cDNA clone reacting significantly with specific IgE antibodies was selected and subcloned and subsequently expressed in E. coli. The amino acids deducted from PCR product of clone showed homology to 60s acidic ribosomal protein of almond. The expressed protein was 11,450 Dalton without leader sequence. Immunoreactivity of the recombinant 60s ribosomal protein (r60sRP) was evaluated with dot blot analysis using pooled and individual sera of allergic patients. The data showed that r60sRP and almond extract (as positive control) possess the ability to bind the IgE antibodies. The results showed that expressed protein is an almond allergen.Whether this r60sRP represents a major allergen of almond needs to be further studied which requires a large number of sera from the almond atopic patients and also need to determine the IgE-reactive frequencies of each individual allergen.

  9. Generation of thermostable Moloney murine leukemia virus reverse transcriptase variants using site saturation mutagenesis library and cell-free protein expression system.

    Science.gov (United States)

    Katano, Yuta; Li, Tongyang; Baba, Misato; Nakamura, Miyo; Ito, Masaaki; Kojima, Kenji; Takita, Teisuke; Yasukawa, Kiyoshi

    2017-12-01

    We attempted to increase the thermostability of Moloney murine leukemia virus (MMLV) reverse transcriptase (RT). The eight-site saturation mutagenesis libraries corresponding to Ala70-Arg469 in the whole MMLV RT (Thr24-Leu671), in each of which 1 out of 50 amino acid residues was replaced with other amino acid residue, were constructed. Seven-hundred and sixty eight MMLV RT clones were expressed using a cell-free protein expression system, and their thermostabilities were assessed by the temperature of thermal treatment at which they retained cDNA synthesis activity. One clone D200C was selected as the most thermostable variant. The highest temperature of thermal treatment at which D200C exhibited cDNA synthesis activity was 57ºC, which was higher than for WT (53ºC). Our results suggest that a combination of site saturation mutagenesis library and cell-free protein expression system might be useful for generation of thermostable MMLV RT in a short period of time for expression and selection.

  10. Paramyosin from the parasitic mite Sarcoptes scabiei: cDNA cloning and heterologous expression.

    Science.gov (United States)

    Mattsson, J G; Ljunggren, E L; Bergström, K

    2001-05-01

    The burrowing mite Sarcoptes scabiei is the causative agent of the highly contagious disease sarcoptic mange or scabies. So far, there is no in vitro propagation system for S. scabiei available, and mites used for various purposes must be isolated from infected hosts. Lack of parasite-derived material has limited the possibilities to study several aspects of scabies, including pathogenesis and immunity. It has also hampered the development of high performance serological assays. We have now constructed an S. scabiei cDNA expression library with mRNA purified from mites isolated from red foxes. Immunoscreening of the library enabled us to clone a full-length cDNA coding for a 102.5 kDa protein. Sequence similarity searches identified the protein as a paramyosin. Recombinant S. scabiei paramyosin expressed in Escherichia coli was recognized by sera from dogs and swine infected with S. scabiei. We also designed a small paramyosin construct of about 17 kDa that included the N-terminal part, an evolutionary variable part of the helical core, and the C-terminal part of the molecule. The miniaturized protein was efficiently expressed in E. coli and was recognized by sera from immunized rabbits. These data demonstrate that the cDNA library can assist in the isolation of important S. scabiei antigens and that recombinant proteins can be useful for the study of scabies.

  11. Purification, reactivity with IgE and cDNA cloning of parvalbumin as the major allergen of mackerels.

    Science.gov (United States)

    Hamada, Y; Tanaka, H; Ishizaki, S; Ishida, M; Nagashima, Y; Shiomi, K

    2003-08-01

    Three species of mackerels (Scomber japonicus, S. australasicus and S. scombrus) are widely consumed and considered to be most frequently involved in incidents of IgE-mediated fish allergy in Japan. In this study, parvalbumin, a possible candidate for the major allergen, was purified from the white muscle of three species of mackerels by gel filtration on Sephadex G-75 and reverse-phase HPLC on TSKgel ODS-120T. All the purified preparations from three species gave a single band of about 11 kDa and were clearly identified as parvalbumins by analyses of their partial amino acid sequences. In ELISA experiments, four of five sera from fish-allergic patients reacted to all the purified parvalbumins, demonstrating that parvalbumin is the major allergen in common with the mackerels. Antigenic cross-reactivity among the mackerel parvalbumins was also established by ELISA inhibition experiments. A cDNA library was constructed from the white muscle of S. japonicus and the cDNA encoding parvalbumin was cloned. The amino acid sequence translated from the nucleotide sequence revealed that the S. japonicus parvalbumin is composed of 108 residues, being a member of beta-type parvalbumins.

  12. The First Human Cloned Embryo.

    Science.gov (United States)

    Cibelli, Jose B.; Lanza, Robert P.; West, Michael D.; Ezzell, Carol

    2002-01-01

    Describes a process known as parthenogenesis which produces cloned, early-stage embryos and human embryos generated only from eggs. Speculates that this technology puts therapeutic cloning within reach. (DDR)

  13. Display of a Maize cDNA library on baculovirus infected insect cells

    Directory of Open Access Journals (Sweden)

    Jones Ian M

    2008-08-01

    Full Text Available Abstract Background Maize is a good model system for cereal crop genetics and development because of its rich genetic heritage and well-characterized morphology. The sequencing of its genome is well advanced, and new technologies for efficient proteomic analysis are needed. Baculovirus expression systems have been used for the last twenty years to express in insect cells a wide variety of eukaryotic proteins that require complex folding or extensive posttranslational modification. More recently, baculovirus display technologies based on the expression of foreign sequences on the surface of Autographa californica (AcMNPV have been developed. We investigated the potential of a display methodology for a cDNA library of maize young seedlings. Results We constructed a full-length cDNA library of young maize etiolated seedlings in the transfer vector pAcTMVSVG. The library contained a total of 2.5 × 105 independent clones. Expression of two known maize proteins, calreticulin and auxin binding protein (ABP1, was shown by western blot analysis of protein extracts from insect cells infected with the cDNA library. Display of the two proteins in infected insect cells was shown by selective biopanning using magnetic cell sorting and demonstrated proof of concept that the baculovirus maize cDNA display library could be used to identify and isolate proteins. Conclusion The maize cDNA library constructed in this study relies on the novel technology of baculovirus display and is unique in currently published cDNA libraries. Produced to demonstrate proof of principle, it opens the way for the development of a eukaryotic in vivo display tool which would be ideally suited for rapid screening of the maize proteome for binding partners, such as proteins involved in hormone regulation or defence.

  14. Construction of a normalized full-length cDNA library of cephalopod Amphioctopus fangsiao and development of microsatellite markers

    Science.gov (United States)

    Feng, Yanwei; Liu, Wenfen; Xu, Xin; Yang, Jianmin; Wang, Weijun; Wei, Xiumei; Liu, Xiangquan; Sun, Guohua

    2017-10-01

    Amphioctopus fangsiao is one of the most economically important species and has been considered to be a candidate for aquaculture. In order to facilitate its fine-scale genetic analyses, we constructed a normalized full-length library successfully and developed a set of microsatellite markers in this study. The normalized full-length library had a storage capacity of 6.9×105 independent clones. The recombination efficiency was 95% and the average size of inserted fragments was longer than 1000 bp. A total of 3440 high quality ESTs were obtained, which were assembled into 1803 unigenes. Of these unigenes, 450 (25%) were assigned into 33 Gene Ontology terms, 576 (31.9%) into 153 Kyoto Encyclopedia of Genes and Genomes pathways, and 275 (15.3%) into 22 Clusters of Orthologous Groups. Seventy-six polymorphic microsatellite markers were identified. The number of alleles per locus ranged from 4 to 17, and the observed and expected heterozygosities varied between 0.167 and 0.967 and between 0.326 and 0.944, respectively. Twelve loci were significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction and no linkage disequilibrium was found between different loci. This study provided not only a useful resource for the isolation of the functional genes, but also a set of informative microsatellites for the assessment of population structure and conservation genetics of A. fangsiao.

  15. Automated cloning methods.; TOPICAL

    International Nuclear Information System (INIS)

    Collart, F.

    2001-01-01

    Argonne has developed a series of automated protocols to generate bacterial expression clones by using a robotic system designed to be used in procedures associated with molecular biology. The system provides plate storage, temperature control from 4 to 37 C at various locations, and Biomek and Multimek pipetting stations. The automated system consists of a robot that transports sources from the active station on the automation system. Protocols for the automated generation of bacterial expression clones can be grouped into three categories (Figure 1). Fragment generation protocols are initiated on day one of the expression cloning procedure and encompass those protocols involved in generating purified coding region (PCR)

  16. Croatian library leaders’ views on (their library quality

    Directory of Open Access Journals (Sweden)

    Kornelija Petr Balog

    2014-04-01

    Full Text Available The purpose of this paper is to determine and describe the library culture in Croatian public libraries. Semi-structured interviews with 14 library directors (ten public and four academic were conducted. The tentative discussion topics were: definition of quality, responsibility for quality, satisfaction with library services, familiarization with user perspective of library and librarians, monitoring of user expectations and opinions. These interviews incorporate some of the findings of the project Evaluation of library and information services: public and academic libraries. The project investigates library culture in Croatian public and academic libraries and their preparedness for activities of performance measurement. The interviews reveal that library culture has changed positively in the past few years and that library leaders have positive attitude towards quality and evaluation activities. Library culture in Croatian libraries is a relatively new concept and as such was not actively developed and/or created. This article looks into the library culture of Croatian libraries, but at the same time investigates whether there is any trace of culture of assessment in them. Also, this article brings the latest update on views, opinions and atmosphere in Croatian public and academic libraries.

  17. Wine Chemical Composition and Radical Scavenging Activity of Some Cabernet Franc Clones.

    Science.gov (United States)

    Popovic-Djordjevic, Jelena; Pejin, Boris; Dramicanin, Aleksandra; Jovic, Sonja; Vujovic, Dragan; Zunic, Dragoljub; Ristic, Renata

    2017-01-01

    Three clones of Cabernet Franc (Nos. 02, 010 and 012) were selected in the last phase of clonal selection in Serbia. Wines made from each clone were assessed for quality parameters and taste during five consecutive vintages (2008-2012) and compared to the standard. The wine quality was determined based on the following parameters: alcohol, total extract, anthocyanins, tannins, pH, titratable acidity, volatile acidity, aldehydes, esters and reducing sugars, relative density, ash, colour, tonality, and tasting score. In the last year of the study, grapes and wines of Cabernet Franc clones and a standard were subjected to a chemical analysis of their phenolic composition, resveratrol and radical scavenging activity. In the last year of the study, grapes and wines of Cabernet Franc clones and a standard were subjected to a chemical analysis of their phenolic composition, resveratrol and radical scavenging activity. Chemical analyses of grapes and wines along with sensory and radical scavenging activity evaluations were done according to the standard procedures. The wines of the clone No. 010 showed some superior properties compared to the other two clones and the standard; in five-year period the average concentration of anthocyanins (179±3.8 mg/L) and polyphenolics (1.85±0.02 g/L) was significantly higher than in wines of clones and the standard, (168-173 mg/L and 1.63-1.74 g/L for anthocyanins and phenolics, respectively). Furthermore, the same clone had a higher alcohol content (13.97±0.03%) in each year of the study, which indicated that it ripened faster than other clones (13.06-13.08 %) and compared to the standard (13.04±0.07%). This finding suggested that the clone No. 010 could possibly have a significant economic impact and further increase popularity of Cabernet Franc in a cooler climate viticultural region. It was also found to have the highest contents of aldehydes (488±1.54 mg/L) and esters (322±0.71 mg/L) compared to aldehydes (452-467 mg/L) and

  18. Cloning and Expression Vector Construction of Glutamate Decarboxylase Gene from Lactobacillus Plantarum

    Directory of Open Access Journals (Sweden)

    B Arabpour

    2016-06-01

    Full Text Available BACKGROUND AND OBJECTIVE: Gamma-aminobutyric acid (GABA is a four-carbon non-protein amino acid used in the treatment of hypertension, diabetes, inflammation, and depression. GABA is synthesized by glutamic acid decarboxylase (GAD enzyme in many organisms, including bacteria. Therefore, cloning of this enzyme is essential to the optimization of GABA production. This study aimed to clone and construct the expression vector of GAD gene from Lactobacillus plantarum PTCC 1058 bacterium. METHODS: In this experimental study, we investigated the morphological, biochemical, genetic and 16s rDNA sequencing of L. plantarum PTCC 1058 strain. Genomic DNA of the bacterium was isolated and amplified using the GAD gene via polymerase chain reaction (PCR. Afterwards, the gene was inserted into the pJET1.2/blunt cloning vector and subcloned in vector pET32a. Plasmid pET32a-gad expression vector was transformed in Escherichia coli BL21 strain, and protein expression was assessed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE. FINDINGS: Morphological, biochemical and genetic analyses of 16s rDNA sequencing indicated that the studied substrain was of the L. plantarum strain. In addition, results of nucleotide sequencing of the fragmented segment via PCR showed the presence of GAD gene. Results of colony PCR and SDS-PAGE analysis confirmed the accuracy of the cloning and gene expression of the recombinant Escherichia coli BL21 strain. CONCLUSION: According to the results of this study, cloning of GAD gene from L. plantarum PTCC 1058 was successful. These cloned genes could grow rapidly in prokaryotic and eukaryotic systems and be used in cost-effective culture media and even non-recyclable waste.

  19. Cloning and Molecular Analyses of a Gibberellin 20-Oxidase Gene Expressed Specifically in Developing Seeds of Watermelon1

    Science.gov (United States)

    Kang, Hong-Gyu; Jun, Sung-Hoon; Kim, Junyul; Kawaide, Hiroshi; Kamiya, Yuji; An, Gynheung

    1999-01-01

    To understand the biosynthesis and functional role of gibberellins (GAs) in developing seeds, we isolated Cv20ox, a cDNA clone from watermelon (Citrullus lanatus) that shows significant amino acid homology with GA 20-oxidases. The complementary DNA clone was expressed in Escherichia coli as a fusion protein, which oxidized GA12 at C-20 to the C19 compound GA9, a precursor of bioactive GAs. RNA-blot analysis showed that the Cv20ox gene was expressed specifically in developing seeds. The gene was strongly expressed in the integument tissues, and it was also expressed weakly in inner seed tissues. In parthenocarpic fruits induced by 1-(2-chloro-4-pyridyl)-3-phenylurea treatment, the expression pattern of Cv20ox did not change, indicating that the GA 20-oxidase gene is expressed primarily in the maternal cells of developing seeds. The promoter of Cv20ox was isolated and fused to the β-glucuronidase (GUS) gene. In a transient expression system, β-glucuronidase staining was detectable only in the integument tissues of developing watermelon seeds. PMID:10517828

  20. Skewed X-inactivation in cloned mice

    International Nuclear Information System (INIS)

    Senda, Sho; Wakayama, Teruhiko; Yamazaki, Yukiko; Ohgane, Jun; Hattori, Naka; Tanaka, Satoshi; Yanagimachi, Ryuzo; Shiota, Kunio

    2004-01-01

    In female mammals, dosage compensation for X-linked genes is accomplished by inactivation of one of two X chromosomes. The X-inactivation ratio (a percentage of the cells with inactivated maternal X chromosomes in the whole cells) is skewed as a consequence of various genetic mutations, and has been observed in a number of X-linked disorders. We previously reported that phenotypically normal full-term cloned mouse fetuses had loci with inappropriate DNA methylation. Thus, cloned mice are excellent models to study abnormal epigenetic events in mammalian development. In the present study, we analyzed X-inactivation ratios in adult female cloned mice (B6C3F1). Kidneys of eight naturally produced controls and 11 cloned mice were analyzed. Although variations in X-inactivation ratio among the mice were observed in both groups, the distributions were significantly different (Ansary-Bradley test, P < 0.01). In particular, 2 of 11 cloned mice showed skewed X-inactivation ratios (19.2% and 86.8%). Similarly, in intestine, 1 of 10 cloned mice had a skewed ratio (75.7%). Skewed X-inactivation was observed to various degrees in different tissues of different individuals, suggesting that skewed X-inactivation in cloned mice is the result of secondary cell selection in combination with stochastic distortion of primary choice. The present study is the first demonstration that skewed X-inactivation occurs in cloned animals. This finding is important for understanding both nuclear transfer technology and etiology of X-linked disorders

  1. Phage Display Breast Carcinoma cDNA Libraries: Isolation of Clones Which Specifically Bind to Membrane Glycoproteins, Mucins, and Endothelial Cell Surface

    National Research Council Canada - National Science Library

    Yamamoto, Fumiichiro

    2000-01-01

    .... Using blood- group H-expressing glycoprotein fraction as bait, we observed enrichment of phage clones expressing sequences from galectin-3, a lectin with an affinity with the blood-group substance...

  2. Thermal reactor benchmark testing of 69 group library

    International Nuclear Information System (INIS)

    Liu Guisheng; Wang Yaoqing; Liu Ping; Zhang Baocheng

    1994-01-01

    Using a code system NSLINK, AMPX master library in WIMS 69 groups structure are made from nuclides relating to 4 newest evaluated nuclear data libraries. Some integrals of 10 thermal reactor benchmark assemblies recommended by the U.S. CSEWG are calculated using rectified PASC-1 code system and compared with foreign results, the authors results are in good agreement with others. 69 group libraries of evaluated data bases in TPFAP interface file are generated with NJOY code system. The k ∞ values of 6 cell lattice assemblies are calculated by the code CBM. The calculated results are analysed and compared

  3. Species-specific challenges in dog cloning.

    Science.gov (United States)

    Kim, G A; Oh, H J; Park, J E; Kim, M J; Park, E J; Jo, Y K; Jang, G; Kim, M K; Kim, H J; Lee, B C

    2012-12-01

    Somatic cell nuclear transfer (SCNT) is now an established procedure used in cloning of several species. SCNT in dogs involves multiple steps including the removal of the nuclear material, injection of a donor cell, fusion, activation of the reconstructed oocytes and finally transfer to a synchronized female recipient. There are therefore many factors that contribute to cloning efficiency. By performing a retrospective analysis of 2005-2012 published papers regarding dog cloning, we define the optimum procedure and summarize the specific feature for dog cloning. © 2012 Blackwell Verlag GmbH.

  4. An MDE Approach for Modular Program Analyses

    NARCIS (Netherlands)

    Yildiz, Bugra Mehmet; Bockisch, Christoph; Aksit, Mehmet; Rensink, Arend

    Program analyses are an important tool to check if a system fulfills its specification. A typical implementation strategy for program analyses is to use an imperative, general-purpose language like Java, and access the program to be analyzed through libraries that offer an API for reading, writing

  5. Assessment of the genetic diversity of natural rubber tree clones of the SINCHI Institutes clone collection, using of morphological descriptors

    International Nuclear Information System (INIS)

    Quesada Mendez, Isaac; Quintero Barrera, Lorena; Aristizabal, Fabio A; Rodriguez Acuna, Olga

    2011-01-01

    Genetic diversity of natural rubber clones of the in SINCHI Institute’s clone collection was assessed. Clones of Hevea brasiliensis (Willd. ex Adr. De Juss.) Muell.Arg., Hevea spp. (H. brasiliensis x H. benthamiana), and three more species of Hevea genus are a part of the collection. Seventy-two materials were characterized with twenty-eight morphological descriptors. They were later used to generate a similarity matrix through the analysis of multi-categorical variables, and to obtain clusters based on the matrix. A low variability between clones of H. brasiliensis and H. spp. was observed, presumably because of the direct descendants of most of the materials from crosses of parental PB 80, PB 5/51, PB 49 and Tjir, exception made of clone GU 1410. Clustering between some materials product of exclusive cross of PB series, a group between clones descendants of parental clones PB 86, and clustering between descendants of parental clones PB 5/51, were observed. Clones from other species of Hevea differ from this big group.

  6. Unified universal quantum cloning machine and fidelities

    Energy Technology Data Exchange (ETDEWEB)

    Wang Yinan; Shi Handuo; Xiong Zhaoxi; Jing Li; Mu Liangzhu [School of Physics, Peking University, Beijing 100871 (China); Ren Xijun [School of Physics and Electronics, Henan University, Kaifeng 4750011 (China); Fan Heng [Institute of Physics, Chinese Academy of Sciences, Beijing 100190 (China)

    2011-09-15

    We present a unified universal quantum cloning machine, which combines several different existing universal cloning machines together, including the asymmetric case. In this unified framework, the identical pure states are projected equally into each copy initially constituted by input and one half of the maximally entangled states. We show explicitly that the output states of those universal cloning machines are the same. One importance of this unified cloning machine is that the cloning procession is always the symmetric projection, which reduces dramatically the difficulties for implementation. Also, it is found that this unified cloning machine can be directly modified to the general asymmetric case. Besides the global fidelity and the single-copy fidelity, we also present all possible arbitrary-copy fidelities.

  7. Survival of Skin Graft between Transgenic Cloned Dogs and Non-Transgenic Cloned Dogs

    Science.gov (United States)

    Kim, Geon A; Oh, Hyun Ju; Kim, Min Jung; Jo, Young Kwang; Choi, Jin; Park, Jung Eun; Park, Eun Jung; Lim, Sang Hyun; Yoon, Byung Il; Kang, Sung Keun; Jang, Goo; Lee, Byeong Chun

    2014-01-01

    Whereas it has been assumed that genetically modified tissues or cells derived from somatic cell nuclear transfer (SCNT) should be accepted by a host of the same species, their immune compatibility has not been extensively explored. To identify acceptance of SCNT-derived cells or tissues, skin grafts were performed between cloned dogs that were identical except for their mitochondrial DNA (mtDNA) haplotypes and foreign gene. We showed here that differences in mtDNA haplotypes and genetic modification did not elicit immune responses in these dogs: 1) skin tissues from genetically-modified cloned dogs were successfully transplanted into genetically-modified cloned dogs with different mtDNA haplotype under three successive grafts over 63 days; and 2) non-transgenic cloned tissues were accepted into transgenic cloned syngeneic recipients with different mtDNA haplotypes and vice versa under two successive grafts over 63 days. In addition, expression of the inserted gene was maintained, being functional without eliciting graft rejection. In conclusion, these results show that transplanting genetically-modified tissues into normal, syngeneic or genetically-modified recipient dogs with different mtDNA haplotypes do not elicit skin graft rejection or affect expression of the inserted gene. Therefore, therapeutically valuable tissue derived from SCNT with genetic modification might be used safely in clinical applications for patients with diseased tissues. PMID:25372489

  8. Knowledge and attitudes toward human cloning in Israel.

    Science.gov (United States)

    Barnoy, Sivia; Ehrenfeld, Malka; Sharon, Rina; Tabak, Nili

    2006-04-01

    The success of mammal cloning in 1997 has brought the issue of human cloning into public discussion. Human cloning has several aspects and potential applications for use in both reproductive and non-reproductive matters. The aim of this study was to evaluate the knowledge and attitudes toward human cloning in Israel. Data from 120 respondents (68 health professionals and 52 non-health professionals), all Jewish, Hebrew speaking with at least 15 years of education each, were collected using two questionnaires that dealt with knowledge and attitudes toward human cloning. Results showed that although health professionals had significantly more knowledge that non-health professionals, all respondents had poor knowledge about cloning. No difference in attitudes was found between the groups. Most respondents opposed human cloning, but more positive attitudes toward non-reproductive cloning were found. The results are discussed in the context of the deficit model. The findings indicate a need to provide information about human cloning to allow people to form their attitudes based on factual knowledge.

  9. Reproductive cloning combined with genetic modification.

    Science.gov (United States)

    Strong, C

    2005-11-01

    Although there is widespread opposition to reproductive cloning, some have argued that its use by infertile couples to have genetically related children would be ethically justifiable. Others have suggested that lesbian or gay couples might wish to use cloning to have genetically related children. Most of the main objections to human reproductive cloning are based on the child's lack of unique nuclear DNA. In the future, it may be possible safely to create children using cloning combined with genetic modifications, so that they have unique nuclear DNA. The genetic modifications could be aimed at giving such children genetic characteristics of both members of the couple concerned. Thus, cloning combined with genetic modification could be appealing to infertile, lesbian, or gay couples who seek genetically related children who have genetic characteristics of both members. In such scenarios, the various objections to human reproductive cloning that are based on the lack of genetic uniqueness would no longer be applicable. The author argues that it would be ethically justifiable for such couples to create children in this manner, assuming these techniques could be used safely.

  10. Improving Library Management by Using Cost Analysis Tools: A Case Study for Cataloguing Processes

    Directory of Open Access Journals (Sweden)

    Lorena Siguenza-Guzman

    2014-02-01

    Full Text Available TTDABC is a relatively new costing management technique, initially developed for manufacturing processes, which is gaining attention in libraries. This is because TDABC is a fast and simple method that only requires two parameters, an estimation of time required to perform an activity and the unit cost per time of supplying capacity. A few case studies have been documented with regard to TDABC in libraries; all of them being oriented to analyse specific library activities such as inter-library loan, acquisition and circulation processes. The primary focus of this paper is to describe TDABC implementation in one of the most important library processes, namely cataloguing. In particular, original and copy cataloguing are analysed through a case study to demonstrate the applicability and usefulness of TDABC to perform cost analysis of cataloguing processes.

  11. Human cloning and 'posthuman' society.

    Science.gov (United States)

    Blackford, Russell

    2005-01-01

    Since early 1997, when the creation of Dolly the sheep by somatic cell nuclear transfer was announced in Nature, numerous government reports, essays, articles and books have considered the ethical problems and policy issues surrounding human reproductive cloning. In this article, I consider what response a modern liberal society should give to the prospect of human cloning, if it became safe and practical. Some opponents of human cloning have argued that permitting it would place us on a slippery slope to a repugnant future society, comparable to that portrayed in Aldous Huxley's novel, Brave New World. I conclude that, leaving aside concerns about safety, none of the psychological or social considerations discussed in this article provides an adequate policy justification for invoking the state's coercive powers to prevent human cloning.

  12. Screening and identification of RhD antigen mimic epitopes from a phage display random peptide library for the serodiagnosis of haemolytic disease of the foetus and newborn.

    Science.gov (United States)

    Wang, Jiao; Song, Jingjing; Zhou, Shuimei; Fu, Yourong; Bailey, Jeffrey A; Shen, Changxin

    2018-01-16

    Identification of RhD antigen epitopes is a key component in understanding the pathogenesis of haemolytic disease of the foetus and newborn. Research has indicated that phage display libraries are useful tools for identifying novel mimic epitopes (mimotopes) which may help to determine antigen specificity. We selected the mimotopes of blood group RhD antigen by affinity panning a phage display library using monoclonal anti-D. After three rounds of biopanning, positive phage clones were identified by enzyme-linked immunosorbent assay (ELISA) and then sent for sequencing and peptides synthesis. Next, competitive ELISA and erythrocyte haemagglutination inhibition tests were carried out to confirm the inhibitory activity of the synthetic peptide. To evaluate the diagnostic performance of the synthetic peptide, a diagnostic ELISA was examined. Fourteen of 35 phage clones that were chosen randomly from the titering plate were considered to be positive. Following DNA sequencing and translation, 11 phage clones were found to represent the same peptide - RMKMLMMLMRRK (P4) - whereas each of the other three clones represented a unique peptide. Through the competitive ELISA and erythrocyte haemagglutination inhibition tests, the peptide (P4) was verified to have the ability to mimic the RhD antigen. The diagnostic ELISA for P4 proved to be sensitive (82.61%) and specific (88.57%). This study reveals that the P4 peptide can mimic RhD antigen and paves the way for the development of promising targeted diagnostic and therapeutic platforms for haemolytic disease of the foetus and newborn.

  13. U.S. consumers attitudes toward farm animal cloning.

    Science.gov (United States)

    Brooks, Kathleen R; Lusk, Jayson L

    2011-10-01

    In January 2008, the United States Food and Drug Administration concluded "meat and milk from cattle, swine, and goat clones or their offspring are as safe to eat as food we eat from those species now" (U.S. FDA, 2010). However, cloning remains a very controversial topic. A web-based survey administered by Knowledge Networks was used to determine U.S. consumers' awareness of and attitudes toward meat and milk from cloned cattle. Findings reveal consumers do not differentiate much between products from cloned animals and products from non-cloned animals. Overall consumers are concerned that animal cloning is an unnatural process and that it will lead to human cloning. Copyright © 2011 Elsevier Ltd. All rights reserved.

  14. Rapid in silico cloning of genes using expressed sequence tags (ESTs).

    Science.gov (United States)

    Gill, R W; Sanseau, P

    2000-01-01

    Expressed sequence tags (ESTs) are short single-pass DNA sequences obtained from either end of cDNA clones. These ESTs are derived from a vast number of cDNA libraries obtained from different species. Human ESTs are the bulk of the data and have been widely used to identify new members of gene families, as markers on the human chromosomes, to discover polymorphism sites and to compare expression patterns in different tissues or pathologies states. Information strategies have been devised to query EST databases. Since most of the analysis is performed with a computer, the term "in silico" strategy has been coined. In this chapter we will review the current status of EST databases, the pros and cons of EST-type data and describe possible strategies to retrieve meaningful information.

  15. Local circulating clones of Staphylococcus aureus in Ecuador.

    Science.gov (United States)

    Zurita, Jeannete; Barba, Pedro; Ortega-Paredes, David; Mora, Marcelo; Rivadeneira, Sebastián

    The spread of pandemic Staphylococcus aureus clones, mainly methicillin-resistant S. aureus (MRSA), must be kept under surveillance to assemble an accurate, local epidemiological analysis. In Ecuador, the prevalence of the USA300 Latin American variant clone (USA300-LV) is well known; however, there is little information about other circulating clones. The aim of this work was to identify the sequence types (ST) using a Multiple-Locus Variable number tandem repeat Analysis 14-locus genotyping approach. We analyzed 132 S. aureus strains that were recovered from 2005 to 2013 and isolated in several clinical settings in Quito, Ecuador. MRSA isolates composed 46.97% (62/132) of the study population. Within MRSA, 37 isolates were related to the USA300-LV clone (ST8-MRSA-IV, Panton-Valentine Leukocidin [PVL] +) and 10 were related to the Brazilian clone (ST239-MRSA-III, PVL-). Additionally, two isolates (ST5-MRSA-II, PVL-) were related to the New York/Japan clone. One isolate was related to the Pediatric clone (ST5-MRSA-IV, PVL-), one isolate (ST45-MRSA-II, PVL-) was related to the USA600 clone, and one (ST22-MRSA-IV, PVL-) was related to the epidemic UK-EMRSA-15 clone. Moreover, the most prevalent MSSA sequence types were ST8 (11 isolates), ST45 (8 isolates), ST30 (8 isolates), ST5 (7 isolates) and ST22 (6 isolates). Additionally, we found one isolate that was related to the livestock associated S. aureus clone ST398. We conclude that in addition to the high prevalence of clone LV-ST8-MRSA-IV, other epidemic clones are circulating in Quito, such as the Brazilian, Pediatric and New York/Japan clones. The USA600 and UK-EMRSA-15 clones, which were not previously described in Ecuador, were also found. Moreover, we found evidence of the presence of the livestock associated clone ST398 in a hospital environment. Copyright © 2016 Sociedade Brasileira de Infectologia. Published by Elsevier Editora Ltda. All rights reserved.

  16. Molecular cloning and construction of the coding region for human acetylcholinesterase reveals a G + C-rich attenuating structure

    International Nuclear Information System (INIS)

    Soreq, H.; Ben-Aziz, R.; Prody, C.A.; Seidman, S.; Gnatt, A.; Neville, L.; Lieman-Hurwitz, J.; Lev-Lehman, E.; Ginzberg, D.; Lapidot-Lifson, Y.; Zakut, H.

    1990-01-01

    To study the primary structure of human acetylcholinesterase and its gene expression and amplification, cDNA libraries from human tissues expressing oocyte-translatable AcChoEase mRNA were constructed and screened with labeled oligodeoxynucleotide probes. Several cDNA clones were isolated that encoded a polypeptide with ≥50% identically aligned amino acids to Torpedo AcChoEase and human butyrylcholinesterase. However, these cDNA clones were all truncated within a 300-nucleotide-long G + C-rich region with a predicted pattern of secondary structure having a high Gibbs free energy downstream from the expected 5' end of the coding region. Screening of a genomic DNA library revealed the missing 5' domain. When ligated to the cDNA and constructed into a transcription vector, this sequence encoded a synthetic mRNA translated in microinjected oocytes into catalytically active AcChoEase with marked preference for acetylthiocholine over butyrylthiocholine as a substrate, susceptibility to inhibition by the AcChoEase inhibitor BW284C51, and resistance to the AcChoEase inhibitor tetraisopropylpyrophosphoramide. Blot hybridization of genomic DNA from different individuals carrying amplified AcChoEase genes revealed variable intensities and restriction patterns with probes from the regions upstream and downstream from the predicted G + C-rich structure. Thus, the human AcChoEase gene includes a putative G + C-rich attenuator domain and is subject to structural alterations in cases of AcChoEase gene amplification

  17. Local cloning of entangled states

    International Nuclear Information System (INIS)

    Gheorghiu, Vlad; Yu Li; Cohen, Scott M.

    2010-01-01

    We investigate the conditions under which a set S of pure bipartite quantum states on a DxD system can be locally cloned deterministically by separable operations, when at least one of the states is full Schmidt rank. We allow for the possibility of cloning using a resource state that is less than maximally entangled. Our results include that: (i) all states in S must be full Schmidt rank and equally entangled under the G-concurrence measure, and (ii) the set S can be extended to a larger clonable set generated by a finite group G of order |G|=N, the number of states in the larger set. It is then shown that any local cloning apparatus is capable of cloning a number of states that divides D exactly. We provide a complete solution for two central problems in local cloning, giving necessary and sufficient conditions for (i) when a set of maximally entangled states can be locally cloned, valid for all D; and (ii) local cloning of entangled qubit states with nonvanishing entanglement. In both of these cases, we show that a maximally entangled resource is necessary and sufficient, and the states must be related to each other by local unitary 'shift' operations. These shifts are determined by the group structure, so need not be simple cyclic permutations. Assuming this shifted form and partially entangled states, then in D=3 we show that a maximally entangled resource is again necessary and sufficient, while for higher-dimensional systems, we find that the resource state must be strictly more entangled than the states in S. All of our necessary conditions for separable operations are also necessary conditions for local operations and classical communication (LOCC), since the latter is a proper subset of the former. In fact, all our results hold for LOCC, as our sufficient conditions are demonstrated for LOCC, directly.

  18. Analysis of a cDNA clone expressing a human autoimmune antigen: full-length sequence of the U2 small nuclear RNA-associated B antigen

    International Nuclear Information System (INIS)

    Habets, W.J.; Sillekens, P.T.G.; Hoet, M.H.; Schalken, J.A.; Roebroek, A.J.M.; Leunissen, J.A.M.; Van de Ven, W.J.M.; Van Venrooij, W.J.

    1987-01-01

    A U2 small nuclear RNA-associated protein, designated B'', was recently identified as the target antigen for autoimmune sera from certain patients with systemic lupus erythematosus and other rheumatic diseases. Such antibodies enabled them to isolate cDNA clone λHB''-1 from a phage λgt11 expression library. This clone appeared to code for the B'' protein as established by in vitro translation of hybrid-selected mRNA. The identity of clone λHB''-1 was further confirmed by partial peptide mapping and analysis of the reactivity of the recombinant antigen with monospecific and monoclonal antibodies. Analysis of the nucleotide sequence of the 1015-base-pair cDNA insert of clone λHB''-1 revealed a large open reading frame of 800 nucleotides containing the coding sequence for a polypeptide of 25,457 daltons. In vitro transcription of the λHB''-1 cDNA insert and subsequent translation resulted in a protein product with the molecular size of the B'' protein. These data demonstrate that clone λHB''-1 contains the complete coding sequence of this antigen. The deduced polypeptide sequence contains three very hydrophilic regions that might constitute RNA binding sites and/or antigenic determinants. These findings might have implications both for the understanding of the pathogenesis of rheumatic diseases as well as for the elucidation of the biological function of autoimmune antigens

  19. Cloning and chromosomal assignment of a human cDNA encoding a T cell- and natural killer cell-specific trypsin-like serine protease

    International Nuclear Information System (INIS)

    Gershenfeld, H.K.; Hershberger, R.J.; Shows, T.B.; Weissman, I.L.

    1988-01-01

    A cDNA clone encoding a human T cell- and natural killer cell-specific serine protease was obtained by screening a phage λgt10 cDNA library from phytohemagglutinin-stimulated human peripheral blood lymphocytes with the mouse Hanukah factor cDNA clone. In an RNA blot-hybridization analysis, this human Hanukah factor cDNA hybridized with a 1.3-kilobase band in allogeneic-stimulated cytotoxic T cells and the Jurkat cell line, but this transcript was not detectable in normal muscle, liver, tonsil, or thymus. By dot-blot hybridization, this cDNA hybridized with RNA from three cytolytic T-cell clones and three noncytolytic T-cell clones grown in vitro as well as with purified CD16 + natural killer cells and CD3 + , CD16 - T-cell large granular lymphocytes from peripheral blood lymphocytes (CD = cluster designation). The nucleotide sequence of this cDNA clone encodes a predicted serine protease of 262 amino acids. The active enzyme is 71% and 77% similar to the mouse sequence at the amino acid and DNA level, respectively. The human and mouse sequences conserve the active site residues of serine proteases--the trypsin-specific Asp-189 and all 10 cysteine residues. The gene for the human Hanukah factor serine protease is located on human chromosome 5. The authors propose that this trypsin-like serine protease may function as a common component necessary for lysis of target cells by cytotoxic T lymphocytes and natural killer cells

  20. Quantum cloning of mixed states in symmetric subspaces

    International Nuclear Information System (INIS)

    Fan Heng

    2003-01-01

    Quantum-cloning machine for arbitrary mixed states in symmetric subspaces is proposed. This quantum-cloning machine can be used to copy part of the output state of another quantum-cloning machine and is useful in quantum computation and quantum information. The shrinking factor of this quantum cloning achieves the well-known upper bound. When the input is identical pure states, two different fidelities of this cloning machine are optimal

  1. Cloning of a quantum measurement

    International Nuclear Information System (INIS)

    Bisio, Alessandro; D'Ariano, Giacomo Mauro; Perinotti, Paolo; Sedlak, Michal

    2011-01-01

    We analyze quantum algorithms for cloning of a quantum measurement. Our aim is to mimic two uses of a device performing an unknown von Neumann measurement with a single use of the device. When the unknown device has to be used before the bipartite state to be measured is available we talk about 1→2 learning of the measurement, otherwise the task is called 1→2 cloning of a measurement. We perform the optimization for both learning and cloning for arbitrary dimension d of the Hilbert space. For 1→2 cloning we also propose a simple quantum network that achieves the optimal fidelity. The optimal fidelity for 1→2 learning just slightly outperforms the estimate and prepare strategy in which one first estimates the unknown measurement and depending on the result suitably prepares the duplicate.

  2. Cloning of a quantum measurement

    Energy Technology Data Exchange (ETDEWEB)

    Bisio, Alessandro; D' Ariano, Giacomo Mauro; Perinotti, Paolo; Sedlak, Michal [QUIT Group, Dipartimento di Fisica ' ' A. Volta' ' and INFN, via Bassi 6, I-27100 Pavia (Italy); QUIT Group, Dipartimento di Fisica ' ' A. Volta' ' via Bassi 6, I-27100 Pavia (Italy) and Institute of Physics, Slovak Academy of Sciences, Dubravska cesta 9, SK-845 11 Bratislava (Slovakia)

    2011-10-15

    We analyze quantum algorithms for cloning of a quantum measurement. Our aim is to mimic two uses of a device performing an unknown von Neumann measurement with a single use of the device. When the unknown device has to be used before the bipartite state to be measured is available we talk about 1{yields}2 learning of the measurement, otherwise the task is called 1{yields}2 cloning of a measurement. We perform the optimization for both learning and cloning for arbitrary dimension d of the Hilbert space. For 1{yields}2 cloning we also propose a simple quantum network that achieves the optimal fidelity. The optimal fidelity for 1{yields}2 learning just slightly outperforms the estimate and prepare strategy in which one first estimates the unknown measurement and depending on the result suitably prepares the duplicate.

  3. Human placental Na/sup +/, K/sup +/-ATPase. cap alpha. subunit: cDNA cloning, tissue expression, DNA polymorphism, and chromosomal localization

    Energy Technology Data Exchange (ETDEWEB)

    Chehab, F.F.; Kan, Y.W.; Law, M.L.; Hartz, J.; Kao, F.T.; Blostein, R.

    1987-11-01

    A 2.2-kilobase clone comprising a major portion of the coding sequence of the Na/sup +/, K/sup +/-ATPase ..cap alpha.. subunit was cloned from human placenta and its sequence was identical to that encoding the ..cap alpha.. subunit of human kidney and HeLa cells. Transfer blot analysis of the mRNA products of the Na/sup +/, K/sup +/-ATPase gene from various human tissues and cell lines revealed only one band (approx. = 4.7 kilobases) under low and high stringency washing conditions. The levels of expression in the tissues were intestine > placenta > liver > pancreas, and in the cell lines the levels were human erythroleukemia > butyrate-induced colon > colon > brain > HeLa cells. mRNA was undetectable in reticulocytes, consistent with the authors failure to detect positive clones in a size-selected ( > 2 kilobases) lambdagt11 reticulocyte cDNA library. DNA analysis revealed by a polymorphic EcoRI band and chromosome localization by flow sorting and in situ hybridization showed that the ..cap alpha.. subunit is on the short is on the short arm (band p11-p13) of chromosome 1.

  4. Human embryo cloning prohibited in Hong Kong.

    Science.gov (United States)

    Liu, Athena

    2005-12-01

    Since the birth of Dolly (the cloned sheep) in 1997, debates have arisen on the ethical and legal questions of cloning-for-biomedical-research (more commonly termed "therapeutic cloning") and of reproductive cloning using human gametes. Hong Kong enacted the Human Reproductive Technology Ordinance (Cap 561) in 2000. Section 15(1)(e) of this Ordinance prohibits the "replacing of the nucleus of a cell of an embryo with a nucleus taken from any other cell," i.e., nucleus substitution. Section 15(1)(f) prohibits the cloning of any embryo. The scope of the latter, therefore, is arguably the widest, prohibiting all cloning techniques such as cell nucleus replacement, embryo splitting, parthenogenesis, and cloning using stem cell lines. Although the Human Reproductive Technology Ordinance is not yet fully operative, this article examines how these prohibitions may adversely impact on basic research and the vision of the Hong Kong scientific community. It concludes that in light of recent scientific developments, it is time to review if the law offers a coherent set of policies in this area.

  5. Cloning the entanglement of a pair of quantum bits

    International Nuclear Information System (INIS)

    Lamoureux, Louis-Philippe; Navez, Patrick; Cerf, Nicolas J.; Fiurasek, Jaromir

    2004-01-01

    It is shown that any quantum operation that perfectly clones the entanglement of all maximally entangled qubit pairs cannot preserve separability. This 'entanglement no-cloning' principle naturally suggests that some approximate cloning of entanglement is nevertheless allowed by quantum mechanics. We investigate a separability-preserving optimal cloning machine that duplicates all maximally entangled states of two qubits, resulting in 0.285 bits of entanglement per clone, while a local cloning machine only yields 0.060 bits of entanglement per clone

  6. Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi

    DEFF Research Database (Denmark)

    Frandsen, Rasmus John Normand; Andersson, Jens A.; Kristensen, Matilde Bylov

    2008-01-01

    Background: The rapid increase in whole genome fungal sequence information allows large scale functional analyses of target genes. Efficient transformation methods to obtain site-directed gene replacement, targeted over-expression by promoter replacement, in-frame epitope tagging or fusion...... of coding sequences with fluorescent markers such as GFP are essential for this process. Construction of vectors for these experiments depends on the directional cloning of two homologous recombination sequences on each side of a selection marker gene. Results: Here, we present a USER Friendly cloning based...

  7. Molecular cloning and expression of the gene encoding the kinetoplast-associated type II DNA topoisomerase of Crithidia fasciculata.

    Science.gov (United States)

    Pasion, S G; Hines, J C; Aebersold, R; Ray, D S

    1992-01-01

    A type II DNA topoisomerase, topoIImt, was shown previously to be associated with the kinetoplast DNA of the trypanosomatid Crithidia fasciculata. The gene encoding this kinetoplast-associated topoisomerase has been cloned by immunological screening of a Crithidia genomic expression library with monoclonal antibodies raised against the purified enzyme. The gene CfaTOP2 is a single copy gene and is expressed as a 4.8-kb polyadenylated transcript. The nucleotide sequence of CfaTOP2 has been determined and encodes a predicted polypeptide of 1239 amino acids with a molecular mass of 138,445. The identification of the cloned gene is supported by immunoblot analysis of the beta-galactosidase-CfaTOP2 fusion protein expressed in Escherichia coli and by analysis of tryptic peptide sequences derived from purified topoIImt. CfaTOP2 shares significant homology with nuclear type II DNA topoisomerases of other eukaryotes suggesting that in Crithidia both nuclear and mitochondrial forms of topoisomerase II are encoded by the same gene.

  8. Research data management and libraries: relationships, activities, drivers and influences.

    Directory of Open Access Journals (Sweden)

    Stephen Pinfield

    Full Text Available The management of research data is now a major challenge for research organisations. Vast quantities of born-digital data are being produced in a wide variety of forms at a rapid rate in universities. This paper analyses the contribution of academic libraries to research data management (RDM in the wider institutional context. In particular it: examines the roles and relationships involved in RDM, identifies the main components of an RDM programme, evaluates the major drivers for RDM activities, and analyses the key factors influencing the shape of RDM developments. The study is written from the perspective of library professionals, analysing data from 26 semi-structured interviews of library staff from different UK institutions. This is an early qualitative contribution to the topic complementing existing quantitative and case study approaches. Results show that although libraries are playing a significant role in RDM, there is uncertainty and variation in the relationship with other stakeholders such as IT services and research support offices. Current emphases in RDM programmes are on developments of policies and guidelines, with some early work on technology infrastructures and support services. Drivers for developments include storage, security, quality, compliance, preservation, and sharing with libraries associated most closely with the last three. The paper also highlights a 'jurisdictional' driver in which libraries are claiming a role in this space. A wide range of factors, including governance, resourcing and skills, are identified as influencing ongoing developments. From the analysis, a model is constructed designed to capture the main aspects of an institutional RDM programme. This model helps to clarify the different issues involved in RDM, identifying layers of activity, multiple stakeholders and drivers, and a large number of factors influencing the implementation of any initiative. Institutions may usefully benchmark their

  9. Research data management and libraries: relationships, activities, drivers and influences.

    Science.gov (United States)

    Pinfield, Stephen; Cox, Andrew M; Smith, Jen

    2014-01-01

    The management of research data is now a major challenge for research organisations. Vast quantities of born-digital data are being produced in a wide variety of forms at a rapid rate in universities. This paper analyses the contribution of academic libraries to research data management (RDM) in the wider institutional context. In particular it: examines the roles and relationships involved in RDM, identifies the main components of an RDM programme, evaluates the major drivers for RDM activities, and analyses the key factors influencing the shape of RDM developments. The study is written from the perspective of library professionals, analysing data from 26 semi-structured interviews of library staff from different UK institutions. This is an early qualitative contribution to the topic complementing existing quantitative and case study approaches. Results show that although libraries are playing a significant role in RDM, there is uncertainty and variation in the relationship with other stakeholders such as IT services and research support offices. Current emphases in RDM programmes are on developments of policies and guidelines, with some early work on technology infrastructures and support services. Drivers for developments include storage, security, quality, compliance, preservation, and sharing with libraries associated most closely with the last three. The paper also highlights a 'jurisdictional' driver in which libraries are claiming a role in this space. A wide range of factors, including governance, resourcing and skills, are identified as influencing ongoing developments. From the analysis, a model is constructed designed to capture the main aspects of an institutional RDM programme. This model helps to clarify the different issues involved in RDM, identifying layers of activity, multiple stakeholders and drivers, and a large number of factors influencing the implementation of any initiative. Institutions may usefully benchmark their activities against the

  10. Human cloning: Eastern Mediterranean Region perspective.

    Science.gov (United States)

    Abdur Rab, M; Khayat, M H

    2006-01-01

    Recent advances in genomics and biotechnology have ushered in a new era in health development. Therapeutic cloning possesses enormous potential for revolutionizing medical and therapeutic techniques. Cloning technology, however, is perceived as having the potential for reproductive cloning, which raises serious ethical and moral concerns. It is important that the Islamic countries come to a consensus on this vital issue. Developing science and technology for better health is a religious and moral obligation. There is an urgent need for Muslim scholars to discuss the issue of stem cell research and cloning rationally; such dialogue will not only consider the scientific merits but also the moral, ethical and legal implications.

  11. Libraries for users services in academic libraries

    CERN Document Server

    Alvite, Luisa

    2010-01-01

    This book reviews the quality and evolution of academic library services. It revises service trends offered by academic libraries and the challenge of enhancing traditional ones such as: catalogues, repositories and digital collections, learning resources centres, virtual reference services, information literacy and 2.0 tools.studies the role of the university library in the new educational environment of higher educationrethinks libraries in academic contextredefines roles for academic libraries

  12. Radiation-induced aneusomic clones in bone marrow of rats

    International Nuclear Information System (INIS)

    Kohno, Sei-Ichi; Ishihara, Takaaki

    1976-01-01

    Wistar rats 3 months old were given a single whole-body X-irradiation with 700 R. They were killed 9.3 months, on average, after irradiation. From the bone marrows of the 23 irradiated rats, 54 clones of cells with radiation-induced chromosome abnormalities ranging from 3.3 to 78.3% in size were obtained. Karyotype analysis at the banding level showed that 43 out of the 54 clones had balanced chromosome constitutions and that the remaining 11 clones were unbalanced. The 43 balanced clones consisted of 33 clones with reciprocal translocations, 6 with inversions and 4 with both translocations and inversions. The 11 unbalanced clones were made up of 7 aneuploid clones and 4 pseudo-diploid clones. Of the 54 clones, 15 were large with frequencies of more than 25%. Contrary to general belief that cells with unbalanced chromosome constitutions have less capacity to proliferate than those with balanced ones, 8 of the 15 large clones, especially all, except 1, of the largest 6 clones were unbalanced, either aneuploid or pseudo-diploid

  13. WIMS nuclear data library and its updating

    Energy Technology Data Exchange (ETDEWEB)

    Bakhtyar, S; Salahuddin, A; Arshad, M

    1995-10-01

    This report gives a brief overview of the status of reactor physics computer code WIMS-D/4 and its library. It presents the details of WIMS-D/4 Library Update Project (WLUP), initiated by International Atomic Energy Agency (IAEA) with the goal of providing updated nuclear data library to the user of WIMS-D/4. The WLUP was planned to be executed in several stages. In this report the calculations performed for the first stage are presented. A number of benchmarks for light water and heavy water lattices proposed by IAEA have been analysed and the results have been compared with the average of experimental values, the IAEA reference values and the average of calculated results from different international laboratories. (author) 8 figs.

  14. WIMS nuclear data library and its updating

    International Nuclear Information System (INIS)

    Bakhtyar, S.; Salahuddin, A.; Arshad, M.

    1995-10-01

    This report gives a brief overview of the status of reactor physics computer code WIMS-D/4 and its library. It presents the details of WIMS-D/4 Library Update Project (WLUP), initiated by International Atomic Energy Agency (IAEA) with the goal of providing updated nuclear data library to the user of WIMS-D/4. The WLUP was planned to be executed in several stages. In this report the calculations performed for the first stage are presented. A number of benchmarks for light water and heavy water lattices proposed by IAEA have been analysed and the results have been compared with the average of experimental values, the IAEA reference values and the average of calculated results from different international laboratories. (author) 8 figs

  15. Mitochondrial DNA heteroplasmy in ovine fetuses and sheep cloned by somatic cell nuclear transfer

    Directory of Open Access Journals (Sweden)

    Müller Mathias

    2007-12-01

    Full Text Available Abstract Background The mitochondrial DNA (mtDNA of the cloned sheep "Dolly" and nine other ovine clones produced by somatic cell nuclear transfer (SCNT was reported to consist only of recipient oocyte mtDNA without any detectable mtDNA contribution from the nucleus donor cell. In cattle, mouse and pig several or most of the clones showed transmission of nuclear donor mtDNA resulting in mitochondrial heteroplasmy. To clarify the discrepant transmission pattern of donor mtDNA in sheep clones we analysed the mtDNA composition of seven fetuses and five lambs cloned from fetal fibroblasts. Results The three fetal fibroblast donor cells used for SCNT harboured low mtDNA copy numbers per cell (A: 753 ± 54, B: 292 ± 33 and C: 561 ± 88. The ratio of donor to recipient oocyte mtDNAs was determined using a quantitative amplification refractory mutation system (ARMS PCR (i.e. ARMS-qPCR. For quantification of SNP variants with frequencies below 0.1% we developed a restriction endonuclease-mediated selective quantitative PCR (REMS-qPCR. We report the first cases (n = 4 fetuses, n = 3 lambs of recipient oocyte/nuclear donor mtDNA heteroplasmy in SCNT-derived ovine clones demonstrating that there is no species-effect hindering ovine nucleus-donor mtDNA from being transmitted to the somatic clonal offspring. Most of the heteroplasmic clones exhibited low-level heteroplasmy (0.1% to 0.9%, n = 6 indicating neutral transmission of parental mtDNAs. High-level heteroplasmy (6.8% to 46.5% was observed in one case. This clone possessed a divergent recipient oocyte-derived mtDNA genotype with three rare amino acid changes compared to the donor including one substitution at an evolutionary conserved site. Conclusion Our study using state-of-the-art techniques for mtDNA quantification, like ARMS-qPCR and the novel REMS-qPCR, documents for the first time the transmission of donor mtDNA into somatic sheep clones. MtDNA heteroplasmy was detected in seven of 12 clones

  16. A Java Bytecode Metamodel for Composable Program Analyses

    NARCIS (Netherlands)

    Yildiz, Bugra Mehmet; Bockisch, Christoph; Rensink, Arend; Aksit, Mehmet; Seidl, Martina; Zschaler, Steffen

    Program analyses are an important tool to check if a system fulfills its specification. A typical implementation strategy for program analyses is to use an imperative, general-purpose language like Java; and access the program to be analyzed through libraries for manipulating intermediate code, such

  17. From Libraries to ‘Libratories’

    Directory of Open Access Journals (Sweden)

    Leo Waaijers

    2006-04-01

    Full Text Available While the eighties of the last century were a time of local automation for libraries and the nineties the decade in which libraries embraced the internet and the WWW, now is the age in which the big search engines and institutional repositories are gaining a firm footing. This heralds a new era in both the evolution of scholarly communication and its agencies themselves, i.e. the libraries. Until now libraries and publishers have developed a digital variant of existing processes and products, i.e. catalogues posted on the Web, scanned copies of articles, e-mail notification about acquisitions or expired lending periods, or traditional journals in a digital jacket. However, the new OAI repositories and services based upon them have given rise to entirely new processes and products, libraries transforming themselves into partners in setting up virtual learning environments, building an institution’s digital showcase, maintaining academics’ personal websites, designing refereed portals and – further into the future – taking part in organising virtual research environments or collaboratories. Libraries are set to metamorphose into ‘libratories’, an imaginary word to express their combined functions of library, repository and collaboratory. In such environments scholarly communication will be liberated from its current copyright bridle while its coverage will be both broader - including primary data, audiovisuals and dynamic models - and deeper, with cross-disciplinary analyses of methodologies and applications of instruments. Universities will make it compulsory to store in their institutional repositories the results of research conducted within their walls for purposes of academic reporting, review committees, and other modes of clarification and explanation. Big search engines will provide access to this profusion of information and organise its mass customisation.

  18. Human Cloning

    National Research Council Canada - National Science Library

    Johnson, Judith A; Williams, Erin D

    2006-01-01

    .... Scientists in other labs, including Harvard University and the University of California at San Francisco, intend to produce cloned human embryos in order to derive stem cells for medical research...

  19. SCHOOL COMMUNITY PERCEPTION OF LIBRARY APPS AGAINTS LIBRARY EMPOWERMENT

    Directory of Open Access Journals (Sweden)

    Achmad Riyadi Alberto

    2017-07-01

    Full Text Available Abstract. This research is motivated by the development of information and communication technology (ICT in the library world so rapidly that allows libraries in the present to develop its services into digital-based services. This study aims to find out the school community’s perception of library apps developed by Riche Cynthia Johan, Hana Silvana, and Holin Sulistyo and its influence on library empowerment at the library of SD Laboratorium Percontohan UPI Bandung. Library apps in this research belong to the context of m-libraries, which is a library that meets the needs of its users by using mobile platforms such as smartphones,computers, and other mobile devices. Empowerment of library is the utilization of all aspects of the implementation of libraries to the best in order to achieve the expected goals. An analysis of the schoolcommunity’s perception of library apps using the Technology Acceptance Model (TAM includes: ease of use, usefulness, usability, usage trends, and real-use conditions. While the empowerment of the library includes aspects: information empowerment, empowerment of learning resources, empowerment of human resources, empowerment of library facilities, and library promotion. The research method used in this research is descriptive method with quantitative approach. Population and sample in this research is school community at SD Laboratorium Percontohan UPI Bandung. Determination of sample criteria by using disproportionate stratified random sampling with the number of samples of 83 respondents. Data analysis using simple linear regression to measure the influence of school community perception about library apps to library empowerment. The result of data analysis shows that there is influence between school community perception about library apps to library empowerment at library of SD Laboratorium Percontohan UPI Bandung which is proved by library acceptance level and library empowerment improvement.

  20. "Goodbye Dolly?" The ethics of human cloning.

    Science.gov (United States)

    Harris, J

    1997-01-01

    The ethical implications of human clones have been much alluded to, but have seldom been examined with any rigour. This paper examines the possible uses and abuses of human cloning and draws out the principal ethical dimensions, both of what might be done and its meaning. The paper examines some of the major public and official responses to cloning by authorities such as President Clinton, the World Health Organisation, the European parliament, UNESCO, and others and reveals their inadequacies as foundations for a coherent public policy on human cloning. The paper ends by defending a conception of reproductive rights of "procreative autonomy" which shows human cloning to be not inconsistent with human rights and dignity. PMID:9451604