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Sample records for cdna inserts flics

  1. Characterization of full-length sequenced cDNA inserts (FLIcs from Atlantic salmon (Salmo salar

    Directory of Open Access Journals (Sweden)

    Lunner Sigbjørn

    2009-10-01

    Full Text Available Abstract Background Sequencing of the Atlantic salmon genome is now being planned by an international research consortium. Full-length sequenced inserts from cDNAs (FLIcs are an important tool for correct annotation and clustering of the genomic sequence in any species. The large amount of highly similar duplicate sequences caused by the relatively recent genome duplication in the salmonid ancestor represents a particular challenge for the genome project. FLIcs will therefore be an extremely useful resource for the Atlantic salmon sequencing project. In addition to be helpful in order to distinguish between duplicate genome regions and in determining correct gene structures, FLIcs are an important resource for functional genomic studies and for investigation of regulatory elements controlling gene expression. In contrast to the large number of ESTs available, including the ESTs from 23 developmental and tissue specific cDNA libraries contributed by the Salmon Genome Project (SGP, the number of sequences where the full-length of the cDNA insert has been determined has been small. Results High quality full-length insert sequences from 560 pre-smolt white muscle tissue specific cDNAs were generated, accession numbers [GenBank: BT043497 - BT044056]. Five hundred and ten (91% of the transcripts were annotated using Gene Ontology (GO terms and 440 of the FLIcs are likely to contain a complete coding sequence (cCDS. The sequence information was used to identify putative paralogs, characterize salmon Kozak motifs, polyadenylation signal variation and to identify motifs likely to be involved in the regulation of particular genes. Finally, conserved 7-mers in the 3'UTRs were identified, of which some were identical to miRNA target sequences. Conclusion This paper describes the first Atlantic salmon FLIcs from a tissue and developmental stage specific cDNA library. We have demonstrated that many FLIcs contained a complete coding sequence (cCDS. This

  2. The FLIC conversion codes

    International Nuclear Information System (INIS)

    Basher, J.C.

    1965-05-01

    This report describes the FORTRAN programmes, FLIC 1 and FLIC 2. These programmes convert programmes coded in one dialect of FORTRAN to another dialect of the same language. FLIC 1 is a general pattern recognition and replacement programme whereas FLIC 2 contains extensions directed towards the conversion of FORTRAN II and S2 programmes to EGTRAN 1 - the dialect now in use on the Winfrith KDF9. FII or S2 statements are replaced where possible by their E1 equivalents; other statements which may need changing are flagged. (author)

  3. Dynamical FLIC fermions

    International Nuclear Information System (INIS)

    Kamleh, W.; Leinweber, D.B.; Williams, A.G.

    2004-01-01

    The use of APE smearing or other blocking techniques in fermion actions can provide many advantages. There are many variants of these fat link actions in lattice QCD currently, such as FLIC fermions. The FLIC fermion formalism makes use of the APE blocking technique in combination with a projection of the blocked links back into the special unitary group. This reunitarisation is often performed using an iterative maximisation of a gauge invariant measure. This technique is not differentiable with respect to the gauge field and thus prevents the use of standard. Hybrid Monte Carlo simulation algorithms. The use of an alternative projection technique circumvents this difficulty and allows the simulation of dynamical fat link fermions with standard HMC and its variants

  4. The FLIC1 conversion code

    International Nuclear Information System (INIS)

    Basher, J.C.

    1964-08-01

    This memorandum describes the FORTRAN II programme, FLIC1, which converts programmes written in one dialect of FORTRAN to another dialect of the same language. Particular translation processes considered are those from existing dialects (FORTRAN II and S2) to EGTRAN1 - the dialect to be used initially on the Winfrith KDF9. The basic version of the programme replaces FII or S2 statements where these are different from, but in one-to-one correspondence with, E1 statements. Other statements which need changing are flagged. (author)

  5. FLIC-overlap fermions and topology

    International Nuclear Information System (INIS)

    Kamleh, W.; Kusterer, D.J.; Leinweber, D.B.; Williams, A.G.

    2003-01-01

    APE smearing the links in the irrelevant operators of clover fermions (Fat-Link Irrelevant Clover (FLIC) fermions) provides significant improvement in the condition number of the Hermitian-Dirac operator and gives rise to a factor of two savings in computing the overlap operator. This report investigates the effects of using a highly-improved definition of the lattice field-strength tensor F μν in the fermion action, made possible through the use of APE-smeared fat links in the construction of the irrelevant operators. Spurious double-zero crossings in the spectral flow of the Hermitian-Wilson Dirac operator associated with lattice artifacts at the scale of the lattice spacing are removed with FLIC fermions composed with an O(α 4 )-improved lattice field strength tensor. Hence, FLIC-Overlap fermions provide an additional benefit to the overlap formalism: a correct realization of topology in the fermion sector on the lattice

  6. Production of glycosylated physiologically normal human α1-antitrypsin by mouse fibroblasts modified by insertion of a human α1-antitrypsin cDNA using a retroviral vector

    International Nuclear Information System (INIS)

    Garver, R.I. Jr.; Chytil, A.; Karlsson, S.

    1987-01-01

    α 2 -Antitrypsin (α 1 AT) deficiency is a hereditary disorder characterized by reduced serum levels of α 1 AT, resulting in destruction of the lower respiratory tract by neutrophil elastase. As an approach to augment α 1 AT levels in this disorder with physiologically normal human α 1 AT, the authors have integrated a full-length normal human α 1 AT cDNA into the genome of mouse fibroblasts. To accomplish this, the retroviral vector N2 was modified by inserting the simian virus 40 early promoter followed by the α 1 AT cDNA. Southern analysis demonstrated that the intact cDNA was present in the genome of selected clones of the transfected murine fibroblasts psi2 and infected NIH 3T3. The clones produced three mRNA transcripts containing human α 1 AT sequences, secreted an α 1 AT molecule recognized by an anti-human α 1 AT antibody, with the same molecular mass as normal human α 1 AT and that complexed with and inhibited human neutrophil elastase. The psi2 produced α 1 AT was glycosylated, and when infused intravenously into mice, it had a serum half-life similar to normal α 1 AT purified from human plasma and markedly longer than that of nonglycosylated human α 1 AT cDNA-directed yeast-produced α 1 AT. These studies demonstrate the feasibility of using a retroviral vector to insert the normal human α 1 AT cDNA into non-α 1 AT-producing cells, resulting in the synthesis and secretion of physiologically normal α 1 AT

  7. Dissolved Nutrients from Submarine Groundwater in Flic en Flac ...

    African Journals Online (AJOL)

    Abstract—The aim of this study was to investigate dissolved nutrients in a submarine groundwater discharge (SGD) in Flic en Flac lagoon on the west coast of the volcanic island of Mauritius. The SGD enters Flic en Flac lagoon through a thin blanket of unconsolidated sediment through a fracture system and is concentrated ...

  8. The FTK to Level-2 Interface Card (FLIC)

    CERN Document Server

    Anderson, John Thomas; The ATLAS collaboration; Drake, Gary; Love, Jeremy; Proudfoot, James; Wang, Rui; Zhang, Jinlong; Auerbach, Benjamin

    2015-01-01

    The FTK to Level-2 Interface Card (FLIC) of the ATLAS Fast TracKer (FTK) trigger upgrade is the final component in the FTK chain of custom electronics. The FTK performs full event tracking using the ATLAS Silicon detectors for every Level-1 accepted event at 100 kHz. The FLIC is a custom Advanced Telecommunications Architecture (ATCA) card that interfaces the upstream FTK system with the ATLAS trigger and data acquisition (TDAQ) system, and allows for event processing on commercial PC blades making use of the 10 GB Ethernet full mesh ATCA back-plane. The FLIC receives data on 8 optical links at a bandwidth of ~1 Gbps per channel, reformats the data to the ATLAS standard record format, and performs the conversion from local to global module identifier using look up tables in SRAM. After processing, the event records are sent out to the TDAQ system using the S-LINK protocol at 2 Gbps, with a latency of O(10 microseconds). The data processing is handled in two Xilinx Virtex-6 FPGAs, with two additional Virtex-6 ...

  9. The FTK to Level-2 Interface Card (FLIC)

    CERN Document Server

    Wang, R.; The ATLAS collaboration; Auerbach, Benjamin; Blair, Robert; Drake, Gary; Love, Jeremy; Proudfoot, James; Anderson, J.; Zhang, Jinlong

    2015-01-01

    The FTK to Level-2 Interface Card (FLIC) of the ATLAS Fast TracKer (FTK) trigger upgrade is the final component in the FTK chain of custom electronics. The FTK performs full event tracking using the ATLAS Silicon detectors for every Level-1(L1) accepted event at 100 kHz. The FLIC is a custom Advanced Telecommunications Architecture (ATCA) card that interfaces the upstream FTK system with the ATLAS trigger and data acquisition (TDAQ) system, and allows for event processing on commercial PC blades making use of the 10 GB Ethernet full mesh ATCA back-plane. The FLIC receives data on 8 optical links at a bandwidth of about 1 Gbps per channel, reformats the data to the ATLAS standard record format, and performs the conversion from local to global module identifier using look up tables in SRAM. After processing, the event records are sent out to the TDAQ system using the S-LINK protocol at 2 Gbps, with a latency of O(10 microseconds). The data processing is handled in two Xilinx Virtex-6 FPGAs, with two additional ...

  10. PCR-based cDNA library construction: general cDNA libraries at the level of a few cells.

    OpenAIRE

    Belyavsky, A; Vinogradova, T; Rajewsky, K

    1989-01-01

    A procedure for the construction of general cDNA libraries is described which is based on the amplification of total cDNA in vitro. The first cDNA strand is synthesized from total RNA using an oligo(dT)-containing primer. After oligo(dG) tailing the total cDNA is amplified by PCR using two primers complementary to oligo(dA) and oligo(dG) ends of the cDNA. For insertion of the cDNA into a vector a controlled trimming of the 3' ends of the cDNA by Klenow enzyme was used. Starting from 10 J558L ...

  11. Specific and sensitive detection of Ralstonia solanacearum in soil on the basis of PCR amplification of fliC fragments

    NARCIS (Netherlands)

    Schonfeld, J.; Heuer, H.; Elsas, van J.D.; Smalla, K.

    2003-01-01

    Ralstonia solanacearum is the causative agent of bacterial wilt in many important crops. A specific and sensitive PCR detection method that uses primers targeting the gene coding for the flagella subunit, fliC, was established. Based on the first fliC gene sequence of R. solanacearum strain K60

  12. PCR-RFLP Analysis of a fliC Gene Fragment in Avian Salmonella Isolates

    Directory of Open Access Journals (Sweden)

    Zohreh Ebrahimvandi

    2014-07-01

    Full Text Available Background: Salmonella are a genus of zoonotic bacteria of worldwide economic and health importance. Members of Salmonella enterica subspecies enterica are mainly associated with warm-blooded vertebrates and are usually transmitted by ingestion of food or watercontaminated by infected feces. Objectives: The aim of this study was to apply a PCR-RFLP method based on the fliC gene to identify the serotypes of Salmonella isolates from Karaj, Iran. Materials and Methods: A total of 30 Salmonella isolates were serotyped by specific antisera. For the PCR-RFLP method based on the fliC gene, extracted DNA was used as the template for amplifying the fliC gene (1500 bp using specific primers. PCR products were subjected to digestion using HhaI restriction endonuclease. Results: This study determined 30 serotypes as Salmonella durban (56.6%, Salmonella uno (23.3%, Salmonella enteritidis (3.3%, Salmonella tinda (3.3%, Salmonella mjimweme (3.3%, Salmonella Thompson (3.3%, Salmonella sIIO8 (3.3 % and Salmonella sIIO7 (3.3%. Observations indicated that HhaI is able to discriminate Salmonella tinda and Salmonella thompson, yet Salmonella enteritidis, Salmonella durban and Salmonella mjimweme had the same pattern with this enzyme. Also Salmonella sIIO8, Salmonella sIIO7 and Salmonella uno showed the same pattern. Thus, regarding the size and the number of resulting fragments from this enzyme, four patterns were obtained for HhaI. Conclusion: A large number of Salmonella serotypes need to be analyzed by the PCR-RFLP method and different enzymes must be used to give reliable results.

  13. 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...

  14. [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.

  15. 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...... 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...

  16. [Construction of chicken embryo fibroblasts cDNA expression library].

    Science.gov (United States)

    Liu, Wei; Gao, Yu-long; Gao, Hong-lei; Wang, Xiao-mei; Xu, Xiu-hong

    2007-06-01

    Chicken embryo fibroblast (CEF) is a primary cellular material to research the infectious bursal disease virus (IBDV). Constructing the cDNA expression library of CEF is the foundation to research cell tropism and find cell receptors of IBDV from CEF. In order to achieve that purpose, a high-quality cDNA expression library of CEF was constructed by Gateway technology, which could avoid using the restriction enzyme for cloning to solve technical limitation of roution method. The mRNA was extracted from chicken embryonic fibroblast. Moreover, single-strand cDNA and double-strand cDNA were synthesized by using biotin-conjugated Oligo (dT) primer in turn. The double-strand cDNA was ligated Adapter and then purified by the cDNA Size Fractionation Columns. After BP recombination reaction, a cDNA entry library was constructed with a titer of 1 x 10(6) cfu/mL, total clones of 1.2 x 10(7) cfu and an average insertion size of about 2243 bp. After LR recombination reaction, the cDNA entry library was transformed into expression library which took on a titer of 5 x 10(5) cfu/mL, total clones of 5.5 x 10(6) cfu and an average insertion size of about 2411bp. The results indicate that the constructed cDNA expression library performs a remarkable high value in both recombination rate and library coverage. As a result, the cDNA expression library, with its good quality, may facilitate to identify the receptors associated with the resistance against IBDV in chicken embryonic fibroblast and to cast new light on the mechanism of cellular tropism. Moreover, it may also provide data of chicken embryonic fibroblast in transcription level and may be helpful to study its biological functions.

  17. cDNA libraries for virus-induced gene silencing.

    Science.gov (United States)

    Todd, Andrea T; Liu, Enwu; Page, Jonathan E

    2010-01-01

    Virus-induced gene silencing (VIGS) exploits endogenous plant antiviral defense mechanisms to posttranscriptionally silence the expression of targeted plant genes. VIGS is quick and relatively easy to perform and therefore serves as a powerful tool for high-throughput functional genomics in plants. Combined with the use of subtractive cDNA libraries for generating a collection of VIGS-ready cDNA inserts, VIGS can be utilized to screen a large number of genes to determine phenotypes resulting from the knockdown/knockout of gene function. Taking into account the optimal insert design for VIGS, we describe a methodology for producing VIGS-ready cDNA libraries enriched for inserts relevant to the biological process of interest.

  18. [Construction and identification of the expression library of album pollen allergens cDNA].

    Science.gov (United States)

    Zhang, Jie; Sun, Xiu-zhen; Yan, Hong; Zhang, Ni; Feng, Xiang-li

    2011-05-01

    To construct and identify the express library of album pollen allergens cDNA. Total RNA were extracted from the album pollen with TRIzol reagent and the mRNA was isolate for the amplify followed. A double stranded cDNA (ds cDNA) was synthesized using primers containing Xho I and Poly(dT) sequence by ZAP Express®cDNA synthesis kit. The ds cDNA was modified and purified by gel chromatography, and then the cDNA fragment with the length of more than 400 bp containing sticky ends was obtained. The cDNA fragment was ligated with Uni-ZAP XR vector and subsequently treated with in vitro packaging using phage by ZAP-cDNA express GigapackIII Gold cloning kit. The express library of album pollen cDNA was constructed by in vitro packaging. The recombination rate and the lengths of fragments inserted of the cDNA library were detected by polymerase chain reaction. The titer and the recombination rate of cDNA expression library constructed were 9.7×10(5) and 100%, respectively. The capacity of the library was 4.85 Pfu. The average length of cDNA fragments inserted was about 1.0 kb. Based on the capacity of cDNA expression library constructed and the length of cDNA insertion fragments, the cDNA expression library constructed is qualified to screening target cDNA clone, laying the foundation for preparation of gene recombinant allergen pollen vaccine.

  19. New Approaches to Attenuated Hepatitis a Vaccine Development: Cloning and Sequencing of Cell-Culture Adapted Viral cDNA.

    Science.gov (United States)

    1987-10-13

    Insert frag- ments from p16 cDNA clones were subcloned into the phage vector Ml3mp8 or Ml3mpl9 qnd subjected to rapid sequencing using the...2), and selected cDNA insert fragments were subcloned into M13 vectors for sequencing. The sequence of the complete genome was determined, with over

  20. 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

  1. [Construction and Identification of the cDNA Expression Library for Human Esophageal Cancer Cells].

    Science.gov (United States)

    Zhang, Zhe; Wu, Xiang-Yu; Feng, Lu; Huang, Shang-Ke; Luo, Min-Na; Shao, Shan; Zhao, Xin-Han

    2016-09-01

    To construct a cDNA phage expression library for human esophageal cancer cells. After the total RNA were obtained from esophageal cancer cells, the mRNA were separated with magnetic beads adsorption method, and the single-strand and double-strand cDNA were synthesized through reverse transcription. With the undesirable cDNA fragments removed, the remaining cDNA (linked with Eco R1 aptamer and phosphorylated its 5'end) combined with the carrier of T7 Select10-3b. The recombinant phage were packaged in vitro for preliminary cDNA library. PCR was used to identify the size of inserted cDNA. The constructed original cDNA phage expression library for human esophageal cancer cells was consisted of 2.01×10⁶ pfu/mL bacteriophages with a recombination rate of 100%. The length of the inserted cDNA fragments were range from 300 bp to 1 500 bp. The cDNA phage expression library of human esophageal cell is successfully constructed to meet the currently recognized standards, and can be well used to screen cDNA-cloned genes of human esophageal cancer antigens by serological analysis of recombinantly expressed cDNA clone (SEREX).

  2. Normalizing cDNA libraries.

    Science.gov (United States)

    Bogdanov, Ekaterina A; Shagina, Irina; Barsova, Ekaterina V; Kelmanson, Ilya; Shagin, Dmitry A; Lukyanov, Sergey A

    2010-04-01

    The characterization of rare messages in cDNA libraries is complicated by the substantial variations that exist in the abundance levels of different transcripts in cells and tissues. The equalization (normalization) of cDNA is a helpful approach for decreasing the prevalence of abundant transcripts, thereby facilitating the assessment of rare transcripts. This unit provides a method for duplex-specific nuclease (DSN)-based normalization, which allows for the fast and reliable equalization of cDNA, thereby facilitating the generation of normalized, full-length-enriched cDNA libraries, and enabling efficient RNA analyses. (c) 2010 by John Wiley & Sons, Inc.

  3. Flagellin gene (fliC) of Thermus thermophilus HB8: characterization of its product and involvement to flagella assembly and microbial motility.

    Science.gov (United States)

    Papaneophytou, Christos P; Papi, Rigini M; Pantazaki, Anastasia A; Kyriakidis, Dimitrios A

    2012-06-01

    Thermus thermophilus HB8 flagellin protein (FliC) is encoded by the TTHC004 (fliC) gene, which is located in the pTT8 plasmid of the bacterium. Flagellin monomer and flagella fibres were isolated from a culture of T. thermophilus grown in rich medium, or in mineral salt medium with sodium gluconate as the carbon source. Western blot immunodetection with anti-FliC revealed a stable complex (FliC)(1)(FliS)(2) of flagellin (FliC, 27.7 kDa) with a homodimer of FliS (FliS, 18.2 kDa) that are encoded by TTHC004 and TTHC003 genes, respectively. The complex is dissociable at low pHs and/or by heat treatment. Glycan staining of purified flagella and treatment with N-glycosidase F suggested that flagellin of T. thermophilus is a glycosylated protein. Size exclusion chromatography revealed that flagellar filaments (FliC) have a molecular mass higher than 200 kDa. The formation of flagella is enhanced after prolonged cultivation time where phosphate and other nutrient were depleted, giving in the bacterium considerable swimming motility in low viscosity media.

  4. Construction of cDNA library of Pyrocystis lunula (Pyrophyta)

    Science.gov (United States)

    Sui, Zhenghong; Kowallik, Klaus V.

    2004-10-01

    Complementary DNA library of a dinoflagellate Pyrocystis lunula was constructed for the purpose of expression sequence tags analysis. The RNA isolated from this alga was about 20µgg-1 net cells, and the band intensity ratio of 28S/18 S in electrophoresis pattern was nearly 1 to 1. Different cDNA/vector molar ratios were exploited in the ligating reaction to be optimized. The clones produced by cDNA/vector molar ratio of 3.75 to 1 were desirable, most of whose inserts were longer than 300 bp. The recombinants insert length of the unfractionation cDNA library was largely shorter than 500 bp. However, in the fractionation library made from high molecule weight cDNA parts, over seventy percent of the recombinants contained inserts longer than 1 kb, some of which were even longer than 3 kb. Operating concerns were discussed at the end.

  5. 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.

  6. Efficient and simpler method to construct normalized cDNA libraries with improved representations of full-length cDNAs

    Science.gov (United States)

    Soares, Marcelo Bento; Bonaldo, Maria de Fatima

    1998-01-01

    This invention provides a method to normalize a cDNA library comprising: (a) constructing a directionally cloned library containing cDNA inserts wherein the insert is capable of being amplified by polymerase chain reaction; (b) converting a double-stranded cDNA library into single-stranded DNA circles; (c) generating single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) by polymerase chain reaction with appropriate primers; (d) hybridizing the single-stranded DNA circles converted in step (b) with the complementary single-stranded nucleic acid molecules generated in step (c) to produce partial duplexes to an appropriate Cot; and (e) separating the unhybridized single-stranded DNA circles from the hybridized DNA circles, thereby generating a normalized cDNA library. This invention also provides a method to normalize a cDNA library wherein the generating of single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) is by excising cDNA inserts from the double-stranded cDNA library; purifying the cDNA inserts from cloning vectors; and digesting the cDNA inserts with an exonuclease. This invention further provides a method to construct a subtractive cDNA library following the steps described above. This invention further provides normalized and/or subtractive cDNA libraries generated by the above methods.

  7. cDNA library preparation.

    Science.gov (United States)

    Kooiker, Maarten; Xue, Gang-Ping

    2014-01-01

    The construction of full-length cDNA libraries allows researchers to study gene expression and protein interactions and undertake gene discovery. Recent improvements allow the construction of high-quality cDNA libraries, with small amounts of mRNA. In parallel, these improvements allow for the incorporation of adapters into the cDNA, both at the 5' and 3' end of the cDNA. The 3' adapter is attached to the oligo-dT primer that is used by the reverse transcriptase, whereas the 5' adapter is incorporated by the template switching properties of the MMLV reverse transcriptase. This allows directional cloning and eliminates inefficient steps like adapter ligation, phosphorylation, and methylation. Another important step in the construction of high-quality cDNA libraries is the normalization. The difference in the levels of expression between genes might be several orders of magnitude. Therefore, it is essential that the cDNA library is normalized. With a recently discovered enzyme, duplex-specific nuclease, it is possible to normalize the cDNA library, based on the fact that more abundant molecules are more likely to reanneal after denaturation compared to rare molecules.

  8. Normalized cDNA libraries

    Science.gov (United States)

    Soares, Marcelo B.; Efstratiadis, Argiris

    1997-01-01

    This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3' noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to moderate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library.

  9. Experimental insertions

    International Nuclear Information System (INIS)

    Sandweiss, J.; Kycia, T.F.

    1975-01-01

    A discussion is given of the eight identical experimental insertions for the planned ISABELLE storage rings. Four sets of quadrupole doublets are used to match the β functions in the insertions to the values in the cells, and the total free space available at the crossing point is 40 meters. An asymmetric beam energy operation is planned, which will be useful in a number of experiments

  10. Peripherally inserted central catheter - insertion

    Science.gov (United States)

    PICC - insertion ... A PICC is a long, thin tube (called a catheter) that goes into your body through a vein in ... into a large vein near your heart. The PICC helps carry nutrients and medicines into your body. ...

  11. N-glycosidase treatment with 18O labeling and de novo sequencing argues for flagellin FliC glycopolymorphism in Pseudomonas aeruginosa.

    Science.gov (United States)

    Khemiri, Arbia; Naudin, Bertrand; Franck, Xavier; Song, Philippe Chan Tchi; Jouenne, Thierry; Cosette, Pascal

    2013-12-01

    In prokaryote organisms, N-glycosylation of proteins is often correlated to cell-cell recognition and extracellular events. Those glycoproteins are potential targets for infection control. To date, many surface-glycosylated proteins from bacterial pathogens have been described. However, N-linked Pseudomonas surface-associated glycoproteins remain underexplored. We report a combined enrichment and labeling strategy to identify major glycoproteins on the outside of microorganisms. More precisely, bacteria were exposed to a mix of biotinylated lectins able to bind with glycoproteins. The latter were then recovered by avidin beads, digested with trypsin, and submitted to mass spectrometry. The targeted mixture of glycoproteins was additionally deglycosylated in the presence of H2(18)O to incorporate (18)O during PNGase F treatment and were also analyzed using mass spectrometry. This approach allowed us to identify a few tens of potential N-glycoproteins, among which flagellin FliC was the most abundant. To detect the possible sites of FliC modifications, a de novo sequencing step was also performed to discriminate between spontaneous deamidation and N-glycan loss. This approach led to the proposal of three potential N-glycosylated sites on the primary sequence of FliC: N26, N69, and N439, with two of these three asparagines belonging to an N-X-(S/T) consensus sequence. These observations suggest that flagellin FliC is a heterogeneous protein mixture containing both O- and N-glycoforms.

  12. Construction of a T7 Human Lung Cancer cDNA Library

    Directory of Open Access Journals (Sweden)

    Wentao YUE

    2008-10-01

    Full Text Available Background and objective Currently, only a limited numbers of tumor markers for non small lung cancer (NSCLC diagnosis, new biomarker, such as serum autoantibody may improve the early detection of lung cancer. Our objective is construction human lung squamous carcinoma and adenocarcinoma T7 phage display cDNA library from the tissues of NSCLC patients. Methods mRNA was isolated from a pool of total RNA extract from NSCLC tissues obtained from 5 adenocarcinomas and 5 squamous carcinomas, and then mRNA was reverse transcribed into double stranded cDNA. After digestion, the cDNA was inserted into T7Select 10-3 vector. The phage display cDNA library was constructed by package reaction in vitro and plate proliferation. Plaque assay and PCR were used to evaluate the library.Results Two T7 phage display cDNA library were established. Plaque assay show the titer of lung squamas carcinoma library was 1.8×106 pfu, and the adenocarcinoma library was 5×106 pfu. The phage titer of the amplified library were 3.2×1010 pfu/mL and 2.5×1010 pfu/mL. PCR amplification of random plaque show insert ratio were 100% (24/24 in adenocarcinoma library and 95.8% in human lung squamas carcinoma library (23/24. Insert range from 300 bp to 1 500 bp. Conclusion Two phage display cDNA library from NSCLC were constructed.

  13. Cloning of human purine-nucleoside phosphorylase cDNA sequences by complementation in Escherichia coli.

    OpenAIRE

    Goddard, J M; Caput, D; Williams, S R; Martin, D W

    1983-01-01

    We have obtained cDNA clones that contain the entire coding region of the human purine-nucleoside phosphorylase (PNP; EC 2.4.2.1) mRNA. The cDNA sequences were generated by reverse transcription of PNP-enriched mRNA obtained by immunoadsorption of HeLa cell polyribosomes with monospecific antibody to human PNP. cDNA molecules that were close in length to PNP mRNA were separated by agarose gel electrophoresis and inserted into the Pst I site of the plasmid pBR322. Plasmid DNA from the pooled c...

  14. [Construction of cDNA library of Magnaporthe grisea with magnetic bead].

    Science.gov (United States)

    Feng, Xu; Xiaoli, Wu; Dewen, Qiu

    2008-06-01

    We constructed cDNA library of Magnaporthe grisea. The good quality cDNA library could facilitate finding proteinaceous elicitors of M. grisea, and elucidating the mechanisms of the M. grisea--rice interaction. The Oligo(dT) combined with the magnetic bead was used to extract mRNA from total RNA of Magnaporthe grisea and as primers to synthesize the first-strand cDNA. Terminal transferase introduced PolyA into 3'terminal of the first cDNA strand, then the PolyA was used for amplifying the second-strand cDNA. Restriction enzyme and adapter were avoided in this research, which could solve technical limitation of the traditional method. Because all reactions were done in one centrifuge tube, this process could reduce the risk of cDNA loss and cross-contamination. The primers designed in this research could clone the amplified cDNAs into expression vector in a desirable orientation. The cDNA library constructed had a high titer of 8.9 x 10(6) cfu/mL, and contained a total clones of 8.9 x 10(7) cfu, with an average inserts size of about 1380 bp. Constructing cDNA library with magnetic bead was a highly efficient method using only small amount of experimental materials within a short period.

  15. An Eimeria vaccine candidate based on Eimeria tenella immune mapped protein 1 and the TLR-5 agonist Salmonella typhimurium FliC flagellin

    Energy Technology Data Exchange (ETDEWEB)

    Yin, Guangwen; Qin, Mei [National Animal Protozoa Laboratory and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China); Liu, Xianyong [National Animal Protozoa Laboratory and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China); Key Laboratory of Zoonosis, China Ministry of Agriculture and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China); Suo, Jingxia; Tang, Xinming; Tao, Geru [National Animal Protozoa Laboratory and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China); Han, Qian [Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061 (United States); Suo, Xun [National Animal Protozoa Laboratory and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China); Key Laboratory of Zoonosis, China Ministry of Agriculture and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China); Wu, Wenxue, E-mail: labboard@126.com [National Animal Protozoa Laboratory and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China); Key Laboratory of Zoonosis, China Ministry of Agriculture and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China)

    2013-10-25

    Highlights: •We found a new protective protein – (IMPI) in Eimeria tenella. •EtIMP1-flagellin fusion protein is an effective immunogen against Eimeria infection. •Flagellin can be as an apicomplexan parasite vaccine adjuvant in chickens. -- Abstract: Immune mapped protein-1 (IMP1) is a new protective protein in apicomplexan parasites, and exits in Eimeria tenella. But its structure and immunogenicity in E. tenella are still unknown. In this study, IMPI in E. tenella was predicted to be a membrane protein. To evaluate immunogenicity of IMPI in E. tenella, a chimeric subunit vaccine consisting of E. tenella IMP1 (EtIMP1) and a molecular adjuvant (a truncated flagellin, FliC) was constructed and over-expressed in Escherichia coli and its efficacy against E. tenella infection was evaluated. Three-week-old AA broiler chickens were vaccinated with the recombinant EtIMP1-truncated FliC without adjuvant or EtIMP1 with Freund’s Complete Adjuvant. Immunization of chickens with the recombinant EtIMP1-truncated FliC fusion protein resulted in stronger cellular immune responses than immunization with only recombinant EtIMP1 with adjuvant. The clinical effect of the EtIMP1-truncated FliC without adjuvant was also greater than that of the EtIMP1 with adjuvant, which was evidenced by the differences between the two groups in body weight gain, oocyst output and caecal lesions of E. tenella-challenged chickens. The results suggested that the EtIMP1-flagellin fusion protein can be used as an effective immunogen in the development of subunit vaccines against Eimeria infection. This is the first demonstration of antigen-specific protective immunity against avian coccidiosis using a recombinant flagellin as an apicomplexan parasite vaccine adjuvant in chickens.

  16. An Eimeria vaccine candidate based on Eimeria tenella immune mapped protein 1 and the TLR-5 agonist Salmonella typhimurium FliC flagellin

    International Nuclear Information System (INIS)

    Yin, Guangwen; Qin, Mei; Liu, Xianyong; Suo, Jingxia; Tang, Xinming; Tao, Geru; Han, Qian; Suo, Xun; Wu, Wenxue

    2013-01-01

    Highlights: •We found a new protective protein – (IMPI) in Eimeria tenella. •EtIMP1-flagellin fusion protein is an effective immunogen against Eimeria infection. •Flagellin can be as an apicomplexan parasite vaccine adjuvant in chickens. -- Abstract: Immune mapped protein-1 (IMP1) is a new protective protein in apicomplexan parasites, and exits in Eimeria tenella. But its structure and immunogenicity in E. tenella are still unknown. In this study, IMPI in E. tenella was predicted to be a membrane protein. To evaluate immunogenicity of IMPI in E. tenella, a chimeric subunit vaccine consisting of E. tenella IMP1 (EtIMP1) and a molecular adjuvant (a truncated flagellin, FliC) was constructed and over-expressed in Escherichia coli and its efficacy against E. tenella infection was evaluated. Three-week-old AA broiler chickens were vaccinated with the recombinant EtIMP1-truncated FliC without adjuvant or EtIMP1 with Freund’s Complete Adjuvant. Immunization of chickens with the recombinant EtIMP1-truncated FliC fusion protein resulted in stronger cellular immune responses than immunization with only recombinant EtIMP1 with adjuvant. The clinical effect of the EtIMP1-truncated FliC without adjuvant was also greater than that of the EtIMP1 with adjuvant, which was evidenced by the differences between the two groups in body weight gain, oocyst output and caecal lesions of E. tenella-challenged chickens. The results suggested that the EtIMP1-flagellin fusion protein can be used as an effective immunogen in the development of subunit vaccines against Eimeria infection. This is the first demonstration of antigen-specific protective immunity against avian coccidiosis using a recombinant flagellin as an apicomplexan parasite vaccine adjuvant in chickens

  17. [Construction of a cDNA library from liver tissue of rhesus monkey, Macaca mulatta].

    Science.gov (United States)

    Qin, Sheng-fang; Tan, Wei-dong; Chen, You-nan; Ding, Yang; Li, Sheng-fu; Li, Hong-xia; Wang, Li; Yang, Rong; Lu, Yan-rong

    2007-06-01

    To screen the target rhesus genes and give some basic genetic evidences to its value as one of the most important animal model in biomedical study, we constructed a cDNA expression library from liver tissue of a healthy rhesus monkey. With Trizol reagent, the total RNA was extracted from healthy rhesus liver tissue. By mutant Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT), the first-strand cDNA was synthesized from purified mRNA, and subsequently the second-strand cDNA was generated via E. coli DNA polymerase I . Then, the EcoR I adapter was added to the synthesized double-strand cDNA, which was subsequently digested by Xho I restriction enzyme and fractionated with CHROMA APIN-400 column. The fractionated cDNA fragments to be longer than 0. 5 kb were ligated into lambda ZAP express vector to form the phagemid cDNA recombinants, which were further packaged into the lambda ZAP cDNA library according to the standard protocol with phage lambda Gold packaging extract. In order to get more stable clones with larger quantity, the primary library was amplified through infecting the host strain XL1-Blue MRF'. Then, the library titre, recombinant rate and length of inserted cDNA were measured, respectively. The capacity of the primary stand or unamplified library was 1. 2X 10(6) pfu. The titers of the unamplified library or the amplified library was 1.1 X 10(6) mixture, pfu/mL or 7. 7 X 10(9) pfu/mL respectively, the percentages of recombinants were 99. 3% and 98. 2%, and the average lengths of the inserts were 2.0 kb and 2. 3 kb, respectively. An excellent cDNA expression library has been constructed successfully, which would lay solid foundation for transplantation study and pre-clinic evaluation of related drugs.

  18. Cloning of fimH and fliC and expression of the fusion protein FimH/FliC from Uropathogenic Escherichia coli (UPEC) isolated in Iran.

    Science.gov (United States)

    Asadi, Karam Mr; Oloomi, M; Habibi, M; Bouzari, S

    2012-06-01

    Urinary tract infection (UTI) is one of the most common infections in the world. The majority of UTIs are caused by Uropathogenic Escherichia coli (UPEC) strains. FimH and FliC are the most important virulence factors of UPEC. To date, any ideal vaccine against UTI has not been approved for human use and we need to test new targets to develop an ideal vaccine against UTI. In this study, we constructed fusion fimH/fliC of UPEC as a novel vaccine candidate against UTI. PCR amplification of fimH and fliC genes of the UPEC isolates was performed by specific primers designed for this purpose. Construction of fimH/fliC hybrid gene was performed by overlap PCR. The fimH, fliC and fimH/fliC were cloned in pET28a vector. The confirmation of expression of the proteins was done by SDS-PAGE and Western blot. The fliC and fimH genes were amplified in all of the UPEC isolates tested. The fimH showed significant homology with the sequences in GenBank. We generated a fusion consisting of the fimH linked to the N-terminal end of fliC. Sequencing of the fusion fimH/fliC showed that fusion was constructed correctly. SDS-PAGE and western blot confirmed the expression of the proteins in optimized condition. Urinary tract infection is a huge burden on healthcare system in many countries. UPEC is isolated in around 80% of UTI cases. Antibiotic therapy resulted in the emergence of antibiotic resistance in UPEC strains. This is the major cause for an increasing requirement for a vaccine to prevent UTI. This work describes for the first time the construction of a novel fusion protein from Iranian UPEC isolates. Further immunological studies are required for evaluation of this protein as a novel and safe vaccine candidate against UTI caused by UPEC.

  19. Development of PCR primers for the detection of Salmonella enterica serovar Choleraesuis based on the fliC gene.

    Science.gov (United States)

    Chiu, Tsai-Hsin; Pang, Jen-Chieh; Hwang, Wen-Zhe; Tsen, Hau-Yang

    2005-08-01

    Salmonella enterica serovar Choleraesuis may cause swine salmonellosis and human infection. Because the conventional method for detection of this Salmonella serovar may take 3 to 5 days, a PCR method for detection was evaluated. By comparing the sequence of the phase 1 flagellin (fliC) gene of Salmonella Choleraesuis with that of other Salmonella serovars and of other bacteria species available in GenBank, two PCR primers (flinC-F and flinC-R) were designed. Using these primers, all 97 Salmonella Choleraesuis strains assayed generated the expected PCR product, with a molecular mass of 963 bp. Except for S. enterica Paratyphi C, Salmonella isolates other than Salmonella Choleraesuis and non-Salmonella isolates, including strains of Enterobacteriaceae, all generated negative PCR results. Salmonella Paratyphi C could be differentiated from Salmonella Choleraesuis through the use of primers designed from the viaB gene. When Salmonella Choleraesuis isolates from swine stool, pork, liver, feed, and human whole blood samples were assayed with a preenrichment step, as low as 1 CFU/g or ml of the original sample could be detected.

  20. SMART amplification combined with cDNA size fractionation in order to obtain large full-length clones

    Directory of Open Access Journals (Sweden)

    Poustka Annemarie

    2004-06-01

    Full Text Available Abstract Background cDNA libraries are widely used to identify genes and splice variants, and as a physical resource for full-length clones. Conventionally-generated cDNA libraries contain a high percentage of 5'-truncated clones. Current library construction methods that enrich for full-length mRNA are laborious, and involve several enzymatic steps performed on mRNA, which renders them sensitive to RNA degradation. The SMART technique for full-length enrichment is robust but results in limited cDNA insert size of the library. Results We describe a method to construct SMART full-length enriched cDNA libraries with large insert sizes. Sub-libraries were generated from size-fractionated cDNA with an average insert size of up to seven kb. The percentage of full-length clones was calculated for different size ranges from BLAST results of over 12,000 5'ESTs. Conclusions The presented technique is suitable to generate full-length enriched cDNA libraries with large average insert sizes in a straightforward and robust way. The representation of full-coding clones is high also for large cDNAs (70%, 4–10 kb, when high-quality starting mRNA is used.

  1. Complete amino acid sequence of human intestinal aminopeptidase N as deduced from cloned cDNA

    DEFF Research Database (Denmark)

    Cowell, G M; Kønigshøfer, E; Danielsen, E M

    1988-01-01

    The complete primary structure (967 amino acids) of an intestinal human aminopeptidase N (EC 3.4.11.2) was deduced from the sequence of a cDNA clone. Aminopeptidase N is anchored to the microvillar membrane via an uncleaved signal for membrane insertion. A domain constituting amino acid 250...

  2. [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.

  3. 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

  4. [Screening of specifically expressed genes in amphioxus neurula by construction of a subtractive cDNA library].

    Science.gov (United States)

    Zhang, Lei; Yang, Yong-Jie; Zhang, Yan-Jun

    2010-12-01

    To screen specifically expressed genes in the development of nerve, muscle, and body axis of amphioxus, Branchiostoma belcheri tsingtauenese. A subtractive cDNA library was constructed from the 12-hour amphioxus neurula cDNA after subtractively hybridized with the 6-hour amphioxus gastrula cDNA. The total RNA was extracted from the 12-hour neurula and 6-hour gastrula, then reverse transcribed into cDNA. The 12-hour neurula cDNA was designated as the experimental group (the tester) and the 6-hour gastrula cDNA as the control group (the driver). The differentially expressed sequences were exponentially amplified using suppression PCR. Background was subtracted and differentially expressed sequences were further enriched. The PCR products were ligated to the T Vector. After transformation of the recombinant plasmid carrying inserted amphioxus cDNA into E.coli host cells, the cDNA library was constructed successfully. Two hundred randomly chosen positive clones were sequenced and some of neurula-specifically expressed genes were obtained. SSH is an effective method for searching differentially expressed genes. The subtractive cDNA library we generated provides a tool for further study of regulatory mechanisms of amphioxus early embryonic development.

  5. Optimized cDNA libraries for virus-induced gene silencing (VIGS using tobacco rattle virus

    Directory of Open Access Journals (Sweden)

    Page Jonathan E

    2008-01-01

    Full Text Available Abstract Background Virus-induced gene silencing (VIGS has emerged as a method for performing rapid loss-of-function experiments in plants. Despite its expanding use, the effect of host gene insert length and other properties on silencing efficiency have not been systematically tested. In this study, we probed the optimal properties of cDNA fragments of the phytoene desaturase (PDS gene for efficient VIGS in Nicotiana benthamiana using tobacco rattle virus (TRV. Results NbPDS inserts of between 192 bp and 1304 bp led to efficient silencing as determined by analysis of leaf chlorophyll a levels. The region of the NbPDS cDNA used for silencing had a small effect on silencing efficiency with 5' and 3' located inserts performing more poorly than those from the middle. Silencing efficiency was reduced by the inclusion of a 24 bp poly(A or poly(G homopolymeric region. We developed a method for constructing cDNA libraries for use as a source of VIGS-ready constructs. Library construction involved the synthesis of cDNA on a solid phase support, digestion with RsaI to yield short cDNA fragments lacking poly(A tails and suppression subtractive hybridization to enrich for differentially expressed transcripts. We constructed two cDNA libraries from methyl-jasmonate treated N. benthamiana roots and obtained 2948 ESTs. Thirty percent of the cDNA inserts were 401–500 bp in length and 99.5% lacked poly(A tails. To test the efficiency of constructs derived from the VIGS-cDNA libraries, we silenced the nicotine biosynthetic enzyme, putrescine N-methyltransferase (PMT, with ten different VIGS-NbPMT constructs ranging from 122 bp to 517 bp. Leaf nicotine levels were reduced by more than 90% in all plants infected with the NbPMT constructs. Conclusion Based on the silencing of NbPDS and NbPMT, we suggest the following design guidelines for constructs in TRV vectors: (1 Insert lengths should be in the range of ~200 bp to ~1300 bp, (2 they should be positioned in

  6. 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)

  7. Construction and characterization of a cDNA library from head kidney of Japanese sea bass (Lateolabrax japonicus).

    Science.gov (United States)

    Shao, Zhan-tao; Cong, Xiao; Yuan, Jin-duo; Yang, Gui-wen; Chen, Ying; Pan, Jie; An, Li-guo

    2009-09-01

    In this paper, a cDNA expression library from head kidney of Japanese sea bass (Lateolabrax japonicus) was constructed for the first time. The first-strand cDNA was synthesized with Moloney Murine Leukaemia virus reverse transcriptase and the double-stranded cDNA was digested by Xho I enzyme. Size fractionation was performed on CHROMA SPIN-400 columns. cDNA fragments longer than 500 bps were ligated into the lambdaZAPExpress vector. The recombinant DNA was packaged in vitro with Gigapack III gold packaging extract. The titers of the primary and amplified library were 1.0 x 10(5) and 5.0 x 10(9) pfu/ml, respectively. To characterize the constructed cDNA library, 15 phage plaques were selected randomly to test the inserted fragments. The results showed that the inserts were mostly longer than 500 bps. To test the utility, the library was screened with primers designed for three immune-related genes of, Myxovirus resistant (Mx), tumor necrosis factor-alpha (TNF-alpha) and Toll-like receptor (TLR). Results of Blastn and alignment showed that they are members of Mx, TNF-alpha and TLR gene families, respectively, which meets our anticipates for this cDNA library as an immune-related one. These results confirmed that the cDNA library constructed will provide a useful tool for gene cloning and expression analysis in immune system of Japanese sea bass.

  8. Construction and primary characterization of Echinococcus multilocularis protoscolex cDNA expression library.

    Science.gov (United States)

    Li, S; Chen, Y

    2001-02-01

    To construct a lambda gt11 cDNA expression library of Echinococcus multilocularis protoscolex isolated in China. Echinococcus multilocularis protoscolex mRNA was extracted using a Quickprep MicromRNA purification kit based on combining of the disruptive and protective properties of guanidinium thiocyanate (GTC) with the speed and selectivity of oligo (dT)-cellulose chromatography in a spum-column with some modification. Purified mRNA (1.8 micrograms) was submitted to reverse transcription using random hexamers [pd(N6)]. The double-strand blunt-ended cDNAs were ligated with an EcoRI/Notl adaptor to form a cohesive EcoRI end. Subsequently the synthesized cDNA was inserted into vector lambda gt11 EcoRI arms. After being packaged in vitro, lambda gt11 was put to an infectious bacteria Echinococcus coli (E. coli) strain Y1090; the recombinants were screened by color selection. PCR amplification was performed to evaluate the size of insertion DNA fragments. The recombinant ratio was nearly 100% and approximately 1 x 10(6) clones could be derived from this lambda gt11 cDNA library. PCR results indicated that the insertion DNAs were about 1.48 kb. A lambda gt11 cDNA expression library consisting of a million recombinant clones has been constructed from Echinococcus multicularis protoscolex mRNA. Further studies on this library are deserved.

  9. Construction of Infectious cDNA Clone of a Chrysanthemum stunt viroid Korean Isolate

    Science.gov (United States)

    Yoon, Ju-Yeon; Cho, In-Sook; Choi, Gug-Seoun; Choi, Seung-Kook

    2014-01-01

    Chrysanthemum stunt viroid (CSVd), a noncoding infectious RNA molecule, causes seriously economic losses of chrysanthemum for 3 or 4 years after its first infection. Monomeric cDNA clones of CSVd isolate SK1 (CSVd-SK1) were constructed in the plasmids pGEM-T easy vector and pUC19 vector. Linear positive-sense transcripts synthesized in vitro from the full-length monomeric cDNA clones of CSVd-SK1 could infect systemically tomato seedlings and chrysanthemum plants, suggesting that the linear CSVd RNA transcribed from the cDNA clones could be replicated as efficiently as circular CSVd in host species. However, direct inoculation of plasmid cDNA clones containing full-length monomeric cDNA of CSVd-SK1 failed to infect tomato and chrysanthemum and linear negative-sense transcripts from the plasmid DNAs were not infectious in the two plant species. The cDNA sequences of progeny viroid in systemically infected tomato and chrysanthemum showed a few substitutions at a specific nucleotide position, but there were no deletions and insertions in the sequences of the CSVd progeny from tomato and chrysanthemum plants. PMID:25288987

  10. [Construction of the subtracted cDNA libraries related to artemisinin-resistance of Plasmodium berghei].

    Science.gov (United States)

    Bei, Zhu-chun; Wang, Jing-yan

    2004-06-01

    To construct the subtracted cDNA libraries related to artemisinin-resistance of Plasmodium berghei using suppression subtractive hybridization PCR (SSH PCR). Total RNA was extracted from the artemisinin-sensitive (NS) and artemisinin-resistant (AR) strains of Plasmodium berghei K173. The cDNA synthesis followed the protocol of super SMART cDNA synthesis kit. Taking the NS as driver, AR as tester and reverse, two subtractions were performed by SSH PCR. Enriched different expressed cDNA was cloned into pMD18-T vector to construct subtractive libraries. The subtracted cDNA libraries of NS-AR and AR-NS contained 395 and 506 positive clones respectively. The PCR results of 108 clones picked randomly from each library showed 100 and 104 positive inserts contained in the plasmids respectively, and distributing in 250-2000 bp. The successful construction of the subtracted cDNA libraries related to artemisinin-resistance of P. berghei enable us to identify the different expressed genes involved in the resistance mechanism.

  11. Clonal study of avian Escherichia coli strains by fliC conserved-DNA-sequence regions analysis Estudo clonal de Escherichia coli aviário por análise de seqüências de DNA conservadas do gene fliC

    Directory of Open Access Journals (Sweden)

    Tatiana Amabile de Campos

    2008-10-01

    Full Text Available The clonal relationship among avian Escherichia coli strains and their genetic proximity with human pathogenic E. coli, Salmonela enterica, Yersinia enterocolitica and Proteus mirabilis, was determined by the DNA sequencing of the conserved 5' and 3'regions fliC gene (flagellin encoded gene. Among 30 commensal avian E. coli strains and 49 pathogenic avian E. coli strains (APEC, 24 commensal and 39 APEC strains harbored fliC gene with fragments size varying from 670bp to 1,900bp. The comparative analysis of these regions allowed the construction of a dendrogram of similarity possessing two main clusters: one compounded mainly by APEC strains and by H-antigens from human E. coli, and another one compounded by commensal avian E. coli strains, S. enterica, and by other H-antigens from human E. coli. Overall, this work demonstrated that fliC conserved regions may be associated with pathogenic clones of APEC strains, and also shows a great similarity among APEC and H-antigens of E. coli strains isolated from humans. These data, can add evidence that APEC strains can exhibit a zoonotic risk.A relação clonal entre linhagens de Escherichia coli de origem aviária e sua proximidade genética com E. coli patogênica para humanos, Salmonella enterica, Yersinia enterocolitica e Proteus mirabilis foi determinada através da utilização das seqüências conservadas 5' e 3' do gene fliC (responsável pela codificação da flagelina. Entre as 30 linhagens comensais de E. coli aviária e as 49 linhagens patogênicas de E. coli para aves (APEC, 24 linhagens comensais e 39 APEC apresentaram o gene fliC, que foi encontrado em tamanhos que variam de 670pb a 1900pb. Um dendrograma representando similaridade genética foi obtido a partir do seqüenciamento das regiões 5' e 3' conservadas do gene fliC das linhagens de E. coli de origem aviária, das seqüências dos antígenos H de E. coli de origem humana, de S. enterica, Y. enterocolitica e de P. mirabilis. A an

  12. Construction and analysis of SSH cDNA library of human vascular endothelial cells related to gastrocarcinoma.

    Science.gov (United States)

    Liu, Yong-Bo; Wei, Zhao-Xia; Li, Li; Li, Hang-Sheng; Chen, Hui; Li, Xiao-Wen

    2003-11-01

    To construct subtracted cDNA libraries of human vascular endothelial cells (VECs) related to gastrocarcinoma using suppression substractive hybridization (SSH) and to analyze cDNA libraries of gastrocarcinoma and VECs in Cancer Gene Anatomy Project (CGAP) database. Human VECs related to gastric adenocarcinoma and corresponding normal tissue were separated by magnetic beads coupled with antibody CD31 (Dynabeads CD31). A few amount of total RNA were synthesized and amplified by SMART PCR cDNA Synthesis Kit. Then, using SSH and T/A cloning techniques, cDNA fragments of differentially expressed genes in human VECs of gastric adenocarcinoma were inserted into JM109 bacteria. One hundred positive bacteria clones were randomly picked and identified by colony PCR method. To analyze cDNA libraries of gastrocarcinoma and VECs in CGAP database, the tools of Library Finder, cDNA xProfiler, Digital GENE Expression Displayer (DGED), and Digital Differential Display (DDD) were used. Forward and reverse subtraction cDNA libraries of human VECs related to gastrocarcinoma were constructed successfully with SSH and T/A cloning techniques. Analysis of CGAP database indicated that no appropriate library of VECs related to carcinoma was constructed. Construction of subtraction cDNA libraries of human VECs related to gastrocarcinoma was successful and necessary, which laid a foundation for screening and cloning new and specific genes of VECs related to gastrocarcinoma.

  13. [Construction and immunoscreening of cDNA library of Babesia orientalis].

    Science.gov (United States)

    Liu, Qin; Zhou, Dan-Na; Zhou, Yan-Qin; Zhang, Ying; He, Lan; Yao, Bao-An; Zhao, Jun-Long

    2009-06-01

    To construct a cDNA library for Babesia orientalis and screen immunologically positive clones. Total RNA of B. orientalis in red blood cells from an infected calf was isolated. cDNA was synthesized by reverse transcriptase, amplified by PCR and ligated into lambdaTriplEx2 vector. The recombined vectors were packaged and the unamplified cDNA library was constructed. The cDNA library was then amplified and immunologically screened with rabbit anti-B. orientalis serum. The recombinant lambdaTriplEx2 of positive clones were converted to the corresponding recombinant pTriplEx2. The inserted fragments were identified by PCR amplification. The plasmids were sequenced and compared against GenBank database by Blast. The titer of the unamplified library was 2.0 x 10(6) pfu/ml. The inserted fragment length of the library ranged from 500 to 3,000 bp, and the recombination efficiency accounted for 98.8%. The titer of the amplified library was 5.8 x 10(8) pfu/ml. Three positive clones were selected by serum immunological screening and named B04, B05, and B41, respectively. The inserted fragments of the B04, B05 and B41 were about 1,300 bp, 1,000 bp, and 2,400 bp, respectively. Sequence analysis revealed that the 3 clones contained open reading frames. Blast results showed that they were highly homologous to the nuclear movement protein gene, the hypothetical protein gene and the heat shock protein 70 (HSP70) gene, respectively. The deduced amino acid sequences of B04, B05 and B41 contained 310, 192 and 647 amino acid residues, with Mr of 34,000, 21,000, and 70,700, respectively. A qualified cDNA library of B. orientalis has been constructed and three positive clones of B. orientalis discovered.

  14. Digital analysis of cDNA abundance; expression profiling by means of restriction fragment fingerprinting

    Directory of Open Access Journals (Sweden)

    Regenbogen Johannes

    2002-03-01

    Full Text Available Abstract Background Gene expression profiling among different tissues is of paramount interest in various areas of biomedical research. We have developed a novel method (DADA, Digital Analysis of cDNA Abundance, that calculates the relative abundance of genes in cDNA libraries. Results DADA is based upon multiple restriction fragment length analysis of pools of clones from cDNA libraries and the identification of gene-specific restriction fingerprints in the resulting complex fragment mixtures. A specific cDNA cloning vector had to be constructed that governed missing or incomplete cDNA inserts which would generate misleading fingerprints in standard cloning vectors. Double stranded cDNA was synthesized using an anchored oligo dT primer, uni-directionally inserted into the DADA vector and cDNA libraries were constructed in E. coli. The cDNA fingerprints were generated in a PCR-free procedure that allows for parallel plasmid preparation, labeling, restriction digest and fragment separation of pools of 96 colonies each. This multiplexing significantly enhanced the throughput in comparison to sequence-based methods (e.g. EST approach. The data of the fragment mixtures were integrated into a relational database system and queried with fingerprints experimentally produced by analyzing single colonies. Due to limited predictability of the position of DNA fragments on the polyacrylamid gels of a given size, fingerprints derived solely from cDNA sequences were not accurate enough to be used for the analysis. We applied DADA to the analysis of gene expression profiles in a model for impaired wound healing (treatment of mice with dexamethasone. Conclusions The method proved to be capable of identifying pharmacologically relevant target genes that had not been identified by other standard methods routinely used to find differentially expressed genes. Due to the above mentioned limited predictability of the fingerprints, the method was yet tested only with

  15. Toward a cDNA map of the human genome

    Energy Technology Data Exchange (ETDEWEB)

    Korenberg, J.R.; Chen, X.N. [Cedars-Sinai Research Institute, Los Angeles, CA (United States); Adams, M.D.; Venter, J.C. [Institute for Genomic Research, Gaithersburg, MD (United States)

    1995-09-20

    Advances in the Human Genome Project are shaping the strategies for identifying the 50,000-100,000 human genes. High-resolution genetic maps of the human genome combined with sequencing herald an era of rapid regional definition of disease genes. However, only once their chromosomes band location is known will the systematic partial sequencing of thousands of random cDNA clones provide the reagents for the rapid assessment of the genes responsible for the inherited disorders. We now present an approach to the rapid determination of map position and therefore to the creation of a transcribed map of the human genome. Sensitive fluorescence in situ hybridization has been combined with high-resolution chromosome banding and random cDNA sequencing to 41 cDNAs with an average insert size of < 2 kb to single human chromosome bands. The results provide 15 new genes, with database and functional information, as candidates for human disease. These include the large extracellular single-related kinase (HUMERK), the ERK activator kinase (PRKMK1), a new member of the RAS oncogene family, protein phosphotase 2 regulatory subunit B alpha isoform (PPP2R2A), and a novel human gene with very high homology to a plant membrane transport family. Further, an analysis of expressed genes associated with pseudogenes showed that by using these techniques, it is possible to detect accurately the transcribed locus within a multigene or processed pseudogene family in most cases. These findings suggest that direct cDNA mapping using fluorescence in situ hybridization provides an accurate and rapid approach to the definition of a transcribed map of the human genome. This low-cost, high-resolution (205 Mb) mapping greatly enhances the speed with which these genes can be subsequently assigned to contigs. This assignment provides a necessary first step in understanding the relationship of the genes to both acquired and inherited human diseases. 16 refs., 1 fig., 3 tabs.

  16. Lattice insertions for POPAE

    International Nuclear Information System (INIS)

    Cho, Y.; Crosbie, E.A.; Diebold, R.; Johnson, D.E.; Ohnuma, S.; Ruggiero, A.G.; Teng, L.C.

    1977-01-01

    Four types of insertions are described for the six 200-m straight sections of POPAE. All have dispersion matched to zero. (1) Injection-ejection insertion--This has proper high-β values and phase advances for horizontal injection and vertical ejection. (2) Phase-adjust insertion--The phase advance in this insertion is adjustable over a range of approximately 100 0 . (3) General-purpose insertion--The β* is adjustable from 2.5. to 200 m and the crossing angle is adjustable from 0 to 11 mrad. (4) High-luminosity insertion--This gives an even lower β + of meter

  17. Virulence genes fliC, toxA and phzS are common among Pseudomonas aeruginosa isolates from diabetic foot infections.

    Science.gov (United States)

    Ertugrul, Bulent M; Oryasin, Erman; Lipsky, Benjamin A; Willke, Ayşe; Bozdogan, Bulent

    2018-04-01

    Outcomes of antibiotic treatment of diabetic foot infections (DFIs) may depend not only on the antimicrobial susceptibility of the aetiologic agents, but also their ability to produce virulence factors. This study aimed to use polymerase chain reaction (PCR) with specific primers to investigate the presence of virulence genes among isolates of Pseudomonas aeruginosa isolates cultured from specimens from diabetic foot and other infections. We examined 63 P. aeruginosa isolates from inpatients at two University Hospitals for the presence of 23 known bacterial virulence genes, including lasI, lasR, lasA, lasB, rhll, rhlR, rhlAB, aprA, fliC, toxA, plcH, plcN, ExoS, ExoT, ExoU, ExoY, phzI, phzII, phzM, phzS, pvdA, pilA and pilB. Seven virulence genes (lasl, lasR, lasB, rhll, rhlR, rhlABand Exo T) were present in each isolate. No isolate expressed or presented aprA gene. We found that fliC (p = .01), toxA (p = .041) and phzS (p < .001) were statistically and significantly more common in diabetic foot isolates, while plcH (p < .001) was significantly more common in other infections. Among clinical isolates of P. aeruginosa from patients with DFIs, three virulence genes that can play important roles in tissue penetration (fliC), tissue damage and survival under anaerobic condition (phzS) and cell death (toxA) were significantly more common than isolates from other infections. The Multilocus sequence typing (MLST) analysis of diabetic foot isolates failed to point/indicate the existence of a specific clone or was not able to characterize/identify a specific clone/clonal complex group. Development of new agents to inhibit the synthesis of these genes may improve outcomes in DFIs treatment.

  18. Cinemática de variáveis de técnica-chave nos flic flacs de ginastas de elite

    Directory of Open Access Journals (Sweden)

    Nicola Lovecchio

    2013-08-01

    Full Text Available INTRODUÇÃO: A ginástica é o esporte de habilidades fechadas mais antigo e espetacular. Contudo, parâmetros técnicos de execução geralmente são somente ensinados por treinadores experientes. Desta maneira, existe uma lacuna de informações objetivas sobre o desempenho de ginastas (referências cinemáticas. OBJETIVO: No presente estudo, tentamos quantificar movimentos de inversão linear e de hiperextensão durante a execução de flic flacs. MÉTODOS: Foi efetuada uma detecção não invasiva de flic flacs com o auxílio de um instrumento óptico eletrônico 3D. Treze marcadores esféricos retrorreflexivos (1 cm de diâmetro foram posicionados no corpo de 9 ginastas experientes: maléolos laterais direito e esquerdo, cabeça da fíbula, trocanter maior, acrômio, olecrano, processo estiloide da ulna e vértex. Na mesma sessão e após um período de aquecimento, cada participante executou 15 repetições de flic flacs. Dez repetições forma analisadas, e os trajetos 3D das 13 manobras medidos. RESULTADOS: Em média, os homens obtiveram altura vertical maior (mulheres, 62% da altura; homens, 58%. O alinhamento dos membros inferiores foi homogêneo entre os ginastas: ângulos posteriores de joelho variaram entre 80° e 118°. Nenhuma abdução de membro inferior foi observada: a largura de joelho foi 7 cm menor do que a largura intertrocanter; a largura de tornozelo foi 8 cm menor do que a largura de joelho. Na saída do movimento, o ângulo tronco-coxa apresentou excelente alinhamento corporal, com valores bem próximos de 180°. As mulheres executaram a fase de apoio das mãos com pulsos mais próximos do que os homens (homens, 134% de largura de ombro; mulheres, 121%. CONCLUSÃO: Os resultados podem fornecer informações para melhor conhecimento, definindo assim, a execução de padrão-ouro obtida de ginastas de elite com poucas lesões.

  19. cDNA sequence of human transforming gene hst and identification of the coding sequence required for transforming activity

    International Nuclear Information System (INIS)

    Taira, M.; Yoshida, T.; Miyagawa, K.; Sakamoto, H.; Terada, M.; Sugimura, T.

    1987-01-01

    The hst gene was originally identified as a transforming gene in DNAs from human stomach cancers and from a noncancerous portion of stomach mucosa by DNA-mediated transfection assay using NIH3T3 cells. cDNA clones of hst were isolated from the cDNA library constructed from poly(A) + RNA of a secondary transformant induced by the DNA from a stomach cancer. The sequence analysis of the hst cDNA revealed the presence of two open reading frames. When this cDNA was inserted into an expression vector containing the simian virus 40 promoter, it efficiently induced the transformation of NIH3T3 cells upon transfection. It was found that one of the reading frames, which coded for 206 amino acids, was responsible for the transforming activity

  20. Construction of sized eukaryotic cDNA libraries using low input of total environmental metatranscriptomic RNA

    OpenAIRE

    Yadav, Rajiv Kumar; Barbi, Florian; Ziller, Antoine; Luis, Patricia; Marmeisse, Roland; Reddy, M Sudhakara; Fraissinet-Tachet, Laurence

    2014-01-01

    Background: Construction of high quality cDNA libraries from the usually low amounts of eukaryotic mRNA extracted from environmental samples is essential in functional metatranscriptomics for the selection of functional, full-length genes encoding proteins of interest. Many of the inserts in libraries constructed by standard methods are represented by truncated cDNAs due to premature stoppage of reverse transcriptase activity and preferential cloning of short cDNAs. Results: We report here a ...

  1. [Construction of phage display cDNA library from adult worms of Schistosoma japonicum].

    Science.gov (United States)

    Sun, Yi; Jia, Ren-chu; Liu, Jin-ming; Yuan, Chun-xiu; Shi, Yao-jun; Lu, Ke; Fu, Zhi-qiang; Sun, Huan; Cai, You-min; Lin, Jiao-jiao

    2007-10-01

    To screen protective antigen genes and construct the T7 phage display library from adult worms of Schistosoma japonicum. Total RNA was extracted from adult worms of S. japonicum by Trizol reagent anti mRNA was isolated from the total RNA. The ds cDNA was synthesized by reverse transcription using random primer. Directional EcoR I/ Hind III linkers were ligated into the ends of ds cDNA and the ds cDNA was digested with EcoR I anti Hind III, which resulted in ds cDNA with EcoR I and Hind III adhering ends. The digested ds cDNA fragments longer than 300 bp in length were fractionated and ligated into T7 Select 10-3b vector. After packaging in citro, the T7 Select 10-3b vector was transformed into BLT5403 to construct the T7 phage display cDNA library. Plaque assay and PCR were used to evaluate the library. Seven known objective genes of S. japonicum were screened by PCR to detect the representation of the library. Primary library capacity was 4.98 x 10(6) pfu, and the titer of amplified library was 3.85 x 10(11) pfu/mL. The PCR identification result of 96 clones picked at random showed that recombination rate was 93.8%, in which 95.6% inserted cDNA fragments were longer than 300 bp in length. All the seven known objective genes of S. japonicum were amplified from the library. The T7 phage display library from adult worms of Schistosoma japonicum was constructed.

  2. Cloning and functional characterization of a testicular TSH receptor cDNA from the African catfish (Clarias gariepinus)

    NARCIS (Netherlands)

    Vischer, H F; Bogerd, J.

    A cDNA encoding a putative thyroid-stimulating hormone receptor (cfTSH-R) was cloned from the testis of the African catfish (Clarias gariepinus). The cfTSH-R showed the highest amino acid sequence identity with the TSH-Rs of other fish species. In addition, an insertion of approximately 50 amino

  3. [Construction and characterization of normalized cDNA library of maize inbred Mo17 from multiple tissues and developmental stages].

    Science.gov (United States)

    Zhang, Z X; Zhang, F D; Tang, W H; Pi, Y J; Zheng, Y L

    2005-01-01

    Comprehensive complementary DNA (cDNA) library is a valuable resource for functional genomics. In this study, we set up a normalized cDNA library of Mo17 (MONL) by saturation hybridization with genomic DNA, which contained expressed genes of eight tissues and organs from inbred Mo17 of maize (Zea mays L.). In this library, the insert sizes range from 0.4 kb to 4 kb and the average size is 1.18 kb. 10.830 clones were spotted on nylon membrane to make a cDNA microarray. Randomly picked 300 clones from the cDNA library were sequenced. The cDNA microarry was hybridized with pooled tissue mRNA probes or housekeeping gene cDNA probes. The results showed the normalized cDNA library comprehensively includes tissue-specific genes in which 71% are unique ESTs (expressed sequence tags) based on the 300 sequences analyzed. Using BLAST program to compare the sequences against online nucleotide databases, 88% sequences were found in ZmDB or NCBI, and 12% sequences were not found in existing nucleotide databases. More than 73% sequences are of unknown function. The library could be extensively used in developing DNA markers, sequencing ESTs, mining new genes, identifying positional cloning and candidate gene, and developing microarrays in maize genomics research.

  4. Construction of sized eukaryotic cDNA libraries using low input of total environmental metatranscriptomic RNA.

    Science.gov (United States)

    Yadav, Rajiv Kumar; Barbi, Florian; Ziller, Antoine; Luis, Patricia; Marmeisse, Roland; Reddy, M Sudhakara; Fraissinet-Tachet, Laurence

    2014-09-03

    Construction of high quality cDNA libraries from the usually low amounts of eukaryotic mRNA extracted from environmental samples is essential in functional metatranscriptomics for the selection of functional, full-length genes encoding proteins of interest. Many of the inserts in libraries constructed by standard methods are represented by truncated cDNAs due to premature stoppage of reverse transcriptase activity and preferential cloning of short cDNAs. We report here a simple and cost effective technique for preparation of sized eukaryotic cDNA libraries from as low as three microgram of total soil RNA dominated by ribosomal and bacterial RNA. cDNAs synthesized by a template switching approach were size-fractionated by two dimensional agarose gel electrophoresis prior to PCR amplification and cloning. Effective size selection was demonstrated by PCR amplification of conserved gene families specific of each size class. Libraries of more than one million independent inserts whose sizes ranged between one and four kb were thus produced. Up to 80% of the insert sequences were homologous to eukaryotic gene sequences present in public databases. A simple and cost effective technique has been developed to construct sized eukaryotic cDNA libraries from environmental samples. This technique will facilitate expression cloning of environmental eukaryotic genes and contribute to a better understanding of basic biological and/or ecological processes carried out by eukaryotic microbial communities.

  5. Molecular cloning and nucleotide sequence of full-length cDNA for sweet potato catalase mRNA.

    Science.gov (United States)

    Sakajo, S; Nakamura, K; Asahi, T

    1987-06-01

    A nearly full-length cDNA clone for catalase (pCAS01) was obtained through immunological screening of cDNA expression library constructed from size-fractionated poly(A)-rich RNA of wounded sweet potato tuberous roots by Escherichia coli expression vector-primed cDNA synthesis. Two additional catalase cDNA clones (pCAS10 and pCAS13), which contained cDNA inserts slightly longer than that of pCAS01 at their 5'-termini, were identified by colony hybridization of another cDNA library. Those three catalase cDNAs contained primary structures not identical, but closely related, to one another based on their restriction enzyme and RNase cleavage mapping analyses, suggesting that microheterogeneity exists in catalase mRNAs. The cDNA insert of pCAS13 carried the entire catalase coding capacity, since the RNA transcribed in vitro from the cDNA under the SP6 phage promoter directed the synthesis of a catalase polypeptide in the wheat germ in vitro translation assay. The nucleotide sequencing of these catalase cDNAs indicated that 1900-base catalase mRNA contained a coding region of 1476 bases. The amino acid sequence of sweet potato catalase deduced from the nucleotide sequence was 35 amino acids shorter than rat liver catalase [Furuta, S., Hayashi, H., Hijikata, M., Miyazawa, S., Osumi, T. & Hashimoto, T. (1986) Proc. Natl Acad. Sci. USA 83, 313-317]. Although these two sequences showed only 38% homology, the sequences around the amino acid residues implicated in catalytic function, heme ligand or heme contact had been well conserved during evolution.

  6. [Construction of a subtracted cDNA library of chronic intermittent hypoxia rabbit liver by suppression subtractive hybridization].

    Science.gov (United States)

    Wu, Yue-tao; Liu, Rui-hong; Yang, Yu; Luo, Ying-quan; Rong, Yao

    2007-12-01

    To construct a subtracted cDNA library of chronic intermittent hypoxia (CIH) rabbit liver by suppression subtractive hybridization (SSH). Twenty-four rabbits were divided into 4 groups: ordinary feeding group, full-fat food group, ordinary feeding in chronic intermittent hypoxia group, and full-fat food in chronic intermittent hypoxia group. The mRNAs were extracted from different rabbit livers and converted into double-strand cDNA. After digestion with restriction enzyme, the cDNA of hyperlipidemia-sensitive rabbit group was subdivided into 2 portions and each one was lighted with different adaptors. Two rounds of both hybridization and suppression PCR obtained the differentially expressed cDNA. The PCR products were inserted into T/A vector to set up the subtractive cDNA library. The clones were selected and amplified by PCR and identified. Based on the pathology of the abdominal aorta and liver, and the amplified library contained 500 positive bacteria clones, including 462 clones, which had inserts from 250 to 700 bp by PCR analysis. A novel rabbit gene, Cthrc1, involved in CHI had been cloned. The GenBank Accession Number is XM_418373. The molecular mechanism of CIH promoting atherogenesis formation is made clear.

  7. [Construction and characterization of a cDNA library from human liver tissue of cirrhosis].

    Science.gov (United States)

    Chen, Xiao-hong; Chen, Zhi; Chen, Feng; Zhu, Hai-hong; Zhou, Hong-juan; Yao, Hang-ping

    2005-03-01

    To construct a cDNA library from human liver tissue of cirrhosis. The total RNA from human liver tissue of cirrhosis was extracted using Trizol method, and the mRNA was purified using mRNA purification kit. SMART technique and CDSIII/3' primer were used for first-strand cDNA synthesis. Long distance PCR was then used to synthesize the double-strand cDNA that was then digested by proteinase K and Sfi I, and was fractionated by CHOMA SPIN-400 column. The cDNA fragments longer than 0.4 kb were collected and ligated to lambdaTripl Ex2 vector. Then lambda-phage packaging reaction and library amplification were performed. The qualities of both unamplified and amplified cDNA libraries was strictly checked by conventional titer determination. Eleven plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. The titers of unamplifed and amplified libraries were 1.03 x 10(6) pfu/ml and 1.36 x 10(9) pfu/ml respectively. The percentages of recombinants from both libraries were 97.24 % in unamplified library and 99.02 % in amplified library. The lengths of the inserts were 1.02 kb in average (36.36 % 1 approximately equals 2 kb and 63.64 % 0.5 approximately equals 1.0 kb). A high quality cDNA library from human liver tissue of cirrhosis was constructed successfully, which can be used for screening and cloning new special genes associated with the occurrence of cirrhosis.

  8. [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.

  9. Sequence diversity in the Dickeya fliC gene: phylogeny of the Dickeya genus and TaqMan® PCR for 'D. solani', new biovar 3 variant on potato in Europe.

    Science.gov (United States)

    Van Vaerenbergh, Johan; Baeyen, Steve; De Vos, Paul; Maes, Martine

    2012-01-01

    Worldwide, Dickeya (formerly Erwinia chrysanthemi) is causing soft rot diseases on a large diversity of crops and ornamental plants. Strains affecting potato are mainly found in D. dadantii, D. dianthicola and D. zeae, which appear to have a marked geographical distribution. Furthermore, a few Dickeya isolates from potato are attributed to D. chrysanthemi and D. dieffenbachiae. In Europe, isolates of Erwinia chrysanthemi biovar 1 and biovar 7 from potato are now classified in D. dianthicola. However, in the past few years, a new Dickeya biovar 3 variant, tentatively named 'Dickeya solani', has emerged as a common major threat, in particular in seed potatoes. Sequences of a fliC gene fragment were used to generate a phylogeny of Dickeya reference strains from culture collections and with this reference backbone, to classify pectinolytic isolates, i.e. Dickeya spp. from potato and ornamental plants. The reference strains of the currently recognized Dickeya species and 'D. solani' were unambiguously delineated in the fliC phylogram. D. dadantii, D. dianthicola and 'D. solani' displayed unbranched clades, while D. chrysanthemi, D. zeae and D. dieffenbachiae branched into subclades and lineages. Moreover, Dickeya isolates from diagnostic samples, in particular biovar 3 isolates from greenhouse ornamentals, formed several new lineages. Most of these isolates were positioned between the clade of 'D. solani' and D. dadantii as transition variants. New lineages also appeared in D. dieffenbachiae and in D. zeae. The strains and isolates of D. dianthicola and 'D. solani' were differentiated by a fliC sequence useful for barcode identification. A fliC TaqMan®real-time PCR was developed for 'D. solani' and the assay was provisionally evaluated in direct analysis of diagnostic potato samples. This molecular tool can support the efforts to control this particular phytopathogen in seed potato certification.

  10. Sequence diversity in the Dickeya fliC gene: phylogeny of the Dickeya genus and TaqMan® PCR for 'D. solani', new biovar 3 variant on potato in Europe.

    Directory of Open Access Journals (Sweden)

    Johan Van Vaerenbergh

    Full Text Available Worldwide, Dickeya (formerly Erwinia chrysanthemi is causing soft rot diseases on a large diversity of crops and ornamental plants. Strains affecting potato are mainly found in D. dadantii, D. dianthicola and D. zeae, which appear to have a marked geographical distribution. Furthermore, a few Dickeya isolates from potato are attributed to D. chrysanthemi and D. dieffenbachiae. In Europe, isolates of Erwinia chrysanthemi biovar 1 and biovar 7 from potato are now classified in D. dianthicola. However, in the past few years, a new Dickeya biovar 3 variant, tentatively named 'Dickeya solani', has emerged as a common major threat, in particular in seed potatoes. Sequences of a fliC gene fragment were used to generate a phylogeny of Dickeya reference strains from culture collections and with this reference backbone, to classify pectinolytic isolates, i.e. Dickeya spp. from potato and ornamental plants. The reference strains of the currently recognized Dickeya species and 'D. solani' were unambiguously delineated in the fliC phylogram. D. dadantii, D. dianthicola and 'D. solani' displayed unbranched clades, while D. chrysanthemi, D. zeae and D. dieffenbachiae branched into subclades and lineages. Moreover, Dickeya isolates from diagnostic samples, in particular biovar 3 isolates from greenhouse ornamentals, formed several new lineages. Most of these isolates were positioned between the clade of 'D. solani' and D. dadantii as transition variants. New lineages also appeared in D. dieffenbachiae and in D. zeae. The strains and isolates of D. dianthicola and 'D. solani' were differentiated by a fliC sequence useful for barcode identification. A fliC TaqMan®real-time PCR was developed for 'D. solani' and the assay was provisionally evaluated in direct analysis of diagnostic potato samples. This molecular tool can support the efforts to control this particular phytopathogen in seed potato certification.

  11. PCBs attenuation and abundance of Dehalococcoides spp., bphC, CheA, and flic genes in typical polychlorinated biphenyl-polluted soil under floody and dry soil conditions.

    Science.gov (United States)

    Hashmi, Muhammad Zaffar; Qin, Zhihui; Yao, Xiaoyan; Ahmed, Zulkifl; Xiaomei, Su; Shen, Chaofeng; Tang, Xianjin

    2016-02-01

    This study investigates PCBs attenuation and the abundance of active polychlorinated-degrading Dehalococcoides spp. biphenyl dioxygenase (bphC), chemotaxis (CheA), and flagellum (flic) genes in floody and dry soil conditions polluted with polychlorinated biphenyls. The results revealed that total PCBs, high chlorinated PCBs (>4 cl), and less chlorinated PCBs (<4 cl) decreased with the passage of time in floody and dry soil conditions. The reduction of total PCBs (13.87%) and less chlorinated PCBs (15.39%) was more in dry soil than floody soil, while high chlorinated PCBs showed more reduction in floody soil (8.06%) than dry soil. Dehaloccoides spp., bphC, CheA, and flic genes indicated temporal dynamics in abundance in floody and dry soil conditions. The highest abundance was 1.6 × 10(9), 3.7 × 10(4), and 3.6 × 10(2) copies in floody and 1.6 × 10(4) copies in dry soil for Dehalococcoides spp., bphC, CheA, and flic, respectively. Multivariate statistics (RDA) revealed that Dehaloccoides spp. were positively influenced by the higher chlorinated PCBs and soil physical properties, CheA gene with floody soil, flic gene with total PCBs and less chlorinated PCBs, and bphC gene was affected with moisture contents and less chlorinated PCBs. This study provides new insight in the attenuation of PCBs and the abundance of active Dehalococcoides spp. and genes in PCBs polluted soil under floody and dry soil conditions.

  12. Tie rod insertion test

    CERN Multimedia

    B. LEVESY

    2002-01-01

    The superconducting coil is inserted in the outer vaccum tank and supported by a set of tie rods. These tie rods are made of titanium alloy. This test reproduce the final insertion of the tie rods inside the outer vacuum tank.

  13. Brushless DC motor drives supplied by PV power system based on Z-source inverter and FL-IC MPPT controller

    International Nuclear Information System (INIS)

    Mozaffari Niapour, S.A.KH.; Danyali, S.; Sharifian, M.B.B.; Feyzi, M.R.

    2011-01-01

    Highlights: → Employing the BLDC motor in water pumping systems. → Utilizing the ZSI as a single-stage power converter in the PV water pumping systems based on BLDC motor. → Improvement of the conventional IC MPPT method with the fuzzy logic control scheme to save more energy from the PV array. → Taking the advantages of the DTC drive of the BLDC motor. → Optimizing the water pumping system speed response characteristic by PSO. - Abstract: This paper discusses operation performance of a water pumping system consist of a brushless dc (BLDC) motor coupled a centrifugal pump and accompanying a Z-source inverter (ZSI) fed by a photovoltaic (PV) array, to be improved. Despite conventional double-stage power converters, this paper proposes utilizing a single-stage ZSI to extract the maximum power of the PV array and supply the BLDC motor simultaneously. Utilizing the ZSI provides some inherent advantages such as high efficiency and low cost, which is very promising for PV systems due to its novel voltage buck/boost capability. In addition, in order to precisely perform the maximum power point tracking (MPPT) of the PV array the fuzzy logic-incremental conductance (FL-IC) MPPT scheme is proposed. The proposed FL-IC MPPT scheme provides enough modification to the conventional IC method to enjoy an appropriate variable step size MPPT control signal for the ZSI. Moreover, direct torque control (DTC) is found more effective in comparison with hysteresis current control with current shaping to drive the BLDC motor, because it benefits from faster torque response, reduced torque ripple, less sensitivity to parameters variations, and simple implementation. In the mean time, due to the frequently variations of the PV power generation; delivered mechanical power to the centrifugal pump is variable. Thus, the BLDC motor should be driven with variable reference speed. In order to improve the speed transient response of the BLDC motor and enhance the energy saving aspect of

  14. Lectin cDNA and transgenic plants derived therefrom

    Science.gov (United States)

    Raikhel, Natasha V.

    2000-10-03

    Transgenic plants containing cDNA encoding Gramineae lectin are described. The plants preferably contain cDNA coding for barley lectin and store the lectin in the leaves. The transgenic plants, particularly the leaves exhibit insecticidal and fungicidal properties.

  15. Cloning, sequencing and expression of cDNA encoding growth ...

    Indian Academy of Sciences (India)

    Using polymerase chain reaction (PCR) primers representing the conserved regions of fish GH sequences the 3′ region of catfish GH cDNA (540 bp) was cloned by random amplification of cDNA ends and the clone was used as a probe to isolate recombinant phages carrying the full-length cDNA sequence. The full-length ...

  16. Cloning, sequencing and expression of cDNA encoding growth ...

    Indian Academy of Sciences (India)

    Unknown

    317. 2.4 cDNA sequencing and analysis. The nucleotide sequence of the cloned H. fossilis GH. cDNA was determined by Sanger's dideoxy chain termi- nation method, using Perkin Elmer bigdye terminator kit in an ABI Prism 377 automated DNA sequencer. All other computational analysis of the GH cDNA was done using.

  17. [Construction and identification of a full-length cDNA library from Spirometra erinaceieuropaei].

    Science.gov (United States)

    Lv, Gang; Lu, Ya-Jun; Fan, Zhi-Gang; Shi, Da-Zhong; Gan, Xiu-Feng; Zhong, Sai-Feng

    2010-10-30

    The full-length pBluescript II SK cDNA library of adult Spirometra erinaceieuropaei was constructed by using the SMART method. Data showed that 95.5% of the library was recombinant and the titer of the library was 1.06 x 10(6). The average insert size of the library was about 1.4 kb. Forty-eight randomly selected clones were sequenced. A set of 36 effective expressed sequence tags (ESTs) with the average size of 674 bp was obtained after excluding clones shorter than 450 bp. The unigenes occupied 58.3% of the 36 ESTs. The rate of full-length cDNAs were 57.7% (15/26). The high-quality of full-length cDNA library could be used for large scale EST sequencing.

  18. Genetical and functional investigation of fliC genes encoding flagellar serotype H4 in wildtype strains of Escherichia coli and in a laboratory E. coli K-12 strain expressing flagellar antigen type H48

    Directory of Open Access Journals (Sweden)

    Schaudinn Christoph

    2005-01-01

    Full Text Available Abstract Background Serotyping of O-(lipopolysaccharide and H-(flagellar antigens is a wideley used method for identification of pathogenic strains and clones of Escherichia coli. At present, 176 O- and 53 H-antigens are described for E. coli which occur in different combinations in the strains. The flagellar antigen H4 is widely present in E. coli strains of different O-serotypes and pathotypes and we have investigated the genetic relationship between H4 encoding fliC genes by PCR, nucleotide sequencing and expression studies. Results The complete nucleotide sequence of fliC genes present in E. coli reference strains U9-41 (O2:K1:H4 and P12b (O15:H17 was determined and both were found 99.3% (1043 of 1050 nucleotides identical in their coding sequence. A PCR/RFLP protocol was developed for typing of fliC-H4 strains and 88 E. coli strains reacting with H4 antiserum were investigated. Nucleotide sequencing of complete fliC genes of six E. coli strains which were selected based on serum agglutination titers, fliC-PCR genotyping and reference data revealed 96.6 to 100% identity on the amino acid level. The functional expression of flagellin encoded by fliC-H4 from strain U9-41 and from our strain P12b which is an H4 expressing variant type was investigated in the E. coli K-12 strain JM109 which encodes flagellar type H48. The fliC recombinant plasmid carrying JM109 strains reacted with both H4 and H48 specific antisera whereas JM109 reacted only with the H48 antiserum. By immunoelectron microscopy, we could show that the flagella made by the fliC-H4 recombinant plasmid carrying strain are constituted of H48 and H4 flagellins which are co-assembled into functional flagella. Conclusion The flagellar serotype H4 is encoded by closely related fliC genes present in serologically different types of E. coli strainswhich were isolated at different time periods and geographical locations. Our expression studies show for the first time, that flagellins of

  19. Comparative analysis of fliC Gene from Salmonella enterica sub-species for biosensor probe design and phylogenetic tree construction

    Directory of Open Access Journals (Sweden)

    Sunderamurthe Satisvar

    2017-06-01

    Full Text Available In tandem to the Salmonellosis infection worldwide, a study was conducted to determine the conserved and nonconserved region in Salmonella enterica sub-species so that it can be used to design probes in biosensors for the detection of Salmonella enterica as a species or sub-species distinctively. The region was selected for this study through fliC gene that is present in all Salmonella sub-species, encodes the Salmonella flagella or flagellin determines the serotype due to its H antigen and provides virulence to the bacteria. All sub-species were analyzed in a group of six and then analyzed individually in groups of two with all possible combinations to determine the overlapping regions. Based on the study, the predominant conserved region for S. enterica sub-species is between 103 and 158, whereas the non-conserved region is from 1245 to 1285. A phylogenetic tree was constructed for the S. enterica sub-species to determine the evolution of the Salmonella sub-species.

  20. cDNA sequence quality data - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available List Contact us Budding yeast cDNA sequencing project cDNA sequence quality data Data detail Data name cDNA sequence quality... data DOI 10.18908/lsdba.nbdc00838-003 Description of data contents Phred's quality score. P...tion Download License Update History of This Database Site Policy | Contact Us cDNA sequence quality

  1. Three human alcohol dehydrogenase subunits: cDNA structure and molecular and evolutionary divergence

    International Nuclear Information System (INIS)

    Ikuta, T.; Szeto, S.; Yoshida, A.

    1986-01-01

    Class I human alcohol dehydrogenase (ADH; alcohol:NAD + oxidoreductase, EC 1.1.1.1) consists of several homo- and heterodimers of α, β, and γ subunits that are governed by the ADH1, ADH2, and ADH3 loci. The authors previously cloned a full length of cDNA for the β subunit, and the complete sequence of 374 amino acid residues was established. cDNAs for the α and γ subunits were cloned and characterized. A human liver cDNA library, constructed in phage λgt11, was screened by using a synthetic oligonucleotide probe that was matched to the γ but not to the β sequence. Clone pUCADHγ21 and clone pUCADHα15L differed from β cDNA with respect to restriction sites and hybridization with the nucleotide probe. Clone pUCADHγ21 contained an insertion of 1.5 kilobase pairs (kbp) and encodes 374 amino acid residues compatible with the reported amino acid sequence of the γ subunit. Clone pUCADHα15L contained an insertion of 2.4 kbp and included nucleotide sequences that encode 374 amino acid residues for another subunit, the γ subunit. In addition, this clone contained the sequences that encode the COOH-terminal part of the β subunit at its extended 5' region. The amino acid sequences and coding regions of the cDNAs of the three subunits are very similar. A high degree of resemblance is observed also in their 3' noncoding regions. However, distinctive differences exist in the vicinity of the Zn-binding cysteine residue at position 46. Based on the cDNA sequences and the deduced amino acid sequences of the three subunits, their structural and evolutionary relationships are discussed

  2. Pattern analysis approach reveals restriction enzyme cutting abnormalities and other cDNA library construction artifacts using raw EST data.

    Science.gov (United States)

    Zhou, Sun; Ji, Guoli; Liu, Xiaolin; Li, Pei; Moler, James; Karro, John E; Liang, Chun

    2012-05-03

    Expressed Sequence Tag (EST) sequences are widely used in applications such as genome annotation, gene discovery and gene expression studies. However, some of GenBank dbEST sequences have proven to be "unclean". Identification of cDNA termini/ends and their structures in raw ESTs not only facilitates data quality control and accurate delineation of transcription ends, but also furthers our understanding of the potential sources of data abnormalities/errors present in the wet-lab procedures for cDNA library construction. After analyzing a total of 309,976 raw Pinus taeda ESTs, we uncovered many distinct variations of cDNA termini, some of which prove to be good indicators of wet-lab artifacts, and characterized each raw EST by its cDNA terminus structure patterns. In contrast to the expected patterns, many ESTs displayed complex and/or abnormal patterns that represent potential wet-lab errors such as: a failure of one or both of the restriction enzymes to cut the plasmid vector; a failure of the restriction enzymes to cut the vector at the correct positions; the insertion of two cDNA inserts into a single vector; the insertion of multiple and/or concatenated adapters/linkers; the presence of 3'-end terminal structures in designated 5'-end sequences or vice versa; and so on. With a close examination of these artifacts, many problematic ESTs that have been deposited into public databases by conventional bioinformatics pipelines or tools could be cleaned or filtered by our methodology. We developed a software tool for Abnormality Filtering and Sequence Trimming for ESTs (AFST, http://code.google.com/p/afst/) using a pattern analysis approach. To compare AFST with other pipelines that submitted ESTs into dbEST, we reprocessed 230,783 Pinus taeda and 38,709 Arachis hypogaea GenBank ESTs. We found 7.4% of Pinus taeda and 29.2% of Arachis hypogaea GenBank ESTs are "unclean" or abnormal, all of which could be cleaned or filtered by AFST. cDNA terminal pattern analysis, as

  3. Pattern analysis approach reveals restriction enzyme cutting abnormalities and other cDNA library construction artifacts using raw EST data

    Directory of Open Access Journals (Sweden)

    Zhou Sun

    2012-05-01

    Full Text Available Abstract Background Expressed Sequence Tag (EST sequences are widely used in applications such as genome annotation, gene discovery and gene expression studies. However, some of GenBank dbEST sequences have proven to be “unclean”. Identification of cDNA termini/ends and their structures in raw ESTs not only facilitates data quality control and accurate delineation of transcription ends, but also furthers our understanding of the potential sources of data abnormalities/errors present in the wet-lab procedures for cDNA library construction. Results After analyzing a total of 309,976 raw Pinus taeda ESTs, we uncovered many distinct variations of cDNA termini, some of which prove to be good indicators of wet-lab artifacts, and characterized each raw EST by its cDNA terminus structure patterns. In contrast to the expected patterns, many ESTs displayed complex and/or abnormal patterns that represent potential wet-lab errors such as: a failure of one or both of the restriction enzymes to cut the plasmid vector; a failure of the restriction enzymes to cut the vector at the correct positions; the insertion of two cDNA inserts into a single vector; the insertion of multiple and/or concatenated adapters/linkers; the presence of 3′-end terminal structures in designated 5′-end sequences or vice versa; and so on. With a close examination of these artifacts, many problematic ESTs that have been deposited into public databases by conventional bioinformatics pipelines or tools could be cleaned or filtered by our methodology. We developed a software tool for Abnormality Filtering and Sequence Trimming for ESTs (AFST, http://code.google.com/p/afst/ using a pattern analysis approach. To compare AFST with other pipelines that submitted ESTs into dbEST, we reprocessed 230,783 Pinus taeda and 38,709 Arachis hypogaea GenBank ESTs. We found 7.4% of Pinus taeda and 29.2% of Arachis hypogaea GenBank ESTs are “unclean” or abnormal, all of which could be cleaned

  4. Construction of C35 gene bait recombinants and T47D cell cDNA library.

    Science.gov (United States)

    Yin, Kun; Xu, Chao; Zhao, Gui-Hua; Liu, Ye; Xiao, Ting; Zhu, Song; Yan, Ge

    2017-11-20

    C35 is a novel tumor biomarker associated with metastasis progression. To investigate the interaction factors of C35 in its high expressed breast cancer cell lines, we constructed bait recombinant plasmids of C35 gene and T47D cell cDNA library for yeast two-hybrid screening. Full length C35 sequences were subcloned using RT-PCR from cDNA template extracted from T47D cells. Based on functional domain analysis, the full-length C35 1-348bp was also truncated into two fragments C351-153bp and C35154-348bp to avoid auto-activation. The three kinds of C35 genes were successfully amplified and inserted into pGBKT7 to construct bait recombinant plasmids pGBKT7-C351-348bp, pGBKT7-C351-153bp and pGBKT7-C35154-348bp, then transformed into Y187 yeast cells by the lithium acetate method. Auto-activation and toxicity of C35 baits were detected using nutritional deficient medium and X-α-Gal assays. The T47D cell ds cDNA was generated by SMART TM technology and the library was constructed using in vivo recombination-mediated cloning in the AH109 yeast strain using a pGADT7-Rec plasmid. The transformed Y187/pGBKT7-C351-348bp line was intensively inhibited while the truncated Y187/pGBKT7-C35 lines had no auto-activation and toxicity in yeast cells. The titer of established cDNA library was 2 × 10 7 pfu/mL with high transformation efficiency of 1.4 × 10 6 , and the insert size of ds cDNA was distributed homogeneously between 0.5-2.0 kb. Our research generated a T47D cell cDNA library with high titer, and the constructed two C35 "baits" contained a respective functional immunoreceptor tyrosine based activation motif (ITAM) and the conserved last four amino acids Cys-Ile-Leu-Val (CILV) motif, and therefore laid a foundation for screening the C35 interaction factors in a BC cell line.

  5. Screening a cDNA Library for Protein–Protein Interactions Directly in Planta[W

    Science.gov (United States)

    Lee, Lan-Ying; Wu, Fu-Hui; Hsu, Chen-Tran; Shen, Shu-Chen; Yeh, Hsuan-Yu; Liao, De-Chih; Fang, Mei-Jane; Liu, Nien-Tze; Yen, Yu-Chen; Dokládal, Ladislav; Sýkorová, Eva; Gelvin, Stanton B.; Lin, Choun-Sea

    2012-01-01

    Screening cDNA libraries for genes encoding proteins that interact with a bait protein is usually performed in yeast. However, subcellular compartmentation and protein modification may differ in yeast and plant cells, resulting in misidentification of protein partners. We used bimolecular fluorescence complementation technology to screen a plant cDNA library against a bait protein directly in plants. As proof of concept, we used the N-terminal fragment of yellow fluorescent protein– or nVenus-tagged Agrobacterium tumefaciens VirE2 and VirD2 proteins and the C-terminal extension (CTE) domain of Arabidopsis thaliana telomerase reverse transcriptase as baits to screen an Arabidopsis cDNA library encoding proteins tagged with the C-terminal fragment of yellow fluorescent protein. A library of colonies representing ∼2 × 105 cDNAs was arrayed in 384-well plates. DNA was isolated from pools of 10 plates, individual plates, and individual rows and columns of the plates. Sequential screening of subsets of cDNAs in Arabidopsis leaf or tobacco (Nicotiana tabacum) Bright Yellow-2 protoplasts identified single cDNA clones encoding proteins that interact with either, or both, of the Agrobacterium bait proteins, or with CTE. T-DNA insertions in the genes represented by some cDNAs revealed five novel Arabidopsis proteins important for Agrobacterium-mediated plant transformation. We also used this cDNA library to confirm VirE2-interacting proteins in orchid (Phalaenopsis amabilis) flowers. Thus, this technology can be applied to several plant species. PMID:22623495

  6. Comparing Leaf and Root Insertion

    Directory of Open Access Journals (Sweden)

    Jaco Geldenhuys

    2010-07-01

    Full Text Available We consider two ways of inserting a key into a binary search tree: leaf insertion which is the standard method, and root insertion which involves additional rotations. Although the respective cost of constructing leaf and root insertion binary search trees trees, in terms of comparisons, are the same in the average case, we show that in the worst case the construction of a root insertion binary search tree needs approximately 50% of the number of comparisons required by leaf insertion.

  7. Characterization of cDNA clones encoding rabbit and human serum paraoxonase: The mature protein retains its signal sequence

    Energy Technology Data Exchange (ETDEWEB)

    Hassett, C.; Richter, R.J.; Humbert, R.; Omiecinski, C.J.; Furlong, C.E. (Univ. of Washington, Seattle (United States)); Chapline, C.; Crabb, J.W. (W.Alton Jones Cell Science Center, Lake Placid, NY (United States))

    1991-10-22

    Serum paraoxonase hydrolyzes the toxic metabolites of a variety of organophosphorus insecticides. High serum paraoxonase levels appear to protect against the neurotoxic effects of organophosphorus substrates of this enzyme. The amino acid sequence accounting for 42% of rabbit paraoxonase was determined. From these data, two oligonucleotide probes were synthesized and used to screen a rabbit liver cDNA library. Human paraoxonase clones were isolated from a liver cDNA library by using the rabbit cDNA as a hybridization probe. Inserts from three of the longest clones were sequenced, and one full-length clone contained an open reading frame encoding 355 amino acids, four less than the rabbit paraoxonase protein. Amino-terminal sequences derived from purified rabbit and human paraoxonase proteins suggested that the signal sequence is retained, with the exception of the initiator methionine residue. Characterization of the rabbit and human paraoxonase cDNA clones confirms that the signal sequences are not processed, except for the N-terminal methionine residue. The rabbit and human cDNA clones demonstrate striking nucleotide and deduced amino acid similarities (greater than 85%), suggesting an important metabolic role and constraints on the evolution of this protein.

  8. [Construction and sequence analysis of a normalized full-length cDNA library of Dendrobium officinale].

    Science.gov (United States)

    Jiang, Min; Wang, Jiang; Wen, Guo-Song; Xu, Shao-Zhong; Zha, Ying-Hong; Rong, Tian-Ju; Qian, Xiong

    2013-02-01

    In order to obtain functional genes, a normalized stems cDNA library was constructed from medicinal plant Dendrobium officinale. SMART (switching mechanism at 5' end of RNA transcript) cDNA synthesis combined with DSN (duplex-specific nuclease) normalization was applied to construct the normalized full-length cDNA library of D. officinale. The titer of cDNA library was about 1.3 x 10(6) cfu x mL(-1) and the average insertion size was about 1.5 kb with high recombination rate (93.9%). Random selected 163 positive clones were sequenced at single side. Bio-information analysis indicated that 147 from 150 high-quality unique sequences matched corresponding homologous proteins, and they participated in various biological processes based on GO (gene ontology). There were 8 clones with complete coding sequence, which presumed to be full-length genes. These results showed preliminarily that we successfully constructed a normalized full-length cDNA library of D. officinale which could be used to screen the functional genes related to metabolic pathways of medicinal ingredients.

  9. [Construction and preliminary analysis of a full-length cDNA library for Paris polyphylla var. yunnanensis].

    Science.gov (United States)

    Zhao, Shuang; Dong, Xu; Ma, Teng

    2014-01-01

    A full-length cDNA library of Paris polyphylla var. yunnanensis was constructed in order to research the genes relating to growing development and the genes regulation of its secondary metabolite biosynthesis. The total RNA was extracted from Paris polyphylla var. yunnanensis using modified Trizol method. The SMART (switching mechanism at 5' end of RNA transcript )technology was appliedl to construct the full-length cDNA library. The library titer,recombinant rate and length of insert fragments were determined,the sequences of the library were analyzed by Blastx and were compared to GenBank database. The capacity of the library was 2. 5 x 107 cfu/mL, the recombinant rate was 98.5% and the average size of the inserted fragment was 1.5 kb. 9 ESTs (Expressed Sequence Tags) were relating to growing development and 5 ESTs were relating to regulation of secondary metabolite biosynthesis among 149 ESTs obtained from 192 clones sequenced. A full-length cDNA library of Paris polyphylla var. yunnanensis is constructed by SMART technology successfully, and the library has enough capacity, high recombinant rate and long insert fragment for the further research to screen and identify the functional genes of Paris polyphylla var. yunnanensis.

  10. [Construction and preliminary screening of a forward-subtracted cDNA library for differentially expressed genes in rat liver of prothrombotic state].

    Science.gov (United States)

    Fang, Ding-Zhi; Liu, Bing-Wen; Shen, Tao; Bai, Huai

    2005-11-01

    To construct and preliminarily screen the forward-subtracted cDNA library of differentially expressed genes in rat liver of prothrombotic state (PTS). The forward-subtracted cDNA library for differentially expressed genes in rat liver of PTS was constructed by suppression subtractive hybridization using cDNAs synthesized from mRNA of PTS rat as Tester and cDNAs from mRNA of control rat as Driver. The products from the last PCR amplification of suppression subtractive hybridization were inserted into a T/A plasmid vectors to transform the Escherichia coli JM109 cells. To produce the library, the transformed cells were incubated at 37 C overnight on a LB agar plate containing ampicillin (50 microg/ml), IPTG and X-gal. Forward-subtracted cDNA probes and reverse-subtracted cDNA probes were prepared by nested PCR amplification, which were labeled with HRP. Positive clones were selected by differential screening in which forward-subtracted and reverse-subtracted cDNA probes were separately hybridized with the membranes slot-blotted by plasmid DNAs amplified and isolated from the library. Inserts in the positive clones were submitted to DNA sequencing. Nucleic acid sequence homology search was performed against the GenBank DNA database (non-redundant, and non-mouse and non-human EST entries) using the Standard nucleotide-nucleotide BLAST [blastn] program via a network connection to the National Center for Biotechnology information. The forward-subtracted cDNA library for differentially expressed genes in rat liver of PTS was successfully constructed. Two differentially expressed cDNA fragments were found after preliminary screening. The forward-subtracted cDNA library for differentially expressed genes in rat liver of PTS was successfully constructed in the present study.

  11. Nanopattern insert molding

    International Nuclear Information System (INIS)

    Kim, S H; Youn, J R; Jeong, J H

    2010-01-01

    A new method was investigated to produce nanopatterns on polymeric surfaces with high resolution, good productivity, and low cost. It has certain advantages when compared with such conventional techniques as nanoimprint lithography (NIL), hot embossing, and injection molding. Polyvinyl alcohol (PVA) was utilized for preparation of the stamp with nanopatterns on its surface. The nanoimprinted PVA film was inserted into the cavity and the polymer melt was injected into the mold. Nanopatterns with pillars smaller than 100 nm were produced on the polymeric surface. The water soluble PVA film was used as the inserted template to overcome the difficulties of releasing the nanopatterned film from the substrate.

  12. Construction of cDNA library and preliminary analysis of expressed sequence tags from Siberian tiger.

    Science.gov (United States)

    Liu, Chang-Qing; Lu, Tao-Feng; Feng, Bao-Gang; Liu, Dan; Guan, Wei-Jun; Ma, Yue-Hui

    2010-10-01

    In this study we successfully constructed a full-length cDNA library from Siberian tiger, Panthera tigris altaica, the most well-known wild Animal. Total RNA was extracted from cultured Siberian tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.30×10(6) pfu/ml and 1.62×10(9) pfu/ml respectively. The proportion of recombinants from unamplified library was 90.5% and average length of exogenous inserts was 1.13 kb. A total of 282 individual ESTs with sizes ranging from 328 to 1,142 bps were then analyzed the BLASTX score revealed that 53.9% of the sequences were classified as strong match, 38.6% as nominal and 7.4% as weak match. 28.0% of them were found to be related to enzyme/catalytic protein, 20.9% ESTs to metabolism, 13.1% ESTs to transport, 12.1% ESTs to signal transducer/cell communication, 9.9% ESTs to structure protein, 3.9% ESTs to immunity protein/defense metabolism, 3.2% ESTs to cell cycle, and 8.9 ESTs classified as novel genes. These results demonstrated that the reliability and representativeness of the cDNA library attained to the requirements of a standard cDNA library. This library provided a useful platform for the functional genomic research of Siberian tigers.

  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. Isolation and sequence analysis of a cDNA clone encoding the fifth complement component

    DEFF Research Database (Denmark)

    Lundwall, Åke B; Wetsel, Rick A; Kristensen, Torsten

    1985-01-01

    clone of 1.85 kilobase pairs was isolated. Hybridization of the mixed-sequence probe to the complementary strand of the plasmid insert and sequence analysis by the dideoxy method predicted the expected protein sequence of C5a (positions 1-12), amino-terminal to the anticipated priming site. The sequence......We have used available protein sequence data for the anaphylatoxin (C5a) portion of the fifth component of human complement (residues 19-25) to synthesize a mixed-sequence oligonucleotide probe. The labeled oligonucleotide was then used to screen a human liver cDNA library, and a single candidate cDNA...... obtained further predicted an arginine-rich sequence (RPRR) immediately upstream of the N-terminal threonine of C5a, indicating that the promolecule form of C5 is synthesized with a beta alpha-chain orientation as previously shown for pro-C3 and pro-C4. The C5 cDNA clone was sheared randomly by sonication...

  15. Construction of cDNA library and preliminary analysis of expressed sequence tags from Siberian tiger

    Science.gov (United States)

    Liu, Chang-Qing; Lu, Tao-Feng; Feng, Bao-Gang; Liu, Dan; Guan, Wei-Jun; Ma, Yue-Hui

    2010-01-01

    In this study we successfully constructed a full-length cDNA library from Siberian tiger, Panthera tigris altaica, the most well-known wild Animal. Total RNA was extracted from cultured Siberian tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.30×106 pfu/ml and 1.62×109 pfu/ml respectively. The proportion of recombinants from unamplified library was 90.5% and average length of exogenous inserts was 1.13 kb. A total of 282 individual ESTs with sizes ranging from 328 to 1,142bps were then analyzed the BLASTX score revealed that 53.9% of the sequences were classified as strong match, 38.6% as nominal and 7.4% as weak match. 28.0% of them were found to be related to enzyme/catalytic protein, 20.9% ESTs to metabolism, 13.1% ESTs to transport, 12.1% ESTs to signal transducer/cell communication, 9.9% ESTs to structure protein, 3.9% ESTs to immunity protein/defense metabolism, 3.2% ESTs to cell cycle, and 8.9 ESTs classified as novel genes. These results demonstrated that the reliability and representativeness of the cDNA library attained to the requirements of a standard cDNA library. This library provided a useful platform for the functional genomic research of Siberian tigers. PMID:20941376

  16. Inserting the CMS solenoid

    CERN Multimedia

    Maximilien Brice

    2005-01-01

    The huge superconducting solenoid for CMS is inserted into the cryostat barrel. CMS uses the world's largest thin solenoid, in terms of energy stored, and is 12 m long, with a diameter of 6 m and weighing 220 tonnes. When turned on the magnet will produce a field strength of 4 T using superconducting niobium-titanium material at 4.5 K.

  17. Learning curves for insertion

    African Journals Online (AJOL)

    Adele

    Introduction. Both the Unique™ LMA, and lately the Cobra™ PLA, is available in most of the larger state hospitals in South Africa. This study's objective is to evaluate and compare the learning curves for insertion of these two single-use airway devices. This is to ascertain which of these two devices is easier and safer to ...

  18. Insertion in Persian

    Science.gov (United States)

    Kambuziya, Aliyeh Kord-e Zafaranlu; Dehghan, Masoud

    2011-01-01

    This paper investigates epenthesis process in Persian to catch some results in relating to vowel and consonant insertion in Persian lexicon. This survey has a close relationship to the description of epenthetic consonants and the conditions in which these consonants are used. Since no word in Persian may begin with a vowel, so that hiatus can't be…

  19. The Composite Insertion Electrode

    DEFF Research Database (Denmark)

    Atlung, Sven; Zachau-Christiansen, Birgit; West, Keld

    1984-01-01

    The specific energy obtainable by discharge of porous insertion electrodes is limited by electrolyte depletion in thepores. This can be overcome using a solid ion conductor as electrolyte. The term "composite" is used to distinguishthese electrodes from porous electrodes with liquid electrolyte...

  20. Construction and characterization of a cDNA library from liver tissue of Chinese Banna minipig inbred line.

    Science.gov (United States)

    Tan, W; Chen, Y; Zhang, L; Lu, Y; Li, S; Zeng, R; Zeng, Y; Li, Y; Cheng, J

    2006-09-01

    A xenograft that performs efficient functions is an essential premise for successful xenotransplantation. Our early study indicated that Chinese Banna minipig inbred line (BMI) was an ideal xenograft donor. However, the activities of some proteins synthesized by the BMI liver are different from the human, which could lead to functional disorders in coagulation, fibrinolysis, and anticoagulation after liver xenotransplantation. Therefore, it is important to investigate the genetic background of protein incompatibility and to provide new strategies for gene manipulation. In this study we constructed a cDNA expression library using BMI liver tissue to obtain an understanding of nucleic acid and protein differences between the two species. We extracted total RNA and purified mRNA of the liver tissue from one of the sixteenth inbred generation of BMI/JS 151 substrain. After double-strand cDNA synthesis, we fractionated it on a CHROMA APIN-400 column; ligated the longer than 500bp cDNA into a ZAP Express Vector; and performed a lambda: phage packaging reaction, library amplification, and titer. We randomly picked 12 plaques and tested the length of inserts. The titers of the primary and amplified libraries were 1.0 x 10(6) pfu/mL and 5.0 x 10(9) pfu/mL, respectively. The percentages of recombinants were 97.0% in the primary library and 98.0% in the amplified library. The lengths of most inserts were between 750 bp and 2.0 kb. Thus, we successfully constructed a cDNA expression library from BMI liver tissue. Using the library, we hope to get a full-length cDNA of some important genes and conduct further studies on porcine liver function in xenotransplantation.

  1. Phage lambda cDNA cloning vectors for subtractive hybridization, fusion-protein synthesis and Cre-loxP automatic plasmid subcloning.

    Science.gov (United States)

    Palazzolo, M J; Hamilton, B A; Ding, D L; Martin, C H; Mead, D A; Mierendorf, R C; Raghavan, K V; Meyerowitz, E M; Lipshitz, H D

    1990-03-30

    We describe the construction and use of two classes of cDNA cloning vectors. The first class comprises the lambda EXLX(+) and lambda EXLX(-) vectors that can be used for the expression in Escherichia coli of proteins encoded by cDNA inserts. This is achieved by the fusion of cDNA open reading frames to the T7 gene 10 promoter and protein-coding sequences. The second class, the lambda SHLX vectors, allows the generation of large amounts of single-stranded DNA or synthetic cRNA that can be used in subtractive hybridization procedures. Both classes of vectors are designed to allow directional cDNA cloning with non-enzymatic protection of internal restriction sites. In addition, they are designed to facilitate conversion from phage lambda to plasmid clones using a genetic method based on the bacteriophage P1 site-specific recombination system; we refer to this as automatic Cre-loxP plasmid subcloning. The phage lambda arms, lambda LOX, used in the construction of these vectors have unique restriction sites positioned between the two loxP sites. Insertion of a specialized plasmid between these sites will convert it into a phage lambda cDNA cloning vector with automatic plasmid subcloning capability.

  2. [Cold induced cDNA library construction of highland barley (Hordeum vulgare L. var. nudum Hk. f.) using suppression subtractive hybridization technology].

    Science.gov (United States)

    He, Tao; Jia, Jing Fen

    2008-12-01

    Cold-induced genes of highland barley (Hordeum vulgare L. var. nudum Hk. f.) were studied using suppression subtractive hybridization (SSH) technique. The cDNA from the materials treated with 4 degrees C was used as "tester", and that from the materials growing in green house (20+/-2 degrees C) as "driver". A subtractive library of highland barley including 640 cDNA clones was constructed in this study. Enzyme digestion of 32 clones chosen randomly from the library indicated that 87.5% of them contained inserts. The cDNA inserts of 16 clones were sequenced. Blast search analyses showed that these cDNAs were homologies to genes encoding the following proteins: metallothionein, protein kinase, ethylene signal transcription factor, bZIP transcription factor, zing finger transcription factor, ribulose-1,5-bisphosphate carboxylase, ribosomal protein, sodium: hydrogen antiporter, catalase, NADPH-cytochrome reductase, ascorbate peroxidase, DNA binding protein, and sugar transporter-like protein. These results indicated that the cDNA clones in the library were related to cold-induced genes, and suggested that the cold-tolerant mechanism of highland barley might be a complicated, interactive system involving multiple approaches and genes. Construction of subtractive cDNA library provided an advantage for further studies to isolate and clone cold-induced genes in highland barley.

  3. Fuel insert shuffler

    International Nuclear Information System (INIS)

    Naser, J.; Colley, R.; Gaiser, J.; Brookmire, T.; Engle, S.

    1987-01-01

    The potential for the use of expert systems in the nuclear power industry is widely recognized. The benefits of such systems include consistency of reasoning during off-normal situations when humans are under great stress, the reduction of time required to perform certain functions and the retention of human expertise in performing specialized functions. As the potential benefits are more and more demonstrated and realized, the development of expert systems becomes a necessary part of the nuclear power industry. The development of the fuel insert shuffle expert system is used as a case study. In fact, it shows that the potential benefits are realizable. Currently, the development of the insert shuffle plan requires three to four man-weeks of effort. Further modifications to this plan are sometimes required dur to either changes in the desired core load pattern or damaged fuel assemblies or inserts. These changes generally require two to four man-days of effort and could be stressful if they are critical path items on the outage schedule

  4. Construction and selection of subtracted cDNA library of mouse hepatocarcinoma cell lines with different lymphatic metastasis potential

    Science.gov (United States)

    Hou, Li; Tang, Jan-Wu; Cui, Xiao-Nan; Wang, Bo; Song, Bo; Sun, Lei

    2004-01-01

    AIM: In order to elucidate the molecular mechanism of lymphatic metastasis of hepatocarcinoma, we detected the difference of gene expression between mouse hepatocarcinoma cell lines Hca-F and Hca-P with different lymphatic metastasis potential. METHODS: cDNA of Hca-F cells was used as a tester and cDNA of Hca-P cells was used as a driver. cDNAs highly expressed in Hca-F cells were isolated by the suppression subtractive hybridization (SSH) method. The isolated cDNA was cloned into T/A cloning vector. The ligation products were transformed into DH5 α competent cells. Individual clones were randomly selected and used for PCR amplification. Vector DNA from positive clones was isolated for sequencing. RESULTS: There were 800 positive clones in amplified subtracted cDNA library. Random analysis of 160 clones with PCR showed that 95% of the clones contained 100-700 bp inserts. Analysis of 20 sequenced cDNA clones randomly picked from the SSH library revealed 4 known genes (mouse heat shock protein 84 ku, DNA helicase, ribosomal protein S13 ,ethanol induced 6 gene) and 3 expressed sequence tags (ESTs). Four cDNAs showed no homology and presumably represent novel genes. CONCLUSION: A subtracted cDNA library of differentially expressed genes in mouse heptocarcinoma cell lines with different lymphatic metastasis potential was successfully constructed with SSH and T/A cloning techniques. The library is efficient and lays a solid foundation for searching new lymphatic metastasis related genes. The expression of mouse heat shock protein gene, DNA helicase and other 4 novel gene may be different between mouse heptocarcinoma cell lines with different lymphatic metastasis potential. PMID:15285011

  5. cDNA structure, genomic organization and expression patterns of ...

    African Journals Online (AJOL)

    Visfatin was a newly identified adipocytokine, which was involved in various physiologic and pathologic processes of organisms. The cDNA structure, genomic organization and expression patterns of silver Prussian carp visfatin were described in this report. The silver Prussian carp visfatin cDNA cloned from the liver was ...

  6. cDNA structure, genomic organization and expression patterns of ...

    African Journals Online (AJOL)

    use

    2011-11-23

    Nov 23, 2011 ... Visfatin was a newly identified adipocytokine, which was involved in various physiologic and pathologic processes of organisms. The cDNA structure, genomic organization and expression patterns of silver Prussian carp visfatin were described in this report. The silver Prussian carp visfatin. cDNA cloned ...

  7. 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.

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

    Science.gov (United States)

    Ling, Peng; Wang, Meinan; Chen, Xianming; Campbell, Kimberly Garland

    2007-06-04

    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. 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. The full-length cDNA library is useful in identifying functional genes of P. striiformis.

  9. Molecular characterization of a Leishmania donovani cDNA clone with similarity to human 20S proteasome a-type subunit

    DEFF Research Database (Denmark)

    Christensen, C B; Jørgensen, L; Jensen, A T

    2000-01-01

    Using plasma from patients infected or previously infected with Leishmania donovanii, we isolated a L. donovanii cDNA clone with similarity to the proteasome a-type subunit from humans and other eukaryotes. The cDNA clone, designated LePa, was DNA sequenced and Northern blot analysis of L...... out of 25 patients with visceral leishmaniasis and four out of 18 patients with cutaneous leishmaniasis contained IgG antibodies which reacted with the purified LePa fusion protein as evaluated in an ELISA. The LePa DNA sequence was inserted into an eukaryotic expression vector and Balb/c mice were...

  10. 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.

  11. Fuel assembly insertion system

    International Nuclear Information System (INIS)

    Barkhurst, D.J.

    1987-01-01

    This patent describes a nuclear reactor facility having fuel bundles: a system for the insertion of a fuel bundle into a position where vertically arranged fuel bundles surround and are adjacent the system comprising, in combination, separate and individual centering devices secured to and disposed on top of each fuel bundle adjacent the position. Each such centering device has a generally box-like cap configuration on the upper end of each fuel bundle and includes: a top wall; first and second side walls, each secured along and upper edge to the top wall; a rear plate attached along opposite vertical edges to the first and second side walls; a front inclined wall joined along an upper edge to the top to the wall and attached along opposite vertical edges first and second side walls; pad means secured to the lower edge of the first and second side walls, the front inclined wall and the rear plate for mounting each centering device on top of an associated fuel bundle; pin means carried by at least two of the pad means engageable with an associated aperature for locating and laterally fixing each centering device on top of its respective fuel bundle. Each front inclined wall of each of the centering devices is orientated on top of its respective fuel bundle to slope upwardly and away from the position where upon downward insertion of a fuel bundle any contact between the lower end of the fuel bundle inserted with a front inclined wall of a centering device will laterally deflect the fuel bundle. Each centering device further includes a central socket means secured to the top wall, and an elongated handling pole pivotally attached to the socket

  12. [The construction of rapid amplification of cDNA ends cDNA libraries from human fetal bone and joint].

    Science.gov (United States)

    Liang, X; Gong, Y; Liu, Q; Li, J; Chen, B; Guo, C

    2001-02-01

    To construct rapid amplification cDNA ends(RACE) cDNA libraries from human fetal bone and joint and provide resources for isolation of bone- and joint-specific development-related genes. Total RNA of bone and joint were extracted with the modified single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. The double-stranded end-blunted cDNA were synthesized using TaKaRa's cDNA synthesis kit and ligated to cassette adaptors. All of the cDNA molecules were amplified by a pair of common primers. A protocol for RACE cDNA library construction from bone and joint was established and two RACE cDNA libraries from human fetal bone and joint were successfully constructed. The protocol of RACE cDNA library construction from limited materials proved to be simple and efficient and the library was suitable for RACE to isolate tissue-specific genes.

  13. Data Insertion in Bitcoin's Blockchain

    Directory of Open Access Journals (Sweden)

    Andrew Sward

    2018-04-01

    Full Text Available This paper provides the first comprehensive survey of methods for inserting arbitrary data into Bitcoin’s blockchain. Historical methods of data insertion are described, along with lesser-known techniques that are optimized for efficiency. Insertion methods are compared on the basis of efficiency, cost, convenience of data reconstruction, permanence, and potentially negative impact on the Bitcoin ecosystem.

  14. 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

  15. cDNA sequence analysis of a 29-kDa cysteine-rich surface antigen of pathogenic Entamoeba histolytica

    International Nuclear Information System (INIS)

    Torian, B.E.; Stroeher, V.L.; Stamm, W.E.; Flores, B.M.; Hagen, F.S.

    1990-01-01

    A λgt11 cDNA library was constructed from poly(U)-Spharose-selected Entamoeba histolytica trophozoite RNA in order to clone and identify surface antigens. The library was screened with rabbit polyclonal anti-E. histolytica serum. A 700-base-pair cDNA insert was isolated and the nucleotide sequence was determined. The deduced amino acid sequence of the cDNA revealed a cysteine-rich protein. DNA hybridizations showed that the gene was specific to E. histolytica since the cDNA probe reacted with DNA from four axenic strains of E. histolytica but did not react with DNA from Entamoeba invadens, Acanthamoeba castellanii, or Trichomonas vaginalis. The insert was subcloned into the expression vector pGEX-1 and the protein was expressed as a fusion with the C terminus of glutathione S-transferase. Purified fusion protein was used to generate 22 monoclonal antibodies (mAbs) and a mouse polyclonal antiserum specific for the E. histolytica portion of the fusion protein. A 29-kDa protein was identified as a surface antigen when mAbs were used to immunoprecipitate the antigen from metabolically 35 S-labeled live trophozoites. The surface location of the antigen was corroborated by mAb immunoprecipitation of a 29-kDa protein from surface- 125 I-labeled whole trophozoites as well as by the reaction of mAbs with live trophozoites in an indirect immunofluorescence assay performed at 4 degree C. Immunoblotting with mAbs demonstrated that the antigen was present on four axenic isolates tested. mAbs recognized epitopes on the 29-kDa native antigen on some but not all clinical isolates tested

  16. cDNA sequence analysis of a 29-kDa cysteine-rich surface antigen of pathogenic Entamoeba histolytica

    Energy Technology Data Exchange (ETDEWEB)

    Torian, B.E.; Stroeher, V.L.; Stamm, W.E. (Univ. of Washington, Seattle (USA)); Flores, B.M. (Louisiana State Univ. Medical Center, New Orleans (USA)); Hagen, F.S. (Zymogenetics Incorporated, Seattle, WA (USA))

    1990-08-01

    A {lambda}gt11 cDNA library was constructed from poly(U)-Spharose-selected Entamoeba histolytica trophozoite RNA in order to clone and identify surface antigens. The library was screened with rabbit polyclonal anti-E. histolytica serum. A 700-base-pair cDNA insert was isolated and the nucleotide sequence was determined. The deduced amino acid sequence of the cDNA revealed a cysteine-rich protein. DNA hybridizations showed that the gene was specific to E. histolytica since the cDNA probe reacted with DNA from four axenic strains of E. histolytica but did not react with DNA from Entamoeba invadens, Acanthamoeba castellanii, or Trichomonas vaginalis. The insert was subcloned into the expression vector pGEX-1 and the protein was expressed as a fusion with the C terminus of glutathione S-transferase. Purified fusion protein was used to generate 22 monoclonal antibodies (mAbs) and a mouse polyclonal antiserum specific for the E. histolytica portion of the fusion protein. A 29-kDa protein was identified as a surface antigen when mAbs were used to immunoprecipitate the antigen from metabolically {sup 35}S-labeled live trophozoites. The surface location of the antigen was corroborated by mAb immunoprecipitation of a 29-kDa protein from surface-{sup 125}I-labeled whole trophozoites as well as by the reaction of mAbs with live trophozoites in an indirect immunofluorescence assay performed at 4{degree}C. Immunoblotting with mAbs demonstrated that the antigen was present on four axenic isolates tested. mAbs recognized epitopes on the 29-kDa native antigen on some but not all clinical isolates tested.

  17. Nucleotide sequence and infectious cDNA clone of the L1 isolate of Pea seed-borne mosaic potyvirus.

    Science.gov (United States)

    Olsen, B S; Johansen, I E

    2001-01-01

    The complete nucleotide sequence of Pea seed-borne mosaic potyvirus isolate L1 has been determined from cloned virus cDNA. The PSbMV L1 genome is 9895 nucleotides in length excluding the poly(A) tail. Computer analysis of the sequence revealed a single long open reading frame (ORF) of 9594 nucleotides. The ORF potentially encodes a polyprotein of 3198 amino acids with a deduced Mr of 363537. Nine putative proteolytic cleavage sites were identified by analogy to consensus sequences and genome arrangement in other potyviruses. Two full-length cDNA clones, p35S-L1-4 and p35S-L1-5, were assembled under control of an enhanced 35S promoter and nopaline synthase terminator. Clone p35S-L1-4 was constructed with four introns and p35S-L1-5 with five introns inserted in the cDNA. Clone p35S-L1-4 was unstable in Escherichia coli often resulting in amplification of plasmids with deletions. Clone p35S-L1-5 was stable and apparently less toxic to Escherichia coli resulting in larger bacterial colonies and higher plasmid yield. Both clones were infectious upon mechanical inoculation of plasmid DNA on susceptible pea cultivars Fjord, Scout, and Brutus. Eight pea genotypes resistant to L1 virus were also resistant to the cDNA derived L1 virus. Both native PSbMV L1 and the cDNA derived virus infected Chenopodium quinoa systemically giving rise to characteristic necrotic lesions on uninoculated leaves.

  18. Construction and characterization of a cDNA library from human liver tissue with chronic hepatitis B.

    Science.gov (United States)

    Chen, Xiao-hong; Chen, Zhi; Yao, Hang-ping; Chen, Feng; Zhu, Hai-hong; Zhou, Hong-juan

    2005-04-01

    To construct a cDNA library from human liver tissue with chronic hepatitis B and check its quality for investigating the expression level of liver tissue infected by hepatitis B virus. This will then be used to find the relevant genes and interesting proteins associated with the development of hepatitis B. The total RNA from liver tissue with chronic hepatitis B was extracted and the mRNA was purified using TRIZOL method. Switching mechanism at 5' end of the RNA transcript (SMART) technique and CDS III/3' primer were used for first-strand cDNA synthesis. Long distance polymerase chain reaction (LD PCR) was then used to synthesize the double-strand cDNA that was then digested by Sfi I and fractionated by CHROMA SPIN-400 column. The longer than 0.4 kb cDNAs were collected and ligated to lambdaTriplEx2 vector. Then lambda phage packaging reaction and library amplification were performed. The qualities of both unamplified and amplified cDNA libraries were strictly checked by conventional titer determination. Fourteen plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. The titers of unamplifed and amplified libraries were 1.94 x 10(6) pfu/ml and 1.49 x 10(9) pfu/ml respectively. The percentages of recombinants from both libraries were 98.15% in unamplified library and 98.76% in amplified library. The lengths of the inserts were 1.23 kb in average, 1-2 kb in 64.29%, and 0.5-1.0 kb in 35.71%. A high quality cDNA library from human liver tissue with chronic hepatitis B was successfully constructed.

  19. Isolation and Cloning of cDNA Fragment of Gene Encoding for Multidrug Resistance Associated Protein from M. affine.

    Directory of Open Access Journals (Sweden)

    Utut Widyastuti Suharsono

    2008-11-01

    Full Text Available Isolation and Cloning of cDNA Fragment of Gene Encoding for Multidrug Resistance Associated Protein from M. affine. M. affine can grow well in acid soil with high level of soluble aluminum. One of the important proteins in the detoxifying xenobiotic stress including acid and Al stresses is a multidrug resistance associated protein (MRP encoded by mrp gene. The objective of this research is to isolate and clone the cDNA fragment of MaMrp encoding MRP from M. affine. By reverse transcription, total cDNA had been synthesized from the total RNA as template. The fragment of cDNA MaMrp had been successfully isolated by PCR by using total cDNA as template and mrp primer designed from A. thaliana, yeast, and human. This fragment was successfully inserted into pGEM-T Easy and the recombinant plasmid was successfully introduced into E. coli DH5α. Nucleotide sequence analysis showed that the lenght of MaMrp fragment is 633 bp encoding 208 amino acids. Local alignment analysis based on nucleotide of mRNA showed that MaMrp fragment is 69% identical to AtMrp1 and 63% to AtMrp from A. thaliana. Based on deduced amino acid sequence, MaMRP is 84% identical to part of AtMRP13, 77% to AtMRP12, and 73% to AtMRP1 from A. thaliana respectively. Alignment analysis with AtMRP1 showed that MaMRP fragment is located in TM1 and NBF1 domains and has a specific amino acid sequence QCKAQLQNMEEE.

  20. Molecular cloning and characterization of a cDNA encoding ...

    African Journals Online (AJOL)

    enoh

    2012-03-29

    Nanjing) co., Ltd. The nucleotide sequences of these primers are as follows: ..... Ebizuka Y (2000). Molecular cloning and characterization of a cDNA for Glycyrrhiza glabra cycloartenol synthase. Biol. Pharm. Bull. 23(2):231-234.

  1. Method for construction of normalized cDNA libraries

    Science.gov (United States)

    Soares, Marcelo B.; Efstratiadis, Argiris

    1998-01-01

    This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3' noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to appropriate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library. This invention also provides normalized cDNA libraries generated by the above-described method and uses of the generated libraries.

  2. Screening of cDNA libraries on glass slide microarrays.

    Science.gov (United States)

    Berger, Dave K; Crampton, Bridget G; Hein, Ingo; Vos, Wiesner

    2007-01-01

    A quantitative screening method was developed to evaluate the quality of cDNA libraries constructed by suppression subtraction hybridization (SSH) or other enrichment techniques. The SSH technique was adapted to facilitate screening of the resultant library on a small number of glass slide microarrays. A simple data analysis pipeline named SSHscreen using "linear models for microarray data" (limma) functions in the R computing environment was developed to identify clones in the cDNA libraries that are significantly differentially expressed, and to determine if they were rare or abundant in the original treated sample. This approach facilitates the choice of clones from the cDNA library for further analysis, such as DNA sequencing, Northern blotting, RT-PCR, or detailed expression profiling using a custom cDNA microarray. Furthermore, this strategy is particularly useful for studies of nonmodel organisms for which there is little genome sequence information.

  3. 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.

  4. Construction and characterization of a goat mammary gland cDNA library.

    Science.gov (United States)

    Han, Xue Feng; Luo, Jun; Wu, Ning; Matand, Kanyand; Yang, Bao Jin; Wu, Hui Juan; Zhang, Li Juan; Wang, Hai Bin

    2008-03-01

    A lactating goat mammary gland cDNA library was constructed by using a modified commercially available cDNA library construction kit protocol. The resulting clones were sequenced and functionally analyzed through cross-species genomic comparison to assess (1) the capacity and functional quality of the constructed library for subsequent research and (2) the efficiency of the procedural modifications. The study resulted in the construction of a high-quality mammary gland cDNA library, which was characterized by (1) the total recombinants number of 1.4 x 10(7) colony-forming units (cfus) that was at least 10 times greater than the number expected from the application of the standard kit protocol, (2) the recombinants rate of 96%, and (3) the average insert size of 1,082 bp. BLAST analysis of sequenced clones against GenBank databases determined 55.7% of clone redundancy, 22 known function gene clusters, and 29 novel gene clusters. The analysis of the primary gene expression profile showed that 59% of the tested clones were genes that coded for milk proteins while 16% of the clones coded for ribosomal, metabolism, immune response, and translation proteins. The remaining 25% of the tested clones were described as novel genes. Cross-species comparison showed that 77% of characterized gene clusters were successfully identified by using resources from other ruminants and unrelated species. This outcome is in consonance with the common belief that the genomic resources that have been generated across species are potentially powerful tools that could be used for enhancing the molecular understanding of less genomically studied species, such as goat.

  5. Facility target insert shielding assessment

    Energy Technology Data Exchange (ETDEWEB)

    Mocko, Michal [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2015-10-06

    Main objective of this report is to assess the basic shielding requirements for the vertical target insert and retrieval port. We used the baseline design for the vertical target insert in our calculations. The insert sits in the 12”-diameter cylindrical shaft extending from the service alley in the top floor of the facility all the way down to the target location. The target retrieval mechanism is a long rod with the target assembly attached and running the entire length of the vertical shaft. The insert also houses the helium cooling supply and return lines each with 2” diameter. In the present study we focused on calculating the neutron and photon dose rate fields on top of the target insert/retrieval mechanism in the service alley. Additionally, we studied a few prototypical configurations of the shielding layers in the vertical insert as well as on the top.

  6. High-Throughput Plasmid cDNA Library Screening

    Energy Technology Data Exchange (ETDEWEB)

    Wan, Kenneth H.; Yu, Charles; George, Reed A.; Carlson, JosephW.; Hoskins, Roger A.; Svirskas, Robert; Stapleton, Mark; Celniker, SusanE.

    2006-05-24

    Libraries of cDNA clones are valuable resources foranalysing the expression, structure, and regulation of genes, as well asfor studying protein functions and interactions. Full-length cDNA clonesprovide information about intron and exon structures, splice junctionsand 5'- and 3'-untranslated regions (UTRs). Open reading frames (ORFs)derived from cDNA clones can be used to generate constructs allowingexpression of native proteins and N- or C-terminally tagged proteins.Thus, obtaining full-length cDNA clones and sequences for most or allgenes in an organism is critical for understanding genome functions.Expressed sequence tag (EST) sequencing samples cDNA libraries at random,which is most useful at the beginning of large-scale screening projects.However, as projects progress towards completion, the probability ofidentifying unique cDNAs via EST sequencing diminishes, resulting in poorrecovery of rare transcripts. We describe an adapted, high-throughputprotocol intended for recovery of specific, full-length clones fromplasmid cDNA libraries in five days.

  7. 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

  8. [Construction of a subtracted cDNA library of differentially expressed genes in human normal liver tissue and primary hepatocellular carcinoma tissue].

    Science.gov (United States)

    Li, J; Xu, X; Han, B; Huang, G; Qian, G; Liang, P; Yang, T

    2001-12-01

    To construct a subtracted cDNA library of differentially expressed genes in human normal liver tissue and primary hepatocellular carcinoma (HCC) tissue. Using the suppression subtractive hybridization (SSH), a novel technique has been described recently. cDNA fragments of missing or low expressing tumor suppressor genes in HCC tissue were isolated using paracancerous normal liver tissue and HCC tissue as targets. Then these cDNA fragments were directly inserted into T/A cloning vector to set up the subtractive library. Amplification of the library was carried out with transformation of E.coli by high voltage electroperforation. One hundred positive bacteria clones were randomly picked and identified using enzyme restriction method. The amplified library contained more than 4,000 positive bacteria clones. Random analysis of 100 clones with enzyme restriction method showed that all clones contained 200-600 bp inserts. A subtracted cDNA library of differentially expressed genes in human normal liver tissue and HCC tissue is constructed successfully with SSH and T/A cloning techniques. The library is efficient and lays solid foundation for screening and cloning new and specific missing or low expressing tumor suppressor genes of HCC.

  9. Differential gene expression profiling in aggressive bladder transitional cell carcinoma compared to the adjacent microscopically normal urothelium by microdissection-SMART cDNA PCR-SSH.

    Science.gov (United States)

    Wang, H T; Ma, F L; Ma, X B; Han, R F; Zhang, Y B; Chang, J W

    2006-01-01

    Identifying novel and known genes that are differentially expressed in aggressive bladder transitional cell carcinoma (BTCC) has important implications in understanding the biology of bladder tumorigenesis and developing new diagnostic and therapeutic agents. In this study we identified the differential gene expression profiles comparing tumor to the adjacent microscopically normal mucosa by manual microdissection on frozen sections. The RNAs extracted from microdissected tissues were amplified by SMART cDNA PCR technology to generate forward subtractive cDNA library by suppressive subtractive hybridization (SSH). We obtained 376 positive clones, one hundred clones of aggressive BTCC subtracted cDNA library were selected at random and inserts were reamplified by PCR. After differential screening by reverse dot blotting, 73 positive clones, that contend inserts putatively upregulated in aggressive BTCC, were further analysed by DNA sequencing, GenBank and EST database searching. Sequencing results showed that 66 clones stand for 23 known genes and 7 clones for three new EST (Genbank number: DN236875, DN236874 and DN236873). In conclusion, microdissection-SMART cDNA PCR-SSH allowed for an efficient way to identify aggressive BTCC-specific differential expressed genes that may potentially be involved in the carcinogenesis and/or progression of aggressive BTCC. These differentially expressed genes may be of potential utility as therapeutic and diagnostic targets for aggressive BTCC.

  10. Construction of a muscle cDNA library of Chinese shrimp Fenneropenaeus chinensis and sequence analysis of the troponin I gene

    Science.gov (United States)

    Li, Jitao; Chen, Ping; Li, Jian; Liu, Ping; He, Yuying; Wang, Qingyin

    2010-03-01

    A muscle cDNA library of Chinese shrimp ( Fenneropenaeus chinensis) was constructed with the SMART™ cDNA Library Construction Kit. The titer of optimal primary library was 7.7×105 pfu mL-1 and that of the amplified library was 3.0×109 pfu mL-1. The percentages of the recombinant clones of primary and amplified libraries were over 98%. The insert sizes were longer than 400 bp with an average of 1000 bp. A positive clone containing a 794 bp insert was sequenced and identified encoding fast skeletal troponin I gene. This library provided a useful resource for the functional genomic research of F. chinensis.

  11. [Cloning, sequencing and subcloning of cDNA coding for group I allergen of Dermatophagoides farinae].

    Science.gov (United States)

    Yang, Qing-gui; Li, Chao-pin

    2004-06-01

    To clone, sequence and subclone the cDNA coding for group 1 allergen of Dermatophagoides farinae (Der f 1). The cDNA of Der f 1 was amplified by RT-PCR and PCR. After purified, the gene fragment was cloned into a vector pMD-18T. The recombinant plasmid pMD-18T-Der f 1 was transformed into E. coli JM109. Positive clones were screened and identified by PCR and digestion with restriction enzyme. The sequence of inserted Der f 1 gene fragment was also detected. Der f 1 was then subcloned into the vector of pET-32a(+). The Der f 1 gene fragment of Dermatophagoides farinae was specifically amplified from RNA by RT-PCR and PCR. The recombinant plasmid pMD-18T-Der f 1 and pET-32a(+)-Der f 1 was constructed and digested by Bam H I and Sac I, the size of gene fragment was 646 bp and in accordance with the expected one. The pET-32a(+)-Der f 1 subcloning has been constructed successfully.

  12. Insertion device and method for accurate and repeatable target insertion

    Energy Technology Data Exchange (ETDEWEB)

    Gubeli, III, Joseph F.; Shinn, Michelle D.; Bevins, Michael E.; Dillon-Townes, Lawrence; Neil, George R.

    2017-07-04

    The present invention discloses a device and a method for inserting and positioning a target within a free electron laser, particle accelerator, or other such device that generates or utilizes a beam of energy or particles. The system includes a three-point registration mechanism that insures angular and translational accuracy and repeatability of positioning upon multiple insertions within the same structure.

  13. Germacrene C synthase from Lycopersicon esculentum cv. VFNT cherry tomato: cDNA isolation, characterization, and bacterial expression of the multiple product sesquiterpene cyclase.

    Science.gov (United States)

    Colby, S M; Crock, J; Dowdle-Rizzo, B; Lemaux, P G; Croteau, R

    1998-03-03

    Germacrene C was found by GC-MS and NMR analysis to be the most abundant sesquiterpene in the leaf oil of Lycopersicon esculentum cv. VFNT Cherry, with lesser amounts of germacrene A, guaia-6,9-diene, germacrene B, beta-caryophyllene, alpha-humulene, and germacrene D. Soluble enzyme preparations from leaves catalyzed the divalent metal ion-dependent cyclization of [1-3H]farnesyl diphosphate to these same sesquiterpene olefins, as determined by radio-GC. To obtain a germacrene synthase cDNA, a set of degenerate primers was constructed based on conserved amino acid sequences of related terpenoid cyclases. With cDNA prepared from leaf epidermis-enriched mRNA, these primers amplified a 767-bp fragment that was used as a hybridization probe to screen the cDNA library. Thirty-one clones were evaluated for functional expression of terpenoid cyclase activity in Escherichia coli by using labeled geranyl, farnesyl, and geranylgeranyl diphosphates as substrates. Nine cDNA isolates expressed sesquiterpene synthase activity, and GC-MS analysis of the products identified germacrene C with smaller amounts of germacrene A, B, and D. None of the expressed proteins was active with geranylgeranyl diphosphate; however, one truncated protein converted geranyl diphosphate to the monoterpene limonene. The cDNA inserts specify a deduced polypeptide of 548 amino acids (Mr = 64,114), and sequence comparison with other plant sesquiterpene cyclases indicates that germacrene C synthase most closely resembles cotton delta-cadinene synthase (50% identity).

  14. ISR Superconducting High luminosity Insertion

    CERN Multimedia

    1981-01-01

    The picture shows two of the eight superconducting quadrupoles of the low-beta insertion at intersection I8.The increase of luminosity produced by this insertion was above a factor 7. At right one can also see the Open- Axial- Field Magnet. The person is Stephan Pichler. See also 7702690X, 8102123, 8010397, 8008332.

  15. Lithium insertion in nanostructured titanates

    NARCIS (Netherlands)

    Borghols, W.J.H.

    2010-01-01

    Upon nano-sizing of insertion compounds several significant changes in Li-insertion behavior have been observed for sizes below approximately 50 nm. Although the origins of the phenomena are interrelated, the changes can be divided in three main observations. (1) The formation of new phases, leading

  16. cDNA library Table - KAIKOcDNA | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available switchLanguage; BLAST Search Image Search Home About Archive Update History Data List Contact us KAIKOcDNA... cDNA library Table Data detail Data name cDNA library Table DOI 10.18908/lsdba.nbd...c00951-005 Description of data contents List of Bombyx mori cDNA libraries. Data file File name: kaiko_cdna_...iption Registered library name Registered name of the partial cDNA library Library synonym Another name for cDNA... Download License Update History of This Database Site Policy | Contact Us cDNA library Table - KAIKOcDNA | LSDB Archive ...

  17. Study on construction of cDNA library of the treated changliver cell and quality analysis

    OpenAIRE

    Juntang, Lin; Pramanik, Jogenananda; Congrui, Wang; Huiyong, Zhang; Huigen, Feng; Baosheng, Yang; Yuchang, Li; Cunshuan, Xu

    2004-01-01

    The study aims to construct cDNA library of Changliver cell by SMART (switching mechanism at 5′ end of RNA transcript) technique and analyze its quality. cDNA of Changliver cell was made with RT-PCR and LD-PCR (long-distance PCR), the cDNA library was constructed with SMART cDNA library construction kit. Through testing, the high quality cDNA library containing whole long cDNA of Changliver cell had been constructed. The titer of the amplified cDNA library was 4.5 × 1010 pfu/ml and the averag...

  18. 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.

  19. Second-strand cDNA synthesis: classical method

    International Nuclear Information System (INIS)

    Gubler, U.

    1987-01-01

    The classical scheme for the synthesis of double-stranded cDNA as it was reported in 1976 is described. Reverse transcription of mRNA with oligo(dT) as the primer generates first strands with a small loop at the 3' end of the cDNA (the end that corresponds to the 5' end of the mRNA). Subsequent removal of the mRNA by alkaline hydrolysis leaves single-stranded cDNA molecules again with a small 3' loop. This loop can be used by either reverse transcriptase or Klenow fragment of DNA polymerase I as a primer for second-strand synthesis. The resulting products are double-stranded cDNA molecules that are covalently closed at the end corresponding to the 5' end of the original mRNA. Subsequent cleavage of the short piece of single-stranded cDNA within the loop with the single-strand-specific S 1 nuclease generate open double-stranded molecules that can be used for molecular cloning in plasmids or in phage. Useful variations of this scheme have been described

  20. Human uroporphyrinogen III synthase: Molecular cloning, nucleotide sequence, and expression of a full-length cDNA

    International Nuclear Information System (INIS)

    Tsai, Shihfeng; Bishop, D.F.; Desnick, R.J.

    1988-01-01

    Uroporphyrinogen III synthase, the fourth enzyme in the heme biosynthetic pathway, is responsible for conversion of the linear tetrapyrrole, hydroxymethylbilane, to the cyclic tetrapyrrole, uroporphyrinogen III. The deficient activity of URO-synthase is the enzymatic defect in the autosomal recessive disorder congenital erythropoietic porphyria. To facilitate the isolation of a full-length cDNA for human URO-synthase, the human erythrocyte enzyme was purified to homogeneity and 81 nonoverlapping amino acids were determined by microsequencing the N terminus and four tryptic peptides. Two synthetic oligonucleotide mixtures were used to screen 1.2 x 10 6 recombinants from a human adult liver cDNA library. Eight clones were positive with both oligonucleotide mixtures. Of these, dideoxy sequencing of the 1.3 kilobase insert from clone pUROS-2 revealed 5' and 3' untranslated sequences of 196 and 284 base pairs, respectively, and an open reading frame of 798 base pairs encoding a protein of 265 amino acids with a predicted molecular mass of 28,607 Da. The isolation and expression of this full-length cDNA for human URO-synthase should facilitate studies of the structure, organization, and chromosomal localization of this heme biosynthetic gene as well as the characterization of the molecular lesions causing congenital erythropoietic porphyria

  1. Construction of cDNA library and preliminary analysis of expressed sequence tags from green microalga Ankistrodesmus convolutus Corda.

    Science.gov (United States)

    Thanh, Tran; Chi, Vu Thi Quynh; Abdullah, Mohd Puad; Omar, Hishamuddin; Noroozi, Mostafa; Ky, Huynh; Napis, Suhaimi

    2011-01-01

    Green microalga Ankistrodesmus convolutus Corda is a fast growing alga which produces appreciable amount of carotenoids and polyunsaturated fatty acids. To our knowledge, this is the first report on the construction of cDNA library and preliminary analysis of ESTs for this species. The titers of the primary and amplified cDNA libraries were 1.1×10(6) and 6.0×10(9) pfu/ml respectively. The percentage of recombinants was 97% in the primary library and a total of 337 out of 415 original cDNA clones selected randomly contained inserts ranging from 600 to 1,500 bps. A total of 201 individual ESTs with sizes ranging from 390 to 1,038 bps were then analyzed and the BLASTX score revealed that 35.8% of the sequences were classified as strong match, 38.3% as nominal and 25.9% as weak match. Among the ESTs with known putative function, 21.4% of them were found to be related to gene expression, 14.4% ESTs to photosynthesis, 10.9% ESTs to metabolism, 5.5% ESTs to miscellaneous, 2.0% to stress response, and the remaining 45.8% were classified as novel genes. Analysis of ESTs described in this paper can be an effective approach to isolate and characterize new genes from A. convolutus and thus the sequences obtained represented a significant contribution to the extensive database of sequences from green microalgae.

  2. Procedure for normalization of cDNA libraries

    Science.gov (United States)

    Bonaldo, Maria DeFatima; Soares, Marcelo Bento

    1997-01-01

    This invention provides a method to normalize a cDNA library constructed in a vector capable of being converted to single-stranded circles and capable of producing complementary nucleic acid molecules to the single-stranded circles comprising: (a) converting the cDNA library in single-stranded circles; (b) generating complementary nucleic acid molecules to the single-stranded circles; (c) hybridizing the single-stranded circles converted in step (a) with complementary nucleic acid molecules of step (b) to produce partial duplexes to an appropriate Cot; (e) separating the unhybridized single-stranded circles from the hybridized single-stranded circles, thereby generating a normalized cDNA library.

  3. Restriction landmark cDNA scanning (RLCS): a novel cDNA display system using two-dimensional gel electrophoresis.

    Science.gov (United States)

    Suzuki, H; Yaoi, T; Kawai, J; Hara, A; Kuwajima, G; Wantanabe, S

    1996-01-01

    We have developed a new method, designated restriction landmark cDNA scanning (RLCS), which displays many cDNA species quantitatively and simultaneously as two-dimensional gel spots. In this method cDNA species of uniform length were prepared for each mRNA species using restriction enzymes. After the restriction enzyme sites were radiolabeled as landmarks, the labeled fragments were subjected to high resolution two-dimensional gel electrophoresis. In analyses of cDNA samples from adult mouse liver and brain (cerebral cortex, cerebellum and brain stem) we detected approximately 500 and >1000 discrete gel spots respectively of various intensities at a time. The spot patterns of the three brain regions were very similar, although not identical, but were quite different from the pattern for the liver. RNA blot hybridization analysis using several cloned spot DNAs as probes showed that differences in intensity of the spots among RLCS profiles correlated well with expression levels of the corresponding mRNA species in the brain regions. Because the spots and their intensities reflect distinct mRNA species and their expression level respectively, the RLCS is a novel cDNA display system which provides a great deal of information and should be useful for systematic documentation of differentially expressed genes. PMID:8628652

  4. [Construction of anti-sense cDNA library of human breast cancer cells during apoptosis induced by trichostatin A and preliminary screening of essential genes].

    Science.gov (United States)

    Ma, Xiao-Li; Wang, Bei-Bei; Wu, Peng; Lu, Yun-Ping; Zhou, Jian-Feng; Ma, Ding

    2009-02-24

    To construct an anti-sense cDNA library of human breast cancer cells to screen essential genes with anti-tumor effects on apoptosis of human breast cancer cells induced by trichostatin A. Poly (A)(+)RNA was extracted from human breast cancer cells of the line MCF-7 treated by trichostatin A for 0, 12, 24, 36, 48, 60, or 72 h. cDNA were synthesized and inserted reversely into PCEP 4 vector to construct an anti-sense cDNA library. HeLa cells were transfected with the library DNA or blank PCEP 4 vector as control group. All the transfected cells were screened by 200 nmol/L trichostatin A and 200 microg/ml hygromycin B. Screening was stopped when the control cells died. Then the surviving cell clones were amplified and Hirt DNA was extracted. Several expressed sequence tags were thus obtained. The data were analyzed by bioinformatics and interested EST fragment was chosen for preliminary functional screening. An anti-sense cDNA library was constructed containing 2 x 10(6) independent clones with an insert efficiency of more than 90%; DNA sequencing and bioinformatic analysis suggested that the No.27 survival clone was zinc transporter LIV1 showing a strong resistance against trichostatin A-induced apoptosis during functional screening. An anti-sense cDNA library with high quantity and quality has been successfully constructed; LIV1 gene may be one of the essential genes with anti-tumor effects on apoptosis induced by trichostatin A.

  5. Percutaneously inserted central catheter - infants

    Science.gov (United States)

    PICC - infants; PQC - infants; Pic line - infants; Per-Q cath - infants ... A percutaneously inserted central catheter (PICC) is a long, very thin, soft plastic tube that is put into a small blood vessel. This article addresses PICCs in ...

  6. Non-insertional Achilles tendinopathy

    Science.gov (United States)

    Pearce, Christopher J.; Tan, Audrey

    2016-01-01

    Non-insertional Achilles tendinopathy is a degenerative condition characterised by pain on activity. Eccentric stretching is the most effective treatment. Surgical treatment is reserved for recalcitrant cases. Minimally-invasive and tendinoscopic treatments are showing promising results. Cite this article: Pearce CJ, Tan A. Non-insertional Achilles tendinopathy. EFORT Open Rev 2016;1:383-390. DOI: 10.1302/2058-5241.1.160024. PMID:28461917

  7. [Intracavitary electrocardiogram during the insertion of peripherally inserted central catheters].

    Science.gov (United States)

    Ortiz-Miluy, Gloria; Sánchez-Guerra, Carmen

    2013-01-01

    To evaluate the applicability, feasibility and accuracy of the IC-ECG with column of saline technique for verifying the final tip position of peripherally inserted central catheters (PICC) by specialist nurses. A total of 99 consecutive PICC were inserted. Patients with no superficial ECG P wave, atrial fibrillation, or a pacemaker were excluded. The IC-ECG technique was performed on 84 patients. A chest x-ray was performed after insertion in all cases, in order to compare images with IC-ECG. The technique showed an applicability of 84.4%, an feasibility of 88%, and an accuracy of 87.8%. The IC-ECG technique for verification of catheter PICC tip locations with column of saline is easy to apply, is cost-effective, is achievable by nurses, and does not involve any risk for patients. The technique involves a learning curve, and it must be performed by qualified health care professionals. The technique is performed during the insertion of the catheter, so verification of the tip is made in situ. It reduces future re-insertions due to wrong positioning of the tip. Copyright © 2013 Elsevier España, S.L. All rights reserved.

  8. Cloning, sequencing and expression of cDNA encoding growth ...

    Indian Academy of Sciences (India)

    Unknown

    cell embryo and the expression was monitored continuously. The expression shown here is in developing embryo and freshly hatched fish. The intensity of green colour indicate the strong expression of EGFP in all the tissues of the embryo/fry. The expression of EGPF indicates the co-expression of catfish GH cDNA and the ...

  9. cDNA cloning, structural analysis, SNP detection and tissue ...

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Genetics; Volume 96; Issue 2. cDNA cloning, structural analysis, SNP detection and tissue ... Abstract. Insulin-like growth factor 1 (IGF1) plays an important role in growth, reproduction, foetal development and cell proliferation. The present study was conducted to clone and sequence the ...

  10. Cloning and characterization of cDNA encoding xyloglucan ...

    African Journals Online (AJOL)

    Jane

    2011-08-22

    Aug 22, 2011 ... construction and restructuring of xyloglucan cross-links, thereby controlling the mechanical properties of cell wall. We cloned complete cDNA of an ..... are marked by horizontal lines. The conserved cysteine residues (amino acids 220, 229, 274 and 288 in P. glaucum) are marked by vertical blue arrows.

  11. cDNA, genomic sequence cloning and overexpression of ribosomal ...

    African Journals Online (AJOL)

    RPS16 of eukaryote is a component of the 40S small ribosomal subunit encoded by RPS16 gene and is also a homolog of prokaryotic RPS9. The cDNA and genomic sequence of RPS16 was cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) using reverse transcription-polymerase chain ...

  12. cDNA cloning, structural analysis, SNP detection and tissue ...

    Indian Academy of Sciences (India)

    THOMAS NAICY

    [Naicy T., Venkatachalapathy T., Aravindakshan T., Raghavan K. C., Mini M. and Shyama K. 2017 cDNA cloning, structural analysis, SNP detection and tissue expression profile of the IGF1 gene in Malabari and Attappady Black goats of India. J. Genet. 96, xx–xx]. Introduction. Insulin-like growth factor 1 (IGF1), an important ...

  13. Construction of yeast surface-displayed cDNA libraries.

    Science.gov (United States)

    Bidlingmaier, Scott; Liu, Bin

    2011-01-01

    Using yeast display, heterologous protein fragments can be efficiently displayed at high copy levels on the Saccharomyces cerevisiae cell wall. Yeast display can be used to screen large expressed protein libraries for proteins or protein fragments with specific binding properties. Recently, yeast surface-displayed cDNA libraries have been constructed and used to identify proteins that bind to various target molecules such as peptides, small molecules, and antibodies. Because yeast protein expression pathways are similar to those found in mammalian cells, human protein fragments displayed on the yeast cell wall are likely to be properly folded and functional. Coupled with fluorescence-activated cell sorting, yeast surface-displayed cDNA libraries potentially allow the selection of protein fragments or domains with affinity for any soluble molecule that can be fluorescently detected. In this report, we describe protocols for the construction and validation of yeast surface-displayed cDNA libraries using preexisting yeast two-hybrid cDNA libraries as a starting point.

  14. A BIOINFORMATIC STRATEGY TO RAPIDLY CHARACTERIZE CDNA LIBRARIES

    Science.gov (United States)

    A Bioinformatic Strategy to Rapidly Characterize cDNA LibrariesG. Charles Ostermeier1, David J. Dix2 and Stephen A. Krawetz1.1Departments of Obstetrics and Gynecology, Center for Molecular Medicine and Genetics, & Institute for Scientific Computing, Wayne State Univer...

  15. Nucleotide sequence of cloned cDNA for human sphingolipid activator protein 1 precursor

    International Nuclear Information System (INIS)

    Dewji, N.N.; Wenger, D.A.; O'Brien, J.S.

    1987-01-01

    Two cDNA clones encoding prepro-sphingolipid activator protein 1 (SAP-1) were isolated from a λ gt11 human hepatoma expression library using polyclonal antibodies. These had inserts of ≅ 2 kilobases (λ-S-1.2 and λ-S-1.3) and both were both homologous with a previously isolated clone (λ-S-1.1) for mature SAP-1. The authors report here the nucleotide sequence of the longer two EcoRI fragments of S-1.2 and S-1.3 that were not the same and the derived amino acid sequences of mature SAP-1 and its prepro form. The open reading frame encodes 19 amino acids, which are colinear with the amino-terminal sequence of mature SAP-1, and extends far beyond the predicted carboxyl terminus of mature SAP-1, indicating extensive carboxyl-terminal processing. The nucleotide sequence of cDNA encoding prepro-SAP-1 includes 1449 bases from the assigned initiation codon ATG at base-pair 472 to the stop codon TGA at base-pair 1921. The first 23 amino acids coded after the initiation ATG are characteristic of a signal peptide. The calculated molecular mass for a polypeptide encoded by 1449 bases is ≅ 53 kDa, in keeping with the reported value for pro-SAP-1. The data indicate that after removal of the signal peptide mature SAP-1 is generated by removing an additional 7 amino acids from the amino terminus and ≅ 373 amino acids from the carboxyl terminus. One potential glycosylation site was previously found in mature SAP-1. Three additional potential glycosylation sites are present in the processed carboxyl-terminal polypeptide, which they designate as P-2

  16. Structure and characterization of a cDNA clone for phenylalanine ammonia-lyase from cut-injured roots of sweet potato

    Energy Technology Data Exchange (ETDEWEB)

    Tanaka, Yoshiyuki; Matsuoka, Makoto; Yamanoto, Naoki; Ohashi, Yuko; Kano-Murakami, Yuriko; Ozeki, Yoshihiro (National Institute of Agro-Environmental Sciences, Ibaraki (Japan) Univ. of Tokyo (Japan))

    1989-08-01

    A cDNA clone for phenylalanine ammonia-lyase (PAL) induced in wounded sweet potato (Ipomoea batatas Lam.) root was obtained by immunoscreening a cDNA library. The protein produced in Escherichia coli cells containing the plasmid pPAL02 was indistinguishable from sweet potato PAL as judged by Ouchterlony double diffusion assays. The M{sub r} of its subunit was 77,000. The cells converted ({sup 14}C)-L-phenylalanine into ({sup 14}C)-t-cinnamic acid and PAL activity was detected in the homogenate of the cells. The activity was dependent on the presence of the pPAL02 plasmid DNA. The nucleotide sequence of the cDNA contained a 2,121-base pair (bp) open-reading frame capable of coding for a polypeptide with 707 amino acids (M{sub r} 77,137), a 22-bp 5{prime}-noncoding region and a 207-bp 3{prime}-noncoding region. The results suggest that the insert DNA fully encoded the amino acid sequence for sweet potato PAL that is induced by wounding. Comparison of the deduced amino acid sequence with that of a PAL cDNA fragment from Phaseolus vulgaris revealed 78.9% homology. The sequence from amino acid residues 258 to 494 was highly conserved, showing 90.7% homology.

  17. Structure and characterization of a cDNA clone for phenylalanine ammonia-lyase from cut-injured roots of sweet potato

    International Nuclear Information System (INIS)

    Tanaka, Yoshiyuki; Matsuoka, Makoto; Yamanoto, Naoki; Ohashi, Yuko; Kano-Murakami, Yuriko; Ozeki, Yoshihiro

    1989-01-01

    A cDNA clone for phenylalanine ammonia-lyase (PAL) induced in wounded sweet potato (Ipomoea batatas Lam.) root was obtained by immunoscreening a cDNA library. The protein produced in Escherichia coli cells containing the plasmid pPAL02 was indistinguishable from sweet potato PAL as judged by Ouchterlony double diffusion assays. The M r of its subunit was 77,000. The cells converted [ 14 C]-L-phenylalanine into [ 14 C]-t-cinnamic acid and PAL activity was detected in the homogenate of the cells. The activity was dependent on the presence of the pPAL02 plasmid DNA. The nucleotide sequence of the cDNA contained a 2,121-base pair (bp) open-reading frame capable of coding for a polypeptide with 707 amino acids (M r 77,137), a 22-bp 5'-noncoding region and a 207-bp 3'-noncoding region. The results suggest that the insert DNA fully encoded the amino acid sequence for sweet potato PAL that is induced by wounding. Comparison of the deduced amino acid sequence with that of a PAL cDNA fragment from Phaseolus vulgaris revealed 78.9% homology. The sequence from amino acid residues 258 to 494 was highly conserved, showing 90.7% homology

  18. Gene Insertion Patterns and Sites

    Science.gov (United States)

    Vain, Philippe; Thole, Vera

    During the past 25 years, the molecular analysis of transgene insertion patterns and sites in plants has greatly contributed to our understanding of the mechanisms underlying transgene integration, expression, and stability in the nuclear genome. Molecular characterization is also an essential step in the safety assessment of genetically modified crops. This chapter describes the standard experimental procedures used to analyze transgene insertion patterns and loci in cereals and grasses transformed using Agrobacterium tumefaciens or direct transfer of DNA. Methods and protocols enabling the determination of the number and configuration of transgenic loci via a combination of inheritance studies, polymerase chain reaction, and Southern analyses are presented. The complete characterization of transgenic inserts in plants is, however, a holistic process relying on a wide variety of experimental approaches. In this chapter, these additional approaches are not detailed but references to relevant bibliographic records are provided.

  19. LOFT voltage insertion calibaration program

    International Nuclear Information System (INIS)

    Tillitt, D.N.; Miyasaki, F.S.

    1975-08-01

    The Loss-of-Fluid Test (LOFT) Facility is an experimental facility built around a ''scaled'' version of a large pressurized water reactor (LPWR). Part of this facility is the Data Acquisition and Visual Display System (DAVDS) as defined by the LOFT System Design Document SDD 1.4.2C. The DAVDS has a 702 data channel recording capability of which 548 are recorded digitally. The DAVDS also contains a Voltage Insertion Calibration Subsystem used to inject precise and known voltage steps into the recording systems. The computer program that controls the Voltage Insertion Calibration Subsystem is presented. 7 references. (auth)

  20. Molecular cloning of growth hormone encoding cDNA of Indian ...

    Indian Academy of Sciences (India)

    A modified rapid amplification of cDNA ends (RACE) strategy has been developed for cloning highly conserved cDNA sequences. Using this modified method, the growth hormone (GH) encoding cDNA sequences of Labeo rohita, Cirrhina mrigala and Catla catla have been cloned, characterized and overexpressed in ...

  1. cDNA cloning and characterization of a mannose-binding lectin from ...

    Indian Academy of Sciences (India)

    Using RNA extracted from Zingiber officinale rhizomes and primers designed according to the conservative regions of monocot mannose-binding lectins, the full-length cDNA of Z. officinale agglutinin (ZOA) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of zoa was 746 bp and contained a ...

  2. Isolation and characterization of a Coffea canephora ERF-like c-DNA

    African Journals Online (AJOL)

    A cDNA corresponding to an ERF gene has been isolated from a Coffea canephora fruit cDNA library. The cDNA was 1,317 nucleotides long and has an open reading frame of 987 bp. The predicted polypeptide showed a great similitude with equivalent proteins from others plant species. The binding domain shows 98.3% ...

  3. Molecular cloning of growth hormone encoding cDNA of Indian

    Indian Academy of Sciences (India)

    A modified rapid amplification of cDNA ends (RACE) strategy has been developed for cloning highly conserved cDNA sequences. Using this modified method, the growth hormone (GH) encoding cDNA sequences of Labeo rohita, Cirrhina mrigala and Catla catla have been cloned, characterized and overexpressed in ...

  4. cDNA cloning and characterization of a mannose-binding lectin

    Indian Academy of Sciences (India)

    Using RNA extracted from Zingiber officinale rhizomes and primers designed according to the conservative regions of monocot mannose-binding lectins, the full-length cDNA of Z. officinale agglutinin (ZOA) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of zoa was 746 bp and contained a ...

  5. cDNA library information - Dicty_cDB | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available (C) 5) sexually fusion-competent KAX3 cells (Gamete phase) (F) cDNA library construction method How to const...dir) 2) Full-length cDNA libraries (oligocapped method)(fl) 3) Gamete-specific subtraction library (sub) cDNA library construction

  6. Concepts for stereoselective acrylate insertion

    KAUST Repository

    Neuwald, Boris

    2013-01-23

    Various phosphinesulfonato ligands and the corresponding palladium complexes [{((PaO)PdMeCl)-μ-M}n] ([{( X1-Cl)-μ-M}n], (PaO) = κ2- P,O-Ar2PC6H4SO2O) with symmetric (Ar = 2-MeOC6H4, 2-CF3C6H4, 2,6-(MeO)2C6H3, 2,6-(iPrO)2C 6H3, 2-(2′,6′-(MeO)2C 6H3)C6H4) and asymmetric substituted phosphorus atoms (Ar1 = 2,6-(MeO)2C6H 3, Ar2 = 2′-(2,6-(MeO)2C 6H3)C6H4; Ar1 = 2,6-(MeO)2C6H3, Ar2 = 2-cHexOC 6H4) were synthesized. Analyses of molecular motions and dynamics by variable temperature NMR studies and line shape analysis were performed for the free ligands and the complexes. The highest barriers of ΔGa = 44-64 kJ/mol were assigned to an aryl rotation process, and the flexibility of the ligand framework was found to be a key obstacle to a more effective stereocontrol. An increase of steric bulk at the aryl substituents raises the motional barriers but diminishes insertion rates and regioselectivity. The stereoselectivity of the first and the second methyl acrylate (MA) insertion into the Pd-Me bond of in situ generated complexes X1 was investigated by NMR and DFT methods. The substitution pattern of the ligand clearly affects the first MA insertion, resulting in a stereoselectivity of up to 6:1 for complexes with an asymmetric substituted phosphorus. In the consecutive insertion, the stereoselectivity is diminished in all cases. DFT analysis of the corresponding insertion transition states revealed that a selectivity for the first insertion with asymmetric (P aO) complexes is diminished in the consecutive insertions due to uncooperatively working enantiomorphic and chain end stereocontrol. From these observations, further concepts are developed. © 2012 American Chemical Society.

  7. [CDNA cloning of human leptin and its expression].

    Science.gov (United States)

    Jia, Zhen-Yu; Fu, Xiao-Min; Jin, Ai-Hua; Cao, Jiang

    2003-07-01

    To clone cDNA of human leptin gene and obtain leptin protein for future study on leptin binding proteins. The cDNA of human leptin with 6 x his-tag was cloned by over-hang extension PCR protocol using human genomic DNA as template, and subcloned into in vitro expression vector pIVEX2.3MCS, and the fusion protein was expressed in vitro by Rapid Translation System (RTS) (RTS500 cycle primer Kit and RTS500 ProteoMaster of Roche company). The apparent molecular weight(19.46 kD) and the immuno-specificity of the fusion protein were confirmed by SDS-PAGE and Western blot, and the expressed fusion protein stayed mainly in the supernatant of the reaction mixture in soluble form. This work provides us solid basis for further study on new leptin-associated proteins.

  8. Construction of cDNA libraries in vaccinia virus.

    Science.gov (United States)

    Smith, Ernest S; Shi, Shuying; Zauderer, Maurice

    2004-01-01

    Poxvirus expression vectors have gained widespread use for expression of foreign proteins and as delivery vehicles for vaccine antigens. We have developed a novel method using the poxvirus as a library vector for functional selection of specific cDNA. Poxviruses have several unique and useful properties as a library vector. Most importantly, because poxviruses are packaged into fully infectious particles in the cell cytoplasm, specific recombinants can be readily recovered even from a very small number of selected cells. Moreover, in contrast to libraries constructed in retrovirus or plasmid-based vectors, recombinant vaccinia virus can be efficiently recovered even from cells that have been induced to undergo apoptosis or cessation of cell growth. In the past, the major obstacle in this application to poxviruses has been the low frequency with which recombinants can be generated. The most commonly used method to construct recombinant poxvirus is homologous recombination. The frequency of recombinants derived in this manner is of the order of 0.1%, sufficient to recover a recombinant of a purified DNA clone in a transfer plasmid, but far too low to permit construction of a representative cDNA library. We have developed a method that generates nearly 100% recombinant vaccinia viruses at good titer. We have termed this method trimolecular recombination. cDNA libraries of as many as 107 or more independent viral recombinants can be constructed by trimolecular recombination. For the first time, large, diverse, and representative cDNA libraries can be screened in a vaccinia virus-based expression vector.

  9. cDNA cloning, structural analysis, SNP detection and tissue ...

    Indian Academy of Sciences (India)

    THOMAS NAICY

    E-mail: naicy@kvasu.ac.in. and conception rate ... transformed into DH5α strain of Escherichia coli and the clones harbouring ... Primer pairs for caprine IGF1 and GAPDH were designed using Primer3 software (table 1). RTq-PCR was conducted in a 25 μL reaction volume containing 50 ng of cDNA and 2× Max- ima SYBR ...

  10. CDNA encoding a polypeptide including a hevein sequence

    Science.gov (United States)

    Raikhel, Natasha V.; Broekaert, Willem F.; Chua, Nam-Hai; Kush, Anil

    1995-03-21

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

  11. cDNA library generation from ribonucleoprotein particles.

    Science.gov (United States)

    Rederstorff, Mathieu; Hüttenhofer, Alexander

    2011-02-01

    Most, if not all, known noncoding RNAs (ncRNAs) are associated with RNA binding proteins, thus forming ribonucleoprotein particles or RNPs. Here we describe a protocol for the generation of a specialized cDNA library from RNPs, thereby increasing the proportion of functional ncRNA species in the library. To that end, cellular extracts are fractionated on 10-30% glycerol gradients. Subsequently, RNP-derived ncRNAs are isolated and 3'-tailed by cytidine triphosphate and poly(A) polymerase; this is followed by 5' adapter ligation by T4 RNA ligase. Reverse transcription of ncRNAs into cDNAs is carried out with an oligo-d(G) anchor primer. The generated cDNA libraries are subsequently submitted to high-throughput sequencing. This RNP selection procedure increases the probability of the presence of biologically relevant ncRNA species in the library compared with libraries generation methods that use size-selected, protein-devoid ncRNAs. The protocol enables the generation of deep-sequencing-compatible cDNA libraries that code for functional ncRNAs within 1 week.

  12. cDNA encoding a polypeptide including a hevein sequence

    Energy Technology Data Exchange (ETDEWEB)

    Raikhel, N.V.; Broekaert, W.F.; Chua, N.H.; Kush, A.

    2000-07-04

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74--79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

  13. cDNA clone for the alpha-chain of human beta-hexosaminidase: deficiency of alpha-chain mRNA in Ashkenazi Tay-Sachs fibroblasts.

    Science.gov (United States)

    Myerowitz, R; Proia, R L

    1984-09-01

    We have isolated a cDNA clone containing sequences complementary to mRNA encoding the alpha-chain of the lysosomal enzyme beta-hexosaminidase. RNA from a human lung fibroblast strain, IMR90, was enriched for beta-hexosaminidase messenger by polysome immunoselection with antiserum against beta-hexosaminidase A. This preparation was used to construct cDNA recombinant plasmids by the Okayama-Berg vector primer procedure. After transformation of Escherichia coli, 385 ampicillin-resistant colonies were obtained, 44 of which contained inserts in the plasmid DNA. Differential hybridization, with cDNA probes prepared from polysomal RNA enriched or depleted for beta-hexosaminidase messenger, was used to screen the recombinant plasmids for sequences encoding beta-hexosaminidase. One clone, p beta H alpha-1, containing a cDNA insert of approximately equal to 240 base pairs, was identified in this manner. The plasmid hybrid-selected a messenger from placental RNA that programed a translation system to synthesize the alpha-chain of beta-hexosaminidase. p beta H alpha-1 hybridized to an mRNA of approximately equal to 1.9 kilobases in preparations enriched separately in messenger for the alpha-chain or for both alpha- and beta-chains (by polysome immunoselection with antiserum against isolated alpha-chain or against beta-hexosaminidase A, respectively). It did not hybridize to an RNA preparation enriched for messenger of beta-chain by immunoselection with antiserum against beta-hexosaminidase B. The 1.9-kilobase mRNA was observed in poly(A)+ RNA preparations from control fibroblasts and from fibroblasts of a Tay-Sachs patient that synthesize an altered alpha-chain; however, it was not seen in similar preparations from fibroblasts of four Ashkenazi Tay-Sachs patients.

  14. Succinyl-CoA:3-oxoacid transferase (SCOT): Human cDNA and genomic cloning and chromosomal mapping of the human gene

    Energy Technology Data Exchange (ETDEWEB)

    Kassovska-Bratinova, S.; Mitchell, G.A. [Hopital Ste-Justine, Montreal, Quebec (Canada); Duncan, A. [Kingston General Hospital, Kingston, Ontario (Canada)] [and others

    1994-09-01

    SCOT (EC 2.8.3.5) mediates the activation and utilization of ketone bodies in extrahepatic tissues, especially brain, heart and kidney. Children with hereditary SCOT deficiency have episodes of severe ketoacidosis. Using a partial human heart SCOT cDNA, hSCOT-G, we detected a single {approximately}3 Kb mRNA in human heart and leukocytes, but not in liver. The length of the mouse SCOT mRNA detected with hSCOT-G in muscle, heart, kidney and brain is {approximately}3 Kb. We mapped the human SCOT gene to chromosome 5p13 by in situ hybridization. To date we have isolated human heart cDNAs spanning 2.9 Kb and including a 1248 bp open reading frame. The 3{prime} nontranslated region of the human SCOT mRNA extends at least 1712 bp, in contrast to the 209 bp sequence reported for pig SCOT cDNA. In one heart cDNA clone we detected a 58 bp insertion 258 bp downstream from the stop codon. We performed RT-PCR using a 5{prime} degenerate-sequence primer designed from the pig SCOT leader peptide sequence and 3{prime} human-specific primers. We obtained a fragment of the expected 320 bp length which strongly hybridizes to an internal oligonucleotide and which we are now characterizing. Human genomic Southern blot analysis with a partial human cDNA as probe suggests that the length of the human SCOT gene is about 40 K. Using hSCOT-G as a probe, we screened a human leukocyte genomic library in EMBL-3 phage and isolated two genomic clones. One of them contains a processed pseudogene. The other contains at least two exons of the human SCOT gene spanning cDNA residues 431 to 734. These findings will be useful for mutation analysis in SCOT-deficient patients.

  15. Germacrene C synthase from Lycopersicon esculentum cv. VFNT Cherry tomato: cDNA isolation, characterization, and bacterial expression of the multiple product sesquiterpene cyclase

    Science.gov (United States)

    Colby, Sheila M.; Crock, John; Dowdle-Rizzo, Barbara; Lemaux, Peggy G.; Croteau, Rodney

    1998-01-01

    Germacrene C was found by GC-MS and NMR analysis to be the most abundant sesquiterpene in the leaf oil of Lycopersicon esculentum cv. VFNT Cherry, with lesser amounts of germacrene A, guaia-6,9-diene, germacrene B, β-caryophyllene, α-humulene, and germacrene D. Soluble enzyme preparations from leaves catalyzed the divalent metal ion-dependent cyclization of [1-3H]farnesyl diphosphate to these same sesquiterpene olefins, as determined by radio-GC. To obtain a germacrene synthase cDNA, a set of degenerate primers was constructed based on conserved amino acid sequences of related terpenoid cyclases. With cDNA prepared from leaf epidermis-enriched mRNA, these primers amplified a 767-bp fragment that was used as a hybridization probe to screen the cDNA library. Thirty-one clones were evaluated for functional expression of terpenoid cyclase activity in Escherichia coli by using labeled geranyl, farnesyl, and geranylgeranyl diphosphates as substrates. Nine cDNA isolates expressed sesquiterpene synthase activity, and GC-MS analysis of the products identified germacrene C with smaller amounts of germacrene A, B, and D. None of the expressed proteins was active with geranylgeranyl diphosphate; however, one truncated protein converted geranyl diphosphate to the monoterpene limonene. The cDNA inserts specify a deduced polypeptide of 548 amino acids (Mr = 64,114), and sequence comparison with other plant sesquiterpene cyclases indicates that germacrene C synthase most closely resembles cotton δ-cadinene synthase (50% identity). PMID:9482865

  16. The first LHC insertion quadrupole

    CERN Document Server

    2004-01-01

    An important milestone was reached in December 2003 at the CERN Magnet Assembly Facility. The team from the Accelerator Technology - Magnet and Electrical Systems group, AT-MEL, completed the first special superconducting quadrupole for the LHC insertions which house the experiments and major collider systems. The magnet is 8 metres long and contains two matching quadrupole magnets and an orbit corrector, a dipole magnet, used to correct errors in quadrupole alignment. All were tested in liquid helium and reached the ultimate performance criteria required for the LHC. After insertion in the cryostat, the superconducting magnet will be installed as the Q9 quadrupole in sector 7-8, the first sector of the LHC to be put in place in 2004. Members of the quadrupole team, from the AT-MEL group, gathered around the Q9 quadrupole at its inauguration on 12 December 2003 in building 181.

  17. ATLAS insertable B-layer

    Czech Academy of Sciences Publication Activity Database

    Marčišovský, Michal

    2011-01-01

    Roč. 633, č. 1 (2011), "S224"-"S225" ISSN 0168-9002. [International workshop on radiation imaging detectors /11./. Praha, 26.06.2009-02.07.2009] R&D Projects: GA MŠk LA08015; GA MŠk LA08032 Institutional research plan: CEZ:AV0Z10100502 Keywords : ATLAS * pixel detector * insertable B-layer Subject RIV: BF - Elementary Particles and High Energy Physics Impact factor: 1.207, year: 2011

  18. Inserting Agility in System Development

    Science.gov (United States)

    2012-07-01

    Agile IT Acquisition, IT Box, Scrum Inserting Agility in System Development Matthew R. Kennedy and Lt Col Dan Ward, USAF With the fast-paced nature... Scrum is a process to manage a product, eXtreme Program- ming (XP) is an agile development methodology focused on software development as a whole. XP...249 A Publication of the Defense Acquisition University http://www.dau.mil Keywords: Agile , Systems Engineering, Information Technology (IT), DoD

  19. Ring insertions as light sources

    International Nuclear Information System (INIS)

    Green, G.K.

    1975-01-01

    Bending magnets can be inserted in the long straight sections of electron storage rings to produce synchrotron radiation. If the design is carefully proportioned, the bending magnets create only a small perturbation of the properties of the ring. The resulting spectra have favorable optical properties as sources for spectroscopy and diffraction studies. The characteristics of the source are discussed, and the geometrical requirements of the magnets are presented

  20. High-efficiency system for the construction of adenovirus vectors and its application to the generation of representative adenovirus-based cDNA expression libraries.

    Science.gov (United States)

    Hillgenberg, Moritz; Hofmann, Christian; Stadler, Herbert; Löser, Peter

    2006-06-01

    We here describe a convenient system for the production of recombinant adenovirus vectors and its use for the construction of a representative adenovirus-based cDNA expression library. The system is based on direct site-specific insertion of transgene cassettes into a replicating donor virus. The transgene is inserted into a donor plasmid containing the viral 5' inverted terminal repeat, the complete viral packaging signal, and a single loxP site. The plasmid is then transfected into a Cre recombinase-expressing packaging cell line that has been infected with a donor virus containing a partially deleted packaging signal flanked by loxP sites. Cre recombinase, by two steps of action, sequentially catalyzes the generation of a nonpackageable donor virus acceptor substrate and the generation of the desired recombinant adenovirus vector. Due to its growth impairment, residual donor virus can efficiently be counterselected during amplification of the recombinant adenovirus vector. By using this adenovirus construction system, a plasmid-based human liver cDNA library was converted by a single step into an adenovirus-based cDNA expression library with about 10(6) independent adenovirus clones. The high-titer purified library was shown to contain about 44% of full-length cDNAs with an average insert size of 1.3 kb. cDNAs of a gene expressed at a high level (human alpha(1)-antitrypsin) and a gene expressed at a relatively low level (human coagulation factor IX) in human liver were isolated from the adenovirus-based library using an enzyme-linked immunosorbent assay-based screening procedure.

  1. HTS Insert Magnet Design Study

    CERN Document Server

    Devaux, M; Fleiter, J; Fazilleau, P; Lécrevisse, T; Pes, C; Rey, J-M; Rifflet, J-M; Sorbi, M; Stenvall, A; Tixador, P; Volpini, G

    2011-01-01

    Future accelerator magnets will need to reach higher field in the range of 20 T. This field level is very difficult to reach using only Low Temperature Superconductor materials whereas High Temperature Superconductors (HTS) provide interesting opportunities. High current densities and stress levels are needed to design such magnets. YBCO superconductor indeed carries large current densities under high magnetic field and provides good mechanical properties especially when produced using the IBAD approach. The HFM EUCARD program studies the design and the realization of an HTS insert of 6 T inside a Nb3Sn dipole of 13T at 4.2 K. In the2HTS insert, engineering current densities higher than 250 MA/m under 19 T are required to fulfill the specifications. The stress level is also very severe. YBCO IBAD tapes theoretically meet these challenges from presented measurements. The insert protection is also a critical because HTS materials show low quench propagation velocities and the coupling with the Nb3Sn magnet make...

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

    Energy Technology Data Exchange (ETDEWEB)

    Zarlenga, D.; Gamble, H.R.

    1987-05-01

    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 TSP 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.

  3. Functional characterization of an acidic SK(3) dehydrin isolated from an Opuntia streptacantha cDNA library.

    Science.gov (United States)

    Ochoa-Alfaro, A E; Rodríguez-Kessler, M; Pérez-Morales, M B; Delgado-Sánchez, P; Cuevas-Velazquez, C L; Gómez-Anduro, G; Jiménez-Bremont, J F

    2012-03-01

    Cactus pears are succulent plants of the Cactaceae family adapted to extremely arid, hot and cold environments, making them excellent models for the study of molecular mechanisms underlying abiotic stress tolerance. Herein, we report a directional cDNA library from 12-month-old cladodes of Opuntia streptacantha plants subjected to abiotic stresses. A total of 442 clones were sequenced, representing 329 cactus pear unigenes, classified into eleven functional categories. The most abundant EST (unigen 33) was characterized under abiotic stress. This cDNA of 905 bp encodes a SK(3)-type acidic dehydrin of 248 amino acids. The OpsDHN1 gene contains an intron inserted within the sequence encoding the S-motif. qRT-PCR analysis shows that the OpsDHN1 transcript is specifically accumulated in response to cold stress, and induced by abscisic acid. Over-expression of the OpsDHN1 gene in Arabidopsis thaliana leads to enhanced tolerance to freezing treatment, suggesting that OpsDHN1 participates in freezing stress responsiveness. Generation of the first EST collection for the characterization of cactus pear genes constitutes a useful platform for the understanding of molecular mechanisms of stress tolerance in Opuntia and other CAM plants.

  4. Preparation of a differentially expressed, full-length cDNA expression library by RecA-mediated triple-strand formation with subtractively enriched cDNA fragments

    NARCIS (Netherlands)

    Hakvoort, T. B.; Spijkers, J. A.; Vermeulen, J. L.; Lamers, W. H.

    1996-01-01

    We have developed a fast and general method to obtain an enriched, full-length cDNA expression library with subtractively enriched cDNA fragments. The procedure relies on RecA-mediated triple-helix formation of single-stranded cDNA fragments with a double-stranded cDNA plasmid library. The complexes

  5. Construction and characterization of a normalized cDNA library.

    OpenAIRE

    Soares, M B; Bonaldo, M F; Jelene, P; Su, L; Lawton, L; Efstratiadis, A

    1994-01-01

    We have developed a simple procedure based on reassociation kinetics that can reduce effectively the high variation in abundance among the clones of a cDNA library that represent individual mRNA species. For this normalization, we used as a model system a library of human infant brain cDNAs that were cloned directionally into a phagemid vector and, thus, could be easily converted into single-stranded circles. After controlled primer extension to synthesize a short complementary strand on each...

  6. Regional insertion: an emergent approach

    International Nuclear Information System (INIS)

    Serra, M.T.F.; Nascimento Teixeira, P. do

    1989-01-01

    The Brazilian Electrical Sector incorporates new variables that expressing the extensive spectrum of environmental impacts in the take of decisions, referring to the viability of realizing a electrical undertaking, attends the several restrictions that are important by the sector and by the society in the environment area and promotes the adequate generation of liquid benefits, consequential of the electrical undertaking. Due to these factors, the Electrical Sector is improving the concept of regional insertion, with the sectorial expansion in long-dated and the created demand in the environmental and social area, focalizing the solution for these questions. (C.G.C.). 1 fig, 2 tabs

  7. Characterization of a cDNA encoding cottonseed catalase.

    Science.gov (United States)

    Ni, W; Turley, R B; Trelease, R N

    1990-06-21

    A 1.7 kb cDNA clone was isolated from our lambda gt11 library constructed from poly(A) RNA of 24-h-old cotyledons. The cDNA encodes a full-length catalase peptide (492 amino acid residues). The calculated molecular mass is 56,800, similar to that determined for purified enzyme (57,000 SDS-PAGE). Among higher plant catalases, this cotton catalase shows the highest amino acid sequence identity (85%) to the subunit of homotetrameric maize CAT 1, a developmental counterpart to the homotetrameric CAT A isoform of cotton seeds. Comparison of sequences from cotton, sweet potato, maize CAT 1, and yeast with bovine catalase revealed that the amino acid residues and regions that are involved in catalytic activity and/or required to maintain basic catalase structure, are highly conserved. The C-terminus region, which has the lowest nucleotide sequence identity between plant and mammalian catalases, does not terminate with a tripeptide, S-K/R/H-L, a putative targeting signal for peroxisomal proteins.

  8. Infectious Maize rayado fino virus from Cloned cDNA.

    Science.gov (United States)

    Edwards, Michael C; Weiland, John J; Todd, Jane; Stewart, Lucy R

    2015-06-01

    A full-length cDNA clone was produced from a U.S. isolate of Maize rayado fino virus (MRFV), the type member of the genus Marafivirus within the family Tymoviridae. Infectivity of transcripts derived from cDNA clones was demonstrated by infection of maize plants and protoplasts, as well as by transmission via the known leafhopper vectors Dalbulus maidis and Graminella nigrifrons that transmit the virus in a persistent-propagative manner. Infection of maize plants through vascular puncture inoculation of seed with transcript RNA resulted in the induction of fine stipple stripe symptoms typical of those produced by wild-type MRFV and a frequency of infection comparable with that of the wild type. Northern and Western blotting confirmed the production of MRFV-specific RNAs and proteins in infected plants and protoplasts. An unanticipated increase in subgenomic RNA synthesis over levels in infected plants was observed in protoplasts infected with either wild-type or cloned virus. A conserved cleavage site motif previously demonstrated to function in both Oat blue dwarf virus capsid protein and tymoviral nonstructural protein processing was identified near the amino terminus of the MRFV replicase polyprotein, suggesting that cleavage at this site also may occur.

  9. Novel insertion mutation in a non-Jewish Caucasian type 1 Gaucher disease patient

    Energy Technology Data Exchange (ETDEWEB)

    Choy, F.Y.M.; Humphries, M.L. [Univ. of Victoria, British Columbia (Canada); Ferreira, P. [Univ. of Alberta, Edmonton (Canada)

    1997-01-20

    Gaucher disease is the most prevalent lysosomal storage disorder. It is autosomal recessive, resulting in lysosomal glucocerebrosidase deficiency. Three clinical forms of Gaucher disease have been described: type 1 (nonneuronopathic), type 2 (acute neuronopathic), and type 3 (subacute neuronopathic). We performed PCR-thermal cycle sequence analysis of glucocerebrosidase genomic DNA and identified a novel mutation in a non-Jewish type 1 Gaucher disease patient. It is a C insertion in exon 3 at cDNA nucleotide position 122 and genomic nucleotide position 1626. This mutation causes a frameshift and, subsequently, four of the five codons immediately downstream of the insertion were changed while the sixth was converted to a stop codon, resulting in premature termination of protein translation. The 122CC insertion abolishes a Cac81 restriction endonuclease cleavage site, allowing a convenient and reliable method for detection using RFLP analysis of PCR-amplified glucocerebrosidase genomic DNA. The mutation in the other Gaucher allele was found to be an A{r_arrow}G substitution at glucocerebrosidase cDNA nucleotide position 1226 that so far has only been reported among type 1 Gaucher disease patients. Since mutation 122CC causes a frameshift and early termination of protein translation, it most likely results in a meaningless transcript and subsequently no residual glucocerebrosidase enzyme activity. We speculate that mutation 122CC may result in a worse prognosis than mutations associated with partial activity. When present in the homozygous form, it could be a lethal allele similar to what has been postulated for the other known insertion mutation, 84GG. Our patient, who is a compound heterozygote 122CC/1226G, has moderately severe type 1 Gaucher disease. Her clinical response to Ceredase{reg_sign} therapy that began 31 months ago has been favorable, though incomplete. 30 refs., 3 figs., 2 tabs.

  10. Isolation and sequence of a cDNA clone for human tyrosinase that maps at the mouse c-albino locus.

    Science.gov (United States)

    Kwon, B S; Haq, A K; Pomerantz, S H; Halaban, R

    1987-01-01

    Screening of a lambda gt11 human melanocyte cDNA library with antibodies against hamster tyrosinase (monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1) resulted in the isolation of 16 clones. The cDNA inserts from 13 of the 16 clones cross-hybridized with each other, indicating that they were from related mRNA species. One of the cDNA clones, Pmel34, detected one mRNA species with an approximate length of 2.4 kilobases that was expressed preferentially in normal and malignant melanocytes but not in other cell types. The amino acid sequence deduced from the nucleotide sequence showed that the putative human tyrosinase is composed of 548 amino acids with a molecular weight of 62,610. The deduced protein contains glycosylation sites and histidine-rich sites that could be used for copper binding. Southern blot analysis of DNA derived from newborn mice carrying lethal albino deletion mutations revealed that Pmel34 maps near or at the c-albino locus, the position of the structural gene for tyrosinase. Images PMID:2823263

  11. Construction of a full-length cDNA library and preliminary analysis of expressed sequence tags from lymphocytes of half-pipe snowboarding athletes.

    Science.gov (United States)

    Zhao, Y H; Zhang, Z B; Zhao, C Q; Zhang, Y; Wang, Y F; Guan, W J; Zhu, Z Q

    2015-10-21

    The genes of top athletes are a valuable genetic resource for the human race, and could be exploited to identify novel genes related to sports ability, as well as other functions. We analyzed the expressed sequence tags from top half-pipe snowboarding athletes using the SMART complementary DNA (cDNA) library construction method to elucidate the characteristics of the athlete genome and the differential expression of the genes it contains. Overall, we established a full-length cDNA library from the lymphocytes of half-pipe snowboarding athletes and analyzed the inserted gene fragments. We also classified those genes according to molecular function, biological characteristics, cellular composition, protein types, and signal paths. A total of 201 functional genes were noted, which were distributed in 27 pathways. TXN, MDH1, ARL1, ARPC3, ACTG1, and other genes measured in sequence may be associated with physical ability. This suggests that the SMART cDNA library constructed from the genetic material from top athletes is an effective tool for preserving genetic sports resources and providing genetic markers of physical ability for athlete selection.

  12. Construction of cDNA expression library of watermelon for isolation of ClWRKY1 transcription factors gene involved in resistance to Fusarium wilt.

    Science.gov (United States)

    Yang, Bing-Yan; Huo, Xiu-Ai; Li, Peng-Fei; Wang, Cui-Xia; Duan, Hui-Jun

    2014-08-01

    Full-length cDNAs are very important for genome annotation and functional analysis of genes. The number of full-length cDNAs from watermelon remains limited. Here we report first the construction of a full-length enriched cDNA library from Fusarium wilt stressed watermelon (Citrullus lanatus Thunb.) cultivar PI296341 root tissues using the SMART method. The titer of primary cDNA library and amplified library was 2.21 x 10(6) and 2.0 x 10(10) pfu/ml, respectively and the rate of recombinant was above 85%. The size of insert fragment ranged from 0.3 to 2.0 kb. In this study, we first cloned a gene named ClWRKY1, which was 1981 bp long and encoded a protein consisting of 394 amino acids. It contained two characteristic WRKY domains and two zinc finger motifs. Quantitative real-time PCR showed that ClWRKY1 expression levels reached maximum level at 12 h after inoculation with Fusarium oxysporum f. sp. niveum. The full-length cDNA library of watermelon root tissues is not only essential for the cloning of genes which are known, but also an initial key for the screening and cloning of new genes that might be involved in resistance to Fusarium wilt.

  13. Preparation of full-length cDNA libraries: focus on metazoans.

    Science.gov (United States)

    Harada, Masako; Hayashizaki, Yoshihide

    2009-01-01

    Critical steps in a cDNA library preparation include efficient cDNA synthesis, selection of full-length cDNAs, normalizing their abundance, and the subtraction of redundant transcripts. The use of trehalose and sorbiol stabilizes the activity of the reverse transcriptase leading to efficient cDNA synthesis and the cap-trapping method is used for efficient full-length cDNA selection. Through the incorporation of additional normalization and subtraction steps that eliminate the size bias and expressed gene frequency, it is possible to attain cDNA libraries that include larger or rarely expressed genes. This chapter describes an efficient method to construct a full-length cDNA library, with a focus on metazoan samples.

  14. Solid-phase cDNA library construction, a versatile approach.

    OpenAIRE

    Roeder, T

    1998-01-01

    A rapid and versatile method for cDNA library construction was developed. It is based on conventional cDNA library synthesis including all enzymatic steps usually required, but is performed on a solid support. The cDNA is immobilised via a biotin residue to streptavidin coupled magnetic beads, which allows rapid and easy to perform changes of buffers and enzymes. Therefore, it combines speed (library construction within a single day) with high quality libraries, making it ideally suited for m...

  15. [Software development in data analysis and mining for cDNA microarray].

    Science.gov (United States)

    Wu, Bin; Wang, Jianguo; Wang, Miqu

    2007-12-01

    Data analysis and mining is a key issue to microarray technology and is usually implemented through software development. This paper summarizes the state-of-art software development in cDNA microarray data analysis and mining. The updated software developments are discussed in three stages: data inquisition from cDNA microarray tests, statistical treatment of cDNA data and data mining from gene network.

  16. Molecular cloning and mammalian expression of human beta 2-glycoprotein I cDNA

    DEFF Research Database (Denmark)

    Kristensen, Torsten; Schousboe, Inger; Boel, Espen

    1991-01-01

    Human β2-glycoprotein (β2gpI) cDNA was isolated from a liver cDNA library and sequenced. The cDNA encoded a 19-residue hydrophobic signal peptide followed by the mature β2gpI of 326 amino acid residues. In liver and in the hepatoma cell line HepG2 there are two mRNA species of about 1.4 and 4.3 kb...

  17. Cloning of partial cDNA encoding differentiation and tumor-associated mucin glycoproteins expressed by human mammary epithelium

    Energy Technology Data Exchange (ETDEWEB)

    Gender, S.J.; Burchell, J.M.; Duhig, T.; Lamport, D.; White, R.; Parker, M.; Taylor-Papadimitriou, J.

    1987-09-01

    Human mammary epithelial cells secrete and express on their cell surfaces complex mucin glycoproteins that are developmentally regulated, tumor-associated, and highly immunogenic. Studies using monoclonal antibodies directed to these glycoproteins suggest that their molecular structures can vary with differentiation stages in the normal gland and in malignancy. To analyze the molecular nature of these glycoproteins, milk mucin was affinity-purifed and deglycosylated with hydrogen fluoride, yielding bands at 68 and 72 kDa on silver-stained gels. Polyclonal and monoclonal antibodies to the stripped core protein were developed and used to screen a lambdagt11 expression library of cDNA made from mRNA of the mammary tumor cell line MCF-7. Seven crossreacting clones were isolated, with inserts 0.1-1.8 kilobases long. RNA blot analysis, using as a probe the 1.8-kilobase insert subcloned in plasmid pUC8 (pMUC10), revealed transcripts of 4.7 and 6.4 kilobases in MCF-7 and T47D mammary tumor cells, whereas normal mammary epithelial cells from pooled milks have additional transcripts. The expression of mRNA correlates with antigen expression as determined by binding of two previously characterized anti-mucin monoclonal antibodies (HMFG-1 and HMFG-2) to seven cell lines. Restriction enzyme analysis detected a restriction fragment length polymorphism when human genomic DNA was digested with EcoRI or HinfI.

  18. Expression analysis of a ''Cucurbita'' cDNA encoding endonuclease

    International Nuclear Information System (INIS)

    Szopa, J.

    1995-01-01

    The nuclear matrices of plant cell nuclei display intrinsic nuclease activity which consists in nicking supercoiled DNA. A cDNA encoding a 32 kDa endonuclease has been cloned and sequenced. The nucleotide and deduced amino-acid sequences show high homology to known 14-3-3-protein sequences from other sources. The amino-acid sequence shows agreement with consensus sequences for potential phosphorylation by protein kinase A and C and for calcium, lipid and membrane-binding sites. The nucleotide-binding site is also present within the conserved part of the sequence. By Northern blot analysis, the differential expression of the corresponding mRNA was detected; it was the strongest in sink tissues. The endonuclease activity found on DNA-polyacrylamide gel electrophoresis coincided with mRNA content and was the highest in tuber. (author). 22 refs, 6 figs

  19. Genetic Diversity of the fliC Genes Encoding the Flagellar Antigen H19 of Escherichia coli and Application to the Specific Identification of Enterohemorrhagic E. coli O121:H19

    Science.gov (United States)

    Beutin, Lothar; Delannoy, Sabine

    2015-01-01

    Enterohemorrhagic Escherichia coli (EHEC) O121:H19 belong to a specific clonal type distinct from other classical EHEC and major enteropathogenic E. coli groups and is regarded as one of the major EHEC serogroups involved in severe infections in humans. Sequencing of the fliC genes associated with the flagellar antigen H19 (fliCH19) revealed the genetic diversity of the fliCH19 gene sequences in E. coli. A cluster analysis of 12 fliCH19 sequences, 4 from O121 and 8 from non-O121 E. coli strains, revealed five different genotypes. All O121:H19 strains fell into one cluster, whereas a second cluster was formed by five non-O121:H19 strains. Cluster 1 and cluster 2 strains differ by 27 single nucleotide exchanges in their fliCH19 genes (98.5% homology). Based on allele discrimination of the fliCH19 genes, a real-time PCR test was designed for specific identification of EHEC O121:H19. The O121 fliCH19 PCR tested negative in 73 E. coli H19 strains that belonged to serogroups other than O121, including 28 different O groups, O-nontypeable H19, and O-rough:H19 strains. The O121 fliCH19 PCR reacted with all 16 tested O121:H19 strains and 1 O-rough:H19 strain which was positive for the O121 wzx gene. A cross-reaction was observed only with E. coli H32 strains which share sequence similarities in the target region of the O121 fliCH19 PCR. The combined use of O-antigen genotyping (O121 wzx) and the detection of O121 fliCH19 allele type contributes to improving the identification and molecular serotyping of EHEC O121:H19 motile and nonmotile strains and variants of these strains lacking stx genes. PMID:25862232

  20. [Construction of the suppression subtractive cDNA libraries of human large cell lung cancer line L9981 before and after transfection with nm23-H1 gene.].

    Science.gov (United States)

    Ye, Sujuan; Feng, Zhihua; Zhu, Wen; Cai, Chunji; Li, Lu; Sun, Liya; Wan, Haisu; Ma, Li; Zhou, Qinghua

    2008-08-20

    It has been proven that nm23-H1 gene is an important metastaticsuppressed gene of lung cancer. In order to screen the differential expression genes related to nm23-H1 , we constructed the suppression subtractive cDNA libraries of human large cell lung cancer line L9981 transfected and untransfected with nm23-H1 gene by suppression subtractive hybridization (SSH) in this study, which lay a solid foundation for further screening and cloning metastatic-related genes of nm23-H1. The forward and reverse suppression subtractive cDNA libraries were constructed in the human large cell lung cancer line L9981 before and after transfection with nm23-H1 gene (L9981 and L9981-nm23-H1) by SSH method. The positive clones were preliminarily screened by bluewhite colony, and precisely identified by PCR. The suppression subtractive cDNA libraries were successfully constructed in the human large cell lung cancer line L9981 transfected and untransfected with nm23-H1 gene (L9981-nm23-H1 and L9981). After the blue-white screening, about three hundred positive clones in the forward subtracted library and four hundred positive clones in the reverse subtracted library were obtained. Ramdom analysis of 96 clones in each library with colony PCR methods showed that 84 clones in the forward subtracted library and 83 clones in the reverse subtracted library contained (300-750) bp inserts. SSH is proved to be an efficient tool for differential expression gene cloning. The forward and reverse suppression subtractive cDNA libraries of human large cell lung cancer line L9981 transfected and untransfected with nm23-H1 gene (L9981-nm23-H1 and L9981) are successfully constructed by SSH and T/A cloning technology. The expression of nm23-H1 gene in the human large cell lung cancer cell lines may affect the differential expression of some metastatic-related genes.

  1. [Screening of high taxol producing fungi by mutagenesis and construction of subtracted cDNA library by suppression subtracted hybridization for differentially expressed genes].

    Science.gov (United States)

    Zhao, Kai; Sun, Lixin; Wang, Xuan; Li, Xiuliang; Wang, Xin; Zhou, Dongpo

    2011-07-01

    To screen mutants with high yield of taxol, and construct cDNA subtractive library of obtained mutant and primary strain HD(1-3). The spores of taxol-producing fungus HD(1-3) were treated by diethyl sulphate (DES), ultraviolet radiation and diethyl sulphate (UV + DES). cDNA subtractive library of taxol producing fungi from the mRNA of obtained mutant with high yield of taxol tester and HD(1-3) driver was constructed by using suppression subtracted hybridization (SSH). The optimal conditions for mutagenesis of strain HD(1-3) were as follows: the spore suspension was treated with 8% DES for 15 min, followed by UV irradiation (30 w, 30 cm distance) for 45 sec under magnetic stirring, a mutant UD(14-1) which was able to produce taxol with high yield and could be stably passed on genetics was found. Its ability to produce taxol was improved from 232.73 +/- 4.61 microg/L (strain HD(1-3)) to 312.81 +/- 7.51 microg/L (strain UD(14-1)). The tilter of the constructed cDNA library was 1.2 x 10(7) cfu/mL, the recombinant rate reached to 75.3% and the length of the inserted fragments was mostly 300 bp-1.0 kb. A mutant UD(14-11) with high yield was obtained, and cDNA subtractive library of the mutant UD(14-11) and strain HD(1-3) was constructed. The study laid solid foundation for isolation of taxol biosynthesis related genes and construction of engineering strains with high yield of taxol by genetic techniques.

  2. 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

  3. Neuropathy associated with etonogestrel implant insertion.

    Science.gov (United States)

    Brown, Matthew; Britton, John

    2012-11-01

    The etonogestrel contraceptive implant (Implanon®) is an effective, long-acting subdermal method of hormonal contraception for women. We describe a case of forearm pain and hypoesthesia associated with the insertion of the Implanon® contraceptive implant in a healthy 26-year-old female. These symptoms were due to direct implant contact with the medial cutaneous nerve of the forearm. The importance of correct insertion technique is discussed. Care should be taken to avoid nerve injury during insertion of subdermal contraceptive implants. An understanding of regional anatomy and the correct insertion technique will prevent insertion-related complications. Nexplanon® has been developed to replace Implanon®. It has a redesigned applicator intended to increase insertion accuracy. Copyright © 2012 Elsevier Inc. All rights reserved.

  4. Hamstring tendons insertion - an anatomical study

    OpenAIRE

    Cristiano Antonio Grassi; Vagner Messias Fruheling; Joao Caetano Abdo; Marcio Fernando Aparecido de Moura; Mario Namba; Joao Luiz Vieira da Silva; Luiz Antonio Munhoz da Cunha; Ana Paula Gebert de Oliveira Franco; Isabel Ziesemer Costa; Edmar Stieven Filho

    2013-01-01

    OBJECTIVE: To study the anatomy of the hamstring tendons insertion and anatomical rela-tionships. METHODS: Ten cadaver knees with medial and anterior intact structures were selected. The dissection was performed from anteromedial access to exposure of the insertion of the flexor tendons (FT), tibial plateau (TP) and tibial tuberosity (TT). A needle of 40 × 12 and a caliper were used to measure the distance of the tibial plateau of the knee flexor tendons insertion at 15 mm from the ...

  5. Insertion devices at the advanced photon source

    International Nuclear Information System (INIS)

    Moog, E.R.

    1996-01-01

    The insertion devices being installed at the Advanced Photon Source cause the stored particle beam to wiggle, emitting x-rays with each wiggle. These x-rays combine to make an intense beam of radiation. Both wiggler and undulator types of insertion devices are being installed; the characteristics of the radiation produced by these two types of insertion devices are discussed, along with the reasons for those characteristics

  6. Construction and analysis of a cDNA library from yellow-fruit ginseng

    African Journals Online (AJOL)

    The total RNA was isolated from yellow-fruit ginseng (Panax ginseng C.A. Meyer) leaf tissue. A cDNA library of panax ginseng leaves was constructed by using pDNR-LIB vector according to the SMART cDNA library construction kit protocol. We obtained 378 high quality sequences (GenBank accession number: ...

  7. cDNA table - RPD | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available switchLanguage; BLAST Search Image Search Home About Archive Update History Data ...ile URL: ftp://ftp.biosciencedbc.jp/archive/rpd/LATEST/rpd_cdna.zip File size: 15 KB Simple search URL http:...age About This Database Database Description Download License Update History of This Database Site Policy | Contact Us cDNA table - RPD | LSDB Archive ...

  8. Development of a simple and powerful method, cDNA AFLP-SSPAG ...

    African Journals Online (AJOL)

    Differential cDNAs were easily obtained from silver stained cDNA-AFLP separated on polyacylamide gels. The cDNA was then reamplified, cloned and fragments were sequenced. Sequenced clones were used as probes in northern dot blot analyses and library screening. Full-length cDNA was cloned from a library ...

  9. Genomic and cDNA cloning of a novel mouse lipoxygenase gene

    NARCIS (Netherlands)

    Willems van Dijk, K.; Steketee, K.; Havekes, L.; Frants, R.; Hofker, M.

    1995-01-01

    A novel 12- and 15-lipoxygenase related gene was isolated from a mouse strain 129 genomic phage library in a screen with a human 15-lipoxygenase cDNA probe. The complete genomic sequence revealed 14 exons and 13 introns covering 7.3 kb of DNA. The splice junctions were verified from the cDNA

  10. 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 ...

  11. 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

  12. Cloning and sequence analysis of H. contortus HC58cDNA gene ...

    African Journals Online (AJOL)

    Phylogenetic analysis revealed close evolutionary proximity of the protein sequence to counterpart sequences in the cathepsin B like proteases, suggesting that HC58cDNA was a member of the papain family. Keywords:Haemonchus contortus, HC58cDNA, cathepsin B like protease, papain family. Kenya Veterinarian Vol.

  13. [A novel vector for construction of a cDNA library].

    Science.gov (United States)

    Fedchenko, V I; Kaloshin, A A; Medvedev, A E

    2010-01-01

    A new original vector pEM-(dT)40(f+) has been prepared. It can be used for cDNA library construction from polyadenylated mRNA, isolated from various sources. The pGEM-(dT)40f(+) is initially transformed into single stranded and then into a linear form and its (dT)40 tail at 3'-end is used as the vector-primer for synthesis of the first strand cDNA. The use of a synthetic oligonucleotide complementary to the vector and recombinant DNA results in vector cyclization and synthesis of the second strand cDNA. This approach significantly simplifies cDNA library construction, it does not require PCR reaction (which can induce artifact mutations in cDNA sequences) and restrictase treatment.

  14. Selective and flexible depletion of problematic sequences from RNA-seq libraries at the cDNA stage.

    Science.gov (United States)

    Archer, Stuart K; Shirokikh, Nikolay E; Preiss, Thomas

    2014-05-26

    A major hurdle to transcriptome profiling by deep-sequencing technologies is that abundant transcripts, such as rRNAs, can overwhelm the libraries, severely reducing transcriptome-wide coverage. Methods for depletion of such unwanted sequences typically require treatment of RNA samples prior to library preparation, are costly and not suited to unusual species and applications. Here we describe Probe-Directed Degradation (PDD), an approach that employs hybridisation to DNA oligonucleotides at the single-stranded cDNA library stage and digestion with Duplex-Specific Nuclease (DSN). Targeting Saccharomyces cerevisiae rRNA sequences in Illumina HiSeq libraries generated by the split adapter method we show that PDD results in efficient removal of rRNA. The probes generate extended zones of depletion as a function of library insert size and the requirements for DSN cleavage. Using intact total RNA as starting material, probes can be spaced at the minimum anticipated library size minus 20 nucleotides to achieve continuous depletion. No off-target bias is detectable when comparing PDD-treated with untreated libraries. We further provide a bioinformatics tool to design suitable PDD probe sets. We find that PDD is a rapid procedure that results in effective and specific depletion of unwanted sequences from deep-sequencing libraries. Because PDD acts at the cDNA stage, handling of fragile RNA samples can be minimised and it should further be feasible to remediate existing libraries. Importantly, PDD preserves the original RNA fragment boundaries as is required for nucleotide-resolution footprinting or base-cleavage studies. Finally, as PDD utilises unmodified DNA oligonucleotides it can provide a low-cost option for large-scale projects, or be flexibly customised to suit different depletion targets, sample types and organisms.

  15. The NF1 gene contains hotspots for L1 endonuclease-dependent de novo insertion.

    Directory of Open Access Journals (Sweden)

    Katharina Wimmer

    2011-11-01

    Full Text Available Long interspersed (L1 and Alu elements are actively amplified in the human genome through retrotransposition of their RNA intermediates by the -100 still retrotranspositionally fully competent L1 elements. Retrotransposition can cause inherited disease if such an element is inserted near or within a functional gene. Using direct cDNA sequencing as the primary assay for comprehensive NF1 mutation analysis, we uncovered in 18 unrelated index patients splicing alterations not readily explained at the genomic level by an underlying point-mutation or deletion. Improved PCR protocols avoiding allelic drop-out of the mutant alleles uncovered insertions of fourteen Alu elements, three L1 elements, and one poly(T stretch to cause these splicing defects. Taken together, the 18 pathogenic L1 endonuclease-mediated de novo insertions represent the largest number of this type of mutations characterized in a single human gene. Our findings show that retrotransposon insertions account for as many as -0.4% of all NF1 mutations. Since altered splicing was the main effect of the inserted elements, the current finding was facilitated by the use of RNA-based mutation analysis protocols, resulting in improved detection compared to gDNA-based approaches. Six different insertions clustered in a relatively small 1.5-kb region (NF1 exons 21(16-23(18 within the 280-kb NF1 gene. Furthermore, three different specific integration sites, one of them located in this cluster region, were each used twice, i.e. NM_000267.3(NF1:c.1642-1_1642 in intron 14(10c, NM_000267.3(NF1:c.2835_2836 in exon 21(16, and NM_000267.3(NF1:c.4319_4320 in exon 33(25. Identification of three loci that each served twice as integration site for independent retrotransposition events as well as 1.5-kb cluster region harboring six independent insertions supports the notion of non-random insertion of retrotransposons in the human genome. Currently, little is known about which features make sites

  16. Marginal adaptation of ceramic inserts after cementation

    NARCIS (Netherlands)

    Ozcan, M; Pfeiffer, P; Nergiz, [No Value

    2002-01-01

    The advantage of using ceramic inserts is to prevent major drawbacks of composite resins such as polymerization shrinkage, wear and microleakage. This in vitro study evaluated the marginal adaptation of two approximal ceramic insert systems after cementation to the cavities opened with ultrasonic

  17. Development of Helios target insertion mechanism

    International Nuclear Information System (INIS)

    Day, R.D.; Cummings, C.E.; Tucker, H.E.

    1979-01-01

    A system for precisely positioning a DT-filled target in the Helios target vacuum chamber is described. The target insertion mechanism (TIM), which is designed to insert either a target or a surrogate sphere into the vacuum chamber through an airlock to prevent loss of vacuum, is discussed in detail and its performance is evaluated

  18. Construction and characterization of a normalized cDNA library.

    Science.gov (United States)

    Soares, M B; Bonaldo, M F; Jelene, P; Su, L; Lawton, L; Efstratiadis, A

    1994-09-27

    We have developed a simple procedure based on reassociation kinetics that can reduce effectively the high variation in abundance among the clones of a cDNA library that represent individual mRNA species. For this normalization, we used as a model system a library of human infant brain cDNAs that were cloned directionally into a phagemid vector and, thus, could be easily converted into single-stranded circles. After controlled primer extension to synthesize a short complementary strand on each circular template, melting and reannealing of the partial duplexes at relatively low C0t, and hydroxyapatite column chromatography, unreassociated circles were recovered from the flow through fraction and electroporated into bacteria, to propagate a normalized library without a requirement for subcloning steps. An evaluation of the extent of normalization has indicated that, from an extreme range of abundance of 4 orders of magnitude in the original library, the frequency of occurrence of any clone examined in the normalized library was brought within the narrow range of only 1 order of magnitude.

  19. Construction and analysis of SSH cDNA library of human vascular endothelial cells related to gastrocarcinoma

    OpenAIRE

    Liu, Yong-Bo; Wei, Zhao-Xia; Li, Li; Li, Hang-Sheng; Chen, Hui; Li, Xiao-Wen

    2003-01-01

    AIM: To construct subtracted cDNA libraries of human vascular endothelial cells (VECs) related to gastrocarcinoma using suppression substractive hybridization (SSH) and to analyze cDNA libraries of gastrocarcinoma and VECs in Cancer Gene Anatomy Project (CGAP) database.

  20. Central Solenoid Insert Technical Specification

    Energy Technology Data Exchange (ETDEWEB)

    Martovetsky, Nicolai N [ORNL; Smirnov, Alexandre [ORNL

    2011-09-01

    The US ITER Project Office (USIPO) is responsible for the ITER central solenoid (CS) contribution to the ITER project. The Central Solenoid Insert (CSI) project will allow ITER validation the appropriate lengths of the conductors to be used in the full-scale CS coils under relevant conditions. The ITER Program plans to build and test a CSI to verify the performance of the CS conductor. The CSI is a one-layer solenoid with an inner diameter of 1.48 m and a height of 4.45 m between electric terminal ends. The coil weight with the terminals is approximately 820 kg without insulation. The major goal of the CSI is to measure the temperature margin of the CS under the ITER direct current (DC) operating conditions, including determining sensitivity to load cycles. Performance of the joints, ramp rate sensitivity, and stability against thermal or electromagnetic disturbances, electrical insulation, losses, and instrumentation are addressed separately and therefore are not major goals in this project. However, losses and joint performance will be tested during the CSI testing campaign. The USIPO will build the CSI that will be tested at the Central Solenoid Model Coil (CSMC) Test Facility at the Japan Atomic Energy Agency (JAEA), Naka, Japan. The industrial vendors (the Suppliers) will report to the USIPO (the Company). All approvals to proceed will be issued by the Company, which in some cases, as specified in this document, will also require the approval of the ITER Organization. Responsibilities and obligations will be covered by respective contracts between the USIPO, called Company interchangeably, and the industrial Prime Contractors, called Suppliers. Different stages of work may be performed by more than one Prime Contractor, as described in this specification. Technical requirements of the contract between the Company and the Prime Contractor will be covered by the Fabrication Specifications developed by the Prime Contractor based on this document and approved by

  1. cDNA amplification by SMART-PCR and suppression subtractive hybridization (SSH)-PCR.

    Science.gov (United States)

    Hillmann, Andrew; Dunne, Eimear; Kenny, Dermot

    2009-01-01

    The comparison of two RNA populations that differ from the effects of a single-independent variable, such as a drug treatment or a specific genetic defect, can identify differences in the abundance of specific transcripts that vary in a population-dependent manner. There are a variety of methods for identifying differentially expressed genes, including microarray, SAGE, qRT-PCR, and DDGE. This protocol describes a potentially less sensitive yet relatively easy and cost-effective alternative that does not require prior knowledge of the transcriptomes under investigation and is particularly applicable when minimal levels of starting material, RNA, are available. RNA input can often be a limiting factor when analyzing RNA from, for example, rigorously purified blood cells. This protocol describes the use of SMART-PCR to amplify cDNA from sub-microgram levels of RNA. The amplified cDNA populations under comparison are then subjected to suppression subtractive hybridization (SSH-PCR), a technique that couples subtractive hybridization with suppression PCR to selectively amplify fragments of differentially expressed genes. The final products are cDNA populations enriched for significantly over-represented transcripts in either of the two input RNA preparations. These cDNA populations may then be cloned to make subtracted cDNA libraries and/or used as probes to screen subtracted cDNA, global cDNA, or genomic DNA libraries.

  2. [cDNA libraries construction and screening in gene expression profiling of disease resistance in wheat].

    Science.gov (United States)

    Luo, Meng; Kong, Xiu-Ying; Liu, Yue; Zhou, Rong-Hua; Jia, Ji-Zeng

    2002-09-01

    A wheat line, Bai Nong 3217/Mardler BC5F4 with resistance to powdery mildew, was used to construct a conventional cDNA library and a suppression subtractive hybridization (SSH) cDNA library from wheat leaves inoculated by Erysiphe graminis DC. Three hundred and eighty-seven non-redundant ESTs from the conventional cDNA library and 760 ESTs from the SSH cDNA library were obtained, and the ESTs similarity analysis using BLASTn and BLASTx were conducted by comparing these ESTs with sequences in GenBank. The results showed that the redundancy of some kinds of genes such as photosynthesis related genes and ribosome related genes was higher in the conventional cDNA library but the varieties and quantities of disease resistance genes were less than those in the SSH cDNA library. The SSH cDNA library was found to have obvious advantages in gene expression profiling of disease resistance such as simple library construction procedure, rich specific DRR (disease-resistance-related) genes and decreased sequencing amount. To acquire genes that were involved in the powdery mildew resistance of wheat, hybridization with high-density dots membranes was used to screen the two libraries. The result showed that the method was relatively simple in operation, and the membranes could be used for many times. But some problems also existed with this screening method. For instance, a large amount of mRNA and radioactive isotope were needed and the hybridization procedure must be repeated several times to obtain stable hybridization results. About 54.1% function-known ESTs in the SSH cDNA library were identified to be DRR genes by screening. There were 247 clones of the SSH cDNA library that had positive signal in the repeated hybridizations with the pathogen uninfected probe. The identified DRR genes distributed in the whole procedure of powdery mildew resistance, but mainly focused on the SAR (systemic of acquired resistance).

  3. Isolation of cDNA clones coding for human tissue factor: primary structure of the protein and cDNA

    International Nuclear Information System (INIS)

    Spicer, E.K.; Horton, R.; Bloem, L.

    1987-01-01

    Tissue factor is a membrane-bound procoagulant protein that activates the extrinsic pathway of blood coagulation in the presence of factor VII and calcium. λ Phage containing the tissue factor gene were isolated from a human placental cDNA library. The amino acid sequence deduced from the nucleotide sequence of the cDNAs indicates that tissue factor is synthesized as a higher molecular weight precursor with a leader sequence of 32 amino acids, while the mature protein is a single polypeptide chain composed of 263 residues. The derived primary structure of tissue factor has been confirmed by comparison to protein and peptide sequence data. The sequence of the mature protein suggests that there are three distinct domains: extracellular, residues 1-219; hydrophobic, residues 220-242; and cytoplasmic, residues 243-263. Three potential N-linked carbohydrate attachment sites occur in the extracellular domain. The amino acid sequence of tissue factor shows no significant homology with the vitamin K-dependent serine proteases, coagulation cofactors, or any other protein in the National Biomedical Research Foundation sequence data bank (Washington, DC)

  4. Generation of cDNA expression libraries enriched for in-frame sequences

    OpenAIRE

    Davis, Claytus A.; Benzer, Seymour

    1997-01-01

    Bacterial cDNA expression libraries are made to reproduce protein sequences present in the mRNA source tissue. However, there is no control over which frame of the cDNA is translated, because translation of the cDNA must be initiated on vector sequence. In a library of nondirectionally cloned cDNAs, only some 8% of the protein sequences produced are expected to be correct. Directional cloning can increase this by a factor of two, but it does not solve the frame problem. We have therefore deve...

  5. Generation of full-length cDNA libraries: focus on plants.

    Science.gov (United States)

    Seki, Motoaki; Kamiya, Asako; Carninci, Piero; Hayashizaki, Yoshihide; Shinozaki, Kazuo

    2009-01-01

    Full-length cDNAs are essential for the correct annotation of transcriptional units and gene products from genomic sequence data and for functional analysis of the genes. Full-length cDNA libraries are very important resources for isolation of the full-length cDNAs. The biotinylated cap trapper method using the trehalose-thermostabilized reverse transcriptase has been developed and has become an efficient method for construction of high-content full-length cDNA libraries. We have constructed full-length cDNA libraries from various plants and animals using this method. The protocol of the method is described in this chapter.

  6. Cloning and sequencing of dolphinfish (Coryphaena hippurus, Coryphaenidae) growth hormone-encoding cDNA.

    Science.gov (United States)

    Peduel, A D; Elizur, A; Knibb, W

    1994-01-01

    The cDNA encoding the preprotein growth hormone from the dolphinfish (Coryphaena hippurus) has been cloned and sequenced. The cDNA was derived by reverse transcription of RNA from the pituitary of a young fish using the method known as Rapid Amplification of cDNA Ends (RACE). An oligonucleotide primer corresponding to the 5' region of Pagrus major and the universal RACE primer enabled amplification using the Polymerase Chain Reaction (PCR). The dolphinfish and yellow-tail, Seriola quineqeradiata, are both members of the sub-order Percoidei (Perciforme) and their GH sequences show a high level of homology.

  7. Insertion profiles of 4 headless compression screws.

    Science.gov (United States)

    Hart, Adam; Harvey, Edward J; Lefebvre, Louis-Philippe; Barthelat, Francois; Rabiei, Reza; Martineau, Paul A

    2013-09-01

    In practice, the surgeon must rely on screw position (insertion depth) and tactile feedback from the screwdriver (insertion torque) to gauge compression. In this study, we identified the relationship between interfragmentary compression and these 2 factors. The Acutrak Standard, Acutrak Mini, Synthes 3.0, and Herbert-Whipple implants were tested using a polyurethane foam scaphoid model. A specialized testing jig simultaneously measured compression force, insertion torque, and insertion depth at half-screw-turn intervals until failure occurred. The peak compression occurs at an insertion depth of -3.1 mm, -2.8 mm, 0.9 mm, and 1.5 mm for the Acutrak Mini, Acutrak Standard, Herbert-Whipple, and Synthes screws respectively (insertion depth is positive when the screw is proud above the bone and negative when buried). The compression and insertion torque at a depth of -2 mm were found to be 113 ± 18 N and 0.348 ± 0.052 Nm for the Acutrak Standard, 104 ± 15 N and 0.175 ± 0.008 Nm for the Acutrak Mini, 78 ± 9 N and 0.245 ± 0.006 Nm for the Herbert-Whipple, and 67 ± 2N, 0.233 ± 0.010 Nm for the Synthes headless compression screws. All 4 screws generated a sizable amount of compression (> 60 N) over a wide range of insertion depths. The compression at the commonly recommended insertion depth of -2 mm was not significantly different between screws; thus, implant selection should not be based on compression profile alone. Conically shaped screws (Acutrak) generated their peak compression when they were fully buried in the foam whereas the shanked screws (Synthes and Herbert-Whipple) reached peak compression before they were fully inserted. Because insertion torque correlated poorly with compression, surgeons should avoid using tactile judgment of torque as a proxy for compression. Knowledge of the insertion profile may improve our understanding of the implants, provide a better basis for comparing screws, and enable the surgeon to optimize compression. Copyright

  8. 5'-end sequences of budding yeast full-length cDNA clones and quality scores - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available List Contact us Budding yeast cDNA sequencing project 5'-end sequences of budding yeast full-length cDNA clones and quality... scores Data detail Data name 5'-end sequences of budding yeast full-length cDNA clones and quality...or-capping method, the sequence quality score generated by the Phred software, and links to SGD, dbEST and U...es. FASTA format. Quality Phred's quality score About This Database Database Desc...g yeast full-length cDNA clones and quality scores - Budding yeast cDNA sequencing project | LSDB Archive ...

  9. Utility Bill Insert for Wastewater Services

    Science.gov (United States)

    Intended for use by wastewater and water supply utilities, one side of the utility bill insert has information for customers that discharge to sanitary sewer systems; the other side is for customers with septic systems.

  10. HB+ inserted into the CMS Solenoid

    CERN Multimedia

    Tejinder S. Virdee, CERN

    2006-01-01

    The first half of the barrel hadron calorimeter (HB+) has been inserted into the superconducting solenoid of CMS, in preparation for the magnet test and cosmic challenge. The operation went smoothly, lasting a couple of days.

  11. Peripherally inserted central catheter - dressing change

    Science.gov (United States)

    PICC - dressing change ... You have a peripherally inserted central catheter (PICC). This is a tube that goes into a vein in your arm. It carries nutrients and medicines into your body. It may also ...

  12. Bulkhead insert for an internal combustion engine

    Science.gov (United States)

    Maki, Clifford E.; Chottiner, Jeffrey Eliot; Williams, Rick L.; Thibault, Mark W.; Ervin, James Douglas; Boileau, James Maurice; McKeough, Bryan

    2017-08-01

    An engine includes a cylinder block defining at least one main bearing bulkhead adjacent to a cylinder, and a crankshaft rotatably housed within the block by a main bearing. A bulkhead insert has a cap portion, and an insert portion provided within the bulkhead. The insert portion has having first and second end regions connected by first and second straps. Each strap having a flanged beam cross section. The first and second ends of the insert portion are configured to connect a main bearing cap column to a cylinder head column. Each of the first and second end regions define at least one protrusion having a surface substantially normal to engine combustion and reactive loads. The cap portion is configured to mate with the first end region at the main bearing cap column and support the main bearing.

  13. Construction and identification of a cDNA library for use in the yeast two-hybrid system from duck embryonic fibroblast cells post-infected with duck enteritis virus.

    Science.gov (United States)

    Gao, Xinghong; Jia, Renyong; Wang, Mingshu; Zhu, Dekang; Chen, Shun; Lin, Meng; Yin, Zhongqiong; Wang, Yin; Chen, Xiaoyue; Cheng, Anchun

    2014-01-01

    To explore and isolate genes related to duck embryonic fibroblast cells (DEFs) post-infected with duck enteritis virus (DEV), a cDNA library was established using SMART (Switching Mechanism At 5' end of the RNA Transcript) technique coupling with a homologous recombination method. The cells were harvested and total RNA was extracted at 48 h post infection. Then the mRNAs were purified and reverse transcribed to first-strand cDNAs using oligo (dT) primers (CDS III). Subsequently, long distance-PCR was performed, the double-stranded cDNAs were purified, and a transformation assay was carried out in that order. Eventually, a high qualitative library was successfully established according to an evaluation on quality. The transformation efficiency was about 2.33 × 10(6) transformants/4.34 μg pGADT7-Rec (>1.0 × 10(6)). The cell density of the library was 1.75 × 10(9) cells/mL (>2 × 10(7) cells/mL). The titer of the primary cDNA library and amplified cDNA library was 6.75 × 10(5) and 2.33 × 10(7) CFU/mL respectively. The numbers for the primary cDNA library and amplified cDNA library were 1.01 × 10(7) and 1.14 × 10(9), respectively, and the recombinant rate was 97.14 %. The sequence results of 27 randomly picked independent clones revealed the insert ranged from 0.323 to 2.017 kb with an average insert size of 0.807 kb. Full-length transcripts of DEV-CHv LORF3, UL26 and UL35 genes were acquired through sequence similarity analysis from the non-redundant nucleic acid or protein database. Five polyA sites were identified in the DEV-CHv genome. Also, a new transcript of 668 bp was found between the IRS gene and US1 gene of the DEV-CHv genome. Thus, we concluded that the constructed cDNA library will be a useful tool in proteomic analysis of interactions between the DEV and host DEFs, and discovery of biomarkers studies on the mechanism of DEV and subsequently exploitation original vaccines and antiviral drugs to prevent or cure diseases.

  14. 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.

  15. Mouse tetranectin: cDNA sequence, tissue-specific expression, and chromosomal mapping

    DEFF Research Database (Denmark)

    Ibaraki, K; Kozak, C A; Wewer, U M

    1995-01-01

    regulation, mouse tetranectin cDNA was cloned from a 16-day-old mouse embryo library. Sequence analysis revealed a 992-bp cDNA with an open reading frame of 606 bp, which is identical in length to the human tetranectin cDNA. The deduced amino acid sequence showed high homology to the human cDNA with 76...... in human. Although additional minor bands of 1.5 and 3.3 kb were found in Northern blots, RT-PCR (reverse transcription polymerase chain reaction) analysis failed to provide evidence that these minor bands are products of the tetranectin gene. Finally, the genetic map location for this gene, Tna...

  16. Cloning and sequencing of complete τ-crystallin cDNA from ...

    Indian Academy of Sciences (India)

    Unknown

    length τ-crystallin cDNA from crocodilian lens and α-enolase from other tissues. ... human (Acc. No. NM_001428). The sequences were used to construct a phylogenetic tree depicting gene lineage, using the clustering program DNAML.

  17. Construction and characterization of a cDNA expression library from the endangered Hu sheep.

    Science.gov (United States)

    Hu, P-F; Li, X-C; Liu, H-K; Guan, W-J; Ma, Y-H

    2014-10-31

    Hu sheep is one of the most important species in China; it is also listed as one of the 78 nationally protected domestic animals by the Chinese government in 2000. The construction of cDNA expression library of Hu sheep is of great significance for protecting individual genomes, generating transgenic sheep, and conducting clinical research using cDNA from Hu sheep. In this study, the total RNA from the ear tissue of Hu sheep was extracted, and a cDNA expression library was constructed using the SMART(TM) technique. The titer of amplified cDNA library was 1.09 x 10(10) PFU/mL, the rate of recombination was above 91.6%, and the average size of fragments was 1.1 kb. This study has an important significance for the preservation of Hu sheep resources at the genome level.

  18. Mouse tetranectin: cDNA sequence, tissue-specific expression, and chromosomal mapping

    DEFF Research Database (Denmark)

    Ibaraki, K; Kozak, C A; Wewer, U M

    1995-01-01

    regulation, mouse tetranectin cDNA was cloned from a 16-day-old mouse embryo library. Sequence analysis revealed a 992-bp cDNA with an open reading frame of 606 bp, which is identical in length to the human tetranectin cDNA. The deduced amino acid sequence showed high homology to the human cDNA with 76......(s) of tetranectin. The sequence analysis revealed a difference in both sequence and size of the noncoding regions between mouse and human cDNAs. Northern analysis of the various tissues from mouse, rat, and cow showed the major transcript(s) to be approximately 1 kb, which is similar in size to that observed...

  19. cDNA - ASTRA | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available ing/transcriptional initiation regionss About This Database Database Description Download License Update History of This Database Site Policy | Contact Us cDNA - ASTRA | LSDB Archive ...

  20. Construction of primary and subtracted cDNA libraries from early embryos.

    Science.gov (United States)

    Rothstein, J L; Johnson, D; Jessee, J; Skowronski, J; DeLoia, J A; Solter, D; Knowles, B B

    1993-01-01

    By modifying current cDNA cloning and electroporation methods, large and representative murine cDNA libraries were synthesized from 10 to 100 ng mRNA isolated from unfertilized egg and preimplantation mouse embryos. High cloning efficiency is essential for complete representation of genes expressed in egg and preimplantation embryos and for the isolation of stage-specific genes using subtractive hybridization. Because the mouse embryo contains no more than 50 pg of poly(A)+ mRNA at any stage of preimplantation development, approximately 5000-10,000 embryos are required to obtain enough mRNA to synthesize libraries using current methods. To obtain a representative library that also includes rare transcripts, the size of the library should be at least 10(6) clones. The average percent conversion of mRNA to single-stranded cDNA was 20-40%, so that a cloning efficiency of nearly 2 x 10(8) cfu/microgram cDNA is required for such a cDNA library. No previous methods have provided directional cloning of cDNA into plasmids with these high efficiencies. The advent of electroporation methods for the introduction of nucleic acids into bacteria has made possible the use of standard plasmid vectors for high-efficiency cDNA cloning. Plasmid vectors are currently available that can accommodate the directional cloning of cDNA such that T7 and T3 RNA polymerase promoter sequences can be used to generate sense and anti-sense transcripts for subtractive hybridization and riboprobe synthesis. The cDNA libraries we derived using this methodology are a reusable and abundant source of genetic information about the control of preimplantation development. Specialized subtractive cDNA libraries enriched for genes expressed exclusively at a predetermined time in development give access to genes expressed in a stage-specific manner. The ability to construct new cDNA libraries from limited amounts of starting material ensures the provision of new and important resources for the identification

  1. Radioactive cDNA microarray (II): Gene expression profiling of antidepressant treatment by human cDNA microarray

    International Nuclear Information System (INIS)

    Lee, Ji Hye; Kang, Rhee Hun; Ham, Byung Joo; Lee, Min Su; Shin, Kyung Ho; Choe, Jae Gol; Kim, Meyoung Kon

    2003-01-01

    Major depressive disorder is a prevalent psychiatric disorder in primary care, associated with impaired patient functioning and well-being. Fluoxetine is a selective serotonin-reuptake inhibitors (SSRIs) and is a commonly prescribed antidepressant compound. Its action is primarily attributed to selective inhibition of the reuptake of serotonin (5-hydroxytryptamine) in the central nervous system. Objectives ; the aims of this study were two-fold: (1) to determine the usefulness for investigation of the transcription profiles in depression patients, and (2) to assess the differences in gene expression profiles between positive response group and negative response groups by fluoxetine treatment. This study included 53 patients with major depression (26 in positive response group with antidepressant treatment, 27 in negative response group with antidepressant treatment), and 53 healthy controls. To examine the difference of gene expression profile in depression patients, radioactive complementary DNA microarrays were used to evaluate changes in the expression of 1,152 genes in total. Using 33p-labeled probes, this method provided highly sensitive gene expression profiles including brain receptors, drug metabolism, and cellular signaling. Gene transcription profiles were classified into several categories in accordance with the antidepressant gene-regulation. The gene profiles were significantly up-(22 genes) and down-(16 genes) regulated in the positive response group when compared to the control group. Also, in the negative response group, 35 genes were up-regulated and 8 genes were down-regulated when compared to the control group. Consequently, we demonstrated that radioactive human cDNA microarray is highly likely to be an efficient technology for evaluating the gene regulation of antidepressants, such as selective serotonin-reuptake inhibitors (SSRIs), by using high-throughput biotechnology

  2. Radioactive cDNA microarray (II): Gene expression profiling of antidepressant treatment by human cDNA microarray

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Ji Hye; Kang, Rhee Hun; Ham, Byung Joo; Lee, Min Su; Shin, Kyung Ho; Choe, Jae Gol; Kim, Meyoung Kon [College of Medicine, Univ. of Korea, Seoul (Korea, Republic of)

    2003-07-01

    Major depressive disorder is a prevalent psychiatric disorder in primary care, associated with impaired patient functioning and well-being. Fluoxetine is a selective serotonin-reuptake inhibitors (SSRIs) and is a commonly prescribed antidepressant compound. Its action is primarily attributed to selective inhibition of the reuptake of serotonin (5-hydroxytryptamine) in the central nervous system. Objectives ; the aims of this study were two-fold: (1) to determine the usefulness for investigation of the transcription profiles in depression patients, and (2) to assess the differences in gene expression profiles between positive response group and negative response groups by fluoxetine treatment. This study included 53 patients with major depression (26 in positive response group with antidepressant treatment, 27 in negative response group with antidepressant treatment), and 53 healthy controls. To examine the difference of gene expression profile in depression patients, radioactive complementary DNA microarrays were used to evaluate changes in the expression of 1,152 genes in total. Using 33p-labeled probes, this method provided highly sensitive gene expression profiles including brain receptors, drug metabolism, and cellular signaling. Gene transcription profiles were classified into several categories in accordance with the antidepressant gene-regulation. The gene profiles were significantly up-(22 genes) and down-(16 genes) regulated in the positive response group when compared to the control group. Also, in the negative response group, 35 genes were up-regulated and 8 genes were down-regulated when compared to the control group. Consequently, we demonstrated that radioactive human cDNA microarray is highly likely to be an efficient technology for evaluating the gene regulation of antidepressants, such as selective serotonin-reuptake inhibitors (SSRIs), by using high-throughput biotechnology.

  3. cDNA fingerprinting of osteoprogenitor cells to isolate differentiation stage-specific genes.

    OpenAIRE

    Candeliere, G A; Rao, Y; Floh, A; Sandler, S D; Aubin, J E

    1999-01-01

    A cDNA fingerprinting strategy was developed to identify genes based on their differential expression pattern during osteoblast development. Preliminary biological and molecular staging of cDNA pools prepared by global amplification PCR allowed discrim-inating choices to be made in selection of expressed sequence tags (ESTs) to be isolated. Sequencing of selected ESTs confirmed that both known and novel genes can be isolated from any developmental stage of interest, e.g. from primitive progen...

  4. Construction and characteristics of 3-end enriched cDNA library from individual embryos of cattle.

    Science.gov (United States)

    Long, Jian-Er; He, Li-Qiang; Cai, Xia; Ren, Zhao-Rui; Huang, Shu-Zhen; Zeng, Yi-Tao

    2006-11-01

    To analyze stage-specific gene expression profiles of pre-implantation embryos and evaluate potential viability, techniques were adapted to generate 3-end enriched cDNA libraries from individual embryos of cattle based on RT-PCR methodology. The reproducibility of constructing a cDNA library was tested by five independent PCR experiments with specific primers for the presence of several rare genes such as DNMT1 (DNA methylation transferase 1), DNMT2, DNMT3A, Oct-4/3 (octmer-binding transcription factor), IFN-iota, IGF-2r (insulin like growth factor 2 receptor), and the housekeeping genes, H2A and beta-actin. Results indicated repeatability and that a proportion of expressed genes in the cDNA library from an individual embryo was not affected by limited PCR amplification. From the cDNA library, 134 clones were randomly selected for sequencing and showed that structure related elements accounted for 33.5% of transcripts and the energy- and metabolism-related genes were also an important component being 11.9% in the cDNA library. Approximately 14% of genes in the library were functionally unknown including greater than 5% of genes that were likely novel because there was no identity in Genbank. The frequency of structure-related genes such as beta-actin and ribosomal proteins in the cDNA library corresponded to other reports and suggested that the cDNA library constructed by RT-PCR might be proportional to the mRNA populations. The cDNA libraries constructed from different stage embryos will provide a powerful tool to explore novel genes relevant to embryogenesis, determine the profiling of stage-specific gene expression, and evaluate the potential viability of embryos.

  5. 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...

  6. Construction of an infectious cDNA clone of foot-and-mouth disease ...

    Indian Academy of Sciences (India)

    A stable, full-length cDNA clone of FMDV type O1BFS 1860 preceded by a bacteriophage T7 polymerase promoter was assembled in a plasmid vector pGEMR-7Zf(–). An ∼8.2 kb PCR product was amplified from the cDNA clone and a full-length RNA was generated from it by in vitro transcription. Transfection of BHK-21 ...

  7. 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 ...

  8. Construction of a Full-Length Enriched cDNA Library and Preliminary Analysis of Expressed Sequence Tags from Bengal Tiger Panthera tigris tigris

    Directory of Open Access Journals (Sweden)

    Changqing Liu

    2013-05-01

    Full Text Available In this study, a full-length enriched cDNA library was successfully constructed from Bengal tiger, Panthera tigris tigris, the most well-known wild Animal. Total RNA was extracted from cultured Bengal tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.28 × 106 pfu/mL and 1.56 × 109 pfu/mL respectively. The percentage of recombinants from unamplified library was 90.2% and average length of exogenous inserts was 0.98 kb. A total of 212 individual ESTs with sizes ranging from 356 to 1108 bps were then analyzed. The BLASTX score revealed that 48.1% of the sequences were classified as a strong match, 45.3% as nominal and 6.6% as a weak match. Among the ESTs with known putative function, 26.4% ESTs were found to be related to all kinds of metabolisms, 19.3% ESTs to information storage and processing, 11.3% ESTs to posttranslational modification, protein turnover, chaperones, 11.3% ESTs to transport, 9.9% ESTs to signal transducer/cell communication, 9.0% ESTs to structure protein, 3.8% ESTs to cell cycle, and only 6.6% ESTs classified as novel genes. By EST sequencing, a full-length gene coding ferritin was identified and characterized. The recombinant plasmid pET32a-TAT-Ferritin was constructed, coded for the TAT-Ferritin fusion protein with two 6× His-tags in N and C-terminal. After BCA assay, the concentration of soluble Trx-TAT-Ferritin recombinant protein was 2.32 ± 0.12 mg/mL. These results demonstrated that the reliability and representativeness of the cDNA library attained to the requirements of a standard cDNA library. This library provided a useful platform for the functional genome and transcriptome research of Bengal tigers.

  9. Construction of a full-length enriched cDNA library and preliminary analysis of expressed sequence tags from Bengal Tiger Panthera tigris tigris.

    Science.gov (United States)

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

    2013-05-24

    In this study, a full-length enriched cDNA library was successfully constructed from Bengal tiger, Panthera tigris tigris, the most well-known wild Animal. Total RNA was extracted from cultured Bengal tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.28 × 106 pfu/mL and 1.56 × 109 pfu/mL respectively. The percentage of recombinants from unamplified library was 90.2% and average length of exogenous inserts was 0.98 kb. A total of 212 individual ESTs with sizes ranging from 356 to 1108 bps were then analyzed. The BLASTX score revealed that 48.1% of the sequences were classified as a strong match, 45.3% as nominal and 6.6% as a weak match. Among the ESTs with known putative function, 26.4% ESTs were found to be related to all kinds of metabolisms, 19.3% ESTs to information storage and processing, 11.3% ESTs to posttranslational modification, protein turnover, chaperones, 11.3% ESTs to transport, 9.9% ESTs to signal transducer/cell communication, 9.0% ESTs to structure protein, 3.8% ESTs to cell cycle, and only 6.6% ESTs classified as novel genes. By EST sequencing, a full-length gene coding ferritin was identified and characterized. The recombinant plasmid pET32a-TAT-Ferritin was constructed, coded for the TAT-Ferritin fusion protein with two 6× His-tags in N and C-terminal. After BCA assay, the concentration of soluble Trx-TAT-Ferritin recombinant protein was 2.32 ± 0.12 mg/mL. These results demonstrated that the reliability and representativeness of the cDNA library attained to the requirements of a standard cDNA library. This library provided a useful platform for the functional genome and transcriptome research of Bengal tigers.

  10. Construction of a Full-Length Enriched cDNA Library and Preliminary Analysis of Expressed Sequence Tags from Bengal Tiger Panthera tigris tigris

    Science.gov (United States)

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

    2013-01-01

    In this study, a full-length enriched cDNA library was successfully constructed from Bengal tiger, Panthera tigris tigris, the most well-known wild Animal. Total RNA was extracted from cultured Bengal tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.28 × 106 pfu/mL and 1.56 × 109 pfu/mL respectively. The percentage of recombinants from unamplified library was 90.2% and average length of exogenous inserts was 0.98 kb. A total of 212 individual ESTs with sizes ranging from 356 to 1108 bps were then analyzed. The BLASTX score revealed that 48.1% of the sequences were classified as a strong match, 45.3% as nominal and 6.6% as a weak match. Among the ESTs with known putative function, 26.4% ESTs were found to be related to all kinds of metabolisms, 19.3% ESTs to information storage and processing, 11.3% ESTs to posttranslational modification, protein turnover, chaperones, 11.3% ESTs to transport, 9.9% ESTs to signal transducer/cell communication, 9.0% ESTs to structure protein, 3.8% ESTs to cell cycle, and only 6.6% ESTs classified as novel genes. By EST sequencing, a full-length gene coding ferritin was identified and characterized. The recombinant plasmid pET32a-TAT-Ferritin was constructed, coded for the TAT-Ferritin fusion protein with two 6× His-tags in N and C-terminal. After BCA assay, the concentration of soluble Trx-TAT-Ferritin recombinant protein was 2.32 ± 0.12 mg/mL. These results demonstrated that the reliability and representativeness of the cDNA library attained to the requirements of a standard cDNA library. This library provided a useful platform for the functional genome and transcriptome research of Bengal tigers. PMID:23708105

  11. Trehalose as a good candidate for enriching full-length cDNAs in cDNA library construction.

    Science.gov (United States)

    Chen, Lei; Cao, Lixue; Zhou, Longhai; Jing, Yudong; Chen, Zuozhou; Deng, Cheng; Shen, Yu; Chen, Liangbiao

    2007-01-10

    It has been reported that the disaccharide trehalose is capable of increasing the thermostability and thermoactivity of reverse transcriptase, and therefore improving the length of cDNA synthesis. However, no test has been done on how the disaccharide trehalose performs in the context of the entire cDNA synthesis processes, or whether it can seamlessly integrate into the commercially available cDNA synthesis kit. In this report, we optimized a protocol to incorporate trehalose in the Stratagene's cDNA library construction kit in order to demonstrate great improvement in cDNA's length (average length of 1.8 kb in the trehalose group versus 1.0 kb in the control). Sequence analysis of the cDNA clones showed that the addition of trehalose did not increase the error rate of the RT products but greatly increase the quantity of full-length in cDNA library.

  12. [Construction of suppression subtracted cDNA library of deltamethrin-resistant Aedes albopictus].

    Science.gov (United States)

    Wu, Jia-hong; Zhao, Tong-yan; Dong, Yan-de

    2006-08-01

    To construct the suppression subtracted cDNA library of deltamethrin-resistant Aedes albopictus. Total RNA was extracted from the deltamethrin-resistant (R-lab) and -sensitive (S-lab) isolates, mRNA was obtained after purification. Double stranded cDNAs were synthesized after reverse transcription. Two subtractions were performed by suppression subtractive hybridization with S-lab as tester and R-lab as driver or S-lab as driver and R-lab as tester. Enriched different expressed cDNA was cloned into pMD18-T vector to construct subtractive libraries. The subtracted cDNA libraries contained 580 and 477 positive clones respectively. The PCR results of 150 clones picked randomly from each library showed that the positive ratio of constructed cDNA libraries was 93%, with a length of cDNA fragments ranged from 150bp to 750bp. The suppression subtracted cDNA library of deltamethrin-resistant Ae. albopictus is constructed.

  13. [cDNA library constructing and specific antigen expression of Streptomyces thermohydroscopicus].

    Science.gov (United States)

    Xu, Lei; Wang, Ling-ling; Liu, Shuo; Ling, Yuan; Ma, Lie; Wang, Qun; Zhang, Li-jiao; He, Xiao-yu; Zhao, Ming-jing; Wang, Xiao-ge

    2012-03-01

    To construct a cDNA library from Streptomyces thermohydroscopicus and screen genes with virulence, obtain the recombinant fusion virulence proteins by prokaryotic expression system. The Streptomyces thermohydroscopicus cDNA library was constructed by switching mechanism at 5'end of RNA transcript approach. A total of 1020 clones randomly selected from the cDNA library were sequenced and these expressed sequence tags (EST) were further analyzed for the screen of antigen-specific genes. The two candidate genes were subcloned into expression vector pET-28a. The recombinants were transformed into BL2 and proteins were expressed by the induction of isopropyl-β-D-1-thiogalactopyranoside (IPTG). A high-quality cDNA library from Streptomyces thermohydroscopicus was constructed and a set of 978 valid sequences were obtained. Clustering and assembly of these cDNA sequences resulted in 347 unique genes, among which 2 potential antigen-specific genes were highly allied with outer membrane lipoprotein (51%) and transferring-binding protein B (42%) from Actinobacillus pleuropneumoniae serotype (APP). The open reading frame (ORF) of the two candidate genes are 1554 bp and 726 bp, which coded two peptides with 517 and 241 amino acids, respectively. The molecular weights of the recombinant fusion proteins were 63 000 and 30 000. The cDNA library of Streptomyces thermohydroscopicus reached the quality requirement of gene library. EST database in the library would greatly facilitate further screening of virulence genes.

  14. 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

  15. Cloning of a cDNA encoding the human cation-dependent mannose 6-phosphate-specific receptor

    International Nuclear Information System (INIS)

    Pohlmann, R.; Nagel, G.; Schmidt, B.

    1987-01-01

    Complementary DNA clones for the human cation-dependent mannose 6-phosphate-specific receptor have been isolated from a human placenta library in λgt11. The nucleotide sequence of the 2463-base-pair cDNA insert includes a 145-base-pair 5' untranslated region, an open reading frame of 831 base pairs corresponding to 277 amino acids, and a 1487-base-pair 3' untranslated region. The deduced amino acid sequence is colinear with that determined by amino acid sequencing of the N-terminus peptide (41 residues) and nine tryptic peptides (93 additional residues). The receptor is synthesized as a precursor with a signal peptide of 20 amino acids. The hydrophobicity profile of the receptor indicates a single membrane-spanning domain, which separates an N-terminal region containing five potential N-glycosylation sites from a C-terminal region lacking N-glycosylation sites. Thus the N-terminal (M/sub r/ = 18,299) and C-terminal (M/sub r/ ≤ 7648) segments of the mature receptor are assumed to be exposed to the extracytosolic and cytosolic sides of the membrane, respectively. Analysis of a panel of somatic cell (mouse-human) hybrids shows that the gene for the receptor is located on human chromosome 12

  16. Systematic mining of salt-tolerant genes in halophyte-Zoysia matrella through cDNA expression library screening.

    Science.gov (United States)

    Chen, Yu; Zong, Junqin; Tan, Zhiqun; Li, Lanlan; Hu, Baoyun; Chen, Chuanming; Chen, Jingbo; Liu, Jianxiu

    2015-04-01

    Though a large number of salt-tolerant genes were identified from Glycophyte in previous study, genes involved in salt-tolerance of halophyte were scarcely studied. In this report, an important halophyte turfgrass, Zoysia matrella, was used for systematic excavation of salt-tolerant genes using full-length cDNA expression library in yeast. Adopting the Gateway-compatible vector system, a high quality entry library was constructed, containing 3 × 10(6) clones with an average inserted fragments length of 1.64 kb representing a 100% full-length rate. The yeast expression library was screened in a salt-sensitive yeast mutant. The screening yielded dozens of salt-tolerant clones harboring 16 candidate salt-tolerant genes. Under salt-stress condition, these 16 genes exhibited different transcription levels. According to the results, we concluded that the salt-tolerance of Z. matrella might result from known genes involved in ion regulation, osmotic adjustment, as well as unknown pathway associated with protein folding and modification, RNA metabolism, and mitochondrial membrane translocase, etc. In addition, these results shall provide new insight for the future researches with respect to salt-tolerance. Crown Copyright © 2015. Published by Elsevier Masson SAS. All rights reserved.

  17. Construction of the subtracted cDNA library of striatal neurons treated with long-term morphine.

    Science.gov (United States)

    Bai, Bo; Liu, Hai-qing; Chen, Jing; Li, Ya-lin; Du, Hui; Lu, Hai; Yu, Peng-li

    2011-03-01

    To construct a morphine tolerance model in primarily cultured striatal neurons, and screen the differentially expressed genes in this model using suppression subtractive hybridization (SSH). Sbtracted cDNA libraries were constructed using SSH from normal primarily cultured striatal neurons and long-term morphine treated striatal neurons (10-5 mol/L for 72 hours). To check reliability of the cell culture model, RT-PCR was performed to detect the cAMP-responsive element-binding protein (CREB) mRNA expression. The subtracted clones were prescreened by PCR. The clones containing inserted fragments from forward libraries were sequenced and submitted to GenBank for homology analysis. And the expression levels of genes of interest were confirmed by RT-PCR. Results CREB mRNA expression showed a significant increase in morphine treated striatal neurons (62.85 ± 1.98) compared with normal striatal neurons (28.43 ± 1.46, P library of striatal neurons treated with long-term morphine is constructed. Mtch1 and Akt1 might be the candidate genes for the development of morphine tolerance.

  18. Retrovirus-mediated gene transfer of a human c-fos cDNA into mouse bone marrow stromal cells.

    Science.gov (United States)

    Roux, P; Verrier, B; Klein, B; Niccolino, M; Marty, L; Alexandre, C; Piechaczyk, M

    1991-11-01

    A cDNA encoding a complete human c-fos protein was isolated and inserted into two different murine MoMuLV-derived recombinant retroviruses allowing expression of c-fos protein in different cell types. One c-fos-expressing retrovirus, chosen for its ability to express high levels of proteins in fibroblast-like cells, was shown to potentiate long-term cultures of mouse bone marrow stromal cells in vitro and therefore constitutes a potential tool for immortalizing such cells. Moreover, when tested in an in vitro differentiation assay, stromal cells constitutively expressing c-fos favor the granulocyte differentiation of hematopoietic precursors. Interestingly, retroviruses expressing v-src and v-abl oncogenes, included as controls in our experiments, do not produce any detectable effects, whereas those expressing polyoma virus middle T antigen facilitate long-term growth in vitro of stromal cells that favor the macrophage differentiation pathway of bone marrow stem cells. Our observation supports the idea that constitutive expression of some oncogenes, including c-fos and polyoma virus middle T antigen, may influence cytokine production by bone marrow stromal cells.

  19. Insertion site assessment of peripherally inserted central catheters: Inter-observer agreement between nurses and inpatients.

    Science.gov (United States)

    Webster, Joan; Northfield, Sarah; Larsen, Emily N; Marsh, Nicole; Rickard, Claire M; Chan, Raymond J

    2018-03-01

    Many patients are discharged from hospital with a peripherally inserted central catheter in place. Monitoring the peripherally inserted central catheter insertion site for clinical and research purposes is important for identifying complications, but the extent to which patients can reliably report the condition of their catheter insertion site is uncertain. The aim of this study was to assess the inter-observer agreement between nurses and patients when assessing a peripherally inserted central catheter site. The study was based on inpatients who were enrolled in a single-centre, randomised controlled trial comparing four different dressing and securement devices for peripherally inserted central catheter sites. A seven-item peripherally inserted central catheter site assessment tool, containing questions about the condition of the dressing and the insertion site, was developed. Assessment was conducted once by the research nurse and, within a few minutes, independently by the patient. Proportions of agreement and Cohen's kappa were calculated. In total, 73 patients agreed to participate. Overall, percentage agreement ranged from 83% to 100% (kappa = .65-.82). For important clinical signs (redness, swelling, ooze, pus and tracking), there were high levels of percentage agreement (99%-100%). The high level of agreement between nurse/patient pairs make the instrument useful for assessing peripherally inserted central catheter-associated signs of localised infection, allergic or irritant dermatitis or dressing dislodgement in a community setting.

  20. Efficacy Testing of H56 cDNA Tattoo Immunization against Tuberculosis in a Mouse Model.

    Science.gov (United States)

    Platteel, Anouk C M; Nieuwenhuizen, Natalie E; Domaszewska, Teresa; Schürer, Stefanie; Zedler, Ulrike; Brinkmann, Volker; Sijts, Alice J A M; Kaufmann, Stefan H E

    2017-01-01

    Tuberculosis (TB), caused by Mycobacterium tuberculosis ( Mtb ), remains a global threat. The only approved vaccine against TB, Mycobacterium bovis bacillus Calmette-Guérin (BCG), provides insufficient protection and, being a live vaccine, can cause disseminated disease in immunocompromised individuals. Previously, we found that intradermal cDNA tattoo immunization with cDNA of tetanus toxoid fragment C domain 1 fused to cDNA of the fusion protein H56, comprising the Mtb antigens Ag85B, ESAT-6, and Rv2660c, induced antigen-specific CD8 + T cell responses in vivo . As cDNA tattoo immunization would be safer than a live vaccine in immunocompromised patients, we tested the protective efficacy of intradermal tattoo immunization against TB with H56 cDNA, as well as with H56_E, a construct optimized for epitope processing in a mouse model. As Mtb antigens can be used in combination with BCG to boost immune responses, we also tested the protective efficacy of heterologous prime-boost, using dermal tattoo immunization with H56_E cDNA to boost BCG immunization in mice. Dermal H56 and H56_E cDNA immunization induced H56-specific CD4 + and CD8 + T cell responses and Ag85B-specific IgG antibodies, but did not reduce bacterial loads, although immunization with H56_E ameliorated lung pathology. Both subcutaneous and intradermal immunization with BCG resulted in broad cellular immune responses, with increased frequencies of CD4 + T effector memory cells, T follicular helper cells, and germinal center B cells, and resulted in reduced bacterial loads and lung pathology. Heterologous vaccination with BCG/H56_E cDNA induced increased H56-specific CD4 + and CD8 + T cell cytokine responses compared to vaccination with BCG alone, and lung pathology was significantly decreased in BCG/H56_E cDNA immunized mice compared to unvaccinated controls. However, bacterial loads were not decreased after heterologous vaccination compared to BCG alone. CD4 + T cells responding to Ag85B- and ESAT-6

  1. Hamstring tendons insertion - an anatomical study

    Directory of Open Access Journals (Sweden)

    Cristiano Antonio Grassi

    2013-09-01

    Full Text Available OBJECTIVE: To study the anatomy of the hamstring tendons insertion and anatomical rela-tionships. METHODS: Ten cadaver knees with medial and anterior intact structures were selected. The dissection was performed from anteromedial access to exposure of the insertion of the flexor tendons (FT, tibial plateau (TP and tibial tuberosity (TT. A needle of 40 × 12 and a caliper were used to measure the distance of the tibial plateau of the knee flexor tendons insertion at 15 mm from the medial border of the patellar tendon and tibial tuberosity to the insertion of the flexor tendons of the knee. The angle between tibial plateau and the insertion of the flexor tendons of the knee (A-TP-FT was calculated using Image Pro Plus software. RESULTS: The mean distance TP-FT was 41 ± 4.6 mm. The distance between the TT-FT was 6.88 ± 1 mm. The (A-TP-FT was 20.3 ± 4.9°. CONCLUSION: In the anterior tibial flexor tendons are about 40 mm from the plateau with an average of 20°.

  2. Test manufacture of a canister insert

    International Nuclear Information System (INIS)

    Raiko, H.

    2004-11-01

    This report describes the insert-manufacturing test of a disposal canister for spent nuclear fuel that was made by Metso Paper Oy, Jyvaeskylae Foundry, in 2003 on contract for Posiva Oy. The test manufacture was a part of the co-operation development programme of encapsulation technology between SKB AB and Posiva Oy. Insert casting was specified according to the current manufacturing specifications of SKB. The canister insert was of BWR-type with integral bottom. This was the first trial manufacture of this type of insert in Finland and, in total, the second test manufacture of insert by Metso Paper. The result fulfilled all the requirements but the material mechanical properties and metallurgical structure of the cast material. The measured tensile strength, ultimate strength and elongation at rupture were lower than specified. The reason for this was revealed in the metallurgical investigation of the cast material. The nodulizing of the graphite was not occurred during the casting process according to the requirements. (orig.)

  3. cDNA sequencing improves the detection of P53 missense mutations in colorectal cancer

    International Nuclear Information System (INIS)

    Szybka, Malgorzata; Kordek, Radzislaw; Zakrzewska, Magdalena; Rieske, Piotr; Pasz-Walczak, Grazyna; Kulczycka-Wojdala, Dominika; Zawlik, Izabela; Stawski, Robert; Jesionek-Kupnicka, Dorota; Liberski, Pawel P

    2009-01-01

    Recently published data showed discrepancies beteween P53 cDNA and DNA sequencing in glioblastomas. We hypothesised that similar discrepancies may be observed in other human cancers. To this end, we analyzed 23 colorectal cancers for P53 mutations and gene expression using both DNA and cDNA sequencing, real-time PCR and immunohistochemistry. We found P53 gene mutations in 16 cases (15 missense and 1 nonsense). Two of the 15 cases with missense mutations showed alterations based only on cDNA, and not DNA sequencing. Moreover, in 6 of the 15 cases with a cDNA mutation those mutations were difficult to detect in the DNA sequencing, so the results of DNA analysis alone could be misinterpreted if the cDNA sequencing results had not also been available. In all those 15 cases, we observed a higher ratio of the mutated to the wild type template by cDNA analysis, but not by the DNA analysis. Interestingly, a similar overexpression of P53 mRNA was present in samples with and without P53 mutations. In terms of colorectal cancer, those discrepancies might be explained under three conditions: 1, overexpression of mutated P53 mRNA in cancer cells as compared with normal cells; 2, a higher content of cells without P53 mutation (normal cells and cells showing K-RAS and/or APC but not P53 mutation) in samples presenting P53 mutation; 3, heterozygous or hemizygous mutations of P53 gene. Additionally, for heterozygous mutations unknown mechanism(s) causing selective overproduction of mutated allele should also be considered. Our data offer new clues for studying discrepancy in P53 cDNA and DNA sequencing analysis

  4. [Construction of cDNA expression library of unfed female Haemaphysalis longicornis and immuno-screening].

    Science.gov (United States)

    Chai, Hui-ping; Liu, Guang-yuan; Zhang, Lin; Gong, Zhen-li; Xie, Jun-ren; Tian, Zhan-cheng; Wang, Lu; Jia, Ning

    2009-02-28

    To construct a cDNA expression library from unfed female tick Haemaphysalis longicornis for screening and cloning potential antigenic genes. Total RNA was isolated from unfed female ticks, mRNA was purified and a library of oligo (dT) -primed cDNA with added directional EcoR I /Hind III linkers was constructed from the purified mRNA. The constructed cDNA was ligated to the EcoR I /Hind III arms of the lambda SCREEN vector. Pure phage stocks were harvested by plaque purification and converted to plasmid subclones by plating phage on host strain BM25.8. Recombinant plasmids that were subcloned to E. coli BM25.8 were isolated and transformed into E. coli JM109. Recombinant plasmids abstracted from JM109 were identified by PCR and sequencing. The recombinant phage DNA was packaged by using phage-marker packaging extracts, resulting in a primary cDNA library with a size of 1.8 x 10(6) pfu. Data showed 100% of the library were recombinant and the titer of the amplified library was 2.4 x 10(9) pfu/ml. Forty-two clones of encoding immunodominant antigens were obtained from the cDNA library. Sequence analysis revealed 12 unique cDNA sequences and the encoded putative proteins showed similarities to H. longicornis tropomyosin mRNA, Rhipicephalus annulatus unknown larval protein mRNA, chromosome 2R of Drosophila melanogaster, mitochondrial DNA of H. flava, clones HqL09 unkown mRNA and Hq05 mRNA of H. qinghaiensis, and myosin alkali light chain protein mRNA. The cDNA expression library from unfed female H. longicornis was successfully constructed and screening of protective genes may provide candidate antigens of the tick.

  5. Severe neonatal marfan syndrome resulting from a De Novo 3-bp insertion into the fibrillin gene on chromosome 15

    Energy Technology Data Exchange (ETDEWEB)

    Milewicz, D.M.; Duvic, M. (Univ. of Texas Medical School, Houston, TX (United States))

    1994-03-01

    Severe neonatal Marfan syndrome has features of the Marfan syndrome and congenital contractural arachnodactyly present at birth, along with unique features such as loose, redundant skin and pulmonary emphysema. Since the Marfan syndrome and congenital contractural arachnodactyly are due to mutations in different genes, it has been uncertain whether neonatal Marfan syndrome is due to mutations in the fibrillin gene on chromosome 15 or in another gene. The authors studied an infant with severe neonatal Marfan syndrome. Dermal fibroblasts were metabolically labeled and found to secrete fibrillin inefficiently when compared with control cells. Reverse transcription and amplification of the proband's fibroblast RNA was used to identify a 3-bp insertion between nucleotides 480-481 or 481-482 of the fibrillin cDNA. The insertion maintains the reading frame of the protein and inserts a cysteine between amino acids 160 and 161 in an epidermal growth-factor-like motif of fibrillin. This 3-bp insertion was not found in the fibrillin gene in 70 unrelated, unaffected individuals and 11 unrelated individuals with the Maran syndrome. The authors conclude that neonatal Marfan syndrome is the result of mutations in the fibrillin gene on chromosome 15 and is part of the Marfan syndrome spectrum. 32 refs., 3 figs.

  6. Construction of the suppression subtractive cDNA libraries of human large cell lung cancer line L9981 before and after transfection with nm23-H1 gene

    Directory of Open Access Journals (Sweden)

    Sujuan YE

    2008-08-01

    Full Text Available Background and objective It has been proven that nm23-H1 gene is an important metastatic-suppressed gene of lung cancer. In order to screen the differential expression genes related to nm23-H1, we constructed the suppression subtractive cDNA libraries of human large cell lung cancer line L9981 transfected and untransfected with nm23-H1 gene by suppression subtractive hybridization (SSH in this study, which lay a solid foundation for further screening and cloning metastatic-related genes of nm23-H1. Methods The forward and reverse suppression subtractive cDNA libraries were constructed in the human large cell lung cancer line L9981 before and after transfection with nm23-H1 gene (L9981 and L9981-nm23-H1 by SSH method. The positive clones were preliminarily screened by blue-white colony, and precisely identified by PCR. Results The suppression subtractive cDNA libraries were successfully constructed in the human large cell lung cancer line L9981 transfected and untransfected with nm23-H1 gene (L9981-nm23-H1 and L9981. After the blue-white screening, about three hundred positive clones in the forward subtracted library and four hundred positive clones in the reverse subtracted library were obtained. Ramdom analysis of 96 clones in each library with colony PCR methods showed that 84 clones in the forward subtracted library and 83 clones in the reverse subtracted library contained (300-750 bp inserts. Conclusion SSH is proved to be an efficient tool for differential expression gene cloning. The forward and reverse suppression subtractive cDNA libraries of human large cell lung cancer line L9981 transfected and untransfected with nm23-H1 gene (L9981-nm23-H1 and L9981 are successfully constructed by SSH and T/A cloning technology. The expression of nm23-H1 gene in the human large cell lung cancer cell lines may affect the differential expression of some metastatic-related genes.

  7. Frameshift mutations in infectious cDNA clones of Citrus tristeza virus: a strategy to minimize the toxicity of viral sequences to Escherichia coli

    International Nuclear Information System (INIS)

    Satyanarayana, Tatineni; Gowda, Siddarame; Ayllon, Maria A.; Dawson, William O.

    2003-01-01

    The advent of reverse genetics revolutionized the study of positive-stranded RNA viruses that were amenable for cloning as cDNAs into high-copy-number plasmids of Escherichia coli. However, some viruses are inherently refractory to cloning in high-copy-number plasmids due to toxicity of viral sequences to E. coli. We report a strategy that is a compromise between infectivity of the RNA transcripts and toxicity to E. coli effected by introducing frameshift mutations into 'slippery sequences' near the viral 'toxicity sequences' in the viral cDNA. Citrus tristeza virus (CTV) has cDNA sequences that are toxic to E. coli. The original full-length infectious cDNA of CTV and a derivative replicon, CTV-ΔCla, cloned into pUC119, resulted in unusually limited E. coli growth. However, upon sequencing of these cDNAs, an additional uridinylate (U) was found in a stretch of U's between nts 3726 and 3731 that resulted in a change to a reading frame with a stop codon at nt 3734. Yet, in vitro produced RNA transcripts from these clones infected protoplasts, and the resulting progeny virus was repaired. Correction of the frameshift mutation in the CTV cDNA constructs resulted in increased infectivity of in vitro produced RNA transcripts, but also caused a substantial increase of toxicity to E. coli, now requiring 3 days to develop visible colonies. Frameshift mutations created in sequences not suspected to facilitate reading frame shifting and silent mutations introduced into oligo(U) regions resulted in complete loss of infectivity, suggesting that the oligo(U) region facilitated the repair of the frameshift mutation. Additional frameshift mutations introduced into other oligo(U) regions also resulted in transcripts with reduced infectivity similarly to the original clones with the +1 insertion. However, only the frameshift mutations introduced into oligo(U) regions that were near and before the toxicity region improved growth and stability in E. coli. These data demonstrate that

  8. ALS insertion device block measurement and inspection

    International Nuclear Information System (INIS)

    Marks, S.; Carrieri, J.; Cook, C.; Hassenzahl, W.V.; Hoyer, E.; Plate, D.

    1991-05-01

    The performance specifications for ALS insertion devices require detailed knowledge and strict control of the Nd-Fe-B permanent magnet blocks incorporated in these devices. This paper describes the measurement and inspection apparatus and the procedures designed to qualify and characterize these blocks. A detailed description of a new, automated Helmholtz coil facility for measurement of the three components of magnetic moment is included. Physical block inspection and magnetic moment measurement procedures are described. Together they provide a basis for qualifying blocks and for specifying placement of blocks within an insertion devices' magnetic structures. 1 ref., 4 figs

  9. Compact insert design for cryogenic pressure vessels

    Energy Technology Data Exchange (ETDEWEB)

    Aceves, Salvador M.; Ledesma-Orozco, Elias Rigoberto; Espinosa-Loza, Francisco; Petitpas, Guillaume; Switzer, Vernon A.

    2017-06-14

    A pressure vessel apparatus for cryogenic capable storage of hydrogen or other cryogenic gases at high pressure includes an insert with a parallel inlet duct, a perpendicular inlet duct connected to the parallel inlet. The perpendicular inlet duct and the parallel inlet duct connect the interior cavity with the external components. The insert also includes a parallel outlet duct and a perpendicular outlet duct connected to the parallel outlet duct. The perpendicular outlet duct and the parallel outlet duct connect the interior cavity with the external components.

  10. Isolation and characterization of the murine alpha-L-iduronidase cDNA

    Energy Technology Data Exchange (ETDEWEB)

    Clarke, L.A.; Zhang, H. Nasir, J. [Univ. of British Columbia, Vancouver (Canada)] [and others

    1994-09-01

    Mucopolysaccharidosis I (MPS I) are a group of disorders caused by deficiency of the lysosomal enzyme alpha-L-iduronidase. The characterization of the human gene and the identification of mutations underlying MPS I in humans has led to the delineation of the molecular basis of this disorder. Model systems are now needed for the evaluation and development of therapeutics for this disorder. Both canine and feline models for MPS type I have been described but only the canine gene has been isolated and characterized. We report here the cloning and expression of the murine alpha-L-iduronidase cDNA. The murine cDNA was obtained by screening a mouse liver cDNA library with a probe from the human cDNA. The full length murine cDNA is 3120 base pairs in length and thus is considerably larger than both the human and canine transcripts. The increase in size is due to a 1.2 kb 3{prime} untranslated region in the murine cDNA that contains a CA dinucleotide repeat. Within the coding region the murine cDNA shows sequences. At the protein level the murine protein shows 77% similarity with the human protein and 75% similarity with the canine protein. There are significant differences in both the start and stop sites with the murine protein 9 amino acids shorter at both the N terminal signal peptide region and the C terminus. Expression of the murine cDNA in COS-1 cells resulted in a 20 fold increase in intracellular alpha-L-iduronidase activity as well as the detection of considerable enzyme activity in the culture medium. Comparison of the reported missense mutations underlying MPS I in humans (A75T, H82P, R89Q, L218P, P533R, Q310X, T366P) has shown conservation of these amino acid residues in the murine protein. The isolation of the murine iduronidase cDNA will now allow for the development of a murine model for MPS I.

  11. Integrative annotation of 21,037 human genes validated by full-length cDNA clones.

    Directory of Open Access Journals (Sweden)

    Tadashi Imanishi

    2004-06-01

    Full Text Available The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/. It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs, identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA

  12. Generation of cDNA expression libraries enriched for in-frame sequences.

    Science.gov (United States)

    Davis, C A; Benzer, S

    1997-03-18

    Bacterial cDNA expression libraries are made to reproduce protein sequences present in the mRNA source tissue. However, there is no control over which frame of the cDNA is translated, because translation of the cDNA must be initiated on vector sequence. In a library of nondirectionally cloned cDNAs, only some 8% of the protein sequences produced are expected to be correct. Directional cloning can increase this by a factor of two, but it does not solve the frame problem. We have therefore developed and tested a library construction methodology using a novel vector, pKE-1, with which translation in the correct reading frame confers kanamycin resistance on the host. Following kanamycin selection, the cDNA libraries contained 60-80% open, in-frame clones. These, compared with unselected libraries, showed a 10-fold increase in the number of matches between the cDNA-encoded proteins made by the bacteria and database protein sequences. cDNA sequencing programs will benefit from the enrichment for correct coding sequences, and screening methods requiring protein expression will benefit from the enrichment for authentic translation products.

  13. [Construction of a yeast two-hybrid cDNA library from the human testis].

    Science.gov (United States)

    Zheng, Ying; Zhang, Lu-Ping; Jia, Xiao-Qin; Wang, Hai-Yan

    2012-04-01

    To construct a human testis cDNA library for yeast two-hybrid screening. Human normal testis mRNA was purified from total RNA, and ds cDNA was synthesized and amplified using primers SMART III and CDS III oligo (dT) as the base of recombination. The purified PCR products and linearized plasmid pGADT7-Rec were co-transformed into the competent yeast Y187 and recombined by yeast homologous recombinase in the yeast cells to form an active cyclic plasmid. All the clones growing on the SD/-Leu plates were harvested to constitute a human testis cDNA library. We constructed a human testis cDNA library with high multiplication and adequate capacity, from which 2.0 x 10(6) recombinants were obtained. The amplified PCR fragments were between 0.3 kb and 4.0 kb in length. The yeast two-hybrid cDNA library of human testis was successfully constructed by the Clontech SMART method, which has prepared a ground for further studies on the molecular mechanism of spermatogenesis.

  14. [Construction of cDNA expression library of salivary gland from Boophilus microplus].

    Science.gov (United States)

    Tian, Zhan-Cheng; Liu, Guang-Yuan; Xie, Jun-Ren; Gong, Zhen-Li

    2008-10-30

    Total RNA were isolated from salivary gland dissected from partially engorged Boophilus microplus. The mRNA was purified. A library of oligo (dT)-primed cDNA with added directional EcoR I/Hind III linkers was constructed from the purified mRNA. The constructed cDNA was ligated to the EcoR I/Hind III arms of the lambda SCREEN vector. The recombinant phage DNA was packaged by phage-marker packaging extracts, resulting in a primary cDNA library with a size of 1.38x10(6) PFU. Data showed 100% of the library were recombinant and the titer of the amplified library was 2x10(9) PFU/ml. A partial cDNA encoding cytochrome oxidase C subunit II of B. microplus was screened from the expression library with rabbit serum against B. microplus salivary gland proteins. The results is suggested that the cDNA expression library has been constructed.

  15. Construction of equalized short hairpin RNA library from human brain cDNA.

    Science.gov (United States)

    Xu, Lei; Li, Jingqi; Liu, Li; Lu, Lixia; Gao, Jingxia; Li, Xueli

    2007-02-20

    Short hairpin RNA (shRNA) library is a powerful new tool for high-throughput loss-of-function genetic screens in mammalian cells. An shRNA library can be constructed from synthetic oligonucleotides or enzymatically cleaved natural cDNA. Here, we describe a new method for constructing equalized shRNA libraries from cDNA. First, enzymatically digested cDNA fragments are equalized by a suppression PCR-based method modified from suppression subtractive hybridization. The efficiency of equalization was confirmed by quantitative real-time PCR. The fragments are then converted into an shRNA library by a series of enzymatic treatments. With this new technology, we constructed a library from human brain cDNA. Sequence analysis showed that most of the randomly selected clones had inverted repeat sequences converted from different cDNA. After transfecting HEK 293T cells and detecting gene expression, three out of eight clones were demonstrated to significantly inhibit their target genes.

  16. Molecular cloning of goat 20alpha-hydroxysteroid dehydrogenase cDNA.

    Science.gov (United States)

    Jayasekara, Walimuni Samantha Nilanthi; Yonezawa, Tomohiro; Ishida, Maho; Yamanouchi, Keitaro; Nishihara, Masugi

    2004-06-01

    20Alpha-hydroxysteroid dehydrogenase (20alpha-HSD), which catalyzes the conversion of progesterone to its inactive form 20alpha-dihydroprogesterone, is expressed in murine placenta and has been suggested to play roles in maintaining pregnancy. To understand the role of 20alpha-HSD during pregnancy in the goat, as a first step, cloning and sequencing of 20alpha-HSD cDNA were performed. The full nucleotide sequence of 20alpha-HSD cDNA was determined on samples obtained from the corpus luteum at the luteal phase of the estrous cycle and the placenta in late pregnancy by RT-PCR and 3' and 5' RACE systems. Cloned 20alpha-HSD cDNA consisted of 1124 bp and belonged to the aldo-keto reductase superfamily. From the start codon to stop codon there were 323 amino acids, the same as in other species. To verify whether the protein derived from goat 20alpha-HSD cDNA had 20alpha-HSD activity, the cDNA was expressed by bacteria. Bacterially expressed goat 20alpha-HSD protein showed 20alpha-HSD enzyme activity. A tissue distribution study demonstrated that 20alpha-HSD was expressed in the placenta, but not in the adrenal gland, liver and spleen during pregnancy. The present study suggests that goat 20alpha-HSD is another member of the aldo-keto reductase superfamily and that it plays a role in the placenta during pregnancy.

  17. [Combining SSH and cDNA microarray for identification of lung cancer related genes].

    Science.gov (United States)

    Fan, Baoxing; Zhang, Kaitai; Da, Jiping; Xie, Ling; Wang, Shengqi; Wu, Dechang

    2003-04-20

    To screen and identify differentially expressed genes among lung cancer tissues, paracancerous pulmonary tissues and some other kinds of tumor tissues using suppression subtractive hybridization (SSH) and cDNA Microarray. One cDNA chip was made by gathering clones of three differentially expressed cDNA libraries which came from BEP2D cell lines during three different malignant transformed phases. Then the clones were hybridizated with cDNA probes which extracted from 15 cases of lung cancer tissues, 5 cases of paracancerous pulmonary tissues and 24 cases of other 8 kinds of tumor tissues respectively. Twenty-six cDNAs were obtained which expressed higher in lung cancer tissues than that in paracancerous pulmonary tissues. Thirty-one cDNAs expressed remarkably higher in paracancerous tissues than those in cancer tissues. Compared with other 8 kinds of tumors, paracancerous tissues had 63 overexpressed cDNAs and lung cancer tissues had 87 overexpressed cDNAs. The combination of SSH and cDNA microarray is rapid and effective for screening and identification of differentially expressed genes in different samples. It may be potentially useful for diagnosis of lung cancer to further study the differentially expressed genes among lung cancer tissues, paracancerous pulmonary tissues and other tumor tissues.

  18. Sequence of a cDNA encoding turtle high mobility group 1 protein.

    Science.gov (United States)

    Zheng, Jifang; Hu, Bi; Wu, Duansheng

    2005-07-01

    In order to understand sequence information about turtle HMG1 gene, a cDNA encoding HMG1 protein of the Chinese soft-shell turtle (Pelodiscus sinensis) was amplified by RT-PCR from kidney total RNA, and was cloned, sequenced and analyzed. The results revealed that the open reading frame (ORF) of turtle HMG1 cDNA is 606 bp long. The ORF codifies 202 amino acid residues, from which two DNA-binding domains and one polyacidic region are derived. The DNA-binding domains share higher amino acid identity with homologues sequences of chicken (96.5%) and mammalian (74%) than homologues sequence of rainbow trout (67%). The polyacidic region shows 84.6% amino acid homology with the equivalent region of chicken HMG1 cDNA. Turtle HMG1 protein contains 3 Cys residues located at completely conserved positions. Conservation in sequence and structure suggests that the functions of turtle HMG1 cDNA may be highly conserved during evolution. To our knowledge, this is the first report of HMG1 cDNA sequence in any reptilian.

  19. Insertion Loss of Personal Protective Clothing

    International Nuclear Information System (INIS)

    Shull, D.J.

    1999-01-01

    The use of personal protective clothing that covers the head is a common practice in many industries. Such personal protective clothing will impact the sound pressure level and the frequency content of sounds to which the wearer will be exposed. The use of such clothing, then, may impact speech and alarm audibility. A measure of the impact of such clothing is its insertion loss. Insertion loss measurements were performed on four types of personal protective clothing in use by Westinghouse Savannah River Company personnel which utilize cloth and plastic hood configurations to protect the head. All clothing configurations tested at least partially cover the ears. The measurements revealed that insertion loss of the items tested was notable at frequencies above 1000 Hz only and was a function of material stiffness and acoustic flanking paths to the ear. Further, an estimate of the clothing's noise reduction rating reveals poor performance in that regard, even though the insertion loss of the test articles was significant at frequencies at and above 1000 Hz.'

  20. Direct random insertion mutagenesis of Helicobacter pylori

    NARCIS (Netherlands)

    de Jonge, Ramon; Bakker, Dennis; van Vliet, Arnoud H. M.; Kuipers, Ernst J.; Vandenbroucke-Grauls, Christina M. J. E.; Kusters, Johannes G.

    2003-01-01

    Random insertion mutagenesis is a widely used technique for the identification of bacterial virulence genes. Most strategies for random mutagenesis involve cloning in Escherichia coli for passage of plasmids or for phenotypic selection. This can result in biased selection due to restriction or

  1. Aspects of insertion in random trees

    NARCIS (Netherlands)

    Bagchi, Arunabha; Reingold, E.M.

    1982-01-01

    A method formulated by Yao and used by Brown has yielded bounds on the fraction of nodes with specified properties in trees bult by a sequence of random internal nodes in a random tree built by binary search and insertion, and show that in such a tree about bounds better than those now known. We

  2. SOLVENTLESS MIGRATORY-INSERTION REACTIONS OF ...

    African Journals Online (AJOL)

    Preferred Customer

    2CH3 and PPh3 as well as CO ligands in the absence of solvent was reported by us. [18]. In this study we report on the influence of steric and electronic effects of the incoming ligand on the insertion reaction and evaluate a green chemistry ...

  3. Patients' experiences of the PICC insertion procedure.

    Science.gov (United States)

    Nicholson, Jackie; Davies, Louise

    Peripherally inserted central catheters (PICCs) are a type of central venous access device used to deliver a variety of intravenous therapies, including chemotherapy. PICCs may be placed by interventional radiologists, anaesthetists or, as is increasingly common, by specialist nurses in the hospital setting. However, little is known about how patients feel regarding the PICC insertion procedure. The aim of this study was to interview patients who had undergone a recent PICC insertion in the chemotherapy day unit to identify their experiences. On analysis of the qualitative data obtained from the semi-structured interview, five themes emerged: the context of cancer; expectations; levels of pain and anxiety; coping strategies; and explanation. The findings of this study support some previously described elements of procedural experiences; however, new understanding has provided implications for practice in the areas of expectations, allaying anxiety levels, supporting individual coping strategies and providing explanation. The major limitation of the study was the homogenous sample of oncology patients with a clear link between the patient experience of the PICC insertion and the context of cancer. The main recommendation for further research would be to repeat this study with a broader patient population.

  4. Expressed sequence tags: normalization and subtraction of cDNA libraries expressed sequence tags\\ normalization and subtraction of cDNA libraries.

    Science.gov (United States)

    Soares, Marcelo Bento; de Fatima Bonaldo, Maria; Hackett, Jeremiah D; Bhattacharya, Debashish

    2009-01-01

    Expressed Sequence Tags (ESTs) provide a rapid and efficient approach for gene discovery and analysis of gene expression in eukaryotes. ESTs have also become particularly important with recent expanded efforts in complete genome sequencing of understudied, nonmodel eukaryotes such as protists and algae. For these projects, ESTs provide an invaluable source of data for gene identification and prediction of exon-intron boundaries. The generation of EST data, although straightforward in concept, requires nonetheless great care to ensure the highest efficiency and return for the investment in time and funds. To this end, key steps in the process include generation of a normalized cDNA library to facilitate a high gene discovery rate followed by serial subtraction of normalized libraries to maintain the discovery rate. Here we describe in detail, protocols for normalization and subtraction of cDNA libraries followed by an example using the toxic dinoflagellate Alexandrium tamarense.

  5. A critical appraisal of medication package inserts

    Directory of Open Access Journals (Sweden)

    Pranjit Narzaree

    2015-10-01

    Full Text Available Introduction: Package Inserts (PIs refers to officially specified document that accompanies a drug for relevant, updated and unbiased information for rational drug use based on regulatory guidelines as per section 6.2 and 6.3 of schedule D of Indian Drug and cosmetic Act 1945. But some studies had shown non-uniformity with suboptimal level of informations which frequently can lead to medication errors. Hence this study was conducted to evaluate the completeness of PIs.Aim: To critically evaluate package inserts of allopathic medicines.Material and Methods: 100 allopathic drug PIs were collected from pharmacies in Rohtak and were checked for the presence of each heading as per schedule D criteria, followed by scrutiny of the information included under the heading. Indian guidelines were also compared with US FDA guidelines for PIs.Scoring of package inserts: The informations were evaluated for completeness and scored as 1 if present otherwise scored as zero for no information or partial information. Scores for each heading were calculated by totaling the scores of all the package inserts. The total scores were expressed as absolute numbers and percentages.Results:  On an average PIs analyzed for the completeness of the criteria scored 10 (Mean± SD = 9.73±2.48 out of 16. Absence of common layout and headings caused inconvenience. In comparison to US FDA guidelines it lacked, disclaimer statement, boxed warning, revision date, approval date, toll-free number etc.Conclusion: PIs don’t seem to be serving effectively because of multiple deficiencies like completeness, uniformity, absence of headings.Keywords: Critical appraisal, package inserts.

  6. ESTs, cDNA microarrays, and gene expression profiling: tools for dissecting plant physiology and development.

    Science.gov (United States)

    Alba, Rob; Fei, Zhangjun; Payton, Paxton; Liu, Yang; Moore, Shanna L; Debbie, Paul; Cohn, Jonathan; D'Ascenzo, Mark; Gordon, Jeffrey S; Rose, Jocelyn K C; Martin, Gregory; Tanksley, Steven D; Bouzayen, Mondher; Jahn, Molly M; Giovannoni, Jim

    2004-09-01

    Gene expression profiling holds tremendous promise for dissecting the regulatory mechanisms and transcriptional networks that underlie biological processes. Here we provide details of approaches used by others and ourselves for gene expression profiling in plants with emphasis on cDNA microarrays and discussion of both experimental design and downstream analysis. We focus on methods and techniques emphasizing fabrication of cDNA microarrays, fluorescent labeling, cDNA hybridization, experimental design, and data processing. We include specific examples that demonstrate how this technology can be used to further our understanding of plant physiology and development (specifically fruit development and ripening) and for comparative genomics by comparing transcriptome activity in tomato and pepper fruit.

  7. Construction and analysis of full-length and normalized cDNA libraries from citrus.

    Science.gov (United States)

    Marques, M Carmen; Perez-Amador, Miguel A

    2012-01-01

    We have developed an integrated method to generate a normalized cDNA collection enriched in full-length and rare transcripts from citrus, using different species and multiple tissues and developmental stages. Interpretation of ever-increasing raw sequence information generated by modern genome sequencing technologies faces multiple challenges, such as gene function analysis and genome annotation. In this regard, the availability of full-length cDNA clones facilitates functional analysis of the corresponding genes enabling manipulation of their expression and the generation of a variety of tagged versions of the native protein. The development of full-length cDNA sequences has the power to improve the quality of genome annotation, as well as provide tools for functional characterization of genes.

  8. Design and Screening of M13 Phage Display cDNA Libraries

    Directory of Open Access Journals (Sweden)

    Yuliya Georgieva

    2011-02-01

    Full Text Available The last decade has seen a steady increase in screening of cDNA expression product libraries displayed on the surface of filamentous bacteriophage. At the same time, the range of applications extended from the identification of novel allergens over disease markers to protein-protein interaction studies. However, the generation and selection of cDNA phage display libraries is subjected to intrinsic biological limitations due to their complex nature and heterogeneity, as well as technical difficulties regarding protein presentation on the phage surface. Here, we review the latest developments in this field, discuss a number of strategies and improvements anticipated to overcome these challenges making cDNA and open reading frame (ORF libraries more readily accessible for phage display. Furthermore, future trends combining phage display with next generation sequencing (NGS will be presented.

  9. COMPARATIVE STUDY OF EARLY POSTPARTUM IUCD INSERTION TO INTERVAL IUCD INSERTION

    Directory of Open Access Journals (Sweden)

    Shibani Devi

    2016-07-01

    Full Text Available INTRODUCTION According to National Family Health Survey-3, Indian women have 13% unmet need for contraception and according to District Level Household & Facility Survey-3, it is 21.3% in the postpartum period. Postpartum intrauterine contraceptive device insertion - both immediately post-placental delivery and somewhat later, but within 48 hours after delivery are options which merit consideration as the woman is likely to have a high motivation for accepting contraception and the healthcare centre provides a convenient setting for insertion of IUCD. AIM Comparison of efficacy and complications of IUCD insertions in post-placental with interval period: 6-month followup. METHOD This perspective study was conducted among 100 women: - 50 women had IUCD inserted within 10 minutes of placental delivery and 50 had insertion more than 6 weeks after delivery. They were followed till 6 months post insertion and were compared regarding early and late complications, continuation rates and expulsion rates. RESULT At the end of six months, we found higher occurrence of lower abdominal pain, heavy menstrual bleeding in case of interval insertion as compared to post-placental insertion which was statistically significant (p value-0.04 & 0.007 respectively. However, the expulsion rates of post-placental IUCD were somewhat elevated (14% compared to interval insertions (2%. Continuation rates at the end of 6 months in both the groups were 82% and 86% respectively which is comparable. CONCLUSION Post-placental IUCD is thus found to be an ideal method to meet the unmet need of postpartum women as it is easily accessible and convenient for both women and their health care providers, is associated with less discomfort and fewer side effects and allow women to obtain safe, long acting, highly effective contraception while still in the health care system.

  10. Gene therapy for bladder pain with gene gun particle encoding pro-opiomelanocortin cDNA.

    Science.gov (United States)

    Chuang, Yao-Chi; Chou, A-K; Wu, P-C; Chiang, Po-Hui; Yu, T-J; Yang, L-C; Yoshimura, Naoki; Chancellor, Michael B

    2003-11-01

    Interstitial cystitis is a bladder hypersensitivity disease associated with bladder pain that has been a major challenge to understand and treat. We hypothesized that targeted and localized expression of endogenous opioid peptide in the bladder could be useful for the treatment of bladder pain. Pro-opiomelanocortin (POMC) is one of such precursor molecules. In this study we developed a gene gun method for the transfer of POMC cDNA in vivo and investigated its therapeutic effect on acetic acid induced bladder hyperactivity in rats. Human POMC cDNA was cloned into a modified pCMV plasmid and delivered into the bladder wall of adult female rats by direct injection or the gene gun. Three days after gene therapy continuous cystometrograms were performed using urethane anesthesia by filling the bladder (0.08 ml per minute) with saline, followed by 0.3% acetic acid. Bladder immunohistochemical testing was used to detect endorphin after POMC cDNA transfer. The intercontraction interval was decreased after intravesical instillation of acetic acid (73.1% or 68.1% decrease) in 2 control groups treated with saline or the gene gun without POMC cDNA, respectively. However, rats that received POMC cDNA via the gene gun showed a significantly decreased response (intercontraction interval 35% decreased) to acetic acid instillation, whereas this antinociceptive effect was not detected in the plasmid POMC cDNA direct injection group. This effect induced by POMC gene gun treatment was reversed by intramuscular naloxone (1 mg/kg), an opioid antagonist. Increased endorphin immunoreactivity with anti-endorphin antibodies was observed in the bladder of gene gun treated animals. The POMC gene can be transferred in the bladder using the gene gun and increased bladder expression of endorphin can suppress nociceptive responses induced by bladder irritation. Thus, POMC gene gun delivery may be useful for the treatment of interstitial cystitis and other types of visceral pain.

  11. Improved coverage of cDNA-AFLP by sequential digestion of immobilized cDNA

    Directory of Open Access Journals (Sweden)

    Karlovsky Petr

    2008-10-01

    Full Text Available Abstract Background cDNA-AFLP is a transcriptomics technique which does not require prior sequence information and can therefore be used as a gene discovery tool. The method is based on selective amplification of cDNA fragments generated by restriction endonucleases, electrophoretic separation of the products and comparison of the band patterns between treated samples and controls. Unequal distribution of restriction sites used to generate cDNA fragments negatively affects the performance of cDNA-AFLP. Some transcripts are represented by more than one fragment while other escape detection, causing redundancy and reducing the coverage of the analysis, respectively. Results With the goal of improving the coverage of cDNA-AFLP without increasing its redundancy, we designed a modified cDNA-AFLP protocol. Immobilized cDNA is sequentially digested with several restriction endonucleases and the released DNA fragments are collected in mutually exclusive pools. To investigate the performance of the protocol, software tool MECS (Multiple Enzyme cDNA-AFLP Simulation was written in Perl. cDNA-AFLP protocols described in the literatur and the new sequential digestion protocol were simulated on sets of cDNA sequences from mouse, human and Arabidopsis thaliana. The redundancy and coverage, the total number of PCR reactions, and the average fragment length were calculated for each protocol and cDNA set. Conclusion Simulation revealed that sequential digestion of immobilized cDNA followed by the partitioning of released fragments into mutually exclusive pools outperformed other cDNA-AFLP protocols in terms of coverage, redundancy, fragment length, and the total number of PCRs. Primers generating 30 to 70 amplicons per PCR provided the highest fraction of electrophoretically distinguishable fragments suitable for normalization. For A. thaliana, human and mice transcriptome, the use of two marking enzymes and three sequentially applied releasing enzymes for each

  12. Display of a maize cDNA library on baculovirus infected insect cells.

    Science.gov (United States)

    Meller Harel, Helene Y; Fontaine, Veronique; Chen, Hongying; Jones, Ian M; Millner, Paul A

    2008-08-12

    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. 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 x 10(5) 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. 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.

  13. Cloning, sequencing, and expression of cDNA for human β-glucuronidase

    International Nuclear Information System (INIS)

    Oshima, A.; Kyle, J.W.; Miller, R.D.

    1987-01-01

    The authors report here the cDNA sequence for human placental β-glucuronidase (β-D-glucuronoside glucuronosohydrolase, EC 3.2.1.31) and demonstrate expression of the human enzyme in transfected COS cells. They also sequenced a partial cDNA clone from human fibroblasts that contained a 153-base-pair deletion within the coding sequence and found a second type of cDNA clone from placenta that contained the same deletion. Nuclease S1 mapping studies demonstrated two types of mRNAs in human placenta that corresponded to the two types of cDNA clones isolated. The NH 2 -terminal amino acid sequence determined for human spleen β-glucuronidase agreed with that inferred from the DNA sequence of the two placental clones, beginning at amino acid 23, suggesting a cleaved signal sequence of 22 amino acids. When transfected into COS cells, plasmids containing either placental clone expressed an immunoprecipitable protein that contained N-linked oligosaccharides as evidenced by sensitivity to endoglycosidase F. However, only transfection with the clone containing the 153-base-pair segment led to expression of human β-glucuronidase activity. These studies provide the sequence for the full-length cDNA for human β-glucuronidase, demonstrate the existence of two populations of mRNA for β-glucuronidase in human placenta, only one of which specifies a catalytically active enzyme, and illustrate the importance of expression studies in verifying that a cDNA is functionally full-length

  14. Construction of cDNA libraries: focus on protists and fungi.

    Science.gov (United States)

    Rodríguez-Ezpeleta, Naiara; Teijeiro, Shona; Forget, Lise; Burger, Gertraud; Lang, B Franz

    2009-01-01

    Sequencing of cDNA libraries is an efficient and inexpensive approach to analyze the protein-coding portion of a genome. It is frequently used for surveying the genomes of poorly studied eukaryotes, and is particularly useful for species that are not easily amenable to genome sequencing, because they are nonaxenic and/or difficult to cultivate. In this chapter, we describe protocols that have been applied successfully to construct and normalize a variety of cDNA libraries from many different species of free-living protists and fungi, and that require only small quantities of cell material.

  15. Development of an HIV-based cDNA expression cloning system.

    Science.gov (United States)

    van Maanen, Marc; Tidwell, Jennie K; Donehower, Lawrence A; Sutton, Richard E

    2003-07-01

    Expression cloning of cDNAs is a powerful tool with which to identify genes based on their specific functional properties. Here we describe the development of a cDNA library transfer system based on the human immunodeficiency virus type-1 (HIV). This system represents an improvement over current oncoretroviral cDNA expression systems in terms of target cell range and the inclusion of a selectable marker. By use of a simple packaging system, we were able to produce high-titer vector stocks from HIV vector-based cDNA libraries and demonstrate highly efficient cDNA expression cloning in three model experiments. First, HOS TK(-) cells, which are null for thymidine kinase (TK) expression, were transduced with an HIV-based cDNA library derived from primary human foreskin fibroblasts (HFFs) and functionally selected for TK expression. In a second experiment, hypoxanthine guanine phosphoribosyltransferase-1-deficient (HPRT(-)) fibroblasts were transduced with a T cell (PM1) line-derived cDNA library and selected for HPRT expression. Both TK (frequency 1 in 5.0 x 10(4)) and HPRT (frequency 1 in 2.0 x 10(4)) cDNAs were readily isolated from these HIV-based cDNA libraries. As a third example, we demonstrated the ability of this vector system to allow functional cDNA library screens to be performed in primary, mitotically inactive cell types. Using senescent HFFs as a target cell population, we were able to isolate SV40 large T antigen cDNA-containing clones (frequency 1 in 2.5 x 10(4)) based on their ability to overcome the senescence-induced block to cell proliferation. Thus, this system can be used to clone relatively low-abundance cDNAs based upon their expression. Because of the ability of HIV-based vectors to transduce primary and nondividing cells efficiently, this vector system will further broaden the range of cell types in which expression cloning studies can be performed.

  16. Medications to ease intrauterine device insertion: a systematic review.

    Science.gov (United States)

    Zapata, Lauren B; Jatlaoui, Tara C; Marchbanks, Polly A; Curtis, Kathryn M

    2016-12-01

    Potential barriers to intrauterine device (IUD) use include provider concern about difficult insertion, particularly for nulliparous women. This study aims to evaluate the evidence on the effectiveness of medications to ease IUD insertion on provider outcomes (i.e., ease of insertion, need for adjunctive insertion measures, insertion success). We searched the PubMed database for peer-reviewed articles published in any language from database inception through February 2016. We included randomized controlled trials (RCTs) that examined medications to ease interval insertion of levonorgestrel-releasing IUDs and copper T IUDs. From 1855 articles, we identified 15 RCTs that met our inclusion criteria. Most evidence suggested that misoprostol did not improve provider ease of insertion, reduce the need for adjunctive insertion measures or improve insertion success among general samples of women seeking an IUD (evidence Level I, good to fair). However, one RCT found significantly higher insertion success among women receiving misoprostol prior to a second IUD insertion attempt after failed attempt versus placebo (evidence Level I, good). Two RCTs on 2% intracervical lidocaine as a topical gel or injection suggested no positive effect on provider ease of insertion (evidence Level I, good to poor), and one RCT on diclofenac plus 2% intracervical lidocaine as a topical gel suggested no positive effect on provider ease of insertion (evidence Level I, good). Limited evidence from two RCTs on nitric oxide donors, specifically nitroprusside or nitroglycerin gel, suggested no positive effect on provider ease of insertion or need for adjunctive insertion measures (evidence Level I, fair). Overall, most studies found no significant differences between women receiving interventions to ease IUD insertion versus controls. Among women with a recent failed insertion who underwent a second insertion attempt, one RCT found improved insertion success among women using misoprostol versus

  17. Mechanistic insights on acrylate insertion polymerization.

    Science.gov (United States)

    Guironnet, Damien; Caporaso, Lucia; Neuwald, Boris; Göttker-Schnetmann, Inigo; Cavallo, Luigi; Mecking, Stefan

    2010-03-31

    Complexes [{(PwedgeO)PdMe}(n)] (1(n); PwedgeO = kappa(2)-P,O-Ar(2)PC(6)H(4)SO(2)O with Ar = 2-MeOC(6)H(4)) are a single-component precursor of the (PwedgeO)PdMe fragment devoid of additional coordinating ligands, which also promotes the catalytic oligomerization of acrylates. Exposure of 1(n) to methyl acrylate afforded the two diastereomeric chelate complexes [(PwedgeO)Pd{kappa(2)-C,O-CH(C(O)OMe)CH(2)CH(C(O)OMe)CH(2)CH(3)}] (3-meso and 3-rac) resulting from two consecutive 2,1-insertions of methyl acrylate into the Pd-Me bond with the same or opposite stereochemistry, respectively, in a 3:2 ratio as demonstrated by comprehensive NMR spectroscopic studies and single crystal X-ray diffraction. These six-membered chelate complexes are direct key models for intermediates of acrylate insertion polymerization, and also ethylene-acrylate copolymerization to high acrylate content copolymers. Studies of the binding of various substrates (pyridine, dmso, ethylene and methyl acrylate) to 3-meso and 3-rac show that hindered displacement of the chelating carbonyl moiety by pi-coordination of incoming monomer significantly retards, but does not prohibit, polymerization. For 3-meso,3-rac + C(2)H(4) right arrow over left arrow 3-meso-C(2)H(4,) 3-rac-C(2)H(4) an equilibrium constant K(353 K) approximately 2 x 10(-3) L mol(-1) was estimated. Reaction of 3-meso, 3-rac with methyl acrylate afforded higher insertion products [(PwedgeO)Pd(C(4)H(6)O(2))(n)Me] (n = 3, 4) as observed by electrospray ionization mass spectrometry (ESI-MS). Theoretical studies by DFT methods of consecutive acrylate insertion provide relative energies of intermediates and transition states, which are consistent with the aforementioned experimental observations, and give detailed insights to the pathways of multiple consecutive acrylate insertions. Acrylate insertion into 3-meso,3-rac is associated with an overall energy barrier of ca. 100 kJ mol(-1).

  18. Comparison of conventional Injection Mould Inserts to Additively Manufactured Inserts using Life Cycle Assessment

    DEFF Research Database (Denmark)

    Hofstätter, Thomas; Bey, Niki; Mischkot, Michael

    2016-01-01

    Polymer Additive Manufacturing can be used to produce soft tooling inserts for injection moulding. Compared to conventional tooling, the energy and time consumption during production are significantly lower. As the life time of such inserts is significantly shorter than the life time of traditional...... of their potential environmental impact and yield throughout the development and pilot phase. Insert geometry is particularly advantageous for pilot production and small production sizes. In this research, Life Cycle Assessment is used to compare the environmental impact of soft tooling by Additive Manufacturing...... (using Digital Light Processing) and three traditional methods for the manufacture of inserts (milling of brass, steel, and aluminium) for injection moulds during the pre-production phase....

  19. Symplectic models for general insertion devices

    International Nuclear Information System (INIS)

    Wu, Y.; Forest, E.; Robin, D. S.; Nishimura, H.; Wolski, A.; Litvinenko, V. N.

    2001-01-01

    A variety of insertion devices (IDs), wigglers and undulators, linearly or elliptically polarized,are widely used as high brightness radiation sources at the modern light source rings. Long and high-field wigglers have also been proposed as the main source of radiation damping at next generation damping rings. As a result, it becomes increasingly important to understand the impact of IDs on the charged particle dynamics in the storage ring. In this paper, we report our recent development of a general explicit symplectic model for IDs with the paraxial ray approximation. High-order explicit symplectic integrators are developed to study real-world insertion devices with a number of wiggler harmonics and arbitrary polarizations

  20. Two cases of anaphylaxis after laminaria insertion.

    Science.gov (United States)

    Kim, Sang-Hoon; Chang, Yun-Hae; Kim, Woo-Kyoung; Kim, Yoon-Keun; Cho, Sang-Heon; Kim, You-Young; Min, Kyung-Up

    2003-01-01

    Anaphylaxis following laminaria insertion rarely occurs but may be a life-threatening condition. Laminaria tents, prepared from natural sea kelp, are commonly used prior to elective termination of pregnancy to achieve cervical dilatation. We report herein two cases of anaphylaxis caused by IgE-mediated hypersensitivity to laminaria. Two women, each of whom had undergone at least one previous abortion where a laminaria had been utilized, developed anaphylactic reaction following laminaria insertion. The reaction included urticaria, nausea, breathing difficulty, and hypotension. The patients subsequently underwent skin testing and measurement of serum specific IgE level to laminaria extract, and were shown to elicit positive responses to laminaria. The implication and impact of laminaria allergy on gynecologic procedures are significant and this allergy should be included in the list of differential diagnoses for hypersensitive reaction in gynecologic procedures. PMID:14676449

  1. High-level expression of human insulin receptor cDNA in mouse NIH 3T3 cells

    Energy Technology Data Exchange (ETDEWEB)

    Whittaker, J.; Okamoto, A.K.; Thys, R.; Bell, G.I.; Steiner, D.F.; Hofmann, C.A.

    1987-08-01

    In order to develop a simple, efficient system for the high-level expression of human insulin receptors in eukaryotic cells, a full-length human kidney insulin receptor cDNA was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH 3T3 cells with this construct, seven cell lines expressing insulin receptors were isolated; two cell lines had more than 10/sup 6/ receptors per cell. The cell line with the highest /sup 125/I-insulin binding (NIH 3T3 HIR3.5) had 6 x 10/sup 6/ receptors with a K/sub d/ of 10/sup -9/ M. This level was not dependent on exposure to metals but could be increased further to 2 x 10/sup 7/ receptors per cell by addition of sodium butyrate to the culture medium. The ..cap alpha.. and ..beta.. subunits had apparent molecular weights of 147,000 and 105,000, respectively (compared to 135,000 and 95,000 in IM-9 human lymphocytes), values identical to those of the ..cap alpha.. and ..beta.. subunits of the insulin receptors of nontransformed NIH 3T3 cells. This size difference was due to altered carbohydrate composition, as N-glycanase digestion reduced the apparent receptor subunit size of the transfected cells and IM-9 lymphocytes to identical values. The alteration in N-linked oligosaccharide composition could not be ascribed to differences in the kinetics of posttranslational processing of the insulin receptors, which was comparable to that of other cells studied. The basal rate of glycogen synthesis in the cells overexpressing insulin receptors was increased 4- to 5-fold compared with controls. Low levels of added insulin (0.1 nM) caused a 50% increase in the rate of glycogen synthesis

  2. High-level expression of human insulin receptor cDNA in mouse NIH 3T3 cells

    International Nuclear Information System (INIS)

    Whittaker, J.; Okamoto, A.K.; Thys, R.; Bell, G.I.; Steiner, D.F.; Hofmann, C.A.

    1987-01-01

    In order to develop a simple, efficient system for the high-level expression of human insulin receptors in eukaryotic cells, a full-length human kidney insulin receptor cDNA was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH 3T3 cells with this construct, seven cell lines expressing insulin receptors were isolated; two cell lines had more than 10 6 receptors per cell. The cell line with the highest 125 I-insulin binding (NIH 3T3 HIR3.5) had 6 x 10 6 receptors with a K/sub d/ of 10 -9 M. This level was not dependent on exposure to metals but could be increased further to 2 x 10 7 receptors per cell by addition of sodium butyrate to the culture medium. The α and β subunits had apparent molecular weights of 147,000 and 105,000, respectively (compared to 135,000 and 95,000 in IM-9 human lymphocytes), values identical to those of the α and β subunits of the insulin receptors of nontransformed NIH 3T3 cells. This size difference was due to altered carbohydrate composition, as N-glycanase digestion reduced the apparent receptor subunit size of the transfected cells and IM-9 lymphocytes to identical values. The alteration in N-linked oligosaccharide composition could not be ascribed to differences in the kinetics of posttranslational processing of the insulin receptors, which was comparable to that of other cells studied. The basal rate of glycogen synthesis in the cells overexpressing insulin receptors was increased 4- to 5-fold compared with controls. Low levels of added insulin (0.1 nM) caused a 50% increase in the rate of glycogen synthesis

  3. Probabilistic phylogenetic inference with insertions and deletions.

    Directory of Open Access Journals (Sweden)

    Elena Rivas

    2008-09-01

    Full Text Available A fundamental task in sequence analysis is to calculate the probability of a multiple alignment given a phylogenetic tree relating the sequences and an evolutionary model describing how sequences change over time. However, the most widely used phylogenetic models only account for residue substitution events. We describe a probabilistic model of a multiple sequence alignment that accounts for insertion and deletion events in addition to substitutions, given a phylogenetic tree, using a rate matrix augmented by the gap character. Starting from a continuous Markov process, we construct a non-reversible generative (birth-death evolutionary model for insertions and deletions. The model assumes that insertion and deletion events occur one residue at a time. We apply this model to phylogenetic tree inference by extending the program dnaml in phylip. Using standard benchmarking methods on simulated data and a new "concordance test" benchmark on real ribosomal RNA alignments, we show that the extended program dnamlepsilon improves accuracy relative to the usual approach of ignoring gaps, while retaining the computational efficiency of the Felsenstein peeling algorithm.

  4. cDNA, genomic cloning and sequence analysis of ribosomal protein ...

    African Journals Online (AJOL)

    Ribosomal protein S4X (RPS4X) is one of the 40S ribosomal proteins encoded by the RPS4X gene. The cDNA and the genomic sequence of RPS4X were cloned successfully from giant panda (Ailuropoda melanoleuca) using reverse transcriptase-polymerase chain reaction (RT-PCR) and touchdown-PCR technology ...

  5. Cloning and sequencing of complete τ-crystallin cDNA from ...

    Indian Academy of Sciences (India)

    Unknown

    brain, heart and gonad, suggesting both to be the product of the same gene. The study thus provides the first report on cDNA sequence of τ-crystallin from a reptilian species and also re-confirms it to be an example of the phenomenon of gene sharing as was demonstrated earlier in the case of peking duck. Moreover, the ...

  6. Strategies to enhance immunogenicity of cDNA vaccine encoded antigens by modulation of antigen processing

    NARCIS (Netherlands)

    Platteel, Anouk C M; Marit de Groot, A; Andersen, Peter; Ovaa, Huib; Kloetzel, Peter M; Mishto, Michele; Sijts, Alice J A M

    2016-01-01

    Most vaccines are based on protective humoral responses while for intracellular pathogens CD8(+) T cells are regularly needed to provide protection. However, poor processing efficiency of antigens is often a limiting factor in CD8(+) T cell priming, hampering vaccine efficacy. The multistage cDNA

  7. Avoiding cross hybridization by choosing nonredundant targets on cDNA arrays

    DEFF Research Database (Denmark)

    Nielsen, Henrik Bjørn; Knudsen, Steen

    2002-01-01

    PROBEWIZ designs PCR primers for amplifying probes for cDNA arrays. The probes are designed to have minimal homology to other expressed sequences from a given organism. The primer selection is based on user-defined penalties for homology, primer quality, and proximity to the 3' end....

  8. Observation of intermittency in gene expression on cDNA microarrays

    CERN Document Server

    Peterson, L E

    2002-01-01

    We used scaled factorial moments to search for intermittency in the log expression ratios (LERs) for thousands of genes spotted on cDNA microarrays (gene chips). Results indicate varying levels of intermittency in gene expression. The observation of intermittency in the data analyzed provides a complimentary handle on moderately expressed genes, generally not tackled by conventional techniques.

  9. Isolation of an ATP synthase cDNA from Sinonovacula constricta ...

    African Journals Online (AJOL)

    Yomi

    2012-01-24

    Jan 24, 2012 ... 2Ningbo City College of Vocational Technology, Ningbo, 315100 People's Republic of China. 3National Marine Environmental Monitoring Center. Dalian, 116023, People's ... The SMART cDNA library of S. constricta was constructed by our laboratory. Random sequencing of the library using T3 primer.

  10. cDNA, genomic sequence cloning and analysis of the ribosomal ...

    African Journals Online (AJOL)

    Ribosomal protein L37A (RPL37A) is a component of 60S large ribosomal subunit encoded by the RPL37A gene, which belongs to the family of ribosomal L37AE proteins, located in the cytoplasm. The complementary deoxyribonucleic acid (cDNA) and the genomic sequence of RPL37A were cloned successfully from giant ...

  11. cDNA sequence of the long mRNA for human glutamine synthase

    NARCIS (Netherlands)

    van den Hoff, M. J.; Geerts, W. J.; Das, A. T.; Moorman, A. F.; Lamers, W. H.

    1991-01-01

    Screening a human liver cDNA library in lambda ZAP revealed several clones for the mRNA of glutamine synthase. The longest clone was completely sequenced and consists of a 109 bp 5' untranslated region, a 1119 bp protein coding region, a 1498 bp 3' untranslated region and a poly(A) tract of 12 bp

  12. Cloning a cDNA for the lysosomal alpha-glucosidase

    NARCIS (Netherlands)

    KONINGS, A.; HUPKES, P.; Versteeg, R.; Grosveld, G.; Reuser, A.; Galjaard, H.

    1984-01-01

    Messenger RNA was isolated from monkey testes and size-fractionated on sucrose gradients. In vitro translation of these mRNA fractions resulted in nascent, labeled alpha-glucosidase that could be precipitated with anti human alpha-glucosidase antiserum. A cDNA library was constructed from the most

  13. 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

  14. cDNA Library Enrichment of Full Length Transcripts for SMRT Long Read Sequencing.

    Science.gov (United States)

    Cartolano, Maria; Huettel, Bruno; Hartwig, Benjamin; Reinhardt, Richard; Schneeberger, Korbinian

    2016-01-01

    The utility of genome assemblies does not only rely on the quality of the assembled genome sequence, but also on the quality of the gene annotations. The Pacific Biosciences Iso-Seq technology is a powerful support for accurate eukaryotic gene model annotation as it allows for direct readout of full-length cDNA sequences without the need for noisy short read-based transcript assembly. We propose the implementation of the TeloPrime Full Length cDNA Amplification kit to the Pacific Biosciences Iso-Seq technology in order to enrich for genuine full-length transcripts in the cDNA libraries. We provide evidence that TeloPrime outperforms the commonly used SMARTer PCR cDNA Synthesis Kit in identifying transcription start and end sites in Arabidopsis thaliana. Furthermore, we show that TeloPrime-based Pacific Biosciences Iso-Seq can be successfully applied to the polyploid genome of bread wheat (Triticum aestivum) not only to efficiently annotate gene models, but also to identify novel transcription sites, gene homeologs, splicing isoforms and previously unidentified gene loci.

  15. Isolation of an ATP synthase cDNA from Sinonovacula constricta ...

    African Journals Online (AJOL)

    Yomi

    2012-01-24

    Jan 24, 2012 ... Complete cDNA sequence of ScATPase and its deduced amino acid sequence. Nucleotides were numbered from the first base at the 5'end. The canonical polyadenylation signal-sequence was italic and underlined. The asterisk indicated the stop codon. The domain for ATP synthase C was underlined.

  16. Detection of reverse transcriptase termination sites using cDNA ligation and massive parallel sequencing

    DEFF Research Database (Denmark)

    Kielpinski, Lukasz J; Boyd, Mette; Sandelin, Albin

    2013-01-01

    of these methods can be increased by applying massive parallel sequencing technologies.Here, we describe a versatile method for detection of reverse transcriptase termination sites based on ligation of an adapter to the 3' end of cDNA with bacteriophage TS2126 RNA ligase (CircLigase™). In the following PCR...

  17. Primary structure of a lipoxygenase from barley grain as deduced from its cDNA sequence

    NARCIS (Netherlands)

    Mechelen, J.R. van; Smits, M.; Douma, A.C.; Rouster, J.; Cameron-Mills, V.; Heidekamp, F.; Valk, B.E.

    1995-01-01

    A full length cDNA sequence for a barley grain lipoxygenase was obtained. It includes a 5' untranslated region of 69 nucleotides, an open reading frame of 2586 nucleotides encoding a protein of 862 amino acid residues and a 3' untranslated region of 142 nucleotides. The molecular mass of the encoded

  18. cDNA cloning and expression analysis of two distinct Sox8 genes in ...

    Indian Academy of Sciences (India)

    2010-08-06

    Aug 6, 2010 ... cDNA cloning and expression analysis of two distinct Sox8 genes in. Paramisgurnus dabryanus (Cypriniformes). XIAOHUA XIA, JIE ZHAO, QIYAN DU and ZHONGJIE CHANG. ∗. Molecular and Genetic Laboratory, College of Life Sciences, Henan Normal University, 46 East of Construction Road,. Xinxiang ...

  19. cDNA, genomic cloning and sequence analysis of ribosomal protein ...

    African Journals Online (AJOL)

    enoh

    2012-03-13

    Mar 13, 2012 ... Ribosomal protein S4X (RPS4X) is one of the 40S ribosomal proteins encoded by the RPS4X gene. The. cDNA and the genomic sequence of RPS4X were cloned successfully from giant panda (Ailuropoda melanoleuca) using reverse transcriptase-polymerase chain reaction (RT-PCR) and touchdown- ...

  20. Construction and characterization of a cDNA library from human ...

    African Journals Online (AJOL)

    The tumor-suppressor gene p53 and its downstream genes consist of a complicated gene network, and the challenge to understand the network is to identify p53 downstream genes. In order to isolate and identify new p53 regulated genes, we constructed and characterized a normalized cDNA library from human brain ...

  1. First-strand cDNA synthesis primed with oligo(dT)

    International Nuclear Information System (INIS)

    Krug, M.S.; Berger, S.L.

    1987-01-01

    The quality of a cDNA library depends on the integrity of the messenger RNA and the fidelity with which it can be reverse transcribed. RNA cannot be cloned directly; in a reaction catalyzed by reverse transcriptase, the RNA, together with a suitable primer and a supply of deoxyribonucleoside triphosphates (dNTPs), must be converted to a double-stranded molecule. The product contains a complementary strand (first, antisense, or minus-strand cDNA) that is hybridized to what remains of the original RNA template. Such DNA-RNA hybrids can be cloned albeit often with lower efficiency than their double-stranded DNA counterparts. Usually the hybrid molecules are treated as intermediates in a scheme aimed at replacing the fragmented RNA with continuous DNA to form a double-stranded cDNA molecule. From this brief summary of cDNA cloning, it should be obvious that, regardless of the strategy, reverse transcriptase does and how it does it in vitro is discussed

  2. GENE EXPRESSION IN THE TESTES OF NORMOSPERMIC VERSUS TERATOSPERMIC DOMESTIC CATS USING HUMAN CDNA MICROARRAY ANALYSES

    Science.gov (United States)

    GENE EXPRESSION IN THE TESTES OF NORMOSPERMIC VERSUS TERATOSPERMIC DOMESTIC CATS USING HUMAN cDNA MICROARRAY ANALYSESB.S. Pukazhenthi1, J. C. Rockett2, M. Ouyang3, D.J. Dix2, J.G. Howard1, P. Georgopoulos4, W.J. J. Welsh3 and D. E. Wildt11Department of Reproductiv...

  3. cDNA cloning and primary structure analysis of invariant chain in ...

    African Journals Online (AJOL)

    cDNA cloning and primary structure analysis of invariant chain in Chinese Pengze crucian carp. X Liu, W Yu, J Li, F Chen, S Liu, C Wu, J Xu. Abstract. Invariant chain (Ii) plays an important role in MHC class II molecules assembly and exogenous peptide presentation in vertebrates. Although mammalian Ii has been ...

  4. Molecular cloning of growth hormone encoding cDNA of Indian ...

    Indian Academy of Sciences (India)

    Unknown

    Evans and Long 1921) and the human growth hormone (GH) encoding cDNA was per- haps the first to be isolated and characterized (Li and. Evans 1944). GH, chorionic somatomamotropin (placental lactogen) and prolactin (PRL) are all a family of ...

  5. Construction and Screening of an Expression cDNA Library from the Triactinomyxon Spores of Myxobolus cerebralis, the causative agent of Salmonid Whirling Diseases

    OpenAIRE

    Soliman, Hatem Mohamed Touhan

    2005-01-01

    The ZAP Express cDNA library was constructed using mRNA extracted from the triactinomyxon spores. First-strand cDNA was synthesized using Moloney Murine leukaemia virus reverse transcriptase. Following second-strand cDNA synthesis, the double-stranded cDNA was digested with Xho I restriction enzyme, cDNA fragments less than 400bp were removed and the remaining cDNA was ligated with the lambda ZAP Express vector. The recombinants were packaged in vitro using Gigapack III gold packaging extract...

  6. KLONING cDNA HORMON PERTUMBUHAN DARI IKAN GURAME (Osphronemus gouramy

    Directory of Open Access Journals (Sweden)

    Estu Nugroho

    2016-11-01

    Full Text Available Penelitian mengenai kloning cDNA pengkode hormon pertumbuhan ikan gurame telah dilakukan. Tujuan dari penelitian ini adalah untuk memperoleh sekuens DNA komplemen hormon pertumbuhan sebagai langkah awal dalam rangka pengembangan teknologi rekayasa genetik ikan gurame. Empat buah kelenjar hifopisa ikan gurame digunakan sebagai bahan bakunya dan dilakukan proses ekstraksi RNA total dari kelenjar hipofisa, dilanjutkan dengan sintesis cDNA, amplifikasi PCR, purifikasi fragmen DNA dari gel, ligasi produk PCR dengan vektor kloning, transformasi dan inkubasi bakteri, seleksi koloni bakteri putih, isolasi plasmid, dan sekuensing. Hasil sekuensing menunjukkan bahwa panjang produk amplifikasi PCR adalah 843 bp yang menyandikan 204 asam amino residu dan mengandung sekuens-sekuens yang konserf untuk gen hormon pertumbuhan (GH. Analisis homologi menunjukkan kesamaan sekuens hasil isolasi antara 52,4%--97,6% dengan gen GH ikan lainnya, dengan persentase homologi tertinggi adalah dengan ikan sepat. Dengan demikian dapat disimpulkan bahwa sekuens hasil isolasi merupakan sekuens gen GH. Dari hasil analisis sekuens terlihat bahwa gen GH ikan gurame secara evolusi adalah konserf. Research on cDNA cloning encoded the gouramy growth hormone was conducted. The aim of the research was to get complementary DNA, cDNA, sequences of growth hormone as an initial step to develop genetic engineering of gouramy fish. Four pituitary glands of the gouramy were taken and then processed with total RNA extraction, and continued with cDNA synthesis, PCR amplification, DNA fragment purification from the gel, PCR product legation with cloning vector, transformation and incubation of bacteria, white colony bacteria selection, plasmid isolation and sequencing analysis. Sequencing result showed that the amplified PCR product length had 834 bp, encoding 204 amino acid residue and contained conserve sequence for GH (growth hormone gen. Homolog analysis showed sequence similarity of

  7. Follicle cell trypsin-like protease HrOvochymase: Its cDNA cloning, localization, and involvement in the late stage of oogenesis in the ascidian Halocynthia roretzi.

    Science.gov (United States)

    Mino, Masako; Sawada, Hitoshi

    2016-04-01

    We previously reported that the sperm trypsin-like protease HrAcrosin and its precursor HrProacrosin participate in fertilization of the ascidian Halocynthia roretzi. The HrProacrosin gene is annotated in the H. roretzi genome database as Harore.CG.MTP2014.S89.g15383; our previously reported sequence of HrProacrosin gene appeared to include four nucleotides inserted near the 3'-end of HrProacrosin, resulting in a frame-shift mutation and a premature termination codon. The gene architecture of HrProacrosin and Harore.CG.MTP2014.S89.g15383 resembles that of Xenopus laevis ovochymase-1/OVCH1 and ovochymase-2/OVCH2, which encode egg extracellular polyproteases. Considering these new observations, we evaluated the cDNA cloning, expression, localization, and function of Harore.CG.MTP2014.S89.g15383, herein designated as HrOvochymase/HrOVCH. We found that HrOVCH cDNA consists of a single open reading frame of 1,575 amino acids, containing a signal peptide, three trypsin-like protease domains, and six CUB domains. HrOVCH was transcribed by the testis and ovary, but the majority of protein exists in ovarian follicle cells surrounding eggs. An anti-HrOVCH antibody inhibited elevation of the vitelline coat at a late stage of oogenesis, during the period when self-sterility is acquired. As trypsin inhibitors are reported to block the acquisition of self-sterility during oogenesis, whereas trypsin induces the acquisition of self-sterility and elevation of the vitelline coat in defolliculated ovarian eggs, we propose that HrOVCH may play a role in the acquisition of self-sterility by late-stage H. roretzi oocytes. © 2016 Wiley Periodicals, Inc.

  8. Construction of cDNA library from intestine, mesentery and coelomocyte of Apostichopus japonicus Selenka infected with Vibrio sp. and a preliminary analysis of immunity-related genes

    Science.gov (United States)

    Liu, Hongzhan; Zheng, Fengrong; Sun, Xiuqin; Cai, Yimei

    2012-06-01

    The aquaculture of sea cucumber Apostichopus japonicus (Echinodermata, Holothuroidea) has grown rapidly during recent years and has become an important sector of the marine industry in Northern China. However, with the rapid growth of the industry and the use of non-standard culture techniques, epidemic diseases of A. japonicus now pose increasing problems to the industry. To screen the genes with stress response to bacterial infection in sea cucumber at a genome wide level, we constructed a cDNA library from A. japonicus Selenka (Aspidochirotida: Stichopodidae) after infecting them with Vibrio sp. for 48 h. Total RNA was extracted from the intestine, mesentery and coelomocyte of infected sea cucumber using Trizol and mRNA was isolated by Oligotex mRNA Kits. The ligated cDNAs were transformed into DH5α, and a library of 3.24×105 clones (3.24×105 cfu mL-1) was obtained with the sizes of inserted fragments ranging from 0.8 to 2.5 kb. Sequencing the cDNA clones resulted in a total of 1106 ESTs that passed the quality control. BlastX and BlastN searches have identified 168 (31.5%) ESTs sharing significant homology with known sequences in NCBI protein or nucleotide databases. Among a panel of 25 putative immunity-related genes, serum lectin isoform, complement component 3, complement component 3-like genes were further studied by real-time PCR and they all increased more than 5 fold in response to Vibrio sp. challenge. Our library provides a valuable molecular tool for future study of invertebrate immunity against bacterial infection and our gene expression data indicates the importance of the immune system in the evolution and development of sea cucumber.

  9. Experimental testing of exchangeable cutting inserts cutting ability

    OpenAIRE

    Čep, Robert; Janásek, Adam; Čepová, Lenka; Petrů, Jana; Hlavatý, Ivo; Car, Zlatan; Hatala, Michal

    2013-01-01

    The article deals with experimental testing of the cutting ability of exchangeable cutting inserts. Eleven types of exchangeable cutting inserts from five different manufacturers were tested. The tested cutting inserts were of the same shape and were different especially in material and coating types. The main aim was both to select a suitable test for determination of the cutting ability of exchangeable cutting inserts and to design such testing procedure that could make it possible...

  10. Gene discovery from Jatropha curcas by sequencing of ESTs from normalized and full-length enriched cDNA library from developing seeds

    Directory of Open Access Journals (Sweden)

    Sugantham Priyanka Annabel

    2010-10-01

    Full Text Available Abstract Background Jatropha curcas L. is promoted as an important non-edible biodiesel crop worldwide. Jatropha oil, which is a triacylglycerol, can be directly blended with petro-diesel or transesterified with methanol and used as biodiesel. Genetic improvement in jatropha is needed to increase the seed yield, oil content, drought and pest resistance, and to modify oil composition so that it becomes a technically and economically preferred source for biodiesel production. However, genetic improvement efforts in jatropha could not take advantage of genetic engineering methods due to lack of cloned genes from this species. To overcome this hurdle, the current gene discovery project was initiated with an objective of isolating as many functional genes as possible from J. curcas by large scale sequencing of expressed sequence tags (ESTs. Results A normalized and full-length enriched cDNA library was constructed from developing seeds of J. curcas. The cDNA library contained about 1 × 106 clones and average insert size of the clones was 2.1 kb. Totally 12,084 ESTs were sequenced to average high quality read length of 576 bp. Contig analysis revealed 2258 contigs and 4751 singletons. Contig size ranged from 2-23 and there were 7333 ESTs in the contigs. This resulted in 7009 unigenes which were annotated by BLASTX. It showed 3982 unigenes with significant similarity to known genes and 2836 unigenes with significant similarity to genes of unknown, hypothetical and putative proteins. The remaining 191 unigenes which did not show similarity with any genes in the public database may encode for unique genes. Functional classification revealed unigenes related to broad range of cellular, molecular and biological functions. Among the 7009 unigenes, 6233 unigenes were identified to be potential full-length genes. Conclusions The high quality normalized cDNA library was constructed from developing seeds of J. curcas for the first time and 7009 unigenes coding

  11. Atomistic Galois insertions for flow sensitive integrity

    DEFF Research Database (Denmark)

    Nielson, Flemming; Nielson, Hanne Riis

    2017-01-01

    Several program verification techniques assist in showing that software adheres to the required security policies. Such policies may be sensitive to the flow of execution and the verification may be supported by combinations of type systems and Hoare logics. However, this requires user assistance...... and to obtain full automation we shall explore the over-approximating nature of static analysis. We demonstrate that the use of atomistic Galois insertions constitutes a stable framework in which to obtain sound and fully automatic enforcement of flow sensitive integrity. The framework is illustrated...... on a concurrent language with local storage and polyadic synchronous communication....

  12. Artists's Conception of Cassini Saturn Orbit Insertion

    Science.gov (United States)

    2002-01-01

    This is an artists concept of Cassini during the Saturn Orbit Insertion (SOI) maneuver, just after the main engine has begun firing. The spacecraft is moving out of the plane of the page and to the right (firing to reduce its spacecraft velocity with respect to Saturn) and has just crossed the ring plane.The SOI maneuver, which is approximately 90 minutes long, will allow Cassini to be captured by Saturn's gravity into a five-month orbit.Cassini's close proximity to the planet after the maneuver offers a unique opportunity to observe Saturn and its rings at extremely high resolution.

  13. Reflex sympathetic dystrophy following pacemaker insertion.

    Science.gov (United States)

    Londhey, Vikram A; Singh, Nishant; Kini, Seema

    2011-09-01

    A 55 year old male presented with pain and swelling over dorsum of right hand and small joints, and loss of sweating over right hand since two months. He was a known case of mitral valve prolapse (MVP) with mitral regurgitation and complete heart block for which pacemaker was implanted 1 year back. Bilateral wrist X-ray was suggestive of pronounced demineralization (osteopenia) in the right hand. He was thus diagnosed to have reflex sympathetic dystrophy syndrome (RSDS) considered to be induced by pacemaker insertion. After treatment with amitryptiline and indomethacin his symptoms dramatically improved.

  14. Compiler-assisted static checkpoint insertion

    Science.gov (United States)

    Long, Junsheng; Fuchs, W. K.; Abraham, Jacob A.

    1992-01-01

    This paper describes a compiler-assisted approach for static checkpoint insertion. Instead of fixing the checkpoint location before program execution, a compiler enhanced polling mechanism is utilized to maintain both the desired checkpoint intervals and reproducible checkpoint 1ocations. The technique has been implemented in a GNU CC compiler for Sun 3 and Sun 4 (Sparc) processors. Experiments demonstrate that the approach provides for stable checkpoint intervals and reproducible checkpoint placements with performance overhead comparable to a previously presented compiler assisted dynamic scheme (CATCH) utilizing the system clock.

  15. Lithium Insertion Chemistry of Some Iron Vanadates

    Energy Technology Data Exchange (ETDEWEB)

    Patoux, Sebastien; Richardson, Thomas J.

    2007-02-02

    Lithium insertion into various iron vanadates has been investigated. Fe{sub 2}V{sub 4}O{sub 13} and Fe{sub 4}(V{sub 2}O{sub 7}){sub 3} {center_dot} 3H{sub 2}O have discharge capacities approaching 200 mAh/g above 2.0 V vs. Li{sup +}/Li. Although the potential profiles change significantly between the first and subsequent discharges, capacity retention is unexpectedly good. Other phases, structurally related to FeVO{sub 4}, containing copper and/or sodium ions were also studied. One of these, {beta}-Cu{sub 3}Fe{sub 4}(VO{sub 4}){sub 6}, reversibly consumes almost 10 moles of electrons per formula unit (ca. 240 mAh g{sup -1}) between 3.6 and 2.0 V vs. Li{sup +}/Li, in a non-classical insertion process. It is proposed that both copper and vanadium are electrochemically active, whereas iron(III) reacts to form LiFe{sup III}O{sub 2}. The capacity of the Cu{sub 3}Fe{sub 4}(VO{sub 4}){sub 6}/Li system is nearly independent of cycling rate, stabilizing after a few cycles at 120-140 mAh g{sup -1}. Iron vanadates exhibit better capacities than their phosphate analogues, whereas the latter display more constant discharge potentials.

  16. Safety analysis for reactivity insertion on ADS

    Energy Technology Data Exchange (ETDEWEB)

    Santos, Marcia S.; Velasquez, Carlos E.; Pereira, Claubia; Veloso, Maria Auxiliadora F.; Costa, Antonella L. [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Departamento de Engenharia Nuclear; Barros, Graiciany de P. [Comissão Nacional de Energia Nuclear (CNEN), Rio de Janeiro, RJ (Brazil)

    2017-07-01

    In this work, an accelerator driven system (ADS) has been studied to fuel regeneration, partitioning and transmutation using fuel removed from LWR-spent fuel. This spent fuel was reprocessed by GANEX and then spiked with thorium. Monteburns 2.0 (MCNP5/ORIGEN 2.1) code was used to simulate burnup and neutronic parameters of the systems. However, these systems might be interrupted for some technical problems during few days or long periods to perform maintenance of accelerator systems without shutdown the fission system. Therefore, in this work, the aim was to investigate the nuclear fuel evolution in three different cases with reactivity insertions and variations in the functionality of the ADS. The evaluation has been performed for a burnup of 3 years, which are the most dangerous years in case of reactivity insertions. Therefore, in the first case the ADS during its burnup the external source (spallation source) is cutting off from the system several times during the first year of burnup. In the second case, it is simulated the reactor at different power from 0 (shutdown) to 1.5 times the 515MWt. The results shows the safety limits of the ADS for different situation. (author)

  17. Polymeric Biodegradable Stent Insertion in the Esophagus

    Directory of Open Access Journals (Sweden)

    Kai Yang

    2016-04-01

    Full Text Available Esophageal stent insertion has been used as a well-accepted and effective alternative to manage and improve the quality of life for patients diagnosed with esophageal diseases and disorders. Current stents are either permanent or temporary and are fabricated from either metal or plastic. The partially covered self-expanding metal stent (SEMS has a firm anchoring effect and prevent stent migration, however, the hyperplastic tissue reaction cause stent restenosis and make it difficult to remove. A fully covered SEMS and self-expanding plastic stent (SEPS reduced reactive hyperplasia but has a high migration rate. The main advantage that polymeric biodegradable stents (BDSs have over metal or plastic stents is that removal is not require and reduce the need for repeated stent insertion. But the slightly lower radial force of BDS may be its main shortcoming and a post-implant problem. Thus, strengthening support of BDS is a content of the research in the future. BDSs are often temporarily effective in esophageal stricture to relieve dysphagia. In the future, it can be expect that biodegradable drug-eluting stents (DES will be available to treat benign esophageal stricture, perforations or leaks with additional use as palliative modalities for treating malignant esophageal stricture, as the bridge to surgery or to maintain luminal patency during neoadjuvant chemoradiation.

  18. [Construction of subtractive cDNA libraries of the sporogony stage of Eimeria tenella by suppression subtractive hybridization].

    Science.gov (United States)

    Han, Hong-Yu; Lin, Jiao-Jiao; Zhao, Qi-Ping; Dong, Hui; Jiang, Lian-Lian; Wang, Xin; Han, Jing-Fang; Huang, Bing

    2007-11-01

    In order to clone and identify differentially expressed genes in the sporogony stage of Eimeria tenella, the cDNAs from unsporulated oocysts and sporulated oocysts of E. tenella were used as driver, respectively, the cDNAs from sporozoites of E. tenella was used tester, Two subtractive cDNA libraries of sporozoites were constructed by using the technique of suppression subtractive hybridization (SSH). the cDNAs from unsporulated oocysts was used driver, the cDNAs from sporulated ooceysts was used tester, one subtractive cDNA library of sporulated oocysts was constructed. PCR amplification revealed that the two subtractive cDNA libraries of sporozoites and one subtractive cDNA library of sporulated oocysts contained approximated 96%, 96% and 98% recombinant clones, respectively. Fifty positive clones were sequenced and analyzed in GenBank with Blast search from three subtractive cDNA libraries, respectively, thirteen unique sequences were found from the subtractive cDNA library of sporulated oocysts, eight ESTs shared significant identity with previously described. A total of forty unique sequences were obtained from the two subtractive cDNA libraries, nine ESTs shared significant identity with previously described, the other sequences represent novel genes of E. tenella with no significant homology to the proteins in Genbank. These results have provided the foundation for cloning new genes of E. tenella and further studying new approaches to control coccidiosis.

  19. [Cloning and sequence analysis of Eg95 cDNA from different stages of Echinococcus granulosus in Xinjiang].

    Science.gov (United States)

    Lin, Ren-yong; Ding, Jian-bing; Wen, Hao; Zhang, Wen-bao; Li, Jun; Lu, Xiao-mei

    2003-01-01

    To study expression and sequence differences of Echinococcus granulosus 95(Eg95) antigen cDNA from different stages of protoscolex, oncosphere and adult worm of E. granulosus from Xinjiang Uighur Aut. Reg. In accordance with the sequence of Eg95 antigen cDNA, the primers of Eg95 were designed. Eg95 antigen cDNAs were amplified by PCR from protoscolex, oncosphere and adult worm cDNA libraries of E. granulosus, respectively and were cloned into pUCm-T plasmid, and sequenced. The sequences were analyzed by DNAman and GenBank/BLAST biosoftware. PCR results showed that Eg95 antigen cDNA was amplified from three stages of E. granulosus cDNA libraries. Sequencing analysis indicated that the Eg95 cDNA length was 402 bp, same as the reported data in GenBank. The Eg95 antigen cDNA was expressed in the different life-cycle stages of E. granulosus in Xinjiang and there was no nucleic acid sequence difference of Eg95 antigen among the protoscolex, oncosphere and adult worm of E. granulosus.

  20. Isolation and characterization of sequences homologous to the tobacco clone axi 1 (auxin independent) from a Vicia sativa nodule cDNA library

    NARCIS (Netherlands)

    Yalçin-Mendi, Y.; Çetiner, S.; Bisseling, T.

    2001-01-01

    In this research, partial nucleotide sequences of the axi 1 gene, which is related to auxin perception and transduction, isolated from Vicia sativa using cDNA library screening were investigated. Four V. sativa cDNA clones representing homologous of the tobacco axi 1 (auxin independent) cDNA clone

  1. cDNA cloning of a major allergen from timothy grass (Phleum pratense) pollen; characterization of the recombinant Phl pV allergen

    NARCIS (Netherlands)

    Vrtala, S.; Sperr, W. R.; Reimitzer, I.; van Ree, R.; Laffer, S.; Müller, W. D.; Valent, P.; Lechner, K.; Rumpold, H.; Kraft, D.

    1993-01-01

    We isolated a cDNA encoding a major grass pollen allergen from a timothy grass (Phleum pratense) pollen expression cDNA library using allergic patients' IgE. The complete cDNA encoded an allergen that binds IgE from about 80% of grass pollen-allergic patients. Significant sequence homology was found

  2. 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.

  3. Cloning and expression of full-length cDNA encoding human vitamin D receptor

    Energy Technology Data Exchange (ETDEWEB)

    Baker, A.R.; McDonnell, D.P.; Hughes, M.; Crisp, T.M.; Mangelsdorf, D.J.; Haussler, M.R.; Pike, J.W.; Shine, J.; O' Malley, B.W. (California Biotechnology Inc., Mountain View (USA))

    1988-05-01

    Complementary DNA clones encoding the human vitamin D receptor have been isolated from human intestine and T47D cell cDNA libraries. The nucleotide sequence of the 4605-base pair (bp) cDNA includes a noncoding leader sequence of 115 bp, a 1281-bp open reading frame, and 3209 bp of 3{prime} noncoding sequence. Two polyadenylylation signals, AATAAA, are present 25 and 70 bp upstream of the poly(A) tail, respectively. RNA blot hybridization indicates a single mRNA species of {approx} 4600 bp. Transfection of the cloned sequences into COS-1 cells results in the production of a single receptor species indistinguishable from the native receptor. Sequence comparisons demonstrate that the vitamin D receptor belongs to the steroid-receptor gene family and is closest in size and sequence to another member of this family, the thyroid hormone receptor.

  4. Construction and analysis of full-length cDNA library of Cryptosporidium parvum.

    Science.gov (United States)

    Yamagishi, Junya; Wakaguri, Hiroyuki; Sugano, Sumio; Kawano, Suguru; Fujisaki, Kozo; Sugimoto, Chihiro; Watanabe, Junichi; Suzuki, Yutaka; Kimata, Isao; Xuan, Xuenan

    2011-06-01

    A full-length cDNA library was constructed from the sporozoite of Cryptosporidium parvum. Normalized clones were subjected to Solexa shotgun sequencing, and then complete sequences for 1066 clones were reconfigured. Detailed analyses of the sequences revealed that 13.5% of the transcripts were spliced; the average and median 5' UTR lengths were 213.5 and 122 nucleotides, respectively. There were 148 inconsistencies out of 562 examined genes between the experimentally described cDNA sequence and the predicted sequence from its genome. In addition, we identified 118 sequences that had little homology against annotated genes of C. parvum as prospective candidates for addable genes. These observations should improve the reliability of C. parvum transcriptome and provide a versatile resource for further studies. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  5. Peptidomics combined with cDNA library unravel the diversity of centipede venom

    DEFF Research Database (Denmark)

    Rong, Mingqiang; Yang, Shilong; Wen, Bo

    2015-01-01

    the extensive diversity of centipede toxins and provide powerful tools to understand the capture and defense weapon of centipede. BIOLOGICAL SIGNIFICANCE: Peptide toxins from venomous animal have attracted increasing attentions due to their extraordinary chemical and pharmacological diversity. Centipedes......UNLABELLED: Centipedes are one of the oldest venomous arthropods using toxin as their weapon to capture prey. But little attention was focused on them and only few centipede toxins were demonstrated with activity on ion channels. Therefore, more deep works are needed to understand the diversity...... of centipede venom. In the present study, we use peptidomics combined with cDNA library to uncover the diversity of centipede Scolopendra subspinipes mutilans L. Koch. 192 peptides were identified by LC-MS/MS and 79 precursors were deduced by cDNA library. Surprisingly, the signal peptides of centipede toxins...

  6. Molecular cloning and characterization of cDNA encoding fibrinolytic enzyme-3 from earthworm Eisenia foetida.

    Science.gov (United States)

    Dong, Guo-Qing; Yuan, Xiao-Ling; Shan, Ya-Jun; Zhao, Zhen-Hu; Chen, Jia-Pei; Cong, Yu-Wen

    2004-04-01

    The earthworm fibrinolytic enzyme-3 (EFE-3, GenBank accession No: AY438622), from the earthworm Eisenia foetida, is a component of earthworm fibrinolytic enzymes. In this study, cDNA encoding the EFE-3 was cloned by RT-PCR. The cDNA contained an open reading frame of 741 nucleotides, which encoded a deduced protein of 247 amino acid residues, including signal sequences. EFE-3 showed a high degree of homology to earthworm (Lumbricus rebullus) proteases F-III-1, F-III-2, and bovine trypsin. The recombinant EFE-3 was expressed in E. coli as inclusion bodies, and the gene encoding the native form of EFE-3 was expressed in COS-7 cells in the medium. Both the refolding product of inclusion bodies and the secreted protease could dissolve the artificial fibrin plate.

  7. Consistent errors in first strand cDNA due to random hexamer mispriming.

    Directory of Open Access Journals (Sweden)

    Thomas P van Gurp

    Full Text Available Priming of random hexamers in cDNA synthesis is known to show sequence bias, but in addition it has been suggested recently that mismatches in random hexamer priming could be a cause of mismatches between the original RNA fragment and observed sequence reads. To explore random hexamer mispriming as a potential source of these errors, we analyzed two independently generated RNA-seq datasets of synthetic ERCC spikes for which the reference is known. First strand cDNA synthesized by random hexamer priming on RNA showed consistent position and nucleotide-specific mismatch errors in the first seven nucleotides. The mismatch errors found in both datasets are consistent in distribution and thermodynamically stable mismatches are more common. This strongly indicates that RNA-DNA mispriming of specific random hexamers causes these errors. Due to their consistency and specificity, mispriming errors can have profound implications for downstream applications if not dealt with properly.

  8. Molecular characterization of MHC-DRB cDNA in water buffalo (Bubalus bubalis

    Directory of Open Access Journals (Sweden)

    Soumen Naskar

    2012-01-01

    Full Text Available In the present study, water buffalo MHC (Bubu-DRB cDNA was cloned and characterized. The 1022 base long-amplified cDNA product encompassed a single open reading frame of 801 bases that coded for 266 amino acids. The Bubu-DRB sequence showed maximum homology with the BoLA-DRB3*0101 allele of cattle. A total of seven amino acid residues were found to be unique for the Bubu-DRB sequence. The majority of amino acid substitutions was observed in the β1 domain. Residues associated with important functions were mostly conserved. Water buffalo DRB was phylogenetically closer to goat DRB*A.

  9. Reverse transcription using random pentadecamer primers increases yield and quality of resulting cDNA

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Dufva, I.H.; Dufva, Hans Martin

    2006-01-01

    Reverse transcription of RNA is an invaluable method for gene expression analysis by real-time PCR or microarray methods. Random primers of varying lengths were compared with respect to their efficiency of priming reverse transcription reactions. The results showed that l5-nucleotide-long random...... that random pentadecamers can replace random hexamers in reverse transcription reactions on both poly(A) RNA and amplified RNA, resulting in higher cDNA yields and quality....... with cDNA generated with random hexamers. The increased efficiency of priming using random pentadecamers resulted in reverse transcription of > 80% of the template aRNA, while random hexamers induced reverse transcription of only 40% of the template aRNA. This suggests a better coverage...

  10. A piezoelectric vibration-based syringe for reducing insertion force

    Science.gov (United States)

    Huang, Y. C.; Tsai, M. C.; Lin, C. H.

    2012-12-01

    Puncturing of the human skin with a needle is perhaps the most common invasive medical procedure. Clinical studies have revealed that tissue deformation and needle deflection are the primary problem for needle misplacement in percutaneous procedures. To avoid this, various techniques for reducing insertion forces during needle insertion have been considered. This paper presents a piezoelectric vibration-based syringe to reduce insertion force. AC power was applied to the piezoelectric elements to vibrate the needle with high frequency and thereby reduce the friction and cutting forces between the needle and tissue. Vibration mode shapes of the needle were observed by finite element analysis and verified by experimental results. Effects of reducing insertion force via the vibrating needle were also confirmed by inserting the needle into the porcine tissues. The proposed syringe, which minimizes the insertion force and overcomes limitations of needle materials, can be widely utilized in robot-assisted needle insertion systems.

  11. Attenuation and cell culture adaptation of hepatitis A virus (HAV): a genetic analysis with HAV cDNA.

    OpenAIRE

    Cohen, J I; Rosenblum, B; Feinstone, S M; Ticehurst, J; Purcell, R H

    1989-01-01

    RNA transcripts of hepatitis A virus (HAV) HM-175 cDNA from attenuated, cell culture-adapted HAV were infectious in cell culture. A full-length HAV cDNA from wild-type HAV (propagated in marmosets in vivo) was constructed. Chimeric cDNAs that contained portions of both wild-type and attenuated genomes were produced. Oligonucleotide-directed mutagenesis was used to engineer a point mutation into the VP1 gene of attenuated HAV cDNA, so that the sequence of this capsid protein would be identical...

  12. Identification of differential genes by suppression subtractive hybridization: I. Preparation of subtracted cDNA or genomic DNA library.

    Science.gov (United States)

    Rebrikov, Denis V

    2008-07-01

    INTRODUCTIONSuppression subtractive hybridization (SSH) is one of the most powerful and popular methods for generating subtracted cDNA or genomic DNA libraries. This technique can be used to compare two mRNA populations and obtain cDNAs representing genes that are either overexpressed or exclusively expressed in one population as compared to another. It can also be used for comparison of genomic DNA populations. This protocol describes the preparation of a subtracted cDNA or genomic DNA library, and includes methods for cDNA synthesis, tester and driver DNA digestion, and adapter ligation.

  13. Construction of an infectious cDNA clone of foot-and-mouth disease ...

    Indian Academy of Sciences (India)

    Prakash

    Foot-and-mouth disease virus (FMDV) serotype O is the most predominant among the endemic serotypes in India. A stable, full-length cDNA clone of FMDV type O1BFS 1860 preceded by a bacteriophage T7 polymerase promoter was assembled in a plasmid vector pGEMR-7Zf(–). An ~8.2 kb PCR product was amplified ...

  14. cDNA cloning and expression analysis of a mannose-binding lectin ...

    Indian Academy of Sciences (India)

    Pinellia pedatisecta agglutinin (PPA) is a very basic protein that accumulates in the tuber of P. pedatisecta. PPA is a hetero-tetramer protein of 40 kDa, composed of two polypeptide chains A (about 12 kDa) and two polypeptides chains B (about 12 kDa). The full-length cDNA of PPA was cloned from P. pedatisecta using ...

  15. Coordinate regulation of stromelysin and collagenase genes determined with cDNA probes

    International Nuclear Information System (INIS)

    Frisch, S.M.; Clark, E.J.; Werb, Z.

    1987-01-01

    Secreted proteinases are required for tumor metastasis, angiogenesis, and tissue remodeling during wound healing and embryonic growth. Thus, the regulation of the genes of secreted proteinases may serve as an interesting model for growth-controlled genes in general. The authors studied the genes of the secreted proteinases stromelysin and collagenase by using molecularly cloned cDNAs from each proteinase. Stromelysin cDNA was cloned by differential screening of a total cDNA library from rabbit synovial cells treated with phorbol 12-myristate 13-acetate, which yielded a clone of 1.2 kilobase pairs; collagenase cDNA was obtained by cloning reverse transcripts of anti-collagenase-immunoadsorbed polysomal mRNA, which yielded a clone of 0.8 kilobase pairs. Stromelysin and collagenase mRNA species of 2.2 and 2.4 kilobases, respectively, were detected on hybridization blots of RNA from phorbol 12-myristate 13-acetate-treated but not untreated rabbit synovial cells. Expression of stromelysin mRNA was also induced in rabbit alveolar macrophages and rabbit brain capillary endothelial cells treated with phorbol 12-myristate 13-acetate. Stromelysin and collagenase mRNA were both induced by phorbol 12-myristate 13-acetate and cytochalasin B at a constant ratio of the two gene products; this suggest coordinate regulation. The fact that induction was blocked after inhibition of protein synthesis by cycloheximide implicates an indirect signal transduction pathway that requires new protein synthesis

  16. Screening for candidate genes related to breast cancer with cDNA microarray analysis

    Directory of Open Access Journals (Sweden)

    Yu-Juan Xiang

    2015-06-01

    Full Text Available Objective: The aim of this study was to reveal the exact changes during the occurrence of breast cancer to explore significant new and promising genes or factors related to this disease. Methods: We compared the gene expression profiles of breast cancer tissues with its uninvolved normal breast tissues as controls using the cDNA microarray analysis in seven breast cancer patients. Further, one representative gene, named IFI30, was quantitatively analyzed by real-time PCR to confirm the result of the cDNA microarray analysis. Results: A total of 427 genes were identified with significantly differential expression, 221 genes were up-regulated and 206 genes were down-regulated. And the result of cDNA microarray analysis was validated by detection of IFI30 mRNA level changes by real-time PCR. Genes for cell proliferation, cell cycle, cell division, mitosis, apoptosis, and immune response were enriched in the up-regulated genes, while genes for cell adhesion, proteolysis, and transport were significantly enriched in the down-regulated genes in breast cancer tissues compared with normal breast tissues by a gene ontology analysis. Conclusion: Our present study revealed a range of differentially expressed genes between breast cancer tissues and normal breast tissues, and provide candidate genes for further study focusing on the pathogenesis and new biomarkers for breast cancer. Keywords: Breast neoplasms, Candidate genes, Microarray

  17. A novel method using edge detection for signal extraction from cDNA microarray image analysis.

    Science.gov (United States)

    Kim, J H; Kim, H Y; Lee, Y S

    2001-06-30

    Gene expression analyses by probes of hybridization from mRNA to cDNA targets arrayed on membranes or activated glass surfaces have revolutionized the way of profiling mega level gene expression. The main remaining problems however are sensitivity of detection, reproducibility and data processing. During processing of microarray images, especially irregularities of spot position and shape could generate significant errors: small regions of signal spots can be mis-included into background area and vice versa. Here we report a novel method to eliminate such obstacles by sensing their edges. Application of edge detection technology on separating spots from the background decreases the probability of the errors and gives more accurate information about the states of spots such as the pixel number, degree of fragmentation, width and height of spot, and circumference of spot. Such information can be used for the quality control of cDNA microarray experiments and filtering of low quality spots. We analyzed the cDNA microarray image that contains 10,368 genes using edge detection and compared the result with that of conventional method which draws circle around the spot.

  18. 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.

  19. Cloning and characterization of human liver cDNA encoding a protein S precursor

    International Nuclear Information System (INIS)

    Hoskins, J.; Norman, D.K.; Beckmann, R.J.; Long, G.L.

    1987-01-01

    Human liver cDNA encoding a protein S precursor was isolated from two cDNA libraries by two different techniques. Based upon the frequency of positive clones, the abundance of mRNA for protein S is ≅ 0.01%. Blot hybridization of electrophoretically fractionated poly(A) + RNA revealed a major mRNA ≅ 4 kilobases long and two minor forms of ≅ 3.1 and ≅ 2.6 kilobases. One of the cDNA clones contains a segment encoding a 676 amino acid protein S precursor, as well as 108 and 1132 nucleotides of 5' and 3' noncoding sequence, respectively, plus a poly(A) region at the 3' end. The cDNAs are adenosine plus thymidine-rich (60%) except for the 5' noncoding region, where 78% of the nucleotides are guanosine or cytosine. The protein precursor consists of a 41 amino acid leader peptide followed by 635 amino acids corresponding to mature protein S. Comparison of the mature protein region with homologous vitamin K-dependent plasma proteins shows that it is composed of the following domains: an amino-terminal γ-carboxyglutamic acid-rich region of 37 amino acids; a 36 amino acid linker region rich in hydroxy amino acids; four epidermal growth factor-like segments, each ≅ 45 amino acids long; and a 387 amino acid carboxyl-terminal domain of unrecognized structure and unknown function

  20. Generation of longer 3' cDNA fragments from massively parallel signature sequencing tags.

    Science.gov (United States)

    Silva, Ana Paula M; Chen, Jianjun; Carraro, Dirce M; Wang, San Ming; Camargo, Anamaria A

    2004-07-06

    Massively Parallel Signature Sequencing (MPSS) is a powerful technique for genome-wide gene expression analysis, which, similar to SAGE, relies on the production of short tags proximal to the 3'end of transcripts. A single MPSS experiment can generate over 10(7) tags, providing a 10-fold coverage of the transcripts expressed in a human cell. A significant fraction of MPSS tags cannot be assigned to known transcripts (orphan tags) and are likely to be derived from transcripts expressed at very low levels (approximately 1 copy per cell). In order to explore the potential of MPSS for the characterization of the human transcriptome, we have adapted the GLGI protocol (Generation of Longer cDNA fragments from SAGE tags for Gene Identification) to convert MPSS tags into their corresponding 3' cDNA fragments. GLGI-MPSS was applied to 83 orphan tags and 41 cDNA fragments were obtained. The analysis of these 41 fragments allowed the identification of novel transcripts, alternative tags generated from polymorphic and alternatively spliced transcripts, as well as the detection of artefactual MPSS tags. A systematic large-scale analysis of the genome by MPSS, in combination with the use of GLGI-MPSS protocol, will certainly provide a complementary approach to generate the complete catalog of human transcripts.

  1. Generation of longer 3′ cDNA fragments from massively parallel signature sequencing tags

    Science.gov (United States)

    Silva, Ana Paula M.; Chen, Jianjun; Carraro, Dirce M.; Wang, San Ming; Camargo, Anamaria A.

    2004-01-01

    Massively Parallel Signature Sequencing (MPSS) is a powerful technique for genome-wide gene expression analysis, which, similar to SAGE, relies on the production of short tags proximal to the 3′end of transcripts. A single MPSS experiment can generate over 107 tags, providing a 10-fold coverage of the transcripts expressed in a human cell. A significant fraction of MPSS tags cannot be assigned to known transcripts (orphan tags) and are likely to be derived from transcripts expressed at very low levels (∼1 copy per cell). In order to explore the potential of MPSS for the characterization of the human transcriptome, we have adapted the GLGI protocol (Generation of Longer cDNA fragments from SAGE tags for Gene Identification) to convert MPSS tags into their corresponding 3′ cDNA fragments. GLGI-MPSS was applied to 83 orphan tags and 41 cDNA fragments were obtained. The analysis of these 41 fragments allowed the identification of novel transcripts, alternative tags generated from polymorphic and alternatively spliced transcripts, as well as the detection of artefactual MPSS tags. A systematic large-scale analysis of the genome by MPSS, in combination with the use of GLGI-MPSS protocol, will certainly provide a complementary approach to generate the complete catalog of human transcripts. PMID:15247327

  2. [Construction and analysis of suppression subtractive cDNA libraries of continuous monoculture Rehmannia glutinosa].

    Science.gov (United States)

    Zhang, Zhongyi; Fan, Huamin; Yang, Yanhui; Li, Mingjie; Li, Juan; Xu, Haixia; Chen, Junying; Chen, Xinjian

    2011-02-01

    To explore the molecular mechanism of continuous monoculture problem by constructing the cDNA libraries of continuous monoculture Rehmannia glutinosa. To use the suppression subtractive hybridization (SSH) technique to construct the forward and reverse subtractive cDNA libraries of continuous monoculture R. glutinosa to adopt blue-white colony screening and PCR to detect the positive clones which would be sequenced and analyzed by bioinformatics. The subtracted cDNA libraries of continuous monoculture R. glutinosa. were successfully constructed, and the result showed that the forward and reverse subtracted libraries obtained 300 positive clones, respectively. The forward and reverse libraries got different ESTs, and produced 232 (forward library) and 214 (reverse library) unique ESTs by sequencing. Based on homology search of BLASTX and BLASTN in NCBI, 200 and 195 of unique ESTs were homologous to known genes in the forward and reverse libraries, respectively. Categories of orthologous group (COG) showed that the forward and reverse libraries got 60 and 61 ESTs with the corresponding gene annotation, involving 21 metabolic pathways. The information of differential expression genes in continuous monoculture R. glutinosa, and their functional annotation of differentially expressed genes indicate that continuous monoculture has a profound effect on expression of the genes in R. glutinosa. Furthermore, the research analyzed several key genes in response to replant problem, which provided a foundation for revealing the molecular mechanism of continuous monoculture R. glutinosa.

  3. CONSTRUCTION AND APPLICATIONS OF A MYCORRHIZAL ARBUSCULAR SPECIFIC cDNA LIBRARY.

    Science.gov (United States)

    Isayenkov, S; Maathuis, F J M

    2016-01-01

    To exploit the potential benefits of mycorrhizas, we need to investigate the processes that occur in these symbiotic interactions, particularly in the arbuscular compartment where nutrients are exchanged between the plant and the fungus. Progress in this area is restricted due to the intricacy and complexity of this plant-fungus interface and many techniques that have been employed successfully in other plants and animal systems cannot be used. An effective approach to study processes in arbuscules is to examine transcript composition and dynamics. We applied laser capture microdissection (LCM) to isolate approximately 3000 arbuscules from Glomus intraradices colonised Me- dicago truncatula roots. Total RNA was extracted from microdissected arbuscules and subjected to T7 RNA polymerase-based linear amplification. Amplified RNA was then usedfor construction of a cDNA library. The presence and level of enrichment of mycorrhiza-specific transcripts was determined by quantitative Real-time and conventional PCR. To improve enrichment a cDNA library subtraction was performed. Complementation of yeast mutants deficient in the uptake of.potassium, phosphate, sulphate, amino acids, ammonium and of a Mn²⁺sensitive strain, demonstrates the functionality of our cDNA library.

  4. Method for RNA extraction and cDNA library construction from microbes in crop rhizosphere soil.

    Science.gov (United States)

    Fang, Changxun; Xu, Tiecheng; Ye, Changliang; Huang, Likun; Wang, Qingshui; Lin, Wenxiong

    2014-02-01

    Techniques to analyze the transcriptome of the soil rhizosphere are essential to reveal the interactions and communications between plants and microorganisms in the soil ecosystem. In this study, different volumes of Al₂(SO₄)₃ were added to rhizosphere soil samples to precipitate humic substances, which interfere with most procedures of RNA and DNA analyses. After humic substances were precipitated, cells of soil microorganisms were broken by vortexing with glass beads, and then DNA and RNA were recovered using Tris-HCl buffer with LiCl, SDS, and EDTA. The crude extract was precipitated and dissolved in RNAse-free water, and then separated by agarose gel electrophoresis. We determined the optimum volume of Al₂(SO₄)₃ for treating rhizosphere soil of rice, tobacco, sugarcane, Rehmannia glutinosa, and Pseudostellaria heterophylla. The crude nucleic acids extract from rice soil was treated with DNase I and then RNA was purified using a gel filtration column. The purified RNA was reverse-transcribed into single-strand cDNA and then ligated with an adaptor at each end before amplifying ds cDNA. The ds cDNA was sub-cloned for subsequent gene sequence analysis. We conducted qPCR to amplify 16S ribosomal DNA and observed highly efficient amplification. These results show that the extraction method can be optimized to isolate and obtain high-quality nucleic acids from microbes in different rhizosphere soils, suitable for genomic and post-genomic analyses.

  5. Effects of heavy metals on Drosophila larvae and a metallothionein cDNA

    Energy Technology Data Exchange (ETDEWEB)

    Maroni, G.; Lastowski-Perry, D.; Otto, E.; Watson, D.

    1986-03-01

    Drosophila melanogaster larvae reared on food containing radioactive cadmium retained over 80% of it, mostly in the intestinal epithelium. The majority of this radioactivity was associated with a soluble protein of less than 10,000 molecular weight. Synthesis of this cadmium-binding protein was induced by the metal as demonstrated by incorporation of radioactive cysteine. Most copper ingested by larvae was also found to associate with a low molecular weight, inducible protein, but some of it was found in an insoluble fraction. A D. melanogaster cDNA clone was isolated based on its more intense hybridization to copies of RNA sequences from copper-fed larvae than from control larvae. This clone showed strong hybridization to mouse metallothionein-I cDNA at reduced stringency. Its nucleotide sequence includes an open-reading segment which codes for a 40 amino acid protein; this protein was identified as metallothionein based on its similarity to the amino-terminal portion of mammalian and crab metalloproteins. The ten cysteine residues present occur in five pairs of near-vicinal cysteines (Cys-X-Cys). This cDNA sequence hybridized to a 400-nucleotide polyadenylated RNA whose presence in the cells of the alimentary canal of larvae was stimulated by ingestion of cadmium or copper; in other tissues this RNA was present at much lower levels.

  6. Sequencing of first-strand cDNA library reveals full-length transcriptomes.

    Science.gov (United States)

    Agarwal, Saurabh; Macfarlan, Todd S; Sartor, Maureen A; Iwase, Shigeki

    2015-01-21

    Massively parallel strand-specific sequencing of RNA (ssRNA-seq) has emerged as a powerful tool for profiling complex transcriptomes. However, many current methods for ssRNA-seq suffer from the underrepresentation of both the 5' and 3' ends of RNAs, which can be attributed to second-strand cDNA synthesis. The 5' and 3' ends of RNA harbour crucial information for gene regulation; namely, transcription start sites (TSSs) and polyadenylation sites. Here we report a novel ssRNA-seq method that does not involve second-strand cDNA synthesis, as we Directly Ligate sequencing Adaptors to the First-strand cDNA (DLAF). This novel method with fewer enzymatic reactions results in a higher quality of the libraries than the conventional method. Sequencing of DLAF libraries followed by a novel analysis pipeline enables the profiling of both 5' ends and polyadenylation sites at near-base resolution. Therefore, DLAF offers the first genomics tool to obtain the 'full-length' transcriptome with a single library.

  7. An efficient and sensitive method for preparing cDNA libraries from scarce biological samples.

    Science.gov (United States)

    Sterling, Catherine H; Veksler-Lublinsky, Isana; Ambros, Victor

    2015-01-01

    The preparation and high-throughput sequencing of cDNA libraries from samples of small RNA is a powerful tool to quantify known small RNAs (such as microRNAs) and to discover novel RNA species. Interest in identifying the small RNA repertoire present in tissues and in biofluids has grown substantially with the findings that small RNAs can serve as indicators of biological conditions and disease states. Here we describe a novel and straightforward method to clone cDNA libraries from small quantities of input RNA. This method permits the generation of cDNA libraries from sub-picogram quantities of RNA robustly, efficiently and reproducibly. We demonstrate that the method provides a significant improvement in sensitivity compared to previous cloning methods while maintaining reproducible identification of diverse small RNA species. This method should have widespread applications in a variety of contexts, including biomarker discovery from scarce samples of human tissue or body fluids. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. Chromosomal Localization of DNA Amplifications in Neuroblastoma Tumors Using cDNA Microarray Comparative Genomic Hybridization

    Directory of Open Access Journals (Sweden)

    Ben Beheshti

    2003-01-01

    Full Text Available Conventional comparative genomic hybridization (CGH profiling of neuroblastomas has identified many genomic aberrations, although the limited resolution has precluded a precise localization of sequences of interest within amplicons. To map high copy number genomic gains in clinically matched stage IV neuroblastomas, CGH analysis using a 19,200-feature cDNA microarray was used. A dedicated (freely available algorithm was developed for rapid in silico determination of chromosomal localizations of microarray cDNA targets, and for generation of an ideogram-type profile of copy number changes. Using these methodologies, novel gene amplifications undetectable by chromosome CGH were identified, and larger MYCN amplicon sizes (in one tumor up to 6 Mb than those previously reported in neuroblastoma were identified. The genes HPCAL1, LPIN1/KIAA0188, NAG, and NSE1/LOC151354 were found to be coamplified with MYCN. To determine whether stage IV primary tumors could be further subclassified based on their genomic copy number profiles, hierarchical clustering was performed. Cluster analysis of microarray CGH data identified three groups: 1 no amplifications evident, 2 a small MYCN amplicon as the only detectable imbalance, and 3 a large MYCN amplicon with additional gene amplifications. Application of CGH to cDNA microarray targets will help to determine both the variation of amplicon size and help better define amplification-dependent and independent pathways of progression in neuroblastoma.

  9. Isolation and characterization of cDNA clones for human erythrocyte β-spectrin

    International Nuclear Information System (INIS)

    Prchal, J.T.; Morley, B.J.; Yoon, S.H.; Coetzer, T.L.; Palek, J.; Conboy, J.G.; Kan, Y.W.

    1987-01-01

    Spectrin is an important structural component of the membrane skeleton that underlies and supports the erythrocyte plasma membrane. It is composed of nonidentical α (M/sub r/ 240,000) and β (M/sub r/ 220,000) subunits, each of which contains multiple homologous 106-amino acid segments. The authors report here the isolation and characterization of a human erythroid-specific β-spectrin cDNA clone that encodes parts of the β-9 through β-12 repeat segments. This cDNA was used as a hybridization probe to assign the β-spectrin gene to human chromosome 14 and to begin molecular analysis of the gene and its mRNA transcripts. RNA transfer blot analysis showed that the reticulocyte β-spectrin mRNA is 7.8 kilobases in length. Southern blot analysis of genomic DNA revealed the presence of restriction fragment length polymorphisms (RFLPs) within the β-spectrin gene locus. The isolation of human spectrin cDNA probes and the identification of closely linked RFLPs will facilitate analysis of mutant spectrin genes causing congenital hemolytic anemias associated with quantitative and qualitative spectrin abnormalities

  10. cDNA encoding a polypeptide including a hev ein sequence

    Energy Technology Data Exchange (ETDEWEB)

    Raikhel, Natasha V. (Okemos, MI); Broekaert, Willem F. (Dilbeek, BE); Chua, Nam-Hai (Scarsdale, NY); Kush, Anil (New York, NY)

    2000-07-04

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

  11. Insertion Device Upgrade Plans at the NSLS

    CERN Document Server

    Tanabe, Toshiya; Harder, David; Lehecka, Michael; Rakowsky, George; Skaritka, John

    2005-01-01

    This paper describes plans to upgrade insertion devices at the National Synchrotron Light Source (NSLS), Brookhaven National Laboratory, U.S.A. The aging wiggler (W80) at X25 is being replaced by a 1 m long in-vacuum mini-gap undulator (MGU-18) optimized for a dedicated macromolecular crystallography program. A new, 1/3 m long, in-vacuum undulator (MGU-13.5), will be installed between a pair of RF cavities at X9, and will serve a new beamline dedicated for small angle x-ray scattering (SAXS). Both MGU’s will have provision for cryocooling the NdFeB hybrid arrays to 150K to raise the field and K-value and to obtain better spectral coverage. Design issues of the devices and other considerations, especially magnetic measurement methods in low temperature will be discussed.

  12. Hand-actuated spring clip insertion tool

    International Nuclear Information System (INIS)

    Cuba, G.W.

    1993-01-01

    A hand-actuated insertion tool includes a handle assembly, an elongated hollow tubular outer support tube, an elongated inner pull rod, and a coupling arrangement. The handle assembly has a first handle member and a second handle member pivoted to a member for movement between unactuated and actuated positions. The tube is attached in a fixed relation to a handle member. The rod is mounted within the tube for sliding movement relative thereto between home and retracted positions. The coupling arrangement pivotally connects the rod to the second handle member such that the rod will undergo sliding movement from the home position to the retracted positions relative to the tube as the second handle member is moved from the unactuated position to the actuated position adjacent to the first handle member. (author)

  13. Impact of Orthodontic Decompensation on Bone Insertion

    Science.gov (United States)

    Guedes, Fabio Pinto; Capelozza Filho, Leopoldino; Garib, Daniela Gamba; Nary Filho, Hugo; Borgo, Evandro José; Cardoso, Mauricio de Almeida

    2014-01-01

    There has always been concern in determining the relationship between orthodontic tooth movement and the consequent biological costs to the periodontium and tooth root. The possibility of evaluating the tooth and bone morphology by CBCT allows more accurate analysis of qualitative and quantitative aspects of these processes. This paper presents a case report of a 20-year-old male patient with Class III malocclusion and hyperdivergent facial pattern, who was surgically treated. A significant amount of labial movement of mandibular incisors was performed during orthodontic treatment before surgery. CBCT was used for evaluation of buccal and lingual bone plates before and after tooth decompensation. The changes in the bone insertion level of maxillary and mandibular incisors in the present case encourage a reflection on the treatment protocol in individuals with dentoskeletal discrepancies. PMID:25436157

  14. Impact of Orthodontic Decompensation on Bone Insertion

    Directory of Open Access Journals (Sweden)

    Fabio Pinto Guedes

    2014-01-01

    Full Text Available There has always been concern in determining the relationship between orthodontic tooth movement and the consequent biological costs to the periodontium and tooth root. The possibility of evaluating the tooth and bone morphology by CBCT allows more accurate analysis of qualitative and quantitative aspects of these processes. This paper presents a case report of a 20-year-old male patient with Class III malocclusion and hyperdivergent facial pattern, who was surgically treated. A significant amount of labial movement of mandibular incisors was performed during orthodontic treatment before surgery. CBCT was used for evaluation of buccal and lingual bone plates before and after tooth decompensation. The changes in the bone insertion level of maxillary and mandibular incisors in the present case encourage a reflection on the treatment protocol in individuals with dentoskeletal discrepancies.

  15. Quench Performance of the LHC Insertion Magnets

    CERN Document Server

    Lasheras, N C; Siemko, A; Ostojic, R; Kirby, G

    2009-01-01

    After final installation in the LHC tunnel, the MQM and MQY quadrupole magnets of the LHC insertions are now being commissioned to their nominal currents. These two types of magnets operate at 1.9 K and 4.5 K and with nominal currents ranging from 3600 A to 5390 A. From the very first acceptance tests of the bare magnets coming from the manufacturers, they have been powered using different cycles, in different configurations, at different temperatures and in different tests facilities. In this paper we present the global results of these powering tests. We aim at separating common from individual features of these groups of magnets. Temperature dependence of the training, temperature margin, and ultimate current can be extracted from these tests. As these magnets are used to match the optics and the dispersion in the machine, the projected ultimate current at which they can be operated is critical in view of operation of LHC.

  16. An update on insertable cardiac monitors

    DEFF Research Database (Denmark)

    Olsen, Flemming J; Biering-Sørensen, Tor; Krieger, Derk W

    2015-01-01

    Continuous cardiac rhythm monitoring has undergone compelling progress over the past decades. Cardiac monitoring has emerged from 12-lead electrocardiograms being performed at the discretion of the treating physician to in-hospital telemetry, Holter monitoring, prolonged external event monitoring...... turning point in the field of arrhythmia management. However, their role in the detection of paroxysmal atrial fibrillation after cryptogenic strokes has yet to evolve. This will be the main focus of this review. Issues surrounding patient selection, clinical relevance and determination of cost......-effectiveness for prolonged cardiac monitoring require further studies. Furthermore, insertable cardiac monitoring has not only the potential to augment diagnostic capabilities but also to improve the management of paroxysmal atrial fibrillation....

  17. COMPLICATIONS NASOGASTRIC TUBE INSERTION AND TREATMENT

    Directory of Open Access Journals (Sweden)

    Dwi Kresnawati

    2013-12-01

    Full Text Available Normal 0 false false false EN-US X-NONE X-NONE MicrosoftInternetExplorer4 Insertion of a nasogastric tube (NGT is an invasive procedure that is often done by increasing medical emergency. There are two indications of this procedure,the indications are for diagnostic and therapeutic interest mainly to access and evaluate the nutritional content of the stomach for patients with suspected gastrointestinal bleeding. Although nasogastric tube insertion simpler, easier and safer than with a pipe orogastrik, but complications can occur during installation such as malposition of NGT, epistaxis, mucosal trauma, aspiration pneumonia, hypoxemia and pneumothorak. So the importance of knowledge, skills, and how treatment for complications arising prevents one way to ensure the proper placement of NGT. /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}

  18. Increasing prion propensity by hydrophobic insertion.

    Directory of Open Access Journals (Sweden)

    Aaron C Gonzalez Nelson

    Full Text Available Prion formation involves the conversion of proteins from a soluble form into an infectious amyloid form. Most yeast prion proteins contain glutamine/asparagine-rich regions that are responsible for prion aggregation. Prion formation by these domains is driven primarily by amino acid composition, not primary sequence, yet there is a surprising disconnect between the amino acids thought to have the highest aggregation propensity and those that are actually found in yeast prion domains. Specifically, a recent mutagenic screen suggested that both aromatic and non-aromatic hydrophobic residues strongly promote prion formation. However, while aromatic residues are common in yeast prion domains, non-aromatic hydrophobic residues are strongly under-represented. Here, we directly test the effects of hydrophobic and aromatic residues on prion formation. Remarkably, we found that insertion of as few as two hydrophobic residues resulted in a multiple orders-of-magnitude increase in prion formation, and significant acceleration of in vitro amyloid formation. Thus, insertion or deletion of hydrophobic residues provides a simple tool to control the prion activity of a protein. These data, combined with bioinformatics analysis, suggest a limit on the number of strongly prion-promoting residues tolerated in glutamine/asparagine-rich domains. This limit may explain the under-representation of non-aromatic hydrophobic residues in yeast prion domains. Prion activity requires not only that a protein be able to form prion fibers, but also that these fibers be cleaved to generate new independently-segregating aggregates to offset dilution by cell division. Recent studies suggest that aromatic residues, but not non-aromatic hydrophobic residues, support the fiber cleavage step. Therefore, we propose that while both aromatic and non-aromatic hydrophobic residues promote prion formation, aromatic residues are favored in yeast prion domains because they serve a dual

  19. cDNA sequences reveal considerable gene prediction inaccuracy in the Plasmodium falciparum genome

    Directory of Open Access Journals (Sweden)

    Valenzuela Jesus G

    2007-07-01

    Full Text Available Abstract Background The completion of the Plasmodium falciparum genome represents a milestone in malaria research. The genome sequence allows for the development of genome-wide approaches such as microarray and proteomics that will greatly facilitate our understanding of the parasite biology and accelerate new drug and vaccine development. Designing and application of these genome-wide assays, however, requires accurate information on gene prediction and genome annotation. Unfortunately, the genes in the parasite genome databases were mostly identified using computer software that could make some erroneous predictions. Results We aimed to obtain cDNA sequences to examine the accuracy of gene prediction in silico. We constructed cDNA libraries from mixed blood stages of P. falciparum parasite using the SMART cDNA library construction technique and generated 17332 high-quality expressed sequence tags (EST, including 2198 from primer-walking experiments. Assembly of our sequence tags produced 2548 contigs and 2671 singletons versus 5220 contigs and 5910 singletons when our EST were assembled with EST in public databases. Comparison of all the assembled EST/contigs with predicted CDS and genomic sequences in the PlasmoDB database identified 356 genes with predicted coding sequences fully covered by EST, including 85 genes (23.6% with introns incorrectly predicted. Careful automatic software and manual alignments found an additional 308 genes that have introns different from those predicted, with 152 new introns discovered and 182 introns with sizes or locations different from those predicted. Alternative spliced and antisense transcripts were also detected. Matching cDNA to predicted genes also revealed silent chromosomal regions, mostly at subtelomere regions. Conclusion Our data indicated that approximately 24% of the genes in the current databases were predicted incorrectly, although some of these inaccuracies could represent alternatively

  20. Posterior cruciate ligament's tibial insertions: topographic anatomy and morphometric study

    Directory of Open Access Journals (Sweden)

    Julio Cesar Gali

    2013-06-01

    Full Text Available OBJECTIVE: To provide anatomical and morphometric basis of the posterior cruciate ligament's tibial insertions in order to assist the creation of anatomical tibial tunnels, in the ligament surgical reconstruction. MATERIAL AND METHODS: The topographic anatomy and morphometry of the posterior cruciate ligament's anterolateral and posteromedial bundles' tibial insertions were analyzed in 24 anatomical knee pieces. The pieces were photographed by a digital camera and the images obtained were studied by the software ImageJ, where the bundles' insertion areas were measured in square millimeters, and the length of structures and the distances between significant points were measured in millimeters. RESULTS: In 54.2% of the knees the insertion' shape was concave; in most pieces (41.6% the form of insertion was oval. The average posterior cruciate ligament's tibial insertion total area was 88.33 ± 21.66 mm2; the average anterolateral bundle's tibial insertion area was 46.79 ± 14.10 mm2 and it was 41.54 ± 9.75 mm2 for the posteromedial bundle. CONCLUSIONS: The anterolateral bundle has a tibial insertion area larger than the posteromedial bundle; the insertion areas of those bundles in our study, were smaller than the ones found in the literature. The variations in the posterior cruciate ligament's tibial insertion area suggest that there should be an indication for anatomical reconstructions of this ligament using single or double tibial tunnels according to individual characteristics.

  1. Identification of a cryptic prokaryotic promoter within the cDNA encoding the 5' end of dengue virus RNA genome.

    Directory of Open Access Journals (Sweden)

    Dongsheng Li

    Full Text Available Infectious cDNA clones of RNA viruses are important research tools, but flavivirus cDNA clones have proven difficult to assemble and propagate in bacteria. This has been attributed to genetic instability and/or host cell toxicity, however the mechanism leading to these difficulties has not been fully elucidated. Here we identify and characterize an efficient cryptic bacterial promoter in the cDNA encoding the dengue virus (DENV 5' UTR. Following cryptic transcription in E. coli, protein expression initiated at a conserved in-frame AUG that is downstream from the authentic DENV initiation codon, yielding a DENV polyprotein fragment that was truncated at the N-terminus. A more complete understanding of constitutive viral protein expression in E. coli might help explain the cloning and propagation difficulties generally observed with flavivirus cDNA.

  2. Isolation and sequence analysis of a chalcone synthase cDNA of Matthiola incana R. Br. (Brassicaceae).

    Science.gov (United States)

    Epping, B; Kittel, M; Ruhnau, B; Hemleben, V

    1990-06-01

    A cDNA clone (pcM12) of the chalcone synthase (CHS) of Matthiola incana R. Br. (Brassicaceae) was isolated from a cDNA library, sequenced and analysed. It comprises the complete coding sequence for the CHS and 5' and 3' untranslated regions. The deduced amino acid sequence shows that the Matthiola incana CHS consists of 394 amino acid residues. Comparison with CHS amino acid sequences of other plants indicates more than 82% homology.

  3. Identification of a Cryptic Bacterial Promoter in Mouse (mdr1a P-Glycoprotein cDNA.

    Directory of Open Access Journals (Sweden)

    Kristen M Pluchino

    Full Text Available The efflux transporter P-glycoprotein (P-gp is an important mediator of various pharmacokinetic parameters, being expressed at numerous physiological barriers and also in multidrug-resistant cancer cells. Molecular cloning of homologous cDNAs is an important tool for the characterization of functional differences in P-gp between species. However, plasmids containing mouse mdr1a cDNA display significant genetic instability during cloning in bacteria, indicating that mdr1a cDNA may be somehow toxic to bacteria, allowing only clones containing mutations that abrogate this toxicity to survive transformation. We demonstrate here the presence of a cryptic promoter in mouse mdr1a cDNA that causes mouse P-gp expression in bacteria. This expression may account for the observed toxicity of mdr1a DNA to bacteria. Sigma 70 binding site analysis and GFP reporter plasmids were used to identify sequences in the first 321 bps of mdr1a cDNA capable of initiating bacterial protein expression. An mdr1a M107L cDNA containing a single residue mutation at the proposed translational start site was shown to allow sub-cloning of mdr1a in E. coli while retaining transport properties similar to wild-type P-gp. This mutant mdr1a cDNA may prove useful for efficient cloning of mdr1a in E. coli.

  4. Evaluation of a New Ultrasonic Insert for Prosthodontic Preparation.

    Science.gov (United States)

    Baldi, Domenico; Menini, Maria; Colombo, Jacopo; Lertora, Enrico; Pera, Paolo

    This paper describes a new ultrasonic instrument (tipholder DB1 with crown prep tip inserts) designed to optimize prosthodontic margin repositioning and finishing. The insert movement was assessed, and it was demonstrated that tipholder DB1 provides its inserts with an elliptical-like movement, making the entire insert surface able to cut. Then, 20 extracted teeth were prepared using tipholder DB1, sonic instruments, and traditional drills. Dental surface roughness produced using each of the three tools was measured using a roughness tester. Results were compared using univariate analysis of variance and Bonferroni post hoc test. The roughness produced using tipholder DB1 with crown prep insert presented no statistically significant differences compared to the roughness produced using sonic instruments and traditional drills. Tipholder DB1 with crown prep inserts is a promising treatment for margin repositioning and finishing.

  5. Does a novel method of PICC insertion improve safety?

    Science.gov (United States)

    Caparas, Jona; Hu, Jian Ping; Hung, Hwei-San

    2014-05-01

    Placing a central venous access device via the internal jugular or subclavian vein entails significant risks to both patient and healthcare worker. The purpose of this randomized, prospective study was to determine whether the accelerated Seldinger technique (AST) offers significant safety advantages over the modified Seldinger technique (MST) for peripherally inserted central catheter insertion. Patients were randomly assigned to undergo introducer sheath insertion by means of either MST or AST. Primary outcome measures included time to completion of introducer sheath insertion, estimated blood loss, and success rate. Secondary outcome measures included vessel-to-air exposure events and unprotected sharps exposure. While both insertion methods proved equivalent for successful vessel cannulation, AST was significantly faster (P = 0.0048) and resulted in less blood loss (P = 0.0295) than MST. Additionally, AST resulted in significantly fewer vessel-to-air exposure events (P PICC peelable introducer sheath insertion.

  6. Salmo salar and Esox lucius full-length cDNA sequences reveal changes in evolutionary pressures on a post-tetraploidization genome

    Directory of Open Access Journals (Sweden)

    Holt Robert A

    2010-04-01

    Full Text Available Abstract Background Salmonids are one of the most intensely studied fish, in part due to their economic and environmental importance, and in part due to a recent whole genome duplication in the common ancestor of salmonids. This duplication greatly impacts species diversification, functional specialization, and adaptation. Extensive new genomic resources have recently become available for Atlantic salmon (Salmo salar, but documentation of allelic versus duplicate reference genes remains a major uncertainty in the complete characterization of its genome and its evolution. Results From existing expressed sequence tag (EST resources and three new full-length cDNA libraries, 9,057 reference quality full-length gene insert clones were identified for Atlantic salmon. A further 1,365 reference full-length clones were annotated from 29,221 northern pike (Esox lucius ESTs. Pairwise dN/dS comparisons within each of 408 sets of duplicated salmon genes using northern pike as a diploid out-group show asymmetric relaxation of selection on salmon duplicates. Conclusions 9,057 full-length reference genes were characterized in S. salar and can be used to identify alleles and gene family members. Comparisons of duplicated genes show that while purifying selection is the predominant force acting on both duplicates, consistent with retention of functionality in both copies, some relaxation of pressure on gene duplicates can be identified. In addition, there is evidence that evolution has acted asymmetrically on paralogs, allowing one of the pair to diverge at a faster rate.

  7. Insertion torque versus mechanical resistance of mini- implants inserted in different cortical thickness

    Directory of Open Access Journals (Sweden)

    Renata de Faria Santos

    2014-06-01

    Full Text Available OBJECTIVE: This study aimed to measure insertion torque, tip mechanical resistance to fracture and transmucosal neck of mini-implants (MI (Conexão Sistemas de PróteseT, as well as to analyze surface morphology. METHODS: Mechanical tests were carried out to measure the insertion torque of MIs in different cortical thicknesses, and tip mechanical resistance to fracture as well as transmucosal neck of MIs. Surface morphology was assessed by scanning electron microscopy (SEM before and after the mechanical tests. RESULTS: Values of mechanical resistance to fracture (22.14 N.cm and 54.95 N.cm were higher and statistically different (P 0.05 to torsional fracture in the tip of MI (22.14 N.cm when 3 mm cortical thickness (16.11 N.cm and dense bone (23.95 N.cm were used. Torsional fracture of the transmucosal neck (54.95 N.cm was higher and statistically different (P < 0.05 from insertion torsional strength in all tested situations. SEM analysis showed that the MIs had the same smooth surface when received from the manufacturer and after the mechanical tests were performed. Additionally, no significant marks resulting from the manufacturing process were observed. CONCLUSION: All mini-implants tested presented adequate surface morphology. The resistance of mini-implants to fracture safely allows placement in 1 and 2-mm cortical thickness. However, in 3-mm cortical thickness and dense bones, pre-drilling with a bur is recommended before insertion.

  8. Construction and evaluation of normalized cDNA libraries enriched with full-length sequences for rapid discovery of new genes from Sisal (Agave sisalana Perr.) different developmental stages.

    Science.gov (United States)

    Zhou, Wen-Zhao; Zhang, Yan-Mei; Lu, Jun-Ying; Li, Jun-Feng

    2012-10-12

    To provide a resource of sisal-specific expressed sequence data and facilitate this powerful approach in new gene research, the preparation of normalized cDNA libraries enriched with full-length sequences is necessary. Four libraries were produced with RNA pooled from Agave sisalana multiple tissues to increase efficiency of normalization and maximize the number of independent genes by SMART™ method and the duplex-specific nuclease (DSN). This procedure kept the proportion of full-length cDNAs in the subtracted/normalized libraries and dramatically enhanced the discovery of new genes. Sequencing of 3875 cDNA clones of libraries revealed 3320 unigenes with an average insert length about 1.2 kb, indicating that the non-redundancy of libraries was about 85.7%. These unigene functions were predicted by comparing their sequences to functional domain databases and extensively annotated with Gene Ontology (GO) terms. Comparative analysis of sisal unigenes and other plant genomes revealed that four putative MADS-box genes and knotted-like homeobox (knox) gene were obtained from a total of 1162 full-length transcripts. Furthermore, real-time PCR showed that the characteristics of their transcripts mainly depended on the tight expression regulation of a number of genes during the leaf and flower development. Analysis of individual library sequence data indicated that the pooled-tissue approach was highly effective in discovering new genes and preparing libraries for efficient deep sequencing.

  9. Construction and Evaluation of Normalized cDNA Libraries Enriched with Full-Length Sequences for Rapid Discovery of New Genes from Sisal (Agave sisalana Perr. Different Developmental Stages

    Directory of Open Access Journals (Sweden)

    Jun-Feng Li

    2012-10-01

    Full Text Available To provide a resource of sisal-specific expressed sequence data and facilitate this powerful approach in new gene research, the preparation of normalized cDNA libraries enriched with full-length sequences is necessary. Four libraries were produced with RNA pooled from Agave sisalana multiple tissues to increase efficiency of normalization and maximize the number of independent genes by SMART™ method and the duplex-specific nuclease (DSN. This procedure kept the proportion of full-length cDNAs in the subtracted/normalized libraries and dramatically enhanced the discovery of new genes. Sequencing of 3875 cDNA clones of libraries revealed 3320 unigenes with an average insert length about 1.2 kb, indicating that the non-redundancy of libraries was about 85.7%. These unigene functions were predicted by comparing their sequences to functional domain databases and extensively annotated with Gene Ontology (GO terms. Comparative analysis of sisal unigenes and other plant genomes revealed that four putative MADS-box genes and knotted-like homeobox (knox gene were obtained from a total of 1162 full-length transcripts. Furthermore, real-time PCR showed that the characteristics of their transcripts mainly depended on the tight expression regulation of a number of genes during the leaf and flower development. Analysis of individual library sequence data indicated that the pooled-tissue approach was highly effective in discovering new genes and preparing libraries for efficient deep sequencing.

  10. Construction and analysis of antennal cDNA library from rice striped stem borer, Chilo suppressalis (Walker) (Lepidoptera: Pyralidae), and expression profiles of putative odorant-binding protein and chemosensory protein genes.

    Science.gov (United States)

    Gong, Zhong-Jun; Liu, Su; Jiang, Yan-Dong; Zhou, Wen-Wu; Liang, Qing-Mei; Cheng, Jiaan; Zhang, Chuan-Xi; Zhu, Zeng-Rong; Gurr, Geoff M

    2015-05-01

    In this study, we constructed a high-quality cDNA library from the antennae of the Chilo suppressalis (Walker) (Lepidoptera: Pyralidae). A total of 1,235 colonies with inserts greater than 0.7 kb were sequenced and analyzed. Homology searching coupled with bioinformatics analysis identified 15 and 7 cDNA sequences, respectively, encoding putative odorant-binding proteins (OBPs) and chemosensory proteins (CSPs). A phylogenetic tree of CsupCSPs showed that each CsupCSP has orthologs in Manduca sexta and Bombyx mori with strong bootstrapping support. One CSP was either very specific or more related to the CSPs of another species than to conspecific CSP. The expression profiles of the OBPs and CSPs in different tissues were measured by real-time quantitative PCR. The results revealed that of the 11 OBP genes, the transcript levels of CsupOBP1, CsupOBP5, and CsupOBP7 were higher in both male and female antennae than those in other tissues. And CsupCSP7 was highly expressed in both male and female antennae. Based on these results, the possible physiological functions of CsupOBPs and CsupCSPs were discussed. © 2015 Wiley Periodicals, Inc.

  11. Nasogastric tubes. 1: Insertion technique and confirming the correct position.

    Science.gov (United States)

    Durai, Rajaraman; Venkatraman, Ramya; Ng, Philip C H

    This is the first in a two-part unit on nasogastric tube management. It discusses the indications, patient preparation, insertion technique and various methods of confirming the tube's position. According to the National Patient Safety Agency, 11 deaths and one case of serious harm occurred due to misplaced nasogastric feeding tubes over a two-year period. It is therefore vital for staff inserting these tubes to know both the correct insertion methods and the procedure for verifying their correct intragastric positioning.

  12. A nurse led peripherally inserted central catheter line insertion service is effective with radiological support

    International Nuclear Information System (INIS)

    Barber, Jonathan M.; Booth, Doris M.; King, Julia A.; Chakraverty, Sam

    2002-01-01

    AIM: Peripherally inserted central catheters (PICC) are increasingly used as a route of chemotherapy administration. Our aims were to assess a collaborative approach to PICC placement, with radiological support for a nurse led line insertion service in a minority of cases, and to determine whether PICC provided a safe and reliable method of chemotherapy administration. MATERIALS AND METHODS: Prospective data on 100 consecutive patients undergoing PICC placement for chemotherapy were collected. Lines were inserted by ward based nurses or under ultrasound guidance by radiologists. End points were successful completion of treatment or patient death. RESULTS: One hundred and forty-four lines were placed for 118 courses of chemotherapy. 107 (74%) were placed by nurses and 37 (26%) by radiologists. Ninety-five percent of patients completed therapy with either one or two lines. Seventy percent of lines were removed on achieving the primary end points. In two additional patients PICC could not be placed radiologically. Twelve patients were unable to complete treatment with PICC alone, nine of these required an alternative administration route. The catheter related sepsis rate was 4.9%. CONCLUSION: The majority of PICC can be successfully placed by trained nurses, reserving image guidance only for more difficult cases. PICC have an acceptable complication profile, and decrease the need for tunnelled central lines. Barber, J.M. et al. (2002)

  13. Insertion torque versus mechanical resistance of mini-implants inserted in different cortical thickness.

    Science.gov (United States)

    Santos, Renata Faria; Ruellas, Antonio Carlos de Oliveira; Fernandes, Daniel Jogaib; Elias, Carlos Nelson

    2014-01-01

    This study aimed to measure insertion torque, tip mechanical resistance to fracture and transmucosal neck of mini-implants (MI) (Conexão Sistemas de PróteseT), as well as to analyze surface morphology. Mechanical tests were carried out to measure the insertion torque of MIs in different cortical thicknesses, and tip mechanical resistance to fracture as well as transmucosal neck of MIs. Surface morphology was assessed by scanning electron microscopy (SEM) before and after the mechanical tests. Values of mechanical resistance to fracture (22.14 N.cm and 54.95 N.cm) were higher and statistically different (P 0.05) to torsional fracture in the tip of MI (22.14 N.cm) when 3 mm cortical thickness (16.11 N.cm) and dense bone (23.95 N.cm) were used. Torsional fracture of the transmucosal neck (54.95 N.cm) was higher and statistically different (P mini-implants tested presented adequate surface morphology. The resistance of mini-implants to fracture safely allows placement in 1 and 2-mm cortical thickness. However, in 3-mm cortical thickness and dense bones, pre-drilling with a bur is recommended before insertion.

  14. Insertion torque versus mechanical resistance of mini-implants inserted in different cortical thickness

    Science.gov (United States)

    Santos, Renata de Faria; Ruellas, Antonio Carlos de Oliveira; Fernandes, Daniel Jogaib; Elias, Carlos Nelson

    2014-01-01

    Objective This study aimed to measure insertion torque, tip mechanical resistance to fracture and transmucosal neck of mini-implants (MI) (Conexão Sistemas de PróteseT), as well as to analyze surface morphology. Methods Mechanical tests were carried out to measure the insertion torque of MIs in different cortical thicknesses, and tip mechanical resistance to fracture as well as transmucosal neck of MIs. Surface morphology was assessed by scanning electron microscopy (SEM) before and after the mechanical tests. Results Values of mechanical resistance to fracture (22.14 N.cm and 54.95 N.cm) were higher and statistically different (P 0.05) to torsional fracture in the tip of MI (22.14 N.cm) when 3 mm cortical thickness (16.11 N.cm) and dense bone (23.95 N.cm) were used. Torsional fracture of the transmucosal neck (54.95 N.cm) was higher and statistically different (P mini-implants tested presented adequate surface morphology. The resistance of mini-implants to fracture safely allows placement in 1 and 2-mm cortical thickness. However, in 3-mm cortical thickness and dense bones, pre-drilling with a bur is recommended before insertion. PMID:25162571

  15. A nurse led peripherally inserted central catheter line insertion service is effective with radiological support

    Energy Technology Data Exchange (ETDEWEB)

    Barber, Jonathan M.; Booth, Doris M.; King, Julia A.; Chakraverty, Sam

    2002-05-01

    AIM: Peripherally inserted central catheters (PICC) are increasingly used as a route of chemotherapy administration. Our aims were to assess a collaborative approach to PICC placement, with radiological support for a nurse led line insertion service in a minority of cases, and to determine whether PICC provided a safe and reliable method of chemotherapy administration. MATERIALS AND METHODS: Prospective data on 100 consecutive patients undergoing PICC placement for chemotherapy were collected. Lines were inserted by ward based nurses or under ultrasound guidance by radiologists. End points were successful completion of treatment or patient death. RESULTS: One hundred and forty-four lines were placed for 118 courses of chemotherapy. 107 (74%) were placed by nurses and 37 (26%) by radiologists. Ninety-five percent of patients completed therapy with either one or two lines. Seventy percent of lines were removed on achieving the primary end points. In two additional patients PICC could not be placed radiologically. Twelve patients were unable to complete treatment with PICC alone, nine of these required an alternative administration route. The catheter related sepsis rate was 4.9%. CONCLUSION: The majority of PICC can be successfully placed by trained nurses, reserving image guidance only for more difficult cases. PICC have an acceptable complication profile, and decrease the need for tunnelled central lines. Barber, J.M. et al. (2002)

  16. Insertion mechanics of bioinspired needles into soft tissues.

    Science.gov (United States)

    Sahlabadi, Mohammad; Khodaei, Seyedvahid; Jezler, Kyle; Hutapea, Parsaoran

    2017-12-22

    Most studies to date confirm that any increase in the needle insertion force increases the damage to the tissue. When it comes to brain tissue, even minor damage can cause a long-lasting traumatic brain injury. Thus there is a great demand for innovative minimally invasive needles among the medical community. In our previous studies a novel bioinspired needle design with specially designed barbs was used to perform insertion tests into Polyvinyl chloride (PVC) tissue-mimicking gels, in which it decreased the insertion force by as much as 25%. In this work, bioinspired needles were designed using a CAD software, and were then manufactured using a 3 D printer. The insertion tests into bovine brain and liver were then performed to further investigate the performance of our bioinspired needles in real tissues. Our results show that there was a 10-25% decrease in the insertion force for insertions into bovine brain, and a 35-45% reduction in the insertion force for insertions into bovine liver using the proposed bioinspired needles. The reduction in the insertion force is due to the decrease in the friction force of the bioinspired needle with the bovine tissues, and its results are consistent with our previous results.

  17. Effect of vibration frequency on biopsy needle insertion force.

    Science.gov (United States)

    Tan, Lei; Qin, Xuemei; Zhang, Qinhe; Zhang, Hongcai; Dong, Hongjian; Guo, Tuodang; Liu, Guowei

    2017-05-01

    Needle insertion is critical in many clinical medicine procedures, such as biopsy, brachytherapy, and injection therapy. A platform with two degrees of freedom was set up to study the effect of vibration frequency on needle insertion force. The gel phantom deformation at the needle cutting edge and the Voigt model are utilized to develop a dynamic model to explain the relationship between the insertion force and needle-tip velocity. The accuracy of this model was verified by performing needle insertions into phantom gel. The effect of vibration on insertion force can be explained as the vibration increasing the needle-tip velocity and subsequently increasing the insertion force. In a series of needle insertion experiments with different vibration frequencies, the peak forces were selected for comparison to explore the effect of vibration frequency on needle insertion force. The experimental results indicate that the insertion force at 500Hz increases up to 17.9% compared with the force at 50Hz. Copyright © 2017 IPEM. Published by Elsevier Ltd. All rights reserved.

  18. Development of collimator insert for linac based stereotactic irradiation

    International Nuclear Information System (INIS)

    Singh, I.R.R.; Brindha, S.; Ravindran, B.P.; Rajshekhar, V.

    1999-01-01

    The aim of this study is to develop collimator inserts of various sizes which are either not commercially available or are expensive to import. The dosimetry parameters such as tissue maximum ratio (TMR), off-axis ratio (OAR) and output factor of the developed collimator insert are compared with that of the commercial collimator insert (Radionics). In order to check the suitability of the collimator insert developed locally for clinical use and to standardize the method of development, a collimator insert of 15 mm identical to the one supplied by Radionics is developed with low-melting alloy (Cerrobend). Moreover for the clinical use of the developed collimator insert, certain acceptance tests are performed which include a collimator concentricity test, beam size check and radiation leakage test. The dose verification is carried out with a thermoluminescent dosimeter ( 7 LiF rods) and an FBX chemical dosimeter in a human-head-shaped Perspex phantom filled with water. The variation between the calculated and measured dose is found to be within +2.4% for 7 LiF rods and -2.0% for the FBX chemical dosimeter thus ensuring the suitability of the developed collimator insert for clinical use. This has encouraged us to standardize the method adapted to develop the collimator insert and to develop collimator inserts of different field sizes. (author)

  19. Intrauterine device insertion in the postpartum period: a systematic review.

    Science.gov (United States)

    Sonalkar, Sarita; Kapp, Nathalie

    2015-02-01

    Given new research on postpartum placement of levonorgestrel and copper intrauterine devices (IUDs), our objective was to update a prior systematic review of the safety and expulsion rates of postpartum IUDs. We searched MEDLINE, CENTRAL, LILACS, POPLINE, Web of Science, and ClinicalTrials.gov databases for articles between the database inception until July 2013. We included studies that compared IUD insertion time intervals and routes during the postpartum period. We used standard abstract forms and the United States Preventive Services Task Force grading system to summarise and assess the quality of the evidence. We included 18 articles. New evidence suggests that a levonorgestrel releasing-intrauterine system (LNG-IUS) insertion within 48 hours of delivery is safe. Postplacental insertion and insertion between 10 minutes and 48 hours after delivery result in higher expulsion rates than insertion 4 to 6 weeks postpartum, or non-postpartum insertion. Insertion at the time of caesarean section is associated with lower expulsion rates than postplacental insertion at the time of vaginal delivery. This review supports the evidence that insertion of an intrauterine contraceptive within the first 48 hours of vaginal or caesarean delivery is safe. Expulsion rates should be further studied in larger randomised controlled trials.

  20. Insertional footprint anatomy of the pectoralis major tendon.

    Science.gov (United States)

    Carey, Paul; Owens, Brett D

    2010-01-01

    Correct anatomical repair of pectoralis major tendon injuries requires accurate identification of the humeral insertion site. In the absence of residual fibers at the insertion, proper placement requires knowledge of the anatomic relationships in the proximal humerus. This anatomic study attempts to measure these relationships and the dimensions of the insertional footprint to assist in surgical repair and reconstruction. Six matched pairs of cadaver proximal humeri were examined. All specimens were found to be in good condition and none were excluded. The proximal to distal length and maximum width of the pectoralis major tendon were measured at the humeral insertion with a digital caliper. The distance from the superomedial corner of the greater tuberosity to the superior aspect of the tendon insertion was also measured. The mean proximal to distal dimension of the pectoralis major insertion was 72.3+/-12.3 mm. The mean maximal thickness at the humeral insertion was 1.4+/-.2 mm. The distance from the superomedial corner of the greater tuberosity to the bony tendon insertion was 42.2+/-8.5 mm. The anatomic location of the humeral insertion of the pectoralis major tendon can be located in repair of pectoralis tendon injuries by referencing the superomedial corner of the greater tuberosity and the lateral lip of the bicipital groove. Findings in the present study will improve knowledge of the surgical anatomy relevant to repair and reconstruction of the pectoralis major tendon. Copyright 2010, SLACK Incorporated.

  1. Single-copy insertion of transgenes in Caenorhabditis elegans

    DEFF Research Database (Denmark)

    Frøkjaer-Jensen, Christian; Davis, M Wayne; Hopkins, Christopher E

    2008-01-01

    developed a method that inserts a single copy of a transgene into a defined site. Mobilization of a Mos1 transposon generates a double-strand break in noncoding DNA. The break is repaired by copying DNA from an extrachromosomal template into the chromosomal site. Homozygous single-copy insertions can...... be obtained in less than 2 weeks by injecting approximately 20 worms. We have successfully inserted transgenes as long as 9 kb and verified that single copies are inserted at the targeted site. Single-copy transgenes are expressed at endogenous levels and can be expressed in the female and male germlines....

  2. The ATLAS Insertable B-Layer Project

    CERN Document Server

    Miucci, A; The ATLAS collaboration

    2014-01-01

    The ATLAS experiment will upgrade its Pixel Detector with the installation of a new pixel layer in 2013-14. The new sub-detector, named Insertable B-layer (IBL), will be installed between the existing Pixel Detector and a new smaller radius beam-pipe at a radius of 3.3 cm. To cope with the high radiation and pixel occupancy due to the proximity to the interaction point, a new read-out chip and two different silicon sensor technologies (planar and 3D) have been de- veloped. Furthermore, the physics performance should be improved through the reduction of pixel size while a low material budget should be imposed. A new mechanical support using lightweight staves and a CO2 based cooling system is used. An overview of the IBL project and the status of the production of staves and the qualification of the assembly procedure, the loaded module electrical integrity and the read-out chain will be presented.

  3. The ATLAS Insertable B-Layer

    CERN Document Server

    Alkire, S P; The ATLAS collaboration

    2014-01-01

    During the first shutdown of LHC, the ATLAS detector is undergoing an upgrade of the Pixel Detector in preparation for high luminosity LHC. This upgrade consists of a new pixel layer, called the Insertable B-Layer (IBL), which is positioned between the former inner layer of the Pixel Detector (B-Layer) and a new, smaller, beam pipe, at a radial distance of 3.3 cm from the beam axis. The realization of the IBL required the development of several new technologies and solutions in order to overcome the challenges introduced by the extreme environment and working conditions, such as the high radiation levels, the high pixel occupancy and the need of an exceptionally low material budget. Two silicon sensor technologies have been adopted for the IBL modules: planar n-in-n and 3D. Both of these are connected via bump bonding to the new generation 130 nm IBM CMOS FE-I4 front-end read-out chip. An overview of the IBL project, module design, the qualification tests, data acquisition, results and performance is presente...

  4. Isolation and characterization of a cDNA from flowers of Cynara cardunculus encoding cyprosin (an aspartic proteinase) and its use to study the organ-specific expression of cyprosin.

    Science.gov (United States)

    Cordeiro, M C; Xue, Z T; Pietrzak, M; Pais, M S; Brodelius, P E

    1994-03-01

    Poly(A)+ RNA isolated from flower buds of Cynara cardunculus has been used to prepare a cDNA library. Screening of the cDNA after expression of cloned DNA with antibodies raised against the large subunit of cyprosin 3 resulted in the isolation of six positive clones. One of these clones (cypro1s; a 1.7 kb Eco RI fragment) codes for cyprosin. The nucleotide sequence contain a 1419 bp open reading frame coding for 473 amino acids (aa) including a putative full-length mature protein (440 aa) and a partial prosequence (33 aa). Cypro1s contains a 162 bp 3' non-coding region followed by a poly(A) tail. The deduced amino acid sequence shows high homology to other plant aspartic proteinases. The homology to mammalian and microbial aspartic proteinases is somewhat lower. Plant aspartic proteinases contain an insert of around 100 aa. We are modelling where this plant-specific insert will appear in the structure of cyprosin. Using cypro1s as a probe in northern blot analysis, the expression of cyprosin in developing flowers and other tissues has been studied. The signal on the northern blot increased for RNA samples from early (flower buds 6 mm in length) to later stages of floral development (flower buds up to 40 mm in length). In late stages of floral development (open flowers 50 mm in length and styles from such flowers) no hybridization signal was visualized showing that the synthesis of mRNA encoding the cyprosin starts in early stages of floral development and switches off at maturation of the flower.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Characterization of a cDNA encoding metallothionein 3 from cotton (Gossypium hirsutum L.).

    Science.gov (United States)

    Jordan, Robin H; Turley, Rickie B; Defauw, Sherri L; Steele, Mark

    2005-04-01

    A cDNA encoding metallothionein (MT) was isolated from a library constructed with poly A(+) RNA purified from 48 h etiolated cotton (Gossypium hirsutum L.) cotyledons. This cDNA encodes a deduced protein with 63 residues and a molecular weight of 6.3 kDa. The protein has 10 cysteines of which 4 are within the CXXCXCXXXXXC amino-terminus motif and six are within the CXCXXXCXCXXCXC carboxyl-terminus motif characteristic of the type III MT (MT3). The cotton MT3 protein sequence is 76.2, 69.8, 66.7, 60.3 and 33.5% identical to MT3 from Carica papaya, Rubus idaeus, Ribes nigrum, Citrus unshiu, and Gossypium hirsutum type I MT, respectively. A fusion protein was constructed by producing PCR primers for the 5' and 3' ends of the cotton MT3 cDNA and ligating the PCR product inframe at the 3' end of a bacterial glutathione S-transferase (GST) gene in the pGEX3 vector. The 5' PCR primer incorporated a segment of the cotton MT3 noncoding region, resulting in an addition of 9 residues to the MT3 (after Factor Xa digestion site) which increased the size of the expressed protein to 72 residues and 7.6 kDa. Expression of the 7.6 kDa protein in bacteria was confirmed by SDS-PAGE. Induction and accumulation of the GST-MT3 protein began inhibiting bacterial growth after 1 h. Addition of Cu (1 muM to 1 mM), 1 mM cysteine, or 1 mM cystine to the media did not rescue growth. Additionally, this protein was evaluated for its ability to bind Cd, Cu, Ni and Zn in the bacterial expression system. We found that cotton MT3 preferentially binds Cu.

  6. A method for the construction of equalized directional cDNA libraries from hydrolyzed total RNA.

    Science.gov (United States)

    Davis, Claytus; Barvish, Zeev; Gitelman, Inna

    2007-10-09

    The transcribed sequences of a cell, the transcriptome, represent the trans-acting fraction of the genetic information, yet eukaryotic cDNA libraries are typically made from only the poly-adenylated fraction. The non-coding or translated but non-polyadenylated RNAs are therefore not represented. The goal of this study was to develop a method that would more completely represent the transcriptome in a useful format, avoiding over-representation of some of the abundant, but low-complexity non-translated transcripts. We developed a combination of self-subtraction and directional cloning procedures for this purpose. Libraries were prepared from partially degraded (hydrolyzed) total RNA from three different species. A restriction endonuclease site was added to the 3' end during first-strand synthesis using a directional random-priming technique. The abundant non-polyadenylated rRNA and tRNA sequences were largely removed by using self-subtraction to equalize the representation of the various RNA species. Sequencing random clones from the libraries showed that 87% of clones were in the forward orientation with respect to known or predicted transcripts. 70% matched identified or predicted translated RNAs in the sequence databases. Abundant mRNAs were less frequent in the self-subtracted libraries compared to a non-subtracted mRNA library. 3% of the sequences were from known or hypothesized ncRNA loci, including five matches to miRNA loci. We describe a simple method for making high-quality, directional, random-primed, cDNA libraries from small amounts of degraded total RNA. This technique is advantageous in situations where a cDNA library with complete but equalized representation of transcribed sequences, whether polyadenylated or not, is desired.

  7. A method for the construction of equalized directional cDNA libraries from hydrolyzed total RNA

    Directory of Open Access Journals (Sweden)

    Gitelman Inna

    2007-10-01

    Full Text Available Abstract Background The transcribed sequences of a cell, the transcriptome, represent the trans-acting fraction of the genetic information, yet eukaryotic cDNA libraries are typically made from only the poly-adenylated fraction. The non-coding or translated but non-polyadenylated RNAs are therefore not represented. The goal of this study was to develop a method that would more completely represent the transcriptome in a useful format, avoiding over-representation of some of the abundant, but low-complexity non-translated transcripts. Results We developed a combination of self-subtraction and directional cloning procedures for this purpose. Libraries were prepared from partially degraded (hydrolyzed total RNA from three different species. A restriction endonuclease site was added to the 3' end during first-strand synthesis using a directional random-priming technique. The abundant non-polyadenylated rRNA and tRNA sequences were largely removed by using self-subtraction to equalize the representation of the various RNA species. Sequencing random clones from the libraries showed that 87% of clones were in the forward orientation with respect to known or predicted transcripts. 70% matched identified or predicted translated RNAs in the sequence databases. Abundant mRNAs were less frequent in the self-subtracted libraries compared to a non-subtracted mRNA library. 3% of the sequences were from known or hypothesized ncRNA loci, including five matches to miRNA loci. Conclusion We describe a simple method for making high-quality, directional, random-primed, cDNA libraries from small amounts of degraded total RNA. This technique is advantageous in situations where a cDNA library with complete but equalized representation of transcribed sequences, whether polyadenylated or not, is desired.

  8. Generation and Analysis of Full-length cDNA Sequences from Elephant Shark (Callorhinchus milii)

    KAUST Repository

    Kodzius, Rimantas

    2009-03-17

    Cartilaginous fishes are the oldest living group of jawed vertebrates and therefore is an important group for understanding the evolution of vertebrate genomes including the human genome. Our laboratory has proposed elephant shark (C. milii) as a model cartilaginous fish genome because of its relatively small genome size (910 Mb). The whole genome of C. milii is being sequenced (first cartilaginous fish genome to be sequenced completely). To characterize the transcriptome of C. milii and to assist in annotating exon-intron boundaries, transcriptional start sites and alternatively spliced transcripts, we are generating full-length cDNA sequences from C. milii.

  9. Cloning and sequencing of Indian Water buffalo (Bubalus bubalis) interleukin-3 cDNA

    KAUST Repository

    Sugumar, Thennarasu

    2011-12-12

    Full-length cDNA (435 bp) of the interleukin-3(IL-3) gene of the Indian water buffalo was amplified by reverse transcriptase-polymerase chain reaction and sequenced. This sequence had 96% nucleotide identity and 92% amino acid identity with bovine IL-3. There are 10 amino acid substitutions in buffalo compared with that of bovine. The amino acid sequence of buffalo IL-3 also showed very high identity with that of other ruminants, indicating functional cross-reactivity. Structural homology modelling of buffalo IL-3 protein with human IL-3 showed the presence of five helical structures.

  10. Cloning and Characterization of a cDNA Encoding a Novel Extracellular Peroxidase from Trametes versicolor

    Science.gov (United States)

    Collins, Patrick J.; O’Brien, Margaret M.; Dobson, Alan D. W.

    1999-01-01

    The white rot basidiomycete Trametes versicolor secretes a large number of peroxidases which are believed to be involved in the degradation of polymeric lignin. These peroxidases have been classified previously as lignin peroxidases or manganese peroxidases (MnP). We have isolated a novel extracellular peroxidase-encoding cDNA sequence from T. versicolor CU1, the transcript levels of which are repressed by low concentrations of Mn2+ and induced by nitrogen and carbon but not induced in response to a range of stresses which have been reported to induce MnP expression. PMID:10049906

  11. Construction of small RNA cDNA libraries for deep sequencing.

    Science.gov (United States)

    Lu, Cheng; Meyers, Blake C; Green, Pamela J

    2007-10-01

    Small RNAs (21-24 nucleotides) including microRNAs (miRNAs) and small interfering RNAs (siRNAs) are potent regulators of gene expression in both plants and animals. Several hundred genes encoding miRNAs and thousands of siRNAs have been experimentally identified by cloning approaches. New sequencing technologies facilitate the identification of these molecules and provide global quantitative expression data in a given biological sample. Here, we describe the methods used in our laboratory to construct small RNA cDNA libraries for high-throughput sequencing using technologies such as MPSS, 454 or SBS.

  12. Construction of small RNA cDNA libraries for high-throughput sequencing.

    Science.gov (United States)

    Lu, Cheng; Shedge, Vikas

    2011-01-01

    Small RNAs (smRNAs) play an essential role in virtually every aspect of growth and development, by regulating gene expression at the post-transcriptional and/or transcriptional level. New high-throughput sequencing technology allows for a comprehensive coverage of smRNAs in any given biological sample, and has been widely used for profiling smRNA populations in various developmental stages, tissue and cell types, or normal and disease states. In this article, we describe the method used in our laboratory to construct smRNA cDNA libraries for high-throughput sequencing.

  13. 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.

  14. The timing of norplant insertion and postpartum depression in teenagers.

    Science.gov (United States)

    Stevens-Simon, C; Kelly, L; Wallis, J

    2000-06-01

    This study tested the hypothesis that teenagers who have Norplant inserted during the puerperium report more depressive symptoms during the first postpartum year than their peers who do not receive Norplant. We studied the prevalence of depressive symptoms in a group of 212 mothers aged 19 years less, in relation to the timing of Norplant insertion. The participants were divided into 3 groups: 100 (47%) had Norplant inserted during the puerperium (early Norplant users); 72 (34%) had Norplant inserted during the next 10 months (late Norplant users); and 40 (19%) used other contraceptives (40% oral contraceptives; 17% condoms; 43% nothing). Depressive symptoms were measured with the Center for Epidemiologic Studies - Depression Scale. Postpartum depression was defined as a scale score >/=16, 6-12 months after Norplant insertion or delivery. Variables examined as potential confounders were identified a priori from a review of the literature and controlled for in analysis of variance. At delivery, members of the 3 contraceptive groups did not differ significantly with regard to age, race, parity, educational, marital, or socioeconomic status. Late Norplant users were, however, more apt to have new boyfriends (p =. 03), to rate the support they received from the baby's father as poor (p =.004), and experience depression prior to Norplant insertion (p =.02). Contrary to the study hypothesis, late rather than early Norplant insertion was associated with postpartum depression. Multivariate analyses identified 3 independent predictors of the severity of depressive symptoms at follow-up (depression prior to Norplant insertion, a new boyfriend at delivery, and late Norplant insertion); R(2) = 41.3%. Contrary to the study hypothesis, puerperal Norplant insertion did not exacerbate postpartum depression. Delaying Norplant insertion may increase the risk of depression during the first postpartum year, particularly in teenagers with other psychosocial risk factors.

  15. Clinical information in drug package inserts in India

    Directory of Open Access Journals (Sweden)

    Shivkar Y

    2009-01-01

    Full Text Available Background: It is widely recognized that accurate and reliable product information is essential for the safe and effective use of medications. Pharmaceutical companies are the primary source of most drug information, including package inserts. Package inserts are printed leaflets accompanying marketed drug products and contain information approved by the regulatory agencies. Studies on package inserts in India, in 1996, had shown that crucial information was often missing and they lacked uniformity. Aim: To assess the presentation and completeness of clinically important information provided in the currently available package inserts in India. Materials and Methods: Package inserts accompanying allopathic drug products marketed by pharmaceutical companies in India were collected. These package inserts were analyzed for the content of clinically important information in various sections. Statistical Analysis: The results were expressed as absolute numbers and percentages. Results: Preliminary analyses revealed that most package inserts did contain information under headings, such as, therapeutic indications, contraindications, undesirable effects, etc., listed in the Drugs and Cosmetics Rules 1945. The findings indicated considerable improvement in package inserts since 1996. However, on critical evaluation it was revealed that clinically important information was not well presented and was often incomplete. Information with regard to pediatric and geriatric use was present in only 44% and 13% of the package inserts, respectively. Only five of the inserts had information on the most frequent adverse drug reactions associated with the drug. Also, information on interactions and overdosage was often missing. Conclusion: Although the package inserts appear to have improved over the past decade there is still a definite need to further refine the clinical information contained, to minimize the risks to patients. This could be brought about by self

  16. Rapid and Efficient cDNA Library Screening by Self-Ligation ofInverse PCR Products (SLIP)

    Energy Technology Data Exchange (ETDEWEB)

    Hoskins, Roger A.; Stapleton, Mark; George, Reed A.; Yu, Charles; Wan, Kenneth H.; Carlson, Joseph W.; Celniker, Susan E.

    2005-04-22

    The production of comprehensive cDNA clone collections is an important goal of the human and model organism genome projects. cDNA sequences are used to determine the structures of transcripts, including splice junctions, polyadenylation sites, and 5' and 3' untranslated regions (UTRs). cDNA collections are also valuable resources for functional studies of genes and proteins. Expressed Sequence Tag (EST)sequencing is the method of choice for recovering cDNAs representing a majority of the transcripts encoded in a eukaryotic genome. However, EST sequencing samples a library at random, so it realizes diminishing returns as the project progresses. To drive cDNA collections toward completion new methods are needed to recover cDNAs representing specific genes and alternative transcripts, including transcripts with low expression levels. We describe a simple and effective inverse-PCR-based method for screening plasmid libraries to recover intact cDNAs for specific transcripts. We tested the method by screening libraries used in our Drosophila EST projects for 153 transcription factor genes that were not yet represented by full-length cDNAs. We recovered target-specific clones for 104 of the genes: 46 exactly match, 30 improve and 28partially match current gene annotations. Successful application of the screening method depends on cDNA library complexity and quality of the gene models. The approach should be effective for improving cDNA collections for other model organisms and the human. It also provides a simple and rapid method for isolating cDNAs of interest in any system for which plasmid cDNA libraries and complete or partial gene sequences are available.

  17. Polymorphism of angiotensin converting enzyme (insertion/deletion ...

    Indian Academy of Sciences (India)

    and electrolyte balance, and blood pressure (Malik et al. 1997) contains a ... of diverse population groups with a variable prevalence of hypertension .... group were in HWE. The 287-bp insertion/deletion polymorphism located in intron 16 of ACE gene was detected by PCR analysis in the three ethnic groups. The insertion ...

  18. Load beam unit replaceable inserts for dry coal extrusion pumps

    Science.gov (United States)

    Saunders, Timothy; Brady, John D.

    2012-11-13

    A track assembly for a particulate material extrusion pump according to an exemplary aspect of the present disclosure includes a link assembly with a roller bearing. An insert mounted to a load beam located such that the roller bearing contacts the insert.

  19. Lack of association of insertion/deletion polymorphism in ...

    African Journals Online (AJOL)

    In the present preliminary study the insertion/deletion polymorphism within angiotensin converting enzyme gene is not likely to be associated with nephropathy in type 2 diabetic patients of Punjabi population of Pakistan. Key words: Angiotensin converting enzymes, insertion/deletion polymorphism, albuminuria and type 2 ...

  20. Inactivation of a transgene due to transposition of insertion ...

    Indian Academy of Sciences (India)

    Agrobacterium strains harbour insertion sequences, which are known to transpose into genomes as well as into Ti plasmids. In this study we report the inactivation of a transgene due to transposition of the A. tumefaciens insertion sequence IS136. The transposition was discovered following transformation of plant tissues, ...

  1. Forward and reverse genetics: The LORE1 retrotransposon insertion mutants

    DEFF Research Database (Denmark)

    Fukai, Eigo; Malolepszy, Anna; Sandal, Niels Nørgaard

    2014-01-01

    The endogenous Lotus retrotransposon 1 (LORE1) transposes in the germ line of Lotus japonicus plants that carry an active element. This feature of LORE1 has been exploited for generation of a large non-transgenic insertion mutant population, where insertions have been annotated using next-generat...

  2. Enhanced citrate production through gene insertion in Aspergillus niger

    DEFF Research Database (Denmark)

    Jongh, Wian de; Nielsen, Jens

    2007-01-01

    The effect of inserting genes involved in the reductive branch of the tricarboxylic acid (TCA) cycle on citrate production by Aspergillus niger was evaluated. Several different genes were inserted individually and in combination, i.e. malate dehydrogenase (mdh2) from Saccharomyces cerevisiae, two...

  3. Ultrasonic Guided Insertion of Central Venous Catheter in Infants ...

    African Journals Online (AJOL)

    Background/Purpose: ultrasound is licensed for application of regional blocks and insertion of vascular access. We aimed to compare ultrasonic guided (USG) and anatomical landmark technique (ALT) for insertion of central venous catheter (CVC) as regard success rate and rate of complications in infants and children.

  4. Nurse-led PICC insertion: is it cost effective?

    Science.gov (United States)

    Walker, Graham; Todd, Alistair

    Repeated attempts to cannulate small veins can cause considerable distress for patients and expend substantial staff time.For longer term venous access, a peripherally inserted central catheter(PICC) may be used instead of a peripheral cannula. Previous studies indicate that insertion of a PICC costs three times more than a cannula but the lifespan is substantially longer. This study aimed to compare insertion cost, patient satisfaction, and infection rates of PICCs for the two main staff groups (trained nurses and radiologists) inserting these devices in a district general hospital. The study took place over 4 months in 2012-13.A questionnaire was attached to all identified PICCs in stock at Raigmore Hospital to collect details of the date of insertion, patient involved, time taken, attendant staff grade and experience level,consumables used and insertion success. The lead author's personal observation of PICC insertion by different staff groups allowed estimation of staff time, costs and success rates. Patient experience and satisfaction was assessed before and after insertion using a patient questionnaire. PICC longevity, infection rates and failures were assessed by review of patient notes. The radiologist group had a statistically significant (pPICCs can be safely performed without x-ray screening in a ward-based environment. This is likely to be the most cost-effective solution for large volume services.

  5. Nurse-led PICC insertion: is it cost effective?

    Science.gov (United States)

    Walker, Graham; Todd, Alistair

    2013-10-24

    Repeated attempts to cannulate small veins can cause considerable distress for patients and expend substantial staff time. For longer term venous access, a peripherally inserted central catheter (PICC) may be used instead of a peripheral cannula. Previous studies indicate that insertion of a PICC costs three times more than a cannula but the lifespan is substantially longer. This study aimed to compare insertion cost, patient satisfaction, and infection rates of PICCs for the two main staff groups (trained nurses and radiologists) inserting these devices in a district general hospital. The study took place over 4 months in 2012-13. A questionnaire was attached to all identified PICCs in stock at Raigmore Hospital to collect details of the date of insertion, patient involved, time taken, attendant staff grade and experience level, consumables used and insertion success. The lead author's personal observation of PICC insertion by different staff groups allowed estimation of staff time, costs and success rates. Patient experience and satisfaction was assessed before and after insertion using a patient questionnaire. PICC longevity, infection rates and failures were assessed by review of patient notes. The radiologist group had a statistically significant (pPICCs can be safely performed without x-ray screening in a ward-based environment. This is likely to be the most cost-effective solution for large volume services.

  6. Measurement of Vein Diameter for Peripherally Inserted Central Catheter (PICC) Insertion: An Observational Study.

    Science.gov (United States)

    Sharp, Rebecca; Cummings, Melita; Childs, Jessie; Fielder, Andrea; Mikocka-Walus, Antonina; Grech, Carol; Esterman, Adrian

    2015-01-01

    Choosing an appropriately sized vein reduces the risk of venous thromboembolism associated with peripherally inserted central catheters. This observational study described the diameters of the brachial, basilic, and cephalic veins and determined the effect of patient factors on vein size. Ultrasound was used to measure the veins of 176 participants. Vein diameter was similar in both arms regardless of hand dominance and side. Patient factors-including greater age, height, and weight, as well as male gender-were associated with increased vein diameter. The basilic vein tended to have the largest diameter statistically. However, this was the case in only 55% of patients.

  7. Turbine airfoil having near-wall cooling insert

    Energy Technology Data Exchange (ETDEWEB)

    Martin, Jr., Nicholas F.; Wiebe, David J.

    2017-09-12

    A turbine airfoil is provided with at least one insert positioned in a cavity in an airfoil interior. The insert extends along a span-wise extent of the turbine airfoil and includes first and second opposite faces. A first near-wall cooling channel is defined between the first face and a pressure sidewall of an airfoil outer wall. A second near-wall cooling channel is defined between the second face and a suction sidewall of the airfoil outer wall. The insert is configured to occupy an inactive volume in the airfoil interior so as to displace a coolant flow in the cavity toward the first and second near-wall cooling channels. A locating feature engages the insert with the outer wall for supporting the insert in position. The locating feature is configured to control flow of the coolant through the first or second near-wall cooling channel.

  8. Construction and biological activities of the first infectious cDNA clones of the genus Foveavirus

    Energy Technology Data Exchange (ETDEWEB)

    Meng, Baozhong, E-mail: bmeng@uoguelph.ca [Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road, Guelph, Ontario, Canada N1G2W1 (Canada); Venkataraman, Srividhya; Li, Caihong; Wang, Weizhou [Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road, Guelph, Ontario, Canada N1G2W1 (Canada); Dayan-Glick, Cathy; Mawassi, Munir [The Plant Pathology Department-The Virology Unit, Plant Protection Institute, Agricultural Research Organization, The Volcani Center, Bet-Dagan 50250 (Israel)

    2013-01-20

    Grapevine rupestris stem pitting-associated virus (GRSPaV, genus Foveavirus, family Betaflexiviridae) is one of the most prevalent viruses in grapevines and is associated with three distinct diseases: rupestris stem pitting, vein necrosis and Syrah decline. Little is known about the biology and pathological properties of GRSPaV. In this work, we engineered a full-length infectious cDNA clone for GRSPaV and a GFP-tagged variant, both under the transcriptional control of Cauliflower mosaic virus 35 S promoter. We demonstrated that these cDNA clones were infectious in grapevines and Nicotiana benthamiana through fluorescence microscopy, RT-PCR, Western blotting and immuno electron microscopy. Interestingly, GRSPaV does not cause systemic infection in four of the most commonly used herbaceous plants, even in the presence of the movement proteins of two other viruses which are known to complement numerous movement-defective viruses. These infectious clones are the first of members of Foveavirus which would allow further investigations into mechanisms governing different aspects of replication for GRSPaV and perhaps related viruses.

  9. Evaluation of the gene-specific dye bias in cDNA microarray experiments.

    Science.gov (United States)

    Martin-Magniette, Marie-Laure; Aubert, Julie; Cabannes, Eric; Daudin, Jean-Jacques

    2005-05-01

    In cDNA microarray experiments all samples are labeled with either Cy3 or Cy5. Systematic and gene-specific dye bias effects have been observed in dual-color experiments. In contrast to systematic effects which can be corrected by a normalization method, the gene-specific dye bias is not completely suppressed and may alter the conclusions about the differentially expressed genes. The gene-specific dye bias is taken into account using an analysis of variance model. We propose an index, named label bias index, to measure the gene-specific dye bias. It requires at least two self-self hybridization cDNA microarrays. After lowess normalization we have found that the gene-specific dye bias is the major source of experimental variability between replicates. The ratio (R/G) may exceed 2. As a consequence false positive genes may be found in direct comparison without dye-swap. The stability of this artifact and its consequences on gene variance and on direct or indirect comparisons are addressed. http://www.inapg.inra.fr/ens_rech/mathinfo/recherche/mathematique

  10. Construction and characterization of a normalized cDNA library of Nannochloropsis oculata (Eustigmatophyceae)

    Science.gov (United States)

    Yu, Jianzhong; Ma, Xiaolei; Pan, Kehou; Yang, Guanpin; Yu, Wengong

    2010-07-01

    We constructed and characterized a normalized cDNA library of Nannochloropsis oculata CS-179, and obtained 905 nonredundant sequences (NRSs) ranging from 431-1 756 bp in length. Among them, 496 were very similar to nonredundant ones in the GenBank ( E ≤1.0e-05), and 349 ESTs had significant hits with the clusters of eukaryotic orthologous groups (KOG). Bases G and/or C at the third position of codons of 14 amino acid residues suggested a strong bias in the conserved domain of 362 NRSs (>60%). We also identified the unigenes encoding phosphorus and nitrogen transporters, suggesting that N. oculata could efficiently transport and metabolize phosphorus and nitrogen, and recognized the unigenes that involved in biosynthesis and storage of both fatty acids and polyunsaturated fatty acids (PUFAs), which will facilitate the demonstration of eicosapentaenoic acid (EPA) biosynthesis pathway of N. oculata. In comparison with the original cDNA library, the normalized library significantly increased the efficiencies of random sequencing and rarely expressed genes discovering, and decreased the frequency of abundant gene sequences.

  11. cDNA sequences of two inducible T-cell genes

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    Kwon, B.S. (Indiana Univ. School of Medicine, Indianapolis (USA) Guthrie Research Institute, Sayre, PA (USA)); Weissman, S.M. (Yale Univ., New Haven, CT (USA))

    1989-03-01

    The authors have previously described a set of human T-lymphocyte-specific cDNA clones isolated by a modified differential screening procedure. Apparent full-length cDNAs containing the sequences of 14 of the 16 initial isolates were sequenced and were found to represent five different species of mRNA; three of the five species were identical to previously reported cDNA sequences of preproenkephalin, T-cell-replacing factor, and a serine esterase, respectively. The other two species, 4-1BB and L2G25B, were inducible sequences found in mRNA from both a cytolytic T-lymphocyte and a helper T-lymphocyte clone and were not previously described in T-cell mRNA; these mRNA sequences encode peptides of 256 and 92 amino acids, respectively. Both peptides contain putative leader sequences. The protein encoded by 4-1BB also has a potential membrane anchor segment and other features also seen in known receptor proteins.

  12. Discovery of Phytophthora infestans Genes Expressed in Planta through Mining of cDNA Libraries

    Science.gov (United States)

    Chaves, Diego; Pinzón, Andrés; Grajales, Alejandro; Rojas, Alejandro; Mutis, Gabriel; Cárdenas, Martha; Burbano, Daniel; Jiménez, Pedro; Bernal, Adriana; Restrepo, Silvia

    2010-01-01

    Background Phytophthora infestans (Mont.) de Bary causes late blight of potato and tomato, and has a broad host range within the Solanaceae family. Most studies of the Phytophthora – Solanum pathosystem have focused on gene expression in the host and have not analyzed pathogen gene expression in planta. Methodology/Principal Findings We describe in detail an in silico approach to mine ESTs from inoculated host plants deposited in a database in order to identify particular pathogen sequences associated with disease. We identified candidate effector genes through mining of 22,795 ESTs corresponding to P. infestans cDNA libraries in compatible and incompatible interactions with hosts from the Solanaceae family. Conclusions/Significance We annotated genes of P. infestans expressed in planta associated with late blight using different approaches and assigned putative functions to 373 out of the 501 sequences found in the P. infestans genome draft, including putative secreted proteins, domains associated with pathogenicity and poorly characterized proteins ideal for further experimental studies. Our study provides a methodology for analyzing cDNA libraries and provides an understanding of the plant – oomycete pathosystems that is independent of the host, condition, or type of sample by identifying genes of the pathogen expressed in planta. PMID:20352100

  13. Discovery of Phytophthora infestans genes expressed in planta through mining of cDNA libraries.

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    Roberto Sierra

    Full Text Available BACKGROUND: Phytophthora infestans (Mont. de Bary causes late blight of potato and tomato, and has a broad host range within the Solanaceae family. Most studies of the Phytophthora--Solanum pathosystem have focused on gene expression in the host and have not analyzed pathogen gene expression in planta. METHODOLOGY/PRINCIPAL FINDINGS: We describe in detail an in silico approach to mine ESTs from inoculated host plants deposited in a database in order to identify particular pathogen sequences associated with disease. We identified candidate effector genes through mining of 22,795 ESTs corresponding to P. infestans cDNA libraries in compatible and incompatible interactions with hosts from the Solanaceae family. CONCLUSIONS/SIGNIFICANCE: We annotated genes of P. infestans expressed in planta associated with late blight using different approaches and assigned putative functions to 373 out of the 501 sequences found in the P. infestans genome draft, including putative secreted proteins, domains associated with pathogenicity and poorly characterized proteins ideal for further experimental studies. Our study provides a methodology for analyzing cDNA libraries and provides an understanding of the plant--oomycete pathosystems that is independent of the host, condition, or type of sample by identifying genes of the pathogen expressed in planta.

  14. Construction of Agrobacterium tumefaciens-mediated tomato black ring virus infectious cDNA clones.

    Science.gov (United States)

    Zarzyńska-Nowak, Aleksandra; Ferriol, Inmaculada; Falk, Bryce W; Borodynko-Filas, Natasza; Hasiów-Jaroszewska, Beata

    2017-02-15

    Tomato black ring virus (TBRV, genus Nepovirus) infects a wide range of economically important plants such as tomato, potato, tobacco and cucumber. Here, a successful construction of infectious full-length cDNA clones of the TBRV genomic RNAs (RNA1 and RNA2) is reported for the first time. The engineered constructs consisting of PCR-amplified DNAs were cloned into binary vector pJL89 immediately downstream of a double cauliflower mosaic virus (CaMV) 35S promoter, and upstream of the hepatitis delta virus (HDV) ribozyme and nopaline synthase terminator (NOS). The symptoms induced on plants agroinoculated with both constructs were indistinguishable from those caused by the wild-type virus. The infectivity of obtained clones was verified by reinoculation to Nicotiana tabacum cv. Xanthi, Chenopodium quinoa and Cucumis sativus. The presence of viral particles and RNA was confirmed by electron microscopy and reverse transcription polymerase chain reaction, respectively. Constructed full-length infectious cDNA clones will serve as an excellent tool to study virus-host-vector interactions. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Phenoloxidase from the sea cucumber Apostichopus japonicus: cDNA cloning, expression and substrate specificity analysis.

    Science.gov (United States)

    Jiang, Jingwei; Zhou, Zunchun; Dong, Ying; Sun, Hongjuan; Chen, Zhong; Yang, Aifu; Gao, Shan; Wang, Bai; Jiang, Bei; Guan, Xiaoyan

    2014-02-01

    Phenoloxidase (PO) is a crucial component of the immune system of echinoderms. In the present study, the full-length cDNA of PO (AjPO) was cloned from coelomocytes of the sea cucumber Apostichopus japonicus using 3'- and 5'-rapid amplification of cDNA ends (RACE) PCR method, which is 2508 bp, with an open reading frame (ORF) of 2040 bp encoding 679 amino acids. AjPO contains a transmembrane domain, and three Cu-oxidase domains with copper binding centers formed by 10 histidines, one cysteine and one methionine respectively. Phylogenetic analysis revealed that AjPO was clustered with laccase-type POs of invertebrates. Using the isolated membrane proteins as crude AjPO, the enzyme could catalyze the substrates catechol, L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine and hydroquinone, but failed to oxidize tyrosine. The results described above collectively proved that AjPO was a membrane-binding laccase-type PO. The quantitative real-time PCR (qRT-PCR) analysis revealed that AjPO mRNA was expressed in muscle, body wall, coelomocytes, tube feet, respiratory tree and intestine with the highest expression level in coelomocytes. AjPO could be significantly induced by lipopolysaccharide (LPS), peptidoglycan (PGN), Zymosan A and polyinosinic-polycytidylic acid (PolyI:C), suggesting AjPO is closely involved in the defense against the infection of bacteria, fungi and double-stranded RNA viruses. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. High-quality RNA preparation from Rhodosporidium toruloides and cDNA library construction therewith.

    Science.gov (United States)

    Yang, Fan; Tan, Haidong; Zhou, Yongjin; Lin, Xinping; Zhang, Sufang

    2011-02-01

    Oleaginous yeast Rhodosporidium toruloides is an excellent microbial lipid producer. Therefore, it is important to develop molecular biology tools to understand the basic mechanism for lipid accumulation and further manipulate the microorganism. High-quality RNA extraction from R. toruloides is particularly challenging due to high level of polysaccharides, lipids, and other secondary metabolites. To obtain an optimal protocol for RNA extraction from R. toruloides, four methods were evaluated. Large difference in RNA yield and quality among these protocols was found. The optimum method was modified RNAiso procedure, where RNA was isolated using liquid nitrogen-RNAiso method with salt precipitation and the addition of β-mercaptoethanol. This method consistently recovered RNA in good quality with high yield. Around 297 μg total RNA per gram of cells was obtained with an average purity measured as A₂₆₀/A₂₈₀ of 2.09. A titer of 10⁵ cfu/ml could be harvested to construct a full-length cDNA library with the RNA sample in this quality. Electrophoresis gel analysis indicated the fragments ranged from 200 bp to 4.0 kb, with the average size of 1000 bp. Randomly picked clones showed the recombination efficiency at 80%. These results showed that RNA of R. toruloides was successfully extracted for the first time using the modified RNAiso method, and the cDNA library was appropriate for screening the genes related to lipid accumulation.

  17. Construction of cDNA library of cotton mutant Xiangmian-18 library during gland forming stage.

    Science.gov (United States)

    Xie, Yong-Fang; Wang, Bo-Chu; Li, Biao; Cai, Ying-Fan; Xie, Lei; Xia, Yu-Xian; Chang, Ping-An; Jiang, Huai-Zhong

    2007-11-15

    Gossypol, a secondary metabolite stored in the glands of cotton, protecting cottonseed from consumption of human and monogastric animal. This ability is unique to the tribe Gossypieae. Although the relationship between gossypol and pigment gland has been studied for a long time, the development mechanism of pigment gland has not been investigated at molecular level. Here we described a simple and efficient method for constructing a normalized cDNA library from a cotton mutant, Xiangmian-18, during its pigments gland forming stage. It combined switching mechanism at 5'-end of RNA transcript (SMART) technique and duplex-specific nuclease (DSN) normalization methods. In a model experiment, double-stranded cDNAs were synthesized from mRNAs, processed by normalization and Sfi I restriction endonuclease, and finally the cDNAs were ligated to pDNR-LIB vector. The ligation mixture was transformed into E. coli JM109 by electroporation. Counting the number of colonies, the titer of the original library was 5.86x10(5)cfu/ml in this library. Electrophoresis gel results indicated the fragments ranged from 800bp to 2kb, with the average size of 1400bp. Random picking clones showed that the recombination rate was 94%. The results showed that the cDNA library constructed successfully was a full-length library with high quality, and could be used to screen the genes related to development of pigments gland cottons.

  18. Construction of Hypericin Gland-Specific cDNA Library via Suppression Subtractive Hybridization.

    Science.gov (United States)

    Singh, Rupesh Kumar; Hou, Weina; Franklin, Gregory

    2016-01-01

    Hypericin, an important determinant of the pharmacological properties of the genus Hypericum, is considered as a major molecule for drug development. However, biosynthesis and accumulation of hypericin is not well understood. Identification of genes differentially expressed in tissues with and without hypericin accumulation is a useful strategy to elucidate the mechanisms underlying the development of the dark glands and hypericin biosynthesis. Suppression Subtractive Hybridization (SSH) is a unique method for PCR-based amplification of specific cDNA fragments that differ between a control (driver) and experimental (tester) transcriptome. This technique relies on the removal of dsDNA formed by hybridization between a control and test sample, thus eliminating cDNAs of similar abundance, and retaining differentially expressed or variable in sequence cDNAs. In our laboratory we applied this method to identify the genes involved in the development of dark glands and accumulation of hypericin in Hypericum perforatum. Here we describe the complete procedure for the construction of hypericin gland-specific subtracted cDNA library.

  19. Radioactive cDNA microarrys for gene expression profiles in antidepressant therapy

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    Lee, M. S.; Han, B. J.; Cha, J. H.; Ryu, Y. M.; Shin, E. K.; Park, J. H.; Park, Y. H.; Kim, M. K. [Korea University Medical College, Seoul (Korea, Republic of)

    2002-07-01

    Using radioactive cDNA microarray, we investigated a pattern of gene regulation under treatment of antidepressant on patients of depressive disoder. Basic microarray technology was performed as previously described in our research. The bioinformatic selection of human cDNAs, which is specifically designed for psychiatry, neurology, and signal transduction, were arrayed on nylon membranes. Using with 33P-labeled probes, this method provided highly sensitive gene expression profiles of our interest including brain receptors, drug metabolism, and cellular signalings. Gene expression profiles were also classified into several categories in accordance with the gene-regulation of antidepressant. The gene profiles of our interest were significantly up- (16 genes, >2.0 of Z-ratio) or down- (24 genes, <-2.0 of Z ratio) regulated when compared the good responsed group with the bad-responsed one. Consequently, we demonstrated that radioactive human cDNA microarray is highly likely to be an efficient technology for evaluating the gene regulation of antidepressants, such as selective serotonin-reuptake inhibitors (SSRIs), by using high-throughput biotechnology.

  20. Novel transcripts of human cytomegalovirus clinical strain found by cDNA library screening.

    Science.gov (United States)

    Ma, Y P; Ruan, Q; Ji, Y H; Wang, N; Li, M L; Qi, Y; He, R; Sun, Z R; Ren, G W

    2011-04-05

    Human cytomegalovirus (HCMV) is a double-stranded DNA virus with the largest genome (~235 kb) of the known human herpes viruses. The coding potential and transcript structures of most HCMV predicted genes have not been identified. New or unknown genes could exist in clinical strains. The SMART (switching mechanism at 5' end of RNA template of reverse transcriptase) technique was used to construct a full-length cDNA library of an HCMV clinical strain in the late expression phase. Randomly selected clones were sequenced. The sequenced expressed sequence tags were used to identify the expression and transcript structures of some predicted and unpredicted genes of HCMV. The transcripts of the UL99, TRL5/IRL5, UL73 to UL75, UL4, and UL115 genes, which were previously detected, were obtained with full-length structures from this library. Some novel transcripts, including several transcripts of UL/b' genes and three antisense transcripts of UL83, UL87 and UL31 were found. The novel transcripts that were found, particularly the antisense transcripts of UL83, UL87 and UL31, showed that the transcription of HCMV genes is more complex than previously predicted. Our study highlights the usefulness of the full-length cDNA library for discovering new genes and transcripts of HCMV.

  1. Purification, characterisation and cDNA cloning of an antimicrobial peptide from Macadamia integrifolia.

    Science.gov (United States)

    Marcus, J P; Goulter, K C; Green, J L; Harrison, S J; Manners, J M

    1997-03-15

    An antimicrobial peptide with no significant amino acid sequence similarity to previously described peptides has been isolated from the nut kernels of Macadcamia integrifolia. The peptide, termed MiAMP1, is highly basic with an estimated pI of 10.1, a mass of 8.1 kDa and contains 76 amino acids including 6 cysteine residues. A cDNA clone containing the entire coding region corresponding to the peptide was obtained. The deduced amino acid sequence of the cDNA indicated a 26-amino-acid signal peptide at the N-terminus of the preprotein. Purified MiAMP1 inhibited the growth of a variety of fungal, oomycete and gram-positive bacterial phytopathogens in vitro. Some pathogens exhibited close to 100% inhibition in less than 1 microM peptide (5 microg/ml). Antimicrobial activity was diminished against most, but not all, microbes in the presence of calcium and potassium chloride salts (1 mM and 50 mM, respectively). MiAMP1 was active against bakers yeast, was inactive against Escherichia coli and was non-toxic to plant and mammalian cells. Analysis of genomic DNA indicated that MiAMP1 was encoded on a single copy gene containing no introns. The MiAMP1 gene may prove useful in genetic manipulations to increase disease resistance in transgenic plants.

  2. De-novo transcriptome sequencing of a normalized cDNA pool from influenza infected ferrets.

    Directory of Open Access Journals (Sweden)

    Jeremy V Camp

    Full Text Available The ferret is commonly used as a model for studies of infectious diseases. The genomic sequence of this animal model is not yet characterized, and only a limited number of fully annotated cDNAs are currently available in GenBank. The majority of genes involved in innate or adaptive immune response are still lacking, restricting molecular genetic analysis of host response in the ferret model. To enable de novo identification of transcriptionally active ferret genes in response to infection, we performed de-novo transcriptome sequencing of animals infected with H1N1 A/California/07/2009. We also included splenocytes induced with bacterial lipopolysaccharide to allow for identification of transcripts specifically induced by gram-negative bacteria. We pooled and normalized the cDNA library in order to delimit the risk of sequencing only highly expressed genes. While normalization of the cDNA library removes the possibility of assessing expression changes between individual animals, it has been shown to increase identification of low abundant transcripts. In this study, we identified more than 19,000 partial ferret transcripts, including more than 1000 gene orthologs known to be involved in the innate and the adaptive immune response.

  3. Primary analysis of the expressed sequence tags in a pentastomid nymph cDNA library.

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    Jing Zhang

    Full Text Available BACKGROUND: Pentastomiasis is a rare zoonotic disease caused by pentastomids. Despite their worm-like appearance, they are commonly placed into a separate sub-class of the subphylum Crustacea, phylum Arthropoda. However, until now, the systematic classification of the pentastomids and the diagnosis of pentastomiasis are immature, and genetic information about pentastomid nylum is almost nonexistent. The objective of this study was to obtain information on pentastomid nymph genes and identify the gene homologues related to host-parasite interactions or stage-specific antigens. METHODOLOGY/PRINCIPAL FINDINGS: Total pentastomid nymph RNA was used to construct a cDNA library and 500 colonies were sequenced. Analysis shows one hundred and ninety-seven unigenes were identified. In which, 147 genes were annotated, and 75 unigenes (53.19% were mapped to 82 KEGG pathways, including 29 metabolism pathways, 29 genetic information processing pathways, 4 environmental information processing pathways, 7 cell motility pathways and 5 organismal systems pathways. Additionally, two host-parasite interaction-related gene homologues, a putative Kunitz inhibitor and a putative cysteine protease. CONCLUSION/SIGNIFICANCE: We first successfully constructed a cDNA library and gained a number of expressed sequence tags (EST from pentastomid nymphs, which will lay the foundation for the further study on pentastomids and pentastomiasis.

  4. The effects of radiation on p53-mutated glioma cells using cDNA microarray technique

    International Nuclear Information System (INIS)

    Ngo, F.Q.H.; Hsiao, Y.-Y.H.

    2003-01-01

    Full text: In this study, we investigated the effects of 10-Gy irradiation on cell-cycle arrest, apoptosis and clonogenic death in the p53-mutated human U138MG (malignant glioblastoma) cell line. In order to evaluate time-dependent events in cellular responses to radiation, we did a time course study by incubating cells ranging from 0.5 to 48 hours after irradiation. Cell-cycle distribution and apoptosis were evaluated by flow cytometry using propidium iodide (PI) and annexin-V plus PI staining. Cell viability and proliferative capacity were studied by colony formation assay. Dual fluorescence cDNA microarray technique was used to examine the differential expression patterns of the irradiated cells. The cDNA microarray chips used contained DNA sequences corresponding to 12,814 human genes. From the flow cytometry data, it can be observed that radiation induced G2/M phase arrest and that late apoptosis was more evident following G2/M arrest. After 36 hours, some cells underwent senescence and the remains continued on with the cell cycle. Microarray analyses revealed changes in the expression of a small number of cell-cycle-related genes (p21, cyclin B1, etc.) and cell-death genes (tumor necrosis factors, DDB2, etc.) suggesting their involvement in radiation-induced cell-cycle arrest and apoptosis. In silico interpretations of the molecular mechanisms responsible for these radiation effects are in progress

  5. High-throughput screening of suppression subtractive hybridization cDNA libraries using DNA microarray analysis.

    Science.gov (United States)

    van den Berg, Noëlani; Crampton, Bridget G; Hein, Ingo; Birch, Paul R J; Berger, Dave K

    2004-11-01

    Efficient construction of cDNA libraries enriched for differentially expressed transcripts is an important first step in many biological investigations. We present a quantitative procedure for screening cDNA libraries constructed by suppression subtractive hybridization (SSH). The methodology was applied to two independent SSHs from pearl millet and banana. Following two-color cyanin dye labeling and hybridization of subtracted tester with either unsubtracted driver or unsubtracted tester cDNAs to the SSH libraries arrayed on glass slides, two values were calculated for each clone, an enrichment ratio 1 (ER1) and an enrichment ratio 2 (ER2). Graphical representation of ER1 and ER2 enabled the identification of clones that were likely to represent up-regulated transcripts. Normalization of each clone by the SSH process was determined from the ER2 values, thereby indicating whether clones represented rare or abundant transcripts. Differential expression of pearl millet and banana clones identified from both libraries by this quantitative approach was verified by inverse Northern blot analysis.

  6. In-frame cDNA library combined with protein complementation assay identifies ARL11-binding partners.

    Directory of Open Access Journals (Sweden)

    Sangkyou Lee

    Full Text Available The cDNA expression libraries that produce correct proteins are essential in facilitating the identification of protein-protein interactions. The 5'-untranslated regions (UTRs that are present in the majority of mammalian and non-mammalian genes are predicted to alter the expression of correct proteins from cDNA libraries. We developed a novel cDNA expression library from which 5'-UTRs were removed using a mixture of polymerase chain reaction primers that complement the Kozak sequences we refer to as an "in-frame cDNA library." We used this library with the protein complementation assay to identify two novel binding partners for ras-related ADP-ribosylation factor-like 11 (ARL11, cellular retinoic acid binding protein 2 (CRABP2, and phosphoglycerate mutase 1 (PGAM1. Thus, the in-frame cDNA library without 5'-UTRs we describe here increases the chance of correctly identifying protein interactions and will have wide applications in both mammalian and non-mammalian detection systems.

  7. Cloning, expression, and mapping of GDP-D-mannose pyrophosphorylase cDNA from tomato (Lycopersicon esculentum).

    Science.gov (United States)

    Zou, Li-Ping; Li, Han-Xia; Ouyang, Bo; Zhang, Jun-Hong; Ye, Zhi-Biao

    2006-08-01

    GDP-D-mannose pyrophosphorylase (GMP, EC 2.7.7.22) catalyzes the synthesis of GDP-D-mannose and represents the first committed step in plant ascorbic acid biosynthesis. Using potato GMP cDNA sequence as a querying probe, 65 highly homologous tomato ESTs were obtained from dbEST of GenBank and the putative cDNA sequence of tomato GMP was assembled. The full-length GMP cDNA of tomato was cloned by RACE-PCR with primers designed according to the assembled cDNA sequence. The full-length cDNA sequence contained a complete open reading frame (ORF) of 1,086 bp, which encoded 361 amino acid residues. This gene was designated as LeGMP (GenBank accession No. AY605668). Homology analysis of LeGMP showed a 96% identity with potato GMP and the deduced amino acid showed 99%, 97%, 91% and 89% homology with GMP from potato, tobacco, alfalfa and Arabidopsis thaliana, respectively. Northern blot analysis showed that LeGMP was constitutively expressed in roots, stems, leaves, flowers and fruits of tomato; but the expression levels varied. LeGMP was mapped to 3-D using 75 tomato introgression lines (ILs), each containing a single homozygous RFLP-defined chromosome segment from the green-fruited species Lycopersicon pennellii.

  8. Construction and characterization of yeast two-hybrid cDNA library derived from LFBK cell line.

    Science.gov (United States)

    Mahajan, Sonalika; Sharma, Gaurav Kumar; Matura, Rakesh; Subramaniam, Saravanan; Mohapatra, Jajati Keshari; Pattnaik, Bramhadev

    2015-05-01

    The cDNA libraries are indispensable and critical tools for performing protein-protein interaction studies. In this study, a high quality yeast two-hybrid cDNA library from the LFBK cell line was constructed and characterized. LFBK cell line was originally derived from the swine kidney cells and is highly susceptible to foot-and-mouth disease virus (FMDV) infection. The total RNA was extracted from the LFBK cells and the switching mechanism at the 5' end of RNA template (SMART) technique was employed for the cDNA synthesis. Subsequently, double stranded cDNA was amplified by long-distance PCR, purified and co-transformed with pGADT7-rec vector in yeast strain Y187. The quality parameters of the constructed library were evaluated to qualify the constructed library. Nucleotide sequencing of the randomly selected clones from the library confirmed the swine genotype of LFBK cell line. The LFBK cDNA library was mated with the 2C protein of FMDV in yeast two-hybrid (YTH) system and several putative interaction partners were identified in the preliminary screening. The LFBK library was observed to be of high quality and could potentially be applied to protein interaction studies between FMDV and the host cells using YTH system. Copyright © 2015 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

  9. A High Performance Platform Based on cDNA Display for Efficient Synthesis of Protein Fusions and Accelerated Directed Evolution.

    Science.gov (United States)

    Naimuddin, Mohammed; Kubo, Tai

    2016-02-08

    We describe a high performance platform based on cDNA display technology by developing a new modified puromycin linker-oligonucleotide. The linker consists of four major characteristics: a "ligation site" for hybridization and ligation of mRNA by T4 RNA ligase, a "puromycin arm" for covalent linkage of the protein, a "polyadenosine site" for a longer puromycin arm and purification of protein fusions (optional) using oligo-dT matrices, and a "reverse transcription site" for the formation of stable cDNA protein fusions whose cDNA is covalently linked to its encoded protein. The linker was synthesized by a novel branching strategy and provided >8-fold higher yield than previous linkers. This linker enables rapid and highly efficient ligation of mRNA (>90%) and synthesis of protein fusions (∼ 50-95%) in various cell-free expression systems. Overall, this new cDNA display method provides 10-200 fold higher end-usage fusions than previous methods and benefits higher diversity libraries crucial for directed protein/peptide evolution. With the increased efficiency, this system was able to reduce the time for one selection cycle to cDNA display method. A three-finger protein library was evolved to isolate superior nanomolar range binding candidates for vascular endothelial growth factor. This method is expected to provide a beneficial impact to accelerated drug discovery and proteome analysis.

  10. Construction of cDNA libraries from Pseudocercospora fijiensis Morelet infected leaves of the cultivars Calcutta 4 and Niyarma Yik

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    Milady Mendoza-Rodríguez

    2004-01-01

    Full Text Available Molecular studies of plant-pathogen interaction are very important for the identification of gene (s related with the pathogenic process, as well as with the plant resistance. These gene (s could be use for the genetic improvement programs in order to obtain resistant cultivars. The aim of this work was to construct complementary DNA (cDNA libraries from infected leaves with Pseudocercospora fijiensis CCIBP-Pf1 isolated of two banana cultivars (a resistant one Calcutta4 and another one susceptible Niyarma Yik. First-strand cDNA synthesis, was made beginning with one microgram of total RNA by using oligo dT primer and cDNA quality was checked by Polimerase chain reaction (PCR with cytochrome b specific primers. Second-strand cDNA synthesis was performed by using the homopolymeric tailing with dC-BamH I + dT-Not I primer combination. Four cDNA libraries of infected plants at different times of infection with the pathogen were obtained. Forty one clones of one of the libraries of Niyarma Yik were sequenced and the obtained sequences correspond with genes related to fungi. Key words: Banana-Mycosphaerella fijiensis interaction,Black Sigatoka, Musa spp.

  11. Peripherally-inserted Central Catheter (PICC Insertion in Neonates: Apnea and Radiocontrasts Complications

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    Eissa Bilehjani

    2015-01-01

    Full Text Available The importance of pain management throughout minimally-invasive procedures such as peripherally-inserted central catheter (PICC insertion is an inevitable fact. However, administration of varied analgesics, especially opioids, in specific age ranges such as neonate could be of immense concern. Most of these patients are on different medications, especially those hospitalized in the ICUs or with critical conditions. Neonates receiving continuous opioid infusions might also require an additional opioid bolus to control the associated pain (1. Importantly, it should be taken into consideration that administration of opioids or even a light sedation would increase the risk of delayed apnea in this group of patients (2. Factors contributing to this complication include ex-premature infants (gestational age<37 weeks, premature infants younger than 46 weeks’ postconceptual age and anemia (hematocrit< 30% (3, 4. Hence, it is recommended to use other supplementary analgesic techniques including local or topical anesthesia, especially in neonates having previously received continuous or bolus opioids. Furthermore, supplementary techniques assisting the confirmation of proper placement such as overpenetrating the radiograph, lateral X-rays and positioning the infant for a lateral oblique radiograph (right side elevated at a 10°–15° angle are frequently used by clinicians to avoid administration of radiocontrasts and their associated complications. In the latter technique, catheter visualization could be enhanced as the PICC will not be superimposed over the mediastinal structures (5, 6.

  12. Postplacental insertion of the levonorgestrel intrauterine device after cesarean delivery vs. delayed insertion: a randomized controlled trial.

    Science.gov (United States)

    Whitaker, Amy K; Endres, Loraine K; Mistretta, Stephanie Q; Gilliam, Melissa L

    2014-06-01

    This trial was designed to compare levonorgestrel intrauterine device (LNG-IUD) use at 1 year after delivery between women randomized to postplacental insertion at the time of cesarean delivery and delayed insertion 4-8 weeks after delivery. This randomized controlled trial was conducted at two urban medical centers. Eligible pregnant women with planned cesarean deliveries were randomized to immediate postplacental insertion during cesarean or delayed insertion after 4-8 weeks. We used intention-to-treat analysis for the primary outcome of LNG-IUD use 12 months after delivery. Forty-two women were randomized, 20 into the postplacental group and 22 in the delayed group. Although confirmed use of the LNG-IUD 12 months after delivery was higher in the postplacental group (60.0% vs. 40.9%, p=.35), this difference was not statistically significance. Expulsion was significantly more common in the postplacental group (20.0% vs. 0%, p=.04). There were significant differences between the two sites in baseline population characteristics, follow-up and expulsion. The trial did not answer the intended question as it was halted early due to slow enrollment. Our results show higher expulsion after postplacental insertion compared to delayed insertion but suggest similar IUD use at 12 months. Moreover, it provides valuable lessons regarding a randomized controlled trial of postplacental LNG-IUD placement due to the challenges of estimating effect size and the nature of the population who might benefit from immediate insertion. Postplacental insertion of an IUD may improve use of highly effective contraception during the postpartum period. While our results suggest higher expulsion after postplacental insertion compared to delayed insertion and similar IUD use at 12 months, our trial was insufficient to definitively test our hypothesis. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Cloning and expression of a cDNA covering the complete coding region of the P32 subunit of human pre-mRNA splicing factor SF2

    DEFF Research Database (Denmark)

    Honoré, B; Madsen, Peder; Rasmussen, H H

    1993-01-01

    We have cloned and expressed a cDNA encoding the 32-kDa subunit (P32) of the human pre-mRNA splicing factor, SF2. This cDNA extends beyond the 5'-end of a previously reported cDNA [Krainer et al., Cell 66 (1991) 383-394]. Importantly, our fragment includes an ATG start codon which was absent from...

  14. Predicting painful or difficult intrauterine device insertion in nulligravid women.

    Science.gov (United States)

    Kaislasuo, Janina; Heikinheimo, Oskari; Lähteenmäki, Pekka; Suhonen, Satu

    2014-08-01

    To assess the relationship of preinsertion vaginal ultrasound assessment and menstrual and gynecologic history as predictors of difficult or painful intrauterine device insertion in nulligravid women. Nulligravid women seeking contraception were invited to participate in this nonrandomized study and given the choice between the levonorgestrel-releasing intrauterine system or a copper-releasing intrauterine device. All 165 enrolled women were interviewed and a pelvic examination, including vaginal ultrasonography, was performed before insertion. Insertion difficulties and pain intensity were recorded and assessed against uterine measurements and background characteristics. Most insertions were assessed as easy (n=144 [89.4%]) and only two (1.2%) failed. Most women had uterine measurements smaller than the studied devices. Odds for difficulties at insertion decreased with every increasing millimeter in total uterine length (odds ratio [OR] 0.86, 95% confidence interval [CI] 0.78-0.96, P=.006) and cervical length (OR 0.85, 95% CI 0.74-0.97, P=.02) and similarly with every decreasing degree of (straighter) flexion angle (OR 0.96, 95% CI 0.94-0.99, P=.005). No absolute threshold measurements could be determined. Still, the majority of insertions in small and flexed uteri were uneventful. Severe insertion pain was common (n=94 [58.4%]). Severe dysmenorrhea was the only predictor of insertion pain (OR 8.16 95% CI 2.56-26.02, P<.001). Ultrasonographic evaluation does not give additional information compared with clinical pelvic examination and sound measure. Although smaller uterine length measurements and steeper flexion angle more often predicted difficulties, the majority of insertions were uneventful in women with small measures. Dysmenorrhea was the only predictor of pain. ClinicalTrials.gov, www.clinicaltrials.gov, NCT01685164. II.

  15. Redefining Zone II: Anatomy of the Flexor Digitorum Superficialis Insertion.

    Science.gov (United States)

    Nicholson, Luke T; Hill, Jeffrey R; McKnight, Braden; Heckmann, Nathanael; Stevanovic, Milan; Ghiassi, Alidad

    2017-10-01

    Flexor zone II is defined as the region spanning the proximal aspect of the A1 pulley to the insertion of the flexor digitorum superficialis (FDS) tendon. Descriptions of the FDS insertion are inconsistent in the literature, but zones of injury are frequently determined with reference to superficial landmarks. The purpose of this study was to describe the footprint of the FDS insertion and define its relationship to the proximal interphalangeal (PIP) skin crease. The FDS insertion on the index, middle, ring, and small fingers was dissected in 6 matched pairs of fresh-frozen cadaveric hands. A Kirschner wire was used to mark the level of the PIP skin crease on bone before measurements of the FDS footprint and its position relative to the PIP skin crease were made using digital calipers. The radial and ulnar FDS slips inserted a mean distance of 3.22 mm from the distal aspect of the PIP skin crease and varied by digit. The mean distal extent of the FDS insertion was 8.29 mm. The mean length of the insertion of each FDS slip was 5.15 mm and the mean width was 1.9 mm. The radial and ulnar FDS slips insert on average 3.22 mm distal to the PIP skin crease and vary by digit. Knowledge of the FDS insertion is clinically relevant when differentiating between flexor zone I and zone II injuries, planning surgical approaches to the finger, and in guiding patient expectations for surgery given the variability in outcome based on zone of injury.

  16. 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

  17. Cloning and characterization of a female genital complex cDNA from the liver fluke Fasciola hepatica.

    Science.gov (United States)

    Zurita, M; Bieber, D; Ringold, G; Mansour, T E

    1987-01-01

    A cDNA clone whose RNA is abundant in the female genital complex of the liver fluke Fasciola hepatica has been isolated from a cDNA library in lambda gt10 by differential screening. The pattern of expression in different fluke tissues and at different stages of miracidium formation suggests that this gene is expressed in the F. hepatica vitelleria. The nucleotide sequence of the cloned cDNA was determined and the primary structure of the putative protein was deduced. The proposed protein is rich in glycine, lysine, and tyrosine and its overall amino acid composition agrees with that reported for the F. hepatica egg shell. The clone has homology with DNA from other trematodes; this homology is higher in organisms in which egg development is similar to that of F. hepatica and suggests that the protein is conserved in organisms in which miracidium formation occurs in fresh water. Images PMID:3470798

  18. 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

  19. Data on internal cDNA amplification and color changes of the proteins derived from Pacific white leg shrimp shell.

    Science.gov (United States)

    Chuang, Pan; Shoichiro, Ishizaki; Yuji, Nagashima; Jialong, Gao; Shugo, Watabe

    2018-02-01

    In this article, we report original data on the designation of the primers for full-length cDNA amplification and the internal cDNA amplification of red color-related pigment-binding protein derived from shrimp shell. Data on the color shifts of different soluble proteins under 100 °C 10 min heat treatment and the effects of heating temperatures (from 30 to 100 °C) on the color changes of crude water-soluble proteins are also included in this report. For further details and experimental findings please refer to the article "Isolation and cDNA cloning of a novel red color-related pigment-binding protein derived from the shell of shrimp, Litopenaeus vannamei " (Chuang et al., 2017) [1].

  20. Data on internal cDNA amplification and color changes of the proteins derived from Pacific white leg shrimp shell

    Directory of Open Access Journals (Sweden)

    Pan Chuang

    2018-02-01

    Full Text Available In this article, we report original data on the designation of the primers for full-length cDNA amplification and the internal cDNA amplification of red color-related pigment-binding protein derived from shrimp shell. Data on the color shifts of different soluble proteins under 100 °C 10 min heat treatment and the effects of heating temperatures (from 30 to 100 °C on the color changes of crude water-soluble proteins are also included in this report. For further details and experimental findings please refer to the article “Isolation and cDNA cloning of a novel red color-related pigment-binding protein derived from the shell of shrimp, Litopenaeus vannamei” (Chuang et al., 2017 [1].

  1. Molecular cloning and sequence analysis of growth hormone cDNA of Neotropical freshwater fish Pacu (Piaractus mesopotamicus

    Directory of Open Access Journals (Sweden)

    Janeth Silva Pinheiro

    2008-01-01

    Full Text Available RT-PCR was used for amplifying Piaractus mesopotamicus growth hormone (GH cDNA obtained from mRNA extracted from pituitary cells. The amplified fragment was cloned and the complete cDNA sequence was determined. The cloned cDNA encompassed a sequence of 543 nucleotides that encoded a polypeptide of 178 amino acids corresponding to mature P. mesopotamicus GH. Comparison with other GH sequences showed a gap of 10 amino acids localized in the N terminus of the putative polypeptide of P. mesopotamicus. This same gap was also observed in other members of the family. Neighbor-joining tree analysis with GH sequences from fishes belonging to different taxonomic groups placed the P. mesopotamicus GH within the Otophysi group. To our knowledge, this is the first GH sequence of a Neotropical characiform fish deposited in GenBank.

  2. Direct trocar insertion vs. Verres needle use for laparoscopic sterilization.

    Science.gov (United States)

    Borgatta, L; Gruss, L; Barad, D; Kaali, S G

    1990-09-01

    A randomized, prospective trial was designed to compare direct trocar insertion with prior peritoneal insufflation with a Verres needle for laparoscopic tubal sterilization. Direct trocar insertion resulted in fewer instrument insertions (21.8% vs. 7.8%) and use of smaller volumes of CO2 (2.67 vs. 2.32 L). Direct trocar use resulted in a decrease in operating time from 9 minutes, 40 seconds in the needle group to 7 minutes, 30 seconds in the trocar group. Minor omental injuries occurred in a small percentage of each group, while serious complications occurred once in each group.

  3. Evaluation of normalization methods for cDNA microarray data by k-NN classification

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Wei; Xing, Eric P; Myers, Connie; Mian, Saira; Bissell, Mina J

    2004-12-17

    Non-biological factors give rise to unwanted variations in cDNA microarray data. There are many normalization methods designed to remove such variations. However, to date there have been few published systematic evaluations of these techniques for removing variations arising from dye biases in the context of downstream, higher-order analytical tasks such as classification. Ten location normalization methods that adjust spatial- and/or intensity-dependent dye biases, and three scale methods that adjust scale differences were applied, individually and in combination, to five distinct, published, cancer biology-related cDNA microarray data sets. Leave-one-out cross-validation (LOOCV) classification error was employed as the quantitative end-point for assessing the effectiveness of a normalization method. In particular, a known classifier, k-nearest neighbor (k-NN), was estimated from data normalized using a given technique, and the LOOCV error rate of the ensuing model was computed. We found that k-NN classifiers are sensitive to dye biases in the data. Using NONRM and GMEDIAN as baseline methods, our results show that single-bias-removal techniques which remove either spatial-dependent dye bias (referred later as spatial effect) or intensity-dependent dye bias (referred later as intensity effect) moderately reduce LOOCV classification errors; whereas double-bias-removal techniques which remove both spatial- and intensity effect reduce LOOCV classification errors even further. Of the 41 different strategies examined, three two-step processes, IGLOESS-SLFILTERW7, ISTSPLINE-SLLOESS and IGLOESS-SLLOESS, all of which removed intensity effect globally and spatial effect locally, appear to reduce LOOCV classification errors most consistently and effectively across all data sets. We also found that the investigated scale normalization methods do not reduce LOOCV classification error. Using LOOCV error of k-NNs as the evaluation criterion, three double

  4. ISOLASI cDNA SUCROSE TRANSPORTER (SUT DARI BATANG TANAMAN TEBU (Saccharum officinarum L.

    Directory of Open Access Journals (Sweden)

    - Slameto

    2010-09-01

    Full Text Available Sucrose Transporter (SUT is kind of protein transporter that control in sucrose translocation. Sucrose Transporter is intermediate in translocation of sucrose from apoplasmic to simplasmic. SUT facilitates sucrose transportation from vascular tissues to parenchyma cells toward in node sugarcane stem. This research was purposed to isolate cDNA SUT from sugarcane stem, and cloned in Escherichia coli strain DH5α. Total RNA of sugarcane stem was isolated by single step method, then add with oligo dT in order to obtain the first strand of SUT cDNA then used as template for PCR. The primer used for PCR is 5’ –ggg ctg att gtg gcc atg tc- ‘3 (SUT-F and 5’ –tgc cct ttg tct ccg gaa cc- ‘3 (SUT-R. PCR was programmed as follow denaturation at 94°C for 2 minutes and 30 second, annealing at 54°C for 30 s, extension at 72°C 2 min and 7 min, and storage at 4°C for unlimited, It was for 30 cycles. Complementary DNA SUT from PCR ligalized to pTOPO bunt-end, then it cloned in to E. coli strain DH5α. The cloning resulted then be sequenced in order to observe the homologues with other nucleotides sequences of some plant using BLASTn program in GENE BANK NCBI and the level of homology determined by Genetyx program. The concentrated of total RNA isolated was 5,024 μg/μl, with purity of 1,85. Complementary DNA SUT fragment from PCR with size 2037 bp appropriated to the both of primer was used. Complementary DNA SUT fragment showed by analyzed some of restriction enzyme e.g. EcoRI, PstI and BamHI. Homologues of this cDNA SUT fragment was 100% to SoSUT 2A of sugarcane stem and 84% to OsSUT of rice plant (Casu et al ., 2003.

  5. The isolation and amplification of full length cDNA of oleosins from ...

    African Journals Online (AJOL)

    STORAGESEVER

    2010-03-29

    Mar 29, 2010 ... Subcloning of DNA by inserting into pCR® 4-TOPO vector was performed using TOPO TA Cloning® Kit for Sequencing. (Invitrogen, USA). The sequencing of plasmid clones was performed using ABI PRISMTM Dye Terminator Cycle Sequencing Ready. Reaction Kit (Applied Biosystems Inc., USA) and an ...

  6. Cloning of the mouse cDNA encoding DNA topoisomerase I and chromosomal location of the gene.

    Science.gov (United States)

    Koiwai, O; Yasui, Y; Sakai, Y; Watanabe, T; Ishii, K; Yanagihara, S; Andoh, T

    1993-03-30

    The mouse cDNA encoding DNA topoisomerase I (TopoI) was cloned and the nucleotide sequence of 3512 bp was determined. The cDNA clone contained an open reading frame encoding a protein of 767 amino acids (aa), which is 2 aa longer than its human counterpart. Overall aa sequence homology between the mouse and human, and between the mouse and yeast (Saccharomyces cerevisiae) sequences was 96% and 42%, respectively. The mouse TopI gene was mapped at position 54.5 on chromosome 2 from linkage analyses of a three-point cross test with Geg, Ada, and a as marker genes.

  7. Increased risk of symptomatic upper-extremity venous thrombosis with multiple peripherally inserted central catheter insertions in pediatric patients.

    Science.gov (United States)

    Gnannt, Ralph; Waespe, Nicolas; Temple, Michael; Amirabadi, Afsaneh; Liu, Kuan; Brandão, Leonardo R; Connolly, Bairbre L

    2018-02-27

    Peripherally inserted central catheters (PICCs) are associated with superficial and deep venous thrombosis of the arm. The purpose of this study was to analyze the sequelae of repeated upper limb PICC insertions in children, in terms of the frequency of upper limb thrombosis in this patient group. The study population included all children who underwent their first successful arm PICC insertion between January 2010 and December 2015. We included subsequent ipsilateral arm PICCs in the analysis. Patients were followed until March 2016 or until any alternative central venous line insertion. For each PICC insertion, we collected demographic variables and line characteristics. We correlated all symptomatic deep and superficial thromboses of the arm with the PICC database. Applying inclusion and exclusion criteria, 2,180 PICCs remained for analysis. We identified first, second, third and fourth PICC insertions in the same arm in 1,955, 181, 38 and 6 patients, respectively. In total there were 57 upper body deep symptomatic thrombotic events. An increasing odds ratio was seen with higher numbers of PICC insertions, which was significant when comparing the first with the third and fourth PICC insertions in the same arm (odds ratio [OR] 6.00, 95% confidence interval [CI] 2.25-16.04, P=0.0004). Double-lumen PICCs were associated with a significantly higher risk of thrombosis than single lumen (OR 2.77, 95% CI 1.72-4.47, P=0.0003). Repetitive PICC insertions in the same arm are associated with an increased risk of symptomatic thrombosis. Double-lumen PICCs are associated with a higher risk of thrombosis compared to single-lumen lines.

  8. 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)

  9. Genes expression by using cDNA Microarray in Whallak-tang

    Directory of Open Access Journals (Sweden)

    Cheol-kyung Sin

    2008-12-01

    Full Text Available Objective : This study was undertaken to determine the effect of Whallak-tang on expression of CD/cytokine Genes. Methods : The expression of CD/Cytokine Genes were examined by cDNA microarray using the human mast cell line(HMC-l. Results : The expression of ATP5F1, FLJ20671, unknown, KIAA0342, OAS2, unknown genes were increased in 200~300% range. The expression of unknown, MDS006, IFITM1, MRPL3, ZNF207, FTH1, FBP1, NRGN, NR1H2, KIAA0747 genes were decreased in 0~33% range. Conclusion : These results would provide important basic data on the possibility of the clinical treatment of Whallaktang in musculoskeletal disease.

  10. Gene expression profile analysis in human hepatocellular carcinoma by cDNA microarray.

    Science.gov (United States)

    Chung, Eun Jung; Sung, Young Kwan; Farooq, Mohammad; Kim, Younghee; Im, Sanguk; Tak, Won Young; Hwang, Yoon Jin; Kim, Yang Il; Han, Hyung Soo; Kim, Jung-Chul; Kim, Moon Kyu

    2002-12-31

    We performed gene expression profiling of normal and hepatocellular carcinoma (HCC) liver tissues using a high-density microarray that contained 3,063 human cDNA. The results of a microarray hybridization experiment from eight different HCC tissues were analyzed and classified by the Cluster program. Among these differentially-expressed genes, the galectin-3, serine/threonine kinase SGK, translation factor eIF-4A, -4B, -3, fibroblast growth factor receptor, and ribosomal protein L35A were up-regulated; the mRNAs of Nip3, decorin, and the insulin-like growth factor binding protein-3 were down-regulated in HCC. The differential expression of these genes was further confirmed by an RT-PCR analysis. In addition, our data suggest that the gene expression profile of HCC varies according to the histological types.

  11. [Development of new SSR markers from EST of SSH cDNA libraries on rose fragrance].

    Science.gov (United States)

    Yan, Hui-Jun; Zhang, Hao; Xie, Ji-Rong; Li, Shu-Fa; Jian, Hong-Ying; Qiu, Xian-Qin; Wang, Qi-Gang; Wang, Ji-Hua; Tang, Kai-Xue

    2009-09-01

    The new SSR markers of rose related fragrance were developed based on the SSH cDNA libraries of rose floral scent mutant. In this study, 10 EST-SSRs (2.6%) from 391 ESTs in the libraries were identified. Six EST-SSRs primers were designed to sequence flanking SSRs. The primer pairs designed were screened on the wild-type Jinyindao, which has flowers full of pleasant scent, and the mutant-type Wangriqinghuai without perceivable floral scent. Five primer pairs were amplified effectively in Jinyindao and Wangriqinghuai, and 3 were polymorphic between Jinyindao and Wangriqinghuai. Eighteen rose cultivars including fragrant roses and nonfragrant roses were identified by the five prime pairs. These results proved that EST-SSR markers are effective markers to identify the polymorphism of the rose.

  12. Optics Solutions for the Collimation Insertion of LHC

    CERN Document Server

    Craddock, M K; Servranckx, R V; Luccio, A U; MacKay, W W

    1999-01-01

    While the two collimation insertions in the LHC must have similar basic layouts and match to almost identical dispersion suppressors to respect the geometry of the existing tunnel, their different roles impose opposite requirements on the normalized dispersion within them. For betatron collimation it must be near zero, while for momentum collimation it must have a peak at the location of the primary collimator, immediately after the dispersion suppressor. The insertion lattice solution found for the latter case requires up to 30% asymmetry in the quadrupole gradients (in line with the current trend in LHC lattice development to break the exact antisymmetry within insertions). To achieve this using twin-aperture warm quadrupoles, the modules making up each quadrupole will be wired in such a way that the two beams still see the same sequence of focusing fields. We describe the optimum setup, exibility and collimation quality for the two insertions.

  13. Laparoscopic insertion of lumbar peritoneal shunts for idiopathic ...

    African Journals Online (AJOL)

    Background: The treatment of idiopathic intracranial hypertension varies from simple observation to the mechanical diversion of cerebrospinal fluid. Objective: To describe our technique and initial experience with the laparoscopic insertion of lumbarperitoneal shunts. Conclusion: Lumbarperitoneal shunts should be ...

  14. Net-bottom Cage Inserts for Water Bird Casualties

    Directory of Open Access Journals (Sweden)

    Jackie Belle

    2017-10-01

    Full Text Available My Bright Idea is a net-bottomed cage insert, which is used to support pelagic avian casualties. The idea was designed and modified by the International Bird Rescue in California (Bird Rescue.

  15. Fireplace insert and its parameters depend on the used glazing

    Science.gov (United States)

    Papučík, Štefan; Čaja, Alexander

    2016-06-01

    The contribution deals with the analysis of the impact of using double glass to change the performance and emission parameters of the fireplace insert. Conventional fireplace inserts are equipped with heat-resistant glass, which is resistant to high temperatures. For this type of inserts are required to be radiant constituent maximized. Prevailing part of heat is into the interior gets just by radiation through the glazed part. The hot water fireplace inserts is the requirement that the radiant constituent to the environment to a minimum. Therefore, instead of a single glass using double glazing which is intended to reduce this part of heat transfer. The temperature in the furnace is increased, and transmitted most of the heat into the water.

  16. Single-copy insertion of transgenes in Caenorhabditis elegans.

    Science.gov (United States)

    Frøkjaer-Jensen, Christian; Davis, M Wayne; Hopkins, Christopher E; Newman, Blake J; Thummel, Jason M; Olesen, Søren-Peter; Grunnet, Morten; Jorgensen, Erik M

    2008-11-01

    At present, transgenes in Caenorhabditis elegans are generated by injecting DNA into the germline. The DNA assembles into a semistable extrachromosomal array composed of many copies of injected DNA. These transgenes are typically overexpressed in somatic cells and silenced in the germline. We have developed a method that inserts a single copy of a transgene into a defined site. Mobilization of a Mos1 transposon generates a double-strand break in noncoding DNA. The break is repaired by copying DNA from an extrachromosomal template into the chromosomal site. Homozygous single-copy insertions can be obtained in less than 2 weeks by injecting approximately 20 worms. We have successfully inserted transgenes as long as 9 kb and verified that single copies are inserted at the targeted site. Single-copy transgenes are expressed at endogenous levels and can be expressed in the female and male germlines.

  17. Automatic needle insertion diminishes pain during growth hormone injection

    DEFF Research Database (Denmark)

    Main, K M; Jørgensen, J T; Hertel, N T

    1995-01-01

    Non-compliance in children receiving growth hormone (GH) treatment is often caused by pain on injection and difficulties in administration of GH. It has been suggested that automatic needle insertion diminishes pain perception. We quantitatively measured pain intensity on injection with two...... prototype pens for GH administration, providing either manual or automatic sc needle insertion, using a combined visual analogue/facial scale and a five-item scale in 18 children. With the automatic pen there was a significantly lower maximum pain score compared with the manual pen (median 28.5 versus 52.......0 mm) as well as a lower mean pain score (mean 13.7 versus 23.5 mm). The five-item scale revealed that automatic needle insertion was significantly less painful than manual insertion and 13 patients chose to continue treatment with the automatic pen. In conclusion, pain during GH injection can...

  18. Insertion device magnet measurements for the Advanced Light Source

    International Nuclear Information System (INIS)

    Marks, S.; Cork, C.; Hoyer, E.; Humphries, D.; Kincaid, B.; Plate, D.; Robb, A.; Schlueter, R.; Wang, C.; Hassenzahl, W.

    1993-05-01

    Allowable magnetic field errors for the 4.6 m long insertion devices for the Advanced Light Source (ALS) are extremely small and are driven by electron beam and radiation requirements. Detailed measurements and adjustments of each insertion device are performed to qualify them for installation in the ALS. To accomplish this, a high speed, precision magnetic measurement facility has been designed and built. Hall probe mapping equipment, capable of completing a 2500 sample, 6 m scan with precision axial position monitoring using a laser interferometer in under one minute, is used to obtain both local and integrated field information. A 5.5 m long, 1 cm wide coil is used to measure the field integral through an entire insertion device. This paper describes magnetic measurement equipment, and results of measurements on IDA, the first of the ALS insertion devices

  19. Development of infectious cDNA clones of Salmonid alphavirus subtype 3

    Directory of Open Access Journals (Sweden)

    Karlsen Marius

    2010-09-01

    Full Text Available Abstract Background Salmonid alphavirus (SAV is a widespread pathogen in European aquaculture of salmonid fish. Distinct viral subtypes have been suggested based on sequence comparisons and some of these have different geographical distributions. In Norway, only SAV subtype 3 have so far been identified. Little is known about viral mechanisms important for pathogenesis and transmission. Tools for detailed exploration of SAV genomes are therefore needed. Results Infectious cDNA clones in which a genome of subtype 3 SAV is under the control of a CMV promoter were constructed. The clones were designed to express proteins that are putatively identical to those previously reported for the SAVH20/03 strain. A polyclonal antiserum was raised against a part of the E2 glycoprotein in order to detect expression of the subgenomic open reading frame (ORF encoding structural viral proteins. Transfection of the cDNA clone revealed the expression of the E2 protein by IFAT, and in serial passages of the supernatant the presence of infectious recombinant virus was confirmed through RT-PCR, IFAT and the development of a cytopathic effect similar to that seen during infection with wild type SAV. Confirmation that the recovered virus originated from the infectious plasmid was done by sequence identification of an introduced genetic tag. The recombinant virus was infectious also when an additional ORF encoding an EGFP reporter gene under the control of a second subgenomic alphavirus promoter was added. Finally, we used the system to study the effect of selected point mutations on infectivity in Chinook salmon embryo cells. While introduced mutations in nsP2197, nsP3263 and nsP3323 severely reduced infectivity, a serine to proline mutation in E2206 appeared to enhance the virus titer production. Conclusion We have constructed infectious clones for SAV based on a subtype 3 genome. The clones may serve as a platform for further functional studies.

  20. Analysis of cDNA libraries from developing seeds of guar (Cyamopsis tetragonoloba (L. Taub

    Directory of Open Access Journals (Sweden)

    Dixon Richard A

    2007-11-01

    Full Text Available Abstract Background Guar, Cyamopsis tetragonoloba (L. Taub, is a member of the Leguminosae (Fabaceae family and is economically the most important of the four species in the genus. The endosperm of guar seed is a rich source of mucilage or gum, which forms a viscous gel in cold water, and is used as an emulsifier, thickener and stabilizer in a wide range of foods and industrial applications. Guar gum is a galactomannan, consisting of a linear (1→4-β-linked D-mannan backbone with single-unit, (1→6-linked, α-D-galactopyranosyl side chains. To better understand regulation of guar seed development and galactomannan metabolism we created cDNA libraries and a resulting EST dataset from different developmental stages of guar seeds. Results A database of 16,476 guar seed ESTs was constructed, with 8,163 and 8,313 ESTs derived from cDNA libraries I and II, respectively. Library I was constructed from seeds at an early developmental stage (15–25 days after flowering, DAF, and library II from seeds at 30–40 DAF. Quite different sets of genes were represented in these two libraries. Approximately 27% of the clones were not similar to known sequences, suggesting that these ESTs represent novel genes or may represent non-coding RNA. The high flux of energy into carbohydrate and storage protein synthesis in guar seeds was reflected by a high representation of genes annotated as involved in signal transduction, carbohydrate metabolism, chaperone and proteolytic processes, and translation and ribosome structure. Guar unigenes involved in galactomannan metabolism were identified. Among the seed storage proteins, the most abundant contig represented a conglutin accounting for 3.7% of the total ESTs from both libraries. Conclusion The present EST collection and its annotation provide a resource for understanding guar seed biology and galactomannan metabolism.

  1. Blind bedside insertion of small bowel feeding tubes.

    LENUS (Irish Health Repository)

    Duggan, SN

    2009-12-01

    The use of Naso-Jejunal (NJ) feeding is limited by difficulty in feeding tube placement. Patients have traditionally required transfer to Endoscopy or Radiology for insertion of small bowel feeding tubes, with clear resource implications. We hypothesised that the adoption of a simple bedside procedure would be effective and reduce cost. Clinical nutrition and nurse specialist personnel were trained in the 10\\/10\\/10 method of blind bedside NJ insertion.

  2. Lithium Insertion In Silicon Nanowires: An ab Initio Study

    KAUST Repository

    Zhang, Qianfan

    2010-09-08

    The ultrahigh specific lithium ion storage capacity of Si nanowires (SiNWs) has been demonstrated recently and has opened up exciting opportunities for energy storage. However, a systematic theoretical study on lithium insertion in SiNWs remains a challenge, and as a result, understanding of the fundamental interaction and microscopic dynamics during lithium insertion is still lacking. This paper focuses on the study of single Li atom insertion into SiNWs with different sizes and axis orientations by using full ab initio calculations. We show that the binding energy of interstitial Li increases as the SiNW diameter grows. The binding energies at different insertion sites, which can be classified as surface, intermediate, and core sites, are quite different. We find that surface sites are energetically the most favorable insertion positions and that intermediate sites are the most unfavorable insertion positions. Compared with the other growth directions, the [110] SiNWs with different diameters always present the highest binding energies on various insertion locations, which indicates that [110] SiNWs are more favorable by Li doping. Furthermore, we study Li diffusion inside SiNWs. The results show that the Li surface diffusion has a much higher chance to occur than the surface to core diffusion, which is consistent with the experimental observation that the Li insertion in SiNWs is layer by layer from surface to inner region. After overcoming a large barrier crossing surface-to-intermediate region, the diffusion toward center has a higher possibility to occur than the inverse process. © 2010 American Chemical Society.

  3. Cervical Lidocaine for IUD Insertional Pain: a Randomized Controlled Trial

    Science.gov (United States)

    McNicholas, Colleen P.; Madden, Tessa; Zhao, Qiuhong; Secura, Gina; Allsworth, Jenifer E.; Peipert, Jeffrey F.

    2012-01-01

    Objective Anticipated pain with intrauterine device (IUD) insertion may be a barrier to widespread use. Our objective was to evaluate the efficacy of intracervical 2% lidocaine gel for pain relief with IUD insertion. Study Design We performed a double-blind, randomized controlled trial of women undergoing IUD insertion. Participants were randomly assigned to 2% lidocaine or placebo gel. Study gel (3ccs) wase placed 3 minutes prior to IUD insertion. Pain scores were measured at various time points using a 10-point visual analog scale. Results Of the 200 participants randomized, 199 completed the study. Pain scores among lidocaine and placebo arms were similar at tenaculum placement (lidocaine and placebo; median 4, range 0–10 p=0.15) as well as with insertion (lidocaine: median 5 range 1–10, placebo: median 6 range 0–10 p=0.16). These results did not differ by parity. Conclusions Topical or intracervical 2% lidocaine gel prior to IUD insertion does not decrease pain scores. PMID:23107081

  4. A new specifically designed forceps for chest drain insertion.

    LENUS (Irish Health Repository)

    Andrews, Emmet

    2012-02-03

    Insertion of a chest drain can be associated with serious complications. It is recommended that the drain is inserted with blunt dissection through the chest wall but there is no specific instrument to aid this task. We describe a new reusable forceps that has been designed specifically to facilitate the insertion of chest drains.A feasibility study of its use in patients who required a chest drain as part of elective cardiothoracic operations was undertaken. The primary end-point was successful and accurate placement of the drain. The operators also completed a questionnaire rating defined aspects of the procedure. The new instrument was used to insert the chest drain in 30 patients (19 male, 11 female; median age 61.5 years (range 16-81 years)). The drain was inserted successfully without the trocar in all cases and there were no complications. Use of the instrument rated as significantly easier relative to experience of previous techniques in all specified aspects. The new device can be used to insert intercostal chest drains safely and efficiently without using the trocar or any other instrument.

  5. Descriptive anatomy of the insertion of the biceps femoris muscle.

    Science.gov (United States)

    Tubbs, R Shane; Caycedo, Francisco J; Oakes, W Jerry; Salter, E George

    2006-09-01

    The biceps femoris is the most lateral component of the so-called hamstring muscles. Classically, this muscle's insertion into the head of the fibula has been described but further details of its anatomy have not been universally appreciated. Additional insertions into the crural fascia and tibia have been described. We dissected 56 cadavers paying especially close attention to the insertion of the biceps femoris muscle. The tendon of this muscle was found to have both medial and lateral slips each with an anterior and posterior component. Further, we found an attachment not only into the lateral condyle of the femur but also the popliteus tendon and arcuate popliteal ligament. Our study has found that the tendon of insertion of the biceps femoris muscle is more complex than described previously and suggests that this tendon may be far more important in knee stability based on the multiple attachment sites found. We hypothesize that there may be a synergistic effect between the biceps femoris and the popliteus muscles based on our findings of an additional attachment of the biceps femoris tendon into the popliteus tendon. This study provides new detailed nomenclature for the description of the tendon of insertion of the biceps femoris muscle and indicates that the current description of the insertion of the tendon of the biceps femoris muscle should be revised. The clinician must have a thorough understanding of this anatomy before correct therapeutic maneuvers can be implemented.

  6. Postplacental intrauterine device insertion at a teaching hospital.

    Science.gov (United States)

    Jatlaoui, Tara C; Marcus, Michele; Jamieson, Denise J; Goedken, Peggy; Cwiak, Carrie

    2014-06-01

    To determine whether postplacental intrauterine device (IUD) insertion can be safely and effectively performed within a teaching program. This was a prospective cohort of 177 subjects planning vaginal delivery enrolled antenatally who desired postplacental IUD insertion of either the copper T380A IUD or levonorgestrel IUS. Insertions were performed primarily by resident physicians following a training session. Follow-up included a 4- to 8-week visit and telephone calls at 3 and 6 months. Ninety-nine subjects underwent successful postplacental IUD insertion of 100 attempts. Seventeen expulsions (17%) were noted: 10 complete and 7 partial. The study identified no differences in outcome by training level; however, the study lacked statistical power to evaluate anything other than large differences. Postplacental IUD insertions can be safely and effectively performed within a training program. A training protocol may safely and feasibly be initiated among physicians, advanced practice clinicians or trainees with no prior experience with postplacental IUD insertion. By initiating this practice, access to highly effective contraception may increase for patients who have difficulty returning for a visit or otherwise receiving effective methods. © 2014.

  7. Templated sequence insertion polymorphisms in the human genome

    Science.gov (United States)

    Onozawa, Masahiro; Aplan, Peter

    2016-11-01

    Templated Sequence Insertion Polymorphism (TSIP) is a recently described form of polymorphism recognized in the human genome, in which a sequence that is templated from a distant genomic region is inserted into the genome, seemingly at random. TSIPs can be grouped into two classes based on nucleotide sequence features at the insertion junctions; Class 1 TSIPs show features of insertions that are mediated via the LINE-1 ORF2 protein, including 1) target-site duplication (TSD), 2) polyadenylation 10-30 nucleotides downstream of a “cryptic” polyadenylation signal, and 3) preference for insertion at a 5’-TTTT/A-3’ sequence. In contrast, class 2 TSIPs show features consistent with repair of a DNA double-strand break via insertion of a DNA “patch” that is derived from a distant genomic region. Survey of a large number of normal human volunteers demonstrates that most individuals have 25-30 TSIPs, and that these TSIPs track with specific geographic regions. Similar to other forms of human polymorphism, we suspect that these TSIPs may be important for the generation of human diversity and genetic diseases.

  8. Construcción de una genoteca de cDNA de fríjol (Phaseolus vulgaris L. para mapeo genético

    Directory of Open Access Journals (Sweden)

    Roca William M.

    1995-06-01

    Full Text Available

    A small cDNA library from beans (Phaseolus vulgaris L. of about 1500 clones was constructed, to further saturate the bean RFLP linkage map. The primarily synthesized cDNAs were amplified by PCR using the adaptors as primers for amplification (Jepson et al., 1991. Inserts in the range of 500 bp were obtained. 93 clones were singled for further analysis. They showed a degree of repeatibility of around 61 %. Around 80% of the unique clones were single copy, and 20% were low copy sequences as expected from a cDNA library. Three pairs of parental bean lines were chosen for their agronomical traits, and evaluated for polymorphism, which was highest as revealed by digestion with EcoRV (77%, followed by DraI (73%, EcoRI (63% and HindIIl (60%. The highest polymorphism was observed between the pair DOR60 and APNI8, 71% for EcoRV and 57% for EcoRI, respectively. Two clones of the two groups with the most repeated clones were analyzed by slot blot hybridization against the other clones and ribosomal DNA, to understand the origin of the repetitions. Only one clone seemed to be of ribosomal origin, as confirmed by the patterns obtained by hybridization to bean genomic DNA digested with HaelIl, implying that the whole group to which it belonged was of ribosomal origin.  It can be explained by the combined utilization of the PCR amplification methodology and the multipleprimers for the synthesis of the first cDNA strand.

    Se construyó una pequeña librería de cDNA de fríjol (phaseolus vulgaris L. de alrededor de 1500 clones, con el fin de incrementar la saturación del mapa de ligamiento de fríjol con marcadores de RFLPs. Para la generación de la librería se utilizó la técnica de amplificación por PCR (Jepson et al., 1991. En ella se utilizan como iniciadores para la reacción los mismos adaptadores empleados para generar los terminales cohesivos del cDNA. Se obtuvieron insertos con un promedio de 500 pares de bases. Se aislaron 93 clones, los

  9. Generation and analysis of a large-scale expressed sequence tags from a full-length enriched cDNA library of Siberian tiger (Panthera tigris altaica).

    Science.gov (United States)

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

    2014-05-15

    In this study, a full-length enriched cDNA library was successfully constructed from Siberian tiger, the world's most endangered species. The titers of primary and amplified libraries were 1.28×10(6)pfu/mL and 1.59×10(10)pfu/mL respectively. The proportion of recombinants from unamplified library was 91.3% and the average length of exogenous inserts was 1.06kb. A total of 279 individual ESTs with sizes ranging from 316 to 1258bps were then analyzed. Furthermore, 204 unigenes were successfully annotated and involved in 49 functions of the GO classification, cell (175, 85.5%), cellular process (165, 80.9%), and binding (152, 74.5%) are the dominant terms. 198 unigenes were assigned to 156 KEGG pathways, and the pathways with the most representation are metabolic pathways (18, 9.1%). The proportion pattern of each COG subcategory was similar among Panthera tigris altaica, P. tigris tigris and Homo sapiens, and general function prediction only cluster (44, 15.8%) represents the largest group, followed by translation, ribosomal structure and biogenesis (33, 11.8%), replication, recombination and repair (24, 8.6%), and only 7.2% ESTs classified as novel genes. Moreover, the recombinant plasmid pET32a-TAT-COL6A2 was constructed, coded for the Trx-TAT-COL6A2 fusion protein with two 6× His-tags in N and C-terminal. After BCA assay, the concentration of soluble Trx-TAT-COL6A2 recombinant protein was 2.64±0.18mg/mL. This library will provide a useful platform for the functional genome and transcriptome research of for the P. tigris and other felid animals in the future. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Insertion torque values and success rates for paramedian insertion of orthodontic mini-implants : A retrospective study.

    Science.gov (United States)

    Di Leonardo, Bruno; Ludwig, Björn; Lisson, Jörg Alexander; Contardo, Luca; Mura, Rossano; Hourfar, Jan

    2018-03-01

    Orthodontic mini-implants (OMIs) are a reliable method to provide temporary orthodontic anchorage. We hypothesized that there is an optimal insertion torque (10 Ncm) will decrease the success rate and increase palatal OMI failure was rejected.

  11. CRISPR/Cas9 cleavages in budding yeast reveal templated insertions and strand-specific insertion/deletion profiles.

    Science.gov (United States)

    Lemos, Brenda R; Kaplan, Adam C; Bae, Ji Eun; Ferrazzoli, Alexander E; Kuo, James; Anand, Ranjith P; Waterman, David P; Haber, James E

    2018-02-13

    Harnessing CRISPR-Cas9 technology provides an unprecedented ability to modify genomic loci via DNA double-strand break (DSB) induction and repair. We analyzed nonhomologous end-joining (NHEJ) repair induced by Cas9 in budding yeast and found that the orientation of binding of Cas9 and its guide RNA (gRNA) profoundly influences the pattern of insertion/deletions (indels) at the site of cleavage. A common indel created by Cas9 is a 1-bp (+1) insertion that appears to result from Cas9 creating a 1-nt 5' overhang that is filled in by a DNA polymerase and ligated. The origin of +1 insertions was investigated by using two gRNAs with PAM sequences located on opposite DNA strands but designed to cleave the same sequence. These templated +1 insertions are dependent on the X-family DNA polymerase, Pol4. Deleting Pol4 also eliminated +2 and +3 insertions, which are biased toward homonucleotide insertions. Using inverted PAM sequences, we also found significant differences in overall NHEJ efficiency and repair profiles, suggesting that the binding of the Cas9:gRNA complex influences subsequent NHEJ processing. As with events induced by the site-specific HO endonuclease, CRISPR-Cas9-mediated NHEJ repair depends on the Ku heterodimer and DNA ligase 4. Cas9 events are highly dependent on the Mre11-Rad50-Xrs2 complex, independent of Mre11's nuclease activity. Inspection of the outcomes of a large number of Cas9 cleavage events in mammalian cells reveals a similar templated origin of +1 insertions in human cells, but also a significant frequency of similarly templated +2 insertions.

  12. [Ultrasound-guided peripherally inserted central catheters (PICC) in cancer patients: success of the insertion, survival and complications].

    Science.gov (United States)

    Moraza-Dulanto, Maria Inmaculada; Garate-Echenique, Lucía; Miranda-Serrano, Erika; Armenteros-Yeguas, Victoria; Tomás-López, María Aranzazu; Benítez-Delgado, Beatriz

    2012-01-01

    To evaluate the results of peripherally inserted central catheters (PICC) inserted by nurses using an ultrasound-guided technique at bed-side. An observational and prospective study was conducted on all the PICC inserted at bed-side by an ultrasound-guided technique at the Araba University Hospital. The technique was introduced in June 2010, and the data collection period ended in November 2011. The main study variables were successful insertion, duration of PICC, incidences related to the catheter, devices reaching end of treatment and reasons for withdrawal. Patient sociodemographic data and PICC technical features were also registered. A total of 165 PICC were inserted, 73 are still in use, with 95.2% inserted in patients from oncology or haematology departments. Insertion was successful in the 89.7% (95% CI: 85.1%-94.3%) of the cases. The study included 16,234 catheter days, with a median dwell time of 92 days by PICC. The most frequent incidence was accidental removal in 0.986 per 1000 catheter days (95% CI=0.970-1.001). The thrombosis rate was 0.308 per 1,000 days (95% CI= 0.299-0.317), and the catheter-associated bloodstream infection rate was 0.062 per 1,000 catheter days (95% CI=0.058-0.065). Ultrasound-guided PICC insertion can be performed at bedside by trained nurses with a high probability of success. PICC, because of its low complication rate and long indwelling catheter survival, is a suitable central venous device for long-term treatment in oncology and haematology patients. Copyright © 2011 Elsevier España, S.L. All rights reserved.

  13. Characterisation of immune responses and protective efficacy in mice after immunisation with Rift Valley Fever virus cDNA constructs

    Directory of Open Access Journals (Sweden)

    Lundkvist Åke

    2009-01-01

    Full Text Available Abstract Background Affecting both livestock and humans, Rift Valley Fever is considered as one of the most important viral zoonoses in Africa. However, no licensed vaccines or effective treatments are yet available for human use. Naked DNA vaccines are an interesting approach since the virus is highly infectious and existing attenuated Rift Valley Fever virus vaccine strains display adverse effects in animal trials. In this study, gene-gun immunisations with cDNA encoding structural proteins of the Rift Valley Fever virus were evaluated in mice. The induced immune responses were analysed for the ability to protect mice against virus challenge. Results Immunisation with cDNA encoding the nucleocapsid protein induced strong humoral and lymphocyte proliferative immune responses, and virus neutralising antibodies were acquired after vaccination with cDNA encoding the glycoproteins. Even though complete protection was not achieved by genetic immunisation, four out of eight, and five out of eight mice vaccinated with cDNA encoding the nucleocapsid protein or the glycoproteins, respectively, displayed no clinical signs of infection after challenge. In contrast, all fourteen control animals displayed clinical manifestations of Rift Valley Fever after challenge. Conclusion The appearance of Rift Valley Fever associated clinical signs were significantly decreased among the DNA vaccinated mice and further adjustment of this strategy may result in full protection against Rift Valley Fever.

  14. A cDNA Clone-Launched Platform for High-Yield Production of Inactivated Zika Vaccine

    Directory of Open Access Journals (Sweden)

    Yujiao Yang

    2017-03-01

    Full Text Available A purified inactivated vaccine (PIV using the Zika virus (ZIKV Puerto Rico strain PRVABC59 showed efficacy in monkeys, and is currently in a phase I clinical trial. High-yield manufacture of this PIV is essential for its development and vaccine access. Here we report an infectious cDNA clone-launched platform to maximize its yield. A single NS1 protein substitution (K265E was identified to increase ZIKV replication on Vero cells (a cell line approved for vaccine production for both Cambodian FSS13025 and Puerto Rico PRVABC59 strains. The NS1 mutation did not affect viral RNA synthesis, but significantly increased virion assembly through an increased interaction between NS1 and NS2A (a known regulator of flavivirus assembly. The NS1 mutant virus retained wild-type virulence in the A129 mouse model, but decreased its competence to infect Aedes aegypti mosquitoes. To further increase virus yield, we constructed an infectious cDNA clone of the clinical trial PIV strain PRVABC59 containing three viral replication-enhancing mutations (NS1 K265E, prM H83R, and NS3 S356F. The mutant cDNA clone produced >25-fold more ZIKV than the wild-type parent on Vero cells. This cDNA clone-launched manufacture platform has the advantages of higher virus yield, shortened manufacture time, and minimized chance of contamination.

  15. Heterologous expression of laccase cDNA from Ceriporiopsis subvermispora yields copper-activated apoprotein and complex isoform patterns

    Science.gov (United States)

    Luis F. Larrondo; Marcela Avila; Loreto Salas; Dan Cullen; Rafael Vicuna

    2003-01-01

    Analysis of genomic clones encoding a putative laccase in homokaryon strains of Ceriporiopsis subvermispora led to the identification of an allelic variant of the previously described lcs-1 gene. A cDNA clone corresponding to this gene was expressed in Aspergillus nidulans and in Aspergillus niger. Enzyme assays and Western blots showed that both hosts secreted active...

  16. Monoterpene biosynthesis in lemon (Citrus limon) cDNA isolation and functional analysis of four monoterpene synthases

    NARCIS (Netherlands)

    Lücker, J.; Tamer, El M.K.; Schwab, W.; Verstappen, F.W.A.; Plas, van der L.H.W.; Bouwmeester, H.J.; Verhoeven, H.A.

    2002-01-01

    Citrus limon possesses a high content and large variety of monoterpenoids, especially in the glands of the fruit flavedo. The genes responsible for the production of these monoterpenes have never been isolated. By applying a random sequencing approach to a cDNA library from mRNA isolated from the

  17. Development of a porcine skeletal muscle cDNA microarray: analysis of differential transcript expression in phenotypically distinct muscles

    Directory of Open Access Journals (Sweden)

    Stear Michael

    2003-03-01

    Full Text Available Abstract Background Microarray profiling has the potential to illuminate the molecular processes that govern the phenotypic characteristics of porcine skeletal muscles, such as hypertrophy or atrophy, and the expression of specific fibre types. This information is not only important for understanding basic muscle biology but also provides underpinning knowledge for enhancing the efficiency of livestock production. Results We report on the de novo development of a composite skeletal muscle cDNA microarray, comprising 5500 clones from two developmentally distinct cDNA libraries (longissimus dorsi of a 50-day porcine foetus and the gastrocnemius of a 3-day-old pig. Clones selected for the microarray assembly were of low to moderate abundance, as indicated by colony hybridisation. We profiled the differential expression of genes between the psoas (red muscle and the longissimus dorsi (white muscle, by co-hybridisation of Cy3 and Cy5 labelled cDNA derived from these two muscles. Results from seven microarray slides (replicates correctly identified genes that were expected to be differentially expressed, as well as a number of novel candidate regulatory genes. Quantitative real-time RT-PCR on selected genes was used to confirm the results from the microarray. Conclusion We have developed a porcine skeletal muscle cDNA microarray and have identified a number of candidate genes that could be involved in muscle phenotype determination, including several members of the casein kinase 2 signalling pathway.

  18. Determination of cDNA and genomic DNA sequences of hevamine, a chitinase from the rubber tree Hevea brasiliensis

    NARCIS (Netherlands)

    Bokma, E; Spiering, M; Chow, KS; Mulder, PPMFA; Subroto, T; Beintema, JJ

    Hevamine is a chitinase from the rubber tree Hevea brasiliensis and belongs to the family 18 glycosyl hydrolases. This paper describes the cloning of hevamine DNA and cDNA sequences. Hevamine contains a signal peptide at the N-terminus and a putative vacuolar targeting sequence at the C-terminus

  19. Construction and characterization of a cDNA library from wheat infected with Fusarium graminearum Fg 2.

    Science.gov (United States)

    Al-Taweel, Khaled; Dilantha Fernando, W G; Brûlé-Babel, Anita L

    2011-01-18

    Total RNA from wheat spikes infected with F. graminearum Fg2 was extracted and the mRNA was purified. Switching Mechanism at 5' end of the RNA Transcript (SMART) technique and CDS Ill/3' primer were used for first-strand cDNA synthesis using reverse transcriptase by RT-PCR. Primer extension polymerase chain reaction was used to construct the double-strand cDNA that was digested by proteinase K, then by Sfi I and fractionated. cDNAs longer than 0.5 kb were collected and ligated to λTriplEx2 vector followed λ phage packaging reaction and library amplification. The qualities of both unamplified and amplified cDNA libraries were strictly checked by conventional titer determination. One hundred and sixty five plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. A high quality cDNA library from wheat spikes that have been infected by F. graminearum was successfully constructed.

  20. Construction and Characterization of a cDNA Library from Wheat Infected with Fusarium graminearum Fg 2

    Directory of Open Access Journals (Sweden)

    Anita L. Brûlé-Babel

    2011-01-01

    Full Text Available Total RNA from wheat spikes infected with F. graminearum Fg2 was extracted and the mRNA was purified. Switching Mechanism at 5' end of the RNA Transcript (SMART technique and CDS Ill/3' primer were used for first-strand cDNA synthesis using reverse transcriptase by RT-PCR. Primer extension polymerase chain reaction was used to construct the double-strand cDNA that was digested by proteinase K, then by Sfi I and fractionated. cDNAs longer than 0.5 kb were collected and ligated to λTriplEx2 vector followed λ phage packaging reaction and library amplification. The qualities of both unamplified and amplified cDNA libraries were strictly checked by conventional titer determination. One hundred and sixty five plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. A high quality cDNA library from wheat spikes that have been infected by F. graminearum was successfully constructed.